The Immune Response to Infection
This page intentionally left blank
The Immune Response to Infection edited
by
Stefan H. E.Kaufmann
Max Planck Institute for Infection Biology Berlin, Germany
Barry T. Rouse
College of Veterinary Medicine, University of Tennessee Knoxville, Tennessee
David L. Sacks
Laboratory of Parasitic Diseases, National Institutes of Health Bethesda, Maryland
W A S H I N G T O N ,
D C
Front cover: Macrophage in the process of engulfing Mycobacterium tuberculosis organisms. Back cover: Left (red): Helicobacter pylori; right (blue): Neisseria gonorrhoeae attached to epithelial cells. All three photos courtesy of Volker Brinkmann and Stefan H.E. Kaufmann (Max Planck Institute for Infection Biology, Berlin, Germany).
Copyright © 2011
ASM Press American Society for Microbiology 1752 N Street, N.W. Washington, DC 20036-2904
Library of Congress Cataloging-in-Publication Data The immune response to infection / edited by Stefan H.E. Kaufmann, Barry T. Rouse, David L. Sacks. p. ; cm. Includes bibliographical references and index. ISBN 978-1-55581-514-1 (alk. paper) 1. Immune response. I. Kaufmann, S. H. E. (Stefan H. E.) II. Rouse, Barry T. III. Sacks, David Lawrence. [DNLM: 1. Adaptive Immunity—immunology. 2. Immunity, Innate—immunology. 3. Host-Pathogen Interactions—immunology. 4. Infection—immunology. QW 541 I326 2011] QR186.I445 2011 616.07'9—dc22 2010019964 All Rights Reserved Printed in the United States of America 10
9
8
7
6
5
4
3
2
1
Address editorial correspondence to: ASM Press, 1752 N St., N.W., Washington, DC 20036-2904, U.S.A. Send orders to: ASM Press, P.O. Box 605, Herndon, VA 20172, U.S.A. Phone: 800-546-2416; 703-661-1593 Fax: 703-661-1501 Email:
[email protected] Online: estore.asm.org
Contents
8 Regulation of Antimicrobial Immunity / 109
Contributors / ix Preface / xv
yASMINE BELKAID, SHARVAN SEHRAWAT, AND BARRy T. ROUSE
The Immune Response to Infection: Introduction / 1
STEFAN H. E. KAUFMANN, BARRy ROUSE, AND DAVID SACKS
9
SECTIon I
SECTIon II
HoST DEFEnSE: GEnERAL / 5 1
Memory and Infection / 121
DAVID MASOPUST AND MARK K. SLIFKA
THE PATHoGEnS / 131
Invertebrate Innate Immune Defenses / 7
LAURE EL CHAMy, CHARLES HETRU, AND JULES HOFFMANN
10
overview of Viral Pathogens / 133
11
overview of Parasitic Pathogens / 143
12
overview of Bacterial Pathogens / 155
13
overview of Fungal Pathogens / 165
14
Prionoses and the Immune System / 173
JONATHAN W. yEWDELL AND JACK R. BENNINK
2 The ontogeny of the Cells of the Innate and the Adaptive Immune System / 21
RICK L. TARLETON AND EDWARD J. PEARCE
FRITz MELCHERS
3 The Evolutionary origins of the Adaptive Immune System of Jawed Vertebrates / 41
PHILIPPE J. SANSONETTI AND ANDREA PUHAR
JIM KAUFMAN
AxEL A. BRAKHAGE AND PETER F. zIPFEL
4 Host Defense (Antimicrobial) Peptides and Proteins / 57 LAURENCE MADERA, SHUHUA MA, AND ROBERT E. W. HANCOCK
JüRGEN A. RICHT AND ALAN yOUNG
5 Reactive oxygen and Reactive nitrogen Intermediates in the Immune System / 69
SECTIon III
InnATE IMMUnITY To MICRoBIAL InFECTIonS / 183
CHRISTIAN BOGDAN
6
Complement in Infections / 85
15
WILHELM J. SCHWAEBLE, yOUSSIF MOHAMMED ALI, NICHOLAS J. LyNCH, AND RUSSELL WALLIS
7
Innate Immunity to Viruses / 185
AKIKO IWASAKI
16 natural Killer Cell Response against Viruses / 197
Immune Defense at Mucosal Surfaces / 97
MARIAN R. NEUTRA AND JEAN-PIERRE KRAEHENBUHL
JOSEPH C. SUN AND LEWIS L. LANIER v
vi
17
Contents
Innate Immunity against Bacteria / 209
THOMAS ARESCHOUG, ANNETTE PLüDDEMANN, AND SIAMON GORDON
18 Innate Immunity to Parasitic Infections / 225
CHRISTOPHER A. HUNTER AND ALAN SHER
SECTIon IV
ACQUIRED IMMUnITY To MICRoBIAL InFECTIonS / 237 19 Acquired Immunity against Virus Infections / 239
EVA SzOMOLANyI-TSUDA, MICHAEL A. BREHM, AND RAyMOND M. WELSH
20 Immune Responses to Persistent Viruses / 255
E. JOHN WHERRy AND PAUL KLENERMAN
21 Acquired Immunity: Acute Bacterial Infections / 269 DENNIS W. METzGER
22 Acquired Immunity: Chronic Bacterial Infections / 279
ANDREA M. COOPER AND RICHARD ROBINSON
23 Acquired Immunity: Fungal Infections / 289 LUIGINA ROMANI
24 Acquired Immunity to Intracellular Protozoa / 301 PHILLIP SCOTT AND ELEANOR M. RILEy
25 Acquired Immunity to Helminths / 313
29 Pathology and Pathogenesis of Malaria / 361
CHANAKI AMARATUNGA, TATIANA M. LOPERA-MESA, JEANETTE G. TSE, NEIDA K. MITA-MENDOzA, AND RICK M. FAIRHURST
30 Pathology and Pathogenesis of Virus Infections / 383 CARMEN BACA JONES AND MATTHIAS VON HERRATH
SECTIon VI
EVASIon AnD SUPPRESSIon oF THE AnTIMICRoBIAL HoST RESPonSE / 391 31 Viral Immune Evasion / 393
LILA FARRINGTON, GABRIELA O’NEILL, AND ANN B. HILL
32 Growing old and Immunity to Viruses / 403 JANKO NIKOLICH-ŽUGICH AND MARCIA A. BLACKMAN
33 Growing old and Immunity to Bacteria / 413 JOANNE TURNER
34 Bacterial Strategies for Survival in the Host / 425
ANNA D. TISCHLER AND JOHN D. McKINNEy
35 Suppression of Immune Responses to Protozoan Parasites / 441 DAVID L. SACKS
36
Immune Evasion by Parasites / 453
JOHN M. MANSFIELD AND MARTIN OLIVIER
DAVID ARTIS AND RICK M. MAIzELS
SECTIon VII SECTIon V
PATHoLoGY AnD PATHoGEnESIS / 325
GEnETICS oF THE AnTIMICRoBIAL HoST RESPonSE / 471
26 Pathology and Pathogenesis of Bacterial Infections / 327
37 Genetics of Antibacterial Host Defenses / 473
WARWICK J. BRITTON AND BERNADETTE M. SAUNDERS
27 Helicobacter pylori: the Role of the Immune Response in Pathogenesis / 337
STEVEN M. HOLLAND
38 Immunogenetics of Host Response to Parasites in Humans / 483 JENEFER M. BLACKWELL
KAREN ROBINSON AND JOHN C. ATHERTON
28 Pathogenesis of Helminth Infections / 347
THOMAS A. WyNN AND JUDITH E. ALLEN
39 Immunogenetics of Virus Pathogenesis / 491
SEAN WILTSHIRE, DAVID I. WATKINS, EMIL SKAMENE, AND SILVIA M. VIDAL
Contents
SECTIon VIII
AUToIMMUnITY AnD CAnCER / 509 40 Viruses, Autoimmunity, and Cancer / 511 MEGHANN TEAGUE GETTS, LIES BOGAERT, W. MARTIN KAST, AND STEPHEN D. MILLER
41 The Role of Bacterial and Parasitic Infections in Chronic Inflammatory Disorders and Autoimmunity / 521 STEFAN EHLERS AND GRAHAM A. W. ROOK
42 Theileria-Induced Leukocyte Transformation: an Example of oncogene Addiction? / 537 MARIE CHAUSSEPIED AND GORDON LANGSLEy
SECTIon IX
IMMUnE InTERVEnTIon / 547 43 Systems Vaccinology: Using Functional Signatures To Design Successful Vaccines / 549 TROy D. QUEREC AND BALI PULENDRAN
44 Meeting the Challenge of Vaccine Design To Control HIV and other Difficult Viruses / 559 BARNEy S. GRAHAM AND CHRISTOPHER WALKER
vii
45 Immune Intervention Strategies against Tuberculosis / 571
PETER ANDERSEN AND STEFAN H. E. KAUFMANN
46
Immune Intervention in Malaria / 587
CAROLE A. LONG AND FIDEL P. zAVALA
47 Targeting Components in Vector Saliva / 599
MARy ANN McDOWELL AND SHADEN KAMHAWI
SECTIon X
THE MAJoR KILLERS (CLInICS, EPIDEMIoLoGY, AnD IMMUnE PARAMETERS) / 609 48 AIDS Vaccines: the Unfolding Story / 611 STEPHEN NORLEy
49 Tuberculosis / 623
GERHARD WALzL, PAUL VAN HELDEN, AND PHILIP R. BOTHA
50 Malaria: Clinical and Epidemiological Aspects / 633 ANDREA A. BERRy, MyAING M. NyUNT, AND CHRISTOPHER V. PLOWE
51 The Epidemiology and Immunology of Influenza Viruses / 643 RAFAEL A. MEDINA, IRENE RAMOS, AND ANA FERNANDEz-SESMA
Index / 653
This page intentionally left blank
Contributors
yOUSSIF MOHAMMED ALI
ANDREA A. BERRy
Department of Infection, Immunity and Inflammation, University of Leicester, MSB, University Road, Leicester LE1 9HN, United Kingdom
Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201
MARCIA A. BLACKMAN
The Trudeau Institute, Saranac Lake, Ny 12983
JUDITH E. ALLEN
Institutes of Evolution, Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3JT, UK
JENEFER M. BLACKWELL
Telethon Institute for Child Health Research, Centre for Child Health Research, University of Western Australia, Subiaco, Western Australia, Australia
CHANAKI AMARATUNGA
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852
LIES BOGAERT
Departments of Molecular Microbiology & Immunology and Obstetrics & Gynecology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033. Department of Surgery and Anesthesiology of Domestic Animals, Faculty of Veterinary Medicine, University of Ghent, Merelbeke, B-9820, Belgium
PETER ANDERSEN
Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark
THOMAS ARESCHOUG
Department of Laboratory Medicine, Division of Medical Microbiology, Lund University, Sölvegatan 23, 22362 Lund, Sweden
CHRISTIAN BOGDAN
Microbiology Institute Clinical Microbiology, Immunology and Hygiene, Friedrich Alexander University Erlangen, Nuremberg, and University Clinic of Erlangen, Wasserturmstraße 3/5, D-91054 Erlangen, Germany
DAVID ARTIS
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104-4539
PHILIP R. BOTHA
Division of Infectious Diseases, Department of Medicine Tygerberg Academic Hospital, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa
JOHN C. ATHERTON
Nottingham Digestive Diseases Centre Biomedical Research Unit, University Hospital, Nottingham, NG7 2UH, United Kingdom
AxEL A. BRAKHAGE
CARMEN BACA JONES
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute (HKI), Friedrich Schiller University Jena, Department Molecular and Applied Microbiology and Department of Infection Biology, Beutenbergstrasse 11a, 07745 Jena, Germany
yASMINE BELKAID
MICHAEL A. BREHM
Center for Type 1 Diabetes Research, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037 Mucosal Immunology Unit, Laboratory of Parasitic Diseases, Division of Intramural Research, National Institute of Health, 4 Center Drive B1-28, Bethesda, MD 20892
Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655
WARWICK J. BRITTON
JACK R. BENNINK
Centenary Institute, Locked Bag No 6, Newtown, 2042; and Discipline of Medicine, Sydney Medical School, University of Sydney (D06), Sydney, 2006, NSW, Australia
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-3209
ix
x
contributors
MARIE CHAUSSEPIED
Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France. Inserm, 1016, Paris, France. Laboratory of Comparative Cell Biology of Apicomplexa, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France
ANDREA M. COOPER
Trudeau Institute, Inc., 154 Algonquin Ave., Saranac Lake, Ny
STEFAN EHLERS
Microbial Inflammation Research, Research Center Borstel, Parkallee 1, D-23845 Borstel, Germany, and Molecular Inflammation Medicine, Institute of Experimental Medicine, Christian-Albrechts-University, Arnold-Heller-Str. 3, D-24105 Kiel, Germany
LAURE EL CHAMy
Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, France
RICK M. FAIRHURST
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852
LILA FARRINGTON
Dept. of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239
ANA FERNANDEz-SESMA
Department of Microbiology and the Emerging Pathogens Institute, Mount Sinai School of Medicine, New york, Ny 10029
MEGHANN TEAGUE GETTS
Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611
SIAMON GORDON
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, Ox1 3RE, United Kingdom
BARNEy S. GRAHAM
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
ROBERT E. W. HANCOCK
Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1z4, Canada
CHARLES HETRU
Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, France
ANN B. HILL
Dept. of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239
JULES HOFFMANN
CHRISTOPHER A. HUNTER
Department of Pathobiology School of Veterinary Medicine, University of Pennsylvania, Rm 313, Hill Pavilion, 380 South University Avenue, Philadelphia, PA 19104-4539
AKIKO IWASAKI
Department of Immunobiology, yale University School of Medicine, New Haven, CT 06520
SHADEN KAMHAWI
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Disease, National Institutes of Health, 12735 Twinbrook Parkway, Rockville, MD 20852
W. MARTIN KAST
Departments of Molecular Microbiology & Immunology and Obstetrics & Gynecology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033. Cancer Research Center of Hawaii, University of Hawaii, Honolulu, HI 96822
JIM KAUFMAN
University of Cambridge Department of Pathology, Tennis Court Road, Cambridge, CB2 1QP, Department of Veterinary Medicine, Madingley Road, Cambridge, CB3 0ES, United Kingdom
STEFAN H. E. KAUFMANN
Department of Immunology, Max Planck Institute for Infection Biology, Chapritéplatz 1, D 10117 Berlin, Germany
PAUL KLENERMAN
Nuffield Dept of Medicine and NIHR Biomedical Research Centre Programme, Peter Medawar Building, University of Oxford, Oxford Ox1 3Sy, UK
JEAN-PIERRE KRAEHENBUHL
Health Sciences eTraining (HSeT) Foundation, CH 1066 Epalinges, Switzerland
GORDON LANGSLEy
Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France. Inserm, 1016, Paris, France. Laboratory of Comparative Cell Biology of Apicomplexa, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France
LEWIS L. LANIER
Department of Microbiology and Immunology and the Cancer Research Institute, University of California, San Francisco, CA 94143
CAROLE A. LONG
Malaria Immunology Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852
TATIANA M. LOPERA-MESA
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852
NICHOLAS J. LyNCH
Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, France
Department of Infection, Immunity and Inflammation, University of Leicester, MSB, University Road, Leicester LE1 9HN, United Kingdom
STEVEN M. HOLLAND
SHUHUA MA
Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1684
Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1z4, Canada
contributors
LAURENCE MADERA
MARTIN OLIVIER
RICK M. MAIzELS
GABRIELA O’NEILL
Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1z4, Canada Centre for Immunity, Infection and Evolution, and Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh EH9 3JT, United Kingdom
JOHN M. MANSFIELD
Department of Microbiology and Immunology, Duff Medical Building, 3775 University, McGill University, Montréal, Québec, Canada. Dept. of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239
EDWARD J. PEARCE
Department of Bacteriology, Microbial Sciences Building, 1550 Linden Drive, University of Wisconsin-Madison, Madison, WI 53716
Trudeau Institute Inc., 154 Algonquin Avenue, Saranac Lake, Ny 12983
DAVID MASOPUST
Center for Vaccine Development, Howard Hughes Medical Institute and University of Maryland School of Medicine, Baltimore, MD 21201
Department of Microbiology, Center for Immunology, University of Minnesota, 2-182 Medical Biosciences Building, 2101 6th St. SE, Minneapolis, MN 55455
MARy ANN McDOWELL
The Eck Institute for Global Health and Infectious Diseases, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556
JOHN D. McKINNEy
Global Heath Institute, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland
RAFAEL A. MEDINA
Department of Microbiology and the Emerging Pathogens Institute, Mount Sinai School of Medicine, New york, Ny 10029
FRITz MELCHERS
Max Planck Institute for Infection Biology, Senior Research Group on Lymphocyte Development, Charitéplatz 1, D 10117, Berlin, Germany
DENNIS W. METzGER
Center for Immunology and Microbial Disease, Albany Medical College, Albany, New york 12208
STEPHEN D. MILLER
Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611
NEIDA K. MITA-MENDOzA
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852
MARIAN R. NEUTRA
Department of Pediatrics, Harvard Medical School, and GI Cell Biology Laboratory, Children’s Hospital, Boston, MA 02115
JANKO NIKOLICH-ŽUGICH
Department of Immunobiology and the Arizona Center on Aging, University of Arizona College of Medicine, Tucson, Az 85718
STEPHEN NORLEy
Robert Koch Institute, 13353 Berlin, Germany
MyAING M. NyUNT
Department of International Health, Global Disease Epidemiology and Control Program, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205
xi
CHRISTOPHER V. PLOWE
ANNETTE PLüDDEMANN
Department of Primary Health Care, University of Oxford Old Road Campus, Oxford, Ox3 7LF, UK
ANDREA PUHAR
Unité de Pathogénie Microbienne Moléculaire, INSERM U786, Institut Pasteur, 75724 Paris Cedex 15, France
BALI PULENDRAN
Emory Vaccine Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329
TROy D. QUEREC
Emory Vaccine Center, Emory University, 954 Gatewood Road, Atlanta, GA 30329
IRENE RAMOS
Department of Microbiology and the Emerging Pathogens Institute, Mount Sinai School of Medicine, New york, Ny 10029
JüRGEN A. RICHT
Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, K224B Mosier Hall, Manhattan, KS 66506-5601
ELEANOR M. RILEy
Department of Infectious and Tropical Diseases, London School of Tropical Medicine and Hygiene, London, UK
RICHARD ROBINSON
Trudeau Institute, Inc., 154 Algonquin Ave., Saranac Lake, Ny
KAREN ROBINSON
Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, United Kingdom
LUIGINA ROMANI
Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy
GRAHAM A. W. ROOK
Centre for International and Medical Microbiology (CIMM), Windeyer Institute for Medical Sciences, University College London (UCL), 46 Cleveland Street, GB - London W1T 4JF, United Kingdom
xii
contributors
BARRy T. ROUSE
JEANETTE G. TSE
DAVID L. SACKS
JOANNE TURNER
Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Ave., Knoxville, TN 37996 Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 126, 4 Center Dr., MSC 0425, Bethesda, MD 20892-0425.
PHILIPPE J. SANSONETTI
Unité de Pathogénie Microbienne Moléculaire, INSERM U786, Institut Pasteur, 75724 Paris Cedex 15, France
BERNADETTE M. SAUNDERS
Centenary Institute, Locked Bag No 6, Newtown, 2042; and Discipline of Medicine, Sydney Medical School, University of Sydney (D06), Sydney, 2006, NSW, Australia
WILHELM J. SCHWAEBLE
Department of Infection, Immunity and Inflammation, University of Leicester, MSB, University Road, Leicester LE1 9HN, United Kingdom
PHILLIP SCOTT
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104
SHARVAN SEHRAWAT
Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Ave., Knoxville, TN 37996
ALAN SHER
Laboratory of Parasitic Diseases NIAID, NIH Building 50, Room 6140, 50 South Drive, MSC-8003, Bethesda, MD 20892-8003
EMIL SKAMENE
Department of Human Genetics, The McGill Life Sciences Complex, Bellini Pavilion, Room 356, 3649 Promenade Sir William Osler, Montreal, QC H3G 0B1, Canada
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852 Center for Microbial Interface Biology, and Department of Internal Medicine, Division of Infectious Diseases, The Ohio State University, 460 West 12th Ave., Columbus, OH 43210
PAUL VAN HELDEN
DST/NRF Centre of Excellence for Biomedical TB Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa
SILVIA VIDAL
Department of Human Genetics, The McGill Life Sciences Complex, Bellini Pavilion, Room 356, 3649 Promenade Sir William Osler, Montreal, QC H3G 0B1, Canada
MATTHIAS VON HERRATH
Center for Type 1 Diabetes Research, La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037
CHRISTOPHER WALKER
Nationwide Children’s Hospital and the Departments of Pediatrics, Pathology, and Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43205
RUSSELL WALLIS
Department of Infection, Immunity and Inflammation, University of Leicester, MSB, University Road, Leicester LE1 9HN, United Kingdom
GERHARD WALzL
MARK K. SLIFKA
Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 NW 185th Ave., Beaverton, OR 97006
DST/NRF Centre of Excellence for Biomedical TB Research, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, University of Stellenbosch, P.O. Box 19063, Tygerberg, 7505, South Africa
JOSEPH C. SUN
DAVID I. WATKINS
Department of Microbiology and Immunology and the Cancer Research Institute, University of California, San Francisco, CA 94143
Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, 6152 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706
EVA SzOMOLANyI-TSUDA
RAyMOND M. WELSH
Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655
Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01655
RICK L. TARLETON
E. JOHN WHERRy
Center for Tropical & Emerging Global Diseases, Coverdell Center for Biomedical Research (Rm 310B), 500 D.W. Brooks Drive, University of Georgia, Athens, GA 30602
Department of Microbiology and Institute for Immunology, University of Pennsylvania, School of Medicine, 421 Corie Blvd., Room 312, Philadelphia, PA 19104
ANNA D. TISCHLER
Department of Human Genetics, The McGill Life Sciences Complex, Bellini Pavilion, Room 356, 3649 Promenade Sir William Osler, Montreal, QC H3G 0B1, Canada
Global Heath Institute, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland
SEAN WILTSHIRE
contributors
THOMAS A. WyNN
Immunopathogenesis Section, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892-8003
JONATHAN W. yEWDELL
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-3209
ALAN yOUNG
Department of Veterinary Science, South Dakota State University, Box 2175, ARW168F, Brookings, SD 57007
xiii
FIDEL P. zAVALA
Department of Molecular Microbiology and Immunity, Bloomberg School of Public Health, Malaria Research Institute, Johns Hopkins University, 615 N. Wolfe St., Baltimore, MD 21205
PETER F. zIPFEL
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute (HKI), Friedrich Schiller University Jena, Department Molecular and Applied Microbiology and Department of Infection Biology, Beutenbergstrasse 11a, 07745 Jena, Germany
This page intentionally left blank
Preface
“Excellent textbooks and review volumes on immunology, virology, parasitology, medical microbiology, and infectious diseases abound. So what gap is this book aimed to fill?” This question was posed in the preface of the ASM Press book Immunology of Infectious Diseases (edited by Stefan H. E. Kaufmann, Alan Sher, and Rafi Ahmed), published in 2002. The explanation provided then still holds true today. Microbiology and immunology, despite their common roots, have matured as distinct disciplines, and infectious diseases are too often viewed from the perspective of either the microbe or the host. A more holistic approach was provided by that book and a second one published by ASM Press in 2004, The Innate Immune Response to Infection (edited by Stefan H. E. Kaufmann, Ruslan Medzhitov, and Siamon Gordon). These books now urgently need updating because the knowledge base in immunology, as well as with all types of infectious agents, has expanded dramatically. The present volume, The Immune Response to Infection, covers all aspects of innate and adaptive immune mechanisms and describes how they interact with pathogens of different types, resulting in either success or failure to control infection and clinical disease. This volume also emphasizes how our understanding of mechanistic events is leading the design and production of more effective prophylactic and therapeutic control measures for infectious agents. Most of the chapters here consider host-pathogen interactions in the context of the broad divisions of the microbial world—either viruses, bacteria, or parasites—
and do not confine their discussion to any individual pathogen. The exceptions are for the agents of the “big three” infectious diseases—HIV/AIDS, tuberculosis, and malaria—which account for almost one-third of human deaths from infections, as well as influenza, which is the focus of much media and public attention. We have also included chapters that consider the detrimental sequelae of infection that are an indirect result of the infectious process, such as chronic inflammation, cancer, and autoimmunity. Finally, all of the chapters emphasize the special attributes that make pathogens difficult to control, and they appraise the prospects of current and future prophylactic and therapeutic vaccines. We hope that this book, which comprises the rich variety of aspects of infection and immunity, helps to further promote the important relationship between immunology and medical microbiology. We express our deep appreciation to the editorial staff of ASM Press, in particular, Greg Payne and Ellie Tupper. We also want to thank our associates Mary Louise Grossman and Lisa Washington for their secretarial help and for their wonderful dedication. Most of all, we thank our colleagues for sacrificing so much of their valuable time to generously share their outstanding expertise with us and with the readers of this book. STEFAN H. E. KAUFMANN, BARRy T. ROUSE, AND DAVID L. SACKS
xv
This page intentionally left blank
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
The Immune Response to Infection: Introduction STEfAN H. E. KAUfMANN, BARRy RoUSE, AND DAvID SACKS
HISTORICAL PERSPECTIVE
immune system. from a coevolutionary perspective, these different stratagems in the struggle for life between microbes and host might seem to offer neither any clear-cut advantage. yet humans have one major advantage: our brain that can envision, design, and develop new countermeasures. In the case of infectious disease these are anti-infectives, mostly antibiotics and vaccines. from the beginning, plagues have written history (fig. 1) (Brock, 1999). However, it took until the late 19th century until we understood what causes plagues and how our body fights against them. Investigations of plagues started almost 500 years bce in ancient Greece, when Thucydides (460–396 bc) assumed contagiousness of the plague in Athens because he realized that those who recovered from disease would not fall ill again. Even though Hippocrates (460–375 bc) and his school recognized that some disease can be transmitted, their main concept viewed disease as an imbalance among the four humors: phlegm, black bile, yellow bile, and blood. Hence their contribution to our understanding of contagious diseases was minimal. Some time later, Diodorus Siculus (1st century bc) impressively described plagues at the times of Pericles and considered that gas arising from the muddy ground was responsible. Bad air is termed miasma in Greek, and a long line of investigators believed in miasma as a cause of infectious diseases. Marcus Terentius varro (116–27 bc) argued against miasma in his treatise on agriculture, where he assumed that minute animals invisible to the naked eye cause severe diseases when they are inhaled through the nose and mouth. This knowledge would have been lost during the Middle Ages were it not continued by the Islamic world. Notably, the Persian Rhazes (864–830 ad), in his treatise about smallpox and measles, precisely described differences and similarities of these two plagues. A major breakthrough, however, came with the work of Girolamo fracastoro (1478–1553 ad), from verona, Italy. In his work About the Contagious Diseases and Their Treatment, he foresaw the major characteristic features of infectious diseases, speculating that small organisms were responsible for transmission. The German Jesuit Athanasius Kircher (1602–1680 ad) was a universally educated man who was also curious about the causes of plague, and was the first to claim to have seen microbes, which cause infectious diseases. This, however, was impossible because the magnification glasses he used were insufficient. What he probably
Microbes were the first living organisms to colonize the world some 3 billion years ago. When our human ancestors entered the scene some millions of years ago, microbes had already occupied every niche on the globe. The human body was no exception to this occupation. Microbes colonized and readily exploited the outer and inner surfaces of our ancestors’ bodies; every now and then they entered into deeper tissue sites where they caused harm—something we now call infectious disease. As long as humans were nomadic and did not settle in larger communities, infectious diseases were restricted to small groups of individuals and could not spread. once humans switched to stable settlements and started to grow crops and tend animals, microbes could better spread from contaminated water, soil, and vegetable sources to animal and human, from animal to human, and from human to human. farming and animal husbandry practices began 10,000 to 20,000 years ago and provided fertile ground for insects, which served as vectors for disease transmission. In fact, we can trace several major infectious diseases of today back to these times. Currently, we witness a resurgence of plagues with the emergence of new diseases such as acquired immunodeficiency syndrome (AIDS) and new strains of influenza virus as well as the reemergence of old diseases such as tuberculosis (TB). In the struggle between microbe and human, the central strategy microbes trust on is rapid replication time paired with frequent mutation so that a few microbes surviving under catastrophic conditions can emerge and conquer a new and previously hostile niche. In contrast, humans developed along a different path based on specialization and complexity. We possess specialized organs that serve as pumps for blood circulation, the heart; as site of metabolite production and detoxification, the liver; or as site for perception and thought, the brain. The combat of infectious agents is the responsibility of a specialized organ, the Stefan H. E. Kaufmann, Max Planck Institute for Infection Biology, Department of Immunology, Charitéplatz 1, 10117 Berlin, Germany. Barry Rouse, Department of Pathobiology, 1414 W. Cumberland Ave, WLS Rm B408, University of Tennessee, Knoxville, TN 37996-0845. David Sacks, Intracellular Parasite Biology Section, Laboratory of Parasitic Diseases, NIAID, Bldg 4, Rm 126, 4 Center Dr., MSC 0425, Bethesda, MD 20892-0425.
1
2
INTRODUCTION
FIGURE 1 Timeline of infectious disease research. The timeline illustrates major breakthroughs in the area of infection and immunity.
observed were some blood cells rather than microbes. The first step from concept to experiment was carried out by the textile merchant Antonius van Leeuwenhoek (1632–1723 ad) from Delft, The Netherlands, who, in his free time, crafted small lenses that allowed him to see real microbes for the first time (which he called “Animalcules”). Thus Leeuwenhoek could be named the father of experimental microbiology, although he had no particular interest in infectious diseases. The next issue to clarify was whether life was generated spontaneously or was derived from living organisms. This question was resolved by the Italian Lazzaro Spallanzani (1729–1799 ad) who showed that after heating, a closed flask containing broth remained free of living organisms, whereas broth in an open flask was soon recolonized by microbes, even after being heated. Louis Pasteur (1822–1895 ad) refined this
experiment by using a flask with an s-shaped neck, which prevented entry of microbes when stored, but allowed exchange of air. Even when left open, the heated broth in these flasks was not colonized. Pasteur also showed that fermentation and putrefaction were not just chemical processes but the function of microorganisms. Pasteur went one step further and opened the way for infectious disease research by showing that putrefaction was the underlying mechanism of diseases that were accompanied by pus formation. Moreover, he laid the foundation for rational vaccine design by attenuating microbes and using them for prevention or therapy of infectious diseases. He is best known for the development of two vaccines, one to prevent anthrax and one for treating rabies. The AustroHungarian Ignaz Semmelweis (1818–1865 ad) emphasized the importance of careful hand washing before diagnosing
The Immune Response to Infection: Introduction
healthy pregnant women. In hospital stations where doctors who had been working in pathology prior to diagnosing pregnant women there was a far higher mortality rate of women and infants at delivery. Semmelweis’s advice for disinfection prior to contact with the patient during delivery was not appreciated by the medical community at the time. The Scottish surgeon Joseph Lister (1827–1912 ad) was more successful in convincing his colleagues to recognize the value of wound disinfection, and, together with Semmelweis, established hygiene as a principle for the prevention of the spread of infectious diseases. The Englishman John Snow (1813–1858 ad) furthered the principles of public health for control of plagues and established the discipline of epidemiology. Robert Koch (1843–1910 ad), by revealing the etiology of TB, anthrax, and cholera, founded the golden age of medical microbiology in Berlin, Germany (Kaufmann & Winau, 2005). Within the next decades, dozens of pathogens were discovered and the etiologies of equally numerous infectious diseases were established. Robert Koch’s school also paved the way for immunology, which was established by his scholars, Emil von Behring (1854–1917 ad) and Paul Ehrlich (1854–1915 ad) (fig. 2). Behring developed the concept of passive vaccination, and Ehrlich built the concepts of antibody specificity and specific immunity in general. Pasteur’s Russian colleague Elie Metchnikoff (1845–1916 ad) described phagocytosis and phagocytes as cellular mediators of nonspecific immunity. While the immunologic concepts introduced by Ehrlich and Metchnikoff provided the scientific basis for an understanding of the mechanisms underlying
3
vaccination, it was Edward Jenner (1749–1823 ad), an English country physician, who had developed the first active vaccine a century before. Jenner used smallpox with low virulence for humans to protect against a highly virulent related human pox. Medical microbiology paved the way for the emergence of immunology as a new discipline. At the same time, it hampered its development because of its focus on exogenous invaders as being exclusively responsible for pathogenesis. This was a necessary step to challenge the prevailing dogma, which considered disease exclusively as result of malfunctioning host cells. Ehrlich and Metchnikoff balanced this out. The host is not a helpless prey for foreign invaders. A highly sophisticated host defense system—the immune system—exists to combat predators. At the same time, not only microbes, but also the host contributes to pathogenesis. The concept of infectious disease as the result of a cross talk between pathogen and host had developed. Even though the Paris and Berlin schools of immunology hotly disputed their opposing theories, we now know how much they complement each other. Both host cells as first described by Metchnikoff and soluble mediators as described by Ehrlich comprise the two pillars of the immune system: Metchnikoff’s nonspecific innate immune system and Ehrlich’s specific, acquired immune system (Kaufmann, 2008). first signs for a synergy between specific and nonspecific immunity emerged when researchers, notably Hans Buchner (1850–1902 ad), Jules Bordet (1870–1961 ad), as well as Ehrlich himself described complement as a soluble factor of innate immunity, which was activated by antibodies.
FIGURE 2 The merger of innate and acquired immunity. The figure depicts the major events that led to the merger of innate and acquired immunity to a global view of the immune response.
4
INTRODUCTION
Complementarities were further emphasized by the Englishman Almroth Wright (1861–1947 ad), who demonstrated the cooperation between antibodies and phagocytes in a process termed opsonization, which allowed engulfment of microbes that resist phagocytosis by means of a capsule (fig. 2). Thus the specific antibodies of Behring and Ehrlich and the nonspecific phagocytes of Metchnikoff could be fused in a more general concept of immunology. It took another half century, however, before the specific mediators of acquired cellular immunity, T lymphocytes, were discovered. This was achieved in the 1960s when J. f. A. P. Miller described the thymusderived lymphocytes. Through the work of Jim Gowans, it was already known that such lymphocytes recirculated through the body and initiated acquired immune responses. The interaction between T lymphocytes and macrophages in defense against intracellular bacteria was described by George B. Mackaness (1924–2007 ad). Communication between T cells and macrophages was facilitated by soluble mediators now called cytokines, as revealed (independently) by Barry R. Bloom and John R. David. Toward the end of the 20th century, the central importance of the innate immune system became clear when it was shown to instruct the acquired immune system and hence play a much more decisive role than originally assumed. Even though numerous researchers contributed to these advances, one of the first who foresaw the intensive cross talk between innate and acquired immunity was Charles A. Janeway (1943–2003 ad). Today, most immunologists agree that cooperation between innate and adaptive immune mechanisms is critical to the success of the immune response. Even though at its birth, immunology was viewed as our defense mechanism against pathogens, it subsequently became the target of more academic research lines focusing on the molecular mechanisms underlying antigen specificity, cell–cell communication, and intracellular signaling processes. The ease with which lymphocytes can be obtained from peripheral blood of humans or lymphoid organs of experimental animals made them preferred targets for many investigators interested in more general biologic issues. In the mid-20th century, diagnosis of infectious diseases had become routine and major intervention measures— notably vaccination to prevent and antibiotics to treat infectious disease—had become broadly available. It is no wonder that the perceived threat of infectious disease had subsided substantially (Kaufmann, 2009). How wrong we were. In fact, globalization has increased the risk of emerging and reemerging plagues, and recent years witness heightened interest in immunity against microbial pathogens. It
soon became clear that the canonical approach to vaccine development had been exhausted. Thus far, vaccines had been developed mostly by trial and error with only minor impact from immunologic knowledge. Principally, these vaccines stimulate antibodies, which fight against pathogens or neutralize their toxic products. This approach, however, is insufficient for design of novel vaccines against agents that are not controlled by antibodies alone. These include the three major killers, malaria, HIv/AIDS, and TB, as well as vaccines against pandemic flu. Here, knowledge in immunology is required for rational vaccine design, since not only antibodies but also multiple subsets of T cells need to be stimulated. Broader immune responses against a variety of antigens are required, which are also directed not only against dominant epitopes, but also against subdominant ones. This needs novel adjuvants designed on the basis of our knowledge about pathogen recognition by the innate immune system, about antigen presentation by antigen-presenting cells through the different presentation pathways, and about stimulatory and inhibitory signalling pathways that control activation and suppression of T lymphocyte populations of distinct functions. It is against this background that we have decided to publish a book on the immunology of infectious diseases, which comprises the whole spectrum of immunology as it relates to infection. It is our goal to describe the cross talk between the different kinds of infectious agents with the different host factors involved in immunity. focus is not only laid on underlying mechanisms, but also at the different sequelae of host–pathogen interactions ranging from sterile eradication of the invader to controlled chronic infection to pathologic corollas of the host–pathogen crosstalk. We also consider the pathogenesis of certain autoimmune disorders and cancers that are induced by infectious agents but then apparently become independent of the infectious process.
REFERENCES Brock, T. D. 1999. Milestones in microbiology: 1546 to 1940. ASM Press, Washington, DC. Kaufmann, S. H. 2008. Immunology’s foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff. Nat. Immunol. 9:705–712. Kaufmann, S. H. E. 2009. The new plagues: pandemics and poverty in a globalized world. Haus Publishing, London. Kaufmann, S. H. E., and F. Winau. 2005. from bacteriology to immunology: the dualism of specificity. Nat. Immunol. 6:1063–1066.
host defense: general
I
This page intentionally left blank
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
1 Invertebrate Innate Immune defenses LAURE EL CHAMY, CHARLES HETRU, AND JULES HOFFMANN
IntrodUCtIon
an attempt to trace the origin of essential elements of the innate immune system.
All metazoans are able to mount an innate immune response. Whereas invertebrate species rely solely on this type of defense, gnathostome vertebrates have developed, in addition, a sophisticated adaptive immune system centered on lymphocytes that are absent from invertebrates. Importantly, these adaptive responses require strong stimulatory signals from cells of the innate immune system, and during infections in vertebrates, both arms of defenses cross talk in their fight against invading microorganisms. Current estimates are that 95% of all metazoan species are invertebrates. Consequently, they lack adaptive immune defenses, which are believed to have appeared in ancestral cartilaginous fish some 450 million years ago and are restricted to vertebrates. In this review, we will focus on invertebrate immune defenses and address the question of when and how they evolved in sponges and in recent forms of arthropods. For lack of space, we will restrict our analysis to invertebrate protostomes and will not consider here immune defenses of deuterostomes such as sea urchins and sea squirts. Recent reviews on these groups are found in Rast and Messier-Solek (2008) and Khalturin et al. (2004). Beyond the long-standing concepts that innate defenses depend on the presence of barrier epithelia, phagocytosis by blood cells, and a variety of circulating proteins, our understanding of the mechanisms of invertebrate immune defenses largely depends on studies performed on the model organism Drosophila melanogaster. Flies are easily induced by septic injuries to mount an immune response and are amenable to large scale genetic analysis to decipher the gene cascades required to mount this response. The overall picture that has evolved on the Drosophila host defense is still unique among invertebrates and we will first present its general outlines. With this platform of information in mind, we will then turn our attention to other arthropod groups and to other invertebrate phyla and provide short overviews of recent advances in the studies of their immune defenses. We will extend our review to currently available data from the simplest metazoan species, the Porifera and Cnidaria, in
a PrototYPICal Innate IMMUne resPonse: the host defense of DROSOPHILA
The Drosophila host defense comprises both humoral and cellular reactions. The hallmark of the humoral response is the challenge-induced synthesis, mainly by the fat-body cells (equivalent of the mammalian liver), of potent antimicrobial peptides (AMPs) and their secretion into the hemolymph (blood), (which is the systemic immune response). AMPs are also produced by epithelial barriers where they contribute to repel the infections, (which is the local immune response). When these barriers are breached, an additional layer of defense is provided by rapidly activated proteolytic cascades leading to coagulation and melanization (i.e., synthesis and deposition of melanin) at the injury site and around the invading microorganisms. The cellular reactions essentially correspond to the phagocytosis of invading microorganisms by professional phagocytes (the so-called plasmatocytes). Encapsulation of parasites by dedicated blood cells (the lamellocytes) can also contribute to the cellular host defense of larvae. Each of these aspects of the Drosophila host defense is presented in the following sections.
the systemic Immune response
Septic injury of Drosophila larvae and adults induces the appearance of an antimicrobial activity in the hemolymph. Seven families of antimicrobial peptides have been identified to date in Drosophila. Their immune-inducibility relies on the activation of transcription factors of the NF-kB family by two signaling cascades, referred to as the Toll and IMD (immune deficiency) pathways. Interestingly, the molecular characterization of these pathways has pointed to significant similarities with NF-kB activating pathways controlling mammalian innate defenses, namely the interleukin 1 receptor/Toll-like receptor (IL-1R/TLR) and tumor necrosis factor-receptor (TNFR) pathways (reviewed in Hoffmann, 2003). The Toll pathway is predominantly activated upon fungal and gram-positive bacterial infections, whereas the IMD pathway is mainly induced by gram-negative bacterial
Laure El Chamy, Charles Hetru, and Jules Hoffman, Institut de Biologie Moléculaire et Cellulaire, 15, Rue René Descartes, 67084 Strasbourg, France.
7
8
host defense: general
challenge, although there are some exceptions to this rule. The JNK (Jun N-terminal kinase) and JAK (Janus kinase)/ STAT (signal transducers and activator of transcription) pathways are also involved in some aspects of the Drosophila immune response, as described below.
Immune effectors
Antimicrobial peptides are the best characterized Drosophila immune effectors. The Drosophila genome encodes at least 25 immune-inducible AMPs that belong to seven families (reviewed in Imler & Bulet, 2005). Although structurally diverse, these peptides are mostly small in size, cationic, and predominantly membrane-active. Whereas Drosomycins and Metchnikowins target mainly fungi, Defensin is active against gram-positive bacteria, and Attacins, Cecropins, Diptericins, and Drosocin predominantly affect gram-negative bacteria. Note that the activity spectra can overlap (e.g., Cecropins have been reported to act against fungi). The combined concentrations of the AMPs can reach up to 400 μM in the hemolymph of infected flies. Some AMPs are very stable and are still detected in the hemolymph several weeks after challenge (e.g., Drosomycins) whereas others (e.g., Cecropins) are rapidly degraded by proteolysis. In addition to the genes encoding AMPs, a large set of genes is up-regulated following microbial infection (reviewed in Lemaitre & Hoffmann, 2007). Many of these can be assigned immune functions such as: regulation of AMP gene expression, phagocytosis, melanization, and production of reactive oxygen species (ROS). Other induced genes are related to iron sequestration. A large fraction of the induced genes have as of yet no known functions. Among these is the Turandot family consisting of eight proteins secreted by the fat body under stress conditions including bacterial infections, heat-shock, or exposure to ultraviolet light. Another group of induced genes encodes the DIMs (Drosophila immune-induced molecules) family, with at least 17 members of generally small sized cystein-containing peptides that do not exhibit antimicrobial activities in conventional in vitro assays.
sensing and signaling
The recent years have seen significant progress in the understanding of the mechanisms underlying microbial detection and the selective activation of the Toll and IMD signaling pathways by various classes of invaders. Our current view is that bacteria are detected through the sensing of distinct forms of peptidoglycan (PGN), whereas fungi are detected through the sensing of b-glucans. Genetic studies have revealed that these tasks are assigned to two families of germ-line encoded receptors, referred to as pattern recognition receptors PRRs: the peptidoglycan recognition proteins (PGRPs) and the glucan binding proteins (GNBPs) (reviewed in Ferrandon et al., 2007). PGRPs are innate immune proteins highly conserved from insects to mammals. These proteins are characterized by a PGRP domain that is evolutionarily related to bacteriophage type II amidases and is implicated in the immune response by either interacting with or degrading microbial PGN. The Drosophila genome encodes 13 PGRP members. Some of these PGRPs have retained the amidase activity while other members have lost crucial amino acid residues that are essential for catalysis and serve as recognition proteins (reviewed in Charroux et al., 2009). A remarkable feature of recognition PGRPs is their ability to discriminate between gram-positive and gram-negative bacteria based on single amino acid substitution in the composition of their respective PGNs and to subsequently initiate distinct signaling pathways. PGN consists of long glycan chains of alternating N-acetylglucosamine and N-acetylmuramic acid residues that are cross-linked to each other
by peptide bridges. The majority of gram-positive bacteria are characterized by a lysine residue at the third position of the stem peptide that is replaced by a meso-diaminopimelic acid (DAP) in the PGN of gram-negative bacteria (and the gram-positive Bacillus species) (Fig. 1). To date, four members of the PGRP family have been assigned PRR functions, namely PGRP-SA and PGRP-SD which mediate Toll pathway activation upon gram-positive bacterial infections, and PGRP-LC and PGRP-LE which trigger IMD signaling upon infections with gram-negative bacteria (reviewed in Charroux et al., 2009). GNBPs, also known as b-GRPs (b-glucan recognition proteins) are related to a group of b-glucanases that are found in bacteria, fungi, plants, and invertebrates. Two domains characterize insect GNBPs: a N-terminal glucan binding domain that binds to b-(1,3)-glucans, and a C-terminal glucanase-like domain (reviewed in Royet et al., 2005). The Drosophila genome encodes three GNBP members, of which GNBP3 was shown to mediate fungal detection upstream of Toll. Unexpectedly, GNBP1 was shown to be required for the sensing of gram-positive bacteria in complex with PGRP-SA (reviewed in Ferrandon et al., 2007).
The Toll Pathway
The Toll pathway was initially discovered for its role in the control of dorsoventral patterning in the Drosophila embryo. A genetic analysis revealed more recently that many components of this pathway are also used for the control of AMP gene expression following fungal and gram-positive bacterial infections (reviewed in Hoffmann, 2003) . Toll is the eponymous member of the conserved family of Tolls and Toll-like receptors (TLRs). Its extracellular domain is characterized by multiple leucine rich repeats (LRRs) and its intracytoplasmic domain shares significant sequence similarities with the corresponding domain of the IL-1R and is referred to as TIR (Toll/IL-1R) domain (reviewed in Leulier & Lemaitre, 2008). Toll signaling in Drosophila drives the expression of immune responsive genes through the activation of two closely related NF-kB transcription factors, DIF in adults, and DIF and/or dorsal in larvae (reviewed in Hoffmann, 2003; Lemaitre & Hoffmann, 2007). Unlike mammalian TLRs, which directly bind microbial elicitors, Drosophila Toll does not act as a prototypical PRR but is activated by an endogenous ligand, the cytokine-like spaetzle. Spaetzle is a cystine-knot polypeptide with structural and functional similarities to mammalian neutrophins (Zhu et al., 2008 and references therein). In the absence of stimuli, spaetzle is present in the hemolymph as an inactive dimeric precursor. Pattern recognition involves circulating PRRs, acting upstream of Toll, which activate a proteolytic cascade leading to the processing of spaetzle to its active Toll ligand form (Fig. 2). The data available to date indicate that recognition of gram-positive bacteria occurs in a protein complex of PGRP-SA and GNBP1; the exact role of the latter is still under investigation. Interestingly, PGRP-SD is required as a third partner in this PRR complex for sensing of some gram-positive bacterial strains. As regards fungal recognition, GNBP3 is the sole PRR that has been identified to date (reviewed in Ferrandon et al., 2007). Binding of microbial ligands to the receptors described above leads, through as yet unknown mechanisms, to the activation of a proteolytic cascade in which four zymogens, ModSP (modular serine protease), grass, spirit, and SPE (spaetzle processing enzyme), have been identified (Buchon et al., 2009 and references therein). ModSP is a modular serine protease characterized by four low-density lipoprotein-receptor class A (LDLa) domains and one complement control protein (CCP) domain at its N-terminus. Genetic analysis has revealed a role for ModSP in integrating signals from the circulating PRRs
1. Invertebrate Innate Immune defenses
9
FIGURE 1 Peptidoglycans as inducers of the Drosophila Toll and IMD signaling pathways. Peptidoglycan (PGN) is a glucopeptide polymer consisting of long chains of alternating N-acetylglucosamine and N-acetylmuramic acid residues connected to each other by short peptide bridges. The nature of the peptide bridge varies depending on the bacterial strains. Most gram-positive bacterial PGN carries a lysine residue at the third amino acid position in the peptide stems (Lys-type PGN), that is replaced by a diaminopimelic acid residue in most gram-negative bacteria (DAP-type PGN). In Drosophila, sensing of Lys-type PGN and DAP-type PGN is mediated by dedicated recognition PGRPs that activate the Toll and IMD pathways, respectively. Catalytic PGRPs have a zinc-dependant amidase activity (scissors). They reduce the immune stimulatory potency of PGN by removing the peptide bridges from the sugar backbone.
and connecting them to the downstream elements of the proteolytic cascade. Grass, spirit, and SPE are characterized by an inhibitory domain, called clip-domain that is only found in arthropods. SPE is the ultimate protease in this cascade and directly cleaves spaetzle to its Toll ligand form. In addition to the detection of microbial moieties a Toll-dependent immune response is also driven by the sensing of microbial virulence factors, notably, by secreted proteases. This reaction is centered on the clip-domain serine protease persephone, which was shown to be activated by secreted fungal subtilisins (Gottar, 2006). Persephone mediates activation of SPE and subsequently that of Toll. This reaction can be mimicked by the injection of purified bacterial proteases, hence the proposal that persephone is a sensor of danger signals (El Chamy, 2008). In agreement with this hypothesis, a recent analysis has revealed that, in the absence of any infection, tracheal melanization triggers the systemic activation of the Toll pathway in a persephone-dependent manner. The current model proposes that a host factor deriv-
ing from the melanization reaction diffuses from the tracheal system into the hemolymph where it triggers persephone activation, further driving the systemic Toll response in the fat body (Tang et al., 2008). Activation of the Toll receptor triggers an intracellular signaling cascade, which has partial similarities with the TLR and IL-1R signaling pathways (Color plate 1 a and b) (reviewed in Hoffmann, 2003; Ferrandon et al., 2007). This process involves the assembly of a multivalent complex, including death-domain-containing proteins around the intracellular TIR domain. In Drosophila, this complex comprises two adaptor molecules: a homologue of myeloid differentiation factor 88 (MyD88), which, in addition to the death domain, has a TIR domain similar to that of Toll and tube, which has a bivalent death domain. Pelle, the third partner, is a member of the IL-1R associated kinase (IRAK) family of serine-threonine kinases. The end result of Toll signaling is the dissociation of the NF-kB transcription factors from the ankyrin repeat inhibitor protein cactus, a homologue of mammalian inhibitor
10
host defense: general
FIGURE 2 Activation of the Drosophila Toll pathway during fungal and gram-positive bacterial infections. The Toll receptor is activated by a proteolytically processed form of the cytokine-like polypeptide spaetzle (present as a dimer in the hemolymph). Microbial cell wall components (right panel) interact with circulating receptors (fungal b-glucans with GNBP3; gram-positive bacterial peptidoglycan (PGN) with a PGRP-SA/GNBP1 complex). This recognition activates, via a still unidentified mechanism, a proteolytic cascade including the zymogen modular serine protease (ModSP) and the serine protease grass, leading to the activation of the spaetzle processing enzyme (SPE), which cleaves pro-spaetzle into its active Toll-ligand form. Alternatively, microbial secreted proteases (left panel) can activate the circulating zymogen persephone, which, directly or indirectly (still unknown), activates SPE to cleave pro-spaetzle.
NF-kB (IkB). This process involves signal-dependent phosphorylation of cactus and its subsequent degradation. By analogy with mammals, it was suggested that kinases related to mitogen activated protein kinase kinase kinase (MAPKKK) and to IkB kinase (IKK) families might be involved in this process. However, such molecules have not been assigned a function in the Toll pathway in Drosophila and the identity of the cactus kinase has remained elusive so far. Liberated DIF and/or dorsal translocate to the nucleus and trigger the expression of hundreds of genes including those encoding some of the AMPs, notably the antifungal drosomycin, which is often used as a read-out of Toll pathway activation. Dorsal and DIF seem to require additional modifications for full biological activity. A protein cassette, which includes dTRAF2 (Drosophila homolog of TNFR-associated factor 2), aPKC (atypical protein
kinase C), and the scaffolding protein Ref2(P) (homologue of mammalian p62), has been suggested to participate in this process. Toll signaling shows slow kinetics, as revealed by transcriptional profiling of AMP gene expression, with maximum peaks at 24 to 48 hours following gram-positive bacterial and fungal infections, respectively. Negative regulators of this pathway have not been investigated in detail. However, following septic injury, the expression of the gene encoding cactus was shown to be up-regulated in a Toll-dependent manner, thus pointing to a negative feedback loop on Toll signaling. In addition to Toll, the Drosophila genome encodes 8 Toll receptors (18 wheeler, Toll 3 to 9). An immune function has so far only been assigned to Toll. Toll 5 and Toll 9 were also proposed to induce AMP gene expression based on
1. Invertebrate Innate Immune defenses
transfection assays in hemocyte-derived Drosophila cell lines but the in vivo significance of these results is not clear at this stage. Conversely, all Drosophila Toll members seem to be required for developmental processes as suggested by their expression patterns during embryogenesis and metamorphosis. These observations were confirmed by genetic analysis for Toll 2 and Toll 8 mutants (reviewed in Leulier & Lemaitre, 2008). This contrasts with the situation in mammals where all TLRs are devoted to immune detection (reviewed in Kawai & Akira, 2009). Further, Drosophila Tolls share sequence characteristics both in the LRR ectodomain and the intracellular TIR domain that distinguish them from mammalian TLRs. Whereas mammalian TLRs (and all identified vertebrate TLRs) contain a single cystein cluster flanking the C-terminal end of the extracellular LRR, Drosophila Tolls contain multiple cystein clusters including two or more of the characteristic C-terminal cluster in addition to a N-terminal cluster. The only exception is Toll 9, which has a single cystein cluster and is more closely related to mammalian TLRs (Fig. 3).
The IMD Pathway
The IMD pathway controls the activation of the third Drosophila NF-kB transcription factor, relish (reviewed in Lemaitre & Hoffmann, 2007). The IMD gene encodes an intracellular death-domain-containing protein with significant similarities to mammalian RIP1 (receptor interacting protein 1), which is associated with the tumor necrosis factor-receptor 1 (TNF1R). As indicated above, the IMD pathway shows striking similarities with the intracellular signaling cascade activated downstream of the TNFR in mammals (reviewed in Hoffmann, 2003; Ferrandon et al., 2007) (Color plate 2 a and b).
11
The IMD pathway is activated through the direct interaction of microbial ligands with the transmembrane receptor PGRP-LC (reviewed in Ferrandon et al., 2007; Charroux et al., 2009). The PGRP-LC receptor displays three distinct isoforms, PGRP-LCa, PGRP-LCx, and PGRP-LCy, resulting from alternative splicing of its transcript. These receptors share identical intracellular domains but present different PGRP exodomains. Our current understanding is that IMD pathway activation is driven by the association of at least two PGRP molecules: PGRP-LCx homodimers mediate the recognition of polymeric DAP-type PGN and PGRP-LCa/ LCx heterodimers sense monomeric PGN. In addition, a secreted form of the PGRP-LE protein, consisting of its PGRP domain, is proposed to enhance the PGRP-LC receptor activity by binding to PGN in circulation and bringing it into close proximity of the cell surface. In the malpighian tubules, the full-length, non-secreted form of PGRP-LE can function as an intracellular receptor and trigger the IMD pathway in a PGRP-LC independent way (reviewed in Charroux et al., 2009; Ferrandon et al., 2007). Upon stimulation of PGRP-LC, the IMD protein is recruited to the intracellular domain of this receptor with which it interacts. The N-terminal domain of PGRP-LC (and that of PGRP-LE) contains a core motif named RHIM domain (for its homology to the RIP homotypic interaction motif) which is crucial for signaling, but not for PGRP-LC and IMD interaction. Thus, signaling via the RHIM domain seems to require a so far unidentified third partner of the PGRP-LC receptor and the IMD adaptor protein. A signaling complex is further established around IMD. FADD, a homologue of mammalian Fas-associated death domain, interacts with IMD via its death domain
FIGURE 3 Characteristic structural domains of Drosophila Toll and Toll9 and of mammalian TLR2.
12
host defense: general
and with the caspase-8 DREDD via its death effector domain (DED). This complex triggers the activation of the MAP3K, TAK1 (transforming growth factor [TGF]-b activated kinase 1), which in turn activates the IKK signalosome. The latter protein complex includes kenny, a regulatory subunit homologous to IKKg/nemo and IRD5, a catalytic subunit homologous of IKKb, which phosphorylates relish on specific serine residues. The precise molecular mechanisms leading to the activation of TAK1 and of the IKK signaling complex remain unclear. However, convincing evidence points to a K63-linked polyubiquitination process involving the ubiquitin conjugating enzyme complex (including the Drosophila homologues of Uev1a and Ubc13), the RING-finger domain protein DIAP2 (Drosophila inhibitor of apoptosis protein 2) and the adaptor protein TAB2 (TAK1 binding protein 2). In contrast to dorsal and DIF, relish carries its own IkB-like inhibitory sequences in the form of several ankyrin repeats located at its C-terminal end which is cleaved, following relish phosphorylation, most probably by the caspase-8 DREDD. The N-terminal fragment bearing the Rel-homology domain translocates to the nucleus and drives the expression of hundreds of genes including diptericin. Although the IKK complex is required for relish cleavage, this process does not depend on its phosphorylation. Instead, phosphorylation is critical for the adequate transcriptional activation of relish target genes (Erturk-Hasdemir, 2009). In addition, a newly identified nuclear protein, akirin is required for the control of relish transcriptional activity. Homology search has led to unravel two akirin homologs in mammals. One of these exerts similar functions in NF-kB -dependent gene expression downstream of TNFR, TLR, and IL-1R (Goto, 2008). It is currently understood that the differential expression profiles of IMD- and Toll-immune induced genes are related to distinct sequence codes of kB-response elements in the promoter regions of the target genes responsive to the various NF-kB family members of Drosophila (for references see recent reviews by Lemaitre & Hoffmann, 2007 and Ferrandon et al., 2007). In contrast to Toll, IMD pathway-dependent signaling is rapid and short-lived. Maximum levels of AMP gene expression are detected around six hours following infection. Negative regulation of the IMD pathway occurs at different levels (reviewed in Aggarwal & Silverman, 2008). First, secreted catalytic members of the PGRP family, namely PGRP-SC and PGRP-LB degrade DAP-type PGN, thus reducing its immune stimulatory potency. In addition, the transmembrane PGRP-LF prevents the inappropriate activation of the IMD pathway by sequestering circulating PGN (reviewed in Charroux et al., 2009). Further, a homologue of the Fas-associating factor 1, caspar, interferes with the DREDD-dependent cleavage of relish. Finally, an intracellular protein, pirk (also designated pims and rudra), is induced in a relish-dependent manner following gram-negative bacterial infections and interferes with the activation of the IMD pathway by interacting with PGRP-LC and IMD (Aggarwal et al., 2008; Lhocine, 2008; Kleino et al., 2008).
The JNK Pathway
The JNK signaling pathway is involved in early developmental processes of the Drosophila embryo where it controls dorsal closure. It is also required for wound healing in adults. Microarray analysis of Drosophila hemocyte-derived cell lines and adult flies indicated an immune induction of the JNK pathway through the upstream components of the IMD signaling cascade (reviewed in Lemaitre & Hoffmann, 2007). Thus, similar to TNFR signaling in mammals, the IMD pathway in Drosophila bifurcates downstream of TAK1,
activating both IKK and JNK signaling. The precise role of the JNK pathway in the Drosophila host defense is not yet firmly established, as genetic analyses are hampered by the early embryonic lethality of JNK pathway null mutants. The transcriptional program triggered by this MAPK signaling cascade is enriched for genes related to cytoskeleton remodeling. These findings are consistent with the role of the JNK pathway in controlling epithelial sheet movement and tissue repair following septic injury and may also be linked to hemocyte activation (all references are included in a recent review by Lemaitre & Hoffmann, 2007). JNK signaling also promotes pro-apoptotic gene expression, an observation that correlates with early lethality of transgenic flies overexpressing IMD and developmental defects associated with the reduction of PGRP-LF expression levels (Georgel, 2001; Maillet et al., 2008). JNK signaling mediates immediate but very short-lived responses (up to 3 hours) following septic injury. Some observations point to negative feedbacks between the IMD/relish and IMD/JNK branches (Aggarwal & Silverman, 2008).
The JAK/STAT Pathway
The JAK/STAT pathway was originally identified for its role in embryonic segmentation and was later shown to control other developmental processes including larval hematopoiesis (reviewed in Agaisse & Perrimon, 2004). The four main components of this pathway in Drosophila are: the cytokine-like ligand Unpaired (Upd), the receptor Domeless (a member of the type-I cytokine receptor family), the Janus Kinase Hopscotch, and the STAT92E transcription factor. Septic injury triggers a transient activation of the JAK/STAT pathway with maximum levels reached 6 hours following infection. The induced genes encode, among others, secreted proteins such as TEP (thiol ester-containing proteins) and Turandot (Tot) family members. TEPs show significant similarities to members of the complement C3/a2macroglobulin family and could function as opsonins as was shown for Anopheles gambiae TEP1 (reviewed in Blandin et al., 2008). The functional relevance of JAK/STAT pathway activation upon septic injury remains unclear, as JAK/STAT-deficient flies are resistant to bacterial infections. An interesting new finding has linked JAK/STAT pathway activation to the resistance to viral infections in Drosophila (reviewed in Kemp & Imler, 2009). Genome-wide expression profile analysis of flies infected by DCV (Drosophila C Virus) has revealed the activation of a dedicated transcriptional program, which is different from that induced following bacterial and fungal infections. Part of the induced genes, such as vir-1, were shown to be JAK/STAT dependent. In addition, Hopscotch mutant flies exhibited accelerated lethality following viral infection, which correlated with an increase in viral loads. These results suggest that the antiviral immune response in Drosophila triggers the production of a cytokine that activates the receptor Domeless and JAK/STAT-dependent induction of a set of genes to control the viral load in infected flies. In mammals, the innate antiviral defense is characterized by the production of interferons (IFN absent from the Drosophila genome), which trigger JAK/STAT pathway transcriptional activation of IFN-stimulated genes (ISGs). Thus, these observations in Drosophila point to an evolutionarily conserved role of the JAK/STAT pathway in the control of viral infections from insects to mammals. It is important to note here that, in addition to the inducible antiviral response, an intrinsic response based on RNA interference (RNAi) also plays a crucial role in the control of viral infection in Drosophila (reviewed in Kemp & Imler, 2009) (Fig. 4). This is reminiscent of the findings in plants and other invertebrates (Caenorhabditis elegans), suggesting that RNAi represents an ancestral response to
1. Invertebrate Innate Immune defenses
13
FIGURE 4 The Antiviral Response in Drosophila. RNA interference and inducible gene reprograming are two major arms of the Drosophila antiviral defense. Viral nucleic acids are recognized by the RNase III enzyme dicer 2, which activates the small interfering RNA (siRNA) pathway leading to the degradation of viral RNA. In addition, dicer 2 can trigger the inducible expression of a variety of genes via a yet unidentified signaling pathway. Among the induced genes is that encoding the vago cystein-rich polypeptide, which is involved in the control of the viral load. Further, a cytokine-mediated response activated upon viral infection leads to the expression of genes via the activation of the JAK/STAT pathway in neighboring cells. The mechanism controlling this antiviral-inducible response is so far unknown.
viral infection. Viral double-stranded RNA is recognized by the RNAse III enzyme dicer 2, which drives the degradation of viral RNA through the small interfering RNA (siRNA) pathway. Intriguingly, dicer 2 (but no other mediators of the siRNA pathway) is also required for the expression of some DCV-inducible genes, such as vago, in the infected cells. Dicer 2 contains an amino-terminal DExD/H box helicase domain, which is important for the inducible vago expression. This domain is similar and is phylogenetically related to the helicase domain of mammalian RIG-I-like receptors, which mediate the sensing of viral nucleic acids in the cytosol and the induction of interferon gene expression (reviewed in Kawai & Akira, 2009). Hence, although Drosophila lacks interferon homologues and the mechanisms of antiviral responses are different between Drosophila and mammals, many similarities can be drawn on sensing (requirement of intracellular DExD/H box helicases) and signaling (cytokine production and JAK/STAT signaling) during viral infection in these evolutionarily distant organisms.
epithelial defense reactions
Epithelial barriers constitute critical interfaces between the host and microorganisms of the environment. Antimicrobial defense reactions deployed at the level of these epithelial layers provide a first tier of defense counteracting microbial invaders. In Drosophila, various spectra of AMPs have been reported in the epidermis, the reproductive system, the respiratory, and the digestive tracts. Whereas some AMPs are constitutively expressed (such as Drosomycin in the salivary glands and Cecropin in the ejaculatory duct), others are induced upon infection, and by contrast to the
systemic response, their regulation is solely dependent on the IMD pathway (including that of Drosomycin in the trachea and of Diptericin in the gut). The development of appropriate food-borne infection models in Drosophila has now uncovered the central role of locally synthesized AMPs in the gut for the survival of pathogenic infections such as Serratia marcescens or Pseudomonas entomophila. Recognition of ingested DAP-type PGN is mediated by the PGRP-LC receptor, triggering the local activation of the IMD pathway, and immune reactivity is attenuated by secreted catalytic PGRPs in the gut lumen, which degrade bacterial PGN into nonstimulatory molecules. It is proposed that this modulation of the IMD pathway protects the gut flora from excessive AMP production. Furthermore, in the absence of gut infection, a selective repression of NF-kB-dependent AMPs is provided by the homeobox gene caudal (Ryu, 2008). In addition to AMPs, ROS are rapidly produced following microbial ingestion and critically contribute to the bactericidal reactions in the gut. ROS production is mediated by dual oxidase (DUOX), a member of the conserved family of NADPH oxidases. DUOX enzymatic activity is Ca2+-dependent and is regulated by the Gaq-Phospholipase C-b (PLC-b) pathway mediating the mobilization of intracellular Ca2+. The identity of the receptor acting upstream of this pathway and the mechanism triggering this oxidative reaction remain unsolved (Ha, 2009). Excessive ROS produced following microbial infection are eliminated by a secreted antioxidant enzyme, the immune regulated catalase (IRC), thereby protecting the host from the deleterious effects of the oxidative burst. (All references for epithelial defense reactions in Drosophila are
14
host defense: general
found in recent reviews by Lemaitre & Hoffmann, 2007 and Ferrandon et al., 2007.)
Melanization
Melanization is a particular feature of invertebrate immune reactions that is immediately activated at the site of cuticular wounding and around invading microorganisms. It relies on the activation of phenoloxidase (PO), a key enzyme that catalyzes the conversion of phenols to quinones, which polymerize nonenzymatically to form melanin. Melanization is generally assumed to contribute to the arthropod defense by facilitating wound healing, sequestrating microorganisms and preventing or retarding their growth, and by the production of toxic intermediates that can kill microbial intruders (for reference see Cerenius & Soderhall, 2004).
Cellular responses Drosophila Blood Cell lineages and functions
Compared to the complexity of blood cell lineages in mammals, the picture is relatively simple in Drosophila as there are no equivalents to lymphoid cells and as hemocyte lineages are restricted to three cell types: plasmatocytes, crystal cells, and lamellocytes. Plasmatocytes represent the predominant population of embryonic and larval hemocytes (95%) and are the unique blood cells found in adults. They are professional phagocytes, which are in charge of the uptake and clearance of both apoptotic cells and microorganisms. Plasmatocytes also contribute to the humoral response by secreting AMPs and, as previously mentioned, they are required for the activation of the systemic response of adults exposed to stress conditions (i.e., JAK/STAT pathway activation). Crystal cells and lamellocytes have no mammalian counterparts and are both involved in defense reactions that are restricted to invertebrates (i.e., melanization for crystal cells and encapsulation for lamellocytes) (reviewed in Meister, 2004).
Phagocytosis
Several studies have revealed the importance of the cellular arm of defense in Drosophila. To give but one example: mutant larvae devoid of hemocytes are filled with microorganisms and cannot reach the adult stage unless reared on germ-free media. Further, adult flies deprived of hemocytes by targeted cell death of hemocyte lineages, are highly susceptible to some bacterial and fungal infections, and this lethality correlates with higher bacterial loads (Charroux & Royet, 2009; Defaye et al., 2009). Phagocytosis is a central theme in innate immunity and is conserved in all metazoans. High-throughput RNAi screens in the embryonic hemocyte-derived S2 cells have deciphered the phagocytic machinery and have identified multiple components of the basic mechanisms of phagocytosis, namely regulators of vesicle transport and actin cytoskeleton remodeling, which are preserved in other animal species. A recent proteomic analysis identified approximately 600 proteins potentially associated with the Drosophila S2 cell phagosome. Of the identified proteins, many are evolutionarily conserved and 70% have mammalian orthologues. By combining several approaches, this study generated a detailed model of the phagosome, which is also valid in mammalian cells. Finally, studies performed in S2 cells and other in vivo and ex vivo analysis using different microorganisms identified diverse phagocytic receptors, some of which have mammalian homologues required for the phagocytic process. (All references are found in a recent review on phagocytosis by Stuart & Ezekowitz, 2008.) These receptors belong to three families
of evolutionarily conserved proteins: scavenger receptors, EGF-like repeat containing receptors, and immunoglobulin repeat containing proteins. Other molecules, including complement-like opsonins are proposed to promote the phagocytic uptake of microorganisms.
Scavenger Receptors
Scavenger receptors represent a large family of structurally heterogeneous receptors, which bind to and internalize a broad array of endogenous molecules and microbial ligands with polyanionic character. Three members of the Drosophila scavenger receptor family were shown to be involved in phagocytosis: croquemort and peste are homologous to mammalian scavenger receptors, and dSR-CI belongs to an insect-specific family of scavenger receptors. Croquemort is required for the phagocytosis of apoptotic cells (in the Drosophila embryo) and is also involved in the phagocytosis of bacteria. In cell culture experiments, peste was shown to be required for the entry of intracellular Mycobacteria and Listeria. Finally, dSR-CI contributes to the phagocytic uptake of both gram-positive and gram-negative bacteria in Drosophila S2 cells.
Receptors Containing Multiple EGF-like Repeats
Receptors containing multiple EGF-like repeats are found in diverse species, including humans, and are emerging as a new family of phagocytic receptors. The best characterized member in Drosophila is eater, a type I transmembrane receptor with 32 typical EGF-like repeats in its extracellular domain. This receptor has a broad reactivity, like scavenger receptors, and directly binds to microbial surfaces. Eater seems to be the major phagocytic receptor in Drosophila, as silencing of its transcript dramatically decreases the phagocytic index of Drosophila S2 cells. Furthermore, eater mutant flies are highly susceptible to infections with both gram-positive and gram-negative bacteria. Another member of this family of EGF-like repeat receptors, nimrod C1, is involved in the uptake of Staphylococcus aureus and may participate to the phagocytosis of Escherichia coli. Finally, draper contains 16 EGF-like repeats and is required for the phagocytosis of apoptotic cells. Interestingly, this receptor contains an ITAM (immunoreceptor tyrosine-based activation motif) in its intracytoplasmic domain.
Immunoglobulin-like Repeat Proteins
Another member of Drosophila phagocytic receptors, Dscam (Down syndrome cell adhesion molecule), belongs to the immunoglobulin superfamily. Dscam is a single pass transmembrane receptor characterized by 10 immunoglobulinlike domains and 6 fibronectin type III domains in its extracellular part. This receptor was identified for its essential functions in neuron wiring and is also expressed in hemocytes. The dscam gene comprises a cluster of variable exons flanked by constant exons which can theoretically generate, by alternative splicing, a large number of distinct protein products. Silencing of the dscam transcript in hemocytes decreases the phagocytic uptake of E. coli by these cells. In addition, some recombinant isoforms of Dscam directly bind to E. coli. Secreted isoforms of Dscam are detected in Drosophila hemolymph and a role of these isoforms in opsonization of pathogens has also been proposed.
Complement-like Opsonins
Other molecules proposed to mediate phagocytosis in Drosophila comprise the secreted family of TEPs. Based on their homology to the complement C3/a2-macroglobulin superfamily, these molecules were anticipated to function either as opsonins to promote phagocytosis and/or as
1. Invertebrate Innate Immune defenses
protease inhibitors. Some data so far support the opsonin function, namely from the studies performed on Anopheles gambiae TEP1 (reviewed in Blandin et al., 2008). The Drosophila genome encodes four expressed TEPs (TEP I to IV) and a closely related gene, Mcr (macroglobulin complement related), which encodes a protein lacking the characteristic thiol ester motif of TEPs. Based on RNAi-mediated gene silencing in Drosophila S2 cells, TEP II and TEP III are proposed to mediate phagocytosis of E. coli and S. aureus respectively, whereas Mcr is proposed to mediate binding and internalization of the yeast Candida albicans. Confirmation of TEP functions during phagocytosis awaits further in vivo studies.
CoMParatIVe analYsIs of the Innate IMMUne sYsteM Insects other than drosophila
Most of the data obtained from the analysis of gene families related to the immune system in other insect species are compatible with the picture that we have described for Drosophila melanogaster. This is namely the case for the two dipteran species, Anopheles gambiae and Aedes aegypti, the hymenopteran Apis mellifera, the lepidopteran, Bombyx mori, and the coleopteran, Tribolium castaneum (Christophides et al., 2004; Waterhouse, 2007; Evans et al., 2006; Tanaka, 2008; Zou et al., 2007). Phylogenetic analysis of the insect immune repertoires (including those of twelve additional Drosophila species [Sackton et al., 2007]) has revealed dynamic changes of genes related to recognition and effector functions, probably as a result of the selective pressure imposed by distinct pathogens encountered by each insect species. These include PRRs (PGRPs and GNBPs) as well as phagocytic receptors (i.e., scavenger receptors) and opsonins (TEPs). With regard to the effector molecules, novelties are noted among insect orders mainly with the emergence of lineage-specific AMPs such as Drosomycin in Drosophila, Gambicin in mosquitoes, and Apisimin and others in bees (Christophides, 2002; Evans et al., 2006; Sackton et al., 2007). In contrast, intracellular signaling mediators are highly conserved in all studied insects. Notably, a nearly complete tool kit of both Toll and IMD pathways has been identified in all analyzed genomes. Some experimental data support the functional conservation of these pathways for immune defenses in the respective insect species. Mosquitoes lack the NF-kB transcription factor dif and rely on a dorsal orthologue, REL1, and on a relish orthologue, REL2. These mosquito REL factors have been reported to control the induced expression of AMPs and the resistance to bacterial infections (Shin et al., 2005; Bian, 2005; Antonova et al., 2009; Meister, 2005). In Anopheles gambiae, REL1 and REL2 are also involved in the resistance to the rodent malaria parasite Plasmodium berghei by controlling the basal expression of the major antiparasitic factors (Blandin et al., 2008). Of the five Toll receptors and the three spaetzle ligands encoded in the genome of the yellow fever mosquito, Aedes aegypti, a role for Toll5A and its putative ligand spaetzle1C is documented in the defense against the entomopathogenic fungus Beauveria bassiana. This receptor ligand complex triggers the activation of the REL1 transcription factor regulating the antifungal immune response (Shin et al., 2006; Shin et al., 2005; Bian, 2005). The Toll pathway, together with the JAK/STAT pathway, has also been linked to the transcriptional program triggered by Dengue virus infection in Aedes aegypti and a contribution of the Toll pathway in the defense against Dengue virus in this species has also been proposed (Xi et al., 2008).
15
In the silkworm Bombyx mori and the beetle Tenebrio molitor, the expression of some AMPs is up-regulated following the injection of a maturated form of spaetzle orthologues (Wang, 2007; Roh et al., 2009). Due to the lack of appropriate genetic tools, functional data for immune-related genes are restrained in the lepidopteran and coleopteran species, as compared to Drosophila. However, these large insects have provided valuable biochemical tools for the investigation of pathogen recognition processes and the characterization of the proteolytic cascades subsequently activated downstream of PRRs (i.e., melanization and the processing of spaetzle orthologues) (reviewed in Iwanaga & Lee, 2005; Roh et al., 2009). Recently, the components of the extracellular pathway leading to the processing of spaetzle were purified from the hemolymph of the beetle Tenebrio molitor. Biochemical assays have allowed the in vitro reconstruction of the proteolytic cascade activated downstream of TmGNBP3, TmPGRP-SA, and TmGNBP1 (Roh et al., 2009). This work led to the identification of the apical protease, DmModSP, acting downstream of PRRs for the activation of the Toll pathway in Drosophila (Buchon et al., 2009).
horseshoe Crab
The horseshoe crab is often referred to as a “living fossil” and first appeared more than 500 million years ago. Its host defense largely relies on granular hemocytes, which constitute 99% of the circulating blood cells. These hemocytes contain secretory granules that store the major elements of the horseshoe crab’s defense arsenal. They include AMPs, serine protease zymogens, the clotting protein coagulogen and several lectins (reviewed in Iwanaga & Lee, 2005). The innate immune system of this chelicerate arthropod recognizes invading pathogens through a combinatorial system involving several lectins with distinct affinities for carbohydrates exposed on the surface of pathogens. Another recognition molecule playing a key role at the frontline of the horseshoe crab antimicrobial defense is the serine protease zymogen Factor C. This factor is mainly found in the hemocyte granules, but is also detected at the hemocyte surface. Factor C is highly sensitive to gram-negative bacterial lipopolysaccharide (LPS) and is autocatalytically activated upon stimulation by this microbial elicitor. This reaction leads to the secretion of the defense molecules from granular hemocytes by exocytosis (reviewed in Kurata et al., 2006). Of the released molecules, Factor C and another serine protease zymogen, factor G, which is responsive to b-(1-3)-glucan, trigger a two-step proteolytic cascade leading to hemolymph coagulation. This reaction consists in the conversion of coagulogen into an insoluble protein, coagulin. Hemolymph clotting is believed to contribute to the animal’s defense by preventing blood leakage and by trapping the pathogen. The immobilized microorganisms can subsequently be recognized by the large number of plasma and hemocyte-delivered lectins and killed by AMPs. Phagocytosis also contributes to the horseshoe crab defense reactions. Homologs of vertebrate complement C3 and C2/ Bf were reported from the horseshoe crab Carcinoscorpius rotundicauda and complement mediated phagocytosis of bacteria by hemocytes was observed (Zhu et al., 2005). These findings imply an early origin of core elements of the complement system. Further evidence for an archaic role of NF-kB signaling cascades in the antimicrobial defense is also provided from the study of this living fossil. Two NF-kB transcription factors, CrNF-kB and CrRelish, and an IkB homologue, CrIkB, have been cloned in C. rotundicauda. CrNF-kB belongs to the dorsal-like group of transcription factors and interacts with CrIkB, which inhibits its nuclear translocation. Bacterial
16
host defense: general
infection causes rapid degradation of CrIkB and subsequent release of CrNF-kB, which activates the expression of immune-related genes such as Factor C (Wang, 2006). Experimental data support the conservation of the CrRelish function and point to its up-regulation following bacterial infection, suggesting an involvement of CrRelish in antibacterial defense (Fan, 2008). A TRAF homolog has also been reported from C. rotundicauda (Wang, 2006). How these signaling cascades are activated is not yet established. A Toll receptor has been identified in the Japanese horseshoe crab Tachypleus tridentatus, called tToll, and is closely related to Drosophila Toll. Its biological function has not been investigated so far and its ligand has not been defined. The expression pattern of tToll does not suggest an immune function (reviewed in Inamori et al., 2004).
Mollusks and annelids
Investigations of the immune reactions of several Annelid and Mollusk species have revealed a potent cellular arm of defense, involving phagocytosis, encapsulation, and production of lytic activities often referred to as natural killer-like (NK) activities. These cellular defenses are combined with humoral reactions involving lysozymes and AMPs (reviewed in Salzet et al., 2006; Cheng-Hua et al., 2009). These reactions are mostly inducible. At present, the mechanisms controlling these responses are still poorly defined. In Mollusks, recognition of nonself relies in particular on lectins. Of special interest is the family of fibrinogenrelated proteins (FREPs) identified from the gastropod Biomphalaria glabrata, an intermediate host of the human parasite Schistosoma mansoni. FREPs were first described as plasma proteins that are induced following trematode infection. They bind to trematode sporocysts and precipitate glucan-bearing molecules released by the parasite. FREPs consist of one or two amino-terminal immunoglobulin superfamily (IgSF) domains and a carboxyterminal fibrinogen domain. Interestingly, for one subfamily, FREP3, it was observed that the IgSF1 is diversified at the genomic level at higher rates than those recorded for control genes. All sequence variants were found to derive from a small set of source sequences by point mutation and recombinatorial processes. Importantly, diverse FREP3 transcripts were also produced. Loker and colleagues, who discovered this phenomenon, have hypothesized that snails have evolved a mechanism capable of diversifying molecules involved in recognition during innate defenses (Zhang et al., 2004). Further, members of the PGRP and GNBP families have also been identified in snails and are proposed to contribute to the microbial detection in these species (reviewed in Bayne, 2009). In the context of immune signaling in Mollusks, genome-based approaches identified several components of NF-kB signaling pathways in various species. In the squid Euprymna scolop, several ESTs (expressed sequence tags) encoding homologues of proteins associated with putative Toll and IMD pathways were identified. These include a Toll receptor; four PGRPs; orthologues of several intracellular signaling mediators including IRAK, TRAF, and the IKK complex; an IkB-like protein; and two NF-kB-like transcription factors (Goodson, 2005). Evidence for the presence of NF-kB signaling pathways have also been reported from other Mollusk species, including an NF-kB transcription factor from the marine gastropod Haliotis diversicolor supertexta, a Toll receptor and a MyD88 homologue in the marine bivalve Chlamys farreri, and orthologues of NF-kB and IKKb from the pacific oyster Crassostrea gigas (Jiang & Wu, 2007; Qiu et al., 2007; Escoubas, 1999; Qiu, 2007; Montagnani et al., 2004).
In Annelids, the molecular mechanisms underlying the antimicrobial defenses are, as yet, poorly understood. The analysis of genome sequence reads of the leech Helobdella robusta and the polychaete Capitella capitata have identified homologues of Toll receptors in both species (Davidson et al., 2008). Surprisingly, the family of TLRs is largely expanded in Capitella with at least 105 members inferred from this analysis. The majority of these receptors are structurally related to vertebrate TLRs (i.e., having a membrane proximal single cystein cluster in their LRR ectodomain). In contrast, of the 16 Toll homologues identified in Helobdella, most show multiple cystein-rich clusters in their LRR ectodomain, as is the case for most insect Toll receptors. Mediators of the NF-kB signaling cascades have also been identified in both species, notably, two dorsal-like transcription factors and most mediators of the Toll signaling (but not yet of the IMD) pathway.
orIgIn of the Innate IMMUne sYsteM: the Innate IMMUne rePertoIre of CnIdarIa and PorIfera
The phylum Cnidaria includes aquatic species such as sea anemones, corals, and jellyfish. These animals provide crucial insights into the early origin of metazoans. The genome sequence is now available for two Cnidarian species: the sea anemone Nematostella vectensis and the fresh water polyp Hydra magnipapillata. Amazingly, analysis of these databases uncovered a complete Toll/TLR pathway in the genome of N. vectensis, indicating that this pathway predates the divergence between Cnidarians and Bilaterians some 600 million years ago (reviewed in Hemmrich et al., 2007). The Nematostella Toll receptor is characterized by multiple cystein clusters in its LRR extracellular domain, suggesting that this form likely represents the ancestral structural organization of Toll receptors. A single NF-kB transcription factor and its IkB inhibitory protein have been identified in the genome of N. vectensis together with many intracellular signaling mediators of the NF-kB signaling pathways, including MyD88, IRAK/Pelle, TRAF, TAK1, and elements of a IKK signalosome. In contrast, and quite intriguingly, the Hydra genome apparently lacks a conventional NF-kB transcription factor and a canonical Toll receptor, although it features two genes encoding transmembrane TIR-domain containing proteins with a short ectodomain deprived of LRR. Hydra mounts an inducible expression of AMPs upon infection and it has been suggested that the TIR-domain transmembrane molecules might function in an atypical signaling complex with separate LRR-containing proteins to control these Cnidarian immune defenses (Bosch, 2009). Other potential immune molecules that have been identified in the genome of the sea anemone are absent from Hydra. These include homologs of C3, Bf, and MASP elements involved in the complement cascades. These observations suggest that the multicomponent complement system was already established in the genome of the common ancestor of Cnidaria and Bilateria (Kimura et al., 2009). The absence of these elements from Hydra likely reflects secondary gene losses during evolution. Evidence for MAPK signaling pathways (including JNK, p38, and AP1) has also been obtained from the analysis of the genomes of both Cnidarian species. Sponges, which belong to the phylum Porifera, represent an even earlier branching lineage than Cnidaria, occupying a basal position near the root of the metazoan tree of life. Investigations of cDNA libraries from the sponge Suberites domuncula and a thorough analysis of the genome of Amphimedon queenslandica have revealed the presence of an impressive armamentarium of genes encoding potential
1. Invertebrate Innate Immune defenses
recognition proteins, including scavenger receptors and several members of NF-kB signaling pathways (reviewed in Hemmrich et al., 2007). Of note, in contrast to the sea anemone NF-kB, the sponge NF-kB is structurally related to Drosophila relish, in that it carries its own inhibitory sequences. The expression pattern of AmqNF-kB during embryonic and larval stages suggests a developmental role, although these observations do not exclude a potential defense function of AmqNF-kB (Gauthier & Degnan, 2008).
ConClUsIons
As illustrated in this presentation, we now have at our disposal a wealth of information on innate defenses in invertebrates ranging from relatively simple organisms to highly evolved animals. Obviously, for many species, we still lack crucial functional data and the cellular and molecular mechanisms underlying the immune reactions still await further analysis in several large invertebrate phyla. In all frankness, we must admit that even our view of the much studied Drosophila immunity is still fragmentary and that it requires intense further investigations. With these caveats in mind, the essential message that we want to convey here is that innate immunity, as we understand it today, has appeared very early in life’s history, probably with the first multicellular organisms. There is clearly a common thread through all invertebrate phyla regarding recognition of microorganisms, activation of intracytoplasmic signaling cascades, and induction of gene programs that culminate in the synthesis of potent defense molecules, among which the antimicrobial peptides feature prominently. Recognition of microorganisms by the invertebrate immune system is a field that has significantly advanced in recent years. A variety of phagocytic receptors have been described, most of which have mammalian counterparts. They interact with an intracellular machinery of vesicular trafficking and cytoskeleton rearrangements, ultimately leading to large phagosomal structures where microorganisms can be disposed of. Again, from our recent information, it appears that most of the players in a few well-studied cases are highly similar to the molecules that govern these processes in mammals. This hardly comes as a surprise as it is largely surmised that phagocytosis evolved very early in evolution for purposes of nutrition, development, and defenses. Intriguingly, some putative phagocytic receptors, such as Dscam in Drosophila and the circulating opsonin-like FREPs in Mollusks, which feature IgSF domains, are encoded by genes that have the potential to generate multiple receptor isoforms. It will be interesting to follow future studies on the functional relevance of these arrangements. Parallels with the now well-established variable lymphocytes receptor (VLR) system of jawless fish (Agnathans) recently described by Cooper and colleagues (Pancer, 2004) appear premature at this stage. Every subfield has its own potential pitfalls. This is also the case in the Toll/TLR context. In the best documented cases, that is Drosophila and mammals, a very important difference is that the Toll receptor becomes activated by a cleaved form of the cytokine spaetzle, whereas mammalian TLRs interact directly with microbial ligands. The expression “pattern recognition receptors,” coined by Janeway (1989) thus perfectly applies to mammalian TLRs but not to that of the Drosophila Toll. This remark underlines that, pending functional studies, we are uncertain how to interpret results on dozens or more Toll/TLRs in various invertebrates. Therefore, it is certainly premature to propose that the increase in number may reflect environmental pressures by microorganisms.
17
One of the beauties of innate immunity, as illustrated by the case of Drosophila, is that the number of microbial ligands that actually activate gene reprogramming is extremely limited: Firm evidence exists to date only for PGNs and b-glucans. More recently, viral RNAs have been added to the list. The receptors, as we see them today, have essentially derived from ancestral enzymes, namely amidases, glucanases, and helicases. In Drosophila at least, the number of documented receptors is less than a dozen proteins to date. Would a larger repertoire be beneficial for this organism? We believe that a larger repertoire of receptors has to be viewed in the context of lymphocyte development, clonal selection, and expansion and memory. In the absence of these features, increasing the repertoire of innate immune receptors might be of little improvement for the defense, but we are ready to reevaluate our view if clear and reproducible data will instruct us otherwise in the future. In the early days of our research on the signaling cascades that led to the expression of immune genes in Drosophila, we were extremely interested by the parallels between these cascades and those of mammalian immune defenses. This is now commonplace, but the data reviewed here strongly suggest that these cascades were in place already at the very earliest stages of evolution. Again, we lack functional data for most of the species in which NF-kB is present, but we anticipate that future studies will show that from sponges to insects, and further to mammals, similar molecular modules direct essentially similar cascades to transcribe gene programs that ultimately are beneficial to the host and deleterious to the invader. Further, as illustrated above, many of the effector molecules appear to be similar in the various phyla and are present in vertebrates as well. An interesting question pertains to the parallel evolutions of developmental regulations and immune defenses. How do we explain that Drosophila has nine Toll receptors and six spaetzle paralogs, three of which have been documented to act as neurotrophins (Zhu et al., 2008), all of which are involved in development and that just one pair, Toll-1 spaetzle, were selected to direct antifungal and some antibacterial defenses? We hope that functional studies in other phyla will help clarify our view on these dual roles in immune and developmental regulation during evolution. Finally, a word of apology to our friends in the growing field of Caenorhabditis elegans defense reactions. C. elegans lacks so many of the features described in this review (namely NF-kB) that we have decided to put this organism apart and refer to excellent recent reviews covering this special field (Schulenburg et al., 2004; Kim & Ausubel, 2005; Shivers et al., 2008). The common view in the community today is that C. elegans has lost most of the conventional traits of the innate immune system of invertebrates as they were described here. In fact, a similar situation might also prevail for the Cnidarian Hydra. Although innate immunity was discovered in invertebrates by Metchnikov nearly 130 years ago, it has long remained a relatively neglected area of research. In the 1990s, molecular genetics have given a potent stimulus to the field, initially in Drosophila and now in many phylogenetically diverse groups. The progress has been astounding over the last 15 years and hundreds of laboratories worldwide are now engaged in research on innate immunity. Moreover, in some precise areas of high significance (e.g., the discovery of antimicrobial peptides, the role of Toll receptors in innate immunity, and the role of peptidoglycan recognition proteins) invertebrate immunity has led the way. We hope that this thread will continue and that this review article has stimulated some interest among potential future students of the field.
18
host defense: general
The authors apologize for their inability to quote many additional contributions due to space limitations. Work in the laboratory of the authors has been generously supported by the French National Research Agency (CNRS), the National Institute of Health (NIH), the European Union, and the University of Strasbourg. We thank our past and present associates in the laboratory for their contributions and for continuous helpful discussions.
REFERENCES Agaisse, H., and N. Perrimon. 2004. The roles of JAK/STAT signaling in Drosophila immune responses. Immunol. Rev. 198:72–82. Aggarwal, K., and N. Silverman. 2008. Positive and negative regulation of the Drosophila immune response. BMB Rep. 41:267–277. Aggarwal, K., F. Rus, C. Vriesema-Magnuson, D. ErturkHasdemir, N. Paquette, and N. Silverman. 2008. Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway. PLoS Pathog. 4:e1000120. Antonova, Y., K. S. Alvarez, Y. J. Kim, V. Kokoza, and A. S. Raikhel. 2009. The role of NF-kappaB factor REL2 in the Aedes aegypti immune response. Insect Biochem. Mol. Biol. 39:303–314. Bayne, C. J. 2009. Successful parasitism of vector snail Biomphalaria glabrata by the human blood fluke (trematode) Schistosoma mansoni: a 2009 assessment. Mol. Biochem. Parasitol. 165:8–18. Bhoj, V. G., and Z. J. Chen. 2009. Ubiquitylation in innate and adaptive immunity. Nature 458:430–437. Bian, G., S. W. Shin, H. M. Cheon, V. Kokoza, and A. S. Raikhel. 2005. Transgenic alteration of Toll immune pathway in the female mosquito Aedes aegypti. Proc. Natl. Acad. Sci. USA 102:13568–13573. Blandin, S. A., E. Marois, and E. A. Levashina. 2008. Antimalarial responses in Anopheles gambiae: from a complement-like protein to a complement-like pathway. Cell Host Microbe 3:364–374. Bosch, T. C., R. Augustin, F. Anton-Erxleben, S. Fraune, G. Hemmrich, H. Zill, P. Rosenstiel, G. Jacobs, S. Schreiber, M. Leippe, M. Stanisak, J. Grötzinger, S. Jung, R. Podschun, J. Bartels, J. Harder, and J. M. Schröder. 2009. Uncovering the evolutionary history of innate immunity: the simple metazoan Hydra uses epithelial cells for host defence. Dev. Comp Immunol. 33:559–569. Buchon, N., M. Poidevin, H. M. Kwon, A. Guillou, V. Sottas, B. L. Lee, and B. Lemaitre. 2009. A single modular serine protease integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway. Proc. Natl. Acad. Sci. USA 106:12442–12447. Cerenius, L., and K. Soderhall. 2004. The prophenoloxidase-activating system in invertebrates. Immunol. Rev. 198:116–126. Charroux, B., T. Rival, K. Narbonne-Reveau, and J. Royet. 2009. Bacterial detection by Drosophila peptidoglycan recognition proteins. Microbes Infect. 11:631–636. Charroux, B., and J. Royet. 2009. Elimination of plasmatocytes by targeted apoptosis reveals their role in multiple aspects of the Drosophila immune response. Proc. Natl. Acad. Sci. USA 106:9797–9802. Cheng-Hua, L., Z. Jian-Min, and S. Lin-Sheng. 2009. A review of advances in research on marine molluscan antimicrobial peptides and their potential application in aquaculture. Molluscan Research 29:17–26. Christophides, G. K., D. Vlachou, and F. C. Kafatos. 2004. Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae. Immunol. Rev. 198:127–148. Christophides, G. K., E. Zdobnov, C. Barillas-Mury, E. Birney, S. Blandin, C. Blass, P. T. Brey, F. H. Collins, A. Danielli, G. Dimopoulos, C. Hetru, N. T. Hoa, J. A. Hoffmann, S. M. Kanzok, I. Letunic, E. A. Levashina, T. G. Loukeris, G. Lycett, S. Meister, K. Michel, L. F. Moita,
H. M. Müller, M. A. Osta, S. M. Paskewitz, J. M. Reichhart, A. Rzhetsky, L. Troxler, K. D. Vernick, D. Vlachou, J. Volz, C. von Mering, J. Xu, L. Zheng, P. Bork and F. C. Kafatos. 2002. Immunity-related genes and gene families in Anopheles gambiae. Science 298:159–165. Davidson, C. R., N. M. Best, J. W. Francis, E. L. Cooper, and T. C. Wood. 2008. Toll-like receptor genes (TLRs) from Capitella capitata and Helobdella robusta (Annelida). Dev. Comp. Immunol. 32:608–612. Defaye, A., I. Evans, M. Crozatier, W. Wood, B. Lemaitre, and F. Leulier. 2009. Genetic ablation of Drosophila phagocytes reveals their contribution to both development and resistance to bacterial infection. J. Innate Immun. 1:322–334. El Chamy, L., V. Leclerc, I. Caldelari, and J. M. Reichhart. 2008. Sensing of ‘danger signals’ and pathogen-associated molecular patterns defines binary signaling pathways ‘upstream’ of Toll. Nat. Immunol. 9:1165–1170. Erturk-Hasdemir, D., M. Broemer, F. Leulier, W. S. Lane, N. Paquette, D. Hwang, C. H. Kim, S. Stöven, P. Meier, and N. Silverman. 2009. Two roles for the Drosophila IKK complex in the activation of Relish and the induction of antimicrobial peptide genes. Proc. Natl. Acad. Sci. USA 106:9779–9784. Escoubas, J. M., L. Briant, C. Montagnani, S. Hez, C. Devaux, and P. Roch. 1999. Oyster IKK-like protein shares structural and functional properties with its mammalian homologues. FEBS Lett. 453:293–298. Evans, J. D., K. Aronstein, Y. P. Chen, C. Hetru, J. L. Imler, H. Jiang, M. Kanost, G. J. Thompson, Z. Zou, and D. Hultmark. 2006. Immune pathways and defence mechanisms in honey bees Apis mellifera. Insect Mol. Biol. 15:645–656. Fan, Z. H., X. M. Wang, J. Lu, B. Ho, and J. L. Ding. 2008. Elucidating the function of an ancient NF-kappaB p100 homologue, CrRelish, in antibacterial defense. Infect. Immun. 76:664–670. Ferrandon, D., J. L. Imler, C. Hetru, and J. A. Hoffmann. 2007. The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat. Rev. Immunol. 7:862–874. Gauthier, M., and B. M. Degnan. 2008. The transcription factor NF-kappaB in the demosponge Amphimedon queenslandica: insights on the evolutionary origin of the Rel homology domain. Dev. Genes Evol. 218:23–32. Georgel, P., S. Naitza, C. Kappler, D. Ferrandon, D. Zachary, C. Swimmer, C. Kopczynski, G. Duyk, J. M. Reichhart, and J. A. Hoffmann. 2001. Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis. Dev. Cell 1:503–514. Goodson, M. S., M. Kojadinovich, J. V. Troll, T. E. Scheetz, T. L. Casavant, M. B. Soares, and M. J. McFall-Ngai. 2005. Identifying components of the NF-kappaB pathway in the beneficial Euprymna scolopes-Vibrio fischeri light organ symbiosis. Appl. Environ. Microbiol. 71:6934–6946. Goto, A. K. Matsushita, V. Gesellchen, L. El Chamy, D. Kuttenkeuler, O. Takeuchi, J. A. Hoffmann, S. Akira, M. Boutros, and J. M. Reichhart. 2008. Akirins are highly conserved nuclear proteins required for NF-kappaB-dependent gene expression in Drosophila and mice. Nat. Immunol. 9:97–104. Gottar, M. V. Gobert, A. A. Matskevich, J. M. Reichhart, C. Wang, T. M. Butt, M. Belvin, J. A. Hoffmann, and D. Ferrandon. 2006. Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell 127:1425–1437. Ha, E. M. K. A. Lee, S. H. Park, S. H. Kim, H. J. Nam, H. Y. Lee, D. Kang, and W. J. Lee. 2009. Regulation of DUOX by the Galphaq-phospholipase Cbeta-Ca21 pathway in Drosophila gut immunity. Dev. Cell 16:386–397. Hemmrich, G., D. J. Miller, and T. C. Bosch. 2007. The evolution of immunity: a low-life perspective. Trends Immunol. 28:449–454. Hoffmann, J. A. 2003. The immune response of Drosophila. Nature 426:33–38.
1. Invertebrate Innate Immune defenses Imler, J. L., and P. Bulet. 2005. Antimicrobial peptides in Drosophila: structures, activities and gene regulation. Chem. Immunol. Allergy 86:1–21. Inamori, K., S. Ariki, and S. Kawabata. 2004. A Toll-like receptor in horseshoe crabs. Immunol. Rev. 198:106–115. Iwanaga, S., and B. L. Lee. 2005. Recent advances in the innate immunity of invertebrate animals. J. Biochem. Mol. Biol. 38:128–150. Janeway, C. A., Jr. 1989. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb. Symp. Quant. Biol. 54 Pt 1:1–13. Jiang, Y., and X. Wu. 2007. Characterization of a Rel\NFkappaB homologue in a gastropod abalone, Haliotis diversicolor supertexta. Dev. Comp. Immunol. 31:121–131. Kawai, T., and S. Akira. 2009. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int. Immunol. 21:317– 337. Kemp, C., and J. L. Imler. 2009. Antiviral immunity in Drosophila. Curr. Opin. Immunol. 21:3–9. Khalturin, K., Z. Panzer, M. D. Cooper, and T. C. Bosch. 2004. Recognition strategies in the innate immune system of ancestral chordates. Mol. Immunol. 41:1077–1087. Kim, D. H., and F. M. Ausubel. 2005. Evolutionary perspectives on innate immunity from the study of Caenorhabditis elegans. Curr. Opin. Immunol. 17:4–10. Kimura, A., E. Sakaguchi, and M. Nonaka. 2009. Multicomponent complement system of Cnidaria: C3, Bf, and MASP genes expressed in the endodermal tissues of a sea anemone, Nematostella vectensis. Immunobiology 214:165–178. Kleino, A., H. Myllymaki, J. Kallio, L. M. Vanha-aho, K. Oksanen, J. Ulvila, D. Hultmark, S. Valanne, and M. Ramet. 2008. Pirk is a negative regulator of the Drosophila Imd pathway. J. Immunol. 180:5413–5422. Kurata, S., S. Ariki, and S. Kawabata. 2006. Recognition of pathogens and activation of immune responses in Drosophila and horseshoe crab innate immunity. Immunobiology 211:237–249. Lemaitre, B., and J. Hoffmann. 2007. The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 25:697–743. Leulier, F., and B. Lemaitre. 2008. Toll-like receptors—taking an evolutionary approach. Nat. Rev. Genet. 9:165–178. Lhocine, N., P. S. Ribeiro, N. Buchon, A. Wepf, R. Wilson, T. Tenev, B. Lemaitre, M. Gstaiger, P. Meier, and F. Leulier. 2008. PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling. Cell Host Microbe 4:147–158. Maillet, F., V. Bischoff, C. Vignal, J. Hoffmann, and J. Royet. 2008. The Drosophila peptidoglycan recognition protein PGRP-LF blocks PGRP-LC and IMD/JNK pathway activation. Cell Host Microbe 3:293–303. Meister, M. 2004. Blood cells of Drosophila: cell lineages and role in host defence. Curr. Opin. Immunol. 16:10–15. Meister, S., S. M. Kanzok, X. L. Zheng, C. Luna, T. R. Li, N. T. Hoa, J. R. Clayton, K. P. White, F. C. Kafatos, G. K. Christophides, and L. Zheng. 2005. Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae. Proc. Natl. Acad. Sci. USA 102:11420–11425. Montagnani, C., C. Kappler, J. M. Reichhart, and J. M. Escoubas. 2004. Cg-Rel, the first Rel/NF-kappaB homolog characterized in a mollusk, the Pacific oyster Crassostrea gigas. FEBS Lett. 561:75–82. Pancer, Z., C. T. Amemiya, G. R. Ehrhardt, J. Ceitlin, G. L. Gartland, and M. D. Cooper. 2004. Somatic diversification of variable lymphocyte receptors in the Agnathan sea lamprey. Nature 430:174–180. Qiu, L., L. Song, W. Xu, D. Ni, and Y. Yu. 2007. Molecular cloning and expression of a Toll receptor gene homologue from Zhikong Scallop, Chlamys farreri. Fish Shellfish Immunol. 22:451–466. Qiu, L., L. Song, Y. Yu, W. Xu, D. Ni, and Q. Zhang. 2007. Identification and characterization of a myeloid differen-
19
tiation factor 88 (MyD88) cDNA from Zhikong scallop Chlamys farreri. Fish Shellfish Immunol. 23:614–623. Rast, J. P., and C. Messier-Solek. 2008. Marine invertebrate genome sequences and our evolving understanding of animal immunity. Biol. Bull. 214:274–283. Roh, K. B., C. H. Kim, H. Lee, H. M. Kwon, J. W. Park, J. H. Ryu, K. Kurokawa, N. C. Ha, W. J. Lee, B. Lemaitre, K. Soderhall, and B. L. Lee. 2009. Proteolytic cascade for the activation of the insect toll pathway induced by the fungal cell wall component. J. Biol. Chem. 284: 19474–19481. Royet, J., J. M. Reichhart, and J. A. Hoffmann. 2005. Sensing and signaling during infection in Drosophila. Curr. Opin. Immunol. 17:11–17. Ryu, J. H., S. H. Kim, H. Y. Lee, J. Y. Bai, Y. D. Nam, J. W. Bae, D. G. Lee, S. C. Shin, E. M. Ha, and W. J. Lee. 2008. Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science 319:777–782. Sackton, T. B., B. P. Lazzaro, T. A. Schlenke, J. D. Evans, D. Hultmark, and A. G. Clark. 2007. Dynamic evolution of the innate immune system in Drosophila. Nat. Genet. 39:1461–1468. Salzet, M., A. Tasiemski, and E. Cooper. 2006. Innate immunity in lophotrochozoans: the annelids. Curr. Pharm. Des. 12:3043–3050. Schulenburg, H., C. L. Kurz, and J. J. Ewbank. 2004. Evolution of the innate immune system: the worm perspective. Immunol. Rev. 198:36–58. Shin, S. W., V. Kokoza, G. Bian, H. M. Cheon, Y. J. Kim, and A. S. Raikhel. 2005. REL1, a homologue of Drosophila dorsal, regulates toll antifungal immune pathway in the female mosquito Aedes aegypti. J. Biol. Chem. 280:16499–16507. Shin, S. W., G. Bian, and A. S. Raikhel. 2006. A toll receptor and a cytokine, Toll5A and Spz1C, are involved in toll antifungal immune signaling in the mosquito Aedes aegypti. J. Biol. Chem. 281:39388–39395. Shivers, R. P., M. J. Youngman, and D. H. Kim. 2008. Transcriptional responses to pathogens in Caenorhabditis elegans. Curr. Opin. Microbiol. 11:251–256. Stuart, L. M., and R. A. Ezekowitz. 2008. Phagocytosis and comparative innate immunity: learning on the fly. Nat. Rev. Immunol. 8:131–141. Tanaka, H., J. Ishibashi, K. Fujita, Y. Nakajima, A. Sagisaka, K. Tomimoto, N. Suzuki, M. Yoshiyama, Y. Kaneko, T. Iwasaki, T. Sunagawa, K. Yamaji, A. Asaoka, K. Mita, and M. Yamakawa. 2008. A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem. Mol. Biol. 38:1087–1110. Tang, H., Z. Kambris, B. Lemaitre, and C. Hashimoto. 2008. A serpin that regulates immune melanization in the respiratory system of Drosophila. Dev. Cell. 15:617–626. Wang, X. W. N. S. Tan, B. Ho, and J. L. Ding. 2006. Evidence for the ancient origin of the NF-kappaB/IkappaB cascade: its archaic role in pathogen infection and immunity. Proc. Natl. Acad. Sci. USA 103:4204–4209. Wang, Y., T. Cheng, S. Rayaprolu, Z. Zou, Q. Xia, Z. Xiang, and H. Jiang. 2007. Proteolytic activation of pro-spatzle is required for the induced transcription of antimicrobial peptide genes in lepidopteran insects. Dev. Comp. Immunol. 31:1002–1012. Waterhouse R. M., E. V. Kriventseva, S. Meister, Z. Xi, K. S. Alvarez, L. C. Bartholomay, C. Barillas-Mury, G. Bian, S. Blandin, B. M. Christensen, Y. Dong, H. Jiang, M. R. Kanost, A. C. Koutsos, E. A. Levashina, J. Li, P. Ligoxygakis, R. M. Maccallum, G. F. Mayhew, A. Mendes, K. Michel, M. A. Osta, S. Paskewitz, S. W. Shin, D. Vlachou, L. Wang, W. Wei, L. Zheng, Z. Zou, D. W. Severson, A. S. Raikhel, F. C. Kafatos, G. Dimopoulos, E. M. Zdobnov, and G. K. Christophides. 2007. Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes. Science 316:1738–1743.
20
host defense: general
Xi, Z., J. L. Ramirez, and G. Dimopoulos. 2008. The Aedes aegypti toll pathway controls dengue virus infection. PLoS Pathog. 4:e1000098. Zhang, S. M., C. M. Adema, T. B. Kepler, and E. S. Loker. 2004. Diversification of Ig superfamily genes in an invertebrate. Science 305:251–254. Zhu, B., J. A. Pennack, P. McQuilton, M. G. Forero, K. Mizuguchi, B. Sutcliffe, C. J. Gu, J. C. Fenton, and A. Hidalgo. 2008. Drosophila neurotrophins reveal a com-
mon mechanism for nervous system formation. PLoS Biol. 6:e284. Zhu, Y., S. Thangamani, B. Ho, and J. L. Ding. 2005. The ancient origin of the complement system. EMBO J. 24:382–394. Zou, Z., J. D. Evans, Z. Lu, P. Zhao, M. Williams, N. Sumathipala, C. Hetru, D. Hultmark, and H. Jiang. 2007. Comparative genomic analysis of the Tribolium immune system. Genome Biol. 8:R177.
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
2 The Ontogeny of the Cells of the Innate and the Adaptive Immune System FRITZ MELCHERS
INTRODUCTION
Recognition of specific antigen by binding at sufficiently high avidity induces clonal lymphocyte proliferation and maturation to effector functions—in the case of T cells to helper, cytotoxic, or regulatory cells; in the case of B cells to antigen receptor-hypermutated, Ig class-switched cells and Ig-secreting plasma cells, which generate and maintain the Ig levels in the extracellular spaces, notably the blood, or, after transport through epithelial layers, in the lumen of the gut.
A single pluripotent hematopoietic stem cell (pHSC) can be the progenitor of all the erythrocytes, megakaryocytes, and blood platelets, of all the cells of the myeloid cell lineages, of all the natural killer (NK) cells, of all the different functional types of T lymphocytes, and of all the sublineages of B lymphocytes. Hence, the ontogeny of these different types of blood cells, the cells of the innate and the adaptive immune system, is very complex. Some of the major molecular steps and cellular stages, including a few of the interactions of the developing hematopoietic cells with the endogenous environment of cooperating cells, as well as with the exogenous environment of antigens and polyclonal activators will be described, mainly with the mouse but also with references to the development in humans. Development begins, shortly after the fertilization of the egg, with the development of embryonic stem (ES) cells; from there, the progenitors of the vascular and hematopoietic cells (called hemangioblasts) are developed; and thereafter is the generation of a pluripotent hematopoietic stem cell. From that point, it continues first in embryonic development and, thereafter, throughout life through the different stages of hematopoietic, myeloid, and lymphoid progenitors, and finally to mature cells of the myeloid cells and of NK cells (i.e., of the innate immune system) and to the development of antigen receptor-expressing lymphocytes of the adaptive immune system and their deposition and use in different parts of the immune system.
The Clonal Selection Hypothesis
The clonal selection hypothesis (Jerne, 1955; Burnet, 1957; Talmage, 1959; Nossal & Lederberg, 1958) proposes that one lymphocyte makes only one structure of an antigenbinding receptor, that this one structure on the surface of one lymphocyte binds one antigen of the wide world of structures (antigens) surrounding these lymphocytes, and that many different antigens bind to many different antigen receptors, but always one antigen to one of the wide world of lymphocytes in the system. To deal with the tolerance of the immune system to its own antigens, Lederberg (1959) extended this hypothesis by proposing that the emerging repertoires of antigen receptors on lymphocytes would first enter a phase—today recognized as immature lymphocytes—in which binding of the body’s own antigens, the autoantigens, would select their specific cells and eliminate them. The surviving lymphocytes would then further differentiate with the next stage of cellular development—now known as mature lymphocytes—in which binding of a foreign antigen would initiate a positive immune response (i.e., the proliferation and maturation of cells to effector functions that the system can use to eliminate the invading antigen). One prediction of this hypothesis was that the second allele of an antigen receptor-encoding gene would not be activated for expression in a single lymphocyte, termed allelic exclusion of the receptor loci. Another expectation was that a single lymphocyte would only rarely find a second, structurally nonidentical antigen to which it could bind to elicit a response. Furthermore, autoantigens would only negatively select and foreign antigens would only positively select cells of the system for a response.
The Adaptive Immune System of Lymphocytes
The major hallmark and specific feature of T and B lymphocytes is their capacity to produce a wide variety of antigenbinding receptors, in T cells, the a/b- or g/dT-cell receptors (TcR), and in B cells, immunoglobulin (Ig) heavy/light (H/L) chain-containing B-cell receptors (BcR), which they deposit for antigen recognition on their surface.
Fritz Melchers, Max Planck Institute for Infection Biology, Senior Research Group on Lymphocyte Development, Charitéplatz 1, D 10117, Berlin, Germany.
21
22
host defense: general
Our current knowledge of the generation and selection of lymphocyte receptor repertoires shows that the clonal selection hypothesis, in its initial definition, is often no longer tenable. Nevertheless, the adaptive immune system manages to engage only a small part of its final, available repertoire of antigen receptors on lymphocytes for the recognition of, and response to, an antigen.
DEVELOPMENT OF HEMATOPOIETIC CELLS AND LYMPHOCYTES Embryonic and Adult Development In Vivo
Three waves of hematopoietic cell developments colonize the mouse embryo (Ling & Dzierzak, 2002; Godin & Cumano, 2002; Cumano et al., 2001). The first begins in extra embryonic tissue (i.e., in the yolk sac at day 7 to 7.5 of development). This so-called primitive hematopoiesis (which shows similarities to hematopoiesis in lower vertebrates) generates primitive erythrocytes (synthesizing “fetal” hemoglobin), megakaryocytes (with lower ploidy) and platelets, and myeloid cells (such as macrophages with a special set of enzymes) (Shivdasani et al., 1995). Lymphocytes are not generated. Although these primitive blood cell lineages are developed at extraembryonic, ventral sides of the embryo, they migrate to the embryonic, dorsal side as soon as blood circulation is established at day 8 to 9 of development with the development of vascular endothelium. At day 8.5 to 9 of mouse embryonic development, the second wave of hematopoiesis is started within the intraembryonic, anterior portion of the aorta-gonad-mesonephros region (Medvinsky & Dzierzak, 1996; Cumano et al., 1996). Undifferentiated, apparently pluripotent stem cells (Ohmura et al., 2001) then migrate through the blood and colonize the rudiments of the thymus and fetal liver. At these sites, the second, so-called definitive wave of hematopoiesis is initiated. It now generates erythrocytes producing adulttype hemoglobin, definitive megakaryocytes and platelets, and adult-type myeloid cells and lymphocytes. In the thymus, several waves of thymopoiesis are initiated. The T cells that are generated in these first, fetal waves of development from day 16 of mouse embryonic development onwards are first g/d and, thereafter, a/b TcRexpressing cells, all with little or no N-region insertions at their V(D)J junctions because of the lack of expression of the enzyme TdT, and they display a restricted repertoire of v-region combinations. They migrate to intraepithelial sites of the body where they might perform first line of defense functions. In the fetal liver of the mouse, between day 13 of embryonic development until birth at day 19, a first, apparently synchronous wave of B-lymphocytes is generated (Strasser et al., 1989). Two other sites of embryonic B-cell development, generating B cells with properties similar to those of the fetal liver, are the embryonic side of the placenta and the omentum (Kincade, 1981; Owen et al., 1976; Melchers, 1979; Gekas et al., 2005, Ottersbach & Dzierzak, 2005; Solvason & Kearney, 1992). Since most of these B cells do not express the enzyme TdT, they rearrange the V(D) J segment of the IgH and IgL chain loci without N-region diversity. The early waves of embryonic B cells preferentially home to the gut-associated lymphoid tissues, belong, in large part, to the BIa-types of B cells, and form a first line of defense and cohabitation against and with the developing intestinal bacterial flora. The third wave of hematopoiesis (i.e., the second definitive hematopoiesis) starts in bone marrow between days 17
and 19 (i.e., at around the birth of a mouse) (Kincade et al., 2002). It initiates a continuous process of hematopoietic cell generation throughout life. B cells develop to form at least three distinct lineages: B1a, B1b, and B2 cells (Melchers, 2009; Tung & Herzenberg, 2007). They are then distributed into at least two major subcompartments of mature, BcR-expressing B-lymphocytes of the mature, peripheral system. Approximately half of them are found in the follicular regions of the spleen and lymph nodes and in the recirculating blood and lymph, a majority of which are the B2-type B cells. The other half is mainly found in the gut-associated lymphoid tissues (e.g., in Peyer’s patches, either in follicular structures near the flat M-cell regions of the gut epithelia, or as single intraepithelial lymphocytes in the lamina propria of the gut). At least a majority of these B cells appear to belong to these so-called BI subpopulations. Most B1a cells develop during the prenatal period in the fetal liver from precursors that lack lg rearrangements and are distinct from those that give rise to B1b and B2 cells (Tung & Herzenberg, 2007). B1a cells persist for some time after birth even though they are derived from precursors that disappear during the neonatal period. B1a cells contribute a large part to the natural lgM antibodies in serum, respond to T cell-independent antigens, express unmutated V regions, contain very few (if any) N-region sequences in their complementarity determining region 3 (CDR3) sequences, and need the spleen to develop (Wardemann et al., 2002). T cells develop in the thymus. Progenitors from bone marrow with myeloid and lymphoid, or only lymphoid potentials migrate to the thymus to continuously generate, until sexual maturity, the different subpopulations of T cells. After sexual maturity, the thymus begins to involute and decreases T-cell production progressively. Even after adult thymectomy the mature T-cell compartments are maintained in size and function, indicating that T-cell maintenance in the system becomes extrathymic. The early stages of thymocytes are CD4 through CD8 and are either CD441CD252 (DN1), CD441CD251 (DN2), CD442CD251 (DN3) or CD442CD252 (DN4) cells in that developmental sequence. At the transition of DN2 to DN3 cells the expression of TcRs decides whether they develop either to a/bTcR- or to g/dTcR-expressing cells. The a/bTcR1 cells become CD41CD81 (DP) cells that are then selected to either the CD41 (SP) or the CD81 (SP) lineages of cells. After exit from the thymus, the a/bT cells mature to either regulatory, helper, or cytotoxic functions. The CD41 a/b T cells further differentiate into helper type 1 (Th1), helper type 2 (Th2), follicular B helper T, helper T-17 (Th17), or regulatory CD41CD251 T cells. The CD81 a/b T cells differentiate into type 1 or type 2 cytotoxic T cells (Tc1,Tc2), or NK T cells (NKT). g/dT cells also differentiate to helper or cytotoxic cells. It will be described below that the specificity of the TcRs determines lineage choice (Kyewski & Klein, 2006; Haks et al., 2005; Hayes et al., 2005; Kreslavsky et al., 2009). In human embryonic development, mature T and B lymphocytes are already detectable at 10 to 12 weeks, hence, well before birth. Therefore, human embryos have mature, antigen-reactive lymphocytes several months before birth, ready to react with a positive memory response to a foreign antigen if challenged by one. However, a more detailed description of prenatal human hematopoietic development is missing, as is a detailed assessment of the postnatal changes in these developments. It is safe to expect that immune systems of mice, as well as of humans, will be different at different times of an aging individual.
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
Development of Hematopoietic Cells from Embryonic Stem (ES) Cells In Vitro
From the 64cell blastocyst stage of the mouse embryo at day 3.5 of development—and from the comparable stage of human embryonic development—ES cell lines have been established in tissue culture that will proliferate for many generations without losing their ability to participate in the in vivo development of all cell lineages when reinjected into a recipient blastocyst and retransplanted into a foster mother, leading to a fully chimeric mouse (Martin, 1981; Evans & Kaufman, 1981). Furthermore, mouse ES cells are capable of homologous recombination (i.e., of introduction of genes) without the help of retroviral transfection at their sites in the chromosomes, thereby allowing proper transgenic changes and repair of selected gene loci (Matise et al., 2000). Furthermore, ES cells can be induced in vitro to differentiate to various cell lineages, notably also to hematopoietic cells and, more specifically, to cells of the innate and the adaptive immune system. These ES cell-differentiated hematopoietic cells, depending on their state of differentiation, can be transplanted in recipients to populate some or all of the hematopoietic compartments. It is surprising that these tissue culture conditions can be chosen in such a way that they will follow the embryonic time program in a wave of development, such that mesodermal cells appear at day 5 (equivalent to 8.5 of embryonic development), pHSC at day 8 to 9 (equivalent to 11.5 to 12.5), and T- and B-lymphocyte progenitors and precursors after day 12 (equivalent to day 15.5) (Nakano et al., 1994; Tsuneto, in preparation). Since it is now also possible to generate ES cell-like iPS (induced pluripotent stem) cells from peripheral, differentiated cells such as epithelial cells or lymphocytes, (Okita et al., 2007; Wernig et al., 2007; Hanna, 2008) and from them transplantation-tolerant hematopoietic cells, all in tissue culture, the study and use of any defined or mutable gene in its function in the development of the innate and the adaptive immune system can be studied. Such studies will help to develop protocols for genetic therapies of defective, disease-inducing gene loci in histocompatible ES-like iPS cells and their subsequent in vitro development to transplantable pHSC, with the hope to cure immunodeficiencies and, hopefully, to interfere with autoimmune diseases.
CELLULAR STAGES OF HEMATOPOIETIC DEVELOPMENT Pluripotent Hematopoietic Stem Cells
Pluripotent hematopoietic stem cells (pHSCs) generate the cells of the innate and the adaptive immune system first as the embryo develops, and thereafter continuously throughout life, not only to build up, but to maintain the numbers of cells (estimated as more than 2 3 109 in an adult mouse, and more than 2 3 1012 in an adult human, of which a large part turns over with half-lives of less than a week. These pHSCs derive from embryonal mesoderm, which also generates the environments of the hematopoietic system. These environments are the vascular endothelial cells that form blood and lymph vessels through which the blood cells travel and the microenvironmental stromal cells, forming hematopoietic “niches” in bone marrow and thymus for the developing progenitors (Jordan & Lemischka, 1990; Spangrude et al., 1991; Smith et al., 1991; Morrison et al., 1995; Osawa et al., 1996a, 1996b). The cooperating stromal cells that help to induce these different hematopoietic lineage differentiations belong, in part, to mesenchymal stem-cell-derived osteoblasts, and, in other parts, to epithelial cells. Osteoblasts have been found
23
to be stimulatory for some early hematopoietic progenitor functions, while differentiated adipocytes are either no longer stimulatory, or even inhibitory (unpublished observations). In part, this may be due to the spectrum of cytokines produced by these different cellular stages of mesenchymal cell differentiation. Thus, in contrast to a series of stromal cell lines derived from wild-type mouse bone marrow the OP9 stromal cell line derived from M-CSF-deficient mouse bone marrow stroma (Nakano et al., 1994) cooperates in early hematopoiesis, even from embryonic stem cells. Five properties characterize pHSCs: 1. Their capacity to renew themselves upon cell division, 2. Their pluripotency to develop into the various differentiation lineages of blood cells, 3. Their chemotactic attraction to home back to specific niches in bone marrow upon transplantation, 4. Their long-term reconstituting ability to again serve as a stem cell when used from the transplanted host in secondary transplantations, and 5. Their potency to protect a transplanted host from death as consequence of lethal irradiation after (socalled bone marrow) transplantation.
self-renewal
pHSCs have the capacity to renew themselves upon cell division. In a steady state of the hematopoietic system selfrenewal will be by asymmetric divisions, generating one further differentiated cell while preserving the other as pHSC when the cell divides.
Intracellular Control Mechanisms
Several factors control the self-renewing proliferation of pHSCs, most notably the Polycomb gene family member bmi-I (Iwama et al., 2004; Park et al., 2003) and the transcription factor HOX B4, (Sauvageau et al., 1995; Helgason et al., 1996; Thorsteinsdottir et al., 1999; Kyba et al., 2002; Antonchuk et al., 2002; Buske et al., 2002; Krosl et al., 2003), both of which are controlled in their gene expression by the nuclear factor NF-Ya (Zhu et al., 2003). The Wntsignaling pathway, including b-catenin (Baba et al., 2005; Duncan et al., 2005; Reya et al., 2003) has been found to be involved in the control of pHSC proliferation, as it does in other stem cell programs. Thus, transgenic expression of NF-Ya, bmi-I, HOX B4, or b-catenin favor long-term proliferation of pHSC. In tissue culture, pHSs can now be expanded and cloned in the presence of appropriate cytokines (e.g., TPO [thrombopoietin], IL3, IL6, and stem cell factor [SCF, the ligand for the receptor tyrosine kinase c-kit]), all on a supportive layer of mesenchymally derived stromal cells. Prolonged, transgenic expression of these genes, however, may inhibit efficient hematopoietic differentiation into the different blood cell lineages (Schiedlmeier et al., 2003).
Extracellular Controls
Mesenchyme-derived as well as epithelial stromal cells provide several pHSC-cooperative, stimulatory interactions, among them SCF, interacting with c-kit on pHSCs (Gilliland & Griffin, 2002); angiopoietin, interacting with Tie-2 on pHSC (Arai et al., 2004; Zhang et al., 2006); and bone morphogenic protein 4 (BMP-4), interacting with its receptor on pHSC (Maeno et al., 1996; Hogan, 1996; Graff, 1997). Also, they chemotactically attract pHSC by the production of CXCL-12 (SDF-1), interacting with its receptor CXCR4 on pHSC (Nagasawa et al., 1996; Egawa et al., 2001; Ma et al., 1999; Ma et al., 1998; Kawabata et al., 1999; Melchers et al., 1999) and Ca-2, which is the Ca11-receptor
24
host defense: general
protein expressed on pHSC, probably sensing Ca11 near the marrow of bones (Adams et al., 2006). Activation of these stromal cells, especially the osteoblasts among them, in hematopoietic cell niches (Wilson & Trumpp, 2006) result in subsequent activation or inhibition of pHSC activities. Furthermore, nor-adrenaline produced by sympathetic nerves in collaboration with actions of the stem-cell-mobilizing cytokine G-CSF induce apoptosis of stromal cells, thereby mobilizing pHSC to leave the marrow and enter blood circulation (Osmond et al., 1985; Rico-Vargas et al., 1995; Medina et al., 1993; Medina et al., 2001; Katayama et al., 2006). Since early progenitors of B lymphocyte development in mice and humans have been shown to decrease in absolute numbers in bone marrow during life (Rolink et al., 1993), it is reasonable to expect that absolute numbers of earlier progenitors and of pHSC also decrease with age, even though they never disappear completely.
Pluripotency
A single pHSC can repopulate all cell lineages of blood and fill their compartments in the body with normal numbers of cells (Jordan & Lemischka, 1990 Spangrude et al., 1991; Smith et al., 1991; Morrison et al., 1995; Osawa et al., 1996a, 1996b). pHSCs give rise to erythrocytes, megakaryocytes, and platelets that are not considered to be parts of the immune system. pHSCs also generate the cells of the innate immune system (i.e., myeloid cells such as monocytes, macrophages, various forms of dendritic cells, granulocytes such as basophils, neutrophils and eosinophils, and osteoclasts). Finally, pHSCs are the origin of lymphoid cells (i.e., the various forms of NK cells and cells of the adaptive immune system, T and B lymphocytes). Pluripotency is extracellularly controlled by the environment of cell contacts, chemokines and cytokines provided by stromal cells, and is intracellularly regulated by hematopoietic cell–autonomous genetic programs (i.e., receptor-mediated signal transductions and regulations of gene expressions).
MYELOID-LYMPHOID PROGENITORS Generation by Environmental Influences
When pHSCs are exposed to the action of FLT3L, the ligand of the membrane-bound tyrosine kinase receptor flt3 they lose their long-term repopulating capacity (Adolfsson et al., 2001). FLT3L is provided by “niches” of stromal cells in the bone marrow. FLT3L induces an apparently irreversible change to short-term repopulating stem cells (ST-pHSC), also called multipotent progenitors (MPP). In tissue cultures on OP9 stromal cells these MPPs can be grown in the presence of FLT3L and SCF. When regrown in tissue cultures with TPO, IL3, IL6, and SCF, they do not regain pHSC potential. Upon transplantation, these cells give rise to one wave of myeloid and lymphoid cell differentiation which is first seen in bone marrow (for myeloid, progenitor lymphoid and B-lymphoid progenitors) and in the thymus (for T-lymphoid progenitors) at 3 to 4 weeks, then as mature myeloid, and T- and B-lymphoid cells in the periphery, such as the spleen, 6 to 8 weeks after transplantation. While myeloid cells disappear again due to their short half-life, long-lived T and B cells remain for more than 4 months in the periphery (e.g., spleen, peritoneum, blood) of the transplanted host.
Induction by Intracellular Controls
GATA-1 controls erythroid development (Pan et al., 2005) while Icaros and PU-I induce myeloid-lymphoid cell development (Klemsz et al., 1990; Hromas et al., 1993; Singh & Pongubala, 2006; Dakic et al., 2005; Nutt et al., 2005).
THE CHOICE BETWEEN MYELOID AND LYMPHOID DEVELOPMENT Environmental Influences
While stromal cell lines derived from wild-type mouse bone marrow most often do not support the development of lymphoid, but do support myeloid cells, it was found that the OP9 stromal cell line derived from M-CSF-deficient mouse bone marrow stroma (Nakano et al., 1994) cooperates in early hematopoiesis, even from embryonic stem cells, to proliferate pHSC with TPO, IL3, IL6 and SCF, and MPP with FLT3L and SCF. When M-CSF is added to such proliferating MPPs, they develop to myeloid cells that cease to proliferate after 1 week. On the other hand, when IL7 is added to the proliferating MPPs they become lymphoid progenitors, with the properties of common lymphoid progenitors (CLP) that can be induced to become either B lymphoid, T lymphoid, or NK cells. Hence, progenitors with the dual capacity to develop to myeloid and to lymphoid cells are induced on OP9 stromal cells, by M-CSF to myeloid, or by IL7 to lymphoid development. It appears that the constitutive production of M-CSF by wild-type stromal cells favors the development of myeloid progenitors, thereby disfavoring lymphoid progenitors. Once MPPs have chosen one of the two ways, the “other” cytokine no longer influences these cells in their continued differentiation (e.g., M-CSF is no longer active on lymphoid, and IL7 no longer on myeloid development). When MPPs or the IL7-induced CLPs are cultured on transgenic NOTCH-ligand DELTA-I expressing OP9 stromal cells, in vitro differentiation and proliferation of thymocytes to the CD4/CD8-double negative state is induced (Schmitt et al., 2004). Finally, osteoclast development is induced on stromal cells expressing transgenic TRANCE (Tsuneto et al., 2005).
Intracellular Controls
High levels of PU.1 induce myeloid and low levels lymphoid cell development (Nutt et al., 2005). Mef2 is required for proper expression of key lymphoid regulators, among them the IL7 receptor. It is in agreement with the expectation that external recognition of IL7 induces lymphoid lineage choice (Stehling-Sun et al., 2009), while at the same time, it represses myeloid development through the downregulation of C/EBPa. It appears to act in concert with PU.1 to modulate the intracellular decisions between myeloid and lymphoid lineage development, maybe differently at different concentrations of complexes containing PU.1 and Mef2c.
EXCLUSIVE B- VERSUS T-LYMPHOID LINEAGE DECISIONS
At least a part of the CD19-negative, CD42/CD8-double negative (DN), IL7-receptor-expressing, receptor-tyrosine kinases c-kit and flk-2-double positive CLP progenitor cells in bone marrow have yet to make the decision to enter either the T- or B-lymphoid lineage pathway of differentiation. Some of these early lymphoid progenitors in bone marrow express both the T-lymphoid lineage specific preTa gene as well as the surrogate L chain-encoding VpreB and 5 genes. Similarly, cellular functions control the proliferative expansion of DN2 to DN3 and 4 and large DP (CD41, CD81) thymocytes once the precursor have migrated to the thymus, and SL chain controls proliferative expansion of pre-BI to large pre-BII cells in the bone marrow. Hence, while these common lymphoid progenitors already express
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
both surrogate chains as well as the rearrangement machinery (i.e., RAG1, RAG2 and TdT), they still have to commit themselves to the expression of only one of the two—either pre-Ta, express CD3 and enter D- to J-rearrangements at the TcRd or b loci, or SL chain, Iga and b and enter D- to J-rearrangements at the IgH locus.
B-Lineage Commitment environmental Influences
We do not know which microenvironmental influences positively stimulate the development of sIgM1 immature B cells from B-lineage-committed progenitors and precursors, in contrast to the other lineages described above in bone marrow. B-progenitors of wild-type mice differentiate spontaneously—without addition of cytokines or exposure to stromal cells—to sIgM1 B-lymphocytes (Rolink et al., 1991). Contrary to the lineage-inducing capacities of other cytokines, IL7 locks DHJH-rearranged pre-BI cells into their early stage of B-lineage differentiation. In the added presence of stromal cells, which provide, for example, stem cell factors (SCF) for interaction with the receptor tyrosine kinase c-kit, these pre-BI cells proliferate but, again, do not differentiate to later stages of B-cell development (Rolink et al., 1995). Removal of IL7, rather than the addition of a different set of cytokines, induces the development of IgM1 B cells.
transcription Controls
Three transcription factors—E2A, EBF, and PAX5—control the initiation and maintenance of B-lymphoid development (Bain et al., 1994; Zhuang et al., 1994; Lin et al., 1995; Sigvardsson et al., 1997; Kee & Murre, 1998; Roessler & Grosschedl, 2006; Busslinger, 2004). Among the three, E2A dominates this control by not only initiating, but also maintaining the expression of EBF1, PAX5, and, thus, the B-cell program. E2A or EBF-deficient mice are arrested in B-cell development, just as PAX5-deficient mice are. However, E2Adeficient progenitor B cells carry no DHJH-rearrangements (Ikawa et al., 2004). By contrast, PAX5-deficiency still allows DH to JH-rearrangements on the IgH chain loci to the same extent (i.e., on both alleles) as PAX5-proficient, wild-type progenitor B cells (Rolink et al., 1999b). Transgenic expression of E2A (Singh & Pongubala, 2006), but not of EBF or PAX-5, or expression of important lineagespecific genes controlled by these early regulators (e.g., the IL7-receptor) (Choi et al., 1996) induce autonomous, environment-independent, B-lineage specific differentiation. Transfection, and subsequent transgenic expression of the E47 component of E2A in nonlymphoid fibroblasts, activates the terminal deoxynucleotidyl transferase (TdT) gene and the IgH chain locus (Choi et al., 1996). When transgenic expression of the rearrangement machinery (i.e., RAG1 and RAG2) is also provided in an embryonic kidney cell line, DH to JH rearrangements are induced at the endogenous IgH chain loci (Romanow et al., 2000). Endogenous rearrangements at the L chain loci in the same nonlymphoid cells require more selective actions of transcriptions factors; in the RAG1/RAG2-transgenic cells, E2A allows for endogenous VK to JK rearrangements, and EBF for V to J rearrangements (Romanow et al., 2000). Finally, myeloid cell differentiation can be autonomously induced by the transgenic expression of high levels of PU-I, or of the gene encoding the receptor for GM-CSF (Singh, 1996) as lymphoid differentiation can be directed by the transgenic expression of the receptor for IL7 (Singh & Pongubala, 2006). Both E2A-deficient, as well as PAX5-deficient progenitor B cells can be grown—like wild-type pre-BI cells—for
25
long periods and cloned on stromal cells in the presence of IL7. In the case of PAX5-deficient pre-B cells, they are cell clones that are genetically identifiable by individual sets of DHJH rearrangements at both IgH chain alleles, just as wildtype, DHJH-DHJH rearranged pre-BI cells are (Rolink et al., 1991; Schaniel et al., 2002a, 2002b). The same is true for RAG-deficient as well as SL-chain-deficient, CD191 pro/ pre-B cells, the former without any DHJH rearranged IgH chain loci, the latter with normally DHJH/DHJH rearranged loci. Hence, the cellular differentiation program of the expression of the tyrosine kinases flk2 and ckit, and of CD19, accompanied by reactivity to IL7 and to stromal cell interactions that provide the ligands for flk2 and ckit and the cytokine IL7, can become disconnected from the DH to JH rearrangement program in progenitor B cells. The PAX5-deficient progenitors can be induced in vitro to (DHJH/DHJH rearranged) macrophages by M-CSF, to (DHJH/DHJH rearranged) dendritic cells by GM-CSF and M-CSF, to (DHJH/DHJH rearranged) granulocytes by GMCSF and G-CSF, to (DHJH/DHJH rearranged) NK cells by IL-2, to (DHJH/DHJH rearranged) osteoclasts by TRANCE expressed on mesenchyme-derived stromal cells, and to (DHJH/DHJH rearranged) thymocytes by NOTCH-ligand DELTA I, again expressed on stromal cells (Rolink et al., 2002a; Radtke et al., 2004; Höflinger et al., 2004). Transplantation of pre-BI cells, grown on stromal cells in the presence of IL7 from day 18 fetal liver, populate exclusively the B1a compartment in spleen, peritoneum, and blood, and do not populate the bone marrow, and only rarely and transiently populate the thymus. In contrast, pre-BI cells derived from bone marrow in SCF/FLT3L/IL7-stimulated cultures on stromal cells populate transiently (i.e., 2 to 4 weeks bone marrow with B-cell progenitors and thymus with T-cell progenitors and, thereafter, spleen and blood with T and B cells, and peritoneum with B cells resembling B1b cells (Vegh et al., 2010). On the other hand, transplantation of PAX5-deficient pre-BI-like cells, grown from bone marrow on stromal cells in the presence of IL7, populate all the myeloid, NK cell, and T cell compartments (with the exception of B-cell compartments in bone marrow), the thymus, and the peripheral lymphoid organs in a long-term repopulating fashion. Hence, such PAX5-deficient pre-BI-like cells can be isolated, regrown, and retransplanted several times into secondary, tertiary, and onward recipients, always repopulating all the different myeloid and lymphoid compartments except the B-lineage compartments (Schaniel et al, 2002).
T-Lineage Commitment Commitment by Internal Controls
Expression of NOTCH-1 accompanies development to the T lineage (Rolink et al., 2002a). Ectopic expression of “active” NOTCH-1 allows early thymocyte development in the absence of a thymus and, at the same time, inhibits B-cell development (Pui et al., 1999). Ectopic expression of Id2 induces differentiation to the NK cell lineage (Yokota et al., 1999; Ikawa et al., 2001; Engel & Murre, 2001) and, again, inhibits B-cell development, possibly by forming heterodimeric complexes with E2A (Engel & Murre, 2001). On the other hand, “inactive” NOTCH-1 inhibits T-cell development at the earliest, DNI stage of T-cell development, but promotes B-cell development even in the thymus (Radtke et al., 1999; Wilson et al., 2001). NOTCH-1 can also be inactivated by Lunatic Fringe (Koch et al., 2001) and by Deltex (Izon, 2002). Again, such activations lead to arrest of T-cell development and stimulation of B-cell development.
26
host defense: general
Whenever activated, NOTCH-1 releases its intracellular domain, which acts on the transcription factor RBPJ to convert it from a repressor to an activator (Tanigaki & Honjo, 2007). Direct targets of the activator are Ptcra, CD25, and Hes-1.Thereafter and throughout a/b TcR T-cell differentiation from DN1 to CD41CD81DP thymocytes, activities of the transcription factors RUNX-1-CFBb, MYB, GATA-3, Icaros, PU.1, ETS-1, ETS-2, SPIB, E2A, HEB, and LEF1 all participate in controls at different developmental points, as described in detail in Rothenberg et al. (2008).
Commitment by external Controls
When CLPs are cultured in vitro not on OP9 stromal cells, but on OP9 stromal cells that express the ligand for NOTCH1 and DELTEX 1 constitutively after transgenic transfection, they develop in the presence of FLT3L and IL7 to the four stages of CD4/CD8-double negative (i.e., DN1, DN2, DN3, and DN4 cells, as well as to CD4/CD8double positive thymocytes) (Schmitt & Zuniga-Pflücket, 2002). In prolonged cultures, cells with a DN2/DN3 phenotype expand. The chemokine CCL25 attracts progenitors expressing its receptor, CCR9, within the thymus.
REVERSIBLE DECISIONS OF EXCLUSIVE B-CELL DIFFERENTIATIONS – THE FLEXIBILITY OF PROGENITOR CELLS
The hierarchical models of hematopoietic cell differentiation propose that commitment to a given cell lineage, with concomitant loss of the respective pluripotency or multipotency, is achieved in irreversible steps. This appears to be the case when FLT3L commits pHSCs to myeloid or lymphoid (i.e., differentiation to MPPs) and also when M-CSF commits MPPs to myeloid (i.e., to CML), and IL7 to lymphoid (i.e., CLP differentiation). By contrast, this appears not to be the case with PAX5deficient pre-BI-like cells. While they are blocked in further B-cell development, they differentiate to all the possible hematopoietic cell lineages (i.e., erythroid, myeloid, NK, and T-lymphoid cells), depending on the external environment of cytokines and niche cell contacts. However, they do so with rates that are too slow to allow protection of the transplanted host from irradiation-induced death. Hence, while they appear to be pluripotent, they are not pHSCs, although they home back to their original sites in bone marrow, retaining the original phenotype of ckit1flk-21CD192 IL7Ra1sca-11 cells (Rolink et al., 1999a; Schaniel et al., 2002b). The same multipotency has also been seen with E2A-deficient pro-B cells (Ikawa et al., 2004). By contrast, wild-type pre-BI cells do not home back to the bone marrow but rather, upon transplantation, give rise to a single wave of B-cell development, mainly to BI type (Rolink et al., 1991). B2-type B cells are developed when CD41 helper T cells, together with CD41CD251 regulatory T cells, are cotransplanted. Hence, PAX5-deficient progenitors are fully functional, pluripotent, long-term reconstituting, and flexible stem cells, but they are not pHSCs since they do not differentiate to all the hematopoietic cell lineages at appropriate rates to allow protection of the host from death by lethal irradiation. The expression of PAX5 can be seen as the final, decisive step to commit hematopoietic progenitors to the B-lineage pathway of development. The importance of PAX5 in this commitment process is further documented by experiments, in which lox-flanked PAX5 has been “knocked into” the PAX5 locus, rendering the locus conditionally inactive. When pre-BI cells of such conditionally PAX5-deficient pre-BI cells are treated with CRE-recombinase, thus deleting the PAX5-locus, these wild-type pre-BI cells revert
back to the PAX5-deficient pro/pre-B-cell phenotype. They become inducible to enter erythroid, myeloid, NK-, and Tlymphoid development (Mikkola et al., 2002). T-lymphoid cells in the thymus derived from the PAX5-inactivated and transplanted pre-BI B cells retain the DHJH/DHJH rearrangements of the original pre-B cells. When mature, sIgM1 B cells of conditionally PAX5deficient mice are rendered PAX-5 deficient by treatment with CRE-recombinase, the cells revert back to a pre-B-like stage (Schebesta et al., 2002). In vitro, such reverted cells regain their IL7-receptor expression and regain the capacity to respond to IL7 by proliferation. Since these cells have already expressed a set of productively VHDHJH/VLJL rearranged IgH/IgL-chain loci (i.e., sIgMs with μH- and kLor L-chains), and since PAX5 deficiency leads to lack of SLP65 expression (Schebesta et al., 2002), the resulting pre-B cells express a polyclonal spectrum of VH-domains on μH chains, and reexpress the SL chain (i.e., reexpress pre-BcRs but cannot signal downregulation of SL chain expression). Hence, they continue to proliferate, stimulated by IL7. Interestingly, the induced PAX5-deficiency turns off the expression of the productively VLJL rearranged Lchain loci (i.e., of L chains), suggesting a tight regulation of L chain expression by PAX5. In vivo transplantation of the CRE-induced, PAX5-deficient pre-B cells leads to repopulation by polyclonal μH chain-expressing thymocytes in the thymus, again without expression of the VLJL rearranged Lchain loci. It remains to be determined whether the CREinduced PAX5 inactivation dedifferentiates these cells to a large pre-BII cell-like stage or to a pro/pre-BI-like stage of B-cell differentiation.
THE GENERATION OF THE ANTIGEN RECEPTOR-EXPRESSING LYMPHOCYTE REPERTOIRES
In order to generate lymphocytes expressing antigen receptors, CLPs have to enter the appropriate primary lymphoid organs and begin to differentiate and rearrange antigen receptor loci. From the moment a lymphocyte precursor expresses the first chain of its antigen-specific receptor, that receptor becomes the dominating control element of all further steps of proliferation or death, and survival and location in the immune system. T- and B-cell antigen receptors (i.e., TCR, BCR) are generated by joining of V, D, and J segments at the TcR and Ig loci in a process ordered in time and cell differentiation stages. First, D to J rearrangements at the TcRb, TcRd, and IgH loci are initiated. For B-lineage cells this happens first during embryonic development in omentum and fetal liver, and later in adult life, in bone marrow, while for the T-lineage this is initiated in the fetal, and then in the adult thymus. Fetal cells appear to originate from progenitors that are distinct from those used after birth and all throughout life. In the B-lineage, D to J-rearrangements are begun in CD19-FLT31B2201c-kit1 pro/pre-B cells and are complete in CD191FLT3-B2201c-kit1pre-BI cells. T-lineage cells begin their D to J rearrangements at the transition from DN1 to DN2 cells, and have completed them in DN2 cells.
V to DJ Rearrangements and Synthesis, and Use of the First Chain of the Heterodimeric Antigen Receptors as Pre-Lymphocyte Receptors
The first functional Ig TcR chains are made when V to DJ rearrangements are initiated that can generate productively rearranged IgH, respectively, TcRb or TcRd loci. In B-lineage cells, V to DJ rearrangements occur in the
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
CD191B2201c-kit-CD251 large pre-BII stage, in the a/b TcR T-lineage at the DN3 stage (Kyewski & Klein, 2006; Melchers, 2005). All T-cell lineages with the possible exception of fetal TcRg/d expressing T cells, are derived from the same intrathymic precursors. All B cells, with the exception of the fetal wave of development in omentum and fetal liver generating B1a cells are derived from the same precursor in bone marrow. Productive TcRg and TcRd rearrangements result in expression of a g/d TcR, which commit cells mostly to the gd lineage (Kyewski & Klein, 2006). Since they do not form a TcRd chain-containing pre-lymphocyte receptor, these T-lineage thymocytes do not expand by proliferation in the thymus as TcRb chain-containing T-lineage cells do. Hence, they make up only 5% of all thymocytes and, consequently, generate correspondingly lower numbers of T cells for the peripheral immune system, at least in the spleen, lymph nodes, and blood. a/b TcR T-cell development and B-cell development show striking similarities. Productive IgH gene and TcRb gene rearrangements generate Igμ-H chains and TcRbchains which are assembled with SL chains—composed of the Vpre-B and 5 proteins, respectively—with pre-Ta chains to form pre-lymphocyte receptors (pre-BcR, pre-TcR) that are deposited on the cell surface of large pre-BII cells, such as DN3 thymocytes. In this way, the Vpre-B part of the SL chain in pre-BcRs (but not pre-TcRs, which lack such Vprobing Vpre-T parts) probe the newly generated IgH chains for their ability to form structurally intact heterodimers, first with the surrogate light chain, and later, in development with IgL chains (Melchers, 2005). Between 50% and 80% of the newly formed IgH chains do not pair well enough to form a pre-BcR on the cell surface, and hence, are excluded from the positive selection for further use in the B-lineage that follows. Initially, all TcRb chains may form a pre-TcR. The newly formed pre-BCRs and TcRb-pre-TcRs induce proliferation of large pre-BII cells, respectively, DN3 thymocytes. In the case of pre-BcRs, the number of induced cell cycles (between 2 and 7) depend on the fitness of the IgH chain/SL chain pairing, while fitness between TcRb chains and pre-Ta chains appear not to influence a larger number of induced cell cycles (7 or more). Mutations that abolish the proper structure of the preBcR (i.e., B-lineage progenitors deficient in VpreB1 and VpreB2 or 5, or VpreB1, VpreB2, and 5), or that interfere with pre-BcR signaling (Melchers, 2005; Melchers et al., 2000), abolish the proliferative expansion of large pre-BII cells, as do mutations that interfere with or abolish pre-TcR functions. Pre-BcRs and pre-TcRs may not even use the complementarity determining regions (CDRs) of the V-domains to bind ligands that could induce proliferation. The newly generated VH-domain repertoire of pre-BcRs is screened for fitness to pair (i.e., to be prepared for the eventual combination with conventional L chains). Thereby unfit H chains are excluded that may have other unwanted properties, such as the formation of self-aggregating immune complexes that might bear the danger of glomerulonephritis and vasculitis (Melchers, 2005). It appears that the non-Ig-portion of the 5-subunit of an SL chain is mandatory for the capacity of pre-BcRs to stimulate pre-BII cell proliferation (Ohnishi & Melchers, 2003). Crosslinking of pre-BCRs via positively charged arginine residues in the non-Ig portion of 5, possibly mediated by repetitive negative charges on molecules such as negatively charged nucleic acids and other molecules, either on the same pre-BII cells or on environmental stromal cells (Ohnishi & Melchers, 2003; Bradl et al., 2003), initiates pre-BCR signaling. Some CDR3 regions of newly generated
27
VH-domains contain argine residences that are apparently capable of mediated pre-BcR signaling functions even in the absence of an SL chain (Doyle et al., 2006). The pre-BCR also induces the downregulation of SL, thereby limiting proliferative expansion of IgH chain-expressing cells through exhaustion of pre-BcR formation. Furthermore, the pre-BcR down regulates recombinase activating gene (RAG1 and 2) expression, thereby preventing VDJ rearrangements on the second allele, possibly because the locus is rendered inaccessible for the rearrangement machinery. This should secure allelic exclusion at the IgH chain locus, so that one B cell produces only one type of H chain. Membrane-bound IgH chains signal the cell to turn off the rearrangement machinery and make the second, DHJH rearranged IgH chain allele inaccessible for further rearrangements. However, it appears that the classical pre-BcR with SL chain is not involved in mediating this allelic exclusion, since SL-triple-deficient (Vpre-B12/2 /vpreB22/252/2) B cells still are allelically excluded (Shimizu et al., 2002; Melchers, 2005). In pre-BII cells where pre-BCRs cannot be formed, these nonproliferating cells nevertheless proceed in differentiation (Grawunder et al., 1993; Rolink et al., 1996; Grawunder et al., 1995). However, their contribution to the developing B-cell compartment is, at best, 20- to 40-fold lower than that of their pre-BCR-expressing counterparts (Rolink et al., 1993b). Deletion of the gene encoding the adaptor protein SLP-65 directly by targeted mutagenesis (Flemming et al., 2003; Jumaa et al., 2003) or indirectly by targeted inactivation of PAX5 controlling SLP-65 expression (Koch et al., 2001) abolishes the capacity of large pre-BII cells to downregulate surrogate L-chain expression. This leads to continued pre-BcR formation in dividing large pre-BII cells so that, in fact, SLP-65-defective mice have a hyperplastic large pre-BII cell compartments. This chronic pre-BII cell proliferation is a breeding ground for secondary mutation so that, in a period of weeks, pre-B cell lymphomas (lymphocytic leukemias) develop in these mice. Do some autoantigens, nevertheless, influence—either positively or negatively—the pre-BcR repertoire development? It has been observed that a subset of autoreactive and polyreactive pre-BCRs expressed in pre-BII-like cells induced a proliferative boost in tissue culture cells, suggesting a positive rather than a negative selection by polyreactive pre-BCRs (Köhler et al., 2008) while other experiments indicated that autoantigens suppress pre-BcR-dependent pre-B-cell development (Keenan et al., 2008). Still other experiments indicate that pre-BCR cross-linking in fetal liver organ culture did not boost pre-B-cell proliferation, nor did it inhibit it (Ceredig et al., 1998). Pre-BCR formation leads to a shift in the originally generated heavy chain repertoire (ten Boekel et. al., 1997) and this shift is delayed in SL chain-deficient bone marrow. It facilitates a loss of positively charged and aromatic amino acid residue-containing polyreactive μH-chains, and thus may represent negative selection of μH-chain repertoires by the pre-BCR (Keenan et al., 2008). It is not clear how much of this negative selection of pre-BCRs is mediated by autoantigens binding to CDRs of μH-chain chains and how much is due to lack of pairing with SL. Productive TcRb rearrangements generate pre-TcRs, that induce proliferation of DN3 thymocytes in a cell autonomous fashion without any apparent requirement for a ligand. As for the B lineage in pre-BII cells, formation of a pre-TcR on the surface of DN3 thymocytes leads to the termination of V to DJ rearrangements on the second TcRb allele. More than 99% of thymocytes and T cells are allelically excluded at their TcRb locus. It is unknown whether
28
host defense: general
TcRb loci can replace their Vb -regions in secondary replacements. Polar residues in the extracellular domain of the pre-TcRa chain, as well as its cytoplasmic tail are likely to contribute to this autonomous signalling (Yamasaki et al, 2006; Aifantis et al., 2002). However, the only conserved portion of the pre-TCRa chain is the transmembrane region and not its polar residues, so that other structural features might contribute to this autonomous signaling. Pre-TcRs terminate further rearrangements at the TcRb locus, but, unlike the pre-BcRs, do not select cells expressing particular TcRb chains by pairing, because pre-Ta does not have a Vpre-B-like structure that would allow such pairing with V regions of TcRb chains. Hence, the distribution of TcRb V regions in unselected and pre-TCR-selected thymocytes is the same (von Boehmer et al., 2003). All pre-TcR-expressing cells commit to the a/b TcR T-lineage with the help of Notch signaling, and they subsequently all undergo TcRa rearrangements.
V to J Rearrangements and Synthesis and Use of the Second Chains of the Heterodimeric Antigen Receptors as TcRs and BcRs in Immature T- and B-Cell Compartments
V to J rearrangements at the TcRa loci occur last in DN4 and CD41/CD81 DP cells, as do rearrangements at the Igk and Igl loci in CD191 B2201c-kit-CD251small preBII and in IgM1 immature B cells. Furthermore, except for most of the embryonically generated V(D)J-joints, N-region sequences of differing lengths are inserted at most joining sites, although this is more evident in human, and less so in murine B-cell development. Most cells with productive TcRd and TcRg rearrangements develop to the gd lineage without allelic exclusion of either TcRd or TcRg chains (Rothenberg et al., 2008). It is in fact not the isotype of the TcR but the signaling strength (Schmitt & Zuniga-Pflücker, 2002; Mikkola et al., 2002) that commits cells to either the d/g or a/b lineage. Some 20% g/d TcR-expressing cells that undergo weak signaling can still enter the a/b lineage by silencing TcRg expression and deleting the TcRd locus by TcRa rearrangement (Nossal & Lederberg, 1958). Little is known about tolerance mechanisms in g/d T cells except that TcR ligands appear to have a role in committing these cells to the g/d lineage (Schmitt & Zuniga-Pflücker, 2002; Mikkola et al., 2002) rather than in causing apoptosis. In fact, TcR ligation results in the generation of a subset of g/d T cells that assume the characteristics of innate lymphocytes in that they express the PLZF transcription factor, which is also is expressed in NKT cells (Schebesta et al., 2002).
Secondary Rearrangements at the IgL and TcRa Loci
A first rearrangement on the Ig L-chain loci can be followed by replacements by secondary VL to JL rearrangements on an already VLJL rearranged L-chain locus (Yamagami et al., 1999a, 1999b). TcRa can enter secondary Va to Ja rearrangements, and they can be extensive since more than 50 Ja segments are available in the mouse for such secondary, tertiary, and other rearrangements (Nossal & Lederberg, 1958). Secondary rearrangements can be made in immature lymphocytes that lack IgL chain, respectively, TcRa chain protein expression as heterodimeric antigen receptors on the cell surface, either as a result of a nonproductive V to J rearrangement, or because the chains do not fit their corresponding partner chain in the heterodimer. They can also be the result of an inappropriately affine binding of a BcR to an autoantigen, in the case of an a/b TcR to MHC-self-peptide. Changing the Ig L chain, respectively TcR a chain in this way has been termed editing. Continued rearrangements at
the IgL chain loci of autoreactive BcR-expressing, immature B cells increase the chance save this cell from apoptosis by a change of the specificity away from autoantigen recognition. By contrast, continued rearrangements at the TcRa locus increase the chance to salvage an immature thymocyte by changing the TcR specificity towards self-MHC/self-peptide recognition. During the process of L-chain editing, evolutionarily selected editor Ig light chains that have low isoelectric points (due, for example, to the presence of aspartate residues) appear to neutralize the DNA binding properties of certain IgH chains; this may facilitate avoidance of diseases mediated by antibody DNA complexes such as SLE (Li et al., 2001). Editing can also lead to the expression of two or even more different L chains (and possibly also two different H chains) in a single immature B cell (Gerdes & Wabl, 2004; Khan et al., 2008; Doyle et al., 2006). If editing results in the expression of one autoreactive and one nonautoreactive BCR, immature B cells can become unreactive to autoantigen due to dilution of the autoreactive with the nonautoreactive BCR. These dual-expressor B cells can enter the mature B cell pool while remaining potentially autoreactive. L chain editing can also lead to polyreactivity. Interestingly, some human B cells coexpress V pre-B and conventional L chains together with H-chains containing CDR3 regions enriched in positively charged and/or aromatic amino acids. Two-thirds of these cells are autoreactive, hence, they appear to have escaped central tolerance. Although human Vpre-B, which has an isoelectric point of 5.67, may act as an “editor” by neutralizing positively charged CDR3 regions, mouse Vpre-B proteins have an isoelectric point of 9.37 and thus may not be able to perform this function.
THE SELECTION OF IMMATURE LYMPHOCYTES BY THEIR ANTIGEN SPECIFICITIES Selection For and Against Autoantigen Reactivities
From the random generation of the antigen-binding regions, especially of the third complementarity region (CDR3) of the V regions of TcRs and BcRs, it is predictable that some of these V regions expressed on BcRs and TcRs of immature T and B cells should be fully autoreactive. In fact, it has been estimated that more than half of all newly generated BCRs are capable of binding autoantigens (Nemazee, 1995). The reactivity or responsiveness of B cells to antigens and autoantigens remains imprecisely defined; however, as the correlation between the binding affinity of an antigen to a BCR and the functional response of the B cell expressing this BCR cannot be given as a quantitative term. Furthermore, such antigen-sensing mechanisms are not necessarily the same in developmentally distinct subsets of lymphocytes. The same limitations in the judgement of quantitations of avidities exist for the interactions of TcRs with MHC-self-peptide or MHC-foreign peptide interactions. There is no reason to expect that the diversities of the originally generated repertoires of TcRb and TcRd chains and, thereafter, of the TcRa and TcRg chains are different from those of the IgH and L chain, since all loci are rearranged by the same rearrangement machinery, including the insertion of N regions by TdT. The originally generated repertoires of TcRs have not been tested for binding to the same collection of autoantigens and foreign antigens as BcRs have. It is, therefore, reasonable to expect that these original TcR repertoires are similarily polyreactive and autoreactive, and many of them should be heterodimer-unfit
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
to pair. The more extensive pre-TcR-driven expansion of TcRb chains may just simply increase the chances to find a pairing TcRa chain later. However, neither polyreactive nor autoreactive a/b TcR matter, because all of them, in contrast to BcRs, are selected to fit only one selected set of autoreactivities, namely recognizing MHCI or MHCII-self-peptide complexes (see below). Only those will be allowed to mature to single-positive (SP) CD41 or CD81 thymocytes, and to leave the thymus and mature in the periphery to effector cytotoxic or helper T cells. It is surprising—and also not in agreement with the hypothesis of clonal selection—that a large part of the originally generated repertoire of IgM1 B cells are most likely are polyreactive (i.e., bind a large collection of structurally seemingly unrelated antigens).
Autoantigen-Presenting Cells in Primary Lymphoid Organs for t Cells
To begin T-cell development 10 to 100 CLP migrate through the blood from the adult bone marrow into the cortical regions of the thymus (Kyewski & Klein, 2006). After commitment to the T lineage they proliferate as CD4-CD8-DN1 and -DN2 thymocytes and, thereby, expand to 5 3 107 cells in 15 to 20 divisions within nearly 2 weeks. In these proliferating cells, D to J rearrangements are initiated and completed. V to DJ rearrangements follow in DN3 and 4 thymocytes. The pre-TcR of the a/b T lineage allows another expansion by more than seven divisions, so that the ratio of a/b versus g/d TcR thymocytes after V to J rearrangements settles at 95% to 5%. However, most of these newly generated thymocytes never leave the thymus; only 1 to 2 3 10106 cells do. Productive TcRa rearrangements in TcRb-expressing cells lead to expression of an a/b TcR on the cell surface if the two chains can form a proper heterodimer. In marked contrast, TcRa rearrangements are only halted when the TcR can bind to MHC I or II self-peptide complexes with sufficient avidities (Borgulya et al., 1992; Casanova et al., 1991). In fact, all mature a/b TcR T cells contain both TcRa alleles in VJ-rearranged forms, and 30% of these thymocytes and T cells express two productive TcRa rearrangements (Padovan et al., 1993). Therefore, the TcRa loci appear to be kept open for secondary rearrangements as long as no MHC-self-peptide complex fits, thereby increasing the chances to positively edit the a/b TcR specificity for self-MHC/self-peptide recognition. However, not all the 30% of T cells express two TcRs on the cell surface (Saito et al., 1989) because pairing preferences exist for some TcRb chains in pairing with certain TcRa chains (Heath et al., 1995). Hence, only 10% of T cells express two different TcRa chains on the cell surface. In summary, a/b TcR T cells are frequently not allelically excluded. The specificity of the a/b TcRs determines whether cells are neglected and die, or are positively or negatively selected (von Boehmer, 2004). a/b TcRs on CD41CD81 thymocytes that recognize MHC class I self-peptide complexes on antigen-presenting cells in the thymus bring these MHCI molecules on the antigen-presenting cells in contact with CD8, which induces their differentiation to naïve CD81 cytotoxic T cells (Tc), on which CD4 has been downregulated. Conversely, other a/b TcRs on other thymocytes that recognize MHC class II self on antigen-presenting cells allow the contact of MHCII with CD4 on thymocytes and induce the differentiation to CD41CD8− helper T cells (Th), or to CD41 CD251 regulatory T cells (Treg) (von Boehmer, 2008). The induction to cytotoxic and helper
29
T-cells differentiation—also called positive selection—is dependent on the avidity of interaction between MHC selfpeptide complexes and a/b TcRs. If these avidities are too high, the thymocytes are driven into programmed cell death (apoptosis), also called negative selection. High avidity interactions can, however, also result in a diversion of developing abTCR1 cells into unconventional lineages involved in innate immunity, such as NK T cells. For the selection of such NK T cells, the selecting ligand is expressed on lymphoid cells (Bendelac et al., 2007).
Generation and Selection of Treg Cells in the Thymus
High affinity TcR ligands can also induce the generation of Treg cells that express the transcription factor Foxp3 and have an essential role in preventing autoimmune disease (Yamagata et al., 2004; Guy-Grand & Vassalli, 2004). Lineage fate of such cells is instructed by TcR binding to agonist ligands expressed on stromal cells within the thymus (Guy-Grand & Vassalli, 2004; Jordan et al., 2001). The Treg cell lineage differentiation process might begin either with CD41CD81 double positive, FOXP3-expressing cells in the thymic cortex (Apostolou et al., 2002), or with CD418− single positive cells in the medulla. It is evident that in developing T cells, the TcR determines lineage fate. The developing thymocytes are confronted with different types of autoantigen-presenting cells: (i) epithelial cells in the subcapsular regions, cortical epithelial cells, and epithelial cells in the medulla (mTEC [medullary thymic epithelial cells]); (ii) hematopoietically derived dendritic cells (DC); and (iii) hematopoietically derived B lymphocytes. All of them can take up, process, and present autoantigens (proteins) as self-peptides in MHC I and II molecules. mTECs and DCs express the highest levels of MHC. Hence, these three types of cells are prime sites for thymocytes probing the specificities of their a/b TcRs on MHC-self-peptide complexes (Kyewski & Klein, 2006). High-avidity interactions lead to negative selection by apoptosis, or to anergy, and short time survival. The ignorance of TcRs with no avidity for MHC-self-peptide complexes does not allow such thymocytes to further differentiate to a state where they could leave the thymus. These thymocytes remain short-lived and die by neglect. Low-avidity interactions lead to positive selection to more mature, CD41 or CD81 cells that are allowed to leave the thymus. Hence, the emerging T-cell repertoire in the peripheral immune system is restricted in the recognition of peptides of foreign antigens by the MHC of the antigenpresenting cells that they have encountered in the thymus. mTECs have the most unusual capacity to transcribe and translate a large variety of autoantigen-encoding gene loci, a capacity that appears to be in part controlled by the AIRE gene product (Peterson et al., 2008). Some thymic DCs also express AIRE and may, therefore, have the same expanded autoantigen-presenting capacity. Therefore, positive and negative selection of helper CD41, cytotoxic CD81, and regulatory CD41CD251 thymocytes does not only include the autoantigens expressed in the normal cells of the thymus; thereby, central tolerance can be generated for all autoantigens.
for B Cells
In immature B cells, surface membrane expression of IgM appears stop further Ig rearrangements. At least two-thirds of such immature B cells are capable of doing so, because two-thirds of the immature, as well as the peripheral, splenic mature B1 and B2 compartments have one of the two IgL chain alleles in germline configuration. They are allelically excluded, since
30
host defense: general
less than 0.5% of all immature or mature B cell express more than one IgH chain allele as BcR on the surface. Quite comparably to T cells, the specificity of the Ig receptor influences the selection into the different B cell lineages, shown in experiments where the transgenic change of a B2-derived Ig receptor to a B1a-derived one changes the selection of the Ig-transgenic cells from B2 to B1a. It is remarkable that almost nothing is known of the autoantigen-presenting modes that select B-cell repertoires and establish central B-cell tolerance in the different B-cell compartments. Genetic deficiencies in the complement components C1q, C4, serum amyloid protein, complement receptor 2, or secreted natural serum IgM lead to systemic autoimmune disease characterized by production of autoantibodies against DNA, and other nuclear antigens suggest that these gene loci may contribute to central B-cell tolerance (Melchers & Rolink, 2006). Two models have been proposed to explain the emergence of autoantigen-reactive B-cell repertoires in these knockout animals. In one model (Carroll, 2004), macrophages expressing the appropriate complement receptors (e.g., C1qR, CR1) efficiently remove apoptotic cells that are bound by natural IgM, C1q, and C4b, thereby preventing accumulation of these cells and subsequent activation of mature B cells. The other model (Melchers & Rolink, 2006), suggests that autoantigens, including those released from apoptotic cells are presented to immature B cells by immune complexes containing C1q, C4b, and IgM on as yet unidentified cells expressing the appropriate receptors (e.g., C1qR, CR1, and possibly FcRμ). In this model, the result of the autoantigen presentation depends on the avidity of interaction of the immune complex-bound autoantigens with the BcR expressed on an immature B cell. If the avidity is very high, it can result in negative selection (i.e., induction of apoptosis and/or anergy in the immature B cell). If the avidity is low, the corresponding immature B cell can be positively selected, maybe into the B1 compartments, if this low autoantigen recognition is maintained in the B1 populated sites. If the BcR on an immature B cell does not bind any autoantigen with even a low avidity this cell is ignored and is allowed to enter (maybe into the B2) conventional B cell compartments. It is notable that, in this view, ignored B cells are allowed to enter the mature, peripheral compartments while T cells must be MHC-self-peptide-selected. A better understanding of the modes of central B-cell tolerance induced by apoptosis, editing, anergy, and ignorance requires identification of the relevant antigen-presenting cell populations. At this first checkpoint of B-cell repertoire development, the contribution of polyreactive BcRs to the total repertoire drops from 60% to 6%. In contrast, in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients with these autoreactive and polyreactive BcRs are not lost at this checkpoint (Carroll, 2004), providing strong evidence of the role of this checkpoint in preventing autoimmune disease.
POSITIVE SELECTION OF B CELLS
Autoreactive, in fact, often polyreactive B cells (Leslie et al., 2001) can be positively selected and appear to accumulate in the BI compartments of the gut-associated lymphoid tissues (i.e., in the follicles below the M cells of the flat epithelium of the gut). Some of them are also found as single, intraepithelial cells. Many positively selected B cells accumulate in the gutassociated lymphoid tissues (GALT), including the lamina propria. They produce IgM and predominantly switch to IgA (Craig & Cebra, 1971). Secreted IgA traverses the epi-
thelia associated with the secretory piece, which it acquires during the migration through the epithelial layer. On the luminal side it binds with low affinity and high crossreactivity to bacteria of the indigenous flora and to food antigens. Hence, the positive selection via low avidity of these (eventually IgA-producing and secreting) BI cells appears to possibly be useful for “neutralizing” interactions of the secreted antibodies with potentially infectious bacteria of the gut flora. B cells within the marginal zone of the spleen also appear to be positively selected. However, it remains to be seen whether this major traffic intersection, in fact, is a selection site for newly generated (as well as possibly of recirculating) B cells, potentially, these B cells could be in contact with cellular debris of dying blood cells and of killed bacteria, which are found at the same sites and which are about to be removed from the circulation. Many of them have a BI phenotype (Hayakawa et al., 1999; Stall et al., 1992). BcRs of BI B cells are often found to cross-react with a variety of auto-antigens and bacterial antigens at low avidities. B1 cells appear mildly activated (i.e., “tickled”) do not proliferate in their GALTs but appear to be long lived. Transplantation of BI cells into secondary hosts, in contrast to the transplantation of conventional B cells is easy and repeatable in subsequent hosts. Even low numbers of BI cells will fully replenish the GALTs. This suggests that the low avidity–autoreactivity, combined with a polyreactivity to several antigens, could be the stimulatory influence for BI cells to replenish their lymphoid sites after transplantation and to attain longevity (Craig & Cebra, 1971; Förster & Rajewsky, 1990). Many positively selected B cells appear to be BI cells, and many are accumulated in the gut. B cells in the GALT, including the lamina propria, produce IgM and predominantly switch to IgA (Craig & Cebra, 1971). Secreted IgA traverses the epithelia associated with the secretory piece, which it acquires during the migration through the epithelial layer. On the luminal side, it binds with low affinity and high cross-reactivity to bacteria of the indigenous flora and to food antigens. Hence, the positive selection via low avidity of these (eventually IgA-producing and secreting) BI cells appears to possibly be useful for “neutralizing” interactions of the secreted antibodies with potentially infectious bacteria of the gut flora. At least in parts this IgA production by gut-associated B cells appears to be quite distinct from the Ig-production elicited in a helper T-cell-dependent, inflammatory response of B cells in germinal centers of peripheral lymph nodes and the spleen. First, while the follicular B cell response beneath the M cells may be helper T-cell-dependent; hence, germinal center-derived, the submucosal, intraepithelial response in the lamina propria can be helper T-cell-independent. Onethird of the IgA might derive from such T-cell-independent B cell responses in these T-cell-independent responses (Rolink et al., 1991). The expression of the Ig class-switchinducing AID (activation-induced cytidine deaminase) gene (Muramatsu et al., 2000; Revy et al., 2000; Shinkura et al., 2004) is normally induced by a CD40 ligand-CD40 interaction. However, the cooperating cell providing the CD40 ligand in the GALT, especially in the intraepithelial spaces is not known, and must not be a helper T cell. Furthermore, these B cell responses could be costimulated by other cell–cell contacts and cytokines that might not be typical for a T-cell-dependent, germinal center response of B cells. Hypermutation of the V regions of the IgM, and later IgA, expressed and secreted by the lamina propria of the intestine BI cells could well occur in the AID-expressing, IgA class switching cells. However, the lack of the selection
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
of better fitting v region mutants by bacterial antigens and the lack of germinal center formation (i.e., exclusive B-cell proliferation) may decrease the probability to generate and select better fitting antibacterial antibodies—in as much as that also should decrease the probability of generating high affinity autoreactive antibodies.
IGNORANCE OF B CELLS
Emerging B cells with no apparent autoreactivity pass the two checkpoints in bone marrow and the spleen as long as they express a surface membrane-deposited BcR. A variety of Ig-transgenic mouse models support this view (Fields & Erikson, 2003). The ignored B cells enter the spleen via the terminal branches of the central arterioles and populate the follicular regions as conventional, B2-type B cells. Initially they are short-lived, with half-lives of 2 to 4 days. They then mature to longer-lived B cells, with half-lives longer than 6 weeks. Many positively selected B cells appear to be BI cells, and many are accumulated in the gut. B cells in the GALT, including the lamina propria, produce IgM and predominantly switch to IgA (Craig & Cebra, 1971). Secreted IgA traverses the epithelia associated with the secretory piece, which it acquires during the migration through the epithelial layer. On the luminal side it binds with low affinity and high cross-reactivity to bacteria of the indigenous flora and to food antigens. Hence, the positive selection via low avidity of these (eventually IgA-producing and secreting) BI cells appears to possibly be useful for “neutralizing” interactions of the secreted antibodies with potentially infectious bacteria of the gut flora. Conventional, B2-type B cells are most often triggered into responses by helper T-cell-dependent antigens. These foreign antigens also stimulate helper T cells to cooperate with the follicular B cells in a response, which takes place mainly in germinal centers within the follicular regions of secondary lymphoid organs. This induces B cells in a CD40 (B)-CD40 ligand (T) cell cytokine (e.g., IL4- or TGF-b-)dependent fashion to switch to IgG, IgE and IgA, and to preferentially hypermutate the V regions of the rearranged IgH- and L-chain genes, leading to affinity maturation of B cells. The switched, hypermutated BcR-expressing B cells have the choice either to mature to Ig-secreting plasma cells or BcR-expressing memory cells. Both of them gain longevity in such a T-cell-dependent response with half-lives changed from a few days to weeks and months of survival in the immune system. The memory B cells and the long-lived plasma cells appear to leave the germinal centers to lodge in special niches in the bone marrow until they are recalled by a secondary challenge of the same antigen. While BI B cells are apparently unable to be stimulated to longevity by BAFF, a member of the TNF family of cytokines, conventional B cells express BAFF-receptor, a member of the TNF-receptor family, and respond to BAFF by polyclonal maturation to long-lived B cells (Rolink & Melchers, 2002). The TNF family ligands BAFF and APRIL and their receptors BCMA, TACI, and BAFF-R control the selection of short-lived immature B cells with no apparent positively (or negatively) selecting specificities for autoantigens to long-lived mature B cells. Experiments of the in vivo administration of soluble BAFF-R ligands and of soluble decoy receptors, the analysis of BAFF-transgenic, BAFF-deficient and BAFF-receptor (BAFF-R)-deficient mice, as well as the in vitro responses of immature and mature B cells of BAFF (all reviewed in Rolink & Melchers, 2002) have shown that immature B cells from bone marrow and the spleen (initially
31
immature as well as transitional B cells) and mature B cells respond to BAFF by polyclonal maturation to long-lived B cells without proliferation. BAFF and BAFF-R deficiencies arrest B-cell development at the transition from immature to transitional B cells. Although the action of BAFF in vitro is polyclonal and independent of BCR occupancy, it remains to be seen whether ligand selection through BCR occupancy plays a role in this selection of the conventional “virgin” antigen-reactive mature B cells. B2 cells can also respond, as B1 cells do, to T-cellindependent antigens (i.e., in the absence of an activation of helper T cells and their cooperative actions on the B cells). In that situation the Ig-secreting cells, which are generated to mainly produce IgM, remain short-lived and do not generate memory B cells. It remains also possible that gut-associated B1 cells can be flexible enough to respond in a T-cell-dependent fashion, form germinal centers, and generate long-lived, Ig class-switched plasma cells and memory B cells. In such cases they are, or become, conventional, B2-type B cells. Germinal center-like lymphoid aggregations have been seen to form near the flat epithelium M cells.
PERIPHERAL B CELLS WITHOUT Ig
In normal B cell development, B-lineage cells that cannot express Ig molecules on their surface are restricted to the primary lymphoid organs and will die there, usually with half-lives of 2 to 4 days. Experimentally induced ablation of surface Ig expression in peripheral mature B cells induces their rapid death (Lam et al., 1997). Therefore, neither immature, mature, memory B cells, nor plasma cells without Ig expression can ever be detected in the peripheral lymphoid organs, and even most B-cell tumors appear to be selected to express Ig. However, several observations, most of them made in vitro, suggest that B-lineage cells from as early as the Blineage committed progenitor B cells and pre-BI cells can differentiate all the way to memory-phenotype and plasma cell-like stages without ever expressing Ig. In kL-chainrearrangements-deficient bone marrow, over 90% of the small pre-BII cells have the proper phenotype, but no Lchain rearrangements, since rearrangements at the L chain loci are so slow that they can only be detected in the remaining 10% of the small pre-BII cells (Yamagami et al., 1999a, 1999b). Most striking is the development of RAG-deficient pre-BI cells (hence with IgH and L-chain loci in nonrearranged, germline configurations) in vitro in the stimulatory presence of CD40-specific monoclonal antibody and IL4 to sμ-sg-switched cells with a mature phenotype (Rolink et al., 1996). Hence, the differentiation of B-lineage cells is a stepwise process driven by cell–cell contacts and cytokines that can occur without the synthesis or surface deposition of Ig. The roles of the pre-BcR and BcR also become apparent from these experiments. They control, positively and negatively, proliferation, anergy, and apoptosis of B-lineage cells and, thereby, ascertain the production of appropriate numbers of cells with selected specificities. Viral infection of B cells by EBV (Epstein-Barr virus) may be another way, by which the requirement of surface Ig deposition on B cells for their survival and use in the peripheral lymphoid organs may be circumvented. The EBV encodes latent membrane proteins (LMP), of which LMP 2A, a multispanning transmembrane protein, expressed in B lymphocytes, can associate with Iga and Igb and share with these two anchor proteins an ITAM motif (Gross et al., 2005; Fruehilng & Longnecker, 1997; Caldwell et al., 1998). In fact, the phosphorylated form of the ITAM
32
host defense: general
motif of LAMP 2A recruits tyrosine kinases and adaptor proteins, which are also used by the BcR complex for signaling. In this way, LMP 2A acts as a BcR analog, supporting the selection into the peripheral mature B-cell compartments of B cells lacking BcRs. High-level transgenic expression of LMP 2A selectively recruits cells into the BI compartments (e.g., into the gut and intestine) while low level expression promotes development of conventional, B2-type, and marginal zone B cells (Casola et al., 2004). Neither of the high-level or low-level LMP2A expressing BcR-negative B-cell populations can be stimulated by T-cell-dependent antigens to develop germinal centers in peripheral lymphoid organs, indicating that BcRs on the surface of B cells are needed for peripheral germinal center responses. By contrast, the low-level, but not the high-level (i.e., the conventional) B2 (and not the B1) cell-directed LMP 2A expression allows germinal centers to be formed in the gut. Hence, the role of BcRs in germinal center formation in the gut might be replaced by an antigen-unspecific mechanism of antigen uptake, which allows processing and presentation by MHC class II molecules to helper T cells. Alternatively, these gut-associated BcR-deficient B cells might be stimulated by bystander helper T cells activated with bacteria of the gut or by bacteria-derived polyclonal activators, such as LPS and lipoprotein (Kawai & Akira, 2009), in the latter case via TLR2, TLR4, and other receptors (Akira & Takeda, 2004), or by other T-lineage cells or NK cells not restricted by the recognition of MHC class/ peptide complexes. In summary, T-cell (or NK-cell) dependent, BcR-independent (and perhaps MHC class II-independent) activation of B cells in germinal centers can lead to GC responses in which the activated B cells (as well as T or NK cells) can be expected to secrete pro-inflammatory cytokines such as IFN-g, IL-1, IL-4, IL-5, IL-6, and TNFa as essential mediators of immune responses. Furthermore, it has been observed in pre-BI cell-transplanted RAG-deficient mice, which contained only pre-BI cell-derived BI cells but no T cells, that one-third of the normal levels of IgA can be produced—apparently T-cellindependently—mainly by cells in the intestinal lamina propria without the formation of germinal centers. It is likely that these cells are also present in BcR-deficient, high-level LMP 2A-expressing lamina propria BI cells. Obviously they cannot produce IgA, but, again, they can be expected to respond to bacteria by the secretion of cytokines that might be of pro- or anti-inflammatory types. In conclusion, the GALT-associated B-cell compartments can develop even when the B cells do not express BcRs, or even MHC class II (i.e., can function in cellular responses of proliferation and differentiation to effector functions in an antigen-independent, and perhaps bacteriadependent way, leading to pro-inflammatory (and maybe also anti-inflammatory) responses. From all of these results, it becomes evident that peripheral mature B cells have important functions beyond their capacity to produce, secrete, class switch, and hypermutate antibodies in the regulation of immune reactions.
LINKING THE DEFENSIVE POWER OF THE INNATE IMMUNE SYSTEM WITH ANTIGEN SPECIFICITY OF THE ADAPTIVE IMMUNE SYSTEM
Since B lymphocytes can be stimulated to develop into cells that no longer deposit their BcRs (Ig) as antigen-specific receptors into the surface membrane but rather secrete this Ig as so-called plasma cells into the blood stream, antigen specificity of a B cell can be transferred to the innate
immune system in three ways. (i) Ig molecules of different IgH chain classes can find receptors for the constant regions of their H chains (so-called Fc receptors) and, thereby, can transfer this antigen specificity (by so-called opsonization) to antigen-nonspecific cells (e.g., cells of the innate immune system, such as macrophages or dendritic cells). (ii) The antigen-unspecific cells of the system can bind antigen–antibody (Ig)-complement immune complexes via complement receptors, again gaining antigen specificity by opsonization. (iii) As mature B cells displaying Ig on their surface, they can attract “their” specific protein antigens with high avidity, take it up, process it, and present it as peptides complexed with MHC class II molecules to helper T cells (i.e., they can act as antigen-specific antigen-presenting cells).
THE TRANSFER OF IMMATURE LYMPHOCYTES FROM PRIMARY LYMPHOID ORGANS TO MATURE COMPARTMENTS IN THE PERIPHERY
Although VH-repertoires expressed in immature B cells in the bone marrow and in immature and mature B cells in the spleen are not significantly different, almost 90% of the newly formed immature B cells never leave the bone marrow (Rolink et al., 1999a). Therefore, they should be subject to additional deletion mechanisms. In humans, there is a further reduction of the percentage of autoreactive cells (40% to 20%) across this transition, while the percent of polyreactive cells remains at the low level (6%) already achieved during the transition from pre-B to immature B cells (Wardemann & Nussenzweig, 2007). Again, SLE and RA patients fail to establish this repertoire change at this second checkpoint. Indicating that several mechanisms for establishing recessive tolerance may contribute to these diseases (Yurasov et al., 2005). Once arrived in the spleen, hardly any cells are lost at the transition from immature, including transitional T1 and T2 cells, to mature B cells. Hence, the mature B1 and B2 cells emerge only after passing through at least two checkpoints, suggesting that several mechanisms may operate to establish tolerance. Both of the checkpoints are not functioning in SLE and RA patients and, thus, may contribute to these diseases. The BcR complex with Iga and Igb is mandatory for transition to the peripheral sIgM1B cell pools—no sIg2 B cells survive normally. The transition from short-lived to long-lived, from AA4.11 to AA4.12, CD212/CD232 to CD211/CD231 cells is controlled by a series of genes, including OBF, btk, and CD40. Thus, btk/CD402 or btk2/ OBF− double deficient mice have a strong defect at the transition from the immature, transitional T1- and T2-type B cells to mature B cells (Rolink et al., 1999a). In addition, cross-linking of surface IgM by anti-IgM-antibodies (possibly a polyclonal example of a cross-linking auto-antigen) induces immature B cells of bone marrow as well as spleen (T1 and T2) to apoptosis. This apoptosis can be prevented by the transgenic expression of the anti-apoptotic transgene bcl-2, and is circumvented by polyclonal activation with LPS and with CD40-specific antibodies or CD40 ligand. Deficiency in the expression of the TNF-family member BAFF (also called B-lys), or its receptor, BAFF-R, blocks the maturation of immature B cells into conventional, B2-type B cells, but not to B1-type B cells (Rolink et al., 2002b; Rolink & Melchers, 2002; Ng et al., 2005; Moore et al., 1999; Schiemann et al., 2001; Gross et al., 2001). In vitro BAFF induces polyclonal maturation of immature B cells from bone marrow and from the spleen (T1 or T2) without proliferation. BAFF has been found in the sera of some SLE
2. the ontogeny of the Cells of the Innate and the adaptive Immune system
patients, as well as in the sera of NZBxNZW SLE autoimmune disease prone mice. In these mice, administration of BAFF-specific antibodies prevents, or at least delays the development of SLE-disease. Therefore, excessive production of BAFF, administered at the sites of negative selection of immature B cells, could “rescue” autoreactive B cells from deletion, leading to autoimmune reactivities in the peripheral mature conventional B-cell repertoires. One major site of BAFF production has been found to be dendritic cells. Hence, in order to interfere with the deletion of autoreactive immature B cells such dendritic cells would have to be localized near the sites of B-cell deletion in bone marrow or the spleen, and would have to be strongly activated to BAFF production to effect rapid maturation of autoreactive immature B cells before they are subjected to negative selection. Such a scenario still needs to be investigated, and the observed partial rescue of B-cell tolerance by T-cell independent antigens may be one experimental way to probe the molecular and cellular requirements for such competition with clonal deletion. In T-cell development, once a/b TcR-expressing thymocytes have been probed as CD41CD81 DP cells for their appropriate fitness to MHC-self-peptide complexes, their first choice, either to become a Th or a Tc cell, has been made. Their further fate, to polarize either into Th1 or Th2 or follicular B helper T or Th17 cells or to Tc1 or Tc2 cells, appears to made as a consequence of a response to MHC-foreign peptide complexes created by introduction or infection of a foreign protein antigen in the secondary lymphoid organs. While Treg cell induction begins in the thymus and appears to be guided by MHCII-self-peptide complex recognition, the rules of their initial selection and their further maturation need to be worked out in detail. The generated repertoires of lymphocytes with BcRs and TcRs, at any time in the life of an individual, are limited by the number of cells generated per day (i.e., 0.1% of the total B- and T-cell pools in a newborn and may be less than 0.001% in an adult), possibly decreasing with age as the frequencies of CLPs and their DJ-rearranged descendants (i.e., FLT3L/IL7/stromal cell-clonal precursors have been seen, at least for the B lineage, to decrease 20- to 100-fold within a few months in mice and from birth to 10 years of age in humans (Ghia et al., 1998, 2000). Since the thymus involutes after sexual maturity, this should also be the case for T-lineage progenitors. This means that in newborn mice with around 5 3 107 B cells as well as T cells, 5 3 104 cells each may be generated per day, while in adult mice, with around 5 3 108 B cells and T cells each, around 5 3 103 B cells and T cells may be generated. Humans, which show striking similarities in the generation of B cells and T cells in bone marrow and the thymus, have approximately 1,000-fold higher numbers of B and T cells and should, therefore, also generate 1,000-fold more cells with newly formed BcRs or TcRs.
FAILURES OF CENTRAL TOLERANCE B Cells
Prolongation of the short half-life of B cells brought about either by a decrease in expression and/or activity of proapoptotic genes or by an increase in pro-survival gene expression (e.g., B lymphocyte-activating factor, BAFF) favors B-cell driven autoimmunity that manifests as SLE-like syndromes (Mackay et al., 1999; Rolink et al., 2002b). Elevated expression of the genes encoding Toll-like receptors (Deane et al., 2007; Shlomchik, 2008) can promote similar autoimmune manifestations.
33
Signaling defects can also impair central tolerance induction in immature B cells. For example, the NZM2410/NZW mouse strain-derived z alleles of Sle1 and its sublocus, Sle1b, as well as the orthologous human locus impair B-cell anergy, receptor editing, and deletion (Liu et al., 2007; Kumar et al., 2006). Members of the SLAM family of costimulatory molecules are candidate Sle1b gene products, among which the Ly1081 isoform of the Ly108 gene is highly expressed in immature B cells (Xie et al., 2007). The Ly108.2 allele, but not the SLE-associated Ly1081 allele, sensitizes immature B cells to deletion and RAG reexpression. Hence, Ly108 is a potential regulator of tolerance checkpoints, as it may censor self-reactive B cells by protecting from autoimmunity. When the z allele of Sle1 is combined with the Fas lpr mutation, lymphoproliferative autoimmunity induced by the phosphatidylinositol-3-kinase-AKT-mTOR pathway is induced. As the signaling pathways in immature B cells will become known in greater detail, more genes that interfere with the proper establishment of B-cell central tolerance are likely to be identified.
T Cells
Lowered expression of pro-apoptotic genes and enhanced expression of pro-survival factors (Takahama et al., 1992; Buch et al., 2002; Bouillet et al., 2002) contribute to a shift towards inclusion of self-peptide/self-MHC-reactive T cells in the mature compartments. The mode of antigen presentation appears to play a major role in human T-cell-dependent autoimmune diseases. Juvenile diabetes contributing susceptibility factors include those that affect the amount of insulin expressed intrathymically (McCaughtry et al., 2008). Consequently, thymic overexpresssion of insulin in diabetes-prone NOD mice reduced the incidence of diabetes (Baldwin et al., 2005). Like in the B-cell lineage, mutations in signal transducing molecules, such as ZAP-70, result in autoimmune disease (Smith et al., 1989). It appears that the ZAP-70 mutations alter the balance of positive and negative selection, allowing certain T cells to escape negative selection and then to be activated by their self-ligands in peripheral lymphoid tissues. It seems possible that these mutations could also disturb the balance of positive selection and Treg cell generation in the CD41 T-cell compartments. In analogy to B cells expressing more than one BCR, it was found that expression of a second TCR in addition to an autoreactive TCR can “dilute” the signaling capacity of the autoreactive TCR such that these cells can escape negative selection (Wack et al., 1996).
CONCLUSIONS
By the time newly generated lymphocytes have been deposited in their peripheral, mature compartments of all the T cells and a large part of the B1 cells have been auto-antigenligand selected (hence their repertoire purged) even before a foreign antigen can select “its” specific lymphocytes for a response. This leaves “holes” in the diversity of these repertoires (i.e., their capacities to recognize a foreign antigen). By contrast, those conventional B cells that are ignored by negative and positive selection should be more diversely reactive, although their short half-life in the system limits them. If these conventional B cells could not enter a helper T-cell-dependent response of Ig class switching and v-region hypermutation, the capacity of most of them to recognize the wide world of foreign antigens might well be much too low to elicit a clonally selected, high-avidity immune response with defensive power.
34
host defense: general
The author has recently written two review articles (Melchers, 2009; von Boehmer & Melchers, 2010), which have covered parts of the contents of this article. He is the recipient of a Reinhard Kosellek grant of the DFG (ME 2467/1-1).
REFERENCES Adams, G. B., K. T. Chabner, I. R. Alley, D. P. Olson, Z. M. Szczepiorkowski, M. C. Poznansky, C. H. Kos, M. R. Pollak, E. M. Brown, and D. T. Scadden. 2006. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 439:599–603. Adolfsson, J., O. J. Borge, D. Bryder, K. Theilgaard-Mönch, I. Astrand-Grundström, E. Sitnicka, Y. Sasaki, and S. E. Jacobsen. 2001. Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(1)c-kit(1) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 15:659–669. Aifantis, I., C. Borowski, F. Gounari, H. D. Lacorazza, J. Nikolich-Zugich, and H. von Boehmer. 2002. A critical role for the cytoplasmic tail of pTalpha in T lymphocyte development. Nat. Immunol. 3:483–488. Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4:499–511. Antonchuk, J., G. Sauvageau, and R. K. Humphries. 2002. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109:39–45. Apostolou, I., A. Sarukhan, L. Klein, and H. von Boehmer. 2002. Origin of regulatory T cells with known specificity for antigen. Nat. Immunol. 3:756–763. Arai, F., A. Hirao, M. Ohmura, H. Sato, S. Matsuoka, K. Takubo, K. Ito, G. Y. Koh, and T. Suda. 2004. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118:149–161. Baba, Y., K. P. Garrett, and P. W. Kincade. 2005. Constitutively active beta-catenin confers multilineage differentiation potential on lymphoid and myeloid progenitors. Immunity 23:599–609. Bain, G., E. C. Maandag, D. J. Izon, D. Amsen, A. M. Kruisbeek, B. C. Weintraub, I. Krop, M. S. Schlissel, A. J. Feeney, and M. van Roon. 1994. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 79:885–892. Baldwin, T. A., M. M. Sandau, S. C. Jameson, and K. A. Hogquist. 2005. The timing of TCR alpha expression critically influences T cell development and selection. J. Exp. Med. 202:111–121. Bendelac, A., P. B. Savage, and L. Teyton. 2007. The biology of NKT cells. Annu. Rev. Immunol. 25:297–336. Borgulya, P., H. Kishi, Y. Uematsu, and H. von Boehmer. 1992. Exclusion and inclusion of alpha and beta T cell receptor alleles. Cell 69:529–537. Bouillet, P., J. F. Purton, D. I. Godfrey, L. C. Zhang, L. Coultas, H. Puthalakath, M. Pellegrini, S. Cory, J. M. Adams, and A. Strasser. 2002. BH3-only Bcl-2 family member Bim is required for apoptosis of autoreactive thymocytes. Nature 415:922–926. Bradl, H., J. Wittmann, D. Milius, C. Vettermann, and H. M. Jäck. 2003. Interaction of murine precursor B cell receptor with stroma cells is controlled by the unique tail of lambda 5 and stroma cell-associated heparan sulfate. J. Immunol. 171:2338–2348. Buch, T., F. Rieux-Laucat, I. Förster, and K. Rajewsky. 2002. Failure of HY-specific thymocytes to escape negative selection by receptor editing. Immunity 16:707–718. Burnet, F. M. 1957. A modification of Jerne’s theory of antibody production using the concept of clonal selection. Austral. J. Sci. 20: 67–69. Buske, C., M. Feuring-Buske, C. Abramovich, K. Spiekermann, C. J. Eaves, L. Coulombel, G. Sauvageau, D. E. Hogge, and R. K. Humphries. 2002. Deregulated expression of HOXB4 enhances the primitive growth activity of human hematopoietic cells. Blood 100:862–868.
Busslinger, M. 2004. Transcriptional control of early B cell development. Annu. Rev. Immunol. 22:55–79. Caldwell, R. G., J. B. Wilson, S. J. Anderson, and R. Longnecker. 1998. Epstein-Barr virus LMP2A drives B cell development and survival in the absence of normal B cell receptor signals. Immunity 9:405–411. Carroll, M. C. 2004. A protective role for innate immunity in systemic lupus erythematosus. Nat. Rev. Immunol. 4:825–831. Casanova, J. L., P. Romero, C. Widmann, P. Kourilsky, and J. L. Maryanski. 1991. T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire. J. Exp. Med. 174:1371–1383. Casola, S., K. L Otipoby, M. Alimzhanov, S. Humme, N. Uyttersprot, J. L. Kutok, M. C. Carroll, and K. Rajewsky. 2004. B cell receptor signal strength determines B cell fate. Nat. Immunol. 5:317–327. Ceredig, R., E. ten Boekel, A. Rolink, F. Melchers, and J. Andersson. 1998. Fetal liver organ cultures allow the proliferative expansion of pre-B receptor-expressing preB-II cells and the differentiation of immature and mature B cells in vitro. Int. Immunol. 10:49–59. Choi, J. K., C. P. Shen, H. S. Radomska, L. A. Eckhardt, and T. Kadesch. 1996. E47 activates the Ig-heavy chain and TdT loci in non-B cells. EMBO J. 15:5014–5021. Craig, S. W., and J. J. Cebra. 1971. Peyer’s patches: an enriched source of precursors for IgA-producing immunocytes in the rabbit. J. Exp. Med. 134:188–200. Cumano, A., F. Dieterlen-Lievre, and I. Godin. 1996. Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell 86:907–916. Cumano, A., J. C. Ferraz, M. Klaine, J. P. Di Santo, and I. Godin. 2001. Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide longterm multilineage reconstitution. Immunity 15:477–485. Dakic, A., D. Metcalf, L. Di Rago, S. Mifsud, L. Wu, and S. L. Nutt. 2005. PU.1 regulates the commitment of adult hematopoietic progenitors and restricts granulopoiesis. J. Exp. Med. 201:1487–1502. Deane, J. A., P. Pisitkun, R. S. Barrett, L. Feigenbaum, T. Town, J. M. Ward, R. A. Flavell, and S. Bolland. 2007. Control of toll-like receptor 7 expression is essential to restrict autoimmunity and dendritic cell proliferation. Immunity 27:801–810. Doyle, C. M., J. Han, M. G. Weigert, and E. T. Prak. 2006. Consequences of receptor editing at the lambda locus: multireactivity and light chain secretion. Proc. Natl. Acad. Sci. USA 103:11264–11269. Duncan, A. W., F. M. Rattis, L. N. DiMascio, K. L. Congdon, G. Pazianos, C. Zhao, K. Yoon, J. M. Cook, K. Willert, N. Gaiano, and T. Reya. 2005. Integration of Notch and Wnt signaling in hematopoietic stem cell maintenance. Nat. Immunol. 6:314–322. Egawa, T., K. Kawabata, H. Kawamoto, K. Amada, R. Okamoto, N. Fujii, T. Kishimoto, Y. Katsura, and T. Nagasawa. 2001. The earliest stages of B cell development require a chemokine stromal cell-derived factor/pre-B cell growth-stimulating factor. Immunity 15:323–334. Engel, I., and C. Murre. 2001. The function of E- and Id proteins in lymphocyte development. Nat. Rev. Immunol. 1:193–199. Evans, M. J., and M. H. Kaufman. 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156. Fields, M. L., and J. Erikson. 2003. The regulation of lupusassociated autoantibodies: immunoglobulin transgenic models. Curr. Opin. Immunol. 15:709–717. Flemming, A., T. Brummer, M. Reth, and H. Jumaa. 2003. The adaptor protein SLP-65 acts as a tumor suppressor that limits pre-B cell expansion. Nat. Immunol. 4:38–43.
2. the ontogeny of the Cells of the Innate and the adaptive Immune system Förster, I., and K. Rajewsky. 1990. The bulk of the peripheral B-cell pool in mice is stable and not rapidly renewed from the bone marrow. Proc. Natl. Acad. Sci. USA 87:4781–4784. Fruehling, S., and R. Longnecker. 1997. The immunoreceptor tyrosine-based activation motif of Epstein-Barr virus LMP2A is essential for blocking BCR-mediated signal transduction. Virology 235:241–251. Gekas, C., F. Dieterlen-Lièvre, S. H. Orkin, and H. K. Mikkola. 2005. The placenta is a niche for hematopoietic stem cells. Dev. Cell. 8:365–375. Gerdes, T., and M. Wabl. 2004. Autoreactivity and allelic inclusion in a B cell nuclear transfer mouse. Nat. Immunol. 5:1282–1287. Ghia, P., F. Melchers, and A. G. Rolink. 2000. Age-dependent changes in B lymphocyte development in man and mouse. Exp. Gerontol. 35:159–165. Ghia, P., E. ten Boekel, A. G. Rolink, and F. Melchers. 1998. B-cell development: a comparison between mouse and man. Immunol. Today. 19:480–485. Gilliland, D. G., and J. D. Griffin. 2002. The roles of FLT3 in hematopoiesis and leukemia. Blood 100:1532–1542. Godin, I., and A. Cumano. 2002. The hare and the tortoise: an embryonic haematopoietic race. Nat. Rev. Immunol. 2:593–604. Graff, J. M. 1997. Embryonic patterning: to BMP or not to BMP, that is the question. Cell 89:171–174. Grawunder, U., D. Haasner, F. Melchers, and A. Rolink. 1993. Rearrangement and expression of kappa light chain genes can occur without mu heavy chain expression during differentiation of pre-B cells. Int. Immunol. 5:1609–1618. Grawunder, U., A. Rolink, and F. Melchers. 1995. Induction of sterile transcription from the kappa L chain gene locus in V(D)J recombinase-deficient progenitor B cells. Int. Immunol. 7:1915–1925. Gross, A. J., D. Hochberg, W. M. Rand, and D. A. ThorleyLawson. 2005. EBV and systemic lupus erythematosus: a new perspective. J. Immunol. 174:6599–6607. Gross, J. A., S. R. Dillon, S. Mudri, J. Johnston, A. Littau, R. Roque, M. Rixon, O. Schou, K. P. Foley, H. Haugen, S. McMillen, K. Waggie, R. W. Schreckhise, K. Shoemaker, T. Vu, M. Moore, A. Grossman, and C. H. Clegg. 2001. TACI-Ig neutralizes molecules critical for B cell development and autoimmune disease. Impaired B cell maturation in mice lacking BLyS. Immunity 15:289–302. Guy-Grand, D., and P. Vassalli. 2004. Immunology. Tracing an orphan’s genealogy. Science 305:185–187. Haks, M. C., J. M. Lefebvre, J. P. Lauritsen, M. Carleton, M. Rhodes, T. Miyazaki, D. J. Kappes, and D. L. Wiest. 2005. Attenuation of gamma deltaTCR signaling efficiently diverts thymocytes to the alpha beta lineage. Immunity 22:595–606. Hanna, J., S. Markoulaki, P. Schorderet, B. W. Carey, C. Beard, M. Wernig, M. P. Creyghton, E. J. Steine, J. P. Cassady, R. Foreman, C. J. Lengner, J. A. Dausman, and R. Jaenisch 2008. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133:250–264. Hayakawa, K., M. Asano, S. A. Shinton, M. Gui, D. Allman, C. L. Stewart, J. Silver, and R. R. Hardy. 1999. Positive selection of natural autoreactive B cells. Science 285:113–116. Hayes, S. M., L. Li, and P. E. Love. 2005. TCR signal strength influences alpha beta/gamma delta lineage fate. Immunity 22:583–593. Heath, W. R., F. R. Carbone, P. Bertolino, J. Kelly, S. Cose, and J. F. Miller. 1995. Expression of two T cell receptor alpha chains on the surface of normal murine T cells. Eur. J. Immunol. 25:1617–1623. Helgason, C. D., G. Sauvageau, H. J. Lawrence, C. Largman, and R. K. Humphries. 1996. Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro. Blood 87:2740–2749.
35
Höflinger, S., K. Kesavan, M. Fuxa, C. Hutter, B. Heavey, F. Radtke, and M. Busslinger. 2004. Analysis of Notch1 function by in vitro T cell differentiation of Pax5 mutant lymphoid progenitors. J. Immunol. 173:3935–3944. Hogan, B. L. 1996. Bone morphogenetic proteins in development. Curr. Opin. Genet. Dev. 6:432–438. Hromas, R., A. Orazi, R. S. Neiman, R. Maki, C. van Beveran, J. Moore, and M. Klemsz. 1993. Hematopoietic lineage- and stage-restricted expression of the ETS oncogene family member PU.1. Blood 82:2998–3004. Ikawa, T., S. Fujimoto, H. Kawamoto, Y. Katsura, and Y. Yokota. 2001. Commitment to natural killer cells requires the helix-loop-helix inhibitor Id2. Proc. Natl. Acad. Sci. USA 98:5164–5169. Ikawa, T., H. Kawamoto, L. Y. Wright, and C. Murre. 2004. Long-term cultured E2A-deficient hematopoietic progenitor cells are pluripotent. Immunity 20:349–360. Iwama, A., H. Oguro, M. Negishi, Y. Kato, Y. Morita, H. Tsukui, H. Ema, T. Kamijo, Y. Katoh-Fukui, H. Koseki, M. van Lohuizen, and H. Nakauchi. 2004. Enhanced selfrenewal of hematopoietic stem cells mediated by the polycomb gene product Bmi-1. Immunity 21:843–851. Izon, D. J., J. C. Aster, Y. He, A. Weng, F. G. Karnell, V. Patriub, L. Xu, S. Bakkour, C. Rodriguez, D. Allman, and W. S. Pear. 2002. Deltex1 redirects lymphoid progenitors to the B cell lineage by antagonizing Notch1. Immunity 16:231–243. Jerne, N. K. 1955. The natural selection theory of antibody formation Proc. Natl. Acad. Sci. USA 41:849–852. Jordan, C. T., and I. R. Lemischka. 1990. Clonal and systemic analysis of long-term hematopoiesis in the mouse. Genes Dev. 4:220–232. Jordan, M. S., A. Boesteanu, A. J. Reed, A. L. Petrone, A. E. Holenbeck, M. A. Lerman, A. Naji, and A. J. Caton. 2001. Thymic selection of CD41CD251 regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2:301–306. Jumaa, H., L. Bossaller, K. Portugal, B. Storch, M. Lotz, A. Flemming, M. Schrappe, V. Postila, P. Riikonen, J. Pelkonen, C. M. Niemeyer, and M. Reth. 2003. Deficiency of the adaptor SLP-65 in pre-B-cell acute lymphoblastic leukaemia. Nature 423:452–456. Katayama, Y., M. Battista, W. M. Kao, A. Hidalgo, A. J. Peired, S. A.Thomas, and P. S. Frenette. 2006. Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow. Cell 124:407–421. Kawabata, K., M. Ujikawa, T. Egawa, H. Kawamoto, K. Tachibana, H. Iizasa, Y. Katsura, T. Kishimoto, and T. Nagasawa. 1999. A cell-autonomous requirement for CXCR4 in long-term lymphoid and myeloid reconstitution. Proc. Natl. Acad. Sci. USA 96:5663–5667. Kawai, T., and S. Akira. 2009. The roles of TLRs, RLRs and NLRs in pathogen recognition. Int. Immunol. 21:317–337. Kee, B. L., and C. Murre. 1998. Induction of early B cell factor (EBF) and multiple B lineage genes by the basic helix-loophelix transcription factor E12. J. Exp. Med. 188:699–713. Keenan, R. A., A. de Riva, B. Corleis, L. Hepburn, S. Licence, T. H. Winkler, and I. L. Mårtensson. 2008. Censoring of autoreactive B cell development by the pre-B cell receptor. Science 321:696–699. Khan, S. N., E. J. Witsch, N. G. Goodman, A. K. Panigrahi, C. Chen, Y. Jiang, A. M. Cline, J. Erikson, M. Weigert, E. T. Prak, and M. Radic. 2008. Editing and escape from editing in anti-DNA B cells. Proc. Natl. Acad. Sci. USA 105:3861–3866. Kincade, P. W. 1981. Formation of B lymphocytes in fetal and adult life. Adv. Immunol. 31:177–245. Kincade, P. W., H. Igarashi, K. L. Medina, T. Kouro, T. Yokota, M. I. Rossi, J. J. Owen, K. P. Garrett, X. H. Sun, and N. Sakaguchi. 2002. Lymphoid lineage cells in adult murine bone marrow diverge from those of other blood cells at an early, hormone-sensitive stage. Semin. Immunol. 14:385–394.
36
host defense: general
Klemsz, M. J., S. R. McKercher, A. Celada, c. van Beveren, and R. A. Maki. 1990. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell 61:113–124. Koch, U., T. A. Lacombe, D. Holland, J. L. Bowman, B. L. Cohen, S. E. Egan, and C. J. Guidos. 2001. Subversion of the T/B lineage decision in the thymus by lunatic fringemediated inhibition of Notch-1. Immunity 15:225–236. Köhler, F., E. Hug, C. Eschbach, S. Meixlsperger, E. Hobeika, J. Kofer, H. Wardemann, and H. Jumaa. 2008. Autoreactive B cell receptors mimic autonomous pre-B cell receptor signaling and induce proliferation of early B cells. Immunity 29:912–921. Kreslavsky, T., A. K. Savage, R. Hobbs, F. Gounari, R. Bronson, P. Pereira, P. P. Pandolfi, A. Bendelac, and H. von Boehmer. 2009. TCR-inducible PLZF transcription factor required for innate phenotype of a subset of gamma delta T cells with restricted TCR diversity. Proc. Natl. Acad. Sci. USA 106:12453–12458. Krosl, J., P. Austin, N. Beslu, E. Kroon, R. K. Humphries, and G. Sauvageau. 2003. In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein. Nat. Med. 9:1428–1432. Kumar, K. R., L. Li, M. Yan, M. Bhaskarabhatla, A. B. Mobley, C. Nguyen, J. M. Mooney, J. D. Schatzle, E. K. Wakeland, and C. Mohan. 2006. Regulation of B cell tolerance by the lupus susceptibility gene Ly108. Science 312:1665–1669. Kyba, M., R. C. Perlingeiro, and G. Q. Daley. 2002. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 109:29–37. Kyewski, B., and L. Klein. 2006. A central role for central tolerance. Annu. Rev. Immunol. 24:571–606. Lam, K. P., R. Kühn, and K. Rajewsky. 1997. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 90:1073–1083. Lederberg, J. 1959. Genes and antibodies. Science 129: 1649–1653. Leslie, D., P. Lipsky, and A. L. Notkins. 2001. Autoantibodies as predictors of disease. J. Clin. Invest. 108:1417–1422. Li, H., Y. Jiang, E. L. Prak, M. Radic, and M. Weigert. 2001. Editors and editing of anti-DNA receptors. Immunity 15:947–957. Lin, H., and R. Grosschedl. 1995. Failure of B-cell differentiation in mice lacking the transcription factor EBF. Nature 376:263–267. Ling, K. W., and E. Dzierzak. 2002. Ontogeny and genetics of the hemato/lymphopoietic system. Curr. Opin. Immunol. 14:186–191. Liu, Y., L. Li, K. R. Kumar, C. Xie, S. Lightfoot, X. J. Zhou, J. F. Kearney, M. Weigert, and C. Mohan. 2007. Lupus susceptibility genes may breach tolerance to DNA by impairing receptor editing of nuclear antigen-reactive B cells. J. Immunol. 179:1340–1352. Ma, Q., D. Jones, P. R. Borghesani, R. A. Segal, T. Nagasawa, T. Kishimoto, R. T. Bronson, and T. A. Springer. 1998. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc. Natl. Acad. Sci. USA 95:9448–9453. Ma, Q., D. Jones, and T. A. Springer. 1999. The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment. Immunity 10:463–471. Mackay, F., S. A. Woodcock, P. Lawton, C. Ambrose, M. Baetscher, P. Schneider, J. Tschopp, and J. L. Browning. 1999. Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations. J. Exp. Med. 190:1697–1710. Maeno, M., P. E. Mead, C. Kelley, R. H. Xu, H. F. Kung, A. Suzuki, N. Ueno, and L. I. Zon. 1996. The role of BMP-4 and GATA-2 in the induction and differentiation of hematopoietic mesoderm in Xenopus laevis. Blood 88:1965–1972.
Martin, G. R. 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78:7634. Matise, M. P., W. Auerbach, and A. L. Joyner. 2001. Production of targeted embryonic stem cell clones, p. 101–132. In A. L. Johner (ed.), Gene Targeting. A Practical Approach, 2nd ed. Oxford University Press, New York. McCaughtry, T. M., T. A. Baldwin, M. S. Wilken, and K. A. Hogquist. 2008. Clonal deletion of thymocytes can occur in the cortex with no involvement of the medulla. J. Exp. Med. 205:2575–2584. Medina, K. L., K. P. Garrett, L. F. Thompson, M. I. Rossi, K. J. Payne, and P. W. Kincade. 2001. Identification of very early lymphoid precursors in bone marrow and their regulation by estrogen. Nat. Immunol. 2:718–724. Medina, K. L., G. Smithson, and P. W. Kincade. 1993. Suppression of B lymphopoiesis during normal pregnancy. J. Exp. Med. 178:1507–1515. Medvinsky, A., and E. Dzierzak. 1996. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 86:897–906. Melchers, F. 1979. Murine embryonic B lymphocyte development in the placenta. Nature 277:219–221. Melchers, F. 2005. The pre-B-cell receptor: selector of fitting immunoglobulin heavy chains for the B-cell repertoire. Nat. Rev. Immunol. 5:578–584. Melchers, F. 2009. Ontogeny of B cells, p. 1–22. In I. T. Magrath (ed.), The Lymphoid Neoplasms, 3rd ed., Hodder Publishers, Abingdon, UK. Melchers, F., A. G. Rolink, and C. Schaniel. 1999. The role of chemokines in regulating cell migration during humoral immune responses. Cell 99:351–354. Melchers, F., and A. R. Rolink. 2006. B cell tolerance—how to make it and how to break it. Curr. Top. Microbiol. Immunol. 305:1–23. Melchers, F., E. ten Boekel, T. Seidl, X. C. Kong, T. Yamagami, K. Onishi, T. Shimizu, A.-G. Rolink, and J. Andersson. 2000. Repertoire selection by pre-B-cell receptors and B-cell receptors, and genetic control of B-cell development from immature to mature B cells. Immunol. Rev. 175:33–46. Mikkola, I., B. Heavey, M. Horcher, and M. Busslinger. 2002. Reversion of B cell commitment upon loss of Pax5 expression. Science 297:110–113. Moore, P. A., O. Belvedere, A. Orr, K. Pieri, D. W. LaFleur, P. Feng, D. Soppet, M. Charters, R. Gentz, D. Parmelee, Y. Li, O. Galperina, J. Giri, V. Roschke, B. Nardelli, J. Carrell, S. Sosnovtseva, W. Greenfield, S. M. Ruben, H. S. Olsen, J. Fikes, and D. M. Hilbert. 1999. BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator. Science 285:260–263. Morrison, S. J., N. Uchida, and I. L. Weissman. 1995. The biology of hematopoietic stem cells. Annu. Rev. Cell. Dev. Biol. 11:35–71. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and T. Honjo. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553–563. Nagasawa, T., T. Nakajima, K. Tachibana, H. Iizasa, C. C. Bleul, O. Yoshie, K. Matsushima, N. Yoshida, T. A. Springer, and T. Kishimoto. 1996. Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin. Proc. Natl. Acad. Sci. USA. 93:14726–14729. Nakano, T., H. Kodama, and T. Honjo. 1994. Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265:1098–1101. Nemazee, D. 1995. Does immunological tolerance explain the waste in the B-lymphocyte immune system? Experiment and theory. Ann. NY Acad. Sci. 764:397–401.
2. the ontogeny of the Cells of the Innate and the adaptive Immune system Ng, L. G., C. R. Mackay, and F. Mackay. 2005. The BAFF/ APRIL system: life beyond B lymphocytes. Mol. Immunol. 42:763–772. Nossal, G. J. V., and J. Lederberg. 1958. Antibody production by single cells. Nature 1419–1420. Nutt, S. L., D. Metcalf, A. D’Amico, M. Polli, and L. Wu. 2005. Dynamic regulation of PU.1 expression in multipotent hematopoietic progenitors. J. Exp. Med. 201:221–231. Ohmura, K., H. Kawamoto, M. Lu, T. Ikawa, S. Ozaki, K. Nakao, and Y. Katsura. 2001. Immature multipotent hemopoietic progenitors lacking long-term bone marrow-reconstituting activity in the aorta-gonad-mesonephros region of murine day 10 fetuses. J. Immunol. 166:3290–3296. Ohnishi, K., and F. Melchers. 2003. The nonimmunoglobulin portion of lambda5 mediates cell-autonomous pre-B cell receptor signaling. Nat. Immunol. 4:849–856. Okita, K., T. Ichisaka, and S. Yamanaka. 2007. Generation of germline-competent induced pluripotent stem cells. Nature 448:313–317. Osawa, M., K. Hanada, H. Hamada, and H. Nakauchi. 1996a. Long-term lymphohematopoietic reconstitution by a single CD34-lowenegative hematopoietic stem cell. Science 273:242–245. Osawa, M., K. Nakamura, N. Nishi, N. Takahasi, Y. Tokuomoto, H. Inoue, and H. Nakauchi. 1996b. In vivo selfrenewal of c-Kit1 Sca-11 Lin(low/-) hemopoietic stem cells. J. Immunol. 156:3207–3214. Osmond, D. G., G. M. Fulop, D. Opstelten, and C. Pietrangeli. 1985. In vivo regulation of B lymphocyte production in the bone marrow: effects and mechanism of action of exogenous stimuli on pre-B cell proliferation and lymphocyte turnover. Adv. Exp. Med. Biol. 186:35–46. Ottersbach, K., and E. Dzierzak. 2005. The murine placenta contains hematopoietic stem cells within the vascular labyrinth region. Dev. Cell. 8:377–387. Owen, J. J., M. C. Raff, and M. D. Cooper. 1976. Studies on the generation of B lymphocytes in the mouse embryo. Eur. J. Immunol. 5:468–473. Padovan, E., G. Casorati, P. Dellabona, S. Meyer, M. Brockhaus, and A. Lanzavecchia. 1993. Expression of two T cell receptor alpha chains: dual receptor T cells. Science 262:422–424. Pan, X., O. Ohneda, K. Ohneda, F. Lindeboom, F. Iwata, R. Shimizu, M. Nagano, N. Suwabe, S. Philipsen, K. C. Lim, J. D. Engel, and M. Yamamoto. 2005. Graded levels of GATA-1 expression modulate survival, proliferation, and differentiation of erythroid progenitors. J. Biol. Chem. 280:22385–22394. Park, I. K., D. Qian, M. Kiel, M. W. Becker, M. Pihalja, I. L. Weissman, S. J. Morrison, and M. F. Clarke. 2003. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 423:302–305. Peterson, P., T. Org, and A. Rebane. 2008. Transcriptional regulation by AIRE: molecular mechanisms of central tolerance. Nat. Rev. Immunol. 8:948–957. Pui, J. C., D. Allman, L. Xu, S. DeRocco, F. G. Karnell, S. Bakkour, J. Y. Lee, T. Kadesch, R. R. Hardy, J. C. Aster, and W. S. Pear. 1999. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 11:299–308. Radtke, F., A. Wilson, S. J. Mancini, and H.r. MacDonald. 2004. Notch regulation of lymphocyte development and function. Nat. Immunol. 5:247–253. Radtke, F., A. Wilson, G. Stark, M. Bauer, J. van Meerwijk, H. R. MacDonald, and M. Aguet. 1999. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10:547–558. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. Catalan, M. Forveille, R. Dufourcq-Labelouse, A. Gennery, I. Tezcan, F. Ersoy, H. Kayserili, A. G. Ugazio, N. Brousse, M. Muramatsu, L. D. Notarangelo, K. Kinoshita, T. Honjo, A. Fischer, and A. Durandy.
37
2000. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the HyperIgM syndrome (HIGM2). Cell 102:565–575. Reya, T., A. W. Duncan, L. Ailles, J. Domen, D. C. Scherer, K. Willert, L. Hintz, R. Nusse, and I. L. Weissman. 2003. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423:409–414. Rico-Vargas, S. A., M. Potter, and D. G. Osmond. 1995. Perturbation of B cell genesis in the bone marrow of pristane-treated mice. Implications for plasmacytoma induction. J. Immunol. 154:2082–2091. Roessler, S., and R. Grosschedl. 2006. Role of transcription factors in commitment and differentiation of early B lymphoid cells. Semin. Immunol. 18:12–19. Rolink, A., P. Ghia, U. Grawunder, D. Haasner, H. Karasuyama, C. Kalberer, T. Winkler, and F. Melchers. 1995. In-vitro analyses of mechanisms of B-cell development. Semin. Immunol. 7:155–167. Rolink, A., D. Haasner, S. Nishikawa, and F. Melchers. 1993a. Changes in frequencies of clonable pre B cells during life in different lymphoid organs of mice. Blood 81:2290–2300. Rolink, A., H. Karasuyama, U. Grawunder, D. Haasner, A. Kudo, and F. Melchers. 1993b. B cell development in mice with a defective lambda 5 gene. Eur. J. Immunol. 23:1284–1288. Rolink, A., A. Kudo, H. Karasuyama, Y. Kikuchi, and F. Melchers. 1991. Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo. EMBO J. 10:327–336. Rolink, A., F. Melchers, and J. Andersson. 1996. The SCID but not the RAG-2 gene product is required for S mu-S epsilon heavy chain class switching. Immunity 5:319–330. Rolink, A. G., T. Brocker, H. Bluethmann, M. H. KoscoVilbois, J. Andersson, and F. Melchers. 1999a. Mutations affecting either generation or survival of cells influence the pool size of mature B cells. Immunity 10:619–628. Rolink, A. G., S. L. Nutt, F. Melchers, and M. Busslinger. 1999b. Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 401:603–606. Rolink, A. G., and F. Melchers. 2002. BAFFled B cells survive and thrive: roles of BAFF in B-cell development. Curr. Opin. Immunol. 14:266–275. Rolink, A. G., C. Schaniel, and F. Melchers. 2002a. Stability and plasticity of wild-type and Pax5-deficient precursor B cells. Immunol. Rev. 187:87–95. Rolink, A. G., J. Tschopp, P. Schneider, and F. Melchers. 2002b. BAFF is a survival and maturation factor for mouse B cells. Eur. J. Immunol. 32:2004–2010. Romanow, W. J., A. W. Langerak, P. Goebel, I. L. WolversTettero, J. J. van Dongen, A. J. Feeney, and C. Murre. 2000. E2A and EBF act in synergy with the V(D)J recombinase to generate a diverse immunoglobulin repertoire in nonlymphoid cells. Mol. Cell 5:343–353. Rothenberg, E. V., J. E. Moore, and M. A. Yui. 2008. Launching the T-cell-lineage developmental programme. Nat. Rev. Immunol. 8:9–21. Saito, T., J. L. Sussman, J. D. Ashwell, and R. N. Germain. 1989. Marked differences in the efficiency of expression of distinct alpha beta T cell receptor heterodimers. J. Immunol. 143:3379–3384. Sauvageau, G., U. Thorsteinsdottir, C. J. Eaves, H. J. Lawrence, C. Largman, P. M. Lansdorp, and R. K. Humphries. 1995. Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev. 9:1753–1765. Schaniel, C., L. Bruno, F. Melchers, and A. G. Rolink. 2002a. Multiple hematopoietic cell lineages develop in vivo from transplanted Pax5-deficient pre-B I-cell clones. Blood 99:472–478.
38
host defense: general
Schaniel, C., M. Gottar, E. Roosnek, F. Melchers, and A. G. Rolink. 2002b. Extensive in vivo self-renewal, longterm reconstitution capacity, and hematopoietic multipotency of Pax5-deficient precursor B-cell clones. Blood 99:2760–2766. Schebesta, M., P. L. Pfeffer, and M. Busslinger. 2002. Control of pre-BCR signaling by Pax5-dependent activation of the BLNK gene. Immunity 17:473–485. Schiedlmeier, B., H. Klump, E. Will, G. Arman-Kalcek, Z. Li, Z. Wang, A. Rimek, J. Friel, C. Baum, and W. Ostertag. 2003. High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD341 cells, but impairs lymphomyeloid differentiation. Blood 101:1759–1768. Schiemann, B., J. L. Gommerman, K. Vora, T. G. Cachero, S. Shulga-Morskaya, M. Dobles, E. Frew, and M. L. Scott. 2001. An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 293:2111–2114. Schmitt, T. M., R. F. de Pooter, M. A. Gronski, S. K. Cho, P. S. Ohashi, and J. C. Zúñiga-Pflücker. 2004. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat. Immunol. 5:410–417. Schmitt, T. M., and J. C. Zúñiga-Pflücker. 2002. Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17:749–756. Shimizu, T., C. Mundt, S. Licence, F. Melchers, and I. L. Mårtensson. 2002. VpreB1/VpreB2/lambda 5 triple-deficient mice show impaired B cell development but functional allelic exclusion of the IgH locus. J. Immunol. 168: 6286–6293. Shinkura, R., S. Ito, N. A. Begum, H. Nagaoka, M. Muramatsu, K. Kinoshita, Y. Sakakibara, H. Hijikata, and T. Honjo. 2004. Separate domains of AID are required for somatic hypermutation and class-switch recombination. Nat. Immunol. 5:707–712. Shivdasani, R. A., E. L. Mayer, and S. H. Orkin. 1995. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature 373:432–434. Shlomchik, M. J. 2008. Sites and stages of autoreactive B cell activation and regulation. Immunity 28:18–28. Sigvardsson, M., M. O’Riordan, and R. Grosschedl. 1997. EBF and E47 collaborate to induce expression of the endogenous immunoglobulin surrogate light chain genes. Immunity 7:25–36. Singh, H. 1996. Gene targeting reveals a hierarchy of transcription factors regulating specification of lymphoid cell fates. Curr. Opin. Immunol. 8:160–165. Singh, H., and J. M. Pongubala. 2006. Gene regulatory networks and the determination of lymphoid cell fates. Curr. Opin. Immunol. 18:116–120. Smith, C. A., G. T. Williams, R. Kingston, E. J. Jenkinson, and J. J. Owen. 1989. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337:181–184. Smith, L. G., I. L. Weissman, and S. Heimfeld. 1991. Clonal analysis of hematopoietic stem-cell differentiation in vivo. Proc. Natl. Acad. Sci. USA 88:2788–2792. Solvason, N., and J. F. Kearney. 1992. The human fetal omentum: a site of B cell generation. J. Exp. Med. 175:397–404. Spangrude, G. J., L. Smith, N. Uchida, K. Ikuta, S. Heimfeld, J. Friedman, and I. L. Weissman. 1991. Mouse hematopoietic stem cells. Blood 78:1395–1402. Stall, A. M., S. Adams, L. A. Herzenberg, and A. B. Kantor. 1992. Characteristics and development of the murine B-1b (Ly-1 B sister) cell population. Ann. NY Acad. Sci. 651:33–43. Stehling-Sun, S., J. Dade, S. L. Nutt, R. P. DeKoter, and F. D. Camargo. 2009. Regulation of lymphoid versus myeloid fate ‘choice’ by the transcription factor Mef2c. Nat. Immunol. 10:289–296.
Strasser, A., A. Rolink, and F. Melchers. 1989. One synchronous wave of B cell development in mouse fetal liver changes at day 16 of gestation from dependence to independence of a stromal cell environment. J. Exp. Med. 170:1973–1986. Takahama, Y., E. W. Shores, and A. Singer. 1992. Negative selection of precursor thymocytes before their differentiation into CD41CD81 cells. Science 258:653–656. Talmage, D. W., 1959. Immunological specificity. Unique combinations of selected natural globulins provide an alternative to the classical concept. Science 129:1643–1648. Tanigaki, K., and T. Honjo. 2007. Regulation of lymphocyte development by Notch signaling. Nat. Immunol. 8:451–456. ten Boekel, E., F. Melchers, and A. G. Rolink. 1997. Changes in the V(H) gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 7:357–368. Thorsteinsdottir, U., G. Sauvageau, and R. K. Humphries. 1999. Enhanced in vivo regenerative potential of HOXB4transduced hematopoietic stem cells with regulation of their pool size. Blood 94:2605–2612. Tsuneto, M., A. Tominaga, H. Yamazaki, M. Yoshino, S. H. Orkin, and S. Hayashi. 2005. Enforced expression of PU.1 rescues osteoclastogenesis from embryonic stem cells lacking Tal-1. Stem Cells 23:134–143. Tung, J. W., and L. A. Herzenberg. 2007. Unraveling B-1 progenitors. Curr. Opin. Immunol. 19:150–155. Vegh, P., J. Winckler, and F. Melchers. 2010. Long-term “in vitro” proliferating mouse hematopoietic progenitor cell lines. Immun. Lett. 130:32–35. von Boehmer, H., I. Aifantis, F. Gounari, O. Azogui, L. Haughn, I. Apostolou, E. Jaeckel, F. Grassi, and L. Klein. 2003. Thymic selection revisited: how essential is it? Immunol. Rev. 191:62–78. von Boehmer, H. 2004. Selection of the T-cell repertoire: receptor-controlled checkpoints in T-cell development. Adv. Immunol. 84:201–238. von Boehmer, H. 2008. Positive and negative selection in Basel. Nat. Immunol. 9:571–573. von Boehmer, H., and F. Melchers. 2010. Checkpoints in lymphocyte development and autoimmune disease. Nat. Immunol. 11:1–8. Wack, A., H. M. Ladyman, O. Williams, K. Roderick, M. A. Ritter, and D. Kioussis. 1996. Direct visualization of thymocyte apoptosis in neglect, acute and steady-state negative selection. Int. Immunol. 8:1537–1548. Wardemann, H., T. Boehm, N. Dear, and R. Carsetti. 2002. B-1a B cells that link the innate and adaptive immune responses are lacking in the absence of the spleen. J. Exp. Med. 195:771–780. Wardemann, H., and M. C. Nussenzweig. 2007. B-cell selftolerance in humans. Adv. Immunol. 95:83–110. Wernig, M., A. Meissner, R. Foreman, T. Brambrink, M. Ku, K. Hochedlinger, B. E. Bernstein, and R. Jaenisch. 2007. In vitro reprogramming of fibroblasts into a pluripotent EScell-like state. Nature 448:318–324. Wilson, A., H. R. MacDonald, and F. Radtke. 2001. Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus. J. Exp. Med. 194:1003–1012. Wilson, A., and A. Trumpp. 2006. Bone-marrow haematopoietic-stem-cell niches. Nat. Rev. Immunol. 6:93–106. Xie, C., R. Patel, T. Wu, J. Zhu, T. Henry, M. Bhaskarabhatla, R. Samudrala, K. Tus, Y. Gong, H. Zhou, E. K. Wakeland, X. J. Zhou, and C. Mohan. 2007. PI3K/AKT/ mTOR hypersignaling in autoimmune lymphoproliferative disease engendered by the epistatic interplay of Sle1b and FASlpr. Int. Immunol. 19:509–522. Yamagami, T., E. ten Boekel, J. Andersson, A. Rolink, and F. Melchers. 1999a. Frequencies of multiple IgL chain gene rearrangements in single normal or kappaL chain-deficient B lineage cells. Immunity 11:317–327.
2. the ontogeny of the Cells of the Innate and the adaptive Immune system Yamagami, T., E. ten Boekel, C. Schaniel, J. Andersson, A. Rolink, and F. Melchers. 1999b. Four of five RAG-expressing JCkappa-/- small pre-BII cells have no L chain gene rearrangements: detection by high-efficiency single cell PCR. Immunity 11:309–316. Yamagata, T., D. Mathis, and C. Benoist. 2004. Self-reactivity in thymic double-positive cells commits cells to a CD8 alpha alpha lineage with characteristics of innate immune cells. Nat. Immunol. 5:597–605. Yamasaki, S., E. Ishikawa, M. Sakuma, K. Ogata, K. SakataSogawa, M. Hiroshima, D. L. Wiest, M. Tokunaga, and T. Saito. 2006. Mechanistic basis of pre-T cell receptormediated autonomous signaling critical for thymocyte development. Nat. Immunol. 7:67–75. Yokota, Y., A. Mansouri, S. Mori, S. Sugawara, S. Adachi, S. Nishikawa, and P. Gruss. 1999. Development of peripheral
39
lymphoid organs and natural killer cells depends on the helixloop-helix inhibitor Id2. Nature 397:702–706. Yurasov, S., H. Wardemann, J. Hammersen, M. Tsuiji, E. Meffre, V. Pascual, and M. C. Nussenzweig. 2005. Defective B cell tolerance checkpoints in systemic lupus erythematosus. J. Exp. Med. 201:703–711. Zhang, C. C., M. Kaba, G. Ge, K. Xie, W. Tong, C. Hug, and H. F. Lodish. 2006. Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells. Nat. Med. 12:240–245. Zhu, J., D. M. Giannola, Y. Zhang, A. J. Rivera, and S. G. Emerson. 2003. NF-Y cooperates with USF1/2 to induce the hematopoietic expression of HOXB4. Blood 102:2420–2427. Zhuang, Y., P. Soriano, and H. Weintraub. 1994. The helixloop-helix gene E2A is required for B cell formation. Cell 79:875–884.
This page intentionally left blank
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
3 The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates JIM KAUFMAN
INTRODUCTION
Thus, the essence of the adaptive immune system has been said to be wide diversity, exquisite specificity, and memory. In contrast, the so-called innate immune system described in mammals lacks the capacity to generate completely new receptors within an individual and lacks memory. There is a division of labor among lymphocytes (discussed in chapter 2 and elsewhere in this book). B cells produce antibodies (Ab), which recognize shapes as the primary defense of the adaptive immune system to extracellular pathogens. Some T cells, those that carry T-cell receptors (TcRs) composed of gamma and delta chains ( TcR), also recognize shapes although these shapes are generally found on a (cell) surface. Other T cells, which carry TcRs composed of alpha and beta chains (ab TcR), recognize only major histocompatibility complex (MHC) molecules carrying molecular pieces (peptides or lipids) from within the cell, thus revealing pathogens that have escaped from antibodies to the inside of a cell. Peptides from pathogens such as viruses replicating in the cytoplasm (or contiguous spaces such as the nucleus) are presented by classical class I molecules to T cells bearing CD8 molecules, whereas pathogens such as bacteria or parasites in intracellular vesicles (such as the endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes) as well as the extracellular space, which is topologically equivalent, give rise to peptides that are presented by classical class II molecules to T cells bearing CD4 molecules. Since the CD4 cells recognize antigens from the extracellular space, they also have a role in regulating B cells that are major effectors of the adaptive immune system dealing with extracellular antigens. Nonclassical class I molecules called CD1 molecules carry lipids from intracellular vesicles to be recognized by a special group of CD8 bearing T cells. The MHC molecules are loaded with their antigens by sophisticated pathways composed of various kinds of molecules, some of which are encoded by genes located in the MHC (discussed in detail in many other chapters of this book). Classical class I molecules bind peptides, which are produced in the cytoplasm by the proteasome (under certain conditions augmented by various inducible components), and pumped into the lumen of the endoplasmic reticulum by the transporter associated with antigen presentation (TAP, a member of the ABC transporter family). The class
The pathogens and tumors confronting an individual are so many, so various, so unrelenting, and so lethal, it is a wonder that any of us survive to discuss the issue. The fact we are here demonstrates that overall and in the long run, the defense systems do work. To do so, they are incredibly complex, with a complicated interplay of many different cell types interacting in many specific locations of the body, utilizing hundreds of cell surface and soluble ligands to bind hundreds of different receptors that respond through various signaling systems to thousands of target genes, all levels of which are controlled by many layers of regulation. This extraordinary complexity is presumably the result of the accumulation of many changes appearing step by step over millions of years, each step arising by accident and, in general, being selected by the need to defend the individual organism from the particular pathogens present at the time. Here we will consider the evolution of only one part of the immune response. The so-called adaptive immune response, examined in great detail for humans (Homo sapiens) and mice (Mus musculus), is conceptually simple. The essential cell is the lymphocyte with a cell surface glycoprotein that functions as an antigen receptor, with each antigen receptor having a binding site with a different protein sequence, which is constructed by somatic changes in the genes for the antigen receptor in the lymphocyte (mediated in part by the recombination-activating genes, RAG1 and RAG2). Each lymphocyte (or clone of lymphocytes) bears a different antigen receptor that can specifically recognize a small group of complementary shapes; but, altogether, antigen receptors of the whole population of lymphocytes can bind an extraordinarily large diversity of shapes. Upon recognition of a molecule with a particular shape, only those clones of lymphocytes that recognize that shape will be stimulated to be activated and to proliferate (so-called clonal selection). Upon a second encounter with the same shape, there will be a faster, greater, and qualitatively different immune response.
Jim Kaufman, University of Cambridge Department of Pathology, Tennis Court Road, Cambridge, CB2 1QP, Department of Veterinary Medicine, Madingley Road, Cambridge, CB3 0ES, United Kingdom.
41
42
HOST DEFENSE: GENERAL
I molecule and TAP are part of a peptide-loading complex (PLC), which includes the dedicated chaperone tapasin (or TAP-binding protein, TAP-BP) in association with ERp57 (a member of the protein disulfide isomerase, PDI, family), the lectin chaperone calnexin and/or calreticulin, and perhaps other molecules (such as endoplasmic reticulum associated aminopeptidase, ERAAP, and PDI itself). Classical class II molecules bind to a trimer of invariant chain (Ii) in the endoplasmic reticulum and are transported (primarily via the cell surface) to a specialized endosomal compartment called MIIC, in which Ii is degraded and then antigenic peptides produced by endosomal proteases are loaded. The class II molecules are stabilized by nonclassical class II molecules that act as dedicated chaperones, DM (as well as DO in some cells) until high affinity peptides are bound. Different CD1 molecules are known to traffic to different endosomal compartments based on signals in their cytoplasmic tails and to be loaded with lipids generated by lipid hydrolases and other degradative enzymes. Of the accessory molecules in these pathways, the genes for some of the inducible components of the proteasome (LMP2 and LMP7), both TAP subunits (TAP1 and TAP2), tapasin, and both chains of DM (DMA and DMB) and DO (DZA and DOB), are located in the MHC. For all immune responses, there is an evolutionary arms race between pathogens and the host (discussed in detail in many other chapters of this book). Pathogens are under selective pressure to change the protective peptides that bind to classical MHC molecules and then stimulate effective T-cell responses, so most pathogens have many variants. In return, and more slowly, the hosts are selected to have multiple MHC molecules, both by having a multigene family and multiple alleles. The most efficient method would appear to be a large multigene family, but each additional MHC molecule increases the possibility that any given T cell will recognize a self-peptide leading to autoimmunity, which is countered (at least in part) by deletion of that T-cell clone in the thymus. Thus, if there are too many MHC molecules, the repertoire of T-cell specificities may be reduced so much that the T-cell response becomes ineffective. In addition, the total number of MHC gene loci may be limited by the number of MHC molecules that can be accommodated on the cell surface. However, there is no obvious reason why the number of MHC alleles should be limited, and, indeed, MHC molecules are among the most polymorphic known, with the human class I locus HLA-B having over 1,000 alleles worldwide. In contrast, the human Ab and TcR loci have large multigene families and further generate variability by gene rearrangement (and other processes) in somatic cells, so they have little allelic polymorphism. Also, the genes involved in processing and loading the peptides (e.g., LMPs, TAPs, tapasin, DM, DO, Ii) are relatively nonpolymorphic in typical mammals, but this becomes a crucial point in the discussion that follows. Although this all sounds quite complicated, the actual adaptive immune system elucidated in typical mammals is, in fact, significantly more complex. Even if we could, it would be beyond the scope of this review to try to explain the emergence of lymphocytes, natural killer (NK) cells, dendritic cells, regulatory T cells, follicular dendritic cells, and the like, with their many interactions, receptors, and signaling cascades. It is far beyond this review to explain the emergence of the various lymphoid tissues, including the thymus and the critical processes of self-tolerance, although some innovative work in this area has been reported recently (Boehm & Bleul, 2007; Bajoghli et al., 2009). It is also beyond our scope to consider all of the various evolutionary novelties and adaptations that have emerged
independently in each of the vertebrate taxa. In this review, we will focus on the question of how the essential components of the adaptive immune system—Ab, TcR, and MHC molecules—arose and began to interact.
THE TIME FRAME: ADAPTIVE IMMUNE SYSTEMS IN JAWED VERTEBRATES AND INVERTEBRATES
There is a long history of speculation about the origins of the adaptive immune system so extensively studied in mammals. There was certainly the possibility of a very ancient origin, given that multicellular organisms have existed for billions of years and presumably always needed protection from pathogens. Moreover, the elements of the adaptive immune system might have evolved originally for other purposes, for instance, development or the nervous system. It was also speculated that the allorecognition system of soft-bodied invertebrates was a primitive form of MHC recognition, although it is now clear that the genes involved in tunicate allorecognition are not closely related to MHC genes (Scofield et al., 1982; De Tomasso et al., 2005). A more recent suggestion is that MHC molecules arose for selection of olfactory specificity in mate recognition (Boehm & Zufall, 2006). Whatever the original function, it appears that the adaptive immune system so extensively studied in mammals arose at the base of the jawed vertebrates over 500 million years ago (Flajnik & Kasahara, 2001, 2010; Litman et al., 2005). In all jawed vertebrates examined thus far, the essential elements of the adaptive immune system are present (Fig. 1). In particular, sharks (as representatives of cartilaginous fish, the most phylogenetically primitive jawed vertebrates) have RAG1 and RAG2, IgM Ab, ab and TcR, and an MHC containing class I and class II genes, as well as genes for processing molecules such as TAPs, tapasin, and proteasome components (Bernstein et al., 1994; Rast et al., 1997; Ohta et al., 2002; Terado et al., 2003). No such genes (with two possible exceptions discussed below) have been found in more phylogenetically primitive organisms, such as the jawless fish (the two kinds of cyclostomes, the hagfish Eptatretus stoutii and the sea lamprey Petromyzon marinus, among several species examined). With the exception of apparent RAG genes in the sea urchin (Fugmann et al., 2006), no such genes were identified in the genomes of other deuterostomes (two chordates, the cephalochordate amphioxus or lancet Branchiostoma floridae and the urochordate tunicate Ciona intestinalis, and an echinoderm, the purple sea urchin, Strongylocentrotus purpuratus), nor in the genomes of any other invertebrate (Azumi et al., 2003; Rast et al., 2006; Putnam et al., 2008; Holland et al., 2008). These data intensified the contrast between vertebrate and invertebrate immune responses, and led to the concept of a “Big Bang,” during which an adaptive immune system suddenly appeared in jawed vertebrates (Schluter et al., 1999). Despite years of functional experiments with invertebrates pointing to recognition of allo- and xenoantigens, none of the essential genetic components of the adaptive immune system in jawed vertebrates were found in the first complete genomic sequences of invertebrates (C. elegans Sequencing Consortium, 1998; Medzhitov & Janeway, 2002): an insect (the fruit fly Drosophila melanogaster) and a nematode worm (Caenorhabditis elegans). Therefore, the notion that invertebrates lack an adaptive immune system was strengthened, and the question became how invertebrates managed to survive without a system that is essential to jawed vertebrates. Early thoughts included the ideas that short lifespans and/or large numbers of offspring made an adaptive immune system
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates
43
FIGURE 1 An idealized tree, showing the relationships of the animals (and their defense systems). Phyla are indicated above the tree with thick lines; common names of species are indicated above the tree with thin lines; defense systems are indicated.
unnecessary, although many invertebrates do not fit this profile, with examples ranging from cockroaches, which live for 3 years like a mouse to some sea urchins, which live for hundreds of years. Another possibility is that invertebrates expand their innate immunity enough to counter their pathogens, which might be (part of) the answer for the purple sea urchin and amphioxus, whose genomes contain hundreds of Toll-like receptor (TLR) and other pattern recognition genes (Fig. 1) (Rast et al., 2006; Huang et al., 2008). A key concept that has relevance no matter what the defense system might be is that pathogens that overcome all of their hosts may face a bleak evolutionary future, and so, in general, the virulence of pathogens is constrained and guided by the particular defenses that their host(s) evolve. In other words, pathogens will not be selected to overcome an adaptive immune system that is not present in their host(s), and therefore, pathogens of invertebrates will not be virulent in the same way as those facing the adaptive immune system of jawed vertebrates. The idea that pathogens evolve with their host means that invertebrate immune systems are not “worse” than vertebrate immune systems, just different, with different benefits and costs. Indeed, jawed vertebrates now have many pathogens, which evade the adaptive immune system, and, on top of that, have autoimmunity that is a consequence of the adaptive immune system, prompting the characterization of “one
moment of ecstasy followed by 400 million years of agony” (Hedrick, 2004). In fact, at least some groups of invertebrates can have adaptive immune systems based on molecules other than Ab, TcR, and MHC (Fig. 1), as first described in some molecular detail for the fibronectin-related proteins (FREPs) from a mollusk, the snail Biomphalaria glabrata. The snail FREP system consists of a multigene family, whose members are composed of immunoglobulin (Ig) V (variable)-like domains and a fibronectin-like domain, apparently undergo somatic mutation within an individual, and have expression correlated with responses to parasites such as schistosomes (Adema et al., 1997; Zhang et al., 2004). In arthropods (such as the insects, the fruit fly Drosophila melanogaster and the mosquito Anopheles gambia, and two species of the crustaceans water flea, Daphnia), a gene related to the human gene Down’s syndrome cell associated molecule (Dscam) is involved in axon guidance in neurons during development, but then is expressed in adult fat body and hemocytes, the latter involved in various kinds of immune response in insects. Alternative splicing, mainly among the extracellular Ig-like domains, gives rise to a large number of variants, and transient silencing of the Dscam gene in mosquitoes has been reported to lead to decreased resistance to the bacterial pathogens and insect malaria (Watson et al., 2005; Dong et al., 2006; Brites et al., 2008).
44
HOST DEFENSE: GENERAL
In jawless fish, like lampreys and hagfish, two variable lymphocyte receptor (VLR) genes, composed mainly of leucine-rich repeats (LRRs) give rise to many variants by gene conversion from nearby LRR pseudogenes. These variants are clonally expressed in lamprey and hagfish lymphocytes. Moreover, there is evidence that the VLR-A gene product is secreted by lymphocytes with some properties in common with vertebrate B cells, in response to and then binding to specific antigens. In contrast, the VLR-B gene product is expressed on the surface of other lymphocytes with some properties in common with T cells. Thus, the adaptive immune system of the jawless fish is founded on a different set of antigen-specific receptor molecules, but otherwise has many features in common with the adaptive immune system of jawed vertebrates, suggesting that they may both have descended from a common ancestor (Pancer et al., 2004b; Cooper & Alder, 2006; Guo et al., 2009). Overall, it seems likely that many, if not most, invertebrates will have some form of adaptive immunity based on different molecular systems, which may allow us at last to understand which features of adaptive immunity are essential in a way that was impossible with just a single example. The postulated “Big Bang,” the apparently sudden appearance of all the essential components of our familiar adaptive immune system, could be explained by another, still somewhat controversial phenomenon, the 2R hypothesis. On the basis of fragmentary data on genome sizes, Susumu Ohno (1970) postulated the following scenario: two rounds of genome-wide duplication at the base of the vertebrates, which, for each gene, would have led to one old gene for the previously essential function and three new
genes (so-called paralogs) available for new functions (socalled neofunctionalization). Such a large number of new genes could allow a burst of innovation, leading to new features (e.g., notochord and vertebrae). Support for this idea came from the analysis of Hox gene clusters, for which there is one in the invertebrate amphioxus and four paralogous regions in vertebrates such as mammals (Garcia-Fernandez & Holland, 1994). Analysis of gene families found in the human MHC on chromosome 6 led to the identification of MHC paralogous regions on chromosomes 1, 9, and 19 (Fig. 2), each one of which has a subset of genes thought to have been present in the primordial cluster (Kasahara et al., 1997; Flajnik & Kasahara, 2001, 2010). For instance, class I genes are found in the MHC while the genes for homologs CD1 and neonatal FcR are found on chromosome 1 and 19, respectively. Similarly, the genes for inducible proteasome components LMP2 and LMP7 are located in the MHC, while the gene for MECL1 in the MHC-paralogous region is on chromosome 9. The complement component genes for C4 are found in the class III region of the human MHC on chromosome 6, and the homologs C5 and C3 are found in the MHC paralogous regions on chromosomes 9 and 19. Many other genes, which are not necessarily involved in the immune system, are also found in these MHC-paralogous regions, including the developmental factors Notch1 on chromosome 9, Notch2 on chromosome 1, Notch3 on chromosome 19, and Notch4 on chromosome 6. Thus, the apparently sudden appearance of the adaptive immune system at the base of the vertebrates could be explained by the sudden appearance of many homologous
FIGURE 2 A model for the evolution of the adaptive immune system of jawed vertebrates, from the primordial MHC (left) through two rounds of genome-wide duplication (middle) and subsequent silencing, deletion, and break up to give MHC paralogous regions (right). Black boxes indicate genes based on data from chickens; open boxes indicate genes based on data from other species. Gga and Hsa, chromosomes from chickens and humans, respectively.
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates
genes, which could adapt to new functions. However, the “Big Bang” might have taken as much as 70 million years, based on the apparent divergence times of jawless fish and the armored fish that led to the jawed vertebrates. Fossils of extinct intermediate forms spanning this time show no sudden jump in the body plan, but instead show a stepwise acquisition of new features (Donoghue & Purnell, 2005). Although there is little scope to observe the evolution of the immune system through fossils, it seems likely that the adaptive immune system of jawed vertebrates also evolved in steps, none of which are separated between those organisms still living. However, these steps might still be inferred by the molecular equivalent of fossil traces.
FIRST ANTIGEN-SPECIFIC RECEPTOR WITH IMMUNOGLOBIN VARIABLE REGIONS: THE T-CELL RECEPTOR
The presence of antigen-specific receptors with highly variable regions (Ab and TcR) is the major difference between the adaptive immune system in jawed vertebrates and the
45
many different kinds of innate immunity. Antigen-specific receptors in innate immunity (e.g., TLRs) can have great specificity for particular antigens, which are found in pathogens and not in the host, but in the vertebrates examined thus far they lack both the enormous diversity and the memory of adaptive immunity. The single most important event in the appearance of the adaptive immune system of vertebrates was undoubtedly the appearance of split Ig V domains with the potential of the separated genomic segments to be rejoined in an imprecise manner (Fig. 3). From the first discoveries of split V genes, it was postulated that insertion(s) by a transposable element was responsible, based on the specific recognition by RAG proteins of recombination signal sequences (RSS) flanking the initial DNA cleavage site (Sakano et al., 1979). More recently, the RAG proteins have been shown to have transposase activity, and a class of transposons have been described whose integrases and recognition sequences are closely related to RAG and RSS sequences (Kapitonov & Jurka, 2005). A (relative of a) potential target gene has been described in the sea lamprey, which encodes a molecule related to TcR
FIGURE 3 A model for the evolution of split variable region-containing antigen specific receptors by insertion of transposons (top), with cartoons indicating the location of the CDRs in a V domain (lower right) and the footprint of the CDRs on an MHC molecule (lower left). In the upper left, open circles indicate domains (Ig V type and C type), lines indicate transmembrane and cytoplasmic regions, filled circles indicate D and J segments of the protein, grey bars indicate membranes; one chain is gray to indicate that the molecule may or may not have been a dimer. In the upper right, boxes indicate genes or gene segments, and dotted lines indicate regions introduced by transposon insertion with the heavy black lines indicating RSS. In the cartoons, cylinders indicate a-helices, arrows indicate b-strands, thin lines indicate other conformations of the protein, the thick line indicates peptide bound to the MHC molecule, and the dotted ovals indicate the footprint of the CDRs from the a-chain and the b-chain of a TcR on a class I molecule.
46
HOST DEFENSE: GENERAL
but with an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail like some NK cell receptors (Pancer et al., 2004a; Yu et al., 2009). Another potential relative of the target gene is the variable region-containing chitin-binding protein (VCBP), found as large diversified families in amphioxus and tunicate genomes (Dishaw et al., 2008). These examples are two of several gene families interpreted to have mixtures of the properties of innate and adaptive immunity, leading to the view of an intimate intertwining of the evolution of innate and adaptive immunity (Kasahara et al., 2004; Flajnik & Du Pasquier, 2004; Litman et al., 2005, 2007; Cannon et al., 2008). A single event that gave rise to all split V genes in jawed vertebrates would mean that one kind of variable antigenspecific receptor came first: either Abs (which recognize three-dimensional shapes and have a high enough affinity to bind soluble antigens), TcRs (which recognize shapes located on cell surfaces), or ab TcRs (which recognize peptide or other antigens on MHC molecules located on cell surfaces). Although in principle, Ab seem like the simplest system to evolve, the fact that joining of the segments of split V genes only generates diversity at complementarity determining region 3 (CDR3) strongly indicates that ab TcRs were the original variable antigen-specific receptor in jawed vertebrates. It was long a mystery why diversity is found in three (or four) loops of variable antigen-specific receptors, but this diversity is generated by joining split V gene segments (J and D, and even multiple D) only at CDR3 (Fig. 3). As first suggested on the basis of a model (Davis & Bjorkman, 1988), many three-dimensional structures of ab TcR bound to peptide-MHC complexes show beyond a doubt that CDR1 and CDR2 contact mainly the MHC molecule, whereas CDR3 contacts mainly the peptide. The original model envisaged a scenario, in which the primordial TcR-like molecule recognized a collection of peptide antigens bound to a single monomorphic MHC class I molecule, so that the TcR needed only to generate diversity at CDR3 in order to recognize different peptide antigens. Later acquisition of MHC polymorphism and polygenism drove the acquisition of diversity in CDR1, CDR2, and elsewhere, primarily by polygenism of ab TcR. After that, TcR evolved from ab TcR by losing the requirement for recognizing antigens in the context of an MHC molecule, and then Ab evolved from ab TcR by losing the requirement to stay bound to a cell membrane. However, an argument that Ab or TcR came first might still be made, based on the fact that CDR3 is in the center of the antigenbinding site and thus might have been particularly important for binding molecular shapes. There are other steps in the emergence of the first variable antigen-specific receptors which remain mysteries, the sequence of events perhaps obscured by further evolution (Flajnik, 2002; Eason et al., 2004; Hsu et al., 2006). TcR locus organization seems relatively stable, perhaps due to the coevolution with the MHC. However, in cartilaginous and some bony fish, certain TcR loci are organized in clusters of V-D-D-J-C, and there are many germline-joined TcR genes expressed early in ontogeny. In contrast, Ab locus organization varies wildly throughout the vertebrates, with various heavy chain and light chain isotypes found in different vertebrate groups. There are even single-chain Ab described in cartilaginous fish (new antigen receptors, NARs) and camels, which are generally thought to be derived rather than an ancestral form. One mystery is that TcR and nearly all Ab are heterodimers of two chains with different numbers of joining segments. The establishment of this pattern must have been very early, because this feature is found in all three classes
of receptor (Fig. 3): one chain (a and chains of TcR, light chains of Ab) only requiring joining of the V segment with a J segment, and the other chains (b and chains of TcR, heavy chains of Ab) requiring joining of a J segment to D segment(s) before joining with the V segment. One possibility is that multiple V segments followed by multiple J segments gives the possibility of sequential rearrangements, whereas once V-D-J joining is complete, all J segments are deleted and rearrangement generally halts. Another mystery is the curious arrangement of TcR loci in most jawed vertebrates: two separate loci for b and chains, but a combined locus for a and , with V, D and J segments as well as C exons upstream of the Ja segments and Ca exons, so that V-Ja joining would delete the D and J segments. The Ab heavy chain loci of (certain) bony fish have a somewhat similar pattern, with the IgM locus embedded in the IgZ locus (although both have J and D segments). An underlying reason for this organization (as well as for germlinejoined TcR expressed early in ontogeny) has been suggested (Danilova & Amemiya, 2009): very young animals have few lymphocytes, and these loci are organized and regulated to restrict diversity to recognition of the most dangerous pathogens during early ontogeny. A third mystery is how and why the RSS are distributed, with the heptamer-nonamer spacing of 12 and 23 nucleotides (roughly the distance of one and two turns of the DNA helix, respectively) flanking different segments in the different Ab and TcR loci. In mammals, these distributions restrict the heavy chains of Ab to one D segment, but allow TcR multiple D segments.
THE PRIMORDIAL MHC MOLECULE: SIMILAR TO A CLASS II MOLECULE
The inference that the first discernible recognition by variable antigen-specific receptors was the recognition of MHClike molecules by TcR suggests that intracellular pathogens were the first major problem tackled by the emerging adaptive immune system. In fact, there are a wide range of MHC-like molecules described in well-studied mammals, ranging from the highly polymorphic peptide-binding classical class I and class II molecules located in the MHC to a host of nonclassical molecules with a variety of polymorphism levels, genomic locations, and functions, some immunological and some not. There are many examples of MHC-like molecules with “closed grooves,” which do not bind peptide or any other ligand (Wilson & Bjorkman, 1998), and some of these molecules function as indicators of stress or infection recognized by T cells (for instance, MIC-A and MIC-B recognition by TcR) (Li et al., 1999). So, it is easy to imagine the beginning of MHC molecules as such a monomorphic MHC-like molecule appearing on the surface of a cell to signal infection through recognition by a nonvariable TcR-like molecule. The first point at which we have some direct evidence, however slight, is the recognition of peptides on an MHC-like molecule by a TcR molecule with variability in CDR3 generated by genomic joining, and that narrows the list of examples to classical class I, classical class II, and CD1 molecules. Based on structure as well as function, these molecules have all descended from a common ancestor, so which one was first? The case for class I molecules coming first has been made repeatedly (Flajnik et al., 1991; Flajnik & Kasahara, 2001), based on the hypothesis that property of peptide binding evolved first in chaperones such as heat shock protein 70 (HSP70, an inducible gene which is located in the MHC of mammals), and that the exon encoding the chaperone domain was then shuffled to the beginning of a gene encoding a transmembrane protein with an extracellular Ig domain. Other arguments that class I molecules came before
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates
class II molecules have included the nearly ubiquitous tissue distribution of class I molecules compared to the relatively restricted expression of class II molecules (at least in mammals), and the direct function of class I molecules in resisting viruses compared to the indirect function of class II molecules in regulating antibody production. A major argument against this scenario is that the structures of chaperones such as HSP70 have now been solved, and they have no obvious relationship to MHC peptide binding domains (Zhu et al., 1996). Moreover, the argument that class II molecules are now specialized in tissue distribution and function is not necessarily true for the original class II molecules. CD1 molecules have a structure like class I molecules but pick up their antigen lipids from deep endosomal compartments like class II molecules, so they are certainly candidates which could give rise to both. Thus far, CD1 genes have been identified in mammals and birds, but not yet in other vertebrates, and the evidence has been interpreted both for and against an ancient origin of CD1 molecules (Miller et al., 2005; Salomonsen et al., 2005). The hypothesis for class II molecules coming first was made initially on the basis of the domain organization and later on the basis of crystallographic structures and gene organizations (Kaufman et al., 1984; Kaufman et al., 1990). This scenario (Fig. 4) envisages a single gene, which encodes a homodimeric molecule with the domain structure of a class II b chain, and then this gene duplicates and diverges to encode a heterodimeric molecule with the domain structure of a class II molecule. The fact that most class II genes are arranged in a/b gene pairs in opposite transcriptional orientation allows an inversion around a central point but
47
with asymmetric breakpoints, which leads in one step to a class I heavy chain gene and a gene encoding the end of a class II a chain, that is the Ig domain with a transmembrane region and a cytoplasmic tail. Finally, a stop codon or splice site mutation leads directly to a b2-microglobulin (b2m) gene. One argument against this simple molecular scenario is that the conformations of the Ig domains in the heterodimers is not the same; the class II a2 domain and b2m are oriented differently from the class II b2 domain and the class I a3 domain, which then requires some explanation that is so far not forthcoming. The class II first scenario assumes that the initial tissue distribution and function of the class II molecule was the same as the class I molecule, except that class II molecules sampled antigen from intracellular vesicles rather than the cytoplasm. Since the extracellular space is topologically equivalent to intracellular vesicles, it was a natural extension of class II function to regulate Ab production by B cells, once they appeared. Regardless of whether it was structurally like class I or class II, the origin of the first MHC-like molecule is of interest. Thus far, no molecule with distinctive feature of MHC molecules, variously described as the “MHC fold,” “peptidebinding domain,” or “open-faced sandwich” has been found in invertebrates. Even though proto-MHC genomic regions have been proposed in organisms as distant from vertebrates as protochordates, nematodes, and fruit flies (Abi-Rached et al., 2002; Danchin et al., 2003; Castro et al., 2004; Danchin & Pontarotti, 2004), no gene with the appropriate features has been identified nearby. Perhaps part of the difficulty might be the fact that there seems to be little or no sequence identity required for generating the MHC fold, as illustrated
FIGURE 4 A model for the evolution of MHC molecules, from a class II b-chain-like homodimer to a class II heterodimer by duplication and divergence, and then to a class I chain with b2m by inversion. On the left, circles indicate domains (open for class II a-chain, gray-filled for class II b-chain, with SS for intrachain disulphide bond), lines indicate transmembrane and cytoplasmic regions, gray bars indicate membranes. On the right, boxes indicate exons (open for coding regions of class II a-chain, gray-filled for coding regions of class II b-chain, black-filled for 3 untranslated region), P indicates promoter region with arrow showing direction of transcription, curved arrow around axis, and lightning bolts indicating an inversion around a central point with asymmetric breakpoints. Adapted from Kaufman, 1988.
48
HOST DEFENSE: GENERAL
by the fact that such homologous domains in class I and CD1 share only 10% sequence identity, so perhaps the gene is present but just as yet unrecognized.
COEVOLUTION OF GENES LED TO THE PRIMORDIAL MHC
The mammalian MHC is a large complex region (MHC Sequencing Consortium, 1999; Kelley et al., 2005) filled with hundreds of genes, pseudogenes, and repetitive elements, typically divided by recombination into regions including a class I region with class I genes and certain other “framework genes,” a class II region with class II genes as well as proteasome and TAP genes, and in between, a class III region with complement component genes, TNF cytokine genes, and many other kinds of genes. The outer boundaries of the MHC can be extended, so that the tapasin gene is found in the “extended class II region,” while TRIM genes and olfactory receptors might be included in the “extended class I region.” However, there is much variation in the boundaries and detailed organization of the MHC between different mammals with the gene organization of the class II and class III regions most similar but with class I genes varying wildly. For instance (Fig. 5), the human classical class I genes (HLA-A, B, and C) are located in the class I region, two mouse classical class I genes (H-2D and L) are located in the class I region but one (H-2K) is located in between the class II region and the extended class II region, while in the rat all classical class I genes (RT1-A, A1, and A2) are located in between the class II region and the extended class II region. This example is mentioned because it has functional implications discussed below, but more detailed descriptions
and the concepts of the framework in the mammalian class I regions are discussed elsewhere (Amadou, 1999). Outside of mammals, the organization of the MHC is reported to be even more various (Kelley et al., 2005). The MHC of the frog Xenopus tropicalis appears to be much the same size as the human MHC, but the MHC of chickens (Gallus gallus) and many other birds seems particularly small and compact, while some passerine birds are thought to have a much larger MHC. Of particular note is the arrangement among bony fish and at least one frog (Xenopus ruwensoriensis), in which the MHC appears to have fragmented (Du Pasquier & Blomberg, 1982; Sammut et al., 2002; Stet et al., 2003). For instance, in zebrafish (Danio rerio) the class I region includes the proteasome and TAP genes and is completely separated (on different chromosomes) from the class II genes found in at least two loci and from class III region genes, which appear to be scattered throughout the genome (Bingulac-Popovic et al., 1997; Sambrook et al., 2005). In the phylogenetically most primitive jawed vertebrates, the cartilaginous fish (as exemplified by the sharks Triakis scyllium and Ginglymostoma cirratum), the class I, TAP, inducible proteasome, class II and class III region (complement components C4 and factor B) genes are found together in a single MHC locus (Ohta et al., 2000, 2003; Terado et al., 2003), so the organization in X. ruwensoriensis and the bony fish appears to be derived. A simple explanation in both cases would be genome-wide duplication followed by silencing and deletion, as discussed in the next section. Amid all of this variability of the MHC in different vertebrates, at least one general rule has emerged about the relationship of genomic organization and function: the coevolution of polymorphic interacting genes that are closely linked in the
FIGURE 5 A cartoon showing the relationship between coevolution of interacting genes and (recombinational) distance in the genome, as illustrated for the class I and TAP genes of the MHC. (Left) The TAP heterodimer and class I molecule(s) are embedded in a membrane. The number of well-expressed class I molecules ranges from three in humans to one in chickens. Similarly, the specificity of interaction with peptide (as indicated by the number and depth of indentations) increases for TAPs and may decrease for class I molecules from humans to chickens. (Right) Different regions of the MHC labelled in bold are separated by thin vertical lines, genes labeled in normal script are indicated by thick vertical lines, and distance between the TAP genes and the most distant class I gene is indicated by the horizontal arrow. Adapted from Kaufman, 1999; B. Walker et al., submitted.
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates
genome. This idea was first suggested (Germain et al., 1985) to explain a curious situation in mouse class II molecules: both the Ab and Aa genes are polymorphic, but the Eb gene is also polymorphic while the Ea gene is not. A hotspot of recombination separates the A region with the Ab, Aa, and the 5 end of the Eb gene (including the exon encoding polymorphic peptide-binding domain) from the E region containing the nonpolymorphic 3 end of the Eb gene and the Ea gene (as well as other genes). The Ab and Aa chains within one haplotype (but rarely from different haplotypes) will interact to make a class II heterodimer, whereas the nonpolymorphic Ea chain will interact with Eb chains from all other haplotypes. The fact that the Ab and Aa genes are rarely separated by recombination means that optimal combinations can coevolve and remain together, whereas the frequent separation of Eb and Ea genes means that one (in this case, the Ea gene) evolves to be an “average best fit” to interact with all of the alleles of the other (in this case, the Eb gene). Once this concept is recognized, examples of it can be seen elsewhere. For instance, of the human class II genes, the DQA and DQB genes are rarely separated by recombination and coevolve as polymorphic partners in haplotype-specific manner; the same is true of DPA and DPB, but DRA is nonpolymorphic and the DRA protein interacts with the proteins encoded by multiple DRB alleles and loci. Human b2m interacts with all classical class I alleles and loci, as well as many different MHC-like proteins including those encoded by CD1 loci gene; the b2m gene is monomorphic and located on a different chromosome from the MHC and class I-like genes. Similarly CD4, CD8, and TcR loci are all nonpolymorphic and located on different chromosomes from the MHC. However, other pressures can cause interacting proteins located on different chromosomes to become polymorphic, a situation which can lead to serious problems of interaction. For instance, NK cell receptors encoded by the killer inhibitory receptor (KIR) complex are individually polymorphic as well as the multigene family expanding and contracting to give “haplotype polymorphism,” all of which are presumably selected by interactions with pathogens. Some combinations of KIR and MHC haplotypes confer poor responses to infectious pathogens, susceptibility to autoimmunity, and reproductive difficulties (Parham, 2005; Trowsdale & Moffett, 2008). In other words, in order for two interacting genes to both become polymorphic without difficulty, in general they must be genetically closely linked so that they can be selected to coevolve. The same concept applies to the genes within the MHC, most clearly shown for the presentation system of class I molecules (Fig. 5). In typical (placental) mammals, the various components involved in peptide processing and loading are located far away from the classical class I genes and are not polymorphic. For instance, in humans, the genes for two inducible components of the proteasome and TAPs are located in the class II region, tapasin is located in the extended class II region, the third inducible proteasome component, ERp57, calnexin and calreticulin are located on different chromosomes from the MHC, and the few variants described for these genes are not reported to result in functional differences. In mice, there is one classical class I gene located in the H-2K region between the class II and the extended class II region, but there is at least one other classical class I gene far away in the class I region (the H-2D region), and the TAP, tapasin, and proteasome genes are nonpolymorphic. However, in laboratory rats (Rattus norvegicus), the only classical class I genes are located in the RT1A region between the class II and the extended class II region, and there is some haplotype-specific coevolution between the class I gene(s) and the TAP2 gene (Momberg et al., 1994; Joly et al., 1998; Hurt et
49
al., 2004). There are two lineages of TAP2, with the TAP2A alleles allowing transport of peptides with any amino acid at the C-terminus, but the TAP2B alleles allowing transport of peptides with a hydrophobic amino acid at the C-terminus. This specificity is generally mirrored in the closely linked class I gene(s), and mismatch of class I and TAP2 specificity found in a natural recombinant led to changes in class I maturation and presentation of peptides to cytotoxic T cells (CTLs). The Syrian hamster (Mesocricetus auratus) may also have this structural and functional oligomorphism (Lobigs et al., 1999). In chickens, the classical MHC (the BF-BL region) has two classical class I genes, but only one is expressed at a high level, apparently due to coevolution with the TAP1, TAP2 and tapasin genes (Kaufman et al., 1995, 1999a, 1999b). Recombination across the chicken MHC is very rare, with no recombinants recovered from thousands of deliberate matings and only one “natural recombinant” known among experimental chicken lines (although there is some evidence for gene conversion or double reciprocal recombination of short stretches within the MHC). No proteasome genes have been found (yet), but the TAP1, TAP2, and tapasin genes are located very close to the two classical class I genes and are highly polymorphic and moderately diverse in sequence, with each haplotype examined (except the natural recombinant) having a unique allele differing in roughly 1% of amino acid residues (Walker et al., submitted; van Hateren et al., submitted). Moreover, they are polymorphic in function, working with a dominantly expressed class I gene in a haplotype-specific manner. Peptide translocation assays show TAP specificity at least three peptide positions, which matches or even exceeds the peptide-binding specificity of the single dominantly expressed class I molecule (Fig. 5). Although such interactions have not yet been shown directly for tapasin, the natural recombinant has been used to show that class I maturation is haplotypedependent through class I residues known in mammals to be important for interaction with tapasin. Most importantly, the coevolution between TAPs, tapasin, and the class I genes leads to a single dominantly expressed class I molecule, which, in turn, has functional consequences (Kaufman et al., 1999a, 1999b; Wallney et al., 2006; Walker et al., submitted; van Hateren et al., submitted). As an example (Fig. 6), peptides isolated from cells of
FIGURE 6 A cartoon showing that coevolution between the peptide-translocation specificity of TAP and the peptide-binding specificity of class I molecules leads to a single dominantly expressed class I molecule, as illustrated with the chicken MHC haplotype B4. Thin lines indicate peptides, a minus sign within a circle indicates negatively charged residue, a plus sign within a circle indicates positively charged residue, a circle indicates a hydrophobic residue. Adapted from Kaufman et al., 1999b.
50
HOST DEFENSE: GENERAL
the B4 haplotype overwhelmingly have negative charges at anchor residues (aspartic acid and/or glutamic acid at positions 2 and 5 and glutamic acid at position 8), and models of the dominantly expressed class I molecule reveal positively charged residues in the peptide-binding domain well positioned to bind such peptides, which are totally lacking in the poorly expressed molecule. The TAP1 in B4 has positive charges in three positions, which are negatively charged in other haplotypes, and overwhelmingly, peptides bearing negative charges at positions 2 and 5 and glutamic acid at position 8 are translocated in B4 cells. In general, the peptide-binding specificity of the dominantly expressed class I molecule converges (or is exceeded by) the peptidetranslocation specificity of the TAP, which means that class I molecules encoded by other loci or haplotypes will receive few, if any, peptides, and thus, will not be utilized much by the adaptive immune system. The important functional consequence of a single dominantly expressed class I molecule is that an MHC haplotype may not be able to respond effectively to a particular pathogen if the class I molecule fails to bind a peptide that confers protection, as compared with a multigene family of MHC molecules, one of which is likely to find a protective peptide (Kaufman et al., 1995; Kaufman, 2008). In fact, the chicken MHC is known for strong associations with resistance to infectious pathogens as well as responses to vaccines, whereas the human MHC is known for strong associations with autoimmune diseases, with the few associations with infectious diseases being relatively weak, and in some cases known to involve interactions with NK cells rather than peptide presentation to T cells. Although a wide range of critical tests are required to establish that this apparent difference is real, it is at least clear for a few examples that peptide presentation by the single dominantly expressed class I molecule can determine whether a chicken lives or dies. Although the data is still patchy, many, if not most, nonmammalian vertebrates examined have the salient features for the class I system first discovered for the chicken MHC: one class I gene expressed at a high level, with polymorphic TAP and tapasin genes next to the class I gene(s), and for one fish, strong genetic associations with resistance to infectious pathogens (Kaufman, 1999). For example, the duck (Anas platyrhynchos) has five class I genes, of which only one is well expressed, adjacent to the polymorphic TAP genes (Mesa et al., 2004; Moon et al., 2005). Similarly, a single classical class I gene is located next to the TAP genes in the frogs Xenopus laevis and X. tropicalis (Ohta et al., 2006). A bony fish, Atlantic salmon (Salmo salar) has a single classical class I gene, with inducible proteasome components, tapasin, and (at least one) TAP genes close by (Grimholt et al., 2002; Lukacs et al., 2007), and cartilaginous fish as exemplified by sharks (Triakis scyllium and Ginglymostoma cirratum) have one (and in some haplotypes, two) class I genes with the proteasome components and TAP genes closely linked (Ohta et al., 2002). The situation for class II genes is less clear, although many birds have elements of the simple class II system of chickens (Jacob et al., 2000; Salomonsen et al., 2003). Thus far, there is little relevant functional data available for nonmammalian vertebrates, but for X. laevis there is an association of the MHC with resistance to a bacterial pathogen (Barribeau et al., 2008), and for Atlantic salmon there are strong genetic associations of the class I region with resistance to a virus and the class II region with resistance to a bacterial pathogen (Grimholt et al., 2003). Taking this data together, there appear to be at least two strategies of MHC genomic organization leading to differences in function. The salient features of the chicken MHC (and those of most nonmammalian vertebrates) are
most likely to represent the ancestral organization, with the typical mammalian (and some nonmammalian vertebrate) MHC organizations being derived. In this view, a large inversion occurred in the lineage leading to mammals, bringing the class III region in between what we now call the mammalian class I and class II regions, but separating the TAP, tapasin, and proteasome genes from the class I genes in the process. In fact, it appears that this event took place in the lineage leading to placental mammals, since at least one marsupial, the American opossum (Monodelphis domestica), has the MHC organization typical of nonmammalian vertebrates (Belov et al., 2006). Independent events leading to the breakup of the ancestral organization of class I, TAP, tapasin, and proteasome genes may have occurred several times. For instance, another marsupial, the Tamar wallaby (Macropus eugenii), has TAP genes in the MHC, but the classical class I genes located at the ends of several other chromosomes (Deakin et al., 2007; Siddle et al., 2009). Some passerine birds have been shown to have a multigene family of expressed classical class I genes, for which individual alleles in warblers and sparrows (great reed warbler Acrocephalus arundinaceus and house sparrow Passer domesticus) confer resistance to infectious pathogens (Westerdahl et al., 2000, 2005; Bonneaud et al., 2006). The genomic sequence of another passerine bird, the zebra finch (Taeniopygia guttata), appears to have classical class I and TAP genes on different chromosomes. The class II B genes of a bony fish, the threespined stickleback (Gasterosteus aculeatus), form a multigene family with expansion and contraction, with evidence that intermediate numbers of loci/alleles are important in mating and pathogen resistance (Wegner et al., 2003). Finally, there may be yet other strategies for MHC organization and function, of which there are two examples that express a large number of classical class I genes at a low level (Sammut et al., 1999; Persson et al., 1999; Miller et al., 2002), with only one expressed class II gene in a salamander, the axolotl (Ambystoma mexicanum), and no obviously expressed class II gene in a bony fish, the Atlantic cod (Gadus morhua). What could be the reason for the presence of a single dominantly expressed MHC molecule in so many nonmammalian vertebrates when a multigene family of MHC molecules allows a much broader response to pathogens (albeit potentially at the expense of greater autoimmunity)? The answer may be that this is the way in which the class I part of the primordial MHC first got started (Kaufman et al., 2008). The primordial class I, TAP, tapasin, and proteasome genes presumably all had functions other than the ones they now fulfill, and then evolved to interact. The simplest and most efficient way for such coevolution to take place would be for the genes to be closely linked, in much the same way that the class I, TAP, and tapasin genes have coevolved as haplotypes in the chicken MHC. Conceptually, this is the same idea proposed for the evolution of metabolic pathways at the beginning of life. The natural recombinant in the chicken MHC may be one example of catching such a coevolution in the act (van Hateren et al., submitted). The Scandinavian B19 haplotype (also known as B19var1) is a recombinant of the B12 and B15 haplotypes, with the site of recombination in the middle of the TAP2 gene. Thus far, all the genes sequenced from BG1 to the middle of TAP2 are identical between B19 and B12, while the end of TAP2 and the dominantly expressed class I gene BF2 are nearly identical between B19 and B15. Transfection of BF2 cDNAs from B15 and B19 into B15 cells showed that BF2*15 matured more rapidly and more completely than BF2*19. Given that the class I peptide motif and the translocation specificity in B15 and B19 cells have the same anchor residues, the only likely
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates
mismatch is with tapasin: BF2*15 interacts well with the tapasin in B15 cells, but BF2*19 expects B12 tapasin. There are only eight differences between BF2*15 and BF2*19, of which five are changes back to the residues found in the BF2*12. Some of these residues are implicated in human class I molecules with tapasin interaction, and swapping these residues between the two molecules shows that they are enough to increase or decrease maturation. Thus, small changes can allow coevolution between genes in a newly formed haplotype, and it is precisely such small changes that are envisaged to allow the primordial class I, TAP, and tapasin genes to coevolve in order to work together as a team. Does this scenario of coevolution fit with the speculations outlined above that the first variable antigen-specific receptor was a TcR recognizing a class II-like molecule? If a class II-like gene came first, then the principles are likely to be the same, although there may have been much repurposing from a molecule sampling the intracellular vesicles for class II-dependent killer cells to a molecule primarily (although not exclusively) involved in regulation of antibody responses. Consistent with coevolution leading to a single dominantly expressed class II molecule in chickens, there are strong associations of the MHC with responses to inactivated vaccines, a single nonpolymorphic class II A gene located outside of the MHC, and two classical class II B genes, only one of which is well expressed, and polymorphic DM genes nearby with which to coevolve in a haplotype-specific manner (Jacob et al., 2000; Salomonsen et al., 2003; Kaufman et al., 2008). Overall, much remains to be clarified about class II genes throughout the rest of the vertebrates, but consistent with the coevolution model, the class II-dependent genetic associations with antibody responses in mammals (immune response or Ir genes) are relatively weak, while associations of the class II region in Atlantic salmon with responses to bacteria are very strong (Benacerraf & Germain, 1978; Grimholt et al., 2003). Whether TcR was the first variable antigen-specific receptor may also involve coevolution within the primordial MHC, as explored in the next section.
THE PRIMORDIAL MHC: THE BIRTHPLACE OF THE ADAPTIVE IMMUNE SYSTEM OF JAWED VERTEBRATES
What did the primordial MHC look like? In various invertebrates, many of the genes in and around the mammalian MHC, including the complement components of the class III region, are found close together in regions dubbed the “proto-MHC” (Abi-Rached et al., 2002; Danchin et al., 2003; Castro et al., 2004; Danchin & Pontarotti, 2004). However, with the possible exceptions of a TcR-like and perhaps a TAP-like gene (Uinuk-ool et al., 2002, 2003; Pancer et al., 2004a), no invertebrate genes have been identified as obvious direct ancestors of the essential components of the adaptive immune system. Nevertheless, genomes in vertebrates provide clues, which suggest that the genes for these essential components were originally all in the same region, allowing them to coevolve into a functioning system. As described above (Fig. 2), MHC paralogous regions have been identified in mammals and are presumed to have arisen from two rounds of genome-wide duplication at the base of the vertebrates (Kasahara et al., 1997; Flajnik & Kasahara, 2001, 2010). They are far from perfect copies of each other, with much evidence of regions breaking up, pieces breaking off, genes deleted, and families expanded, and most importantly, members of gene families with new functions. In humans, the classical class I (and some nonclassical) class I genes are located in the MHC on chromosome 6, the nonclassical class I
51
genes for CD1 molecules are located in the MHC-paralogous region on chromosome 1, and the nonclassical class I gene encoding an Fc receptor is located in the MHC-paralogous region on chromosome 19, but no class I-like gene is located in the MHC-paralogous region on chromosome 9. Also, homologs of certain complement component genes are found in the MHC-paralogous regions (C3, C4, and C5 on chromosome 9, 6, and 19), but the last homolog (the protease inhibitor a2-macroglobulin) is found in the NK receptor cluster (NKC) on chromosome 12, rather than in the MHCparalogous region on chromosome 1. These are just a few of the many examples taken to illustrate regions breaking up and/or pieces breaking off (a2-macroglobulin in the NKC), genes deleted (class I-like genes on chromosome 1), families expanded (many class I genes on chromosome 6, several CD1 genes on chromosome 1), and genes with new functions (Fc receptor gene on chromosome 19). Examination of the chicken MHC shows some of the same genes (Fig. 2), but many expected genes are located elsewhere in the genome (or deleted from the genome entirely) and other unexpected genes are present, overall giving an alternative view of the dynamics of the MHC paralogous regions and allowing some insight into the primordial MHC (Kaufman et al., 1999a; Salomonsen et al., 2005; Rogers et al., 2005, 2008). For instance, a pair of lectin-like genes were identified in the original sequence of the chicken MHC (the BF-BL region) and, on the basis of genomic organization and expression patterns, shown to correspond to pairs of genes in the NKC (particularly the NK receptor gene NKR-P1 and its ligand LLT1 in humans, or Clr in mouse and rat). In contrast, only two lectin-like genes were identified in the chicken region corresponding to the NKC. Later, two genes encoding chicken CD1 were located immediately adjacent to the sequenced portion of the chicken MHC. The simplest explanation for the presence of both NK receptors and CD1 in the chicken MHC is that they were both present in the primordial MHC before the two rounds of genome-wide duplication, and were silenced in different genomic regions in the lineage leading to birds versus the lineage leading to mammals. With this idea in mind, traces of other gene families present in the MHC become understandable. An example recognized early on (Kasahara et al., 1997) is the presence of lectin-like genes CD23 and DC-SIGN in the MHC-paralogous region on human chromosome 19 and many other lectin-like genes, expressed in NK/T cells and myeloid cells, located in the NKC on human chromosome 12. Similarly, the lymphocyte receptor region (LRC) on human chromosome 19 contains many homologous genes, including the KIR genes expressed primarily on NK cells, and a variety of homologs (including an Fc receptor) expressed on cells of the myeloid lineage (Martin et al., 2002). A homolog called NKp30, a member of the natural cytotoxicity receptor (NCR) genes, is located in the human MHC (Moretta et al., 2001). Conversely, two class I-like Mill genes are found just outside the LRC in mice (Kasahara et al., 2002). This fits well with the suggestion that the regions syntenic to the MHC, the NKC and the KIR genes were originally next to each other based on the arrangement in the tunicate Ciona intestinalis (Olinski et al., 2006; Zucchetti et al., 2009). A most intriguing example is a gene related to TcR genes found on the edge of the MHC of the frog X. tropicalis (Ohta et al., 2006). Such examples suggest that all of these gene families were located together in the primordial MHC before the two rounds of genome-wide duplication. The two concepts of paralogous regions and coevolution taken together make a powerful prediction (Kaufman et al., 2008). The presence of MHC, NK receptor, and TcR gene
52
HOST DEFENSE: GENERAL
families in the same genomic locations suggests that genes for receptors and ligands originated in the same genomic region (Fig. 2), presumably to allow coevolution so that they would function together, much in the same way as was outlined above for coevolution of disparate genes to form the class I presentation pathway. In this view, the adaptive immune system of jawed vertebrates arose in a single genomic region, the primordial MHC, which has been breaking apart ever since.
CONCLUSIONS
Some 50 years of research have shown that the adaptive immune system of jawed vertebrates appeared once at least 500 million years ago. Many important events involve drastic changes in the genome, including transposon insertion to create split V genes, horizontal transfer of recombination activating genes (RAGs), inversions and translocations of DNA to create MHC genes, genome-wide duplications to create genes for new functions, and further reorganizations to alter MHC function. Throughout the evolution of this adaptive immune system, coevolution of linked genes was important for different genes to begin to work together, a process still evident in the coevolution of antigen presentation systems in most nonmammalian vertebrates. The best guess is that the birthplace of this adaptive immune system was the primordial MHC, which has been falling apart ever since. Thanks to John Chattaway, Aimee Parker, Andrew Chan, and Hannah Siddle for reading the manuscript, and to the Wellcome Trust (089305/Z/09/Z) for support.
REFERENCES Abi-Rached, L., A. Gilles, T. Shiina, P. Pontarotti, and H. Inoko. 2002. Evidence of en bloc duplication in vertebrate genomes. Nat. Genet. 31:100–105. Adema, C. M., L. A. Hertel, R. D. Miller, and E. S. Loker. 1997. A family of fibrinogen-related proteins that precipitates parasite-derived molecules is produced by an invertebrate after infection. Proc. Natl. Acad. Sci. USA 94: 8691–8696. Amadou, C. 1999. Evolution of the Mhc class I region: the framework hypothesis. Immunogenetics 49:362–367. Azumi, K., R. De Santis, A. De Tomaso, I. Rigoutsos, F. Yoshizaki, M. R. Pinto, R. Marino, K. Shida, M. Ikeda, M. Arai, Y. Inoue, T. Shimizu, N. Satoh, D. S. Rokhsar, L. Du Pasquier, M. Kasahara, M. Satake, and M. Nonaka. 2003. Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: “waiting for Godot”. Immunogenetics 55:570–581. Bajoghli, B., N. Aghaallaei, I. Hess, I. Rode, N. Netuschil, B. H. Tay, B. Venkatesh, J. K. Yu, S. L. Kaltenbach, N. D. Holland, D. Diekhoff, C. Happe, M. Schorpp, and T. Boehm. 2009. Evolution of genetic networks underlying the emergence of thymopoiesis in vertebrates. Cell 138:186–197. Barribeau, S. M., J. Villinger, and B. Waldman. 2008. Major histocompatibility complex based resistance to a common bacterial pathogen of amphibians. PLoS One 3:e2692. Belov, K., J. E. Deakin, A. T. Papenfuss, M. L. Baker, S. D. Melman, H. V. Siddle, N. Gouin, D. L. Goode, T. J. Sargeant, M. D. Robinson, M. J. Wakefield, S. Mahony, J. G. Cross, P. V. Benos, P. B. Samollow, T. P. Speed, J. A. Graves, and R. D. Miller. 2006. Reconstructing an ancestral mammalian immune supercomplex from a marsupial major histocompatibility complex. PLoS Biol. 4:e46. Benacerraf, B., and R. N. Germain. 1978. The immune response genes of the major histocompatibility complex. Immunol. Rev. 38:70–119.
Bernstein, R. M., S. F. Schluter, D. F. Lake, and J. J. Marchalonis. 1994. Evolutionary conservation and molecular cloning of the recombinase activating gene 1. Biochem. Biophys. Res. Commun. 205:687–692. Bingulac-Popovic, J., F. Figueroa, A. Sato, W. S. Talbot, S. L. Johnson, M. Gates, J. H. Postlethwait, and J. Klein. 1997. Mapping of mhc class I and class II regions to different linkage groups in the zebrafish, Danio rerio. Immunogenetics 46:129–134. Boehm, T., and C. C. Bleul. 2007. The evolutionary history of lymphoid organs. Nat. Immunol. 8:131–135. Boehm, T., and F. Zufall. 2006. MHC peptides and the sensory evaluation of genotype. Trends Neurosci. 29:100–107. Bonneaud, C., J. Perez-Tris, P. Federici, O. Chastel, and G. Sorci. 2006. Major histocompatibility alleles associated with local resistance to malaria in a passerine. Evolution 60:383–389. Brites, D., S. McTaggart, K. Morris, J. Anderson, K. Thomas, I. Colson, T. Fabbro, T. J. Little, D. Ebert, and L. Du Pasquier. 2008. The Dscam homologue of the crustacean Daphnia is diversified by alternative splicing like in insects. Mol. Biol. Evol. 25:1429–1439. Cannon, J. P., R. N. Haire, A. T. Magis, D. D. Eason, K. N. Winfrey, J. A. Hernandez Prada, K. M. Bailey, J. Jakoncic, G. W. Litman, and D. A. Ostrov. 2008. A bony fish immunological receptor of the NITR multigene family mediates allogeneic recognition. Immunity 29:228–237. C. elegans Sequencing Consortium. 1998. Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282:2012–2008. Castro, L. F., R. F. Furlong, and P. W. Holland. 2004. An antecedent of the MHC-linked genomic region in amphioxus. Immunogenetics 55:782–784. Cooper, M. D., and M. N. Alder. 2006. The evolution of adaptive immune systems. Cell 124:815–822. Danchin, E. G., L. Abi-Rached, A. Gilles, and P. Pontarotti. 2003. Conservation of the MHC-like region throughout evolution. Immunogenetics 55:141–148. Danchin, E. G., and P. Pontarotti. 2004. Towards the reconstruction of the bilaterian ancestral pre-MHC region. Trends Genet. 20:587–591. Danilova, N. and C. Amemiya. 2009. Going adaptive: the saga of antibodies. Ann. NY Acad. Sci. 1168:130–155. Davis, M. M., and P. J. Bjorkman. 1988. T-cell antigen receptor genes and T-cell recognition. Nature 334:395–402. De Tomaso, A. W., S. V. Nyholm, K. J. Palmeri, K. J. Ishizuka, W. B. Ludington, K. Mitchel, and I. L. Weissman. 2005. Isolation and characterization of a protochordate histocompatibility locus. Nature 438:454–459. Deakin, J. E., H. V. Siddle, J. G. Cross, K. Belov, and J. A. Graves. 2007. Class I genes have split from the MHC in the tammar wallaby. Cytogenet. Genome Res. 116:205–211. Dishaw, L., M. Mueller, N. Gwatney, J. Cannon, R. Haire, R. Litman, C. Amemiya, T. Ota, L. Rowen, G. Glusman, and G. Litman. 2008. Genomic complexity of the variable region-containing chitin-binding proteins in amphioxus. BMC Genet. 9:e78 Dong, Y., H. E. Taylor, and G. Dimopoulos. 2006. AgDscam, a hypervariable immunoglobulin domain-containing receptor of the Anopheles gambiae innate immune system. PLoS Biol. 4:e229. Donoghue, P. C., and M. A. Purnell. 2005. Genome duplication, extinction and vertebrate evolution. Trends Ecol. Evol. 20:312–319. Du Pasquier, L., and B. Blomberg. 1982. The expression of antibody diversity in natural and laboratory-made polyploid individuals of the clawed toad Xenopus. Immunogenetics 15:251–260. Eason, D. D., J. P. Cannon, R. N. Haire, J. P. Rast, D. A. Ostrov, and G. W. Litman. 2004. Mechanisms of antigen receptor evolution. Semin. Immunol. 16:215–226. Flajnik, M. F. 2002. Comparative analyses of immunoglobulin genes: surprises and portents. Nat. Rev. Immunol. 2:688–698.
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates Flajnik, M. F., C. Canel, J. Kramer, and M. Kasahara. 1991. Which came first, MHC class I or class II? Immunogenetics 33:295–300. Flajnik, M. F., and L. Du Pasquier. 2004. Evolution of innate and adaptive immunity: can we draw a line? Trends Immunol. 25:640–644. Flajnik, M. F., and M. Kasahara. 2010. Origin and evolution of the adaptive immune system: genetic events and selective pressures. Nat. Rev. Genet. 11:47–59. Flajnik, M. F., and M. Kasahara. 2001. Comparative genomics of the MHC: glimpses into the evolution of the adaptive immune system. Immunity 15:351–362. Fugmann, S. D., C. Messier, L. A. Novack, R. A. Cameron, and J. P. Rast. 2006. An ancient evolutionary origin of the Rag1/2 gene locus. Proc. Natl. Acad. Sci. USA 103: 3728–3733. Garcia-Fernandez, J., and P. W. Holland. 1994. Archetypal organization of the amphioxus Hox gene cluster. Nature 370:563–566. Germain, R. N., D. M. Bentley, and H. Quill. 1985. Influence of allelic polymorphism on the assembly and surface expression of class II MHC (Ia) molecules. Cell 43:233–242. Grimholt, U., F. Drablos, S. M. Jorgensen, B. Hoyheim, and R. J. Stet. 2002. The major histocompatibility class I locus in Atlantic salmon (Salmo salar L.): polymorphism, linkage analysis and protein modelling. Immunogenetics 54:570–581. Grimholt, U., S. Larsen, R. Nordmo, P. Midtlyng, S. Kjoeglum, A. Storset, S. Saebo, and R. J. Stet. 2003. MHC polymorphism and disease resistance in Atlantic salmon (Salmo salar); facing pathogens with single expressed major histocompatibility class I and class II loci. Immunogenetics 55:210–219. Guo, P., M. Hirano, B. R. Herrin, J. Li, C. Yu, A. Sadlonova, and M. D. Cooper. 2009. Dual nature of the adaptive immune system in lampreys. Nature 459:796–801. Hedrick, S. M. 2004. The acquired immune system: a vantage from beneath. Immunity 21:607–615. Holland, L. Z., R. Albalat, K. Azumi, E. Benito-Gutierrez, M. J. Blow, M. Bronner-Fraser, F. Brunet, T. Butts, S. Candiani, L. J. Dishaw, D. E. Ferrier, J. GarciaFernandez, J. J. Gibson-Brown, C. Gissi, A. Godzik, F. Hallbook, D. Hirose, K. Hosomichi, T. Ikuta, H. Inoko, M. Kasahara, J. Kasamatsu, T. Kawashima, A. Kimura, M. Kobayashi, Z. Kozmik, K. Kubokawa, V. Laudet, G. W. Litman, A. C. McHardy, D. Meulemans, M. Nonaka, R. P. Olinski, Z. Pancer, L. A. Pennacchio, M. Pestarino, J. P. Rast, I. Rigoutsos, M. Robinson-Rechavi, G. Roch, H. Saiga, Y. Sasakura, M. Satake, Y. Satou, M. Schubert, N. Sherwood, T. Shiina, N. Takatori, J. Tello, P. Vopalensky, S. Wada, A. Xu, Y. Ye, K. Yoshida, F. Yoshizaki, J. K. Yu, Q. Zhang, C. M. Zmasek, P. J. de Jong, K. Osoegawa, N. H. Putnam, D. S. Rokhsar, N. Satoh, and P. W. Holland. 2008. The amphioxus genome illuminates vertebrate origins and cephalochordate biology. Genome Res. 18:1100–1111. Hsu, E., N. Pulham, L. Rumfelt, and M. Flajnik. 2006. The plasticity of immunoglobulin gene systems in evolution. Immunol. Rev. 210:8–26. Huang, S., S. Yuan, L. Guo, Y. Yu, J. Li, T. Wu, T. Liu, M. Yang, K. Wu, H. Liu, J. Ge, H. Huang, M. Dong, C. Yu, S. Chen, and A. Xu. 2008. Genomic analysis of the immune gene repertoire of amphioxus reveals extraordinary innate complexity and diversity. Genome Res. 18:1112–1126. Hurt, P., L. Walter, R. Sudbrak, S. Klages, I. Muller, T. Shiina, H. Inoko, H. Lehrach, E. Gunther, R. Reinhardt, and H. Himmelbauer. 2004. The genomic sequence and comparative analysis of the rat major histocompatibility complex. Genome Res. 14:631–639. Jacob, J. P., S. Milne, S. Beck, and J. Kaufman. 2000. The major and a minor class II beta-chain (B-LB) gene flank the Tapasin gene in the B-F /B-L region of the chicken major histocompatibility complex. Immunogenetics 51:138–147.
53
Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20:197–216. Joly, E., A. F. Le Rolle, A. L. Gonzalez, B. Mehling, J. Stevens, W. J. Coadwell, T. Hunig, J. C. Howard, and G. W. Butcher. 1998. Co-evolution of rat TAP transporters and MHC class I RT1-A molecules. Curr. Biol. 8:169–172. Kapitonov, V. V., and J. Jurka. 2005. RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons. PLoS Biol. 3:e181. Kasahara, M., J. Nakaya, Y. Satta, and N. Takahata. 1997. Chromosomal duplication and the emergence of the adaptive immune system. Trends Genet. 13:90–92. Kasahara, M., T. Suzuki, and L. Du Pasquier. 2004. On the origins of the adaptive immune system: novel insights from invertebrates and cold-blooded vertebrates. Trends Immunol. 25:105–111. Kasahara, M., Y. Watanabe, M. Sumasu, and T. Nagata. 2002. A family of MHC class I-like genes located in the vicinity of the mouse leukocyte receptor complex. Proc. Natl. Acad. Sci. USA 99:13687–13692. Kaufman, J. 1988. Vertebrates and the evolution of the Major Histocompatibility Complex class I and class II molecules. Verh. Dtsch. Zool. Ges. 81:131–144. Kaufman, J. 1999. Co-evolving genes in MHC haplotypes: the “rule” for nonmammalian vertebrates? Immunogenetics 50:228–236. Kaufman, J. 2008. The avian MHC, 161–183. In T. F. Davison, B. Kaspers, and K. A. Schat (eds.), The Immunology of Birds, Elsevier, Oxford. Kaufman, J., J. Jacob, I. Shaw, B. Walker, S. Milne, S. Beck, and J. Salomonsen. 1999. Gene organisation determines evolution of function in the chicken MHC. Immunol. Rev. 167:101–117. Kaufman, J., S. Milne, T. W. Gobel, B. A. Walker, J. P. Jacob, C. Auffray, R. Zoorob, and S. Beck. 1999. The chicken B locus is a minimal essential major histocompatibility complex. Nature 401:923–925. Kaufman, J., K. Skjoedt, and J. Salomonsen. 1990. The MHC molecules of nonmammalian vertebrates. Immunol. Rev. 113:83–117. Kaufman, J., H. Volk, and H. J. Wallny. 1995. A “minimal essential Mhc” and an “unrecognized Mhc”: two extremes in selection for polymorphism. Immunol. Rev. 143:63–88. Kaufman, J. F., C. Auffray, A. J. Korman, D. A. Shackelford, and J. Strominger. 1984. The class II molecules of the human and murine major histocompatibility complex. Cell 36:1–13. Kelley, J., L. Walter, and J. Trowsdale. 2005. Comparative genomics of major histocompatibility complexes. Immunogenetics 56:683–695. Li, P., S. T. Willie, S. Bauer, D. L. Morris, T. Spies, and R. K. Strong. 1999. Crystal structure of the MHC class I homolog MIC-A, a gamma delta T cell ligand. Immunity 10:577–584. Litman, G. W., J. P. Cannon, and L. J. Dishaw. 2005. Reconstructing immune phylogeny: new perspectives. Nat. Rev. Immunol. 5:866–879. Litman, G. W., L. J. Dishaw, J. P. Cannon, R. N. Haire, and J. P. Rast. 2007. Alternative mechanisms of immune receptor diversity. Curr. Opin. Immunol. 19:526–534. Lobigs, M., A. Mullbacher, R. V. Blanden, G. J. Hammerling, and F. Momburg. 1999. Antigen presentation in syrian hamster cells: substrate selectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules. Immunogenetics 49:931–941. Lukacs, M. F., H. Harstad, U. Grimholt, M. Beetz-Sargent, G. A. Cooper, L. Reid, H. G. Bakke, R. B. Phillips, K. M. Miller, W. S. Davidson, and B. F. Koop. 2007. Genomic organization of duplicated major histocompatibility complex class I regions in Atlantic salmon (Salmo salar). BMC Genomics 8:e251. Martin, A. M., J. K. Kulski, C. Witt, P. Pontarotti, and F. T. Christiansen. 2002. Leukocyte Ig-like receptor complex (LRC) in mice and men. Trends Immunol. 23:81–88.
54
HOST DEFENSE: GENERAL
Medzhitov, R. 2009. Approaching the asymptote: 20 years later. Immunity 30:766–775. Mesa, C. M., K. J. Thulien, D. A. Moon, S. M. Veniamin, and K. E. Magor. 2004. The dominant MHC class I gene is adjacent to the polymorphic TAP2 gene in the duck, Anas platyrhynchos. Immunogenetics 56:192–203. MHC Sequencing Consortium. 1999. Complete sequence and gene map of a human major histocompatibility complex. Nature 401:921–923. Miller, K. M., K. H. Kaukinen, and A. D. Schulze. 2002. Expansion and contraction of major histocompatibility complex genes: a teleostean example. Immunogenetics 53:941–963. Miller, M. M., C. Wang, E. Parisini, R. D. Coletta, R. M. Goto, S. Y. Lee, D. C. Barral, M. Townes, C. Roura-Mir, H. L. Ford, M. B. Brenner, and C. C. Dascher. 2005. Characterization of two avian MHC-like genes reveals an ancient origin of the CD1 family. Proc. Natl. Acad. Sci. USA 102:8674–8679. Momburg, F., J. Roelse, J. C. Howard, G. W. Butcher, G. J. Hammerling, and J. J. Neefjes. 1994. Selectivity of MHCencoded peptide transporters from human, mouse and rat. Nature 367:648–651. Moon, D. A., S. M. Veniamin, J. A. Parks-Dely, and K. E. Magor. 2005. The MHC of the duck (Anas platyrhynchos) contains five differentially expressed class I genes. J. Immunol. 175:6702–6712. Moretta, A., C. Bottino, M. Vitale, D. Pende, C. Cantoni, M. C. Mingari, R. Biassoni, and L. Moretta. 2001. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu. Rev. Immunol. 19:197–223. Ohno, S. 1970. Evolution by gene duplication. Springer Verlag. Ohta, Y., W. Goetz, M. Z. Hossain, M. Nonaka, and M. F. Flajnik. 2006. Ancestral organization of the MHC revealed in the amphibian Xenopus. J. Immunol. 176:3674–3685. Ohta, Y., E. C. McKinney, M. F. Criscitiello, and M. F. Flajnik. 2002. Proteasome, transporter associated with antigen processing, and class I genes in the nurse shark Ginglymostoma cirratum: evidence for a stable class I region and MHC haplotype lineages. J. Immunol. 168:771–781. Ohta, Y., K. Okamura, E. C. McKinney, S. Bartl, K. Hashimoto, and M. F. Flajnik. 2000. Primitive synteny of vertebrate major histocompatibility complex class I and class II genes. Proc. Natl. Acad. Sci. USA 97:4712–4717. Ohta, Y., S. J. Powis, R. L. Lohr, M. Nonaka, L. D. Pasquier, and M. F. Flajnik. 2003. Two highly divergent ancient allelic lineages of the transporter associated with antigen processing (TAP) gene in Xenopus: further evidence for co-evolution among MHC class I region genes. Eur. J. Immunol. 33:3017–3027. Olinski, R. P., Lundin, L. G. & Hallbook, F. 2006. Conserved synteny between the Ciona genome and human paralogons identifies large duplication events in the molecular evolution of the insulin-relaxin gene family. Mol. Biol. Evol. 23:10–22. Pancer, Z., C. T. Amemiya, G. R. Ehrhardt, J. Ceitlin, G. L. Gartland, and M. D. Cooper. 2004. Somatic diversification of variable lymphocyte receptors in the agnathan sea lamprey. Nature 430:174–180. Pancer, Z., W. E. Mayer, J. Klein, and M. D. Cooper. 2004. Prototypic T cell receptor and CD4-like coreceptor are expressed by lymphocytes in the agnathan sea lamprey. Proc. Natl. Acad. Sci. USA 101:13273–13278. Parham, P. 2005. MHC class I molecules and KIRs in human history, health and survival. Nat. Rev. Immunol. 5:201–214. Persson, A. C., R. J. Stet, and L. Pilstrom. 1999. Characterization of MHC class I and beta(2)-microglobulin sequences in Atlantic cod reveals an unusually high number of expressed class I genes. Immunogenetics 50:49–59. Putnam, N. H., T. Butts, D. E. Ferrier, R. F. Furlong, U. Hellsten, T. Kawashima, M. Robinson-Rechavi, E. Shoguchi, A. Terry, J. K. Yu, E. L. Benito-Gutierrez, I. Dubchak, J. Garcia-Fernandez, J. J. Gibson-Brown, I. V. Grigoriev, A. C. Horton, P. J. de Jong, J. Jurka, V. V.
Kapitonov, Y. Kohara, Y. Kuroki, E. Lindquist, S. Lucas, K. Osoegawa, L. A. Pennacchio, A. A. Salamov, Y. Satou, T. Sauka-Spengler, J. Schmutz, I. T. Shin, A. Toyoda, M. Bronner-Fraser, A. Fujiyama, L. Z. Holland, P. W. Holland, N. Satoh, and D. S. Rokhsar. 2008. The amphioxus genome and the evolution of the chordate karyotype. Nature 453:1064–1071. Rast, J. P., M. K. Anderson, S. J. Strong, C. Luer, R. T. Litman, and G. W. Litman. 1997. alpha, beta, gamma, and delta T cell antigen receptor genes arose early in vertebrate phylogeny. Immunity 6:1–11. Rast, J. P., L. C. Smith, M. Loza-Coll, T. Hibino, and G. W. Litman. 2006. Genomic insights into the immune system of the sea urchin. Science 314:952–956. Rogers, S. L., T. W. Gobel, B. C. Viertlboeck, S. Milne, S. Beck, and J. Kaufman. 2005. Characterization of the chicken C-type lectin-like receptors B-NK and B-lec suggests that the NK complex and the MHC share a common ancestral region. J. Immunol. 174:3475–3483. Rogers, S. L., B. C. Viertlboeck, T. W. Gobel, and J. Kaufman. 2008. Avian NK activities, cells and receptors. Semin. Immunol. 20:353–360. Sakano, H., K. Huppi, G. Heinrich, and S. Tonegawa. 1979. Sequences at the somatic recombination sites of immunoglobulin light-chain genes. Nature 280:288–294. Salomonsen, J., D. Marston, D. Avila, N. Bumstead, B. Johansson, H. Juul-Madsen, G. D. Olesen, P. Riegert, K. Skjodt, O. Vainio, M. V. Wiles, and J. Kaufman. 2003. The properties of the single chicken MHC classical class II alpha chain (B-LA) gene indicate an ancient origin for the DR/E-like isotype of class II molecules. Immunogenetics 55:605–614. Salomonsen, J., M. R. Sorensen, D. A. Marston, S. L. Rogers, T. Collen, A. van Hateren, A. L. Smith, R. K. Beal, K. Skjodt, and J. Kaufman. 2005. Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC. Proc. Natl. Acad. Sci. USA 102:8668–8673. Sambrook, J. G., F. Figueroa, and S. Beck. 2005. A genomewide survey of Major Histocompatibility Complex (MHC) genes and their paralogues in zebrafish. BMC Genomics 6:e152. Sammut, B., L. Du Pasquier, P. Ducoroy, V. Laurens, A. Marcuz, and A. Tournefier. 1999. Axolotl MHC architecture and polymorphism. Eur. J. Immunol. 29:2897–2907. Sammut, B., A. Marcuz, and L. D. Pasquier. 2002. The fate of duplicated major histocompatibility complex class Ia genes in a dodecaploid amphibian, Xenopus ruwenzoriensis. Eur. J. Immunol. 32:1593–1604. Schluter, S. F., R. M. Bernstein, H. Bernstein, and J. J. Marchalonis. 1999. ‘Big Bang’ emergence of the combinatorial immune system. Dev. Comp. Immunol. 23:107–111. Scofield, V. L., J. M. Schlumpberger, L. A. West, and I. L. Weissman. 1982. Protochordate allorecognition is controlled by a MHC-like gene system. Nature 295:499–502. Siddle, H. V., J. E. Deakin, P. Coggill, E. Hart, Y. Cheng, E. S. Wong, J. Harrow, S. Beck, and K. Belov. 2009. MHClinked and un-linked class I genes in the wallaby. BMC Genomics 10:e310. Stet, R. J., C. P. Kruiswijk, and B. Dixon. 2003. Major histocompatibility lineages and immune gene function in teleost fishes: the road not taken. Crit. Rev. Immunol. 23:441–471. Terado, T., K. Okamura, Y. Ohta, D. H. Shin, S. L. Smith, K. Hashimoto, T. Takemoto, M. I. Nonaka, H. Kimura, M. F. Flajnik, and M. Nonaka. 2003. Molecular cloning of C4 gene and identification of the class III complement region in the shark MHC. J. Immunol. 171:2461–2466. Trowsdale, J., and A. Moffett. 2008. NK receptor interactions with MHC class I molecules in pregnancy. Semin. Immunol. 20:317–320.
3. The Evolutionary Origins of the Adaptive Immune System of Jawed Vertebrates Uinuk-Ool, T., W. E. Mayer, A. Sato, R. Dongak, M. D. Cooper, and J. Klein. 2002. Lamprey lymphocyte-like cells express homologs of genes involved in immunologically relevant activities of mammalian lymphocytes. Proc. Natl. Acad. Sci. USA 99:14356–14361. Uinuk-Ool, T. S., W. E. Mayer, A. Sato, N. Takezaki, L. Benyon, M. D. Cooper, and J. Klein. 2003. Identification and characterization of a TAP-family gene in the lamprey. Immunogenetics 55:38–48. Van Hateren, A., A. Williams, J. Jacob, T. Elliot, and J. Kaufman. 2010. Co-evolution in the chicken MHC: haplotype-specific interaction of the polymorphic tapasin and dominantly-expressed class I molecules, submitted. Wallny, H. J., D. Avila, L. G. Hunt, T. J. Powell, P. Riegert, J. Salomonsen, K. Skjodt, O. Vainio, F. Vilbois, M. V. Wiles, and J. Kaufman. 2006. Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens. Proc. Natl. Acad. Sci. USA 103:1434–1439. Watson, F. L., R. Puttmann-Holgado, F. Thomas, D. L. Lamar, M. Hughes, M. Kondo, V. I. Rebel, and D. Schmucker. 2005. Extensive diversity of Ig-superfamily proteins in the immune system of insects. Science 309:1874–1878. Wegner, K. M., M. Kalbe, J. Kurtz, T. B. Reusch, and M. Milinski. 2003. Parasite selection for immunogenetic optimality. Science 301:1343.
55
Westerdahl, H., J. Waldenstrom, B. Hansson, D. Hasselquist, T. von Schantz, and S. Bensch. 2005. Associations between malaria and MHC genes in a migratory songbird. Proc. Biol. Sci. 272:1511–1518. Westerdahl, H., H. Wittzell, and T. von Schantz. 2000. Mhc diversity in two passerine birds: no evidence for a minimal essential Mhc. Immunogenetics 52:92–100. Wilson, I. A., and P. J. Bjorkman. 1998. Unusual MHClike molecules: CD1, Fc receptor, the hemochromatosis gene product, and viral homologs. Curr. Opin. Immunol. 10:67–73. Yu, C., G. R. Ehrhardt, M. N. Alder, M. D. Cooper, and A. Xu. 2009. Inhibitory signaling potential of a TCR-like molecule in lamprey. Eur. J. Immunol. 39: 571–579. Zhang, S. M., C. M. Adema, T. B. Kepler, and E. S. Loker. 2004. Diversification of Ig superfamily genes in an invertebrate. Science 305:251–254. Zhu, X., X. Zhao, W. F. Burkholder, A. Gragerov, C. M. Ogata, M. E. Gottesman, and W. A. Hendrickson. 1996. Structural analysis of substrate binding by the molecular chaperone DnaK. Science 272:1606–1614. Zucchetti, I., R. De Santis, S. Grusea, P. Pontarotti, and L. Du Pasquier. 2009. Origin and evolution of the vertebrate leukocyte receptors: the lesson from tunicates. Immunogenetics 61:463–481.
This page intentionally left blank
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
4 Host Defense (Antimicrobial) Peptides and Proteins LAURENCE MADERA, SHUHUA MA, AND ROBERT E. W. HANCOCK
DEFENSINS
INTRODUCTION
Defensins are a family of small cationic peptides ranging from 3 to 5 kDa in size and possessing a -sheet structure containing three intramolecular disulfide bonds between six conserved cysteine residues (Froy, 2005). Beyond this pattern, peptides within the defensin family are highly variable. In mammals, over 50 defensin members have been identified (Yang et al., 2001). Defensins members are categorized into three families based on their sizes and patterns of disulfide bonding: the -, -, and (among the higher primates only) -defensins (Brown et al., 2007). Humans only possess the - and -defensin subfamilies (Brown et al., 2007). To date, six -defensins, HNP1–4 and HD5–6, and six -defensins, hBD1–6, have been identified in humans (Yamasaki & Gallo, 2008; Brown et al., 2007).
The innate immune response is an ancient defense system against pathogens that is evolutionarily conserved among plants, invertebrates, and vertebrate animals. It is characterized by a rapid, highly effective response against foreign microbial invaders. The effectiveness of innate immunity stems from many multifaceted processes working in concert to rapidly eliminate a threat. The detection of pathogens by sentry cells immediately leads to the triggering of inflammation, the effector arm of innate immunity. Inflammation leads to increased trafficking of immune cells, including phagocytes, antigen-presenting cells, and mast cells, toward the site of infection where they initiate microbial killing or further potentiate the inflammatory response, reinforcing cell recruitment. Pathogen clearance is mediated by a complex set of strategies, ranging from the ingestion of microbes by phagocytes to the production of antimicrobial molecules, including reactive chemical species or lytic compounds. Another strategy is the production of cationic host defence (antimicrobial) proteins and peptides, compounds that play an important role in innate immunity, not only as antimicrobial agents, but also as immune regulators.
Localization and Regulation
Defensins are found widely within the body depending on the family and type of defensin involved. In general, defensins are found in the granules of leukocytes and in the epithelia of the human body (Menendez & Finlay, 2007). HNP1–4 -defensins are prominent in the granules of neutrophils, from which they were first isolated (Yang et al., 2001), and also exist in the granules of other leukocytes including monocytes, lymphocytes and natural killer (NK) cells, and in intestinal Paneth cell granules, where they are secreted into intestinal crypts (Froy, 2005). The enteric -defensins HD5 and HD6 are found in the granules of epithelial cells, including Paneth cells and the cells of the urogenital tract. In contrast, -defensins are expressed in epithelial cells that line the esophagus, intestines, skin, kidney, pancreas, and gut (Brown et al., 2007), whereas hBD5 and hBD6 are found in the epididymis and airway epithelia (Froy, 2005). Unlike -defensins, epithelial -defensins are not stored in cytoplasmic granules (Brown et al., 2007). The production of defensins by the body is variable and depends on many factors, including the defensin class, cell type, and environmental stimuli. -Defensins are constitutively expressed in cell granules (Menendez & Finlay, 2007). In contrast, many -defensins are inducible by a variety of environmental factors, including microbial components and/or host inflammatory mediators, indicating that -defensins might play a role in the host antimicrobial or inflammatory response (see Table 1).
CATIONIC HOST DEFENSE PEPTIDES
Cationic host defense peptides (HDPs) encompass a diverse family of peptides that play a role in the antimicrobial innate immune response of many species. They are generally defined as peptides 12 to 50 amino acids long, containing up to 50% hydrophobic residues and 2 to 9 positively charged arginine or lysine residues (Brown & Hancock, 2006). The two major classes of HDPs found in mammals are the defensins and cathelicidins, which are produced by immune cells, including leukocytes and epithelial cells. Originally thought to play a direct antimicrobial role against pathogens, it has become evident that cationic peptides also modulate the many functions of innate immunity (Fig. 1). Laurence Madera, Shuhua Ma, and Robert E. W. Hancock, Department of Microbiology and Immunology, Centre for Microbial Diseases & Immunity Research, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
57
58
HOST DEFENSE: GENERAL
FIGURE 1 The multiple roles of host defense peptides in the immune response.
Numerous studies have shown -defensin induction in epithelial cells in response to bacterial infections (Froy, 2005; Menendez & Finlay, 2007). Specific bacterial components, including lipopolysaccharide (LPS) of gram negative bacteria and peptidoglycan of bacterial cell walls, lead to production of -defensins (Menendez & Finlay, 2007). In addition, host factors produced in response to infections play a large role in -defensin induction, especially cytokines that are cell-to-cell signalling mediators essential in the coordination of the immune response. Interleukin 1 (IL-1) and tumor necrosis factor (TNF-), cytokines that promote acute inflammation, are strong inducers of -defensin expression (Yamasaki & Gallo, 2008). Supporting the link between -defensin induction and infection is the finding that regulation of -defensin expression is dependent on signaling pathways activated in an immune response. In epithelial cells, hBD2 induction is dependent on the transcription factors NF-B and AP-1, both essential for the transcription of many inflammatory mediators (Froy, 2005). This is supported by the finding that hBD2 expression in an atopic dermatitis model is downregulated by IL-10, a suppressor of NFB activity (Menendez & Finlay, 2007). The induction of hBD3 is also dependent on the activation of the mitogen activated protein kinase (MAPK) signalling pathways involving p38 kinase and ERK-1/2, central mediators of many cellular processes including inflammation (Menendez & Finlay, 2007). -Defensins can be induced by TLRs, which are a major subset of pathogen recognition receptors that detect microbes. For example, in the lung, activation of TLR2, TLR3, TLR4, and TLR9 can lead to hBD2 and hBD3 expression, whereas in the intestine,
activation of TLR4 and the complex of TLR2 and TLR6 leads to hBD2 induction (Froy, 2005). There is a great deal of evidence that -defensin regulation is linked to the detection and progression of a microbial infection. However, the variety of roles that defensins play in the immune response is far less understood.
Antimicrobial Activity
Defensins have generated a large amount of interest due to their modest in vitro antimicrobial activity against a wide range of microorganisms, including enveloped viruses, fungi, and bacteria (Brown et al., 2007). This direct antimicrobial is likely very significant under circumstances where defensins are found in high concentrations, such as the crypts of the intestine and the granules of neutrophils where milligram per milliliter concentrations are found. Defensins also possess antiviral properties, such as against adenovirus, papilloma virus, herpes simplex virus (HSV), and human immunodeficiency virus (HIV) (Yamasaki & Gallo, 2008), and have modest antimicrobial activity against the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa (Chen et al., 2006). hBD3 is more potent and has a broader spectrum of antimicrobial activity against organisms including fungi and gram-positive bacteria, such as Staphylococcus aureus, possibly due to its higher net charge relative to hBD1 and hBD2 (Brown et al., 2007). Although the mechanism of antimicrobial activity by defensins has not been fully elucidated, it is believed that the positive charge of defensins allows them to bind to negatively charged molecules on microbial membranes, such as LPS and lipoteichoic acid (LTA) in gram-negative
4. Host Defense (Antimicrobial) Peptides and Proteins
59
TABLE 1 Human cationic host defense peptides and their regulation and functions in the immune response Host defense peptide
Regulation
Immune functions
-Defensins
Constitutive
Binds and neutralizes bacterial toxins
-Defensins
Inducible in epithelia by bacterial infection: S. aureus, Campylobacter jejuni, H. pylori, and Mycobacterium bovis Inducible by LPS and peptidoglycan Inducible by inflammatory cytokines IL-17, IL-1, and TNF- Suppressed by anti-inflammatory cytokine IL-10
Chemotactic for T-lymphocytes, immature dendritic cells, mast cells, and monocytes Promotes chemokine production and mast cell degranulation Promotes production of inflammatory cytokines Promotes T-cell proliferation Promotes wound healing and regulation of the extracellular matrix
hCAP18/LL-37
Constitutive in leukocyte granules; Inducible in epithelia by bacterial infection H. pylori Inducible in epithelia by inflammatory cytokines IL-1 and IL-6 Suppressed by Shigella.
Chemotactic for monocytes, neutrophils, and T-lymphocytes Promotes chemokine production and induces mast cell degranulation Inhibits neutrophil apoptosis Suppresses of LPS-induced inflammation Regulates dendritic cell maturation and TH polarization of adaptive immunity; Promotes wound healing and angiogenesis
and gram-positive species respectively. This defensin interaction with the membrane leads to translocation across the outer membrane and access to this membrane where either the disruption of membrane integrity results in increased membrane permeability or action on other targets leads to death. The importance of defensin-mediated bacterial killing during an immune response is a point of contention, except under the previously mentioned circumstances when defensin concentrations are very high, since it has been demonstrated that in vitro defensin antimicrobial activity is dramatically reduced in a physiological environment. For example, the addition of 100 mM NaCl, approximately equivalent to the concentration found in airway fluid and blood, reduces hBD2 activity 5-fold to 500-fold and completely abolishes hBD1 activity against gram-negative bacteria (Bowdish et al., 2006). In addition, defensins are sensitive to the presence of divalent cations. It has been shown that -defensin activity is compromised by Mg21 and Ca21 at concentrations as low as 0.5 mM (Bowdish et al., 2006) even though many bodily fluids (including airway fluids, plasma, and sputum) contain Mg21 and Ca21 at concentrations of around 1 mM. Under many circumstances the physiological concentrations of defensins are too low to manifest any significant antimicrobial activity. hBD1 displays antimicrobial activity at concentrations around 1 μg/ml only under low ionic strength conditions that do not exist in vivo; however, the concentrations of hBD1 found in bronchoalveolar fluid is 10-fold lower, at 0.1 μg/ml (Bowdish et al., 2005). Thus, it is likely that defensins play a direct antimicrobial role in a limited number of environments, such as in the phagocytic granules of neutrophils and intestinal crypts where defensin concentrations can reach up to milligrams per milliliter, overcoming any ionic antagonism, or under circumstances where the ionic strength is relatively low (Froy, 2005). The antiviral effects of human defensins are far less understood. Defensins have been postulated to bind to viral
surface molecules, potentially inhibiting their entry and invasion mechanisms. Human -defensins have been shown to bind to viral glycoproteins, molecules integral to the viral invasion process, such as HSV glycoprotein B and HIV gp120 (Yamasaki & Gallo, 2008; Klotman & Chang, 2006). However the antiviral activity of defensins also varies with the physiological environment. For example, HNP-1 is able to directly neutralize HSV and HIV virions in a serum-free environment, an effect that is abolished with the addition of serum (Klotman & Chang, 2006). Interestingly, in the presence of serum, HNP-1 acts on the host cell, inhibiting HIV viral growth following the invasion and reverse transcription stages. Indeed, defensin antiviral activity can be attributed to many cell-mediated effects, including the regulation of host surface receptors used by viruses for invasion, or modulating cellular signaling pathways required for viral growth (Jenssen et al., 2006; Klotman & Chang, 2006). Although in physiological conditions and peptide concentrations the ability to act directly against pathogens is limited, recent studies show that defensins are still able to coordinate many aspects of the host immune response.
Immunomodulation
The pathogenesis of a microbe can be divided into a series of stages, which include the initial invasion by the microbe, the inflammatory innate immune response, the transition into the adaptive immune response, and the resolution of infection (or the establishment of chronic infections, or death of the host). It is becoming evident that defensins play a major role in coordinating each of these processes (see Table 1). Defensins have potent neutralizing functions against certain bacterial toxins, limiting pathogenesis at the early stages of infection. For example, HNP1–3 are able to bind and sequester the lethal factor (LF) of Bacillus anthracis from host cells, thus preventing potential cell death, and can also competitively inhibit diphtheria toxin and P. aeruginosa exotoxin A, preventing the ADP-ribosylation of host elongation factor-2 (Brown et al.,
60
HOST DEFENSE: GENERAL
2007). HNP1-3 is also able to inhibit the binding of S. aureus staphylokinase to host plasminogen, preventing fibrinolysis and the spread of infection (Brown et al., 2007). Defensins also play a role in cell recruitment, influencing the ability of immune effector cells to migrate to the site of infection, which is a mainstay of the innate immune system and impacts all subsequent responses. The -defensins are directly chemotactic for naïve CD4 and CD8 T-lymphocytes as well as immature dendritic cells and monocytes (Yang et al., 2001). hBD1 and hBD2 are also chemotactic for immature dendritic cells and memory T-lymphocytes, while hBD3 is capable of attracting immature dendritic cells, mast cells, and monocytes (Menendez & Finlay, 2007; Yang at al., 2001). Similarly, hBD4 can directly chemoattract mast cells and monocytes (Froy, 2005). As defensins are secreted (e.g., through phagocyte degranulation) or induced at infectious sites, it is likely that defensins enhance the ability to chemoattract immune effector cells to the site of infection. In addition to directly promoting cellular mobilization, defensins induce the production of cytokines and chemokines, which potentiate cell recruitment. For example, -defensins can induce the production of neutrophil chemokine IL-8 in alveolar macrophages and lung epithelial cells as well as cause the degranulation of mast cells and histamine release, promoting permeabilization of blood vessels and further cell recruitment (Yang et al., 2001). hBD3 and hBD4 can also cause degranulation of mast cells (Menendez & Finlay, 2007). Defensins also induce the production of inflammatory cytokines by immune cells. For example, -defensins induce the production of proinflammatory cytokines IL-6 and IFN (gamma interferon) by T lymphocytes and IL-1 and TNF- by human monocytes (Froy, 2005). The -defensins hBD1 and hBD4 induce keratinocytes to produce IL-18, a cytokine that promotes the recruitment of neutrophils and the activation of cell-mediated immunity (Yamasaki & Gallo, 2008). Defensins can also promote the production of regulatory cytokines to balance the inflammatory response. In particular, -defensins stimulate the production of the immunosuppressive cytokine IL-10 in T lymphocytes (Froy, 2005). Defensins also contribute to the adaptive immune response by influencing lymphocyte activity. For example, in murine models, HNPs enhance the proliferation of CD4 T-lymphocytes stimulated with CD3 activating antibodies (Lillard et al., 1999). Correlated in part with this finding, HNPs enhance antigen-specific humoral and cellular responses in mice treated with antigen, indicating an adjuvant role for defensins (Bowdish et al., 2006). Defensins also contribute to wound healing; in particular, hBD2–4 promote the migration of keratinocytes and enhance their proliferation, suggesting that -defensins promote reepithelialization of a wound area, while HNP1 promotes extracellular matrix formation by increasing levels of pro-alpha collagen and decreasing levels of matrix metalloproteinase-1, an enzyme that cleaves the extracellular matrix (Yamasaki & Gallo, 2008). Thus it is known that defensins extensively regulate many immunological processes, but the overall importance of defensins in immunity during an infection or inflammation is still inadequately understood.
Defensins in Human Disease
Attempts to determine the exact roles of each defensin in the overall immune response by knockout studies have not been successful due to the functional redundancy by other defensins and immune mechanisms. Indeed, mice for example contain at least 54 -defensins and 44 -defensins (Amid et al., 2009). However, a substantial number of immunological disorders are correlated with defensin dysregulation, providing tantalizing clues as to the roles of defensins in immunity.
Defensins are upregulated in inflammatory diseases of the bowel, lung, and skin. For example, hBD2 is significantly upregulated in cases of psoriasis, an autoimmune disease of the skin (Yamasaki & Gallo, 2008). Although no causal relationship has been established between defensin dysregulation and inflammatory disease, either defensins or their regulation may be essential for inflammatory homeostasis. Interestingly, HD5–6 downregulation correlates with ileal Crohn’s disease, an inflammatory disorder of the gastrointestinal tract, while a genetic polymorphism leading to low copy number of hBD2 associates with colonic Crohn’s disease. It is hypothesized that the enteric defensins are involved in maintaining control over the normal flora and thus establishing homeostasis in the gut (Menendez & Finlay, 2007), although it is equally possible that their immunomodulatory role is influential. Altered defensin levels are also correlated with differential susceptibility to infections. Psoriatic patients with elevated defensin levels have decreased susceptibility to infection relative to atopic dermatitis patients with reduced defensin levels (Yamasaki & Gallo, 2008). Animal studies also support the role of defensins in the clearance of pathogens. (i) In a mouse model of peritoneal Klebsiella pneumoniae infection, very small doses of HNP1 (4 ng to 4 μg) caused protection that was more likely due to an increase in leukocyte accumulation than to direct antimicrobial activity. Similar results were observed in S. aureus thigh infections (Welling et al., 1998). (ii) Administration of porcine -defensin BD-1 in piglets protected them against a Bordetella pertussis challenge (Menendez & Finlay, 2007). (iii) Overexpression of rat -defensin BD-2 in the lungs increased protection against P. aeruginosa induced pneumonia (Menendez & Finlay, 2007). (iv) BD-1 knockout mice showed delayed clearance of Haemophilus influenzae in the lungs and increased levels of Staphylococcus sp. in the bladder (Menendez & Finlay, 2007).
CATHELICIDINS
Cathelicidins are a family of peptides produced from a precursor polypeptide containing a conserved N-terminal signal and propeptide sequence encompassing approximately 100 amino acids (termed the cathelin domain), as well as a highly variable C-terminal peptide domain (Zanetti, 2004). The N-terminal signal sequence allows for targeting of the polypeptide to the endoplasmic reticulum for intracellular storage (Zanetti, 2004). The cathelin domain is essential for the storage of the polypeptide, preventing its degradation by proteases and inhibiting the biological activity of the Cterminal peptide domain (Zanetti, 2004). During excretion of the precursor polypeptide from cells, the C-terminal peptide domain is cleaved, resulting in a mature cathelicidin of 12 to 80 amino acids in length (Zanetti, 2004). In mammals, more than 50 cathelicidin members have been identified according to UniProt, a comprehensive database of protein sequence and function (Uniprot Consortium, 2008). Among the different members of the cathelicidins family, there is very little structural homology in the mature cathelicidin peptide domain. The mature peptide can fold into a variety of secondary structures, including -helices, -hairpins, and looped structures (Brown et al., 2007). In humans, only one cathelicidin has been found, an 18-kDa precursor human cationic antimicrobial protein, or hCAP18, which is processed into a variety of molecules, including prominently the 37-amino-acid peptide LL-37 (Zanetti, 2004).
Localization and Regulation
In the human body, hCAP18 is found in a range of cellular environments. Although originally isolated and identified in the granules of neutrophils, it can also be found in the
4. Host Defense (Antimicrobial) Peptides and Proteins
granules of many leukocytes including NK cells, monocytes, mast cells, B-lymphocytes, and T-lymphocytes (Dürr et al., 2006). It is also constitutively expressed in epithelial cells, including those of the airways, mouth, esophagus, and intestine, and is inducible in keratinocytes (Zanetti, 2004). In addition, LL-37 is a component of most secreted bodily fluids, including sweat, gastric juices, airway fluids, seminal plasma, and breast milk (Dürr et al., 2006). Although hCAP18 is normally expressed in many intracellular environments, the levels of hCAP18 can vary and are regulated during inflammation and infection. Regulation of hCAP18 can occur at different stages, including the expression and production of the precursor polypeptide and the cleavage of the mature C-terminal peptide domain. Increased expression of hCAP18 is regularly observed during inflammation (its production during infection may indeed be related to this); hCAP18 can be modestly induced in response to the presence of host inflammatory cytokines, IL-1, and IL-6. Also, LL-37 is substantially induced in myeloid cells by the hormonal form of vitamin D3 [1,25(OH)2D3] and, interestingly, critically ill septic patients had significantly lower plasma 25(OH)D concentrations compared to healthy controls (Jeng et al., 2009). Processing of hCAP18 into the active peptide LL-37 can also be regulated. In humans, the enzymes responsible for hCAP18 processing vary between cellular environments. In neutrophil granules, hCAP18 is processed by the serine protease proteinase 3, whereas in seminal fluid and under acidic concentrations such as those found in vaginal fluid, gastricsin (Pepsinogen C) is responsible for cathelicidin processing (Zanetti, 2004). On the skin surface, the stratum corneum tryptic enzyme SCTE processes hCAP18 into LL-37 (Yamasaki & Gallo, 2008). In vitro studies using bovine neutrophil cathelicidins Bac5 and Bac7 demonstrate that the addition of small amounts of elastase results in the complete processing of their pro-forms within minutes (Zanetti, 2004). The addition of elastase inhibitors prevents the maturation and activation of these cathelicidins (Zanetti, 2004). This suggests that the maturation of cathelicidin precursors is intricately linked to the regulation of their cognate processing enzyme. Interestingly, cathelicidin levels can also be directly influenced by microbial pathogens. Cleavage of hCAP18 into LL-37 can be triggered by elastase-producing pathogens such as P. aeruginosa (Zanetti, 2004). In contrast, gut epithelial biopsies of Shigellainfected patients show low expression of hCAP18, suggesting a possible Shigella virulence strategy (Zanetti, 2004). In vitro studies support this finding by demonstrating that hCAP18 expression is downregulated in monocytes and epithelial cell lines infected with Shigella (Zanetti, 2004). It is evident that microbial infections directly regulate hCAP18/LL-37 levels via many complex avenues. In turn, hCAP18/LL-37 plays a multifunctional role in the immune response against pathogens.
Antimicrobial Activity
In a dilute medium such as phosphate buffer, the mature human cathelicidin peptide LL-37 exhibits broad-spectrum in vitro antimicrobial activity against many pathogens, although it is arguable as to whether this activity is relevant in vivo. LL-37 is antimicrobial against gram-positive bacteria, including Streptococcus sp., Listeria monocytogenes, and S. aureus, and gram-negative bacteria, including E. coli, P. aeruginosa, and Salmonella enterica serovar Typhimurium (Dürr et al., 2006), and the fungus Candida albicans (Dürr et al., 2006). LL-37 also displays moderate antiviral properties, being able to slow the growth of vaccinia virus, but having only a mild effect on HSV infection (Dürr et al., 2006). However, as
61
with the defensins, LL-37 activity is sensitive to the presence of monovalent and divalent cations and its direct antimicrobial effects are severely abrogated under physiological conditions. In the presence of 100 mM sodium, LL-37 antimicrobial activity is reduced by twofold to eightfold, whereas in cell culture medium containing physiological concentrations of salts, 1 mM Mg21 and Ca21 and 150 mM NaCl, LL-37 at concentrations of up to 100 μg/ml showed no effect against S. aureus or serovar Typhimurium (Bowdish et al., 2006). To put this in context, LL-37 is normally secreted in the bronchoalveolar fluid of infants at approximately 5 μg/ml, or 10 to 15 μg/ml in children with severe inflammatory disorders (Bowdish et al., 2005). Other host factors can also interfere with the antimicrobial activity of LL-37. Apolipoprotein 1, a human serum protein, has been shown to inhibit the antimicrobial actions of LL-37 (Bowdish et al., 2006). Glycosaminoglycans like heparin strongly inhibit activity. Indeed, the direct antimicrobial role of LL-37 is likely limited to conditions where its concentration is sufficient to overcome the previous limitations, for example, in seminal fluid or in neutrophil granules (Zanetti, 2004). Although many physiological environments do not harbor the optimal in vitro conditions for LL-37 antimicrobial activity, under these discussed conditions, LL-37 is a multifunctional modulator of the host immune response.
Immunomodulation
LL-37 plays multiple roles during an immune response (see Table 1). It is a potent enhancer of immune cell trafficking. LL-37 is directly chemotactic for monocytes, neutrophils, and T-lymphocytes by engaging the G-protein coupled receptor, FPRL-1 (Yamasaki & Gallo, 2008). LL-37 also enhances immune cell recruitment by other routes (e.g., mast cells are directly chemoattracted through a separate Gprotein coupled receptor). LL-37 also induces chemokine production. Murine models have demonstrated that the administration of LL-37 increases levels of the monocyte chemoattractant protein 1 (MCP-1) (Dürr et al., 2006). In vitro studies show increased production of chemokines such as CXCL-1, MCP-1, MCP-3, and IL-8 (among others) in LL-37-stimulated human peripheral blood mononuclear cells (Bowdish et al., 2006; Mookherjee et al., 2009a). LL-37 can also play a balancing role in the immune response, having both pro- and anti-inflammatory functions. LL-37 can promote inflammation and cell-mediated immunity by inducing IL-18 in human keratinocytes, as well as by enhancing the processing of IL-1 in LPS-stimulated human monocytes (Yamasaki & Gallo, 2008). The presence of LL-37 also leads to mast cell degranulation resulting in the release of chemoattractants, prostaglandins, and histamine release (Dürr et al., 2006). LL-37 can also work in tandem with host mediators to selectively modulate inflammation and cell trafficking. Synergy between LL-37 and IL-1 in human peripheral blood cells has been observed, leading to a significant increase of cytokine production, including IL-6 and IL-10, and production of chemokines MCP-1 and MCP-3 (Yu et al., 2007). LL-37 also potentiates the production of proinflammatory cytokines TNF- and IL-6 in peripheral blood cells stimulated by unmethylated CpG DNA, a DNA motif frequently found in the bacterial genome and an activator of human TLR9 (Yu et al., 2007). In contrast, LL-37 is a strong suppressor of LPS-induced inflammation, preventing host damage from a systemic inflammatory response. It inhibits LPS-mediated induction of inflammatory mediators, including TNF- and reactive oxygen species in macrophages, and IL1-, TNF-, IL-6, and IL-8 in human peripheral blood cells (Mookherjee et al., 2006). Consequently, LL-37 is able
62
HOST DEFENSE: GENERAL
to increase the survival of rats in LPS-induced sepsis models (Dürr et al., 2006). Although the LPS-suppressing effects of LL-37 were originally attributed to its capacity to bind LPS, various studies have shown that LL-37 inflammatory regulation is much more complex and likely cell mediated, especially since it can act 30 to 60 min after LPS treatment of cells. Indeed, LL-37 can also limit the production of IL-1, TNF-, IL-6, and IL-8 in TLR2-stimulated human peripheral blood cells (Mookherjee, 2006). These observations suggest that LL-37 might play an important role in coordinating the inflammatory reaction in response to different pathogens. LL-37 also plays a role at the transition between the innate and adaptive immune system and has been demonstrated to have adjuvant activity. It can increase coreceptor CD86 expression and endocytic activity (Brown & Hancock, 2006). LL-37 promotes the polarization of human monocyte-derived dendritic cells to Th-1 type responses by enhancing the secretion of the Th-1 cytokines IL-12 and IL-6 and decreasing the levels of the Th-2 cytokine IL-4, as well as proinflammatory cytokines, in LPS-stimulated dendritic cells (Brown & Hancock, 2006). In addition to its immune regulatory effects, LL-37 is a promoter of wound healing. Epithelial wounds typically display increased levels of local LL-37, which is inducible by various growth factors, leading to increased attraction of keratinocytes (Dürr et al., 2006). Indeed, LL-37-specific antibodies inhibited epithelial healing in a human skin wound model (Yamasaki & Gallo, 2008). LL-37 also promotes angiogenesis in epithelia, which may play a role in reepithelialization (Yamasaki & Gallo, 2008), while its ability to modulate inflammatory activity may also assist in wound healing.
hCAP18/LL-37 in Human Disease
Efforts to characterize the overall role of LL-37 in the immune system have been based on in vivo models and correlational studies linking LL-37 dysregulation to immune disorders. Some immune disorders suggest LL-37 is required for the proper clearance of pathogens, although it is uncertain whether these studies reflect direct antimicrobial activity, as several authors assert, or immunomodulatory activities. As with hBD2, lower expression of hCAP18 is found in atopic dermatitis patients relative to psoriatic patients, which may explain their differential susceptibilities to infection. Patients with morbus Kostmann, a neutropenic disorder, are treated with cytokine G-CSF to restore circulating neutrophils with normal oxidative responses. However, these neutrophils are deficient in LL-37 and this may correlate with the chronic periodontal disease and excessive oral inflammation observed in these patients (Brown et al., 2007). Murine models show that knocking out CRAMP, a murine cathelicidin structurally related to LL-37, increases the susceptibility to skin infections by group A Streptococcus (Zanetti, 2004).
MECHANISMS OF ANTIMICROBIAL KILLING
HDPs possess in vitro antimicrobial activity, ranging from modest (many defensins and LL-37) to potent (e.g., pig protegrin), against an extensive range of microbes including bacterial, viral, and fungal pathogens. However, the mechanisms utilized by HDPs to elicit their direct and broad-spectrum actions are quite complex and likely involve multiple targets. For bacteria, it is known that HDPs interact with negatively charged components on microbial membranes, such as LPS or LTA, and then translocate to the cytoplasmic membrane. Although it is known that HDP interaction with the membrane is a prerequisite for killing, there are many possible results leading to bacterial death, namely the disruption of membrane integrity, interference with essential processes
dependent on membrane-bound enzymes (cell division, cell wall synthesis), or translocation across the membrane to access cytosolic targets. Four models have been proposed to address peptide-membrane interactions, as reviewed by Jenssen et al. in 2006: (i) the toroidal pore, (ii) barrel-stave, (iii) carpet, and (iv) aggregate models. In the toroidal pore model, peptides are thought to intimately associate with the head groups of lipids and reaggregate into the bacterial membrane in a perpendicular orientation, resulting in the formation of a peptide-lipid pore, thus increasing membrane permeability. Certain peptides that form -helical structures in a membrane setting, such as LL-37, have been proposed to act in this fashion. In the barrel-stave model, peptides cluster on the membrane, also in a perpendicular fashion. In this case, the hydrophobic regions of the peptides interact with fatty acyl chains of membrane lipids, and their hydrophilic regions face one another, forming a pore in the microbial membrane. The carpet model, unlike the previous two, suggests that peptides remain aggregated on the membrane surface in a parallel fashion, resulting in localized peptide clusters. At high peptide concentrations the aggregates form micelles with areas of the membrane, leading to a disruption of membrane integrity. None of these models, however, really considers peptide translocation, which is more common than previously supposed, having been experimentally observed with many HDPs. One model to explain both membrane disruption and translocation is the aggregate model. In this model, peptides aggregate on the membrane surface and at a critical concentration form complexes (mixed micelles) with the membrane lipids, permitting increased membrane permeability (which for some peptides might cause death) and allowing for the translocation of peptides across the bilayer as the aggregates collapse. It must be noted that only some of these models may be relevant to antimicrobial killing depending on the peptide and the cellular environment. The mode of action against fungi is quite poorly understood; however, it has been observed that peptides interact with fungal cell walls and membranes as well as acting on internal targets including respiratory processes (Jenssen et al., 2006). A wide variety of studies have revealed mechanisms of antibacterial killing that are unrelated to membrane disruption. Indeed, it seems likely that many peptides have multiple targets, explaining in part the low rates of resistance development to these HDPs. Some peptides inhibit microbial growth without any major disruption of the membrane by acting on intracellular targets. The -helical peptides of some species, such as fish pleurocidin and frog dermaseptin, apparently disrupt bacterial growth by interfering with nucleic acid and/or protein synthesis (Jenssen et al., 2006). The -sheet defensin HNP-1 inhibits nucleic acid synthesis in E. coli, and other peptides, including bovine indolicidin, also inhibit protein synthesis (Jenssen et al., 2006). It is hypothesized that the cationic properties of the peptides allow for their interaction with a range of negatively charged enzymes and molecules leading to disruption of their functions. Binding to membrane components of enveloped viruses and host cells is a major antiviral mechanism of peptides, but a wide range of cellular targets or actions on different stages of the viral life cycle have been reported for many viruses (Jenssen et al., 2006).
MECHANISMS OF IMMUNOMODULATION
HDPs, in addition to their direct but often weak microbial killing ability, regulate an immense range of host immunological functions during an infection. Through selective stimulation of innate immune mechanisms, they likely have a strong impact on the effectiveness of these processes in killing
4. Host Defense (Antimicrobial) Peptides and Proteins
microbes. Due to limitations on the direct microbial killing activities of peptides in a physiological environment at moderate concentrations (e.g., on the mucosa), it seems likely that the primary function of HDPs during an infection is modulation of the immune response. The question is thus how HDPs exert their far-reaching control over the host immune system. Although immunomodulation by HDPs is a relatively new field, researchers have begun to elucidate their regulatory and immune effector mechanisms. Nevertheless, the mechanisms utilized by peptides to regulate immunity may be as numerous as their downstream effects, and it is now clear that there is enormous complexity involved in the selective action of peptides on immune cells, as revealed in a recent systems biology study of LL-37 (Mookherjee et al., 2009a). HDPs are potent modulators of cellular signalling cascades, particularly those that are involved in the regulation of immunity. HDPs regulate immune signalling pathways to promote chemotaxis, through the production of chemokines, as well as regulating downstream immune responses by altering cytokine levels and other cellular functions. MAP-kinase pathways are induced by HDPs. For example, LL-37 induces the phosphorylation of MAP-kinase constituents to modulate the production of chemokines and cytokines (Mookherjee et al., 2009a). Inhibitor experiments show that LL-37-induced transcription of chemokines IL-8, MCP-1, and MCP-3 in human peripheral blood cells is dependent on MAP-kinase signaling while phosphorylation of p38 and ERK-1/2 in response to LL-37 has been directly demonstrated (Bowdish et al., 2006; Mookherjee et al., 2009a). hBD3 and hBD4 also induce the phosphorylation of p38 and ERK-1/2 in human mast cells, correlating with increased mast cell degranulation (Menendez & Finlay, 2007). HNPs also induce the production of IL-8 in monocytes in a p38-dependent manner, and in lung cells, dependent on the activity of the phosphatidylinositol-3-kinase (PI3-K) pathway, another regulator of cell growth and inflammation (Syeda et al., 2008). In keratinocytes, hBD2-4 also induced the phosphorylation of STAT-1 and STAT-3, signalling mediators utilized by cytokines to induce further downstream immune responses (Menendez & Finlay, 2007). Other pathways engaged include the transient but obligate engagement of the NF-B/IB and PI3-K pathways. HDPs are also able to act as relatively weak chemokine analogues, binding to chemokine receptors of host cells at micromolar concentrations, as opposed to nanomolar concentrations for most conventional chemokines to initiate chemotaxis. hBD1 and hBD2 chemoattract immature dendritic cells and memory T-lymphocytes by engaging the chemokine receptor CCR6 (Yamasaki & Gallo, 2008; Yang et al., 2001). LL-37, in a similar fashion, attracts monocytes, neutrophils, and T-lymphocytes by binding to the receptor FPRL-1 (Yang et al., 2001). LL-37, in addition to promoting cell recruitment, acts to modulate inflammation by antagonizing or acting cooperatively with various inflammatory signalling mediators. LL-37 is a potent suppressor of TLR4 activation by LPS and inhibits the downstream nuclear translocation of NF-B subunits p50 and p65 in LPS-stimulated human monocytes, which form a transcription complex essential for the expression of many inflammatory mediators (Mookherjee et al., 2006). It demonstrates rather mixed effects with molecules that signal through other TLRs, suppressing stimulation of TLR2 by LTA, demonstrating mixed responses (antagonism or enhancement depending on the read-out) with CpG oligonucleotides (a TLR9 agonist) and flagellin (a TLR5 agonist) while acting synergistically with IL-1 through the related IL-1 receptor (Mookherjee et al., 2006). HDPs also use growth-related signalling pathways to mediate wound
63
healing. LL-37 is an activator of the epidermal growth factor receptor (EGFR) in keratinocytes, leading to increased keratinocyte migration at wound sites (Yamasaki & Gallo, 2008). Analogously, hBD2-4 increases EGFR activity, correlating to an increase in keratinocyte proliferation (Menendez & Finlay, 2007). LL-37 also utilizes FPRL-1 signal activity to induce angiogenesis (Yamasaki & Gallo, 2008). LL-37 is also a potent inhibitor of human neutrophil apoptosis, signaling through P2X7 receptors and G-protein coupled receptors other than FPRL-1 and involving the activation of PI3-K but not ERK-1/2 MAP-kinase. Of note, although a number of extracellular receptors have been described, LL-37 and the synthetic innate defense regulator peptides are able to freely penetrate into host cells, in a process that mirrors that of so-called cellpenetrating peptides like HIV-1 Tat. Importantly, uptake into cells is absolutely required for chemokine induction by monocytes and epithelial cells. The recent findings of intracellular receptors including GAPDH (Mookherjee et al., 2009b) and others (unpublished data) indicates that LL-37 acts intracellularly through hub proteins to impact on signaling pathways. Although headway is being made in discovering how HDPs regulate cellular signaling, many uncertainties remain. For example, as mentioned above, LL-37 exerts paradoxical effects on the inflammatory response, limiting the activity of certain inflammatory stimulants, such as LPS, but acting synergistically with other stimulants, such as IL-1. Gene expression studies of monocytes show that LL-37 itself induces hundreds of genes while leading to the suppression of a portion of LPS-induced gene expression (while a significant portion remains unchanged) (Mookherjee et al., 2006; Mookherjee et al., 2009a). Thus LL-37 is an authentic modulator of immunity as opposed to an antagonist or immune stimulant. It seems that HDPs balance inflammatory responses, selectively strengthening protective immune response while preventing excessive inflammation. The ability of peptides to utilize multiple receptors and signaling mechanisms to maintain that balance is a major topic of interest in current host defense peptide research.
HOST DEFENSE PROTEINS
Host defense and antimicrobial proteins are another important component of the innate immune response. We utilize the term protein here to describe larger molecules of more than 100 amino acids with a well-defined tertiary structure. Several of these can be digested down to active fragments, some of which have the above-described features and activities of HDPs and thus are likely related to these peptides. Host defense proteins include more than 700 structurally diverse members expressed extensively in both plants and animals (Hall et al., 2002). Humans possess a wide range of host defense proteins that perform many antimicrobial and immunomodulatory functions (see Table 2).
BACTERICIDAL PERMEABILITY-INCREASING PROTEIN (BPI)
BPI is a highly positively charged, 55- to 60-kDa protein that is mainly found in the primary (azurophilic) granules of neutrophils in humans, rabbits, and cows (Canny & Levy, 2008). In humans, BPI is expressed and synthesized in the bone marrow in the granules of neutrophil precursors, called granulocytes, prior to neutrophil maturation (Weiss, 2007). To a lesser extent, BPI is also expressed in eosinophils and the epithelial cells of the gastrointestinal and respiratory tracts, where it acts as a local defense agent against gram-negative bacteria (Schultz, 2007;
64
HOST DEFENSE: GENERAL TABLE 2 Human host defense peptides and proteins Polypeptide -Defensins HNP1–4
Mass (kDa) 3–4
Granules of neutrophils, monocytes, lymphocytes, NK cells, and Paneth cells Granules of various epithelial cells
HD5–6 -Defensins hBD1–4 hBD5–6 Cathelicidin: hCAP18 (LL-37)
Bactericidal permeabilityincreasing (BPI) protein
Localization
4–5 Various epithelial cells Paneth cells; epithelial cells of epididymis and airways 18 (4.5)
55–60
Granules of neutrophils, monocytes mast cells, B-lymphocytes, and T-lymphocytes; various epithelial cells; bodily fluids (sweat, gastric juice, airway fluids, seminal plasma, and breast milk) Granules of neutrophils, eosinophils, epithelium
Lactoferrin
80
Neutrophils; various epithelia; bodily fluids (airway fluid, cervical mucus, breast milk, tears, and saliva)
Lysozyme
14
Neutrophils, monocytes, macrophages, keratinocytes, Paneth cells, pneumocytes, secretory cells of the apocrine gland, various epithelia; bodily fluids (tears, saliva, breast milk, respiratory fluid)
Peroxidases
150
Neutrophils, monocytes, eosinophils, epithelia, milk, saliva
Secretory leukoprotease inhibitor (SLPI)
12
Macrophages, various epithelia
Serprocidins (cathepsin G, elastase, proteinase 3, azurocidin/CAP37) Psoriasin (S100A7)
25–30
11
Neutrophils
Keratinocytes
Brown et al., 2007). BPI is also found on the surfaces of neutrophils and monocytes. However, it is hypothesized that this may be due to the degranulation of adjacent activated neutrophils. The level of BPI remains relatively unchanged. In the peripheral blood of humans, neutrophils store BPI in their primary granules with minimal de novo protein expression. In dermal fibroblasts, the expression of BPI is only slightly increased by IL-4, whereas it was not elevated by other cytokines including TNF-, IFN-, IL-4, and IL-1 in CaCo-2 and other epithelial cells (Schultz, 2007). Interestingly, the pattern of expression of BPI is different from that of other azurophilic granule proteins. This is presumably due to the observation that the BPI promoter binds transcription factors CCAAT/ enhancer binding proteins and (C/EBP, C/EBP) rather than C/EBP, which regulates the expression of other azurophilic granule proteins (Weiss, 2007). Like other lipid-binding proteins, BPI has high binding affinity to LPS. Structurally, human BPI is very similar to LPS-binding protein (LBP). BPI has two similar-sized domains separated by a proline-rich linker. The C-terminal domain is responsible for the binding of BPI to bacteria and facilitating its phagocytosis. The N-terminal domain is responsible for the interaction between BPI and LPS. The cationic N-terminal region of BPI also contributes to BPI-LPS binding through electrostatic interactions with the anionic lipid A region of LPS (Brown et al., 2007). It is thought that the ability of BPI to bind LPS is the basis for its observed antibacterial and endotoxin-neutralizing effects.
Antimicrobial Activity
BPI has modest activity against gram-negative bacteria and has been presumed to act primarily via its interaction with
LPS on the bacterial outer membrane (Schultz, 2007). BPI is thought to mediate its antimicrobial effects by increasing membrane permeability via a process analogous to selfpromoted uptake, involving binding of LPS by BPI leads to its insertion into the outer membrane of intact gramnegative bacteria, stimulating the rearrangement of outer membrane lipids and displacing LPS-bound divalent cations Mg21 and Ca21 (Canny & Levy, 2008). However, since this cannot per se lead to the killing of cells, we assume that it must translocate across the outer membrane and attack other targets in a manner analogous to HDPs. Disruption of the outer membrane increases the susceptibility of microbial phospholipids in the membrane to the metabolic enzyme, phospholipase A (Schultz, 2007). Some gram-negative bacterial species are very resistant to the antimicrobial effects of BPI, specifically those with structural modifications of their LPS, such as those with long LPS chains or those with structural diversity within the lipid A region of LPS (Brown et al., 2007).
Immunomodulation
The ability of BPI to bind LPS is probably even more influential in the host innate immune response. BPI, in contrast to its structural relative LBP, neutralizes the inflammatory response elicited by bacterial endotoxin. BPI is able to reduce LPS-mediated endothelial damage and the production of nitric oxide and inflammatory cytokines, including TNF-, IL-1, IL-6, IL-8, and IL-10 (Van Amersfoort et al., 2007). It also inhibits the binding of LPS to blood vessels, thus preventing circulatory disturbances during infections caused by gram-negative bacteria (Brown et al., 2007). In addition, BPI also exhibits other immunomodulatory prop-
4. Host Defense (Antimicrobial) Peptides and Proteins
erties, including the suppression of IL-1-induced nitric oxide synthase (iNOS) expression in endothelial cells. BPI can also promote the induction of T-cell-dependent immunity by presenting bound gram-negative bacterial components, such as outer membrane vesicles, to dendritic cells. These dendritic cells in turn trigger adaptive immunity (Schultz, 2007).
Therapeutic Potential
Efforts to harness the LPS-neutralizing effects of BPI as potential therapeutics against sepsis models have not proven effective. Recombinant BPI has been tested as an experimental treatment for meningococcal and other forms of sepsis caused by gram-negative bacteria. Unfortunately, efficacy was not demonstrated in Phase III clinical trials. In contrast, in a rabbit model of peritoneal inflammation, extracellular BPI, secreted into inflammatory exudates, showed potent bactericidal activity against many gram-negative bacteria. In addition, some defensins, cathelicidins, and low concentrations of complement potentiated the activity of BPI (Brown et al., 2007).
LYSOZYME
Lysozymes, also called muramidases, are small, abundant cationic enzymes that are widely distributed in plants and animals. They primarily cleave the 1, 4-glycosidic linkage between N-acetyl muramate and N-acetyl glucosamine residues in peptidoglycan, an essential component in bacterial cell walls (Brown et al., 2007; Niyonsaba & Ogawa, 2005). Different species have varying copy numbers of the lysozyme gene, ranging from 1 in humans to 10 in sheep and cows. In humans, lysozymes are expressed in polymorphonuclear leukocytes, monocytes, macrophages, keratinocytes, Paneth cells of the small intestine, tracheal epithelial cells, pneumocytes, and apocrine gland secretory cells (Niyonsaba & Ogawa, 2005). In addition, lysozyme is abundantly found in many bodily secretions, including tears, saliva, breast milk, and respiratory fluid (Brown et al., 2007). The lysozyme protein is divided into two domains connected by a long -helix with six active sites, termed A, B, C, D, E, and F. These active sites are required to bind six consecutive sugar residues, facilitating peptidoglycan binding and processing (Niyonsaba & Ogawa, 2005).
Antimicrobial Activity
Between different species, lysozymes have conserved functions including bacteriolytic and antifungal activities and antitumor and anti-inflammatory properties (Brown et al., 2007). Lysozymes by themselves are able to kill a very restricted range of bacteria by increasing osmotic stress via its disruption of peptidoglycan. Peptidoglycan is a structural glycosidic polymer and is most prominent in the cell wall of gram-positive bacteria, where it maintains shape, stability, and osmotic resistance. Gram-negative bacteria are generally more resistant to lysozyme activity due to the stabilizing effect of the outer membrane (Masschalck & Michiels, 2003). While certain gram-positive bacterial organisms, such as Micrococcus lysodeikticus and Bacillus species, are susceptible to low lysozyme levels, most pathogens can persist in environments with low lysozyme concentrations (Brown et al., 2007). Higher levels of lysozyme are antibacterial, but it has been suggested that this is due to its cationic nature rather than its enzymatic activity (Brown et al., 2007). The antibacterial activity of lysozyme is much more effective in combination with other host defense factors, including complement and cationic peptides, suggesting that lysozyme acts in concert with other host defense mechanisms to aid in pathogen clearance.
65
LACTOFERRIN
Lactoferrin is an 80-kDa iron-binding plasma protein that belongs to the transferrin protein family, which includes serum transferrin, ovotransferrin, melanotransferrin, and the inhibitor of carbonic anhydrase (González-Chávez et al., 2009). It is produced by many animals including humans, cows, goats, horses, dogs, several rodents, and fish (González-Chávez et al., 2009). In humans, lactoferrin is expressed in the secretory granules of neutrophils and has been detected in epithelial cells and mucosal secretions including airway fluid, cervical mucus, breast milk, tears, and saliva. It is the second most abundant protein in milk after casein. Lactoferrin consists of two similar ferric-ion-binding domains; these permit lactoferrin to deprive iron from bacteria and inhibit bacterial growth (Brown et al., 2007). Lactoferrin can be proteolyzed to the cationic peptide lactoferricin by gastric pepsin in the milk of animals. Lactoferricin has moderate activities against bacteria, protozoa, viruses, and fungi due to its cationic bactericidal nature, but not iron absorption, in analogy with cationic host defense peptides (Jenssen & Hancock, 2009). Other host defense functions of lactoferrin unrelated to iron absorption include blocking biofilm development by P. aeruginosa, binding to specific surface receptors, inhibiting the attachment of Helicobacter pylori to gastric epithelial cells, and facilitating clearance of infected host cells by enhancing pro-apoptotic signals (Brown et al., 2007).
CHEMOKINES
Chemokines are an extensive family of small chemotactic cytokine proteins of 7 to 10 kDa in size and include approximately 50 members in humans. Most chemokines are also cationic proteins and thus possess modest antibacterial properties in dilute medium. However, chemokines play an essential role in the host immune response primarily by directing the movement of immune effector leukocytes (including neutrophils, lymphocytes, macrophages, dendritic cells, and monocytes) to the site of infection. According to their functions, chemokines can be divided into two classes: inflammatory and homeostatic. Homeostatic chemokines are constitutively expressed, whereas the expression of inflammatory chemokines is only induced upon activation by the triggering of circulating leukocytes by inflammatory mediators such as TNF-, IFN-, microbial components, or trauma and other danger signals (Allen et al., 2007). Chemokines direct cell movement and migration by acting as soluble chemoattractants or adhering to tissues by binding to their receptors. There are about 20 different chemokine receptors, all of which are G-protein coupled receptors. Based on the patterns of cysteine residues in their ligands, chemokines and their receptors are classified into four families: CXC, CC, C, and CX3C, where C represents cysteine and X/X3 stands for one or three noncysteine amino acids (Allen et al., 2007). Following chemokine receptor binding, a series of events, including cytoskeletal rearrangement and the regulation of cell surface adhesion molecules, lead to cell motility and chemotaxis (Moser et al., 2004). In response to the presence of foreign antigens, tissue damage, and other physiological insults, chemokines play important roles in attracting and activating specific leukocytes to the affected sites and in stimulating strong immune responses against pathogens. Recently, chemokines, in addition to their roles in inflammation and infection, were shown to be involved in autoimmune diseases and cancer (Ward & Marelli-Berg, 2009). It should be noted that chemokines possess characteristics similar to a group of mediators that have been termed alarmins. Alarmins are secreted in response to inflammation and
66
HOST DEFENSE: GENERAL
act as warning signals of tissue damage or invading pathogens (Oppenheim & Yang, 2005). Alarmins possess adjuvant properties, activating and maturing antigen-presenting cells, namely dendritic cells, and enhancing their antigenspecific immune responses. Similar to chemokines, alarmins utilize Gi protein-coupled receptors (including chemokine receptors) on antigen-presenting cells (including dendritic cells, monocytes, and macrophages). However, while the actions of alarmins are limited to their enhancement of antigen-presenting cell function, chemokines play a diverse role in directing the responses of both innate and adaptive immunity. It has been proposed that defensins and cathelicidins are alarmins, but we are uncertain if this adequately describes their function.
CONCLUSIONS
Host defense peptides and proteins are major components in the arsenal of our immune system. Although host defense peptides and proteins were studied initially for their direct antimicrobial effects, it is becoming clearer that they are multifunctional regulatory agents, responsible for the coordination and development of a wide range of immune response actions. The ability of host defense peptides and proteins to mediate a wide array of functions, including cell recruitment, wound healing, and inflammatory regulation to name a few, makes them exciting candidates for the development of novel therapeutics based on immunomodulation. By unlocking the functional mechanisms of these peptides and proteins, researchers will be able to design novel treatments that serve to fine-tune the host immune response. Immunomodulation will transform the way we respond to a wide range of immune-related diseases, ranging from novel, broad range anti-infective therapies to immune regulators that tame a dysfunctional immune response in disorders such as chronic inflammation and autoimmunity. The cationic peptide research of the authors is supported by grants from the Grand Challenges in Global Health Research program and the Canadian Institutes for Health Research (CIHR). S.M. is supported by a postdoctoral fellowship from Wyeth/CIHR while R.E.W.H. holds a Canada Research chair.
REFERENCES Allen, S. J., S. E. Crown, and T. M. Handel. 2007. Chemokine: receptor structure, interactions, and antagonism. Annu. Rev. Immunol. 25:787–820. Amid, C., L. M. Rehaume, K. L. Brown, J. G. Gilbert, G. Dougan, R. E. Hancock, J. L. Harrow. 2009. Manual annotation and analysis of the defensin gene cluster in the C57BL/6J mouse reference genome. BMC Genomics 10:606. Bowdish, D. M. E., D. J. Davidson, and R. E. W. Hancock. 2005. A re-evaluation of the role of host defence peptides in mammalian immunity. Curr. Protein Pept. Sci. 6:35–51. Bowdish, D. M. E., D. J. Davidson, and R. E. W. Hancock. 2006. Immunomodulatory properties of Defensins and Cathelicidins. Curr. Top. Microbiol. Immunol. 306:27–66. Brown, K. L., and R. E. W. Hancock. 2006. Cationic host defense (antimicrobial) peptides. Curr. Opin. Microbiol. 18:24–30. Brown, K. L., N. Mookherjee, and R. E. W. Hancock. 2007. Antimicrobial, host defence peptides, and proteins. In Encyclopedia of Life Sciences (ELS). John Wiley and Sons, Ltd., Chichester, UK. doi: 10.1002/9780470015902.a0001212.pub2 Canny, G., and O. Levy. 2008. Bactericidal/permeabilityincreasing protein (BPI) and BPI homologs at mucosal sites. Trends Immunol. 29:541–547. Chen, X., Z. Xu, L. Peng, X. Fang, X. Yin, N. Xu, and P. Cen. 2006. Recent advances in the research and development of human defensins. Peptides. 27:931–940.
Dürr, U. H. N., U. S. Sudheendra, and A. Ramamoorthy. 2006. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim. Biophys. Acta 1758: 1408–1425. Froy, O. 2005. Regulation of mammalian defensin expression by Toll-like receptor-dependent and independent signalling pathways. Cell. Microbiol. 7:1387–1397. González-Chávez, S. A., S. Arévalo-Gallegos, and Q. RascónCruz. 2009. Lactoferrin: structure, function and applications. Int. J. Antimicrob. Agents 33:301.e1–8. Hall, S. H., K. G. Hamil, and F. S. French. 2002. Host defense proteins of male reproductive tract. J. Androl. 23:585–597. Jeng, L., A. V. Yamshichkov, S. E. Judd, H. M. Blumber, G. S. Martin, T. R. Ziegler, and V. Tangpricha. 2009. Alterations in vitamin D status and anti-microbial peptide levels in patients in the intensive care unit with sepsis. J. Transl. Med. 7:28. Jenssen, H., P. Hamill, and R. E. W. Hancock. 2006. Peptide antimicrobial agents. Clin. Microbiol. Rev. 19:491–511. Jenssen, H., and R. E.W. Hancock. 2009. Antimicrobial properties of lactoferrin. Biochimie 91:19–29. Klotman, M. E., and T. L. Chang. 2006. Defensins in innate antiviral immunity. Nat. Rev. Immunol. 6:447–456. Lillard, J. W., Jr., N. Prosper, O. Chertov, J. J. Oppenheim, and J. R. McGhee. 1999. Mechanisms for induction of acquired host immunity by neutrophil peptide defensins. Proc. Natl. Acad. Sci. 96:651–656. Masschalck, B., and C. W. Michiels. 2003. Antimicrobial properties of lysozyme in relation to foodborne vegetative bacteria. Crit. Rev. Microbiol. 29:191–214. Menendez, A., and B. Finlay. 2007. Defensins in the immunology of bacterial infections. Curr. Opin. Immunol. 19:385–391. Mookherjee, N., K. Brown, D. M. E. Bowdish, S. Doria, R. Falsafi, K. Hokamp, F. M. Roche, R. Mu, G. H. Doho, J. Pistolic, J. P. Powers, J. Bryan, F. S. L. Brinkman, and R. E. W. Hancock. 2006. Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37. J. Immunol. 176:2455–2464. Mookherjee, N., P. Hamill, J. Gardy, D. Blimkie, R. Falsafi, A. Chikatamarla, D. J. Arenillas, S. Doria, T. R. Kollmann, and R. E. W. Hancock. 2009a. Systems biology evaluation of immune responses induced by human host defence peptide LL-37 in mononuclear cells. Mol. Biosystems 5:483–496. Mookherjee, N., D. N. Lippert, P. Hamill, R. Falsafi, A. Nijnik, J. Kindrachuk, J. Pistolic, J. Gardy, P. Miri, M. Naseer, L. J. Foster, and R. E. W. Hancock. 2009b. Intracellular receptor for human host defense peptide LL-37 in monocytes. J. Immunol. 183:2688–2696. Moser, B., M. Wolf, A. Walzl, and P. Loetscher. 2004. Chemokines: multiple levels of leukocyte migration control. Trends Immunol. 25:75–84. Niyonsaba, F., and H. Ogawa. 2005. Protective roles of the skin against infection: implication of naturally occurring human antimicrobial agents -defensins, cathelicidin LL-37 and lysozyme. J. Dermatol. Sci. 40:157–168. Oppenheim, J. J., and D. Yang. 2005. Alarmins: chemotactic activators of immune responses. Curr. Opin. Immunol. 17:359–365. Schultz, H. 2007. From infection to autoimmunity: a new model for induction of ANCA against the bactericidal/permeability increasing protein (BPI). Autoimmun. Rev. 6:223–227. Syeda, F., H. Y. Liu, E. Tullis, M. Liu, A. S. Slutsky, and H. Zhang. 2008. Differential signaling mechanisms of HNPinduced IL-8 production in human lung epithelial cells and monocytes. J. Cell. Physiol. 214:820–827. Uniprot Consortium. 2008. The Universal Protein Resource (UniProt). Nucleic Acids Res. 36:D190–195. Van Amersfoort, E. S., T. J. C. Van Berkel, and J. Kuiper. 2003. Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock. Clin. Microbiol. Rev. 16:379–414. Ward, S. G., and F. M. Marelli-Berg. 2009. Mechanisms of chemokine and antigen-dependent T-lymphocyte navigation. Biochem. J. 418:13–27.
4. Host Defense (Antimicrobial) Peptides and Proteins Weiss, H. J. P. 2007. The bactericidal/permeability-increasing protein (BPI) in infection and inflammatory disease. Clin. Chim. Acta 384:12–23. Welling, M. M., P. S. Hiemstra, M. T. van den Barselaar, A. Paulusma-Annema, P. H. Nibbering, E. K. Pauwels, and W. Calame. 1998. Antibacterial activity of human neutrophil defensins in experimental infections in mice is accompanied by increased leukocyte accumulation. J. Clin. Invest. 102:1583–1590. Yamasaki, K., and R. L. Gallo. 2008. Antimicrobial peptides in human skin disease. Eur. J. Dermatol. 18:11–21.
67
Yang, D., O. Chertov, and J. J. Oppenheim. 2001. Participation of mammalian defensins and cathelicidins in anti-microbial immunity: receptors and activities of human defensins and cathelicidin (LL-37). J. Leukoc. Biol. 69:691–697. Yu, J., N. Mookherjee, K. Wee, D. M. E. Bowdish, J. Pistolic, Y. Li, L. Rehaume, and R. E. W. Hancock. 2007. Host defense peptide LL-37, in synergy with inflammatory mediator IL-1, augments immune responses by multiple pathways. J. Immunol. 179:7684–7691. Zanetti, M. 2004. Cathelicidins, multifunctional peptides of the innate immunity. J. Leukoc. Biol. 75:39–48.
This page intentionally left blank
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
5 Reactive Oxygen and Reactive Nitrogen Intermediates in the Immune System CHRISTIAN BOGDAN
INTRODUCTION
a broad spectrum of mechanisms that impede the replication or lead to the killing of the pathogen (Table 1 and Fig. 1). Some of these mechanisms, such as the phagocyte NADPH oxidase (Phox) or the type 2 nitric oxide synthase (NOS2 or iNOS), are oxygen-dependent and require pathogen recognition receptor- or cytokine-mediated gene expression, whereas others are oxygen-independent and are immediately available because of preformed proteins (e.g., defensins, proteases). In general, the different effector mechanisms are not reserved for certain pathogens and they do not operate in isolation, but are frequently interdependent. For example, the acidification of the phagolysosome enhances the activity of many proteases, which are characterized by an acidic pH optimum; the myeloperoxidase (MPO) converts H2O2 (which is generated from O22 by superoxide dismutases [SOD]) into highly toxic halides; the MPO participates in the interferon (IFN)--mediated priming of phagocytes for Phox-dependent ROI production; ROIs and RNIs react with each other, which leads to the generation of new effector oxidants (e.g., peroxynitrite); and the withdrawal of iron from the pathogen allows the phagocyte to generate ROIs via the iron-catalyzed Haber-Weiss reaction (reviewed in Bogdan, 2004; Adachi et al., 2006). It should also be noted that for certain pathogens (e.g., Listeria monocytogenes, non-tuberculous Mycobacteria, and Anaplasma phagocytophilum) the exact mechanism(s) that lead to their killing by activated phagocytes still remain to be identified (von Loewenich et al., 2004; Birkner et al., 2008; Gomes et al., 2008).
Inorganic molecules such as nitric oxide (NO), superoxide (O22), or hydrogen peroxide (H2O2) are central secretory products of activated phagocytes. A simplified view presented in most immunology textbooks considers reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNI) merely as toxic defense and effector molecules of phagocytes that either help to kill viral and microbial pathogens, or, when produced chronically or in exuberant amounts, can cause severe tissue damage during infections and antigen-driven inflammatory processes. The mechanistic basis for the potent antimicrobial capacity (and for the adverse effects) of ROIs and RNIs is their rapid and high production, their small molecular size and transmembrane diffusibility, and their avid reactivity with organic targets (Bogdan et al., 2000; Bogdan, 2004; Fang, 2004; Segal, 2005). From a broader perspective, however, ROIs and RNIs also serve as important signaling molecules in various organ systems, including the immune system (Bogdan et al., 2000; Bogdan, 2001b; Forman & Torres, 2001; Hancock et al., 2001; Reth, 2002; Bedard & Krause, 2007; Brown & Griendling, 2009). Numerous examples for beneficial as well as counterprotective regulatory functions of RNIs and ROIs have been discovered during the past 20 years (Bogdan, 2004), which not only illustrate how difficult it is to judge their true role during infections, but also raise scepticism about the proposed use of antioxidants or RNI/ROI donors for the treatment of inflammatory or infectious diseases, respectively. In this chapter, I will review the sites of production and the functional diversity of ROIs and RNIs within the immune system with a special emphasis on their host-protective effector and regulator potential.
REACTIVE OXYGEN INTERMEDIATES Enzymatic Systems for the Generation of ROIs
THE PHAGOCYTE AND ANTIMICROBIAL DEFENSE: AN OVERVIEW
ROIs, ROSs, or reactive oxygen metabolites (ROMs) are interchangeable collective terms for all inorganic oxidants consisting of oxygen, hydrogen, and possibly a halogen. Important examples of ROIs are free oxygen radicals (e.g., O22, hydroxyl radical [OH], peroxyl radical [RO2], perhydroxyl radical [HOO]); relatively stable nonradical oxidants (e.g., hypochlorous acid [HOCl], ozone [O3], and H2O2); and labile oxidants with excited electrons (e.g., singlet oxygen [1O2]). There are multiple enzyme systems that generate ROIs. These include: (i) the electron transport chain in mitochondria, the
Following an encounter, entry or uptake of viruses, bacteria, protozoa, or fungi granulocytes and macrophages can resort to Christian Bogdan, Microbiology Institute, Clinical Microbiology, Immunology and Hygiene, Friedrich Alexander University, Erlangen, Nuremberg, and University Clinic of Erlangen Wasserturmstraße 3/5, D-91054 Erlangen, Germany.
69
70
HOST DEFENSE: GENERAL TABLE 1
Antimicrobial effector mechanisms of phagocytes Constitutive/rapidly available
Oxygen independent
Oxygen-dependent
Induced by cytokines and/or crosslinking of PRR
1. Hydrolytic and proteolytic enzymes - lysozyme C* - esterase, gelatinase , matrix metalloproteases (MMP) - serprocidins (azuricidin/ CAP37*, proteinase 3*, elastase*, cathepsin G)
1. Phagosomal acidification ATPase-dependent proton pump
2. Antimicrobial peptides - a-defensins* - b-defensins - bactericidal/permeability increasing protein (BPI)* - cathelicidin (CAP18)(*) and cathelicidin-derived C-terminal peptide (LL-37) - pentraxin (PTX-3)(*) 3. Antimicrobial proteins - histones*
2. Phagolysosomal fusion - small GTPases (47 kDa IRGs, e.g. Irgm1/LRG-47)
4. Depletion of micronutrients and trace elements - lacto(trans)ferrin (iron) - calprotectin (also called MRP8/14 or S100A8/A9 complex)* (Zn21, Mn21) 1. Oxidases - NADPH phagocyte oxidase (Phox, NOX2) - myeloperoxidase (MPO)* 2. Iron catalyzed formation of ROI - Haber-Weiss reaction - Fenton reaction 3. Antibody catalyzed formation of ROI - catalytic antibodies
3. Amino acid depletion - tryptophan depletion (IDO) - arginine depletion (arginase, iNOS/NOS2) 4. Depletion of micronutrients and trace elements - lipocalin-2 (iron)
Oxidases/Oxidoreductases - NADPH phagocyte oxidase (Phox, NOX2) - inducible or type 2 nitric oxide synthase (iNOS or NOS2)
CAP, cationic protein; IDO, indoleamine-2,3-dioxygenase; IRG, immunity-related GTPase; Irgm1, immunity-related GTPase family M member 1; LRG-47, lipopolysaccharide- and interferon-regulated GTPase of 47 kDa; MRP, myeloid-related protein; PRR, pattern recognition receptor; S100 proteins, family of calcium-binding proteins of low molecular weight (9–13 kDa) * These molecules are part of the proteome of “neutrophil extracellular traps” (NETs), which are web-like structures consisting of chromatin (DNA and histones) and granule proteins and are extruded during the death of granulocytes (Urban et al., 2009).
monooxygenases, and oxidases in peroxisomes, and the cytochrome p450 oxidoreductases, all of which generate ROIs as by-products; (ii) the widely expressed xanthine oxidase system, which transforms hypoxanthine to xanthine, and further to uric acid and thereby generates H2O2 from H2O and 3O2; (iii) the generation of ROIs by the pseudoperoxidase or phenoloxidase activity of hemoglobin or hemocyanin, respectively, after contact with microbial pathogens; (iv) the family of NADPH-dependent oxidases (NOX)/dual oxidases (DUOX), which transport electrons from cytoplasmic NADPH to generate O22 (NOX1, 2, 3, 4, and 5) or H2O2 (DUOX) (Bedard & Krause, 2007; Kawahara et al., 2007); (v) and the MPO in the primary (azurophilic) granules of neutrophils and the lysosomes of blood monocytes, which converts H2O2 in the presence of halide anions (Cl2 Br2 I2) to a variety of toxic
products including hypohalous acids (e.g., HOCl), halogens (e.g., Cl2), and other oxidants (e.g., chloramines, hydroxyl radical [OH2], singlet oxygen [1O2]) (Klebanoff, 1999).
Generation of ROIs by Phenoloxidases and Respiratory Proteins
An ancient and highly conserved system for the generation of ROIs is the pro-phenoloxidase (pro-PO)/phenoloxidase (PO)/semiquinone/quinone system that is already found in primitive invertebrates. Inactive pro-PO, which is present in the hemolymph of many arthropods, is cleaved into active PO by a serine protease, which is triggered by pathogenassociated molecular patterns (PAMPs) interacting with the respective pattern-recognition receptors (PRR). The PO converts phenols into quinones, which either polymerize
5. ROIs and RNIs in the Immune System
71
FIGURE 1 Schematic overview of antimicrobial effector mechanisms of neutrophils and other phagocytes. In resting neutrophils gp91phox (NOX2) and p22phox are located in the plasma membrane (depicted) as well as in the membrane of secondary and tertiary granules (not depicted), all of which can contribute to the formation of phagosomes. Upon stimulation (e.g. exposure to pathogens [black oval], crosslinking of Fc-receptors by opsonized particles) the Rho-GTPase Rac (Rac1 or Rac2 depending on the cell-type and species) and p47phox in the cytosol become activated by CARD9-mediated release of GDP-dissociation inhibitor (GDI) and subsequent guanine nucleotide exchange factor (GEF)-mediated exchange of GDP for GTP and by phosphorylation (asteriks), respectively, and translocate to the membrane. Subsequent translocation of p67phox and p40phox leads to the enzymatically active phagocyte NADPH oxidase complex, which generates O22 on the cell surface or within the phagosome. The O22 is converted into H2O2, either spontaneously by the acidic pH in phagolysosomes, or by host- or pathogen-derived superoxide dismutases (SOD). The iron-dependent Haber-Weiss-reaction (HWR), catalase (Cat) and myeloperoxidase (MPO) help to generate further species of ROI, all of which contribute to the killing of the pathogens (dashed oval). Inducible nitric oxide synthase (iNOS or NOS2), which is found in the cytosol (not depicted) as well as in a vesicular compartment (nitroxosomes) of macrophages and granulocytes, generates citrulline and nitric oxide (NO) from the amino acid l-arginine and molecular oxygen. Neutrophils contain large numbers of primary, secondary and tertiary granules, which are loaded with a broad spectrum of antimicrobially active compounds (see Table 1 for abbreviations and details).
into melanin or enter redox cycles with the highly reactive semiquinone, leading to the generation of O22. The deposition of melanin impedes the replication of microbes, whereas the formation of semiquinones and O22 contributes to the killing of microbes (Cerenius & Söderhall, 2004; Park et al., 2007). Recently, it was discovered that organisms of the subgenus Chelicerata (e.g., horseshoe crabs, ticks, and mites), which lack the hemolymphatic pro-PO system, utilize the oxygen-carrier protein hemocyanin for the generation of ROIs. Exposure of purified hemocyanin, first to microbial proteases (e.g., elastase of Pseudomonas aeruginosa, subtilisin A of Staphylococcus aureus) and then to PAMPs (e.g., lipopolysaccharide [LPS], lipoteichoic acid [LTA]), led to a substantial induction of PO activity and O2− production (Jiang et al., 2007). A similar observation was
made with the mammalian respiratory protein hemoglobin, which is frequently released from erythrocytes during bacteremic infections with pathogens that express hemolysins. When human Fe31 hemoglobin (methemoglobin) interacted with microbial proteases and PAMPs, its pseudoperoxidase activity (which leads to the formation of O22 from H2O2) was strongly enhanced (Jiang et al., 2007). The advantages of generating antimicrobially active ROIs by respiratory proteins are obvious. First, the ROIs release is instantaneously available because it does not rely on the activation of immune cells, enzymes, or signaling cascades of the hosts. Second, it is unlikely to cause widespread tissue damage, because it will only occur in close proximity to the pathogen due to the need for direct activation of the respiratory proteins by microbial proteases and PAMPs (Bogdan, 2007a).
72
HOST DEFENSE: GENERAL
Generation of ROIs by the NOX/DUOX Family
The NOX/DUOX family of NADPH-dependent oxidases is evolutionarily highly conserved. In humans, it consists of five different isoforms of NOX (NOX1 to 5) and two isoforms of DUOX (DUOX1 and DUOX2). The NOX/DUOX isoforms differ in their cytosolic subunits, their primary tissue expression pattern and their known key functions in physiology and pathophysiology (Table 2). Independent of the exact enzymatic source, ROIs generated by NOX/DUOX exert three different categories of effects (i) modulation of cellular functions; (ii) regulation of cell differentiation and growth and of tissue repair; and (iii) induction of cell death and tissue destruction. From a molecular point of view these effects result from the oxidation of proteins, lipids, carbohydrates, and/or nucleic acids that either alter intra- or intercellular signaling processes, including gene transcription, or cause direct irreversible damage of cell membranes, organelles, cytoskeletal structures, or enzymes (Bedard & Krause, 2007; Brown & Griendling, 2009). With the exception of DUOX, which was recently implicated in the antimicrobial defense of mucosal epithelia cells (Geiszt et al., 2003; Donko et al., 2005) and the recruitment of leukocytes to wounds (Niethammer et al., 2009), the NOX2/Phox is the only member of the NOX/DUOX family for which a strong antimicrobial and regulatory function in the innate and adaptive immune system has been documented. Therefore, the following summary of the production and function of ROIs will focus on NOX2/Phox.
Enzymology, Subcellular Localization, and Regulation of Phox
NOX2, also termed gp91phox or CYBB (b-subunit of the flavocytochrome b558) is a NADPH:O2-oxidoreductase that transfers electrons from the substrate NADPH (electron donor) to the substrate molecular oxygen (electron acceptor) and generates NADP1, protons, and superoxide anions (O22) (see Fig. 1). 2 O2 1 NADPH → 2 O22 1 NADP1 1 H1 The rapid oxygen consumption that leads to the prompt production of large amounts of ROIs has been termed “respiratory burst” or “oxidative burst.” It results from the assembly of a multicomponent protein complex, which occurs in the plasma membrane or in intracellular membranes (Clark, 1999).
Formation and Localization of Active Phox
The NOX2 itself, which forms the catalytic core of the multiprotein Phox complex, contains two hemes as prosthetic groups and binding sites for the substrate NADPH and the cosubstrate FAD. NOX2 is constitutively associated with a 22 kDa protein (p22phox, a-subunit of flavocytochrome b558 [CYBA]), which stabilizes NOX2 and is thereby indispensable for NOX2 expression. The NOX2/p22phox heterodimer is termed flavocytochrome b558 (Nauseef, 2008b; 2008a). In resting human neutrophils, 70% to 90% of the flavocytochrome b558 is localized in the secondary (specific) and tertiary (gelatinase) granules, and 10% to 30% in the plasma membrane (Jesaitis et al., 1990; Kjeldsen et al., 1994). In mouse bone marrow-derived macrophages, flavocytochrome b558 is found in Rab11-positive recycling endosomes, in Rab5-positive early endosomes, and in the plasma membrane (Casbon et al., 2009). Phox is also detectable in certain lysosome-related organelles and phagosomes of mouse and human dendritic cells (Vulcano et al., 2004; Savina et al., 2009). Upon stimulation of phagocytes by phorbol ester, chemoattractants, pathogens, microbial products, or antibody-opsonized particles, at least four cytosolic proteins(p40phox, p47phox, p67phox, and the small Rho-GTPase Rac1 or Rac2) are
translocated to the membrane, where they associate with the flavocytochrome to form the active Phox (see Fig. 1). The membrane translocation of the p40-p47-p67phox complex and its interaction with p22phox and NOX2 requires the phosphorylation of a number of proteins, notably of p47phox, which, in its nonphosphorylated form, assumes an autoinhibited state (Lambeth et al., 2007; Nauseef, 2008a). Different isoforms of phosphoinositide 3-kinase (PI3K) are critical for the early (PI3K) and late phase (PI3Kd) production of phosphatidylinositol-3,4,5-trisphosphate (PtdIns[3,4,5]P3) in phagocytes following priming and stimulation. PtdIns(3,4,5)P3 regulates various protein kinases (e.g., AKT, PDK1, and certain protein kinase C [PKC] isoforms), which then phosphorylate the autoinhibitory domain of p47phox (Rommel et al., 2007). The membrane translocation of the GTPase Rac1 requires the caspase-recruitment domain (CARD) containing protein 9 (CARD9), which, upon phagocytosis, associates with the GDP-dissociation inhibitor (GDI) LyGDI, and thereby interrupts the blocking effect of LyGDI on GDP-Rac (Wu et al., 2009). The Ras-related GTPases Rap1a and 1b (not depicted in Fig. 1) can also associate with the Phox complex, but they are not essential for the oxidative burst (Li et al., 2007). Depending on the cell type, the active Phox complex assembles on the plasma membrane, on the early or recycling endosomes, or on secondary or tertiary granules. Each of these compartments can supply membrane to the forming phagosomes, which, therefore, are prone to express a strong activity of Phox. O22 can be released by activated neutrophils through exocytosis of intracellular granules carrying active Phox (Kobayashi et al., 1998). Also, following cell death, Phox subunits are found on so-called neutrophil extracellular traps (NETs) (Munafo et al., 2009), which are formed by webs of DNA decorated with histones and various antimicrobial effector proteins (Urban et al., 2009). It is evident that the release of O22 into phagosomes is suitable to combat phagocytosed pathogens, whereas the extracellular accumulation of ROIs might also cause severe tissue damage.
Regulation
The oxidative burst of professional phagocytes (neutrophils and macrophages) is efficiently triggered by Fc- or complement-receptor-mediated phagocytosis of pathogens, by internalization of immune complexes, by chemoattractants (e.g., IL-8, leukotriene B4, C5a, platelet-activating factor), exogenous arachidonic acid, and by certain microbial products such as bacterial peptides (e.g., N-formylmethionyl-leucyl-phenylalanine [fMLP]) and fungal cell wall components (e.g., zymosan). Experimentally, phorbol esters (e.g., phorbol 12-myristate 13-acetate [PMA]), IgG-coated latex particles, or sheep red blood cells are used to activate Phox (Babior, 1999; Yamauchi et al., 2004; Kim & Dinauer, 2006). Although resting phagocytes constitutively express the membrane-bound and cytosolic components of Phox, the activity of Phox is modulated by multiple factors, such as the micromilieu in the tissue, soluble mediators of the immune system, or microbial organisms. Known regulators of Phox are: (i) the concentrations of molecular oxygen, protons (pH value), and NADPH, as seen by the strongly reduced ROI production of phagocytes under hypoxic or acidic conditions (Gabig et al., 1979) or in the presence of an inhibitor of the NADPH-generating hexose-monophosphate shunt (Borregaard et al., 1984; Adachi et al., 2006); (ii) the store-operated calcium entry (SOCE), where the initial phospholipase C- and inositol-triphosphate-dependent depletion of intracellular calcium store causes the subsequent influx of extracellular Ca21 ions, which then mediate the
TABLE 2
NADPH-dependent oxidases (NOX/DUOX family) (Bedard and Krause, 2007; Kawahara et al., 2007; Lambeth et al., 2007)
Family member (catalytic core)
Membrane-bound subunits
NOX1
Cytosolic subunits
Cell/tissue distribution (selective examples) prototypic cell type in bold
Functions in physiology (selective examples)
Functions in pathology (selective examples)
NOXO1 3), NOXA1 4)
Colon epithelium, prostate, vascular smooth muscle cells, endothelial cells, osteoclasts, keratinocytes, microglia
Proliferation of smooth muscle and colonic epithelial cells (?); angiogenesis; osteoclast differentiation
Prostate cancer (?), colonic cancer (?), inflammatory bowel disease (?), neurotoxicity (?)
p22phox
Rac1 or Rac2, p40phox, p47phox, p67phox
Phagocytes (neutrophils, monocytes, macrophages, dendritic cells, B and T cells, hepatocytes, endothelial cells, keratinocytes, neurons
Antimicrobial defense, anti-inflammation, signaling in immune cells; cognitive functions (memory, learning); osteoclast activity; tissue repair
Apoptosis, tissue damage; hypertension; inflammatory diseases; atherosclerosis; neurodegeneration; liver injury/ cancer
NOX3
p22phox
NOXO1 NOXA1(?) Rac (?)
Inner ear (cochlear and vestibular sensory epithelia), fetal tissues
Formation of otoconia crystals in the endolymph of the inner ear
Age-, noise- or drug-induced hearing loss?
NOX4
p22phox
Rac (?)
Kidney cortex and tubular epithelium, osteoclasts; endothelial, smooth muscle and hematopoietic stem cells, hepatocytes, keratinocytes, fibroblasts; neurons, ovaries
Growth of smooth muscle and endothelial cells (?); enhanced adipocyte differentiation (?); osteoclast activity; wound healing; oxygen sensing (?)
Cellular senescence; diabetic nephropathy (?); pulmonary hypertension and fibrosis; growth of melanoma cells (?)
NOX5
None
None (regulation by Ca21 fluxes)
Testis (spermatocytes), spleen (mantle zone [B cells] and periarterial lymphoid sheath [T cells]), ovaries
Sperm development and function; meiotic maturation of oocytes (?)
Prostate cancer (?)
DUOX1
p22phox (?) DUOXA11)
None
Epithelial cells of the thyroid (thyrocytes) and airways, prostate
Prerequisite for thyroid hormone synthesis; mucosal antimicrobial defense; wound healing
DUOX2
p22phox (?) DUOXA22)
None (regulation by Ca21 fluxes)
Epithelial cells of the thyroid, salivary glands, airways, and gastrointestinal tract
73
1), 2) DUOX maturation proteins located in the endoplasmatic reticulum (ER), which are crucial for preventing the ER-retention of DUOX1 and DUOX2. 3) NOXA1 (NOX activator 1 5 p67phox homolog). 4) NOXO1, NOX organizer 1 (p47phox homolog).
5. ROIs and RNIs in the Immune System
NOX2
74
HOST DEFENSE: GENERAL
translocation of Rac, enhance the formation of the active Phox complex (by recruiting the calcium-binding myeloidrelated proteins MRP8 and MRP14), and activate PKC for the phosphorylation of cytosolic Phox components (Brechard & Tschirhart, 2008); (iii) the cytokines tumor necrosis factor (TNF) and IFN- and the LPS of gramnegative bacteria (Ding et al., 1988; Amezaga et al., 1992; Adachi et al., 2006); or (iv) intracellular pathogens such as A. phagocytophilum, which reside in neutrophils and partially shut down the oxidative burst (Garcia-Garcia et al., 2009; Bussmeyer et al., 2010). Pretreatment of macrophages or neutrophils with TNF, IFN-, or LPS (so-called “priming”) potentiates the production of O22 in response to a triggering agent. Part of the enhanced oxidative burst of IFN-- or TNF-primed phagocytes is likely to result from an increased mRNA expression of gp91phox, p47phox, p67phox, and/or p22phox. The transcription factors PU-1, activated protein (AP)-1, interferon-regulatory factor (IRF)-1, and interferon consensus sequence-binding protein were shown to participate in the upregulation of p67phox and/or gp91phox mRNA (reviewed in Bogdan, 2004). Recently, an entirely novel mechanism for the priming, as well as the activation of neutrophils by TNF for enhanced Phox activity was proposed. Yazdanpanah et al. (2009) reported that in mouse and human fibroblasts riboflavin kinase (RFK), which causes the conversion of vitamin B2 (riboflavin) into flavin mononucleotide (FMN) and—via the flavin adenin dinucleotide (FAD) synthetase—further into FAD, is a TNF responsive enzyme. Stimulation by TNF had three major consequences. First, RFK was rapidly recruited to the TNF receptor 1 (TNFR1) complex. Second, within the same time frame, p22phox, NOX1, NOX2, and Rac1 became detectable in the TNFR1 receptosome, provided RFK was present. Third, the cellular levels of FMN and FAD increased. RFK was found to bind to the TNFR1 death domain and to p22phox. This bridging function of RFK was essential for TNFinduced ROI production. Yazdanpanah et al. (2009) argued that the increased activity of RFK in close proximity to Phox leads to greater FAD saturation of NOX1 and NOX2 and accounts for the priming and activating effect of TNF. For a long time, the mechanism(s) by which microbial ligands of Toll-like receptors (TLR)—such as the TLR4agonist LPS—prime and activate Phox have been unclear. Detailed biochemical analyses and the study of patients with defined immunodeficiencies revealed that the IL-1 receptor-associated kinase (IRAK) 4 and the NF-kB essential modulator (NEMO or IkB kinase ), two kinases downstream of TLR4, are critical for the phosphorylation and/or membrane translocation of p47phox or p67phox, respectively (Pacquelet et al., 2007; Singh et al., 2009). In contrast, the TLR9-induced oxidative burst of neutrophils was normal in the absence of IRAK4, most likely because cross-linking of TLR9 led to PI3K-mediated phosphorylation of cytosolic Phox components (Hoarau et al., 2007).
Function of ROIs: Antimicrobial Activity
One of the key functions of ROIs in the immune system is their contribution to the control of infectious pathogens by neutrophils, macrophages, and other phagocytic cells (Bogdan et al., 2000; Bogdan, 2004; Fang, 2004; Segal, 2005). The antimicrobial activity of Phox can be due to five different categories of mechanisms. •
ROIs might exert direct toxic effects on viruses, bacteria, protozoa, and fungi, which can be damaged via oxidation, peroxidation, hydroxylation, or chlorination of proteins, lipids, nucleic acids, iron-sulfur clusters, or heme prostetic groups (reviewed in Klebanoff, 1999; Bogdan, 2004; Fang, 2004).
•
•
•
•
The Phox promotes the activity of serine proteases with a neutral pH optimum (e.g., cathepsin G and elastase). The release of the anionic superoxide (O22) into the phagosomal vacuole necessitates charge compensation. This occurs partly by the influx of K1 ions, partly by the reaction of O22 with protons that are pumped into the vacuole by the H1/ATPase, or are derived from acidic granules fusing with the phagosome. These events raise the intravacuolar tonicity and pH (Segal et al., 1981; Reeves et al., 2002). The hypertonic K1 displaces the cationic granule proteases (cathepsin G, elastase) from the anionic proteoglycan granule matrix, whereas the neutral to alkaline environment provides optimal pH conditions for their proteolytic activity (Segal, 2005). ROIs, notably H2O2, can cause a novel form of cell death (“NETosis”) that is characterized by the extrusion of “neutrophil extracellular traps” (NETs) (Fuchs et al., 2007), which fulfill antimicrobial functions (see above and Table 1). ROIs help to block the phagosomal escape of intracellular bacteria (e.g., L. monocytogenes) into the cytosol (Myers et al., 2003). Several of the signaling functions of ROIs (see below) undoubtedly contribute to the development of a protective immune response and thereby also to the control of infectious pathogens.
The function of Phox for the defense against bacterial, protozoan, or fungal pathogens is best documented by the analysis of the course of infections in transgenic mouse models. The analysis of p47phox-, gp91phox-, Rac2-, or MPO-deficient mouse strains revealed that depending on the type of pathogen, ROIs were dispensable (e.g., A. phagocytophilum) or played a contributory (e.g., Leishmania donovani, L. monocytogenes) or essential role (e.g., Aspergillus fumigatus, S. aureus) for combatting the pathogen and/or for resolution of the inflammation (reviewed in Bogdan, 2004; Fang, 2004; Birkner et al., 2008).
Function of ROIs: Signaling and Immunomodulation
The signaling function of H2O 2 and other ROIs has been subject of earlier detailed reviews (Reth, 2002; Nathan, 2003; Gloire et al., 2006; Stone & Yang, 2006; Brown & Griendling, 2009). Numerous in vitro and in vivo studies have demonstrated that ROIs regulate the differentiation, proliferation, function, and death of many cell types. From a molecular point of view, ROIs alter the redox status of the cells, for instance, by lowering the content of thiols such as glutathione, which secondarily affects the function of many enzymes (Forman & Torres, 2001). Therefore, it was not surprising to see that ROIs, in particular H2O 2, modulate various signaling pathways (e.g., phosphatases, phospholipases, Janus kinases [JAK], signal transducers and activators of transcription [STATs], mitogen activated protein kinases [MAPK], and ion channels) and transcription factors (e.g., NF-kB). The activation of Phox and the production of ROI turned out be essential for certain steps in the signaling cascade of TLRs and NOD-like receptors, which sense extracellular or intracellular pathogens respectively (Ogier-Denis et al., 2008; Martinon et al., 2009). As PRR also trigger Phox (see above), the inflammatory processes following the detection of pathogens and the oxygen-dependent antimicrobial effector mechanisms are interdependent events. Some more recently recognized effects of ROIs in the immune system will follow.
5. ROIs and RNIs in the Immune System
Differentiation of Immune Cells
In Drosophila melanogaster, multipotent progenitor cells in the medullary zone of the primary lobe of the lymph gland give rise to three different types of mature hemocytes: plasmatocytes, which support wound healing, phagocytose bacteria, and form extracellular aggregates to entrap bacteria; crystal cells, which express phenol oxidase activity; and lamellocytes, which are crucial for the encapsulation reaction similar to the granuloma formation in vertebrates. OwusuAnsah and Banerjee (2009) observed ROI production by the multipotent progenitor cells and found that the production of ROI was critical for the development of the mature hemocytes through a signaling pathway that involved the activation of the Jun N-terminal kinase (JNK) and of the Forkhead box protein O (FoxO) as well as the downregulation of polycomb proteins. In mammals, there is compelling evidence that the ability of macrophages to fuse and to form multinucleated giant cells or osteoclasts is dependent on Phox-generated ROI (Quinn & Schepetkin, 2009).
Wound Healing
ROI and various redox-sensitive events contribute to the wound healing response (Sen & Roy, 2008), but the exact source(s) of ROI that drive the dermal tissue repair remain largely unknown. Recently, Niethammer et al. (2009) demonstrated that in locally injured zebra fish larvae, a tissue-scale gradient of H2O2 generated by DUOX acivity in epithelial cells is required for the directional migration and infiltration of leukocytes into the wound. It is currently unclear whether this phenotype results from a direct chemotactic effect of H2O2 or is due to the release of a downstream chemoattractant.
Regulation of Antigen Presentation
The activation of Phox following endocytosis of microbes or antigenic particles antagonizes the acidification of the phagosomes by acidic granules (neutrophils) or by the vacuolar ATPase-dependent H1 pump (dendritic cells) because the generated O22 anions essentially consume protons. In neutrophils, the Phox-mediated neutralization or alkalinization of the phagocytic vacuole catalyzes the activity of serine proteases delivered to the phagosome by acidic granules (see above). In contrast, in dendritic cells, a neutral or alkaline pH limits the degradation of T cell-stimulatory peptides and epitopes by lysosomal cysteine and aspartyl proteases with an acidic pH optimum (e.g., cathepsin D). Consequently, in mouse and human dendritic cells, the Rab27a-dependent recruitment of “inhibitory lysosome-related endosomes,” which contain membrane subunits of Phox, to the phagosome and the Rac2-mediated assembly of Phox in the phagosomal membrane are prerequisites for MHC class I-restricted presentation of exogenous (phagosomal) antigens (so-called cross-presentation) (Savina et al., 2009).
Regulation of T Cell Survival and Function
The activation of T lymphocytes via T-cell receptor stimulation is associated with an early onset and sustained production of H2O2 and a delayed release of O22. mRNA and/ or protein analyses revealed the unexpected presence of p22phox, p47phox, p67phox, and gp91phox in primary mouse and human T-cell blasts (Jackson et al., 2004). Whereas the early production of H2O2 and the late-phase release of O22 were independent of NADPH oxidase, the sustained generation of H2O2 required Phox components as well as Fas/FasL interactions (Jackson et al., 2004). TCR-triggered ROI release is likely to contribute to T-cell homeostasis, because p47phox- or gp91phox-deficient T cells showed an improved survival in vivo, an enhanced activation of Erk
75
kinase, and a relative increase of T-helper type-1 cytokine secretion (Jackson et al., 2004; Donaldson et al., 2009; Purushothaman & Sarin, 2009). Mice deficient in p47phox developed lymph node hyperplasia (with increased numbers of B and T cells) even in the absence of infection (Donaldson et al., 2009) and an increased severity of T-cell-driven autoimmune diseases (Gelderman et al., 2007a; Gelderman et al., 2007b; Hultqvist et al., 2009). There is also evidence for the expression of NOX1 in naive CD41 T lymphocytes, which might participate in IL-4 receptor signaling and the inactivation of receptor-associated protein tyrosine phosphatases (Sharma et al., 2008).
Anti-Inflammatory Function of ROIs
Whereas the pro-inflammatory and tissue-destructive functions of ROI are known from infectious, autoimmune, or degenerative disease models (Snelgrove et al., 2006; Sorce & Krause, 2009), the anti-inflammatory role of ROIs, which was first identified as an effector mechanism of suppressor macrophages (Metzger et al., 1980) and later rediscovered in myeloid-derived suppressor cells (reviewed in Bronte & Zanovello, 2005; Nagaraj et al., 2009), has only recently received new attention (Hultqvist et al., 2009). Patients with congenital defects of Phox components suffer from chronic granulomatous disease (CGD), which is characterized by recurrent bacterial and fungal infections, but also by sterile hyperinflammatory responses and an increased incidence of autoimmune diseases. In vitro and in vivo analyses suggest that multiple mechanisms account for the enhanced inflammation that is seen in the absence of functional Phox (Schappi et al., 2008). These include (i) a reduced spontaneous apoptosis of CGD neutrophils (Brown et al., 2003); (ii) a defective externalization of phosphatidyl-serine in CGD neutrophils, which causes an impaired recognition and removal (efferocytosis) of apoptotic neutrophils by macrophages (Fernandez-Boyanapalli et al., 2009; Sanmun et al., 2009); (iii) an inefficient indoleamine-2,3-dioxygenase-catalyzed and superoxide-dependent conversion of tryptophan into 3-hydroxykynurenine, which leads to an expansion of Th17 cells and d T cells and to a lack of regulatory T cells (Romani et al., 2008); (iv) a missing oxidative inactivation of proinflammatory mediators (e.g., MRP8/S100A8 protein) (Harrison et al., 1999); (v) a general deficiency of ROI-dependent signaling, which leads to a preponderance of proinflammatory stimuli (e.g., Ca21, IL-8) and a lack of antiinflammatory mediators (e.g., adenosine, cAMP, prostaglandin D2, transforming growth factor-b) (Geiszt et al., 1997; Brown et al., 2003; Lekstrom-Himes et al., 2005; Rajakariar et al., 2009).
REACTIVE NITROGEN INTERMEDIATES Pathways for the Generation of RNIs
The terms RNI, RNS, or reactive nitrogen metabolites (RNM) are used for all inorganic or organic compounds that consist of nitrogen and oxygen, exhibit a nitrating or nitrosating capacity and may carry a halogen or sulfur group. Examples are the radicals nitric oxide (also called nitrogen monoxide, NO) and nitrogen dioxide (NO2), the nitrosonium cation (NO1), the anions nitroxyl (NO2) and peroxynitrite (ONOO2), the dinitrogen trioxide (N2O3), nitryl chloride (NO2Cl) and various S-nitrosothiols (e.g., S-nitrosocysteine). The term RNI also covers higher oxidation products of NO such as nitrite (NO22) and nitrate (NO32). The key enzyme system to generate RNIs are the three different isoforms of nitric oxide synthase (NOS). All NOS isoforms are homodimeric enzymes that, in the presence of
76
HOST DEFENSE: GENERAL
molecular oxygen, convert the amino acid l-arginine into Nv-hydroxy-l-arginine and further into citrulline and NO, which initially is complexed to ferric heme (Fig. 2). The formation of the NOS homodimer and the complex oxidoreductase reaction require the binding of calmodulin, the incorporation of iron protoporphyrin IX (heme) and zinc, and the presence of multiple cofactors and cosubstrates ([6R]-tetrahydrobiopterin [BH4], NADPH, FMN, FAD, thiol) (Stuehr et al., 2009). Two of the NOS isoforms (neuronal NOS, nNOS, or NOS1; endothelial NOS, eNOS, or NOS3) are constitutively expressed in many cell types and tissues. They are mainly but not exclusively subject to posttranslational regulation by Ca21 fluxes and primarily fulfill homeostatic functions in the cardiovascular system (eNOS) and the musculoskeletal and central nervous system (nNOS). Due to their Ca21-dependency and constitutive expression these two isoforms are frequently termed cNOS. There is evidence for the expression and function of nNOS and eNOS in inflamed tissues (Rabelink & Luscher, 2006; Zhou & Zhu, 2009) and in cells of the immune system (e.g., T lymphocytes) (Bogdan, 2001a; Ibiza et al., 2008). A third isoform of NOS has been termed inducible nitric oxide synthase (iNOS or NOS2), because it is not expressed in strictly resting cells, but only after exposure to pathogens, microbial ligands, and/or cytokines. iNOS is readily inducible in mouse and human cells that typically encounter microbial pathogens and respond to pro-inflammatory stimuli (e.g., macrophages, dendritic cells, hepatocytes, and epithelial and endothelial cells) and is widely expressed during infectious, autoimmune, malignant, and other inflammatory processes. Whereas the expression of iNOS by human macrophages in vivo is indisputable (Fang & Nathan, 2007), its inducibility in human monocytes and macrophages in vitro is a matter of debate (Bogdan, 2000, 2001a). The expression of iNOS in primary T lymphocytes is highly controversial because most studies lack the analysis of iNOS protein in single T cells (Bogdan, 2000, 2001a; Vig et al., 2004; Choy & Pober, 2009).
Another enzyme that can participate in the generation of RNI is MPO. NO22 converts the inactive form of MPO (compound II, MPO-Fe[IV]) into the native enzyme (ground state, MPO-Fe[III]), thereby releasing the potent nitrating NO2 species. Furthermore, HOCl can also react with NO22, leading to the formation of NO2Cl, which shows a strong chlorinating reactivity towards aromates (Eiserich et al., 2002). RNI can also originate (i) from the chemical reaction between O22 and NO, which leads to the generation of peroxynitrite (ONOO2) (Ferrer-Sueta & Radi, 2009); (ii) from nitrite anions (NO22), which under acidic conditions or in the presence of desoxyhemoglobin release NO; or (iii) from the nitrite reductase and anhydrase reaction of methemoglobin, which catalyzes the reaction between NO22 and NO to form N2O3 (Basu et al., 2007). With the exception of the generation of peroxynitrite, the footprint of which (i.e., tyrosine nitration) is frequently seen in inflamed tissues, the (patho)physiological role of these iNOS-independent routes of generation of RNI remains to be established. The rest of this chapter will therefore exclusively deal with the expression and function of iNOS.
Subcellular Localization and Regulation of iNOS
The expression of iNOS mRNA and protein has been extensively studied in macrophages and other phagocytes, but also in hepatocytes, cardiac myocytes, endothelial cells, epithelial cells, and keratinocytes (Bogdan, 2000). In macrophages, iNOS protein and activity has been detected in three different locations: in the cytosol; in a membranous (particulate) compartment consisting of 50 to 80 nm vesicles, which were operationally termed “nitroxosomes” and await further characterization; and in the cortical actin cytoskeleton immediately beneath the plasma membrane (reviewed in Bogdan, 2000, 2001a, 2004). In human embryonic kidney cells, primary human bronchial epithelial cells, bladder papilloma cells, cardiac myocytes, and vascular smooth muscle cells, iNOS mRNA or protein was not only diffusely distributed in the cytosol, but also found in perinuclear inclusion-like bodies which were coined “physiologic aggresomes” (Jones et al., 2007; Pandit et al., 2009).
Positive and Negative Regulators of iNOS
FIGURE 2 Generation of nitric oxide from l-arginine by nitric oxide synthases (NOS). Only those cofactors are depicted, which are directly involved in the flux of electrons. The initial product of the reaction is not free NO, but a ferric heme-NO complex (not depicted). For further details see text.
iNOS is strongly and rapidly induced by proinflammatory cytokines, notably by IFN-ab and IFN-, in combination with a TLR ligand (e.g., LPS, flagellin, mycobacterial proteins and lipoarabinomannan, H. pylori urease, hemozoin, Trypanosoma cruzi, filarial cystatins; viral, bacterial, or protozoan DNA, unmethylated CpG desoxyoligonucleotides, double stranded RNA) or with a ligand or cross-linker of a costimulatory cell surface receptor (e.g., CD40 ligand, antiCD8, anti-Fc receptor IIb [anti-CD23]). Additional factors that have been reported to coinduce iNOS in macrophages are environmental and milieu factors (e.g., hypoxia, acidic pH, hyperthermia, ionizing radiation, monosodium urate crystals), hormones (e.g., estrogens), and the activation of certain ion channels (e.g., potassium channel). Conversely, a number of cytokines (e.g., transforming growth factor-b, IL-4, IL-13), nonproteinaceous soluble mediators (e.g., peroxisome proliferator-activated receptor- agonists), glucocorticoids, infectious pathogens, or their products (e.g., LPS, leishmanial lipophosphoglycan, Candida albicans, Francisella tularensis) downregulated or counteracted the induction of iNOS in macrophages (Bogdan, 2000; Nathan & Shiloh, 2000; Bogdan, 2001a, 2004; Fang, 2004; Dai et al., 2008; Parsa et al., 2008; Fernandez-Arenas et al., 2009). From a mechanistic point of view, the expression of iNOS in macrophages is regulated at the level of gene transcription, mRNA stability, mRNA translation, protein stability, and enzyme activity.
5. ROIs and RNIs in the Immune System
iNOS Gene Transcription
Potent regulators of iNOS gene transcription in macrophages are STAT1 and STAT2, IRF-1, NF-kB and AP-1 (activated protein-1), which are activated by IFN-, IFN-ab, TNF, or TLR ligands (Bogdan, 2001a). In many cell types, additional transcription factors (e.g., octamer factor, peroxisome proliferator-activated receptors, hypoxia-inducible factor-1, nuclear factor IL-6, zinc finger Kruppel-like factor 4) contribute to the activation of the iNOS promotor. Furthermore, the induction of iNOS is modulated by the various enzymes that intersect with the signaling cascades of the JAK/STAT, the TLR, and/or the NF-kB pathway (e.g., PKC; PI3K; protein tyrosine phosphatases; phosphatase and tensin homologue deleted on chromosome 10 [Pten]; MAPK, such as extracellular signal-regulated kinase 1 and 2 [ERK1/2], suppressors of cytokine signaling [SOCS]) (Bogdan, 2001a; Kleinert et al., 2004; Feinberg et al., 2005).
iNOS mRNA and Protein
The analysis of this level of iNOS regulation was pioneered by Vodovotz et al. (1993), who found that TGF-b downregulates the stability of iNOS mRNA as well as the synthesis and stability of iNOS protein. Since then, several proteins have been described that bind to AU-rich elements within in the 3-untranslated region of the iNOS mRNA and either stabilize (e.g., tristetraprolin; embryonic lethal abnormal vision-like protein, HuR; human polypyrimidine tract binding protein, PTB [also termed heterogenous nuclear ribonucleoprotein I hnRNP I]) or destabilize the iNOS mRNA (e.g., KH-type splicing regulatory protein [KSRP], mouse PTB) (Korhonen et al., 2007; Soderberg et al., 2007, and references therein). The reduced iNOS protein stability in TGF-b-treated macrophages most likely results from enhanced ubiquitination and proteasomal degradation (Kolodziejski et al., 2002; Mitani et al., 2005; Takaki et al., 2006). Conversely, analogs of cyclic adenosine monophosphate (cAMP) or activators of cAMP synthesis inhibited the ubiquitination of iNOS in macrophages and thereby enhanced the stability of the iNOS protein (Won et al., 2004). In inflammatory mouse macrophages, in a mouse keratinocyte cell line, and in neonatal rat astrocytes, it was observed that the synthesis of iNOS protein is dependent on the availability of l-arginine. A deficiency of extracellular arginine ( 10 M) led to a striking reduction of iNOS protein without altering the level of iNOS mRNA (reviewed in König et al., 2009). Arginine depletion can result from the IL-4-, IL-10-, IL-13-, or TGF-b-mediated induction and activation of arginase, which converts arginine into ornithine and urea (Morris, 2009) and might underlie the counterprotective effect of type-2 T-helper cell responses during infections with pathogens that are controlled by iNOS. The molecular mechanism underlying this unique form of posttranscriptional regulation of iNOS is currently being investigated in the laboratory of the author. If the arginine depletion results from a hyperstimulation of arginase, it is conceivable that spermine, a product of the ornithine metabolism, accounts for the inhibition of iNOS protein synthesis (Bussiere et al., 2005). Recently, the first evidence was presented that small noncoding RNAs (so-called microRNAs), which are known to suppress gene expression at the posttranscriptional level, might be involved in the regulation of iNOS (Dai et al., 2008). MicroRNAs might directly block iNOS protein translation and/or degrade iNOS mRNA, which has not yet been demonstrated, or modulate the expression of iNOS indirectly via downregulation of iNOS regulatory genes (Wang et al., 2009).
77
iNOS Enzyme Activity
Considering the known enzymology of iNOS (Stuehr et al., 2009), the activity of iNOS to generate NO is dependent on the presence of the substrate l-arginine and of multiple structural and enzymatic cofactors (see above). Like all heme-containing flavoproteins, iNOS is quite susceptible to inhibition by oxidative or nitrosative stress (Sun et al., 2009). In the presence of high amounts of NO, iNOS loses its capacity to generate NO but instead functions as a NADPH-dependent NO-oxygenase (Stuehr et al., 2009). The downregulation of iNOS activity in the presence of exogenous NO, peroxynitrite or ROI might serve as a feedback control in inflamed tissues. Enzymatically inactive iNOS protein can be sequestered in cytoplasmic inclusion-like bodies (aggresomes) (Pandit et al., 2009).
Functions of iNOS: Conceptual Framework
The functions of iNOS in the immune system result from the three possible consequences of the iNOS reaction: (i) the depletion of arginine; (ii) the intermediate generation of Nv-hydroxy-l-arginine, a known inhibitor of host cell as well as parasite arginases; and (iii) the generation of NO (Fig. 3). The highly reactive NO and the NO-derived congeners have received the greatest attention. Due to their multiple reaction partners (see below) they exert a great variety of effects on immune cells, host organs, and on infectious pathogens, ranging from benign signaling events to overt toxicity, such as microbial death, tissue damage, or vasodilatory shock (Bogdan, 2001b, 2001a; Nathan, 2003). As arginine is a semiessential amino acid for the mammalian host organism and is strictly required for the growth of some pathogens (Gaur et al., 2007; Reguera et al., 2009), iNOS-mediated arginine depletion could not only impair the function of immune cells (Rodriguez et al., 2007), but also contribute to the control of infectious pathogens (see below). The question, whether the iNOS intermediate Nv-hydroxy-l-arginine is relevant as an inhibitor of host or parasite arginases in vivo, is a matter of debate. Considering the fact that Nv-hydroxy-l-arginine does not accumulate, but rapidly enters the second step of the iNOS reaction (Stuehr et al., 2004), strongly argues against this possibility. However, independent of the underlying mechanism, iNOS/NO routinely assumes a bipartite role (i.e., cytotoxic effector function versus signaling and immunomodulatory function) in all major disease categories (i.e., in infectious, autoimmune/inflammatory, malignant, or cardiovascular diseases) (Fig. 3).
Functions of iNOS: Antimicrobial Activity
The antimicrobial effects of iNOS/NO fall into four major categories.
Direct (Cyto)toxic Effects of NO
NO acts on (i) structural components; (ii) the genome and the replication, transcription, and translation machinery; (iii) metabolic products and nutrient pathways; and (iv) virulence factors of infectious pathogens. Major molecular targets of NO are DNA molecules, where NO can cause mutations, oligonucleosomal fragmentation and inhibition of synthesis and repair; structural proteins, which are altered by S-nitrosylation, ADP-ribosylation, or tyrosine nitration; enzymes and metalloproteins, which can be inactivated by disruption of Fe-S clusters, zinc fingers, or heme groups; and membrane lipids that can be modified by peroxidation (Bogdan, 2001b, 2004; Fang, 2004; Bogdan, 2007b; Jones-Carson et al., 2008). Paradoxically, the toxic effects of NO can be expedited by microbial porins, which facilitate the penetration of NO through the bacterial cell wall (Fabrino et al., 2009).
78
HOST DEFENSE: GENERAL
FIGURE 3 Conceptual framework for iNOS-dependent effects in the immune system (for details see text).
NO is capable of reacting with O22 generated by Phox or by the pathogen itself, which leads to the formation of peroxynitrite (ONOO2), an RNI species with major nitrating capacity (Ferrer-Sueta & Radi, 2009). The cytotoxic function of NO and ONOO2 is not only restricted to pathogens, but may also extend to host cells. This explains why iNOS-deficient mice might exhibit less severe disease and a higher survival rate after infection with certain pathogens (reviewed in Bogdan, 2000, 2001a).
NO-Independent Antimicrobial Effect of iNOS
In the absence of l-arginine or arginase (which generates ornithin from l-arginine and, via the ornithin decarboxylase, the growth-promoting polyamines) pathogens such as trypanosomes, Leishmania, Giardia lamblia, or Schistosoma mansoni fail to proliferate or survive (Bogdan, 2004; Gaur et al., 2007; Muleme et al., 2009). The presence of an arginine biosynthetic pathway in obligatively intracellular bacteria such as Ehrlichia chaffensis might rescue these bacteria after arginine depletion by iNOS (Hotopp et al., 2006). Nv-hydroxy-l-arginine is a potent inhibitor of arginases, including arginases of eukaryotic pathogens (Kropf et al., 2005), which has led to the suggestion that it might exert antileishmanial activity (Iniesta et al., 2001). To date, however, there is no in vivo evidence for this hypothesis.
NO-Mediated Inhibition of Microbial Evasion and Resistance Mechanisms
Microbial pathogens express a number of virulence and resistance factors, which protect them against host effector molecules. Some of these evasion mechanisms, such as the expression of RNI or ROI detoxification systems, are specifically upregulated in the presence of oxidative or nitrosative
stress or by NO derived from microbial NOS (Nathan & Shiloh, 2000; Nathan, 2003; Bogdan, 2004; Fang, 2004; Shatalin et al., 2008; Das et al., 2009; Laver et al., 2009; Purwantini & Mukhopadhyay, 2009). On the other hand, NO and its congeners also convey signals to the host cells, which allow them to overcome at least some of the defense strategies of the microorganisms. Paradigmatic examples for this kind of NO-mediated counterstrike are (i) the dispersal of biofilms formed by Pseudomonas aeruginosa, which otherwise protect these bacteria from the action of phagocytes, antimicrobial peptides, and antibiotics (Barraud et al., 2006); (ii) the inhibition of the phagosomal escape of Listeria monocytogenes into the cytosol (Myers et al., 2003); (iii) the reversal of the blockade of phagosomal maturation and phagolysosomal fusion in macrophages infected with Leishmania donovani or Mycobacteria tuberculosis (Winberg et al., 2007; Axelrod et al., 2008); and (iv) the inhibition of the gene expression, protein synthesis, and function of effector molecules and toxins of Salmonella enterica serovar Typhimurium or enterohemorrhagic Escherichia coli (McCollister et al., 2005; Vareille et al., 2007).
Paving of a Protective Immune Response by NO
NO that is released by iNOS-positive phagocytes feeds back on the producer cell as well as on other immune cells. Some of the ensuing effects contribute to the development of a protective immune response against infectious pathogens and can therefore be viewed as a fourth category of antimicrobial effects of NO (Bogdan, 2000, 2001a, 2004). Notable activities of NO along this line of thinking are the proposed type 1 T-helper cell-stimulatory effect of NO (Niedbala et al., 2002), the enhancement of antigen processing and presentation by dendritic cells (Duan et al., 2008; Huang et al., 2008),
5. ROIs and RNIs in the Immune System
and the possible protection of immune cells from premature death due to the antiapoptotic function of NO (Brune, 2003; Choy & Pober, 2009).
Functions of iNOS: Signaling and Immunomodulation
As already indicated above NO and other RNI have a strong regulatory effect on cells of the immune system. This is due to the fact that immune cells are phenotypically highly versatile and undergo phases of lineage differentiation, proliferation, functional diversification, and regulated cell death during inflammatory processes. Many of the known molecular targets of NO have been studied in detail in immune cells and play an important role in shaping of the immune response. These include soluble guanyl cyclase; ion channels; heterotrimeric GTP-binding proteins; phospholipases; NOTCH1 receptor; TLR adaptor molecules (e.g., MyD88 [myeloid differentiation molecule 88]); JAK and MAP kinases; Src kinases; members of the STAT family; caspases; protein phosphatases; metalloproteases; transcription factors (e.g., NF-kB, AP-1, Sp1, HIF-1[hypoxia inducible factor-1]); cell-cycle proteins (e.g., cyclin D1); histone (de)acetylases; DNA methylases; regulators of mRNA stability and translation (e.g., iron regulatory protein-1 and -2); and enzymes that catalyze the posttranslational processing of cytokines (e.g., IL-1b converting enzyme, TNF-converting enzyme) (Bogdan, 2000, 2001b, 2001a; Nathan, 2003; Bogdan, 2004; Into et al., 2008; Kim et al., 2008). In the following, some more recently published effects of iNOS/NO on dendritic cells and T lymphocytes will be summarized.
Dendritic Cells and Antigen Presentation
The ability to express iNOS in response to cytokines and TLR ligands is a general feature of mouse myeloid dendritic cells in vitro and during chronic inflammations in vivo (De Trez et al., 2009) and does not constitute a separate subpopulation of dendritic cells as previously suggested (Serbina et al., 2003). In dendritic cells, endogenous or exogenous NO promotes the depolymerization, processing, and presentation of bacterial carbohydrate antigens by the MHC class II pathway and the induction of MHC class II and of costimulatory molecules (CD80 and CD86) on the plasma membrane. iNOS interacted with the MHC class II-associated invariant chain (CD74) and NO inhibited the caspase-dependent degradation of CD74, which supported the traffic of MHC class II molecules to the cell surface (Duan et al., 2008; Huang et al., 2008). An interesting issue is the question of whether RNI (e.g., NO, ONOO2) and ROI (e.g., HOCl) can alter the immunogenicity of antigens via chemical modification (e.g., oxidation of carbohydrate side-chains, tyrosine nitration, formation of protein aldehydes or chloramines). Recent data suggest that RNI and ROI might function as natural adjuvants causing the generation of neoantigens, which are more efficiently endocytosed (Prokopowicz et al., 2009). It is possible that RNI- or ROI-generated neoantigens contribute to epitope spreading in chronic inflammatory and autoimmune diseases (Griffiths, 2008).
T Lymphocytes
Numerous studies have shown that the proliferation and cytokine production of T lymphocytes in response to specific antigens or mitogens is controlled by the expression of l-arginine-metabolizing enzymes (iNOS and arginase) in myeloid cells. Three different mechanisms were reported: (i) NO derived from iNOS suppressed the IL-2 receptor signaling pathways (JAK/STAT, Ras/MAPK, PI3K/Akt) and the production of IL-2 (Bronte & Zanovello, 2005); (ii) the activity of iNOS or arginase depleted l-arginine, which subsequently caused a strong downregulation of
79
CD3z, the main signal-transduction component of the T-cell receptor (Rodriguez et al., 2003); and (iii) the consumption of l-arginine by arginase deprived iNOS of its substrate, which led to a reduced production of NO. As NO normally scavenges O22, the arginine depletion resulted in an accumulation of H2O2 (derived from O22), which suppressed T-cell proliferation (Kusmartsev et al., 2004). These and other data demonstrated that not only Phox, but also iNOS and arginase are key effector pathways of suppressor macrophages and myeloid-derived suppressor cells (Marigo et al., 2008; Nagaraj et al., 2009). They form a feedback control of T-cell activation, contribute to the resolution of inflammatory processes, and account for the suppression of T-cell responses in certain nonhealing infectious diseases and malignancies (Marigo et al., 2008; Modolell et al., 2009). A down regulation of T-cell responses was also observed in the presence of dendritic cells that expressed increased amounts of iNOS and NO in response to IFN- after exposure to apoptotic cells (Ren et al., 2008). Inflamed endothelium blocked the adhesion of Th1 cells in an iNOS/NO-dependent manner, which might represent another mechanism to terminate a T-cell response in situ (Norman et al., 2008). NO has the ability to deviate the differentiation of T lymphocytes. Depending on the concentration of NO and the presence of cofactors, it was reported to drive the development of Th1 cells (Niedbala et al., 2002), Th2 cells (Daniel et al., 2006), or regulatory T cells (Niedbala et al., 2007).
PERSPECTIVE
The role of ROI and RNI in the immune system (as well as other organ systems) is unpleasantly ambiguous: They function as aggressive oxidants that kill microbial intruders and, at the same time, cause collateral damage to host tissues; and they serve as signaling molecules that not only control and tune the immune system, but also alarm the infectious pathogen to switch on mechanisms for protection against the host defense machinery. Therefore, ROI and RNI are potentially beneficial and detrimental at the same time, depending on their concentration, on the tissue microenvironment and the time course of generation, and certainly also on the chemistry of the individual products. Consequently, the frequently advertized consumption of antioxidants for protection against aging, carcinogenesis, or tissue degeneration has to be judged as equally problematic or ineffective as the systemic application of nonselective RNI- or ROI-donors for the treatment of infectious or autoimmune diseases (Bailey et al., 2007; Gaziano et al., 2009; Lippman et al., 2009). On the other hand, host cellor pathogen-specific targeting or activation of inhibitors of iNOS, Phox, or SOD, or of RNI- or ROI-donors might open up new avenues for future therapeutic approaches. Progress in this direction has recently been made (Singh et al., 2008; Valdez et al., 2008). I wish to apologize to all researchers whose work could only be cited in form of review articles due to space restrictions. The preparation of this chapter and the conduct of some of the studies reviewed were supported by grants to C. B. from the Deutsche Forschungsgemeinschaft (Bo996/3-3, SFB643 A6) and from the IZKF Erlangen (Project A24).
REFERENCES Adachi, Y., A. L. Kindzelskii, A. R. Petty, J. B. Huang, N. Maeda, S. Yotsumoto, Y. Aratani, N. Ohno, and H. R. Petty. 2006. IFN-gamma primes RAW264 macrophages and human monocytes for enhanced oxidant production in response to CpG DNA via metabolic signaling: roles of TLR9 and myeloperoxidase trafficking. J. Immunol. 176:5033–5040.
80
HOST DEFENSE: GENERAL
Amezaga, M. A., F. Bazzoni, C. Sorio, F. Rossi, and M. A. Cassatella. 1992. Evidence for the involvement of distinct signal transduction pathways in then regulation of constitutive and interferon-g-dependent gene expression of NADPH oxidase components (gp91phox, p47phox and p22phox) and high-affinity receptor for IgG (FcgRI) in human polymorphonuclear leukocytes. Blood 79:735–744. Axelrod, S., H. Oschkinat, J. Enders, B. Schlegel, V. Brinkmann, S. H. Kaufmann, A. Haas, and U. E. Schaible. 2008. Delay of phagosome maturation by a mycobacterial lipid is reversed by nitric oxide. Cell Microbiol. 10:1530–1545. Babior, B. M. 1999. NADPH oxidase: an update. Blood 93:1464–1476. Bailey, A., T. W. Pope, S. A. Moore, and C. L. Campbell. 2007. The tragedy of TRIUMPH for nitric oxide synthesis inhibition in cardiogenic shock: where do we go from here? Am. J. Cardiovasc. Drugs 7:337–345. Barraud, N., D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb. 2006. Involvement of nitric oxide in biofilm dispersal of Pseudomonas aeruginosa. J. Bacteriol. 188:7344–7353. Basu, S., R. Grubina, J. Huang, J. Conradie, Z. Huang, A. Jeffers, A. Jiang, X. He, I. Azarov, R. Seibert, A. Mehta, R. Patel, S. B. King, N. Hogg, A. Ghosh, M. T. Gladwin, and D. B. Kim-Shapiro. 2007. Catalytic generation of N2O3 by the concerted nitrite reductase and anhydrase activity of hemoglobin. Nat. Chem. Biol. 3:785–794. Bedard, K., and K. H. Krause. 2007. The NOX family of ROSgenerating NADPH oxidases: physiology and pathophysiology. Physiol. Rev. 87:245–313. Birkner, K., B. Steiner, C. Rinkler, Y. Kern, P. Aichele, C. Bogdan, and F. D. von Loewenich. 2008. The elimination of Anaplasma phagocytophilum requires CD41 T cells, but is independent of Th1 cytokines and a wide spectrum of effector mechanisms. Eur. J. Immunol. 38:3395–3410. Bogdan, C. 2000. The function of nitric oxide in the immune system, p. 443–492. In B. Mayer (ed.), Handbook of Experimental Pharmacology, Volume: Nitric Oxide. Springer, Heidelberg, Germany. Bogdan, C. 2001a. Nitric oxide and the immune response. Nat. Immunol. 2:907–916. Bogdan, C. 2001b. Nitric oxide and the regulation of gene expression. Trends in Cell. Biol. 11:66–75. Bogdan, C. 2004. Reactive oxygen and reactive nitrogen metabolites as effector molecules against infectious pathogens, p. 357–396. In S. H. E. Kaufmann, R. Medzhitov, and S. Gordon (ed.), The Innate Immune Response to Infection. ASM Press, Washington, D.C. Bogdan, C. 2007a. Oxidative burst without phagocytes: the role of respiratory proteins. Nat. Immunol. 8:1029–1031. Bogdan, C. 2007b. Phagocyte effector functions against Leishmania parasites, p. 193–206. In E. D. A. R. Gazzinelli (ed.), Protozoans in Macrophages. Landes Bioscience, Austin, TX. Bogdan, C., M. Röllinghoff, and A. Diefenbach. 2000. Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr. Opin. Immunol. 12:64–76. Borregaard, N., J. H. Schwartz, and A. I. Tauber. 1984. Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst. J. Clin. Invest. 74:455–459. Brechard, S., and E. J. Tschirhart. 2008. Regulation of superoxide production in neutrophils: role of calcium influx. J. Leukoc. Biol. 84:1223–1237. Bronte, V., and P. Zanovello. 2005. Regulation of immune responses by l-arginine metabolism. Nat. Rev. Immunol. 5:641–653. Brown, D. I., and K. K. Griendling. 2009. Nox proteins in signal transduction. Free Radic. Biol. Med. 47:1239–1253. Brown, J. R., D. Goldblatt, J. Buddle, L. Morton, and A. J. Thrasher. 2003. Diminished production of antiinflammatory mediators during neutrophil apoptosis and
macrophage phagocytosis in chronic granulomatous disease (CGD). J. Leukoc. Biol. 73:591–599. Brune, B. 2003. Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ. 10:864–869. Bussiere, F. I., R. Chaturvedi, Y. Cheng, A. P. Gobert, M. Asim, D. R. Blumberg, H. Xu, P. Y. Kim, A. Hacker, R. A. Casero, Jr., and K. T. Wilson. 2005. Spermine causes loss of innate immune response to Helicobacter pylori by inhibition of inducible nitric-oxide synthase translation. J. Biol. Chem. 280:2409–2412. Bussmeyer, U., A. Sarkar, K. Broszat, T. Ludemann, S. Moller, G. van Zandbergen, C. Bogdan, M. Behnen, J. S. Dumler, F. D. von Loewenich, W. Solbach, and T. Laskay. 2010. Impairment of gamma interferon signaling in human neutrophils infected with Anaplasma phagocytophilum. Infect. Immun. 78:358–363. Casbon, A. J., L. A. Allen, K. W. Dunn, and M. C. Dinauer. 2009. Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes. J. Immunol. 182:2325–2339. Cerenius, L., and K. Söderhall. 2004. The prophenoloxidase-activating system in invertebrates. Immunol. Reviews 198:116–126. Choy, J. C., and J. S. Pober. 2009. Generation of NO by bystander human CD8 T cells augments allogeneic responses by inhibiting cytokine deprivation-induced cell death. Am. J. Transplant 9:2281–2291. Clark, R. A. 1999. Activation of the neutrophil respiratory burst oxidase. J. Infect. Dis. 179:S309–S317. Dai, R., R. A. Phillips, Y. Zhang, D. Khan, O. Crasta, and S. A. Ahmed. 2008. Suppression of LPS-induced Interferongamma and nitric oxide in splenic lymphocytes by select estrogen-regulated microRNAs: a novel mechanism of immune modulation. Blood 112:4591–4597. Daniel, T., M. Alexander, W. J. Hubbard, I. H. Chaudry, M. A. Choudhry, and M. G. Schwacha. 2006. Nitric oxide contributes to the development of a post-injury Th2 T-cell phenotype and immune dysfunction. J. Cell Physiol. 208:418–427. Das, P., A. Lahiri, A. Lahiri, and D. Chakravortty. 2009. Novel role of the nitrite transporter NirC in Salmonella pathogenesis: SPI2-dependent suppression of inducible nitric oxide synthase in activated macrophages. Microbiology 155:2476–2489. De Trez, C., S. Magez, S. Akira, B. Ryffel, Y. Carlier, and E. Muraille. 2009. iNOS-producing inflammatory dendritic cells constitute the major infected cell type during the chronic Leishmania major infection phase of C57BL/6 resistant mice. PLoS Pathog. 5:e1000494. Ding, A. H., C. F. Nathan, and D. J. Stuehr. 1988. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J. Immunol. 141:2407–2412. Donaldson, M., A. Antignani, J. Milner, N. Zhu, A. Wood, L. Cardwell-Miller, C. M. Changpriroa, and S. H. Jackson. 2009. p47phox-deficient immune microenvironment signals dysregulate naive T-cell apoptosis. Cell Death Differ. 16: 125–138. Donko, A., Z. Peterfi, A. Sum, T. Leto, and M. Geiszt. 2005. Dual oxidases. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 360:2301–2308. Duan, J., F. Y. Avci, and D. L. Kasper. 2008. Microbial carbohydrate depolymerization by antigen-presenting cells: deamination prior to presentation by the MHCII pathway. Proc. Natl. Acad. Sci. USA 105:5183–5188. Eiserich, J. P., S. Baldus, M.-L. Brennan, W. Ma, C. Zhang, A. Tousson, L. Castro, A. J. Lusis, W. M. Nauseef, C. R. White, and B. A. Freeman. 2002. Myeloperoxidase, a leukocytederived vascular NO synthase. Science 296:2391–2394. Fabrino, D. L., C. K. Bleck, E. Anes, A. Hasilik, R. C. Melo, M. Niederweis, G. Griffiths, and M. G. Gutierrez. 2009. Porins facilitate nitric oxide-mediated killing of mycobacteria. Microbes Infect. 11:868–875.
5. ROIs and RNIs in the Immune System Fang, F. C. 2004. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat. Rev. Immunol. 2:820–832. Fang, F. C., and C. F. Nathan. 2007. Man is not a mouse: reply. J. Leukoc. Biol. 81:580. Feinberg, M. W., Z. Cao, A. K. Wara, M. A. Lebedeva, S. Senbanerjee, and M. K. Jain. 2005. Kruppel-like factor 4 is a mediator of proinflammatory signaling in macrophages. J. Biol. Chem. 280:38247–38258. Fernandez-Arenas, E., C. K. Bleck, C. Nombela, C. Gil, G. Griffiths, and R. Diez-Orejas. 2009. Candida albicans actively modulates intracellular membrane trafficking in mouse macrophage phagosomes. Cell Microbiol. 11:560–589. Fernandez-Boyanapalli, R. F., S. C. Frasch, K. McPhillips, R. W. Vandivier, B. L. Harry, D. W. Riches, P. M. Henson, and D. L. Bratton. 2009. Impaired apoptotic cell clearance in CGD due to altered macrophage programming is reversed by phosphatidylserine-dependent production of IL-4. Blood 113:2047–2055. Ferrer-Sueta, G., and R. Radi. 2009. Chemical biology of peroxynitrite: kinetics, diffusion, and radicals. ACS Chem. Biol. 4:161–177. Forman, H. J., and M. Torres. 2001. Redox signaling in macrophages. Mol. Asp. Med. 22:189–216. Fuchs, T. A., U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. Wahn, Y. Weinrauch, V. Brinkmann, and A. Zychlinsky. 2007. Novel cell death program leads to neutrophil extracellular traps. J. Cell Biol. 176:231–241. Gabig, T. G., S. I. Bearman, and B. M. Babior. 1979. Effects of oxygen tension and pH on the respiratory burst of human neutrophils. Blood 53:1133–1139. Garcia-Garcia, J. C., N. C. Barat, S. J. Trembley, and J. S. Dumler. 2009. Epigenetic silencing of host cell defense genes enhances intracellular survival of the rickettsial pathogen Anaplasma phagocytophilum. PLoS Pathog. 5:e1000488. Gaur, U., S. C. Roberts, R. P. Dalvi, I. Corraliza, B. Ullman, and M. E. Wilson. 2007. An effect of parasite-encoded arginase on the outcome of murine cutaneous leishmaniasis. J. Immunol. 179:8446–8453. Gaziano, J. M., R. J. Glynn, W. G. Christen, T. Kurth, C. Belanger, J. MacFadyen, V. Bubes, J. E. Manson, H. D. Sesso, and J. E. Buring. 2009. Vitamins E and C in the prevention of prostate and total cancer in men: the Physicians’ Health Study II randomized controlled trial. Jama 301:52–62. Geiszt, M., A. Kapus, K. Nemet, L. Farkas, and E. Ligeti. 1997. Regulation of capacitative Ca21 influx in human neutrophil granulocytes. Alterations in chronic granulomatous disease. J. Biol. Chem. 272:26471–26478. Geiszt, M., J. Witta, J. Baffi, K. Lekstrom, and T. L. Leto. 2003. Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense. FASEB J. 17:1502–1504. Gelderman, K. A., M. Hultqvist, L. M. Olsson, K. Bauer, A. Pizzolla, P. Olofsson, and R. Holmdahl. 2007a. Rheumatoid arthritis: the role of reactive oxygen species in disease development and therapeutic strategies. Antioxid. Redox. Signal 9:1541–1567. Gelderman, K. A., M. Hultqvist, A. Pizzolla, M. Zhao, K. S. Nandakumar, R. Mattsson, and R. Holmdahl. 2007b. Macrophages suppress T cell responses and arthritis development in mice by producing reactive oxygen species. J. Clin. Invest. 117:3020–3028. Gloire, G., S. Legrand-Poels, and J. Piette. 2006. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem. Pharmacol. 72:1493–1505. Gomes, M. S., S. Sousa Fernandes, J. V. Cordeiro, S. Silva Gomes, A. Vieira, and R. Appelberg. 2008. Engagement of Toll-like receptor 2 in mouse macrophages infected with Mycobacterium avium induces non-oxidative and TNFindependent anti-mycobacterial activity. Eur. J. Immunol. 38:2180–2189.
81
Griffiths, H. R. 2008. Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease? Autoimmun. Rev. 7:544–549. Hancock, J. T., R. Desikan, and S. J. Neill. 2001. Role of reactive oxygen species in cell signalling pathways. Biochem. Soc. Trans. 29:345–350. Harrison, C. A., M. J. Raftery, J. Walsh, P. Alewood, S. E. Iismaa, S. Thliveris, and C. L. Geczy. 1999. Oxidation regulates the inflammatory properties of the murine S100 protein S100A8. J. Biol. Chem. 274:8561–8569. Hoarau, C., B. Gerard, E. Lescanne, D. Henry, S. Francois, J. J. Lacapere, J. El Benna, P. M. Dang, B. Grandchamp, Y. Lebranchu, M. A. Gougerot-Pocidalo, and C. Elbim. 2007. TLR9 activation induces normal neutrophil responses in a child with IRAK-4 deficiency: involvement of the direct PI3K pathway. J. Immunol. 179:4754–4765. Hotopp, J. C., M. Lin, R. Madupu, J. Crabtree, S. V. Angiuoli, J. Eisen, R. Seshadri, Q. Ren, M. Wu, T. R. Utterback, S. Smith, M. Lewis, H. Khouri, C. Zhang, H. Niu, Q. Lin, N. Ohashi, N. Zhi, W. Nelson, L. M. Brinkac, R. J. Dodson, M. J. Rosovitz, J. Sundaram, S. C. Daugherty, T. Davidsen, A. S. Durkin, M. Gwinn, D. H. Haft, J. D. Selengut, S. A. Sullivan, N. Zafar, L. Zhou, F. Benahmed, H. Forberger, R. Halpin, S. Mulligan, J. Robinson, O. White, Y. Rikihisa, and H. Tettelin. 2006. Comparative genomics of emerging human ehrlichiosis agents. PLoS Genet. 2:e21. Huang, D., D. T. Cai, R. Y. Chua, D. M. Kemeny, and S. H. Wong. 2008. Nitric-oxide synthase 2 interacts with CD74 and inhibits its cleavage by caspase during dendritic cell development. J. Biol. Chem. 283:1713–1722. Hultqvist, M., L. M. Olsson, K. A. Gelderman, and R. Holmdahl. 2009. The protective role of ROS in autoimmune disease. Trends Immunol. 30:201–208. Ibiza, S., A. Perez-Rodriguez, A. Ortega, A. Martinez-Ruiz, O. Barreiro, C. A. Garcia-Dominguez, V. M. Victor, J. V. Esplugues, J. M. Rojas, F. Sanchez-Madrid, and J. M. Serrador. 2008. Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells. Proc. Natl. Acad. Sci. USA 105:10507–10512. Iniesta, V., L. C. Gomez-Nieto, and I. Corraliza. 2001. The inhibition of arginase by Nw-hydroxy-l-arginine controls the growth of Leishmania inside macrophages. J. Exp. Med. 193:777–783. Into, T., M. Inomata, M. Nakashima, K. Shibata, H. Hacker, and K. Matsushita. 2008. Regulation of MyD88-dependent signaling events by S nitrosylation retards toll-like receptor signal transduction and initiation of acute-phase immune responses. Mol. Cell. Biol. 28:1338–1347. Jackson, S. H., S. Devadas, J. Kwon, L. A. Pinto, and M. S. Williams. 2004. T cells express a phagocyte-type NADPH oxidase that is activated after T cell receptor stimulation. Nat. Immunol. 5:818–827. Jesaitis, A. J., E. S. Buescher, D. Harrison, M. T. Quinn, C. A. Parkos, S. Livesey, and J. Linner. 1990. Ultrastructural localization of cytochrome b in the membranes of resting and phagocytosing human granulocytes. J. Clin. Invest. 85:821–835. Jiang, N., N. S. Tan, B. Ho, and J. L. Ding. 2007. Respiratory proteins generate ROS as an antimicrobial strategy. Nat. Immunol. 8:1114–1122. Jones-Carson, J., J. Laughlin, M. A. Hamad, A. L. Stewart, M. I. Voskuil, and A. Vazquez-Torres. 2008. Inactivation of [Fe-S] metalloproteins mediates nitric oxide-dependent killing of Burkholderia mallei. PLoS One 3:e1976. Jones, R. J., D. Jourd’heuil, J. C. Salerno, S. M. Smith, and H. A. Singer. 2007. iNOS regulation by calcium/calmodulindependent protein kinase II in vascular smooth muscle. Am. J. Physiol. Heart Circ. Physiol. 292:H2634–H2642. Kawahara, T., M. T. Quinn, and J. D. Lambeth. 2007. Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes. BMC Evol. Biol. 7:109.
82
HOST DEFENSE: GENERAL
Kim, C., and M. C. Dinauer. 2006. Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid. J. Leukoc. Biol. 79:223–234. Kim, M. Y., J. H. Park, J. S. Mo, E. J. Ann, S. O. Han, S. H. Baek, K. J. Kim, S. Y. Im, J. W. Park, E. J. Choi, and H. S. Park. 2008. Downregulation by lipopolysaccharide of Notch signaling, via nitric oxide. J. Cell Sci. 121:1466–1476. Kjeldsen, L., H. Sengelov, K. Lollike, M. H. Nielsen, and N. Borregaard. 1994. Isolation and characterization of gelatinase granules from human neutrophils. Blood 83:1640–1649. Klebanoff, S. J. 1999. Oxygen metabolites from phagocytes, p. 721–768. In J. I. Gallin, and R. Snyderman (eds.), Inflammation: Basic Principles and Clinical Correlates. Lippincott Williams & Wilkins, Philadelphia, PA. Kleinert, H., A. Pautz, K. Linker, and P. M. Schwarz. 2004. Regulation of the expression of inducible nitric oxide synthase. Eur. J. Pharmacol. 500:255–566. Kobayashi, T., J. M. Robinson, and H. Seguchi. 1998. Identification of intracellular sites of superoxide production in stimulated neutrophils. J. Cell Science 111:81–91. Kolodziejski, P., A. Musial, J.-S. Koo, and N. T. Eissa. 2002. Ubiquitination of inducible nitric oxide synthase is required for its degradation. Proc. Natl. Acad. Sci. USA 99:12315–12320. König, T., C. Bogdan, and U. Schleicher. 2009. Translational repression of inducible NO synthase in macrophages by larginine depletion is not associated with an increased phosphorylation of eIF2alpha. Immunobiology 214:822–827. Korhonen, R., K. Linker, A. Pautz, U. Forstermann, E. Moilanen, and H. Kleinert. 2007. Post-transcriptional regulation of human inducible nitric-oxide synthase expression by the Jun N-terminal kinase. Mol. Pharmacol. 71:1427–1434. Kropf, P., J. M. Fuentes, E. Fahnrich, L. Arpa, S. Herath, V. Weber, G. Soler, A. Celada, M. Modolell, and I. Müller. 2005. Arginase and polyamine synthesis are key factors in the regulation of experimental leishmaniasis in vivo. FASEB J. 19:1000–1002. Kusmartsev, S., Y. Nefedova, D. Yoder, and D. I. Gabrilovich. 2004. Antigen-specific inhibition of CD81 T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species. J. Immunol. 172:989–999. Lambeth, J. D., T. Kawahara, and B. Diebold. 2007. Regulation of Nox and Duox enzymatic activity and expression. Free Radic. Biol. Med. 43:319–331. Laver, J. R., T. M. Stevanin, S. L. Messenger, A. D. Lunn, M. E. Lee, J. W. Moir, R. K. Poole, and R. C. Read. 2009. Bacterial nitric oxide detoxification prevents host cell S-nitrosothiol formation: a novel mechanism of bacterial pathogenesis. FASEB J. 24:286–295. Lekstrom-Himes, J. A., D. B. Kuhns, W. G. Alvord, and J. I. Gallin. 2005. Inhibition of human neutrophil IL-8 production by hydrogen peroxide and dysregulation in chronic granulomatous disease. J. Immunol. 174:411–417. Li, Y., J. Yan, P. De, H. C. Chang, A. Yamauchi, K. W. Christopherson, 2nd, N. C. Paranavitana, X. Peng, C. Kim, V. Munugalavadla, R. Kapur, H. Chen, W. Shou, J. C. Stone, M. H. Kaplan, M. C. Dinauer, D. L. Durden, and L. A. Quilliam. 2007. Rap1a null mice have altered myeloid cell functions suggesting distinct roles for the closely related Rap1a and 1b proteins. J. Immunol. 179:8322–8331. Lippman, S. M., E. A. Klein, P. J. Goodman, M. S. Lucia, I. M. Thompson, L. G. Ford, H. L. Parnes, L. M. Minasian, J. M. Gaziano, J. A. Hartline, J. K. Parsons, J. D. Bearden, III, E. D. Crawford, G. E. Goodman, J. Claudio, E. Winquist, E. D. Cook, D. D. Karp, P. Walther, M. M. Lieber, A. R. Kristal, A. K. Darke, K. B. Arnold, P. A. Ganz, R. M. Santella, D. Albanes, P. R. Taylor, J. L. Probstfield, T. J. Jagpal, J. J. Crowley, F. L. Meyskens, Jr., L. H. Baker, and C. A. Coltman, Jr. 2009. Effect of selenium and vitamin E on risk of prostate cancer and other cancers: the Selenium and Vitamin E Cancer Prevention Trial (SELECT). Jama 301:39–51.
Marigo, I., L. Dolcetti, P. Serafini, P. Zanovello, and V. Bronte. 2008. Tumor-induced tolerance and immune suppression by myeloid derived suppressor cells. Immunol. Rev. 222:162–179. Martinon, F., A. Mayor, and J. Tschopp. 2009. The inflammasomes: guardians of the body. Annu. Rev. Immunol. 27:229–265. McCollister, B. D., T. J. Bourret, R. Gill, J. Jones-Carson, and A. Vazquez-Torres. 2005. Repression of SPI2 transcription by nitric oxide-producing, IFNgamma-activated macrophages promotes maturation of Salmonella phagosomes. J. Exp. Med. 202:625–635. Metzger, Z., J. T. Hoffeld, and J. J. Oppenheim. 1980. Macrophage-mediated suppression I. Evidence for the participation of both hydrogen peroxide and prostaglandins in suppression of murine lymphocyte proliferation. J. Immunol. 124:983–988. Mitani, T., M. Terashima, H. Yoshimura, Y. Nariai, and Y. Tanigawa. 2005. TGF-beta1 enhances degradation of IFNgamma-induced iNOS protein via proteasomes in RAW 264.7 cells. Nitric Oxide 13:78–87. Modolell, M., B. S. Choi, R. O. Ryan, M. Hancock, R. G. Titus, T. Abebe, A. Hailu, I. Muller, M. E. Rogers, C. R. Bangham, M. Munder, and P. Kropf. 2009. Local suppression of T cell responses by arginase-induced l-arginine depletion in nonhealing leishmaniasis. PLoS Negl. Trop. Dis. 3:e480. Morris, S. M., Jr. 2009. Recent advances in arginine metabolism: roles and regulation of the arginases. Br. J. Pharmacol. 157:922–930. Muleme, H. M., R. M. Reguera, A. Berard, R. Azinwi, P. Jia, I. B. Okwor, S. Beverley, and J. E. Uzonna. 2009. Infection with arginase-deficient Leishmania major reveals a parasite number-dependent and cytokine-independent regulation of host cellular arginase activity and disease pathogenesis. J. Immunol. 183:8068–8076. Munafo, D. B., J. L. Johnson, A. A. Brzezinska, B. A. Ellis, M. R. Wood, and S. D. Catz. 2009. DNAse I inhibits a late phase of reactive oxygen species production in neutrophils. J. Innate Immun. 1:527–542. Myers, J. T., A. W. Tsang, and J. A. Swanson. 2003. Localized reactive oxygen and nitrogen intermediates inhibit escape of Listeria monocytogenes from vacuoles in activated macrophages. J. Immunol. 171:5447–5453. Nagaraj, S., M. Collazo, C. A. Corzo, J. I. Youn, M. Ortiz, D. Quiceno, and D. I. Gabrilovich. 2009. Regulatory myeloid suppressor cells in health and disease. Cancer Res. 69:7503– 7506. Nathan, C. 2003. Specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling. J. Clin. Invest. 111:769–778. Nathan, C., and M. U. Shiloh. 2000. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc. Natl. Acad. Sci. USA 97:8841–8848. Nauseef, W. M. 2008a. Biological roles for the NOX family NADPH oxidases. J. Biol. Chem. 283:16961–16965. Nauseef, W. M. 2008b. Nox enzymes in immune cells. Semin. Immunopathol. 30:195–208. Niedbala, W., B. Cai, H. Liu, N. Pitman, L. Chang, and F. Y. Liew. 2007. Nitric oxide induces CD41CD251 Foxp3 regulatory T cells from CD41CD25 T cells via p53, IL-2, and OX40. Proc. Natl. Acad. Sci. USA 104:15478–15483. Niedbala, W., X.-q. Wei, C. Campbell, D. Thomson, M. Komai-Koma, and F. Y. Liew. 2002. Nitric oxide preferentially induces type 1 T cell differentiation by selectively up-regulating IL-12 receptor b2 expression via cGMP. Proc. Natl. Acad. Sci. USA 99:16186–16191. Niethammer, P., C. Grabher, A. T. Look, and T. J. Mitchison. 2009. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebra fish. Nature 459:996–999. Norman, M. U., L. Zbytnuik, and P. Kubes. 2008. Interferongamma limits Th1 lymphocyte adhesion to inflamed endothelium: a nitric oxide regulatory feedback mechanism. Eur. J. Immunol. 38:1368–1380.
5. ROIs and RNIs in the Immune System Ogier-Denis, E., S. B. Mkaddem, and A. Vandewalle. 2008. NOX enzymes and Toll-like receptor signaling. Semin. Immunopathol. 30:291–300. Owusu-Ansah, E., and U. Banerjee. 2009. Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation. Nature 461:537–541. Pacquelet, S., J. L. Johnson, B. A. Ellis, A. A. Brzezinska, W. S. Lane, D. B. Munafo, and S. D. Catz. 2007. Crosstalk between IRAK-4 and the NADPH oxidase. Biochem. J. 403:451–461. Pandit, L., K. E. Kolodziejska, S. Zeng, and N. T. Eissa. 2009. The physiologic aggresome mediates cellular inactivation of iNOS. Proc. Natl. Acad. Sci. USA 106:1211–1215. Park, J.-W., C.-H. Kim, J.-H. Kim, B.-R. Je, K.-B. Roh, S.-J. Kim, H.-H. Lee, J.-H. Ryu, J.-H. Lim, B.-H. Oh, W.-J. Lee, and L. Bok-Luel. 2007. Clustering of peptidoglycan recognition protein-SA is required for sensing lysinetype peptidoglycan in sects. Proc. Natl. Acad. Sci. USA 104:6602–6607. Parsa, K. V., J. P. Butchar, M. V. Rajaram, T. J. Cremer, J. S. Gunn, L. S. Schlesinger, and S. Tridandapani. 2008. Francisella gains a survival advantage within mononuclear phagocytes by suppressing the host IFNgamma response. Mol. Immunol. 45:3428–3437. Prokopowicz, Z. M., F. Arce, R. Biedron, C. L. Chiang, M. Ciszek, D. R. Katz, M. Nowakowska, S. Zapotoczny, J. Marcinkiewicz, and B. M. Chain. 2009. Hypochlorous acid: a natural adjuvant that facilitates antigen processing, cross-priming, and the induction of adaptive immunity. J. Immunol. 184:825–835. Purushothaman, D., and A. Sarin. 2009. Cytokine-dependent regulation of NADPH oxidase activity and the consequences for activated T cell homeostasis. J. Exp. Med. 206:1515–1523. Purwantini, E., and B. Mukhopadhyay. 2009. Conversion of NO2 to NO by reduced coenzyme F420 protects mycobacteria from nitrosative damage. Proc. Natl. Acad. Sci. USA 106:6333–6338. Quinn, M. T., and I. A. Schepetkin. 2009. Role of NADPH oxidase in formation and function of multinucleated giant cells. J. Innate Immun. 1:509–526. Rabelink, T. J., and T. F. Luscher. 2006. Endothelial nitric oxide synthase: host defense enzyme of the endothelium? Arterioscler Thromb. Vasc. Biol. 26:267–271. Rajakariar, R., J. Newson, E. K. Jackson, P. Sawmynaden, A. Smith, F. Rahman, M. M. Yaqoob, and D. W. Gilroy. 2009. Nonresolving inflammation in gp91phox-/- mice, a model of human chronic granulomatous disease, has lower adenosine and cyclic adenosine 5-monophosphate. J. Immunol. 182:3262–3269. Reeves, E. P., H. Lu, H. L. Jacobs, C. G. M. Messina, S. Bolsover, G. Gabella, E. O. Potma, A. Warley, J. Roes, and A. W. Segal. 2002. Killing activity of neutrophils is mediated through activation of proteases by K1 flux. Nature 416:291. Reguera, R. M., R. Balana-Fouce, M. Showalter, S. Hickerson, and S. M. Beverley. 2009. Leishmania major lacking arginase (ARG) are auxotrophic for polyamines but retain infectivity to susceptible BALB/c mice. Mol. Biochem. Parasitol. 165:48–56. Ren, G., J. Su, X. Zhao, L. Zhang, J. Zhang, A. I. Roberts, H. Zhang, G. Das, and Y. Shi. 2008. Apoptotic cells induce immunosuppression through dendritic cells: critical roles of IFN-gamma and nitric oxide. J. Immunol. 181:3277–3284. Reth, M. 2002. Hydrogen peroxide as second messenger in lymphocyte activation. Nat. Immunol. 3:1129–1134. Rodriguez, P. C., D. G. Quiceno, and A. C. Ochoa. 2007. l-arginine availability regulates T-lymphocyte cell-cycle progression. Blood 109:1568–1573. Rodriguez, P. C., A. H. Zea, J. DeSalvo, K. S. Culotta, J. Zabaleta, D. G. Quiceno, J. B. Ochoa, and A. C. Ochoa. 2003. l-arginine consumption by macrophages modulates the expression of CD3z chain in T lymphocytes. J. Immunol. 17:1232–1239.
83
Romani, L., F. Fallarino, A. De Luca, C. Montagnoli, C. D’Angelo, T. Zelante, C. Vacca, F. Bistoni, M. C. Fioretti, U. Grohmann, B. H. Segal, and P. Puccetti. 2008. Defective tryptophan catabolism underlies inflammation in mouse chronic granulomatous disease. Nature 451:211–215. Rommel, C., M. Camps, and H. Ji. 2007. PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond? Nat. Rev. Immunol. 7:191–201. Sanmun, D., E. Witasp, S. Jitkaew, Y. Y. Tyurina, V. E. Kagan, A. Ahlin, J. Palmblad, and B. Fadeel. 2009. Involvement of a functional NADPH oxidase in neutrophils and macrophages during programmed cell clearance: implications for chronic granulomatous disease. Am. J. Physiol. Cell Physiol. 297:C621–C631. Savina, A., A. Peres, I. Cebrian, N. Carmo, C. Moita, N. Hacohen, L. F. Moita, and S. Amigorena. 2009. The small GTPase Rac2 controls phagosomal alkalinization and antigen crosspresentation selectively in CD8(1) dendritic cells. Immunity 30:544–555. Schappi, M. G., V. Jaquet, D. C. Belli, and K. H. Krause. 2008. Hyperinflammation in chronic granulomatous disease and anti-inflammatory role of the phagocyte NADPH oxidase. Semin. Immunopathol. 30:255–271. Segal, A. W. 2005. How neutrophils kill microbes. Annu. Rev. Immunol. 23:197–223. Segal, A. W., M. Geisow, R. Garcia, A. Harper, and R. Miller. 1981. The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH. Nature 290:406–409. Sen, C. K., and S. Roy. 2008. Redox signals in wound healing. Biochim. Biophys. Acta 1780:1348–1361. Serbina, N. V., T. P. Salazar-Mather, C. A. Biron, W. A. Kuziel, and E. G. Pamer. 2003. TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection. Immunity 19:59–70. Sharma, P., R. Chakraborty, L. Wang, B. Min, M. L. Tremblay, T. Kawahara, J. D. Lambeth, and S. J. Haque. 2008. Redox regulation of interleukin-4 signaling. Immunity 29:551–564. Shatalin, K., I. Gusarov, E. Avetissova, Y. Shatalina, L. E. McQuade, S. J. Lippard, and E. Nudler. 2008. Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages. Proc. Natl. Acad. Sci. USA 105:1009–1013. Singh, A., K. A. Zarember, D. B. Kuhns, and J. I. Gallin. 2009. Impaired priming and activation of the neutrophil NADPH oxidase in patients with IRAK4 or NEMO deficiency. J. Immunol. 182:6410–6417. Singh, R., U. Manjunatha, H. I. Boshoff, Y. H. Ha, P. Niyomrattanakit, R. Ledwidge, C. S. Dowd, I. Y. Lee, P. Kim, L. Zhang, S. Kang, T. H. Keller, J. Jiricek, and C. E. Barry, III. 2008. PA-824 kills nonreplicating Mycobacterium tuberculosis by intracellular NO release. Science 322:1392–135. Snelgrove, R. J., L. Edwards, A. J. Rae, and T. Hussell. 2006. An absence of reactive oxygen species improves the resolution of lung influenza infection. Eur. J. Immunol. 36:1364–1373. Soderberg, M., F. Raffalli-Mathieu, and M. A. Lang. 2007. Identification of a regulatory cis-element within the 3-untranslated region of the murine inducible nitric oxide synthase (iNOS) mRNA; interaction with heterogeneous nuclear ribonucleoproteins I and L and role in the iNOS gene expression. Mol. Immunol. 44:434–442. Sorce, S., and K. H. Krause. 2009. NOX enzymes in the central nervous system: from signaling to disease. Antioxid. Redox. Signal 11:2481–2504. Stone, J. R., and S. Yang. 2006. Hydrogen peroxide: a signaling messenger. Antioxid. Redox. Signal 8:243–270. Stuehr, D. J., J. Santolini, Z. Q. Wang, C. C. Wei, and S. Adak. 2004. Update on mechanism and catalytic regulation in the NO synthases. J. Biol. Chem. 279:36167–36170. Stuehr, D. J., J. Tejero, and M. M. Haque. 2009. Structural and mechanistic aspects of flavoproteins: electron transfer through the nitric oxide synthase flavoprotein domain. Febs. J. 276:3959–3974.
84
HOST DEFENSE: GENERAL
Sun, J., L. J. Druhan, and J. L. Zweier. 2009. Reactive oxygen and nitrogen species regulate inducible nitric oxide synthase function shifting the balance of nitric oxide and superoxide production. Arch. Biochem. Biophys. 494:130–137. Takaki, H., Y. Minoda, K. Koga, G. Takaesu, A. Yoshimura, and T. Kobayashi. 2006. TGF-beta1 suppresses IFN-gammainduced NO production in macrophages by suppressing STAT1 activation and accelerating iNOS protein degradation. Genes Cells 11:871–882. Urban, C. F., D. Ermert, M. Schmid, U. Abu-Abed, C. Goosmann, W. Nacken, V. Brinkmann, P. R. Jungblut, and A. Zychlinsky. 2009. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathog. 5:e1000639. Valdez, C. A., J. E. Saavedra, B. M. Showalter, K. M. Davies, T. C. Wilde, M. L. Citro, J. J. Barchi, Jr., J. R. Deschamps, D. Parrish, S. El-Gayar, U. Schleicher, C. Bogdan, and L. K. Keefer. 2008. Hydrolytic Reactivity Trends among Potential Prodrugs of the O(2)-Glycosylated Diazeniumdiolate Family. Targeting Nitric Oxide to Macrophages for Antileishmanial Activity. J. Med. Chem. 51:3961–3970. Vareille, M., T. de Sablet, T. Hindre, C. Martin, and A. P. Gobert. 2007. Nitric oxide inhibits Shiga-toxin synthesis by enterohemorrhagic Escherichia coli. Proc. Natl. Acad. Sci. USA 104:10199–10204. Vig, M., S. Srivastava, U. Kandpal, H. Sade, V. Lewis, A. Sarin, A. George, V. Bal, J. M. Durdik, and S. Rath. 2004. Inducible nitric oxide synthase in T cells regulates T cell death and immune memory. J. Clin. Invest. 113:1734–1742. Vodovotz, Y., C. Bogdan, J. Paik, Q.-w. Xie, and C. Nathan. 1993. Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor-b. J. Exp. Med. 178:605–613. von Loewenich, F. D., D. G. Scorpio, U. Reischl, J. S. Dumler, and C. Bogdan. 2004. Control of Anaplasma phagocytophilum, an obligate intracellular pathogen, in the absence of inducible nitric oxide synthase, phagocyte
NADPH oxidase, tumor necrosis factor, Toll-like receptor (TLR) 2 and 4, or the TLR adaptor molecule MyD88. Eur. J. Immunol. 34:1789–1797. Vulcano, M., S. Dusi, D. Lissandrini, R. Badolato, P. Mazzi, E. Riboldi, E. Borroni, A. Calleri, M. Donini, A. Plebani, L. Notarangelo, T. Musso, and S. Sozzani. 2004. Toll receptor-mediated regulation of NADPH oxidase in human dendritic cells. J. Immunol. 173:5749–5756. Wang, X., Q. Zhao, R. Matta, X. Meng, X. Liu, C. G. Liu, L. D. Nelin, and Y. Liu. 2009. Inducible nitric-oxide synthase expression is regulated by mitogen-activated protein kinase phosphatase-1. J. Biol. Chem. 284:27123–27134. Winberg, M. E., B. Rasmusson, and T. Sundqvist. 2007. Leishmania donovani: inhibition of phagosomal maturation is rescued by nitric oxide in macrophages. Exp. Parasitol. 117:165–170. Won, J. S., Y. B. Im, A. K. Singh, and I. Singh. 2004. Dual role of cAMP in iNOS expression in glial cells and macrophages is mediated by differential regulation of p38MAPK/ATF-2 activation and iNOS stability. Free Radic. Biol. Med. 37:1834–1844. Wu, W., Y. M. Hsu, L. Bi, Z. Songyang, and X. Lin. 2009. CARD9 facilitates microbe-elicited production of reactive oxygen species by regulating the LyGDI-Rac1 complex. Nat. Immunol. 10:1208–1214. Yamauchi, A., C. Kim, S. Li, C. C. Marchal, J. Towe, S. J. Atkinson, and M. C. Dinauer. 2004. Rac2-deficient murine macrophages have selective defects in superoxide production and phagocytosis of opsonized particles. J. Immunol. 173:5971–5979. Yazdanpanah, B., K. Wiegmann, V. Tchikov, O. Krut, C. Pongratz, M. Schramm, A. Kleinridders, T. Wunderlich, H. Kashkar, O. Utermohlen, J. C. Bruning, S. Schutze, and M. Kronke. 2009. Riboflavin kinase couples TNF receptor 1 to NADPH oxidase. Nature 460:1159–1163. Zhou, L., and D. Y. Zhu. 2009. Neuronal nitric oxide synthase: structure, subcellular localization, regulation, and clinical implications. Nitric Oxide 20:223–230.
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
6 Complement in Infections WILHELM J. SCHWAEBLE, YOUSSIF MOHAMMED ALI, NICHOLAS J. LYNCH, AND RUSSELL WALLIS
INTRODUCTION
THE COMPLEMENT SYSTEM
The complement system provides a fundamental component of the body’s immune response to invading microorganisms. The description of the bacteriolytic activity of serum or plasma by Jules Bordet led to the discovery of the complement system nearly 120 years ago. Bordet observed that bacteriolytically active serum essentially requires two components, one of which is present prior to immunization and is heat-labile, which he called alexion, and the other component, which is generated during the immunization process and is heat-stable. This component was later described as “amboceptor” by Paul Ehrlich and is now known to be an immunoglobulin, of the subclasses Igm, or IgG1, or IgG2 or IgG3. He demonstrated in 1898 that the same basic mechanisms that compose the bacteriolytic activity of immune sera are responsible for the hemolytic activity of serum towards erythrocytes of other species, a methodology that was used to analyze the biological activities of complement for decades to come. The observation that the bacteriolytic activity of immune serum is lost following heat inactivation (56°C for 30 min), but can be restored upon addition (complementation) of smaller quantities of fresh nonimmune serum, prompted Paul Ehrlich in the late 1890s to give the complement system its present name. Up to the present day, the bactericidal (or hemolytic) activity of complement mediated through the formation of a lytic membrane attack complex (MAC) (following the activation of the terminal complement components C5 to C9), is still the most widely known biological activity of the complement system. It is, however, by far not the most effective physiological mechanism by which complement contributes to the clearance of invading microorganisms from the body. This chapter highlights the various roles of the complement system in the orchestration of the immune response towards microbial infections, gives examples of microbial strategies to evade complement-mediated clearance, and discusses how acquired and inherited complement deficiencies may predispose an organism to infectious disease.
Composition and Organization of the Complement System
The complement system is composed of more than 32 components in plasma or on the cell surface, including positive and negative regulators. Activation of the complement system encompasses a series of carefully regulated initiation, amplification, and deactivation steps to adapt to specific physiological requirements (Whaley & Schwaeble, 1997). Complement activation acts through a cascade of sequential activation steps. Many complement components are zymogens that are present in plasma in their proenzymatic form and may be cleaved during the activation step and thereby converted into their enzymatically active state to form part of a multimolecular enzyme complex that cleaves and activates other complement components further downstream. Complement is activated by three pathways (Fig. 1): the classical pathway, the alternative pathway, and the lectin pathway (Schwaeble et al., 2002), all of which lead to the activation of C3, the key component of complement, and the subsequent formation of the cytolytic MAC. Following complement activation, the biologically active peptides C3a and C5a (released as a result of activation of C3 and C5) elicit a number of proinflammatory effects such as chemotaxis of leukocytes; degranulation of phagocytic cells, mast cells, and basophils; smooth muscle contraction; and increase of vascular permeability. In response to complement activation products, the inflammatory response is further amplified by subsequent generation of toxic oxygen radicals, induction of synthesis, and release of arachidonic acid metabolites and cytokines (Kirschfink & Mollnes, 2003).
The Classical Pathway
The classical pathway is triggered by C1, a multimolecular initiation complex. There is strong evidence that C1 is a major player in both the innate and the adaptive antimicrobial host defense and for the role of C1 in the maintenance of immune tolerance. The C1 complex (approximately 790 kDa) comprises a recognition subcomponent, C1q, and a heterotetramer
Wilhelm J. Schwaeble, Youssif Mohammed Ali, Nicholas J. Lynch and Russell Wallis, Department of Infection, Immunity and Inflammation, University of Leicester MSB, University Road, Leicester LE1 9HN, United Kingdom.
85
86
host defense: general
FIGURE 1 A simplified diagram of the three complement activation pathways: the classical, the lectin pathway, and the alternative pathway. Both the classical and the lectin pathway share the identical C3 and C5 convertase complexes formed after cleavage of C4 by either activated C1s (classical pathway) or activated MASP-2 (lectin pathway) (see 1) and subsequent cleavage and activation of C4b-bound C2 through activated C1s or MASP-2 (see 2). Activation of the alternative pathway is tightly controlled by membrane associated complement regulatory components and the competition of the main fluid phase antagonists factor B and factor H for binding C3b or hydrolsed C3. The affinity of factor B to bind C3b is higher on “activating “surfaces, and the half-life of C3bB, C3Bb, and C3Bb(C3b)n complexes, significantly increased by the action of properdin, allows the alternative pathway amplification loop to be formed. The alternative pathway activation loop allows C3b to be used to (i) generate more alternative pathway C3 convertases and (ii) maximize complement opsonization of activating (microbial) surfaces, or (iii) switch substrate specificity of the C3 convertases to cleave C5 through deposition of multiple C3b molecules in close proximity. This flow diagram summarizes the synergisms between the three activation pathways and points out that the initial C3b required for the alternative pathway activation loop to form can either be provided via the classical or the lectin pathway, or via spontaneous hydrolysis of C3 (C3(H2O), which, like C3b, can bind to factor B in the absence of activation of the latter two pathways.
of the C1r and C1s zymogens forming a C1q:C1r2:C1s2 complex (Fig. 2). C1q is composed of six identical subunits joined together through their collagen-like stalks that end in globular heads. Each subunit is a heterotrimer of three polypeptide chains, the C1qA-, C1qB-, and C1qC-chains, encoded by tandem genes forming the C1q cluster. Classical pathway activation is initiated either by binding of C1q to a
bacterial surface component or indirectly by binding to immune complexes. Binding of C1q to complement activators causes a conformational change in the collagenous region of C1q, leading to the autoactivation of C1r, which subsequently activates C1s. C1s then cleaves C4 and C4b bound C2, leading to the formation of the C3 convertase (C4b2a) (Arlaud et al., 2002).
6. Complement in Infections
87
FIGURE 2 Structure of the classical and lectin pathway recognition components. The basic subunits are trimers, with N-terminal collagen-like and C-terminal globular domains. In C1q, the subunit is a heterotrimer of C1qA-, B-, and C-chains and the globular heads are antibody Fc binding domains. For MBL and the ficolins, the subunits are homotrimers and the globular domains are C-type lectin and fibrinogen-like domains, respectively. Subunits assemble, via their collagenous domains into higher order oligomers, typically trimers and tetramers for MBL and the ficolins, and hexamers for C1q.
Activation of C3 exposes its reactive thiol ester group, which covalently bonds the larger cleavage product (C3b) to the activating surface. Accumulation of C3b results in the formation of C4b2a(C3b)n, the classical pathway C5 convertase.
The Lectin Pathway the lectin Pathway recognition Molecules
The lectin pathway is activated by carbohydrate recognition molecules that bind to polysaccharide on the surface of a pathogen. Two kinds of recognition molecules have been described, mannan-binding lectin (MBL) and the ficolins. The structure of these macromolecules is very similar to that of C1q. The basic subunit is a homotrimer, with an N-terminal collagen-like domain and a C-terminal carbohydrate recognition domain (CRD). These homotrimers associate via their collagenous regions to form the higherorder oligomers found in plasma. In MBL, the CRD is a C-type lectin domain, whereas in the ficolins the CRD is formed by a fibrinogen like domain (Fig. 2). MBL is present in serum as a mixture of higher oligomers (trimers, tetramers, and hexamers), and the biological activity of MBL increases with the number of oligomers (Wallis et al., 2005). Rodents and other mammals produce two different types of MBL, MBL-A and MBL-C, encoded by two separate genes, MBL1 and MBL2. In humans, MBL1 is a pseudogene and not translated, while MBL2 encodes the human orthologue to murine MBL-C.
Two of the human ficolins (L-ficolin and H-ficolin) are serum proteins that activate the lectin pathway by direct recognition of acetylated sugar moieties on the surface of pathogens. The third type of human ficolins that may activate the lectin pathway is M-ficolin, which is found on the surface of neutrophils and monocytes where it may act as a phagocytic receptor. In addition, M-ficolin forms complexes with the lectin pathway serine proteases, MASP-2 and MASP-1, and activates the lectin pathway of complement on N-acetyl glucosamine coated microtiter plates. M-ficolin was also shown to bind to certain types of bacteria such as S. aureus (Liu et al., 2005). Unlike humans, mice have only two types of ficolins, ficolin A (which is found in the serum and resembles human L-ficolin) and ficolin B (which is expressed by phagocytes and their precursors in bone marrow). In contrast to human M-ficolin, murine ficolin B does not bind to MASP-2 and does not trigger complement activation via the lectin pathway (Endo et al., 2005).
the Classical Pathway and the lectin Pathway specific serine Proteases
The protease subcomponents of the classical and the lectin activation pathway are homologous proteins that are composed modular subunits or domains, an N-terminal CUB domain (CUBI), followed by an EGF-like domain, followed by another CUB domain (CUBII), followed by two complement control protein domains (CCP1 and CCP2) and
88
host defense: general
the C-terminal serine protease (SP) domain (Schwaeble et al., 2002). In the presence of Ca21, the C1r subcomponents form a centrally located C1r dimer via binding interactions between their C-terminal SP domains and the CCP domains of their respective C1r binding partner. The N-terminal CUBI, EGF, and CUBII domains of C1r bind to the corresponding CUBI, EGF, and CUBII domains of C1s to form the heterotetramer (C1s-C1r-C1r-C1s) of approximately 340 kDa. The interaction with C1q is also Ca21-dependent. When C1 binds to a target via its globular heads, C1r activates through autocleavage. C1r then cleaves and activates its only substrate C1s. To ensure only localized activation, the SP domains of C1s remain attached to the complex via single disulfide bonds to their respective N-terminal fragments (see Color plate 3). The first substrate of C1s is C4. Once cleaved it rapidly attaches to the pathogen surface via a reactive thioester bond. C2 then binds to the C4b fragment and is also cleaved by C1s to form the C3 convertase (C4b2a), the next component of the pathway. Thus, C1 activation and subsequent steps in the reaction cascade are limited to the surfaces of pathogens and other targets. The organization of the lectin activation pathway differs considerably from that of the classical pathway. Three lectin pathway specific serine proteases, termed mannanbinding lectin-associated serine proteases (MASPs) have been described (i.e., MASP-1, MASP-2, and MASP-3) (Color plate 3). In addition to these serine proteases, an enzymatically inactive truncated form of MASP-2 of 19 kDa, called MAp19 or sMAP, is a component of the multimolecular lectin pathway activation complexes (Schwaeble et al., 2002; Stover et al., 1999). MASP-1 was described as a C1s-like serine protease associated with MBL by the pioneering work of Matsushita and Fujita (1992). In 1997, the second MBL-associated serine protease (named MASP-2) was isolated from the MBL/MASPs complex (Thiel et al., 1997). All MASPs form homodimers, which associate through binding interactions between the CUBI, EGF, and CUBII domains of each partner. In contrast to the essential requirement for the classical pathway serine protease C1s to be activated by C1r, MASP-2 on its own was shown to be sufficient to form a complex with MBL and cleave C4 and C4b-bound C2 to generate a C3 convertase (C4b2a). As in the classical pathway, the C4b2a complex converts C3 into C3a and C3b and accumulation of covalently bound C3b in close proximity subsequently leads to the formation of a C5 convertase complex C3bBb(C3b)n. Unlike MASP-2, MASP-1 is not able to cleave C4, but may cleave C2 (Rossi et al., 2001). This may explain why MASP-1 can significantly accelerate the turnover rate of lectin pathway activation (Takahashi et al., 2008). A third lectin pathway specific serine protease, MASP-3, was shown to be generated by an alternative splice process as an alternative gene product of a single structural MASP-1 gene. The substrate and physiological role of MASP-3, however, remains a mystery and since it shows no enzymatic activity that may support activation via the lectin pathway, it is considered to regulate lectin pathway functional activity by competing with MASP-2 for the binding sites of the lectin pathway recognition subcomponents (Dahl et al., 2001). A similar regulatory function has recently been demonstrated for an enzymatically inactive protein of 19 kDa, called MAp19 or sMAP (Stover et al., 1999), encoded by an alternative splice mechanism of the MASP2 gene. Like MASP-3, MAp19 binds to the serine protease interaction sites of the lectin pathway recognition complexes and may regulate lectin pathway functional activity (Iwaki et al., 2006).
MBl/MasPs Complexes
The binding between MBL and MASPs was described by several studies. Analysis of the MBL/MASPs complex from human serum revealed that MASP-1 and MAp19 have high binding affinity with trimeric MBL oligomers, whereas MASP-2 and MASP-3 mainly associated with tetrameric MBL oligomers (Dahl et al., 2001). In contrast to this finding, Teillet et al. (2005) postulated that MASPs and MAp19 have the same affinity towards trimeric and tetrameric MBL oligomers. This observation was confirmed by another study, which showed that MASPs can bind efficiently with a dimer form of rat MBL-A. MASP-1, MASP-2, MASP-3, and MAp19 form homodimers through binding via CUBI-EGF-CUBII domains in a calcium-dependant manner. The MASPs and MAp19 binding to MBL or ficolins occurs through a CUBI-EGF moiety and the strength of binding increased by the CUBII domain (Chen & Wallis, 2001). Both the serine proteases of the classical pathway as well as the serine protease of the lectin pathway bind to the same amino acid motif within the collagenous region of their respective recognition molecules. The C1 complex, however, readily dissociates at high salt concentrations, while the MBL/MASPs complex stays intact even at salt concentrations as high as 500 mM, a characteristic physiological feature used to discriminate between the two activation routes.
The Alternative Pathway
Factor B, factor D, and properdin (factor P) are specific components of the alternative pathway of complement activation. Unlike the classical and the lectin pathway, the alternative pathway is initiated through a spontaneous steady state hydrolysis of C3 to form C3(H2O), which in turn binds to factor B to form a C3(H2O)B zymogen complex. In this complex, factor B is cleaved by factor D releasing a Ba fragment while Bb remains attached to the complex formed with C3(H2O). The newly formed complex C3(H2O) Bb forms a C3 convertase enzyme and cleaves C3 into C3a and C3b. Once C3b is generated, it will bind to the surface of pathogens where it can bind to another molecule of factor B and form a new alternative pathway C3 convertase C3bBb. A new viewpoint is that properdin can bind to the surface of pathogens and initiate the alternative pathway activation by stabilization of C3bBb convertase (Kemper & Hourcade, 2008). The alternative pathway can also act as an amplification loop where C3b generated by either the classical or the lectin pathway binds to factor B, which (upon binding to C3b) is subsequently cleaved by factor D to form the alternative pathway convertase C3bBb (Schwaeble & Reid, 1999). Similar to the C3 convertase of the classical and the lectin activation pathway (i.e., C4b2a, see above), the alternative pathway convertase will switch its substrate specificity from cleaving C3 to cleaving C5 upon binding of multiple C3b molecules in close proximity.
The Membrane Attack Complex
C3 convertases produced by either of the three complement activation pathways convert C3 into C3b and C3a. Accumulation of C3b in close proximity of either C4b2a or C3bBb complexes switches the substrate specificity of these C3 convertases from C3 to C5 forming the C5 convertase complexes C4b2a(C3b)n or C3bBb(C3b)n, which cleaves C5 into C5b and C5a. C5b then binds to the cell surface and reacts with C6, C7, and C8 to form a C5b to 8 complex that leads to polymerization of C9, which inserts into the lipid bilayer of the bacterial cell membrane and initiates the MAC formation leading to cell lysis (Podack et al., 1982).
6. Complement in Infections
CONTROL OF COMPLEMENT ACTIVATION
The complement activation is tightly regulated by membrane-bound and fluid-phase regulatory components to avoid runaway activation of the enzymatic cascade that could lead to excess host tissue damage, inflammation, and depletion of complement components. Key events at the center of the cascade are carefully controlled by five closely related complement control proteins, all of which are encoded by genes located in the RCA (regulator of complement activation) cluster on chromosome 1q32 in humans. The complement regulators encoded in the RCA cluster are all composed of structural subunits of approximately 60 amino acids, called short consensus repeats (SCRs) or complement control protein (CCP) motifs. These include the membrane-bound regulators, complement receptor types 1 and 2 (CR1 and CR2), decay-accelerating factors (DAF or CD55), and membrane cofactor proteins (MCP or CD46). CR2 only binds the inactive forms of the C3 protein (i.e., C3d and C3dg); it has minimal complement regulatory functions but functions primarily as a member of the B-cell coreceptor complex and regulates the cell cycle progression during Bcell activation. CR1, MCP, and the fluid-phase regulators, factor H and C4 binding protein (C4bp), act as cofactors for factor I-mediated conversion of membrane-bound C3b and C4b into their inactivated forms, which no longer serve as components of active convertase complexes. In addition to factor I cofactor activity, CR1, C4bp, and factor H shorten the half-life of the C3 and C5 convertases by binding to complex-bound C3b and C4b and dissociating these complexes (decay-accelerating activity). This function is also mediated by DAF, which in contrast to the above, has no cofactor activity for factor I (Table 1). A truncated, 43kDa alternative splice variant of the factor H gene, now called factor H-like protein 1 (FHL-1), is also found in serum and has similar regulatory activity to factor H (Schwaeble et al., 1987). Activation of the classical and lectin pathways is controlled by C1 inhibitor (C1-INH), a serine protease inhibitor (SERPIN) that binds to C1r and C1s complexes and MASP-1 and MASP-2 complexes to prevent spontaneous activation of the proenzymes. C1-INH also removes activated serine proteases from the recognition subcomponents, blocking further activation (Chen et al., 1998).
TABLE 1
89
Clusterin and S protein are regulators for the terminal activation cascade of complement. They bind to the C5b-7 complex and prevent the insertion of C8 and C9, leading to the inhibition of the MAC formation. CD59 is another regulator, which inhibits binding of C8 and C9 to the C5b-7 complex, preventing formation of the MAC.
THE BIOLOGICAL EFFECTS OF COMPLEMENT ACTIVATION
Complement activation leads to a multitude of biological activities, including opsonization, initiation of a proinflammatory response, immune complex clearance, and direct killing of cells via the MAC. Opsonization of pathogens is mediated by the major opsonin C3b or iC3b (the hemolytically inactive cleavage product of C3b) and, to a lesser extent, C4b. C3b coats the surface of microorganisms and enhances their phagocytosis by leukocytes via binding to complement receptor type 1 (CR1) and type 3 (CR3). L-ficolin and MBL have been reported to initiate phagocytosis directly by binding to pathogens and enhance phagocytosis by binding to collectin receptors on the surface of the phagocytes (Jack et al., 2001). In other instances, complement can mediate direct killing of bacteria, especially gram-negative bacteria via the formation of the MAC, which leads to destruction of cytoplasmic membrane and cell lysis. During complement activation, proinflammatory anaphylatoxins such as C5a and C3a are released. These increase vascular permeability and promote formation of inflammatory exudates, which in turn enhance the recruitment of inflammatory mediators and inflammatory cells to the site of injury. This ensures efficient elimination of invading pathogens or other inflammatory factors. Increased vascular permeability facilitates extravasation of leukocytes to the site of inflammation and anaphylatoxins, especially C5a, act as potent chemotactic factors that stimulate leukocyte migration. In addition, C5a was found to increase the synthesis of other chemotactic agents, like ecosanoides and chemokines. Schindler et al. (1990) reported that C5a stimulates the expression of interleukin-1 (IL-1) and tumor necrosis factor (TNF) genes. Both C5a and C3a stimulate the production of monocyte chemotactant protein-1 and macrophage inflammatory protein-2 from mouse endothelial cells (Laudes et al., 2002).
Regulatory components of complement activation and their known interaction with pathogens
Complement regulatory protein
Physiological targets
Pathogens binding complement regulatory proteins (known surface receptors in parentheses)
Fluid Phase Factor H
C3b, C3bBb, C3bBb(C3b)n
Factor H-like protein 1 (FHL-1) As above C4 binding protein (C4bp) C4b, C4b2a, C4b2a(C3b)n
Borrelia burgdorferi (BbCRASPs); C. albicans (CaGpm1p); S. pyogenes (m-protein, Fba & Scl1); S. pneumoniae (PspC) As above S. pyogenes (m-protein); S. pneumoniae (PspC); C. albicans
Membrane Bound Complement receptor 1 (CR1)
C3b, C3bBb, C3bBb(C3b)n C4b, C4b2a, C4b2a(C3b)n MCP C3b, C4b S. pyogenes (m-protein); HIV Decay-accelerating factor (DAF) C3bBb, C3bBb(C3b)n, C4b2a, C4b2a Protectin (CD59) C8, C9 HIV, CMV, vaccinia virus Clusterin C8, C9 Vitronectin (S-protein) C8, C9
90
host defense: general
Complement also plays a major role in the clearance of apoptotic and necrotic cells as well as immune complexes. The globular head of C1q binds to the surface of apoptotic cells and facilitates the uptake of this complex by macrophages (Taylor et al., 2000). Opsonization of apoptotic cells with iC3b leads to recognition of these cells by CR3 and CR4 receptors on the surface of phagocytes with subsequent engulfment of these cells (Mevorach et al., 1998). Complement also inhibits the precipitation of immune complexes and enhances its solubility by binding to C1q, C3b, and C4b. This binding inhibits further increase in the size of the immune complexes. These complexes bind to CR1 on the surface of erythrocytes, which transfer them into the liver and the spleen where they are cleared from the circulation by the resident macrophages.
COMPLEMENT DEFICIENCIES
An intact complement system plays a major role in protection against infectious and noninfectious diseases. Complement deficiencies are associated with recurrent invasive bacterial infections and the development of autoimmune diseases.
Classical Pathway Deficiencies
A deficiency in any component of the classical pathway is associated with an increased risk of immunological disease and recurrent bacterial infections. Individuals with deficiencies of early complement components are more susceptible to autoimmune diseases. For example, C1q and C1s deficiency is associated with an increased risk of developing autoimmune diseases, such as systemic lupus erythematosus (SLE), mainly due to the impaired clearance of immune complexes and removal of apoptotic cells from the circulation (Carroll, 2004), and C2 deficiency was found to be a predisposing factor for atherosclerosis. Children deficient in C2 are susceptible to recurrent pneumococcal infections (Jönsson et al., 2005). C1q deficiency increases the risk of recurrent invasive bacterial infections and polymicrobial peritonitis (Celik et al., 2001). C3 and factor B deficiencies significantly increase the risk of Streptococcus pneumoniae and Pseudomona aeruginosa infection (Brown et al., 2002).
Lectin-Pathway Deficiency Deficiencies of the lectin pathway activation molecules are associated with an increased risk of recurrent bacterial infections and nonbacterial disorders. MBL deficiencies are due to three different mutations in exon 1 of the mbl2 gene located on human chromosome 10. These mutations cause single amino acid substitution at codons 52, 54, and 57. A single nucleotide polymorphism (SNP) at codon 52 leads to the substitution of glycine with aspartic acid at position 34 of the mature protein. Another point mutation at codon 54 leads to substitution of glycine with glutamic acid at position 37 of the mature protein. These two mutations disrupt the collagenous region and impair the interactions between MBL subunits during biosynthesis (Wallis & Cheng, 1999). The third mutation at codon 57 is due to the substitution of cysteine with arginine at position 32 of the mature protein. This mutation leads to the disruption of the oligomerization of MBL polypeptide chains (Wallis et al., 2004). Several polymorphisms within the promoter region of the MBL2 gene were shown to contribute to the large interindividual variations in serum MBL levels. Of those, three polymorphisms H/L, X/Y, and P/Q (at positions 2550, 2221, and 14, respectively) were the most frequently occurring promoter SNPs (Turner, 2003).
Inherited MBL deficiencies are common and most individuals with these deficiencies are healthy and show no obvious predisposition to recurrent infections or morbidity when compared with MBL-sufficient control populations (Dahl et al., 2004). Nevertheless, MBL deficiencies are found to be associated with an increased susceptibility for severe bacterial infections in children and adults especially when combined with other immunodeficiencies such as HIV infection or post-chemotherapy. MBL deficiency was also associated with high risk of developing arterial thrombosis and other cardiovascular disorders. MBL deficiency is also associated with a high risk for developing sepsis in pediatric patients. Patients with MBL deficiencies are more prone to develop rheumatoid arthritis and persistent inflammatory conditions (Turner, 2003). For L-ficolin, another recognition component of the lectin pathway, there is some evidence that low plasma levels play a significant role in recurrent respiratory tract infections in children (Atkinson et al., 2004).
Alternative Pathway Deficiency
Deficiencies of factor H and/or factor I (inherited or acquired) are associated with a typical hemolytic-uremic syndrome (aHUS), which results in thrombotic microangiopathy associated with diarrhea. Deficiencies of properdin, the only positive regulator of complement activation (which ensures the amplification of complement activation by increasing the half-life of the alternative pathway C3 and C5 convertases), result in an increased risk of infection, in particular, meningococcal infections (Schwaeble & Reid, 1999). In a mouse model of properdin deficiency, it was found that the severity of polymicrobial peritonitis was significantly increased in deficient mice when compared to their wild-type littermates (Stover et al., 2008).
THE ROLE OF COMPLEMENT IN BACTERIAL INFECTION Streptococcus pneumoniae as a Model Organism
S. pneumoniae infection is the major cause of pneumonia, otitis media, septicemia, and meningitis in the United Kingdom and it causes substantial morbidity and mortality, especially in young children and elderly patients . 65 years old (Kyaw et al., 2003). Historically, in 1900, S. pneumoniae was the major cause for children’s death in the United States, killing more than 47 of every 1,000 children per year. The increase in living standards and the improvement of nutrition limited the number of deaths even before the prevalence of effective antibiotic treatment. However, in developing regions such as Africa and Asia, pneumonia is still a major cause of the high mortality seen in children, accounting for approximately 25% of deaths in children under the age of 5 years, with more than 1.2 million infant deaths every year (Kadioglu et al., 2008). This high rate of mortality is associated with very poor living standards, malnutrition, and ineffective medical care. Older people are also subjected to the risk of S. pneumoniae infection, with a mortality rate of approximately 20% in patients older than 65 years and increasing to 40% in patients older than 85 years (Kadioglu et al., 2008). Pneumococci colonize the nasopharynx of young children, and the incidence of colonization may reach up to 40%. Several studies have reported a link between a high risk of acute and recurrent otitis media and the rate of colonization in the nasopharynx (Marchisio et al., 2003). Certain diseases like diabetes, asplenia, chronic lung diseases, and AIDS can increase the risk of pneumococcal infection.
6. Complement in Infections
Capsular Polysaccharides of S. pneumoniae
The capsular polysaccharides of S. pneumoniae (CPS) forms the outermost layer surrounding pneumococci. The thickness of the CPS is about 200 to 400 nm. To date, 91 different serological CPS serotypes have been identified (Kadioglu et al., 2008). The capsule is the major virulence factor of S. pneumoniae and encapsulated strains are generally more virulent than noncapsulated strains. Moreover, pneumococcal mutants that lack the polysaccharides capsule were less virulent than the parent strains, indicating that virulence is determined by the capsule type (Kelly et al., 1994). The thickness and the chemical structure of the CPS determine the differential ability of various serotypes to survive in blood and to cause invasive diseases. Pneumococci from different serotypes differ in their ability to cause diseases. This difference between serotypes in pathogenesis is often due to the chemical structure of CPS. Capsular polysaccharides that activate the complement system and allow the deposition of C3b, but prevent its further processing into iC3b and C3d, are more susceptible to phagocytosis and are poorly immunogenic as they can be easily cleared. In contrast, serotypes that have C3b deposited on their capsules and are quickly processed into iC3b and C3d are more resistant to phagocytosis and induce a strong humoral immune response.
Pneumococcal Cell Wall Components
Cell wall peptidoglycans and cell wall polysaccharides (CWPS) are potent inflammatory components and induce inflammatory reactions similar to those associated with S. pneumoniae infection. For example, mice injected with purified peptidoglycans or CWPS showed an inflammatory response similar to that observed after infection with S. pneumoniae and typical symptoms of pneumococcal disease, such as otitis media. These cell wall components activate the complement system and contribute to the generation of the complement anaphylatoxins C3a and C5a, which in turn enhance vascular permeability, induce mast cell degranulation, and lead to neutrophil recruitment into the inflammatory site, as described above.
Pneumolysin
Pneumolysin is a potent virulence factor produced by all serotypes of S. pneumoniae. Pneumolysin is released as a 52 kDa soluble monomer. It binds cholesterol-containing membranes and the monomeric subunits oligomerize to form a pore in the target cell membrane that leads to cell death. Pneumolysin also inhibits ciliary beating of respiratory epithelium and phagocytosis, induces cytokine synthesis, and mediates CD41T cell activation and chemotaxis (Kadioglu et al., 2004). Pneumolysin activates the classical pathway of complement in the lung after intranasal infection and induces an inflammatory response that delays the onset of bacteremia in mice. Intranasal infection of mice with the S. pneumoniae D39 mutant strain deficient in pneumolysin production showed better clearance of the pneumolysin mutant strain after 24 hours in comparison to the wild-type strain. This finding suggests that early activation of complement facilitates the clearance of pneumococci from the lung directly after infection (Jounblat et al., 2003). In contrast, prolonged activation of complement via pneumolysin during the course of pneumonia leads to severe inflammatory response, which exacerbates lung tissue damage (Kadioglu et al., 2000).
Pneumococcal Cell-surface Proteins
Amongst the cell surface proteins of S. pneumoniae, the pneumococcal surface protein A (PspA) and C (PspC) play key roles in the microbial defense against complement attack (Bergmann & Hammerschmidt, 2006).
91
PspA is expressed by all clinical isolates and appears to play a major role in the pathogenesis of S. pneumoniae. PspA interferes with complement activation and C3 deposition on the pneumococcal surface and hence it protects the bacteria from complement-mediated phagocytosis. Mice infected with PspA-deficient S. pneumoniae showed a longer survival time when compared to mice infected with the wild-type strain. Furthermore, immunization of mice with PspA prior to intravenous infection with S. pneumoniae protected the mice from the lethal sepsis and prolonged the survival time, in comparison to the nonimmunized controls (Roche et al., 2003). PspA conjugated with capsular polysaccharides (CPS) from pneumococcus serotype 23F significantly increased the immunogenicity of the capsular polysaccharides, resulting in a high antibody titer response against both CPS and PspA. When challenged with S. pneumoniae expressing PspA, mice showed a significant improvement in survival time in comparison to mice challenged with PspA alone or in combination with CPS. Increased protection in immunized mice was due to the high capacity of the produced antibodies to bind S. pneumoniae and enhance complement deposition (Csordas et al., 2008). PspC is a major virulence factor of S. pneumoniae and contributes to many different biological functions. It binds to IgA, complement component C3, and factor H. Binding to factor H protects the bacterium from complement attack and complement-mediated phagocytosis. PspC was also shown to bind to the classical and lectin pathway regulatory component C4-binding protein to protect the bacterial surface from complement attack. In addition, PspC is involved in the adherence of pneumococci to lung tissues and enhances colonization of the nasopharynx. PspC is upregulated when pneumococci adhere to the host epithelial cells in the respiratory tract and its expression is required for subsequent tissue invasion. Immunization of mice with purified PspC protects the mice from bacterial colonization and S. pneumoniae infection. Furthermore, mutants deficient in PspC have a reduced ability to colonize the nasopharynx and cause lung infection when compared to the wild-type strains. In a model of pneumococcal sepsis, mice infected with serotype 2 and serotype 3 mutant strains deficient in PspC showed a significant increase in survival as compared to the wild-type strains (Iannelli et al., 2004).
host defense against S. pneumoniae
An effective immune defense against S. pneumoniae infection depends on the collaboration between humoral and cell-mediated immunity. Mucosal immunity plays a key role in the protection against respiratory pathogens; the mucous covering the epithelial cells prevents adhesion of microbes and facilitates their removal by the ciliary movement. Extracellular killing of inhaled bacteria has been reported by several antimicrobial factors in the lung lavage, including lysozymes, iron-binding proteins, and fibronectin with subsequent clearance of bacteria by phagocytosis. Breakdown of mucosal defenses results in S. pneumoniae colonization of the upper respiratory tract and nasopharynx, increasing the risk of pneumococcal infection, which may eventually lead to bacteremia (Lamblin & Roussel, 1993). Many complement components are synthesized by alveolar macrophages and lung epithelial cells, and complement activation plays a major role in the host defense against invasive S. pneumoniae infection. Activation of complement C3 and its deposition on the surface of S. pneumoniae significantly facilitates phagocytosis by alveolar leukocytes. Opsonophagocytosis and killing of pathogens by resident alveolar macrophages are the major mechanisms leading to the clearance of invading bacteria from the lung. Because resident macrophages cannot phagocytose most strains of S. pneumoniae,
92
host defense: general
recruited PMNs (polymorphonuclear leukocytes) play the main role in clearing all strains of S. pneumoniae from the alveolar area. Opsonization of bacteria is initiated by antibodies against pneumococcal capsular polysaccharides, complement deposition, or soluble lung surfactant proteins such as surfactant protein A (SP-A), which enhance phagocytosis of S. pneumoniae in nonimmunized subjects (Kuronuma et al., 2004). Complement activation enhances bacterial clearance through opsonization and phagocytosis. C-reactive protein (CRP) is a human acute phase protein that is mainly produced in the liver. Its levels are dramatically increased in response to infection or tissue damage. CRP binds to phosphocholine residues on S. pneumoniae capsular polysaccharides in a Ca12-dependant manner and appears to activate the classical pathway of complement in human serum lacking specific S. pneumoniae antibodies (Kaplan & Volanakis, 1974). In contrast to human CRP, murine CRP is not an acute phase protein so transgenic mice expressing human CRP have been used as a model for testing the role of the human CRP in infection studies. Such transgenic mice had a significant lower mortality than nontransgenic mice. The higher resistance to infection was also associated with a significant reduction in bacteremia (Szalai et al., 1995). In another mouse model of infection, passive administration of human CRP showed a significant protective role against S. pneumoniae, with improved survival times and a significantly lower bacteremia. Interestingly, although C1q binds to CRP, leading to complement activation, the protective role of CRP appears to be complement independent. This finding was confirmed by using a mutant form of CRP that does not bind to C1q and so cannot activate the classical pathway. Passive administration of the recombinant mutant or wild-type CRP showed the same level of protection in mice following experimental S. pneumoniae infection (Suresh et al., 2006). Serum amyloid protein (SAP) is another acute phase protein that plays an important role in protection against S. pneumonia infection. SAP binds to pneumococci, increases complement deposition via the classical pathway, and thus facilitates phagocytosis. Mice deficient in SAP are defective in clearing S. pneumoniae and have significantly higher rates of mortality. Reconstitution of these mice with human SAP reduced the disease severity during the course of infection and the mice showed a clear improvement in controlling the infection with a significant increase in survival (Yuste et al., 2007).
the role of Complement in fighting S. pneumoniae Infection
As soon as pneumococci invade host tissues, the innate immune response is activated, leading to deposition of opsonins on the surface of S. pneumoniae and inducing phagocytosis. Complement activation is one of the first host defense mechanisms and the role of complement in the defense against S. pneumoniae infection has been known for nearly 100 years. During that time, numerous studies have characterized the roles of the complement system in pneumococcal infection. Recently, several gene-targeted mice with deficiencies in one or more complement components have been used to assess which pathway of the complement system is most important for protection against S. pneumoniae. C1q-deficient mice were found to be more susceptible to infection with S. pneumoniae after intranasal infection than the wild-type mice, confirming that the classical pathway has a protective role against S. pneumoniae infection. Natural IgM antibodies against capsular polysaccharides can activate the classical pathway. Mice deficient in IgM are more susceptible to
infection, with a higher bacterial load in blood and in lung tissues in comparison to the wild-type controls, and mice deficient in factor B also showed a significantly higher level of bacteria in lung tissues and in blood in comparison to the wild-type. However, when compared to the classical pathway, the degree of protection provided by the alternative activation pathway appeared to be of lesser significance. Studies are currently under way to assess the role of the lectin pathway in S. pneumoniae infection. As expected from its central role in complement activation, mice lacking C3 are most severely compromised in fighting infections. This deficiency affects the innate as well as the adaptive immune response to S. pneumoniae throughout the course of infection (Brown et al., 2002). Regardless of which pathway contributes most toward complement activation, deposition of C3b on the surface of bacteria is a key step in the downstream complement activation cascade leading to C3b-mediated phagocytosis and clearance of pathogens, as well as the formation of MAC, leading to a lysis of bacteria. As a consequence, the pneumococcus has developed several mechanisms to resist complement attack. (i) The capsular polysaccharides are a major factor in protecting pneumococci from complement attack and opsonophagocytosis (Jarva et al., 2002). (ii) The pneumococcus modulates alternative pathway activation by recruitment of factor H onto its surface to prevent complement-dependent opsonization using host factor H to shift the balance in the favor of the inhibitory regulatory mechanism. (iii) Pneumococcal surface protein A (PspA) was recently identified as a key player in the inhibition of complement deposition. This protein is highly electronegative and has an inhibitory effect on C4b deposition to the bacterial surface (Li et al., 2007). (iv) Pneumolysin also has a protective role against complement-mediated clearance. Mutagenic strains deficient in pneumolysin showed increased C3 deposition on the bacterial cell surface, highlighting the important role of pneumolysin in the pathogenesis of S. pneumoniae infection (Yuste et al., 2005).
The Roles of Complement Activation in the Pathophysiology of Polymicrobial Peritonitis
Intra-abdominal infection is a severe medical complication associated with a high level of morbidity and mortality. A breakdown of the barriers between the gut lumen and the peritoneum subsequently may lead to polymicrobial infections of the peritoneum with mixed aerobic and anaerobic commensal bacterial flora in the intestine. Polymicrobial peritonitis is a life-threatening condition frequently occurring after ruptured appendicitis or rupture of colonic diverticulum (Stover et al., 2008). Polymicrobial peritonitis is also one of the major complications of continuous peritoneal dialysis. Sepsis is characterized by massive infiltration of neutrophils into the peritoneum where they help in the clearance of the invading pathogens. When the innate immune defense fails to overcome the bacterial infection, the pathogens find their way into the blood stream and disseminate through host organs, inducing an exaggerated inflammatory response. During sepsis, macrophages, neutrophils, lymphocytes, and endothelial cells produce powerful inflammatory mediators, including TNF-a, IL-6, IL-1, and IL-8, in addition to acute phase proteins such as Creactive protein. At the same time, the complement system is shifted towards activation and produces potent proinflammatory mediators such as C5a, which stimulate the production of the cytokines and chemokines. The proinflammatory mediators produced in the early stages of sepsis recruit and activate the phagocytic cells (neutrophils and macrophages), which become hyperactive and produce reactive oxygen species (e.g., H2O2), which are effective in killing bacteria, but at the same time may cause tissue injury. The next step of sepsis
6. Complement in Infections
is characterized by production of anti-inflammatory mediators such as IL-10, transforming growth factor-b and IL-13, which down regulate the production of the proinflammatory mediators. The innate immune response then becomes hypoactive, and finally, the immune defense is suppressed, leading to the final stage of septic shock (Riedemann et al., 2003). The treatment of sepsis depends on blocking the complement anaphylatoxin C5a and its receptor C5aR. The anaphylatoxin C5a is continuously produced during complement activation and promotes potent proinflammatory effects. C5a activates phagocytic cells to release the granular enzymes and stimulates superoxide anion release from neutrophils. In addition, it causes vasodilation, increases vascular permeability, and causes T-cell apoptosis. Excessive production of C5a can lead to uncontrolled proinflammatory response and tissue damage that can lead to multiorgan failure. C5a receptors are highly expressed by many cell types in all organs such as the lungs, the liver, the kidney, and the heart. C5 mRNA expression is up regulated during sepsis. In a mouse model of CLP, blocking C5a receptors with specific polyclonal antibodies significantly improved the survival of mice in sepsis. In addition, the serum level of TNF-a and IL-6 and the bacterial loads in the blood and in different organs were significantly lower than in control mice (Riedemann et al., 2003). Mice deficient in C3 and C5 were found to be more susceptible to sepsis when compared to mice deficient in C3 only, with a significant higher rate of mortality and septicemia in mice with combined deficiency. C3 deficient mice suffered from impaired bacterial clearance due to the absence of C3b (which is the major opsonin) resulting in an absence of opsonophagocytosis. A recent report demonstrated that C3-deficient mice were able to activate C5 in the absence of C3 through a new complement activation pathway where thrombin could directly cleave C5 and release biologically active anaphylatoxin, C5a. On the other hand, mice deficient in C5 were found to have a significantly higher degree of bacteremia when compared to wild-type control. This septicemia may be due to the inability of C5-deficient mice to generate C5b, which is important for the formation of the MAC (Flierl et al., 2008). In a rat model of CLP, preimmunization of rats with antibodies against C5a improved the survival of septic rats and decreased the bacterial load in the blood and in different organs. Based on the previous data, blocking C5a and/or C5aR could be a useful therapeutic intervention during sepsis, as this treatment would still allow formation of the MAC. At the same time, blocking C5a or its receptors may decrease the inflammatory storm during sepsis, especially after successful clinical trials showed that anti-C5a treatment can decrease complement-mediated inflammatory responses following ischemia and reperfusion of the heart. In addition, reduced tissue damage in a model of intestinal ischemia/ reperfusion in rats was also observed after pretreatment of rats with anti-C5a (Wada et al., 2001).
Inhibition of Complement by Microbial Virulence Factors
Numerous microbial pathogens express virulence factors that sequester host complement control proteins to the microbial surface, effectively disguising the organism as host tissue and so avoiding complement-mediated killing. Well-documented examples include Borrelia burgdorferi, the causative agent of Lyme disease, and the human pathogenic yeast, Candida albicans, which bind factor H and FHL-1 via borrelial complement regulator-acquiring proteins (BbCRASPs) and C. albicans phosphoglycerate mutase (CaGpm1p), respectively (Siegel et al., 2008; Poltermann et al., 2007). C. albicans also sequesters host C4bp, which retains its complement regulatory activity, although the C4bp receptor has not yet been identified.
93
Streptococcus pyogenes (group A Streptococcus or GAS), the most common cause of acute pharyngitis, binds multiple complement regulatory proteins, including factor H, FHL-1, C4bp, and MCP. Binding is normally attributed to the streptococcal M-protein, although factor H, FHL-1, and C4bp will bind to GAS in the absence of M-protein. Other bacterial surface receptors have been identified for factor H and FHL-1, including fibronectin-binding protein a (Fba) and streptococcal collagen protein 1 (Scl1). Interaction of FHL-1 and Fba promotes intracellular invasion of epithelial cells by GAS (Oliver et al., 2008).
Complement Opsonization of Intracellular Bacterial Parasites
The surface of Mycobacterium avium activates complement via each of the three pathways and the deposition of C3b significantly enhances the uptake of M. avium by macrophages in vitro. It was therefore considered that complement opsonization may provide a critical entry route for mycobacteria to infect and survive in host macrophages. A recent in vivo study, however, has shown that mice deficient of C3 show a similar rate of macrophage ingestion of M. avium when compared to their C3 sufficient littermate controls (Bolhsson et al., 2001). This points to the conclusion that complement opsonization may not be essential for the infectivity of M. avium and possibly other mycobacteria in vivo.
THE ROLE OF COMPLEMENT IN VIRAL INFECTION
Over the past 80 years, there have been numerous reports indicating that the complement system, either independently or in the presence of an antibody, plays an important role in the immune defense against viral infection. The two main mechanisms by which complement may contribute to viral neutralization are either enhancement of viral phagocytosis through complement opsonization or through direct viral lysis. Many viruses have lipoprotein membranes and are therefore susceptible to complement-mediated lysis, which neutralizes viral infectivity. Viruses have, therefore, developed several strategies to protect themselves from complement attack, which contribute to viral virulence. For example, glycoprotein C of herpes simplex virus 1 (HSV-1) blocks the interaction of C5 and properdin with C3b, preventing the formation of C5 convertase (Friedman et al., 2000). Complement deposition on HIV-1 is generally thought to counteract the immune response by enhancing viral entry into host cells. However, HIV-1 is susceptible to complement-mediated lysis, and whether complement deposition is beneficial or detrimental to the virus depends upon the extent of opsonization. HIV-1 sequesters host complement-control proteins, including MCP and CD59 to regulate opsonization, and therefore protecting itself from lysis (Stoiber et al., 2008).
COMPLEMENT RECEPTOR TYPE 2 AND MEMBRANE REGULATORS AS ENTRY PORTS OF VIRAL INFECTION
The demonstration that the Epstein-Barr virus (EBV) utilizes CD21 or complement receptor type 2 (CR2), a complement receptor with a very distinct expression profile, explains why EBV exclusively infects specific cell types, such as B cells and follicular dendritic cells, where CR2 is expressed (Jacobson & Weis, 2008). Other viruses were shown to utilize membrane-attached regulators with much wider tissue distribution as ports of cellular entry and infection, such as coxsackievirus B3 and hantavirus binding to DAF or the measles virus binding to MCP or CD46 (Maisner et al., 1994).
94
host defense: general
REFERENCES Arlaud, G. J., C. Gaboriaud, N. M. Thielens, M. BudayovaSpano, V. Rossi, and J. C. Fontecilla-Camps. 2002. Structural biology of the C1 complex of complement unveils the mechanisms of its activation and proteolytic activity. Mol. Immunol. 39:383–394. Atkinson, A. P., M. Cedzynski, J. Szemraj, A. St. Swierzko, L. Bak-Romaniszyn, M. Banasik, K. Zeman, M. Matsushita, M. L. Turner, and D. C. Kilpatrick. 2004. L-ficolin in children with recurrent respiratory infections. Clin. Exp. Immunol. 138:517–520. Bergmann, S., and S. Hammerschmidt. 2006. Versatility of pneumococcal surface proteins. Microbiology 152:295–303. Bohlson, S. S., J. A. Strasser, J. J. Bower, and J. S. Schorey. 2001. Role of complement in Mycobacterium avium pathogenesis: in vivo and in vitro analyses of the host response to infection in the absence of complement component C3. Infect. Immun. 69:7729–7735. Brown, J. S., T. Hussell, S. M. Gilliland, D. W. Holden, J. C. Paton, M. R. Ehrenstein, M. J. Walport, and M. Botto. 2002. The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice. Proc. Natl. Acad. Sci. USA. 99:16969–16974. Carroll, M. C. 2004. A protective role for innate immunity in systemic lupus erythematosus. Nat. Rev. Immunol. 4:825–831. Celik, I., C. Stover, M. Botto, S. Thiel, S. Tzima, D. Kunkel, M. Walport, W. Lorenz, and W. Schwaeble. 2001. Role of the classical pathway of complement activation in experimentally induced polymicrobial peritonitis. Infect. Immun. 69:7304–7309. Chen, C. B., and R. Wallis. 2001. Stoichiometry of complexes between mannose-binding protein and its associated serine proteases. Defining functional units for complement activation. J. Biol. Chem. 276:25894–25902. Chen, C. H., C. F. Lam, and R. J. Boackle. 1998. C1 inhibitor removes the entire C1qr2s2 complex from anti-C1Q monoclonal antibodies with low binding affinities. Immunology 95:648–654. Csordas, F. C., C. T. Perciani, M. Darrieux, V. M. Goncalves, J. Cabrera-Crespo, M. Takagi, M. E. Sbrogio-Almeida, L. C. Leite, and M. M. Tanizaki. 2008. Protection induced by pneumococcal surface protein A (PspA) is enhanced by conjugation to a Streptococcus pneumoniae capsular polysaccharide. Vaccine 26:2925–2929. Dahl, M., A. Tybjaerg-Hansen, P. Schnohr, and B. G. Nordestgaard. 2004. A population-based study of morbidity and mortality in mannose-binding lectin deficiency. J. Exp. Med. 199:1391–1399. Dahl, M. R., S. Thiel, M. Matsushita, T. Fujita, A. C. Willis, T. Christensen, T. Vorup-Jensen, and J. C. Jensenius. 2001. MASP-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway. Immunity 15:127–135. Endo, Y., N. Nakazawa, Y. Liu, D. Iwaki, M. Takahashi, T. Fujita, M. Nakata, and M. Matsushita. 2005. Carbohydrate-binding specificities of mouse ficolin A, a splicing variant of ficolin A and ficolin B and their complex formation with MASP-2 and sMAP. Immunogenetics 57:837–844. Flierl, M. A., D. Rittirsch, B. A. Nadeau, D. E. Day, F. S. Zetoune, J. V. Sarma, M. S. Huber-Lang, and P. A. Ward. 2008. Functions of the complement components C3 and C5 during sepsis. FASEB J. 22:3483–3490. Friedman, H. M., L. Wang, M. K. Pangburn, J. D. Lambris, and J. Lubinski. 2000. Novel mechanism of antibodyindependent complement neutralization of herpes simplex virus type 1. J. Immunol. 165:4528–4536. Iannelli, F., D. Chiavolini, S. Ricci, M. R. Oggioni, and G. Pozzi. 2004. Pneumococcal surface protein C contributes to sepsis caused by Streptococcus pneumoniae in mice. Infect. Immun. 72:3077–3080.
Iwaki, D., K. Kanno, M. Takahashi, Y. Endo, N. J. Lynch, W. J. Schwaeble, M. Matsushita, M. Okabe, and T. Fujita. 2006. Small mannose-binding lectin-associated protein plays a regulatory role in the lectin complement pathway. J. Immunol. 177:8626–8632. Jack, D. L., N. J. Klein, and M. W. Turner. 2001. Mannosebinding lectin: targeting the microbial world for complement attack and opsonophagocytosis. Immunol. Rev. 180:86–99. Jacobson, A. C., and J. H. Weis. 2008. Comparative functional evolution of human and mouse CR1 and CR2. J. Immunol. 181:2953–2959. Jarva, H., R. Janulczyk, J. Hellwage, P. F. Zipfel, L. Bjorck, and S. Meri. 2002. Streptococcus pneumoniae evades complement attack and opsonophagocytosis by expressing the pspC locus-encoded Hic protein that binds to short consensus repeats 8-11 of factor H. J. Immunol. 168:1886–1894. Jönsson, G., L. Truedsson, G. Sturfelt, V. A. Oxelius, J. H. Braconier, and A. G. Sjoholm. 2005. Hereditary C2 deficiency in Sweden: frequent occurrence of invasive infection, atherosclerosis, and rheumatic disease. Medicine 84:23–34. Jounblat, R., A. Kadioglu, T. J. Mitchell, and P. W. Andrew. 2003. Pneumococcal behavior and host responses during bronchopneumonia are affected differently by the cytolytic and complement-activating activities of pneumolysin. Infect. Immun. 71:1813–1819. Kadioglu, A., W. Coward, M. J. Colston, C. R. Hewitt, and P. W. Andrew. 2004. CD4-T-lymphocyte interactions with pneumolysin and pneumococci suggest a crucial protective role in the host response to pneumococcal infection. Infect. Immun. 72:2689–2697. Kadioglu, A., N. A. Gingles, K. Grattan, A. Kerr, T. J. Mitchell, and P. W. Andrew. 2000. Host cellular immune response to pneumococcal lung infection in mice. Infect. Immun. 68:492–501. Kadioglu, A., J. N. Weiser, J. C. Paton, and P. W. Andrew. 2008. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat. Rev. Microbiol. 6:288–301. Kaplan, M. H., and J. E. Volanakis. 1974. Interaction of C-reactive protein complexes with the complement system. I. Consumption of human complement associated with the reaction of C-reactive protein with pneumococcal C-polysaccharide and with the choline phosphatides, lecithin and sphingomyelin. J. Immunol. 112:2135–2147. Kelly, T., J. P. Dillard, and J. Yother. 1994. Effect of genetic switching of capsular type on virulence of Streptococcus pneumoniae. Infect. Immun. 62:1813–1819. Kemper, C., and D. E. Hourcade. 2008. Properdin: New roles in pattern recognition and target clearance. Mol. Immunol. 45:4048–4056. Kirschfink, M., and T. E. Mollnes. 2003. Modern complement analysis. Clin. Diagn. Lab. Immunol. 10:982–989. Kuronuma, K., H. Sano, K. Kato, K. Kudo, N. Hyakushima, S. Yokota, H. Takahashi, N. Fujii, H. Suzuki, T. Kodama, S. Abe, and Y. Kuroki. 2004. Pulmonary surfactant protein A augments the phagocytosis of Streptococcus pneumoniae by alveolar macrophages through a casein kinase 2-dependent increase of cell surface localization of scavenger receptor A. J. Biol. Chem. 279:21421–21430. Kyaw, M. H., P. Christie, S. C. Clarke, J. D. Mooney, S. Ahmed, I. G. Jones, and H. Campbell. 2003. Invasive pneumococcal disease in Scotland, 1999–2001: use of record linkage to explore associations between patients and disease in relation to future vaccination policy. Clin. Infect. Dis. 37:1283–1291. Lamblin, G., and P. Roussel. 1993. Airway mucins and their role in defence against micro-organisms. Respir. Med. 87:421–426. Laudes, I. J., J. C. Chu, M. Huber-Lang, R. F. Guo, N. C. Riedemann, J. V. Sarma, F. Mahdi, H. S. Murphy, C. Speyer, K. T. Lu, J. D. Lambris, F. S. Zetoune, and P. A. Ward. 2002. Expression and function of C5a receptor in mouse microvascular endothelial cells. J. Immunol. 169:5962–5970.
6. Complement in Infections Li, J., D. T. Glover, A. J. Szalai, S. K. Hollingshead, and D. E. Briles. 2007. PspA and PspC minimize immune adherence and transfer of pneumococci from erythrocytes to macrophages through their effects on complement activation. Infect. Immun. 75:5877–5885. Liu, Y., Y. Endo, D. Iwaki, M. Nakata, M. Matsushita, I. Wada, K. Inoue, M. Munakata, and T. Fujita. 2005. Human M-ficolin is a secretory protein that activates the lectin complement pathway. J. Immunol. 175:3150–3156. Maisner, A., J. Schneider-Schaulies, M. K. Liszewski, J. P. Atkinson, and G. Herrler. 1994. Binding of measles virus to membrane cofactor protein (CD46): importance of disulfide bonds and N-glycans for the receptor function. J. Virol. 68:6299–6304. Marchisio, P., L. Claut, A. Rognoni, S. Esposito, D. Passali, L. Bellussi, L. Drago, G. Pozzi, S. Mannelli, G. Schito, and N. Principi. 2003. Differences in nasopharyngeal bacterial flora in children with nonsevere recurrent acute otitis media and chronic otitis media with effusion: implications for management. Pediatr. Infect. Dis. J. 22:262–268. Matsushita, M., and T. Fujita. 1992. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. J. Exp. Med. 176:1497–1502. Mevorach, D., J. O. Mascarenhas, D. Gershov, and K. B. Elkon. 1998. Complement-dependent clearance of apoptotic cells by human macrophages. J. Exp. Med. 188:2313–2320. Oliver, M. A., J. M. Rojo, S. Rodriguez de Cordoba, and S. Alberti. 2008. Binding of complement regulatory proteins to group A Streptococcus. Vaccine 8:I75–I78. Podack, E. R., H. J. Muller-Eberhard, H. Horst, and W. Hoppe. 1982. Membrane attach complex of complement (MAC): three-dimensional analysis of MAC-phospholipid vesicle recombinants. J. Immunol. 128:2353–2357. Poltermann, S., A. Kunert, M. von der Heide, R. Eck, A. Hartmann, and P. F. Zipfel. 2007. Gpm1p is a factor H-, FHL-1-, and plasminogen-binding surface protein of Candida albicans. J. Biol. Chem. 282:37537–37544. Riedemann, N. C., R. F. Guo, and P. A. Ward. 2003. Novel strategies for the treatment of sepsis. Nat. Med. 9:517–524. Roche, H., B. Ren, L. S. McDaniel, A. Hakansson, and D. E. Briles. 2003. Relative roles of genetic background and variation in PspA in the ability of antibodies to PspA to protect against capsular type 3 and 4 strains of Streptococcus pneumoniae. Infect. Immun. 71:4498–4505. Rossi, V., S. Cseh, I. Bally, N. M. Thielens, J. C. Jensenius, and G. J. Arlaud. 2001. Substrate specificities of recombinant mannan-binding lectin-associated serine protease-1 and -2. J. Biol. Chem. 276:40880–40887. Schindler, R., J. A. Gelfand, and C. A. Dinarello. 1990. Recombinant C5a stimulates transcription rather than translation of interleukin-1 (IL-1) and tumor necrosis factor: translational signal provided by lipopolysaccharide or IL-1 itself. Blood 76:1631–1638. Schwaeble, W., M. R. Dahl, S. Thiel, C. Stover, and J. C. Jensenius. 2002. The mannan-binding lectin-associated serine proteases (MASPs) and MAp19: four components of the lectin pathway activation complex encoded by two genes. Immunobiology 205:455–466. Schwaeble, W., J. Zwirner, T. F. Schulz, R. P. Linke, M. P. Dierich, and E. H. Weiss. 1987. Human complement factor H: expression of an additional truncated gene product of 43 kDa in human liver. Eur. J. Immunol. 17:1485–1489. Schwaeble, W. J., and K. B. Reid. 1999. Does properdin crosslink the cellular and the humoral immune response? Immunol. Today 20:17–21. Siegel, C., J. Schreiber, K. Haupt, C. Skerka, V. Brade, M. M. Simon, B. Stevenson, R. Wallich, P. F. Zipfel, and P. Kraiczy. 2008. Deciphering the ligand-binding sites in the Borrelia burgdorferi complement regulator-acquiring surface protein 2 required for interactions with the human immune regulators factor H and factor H-like protein 1. J. Biol. Chem. 283:34855–34863.
95
Stoiber, H., A. Soederholm, D. Wilflingseder, S. Gusenbauer, A. Hildgartner, and M. P. Dierich. 2008. Complement and antibodies: a dangerous liaison in HIV infection? Vaccine 8:I79–I85. Stover, C. M., J. C. Luckett, B. Echtenacher, A. Dupont, S. E. Figgitt, J. Brown, D. N. Mannel, and W. J. Schwaeble. 2008. Properdin plays a protective role in polymicrobial septic peritonitis. J. Immunol. 180:3313–3318. Stover, C. M., S. Thiel, M. Thelen, N. J. Lynch, T. Vorup-Jensen, J. C. Jensenius, and W. J. Schwaeble. 1999. Two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene. J. Immunol. 162:3481–3490. Suresh, M. V., S. K. Singh, D. A. Ferguson Jr, and A. Agrawal. 2006. Role of the property of C-reactive protein to activate the classical pathway of complement in protecting mice from pneumococcal infection. J. Immunol. 176:4369–4374. Szalai, A. J., D. E. Briles, and J. E. Volanakis. 1995. Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice. J. Immunol. 155:2557–2563. Takahashi, M., D. Iwaki, K. Kanno, Y. Ishida, J. Xiong, M. Matsushita, Y. Endo, S. Miura, N. Ishii, K. Sugamura, and T. Fujita. 2008. Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway. J. Immunol. 180:6132–6138. Taylor, P. R., A. Carugati, V. A. Fadok, H. T. Cook, M. Andrews, M. C. Carroll, J. S. Savill, P. M. Henson, M. Botto, and M. J. Walport. 2000. A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo. J. Exp. Med. 192:359–366. Teillet, F., B. Dublet, J. P. Andrieu, C. Gaboriaud, G. J. Arlaud, and N. M. Thielens. 2005. The two major oligomeric forms of human mannan-binding lectin: chemical characterization, carbohydrate-binding properties, and interaction with MBL-associated serine proteases. J. Immunol. 174:2870–2877. Thiel, S., T. Vorup-Jensen, C. M. Stover, W. Schwaeble, S. B. Laursen, K. Poulsen, A. C. Willis, P. Eggleton, S. Hansen, U. Holmskov, K. B. Reid, and J. C. Jensenius. 1997. A second serine protease associated with mannan-binding lectin that activates complement. Nature 386:506–510. Turner, M. W. 2003. The role of mannose-binding lectin in health and disease. Mol. Immunol. 40:423–429. Wada, K., M. C. Montalto, and G. L. Stahl. 2001. Inhibition of complement C5 reduces local and remote organ injury after intestinal ischemia/reperfusion in the rat. Gastroenterology 120:126–133. Wallis, R., and J. Y. Cheng. 1999. Molecular defects in variant forms of mannose-binding protein associated with immunodeficiency. J. Immunol. 163:4953–4959. Wallis, R., N. J. Lynch, S. Roscher, K. B. Reid, and W. J. Schwaeble. 2005. Decoupling of carbohydrate binding and MASP-2 autoactivation in variant mannose-binding lectins associated with immunodeficiency. J. Immunol. 175:6846–6851. Wallis, R., J. M. Shaw, J. Uitdehaag, C. B. Chen, D. Torgersen, and K. Drickamer. 2004. Localization of the serine protease-binding sites in the collagen-like domain of mannose-binding protein: indirect effects of naturally occurring mutations on protease binding and activation. J. Biol. Chem. 279:14065–14073. Whaley, K., and W. Schwaeble. 1997. Complement and complement deficiencies. Semin. Liver Dis. 17:297–310. Yuste, J., M. Botto, S. E. Bottoms, and J. S. Brown. 2007. Serum amyloid P aids complement-mediated immunity to Streptococcus pneumoniae. PLoS Pathog. 3:1208–1219. Yuste, J., M. Botto, J. C. Paton, D. W. Holden, and J. S. Brown. 2005. Additive inhibition of complement deposition by pneumolysin and PspA facilitates Streptococcus pneumoniae septicemia. J. Immunol. 175:1813–1819.
This page intentionally left blank
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
7 Immune Defense at Mucosal Surfaces MARIAN R. NEUTRA AND JEAN-PIERRE KRAEHENBUHL
INTRODUCTION
GENERAL ORGANIZATION OF THE MUCOSAL IMMUNE SYSTEM
The mucosal surfaces of the body together represent a vast surface area separated from the outside world only by delicate epithelial barriers. The epithelia covering mucosal tissues face a daunting task. This is illustrated dramatically in the intestine, whose luminal microbial populations form complex ecosystems that are essential for digestion, epithelial maintenance, normal immune function, and protection against pathogens (Backhed et al., 2005; Pédron & Sansonetti, 2008). In the human colon, for example, hundreds of commensal bacterial species are present, at densities up to 1012 per gram of luminal contents (MacPherson & Harris, 2004). Thus, it is not surprising that mucosal tissues are extremely immunologically active; for example, the number of antibody-producing B cells in the lamina propria of the intestine is estimated to be greater than in any other organ in the body including the spleen, thymus, and lymph nodes (Brandtzaeg et al., 2005). Although antigen-specific effector and memory lymphocytes are present throughout mucosal tissues, the initial inductive events that lead to their formation occur primarily in organized mucosa-associated lymphoid tissues (MALT), located either within the mucosa or in the lymph nodes that drain the mucosa (McGhee et al., 2005). The basic mechanisms of antigen processing and presentation and lymphocyte differentiation that underlie mucosal immune responses are essentially the same as in the systemic immune system. However, the mucosal immune system faces unique challenges. Foreign antigens must be sampled by immunologically competent cells without compromising the epithelial barrier. In addition, epithelial and antigenpresenting cells must distinguish and respond to potential antigenic and pathogenic threats in an environment rich in commensal microorganisms and nutrients. This chapter will review our current understanding of how this complex task is accomplished, with particular focus on mucosal immunity in the intestine.
Organized Mucosa-Associated Lymphoid Tissues: Local Inductive Sites
Organized MALT, recognized microscopically by the presence of lymphoid follicles, are sites in which immune responses are generated. The best-known example is the Peyer’s patches of the intestine, where follicles are aggregated in large clusters that are visible to the naked eye. The composition and function of these tissues have been described in detail and extensively reviewed (Brandtzaeg et al., 2005; MacPherson et al., 2008; Cerutti, 2008). Mucosal follicles, like those of lymph nodes, consist primarily of a cluster of immature B cells, often including a germinal center supported by a network of follicular dendritic cells. Mucosal follicles are flanked by interfollicular T-cell areas that contain a distinct population of mature, antigen-presenting dendritic cells, naive or antigen-sensitized T cells, and high endothelial venules that allow entry and exit of migrating cells (Kelsall, 2008). Each mucosal follicle is separated from the overlying epithelium by a subepithelial “dome” region filled with T and B cells, along with numerous dendritic cells (Brandtzaeg et al., 2005; Kelsall, 2008). Cells of the dome region function in close collaboration with the follicle-associated epithelium (FAE), which delivers antigens and microorganisms from the lumen into the subepithelial tissue. These sites are organized to allow antigen sampling in an environment where incoming foreign materials and pathogens can be immediately captured, processed, and presented for induction of appropriate immune responses (Neutra et al., 2001). Major organized MALT structures appear during fetal life at predetermined sites positioned to respond to incoming antigens and pathogens that challenge the mucosa immediately after birth. They persist throughout life in the distal small intestine (Peyer’s patches), tonsils and adenoids, appendix, and (in some species) the large intestine. In humans, the oral pharynx is monitored by a ring of mucosal lymphoid tissues that include the palatine tonsils, lingual tonsils, and adenoids. In rodents (but not humans) a strip of organized lymphoid tissue lies along the base of the nasal cavity. Solitary or isolated lymphoid follicles (ILF) and
Marian R. Neutra, Department of Pediatrics, Harvard Medical School, GI Cell Biology Laboratory, Children’s Hospital, Boston, MA 02115. Jean-Pierre Kraehenbuhl, Health Sciences eTraining (HSeT) Foundation, CH 1066 Epalinges, Switzerland.
97
98
HOST DEFENSE: GENERAL
associated T-cell clusters, only visible microscopically, occur in many mucosal locations and are common throughout the gastrointestinal tract (Wang et al., 2008). They appear only after birth, induced by exposure to antigens and microorganisms. In the human digestive tract, ILFs are most frequent where microbial populations are abundant, such as in the large intestine, cecum, and rectum (McGhee et al., 2005). ILFs in the trachea and bronchi are generally present only under conditions of antigenic challenge (Bienenstock & Clancy, 2005). ILFs are also common at mucocutaneous transitions such as the anal-rectal junction and near the ducts of secretory glands that empty onto mucosal surfaces. The initial event leading to an ILF is not clear, but local antigen entry or epithelial signals are likely to be involved. An early stage of ILF formation is recognizable as a small cluster of T lymphocytes near an intestinal crypt, first described as “cryptopatches” in mice. ILF formation involves a distinct sequence of events including entry of migratory dendritic cells and distinct lymphocyte populations and induction of a follicle-associated epithelium (Wang et al., 2008). The “mature” ILF structure is functionally analogous to Peyer’s patches, capable of antigen sampling and induction of immune responses.
Mucosal Tissues as Immune Effector Sites The Lamina Propria: A Diffuse Lymphoid Tissue
Most mucosal cells involved in immune defense are distributed diffusely throughout the subepithelial connective tissue and are directly or indirectly responsible for immune effector functions. Mucosal effector lymphocytes include terminally differentiated, antibody-producing B cells, helper and regulatory CD41 and cytotoxic CD81 T cells, and NK cells (Brandtzaeg et al., 2005; Lefrançois & Puddington, 2006). Memory B and T cells are also stationed in the mucosa to provide rapid effector responses to local antigen reexposure. Indeed, the great majority of T cells in the lamina propria are memory/effector cells. Mucosal macrophages phagocytose pathogens and macromolecular debris while dendritic cells monitor the mucosa for incoming antigens (Kelsall, 2008; Coombes & Powrie, 2008). Collectively, these cells control the populations of beneficial commensal microorganisms associated with the mucosa, prevent colonization and entry of pathogens, and destroy pathogens and infected cells if invasion occurs.
Intraepithelial Lymphocytes and Immune Surveillance
Throughout the small and large intestines, a specialized population of lymphocytes resides in the lateral intercellular spaces between epithelial cells. These intraepithelial lymphocytes (IELs) are primarily T cells that express Tcell receptors (TCRs) capable of recognizing foreign antigens presented on epithelial cell surfaces (Kunisawa et al., 2007; Ishikawa et al., 2007). IELs form a heterogeneous cell population that consists of both CD41 and CD81 cells (Lefrançois & Puddington, 2006). In the upper airways most IELs are CD41 cells, while in the gut CD81 cells expressing either a or T-cell receptors represent the major IEL population. In rodents, T cells predominate, but in the human intestine most IELs express a TCRs; the frequency of these subtypes varies among species and between the small and large intestine. The TCRs of IELs detect antigens presented on epithelial cells, either by classical MHC I molecules (Das & Janeway, 2003; Shastri et al., 2005) or nonclassical MHC molecules such as CD1 (Cohen et al., 2009). Intestinal IELs are oligoclonal and, like skin dendritic T lymphocytes, they probably participate in epithelial monitoring and repair processes rather than in immune
defense. Infection of epithelial cells may result in presentation of peptides on MHC I and/or presentation of microbial lipid antigens on CD1d (Nieuwenhuis et al., 2009). Some IELs also express receptors typical of NK T cells that recognize a nonclassical MHC molecule called MICA/B (MHC class I-related gene A/B) expressed on damaged epithelial cells (Kunisawa et al., 2007). There is evidence that stimulated IELs can induce epithelial apoptosis, dampen the responses of mucosal CD4 cells, and secrete factors that stimulate epithelial cell proliferation. IELs, like other mucosal lymphocytes, migrate into the intestinal mucosa by adhering to adhesion molecules on mucosal venules and by following chemokine gradients generated by epithelial cells, as discussed below. Additional adhesion molecules are involved in the retention of IELs in their intraepithelial niche. IELs express the integrin aE 7 that binds to E-cadherin, an adhesin that holds the lateral membranes of epithelial cells together though calcium dependent, homotypic interactions (Agace et al., 2000). In E–cadherin-deficient mice, IELs are reduced but not absent, indicating that alternate docking mechanisms exist. Although the functions of IELs are not completely understood, they appear to play an important role in epithelial maintenance by eliminating damaged or infected epithelial cells and promoting epithelial cell replication and replacement.
SAMPLING OF ANTIGENS AND PATHOGENS AT MUCOSAL SURFACES Barriers to Antigens and Microorganisms at Mucosal Surfaces
Luminal microorganisms are generally excluded from close contact with epithelial cell surfaces by the interplay of mucous and fluid secretions, antimicrobial peptides, and mucosal clearance mechanisms such as ciliary activity and peristaltic movements. Secretory mucins, the products of genes such as muc2, are large, heavily glycosylated, and negatively charged glycoproteins that are secreted onto mucosal surfaces by goblet cells. Mucins form highly hydrated, loose gels that allow diffusion of large molecules including immunoglobulins (Cone, 2005). However, microorganisms coated with antibody are immobilized in mucus gels due to multiple low-affinity interactions of immunoglobulins with mucins, which promotes entrapment and clearance. The luminal side of the mucus layer is continually eroded and dispersed into the lumen as it is replenished from the epithelium below. Defensins, and other antibacterial proteins such as lysozyme, are secreted by Paneth cells in the crypts (Bevins, 2006; Ouellette, 2006). In addition, S-IgA exported by crypt cells provides nonspecific defense by adhering to lectin-like determinants on certain microorganisms through its carbohydrate side chains or through other binding mechanisms, to block adherence and to entrap potential pathogens (Phalipon & Corthesy, 2003). Surface specializations of epithelial cells are also important for mucosal protection against microorganisms. In the intestines, the apical plasma membranes of enterocytes have closely packed microvilli coated with a thick layer of integral membrane mucins called the filamentous brush border glycocalyx. This coat serves as a diffusion barrier that prevents contact of most microorganisms with integral components of the enterocyte plasma membrane and impedes access to the small intermicrovillar membrane domains involved in endocytosis (Frey et al., 1996; Neutra & Kraehenbuhl, 2005). These membrane-associated mucins adsorb pancreatic proteases and contain enterocyte enzymes involved in terminal digestion, as well as intestinal alkaline phosphatase; together, they protect against microbial contact
7. Immune Defense at Mucosal Surfaces
and degrade microbial products such as lipopolysaccharide (LPS). Membrane mucins and enzymes are continually shed from the surfaces of enterocytes and replaced, presumably to release attached microbes and renew the epithelium’s protective coat.
Epithelial Cells as Sensors of the Mucosal Environment
Epithelial cells are active participants in both innate and adaptive mucosal immune responses, providing the mucosal immune system with a constant stream of information about the contents of the lumen. Epithelial cells respond to microbes and toxins by sending cytokine and chemokine signals to underlying mucosal cells to trigger innate, nonspecific defenses and to promote adaptive immune responses in a variety of ways (Backhed et al., 2005; Rimoldi et al., 2005; Kagnoff, 2006; Rakoff-Nahoum & Medzhitov, 2008). Epithelial cells can detect the presence of microbial components through receptors that recognize common molecular components unique to microorganisms such as flagellin, proteoglycans, lipopolysaccharides, and viral or bacterial nucleic acid sequences. These “pattern-recognition receptors” include Toll-like receptors (TLRs) on epithelial cell membranes and nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) in the enterocyte cytosol (Rakoff-Nahoum & Medzhitov, 2008). In intestinal epithelial cells, TLRs and NLRs (NOD1 and NOD2) influence gene expression through a complex system of intracellular signaling pathways governed by adaptors and enzymes that activate specific transcription factors, which in turn promote expression of proinflammatory and/or immunomodulatory cytokines and chemokines. While maintaining the ability to respond vigorously to pathogens, normal intestinal epithelial cells prevent unwanted inflammatory responses to commensal organisms and nutrients by negatively regulating intracellular signal-
99
ing pathways through cytoplasmic factors such as A20 (a ubiquitin-modifying enzyme), SOCS1 (a suppressor of cytokine signaling), and PPAR gamma (an inhibitor of NFB activity) (Honda & Takeda, 2009). Thus epithelial cells can mount diverse responses to microbial components including inflammation, enhancement of adaptive immunity, or epithelial cell proliferation. Indeed, normal maintenance and renewal of the intestinal epithelium is dependent on controlled TLR-mediated responses (Rakoff-Nahoum & Medzhitov, 2008) and disruption of the flora (e.g., by antibiotics) leads to increased vulnerability to pathogens. Epithelial cells also respond to TLR ligands by releasing chemokines such as CCL20 and CCL25 that attract DCs and lymphocytes into the mucosa (Sierro et al., 2001; Vijay-Kumar & Gewirtz, 2008) and cytokines such as BAFF and APRIL that induce B-cell proliferation and IgA class switching at mucosal effector sites (Shang et al., 2008; Cerutti, 2008). Thus, the epithelium actively contributes to the maintenance of the mucosal barrier and promotes innate and adaptive mucosal protection.
Collaboration of Epithelial Cells with Dendritic Cells in Antigen Sampling
Induction of mucosal immune responses is complicated by the fact that antigens and microorganisms on mucosal surfaces are separated from cells of the mucosal immune system by epithelial barriers. Thus, the mucosal immune system must obtain samples of the external environment without compromising the integrity and protective functions of the epithelium (Neutra et al., 2001). The cellular organization of the local epithelial barrier determines the way in which samples are obtained, but in all cases, a close collaboration between epithelial cells and DCs is essential (Fig. 1). In stratified epithelia that lack tight junctions, such as those in the oral cavity, vagina, or anal canal, motile dendritic (or Langerhans) cells invade the epithelium to capture
FIGURE 1 Antigen sampling across epithelial barriers. Antigen sampling strategies at mucosal surfaces involve collaborations between epithelial and dendritic cells (DCs). The diverse mechanisms involved are adapted to the nature of the local epithelial barrier. At most mucosal surfaces where the epithelium is stratified, pseudostratified, or simple columnar, DCs are stationed immediately under the epithelium, migrate into the epithelial layer, and may extend dendrites into the lumen to capture antigens. These DCs generally travel to the nearest draining lymph node to present antigen to T cells. At sites of organized mucosal lymphoid tissues, specialized M cells in the lymphoid follicle-associated epithelium deliver antigens by transcytosis across the epithelial barrier, directly to intraepithelial and subepithelial DCs. These DCs then migrate to adjacent mucosal Tcell areas to present antigen. (Reproduced from Neutra & Kozlowski, 2006).
100
HOST DEFENSE: GENERAL
foreign macromolecules or microorganisms in the intercellular spaces. Antigen-loaded DCs then migrate to T-cell areas of local mucosal lymphoid tissues (as in the tonsil) or distant lymph nodes (such as those that drain the genital tract). In simple epithelia such as those lining the intestine where intercellular spaces are sealed by tight junctions, samples of luminal material are obtained using multiple strategies. In the follicle-associated epithelium overlying organized MALT, epithelial M cells efficiently transport samples of luminal material directly across the barrier. In other locations, epithelial cells may take up small amounts of antigen, or DCs may extend dendrites through the tight junctions to capture particles or microbes on the mucosal surface. These strategies are described in more detail below.
Specialized Antigen Sampling at Organized Mucosal Inductive Sites
Throughout the intestines, multiple crypts provide a continuous supply of fresh epithelial cells to each villus or intercrypt area. Where mucosal lymphoid follicles occur, epithelial cells emerging from the adjacent follicle-associated crypts migrate onto the dome formed by the underlying lymphoid follicle to form the FAE (Kraehenbuhl & Neutra, 2000). Gene expression in cells of the FAE is strongly influenced by factors produced by cells of the underlying follicle, with the result that the FAE differs dramatically from the villus epithelium (Debard et al., 2001). New sites of FAE and M cells appear in vivo where ILFs assemble in response to microbial challenge (Wang et al., 2008) or after injection of Peyer’s patch cells into the mucosa, and enterocytes in intestinal adenocarcinoma cell monolayers grown in vitro acquire M–cell-like features when cocultured with B cells or certain cytokines (Kernéis et al., 1997; Kraehenbuhl & Neutra, 2000). Thus, the distinctive patterns of FAE gene expression and M cell differentiation appear to be dependent on cells of the underlying follicle. The entire FAE presents a distinct biochemical “face” to the lumen and allows macromolecules, particles, and microorganisms relatively easy access to these infrequent areas (Neutra & Kraehenbuhl, 2005). Whereas the villus epithelium is dominated by absorptive enterocytes, mucin-secreting goblet cells, and enteroendocrine cells, the FAE contains few or no goblet or enteroendocrine cells but does contain M cells and enterocytes displaying a FAE-specific phenotype. Follicleassociated cells produce very low levels of digestive enzymes and little or no mucus. Follicle-associated crypts lack defensinand lysozyme-producing Paneth cells and do not secrete IgA into the lumen, as they lack polymeric immunoglobulin receptors. Gene expression profiling studies have revealed that the FAE expresses distinctive receptors, extracellular matrix and matrix-interacting proteins (Lo et al., 2004; Anderle et al., 2005; Hase et al., 2006), and chemokines. Gene expression in the FAE may also be modulated by the microorganisms that contact the FAE or are transported by M cells into the mucosa. For example, CCL20, a chemokine that attracts DCs and lymphocytes that express the chemokine receptor CCR6, is constitutively expressed in the small intestine only in the FAE in mice and humans, but expression is upregulated by TLR ligands (Sierro et al., 2001) and becomes more widespread during intestinal infection (Kelsall, 2008). Cells of the mouse FAE also express CCL9 (analogous to CCL23 in humans) that attracts CCR1-expressing myeloid DCs (Kelsall, 2008) and CXCL16 that attracts CXCR6-expressing B and T lymphocytes into Peyer’s patches (Hase et al., 2006). The major function of M cells in the FAE is to deliver samples of particulate foreign material and microorganisms by transepithelial transport from the lumen to organized mucosal lymphoid tissues (Neutra et al., 2001; Neutra & Krae-
henbuhl, 2005). M cells create an intraepithelial “pocket” that provides a sequestered space for activated or memory B and T lymphocytes and occasional DCs. The pocket dramatically shortens the transcytotic pathway and provides for rapid delivery of luminal samples to intraepithelial and subepithelial cells. The apical membranes of M cells are designed to allow adherence and uptake of antigens and microorganisms by phagocytosis or endocytosis. In addition, certain pathogens can induce macropinocytotic engulfment involving disruption of the apical cytoskeletal organization, although this may lead to M-cell death. Access of microbes and particles to M-cell membranes is facilitated by the lack of an organized brush border and absence of the thick, protective glycocalyx of integral membrane mucins that blankets the microvillus tips on enterocytes (Frey et al., 1996). The enhanced accessibility of M-cell membranes promotes M-cell-specific adherence of viruses and particles, whether or not the participating receptors are unique to M cells (Mantis et al., 2000). In some species, M cells express proteins that could potentially be exploited as receptors for microorganisms, including beta 1 integrin, ICAM-1, annexin, and peptidoglycan-binding protein. They also display carbohydrate structures that differ from those of neighboring epithelial cells, although M-cell glycosylation patterns vary among species, between different mucosal regions, and even within the same FAE (Neutra & Kraehenbuhl, 2005). Luminal IgA or S-IgA (but not IgG or IgM) adheres specifically to apical membranes of M cells in experimental animals and humans via an unknown mechanism (Mantis et al., 2002), an interaction that may promote immune sampling of IgA-coated microbes or luminal antigen-IgA complexes (Kadaoui & Corthesy, 2007). Antigens and pathogens released into the M-cell pocket immediately contact intraepithelial B and CD41 T cells that display the antigen CD45RO typical of activated or memory cells. The B cells express MHC class II, suggesting that they are capable of antigen presentation (Brandtzaeg et al., 2005). DCs also migrate into the pocket from the subepithelial dome region, and DC migration toward and into the FAE is enhanced when the mucosa is exposed to TLR ligands or enterotoxins, most likely due to release of chemokines from the epithelium (Sierro et al., 2001; Chabot et al., 2008). These DCs, along with the intrapocket and subepithelial DCs and lymphocytes, are well-positioned for efficient exposure or reexposure to incoming antigens, but whether the cells within the M cell pocket have unique functions is unknown.
Antigen Sampling at Widespread Mucosal Effector Sites
Although the tight junctions that join cells of the absorptive intestinal epithelium are impermeable to macromolecules, soluble antigens and bacteria are routinely sampled by lamina propria DCs. This was previously thought to be due to limited, nonspecific transcellular transport by villus enterocytes. It is now clear that more specific mechanisms are in place for widespread antigen sampling. For example, epithelial cells in the intestines and airways of human adults express the neonatal Fc receptor FcRn that recognizes IgGantigen complexes, providing a potential mechanism for sampling of luminal antigens or microorganisms that have been previously encountered (Yoshida et al., 2006). In addition, subepithelial DCs can directly sample luminal contents by sending extensions that reach between epithelial cells, through the tight junctions, and into the intestinal lumen (Rescigno et al., 2001; Neiss & Reinecker, 2006). Although the frequency of this phenomenon is not clear, the fact that noninvasive Salmonella enterica serovar Typhimurium and
7. Immune Defense at Mucosal Surfaces
Escherichia coli were taken up in this way after oral administration to mice suggests that this may be the way that lamina propria DCs routinely sample commensal microorganisms (MacPherson & Uhr, 2004). Indeed, commensal bacteria have been detected within LP DCs. However, commensal bacteria are also observed in the DCs of Peyer’s patches and it is likely that sampling of the endogenous flora occurs in both locations (Kelsall, 2008).
EXPLOITATION OF ANTIGEN SAMPLING MECHANISMS BY PATHOGENS
Mucosal mechanisms for sampling of antigens and pathogens, intended by the host for immune protection and surveillance, creates pathways across the epithelial barrier that can be exploited by pathogens to initiate infection (Neutra et al., 2002). Although the rigid, glycocalyx-coated brush border of enterocytes presents a barrier to endocytosis of particles, certain bacterial and parasitic pathogens have evolved elaborate mechanisms for establishing infection on the epithelial surface, within enterocytes, or in the mucosa. Some adhere and form dense colonies on enterocyte apical surfaces (e.g., Vibrio cholerae), and others co-opt the host cell’s membrane and cytoskeletal machinery to induce novel structures on enterocyte apical poles (e.g., attaching/effacing E. coli and Cryptosporidia) or induce macropinocytosis (e.g., Salmonella). M cells, in contrast, are easily exploited as they are designed to constitutively transcytose adherent particles such as bacteria and viruses and deliver them to phagocytic DCs for degradation and antigen presentation. Although the transport vesicles in M cells can acidify their content and contain proteases, M-cell transport is rapid and pathogens can survive and go on to infect the epithelium itself; target cells in the mucosa; or cells in draining lymph nodes, spleen, or liver. Enteric bacteria that exploit M-cell transport have diverse pathogenic strategies that are described in detail elsewhere in this volume. Briefly, M–cell-transported Shigella invades local epithelial cells through their basolateral surfaces, and phagocytosis of Shigella by macrophages and DCs causes release of proinflammatory cytokines, massive inflammation, and widespread disruption of the epithelial barrier (Sansonetti, 2006). Enteric Yersinia selectively adheres to M cells via its outer membrane protein invasin that recognizes apical M-cell beta1 integrins. After transport, it avoids phagocytosis by injecting a set of Yop proteins directly into the phagocyte cytoplasm via a type III secretion system, which disrupt the cytoskeleton and lead to phagocyte apoptosis (Handley et al., 2005). Salmonella adheres preferentially (but not exclusively) to M cells and injects proteins via a type III secretion system to disrupt actin networks and induce macropinocytosis (Ellermeier & Slouch, 2007). It is phagocytosed by subepithelial DCs, but survives by inducing formation of a nondegradative phagosome and uses migration of phagocytes out of the mucosa to facilitate systemic dissemination (Vasquez-Torres et al., 1999). Studies in experimental animals have shown that the M-cell pathway can be exploited for intestinal invasion by other bacterial pathogens, including Campylobacter jejuni in rabbits, Mycobacterium paratuberculosis in calves, and BCG (bacillus Calmette-Guerin) in rabbits (Neutra et al., 2002). Transport of several pathogenic viruses by M cells has been documented. Viruses lack elaborate signaling or injection systems and generally depend on key surface proteins to adhere to host cells. Enteric viruses are designed to survive the proteolytic environment of the intestine, and adherence to M cells is sufficient to assure endocytosis and transcytosis, as M cells constitutively transport particles of this size. For
101
example, two closely related viral pathogens—reovirus in mice and poliovirus in humans—have been shown to selectively adhere to M cells and to use this as an invasion route (Neutra et al., 2002). Reovirus exploits proteases in the intestinal lumen for reconfiguration of the viral outer caspsid and extension of the attachment protein sigma 1, which mediates M-cell adherence. The sigma 1 protein of reovirus type 1 recognizes a specific sialic acid-containing trisaccharide that is present on all intestinal epithelial cell apical surfaces but is accessible to virus-sized particles only on M cells (Helander et al., 2003). Mucosal transmission of SIV (simian immunodeficiency virus) in nonhuman primates, and presumably of HIV (human immunodeficiency virus) in humans, can occur without epithelial damage by the oral, rectal, and genital mucosa (Haase, 2005). The molecular basis of HIV adherence to epithelia in vivo is not established, but studies using neoplastic intestinal epithelial cell lines in culture have shown that a combination of the ubiquitous glycolipid galactosylceramide and the chemokine receptor CCR5 can mediate HIV adherence and uptake (Fotopoulos et al., 2002). Epithelial cells themselves are not productively infected by HIV. Evidence from SIV studies in nonhuman primates indicates that depending on the nature of the local mucosal epithelium, HIV may exploit multiple mucosal antigen sampling mechanisms at different mucosal sites, including vesicular transepithelial transport by M cells, direct infection of intraepithelial T cells, and capture by intraepithelial or subepithelial DCs (Haase, 2005). As mucosal antigenpresenting cells interact with local CD41 T cells, they unwittingly infect and ultimately disable the very cells needed to mount an effective immune response. Infection of local target cells and dissemination of virus to regional lymph nodes and other tissues occurs rapidly after deposition of virus on mucosal surfaces. Whether transmitted mucosally or injected, HIV and SIV preferentially replicate in mucosal tissues such as the intestinal mucosa (Veazey et al., 2001) due to the abundance of activated T cells expressing CD4, CCR5, and a4/7 integrin, all of which are exploited as receptors for the HIV envelope glycoprotein gp120 (Brenchley & Douek, 2008).
INDUCTION OF MUCOSAL IMMUNE RESPONSES
The events involved in induction and maintenance of secretory IgA responses involve coordinated interactions among many cell types (Cerutti, 2008). The sites at which these interactions can occur are distinct and have distinct outcomes. In all cases, foreign antigens must be transported across epithelial barriers and captured by mucosal dendritic cells, as discussed in detail above. Distinct subpopulations of mucosal DCs play important roles not only in capturing antigens but also in orchestrating the nature of the resulting mucosal immune responses (Kelsall, 2008).
T-Cell Dependent Responses in Organized MALT
Initiation of primary mucosal immune responses to Tdependent (such as protein) antigens requires processing and presentation of antigen to T cells by DCs in organized lymphoid tissues. DCs are influenced by the microenvironment of mucosal tissues to assume unique phenotypes and functions, distinct from DCs in nonmucosal sites. Mucosal DCs are further differentiated with multiple subtypes in organized lymphoid tissues and in the lamina propria (Kelsall, 2008). Immature dendritic cells are present in large numbers in the subepithelial dome region and in smaller numbers within the FAE. Mucosal exposure to live bacteria, bacterial toxins, and
102
HOST DEFENSE: GENERAL
TLR ligands induce a rapid influx of DCs from the subepithelial dome region into the FAE, a response that enhances antigen capture. Peyer’s patch DCs (but not lamina propria DCs) express CCR6, a membrane receptor attracted by the chemokine CCL20 constitutively expressed by FAE cells. Peyer’s patch DCs rapidly capture macromolecules, commensal or pathogenic microorganisms, and particles transported by M cells, as well as virus-infected, apoptotic epithelial cells. This triggers DC maturation and expression of CCR7, a receptor that drives them to nearby interfollicular T-cells zones where the cognate chemokines are expressed (Kelsall, 2008). Here, the DCs initiate a process leading to generation of antigen-specific T- and B-cell clones, described below. The extent to which Peyer’s patch DCs migrate to MLN is not clear, but evidence indicates that induction of mucosal effector cells can occur entirely within the mucosa. In contrast, the DCs that migrate to MLN come primarily from the lamina propria (MacPherson & Uhr, 2004). In the interfollicular areas, a constant stream of naïve T cells are available for testing and possible engagement via appropriate T-cell receptors, coreceptors, and the costimulatory molecules required for T-cell activation. Activated CD41 T-helper cells in Peyer’s patches have immediate access to B cells in the adjacent follicle. Many complex interactions occur in this unique environment; in summary, both interfollicular and follicular T cells and DCs promote B-cell immunoglobulin class switch recombination by producing cytokines, including TGF-beta, IL-6, and IL-10, and by engaging CD40-expressing B cells via T-cell CD40 ligand (Cerutti, 2008; Fazilleau et al., 2009; Reinhardt et al., 2009). The follicular environment also inhibits proinflammatory Th1 responses. Antigen-specific B cells proliferate locally, clonally expanding the follicle to produce large numbers of antigen-specific IgA lymphoblasts. Proliferating B cells expressing high-affinity receptors are selected for survival by antigen-presenting follicular DCs (Cerutti, 2008).
Homing of Lymphocytes to Mucosal Tissues
Within a few days, antigen-specific B and T cells migrate out of the organized mucosal lymphoid tissues and enter the circulation. Before they do, however, mucosal DCs induce expression of mucosa-specific cell surface adhesion molecules called “homing receptors” on both B and T cells. This occurs only in mucosa-associated tissues due to the local production of retinoic acid by epithelial cells and DCs from dietary precursors (Mora et al., 2006). Homing receptors allow mucosally induced lymphocytes to selectively adhere to counterreceptors or “addressins” on mucosal endothelial cells, particularly in the region where the antigen or pathogen was initially encountered. For example, IgA1 B cells generated in organized mucosal lymphoid tissues of the intestine preferentially migrate back into the intestinal mucosa because they express the “homing receptor” a4/7 integrin that interacts strongly with MADCAM1, an addressin expressed by venules in the small and large intestine (and lactating mammary gland) but not in other mucosal tissues. In contrast, a4/1 integrin expression by lymphocytes appears to direct them to the airways (Kunkel & Butcher, 2003; Sigmundsdottir & Butcher, 2008). This system is responsible for the regional nature of mucosal immune responses demonstrated in mice, nonhuman primates, and humans (Neutra & Kozlowski, 2006). Mucosal DCs and lymphocytes are also programmed to express mucosa-specific receptors that recognize chemokines produced by epithelial cells. Chemokine gradients in the lamina propria guide the migration of effector B and T cells as well as DCs toward epithelial surfaces. Some of
these chemokine/receptor systems are common to many mucosal tissues. For example, CCR10, which is expressed by most IgA B cells that originated in MALT, recognizes the chemokine CCL28 that is secreted by epithelial cells in many locations, including the small and large intestines, salivary glands, tonsils, respiratory tract, and lactating mammary gland (Kunkel & Butcher, 2003). This recognition promotes widespread secretion of specific IgA antibodies after mucosal immunization at one site, a phenomenon originally called the “common mucosal immune system” (Woof & Mestecky, 2005). Other chemokines are organ or region-specific; for example, circulating a4/71 B lymphocytes activated in the small intestine express CCR9, which attracts them to CCL25, a chemokine secreted by epithelia throughout the small (but not the large) intestine. Conversely, B lymphocytes that express CCR10 are attracted by the chemokine CCL28 produced in by epithelial cells in the large intestine. Other receptor/chemokine pairs are adapted to the local microenvironment; for example, expression of the epithelial chemokine CCL20 is responsive to the presence of luminal flora. Mucosal immunization can also induce IgA and IgG in serum. This is due in part to migration of antigen-loaded mucosal DCs to mesenteric lymph nodes, where both mucosal and systemic homing receptors are induced, and to systemic inductive sites such as lymph nodes and the spleen (MacPherson et al., 2008). In addition, a fraction of the B cells activated in the mucosa or in draining lymph nodes express the peripheral homing receptors, a4/1 and l-selectin (Kunkel & Butcher, 2003). By contrast, systemic immunization is generally ineffective for induction of mucosal IgA antibody responses because DCs that capture antigen at injection sites are not exposed to retinoic acid locally or in nonmucosal lymph nodes and fail to induce expression of mucosal homing or chemokine receptors on lymphocytes (Mora et al., 2006).
T-Cell Independent Responses in Widespread Mucosal Tissues
From the above, it is evident that arming the mucosa with antigen-specific, high-affinity effector and memory T cells and antibody-producing B cells takes time. Five to seven days are required for initial induction in organized mucosal lymphoid tissues, directed migration, and terminal differentiation of cells in widespread mucosal sites. A more rapid but less specific immune protection mechanism is present throughout the mucosa, where a subset of B cells can rapidly produce IgA (and to a lesser extent IgM and IgG) without CD4 T-cell help (Fagarasan & Honjo, 2003; MacPherson et al., 2008; Cerutti, 2008). In mice, these B-1 cells migrate to the intestine from the peritoneal cavity, and comparable cells of unknown origin are present in humans. B-1 cells express unmutated, polyreactive surface IgA antibodies that recognize bacterial components—primarily complex carbohydrates—with low affinity. When presented with appropriate ligands, such as common carbohydrate structures of commensal bacteria, they can be induced to produce polymeric IgA antibodies that are secreted into the lumen, as described above. These antibodies control mucosa-associated populations of commensals and provide some protection against various pathogens such as Salmonella serovar Typhimurium. They also are essential for keeping bacteria and the immune responses that control them confined to the mucosa (MacPherson & Harris, 2004). When T–cell-independent antigens contact the mucosa, multiple innate immune pathways are activated to promote IgA class switching by B-1 cells. Some antigens, such as LPS, activate epithelial TLRs (Shang et al., 2008)
7. Immune Defense at Mucosal Surfaces
and thereby induce epithelial–cell-derived cytokines, such as BAFF and APRIL that promote B-cell isotype switch and antibody production, as with high-affinity B cells derived from Peyer’s patches (Schneider, 2005; Cerutti, 2008). Other microbial oligosaccharides directly activate B cells through their membrane immunoglobulins or B-cell receptors and TLRs (Peng, 2005; Bergtold et al., 2005). Such antigens also signal through the DCs that are stationed in the mucosa or reach through the epithelium. DCs endocytose T cell-independent antigens from the environment into a nondegradative endocytic pathway, recycle antigen to the plasma membrane, and present it to B cells (Trombetta & Mellman, 2005). DCs also stimulate IgA class switch and antibody production by secreting CD40L, BAFF, and APRIL (Schneider, 2005; Cerutti, 2008).
Terminal B-Cell Differentiation and IgA Secretion
B cells from Peyer’s patches and other organized MALT complete their differentiation only after arrival in the lamina propria. Here they become IgA antibody-secreting plasma cells through the influence of cytokines including BAFF, APRIL, IL-6, and IL-10 produced by epithelial cells and local DCs, as described above. On arrival in the intestinal lamina propria, most IgA1B cells in humans produce dimeric IgA1, a subclass characterized by an extended, 0-glycosylated hinge region containing proteolytic sites vulnerable to specific microbial IgA proteases (Russell & Kilian, 2005; Woof & Mestecky, 2005; MacPherson et al., 2008). Under the influence of BAFF and APRIL from epithelial cells, a final class switch recombination event occurs and IgA plasma cells switch to production of IgA2, a form that lacks the vulnerable hinge region and is more protease resistant. Commensal bacteria are required for optimal IgA production because epithelial cells express these cytokines in response to ligation of their TLRs (Shang et al., 2008). Most IgA1 and IgM1 mucosal plasma cells express a short J-chain polypeptide that mediates dimerization of IgA and pentamerization of IgM. The J chain also mediates binding to the polymeric immunoglobulin receptor (pIgR) in epithelial basolateral membranes (Woof & Mestecky, 2005). The vesicular transport process by which pIgR exports IgA and IgM into the lumen has been studied in detail and represents the classic model for transcytosis across a polarized epithelium (Kaetzel, 2005). The pIgR is a transmembrane glycoprotein with a large extracellular ligand-binding portion, a single membrane-spanning alpha helix, and a cytoplasmic tail that governs the intracellular itinerary of the molecule. The large, highly glycosylated extracellular region called “secretory component” (SC) is comprised of five Ig-like domains and a flexible region containing a protease cleavage site. pIgR is constitutively expressed by mucosal epithelial cells in the intestinal crypts and in glands of other mucosal tissues, but its level of expression can be modulated by cytokines and other mediators (Kaetzel, 2005; MacPherson et al., 2008). pIgR binds J–chain-containing dimeric IgA and pentameric IgM at the basolateral membrane, internalizes the ligand–pIgR complex (as well as unoccupied receptor) by clathrin-mediated endocytosis, and traffics it to a common endosomal compartment where it is sorted and delivered to apical recycling endosomes. Fusion of the apical recycling endosome with the apical plasma membrane and cleavage of the extracytoplasmic portion of pIgR by an apical membrane endoprotease releases the Ig-SC complex into the lumen as secretory IgA (S-IgA). SC enhances the lowaffinity interactions of Igs with mucins and increases the protease resistance and stability of these immunoglobulins
103
in the protease-rich intestinal environment (Phalipon & Corthesy, 2003; Russell & Kilian, 2005; Woof & Mestecky, 2005). S-IgA is by far most abundant in normal secretions, but S-IgM can provide compensatory protection in the immature gut and in selective IgA deficiency (Brandtzaeg et al., 2005).
Regulation of Mucosal Immune Responses
Small doses of soluble foreign proteins and peptides in the gut lumen generated during normal digestion, as well as from dying epithelial cells and some commensals, are sampled by the mucosal immune system but fail to induce immune responses. Repeated, low oral doses of soluble antigens generally result in suppression of systemic immune responses to subsequent injection of the antigen, a phenomenon termed “oral tolerance.” Potential mechanisms responsible for oral tolerance include induction of T-cell anergy, T-cell deletion, and the induction of CD41 regulatory T cells (Tregs) (Faria & Weiner, 2005). High oral doses of antigen tend to lead to anergy and deletion, while low, repeated doses induce the generation of Tregs. Tregs are induced in the mucosa or in MLN by exposure to a subset of antigen-presenting DCs, present in both Peyer’s patches and lamina propria, which express aE integrin (CD103). There is evidence that DCs from Peyer’s patches are involved in the generation of Tregs, but other evidence implicates lamina propria DCs that constitutively traffic to the MLN (Worbs et al., 2006; Kelsall, 2008). Lamina propria DCs capture antigens from commensal microorganisms, apoptotic epithelial cells, or soluble proteins derived from digested food. The DCs then mature and migrate to the MLN in a CCR7-dependent manner (Kelsall, 2008). At sites of mucosal antigen uptake or in MLN, induction of Tregs occurs simultaneously with induction of mucosal effector lymphocytes, as both are activated by mucosa-specific CD1031 DCs that produce retinoic acid and TGF- (Sakaguchi & Powrie, 2007). The genetic program in Tregs that is responsible for the cells’ suppressive functions, including production of IL-10, is controlled by expression of a transcription factor called forkhead box P3 (Foxp3). Adoptive transfer of antigen-specific Tregs to naïve mice results in transfer of antigen tolerance (Faria & Weiner, 2005). A significant number of the CD41 T cells in the normal intestinal lamina propria produce the cytokine IL-17, along with IL-21 and IL-22 (Zhou et al., 2009). These cytokines have diverse effects on neighboring cells, including production of antimicrobial peptides, attraction of neutrophils, and cell proliferation. Th17 cells are important effector cells in host defense against extracellular bacteria and fungi. Th17 cells are consistently associated with the presence of commensal bacteria, and are not present in organized lymphoid tissues (Peyer’s patches, lymph nodes, or spleen). There is evidence that a subset of lamina propria DCs (CD11c1, CD11b high) can be stimulated by flagellin to induce Th17 cells. Under normal conditions these cells appear to protect the mucosa against inflammatory damage, but Th17 cells can also enhance inflammation (Louten et al., 2009).
MECHANISMS OF MUCOSAL PROTECTION Secretory IgA
S-IgA plays multiple roles in mucosal defense (Phalipon & Corthesy, 2003; Russell & Kilian, 2005). It promotes entrapment of antigens or microorganisms in the mucus, preventing direct contact of pathogens with the mucosal surface, a mechanism called “immune exclusion” (Fig. 2). Antigen-specific S-IgA may directly block or sterically
104
HOST DEFENSE: GENERAL
hinder the microbial surface molecules that mediate epithelial attachment or intercept incoming pathogens within epithelial-cell vesicular compartments during pIgR-mediated transport. Within the mucosa, dimeric IgA synthesized by local IgA plasma cells may neutralize pathogens by inhibiting adherence to target cells, mediate transport of pathogens back into the lumen via pIgR, opsonize microbes and particles to promote uptake and killing by phagocytes expressing Fc u/alpha receptors, or mediate antibody-dependent cellular cytoxicity (ADCC) that leads to destruction of local infected cells. S-IgA is also important for maintaining the complex ecosystem of commensal bacteria that is crucial for digestion and mucosal homeostasis (Peterson et al., 2007).
While high-affinity S-IgAs can block or neutralize microbial toxins and pathogens, low-affinity S-IgAs keep commensal populations in balance and prevent commensal bacteria from breaching the mucosal barrier and evoking systemic immune responses (MacPherson et al., 2008; Fagarasan & Honjo, 2003). Conversely, the presence of commensal organisms is required for maintenance of the mucosal immune system (Backhed et al., 2005). Importantly, intestinal IgA protects without inducing damaging mucosal inflammation, in part because it not only prevents commensals and pathogens from contacting the mucosa, but also because of its inability to fix and activate the complement cascade (Russell & Kilian, 2005; Woof & Mestecky, 2005).
FIGURE 2 Mechanisms of immune protection at mucosal surfaces. Mucosal immune protection depends on the contributions of multiple cell types. Antigen-specific effector B and T cells that bear mucosal homing receptors recognize addressins on mucosal high endothelial venules and enter the mucosa. In response to epithelial and dendritic cell signals, the B cells terminally differentiate to become mucosal plasma cells. Most plasma cells produce dimeric IgA that is exported into secretions as S-IgA to intercept antigens and pathogens and prevent mucosal invasion. IgA, as well as IgG from local plasma cells or blood, can also neutralize pathogens within the mucosa. Local cytotoxic T cells and antibodies collaborate to kill infected cells. Pathogens are also captured by dendritic cells (DC) and macrophages (Mf), and carried to draining lymph nodes. (Reproduced from Neutra & Kozlowski, 2006).
7. Immune Defense at Mucosal Surfaces
Mucosal IgG
IgG is also present within mucosal tissues, mostly derived from serum. However, IgG synthesis also occurs locally, especially after local mucosal immunization. IgG- as well as IgAsecreting plasma cells are present in the female genital tracts of macaques and humans (Woof & Mestecky, 2005), and significant concentrations of IgG are present in human cervical/ vaginal and rectal secretions (Neutra & Kozlowski 2006). In the human intestine, 5% to 15% of mucosal plasma cells secrete IgG, but IgG is susceptible to degradation by luminal intestinal and bacterial proteases, and in large intestinal secretions, IgG concentrations are generally 30- to 100-fold lower than those of S-IgA (Woof & Mestecky, 2005). IgG was previously thought to enter the lumen only by leakage, but it is now known that adult human epithelial cells express the pH-dependent Fc receptors that mediate transepithelial IgG transport in neonatal rodents (FcRn), (Yoshida et al., 2006), suggesting a possible IgG secretion mechanism. In any case, there is evidence that IgG as well as S-IgA in secretions can block mucosal infection, neutralize pathogens that enter the mucosa, and prevent systemic spread.
Effector and Memory T Cells
Cytotoxic T lymphocytes (CTLs) in mucosal tissues cannot prevent pathogen entry, but they may play a crucial role in clearance or containment of mucosal viral infections. CD81 lamina propria T cells exhibit potent cytotoxicity against specific pathogen-infected target cells in vitro and convey effective host protection upon adoptive transfer to recipient mice that were orally or systemically infected with pathogens (including viruses and bacteria). For example, Th-1 CD41 lamina propria T cells with specific reactivity against astrovirus, a common and important gastroenteritis virus, have been identified in normal human intestines and are presumably important in intestinal defense against recurrent astroviral infections. Th-2 CD41 lamina propria T cells play a decisive role in driving productive immunity against the intracellular pathogen Toxoplasma gondii, notably by promoting the development of IgA-producing plasma cells. Cytotoxic T cells in mucosal tissues are likely to be important in limiting mucosal replication of HIV (Belyakov & Ahlers, 2008). Mucosally immunized (but not systemically immunized) mice were protected against infection after mucosal challenge with a recombinant vaccinia virus expressing HIV envelope glycoprotein 160 (gp160), but protection was abrogated by treatment of the mice with anti-CD8 antibodies. Immunologically active mucosal tissues, such as the intestinal tract, contain abundant CD41 T cells that are targets for HIV, and as a result, the intestinal mucosa becomes a reservoir of HIV infection regardless of the site of initial viral entry (Veazey et al., 2001). Both CTLs and antibodies within mucosal tissues may contribute to preventing the establishment of such reservoirs (Brenchley & Douek, 2008).
IMPLICATIONS FOR MUCOSAL VACCINES
Many mucosal vaccines and delivery systems have been developed and tested in animals but very few have been approved for human use (Holmgren & Czerkinsky, 2005). Progress has been slow in part because vaccines given orally or deposited directly on mucosal surfaces face the same gauntlet of host defenses as do microbial pathogens: being diluted in mucosal secretions, captured in mucus gels, attacked by proteases and nucleases, and excluded by epithelial barriers (Neutra & Kozlowski, 2006). Thus the exact dose of mucosally administered antigen that actually crossed the epithelial barrier cannot be precisely determined but can only be estimated (Corbett et al., 2008). An additional challenge
105
is that mucosal tissues are adapted to the presence of foreign antigens, such as microorganisms and their products. As a result, vaccines that consist of soluble macromolecules and protein subunit antigens, which may produce vigorous immune responses if injected into a sterile environment such as muscle, are often “ignored” when mucosally administered. For mucosal vaccines to be distinguished from harmless substances and nutrients, they must raise alarms in the mucosa by containing adjuvants or molecular patterns that activate innate signaling pathways in epithelial cells and underlying antigen-presenting cells (Elson & Dertzbaugh, 2005). Thus, mucosal vaccines are most effective when they mimic successful mucosal pathogens in the following key respects: they are live mucosa-tropic vectors or microbe-sized particles that adhere to mucosal surfaces, preferably to the FAE, and are avidly endocytosed by mucosal DCs. Mucosal vaccine protocols also must be adapted to the receptor-mediated recognition systems described above that dictate immune cell migration. These systems serve not only to populate the mucosa with effector cells, but also to focus the immune response at the site where antigen or pathogen was initially encountered. Although region-specific cell homing mechanisms have been most extensively defined in mice (Sigmundsdottir & Butcher, 2008), the regional nature of mucosal immune responses has been clearly demonstrated in nonhuman primates and in humans as well (Neutra & Kozlowski, 2006). Responses to mucosal vaccines are greatly amplified by local boosts, due to the abundant populations of memory T and B cells in mucosal tissues that allow for local amplification of effector responses on reexposure to an antigen. Local exposure to an antigen and/or adjuvant evokes cytokines and chemokines from epithelia, DCs, and other cells and up-regulates expression of addressins on local endothelia, increasing the numbers and enhancing the functions of mucosal effector cells (Kunkel & Butcher, 2003; Sigmundsdottir & Butcher, 2008). The value of repeated mucosal antigen exposure was reinforced by the observation that CD81 T cells activated by mucosal immunization initially had a widespread migration pattern, but over the long term, memory CD81 T cells were preferentially located in the region where antigen was originally encountered (Qimron et al., 2004).
CONCLUSIONS
In conclusion, the recent and ongoing expansion of new information about the complex interactions of mucosal tissues with the commensal flora and microbial pathogens, and the mechanisms of mucosal immune protection, promise to provide the tools needed to exploit the full potential of mucosal vaccines.
REFERENCES Agace, W. W., J. M. Higgins, B. Sadasivan, M. B. Brenner, and C. M. Parker. 2000. T-lymphocyte-epithelial-cell interactions: integrin alpha(E)(CD103)beta(7), LEEP-CAM and chemokines. Curr. Opin. Cell Biol. 12:563–568. Anderle, P., M. Rumbo, F. Sierro, R. Mansourian, P. Michetti, M. Roberts, and J. P. Kraehenbuhl. 2005. Novel markers of the human follicle-associated epithelium identified by genomic profiling and microdissection. Gastroenterology 129: 321–327. Backhed, F., R. E. Ley, J. L. Sonnenburg, D. A. Peterson, and J. I. Gordon. 2005. Host-bacterial mutualism in the human intestine. Science 307:1915–1920. Belyakov, I. M., and J. D. Ahlers. 2008. Functional CD81 CTLs in mucosal sites and HIV infection: moving forward toward a mucosal AIDS vaccine. Trends Immunol. 29:574–585.
106
HOST DEFENSE: GENERAL
Bergtold, A., D. D. Desai, A. Gavhane, and R. Clynes. 2005. Cell surface recycling of internalized antigen permits dendritic cell priming of B cells. Immunity 23:503–514. Bevins, C. L. 2006. Paneth cell defensins: key effector molecules of innate immunity. Biochem. Soc. Trans. 34:263–266. Bienenstock, J., and R. L. Clancy. 2005. Bronchus-associated lymphoid tissues, p. 375–384. In J. Mestecky, J. Bienenstock, M. Lamm, L. Mayer, J. McGhee, and W. Strober (ed.), Mucosal Immunology, 3rd ed. Academic Press, San Diego, CA. Brandtzaeg, P., H. S. Carlsen, and I. N. Farstad. 2005. The human mucosal B cell system. p. 617-654. In J. Mestecky, J. Bienenstock, M. Lamm, L. Mayer, J. McGhee, and W. Strober (eds.), Mucosal Immunology, 3rd ed. Academic Press, San Diego, CA. Brenchley, J. M., and D. C. Douek. 2008. HIV infection and the gastrointestinal immune system. Mucosal Immunol. 1:23–30. Cerutti, A. 2008. The regulation of IgA class switching. Nat. Rev. Immunol. 8:421–434. Chabot, S. M., M. Shawi, T. Eaves-Pyles, and M. R. Neutra. 2008. Flagellin modulates functions of the follicle-associated epithelium to facilitate uptake of particles into organized mucosal lymphoid tissues. J. Infect. Dis. 198: 907–910. Cohen, N. R., S. Garg, and M. B. Brenner. 2009. Antigen presentation by CD1 lipids, T cells, and NKT cells in microbial immunity. Adv. Immunol. 102:1–94. Cone, R. A. 2005. Mucus. p. 49–72. In J. Mestecky, J. Bienenstock, M. Lamm, L. Mayer, J. McGhee, and W. Strober (ed.), Mucosal Immunology, 3rd ed. Academic Press, San Diego, CA. Coombes, J. L., and F. Powrie. 2008. Dendritic cells in intestinal immune regulation. Nat. Rev. Immunol. 8:435–446. Corbett, M., W. M. Bogers, J. L. Heeney, S. Gerber, C. Genin, A. Didierlaurent, H. Oostermeijer, R. Dubbes, G. Braskamp, S. Lerondel, C. E. Gomez, M. Esteban, R. Wagner, I. Kondova, P. Mooij, S. Balla-Jhagjhoorsingh, N. Beenhakker, G. Koopman, S. van der Burg, J. P. Kraehenbuhl, and A. Le Pape. 2008. Aerosol immunization with NYVAC and MVA vectored vaccines is safe, simple, and immunogenic. Proc. Natl. Acad. Sci. USA 105:2046–2051. Das, G., and C. A. Janeway, Jr. 2003. MHC specificity of iIELs. Trends Immunol. 24:88–93. Debard, N., F. Sierro, J. Browning, and J. P. Kraehenbuhl. 2001. Effect of mature lymphocytes and lymphotoxin on the development of the follicle-associated epithelium and M cells in mouse Peyer’s patches. Gastroenterology 120:1173–1182. Ellermeier, J. R., and J. M. Slauch. 2007. Adaptation to the host environment: regulation of the SPI1 type III secretion system in Salmonella enterica serovar Typhimurium. Curr. Opin. Microbiol. 10:24–29. Elson, C. O., and M. T. Dertzbaugh. 2005. Mucosal adjuvants. p. 967–986. In J. Mestecky, J. Bienenstock, M. Lamm, L. Mayer, J. McGhee, and W. Strober (ed.), Mucosal Immunology, 3rd ed. Academic Press, San Diego, CA. Fagarasan, S., and T. Honjo. 2003. Intestinal IgA synthesis: regulation of front-line body defences. Nat. Rev. Immunol. 3:63–72. Faria, A.M., and H. L. Weiner. 2005. Oral tolerance. Immunol. Rev. 206:232–259. Fazilleau, N., L. Mark, L. J. McHeyzer-Williams, and M. G. McHeyzer-Williams. 2009. Follicular helper T cells: lineage and location. Immunity 30:324–335. Fotopoulos, G., A. Harari, P. Michetti, D. Trono, G. Pantaleo, and J. P. Kraehenbuhl. 2002. Transepithelial transport of HIV-1 by M cells is receptor mediated. Proc. Natl. Acad. Sci. USA 99:9410–9414. Frey, A., K. T. Giannasca, R. Weltzin, P. J. Giannasca, H. Reggio, W. I. Lencer, and M. R. Neutra. 1996. Role of the glycocalyx in regulating access of microparticles to apical plasma membranes of intestinal epithelial cells—implications for microbial attachment and oral vaccine targeting. J. Exp. Med. 184:1045–1059.
Haase, A. T. 2005. Perils at mucosal front lines for HIV and SIV and their hosts. Nat. Rev. Immunol. 5:783–792. Handley, S. A., R. D. Newberry, and V. L. Miller. 2005. Yersinia enterocolitica invasin-dependent and invasinindependent mechanisms of systemic dissemination. Infect. Immun. 73:8453–8455. Hase, K., T. Murakami, H. Takatsu, T. Shimaoka, M. Iimura, K. Hamura, K. Kawano, S. Ohshima, R. Chihara, K. Itoh, S. Yonehara, and H. Ohno. 2006. The membranebound chemokine CXCL16 expressed on follicle-associated epithelium and M cells mediates lympho-epithelial interaction in GALT. J. Immunol. 176:43–51. Helander, A., K. J. Silvey, N. J. Mantis, A. B. Hutchings, K. Chandran, W. T. Lucas, M. L. Nibert, and M. R. Neutra. 2003. The viral sigma1 protein and glycoconjugates containing alpha2-3-linked sialic acid are involved in type 1 reovirus adherence to M cell apical surfaces. J. Virol. 77:7964–7977. Holmgren, J., and C. Czerkinsky. 2005. Mucosal immunity and vaccines. Nat. Med. 11:S45–S53. Honda, K., and K. Takeda. 2009. Regulatory mechanisms of immune responses to intestinal bacteria. Mucosal Immunology 2:187–196. Ishikawa, H., T. Naito, T. Iwanaga, H. Takahashi-Iwanaga, M. Suematsu, T. Hibi, and M. Nanno. 2007. Curriculum vitae of intestinal intraepithelial T cells: their developmental and behavioral characteristics. Immunol. Rev. 215:154–165. Kadaoui, K. A., and B. Corthesy. 2007. Secretory IgA mediates bacterial translocation to dendritic cells in mouse Peyer’s patches with restriction to mucosal compartment. J. Immunol. 179:7751–7757. Kaetzel, C. S. 2005. The polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces. Immunol. Rev. 206:83–99. Kagnoff, M. F. 2006. Microbial-epithelial cell crosstalk during inflammation: the host response. Ann. N. Y. Acad. Sci. 1072:313–320. Kelsall, B. 2008. Recent progress in understanding the phenotype and function of intestinal dendritic cells and macrophages. Mucosal Immunology 1:460–469. Kernéis, S., A. Bogdanova, J. P. Kraehenbuhl, and E. Pringault. 1997. Conversion by Peyer’s patch lymphocytes of human enterocytes into M cells that transport bacteria. Science 277:948–952. Kraehenbuhl, J. P., and M. R. Neutra. 2000. Epithelial M cells: differentiation and function. Annu. Rev. Cell Dev. Biol. 16:301–332. Kunisawa, J., I. Takahashi, and H. Kiyono. 2007. Intraepithelial lymphocytes: their shared and divergent immunological behaviors in the small and large intestine. Immunol. Rev. 215:136–153. Kunkel, E. J., and E. C. Butcher. 2003. Plasma-cell homing. Nat. Rev. Immunol. 3:822–829. Lefrançois L., and L. Puddington. 2006. Intestinal and pulmonary mucosal T cells: local heroes fight to maintain the status quo. Annu. Rev. Immunol. 24:681–704. Lo, D., W. Tynan, J. Dickerson, M. Scharf, J. Cooper, D. Byrne, D. Brayden, L. Higgins, C. Evans, and D. J. O’Mahony. 2004. Cell culture modeling of specialized tissue: identification of genes expressed specifically by follicleassociated epithelium of Peyer’s patch by expression profiling of Caco-2/Raji co-cultures. Internat. Immunol. 16:91–99. Louten, J., K. Boniface, and R. de Waal Malefyt. 2009. Development and function of TH17 cells in health and disease. J. Allergy Clin. Immunol. 123:1004–1011. MacPherson, A. J., and T. Uhr. 2004. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science 303:1662–1665. MacPherson, A. J., K. D. McKoy, F. E. Johansen, and P. Brandtzaeg. 2008. The immune geography of IgA induction and function. Mucosal Immunol. 1:11–22.
7. Immune Defense at Mucosal Surfaces MacPherson, A. J., and N. L. Harris. 2004. Interactions between commensal intestinal bacteria and the immune system. Nat. Rev. Immunol. 4:478–485. Mantis, N. J., A. Frey, and M. R. Neutra. 2000. Accessibility of glycolipid and oligosaccharide epitopes on apical surfaces of rabbit villus and follicle-associated epithelium. Am. J. Physiol. (Gastrointest. Liver Physiol.) 278:G915–G923. Mantis, N. J., M. C. Cheung, K. R. Chintalacharuvu, J. Rey, B. Corthesy, and M. R. Neutra. 2002. Selective adherence of IgA to murine Peyer’s patch M cells: evidence for a novel IgA receptor. J. Immunol. 169:1844–1851. McGhee, J. R., M. E. Lamm, and W. Strober. 2005. Inductive and effector tissues and cells of the mucosal immune system, p. 371–375. In J. Mestecky, J. Bienenstock, M. Lamm, L. Mayer, J. McGhee, and W. Strober (ed.), Mucosal Immunology, 3rd ed. Academic Press, San Diego, CA. Mora, J. R., M. Iwata, B. Eksteen, S. Y. Song, T. Junt, B. Senman, K. L. Otipoby, A. Yokota, H. Takeuchi, P. RicciardiCastagnoli, K. Rajewsky, D. H. Adams, and U. H. von Andrian. 2006. Generation of gut-homing IgA-secreting B cells by intestinal dendritic cells. Science 314:1157–1160. Neutra, M. R., and J. P. Kraehenbuhl. 2005. Cellular and molecular basis for antigen transport across epithelial barriers, p. 111–113 In J. Mestecky, J. Bienenstock, M. Lamm, L. Mayer, J. McGhee, and W. Strober (ed.), Mucosal Immunology, 3rd ed. Academic Press, San Diego, CA. Neutra, M. R., N. J. Mantis, and J. P. Kraehenbuhl. 2001. Collaboration of epithelial cells with organized mucosal lymphoid tissues. Nat. Immunol. 2:1004–1009. Neutra, M. R., and P. A. Kozlowski. 2006. Mucosal vaccines: the promise and the challenge. Nat. Rev. Immunol. 6:148–158. Neutra, M. R., P. Sansonetti, and J. P. Kraehenbuhl. 2002. M cells and microbial pathogens, p. 141–156. In M. J. Blaser, P. D. Smith, J. I. Ravdin, H. B. Greenberg, and L. Guerrant (ed.), Infections of the Gastrointestinal Tract. Raven Press, New York. Niess, J. H., and H.-C. Reinecker. 2006. Dendritic cells in the recognition of intestinal microbiota. Cell. Microbiol. 8:558–564. Nieuwenhuis, E. E., T. Matsumoto, D. Lindenbergh, R. Willemsen, A. Kaser, Y. Simons-Oosterhuis, S. Brugman, K. Yamaguchi, H. Ishikawa, Y. Aiba, Y. Koga, J. N. Samsom, K. Oshima, M. Kikuchi, J. C. Escher, M. Hattori, A. B. Onderdonk, and R. S. Blumberg. 2009. Cd1d-dependent regulation of bacterial colonization in the intestine of mice. J. Clin. Invest. 119:1241–1250. Ouellette, A. J. 2006. Paneth cell alpha-defensin synthesis and function. Curr. Top. Microbiol. Immunol. 306:1–25. Pédron, T., and P. Sansonetti. 2008. Commensals, bacterial pathogens and intestinal inflammation: an intriguing ménage à trois. Cell Host Microbe 3:344–347. Peng, S. L. 2005. Signaling in B cells via Toll-like receptors. Curr. Opin. Immunol. 17:230–236. Peterson, D. A., N. P. McNulty, J. L. Guruge, and J. I. Gordon. 2007. IgA response to symbiotic bacteria as a mediator of gut homeostasis. Cell Host Microbe 2:328–339. Phalipon, A., and B. Corthesy. 2003. Novel functions of the polymeric Ig receptor: well beyond transport of immunoglobulins. Trends Immunol. 24:55–58. Qimron, U., L. Paul, E. Bar-Haim, N. Bloushtain, L. Eisenbach, H. F. Staats, and A. Porgador. 2004. Non-replicating mucosal and systemic vaccines: quantitative and qualitative differences in the Ag-specific CD81 T cell population in different tissues. Vaccine 22:1390–1394. Rakoff-Nahoum, S., and R. Medzhitov. 2008. Innate immune recognition of the indigenous microbial flora. Mucosal Immunol. 1:S10–S14. Reinhardt, R. L., H. E. Liang, and R. M. Locksley. 2009. Cytokine-secreting follicular T cells shape the antibody repertoire. Nat. Immunol. 10:385–393. Rescigno, M., M. Urbano, B. Valzasina, M. Francolini, G. Rotta, R. Bonasio, F. Granucci, J. P. Kraehenbuhl, and
107
P. Ricciardi-Castagnoli. 2001. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol. 2:361–367. Rimoldi, M., M. Chieppa, V. Salucci, F. Avogadri, A. Sonzogni, A, G. M. Sampietro, A. Nespoli, G. Viale, P. Allavena, and M. Rescigno. 2005. Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells. Nat. Immunol. 6:507–514. Russell, M. W., and M. Kilian. 2005. Biological activities of IgA, p. 267–290. In J. Mestecky, J. Bienenstock, M. Lamm, L. Mayer, J. McGhee, and W. Strober (ed.), Mucosal Immunology, 3rd ed. Academic Press, San Diego, CA. Sakaguchi, S., and F. Powrie. 2007. Emerging challenges in regulatory T cell function and biology. Science 317:627–629. Sansonetti, P. J. 2006. Rupture, invasion and inflammatory destruction of the intestinal barrier by Shigella: the yin and yang of innate immunity. Can. J. Infect. Dis. Med. Microbiol. 17:117–119. Schneider, P. 2005. The role of APRIL and BAFF in lymphocyte activation. Curr. Opin. Immunol. 17:282–289. Shang L., M. Fukata, N. Thirunarayanan, A. P. Martin, P. Arnaboldi, D. Maussang, C. Berin, J. C. Unkeless, L. Mayer, M. T. Abreu, and S. A. Lira. 2008. Toll-like receptor signaling in small intestinal epithelium promotes B-cell recruitment and IgA production in lamina propria. Gastroenterology 135:529–538. Shastri, N., S. Cardinaud, S. R. Schwab, T. Serwold, and J. Kunisawa. 2005. All the peptides that fit: the beginning, the middle, and the end of the MHC class I antigenprocessing pathway. Immunol. Rev. 207:31–41. Sierro, F., B. Dubois, A. Coste, D. Kaiserlian, J. P. Kraehenbuhl, and J. C. Sirard. 2001. Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells. Proc. Natl. Acad. Sci. USA. 98:13722–13727. Sigmundsdottir, H., and E. C. Butcher. 2008. Environmental cues, dendritic cells and the programming of tissue-selective lymphocyte trafficking. Nat. Immunol. 9:981–987. Trombetta, E. S., and I. Mellman. 2005. Cell biology of antigen processing in vitro and in vivo. Annu. Rev. Immunol. 23:975–1028. Vasquez-Torres, A., J. Jones-Carson, A. J. Baumler, S. Falkow, R. Valdivia, W. Brown, M. Le, R. Berggren, W. T. Parks, and F. Fang. 1999. Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes. Nature 401:804–808. Veazey, R. S., P. A. Marx, and A. A. Lackner. 2001. The mucosal immune system: primary target for HIV infection and AIDS. Trends Immunol. 22:626–633. Vijay-Kumar, M., and A. T. Gewirtz. 2008. Guardians of the gut: newly appreciated role of epithelial toll-like receptors in protecting the intestine. Gastroenterology 135:351–354. Wang, C., J. S. McDonough, K. G. McDonald, C. Huang, and R. D. Newberry. 2008. Alpha4beta7/MAdCAM-1 interactions play an essential role in transitioning cryptopatches into isolated lymphoid follicles and a nonessential role in cryptopatch formation. J. Immunol. 181:4052–4061. Woof, J. M., and J. Mestecky. 2005. Mucosal immunoglobulins. Immunol. Rev. 206:64–82. Worbs, T., U. Bode, S. Yan, M. W. Hoffmann, G. Hintzen, G. Bernhardt, R. Förster, and O. Pabst. 2006. Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells. J. Exp. Med. 203:519–527. Yoshida, M., A. Masuda, T. T. Kuo, K. Kobayashi, S. M. Claypool, T. Takagawa, H. Kutsumi, T. Azuma, W. I. Lencer, and R. S. Blumberg. 2006. IgG transport across mucosal barriers by neonatal Fc receptor for IgG and mucosal immunity. Springer Semin. Immunopathol. 28:397–403. Zhou, L., M. M. Chong, and D. R. Littman. 2009. Plasticity of CD41 T cell lineage differentiation. Immunity 30:646–655.
This page intentionally left blank
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
8 Regulation of Antimicrobial Immunity YASMINE BELKAID, SHARVAN SEHRAWAT, AND BARRY T. ROUSE
INTRODUCTION
represented by CD41 T cells that develop in the thymus and express the signature transcription factor Foxp3 that is involved in controlling regulatory activities of the cells (Belkaid & Tarbell, 2009) . The major role of these Foxp31 natural T regulatory cells (nTreg) is to prevent the occurrence and modulate the expression of autoimmunity, but as will be discussed, nTregs also influence responses to several pathogens. The inducible regulatory cell types are highly heterogeneous in origin, phenotype, and the means by which they exert regulation. These inducible regulators are perhaps the most influential cell types that affect the responses to pathogens, particularly those that persist and cause chronic reactions (Belkaid & Tarbell, 2009). The inducible Treg populations include IL-10 producing CD41 T cells (often referred to as Tr1 cells), TGF-b producing CD41 T cells, CD81 regulatory cells, NK T cells, as well as CD41 T cells that convert to become Foxp31 and regulatory cells (Shevach, 2006). The latter cells usually develop in appropriate environments from naïve precursors but in some instances may develop from functionally differentiated CD41 T cells. This chapter focuses on inducible Foxp31 Tregs since it seems likely that this cell type plays a dominant role in contributing to the maintenance of homeostasis and in limiting collateral tissue damage caused by chronic effector cell responses to pathogens. Moreover, unlike other subtypes of inducible regulators, the Foxp3 transcription factor expression provides a convenient means of distinguishing these cells from nonregulatory cells. However, since there are no convenient phenotypic markers to distinguish between natural and inducible Foxp31 Tregs, it is usually not possible to decide, especially in human systems, if a regulatory effect is mediated by cells derived from nTreg or if they represent converts from nonregulatory cells. Further complicating the story with studies on human tissues is that activated human T cells may temporarily up regulate Foxp31 and not adopt a regulatory function.
Yasmine Belkaid, Mucosal Immunology Unit, Laboratory of Parasitic Diseases, Division of Intramural Research, National Institute of Health, 4 Center Drive B1-28, Bethesda, MD 20892. Sharvan Sehrawat and Barry T. Rouse, Department of Pathobiology, College of Veterinary Medicine, University of Tennessee, 1414 Cumberland Ave., Knoxville, TN 37996.
HOW DOES MICROBE HOST INTERACTION TRIGGER RESPONSES OF NATURAL Treg?
Since vertebrates have developed numerous powerful defense mechanisms, they can survive over the long term in the face of constant exposure to multiple types of microbes that could damage or destroy them. However, many of these defenses are potentially damaging, particularly if they are long acting; so they must be controlled to limit the chance of causing collateral damage to host tissues. Such control becomes of particular relevance in situations where microbes have devised strategies that permit them to persist in the body for lengthy periods of time. Regulation of exuberant reactions to microbes is achieved in many ways and includes not only host control measures, but also manipulation of potentially harmful host responses by the microbes themselves in some cases (this topic is discussed in chapter 31). Clearly, the interests of the microbes are best served by not devastating nor destroying their host since these outcomes will limit their own chance for survival and transmission. Regulation and maintenance of a homeostatic environment by the host is achieved in many ways. These include the production of proteins such as the cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-b) that counteract tissue damaging inflammatory events, as well as the existence of dedicated cell types that can inhibit the activity of the effector cells that orchestrate tissue damage. We now refer to such dedicated cells as regulatory cells, which are heterogeneous in origin and phenotype, as well as with regard to the mechanisms by which they perform regulatory activities. Conceptually, there are two main subtypes of regulatory cells that affect host microbe interactions: cells that are already present and ready to operate at the time of infection and cells that become induced and adopt a regulatory function as a consequence of the microbial infection. These two categories of regulatory cells are commonly referred to as naturally occurring and inducible regulatory T cells. The former cell type is mainly
At the onset of infection, cells are already present that can act as regulators. The most abundant of these are the CD41Foxp31 nTreg, which account for around 8% to 10% 109
110
HOST DEFENSE: GENERAL
of CD41 T cells in the circulation and lymphoid tissues. The nTreg are especially abundant at sites of initial infection, such as the skin and mucosal surfaces. In fact, it is estimated that around 80% of the total nTreg population is present in the skin. As already mentioned, the majority of nTreg appears to be self-reactive and mainly serves to prevent and control autoimmunity. A minor fraction of cells, however, could express T-cell receptors (TCRs) that recognize microbial antigens especially if these are cross-reactive with selfantigens. The issue of what proportion of nTreg have TCRs that recognize microbial antigens has not been fully evaluated and merits further study. However, it is well-known that infection with at least some microbes can expand and activate the nTreg population, and if this population is removed prior to infection, the pattern of events that occur and the magnitude of the immune response generated may differ dramatically (Belkaid et al., 2002; Suvas et al., 2003). Such is the case, for example, with herpes simplex virus (HSV) and some parasitic infections as is further discussed later in this chapter. Assuming TCR stimulation may be an unusual step during Treg responses to microbes, the fact that nTreg can be expanded and activated following infection means that alternative forms of stimulation must be occurring. Logical candidates for this are the pathogen-associated molecular patterns (PAMPs) expressed by microbes that engage receptors either on nTreg themselves or on intermediary cells such as those that compose the innate immune system (Belkaid & Tarbell, 2009). The latter, in turn, will activate nTreg directly or will generate nTreg activators. The PAMPs that have received the most study are ligands for TLRs, most of which are present on nTreg. The source of TLR ligands may be the pathogen but some ligands can also be endogenously derived as a result of tissue damage at the infection site. Examples include self-DNA/RNA, some heat shock proteins, and the extracellular matrix component hyaluronidase. Various studies have shown that several TLR ligands can act on nTreg, but the outcome is variable and somewhat controversial. This topic has been recently reviewed by others (Conroy et al., 2008). It seems that in some instances, TLR stimulation results in activation and expansion of nTreg, whereas engaging other TLRs result in an inhibitory effect on nTreg function. Many studies have focused on TLR2 and TLR4 stimulation. For example, with TLR2, the majority opinion is that TLR2 and TLR4 stimulation causes nTreg to proliferate, although the suppressor functions of individual cells may be diminished (reviewed in Conroy et al., 2008). With TLR4 stimulation, some have reported that the proliferative and suppressive function of nTregs are enhanced, whereas others indicate that the main effect of TLR4 engagement is to enhance the activity of IL-10, producing (Foxp32) inducible Treg (Conroy et al., 2008). The TLR5 stimulating molecule, flagellin—usually derived in the body from enteric bacteria—was also shown to enhance Foxp3 expression and to increase the suppressive function of nTreg (Crellin et al., 2005). In contrast, other TLR ligands appear to mainly suppress regulatory T-cell activity. This has been reported for both TLR8 and TLR9 stimulation. As will be further discussed, it seems that activation of TLR9 by DNA derived from gut flora can also inhibit the conversion of conventional T cells into Foxp31 Treg (Hall et al., 2008). We must conclude that the role of TLR stimulation of nTreg is complex and remains an unfolding story. For instance, few (if any) studies have addressed neither the issue of the quantity of microbe-derived TLR activators that would be available in vivo, nor the outcome of combined stimulation with multiple TLR ligands, as many microbes may provide. Clearly the topic requires more investigation.
There are also other non-TCR mediated mechanisms by which microbes could trigger regulation by nTreg. In some instances, this could include the production of proteins by the pathogen that activate nTreg by binding to a receptor, which the cells express. Recently, for example, the HIV (human immunodeficiency virus) protein gp120 was shown to activate human nTreg via its ability to bind to the CD4 molecule (Becker et al., 2009). Additionally, cytokines generated in the microenvironment of infection could also boost nTreg activity. These include IL-2 (which is essential for nTreg survival and expansion), TNF-a, and TGF-b. Curiously, an injection of IL-2 complexed to anti-IL-2 antibody happens to be a potent means of expanding nTreg in vivo, although such a situation would not be expected to occur in physiological circumstances (Webster et al., 2009). Under conditions of microbial infections, there are many host-derived molecules as well as related molecules derived from microbes themselves that have potent effects on many cells involved in both innate and adaptive immunity. Of particular interest are the family of galectin molecules that are up regulated or secreted during inflammatory responses. Galectins either form lattices on the surface of cells by binding to cell surface glycoproteins and glycolipids or to specific receptors. The outcome is variable but includes apoptosis, proliferation, and changes of function (Rabinovich & Toscano, 2009). Perhaps of most potential interest in terms of this review are the galectins-1 and -9, both of which are secreted during microbe-induced or other inflammatory response. It is not clear if the infected cells themselves are the source of the galectins or if they come from other cells that respond to cytokines induced by the infection, such as IFN-g, which are produced by responding NK cells or later on by T cells. Galectin-9 was shown to modulate the function of T cells that are involved in inflammatory reactions such as those caused by HSV infection of the eye (Sehrawat et al., 2009). Galectin-9 mainly acts by binding to its receptor TIM-3, which is up regulated on activated cells but is also expressed by a high proportion of nTreg (Sehrawat et al., 2009; Zhu et al., 2005). Whereas galectin-9 binding to activated effector cells causes them to undergo apoptosis, its binding to TIM-3 on nTreg may lead to the expansion and activation of these cells, as we have shown with HSV-induced ocular lesions. Both events serve to resolve the ocular lesions. Galectin-1 functions in a similar way to galectin-9, although different recognition systems are involved and it is not clear if galectin-1 similarly acts to expand and activate nTreg. However, galectin-1 has been reported to expand IL-10-producing Treg (Rabinovich & Toscano, 2009). We can conclude that microbial infections could trigger the expansion and activation of nTreg in several ways, some of which may not involve engagement of the nTregspecific TCR, although this latter event might facilitate responses. These various stimulatory processes are summarized in Fig. 1.
ROLE OF INDUCIBLE REGULATORY CELLS DURING INFECTIONS Induction of Tr1 Cells
One of the first types of regulatory cells observed to influence the outcome of infections were called Tr1 cells. These were derived from nonregulatory precursors by antigen stimulation in the presence of the immunoregulatory cytokine, IL-10. The cytokine alone can inhibit the immune responses (by both Th1 cells and Th2 cells) to many pathogens in experimental models and in human infectious
8. Regulation of Antimicrobial Immunity
111
FIGURE 1 Microbes or their products may modulate Treg responses in multiple ways. A few Treg with TCR specificity to pathogen antigens proliferate in response to the infection. Alternatively, cross-reactive pathogen antigens or self-antigens, released as a result of tissue damaging inflammatory reactions, could stimulate self-reactive Treg. Stimulation independent of TCR could be provided by the microbial products such as TLR ligands or other host-derived products, such as cytokines (TGF-b, IL-2, IFN-g, TNF-a, etc.), galectins (galectin-1, 9, and 10), heat shock proteins (Hsp-60, Hsp-60 peptide p277, Hsp-gp96, etc.), cellular metabolites (retinoic acid) as a result of infection.
diseases such as tuberculosis, malaria, hepatitis C, filariasis, leishmaniasis, and schistosomiasis (Moore et al., 2001). The most remarkable example of this control is illustrated by its crucial role during acute infection of mice with Toxoplasma gondii. In this model, IL-10 production by T cells is the key regulator of effector-cell responses, as IL-10-deficient mice can control parasite number, but they succumb to lethal immunopathology driven by unrestrained effector-cell responses (Gazzinelli et al., 1996). Besides T cells, IL-10 can be produced by numerous cell types, including B cells, NK cells, macrophages, and DCs (Moore et al., 2001). IL-10 can also be produced by nTreg and, in some cases, is associated with their function; however, in most cases, the inducible Tr1-cell population is the relevant source of this cytokine during infection. During various infections, Tr1 cells develop from conventional T cells after encountering certain signals, such as exposure to deactivated or immature APCs (antigen-presenting cells), or repeated exposure to antigen or IL-10 itself. Of note, these conditions prevail during chronic infection in which APC functions are often targeted by the pathogen, and cells of the immune system are chronically exposed to microbial antigens. Consistent with a role for these cells in human disease, Tr1-cell clones can be isolated from patients who are chronically infected with HCV (hepatitis C virus) (MacDonald et al., 2002). Interestingly, these regulatory clones had similar viral antigen specificity to protective Th1-cell clones isolated from the same patient (MacDonald et al., 2002). Defined microbial products can manipulate DCs in a way that induces Treg populations (Mills & McGuirk, 2004). For example, filamentous hemagglutinin (FHA) from Bordetella pertussis was shown to induce IL-10 production by DCs; these DC favored the differentiation of naïve T cells into Tr1 cells. Similarly, Tr1 cells can be generated from naïve T cells in the presence of DCs stimulated with phosphatidylserine from Schistosoma mansoni. Although Tr1 cells define a population of T cells that can produce IL-10 and/or TGF-b, some IL-10-producing T cells can also produce IFN-g. In the context of an infectious disease, IFN-g/IL-10 double producers were first described in the bronchoalveolar lavage of patients with tuberculosis and
in individuals chronically infected with Borrelia burgdorferi. Indeed, in many chronic infections, in humans and experimental animals, the presence of CD41 T cells that produce high levels of both IL-10 and IFN-g have been documented. Recently it was shown that IFN-g and IL-10-producing CD41 T cells emerge during experimental infection with T. gondii and in a model of nonhealing leishmaniasis (Anderson et al., 2007; Jankovic et al., 2007), and that these cells share many features with Th1 cells and were the main source of protective IL-10. These T cells were identified as activated T-bet1 Th1 cells and were distinct from Th2 cells, nTregs, or other subsets of inducible regulatory T cells. Unlike IFN-g production, IL-10 production was transient, observed in only a fraction of the IFN-g-producing cells and was produced more rapidly by recently activated T cells than by resting T cells (Jankovic et al., 2007). The instability of IL-10 synthesis, which was noted only when the Th1 cells were fully activated, is probably necessary to prevent sustained suppression of effector functions. Thus, it appears that, in some cases, cells with regulatory properties could arise from fully differentiated Th1 cells as a negative feedback loop. It is likely that numerous previous studies of Tr1 cells were, in fact, incriminating similar populations. These IFN-g and IL-10-producing T cells may represent a dominant regulatory response to infections that induce highly polarized Th1-cell responses. Chronic infections may require an additional layer of regulation, which would be provided by converted Foxp31 regulatory T cells. This hypothesis is supported by the observation that during infection, the downstream effects of inflammatory responses are also often associated with anti-inflammatory processes, including TGF-b production. Furthermore, some pathogens target sites in which TGF-b is highly produced, such as the gastrointestinal tract, the skin, and the eye, which may assist in the conversion in vivo. TGF-b can be also produced by infected cells or by cells the microorganisms are in contact with, or can arise as a result of an inflammatory process. For example, the trypomastigote stage of Trypanosoma cruzi induces TGF-b and IL-10 secretion by DCs. Compelling data in a mouse model of malaria suggest that TGF-b and regulatory T cells are central
112
HOST DEFENSE: GENERAL
regulators of immunopathology and parasite expansion. During late infection with Plasmodium yoelii infection, DCs migrate to the spleen of infected mice and secrete TGF-b together with IL-10 and PGE2 (Ocana-Morgner et al., 2007). Following experimental malaria infection of human volunteers, enhanced TGFb and Foxp31 Treg responses in PBMCs (peripheral blood mononuclear cells) correlate with a faster parasitic growth rate (Walther et al., 2005). Cells with nTreg characteristics are rapidly induced following blood stage infection and are associated with a decrease of proinflammatory cytokines and antigen-specific responses. Monocytes are a likely source of the early TGF-b production in this infection (Walther et al., 2005). Some nematodes can themselves express homologs of TGF-b (Gomez-Escobar et al., 1997). Compelling experimental data support the idea that Foxp31 Treg can be induced during Heligmosomoides polygyrus infection. The relative contribution of these converted Tregs to peripheral tolerance and the outcome of infections as well as how pathogens can utilize or interfere with this pathway to favor their own survival remains to be addressed. Currently, in the absence of definitive markers to distinguish endogenous versus converted Foxp31 regulatory T cells, these questions will remain difficult to answer.
ROLE OF Treg DURING ACUTE INFECTIONS
In this chapter, we consider acute infections to be those where clinical responses occur within a few days of infection with the disease course short ending in death or full recovery. Acute infections often cause lesions at entry sites such as mucosal surfaces and, if the agent persists, it does so silently. Only a few reports have focused on Treg during acute infections and some have concluded that regulatory cells have little influence on the outcome. Most notably, lymphocytic choriomeningitis virus, the favorite agent of viral immunologists, does not seem to involve any participation by nTreg (Rouse et al., 2006). With some acute infections, however, the Foxp31 Treg population is expanded and activated. Moreover, if this response is suppressed prior to infection, the outcome is different and the magnitude of the resulting immune response is increased. This is true for experimental HSV, respiratory syncytial virus (RSV), and influenza virus infections in mice, as well as with several acute parasitic infections (Belkaid & Tarbell, 2009). Many bacterial infections are acute in nature, but there is scant information as to the relevance of Treg responses in such infections. With Listeria monocytogenes, some results report no effect mediated by Tregs (Fontenot et al., 2005), whereas others indicate that this infection may cause the induction of pathogen-specific Treg responses (Ertelt et al., 2009). Why so few acute infections appear to induce Treg responses has not been adequately explained. It could be, in part, because the rapid disease course provides no time for activated or induced Treg to influence the course of events. This scenario might be especially relevant for the responses of regulatory cells that need to be generated from conventional precursor populations. Additionally, many acute infections induce strong inflammatory reactions that include Th1, Th2, and Th17 polarizing cytokines, all of which may interfere with the induction, expansion, and effector function of Foxp31 Treg. Furthermore, inflammatory cytokines may cause dendritic cell maturation, which renders them ineffective activators and inducers of regulatory T cells. In those acute infections where a dominance of IL-10 and TGF-b prevails, a more common circumstance in some locations, and perhaps under certain doses of infection, Treg stimulation is favored. This scenario may be more common with microbes involved in chronic diseases.
An acute infection that does cause activation and expansion of Foxp31 Treg is HSV infection of mice and humans. Initial studies showed that animals depleted of Tregs with PC61 MAb (which selectively depletes nTreg) became more susceptible to infection but developed heightened immune responses (Suvas et al., 2004, 2003). More recently, acute HSV infection was performed in a valuable new transgenic mouse model where all Foxp3 T cells could be specifically depleted and other cell types would be unaffected. Using this model, the Rudensky group not only confirmed that animals without nTregs make better HSV-specific Tcell responses, but also showed that disease (in their model, lethal encephalitis, fortunately a very rare outcome in the natural human host) was accelerated (Lund et al., 2008). They attributed the accelerated disease in the absence of Treg to changes in migration patterns of effector NK and T cells. When Foxp31 T cells were missing, there was a delay in the time when the effectors arrived at the infection site because of their prolonged retention in the lymph nodes as a result of chemokines building up. The virus progressed to the brain, causing encephalitis, a predominantly virological event. Accordingly, nTreg may facilitate the movement of effectors to infection sites by some as of yet unknown mechanism. A similar situation was reported recently for RSV infection (Ruckwardt et al., 2009). Here too, Treg depletion resulted in elevated immune responses in lymphoid tissues, but there was a delay in clearance of the virus from the lung infection site. They attributed this to the retention of protective CD81 T cells in the lymph node. Conceivably, understanding how Treg influence protective immunity to RSV could help explain the lack of CD81 T-cell responses in infants, which results in severe primary disease. It will be important to determine how acute infections trigger the expansion and activation of Foxp31 T cells in those infections where it does occur. With acute HSV infection, the expansion of the Foxp31 T cells was polyclonal rather than antigen specific. Possible mechanisms to explain this expansion were discussed in a previous section. However, in the case of HSV, an additional mechanism may come into play. Thus nTreg express the molecule HVEM (herpes virus entry mediator), a receptor involved in HSV entry into cells. Previous reports demonstrated that engagement of HVEM with some ligands causes T-cell expansion and activation (Tao et al., 2008). Curiously, the major glycoprotein of HSV, gD, also binds HVEM and this might represent a means by which Treg could be stimulated and expanded. The above discussion has focused on the possible influence of Foxp31 Treg in acute microbial infections. However, induced Treg, particularly the IL-10 producing Tr1 regulators, may also influence some acute infections. As explained previously, these cells are induced from nonregulatory precursors when antigen is stimulated under appropriate conditions, which include the presence of IL-10. These regulatory cells may be necessary to control responses to acute parasitic infections such as Toxoplasma gondii and murine malaria (Couper et al., 2009). With T. gondii, animals unable to produce IL-10 (and hence generate Tr1 cells) can control parasite growth but they ultimately succumb to lethal effector T-cell-mediated immunopathological lesions. An acute bacterial infection where Tr1 cells influence the outcome is Bordetella pertussis. In this infection, the filamentous hemagglutinin (FHA) of the bacterium induces dendritic cells to produce IL-10, which then act to elicit the Tr1 response (McGuirk et al., 2002). Some other pathogens may cause Tr1 induction by producing a homolog of IL-10, but these are chronic infectious agents and are discussed in another section.
8. Regulation of Antimicrobial Immunity
Many infections that end up with chronicity initially have an acute phase during which critical events occur that influence the subsequent pattern of disease. One factor thought to play a part is the magnitude and efficacy of initial Treg responses. In hepatitis C infection, a minority of infected persons fully controls their infection and the virus is eliminated. Unfortunately, most suffer chronic disease that can, in some, end in liver failure. Since Foxp31 T cells are increased in patients infected with HCV, and their removal from antigen stimulated PBL results in enhanced responses in vitro, some have suggested that the magnitude of the initial Treg response (natural or induced) might impact the subsequent pattern of chronic disease (Rouse et al., 2006). Thus, conceivably, those that generate a rapid Treg response may go on to develop chronic disease because the protective components of immunity are constrained. Although HCV infection does expand the Treg population, no compelling evidence shows that the extent of one or more types of Treg responses correlates with the eventual outcome of infection. However, the necessary long-term studies measuring Treg responses in the blood, and more relevantly the liver, have yet to be performed. Investigation with acute lentivirus infections in primates is another example indicating that the Treg response could be a critical event. As is well-known, Old World monkeys resist SIV (simian immunodeficiency virus) induced disease, but New World monkeys are very susceptible (Pereira et al., 2007). Levels of virus replication in the different species is approximately the same, but susceptible animals succumb to CD41 T-cell depletion that is the consequence of their hyperactivation. Several explanations have been suggested to explain the differential outcome, but some have observed that African green monkeys develop a more rapid Treg response than do macaques, and that this may serve to inhibit the T-cell hyperactivation (Rouse et al., 2006). There are also reports that the extent of Treg induction may influence the outcome in HIV infection, although this idea is yet to be substantiated (discussed subsequently).
ROLE OF Treg DURING CHRONIC AND PERSISTENT INFECTIONS
Even when Treg successfully preserve homeostasis in the host by controlling excessive immune responses, one consequence of such control is enhanced pathogen survival and, in some cases, long-term pathogen persistence (Table 1). For example, in a resistant mouse model of Leishmania infection, mice remained chronically infected at the site of primary infection. Treg that accumulate at the site of infection regulate the function of local effector cells, which prevents efficient elimination of the parasite (Belkaid et al., 2002). Similarly, in the context of African trypanosomiasis, IL-10 producing Treg can limit the pathogenic consequences of the immune response against the microorganism without
compromising parasite persistence. In its reservoir host, Treg can also favor the persistence of various pathogens such as Mycobacterium tuberculosis, mouse mammary tumor virus, or the pathogenic Seoul virus (Belkaid & Tarbell, 2009). In some cases, Treg can control the fine balance established between the pathogen and its host, mediating an equilibrium that can become mutually beneficial. In other cases, regulatory control is too excessive, allowing the pathogen to replicate without restraint and overwhelm the host, thereby compromising host survival. In a mouse model of malaria, for example, depletion of Treg protected mice from death caused by the lethal strain of Plasmodium yoelii by restoring a vigorous effector immune response that eradicated the parasites (Hisaeda et al., 2004). Following experimental infection with a hypervirulent strain of M. tuberculosis, the early Th1 response was rapidly reduced, correlating with the rapid emergence of IL-10-producing Foxp31 cells (Ordway et al., 2007). Filarial disease, caused by infection with a filarial nematode, is associated with a profound systemic suppression of the host immune system (Belkaid & Tarbell, 2009). Related to this balance of protective versus damaging pathogen-directed immune response is the balance between immune activation and the development of autoimmunity. Immune responses in the context of infection can have varying effects on the potential development of autoimmunity. On the one hand, the damage to self-tissue, the activation of innate immunity, and the cross-reaction between self and foreign antigens can increase the probability of developing self-specific immune responses. On the other hand, pathogens, via TLR ligands and other mechanisms, can enhance immune-suppressive cell types such as Treg, which can result in an infection being negatively correlated with autoimmunity. For example, rates of both allergy and autoimmunity are lower in developing countries with higher rates of infectious disease (Bach, 2002). There is a growing body of literature supporting the idea that microorganism persistence is, in some cases, necessary for the maintenance of protective immunity. In a situation of chronic infection or high exposure to microbial antigens, such as in the case of plasmodium during the transmission season, the generation of potent effector/memory cells could lead to pathogenic consequences. In a model of Leishmania major infection, parasite persistence, as a result of immune suppression by Treg, was necessary for the maintenance of protective immunity against the parasite (Belkaid et al., 2002; Suvas et al., 2004). Another example of this entente between the host and the pathogen is provided by ocular infection of mice with HSV1. When infected with a low dose of the virus, Treg protected mice from the CD41 T-cellmediated pathology, a situation that is compatible with the establishment of immunity to reinfection (Suvas et al., 2004). Similarly, in Candida albicans infection in mice, reduction of the number of Treg led to a better control of the primary infection but enhanced pathology as well as a loss of immunity
TABLE 1 Tregs in chronic infection Host-microbe interaction parameters T-effectors vs Tregs Predominant cytokines Pathogen burden
Outcome Immunosuppression Tregs T-effectors IL-10, TGF-b Prolonged persistence
113
Immunity
Immune-mediated pathology
T-effectors 5 Treg T-effectors Treg IL-2, IFN-g, IL-12, IFN-g, TNF-a, IL-12, IL-17 IL-17, IL-10, TGF-b Timely control Controlled
114
HOST DEFENSE: GENERAL
to reinfection. Immunity to reinfection can be recapitulated when Treg are transferred back (Montagnoli et al., 2002). Thus, a role for Treg in the maintenance of immunity may be common to other chronic infections in which poor quality effectors are generated and pathogen persistence is required to sustain protective responses. A large number of studies support the idea that Treg play a role in the control of persistent infection in humans. In the human population, however, reliable identification of Treg is complicated by the fact that Foxp3 expression does not always correlate with regulatory properties, and activated T cells can transiently express it. Likewise, CD25 or other Treg markers cannot be used to discriminate reliably between Treg and highly activated T cells. Because peripheral blood is the most accessible compartment, most human studies that evaluate Treg functions or numbers are done using this compartment. However, these measurements may not be representative, because in some chronic infections Treg accumulate in infected tissues and consequently are reduced in the blood compartment. Despite these caveats, some reports provide convincing evidence for Foxp3-expressing Treg playing a role in a large number of human infections (Belkaid & Tarbell, 2009). In pulmonary tuberculosis patients, the frequencies of Treg are significantly higher in the blood and at the site of infection compared with normal individuals (Chen et al., 2007b). Furthermore, the frequency of Treg in pleural fluid inversely correlates with local M. tuberculosis-specific immunity. These Treg display an activation phenotype, and, in vitro, the addition of Treg back to cultures significantly suppresses the antigen-specific production of IFN-g by effector T cells. Conversely, the removal of Treg significantly enhances effector responses against bacteria by ab and gd T cells (Belkaid & Tarbell, 2009). Some data suggest that differences in susceptibility to infection may rely on a functional deficit of Treg. Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that the Fulani people are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated with classic malaria-resistance genes, and analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as to nonmalarial antigens, revealed higher immune reactivity in the Fulani group (Torcia et al., 2008). Microarray analysis on RNA from Treg indicated obvious differences between ethnic groups, with 23% of genes, including TGF-b, TGF-b receptors, CTLA4, and Foxp3, less expressed in the Fulani people as compared with the Mossi people and European donors not exposed to malaria. As further indications of low Treg activity, the Fulani showed lower serum levels of TGF-b. Furthermore, the proliferative response of the Fulani to malaria antigens was not affected by the depletion of Treg, whereas that of the Mossi was significantly increased. The results suggest that the higher resistance to infection in a defined ethnic group may derive from a functional deficit of Treg. Several studies also suggest that Treg may limit the ability of adaptive T-cell response to control HIV and SIV replication. In particular, anti-HIV responses were increased after in vitro removal of Treg from peripheral leukocytes and from lymph nodes of HIV-infected patients (Sempere et al., 2007). Several groups have shown that Treg frequencies are decreased in the peripheral blood of HIV-infected patients (Sempere et al., 2007). This observation could suggest that Treg, just as with conventional T cells, are progressively lost during HIV infection. Furthermore, cells from infected individuals that show strong HIV-specific Treg function in vitro had significantly lower
levels of plasma viremia and higher CD41-to-CD81 T cell ratios than did individuals with undetectable Treg activity. However, the observation that the expression of Treg is increased in lymphoid or mucosal tissues from HIV-infected patients and SIV-infected macaques suggests that the redistribution of Treg in infected tissues could account for the decreased frequency of Treg in the blood. Importantly, Treg purified from the lymph node of HIV patients maintain their suppressive capacity against antiviral responses (Sempere et al., 2007). The high frequency of Foxp31 Treg was much higher in the duodenal mucosa of patients infected with HIV as compared to healthy controls (Sempere et al., 2007). These findings suggest that Treg, by suppressing virus-specific immunity, may contribute to uncontrolled viral replication, therefore playing a detrimental role in HIV infection. However, there is a strong negative correlation between the level of T-cell activation and the frequency of Treg, which is most prominent during the early chronic stage of the disease, suggesting a role for these cells in the control of immune activation (Ndhlovu et al., 2008). Furthermore, the preservation of Treg in the PBMCs of HIV-infected elite controllers correlates with low CD41 T cell activation, supporting the idea that it may be one mechanism associated with the nonprogressive nature of HIV infection in elite controllers) (Chase et al., 2008). Thus, although there is a consensus on the capacity of Treg to suppress immune responses during HIV infection, the overall consequence on the development of the disease remains uncertain and is likely to be tightly dependent on the stage of the infection or the site targeted. Treg appear also to play a role in the control of chronic viral hepatitis (Rouse et al., 2006). Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the most common causes of liver disease worldwide, and failure to control infection with either virus results in immune-mediated acute and chronic necroinflammatory liver disease. In patients with chronic infection with HBV, the frequencies of Foxp31 Treg are highly increased, both in the periphery and in the liver, and correlates with viral load (Belkaid & Tarbell, 2009; Rouse et al., 2006). More recently, a positive correlation was shown between IL-10-producing Foxp31 and Treg, which expanded in response to viral antigen and viral load (Barboza et al., 2007). Furthermore, antigen-specific suppression of effector responses in vitro suggests that the expansion of antigenspecific Treg during this infection may contribute to the associated liver pathology (reviewed in Alatrakchi & Koziel, 2009). HCV-associated liver disease also seems to involve Treg, which could impede immune defense against the virus. Individuals who are chronically infected with HCV have a higher number of Treg in the blood compared with those whose HCV infection spontaneously resolved or with those who are not infected (Alatrakchi & Koziel, 2009). Depletion of Treg enhances antigen-specific CD81 T responses in vitro (Alatrakchi & Koziel, 2009). During HCV infection, Treg display enhanced suppressive function when assessed in vitro during both the acute and chronic stages of the infection, compared with uninfected donors or with patients with spontaneous resolution. Tregs also accumulate in the liver of patients with chronic HCV. The inverse correlation between the HCV-specific TGF-b response by Treg and liver damage could support the idea that Treg also have a role in controlling chronic inflammatory responses and liver damage in HCV carriers (Alatrakchi & Koziel, 2009). Interestingly, patients who are chronically infected with HCV and go on to develop autoimmunity have fewer peripheral Treg (Boyer et al., 2004). However, the link between chronic infection, autoimmune disorders, and dysregulation of Treg function requires further analysis.
8. Regulation of Antimicrobial Immunity
NEGATIVE CONTROL OF Treg INDUCTION BY PATHOGENS
It is becoming clear that the status of activation of DCs as well as inflammatory mediators modulates the capacity of antigen-presenting cells to induce Treg de novo. For instance, IL-6 and TGF-b in tandem can direct the production of IL-17-secreting T cells over Treg (Bettelli et al., 2006; Veldhoen et al., 2006), and Th1 and Th2 effector cytokines have an antagonistic effect on Treg conversion (Wei et al., 2007). In addition, exposure to activated DCs or strong costimulation limits the induction of Foxp31 Treg in favor of the induction of effector responses (Benson et al., 2007; Kretschmer et al., 2005; Wang et al., 2008). A correlate of this observation would be that inflammation triggered by infection can antagonize Treg induction. Indeed, acute infection in mice with Listeria monocytogenes failed to induce Foxp3 by conventional CD41 T cells (Fontenot et al., 2005). Furthermore, the constitutive induction of Treg in the gastrointestinal tract following exposure to oral antigens is inhibited following oral infection with the protozoan T. gondii (Oldenhove et al., 2009). Previous reports examining both gut and lung inflammation support the idea that restricted or defective Treg conversion can enhance immunopathology (Curotto de Lafaille et al., 2008; Izcue et al., 2008), supporting the idea that interference with constitutive Treg induction during infection may directly contribute to the pathological process. Furthermore, the observations also raise the possibility that exposure to antigen at a time of acute infection may impair the acquisition of tolerance against innocuous antigens.
CONTROL OF Treg PLASTICITY DURING INFECTION
Recent findings suggest that acquisition of transcription factors in addition to Foxp3 can confer unique functional characteristics to Treg (Zheng et al., 2009). For instance, expression of T-bet by Treg was recently shown to favor their proper control of Th1 environments (Koch et al., 2009). Similarly, in the context of a virulent T. gondii infection, Treg express high levels of the Th1 transcription factor T-bet (Oldenhove et al., 2009). Intriguingly, in the context of Th1 settings, T-bet expression by Treg leads to up regulation of CXCR3 and preferential homing to infected sites (Koch et al., 2009). Thus, the appropriation of T-effector lineage transcription factors by Treg suggests that under nonpathogenic situations, acquisition of these elements controlled by local DCs may represent a necessary layer of their regulation. On the other hand, under highly pathogenic situations such as following a lethal T. gondii infection, T-bet expression led to the production of IFN-g, a cytokine responsible for both effector and pathogenic responses during T. gondii infection (Oldenhove et al., 2009). Although T-cell lineage subsets were initially believed to represent stable populations of cells, recent evidence shows that lymphocytes maintain a certain degree of plasticity with respect to their capacity to produce cytokines (Lee et al., 2009; Wei et al., 2009). Cells expressing both Foxp3 and IL-17 can be found in mucosal tissue or in vitro cultures (Lee et al., 2009; Wei et al., 2009). Genome-wide mapping of H3K4me3 and H3K27me3 performed in ex vivo Treg revealed markers of both repression and induction at the tbx21 locus. However, the Ifng locus did not show any sign of induction or repression (Wei et al., 2009), suggesting that Ifng is poised for transcriptional activation. This would suggest that, in the presence of high levels of inflammatory mediators, T-bet expression might reach a threshold that superimposes an effector program on
115
Treg. Given their high degree of self-reactivity, it is plausible that Treg can contribute to tissue damage or can lose suppressive capacity if armed with effector cytokines. Indeed, a recent report highlighted that Foxp3 instability and acquisition of IFN-g can favor the development of autoimmune diabetes (Zhou et al., 2009).
CONTROL OF ENDOGENOUS Treg DURING INFECTIONS
Overcoming regulation is a prerequisite to the expression of effector responses. Mechanisms associated with negative control of Treg include direct Treg stimulation, as well as strong costimulation of effector T cells mediated by local DCs. Over the past few years, a strong body of literature suggests that a primary function of IL-2 is to promote the expansion and survival of Treg (Malek & Bayer, 2004). Intriguingly, IL-2 production declines upon differentiation into T-effectors, implying that in the context of a highly polarized Th1 response, IL-2 may become limiting. Accordingly, following oral infection with T. gondii, the emergence of a Th1 response was associated with a dramatic reduction of IL-2 production by CD41 T cells in the gastrointestinal tract and in the periphery. Previous work demonstrated that T-bet acquisition, as well as exposure of T cells to IL-27, IL-12, or IL-2 itself, could limit IL-2 production (Hwang et al., 2005; Villarino et al., 2007). In addition, T-effectors, via their consumption of growth factor, may also deprive the host from exogenous IL-2. A link between defective IL-2 production and Treg dysfunction has been previously proposed as one of the mechanisms associated with breakdown of tolerance in nonobese diabetic mice (Tang et al., 2008).
MANIPULATING Treg TO CONTROL INFECTIOUS DISEASE
As discussed already, regulatory cell responses to microbes can be a double-edged sword. They can compromise immune efficacy or they can act to diminish the collateral tissue damage caused by exuberant antimicrobial reactivity, as commonly occurs in chronic infections. With the latter, however, the situation may be complex since several types of regulators with different functions may be involved during the course of a chronic infection. Moreover, even effector cells that formerly helped orchestrate lesions may take on a regulatory function under appropriate conditions. When it comes to devising therapies, there are circumstances where inhibiting the excessive activity of regulatory cells would favor microbial control, and other situations, such as chronic tissue damage, where expanding the influence of regulators would be beneficial. From a practical perspective, we may be closer to achieving the latter effect, and several approaches are quite well-developed to control chronic inflammatory reactions associated with autoimmune diseases and transplantation rejection. Several experimental infections are controlled more effectively if the influence of Treg is diminished. This can be achieved either by depressing Treg numbers or by blunting their function. The usual approach is to use a MAb that mainly targets Treg such as anti-CD25, but with mice that can be constructed that allow specific deletion of Foxp31 Treg, as discussed previously (Lund et al., 2008). The removal of Treg would not seem to be a practical method by which to manage the response to an ongoing infection, but it could prove useful to improve the efficacy of some vaccines. Thus, as initially shown with vaccines against HSV in mice, Treg depletion during vaccination resulted in improved primary and memory T-cell responses and increased
116
HOST DEFENSE: GENERAL
immunity (Rouse et al., 2006). Subsequently, more extensive studies were performed with vaccines against murine malaria (reviewed in Belkaid & Tarbell, 2009). Confirming the HSV studies, depleting Treg at the time of vaccination resulted in heightened CD81 T-cell responses that were more broadly reactive in terms of epitope specificity. The Treg-depleted vaccinees also showed more durable immunity to a malaria challenge. Depleting Treg also resulted in better vaccination responses to influenza, vaccinia, and hepatitis B viruses, as well as to M. tuberculosis. Unfortunately, in the latter instance, although immune responses were accelerated and elevated in Treg-depleted animals, the level of protective immunity to virulent M. tuberculosis was not improved (McGuirk et al., 2002). The observation that Treg depression may improve the immunogenicity of some vaccines raises some interesting issues. These include understanding how the effect actually
occurs, how it can be facilitated, and whether or not the approach could be exploited with a vaccine regime in humans or animals. As to mechanisms of action, it is not clear if the enhancement effect is a consequence of removing Treg or of blocking their response to IL-2, so as to make the latter more available to drive the expansion of effector or memory T cells. Alternatively, the outcome may be the consequence of effects on antigen presentation. Thus, stimulated Treg, perhaps via the cytokines they produce, can influence the function of dendritic cells, causing them to down regulate costimulatory molecules, such as CD80 and CD86, and suppress their production of IL-6. The effects on DCs make them less effective inducers of the T cells involved in protective immunity. Since Treg may also influence some functions of effector cells, reducing Treg activity may contribute to enhanced effector cell activity and may perhaps influence their differentiation into more
TABLE 2 Strategies to boost Treg responses Strategy
Effect
Cytokines IL-2
Proliferation
IL-2 and anti-IL-2 complex TGF-b
Proliferation Induction
TNF-a IFN-g
Proliferation Induction
References Reviewed in Brusko et al., 2008 Webster et al., 2009 Reviewed in Belkaid & Tarbell, 2009 Chen et al., 2007a Chang et al., 2009
Other host proteins and products Galecin-1
Proliferation
Galectin-9 Galectin-10
Proliferation and induction Proliferation
FLT-3 ligand Vasoactive intestinal peptide Progesterone VEGF Retinoic acid
Proliferation Induction Induction Proliferation and induction Induction
Thrombospondin-1
Induction
Reviewed in Rabinovich & Toscano, 2009 Sehrawat et al., 2009 Reviewed in Rabinovich & Toscano, 2009 Swee et al., 2009 Pozo et al., 2009 Mjosberg et al., 2009 Wada et al., 2009 Reviewed in Belkaid & Tarbell, 2009 Grimbert et al., 2006
Anti-CD154 Activation-inducible TNFR-a-Fc (AITR-Fc) CTLA-4-Ig
Proliferation Proliferation
Rigby et al., 2008 Sonawane et al., 2009
Proliferation
Anti-CD3 F(ab)2
Proliferation
Reviewed in Brusko et al., 2008 Reviewed in Brusko et al., 2008
Antibodies and fusion proteins
Chemical compounds Rapamycin FTY720 Corticosteroids
Treg spared from killing Induction Induction
Basu et al., 2008 Sehrawat & Rouse, 2008 Esparza & Arch, 2006
HDAC inhibitors Tacrolimus Glatiramer acetate
Proliferation and induction Induction Induction
Tao et al., 2007 Wang et al., 2009 Cui et al., 2009
8. Regulation of Antimicrobial Immunity
functionally effective memory cells. These issues have not been fully evaluated, nor has the outcome of Treg depletion of later phases of the primary or recall response on the efficiency of vaccination. Nevertheless, a take-home lesson from studies showing that Treg can interfere with the efficacy of vaccines is to choose immunization approaches that minimally stimulate one or more types of regulatory cells. In support of this notion, persons who were nonresponsive to standard hepatitis B vaccination possessed higher numbers of antigen specific Foxp31 Treg in their blood than did responder vaccines (Moore et al., 2005). Vaccine approaches that minimize the chances of inducing Treg include choosing appropriate adjuvants (preferably pro-inflammatory), avoiding multiple low dose immunization schemes, and not using sites for immunization (e.g., the gut) that favor the induction of Treg responses. The topic of practicality is also an issue for consideration. It seems highly likely that administering a MAb to the CD25 receptor, which is available for clinical use in humans to control transplant rejections, would be economically unrealistic for human vaccines even if the approach is shown to work and lacks side effects. However, if we unravel the molecular mechanisms responsible for Treg function, chemical and biological approaches could conceivably be devised that manipulate Treg activity in a negative direction and perhaps enhance vaccine efficacy. Possible targets include adenosine and cAMP (cyclic adenosine monophosphate), which are involved in the suppressor function of Treg. A major function of several types of Treg may be to reduce the tissue damage that results from a chronic inflammatory response to infections. Consequently, enhancing the number or function of Treg would be therapeutically useful. Conceptually, there are a number of ways for achieving this objective (Table 2). These include expanding Treg populations in vitro and using them for adoptive transfer in vivo, using chemical or biological procedures that expand and increase the function of Treg in vivo, or causing uncommitted T cells in vivo to differentiate to become regulatory in function. The field of expanding and manipulating Treg to control immunoinflammatory lesions is led by groups interested in autoimmune disease and transplantation, and little has been done so far to control chronic microbial-induced inflammatory lesions (Brusko et al., 2008). We have evaluated whether or not manipulating Treg activity represents a useful way to modulate immunoinflammatory lesions caused by ocular infection with HSV. Accordingly, it was shown in this system that lesion severity became enhanced if animals were depleted of Treg, and that the inflammatory effects of adoptive transfers of HSV primed T cells into RAG−/− recipients was modulated by co-transfers of polyspecific T cells (Suvas et al., 2004). Hence, in this system, increasing the participation of Treg was expected to reduce disease in an experimental model of an important cause of human blindness. One way to increase the participation of Treg is to generate such cells in vitro and adoptively transfer them into animals ocularly infected with HSV to see if this affected lesion severity. The procedure was successful, but only when transfers were given early in the disease process (Sehrawat et al., 2008). Curiously, the Treg population responsible for lesion suppression were not specific to HSV antigens, indicating that Treg can act nonspecifically in a bystander way in vivo, at least if they are activated. An adoptive transfer approach to control chronic microbial reactions represents an unlikely method to use in clinical situations. It seems more likely that procedures that cause conventional T cells to convert in vivo to become
117
regulatory will be more realistic (see Table 2). One way of achieving this would be to use the fungal metabolite drug FTY720, because this causes conventional T cells to become FoxP31 and regulatory in function both in vitro and in vivo (Sehrawat & Rouse, 2008). However, this approach alone might not be that effective since, under the influence of the drug, Treg did not divide extensively. Therefore, a combination therapy such as the use of cytokines (e.g., IL-2) might prove to be an effective approach to expand the converted Treg (Ertelt et al., 2009). Another approach showing promise is the observation that certain galectin molecules may act as activators of Treg. For example, galectin-9 administration to animals with developing herpetic ocular lesions succeeds in reducing lesion severity in part because populations of FoxP31 are expanded (Sehrawat et al., 2009). Furthermore, blocking galectin-9 from binding to its receptor TIM-3 with simple sugars diminished the suppressive activity of Treg. Consequently, in situations where inhibited Treg response is desired, the use of simple sugars such as lactose might prove useful. In conclusion, although management of microbe-induced chronic lesions is still in the early stages, some results do show promise. We anticipate the rapid discovery of new approaches.
REFERENCES Alatrakchi, N., and M. Koziel. 2009. Regulatory T cells and viral liver disease. J. Viral Hepat. 16:223–229. Anderson, C. F., M. Oukka, V. J. Kuchroo, and D. Sacks. 2007. CD4(1)CD25(2) Foxp3(2) Th1 cells are the source of IL-10-mediated immune suppression in chronic cutaneous leishmaniasis. J. Exp. Med. 204:285–297. Bach, J. F. 2002. The effect of infections on susceptibility to autoimmune and allergic diseases. N. Engl. J. Med. 347:911–920. Barboza, L., S. Salmen, L. Goncalves, M. Colmenares, D. Peterson, H. Montes, R. Cartagirone, C. Gutierrez Mdel, and L. Berrueta. 2007. Antigen-induced regulatory T cells in HBV chronically infected patients. Virology 368:41–49. Basu, S., T. Golovina, T. Mikheeva, C. H. June, and J. L. Riley. 2008. Cutting edge: Foxp3-mediated induction of pim 2 allows human T regulatory cells to preferentially expand in rapamycin. J. Immunol. 180:5794–5798. Becker, C., C. Taube, T. Bopp, K. Michel, J. Kubach, S. Reuter, N. Dehzad, M. F. Neurath, K. Reifenberg, F. J. Schneider, E. Schmitt, and H. Jonuleit. 2009. Protection from graft-versus-host disease by HIV-1 envelope protein gp120-mediated activation of human CD41CD251 regulatory T cells. Blood 114:1263–1269. Belkaid, Y., C. A. Piccirillo, S. Mendez, E. M. Shevach, and D. L. Sacks. 2002. CD41CD251 regulatory T cells control Leishmania major persistence and immunity. Nature 420:502–507. Belkaid, Y., and K. Tarbell. 2009. Regulatory T cells in the control of host-microorganism interactions. Annu. Rev. Immunol. 27:551–589. Benson, M. J., K. Pino-Lagos, M. Rosemblatt, and R. J. Noelle. 2007. All-trans retinoic acid mediates enhanced T reg cell growth, differentiation, and gut homing in the face of high levels of co-stimulation. J. Exp. Med. 204: 1765–1774. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, and V. K. Kuchroo. 2006. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441:235–238. Boyer, O., D. Saadoun, J. Abriol, M. Dodille, J. C. Piette, P. Cacoub, and D. Klatzmann. 2004. CD41CD251 regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis. Blood 103:3428–3430.
118
HOST DEFENSE: GENERAL
Brusko, T. M., A. L. Putnam, and J. A. Bluestone. 2008. Human regulatory T cells: role in autoimmune disease and therapeutic opportunities. Immunol. Rev. 223:371–390. Chang, J. H., Y. J. Kim, S. H. Han, and C. Y. Kang. 2009. IFN-gamma-STAT1 signal regulates the differentiation of inducible Treg: potential role for ROS-mediated apoptosis. Eur. J. Immunol. 39:1241–1251. Chase, A. J., H. C. Yang, H. Zhang, J. N. Blankson, and R. F. Siliciano. 2008. Preservation of FoxP31 regulatory T cells in the peripheral blood of human immunodeficiency virus type 1-infected elite suppressors correlates with low CD41 T-cell activation. J. Virol. 82:8307–8315. Chen, X., M. Baumel, D. N. Mannel, O. M. Howard, and J. J. Oppenheim. 2007a. Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD41CD251 T regulatory cells. J. Immunol. 179:154–161. Chen, X., B. Zhou, M. Li, Q. Deng, X. Wu, X. Le, C. Wu, N. Larmonier, W. Zhang, H. Zhang, H. Wang, and E. Katsanis. 2007b. CD4(1)CD25(1)FoxP3(1) regulatory T cells suppress Mycobacterium tuberculosis immunity in patients with active disease. Clin. Immunol. 123:50–59. Conroy, H., N. A. Marshall, and K. H. Mills. 2008. TLR ligand suppression or enhancement of Treg cells? A double-edged sword in immunity to tumours. Oncogene 27:168–180. Couper, K. N., P. A. Lanthier, G. Perona-Wright, L. W. Kummer, W. Chen, S. T. Smiley, M. Mohrs, and L. L. Johnson. 2009. Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection. J. Immunol. 182:3985–3994. Crellin, N. K., R. V. Garcia, O. Hadisfar, S. E. Allan, T. S. Steiner, and M. K. Levings. 2005. Human CD41 T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD41CD251 T regulatory cells. J. Immunol. 175:8051–8059. Cui, G., Y. Zhang, Z. Gong, J. Z. Zhang, and Y. Q. Zang. 2009. Induction of CD41CD251Foxp31 regulatory T cell response by glatiramer acetate in type 1 diabetes. Cell. Res. 19:574–583. Curotto de Lafaille, M. A., N. Kutchukhidze, S. Shen, Y. Ding, H. Yee, and J. J. Lafaille. 2008. Adaptive Foxp31 regulatory T cell-dependent and -independent control of allergic inflammation. Immunity 29:114–126. Ertelt, J. M., J. H. Rowe, T. M. Johanns, J. C. Lai, J. B. McLachlan, and S. S. Way. 2009. Selective priming and expansion of antigen-specific Foxp32 CD41 T cells during Listeria monocytogenes infection. J. Immunol. 182: 3032–3038. Esparza, E. M., and R. H. Arch. 2006. Signaling triggered by glucocorticoid-induced tumor necrosis factor receptor family-related gene: regulation at the interface between regulatory T cells and immune effector cells. Front. Biosci. 11:1448–1465. Fontenot, J. D., J. P. Rasmussen, L. M. Williams, J. L. Dooley, A. G. Farr, and A. Y. Rudensky. 2005. Regulatory T cell lineage specification by the forkhead transcription factor foxp3. Immunity 22:329–341. Gazzinelli, R. T., M. Wysocka, S. Hieny, T. Scharton-Kersten, A. Cheever, R. Kuhn, W. Muller, G. Trinchieri, and A. Sher. 1996. In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD41 T cells and accompanied by overproduction of IL-12, IFN-gamma and TNFalpha. J. Immunol. 157:798–805. Gomez-Escobar, N., A. van den Biggelaar, and R. Maizels. 1997. A member of the TGF-beta receptor gene family in the parasitic nematode Brugia pahangi. Gene 199:101–109. Grimbert, P., S. Bouguermouh, N. Baba, T. Nakajima, Z. Allakhverdi, D. Braun, H. Saito, M. Rubio, G. Delespesse, and M. Sarfati. 2006. Thrombospondin/CD47 interaction: a pathway to generate regulatory T cells from
human CD41 CD252 T cells in response to inflammation. J. Immunol. 177:3534–3541. Hall, J. A., N. Bouladoux, C. M. Sun, E. A. Wohlfert, R. B. Blank, Q. Zhu, M. E. Grigg, J. A. Berzofsky, and Y. Belkaid. 2008. Commensal DNA limits regulatory T cell conversion and is a natural adjuvant of intestinal immune responses. Immunity 29:637–649. Hisaeda H, M. Y., Iwakawa D, Okada H, Himeno K, Kishihara K, Tsukumo S, Yasutomo K. 2004. Escape of malaria parasites from host immunity requires CD4(1)CD25(1) regulatory T cells. Nat. Med. 10:29–30. Hwang, E. S., J. H. Hong, and L. H. Glimcher. 2005. IL-2 production in developing Th1 cells is regulated by heterodimerization of RelA and T-bet and requires T-bet serine residue 508. J. Exp. Med. 202:1289–1300. Izcue, A., S. Hue, S. Buonocore, C. V. Arancibia-Carcamo, P. P. Ahern, Y. Iwakura, K. J. Maloy, and F. Powrie. 2008. Interleukin-23 restrains regulatory T cell activity to drive T cell-dependent colitis. Immunity 28:559–570. Jankovic, D., M. C. Kullberg, C. G. Feng, R. S. Goldszmid, C. M. Collazo, M. Wilson, T. A. Wynn, M. Kamanaka, R. A. Flavell, and A. Sher. 2007. Conventional T-bet(1) Foxp3(2) Th1 cells are the major source of host-protective regulatory IL-10 during intracellular protozoan infection. J. Exp. Med. 204:273–283. Koch, M., A., G. Tucker-Heard, N. Perdue, R., J. Killebrew, R., K. B. Urdahl, and D. Campbell, J. 2009. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat. Immunol. 10:1–7. Kretschmer, K., I. Apostolou, D. Hawiger, K. Khazaie, M. C. Nussenzweig, and H. von Boehmer. 2005. Inducing and expanding regulatory T cell populations by foreign antigen. Nat. Immunol. 6:1219–1227. Lee, Y. K., H. Turner, C. L. Maynard, J. R. Oliver, D. Chen, C. O. Elson, and C. T. Weaver. 2009. Late developmental plasticity in the T helper 17 lineage. Immunity 30:92–107. Lund, J. M., L. Hsing, T. T. Pham, and A. Y. Rudensky. 2008. Coordination of early protective immunity to viral infection by regulatory T cells. Science 320:1220–1224. MacDonald, A. J., M. Duffy, M. T. Brady, S. McKiernan, W. Hall, J. Hegarty, M. Curry, and K. H. Mills. 2002. CD4 T helper type 1 and regulatory T cells induced against the same epitopes on the core protein in hepatitis C virusinfected persons. J. Infect. Dis. 185:720–727. Malek, T. R., and A. L. Bayer. 2004. Tolerance, not immunity, crucially depends on IL-2. Nat. Rev. Immunol. 4:665–674. McGuirk, P., C. McCann, and K. H. Mills. 2002. Pathogenspecific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J. Exp. Med. 195:221–231. Mills, K. H., and P. McGuirk. 2004. Antigen-specific regulatory T cells—their induction and role in infection. Semin. Immunol. 16:107–117. Mjosberg, J., J. Svensson, E. Johansson, L. Hellstrom, R. Casas, M. C. Jenmalm, R. Boij, L. Matthiesen, J. I. Jonsson, G. Berg, and J. Ernerudh. 2009. Systemic reduction of functionally suppressive CD4dimCD25highFoxp31 Tregs in human second trimester pregnancy is induced by progesterone and 17beta-estradiol. J. Immunol. 183:759–769. Montagnoli, C., A. Bacci, S. Bozza, R. Gaziano, P. Mosci, A. H. Sharpe, and L. Romani. 2002. B7/CD28-dependent CD41CD251 regulatory T cells are essential components of the memory-protective immunity to Candida albicans. J. Immunol. 169:6298–6308. Moore, A. C., A. Gallimore, S. J. Draper, K. R. Watkins, S. C. Gilbert, and A. V. Hill. 2005. Anti-CD25 antibody enhancement of vaccine-induced immunogenicity: increased durable cellular immunity with reduced immunodominance. J. Immunol. 175:7264–7273.
8. Regulation of Antimicrobial Immunity Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O’Garra. 2001. Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19:683–765. Ndhlovu, L. C., C. P. Loo, G. Spotts, D. F. Nixon, and F. M. Hecht. 2008. FOXP3 expressing CD127lo CD41 T cells inversely correlate with CD381 CD81 T cell activation levels in primary HIV-1 infection. J. Leukoc. Biol. 83:254–262. Ocana-Morgner, C., K. A. Wong, F. Lega, J. Dotor, F. Borras-Cuesta, and A. Rodriguez. 2007. Role of TGF-beta and PGE2 in T cell responses during Plasmodium yoelii infection. Eur. J. Immunol. 37:1562–1574. Oldenhove, G., N. Bouladoux, E. A. Wohlfert, J. A. Hall, D. Chou, L. Dos Santos, S. O’Brien, R. Blank, E. Lamb, S. Natarajan, R. Kastenmayer, C. Hunter, M. E. Grigg, and Y. Belkaid. 2009. Decrease of Foxp31 Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity 31:772–786. Ordway, D., M. Henao-Tamayo, M. Harton, G. Palanisamy, J. Troudt, C. Shanley, R. J. Basaraba, and I. M. Orme. 2007. The hypervirulent Mycobacterium tuberculosis strain HN878 induces a potent TH1 response followed by rapid down-regulation. J. Immunol. 179:522–531. Pereira, L. E., F. Villinger, N. Onlamoon, P. Bryan, A. Cardona, K. Pattanapanysat, K. Mori, S. Hagen, L. Picker, and A. A. Ansari. 2007. Simian immunodeficiency virus (SIV) infection influences the level and function of regulatory T cells in SIV-infected rhesus macaques but not SIVinfected sooty mangabeys. J. Virol. 81:4445–4456. Pozo, D., P. Anderson, and E. Gonzalez-Rey. 2009. Induction of alloantigen-specific human T regulatory cells by vasoactive intestinal peptide. J. Immunol. 183:4346–4359. Rabinovich, G. A., and M. A. Toscano. 2009. Turning “sweet” on immunity: galectin-glycan interactions in immune tolerance and inflammation. Nat. Rev. Immunol. 9:338–352. Rigby, M. R., A. M. Trexler, T. C. Pearson, and C. P. Larsen. 2008. CD28/CD154 blockade prevents autoimmune diabetes by inducing nondeletional tolerance after effector t-cell inhibition and regulatory T-cell expansion. Diabetes 57:2672–2683. Rouse, B. T., P. P. Sarangi, and S. Suvas. 2006. Regulatory T cells in virus infections. Immunol. Rev. 212:272–286. Ruckwardt, T. J., K. L. Bonaparte, M. C. Nason, and B. S. Graham. 2009. Regulatory T cells promote early influx of CD81 T cells in the lungs of respiratory syncytial virusinfected mice and diminish immunodominance disparities. J. Virol. 83:3019–3028. Sehrawat, S., and B. T. Rouse. 2008. Anti-inflammatory effects of FTY720 against viral-induced immunopathology: role of drug-induced conversion of T cells to become Foxp31 regulators. J. Immunol. 180:7636–7647. Sehrawat, S., A. Suryawanshi, M. Hirashima, and B. T. Rouse. 2009. Role of Tim-3/galectin-9 inhibitory interaction in viral-induced immunopathology: shifting the balance toward regulators. J. Immunol. 182:3191–3201. Sehrawat, S., S. Suvas, P. P. Sarangi, A. Suryawanshi, and B. T. Rouse. 2008. In vitro-generated antigen-specific CD41 CD251 Foxp31 regulatory T cells control the severity of herpes simplex virus-induced ocular immunoinflammatory lesions. J. Virol. 82:6838–6851. Sempere, J. M., V. Soriano, and J. M. Benito. 2007. T regulatory cells and HIV infection. AIDS Rev. 9:54–60. Shevach, E. M. 2006. From vanilla to 28 flavors: multiple varieties of T regulatory cells. Immunity 25:195–201. Sonawane, S. B., J. I. Kim, M. K. Lee, S. H. Lee, P. E. Duff, D. J. Moore, M. M. Lian, S. Deng, Y. Choi, H. Yeh, A. J. Caton, and J. F. Markmann. 2009. GITR blockade facilitates Treg mediated allograft survival. Transplantation 88:1169–1177. Suvas, S., A. K. Azkur, B. S. Kim, U. Kumaraguru, and B. T. Rouse. 2004. CD41CD251 regulatory T cells control the severity of viral immunoinflammatory lesions. J. Immunol. 172:4123–4132.
119
Suvas, S., U. Kumaraguru, C. D. Pack, S. Lee, and B. T. Rouse. 2003. CD41CD251 T cells regulate virus-specific primary and memory CD81 T cell responses. J. Exp. Med. 198:889–901. Swee, L. K., N. Bosco, B. Malissen, R. Ceredig, and A. Rolink. 2009. Expansion of peripheral naturally occurring T regulatory cells by Fms-like tyrosine kinase 3 ligand treatment. Blood 113:6277–6287. Tang, Q., J. Y. Adams, C. Penaranda, K. Melli, E. Piaggio, E. Sgouroudis, C. A. Piccirillo, B. L. Salomon, and J. A. Bluestone. 2008. Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction. Immunity 28:687–697. Tao, R., E. F. de Zoeten, E. Ozkaynak, C. Chen, L. Wang, P. M. Porrett, B. Li, L. A. Turka, E. N. Olson, M. I. Greene, A. D. Wells, and W. W. Hancock. 2007. Deacetylase inhibition promotes the generation and function of regulatory T cells. Nat. Med. 13:1299–1307. Tao, R., L. Wang, K. M. Murphy, C. C. Fraser, and W. W. Hancock. 2008. Regulatory T cell expression of herpesvirus entry mediator suppresses the function of B and T lymphocyte attenuator-positive effector T cells. J. Immunol. 180:6649–6655. Torcia, M. G., V. Santarlasci, L. Cosmi, A. Clemente, L. Maggi, V. D. Mangano, F. Verra, G. Bancone, I. Nebie, B. S. Sirima, F. Liotta, F. Frosali, R. Angeli, C. Severini, A. R. Sannella, P. Bonini, M. Lucibello, E. Maggi, E. Garaci, M. Coluzzi, F. Cozzolino, F. Annunziato, S. Romagnani, and D. Modiano. 2008. Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria. Proc. Natl. Acad. Sci. USA 105:646–651. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. Stockinger. 2006. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 24:179–189. Villarino, A. V., C. M. Tato, J. S. Stumhofer, Z. Yao, Y. K. Cui, L. Hennighausen, J. J. O’Shea, and C. A. Hunter. 2007. Helper T cell IL-2 production is limited by negative feedback and STAT-dependent cytokine signals. J. Exp. Med. 204:65–71. Wada, J., H. Suzuki, R. Fuchino, A. Yamasaki, S. Nagai, K. Yanai, K. Koga, M. Nakamura, M. Tanaka, T. Morisaki, and M. Katano. 2009. The contribution of vascular endothelial growth factor to the induction of regulatory T-cells in malignant effusions. Anticancer Res. 29:881–888. Walther, M., J. E. Tongren, L. Andrews, D. Korbel, E. King, H. Fletcher, R. F. Andersen, P. Bejon, F. Thompson, S. J. Dunachie, F. Edele, J. B. de Souza, R. E. Sinden, S. C. Gilbert, E. M. Riley, and A. V. Hill. 2005. Upregulation of TGF-beta, FOXP3, and CD41CD251 regulatory T cells correlates with more rapid parasite growth in human malaria infection. Immunity 23:287–296. Wang, L., K. Pino-Lagos, V. C. de Vries, I. Guleria, M. H. Sayegh, and R. J. Noelle. 2008. Programmed death 1 ligand signaling regulates the generation of adaptive Foxp31CD41 regulatory T cells. Proc. Natl. Acad. Sci. USA 105:9331–9336. Wang, Z., B. Shi, H. Jin, L. Xiao, Y. Chen, and Y. Qian. 2009. Low-dose of tacrolimus favors the induction of functional CD4(1)CD25(1)FoxP3(1) regulatory T cells in solidorgan transplantation. Int. Immunopharmacol. 9:564–569. Webster, K. E., S. Walters, R. E. Kohler, T. Mrkvan, O. Boyman, C. D. Surh, S. T. Grey, and J. Sprent. 2009. In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression. J. Exp. Med. 206:751–760. Wei, G., L. Wei, J. Zhu, C. Zang, J. Hu-Li, Z. Yao, K. Cui, Y. Kanno, T. Y. Roh, W. T. Watford, D. E. Schones, W. Peng, H. W. Sun, W. E. Paul, J. J. O’Shea, and K. Zhao. 2009. Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD41 T cells. Immunity 30:155–167.
120
HOST DEFENSE: GENERAL
Wei, J., O. Duramad, O. A. Perng, S. L. Reiner, Y. J. Liu, and F. X. Qin. 2007. Antagonistic nature of T helper 1/2 developmental programs in opposing peripheral induction of Foxp31 regulatory T cells. Proc. Natl. Acad. Sci. USA 104:18169–18174. Zheng, Y., A. Chaudhry, A. Kas, P. deRoos, J. M. Kim, T. T. Chu, L. Corcoran, P. Treuting, U. Klein, and A. Y. Rudensky. 2009. Regulatory T-cell suppressor program coopts transcription factor IRF4 to control T(H)2 responses. Nature 458:351–356.
Zhou, X., S. L. Bailey-Bucktrout, L. T. Jeker, C. Penaranda, M. Martinez-Llordella, M. Ashby, M. Nakayama, W. Rosenthal, and J. A. Bluestone. 2009. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat. Immunol. 10:1000–1007. Zhu, C., A. C. Anderson, A. Schubart, H. Xiong, J. Imitola, S. J. Khoury, X. X. Zheng, T. B. Strom, and V. K. Kuchroo. 2005. The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat. Immunol. 6:1245–1252.
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
9 Memory and Infection DAVID MASOPUST AnD MARK K. SLIFKA
INTRODUCTION
on the host immune system can be exceedingly long-lived and effective at preventing clinical disease upon reexposure. For example, in 1846, the isolated populace of the remote Faroe Islands (lying 200 miles off the northern coast of Scotland) experienced its first measles outbreak in 65 years. Remarkably, those denizens over the age of 65 years whom had survived infection during the previous outbreak were immune from disease (Slifka & Ahmed, 1996b). This observation demonstrates that protective immunity can persist for the life of the host without periodic reexposure from other infected individuals. Similar natural examples derived from epidemiological analyses of other geographically isolated communities demonstrate that protective immunity against other pathogens, including polio and yellow fever viruses, also persist for the life of the host (Slifka & Ahmed, 1996b). While immunity to particular agents will be covered in other chapters within the text, this chapter will review the basic principles of adaptive immunological memory.
The principal function of the immune system is to protect the host from infection. After an infection is cleared, the host retains a state of heightened readiness in the event of reexposure to the same or to a closely related pathogen. This “memory” of past infections, a cardinal feature of the vertebrate adaptive immune system, is mediated by longlived changes in the population of host B and T lymphocytes induced by previous pathogen exposure. In response to stimulation, B lymphocytes differentiate into antibody secreting cells, and T lymphocytes elaborate cytokines, regulate the innate and adaptive immune response, and kill infected host cells. Each naïve lymphocyte exhibits a unique specificity conferred by the restricted expression of a single lymphocyte receptor. Prior to initial infection, the number of B and T cells specific for a particular pathogen are very rare (approximately 1 specific cell per 104 to 106 lymphocytes). Upon infection, antigen-specific lymphocytes undergo a dramatic program of proliferation resulting in up to a 10,000-fold amplification in clonal abundance, and acquire effector functions that help eliminate infection. Following pathogen clearance, most specific lymphocytes die, perhaps creating space for future immune responses to unrelated infections. However, 5% to 10% of specific lymphocytes escape death, and persist in the host long after the infection is eliminated. Thus, as a result of infection, the host’s immune system retains a much higher frequency of lymphocytes specific for that particular infection. Subsets of memory lymphocytes exhibit important functional differences from their naïve counterparts and also have a more pervasive anatomic distribution. Thus, the vertebrate immune system is educated by the unique infectious experience of the individual, which results in long-lived changes in pathogen-specific lymphocyte quantity, quality, and location, all of which contribute to an increase in the speed, robustness, and efficacy of responses upon secondary exposure. This imprint of infectious history
DYNAMICS OF T-CELL RESPONSES
Primary T-cell responses are fairly slow to develop for three reasons. First, the frequency of naïve T cells specific for a particular antigen is exceedingly rare. Second, naïve T cells do not constitutively express effector molecules that promote pathogen clearance. And third, naïve T cells primarily recirculate from blood and lymph to secondary lymphoid organs; yet many infections occur outside of lymphoid tissue. Thus, before T cells can optimally contribute to eradication of primary infections, they must overcome limitations of quantity, quality, and location. Upon primary infection, rare cognate antigen-specific T cells first interact with professional antigen-presenting cells that have acquired foreign antigen and have localized within the T-cell zones of secondary lymphoid tissue. This interaction precipitates a program of activation, and is manifest by a remarkable period of proliferation, and changes in membrane organization, gene expression, homing, and the up regulation of effector molecules that promote pathogen clearance. Within days, antigen-specific naïve T cells expand between 100 to 10,000-fold, depending on the duration and density of antigen presentation, costimulation, and other variables (Masopust et al., 2004). Effector CD4
David Masopust, Department of Microbiology, Center for Immunology, University of Minnesota, 2-182 Medical Biosciences Building, 2101 6th St. SE, Minneapolis, Mn 55455. Mark K. Slifka, Vaccine and Gene Therapy Institute, Oregon Health & Science University, 505 nW 185th Avenue, Beaverton, OR 97006.
121
122
host defense: general
T cells elaborate cytokines and costimulatory molecules that communicate information to cells of the innate and adaptive immune system, promoting affinity maturation and class switching among B cells, CD8 T-cell differentiation, macrophage activation, and many other functions. CD8 T cells secrete cytokines that promote an antiviral state and kill infected host cells via degranulation of perforin and granzymes. It should be noted that many T-cell effector functions are contact dependent. Most effector T cells down regulate CD62L and CCR7, the homing molecules that promote migration to secondary lymphoid organs. In turn, they up regulate receptors that allow them to migrate to nonlymphoid tissues, which often serve as the predominant site of infection. For instance, priming within the mesenteric lymph node induces 47 and CCR9 expression, which facilitates migration to the parenchyma of the small intestine. In contrast, priming within the inguinal lymph node induces expression of skin homing molecules. Thus, gut and skin homing receptor expression is partly coupled to the location of T-cell activation (Butcher & Picker, 1996; von Andrian & Mackay, 2000), which may preferentially target responses to sites of infection. This process of expansion, differentiation, and redistribution allows effectors to optimally contribute to pathogen clearance. In the event that the pathogen is eliminated, most T cells die en masse over a period of a few weeks, which allows room for future immune responses. However, a fraction of T cells escape this death program (typically 5% to 10%) and are maintained long after pathogen clearance. Thus, the population of host lymphocytes retains a permanent imprint, or memory, of past infections. Memory T-cell populations exhibit many properties that distinguish them from their naïve counterparts, including increased clonal abundance, reduced activation requirements in the event of pathogen reexposure, constitutive maintenance or more rapid up regulation of effector gene expression, and more widespread anatomic distribution (Masopust et al., 2004). Collectively, these properties contribute to protection from reinfection by promoting faster, larger, and more efficacious recall responses (Fig. 1). Defining the developmental cues that regulate (i) commitment to the long-lived memory T-cell lineage, (ii) heterogeneity in memory T-cell differentiation state, and (iii) T-cell homing remain some of the biggest challenges in understanding T-cell immunobiology.
COMMITMENT TO MEMORY LINEAGE
How does a small fraction of activated T cells form long-lived memory cells while others die en masse? When does this fate decision occur? It had long been debated whether memory T cells transit through an effector stage, or whether effector cells constitute a distinct terminally differentiated lineage fated for death. Recent data demonstrates that both effector and memory CD8 T cells can be generated from a single naïve cell, suggesting that this fate decision is made subsequent to activation (Stemberger et al., 2007). Reiner and colleagues recently observed that T cells divide asymmetrically during their first division (Chang et al., 2007). Based on elegant phenotypic analyses and cell transfer studies, they proposed that the fate decision between becoming long-lived memory cells and short-lived effector cells is made upon the first division following activation (Chang et al., 2007). However, several recent studies demonstrated that both CD4 and CD8 T cells express equivalent levels of certain effector molecules, such as granzyme B and interferon gamma, regardless of whether they will differentiate into memory T cells or are only shortlived effector cells (Bannard et al., 2009; Harrington et al., 2008; Lohning et al., 2008). While it remains possible that the memory fate decision is made shortly following activation, it apparently does not preclude the expression of these effector molecules. Indeed, disruption of the proapoptotic molecule, bim, allows most effector CD8 T cells to become long-lived memory cells, which argues against the possibility that memory T cells are selected solely on the basis of functionality (Prlic & Bevan, 2008). Cytokine availability can have a substantial impact on T-cell survival during the contraction phase. Increasing the availability of IL-2 or IL-15 prior to contraction results in higher numbers of memory T cells (Blattman et al., 2003; Yajima et al., 2002). Moreover, there is heterogeneity in expression of IL-7R prior to the contraction of a recently activated CD8 T-cell population. A small subset of IL-7Rhi KLRG1lo effector cells are present at the peak of clonal expansion, and these preferentially survive and become memory cells (Hand & Kaech, 2009). Transfer of activated T cells to an IL-7 deficient recipient results in poor memory development, but interestingly, over-expression of IL-7R on all T cells is not sufficient to rescue dying effector cells. These data are consistent with the hypothesis that the contraction may be partially regulated via a Darwinian competition for limited cytokine resources. Collectively, it appears that
FIGURE 1 Dynamics of primary versus secondary T-cell response. Primary T-cell responses are slow to develop, result in the selective expansion of pathogen specific clones, and establish a long-lived increase in the frequency of pathogen specific T cells (memory). Secondary (recall) responses are faster, larger, and more efficacious, and induce a long-lived boost in the number of memory T cells.
9. Memory and Infection
commitment to the memory lineage has at least partially occurred by the peak of clonal expansion, but that extrinsic factors also regulate survival. Dissecting the parameters that influence this process, including inflammation, the dynamics of antigen presentation and costimulation, and competition for cytokines, is still a subject of intense investigation (Harty & Badovinac, 2008).
HETEROGENEITY AMONG MEMORY T-CELL POPULATIONS
Seminal work by Lanzavecchia and colleagues revealed that memory T cells within human blood exhibited significant phenotypic and functional heterogeneity (Sallusto et al., 1999). They reported that antigen-experienced T cells that retained (or reexpressed) homing molecules required for trafficking to secondary lymphoid tissue, deemed “central” memory T cells (TCM), required an extensive period of restimulation before expressing certain effector molecules. In contrast, “effector” memory T cells (TEM) lacked CCR7 and CD62L, but constitutively expressed molecules associated with certain T-cell effector functions, such as perforin. These data suggested that memory T-cell phenotypic properties varied with trafficking potential. Papers that soon followed the Lanzavecchia study also revealed that large numbers of memory T cells were located within nonlymphoid tissues (Masopust et al., 2001; Reinhardt et al., 2001). Consistent with the prediction of Lanzavecchia and colleagues, these nonlymphoid populations expressed more rapid effector functions than their counterparts located within secondary lymphoid tissues (Masopust et al., 2001; Reinhardt et al., 2001). Collectively, these data pointed to the existence of a specialized subset of memory T cells that occupied common portals of pathogen entry, and were immediately poised to respond in the event of reinfection. In contrast, the population that had traditionally been studied in animal models more carefully, TCM within lymphoid tissue, was a resting subset that responded more slowly in the event of reinfection by undergoing a second round of proliferation and only acquiring certain effector functions over a period of a few days (Wherry et al., 2003). The developmental relationship between each subset remains unclear, and evidence supports both the notions that TCM derive from TEM and that TEM derive from TCM (Champagne et al., 2001; Sallusto et al., 2004; Wherry et al., 2003). T-cell priming is associated with many variables, including antigen quantity and duration, TCR affinity, inflammatory milieu, identity of antigen presenting cell, and degree of costimulation, all of which contribute to heritable changes in T-cell gene expression. Data now indicate that events during priming may regulate the distribution of memory T cells into each subset, and increases in the overall strength of stimulation preferentially result in the generation of TEM. This issue has obvious ramifications for vaccination, and suggests that nonreplicating or subunit vaccines may result predominantly in TCM. One of the great remaining challenges is to determine the importance of each subset in contributing to protection from reinfection. It is likely that the answer to this question will vary depending on the pathogenesis and tropism of the infection in question (Bachmann et al., 2005; Wherry et al., 2003). While TEM are often referred to as a homogenous population, it should be noted that they exhibit a broad range of phenotypes, often coupled with their anatomic location (Woodland & Kohlmeier, 2009). For instance, virus-specific TEM within the epithelium of the small intestine exhibit unique phenotypic and functional signatures that are not found in other tissues (Table 1; Masopust et al., 2006). Similar observations have been made for cells isolated
123
TABLE 1 T-cell properties vary with differentiation state and anatomic locationa Property
naïve
Effector
CD44 CD62L e/7 CD69 Ly6C CTL Turnover
/
Blood
IEL
TCM
TEM
TEM
varies
/
a Virus-specific mouse CD8 T cells were examined before (naïve), during (effector), or after (memory) an acute infection (Masopust et al., 2006). Abbreviations: TCM, central memory; TEM, effector memory; IEL, intestinal epithelial lymphocytes (T cells within epithelium of the small intestine); CTL, cytotoxic T lymphocytes (T cells that kill infected targets in short-term in vitro assays).
from the brain and the lung airways (Masopust et al., 2004; Woodland & Kohlmeier, 2009). It remains likely that the tissue microenvironment itself plays a role in shaping memory T-cell differentiation state. This issue is important, because analyses of TEM isolated from blood will not account for the full phenotypic complexity of the large numbers of memory T cells residing in nonlymphoid organs.
MAINTENANCE AND LONGEVITY OF MEMORY T CELLS
Does immunological memory need constant reminding? One long-standing controversy in the field of T-cell memory was whether it could be maintained in the absence of constitutive stimulation by cognate antigen. Resolution of this argument had practical ramifications for the longevity of naturally acquired T-cell memory specific for pathogens that are eliminated, as well as implications for whether vaccines should provide persistent depots of antigen or require periodic boosting. After much debate, it was shown that memory CD8 and CD4 T cells persisted in mice that lacked MHC molecules, providing convincing evidence that cognate antigen was not required for memory T-cell maintenance (Murali-Krishna et al., 1999; Swain et al., 1999). However, there are numerous situations in which memory T cells may be constitutively stimulated by cognate Ag, most obviously in the setting of chronic infections, such as those caused by human immunodeficiency virus (HIV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV). What has become clear is that chronic antigen exposure has a pronounced effect on T-cell differentiation state and the requirements for survival (Wherry & Ahmed, 2004). Low levels of repeated stimulation promote the preferential development of TEM, whereas high levels of repeated stimulation lead to functional exhaustion and even deletion of T cells specific for chronic viruses. In settings wherein the infection is cleared, memory T cells are maintained through contact with IL-7 and IL-15 (Surh & Sprent, 2008). Memory CD8 T cells that are transferred to IL-15 deficient mice die rapidly. Moreover, IL-15 deficient mice have a marked reduction in memory phenotype CD8 T cells, although overexpression of IL-7 compensates for this defect. IL-15 also supports a slow basal level of antigen-independent turnover (termed homeostatic proliferation) among memory T cells. Thus, homeostatic proliferation is linked to survival. Competition for available
124
host defense: general
cytokines may regulate the numbers of total memory T cells present within the host, ensuring that repeated infections do not result in a permanent expansion in the size of the adaptive immune system (Selin & Welsh, 2004). However, TEM undergo much less homeostatic proliferation than TCM (Wherry et al., 2003). Moreover, heterologous prime-boost vaccination can result in a tremendous expansion of the TEM pool within nonlymphoid tissues without inducing significant attrition among preexisting TCM (Vezys et al., 2009). These data raise the possibility that TCM, which largely populate lymphoid tissues, may be regulated differently from TEM, which are preferentially located within other anatomical compartments. The longevity of T-cell memory is still the subject of some debate. Experimental infection of mice with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV), which is rapidly cleared from the host, results in a remarkably stable memory CD8 T-cell pool for more than 900 days. In contrast, virus-specific memory CD4 T cells decayed 50-fold over the same period (Homann et al., 2001), suggesting differences in the regulation of these subsets. Longer studies in mice are not feasible given their relatively brief lifespan. However, evidence suggests that certain heterologous infections may lead to the rapid attrition of preexisting memory CD8 T cells, and it remains possible that the longevity of CD8 T-cell memory in mice is an artifact of their maintenance under specific pathogen-free conditions (Selin & Welsh, 2004). Confounding these issues further, the longevity of memory T cells may depend on the nature of the priming environment. In one striking example, infection of CD4 T-cell-deficient mice with Listeria monocytogenes results in an effective primary CD8 T-cell response. However, this situation results in essentially no memory CD8 T cells that have the capacity to respond and protect the host in the event of a second infection (Prlic et al., 2007). With these issues in mind, it is essential to evaluate the longevity of T-cell memory to naturally acquired infections in humans. However, these studies have several limitations. They require a host population that is not periodically reexposed to the infection for which memory is being measured. Long-term longitudinal studies are difficult. Moreover, they are often restricted to analyses of blood, and many assays will only measure the frequency rather than the total number of recirculating memory T cells. With these caveats in mind, it was recently reported that primary human cytomegalovirus infection resulted in a long-lived increase in the total number of memory CD8 T cells, and induced a reduction in the frequency, but not the number, of preexisting memory CD8 T cells specific for influenza or EBV in blood (van Leeuwen et al., 2006). T-cell memory was recently analyzed in individuals infected with Puumala virus (PUUV), a member of the Hantavirus genus that causes mild hemorrhagic fever. Without evidence of viral persistence or reexposure, fairly robust levels of CD8 T-cell memory were detected in blood for up to 15 years after infection (Van Epps et al., 2002). Between 0.1% and 0.9% of CD8 and CD4 T cells were specific for measles virus among adults aged 25 to 38 years that had a natural history of childhood infection (nanan et al., 2000). In addition, a nonrandomized, cross-sectional analysis of T-cell immunity was performed on volunteers examined 1 month to 75 years after smallpox vaccination (Hammarlund et al., 2003). Remarkably, memory T cells could be detected up to 75 years following a single immunization, although the halflife of both CD4 and CD8 T cells appeared to range between 8 and 15 years, and memory CD8 T cells could not be detected in about 50% of individuals. Collectively, these data indicate that T-cell memory likely can persist for the life of an individual following a single exposure. However, it should
be stressed that certain infections may significantly erode T-cell memory, and the longevity of memory may depend on the nature of the primary infection or vaccine. More studies on the longevity of T-cell memory to different agents will be a welcome addition to the field.
INDUCTION OF T-CELL-DEPENDENT HUMORAL IMMUNITY
Development of antigen-specific antibody responses can be either T-cell-independent or T-cell-dependent. T-cellindependent responses are typically mounted against nonprotein antigens and T cell help cannot be provided because without protein, there is no peptide presentation to elicit a CD4 helper T-cell response. Under these conditions, the resulting T-independent antibody response is often shortlived, lasting only a few weeks or months. In contrast, T-cell-dependent antibody responses contain protein antigens, elicit CD4 T-cell help, and induce antibody responses that undergo somatic hypermutation/affinity maturation and are long-lived—in some cases lasting for decades (Amanna et al., 2007). The majority of vaccines and viral infections trigger high affinity, antigen-specific B-cell responses that are CD4 T-cell-dependent. This process begins in the germinal center (GC), which represents a specialized lymphoid compartment wherein B cells become activated to proliferate, somatically mutate, and differentiate into memory B cells (MBC) and antibody-secreting plasma cells. Within the GC, it is believed that B cells compete for antigen in the form of antigen-antibody immune complexes on the surface of follicular dendritic cells (MacLennan, 1994) and only B cells with the highest affinity are able to effectively compete and be selected for further proliferation and differentiation into long-lived antigen-specific B-cell populations. Although antigen must play an important role in the selection process, mice that lack the ability to form immune complexes can still form a normal GC and develop effective antibody responses (Hannum et al., 2000). This apparent paradox may be explained by other studies that indicate competition for CD4 T-cell help may be an even more important factor in selection of high-affinity B cells during the GC reaction (Allen et al., 2007; Schwickert et al., 2007). One recent study has elegantly shown that B cells that form conjugates with cytokine-secreting follicular T cells in vivo are enriched for the expression of activation-induced cytidine deaminase (AID), an enzyme required for somatic hypermutation and isotype-class switching, and these B cells showed a higher rate of IgG mutations (Reinhardt et al., 2009). Interestingly, this study also demonstrated that B cells conjugated to IL-4-secreting CD4 T cells had class-switched to IgG1, whereas B cells conjugated to IFn-secreting CD4 T cells had class-switched to IgG2a, providing direct evidence that these specific cytokines are intimately involved with IgG class-switching of B cells in the GC. This is an important observation because it indicates that class-switching events are not determined by broad diffusion of T-cell-elicited cytokines throughout the GC, but instead represent compartmentalized events that are dictated by specific B cell-T cell interactions. Interestingly, not only are B cells dependent upon CD4 T-cell help for induction of effective, long-lived humoral immunity, but there is also growing evidence that CD4 T cells depend on help from B cells for the induction of long-lived T-cell memory. Some of the first evidence indicating a role for B cells in the development of functional T cells was demonstrated through adoptive transfer experiments showing that CD4 T cells from B-cell-deficient mice were defective in antiviral activity (Homann et al., 1998).
9. Memory and Infection
At that time, it was unclear whether the fate of the CD4 T cells was due to a lack of an intact antibody response and immune complex deposition in lymphoid tissues or whether the B cells themselves were necessary for development of effective T-cell responses. This issue was revisited in a recent study that compared antiviral T-cell responses in B-cell-deficient mice versus B-cell-transgenic mice that had an intact B-cell compartment, but were unable to mount an antiviral antibody response. In contrast to the B-cell-deficient mice that had defective T-cell responses following acute or chronic LCMV infection, the transgenic mice with normal B-cell numbers developed normal antiviral T-cell responses (Whitmire et al., 2009). This indicates that immune complex formation is not a necessary requirement for developing effective T-cell memory. Instead, this indicates that the B cells themselves must play other important roles in host immunity besides antibody production and that these cells are directly required for optimal induction of antiviral T-cell responses.
DIFFERENTIAL LOCALIZATION OF MEMORY B CELLS AND PLASMA CELLS
During and shortly following the GC reaction, MBCs and plasma cells (PCs) migrate out of the follicle and into surrounding lymphoid tissue or into the circulation. Following vaccination or systemic viral infection, about 10% to 20% of fully differentiated PCs will reside in the red pulp of the spleen, whereas 80% to 90% of the PC population will migrate out of the lymphoid environment and settle into the bone marrow compartment (Slifka & Ahmed, 1998a; Slifka & Ahmed, 1998b). Unlike PC migration to the bone marrow, the majority of MBCs will either remain in lymphoid compartments (e.g., in the marginal zone [Sanz et al., 2008]) or will recirculate in the bloodstream during
125
the course of continued immunosurveillance. At nearly a year after recovery from acute LCMV infection of mice, the spleen and lymph nodes continue to maintain the highest frequency of virus-specific MBCs, with the spleen containing approximately 100-fold more MBCs than in the peripheral blood when calculated per total organ or per total blood volume, respectively (Slifka & Ahmed, 1998a). Antibody-secreting cells (ASCs) can be found in the circulation, but only for a brief period of time after vaccination or infection (Bernasconi et al., 2002; Dilosa et al., 1991; Wrammert et al., 2008), and it is unclear if these represent fully differentiated, nondividing PCs or activated B cells (i.e., plasmablasts) that secrete antibody but retain the ability to divide (Claflin & Smithies, 1967). The differential migration of MBCs and PCs/plasmablasts is most likely accomplished by changes in the expression pattern of different chemokine receptors and other adhesion molecules (Tarlinton et al., 2008), but it remains unknown why PCs specifically migrate to the bone marrow compartment. This remains an area of active investigation.
DURATION OF ANTIGEN-SPECIFIC SERUM ANTIBODY RESPONSES
Induction of a durable neutralizing antibody response represents the basis of many successful vaccines and is important to the maintenance of protective immunity against a wide range of pathogens. The duration of serum antibody production following infection by measles, mumps, rubella, vaccinia, EBV, and varicella-zoster virus (VZV; i.e., chickenpox) was compared to the humoral immunity induced by vaccination with nonreplicating antigens, including tetanus toxoid and diphtheria toxoid by longitudinal analysis (Fig. 2; see Table 2). In this study (Amanna et al., 2007), over 600 serum samples from 45 subjects were used to measure serum
FIGURE 2 Duration of serological memory is dictated largely by the antigen under study. In this illustration (adapted from Amanna et al., 2007), the durability of antigen-specific antibody responses following infection or vaccination are compared over time. Antibody responses to EBV, measles, mumps, rubella, and vaccinia last a lifetime with little to no decrease in titer. Antibody responses to VZV decline more rapidly than antibody responses to other viral infections but remain more durable than the antibody responses to protein antigens such as tetanus or diphtheria. In general, the durability of serum antibody responses differ greatly depending on the antigen under study, and if the underlying mechanisms involved with determining the persistence of antibody can be elucidated, this will have important implications in terms of optimizing future vaccine design.
126
host defense: general
antibody titers longitudinally for an average of 15 years, with serological memory in some subjects examined for up to 26 years. Following acute infection with measles, mumps, rubella, or vaccinia viruses, serum antibody responses were maintained with half-lives of over 90 years. This indicates that despite clearance of the pathogen within a few weeks after infection, immunological memory to these viruses is essentially maintained for life. Unlike acute viral infections in which the initiating pathogen is eventually cleared, chronic or latent viral infections have the potential to “boost” the immune system via persistent antigenic stimulation or periodic reactivation from latency. Analysis of EBV responses indicated that 4 of the 45 subjects showed evidence of serological “boosting,” but these immunological spiking events appeared to be relatively rare (0.6 events per 100 person-years). Examination of VZV-specific immunity revealed a higher frequency of reactivation/reexposure events with 10 out of 45 subjects (22%) demonstrating measurable spikes in serum antibody responses (1.6 events per 100 person-years). However, the duration of immunity to VZV during the intervening periods of time between serospikes revealed an antibody half-life of approximately 50 years, whereas immunity against EBV did not show a statistically significant decline (estimated half-life of 11,552 years). Although this latter result might be expected following infection by a virus that is never fully cleared from the infected host, the relatively short antibody half-life following VZV infection was unexpected and suggests that when this particular virus goes into latency, it may truly be sequestered from the immune system. Analysis of humoral immunity against tetanus and diphtheria toxins revealed that not all antibody responses last a lifetime. Indeed, antibody responses against tetanus were maintained with an estimated half-life of only 11 years, whereas antibody responses against diphtheria were maintained with a half-life of 19 years (see Fig. 2; Table 2). This is a sharply different outcome from what is observed following acute or chronic viral infection and indicates that the nature of the antigen can have a profound effect on the duration of immunological memory. It is unknown if these differences are simply a reflection of the different stimulatory microenvironments (nonreplicating antigen/adjuvant versus replicating antigen/inflammation) or if differences in the structural components of the antigen (repetitive versus nonrepetitive B-cell epitopes) impact the duration of immunological memory.
TABLE 2 Duration of serum antibody production after infection or vaccinationa Antigen Tetanus Diphtheria VZVb Vaccinia Rubella Mumps Measles EBVc
Antibody half-life in years (95% confidence interval) 11 (10–14) 19 (14–33) 50 (30–153) 92 (46–) 114 (48–) 542 (90–) 3,014 (104–) 11,552 (63–)
a Duration of antigen-specific antibody responses was determined using a mixed effects model of longitudinal analysis. Adapted from Amanna et al. (2007). b VZV, varicella-zoster virus c EBV, Epstein-Barr virus
The nature of the antigen or infection appears to play a critical role in determining the duration of the resultant humoral immune response, but host genetics appear to also be involved in this process. When the duration of immunity against multiple antigens was compared within specific subjects, individual variations in serological memory were identified (Amanna et al., 2007). For instance, although most subjects demonstrated uniform long-lived immune responses against viral infections and shorter immune responses to tetanus and diphtheria, there were exceptions. For instance, although one subject mounted a normal antibody response to EBV (with no evidence of decline), the antibody responses to all seven other antigens were similar and ranged from a half-life of 14 to 31 years. This and other individual variations indicate that there may be a genetic element involved in the determination of immunological memory in humans following vaccination or infection. Identification of the specific mechanisms that govern the development of durable antigen-specific antibody responses represents one of the most important aspects of vaccinology and a better understanding of these factors could lead to better optimization of vaccine design.
QUANTITATION OF ANTIGEN-SPECIFIC B CELLS
As mentioned before, humoral immunity is maintained by two types of B cells—MBCs and PCs. The duration of the PC response is most commonly measured indirectly by quantitation of antigen-specific serum antibody levels or directly through the use of the ELISPOT assay, which enumerates the number of individual ASCs/PCs in a given cell population. Although analysis of MBC populations has been performed routinely in animal models, the relatively rare frequency of MBCs in human PBMC samples have made it more difficult to measure this important B-cell population following vaccination or infection of human subjects. Fortunately, several developments in MBC quantitation have made it easier and more feasible to enumerate antigen-specific MBCs in clinical samples. One approach to MBC quantitation is to use polyclonal in vitro stimulation to induce MBC expansion and differentiation into antigen-specific ASCs that can be measured using an ELISPOT assay (Crotty et al., 2004). Advantages of this approach include the ability to measure MBCs of virtually any antigen specificity and with the brief in vitro expansion step, only a relatively small number of PBMCs are required to measure MBC responses. The main disadvantages of this approach is that it is possible that some MBCs may expand or differentiate at different rates (Slifka & Ahmed, 1996a) and the final quantitation of MBCs is determined relative to the total number of IgG ASCs. For this reason, the frequency of antigen-specific MBCs in the starting population is based on an approximation that is based on the expanded number of total IgG-secreting B cells in the sample. Another approach to measuring MBC numbers is to perform limiting dilution analysis (Amanna et al., 2007; Amanna & Slifka, 2006; Pinna et al., 2009). The advantage of this approach is that the limiting dilution step provides quantitation of proliferation or differentiation of individual MBC clones and can be used to measure rare MBC populations specific for up to 8 to 10 different antigens in a single assay (Amanna & Slifka, 2006). The main disadvantages of this approach is that it is labor intensive, involves purifying B-cell populations (in some cases [Amanna & Slifka, 2006] but not others [Pinna et al., 2009]) and this approach often requires a large number of PBMCs in order to identify very rare antigen-specific MBCs. A third approach to MBC quantitation is based on staining the B cells
9. Memory and Infection
with fluorochrome-labeled antigens and measuring antigenspecific MBCs by flow cytometry (Amanna & Slifka, 2006; Leyendeckers et al., 1999; Scibelli et al., 2005; Townsend et al., 2001). The main advantage of this approach is that it allows antigen-specific quantitation of MBC frequencies directly ex vivo and with multiparameter flow cytometry, it is possible to measure a large number of phenotypic markers on antigen-specific B cells at different stages of activation or differentiation following vaccination or infection. The main disadvantage is that accurate quantitation of rare antigenspecific MBCs requires a large number of PBMCs and not all antigens are amenable to flow cytometry techniques, especially if they are highly cross-reactive, have high nonspecific binding (e.g., some complex or highly repetitive antigens demonstrate more nonspecific staining), or are difficult to conjugate to available fluorochromes. However, with this toolbox of available MBC quantitation techniques in hand, we are beginning to learn more about the relationship between MBC and PC populations and can now determine how long antigen-specific MBC responses can be maintained.
LIFESPAN OF MEMORY B-CELL POPULATIONS
Although there is substantial evidence describing longterm immunity following infection or vaccination (Slifka & Ahmed, 1996b), there is relatively little known about the kinetics and duration of B-cell memory. One study demonstrated that vaccinia-specific B-cell memory following smallpox vaccination is quite durable, with MBC numbers maintained for over 50 years after infection with little to no evidence of decline (Crotty et al., 2003). Likewise, analysis of B-cell memory against vaccinia, measles, mumps, rubella, EBV, VZV, tetanus, and diphtheria also indicates that B-cell memory can be maintained for decades or possibly even for life after infection or vaccination (Amanna et al., 2007). Another study measuring MBC responses to VZV, tetanus, measles, influenza, and Toxoplasma gondii in one subject over the course of 17 years has also provided further anecdotal evidence that MBC populations can be maintained for long periods of time. For instance, measles-specific MBCs were identified 50 years after primary infection (Pinna et al., 2009). Interestingly, vaccinia-specific T-cell memory declines slowly over time with an estimated half-life of 8 to 15 years (Crotty et al., 2003; Hammarlund et al., 2003) whereas MBC responses appear to be stable (Amanna et al., 2007; Crotty et al., 2003). This leads one to question whether B-cell memory might be more long-lived than T-cell memory and further studies will be needed to determine if these differences are identified in other model systems.
MODELS EXPLAINING LONG-LIVED HUMORAL IMMUNITY
There are many models that have been proposed to describe how long-term humoral immunity is maintained. The oldest models proposed that persistent antigen (e.g., chronic or latent infection) or environmental reexposure would be required to maintain serum antibody responses by repeatedly stimulating MBCs to proliferate and differentiate into short-lived PCs, which would then maintain antibody levels (Slifka & Ahmed, 1996b). Although this could indeed be the case for some types of viral infections, it is clearly not a universal requirement since antibody responses to the vaccinia virus can be maintained for up to 75 years after infection. This occurs in the absence of persistent or latent infection or reexposure to vaccinia or other
127
potentially cross-reactive Orthopoxviruses (Hammarlund et al., 2003). Another popular model proposes that persisting antigen in the form of immune complexes on the surface of follicular dendritic cells (FDC) is responsible for maintaining long-lived humoral immunity by continuous reactivation of MBCs into short-lived PCs (Slifka & Ahmed, 1996b). Although antigen can be maintained for several weeks or months on the surface of FDCs, it is unlikely that noncovalent protein interactions can be maintained in an aqueous environment for decades without some loss in protein levels over time. Since antibody responses to measles, mumps, rubella, and vaccinia are maintained with half-lives of over 90 years (Amanna et al., 2007), the persistent antigen theory of long-term humoral immunity has fallen out of favor. MBCs can be polyclonally activated in vitro to proliferate and differentiate into ASCs and another model suggests that nonspecific B-cell activation through heterologous infections or vaccinations may represent the mechanism used to sustain humoral immunity for decades (Bernasconi et al., 2002). Although an intriguing theory, it does not explain why antibody responses to different antigens have dramatically different half-lives, especially since one would assume that polyclonal activation should trigger equally long-lived responses by MBCs of all specificities. Clinical data also does not support this model. For instance, when human subjects were infected with vaccinia virus, there was a rapid anamnestic antibody response mounted against vaccinia, whereas serological memory to other viral infections remained essentially unchanged despite the presence of an active viral infection and activation of virus-specific T cells (Amanna et al., 2007). Moreover, each of the models that are based on MBC-based repopulation of short-lived PCs rely on a correlation between MBC numbers and antibody levels to be maintained. MBC numbers often do not correlate with antibody titers (Amanna et al., 2007), and booster vaccination induces higher MBC numbers without appreciable change in long-term serum antibody titers (nanan et al., 2001), indicating that it is unlikely that MBC-dependent models of humoral immunity provide an accurate depiction of the mechanisms involved with maintaining serum antibody responses. Other models of long-term humoral immunity are based on the premise that PCs are long-lived cells that are independently regulated from MBCs and do not require continuous replenishment from the MBC pool. Support for these models can be found in animal studies (Ahuja et al., 2008; DiLillo et al., 2008; Manz et al., 1997; Slifka et al., 1998), as well as limited clinical studies (Cambridge et al., 2003; Edwards et al., 2004) in which antigen-specific antibody responses are maintained for prolonged periods of time after MBC depletion. One model based on the long-lived PC theory suggests that the duration of serum antibody responses is determined by competition between newly formed PCs competing with preexisting PCs for niches within the bone marrow compartment (Fairfax et al., 2008; Radbruch et al., 2006). Similar to the MBC-based models, this particular model fails to predict the different antibody half-lives induced by different antigens since one would assume that newly developed PCs would outcompete the preexisting PCs regardless of their antigen specificity. Moreover, based on this model one would predict that as one reaches advanced age and the finite niches for space in the bone marrow become more constrained, then preexisting antibody titers to specific antigens should begin to decline more rapidly. This is not the case, since longitudinal analysis of antibody responses to childhood diseases such as measles, mumps, and rubella are equally stable regardless of whether the subjects are in
128
host defense: general
their 20s or in their 60s (Amanna et al., 2007). A model that we propose is that the lifespan of PCs is determined during the initiation of the humoral immune response. During PC formation, the cells are programmed with a different lifespan depending on the stimulation conditions and microenvironment encountered during or just prior to their terminal differentiation into antibody-secreting PCs. Based on this model, weaker B-cell signaling would result in PCs with an intermediate or shorter lifespan, whereas strong Bcell signaling during the induction of the PC response would lead to more longer-lived PCs. An example of strong B-cell signaling would include stimulation by a highly repetitive antigen, which would enhance “capping” of surface BcR and lead to increased calcium flux. Further addition of costimulation by antigen-specific CD4 T cells via CD40:CD40L interactions might also lead to an increase in combined signal strength and the induction of long-lived PCs. Since PCs lose surface Ig (i.e., no longer able to detect antigen), down regulate MHC class II expression (i.e., can no longer obtain T-cell help), and become terminally differentiated cells (i.e., no longer able to divide), we believe that the programmed lifespan model provides the most reasonable representation of the clinical findings demonstrating antibody responses of different duration. It is as of yet unknown what specific factors are directly involved with the development of PCs with different predetermined life spans, but identification of these factors will be critical for developing improved adjuvants and vaccines.
CONCLUSIONS
Following infection, protective immunological memory in the form of memory T cells, memory B cells, and plasma cells can be maintained for years, and in some cases, for life. The remarkable durability of immune memory has been the focus of intense investigation and we have learned much about the factors involved with this process. Although memory T cells do not necessarily require cognate antigen to be maintained, they do require exposure to different cytokines such as IL-2, IL-7, and IL-15 in order to optimally survive the contraction phase of the immune response and to persist for long periods of time after an infection has been cleared. Following commitment to the memory cell lineage, memory T-cell populations demonstrate dynamic heterogeneity in terms of phenotype, function, and anatomical distribution, which together are likely to promote a broad arsenal of preexisting T lymphocytes capable of responding to a variety of pathogens and potential routes of infection. Similar to the maintenance of T-cell memory, maintenance of humoral immunity in the form of memory B cells, plasma cells, and serum antibody appears to be very longlived even in the absence of reexposure or reinfection. Induction of persistent antibody responses typically requires T-cell help and exposure to stimulatory cytokines. Moreover, the duration of the humoral immune response appears to be further dictated by the nature of the antigenic exposure. Antibody responses to antigens such as tetanus or diphtheria toxoids will result in slowly declining antibody responses that last for decades, whereas immunity against viral infections such as measles, mumps, and rubella will often last a lifetime. The underlying mechanisms involved with inducing and maintaining humoral immunity have not been fully established, but there are several models that have been proposed. Future studies on immunological memory will likely shed new light on the subject of cellular and humoral immunity, and as we learn more about the requirements for developing protective immune
responses, we will improve our ability to develop better and more effective vaccines.
REFERENCES Ahuja, A., S. M. Anderson, A. Khalil, and M. J. Shlomchik. 2008. Maintenance of the plasma cell pool is independent of memory B cells. Proc. Natl. Acad. Sci. USA 105:4802–4807. Allen, C. D., T. Okada, H. L. Tang, and J. G. Cyster. 2007. Imaging of germinal center selection events during affinity maturation. Science 315:528–531. Amanna, I. J., and M. K. Slifka. 2006. Quantitation of rare memory B cell populations by two independent and complementary approaches. J. Immunol. Methods 317:175–185. Amanna, I. J., N. E. Carlson, and M. K. Slifka. 2007. Duration of humoral immunity to common viral and vaccine antigens. N. Engl. J. Med. 357:1903–1915. Bachmann, M. F., P. Wolint, K. Schwarz, and A. Oxenius. 2005. Recall proliferation potential of memory CD8 T cells and antiviral protection. J. Immunol. 175:4677–4685. Bannard, O., M. Kraman, and D. T. Fearon. 2009. Secondary replicative function of CD8 T cells that had developed an effector phenotype. Science 323:505–509. Bernasconi, N. L., E. Traggiai, and A. Lanzavecchia. 2002. Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298:2199–2202. Blattman, J. N., J. M. Grayson, E. J. Wherry, S. M. Kaech, K. A. Smith, and R. Ahmed. 2003. Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo. Nat. Med. 9:540–547. Butcher, E. C., and L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60–66. Cambridge, G., M. J. Leandro, J. C. Edwards, M. R. Ehrenstein, M. Salden, M. Bodman-Smith, and A. D. Webster. 2003. Serologic changes following B lymphocyte depletion therapy for rheumatoid arthritis. Arthritis Rheum. 48:2146–2154. Champagne, P., G. S. Ogg, A. S. King, C. Knabenhans, K. Ellefsen, M. Nobile, V. Appay, G. P. Rizzardi, S. Fleury, M. Lipp, R. Forster, S. Rowland-Jones, R. P. Sekaly, A. J. McMichael, and G. Pantaleo. 2001. Skewed maturation of memory HIV-specific CD8 T lymphocytes. Nature 410:106–111. Chang, J. T., V. R. Palanivel, I. Kinjyo, F. Schambach, A. M. Intlekofer, A. Banerjee, S. A. Longworth, K. E. Vinup, P. Mrass, J. Oliaro, N. Killeen, J. S. Orange, S. M. Russell, W. Weninger, and S. L. Reiner. 2007. Asymmetric T lymphocyte division in the initiation of adaptive immune responses. Science 315:1687–1691. Claflin, A. J., and O. Smithies. 1967. Antibody-producing cells in division. Science 157:1561–1562. Crotty, S., R. D. Aubert, J. Glidewell, and R. Ahmed. 2004. Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system. J. Immunol. Methods 286:111–122. Crotty, S., P. Felgner, H. Davies, J. Glidewell, L. Villarreal, and R. Ahmed. 2003. Cutting edge: long-term B cell memory in humans after smallpox vaccination. J. Immunol. 171: 4969–4973. DiLillo, D. J., Y. Hamaguchi, Y. Ueda, K. Yang, J. Uchida, K. M. Haas, G. Kelsoe, and T. F. Tedder. 2008. Maintenance of long-lived plasma cells and serological memory despite mature and memory B cell depletion during CD20 immunotherapy in mice. J. Immunol. 180:361–371. Dilosa, R. M., K. Maeda, A. Masuda, A. K. Szakal, and J. G. Tew. 1991. Germinal center B cells and antibody production in the bone marrow. J. Immunol. 146:4071–4077. Edwards, J. C., L. Szczepanski, J. Szechinski, A. FilipowiczSosnowska, P. Emery, D. R. Close, R. M. Stevens, and T. Shaw. 2004. Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N. Engl. J. Med. 350:2572–2581.
9. Memory and Infection Fairfax, K. A., A. Kallies, S. L. Nutt, and D. M. Tarlinton. 2008. Plasma cell development: from B-cell subsets to longterm survival niches. Semin. Immunol. 20:49–58. Hammarlund, E., M. W. Lewis, S. G. Hansen, L. I. Strelow, J. A. Nelson, G. J. Sexton, J. M. Hanifin, and M. K. Slifka. 2003. Duration of antiviral immunity after smallpox vaccination. Nature Medicine 9:1131–1137. Hand, T. W., and S. M. Kaech. 2009. Intrinsic and extrinsic control of effector T cell survival and memory T cell development. Immunol. Res. 45:46–61. Hannum, L. G., A. M. Haberman, S. M. Anderson, and M. J. Shlomchik. 2000. Germinal center initiation, variable gene region hypermutation, and mutant B cell selection without detectable immune complexes on follicular dendritic cells. J. Exp. Med. 192:931–942. Harrington, L. E., K. M. Janowski, J. R. Oliver, A. J. Zajac, and C. T. Weaver. 2008. Memory CD4 T cells emerge from effector T-cell progenitors. Nature 452:356–360. Harty, J. T., and V. P. Badovinac. 2008. Shaping and reshaping CD8 T-cell memory. Nat. Rev. Immunol. 8:107–119. Homann, D., L. Teyton, and M. B. Oldstone. 2001. Differential regulation of antiviral T-cell immunity results in stable CD8 but declining CD4 T-cell memory. Nat. Med. 7:913–919. Homann, D., A. Tishon, D. P. Berger, W. O. Weigle, M. G. von Herrath, and M. B. Oldstone. 1998. Evidence for an underlying CD4 helper and CD8 T-cell defect in B-cell- deficient mice: failure to clear persistent virus infection after adoptive immunotherapy with virus-specific memory cells from muMT/muMT mice. J. Virol. 72:9208–9216. Leyendeckers, H., M. Odendahl, A. Lohndorf, J. Irsch, M. Spangfort, S. Miltenyi, N. Hunzelmann, M. Assenmacher, A. Radbruch, and J. Schmitz. 1999. Correlation analysis between frequencies of circulating antigen- specific IgGbearing memory B cells and serum titers of antigen- specific IgG. Eur. J. Immunol. 29:1406–1417. Lohning, M., A. N. Hegazy, D. D. Pinschewer, D. Busse, K. S. Lang, T. Hofer, A. Radbruch, R. M. Zinkernagel, and H. Hengartner. 2008. Long-lived virus-reactive memory T cells generated from purified cytokine-secreting T helper type 1 and type 2 effectors. J. Exp. Med. 205:53–61. MacLennan, I. C. 1994. Germinal centers. Annu. Rev. Immunol. 12:117–139. Manz, R. A., A. Thiel, and A. Radbruch. 1997. Lifetime of plasma cells in the bone marrow. Nature 388:133–134. Masopust, D., S. M. Kaech, E. J. Wherry, and R. Ahmed. 2004. The role of programming in memory T-cell development. Curr. Opin. Immunol. 16:217–225. Masopust, D., V. Vezys, A. L. Marzo, and L. Lefrancois. 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291:2413–2417. Masopust, D., V. Vezys, E. J. Wherry, D. L. Barber, and R. Ahmed. 2006. Cutting edge: gut microenvironment promotes differentiation of a unique memory CD8 T cell population. J. Immunol. 176:2079–2083. Murali-Krishna, K., L. L. Lau, S. Sambhara, F. Lemonnier, J. Altman, and R. Ahmed. 1999. Persistence of memory CD8 T cells in MHC class I-deficient mice. Science 286:1377–1381. Nanan, R., D. Heinrich, M. Frosch, and H. W. Kreth. 2001. Acute and long-term effects of booster immunisation on frequencies of antigen-specific memory B-lymphocytes. Vaccine 20:498–504. Nanan, R., A. Rauch, E. Kampgen, S. Niewiesk, and H. W. Kreth. 2000. A novel sensitive approach for frequency analysis of measles virus-specific memory T-lymphocytes in healthy adults with a childhood history of natural measles. J. Gen. Virol. 81:1313–1319. Pinna, D., D. Corti, D. Jarrossay, F. Sallusto, and A. Lanzavecchia. 2009. Clonal dissection of the human memory B-cell repertoire following infection and vaccination. Eur. J. Immunol. 39:1260–1270.
129
Prlic, M., and M. J. Bevan. 2008. Exploring regulatory mechanisms of CD8 T cell contraction. Proc. Natl. Acad. Sci. USA 105:16689–16694. Prlic, M., M. A. Williams, and M. J. Bevan. 2007. Requirements for CD8 T-cell priming, memory generation and maintenance. Curr. Opin. Immunol. 19:315–319. Radbruch, A., G. Muehlinghaus, E. O. Luger, A. Inamine, K. G. Smith, T. Dorner, and F. Hiepe. 2006. Competence and competition: the challenge of becoming a long-lived plasma cell. Nat. Rev. Immunol. 6:741–750. Reinhardt, R. L., A. Khoruts, R. Merica, T. Zell, and M. K. Jenkins. 2001. Visualizing the generation of memory CD4 T cells in the whole body. Nature 410:101–105. Reinhardt, R. L., H. E. Liang, and R. M. Locksley. 2009. Cytokine-secreting follicular T cells shape the antibody repertoire. Nat. Immunol. 10:385–393. Sallusto, F., J. Geginat, and A. Lanzavecchia. 2004. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu. Rev. Immunol. 22:745–763. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708–712. Sanz, I., C. Wei, F. E. Lee, and J. Anolik. 2008. Phenotypic and functional heterogeneity of human memory B cells. Semin. Immunol. 20:67–82. Schwickert, T. A., R. L. Lindquist, G. Shakhar, G. Livshits, D. Skokos, M. H. Kosco-Vilbois, M. L. Dustin, and M. C. Nussenzweig. 2007. In vivo imaging of germinal centres reveals a dynamic open structure. Nature 446:83–87. Scibelli, A., R. G. van der Most, J. A. Turkstra, M. P. Ariaans, G. Arkesteijn, E. J. Hensen, and R. H. Meloen. 2005. Fast track selection of immunogens for novel vaccines through visualisation of the early onset of the B-cell response. Vaccine 23:1900–1909. Selin, L. K., and R. M. Welsh. 2004. Plasticity of T cell memory responses to viruses. Immunity 20:5–16. Slifka, M. K., and R. Ahmed. 1996a. Limiting dilution analysis of virus-specific memory B cells by an ELISPOT assay. J. Immunol. Methods 199:37–46. Slifka, M. K., and R. Ahmed. 1996b. Long-term humoral immunity against viruses: revisiting the issue of plasma cell longevity. Trends Microbiol. 4:394–400. Slifka, M. K., and R. Ahmed. 1998a. B cell responses and immune memory. Dev. Biol. Stand. 95: 105–115. Slifka, M. K., and R. Ahmed. 1998b. Long-lived plasma cells: a mechanism for maintaining persistent antibody production. Curr. Opin. Immunol. 10:252–258. Slifka, M. K., R. Antia, J. K. Whitmire, and R. Ahmed. 1998. Humoral immunity due to long-lived plasma cells. Immunity 8:363–372. Stemberger, C., K. M. Huster, M. Koffler, F. Anderl, M. Schiemann, H. Wagner, and D. H. Busch. 2007. A single naive CD8 T cell precursor can develop into diverse effector and memory subsets. Immunity 27:985–997. Surh, C. D., and J. Sprent. 2008. Homeostasis of naive and memory T cells. Immunity 29:848–862. Swain, S. L., H. Hu, and G. Huston. 1999. Class IIindependent generation of CD4 memory T cells from effectors. Science 286:1381–1383. Tarlinton, D., A. Radbruch, F. Hiepe, and T. Dorner. 2008. Plasma cell differentiation and survival. Curr. Opin. Immunol. 20:162–169. Townsend, S. E., C. C. Goodnow, and R. J. Cornall. 2001. Single epitope multiple staining to detect ultralow frequency B cells. J. Immunol. Methods 249:137–146. Van Epps, H. L., M. Terajima, J. Mustonen, T. P. Arstila, E. A. Corey, A. Vaheri, and F. A. Ennis. 2002. Long-lived memory T lymphocyte responses after hantavirus infection. J. Exp. Med. 196:579–588.
130
host defense: general
van Leeuwen, E. M., J. J. Koning, E. B. Remmerswaal, D. van Baarle, R. A. van Lier, and I. J. ten Berge. 2006. Differential usage of cellular niches by cytomegalovirus versus EBV- and influenza virus-specific CD8 T cells. J. Immunol. 177:4998–5005. Vezys, V., A. Yates, K. A. Casey, G. Lanier, R. Ahmed, R. Antia, and D. Masopust. 2009. Memory CD8 T-cell compartment grows in size with immunological experience. Nature 457:196–199. von Andrian, U. H., and C. R. Mackay. 2000. T-cell function and migration. Two sides of the same coin. N. Engl. J. Med. 343:1020–1034. Wherry, E. J., and R. Ahmed. 2004. Memory CD8 T-cell differentiation during viral infection. J. Virol. 78:5535–5545. Wherry, E. J., V. Teichgraber, T. C. Becker, D. Masopust, S. M. Kaech, R. Antia, U. H. Von Andrian, and R. Ahmed. 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 4:225–234.
Whitmire, J. K., M. S. Asano, S. M. Kaech, S. Sarkar, L. G. Hannum, M. J. Shlomchik, and R. Ahmed. 2009. Requirement of B cells for generating CD4 T cell memory. J. Immunol. 182:1868–1876. Woodland, D. L., and J. E. Kohlmeier. 2009. Migration, maintenance and recall of memory T cells in peripheral tissues. Nat. Rev. Immunol. 9:153–161. Wrammert, J., K. Smith, J. Miller, W. A. Langley, K. Kokko, C. Larsen, N. Y. Zheng, I. Mays, L. Garman, C. Helms, J. James, G. M. Air, J. D. Capra, R. Ahmed, and P. C. Wilson. 2008. Rapid cloning of high-affinity human monoclonal antibodies against influenza virus. Nature 453:667–671. Yajima, T., H. Nishimura, R. Ishimitsu, T. Watase, D. H. Busch, E. G. Pamer, H. Kuwano, and Y. Yoshikai. 2002. Overexpression of IL-15 in vivo increases antigen-driven memory CD8 T cells following a microbe exposure. J. Immunol. 168:1198–1203.
THE PATHOGENS
II
This page intentionally left blank
The Immune Response to Infection Edited by S. H. E. Kaufmann, B. T. Rouse, and D. L. Sacks © 2011 ASM Press, Washington, DC
10 Overview of Viral Pathogens JONATHAN W. YEWDELL AND JACK R. BENNINK
and clues for understanding the workings of the innate immune system. Vertebrates have gone to great lengths to protect themselves from viruses (and also other pathogens), evolving intricate systems of innate and adaptive immunity. There are at least four practical reasons for immunologists to have a good working knowledge of virology. (i) Viruses remain a major cause of human morbidity and mortality, and pose a constant threat of devastating pandemics (Table 1) (i.e., imagine for a second, if HIV was efficiently transmitted by the aerosol route). (ii) Viruses are important vectors for vaccines against viral and nonviral pathogens and neoplasia. (iii) Viruses are evolution’s gift to gene therapists. (iv) Viruses are extremely useful experimental tools and probes for understanding the biology of cells and organisms. Given the brevity of this chapter, we have set only the modest goal of providing a simple introduction to viruses, with the hope of piquing the reader’s interest to study the subject in more depth. We encourage readers to explore in detail The Principles of Virology (Flint et al., 2008), a marvelous textbook with superb diagrams and figures that provides many profound insights into the biology and evolution of viruses. There are a number of other excellent texts that cover more specialized aspects of virology or virus–host interactions (see Coffin et al., 1997; Ewald, 1994; Knipe & Howley, 2006; Granoff & Webster, 1999; McCance, 1998; Plotkin et al., 2008; Richman et al., 2009). In addition, throughout the text we refer to textbooks and reviews as jumping off points for those who would like to chew on a given topic.
INTRODUCTION
Viruses are small segments of nucleic acid wrapped in a protein or lipoprotein shell with the ability to penetrate host cells and hijack the host machinery to generate progeny. At their simplest, viruses consist of a few thousand nucleotides that encode a nucleic acid polymerase and several proteins that comprise the virion. At their most complex, viruses contain several hundred genes encoding numerous viral structural proteins, proteins that alter cellular metabolism to favor virus replication (or enable virus to persist in a latent state), and proteins that modify the innate and adaptive host immune responses to favor virus transmission. Viruses are always completely dependent on cells for the production of energy, raw materials (e.g., amino acids, lipids, sugars), and protein synthesis. Viruses are an inescapable product of evolution. Every living species has its own unique set of viruses capable of infecting species members. Host–virus sets overlap considerably, and even dynamically. Viruses exhibit an extremely high mutation rate relative to their hosts, and are constantly changing genetically, with the consequence that their host range is in constant flux. Many hundreds of viruses are known to be capable of infecting humans, with probably thousands more that remain to be discovered. Viruses, like all life-forms, are selected simply to replicate, and typically bear no malice to their hosts. Indeed, efficiently killing hosts is a suicidal mechanism for viruses. Over time, evolution selects for a balance between viruses and their hosts. On the other hand, the genetic variability of viruses means that mutants with enhanced potential for causing mayhem are constantly generated. Moreover, encounters between species enable the introduction of new viruses, with the possibility of tremendous lethality for the new host until a new equilibrium is reached. Consequently, all species—from Escherichia coli to redwoods to blue whales—evolve strategies for controlling virus replication. How this is achieved by simple organisms is well beyond the scope of this chapter, except to point out that this area of research provides essential systems
STRUCTURAL PROPERTIES
Virions range in size from an 18 nm diameter (parvoviruses) to an 750 nm diameter (mimiviruses), amounting to a 40-fold difference in diameter and 72,000-fold difference in volume. They may be comprised of just a few different proteins to over 900 proteins, and their genomes range in size from 3.2 kilobases (hepadnaviruses) to 1.2 million bases (Table 2) (Chiu et al., 1997). For the purposes of the immune system, the most important structural distinction between viruses lies in their outer covering. Naked viruses possess a protein shell while
Jonathan W. Yewdell and Jack R. Bennink, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-3209.
133
134
The paThogens TABLE 1 Leading virus-associated diseasesa Cause
Deaths
DALYsb
Respiratory (pneumonia & influenza) Diarrheac (all causes) AIDS Measles Chronic hepatitis Bd Chronic hepatitis C Dengue Japanese encephalitis virus
4,259,000 2,163,000 2,040,000 424,000 105,000 54,000 18,000 11,000
97,786,000 72,777,000 58,513,000 14,853,000 2,068,000 955,000 670,000 681,000
c
Carriers
33,000,000 350,000,000 170,000,000
a
Data compiled from WHO (1999, 2008) and UNAIDS (2008). DALYs, disability-adjusted life years (disability years due to a disease). c Includes nonviral infectious disease mortality. d Chronic hepatitis B contributes to these deaths from liver cirrhosis and cancer. b
enveloped viruses possess an envelope derived from the host cell membrane (usually the plasma membrane, but some viruses use membranes from the endoplasmic reticulum [ER] or Golgi complex, and sometimes do so in a highly complex manner). Viral envelopes are comprised of cellular lipids and one to many viral proteins. Most enveloped viruses contain minimal amounts of host proteins, but others are more promiscuous, and relatively large amounts of host proteins may be present, including class I or II molecules of the major
histocompatibility complex (MHC). The presence of such polymorphic host molecules provides additional potential targets for immune recognition. The structures of both naked and enveloped viruses are highly repetitive, providing the immune system an opportunity for a high avidity interaction on the basis of multivalent recognition by low affinity receptors. Enveloped viruses usually require membrane integrity for infection, making them vulnerable to the complement system or membrane
TABLE 2 Virus classification Virus family
Genomea
Coat
Adenoviridae Arenaviridae Astroviridae Baculoviridae Birnaviridae Bornaviridae Bunyaviridae Calciviridae Circoviridae Coronaviridae Filoviridae Flaviviridae Hepadnaviridae Herpesviridae Iridoviridae Mimiviridae Orthomyxoviridae Papovaviridae Paramyxoviridae Parvoviridae Picornaviridae Poxviridae Reoviridae Retroviridae Rhabdoviridae Togaviridae
DS DNA, 36–38 Kb NS RNA, 10–14 Kb PS RNA, 7.2–7.9 Kb DS DNA, 100 Kb DS RNA, 7 Kb NS RNA, 8.9 Kb NS RNA, 13.5–21 Kb PS RNA, 8 Kb NS DNA, 3–4 Kb PS RNA, 16–21 Kb NS RNA, 12.7 Kb PS RNA, 10 Kb DS/NS DNA, 3.2 Kb DS DNA, 120–200 Kb DS DNA, 150–350 Kb DS DNA, 1,200 Kb NS RNA, 13.6 Kb DS DNA, 45–55 Kb NS RNA, 16–20 Kb NS DNA, 18–26 Kb PS RNA, 7.2–8.4 Kb DS DNA, 170–200 Kb DS RNA, 22–27 Kb PS RNA, 3.5–9 Kb NS RNA, 13–16 Kb PS RNA, 12 Kb
Naked Enveloped Naked Enveloped Naked Enveloped Enveloped Naked Naked Enveloped Enveloped Enveloped Enveloped Enveloped Naked/Enveloped Naked Enveloped Naked Enveloped Naked Naked Enveloped Naked Enveloped Enveloped Enveloped
a
Abbreviations: DS, double stranded; PS, positive single stranded; NS, negative single stranded.
10. overview of Viral pathogens
disrupting peptides (and detergents). Enveloped viruses also tend to be more fragile than nonenveloped (naked) viruses, making their transmission more dependent on intimate contact between hosts. The extreme fragility of some enveloped viruses (like HIV) limits their transmission to body fluid exchange. The surface proteins of both naked and enveloped viruses are typically highly resistant to proteases present in extracellular fluids, and when evolution dictates, even to the harsh conditions of the upper gastrointestinal tract. The outer virion shell protects the viral nucleic acid, which is complexed to viral structural proteins that enable its packaging into the virion during virus biogenesis. Viruses are grouped into six categories based on the nucleic acid present in infectious virions and the immediate nucleic acid product produced in infected cells. They are: negative strand () RNA (noncoding) transcribed into () RNA, () RNA transcribed into () RNA, () RNA transcribed into () DNA, double-stranded RNA, single-stranded DNA, and double-stranded DNA. Many viruses carry their own nucleic acid polymerases to initiate the infectious cycle, which can occur either in the cytosol or in the nucleus. A common feature of animal viruses is their high particle-to-infectivity ratio, (i.e., the number of particles required to initiate an infection). With very few exceptions, this is greater than 10, commonly greater than 100, and occasionally even greater than 1,000. The extent to which this reflects structural defects in a high percentage of viruses versus a low probability that competent virions have for initiating a successful infection is a question that has dogged virology from its origins. As this is invariably determined in vitro, it is unclear whether the particle to infectivity ratio is lower in vivo, as seems likely. If structural defects are common, they are relatively minor ones, at least as far as the humoral immune system is concerned, as there is little evidence to suggest that distinct antibody responses are elicited by infectious versus noninfectious virions. Indeed, the induction of antibody responses by purposely inactivated viruses is a principal strategy of antiviral vaccination. The prodigious reproductive potential of viruses gives them extreme latitude in producing mutants—given hundreds of offspring each, with a generation time of less than 12 hours, it is advantageous to possess a high mutation rate—many orders of magnitude higher than multicellular organisms such as ourselves. In particular, RNA viruses encode polymerases that generate errors just north of the error catastrophe rate. High mutations rates, in conjunction with selection pressure for high packaging efficiency, makes multiple copies of genes in a virion a liability, and only retroviruses are known to deliberately incorporate more than a single copy of their genomes into virions.
STRATEGIES OF VIRUS ENTRY Organisms
Virus survival requires transmission between hosts. The route of transmission is a critical aspect of viral biology since it dictates (or vice versa) in what cells the infection of host is initiated and what cells must produce the virus for transmission to occur. The transmission of a given virus is usually restricted to a single anatomical location, which provides an opportunity to focus immunity on a given pathogen to a limited number of sites in an organism (see Table 3). For example, influenza virus and other respiratory viruses that replicate in the columnar epithelium of the
135
upper airway are transmitted strictly through this portal, and a local immune response in the upper airway is sufficient to block infection. By contrast, enteric viruses like rotavirus only infect via the gastrointestinal track. Vaccines to these agents should therefore focus on generating effective immune responses in the respiratory and gastrointestinal systems, respectively. Similarly, virus dissemination can be prevented by local immunity at the site of transmission. Thus, HIV infected individuals could potentially be removed from the virus-transmission cycle without a global eradication of virus replication by immune removal of the virus from organs of dissemination (Flint et al. 2008; Mims et al., 2001). As the largest single organ in humans and the most external, the skin is an obvious target for initiation of viral infections. Sufficiently obvious, in fact, that the cornified epithelium is an extremely effective barrier against virus transmission, and only a few viruses have managed the trick of penetrating this barrier on their own. Transmission through the skin can be achieved by physical introduction past the keratinocytes either by natural (insect or animal bites) or artificial (hypodermic) means. In the former case, transmission may be a natural part of the virus life cycle, or may be unrelated to virus evolution (but still detrimental to unlucky individuals infected with West Nile virus, for example). Mammalian (or avian) cells differ considerably from insect cells, and the ability to productively infect members of both phyla strongly implies that cross-species transmission is important to viral survival.
Cells
Viral replication requires delivery of the viral nucleic acid to the cytosol, where it can either immediately initiate the replication cycle or do so after transport to the nucleus. Penetration can occur in two locales: either directly at the plasma membrane or after internalization into an endosome or other internal cellular membrane system (e.g., some viruses are thought to penetrate cells from the endoplasmic reticulum). Enveloped viruses penetrate by fusing their membranes with cell membranes, while nonenveloped viruses penetrate by disrupting cell membranes. In either case, viruses must first adhere to the target cell by binding its receptor to a cellular ligand, which can be any molecule on the cell surface. The specificity of viral receptors ranges from highly specific (e.g., a ligand expressed in a single cell type in a single tissue), to completely promiscuous (e.g., a molecule expressed by every cell; sialic acid is a receptor for many viruses). In the former case, the receptor is usually responsible for viral tropism, while in the latter, viral tropism will depend on other factors (such as route of entry into the host, ability to penetrate the cell, or replicate once inside). Viral penetration is usually a complicated affair that entails conformational alterations in viral surface proteins and interactions with additional cell surface proteins. There are ample opportunities for the innate and adaptive arms of the immune system to block penetration by preventing virus binding to host cells, or by preventing downstream events in the penetration and uncoating process (Berger, 1997; Doms, 2000; Kasamatsu & Nakanishi, 1998; Klasse et al., 1998; McDermott & Murphy, 2000; Norkin, 1995; Sodeik, 2000; Whittaker et al., 2000). The requirement of viral genomes to reach the cytosol provides an early opportunity for recognition by the adaptive immune system in the absence of viral gene expression. MHC class I binding antigenic peptides can be generated from viral proteins that accompany viral genomes into the cytosol. As peptide generation is often quite inefficient, this usually requires that thousands of copies of the source protein
136
The paThogens
be delivered, which limits this pathway to high abundance of proteins from large viruses. There is evidence, however, that this skews CD8 T-cell responses in some systems.
OUTCOMES OF CELLULAR INFECTION
Viral penetration of cells has four potential outcomes (with all gradations in between). First, the cell may be completely inhospitable and the viral proteins and nucleic acids are disposed of with minimal perturbation of the host cells. Second, viral replication initiates but fails to produce infectious progeny with consequences ranging from minimal transient perturbations in cellular physiology to cell death. Third, viral replication results in the generation of infectious progeny and cells are either killed immediately or remain persistently infected and continue to function as they produce progeny viruses. Fourth, the virus enters a latent state and essentially disappears until it is triggered to reactivate with the production of infectious progeny. These outcomes are not mutually exclusive, and in a given individual harboring a virus, these processes likely occur simultaneously, depending on the nature of the cell infected and the exact conditions of infection (e.g., number of viruses infecting the cell, known as the multiplicity of infection [MOI]); exposure of the cell to cytokines; cell cycle status). As always, evolution only selects for the transmission of virus between hosts (the selection of viruses replicating within any given organism is only an intermediate step). In some circumstances, even nonproductive infection in certain cell types may be evolutionarily selected if it facilitates virus transmission. For example, a virus may nonproductively infect immune cells, disabling the cells and thereby enhancing viral growth in productively infected cells. On the other hand, most examples of nonproductive infection are not a result of evolutionary selection but simply an example of the law of biological entropy. There are infinitely more ways for things to go wrong than to go right. The means viruses use to achieve these outcomes varies tremendously with the virus, but all viruses must fulfill several basic tasks: (i) produce mRNA to generate viral proteins on cellular ribosomes; (ii) replicate their genome; (iii) assemble their genomes with viral (and sometimes cellular) proteins, and release progeny from the cells (Doms et al., 1993; Garoff et al., 1998); and (iv) modify host cell metabolism (including avoiding innate cell antiviral responses) to optimize viral replication. A productive viral infection usually results in the generation of hundreds to thousands (or more) progeny. The shortest infectious cycles can be completed in 4 hours. This capacity for replication provides a serious challenge to the immune system, since just three or four infectious cycles over the course of a single day can be sufficient to produce enough of the virus to infect every cell in a target organ. The host must therefore have an immediate response for such rapid cytopathic viruses, and this is, no doubt, one of the major selective forces for the evolution of the innate immune system, which must operate solo for several days (at least) until activated antigen-specific lymphocytes can generate sufficient numbers to mount an effective antiviral offensive.
Coordinated Gene Expression
Following cellular penetration, viruses usually have a precise plan of attack that entails the temporally coordinated transcription/expression of subsets of their genes. During any given segment of the infectious cycle, different viral gene products are often expressed in widely different quantities depending on the function of the gene product. This
plan can vary from cell type to cell type, and abortive infections often result from failing to successfully negotiate the transition from one stage to another. Often, the plan of expression is based on genome replication, and viral genes can be classified as early (pre-genome replication) versus late (post-genome replication). The time of genome replication varies widely, but typically occurs between 2 and 12 hours post-infection. At the great danger of over-simplification, the enzymes required for viral replication and for modifying cellular functions are expressed immediately upon penetration, while many of the proteins that make up virions (viral “structural” proteins) are produced late in the infectious cycle. For the adaptive immune system, the most important aspect of coordinated gene expression is that not all infected cells express the full range of viral gene products, and antigens presented by infected professional antigen presenting cells may be limited to early gene products.
GENETIC INSTABILITY OF VIRUSES
Genetic instability is the sine qua non of viruses. Indeed, the mutation rate of RNA viruses is sufficiently high that despite possessing a tiny genome relative to other life-forms, virtually every individual in a population is still unique. DNA viruses are generally more stable, but still can exhibit mutation rates several orders of magnitude higher than host cells. Therefore, it is necessary to think of viruses as dynamic populations of related genomes and not as monolithic entities (Domingo & Holland, 1997; Koonin, 1992; Shadan & Villarreal, 1996). Although virus recombination is probably a relatively infrequent occurrence, it still is probably very important for most viruses, which have a haploid genome, and can therefore profit by a mechanism that enables escape from lethal mutations. Indeed, it is often observed that serial plaque purification of viruses ultimately results in the generation of poorly replicating viruses, pointing to the importance of recombination in maintaining a functional genome. A number of RNA viruses foster recombination by possessing segmented genomes. Recombination also occurs by polymerases shifting from one genome to another (predominantly RNA viruses) and by strand breakage and rejoining (predominantly DNA viruses). The genetic instability of RNA viruses, in particular, makes them elusive targets for the immune system. For many viruses, mutants that escape neutralization by monoclonal antibodies arise with a frequency of 105 or higher. This can result in antigenic drift, in which the humoral immune system selects for neutralization-resistant mutants. This, of course, is a significant problem in influenza vaccination. The influenza virus, as a segmented virus, also undergoes antigenic shift, in which the viral surface proteins are exchanged in a recombination event. This can result in the introduction of novel surface virion proteins from animal influenza viruses and in devastating epidemics. Hepatitis C virus (HCV), an increasingly important human pathogen, is so highly variable that multiple serotypes are typically isolated from the same individual. For genetic instability to be of any use to the virus, the resulting gene products must be sufficiently plastic in order to utilize the mutations to produce a novel (and useful) phenotype. This is not uniform among viral proteins, and it is commonly found that variation rates among viral proteins vary hugely. At the risk of over-simplification, the extracellular domains of membrane proteins of envelope viruses demonstrate the greatest flexibility for accepting mutations, while proteins involved in protein–protein interactions (e.g., the coat proteins of naked viruses or viral replication proteins) are less tolerant of mutations.
10. overview of Viral pathogens
Capturing Host Genes
Another potential source of genetic variability is the capture of host genes. This happens most often with retroviruses due to their unique life cycle, which entails integration into the host genome (the retroviral capture and mutation of host cell genes involved in cellular proliferation led to the identification of oncogenes as altered cellular genes). Small viruses are under heavy selection pressure for minimizing genome size, which make host genes difficult to swallow. However, for the large DNA viruses, such as Herpesviridae and Poxviridae family members, insertion of even large amounts of foreign genes has no discernible effect on virus function, so there is a large potential for capturing host genes. These viruses often possess sophisticated programs for manipulating host immunity, and clearly these genes are often derived from the host. Such genes are, however, usually distantly related to host genes, so these events are probably extremely infrequent. Thus, for all but retroviruses, the capture of host genes is an important factor in their early evolution but less important over a historical time frame (Alcami & Koszinowski, 2000; Becker, 1996; McFadden et al., 1998; Tortorella et al., 2000; Weinberg, 1997).
TISSUE TROPISM
A critical aspect of viral infections is that replication is usually limited to anatomical locations or cell types (Table 3). There are a number of contributing factors: (i) physical isolation of viruses due to anatomic barriers (e.g., enteric viruses may never have the opportunity to escape the GI system even though they would be capable of infecting other cell types), (ii) the specificity of viral receptors and limited distribution of cellular factors required for viral penetration (e.g., HIV requires cellular expression of CD4, which is limited to a small subset of cells and also requires host cells to express certain chemokine receptors to enable fusion of viral and cellular membranes), and (iii) even given entry to the cytosol, viruses can be rather finicky about replicating and very few viruses (if any) have the ability to productively replicate in every cell type in the body. This implies a requirement for nonhousekeeping cellular genes for productive infection, but identifying cellular gene products that positively (or negatively) influence virus infection is extremely difficult in practice. One aspect of viral tropism is particularly important to the immune system. If viruses are unable to infect professional antigen presenting cells, an alternative mechanism must be used for presenting viral antigens to naïve CD8 T cells. It is thought that under these circumstances presentation occurs via cross-priming, a process in which professional antigen presenting cells acquire viral proteins produced by infected cells and process them for presentation to T cells.
Kinetic Aspects of Infection
Many viruses replicate in a host by a program that requires the dissemination of virus from one organ to another. Virus dissemination can occur via either the blood or lymph. As lymphatics are more accessible to extracellular particles than capillaries, this is a more likely route of dissemination (the lymphatics function to collect foreign material from the periphery to the lymph nodes). Viruses also have an opportunity to reach the blood via the lymphatics if they manage to avoid sequestration in nodes on the way to the venous system. Virions probably have a difficult time spreading in this direct manner, and in many cases virus spread is mediated by infected white blood cells (erythrocytes cannot be infected as they lack essential biosynthetic processes, including protein synthesis). As white blood cells have the intrinsic ability to leave the blood or lymph and infiltrate tissues (diapedesis),
137
they can provide a direct route for virus entry into organs. Lacking a cellular carrier, viruses must rely on other ways to breach the barriers that guard the tissues. Some viruses are capable of being transcytosed by endothelial cells into the underlying tissues. In tissues with sinusoids (e.g., adrenals, bone marrow, liver, spleen), the virus may be transcytosed by resident macrophages that line the sinusoids. In other organs (e.g., kidney, pancreas, gut), many capillaries are fenestrated, providing a direct route for viral penetration. Finally, some connective tissues, muscle, and the central nervous system (CNS), are highly resistant to direct penetration, since the endothelial cells of the capillaries are backed by a formidable basement membrane. In this case, virus penetration from the blood probably occurs by diapedesis. The pattern of virus spread can be dependent on the mechanism of virus maturation. Enveloped viruses can dictate whether they mature from the apical or lumenal surface of polarized epithelial cells by taking advantage of the signals the cells use to maintain cell polarization. Respiratory viruses often mature from the apical surface of epithelial cells, limiting their spread to the surface of the airway. Presumably this is a way of increasing their chance of transmission by maximizing their concentration in respiratory fluids. Other viruses with less restricted tissue tropism mature from the basolateral surface. This increases their chances of hematogenous spread. Viruses capable of infecting neurons (neurotropic viruses) take advantage of the transport capacity of neurons to travel from the periphery to the ganglia or the CNS. Such traffic can be bidirectional. Herpesviruses, geniuses of viral latency, persist in a latent state in ganglia or in the spine, and upon reactivation are transported to the periphery where they bud from sensory nerve termini to be transmitted to new hosts via intimate contact. Rabies virus travels to the CNS from sensory neurons in the vicinity of an animal bite, multiplying in the CNS and then leaving via efferent neurons to the salivary glands, where virus is manufactured for saliva-borne transmission. Incredibly, viral replication in the CNS is sufficiently cell specific to induce the typical rabid behavior essential to transmission. All of this is accomplished by the information encoded by just 12,000 nucleotides. Another mode of virus spread is from mother to fetus. For noncytopathogenic viruses, this can ensure virus maintenance in a species. In the case of retroviruses, which integrate into the genome, the line between virus and host becomes blurred as viral genes are transmitted in the
