Protozoans in Macrophages

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Denkers • Gazzinelli

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Protozoans in Macrophages

Denkers ISBN 978-1-58706-150-9

9 781587 061509

Eric Y. Denkers and Ricardo T. Gazzinelli

Protozoans in Macrophages

MEDICAL INTELLIGENCE UNIT

Protozoans in Macrophages Eric Y. Denkers, Ph.D. Department of Microbiology and Immunology College of Veterinary Medicine Cornell University, Ithaca, New York, U.S.A.

Ricardo T. Gazzinelli, Ph.D. Laboratory of Immunopathology Rene Rachou Research Center – FIOCRUZ Belo Horizonte, MG, Brazil and Division of Infectious Diseases and Immunology University of Massachusetts Medical School Worchester, Massachusetts, U.S.A. LANDES BIOSCIENCE AUSTIN, TEXAS U.S.A.

PROTOZOANS IN MACROPHAGES Medical Intelligence Unit Landes Bioscience Copyright ©2007 Landes Bioscience All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: Landes Bioscience, 1002 West Avenue, Second Floor, Austin, Texas 78701 U.S.A. Phone: 512/ 637 6050; Fax: 512/ 637 6079 www.landesbioscience.com ISBN: 978-1-58706-150-9 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data Library of Congress Cataloging-in-Publication Data Protozoans in macrophages / [edited by] Eric Y. Denkers, Ricardo T. Gazzinelli. p. ; cm. -- (Medical intelligence unit) Includes bibliographical references and index. ISBN 978-1-58706-150-9 1. Protozoa, Pathogenic. 2. Macrophages. I. Denkers, Eric Y. II. Gazzinelli, Ricardo T. III. Series: Medical intelligence unit (Unnumbered : 2003) [DNLM: 1. Macrophages--parasitology. 2. Protozoa--pathogenicity. 3. Immune System-physiopathology. 4. Protozoan Infections--immunology. QX 50 P9675 2007] QR251.P795 2007 616.9'3601--dc22 2007021766

CONTENTS Preface .................................................................................................. xi Introduction: Macrophage Biology ........................................................ 1 D.M.E. Bowdish and Siamon Gordon What Is a Macrophage? ......................................................................... 1 Macrophage Origin ............................................................................... 2 Activation States of Macrophages .......................................................... 3 Types of Macrophages ........................................................................... 4 Recognition and Destruction by Macrophages and Subversion by Pathogens ..................................................................................... 9 1. Invasion and Intracellular Survival by Toxoplasma ............................... 16 L. David Sibley, Audra Charron, Sebastian Håkansson and Dana Mordue Life Cycle and Basic Biology ............................................................... 16 Actin-Based Motility and Cell Invasion ............................................... 17 Host Cell Recognition and Entry ........................................................ 18 Vacuole Modification and Intracellular Survival .................................. 21 2. Macrophages, Alternative Niches for Intracellular Growth of Trypanosoma cruzi ........................................................................... 25 Julio Scharfstein and Marcos André Vannier dos Santos Mechanisms of Cellular Invasion by Insect-Borne Metacyclic Trypomastigotes ............................................................ 26 Lessons from Cellular Invasion by Mammalian Stages of T. cruzi ........ 27 Macrophage Signaling Pathways Involved in Uptake of Trypomastigotes .......................................................................... 29 Macrophages as Niches for T. cruzi Growth: Lessons from in Vivo Infection Models ............................................................................. 30 3. Macrophage – Leishmania Interactions: Complexities and Uncertainties from the Study of Leishmaniasis in Vivo ................. 38 Paul M. Kaye Diversity of Mononuclear Phagocytes .................................................. 39 Host Cells for Leishmania .................................................................... 40 Remodeling the Environment ............................................................. 42 Mononuclear Phagocyte Differentiation .............................................. 43 T Cell Priming .................................................................................... 44 4. Innate Recognition, Cell Signaling and Pro-Inflammatory Response during Infection with Trypanosoma cruzi ............................................. 49 Catherine Ropert and Ricardo T. Gazzinelli T. cruzi and Chagas Disease ................................................................ 50 Evidence for the Activation of the Innate Immune System from Vertebrate Hosts Infected with T. cruzi .................................. 51 Pro-Inflammatory Activity of GPI Anchors from T. cruzi: Relationship of Structure and Function ........................................... 52

Other Molecules from T. cruzi Involved in Activation of Cells from Innate Immune System ........................................................... 54 In Vitro and in Vivo Role of MyD88 and TLRs in Induction of Pro-Inflammatory Cytokines and Host Resistance to Infection with T. cruzi .................................................................................... 54 Signalling Pathway and Tolerance Induced by tGPI-Mucins: An Alternative Way to Control Excessive Inflammatory Response ... 57 5. Modulation of Positive Signaling and Proinflammatory Responses by Hemozoin, a Plasmodium Metabolic Waste .................................... 67 Martin Olivier and Maritza Jaramillo Hemozoin and Macrophages ............................................................... 67 Hemozoin Synthesis ............................................................................ 68 Hemozoin Accumulation and Disease Diagnostic-Prognosis ............... 69 Inhibition of Immune Cell Functions .................................................. 69 Activation of Immune Responses ......................................................... 71 Signaling Events Regulating HZ-Induced Inflammatory Response ...... 72 6. Pro-Inflammatory Responses and Cell Signaling during Malaria Infection: The Parasite Glycosylphosphatidylinositol Ligand ............... 84 D. Channe Gowda Pro-Inflammatory Responses ............................................................... 86 Malaria Pro-Inflammatory Factor ........................................................ 87 7. Pro-Inflammatory Responses in Macrophages during Toxoplasma gondii Infection .................................................................................... 99 Katherine S. Masek and Christopher A. Hunter Macrophages Effector Functions ....................................................... 100 Production of IL-12 .......................................................................... 101 Innate Recognition of T. gondii ......................................................... 101 Pro-Inflammatory Signaling Events in Macrophages ......................... 102 8. Down-Modulation of Proinflammatory Signal Transduction in Toxoplasma gondii-Infected Macrophages ...................................... 108 Barbara A. Butcher, Leesun Kim, Chiang W. Lee and Eric Y. Denkers Transcriptional Profiling in the Toxoplasma Infected Cell .................. 109 Interference with NFκB Signaling ..................................................... 110 Effect of Toxoplasma Infection on MAPK Signaling .......................... 111 Induction of STAT3 Signaling by Toxoplasma ................................... 112 Kinetics of Toxoplasma Invasion and Disruption of Host Signaling Pathways ........................................................... 112 9. Avoidance of Innate Immune Mechanisms by the Protozoan Parasite, Leishmania spp ................................................................................... 118 David M. Mosser and Suzanne A. Miles Life Cycle .......................................................................................... 118 Receptor Mediated Phagocytosis ....................................................... 118

Leishmania and Innate Immunity ...................................................... 120 Suppression of IL-12 and the Th1 Response ..................................... 122 Alterations in Signal Transduction following Leishmania Infection ... 123 IL-10 Induction by IgG-Opsonized Amastigotes ............................... 124 10. Survival Strategies of Toxoplasma gondii: Interference with Regulatory and Effector Functions of Macrophages ................... 130 Carsten G.K. Lüder Inhibition of MHC Class II Expression and Antigen Presentation .... 131 Interference with iNOS Expression and NO-Mediated Anti-Toxoplasma Activity ............................................................... 132 Modulation of Macrophage Apoptosis ............................................... 133 11. Targeting SHP-1 to Prevent Macrophage Activation Promotes Leishmania Pathogenesis .................................................................... 139 Devki Nandan and Neil E. Reiner Leishmania and Macrophage Deactivation ......................................... 140 Disruption of Host Cell Signaling Pathways ...................................... 142 Leishmania-Induces Negative Signaling ............................................. 142 Mechanisms of SHP-1 Activation by Leishmania ............................... 143 12. Negative Signaling and Modulation of Macrophage Function in Trypanosoma cruzi Infection .......................................................... 149 Flávia L. Ribeiro-Gomes, Marcela F. Lopes and George A. DosReis Distinct Programs of Macrophage Activation .................................... 149 Protective Mechanisms against T. cruzi Induced by IFN-γ ................ 150 Evasion of Innate Macrophage Defenses ............................................ 151 Evasion of Activated Macrophages ..................................................... 152 Prospects for the Future..................................................................... 155 13. Effector Functions of Macrophages in Plasmodium Parasite Infections .............................................................................. 160 Mariela Segura, Rebecca Ing, Zhong Su, Neeta Thawani and Mary M. Stevenson The Role of the Spleen in Host Defense against Malaria ................... 162 Functional Properties of Macrophages during Blood-Stage Malaria Infection ........................................................................... 163 Macrophage Interactions with Plasmodium Blood-Stage Parasites in Humans .................................................................................... 163 Opsonin-Independent Adhesion and Phagocytosis ............................ 164 Opsonin-Dependent Parasite Clearance by Human Monocytes ........ 165 Cytokine Production by Activated Monocytes/Macrophages ............. 166 Macrophage-Derived Mediators of Cytotoxicity ................................ 168 Macrophage Interactions with Plasmodium Blood-Stage Parasites in Mouse Models ........................................................................... 169 Opsonin-Independent Adhesion and Phagocytosis ............................ 169 Opsonin-Dependent Parasite Clearance by Mouse Macrophages ....... 170

Cytokine Production by Activated Macrophages ............................... 170 Macrophage-Derived Mediators of Cytotoxicity ................................ 171 Functional Properties of Macrophages during Liver-Stage Malaria .... 172 Role of Dendritic Cells as Phagocytic Cells during Malaria Infection ............................................................... 172 14. Innate Control of Toxoplasma gondii through Macrophage-Based Effector Mechanisms .......................................................................... 184 Gregory A. Taylor Reactive Oxygen Intermediates .......................................................... 185 Nitric Oxide ...................................................................................... 185 Iron Deprivation ............................................................................... 186 Tryptophan Degradation ................................................................... 186 p47 GTPases ..................................................................................... 187 15. Phagocyte Effector Functions against Leishmania Parasites ................ 193 Christian Bogdan NADPH Oxidase .............................................................................. 195 Inducible Nitric Oxide Synthase ....................................................... 197 16. Effector Mechanisms of Macrophages Infected with Trypanosoma cruzi ..................................................................... 207 Fredy R.S. Gutierrez, Flavia S. Mariano, Isabel K.F. Miranda-Santos Bogdan and João S Silva The Clinical Outcome of Infection with Trypanosoma cruzi and Its Preference for an Intracellular Habitat ............................... 207 Triggering the Macrophage to Kill Trypanosoma cruzi ....................... 208 Killing of Trypanosoma cruzi by the Lysosome ................................... 210 Impact of Humoral Effector Mechanisms on the Killing Functions of the Macrophage ........................................................ 211 Killing of Trypanosoma cruzi by Free Radicals: Reactive Oxygen Species, Nitric Oxide and Peroxynitrite .............. 211 Quantitative Effects of NO upon Growth of Trypanosoma cruzi and in the Pathology of Chagas’ Disease ...... 213 Killing of Trypanosma cruzi by Depletion of Tryptophan .................. 214 Control of Macrophage Effector Responses by Trypanosoma cruzi and by the Host ........................................... 214 Index .................................................................................................. 221

EDITORS Eric Y. Denkers Department of Microbiology and Immunology College of Veterinary Medicine Cornell University, Ithaca, New York, U.S.A. Chapter 8

Ricardo T. Gazzinelli Laboratory of Immunopathology Rene Rachou Research Center – FIOCRUZ Belo Horizonte, MG, Brazil and Division of Infectious Diseases and Immunology University of Massachusetts Medical School Worchester, Massachusetts, U.S.A. Chapter 4

CONTRIBUTORS Christian Bogdan Department of Microbiology and Hygiene Institute of Medical Microbiology and Hygiene University of Freiburg Freiburg, Germany Chapter 15

D.M.E. Bowdish Sir William Dunn School of Pathology University of Oxford Oxford, OX1 3RE Introduction

Audra Charron Department of Molecular Microbiology Washington University St. Louis, Missouri, U.S.A. Chapter 1

George A. DosReis Carlos Chagas Filho Institute of Biophysics Federal University of Rio de Janeiro Rio de Janeiro, Brazil and Institute for Investigation in Immunology Millenium Institutes, Brazil

Barbara A. Butcher Department of Microbiology and Immunology College of Veterinary Medicine Cornell University Ithaca, New York, U.S.A.

Chapter 12

Chapter 8

Introduction

Siamon Gordon Sir William Dunn School of Pathology University of Oxford Oxford, OX1 3RE

D. Channe Gowda Department of Biochemistry and Molecular Biology Pennsylvania State University College of Medicine Hershey, Pennsylvania, U.S.A.

Paul M. Kaye The Hull York Medical School Department of Biology Immunology and Infection Unit York, U.K. Chapter 3

Chapter 6

Fredy R.S. Gutierrez Department of Biochemistry and Immunology School of Medicine of Ribeirão Preto University of São Paulo São Paulo, Brazil

Leesun Kim Department of Microbiology and Immunology College of Veterinary Medicine Cornell University Ithaca, New York, U.S.A. Chapter 8

Chapter 16

Sebastian Håkansson Department of Molecular Microbiology Washington University St. Louis, Missouri, U.S.A. Chapter 1

Chiang W. Lee Department of Microbiology and Immunology College of Veterinary Medicine Cornell University Ithaca, New York, U.S.A. Chapter 8

Christopher A. Hunter Department of Pathobiology University of Pennsylvania Philadelphia, Pennsylvania, U.S.A. Chapter 7

Rebecca Ing Centre for the Study of Host Resistance Research Institute of the McGill University Health Centre McGill University Montreal, Quebec, Canada Chapter 13

Maritza Jaramillo The Research Institute of McGill University Health Centre and the Centre for the Study of Host Resistance Departments of Medicine, Microbiology and Immunology Montréal, Québec, Canada Chapter 5

Marcela F. Lopes Carlos Chagas Filho Institute of Biophysics Federal University of Rio de Janeiro Rio de Janeiro, Brazil and Institute for Investigation in Immunology Millenium Institutes, Brazil Chapter 12

Carsten G.K. Lüder Institute for Medical Microbiology Georg-August-University Göttingen, Germany Chapter 10

Flavia S. Mariano Department of Biochemistry and Immunology School of Medicine of Ribeirão Preto University of São Paulo São Paulo, Brazil Chapter 16

Katherine S. Masek Department of Pathobiology University of Pennsylvania Philadelphia, Pennsylvania, U.S.A. Chapter 7

Suzanne A. Miles Department of Cell Biology and Molecular Genetics University of Maryland College Park, Maryland, U.S.A.

Martin Olivier The Research Institute of McGill University Health Centre and the Centre for the Study of Host Resistance Departments of Medicine, Microbiology and Immunology Montréal, Québec, Canada Chapter 5

Isabel K.F. Miranda-Santos Bogdan Department of Biochemistry and Immunology School of Medicine of Ribeirão Preto University of São Paulo São Paulo, Brazil

Neil E. Reiner Department of Medicine (Division of Infectious Diseases) and the Department of Microbiology and Immunology The University of British Columbia Vancouver Coastal Health Research Institute Vancouver, British Columbia, Canada

Chapter 16

Chapter 11

Dana Mordue Department of Molecular Microbiology Washington University St. Louis, Missouri, U.S.A.

Flávia L. Ribeiro-Gomes Carlos Chagas Filho Institute of Biophysics Federal University of Rio de Janeiro Rio de Janeiro, Brazil, and Institute for Investigation in Immunology Millenium Institutes, Brazil

Chapter 9

Chapter 1

David M. Mosser Departments of Cell Biology and Molecular Genetics University of Maryland College Park, Maryland, U.S.A. Chapter 9

Devki Nandan Department of Medicine (Division of Infectious Diseases) The University of British Columbia Vancouver Coastal Health Research Institute Vancouver, British Columbia, Canada Chapter 11

Chapter 12

Catherine Ropert Departamento de Bioquímica e Imunologia Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Belo Horizonte, MG, Brazil Chapter 4

Julio Scharfstein Centro de Pesquisas Gonçalo Moniz Fundação Oswaldo Cruz, FIOCRUZ Salvador, Bahia, Brazil Chapter 2

Mariela Segura Centre for the Study of Host Resistance Research Institute of the McGill University Health Centre McGill University Montreal, Quebec, Canada Chapter 13

L. David Sibley Department of Molecular Microbiology Washington University St. Louis, Missouri, U.S.A. Chapter 1

João S. Silva Department of Biochemistry and Immunology School of Medicine of Ribeirão Preto University of São Paulo São Paulo, Brazil Chapter 16

Mary M. Stevenson Centre for the Study of Host Resistance Research Institute of the McGill University Health Centre Montreal, Quebec, Canada Chapter 13

Zhong Su Centre for the Study of Host Resistance Research Institute of the McGill University Health Centre McGill University Montreal, Quebec, Canada Chapter 13

Gregory A. Taylor Departments of Medicine, Immunology and Molecular Genetics Microbiology Division of Geriatrics Center for the Study of Aging and Human Development Duke University Medical Center and GRECC, VA Medical Center Durham, North Carolina, U.S.A. Chapter 14

Neeta Thawani Centre for the Study of Host Resistance Research Institute of the McGill University Health Centre McGill University Montreal, Quebec, Canada Chapter 13

Marcos André Vannier dos Santos Centro de Pesquisas Gonçalo Moniz Fundação Oswaldo Cruz, FIOCRUZ Salvador, Bahia, Brazil Chapter 2

PREFACE The term “macrophage” was coined by the 19th century Russian immunologist Elie Metchnikov at the dawn of modern immunology to describe cells he saw under the microscope engulfing and destroying microorganisms. Since this first glimpse of innate immunity in action, we have progressed far towards an understanding of the fascinating biology of macro phages. But their full complexity continues to be a source of discovery today. In addition to phagocytic activity, we now know macrophages produce toxic molecules such as reactive oxygen and nitrogen intermediates that can not only cause death of invading microbes, but may also contribute to host tissue pathology. Macrophages are a source of proinflammatory cytokines such as TNF-α and IL-6. These potent mediators can protect against infection or, in some cases, promote pathology. Newly discovered molecules, such as the 47 kDa IFN-γ-inducible GTPases, are also important microbicidal effector molecules — although how they work remains uncertain. The ability of macrophages to produce cytokines such as IL-12, and to express MHC class II and co-stimulatory molecules such as B7.1/B7.2 (CD80/ CD86), licenses these cells as card-carrying professional antigen-presenters. As such, they can participate in the generation of T cell-mediated immunity that is so critical for resistance to microbial pathogens, including the protozoa. Since macrophages come armed with these potent anti-infection properties, microbial pathogens might be expected to avoid their encounter. Yet, many microbes, including several protozoan species, actively target these cells for infection. Such intracellular pathogens must first have a tactic for entering the cell. This can be through active invasion (Toxoplasma gondii), but it can also involve exploitation of endocytic uptake pathways uptake pathways (Leishmania, Trypanosoma). Once inside, they must deploy strategies to survive and replicate within this potentially hostile intracellular environment. Some protozoa create a specialized parasitophorous vacuole (Toxoplasma), others live within acidified endosomes (Leishmania), and still others escape into the host cell cytoplasm (T. cruzi). There is newly emerging evidence to suggest that from within the cell, as well as from without, protozoans manipulate macrophage responses, activating some intracellular signal transduction pathways and subverting others. Using as a theme the encounter between protozoan parasites and macrophages, this volume brings together cell biologists, immunologists and protozoologists to review current developments in this broad and dynamic research area. Discussed are ways protozoans establish their intracellular niche, how they activate macrophage effector functions, what these functions are, and means by which several protozoans subvert macrophage activity. What emerges is a picture of the macrophage as a key cell type in the host response to protozoan infection. How these cells respond, and how their responses can be subverted, are likely to be critical determinants in the outcome of protozoan infection.

Millions of lives are lost every year to protozoan infections, most importantly those caused by Plasmodium, but also Trypanosoma and Leishmania spp. Other protozoans, such as Toxoplasma, are so exquisitely adapted to their host that they rarely cause disease, except during host immunodeficiency. We hope that by working towards an understanding of the biology of protozoans in macrophages we will ultimately be capable of treating and preventing disease and mortality caused by this major class of microbial pathogens. Eric. Y. Denkers Ricardo T. Gazzinelli

Acknowledgments We are extremely grateful to the authors of the chapters that follow for taking time from their busy schedules to contribute to the success of this volume.

INTRODUCTION

Macrophage Biology D.M.E. Bowdish and Siamon Gordon*

Abstract

T

he importance of macrophages in the host response to infection has been recognised for decades. However, the macrophage has a range of phenotypes, functions and activation states and consequently the study of macrophage biology is complicated by the heterogeneity of these cells. An understanding of basic macrophage biology is required to understand the mechanisms of evasion, invasion and subversion of macrophage defences by protozoan pathogens. Herein we review the origins of macrophages, differences in macrophage phenotypes, mechanisms of macrophage based killing and subversion of this killing by pathogens.

What Is a Macrophage? Phenotypically and functionally tissue macrophages are an extremely heterogeneous group of cells derived from circulating monocytes. They range in appearance from the dendritic-like microglial cells to the less aborised Kupffer cell. Fortunately, in humans there exists an intracellular membrane marker by which the majority of macrophages can by identified called CD68 (macrosialin in mouse). It has long been known that macrophages are an important component of the innate immune response, but it is increasingly apparent that they are involved in tissue homeostasis, regulation of haematopoiesis, chronic inflammation, atherosclerosis, wound repair and tissue remodelling, as well as killing of invading micro-organisms. Although macrophage function depends, at least in part, on location, developmental state and in vitro culture conditions, there are some properties that are conserved amongst almost all macrophage populations studied to date. One of the most distinctive properties of macrophages is their ability to ingest particles via phagocytosis. Macrophages are able to recognise both pathogens and noninfectious agents using a variety of germ line-encoded pattern recognition receptors including lectins, toll-like receptors, and receptors for N-formyl methionine containing peptides. Macrophages are involved in safe apoptotic cell clearance and remove small numbers of potentially dangerous micro-organisms via phagocytosis without inducing a strong pro-inflammatory response. Should they fail to clear perceived threats, an acute inflammatory response is mounted. This results in the secretion of a variety of cytokines, chemokines and antimicrobial agents. Secretion of these mediators can result in autocrine activation of the macrophage by binding of cytokines to cytokine receptors or recruitment of cells involved in the adaptive immune response via secretion of chemokines. The macrophage destroys invading micro-organisms using an arsenal of antimicrobial effector mechanisms that encompass enzymatic degradation, oxidation, nutrient limitation and antimicrobial peptides. Upon internalisation and digestion of the pathogen, the macrophage presents foreign antigens to primed T lymphocytes, thus amplifying the adaptive immune response. When macrophage-based clearance is insufficient, prolonged or chronic inflammation may occur. Macrophages *Corresponding Author: S. Gordon—Sir William Dunn School of Pathology University of Oxford,10 South Parks Road, Oxford, OX1 3RE. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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Protozoans in Macrophages

are intimately linked with a number of chronic infections and inflammatory conditions such as the formation of atherosclerotic plaques, and conditions such as rheumatoid arthritis. The role of macrophages in host defence has been extensively reviewed in references 1-5. Herein we describe the origins of macrophages, differences in macrophage phenotypes, mechanisms of macrophage based killing and subversion of this killing by intracellular parasites.

Macrophage Origin Although it is clear that myeloid cells are derived from precursor cells found in the bone marrow, their developmental pathway is not entirely resolved. Confounding this is the fact that studies of leukocyte development are much more easily performed in the mouse and thus there is some confusion as to differences between human and mouse leukocyte development. It is generally accepted that CD34+ precursors give rise to monocytes, granulocytes, erythrocytes and thrombocytes. Cells that express both CD34 and the receptor for macrophage colony stimulating factor (M-CSFR) give rise exclusively to myelomonocytic cells.6 Circulating CD34+ monocytes are found in the blood and it is these circulating monocytes that give rise to tissue macrophages. In human peripheral blood there are at least four subsets of monocytes that are characterised by their levels of expression of CD64 (FCγRII), CD14 and CD16 (FCγRIII). In response to stimuli these monocytes give rise to terminally differentiated cells, macrophages and dendritic cells. Monocytes that express high levels of CD14, CD64 and little or no CD16 (CD14+/CD64+/ CD16-) comprise greater than 80% of the circulating monocyte population in healthy individuals. These monocytes produce high levels of pro-inflammatory cytokines when they are stimulated with bacterial components. Their response to a variety of chemokines in vitro is presumed to be an important factor in migration to peripheral tissues during the course of infection and inflammation; subsequently they are able to differentiate into macrophages with excellent anti-microbial activity and the capacity to interact with both B and T lymphocytes.7 Monocytes expressing CD16 as well as CD64 and CD14 (CD14+/CD64+/CD16+) constitute less than 10% of circulating monocytes in humans. These cells produce high levels of pro-inflammatory cytokines, low levels of IL-10, have a very high phagocytic capacity and participate in antibody dependent cellular cytotoxicity (ADCC). They are believed to be precursors to “resident” macrophages.8 Upon in vitro culture with cytokines, CD14+/CD64+/ CD16+ expressing cells differentiate into either macrophages or dendritic cells with a distinctive DC1 phenotype and are increased in patients with Kawasaki disease and influenza and decreased in patients with rheumatoid arthritis.9,10 Monocytes that do not express CD64 but have low to intermediate levels of CD14 and high levels of CD16 (CD14dim/CD64-/CD16+ or CD14low/CD64low/CD16+) constitute less than 10% of circulating monocytes. These monocytes have increased costimulatory activity and CD45 expression, but produce very little type 1 interferon or other pro-inflammatory cytokines. They have weaker phagocytic responses and ADCC but display an enhanced ability to interact with T or B lymphocytes and express higher chemotactic activity than their CD14+/ CD16+ counterparts, especially in response to the endothelial cell tethered chemokine, fractalkine. The ability of these cells to transmigrate in response to fractalkine (due to expression of CX3CL1, which is not expressed on CD16- cells) indicates that they may be precursors to tissue macrophages. These cells differentiate into myeloid antigen presenting cells and are believed to play a part in the Th1 response in vivo.11 Elevated numbers of these cells are found in HIV infected patients, and those suffering from pararheumatic systemic vasculitis and sepsis.12 Although these characterisations are helpful for making broad generalisations, there are a number of monocyte/macrophage subsets that aren’t easily classified. For example, macrophages of the intestinal mucosa have a distinct receptor expression profile that does not include CD14, complement receptors, or Fc receptors (see below).13 This illustrates the conclusion that there are no clear guidelines to identify and classify subsets of macrophages. For an thorough review on monocyte and macrophage heterogeneity see reference 14.

Macrophage Biology

3

Activation States of Macrophages Macrophage biology is plagued by confusion concerning the terminology of macrophage activation states. In the absence of pro-inflammatory or infectious stimuli macrophages have a number of homeostatic functions including the engulfment of apoptotic cells, erythrocyte clearance, and constitutive tissue repair. The macrophage’s response to infection must be tailored to the microbial threat and it has been discovered, primarily from in vitro studes, that the type of microbial or inflammatory stimulus results in the production of macrophages with varied functions. To date four major classes of immunologically acquired macrophage activation have been proposed, classical, innate, alternative, and deactivated.

Classical Activation The concept of macrophage activation came about as a result of the observation that macrophages treated with bacterial components and interferon-γ (IFN-γ) developed an enhanced ability to destroy a wide range of ingested pathogens. IFN-γ is produced by CD4+ and CD8+ T lymphocytes, NK cells and possibly by the infected macrophages themselves. IFN-γ alone does not confer this ability; rather it primes the macrophages for activation. The second signal is a bacterial component, generally LPS. Some studies suggest that it is the bacterial stimulation of TNF-α that provides the secondary signal rather than the LPS itself, although this has not been completely characterised.15 Classically activated macrophages have an increased ability to present antigen due to an enhanced expression of MHC class II and CD80/CD86 (B7.1/ B7.2) and increased production of iNOS. They have an enhanced ability to destroy intracellular pathogens due to an increased respiratory burst, and acquire the ability to mediate diverse inflammatory effects in the host by secreting a variety of cytokines. The importance of IFN-γ in parasite infection was demonstrated in vivo when it was found that antibody mediated neutralisation of IFN-γ in infected mice caused them to die more rapidly and have increased parasite loads.16-19 Subsequent experiments with mice defective in expression of IFN-γ or its receptor demonstrated that they were more susceptible to a variety of intracellular bacterial or protozoan pathogens.20-23 IFN-γ-induced activation is a contributor to the pathology of rheumatoid arthritis, delayed-type hypersensitivity and may contribute to atherosclerosis.24-26

Innate Activation Classical activation requires two steps, exposure to IFN-γ and to a bacterial products and results in a macrophage with altered phenotypic and functional properties. It has recently been shown that exposure to bacterial components, such as LPS or CpG, alone results in macrophages with altered phenotypes and functional properties. For example, it has been demonstrated that macrophages treated with LPS or CpG have an enhanced ability to produce IL-12 in response to a second exposure to LPS due to expression of the macrophage receptor with collagenous structure (MARCO).27 TLR agonist-induced expression of MARCO has also been linked to an enhanced ability of the macrophage to bind and clear Neisseria.28 A complete description of the receptors involved in innate activation and a full description of the functional properties of these cells has yet to be completed.

Alternative Activation Early on it was observed that the antigen presenting cells obtained from mice with experimental nematode infections (in which there is a Th2 cytokine environment) were able to process and present antigen without inducing T cell proliferation.29 Subseqently it was found that exposure to the Th2 associated cytokines, IL-13 and IL-4, resulted in macrophages with enhanced expression of the mannose receptor and MHC class II, but which were not able to induce T cell proliferation. Increased expression of the mannose receptor is associated with endocytosis and antigen presentation, although perhaps less efficiently than classically activated cells.30 There is also an increased flow of internalised particles and ligands to lysosomes. It has been demonstrated that alternatively activated macrophages are important in clearance of parasitic and

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Protozoans in Macrophages

extracellular pathogens, but unlike classically activated macrophages they do not display an increased oxidative burst and thus are not as efficient in killing intracellular pathogens.30,31 The importance of alternatively activated macrophages in parasitic and protozoan infections is now well established. In vivo models of Schistosoma mansoni, trypanosome and Leishmania infection demonstrate that there is a complex interplay between the production of Th1 and Th2 cytokines and the subsequent development of macrophage subsets. It appears that an initial Th1 response (characterised by elevated levels of IFN-γ and IL-12) is required to control the initial stages of infection by T. cruzi, T. brucei, and S. mansoni, however the cytokine balance shifts during the course of disease to a Th2 response.32-34 It is generally believed that this shift to a Th2 bias is required for clearance and resolution of the infection as animals defective in producing Th2 cytokines and thus alternatively activated macrophages do not survive. There is also evidence that for some protozoan pathogens the shift to a Th2 mediated response may result in dissemination of the parasite throughout the host.35 Alternatively activated macrophages do not make substantial amounts of nitric oxide (NO) because of their induction of arginase, an enzyme that counteracts the harmful effects of NO. Arginase does contribute to polyamine and proline biosynthesis, and promotes cell growth, collagen formation, and tissue remodelling. It has been proposed that this subclass of macrophages may play a primary role in wound repair, angiogenesis, fibrogenesis, synthesis of the extracellular matrix and granuloma formation.36 Alternatively activated macrophages also appear to have an anti-inflammatory function, and they have been demonstrated to decrease T cell proliferation and produce the anti-inflammatory cytokine IL-1 receptor antagonist and IL-10.37 Consistent with this analysis, these cells have a slight decrease in LPS-induced respiratory burst and cytokine production compared to classically activated macrophages. Alternatively activated macrophages are important in Th2- mediated diseases such as asthma, allergy and in the resolution of infectious disease and parasitic infection. The process of alternative activation has been reviewed in reference 38.

Deactivation Activated macrophages have potent biological functions that are essential for the host’s response to infection. However, once infection is resolved it is essential to end the pro-inflammatory program. Exposure to a number of anti-inflammatory molecules such as cytokines (e.g., IL-10, TGF-β), receptor ligation (e.g., CD200 - CD200R), steroids, or uptake of apoptotic cells can induce a “deactivated” phenotype. These cells can be identified by the expression of CD16339 and they have a reduced expression of MHC class II, a decreased respiratory burst and pro-inflammatory cytokine production, as well as enhanced anti-inflammatory cytokine production.

Types of Macrophages Macrophage subpopulations can be divided a number of ways. There are phenotypic and functional distinctions between macrophages found at different locations throughout the body and between resident and recruited macrophages. The distinction between resident and recruited macrophages is particularly murky due to the difficulty in distinguishing the two sets in vivo. Although it has long been known that circulating monocytes migrate to the tissues where they become macrophages40 there is some debate over the role of newly recruited monocytes in the development of resident cells. Originally it was believed that tissue macrophages were derived and replenished exclusively from circulating monocytes, however, transplantation studies in both mice and humans indicate that the replenishment of resident tissue macrophages with donor macrophages is extremely slow. This could occur because of very low levels of recruitment and replenishment by circulating monocytes or because the tissue macrophages of the recipient are capable of self-renewal.41,42 Similar results were found for epidermal Langerhan’s cells.43 Thus it is believed that early in foetal or embryonic development the tissues are populated with cells derived from circulating monocytes. These cells mature into resident macrophages and under steady state conditions replenishment from circulating cells is low. When these

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cells are activated by infection or inflammation they are able to enter the draining lymph nodes with the appropriate chemotactic stimulus and move to B and T lymphocyte areas to present pathogens. Under such conditions monocytes enter the tissues to replenish the activated macrophages and these cells become “recruited” macrophages. The monocytes that are recruited to the sites of infection or inflammation may be different from those which replenish resident cells under steady state conditions. Tissue macrophages generally have stellate morphology and high endocytic ability (including nonspecific uptake of particles and Fc receptor-mediated uptake). Although they proliferate very slowly they have active RNA and protein synthesis. Resident tissue macrophages have important homeostatic functions and clear protein aggregates (e.g., protease-inhibitor complexes), physiological molecules (e.g., lysosomal hydrolases), denatured molecules (e.g., modified lipoproteins) and apoptotic cells from the intracellular spaces in either an immunologically silent or tolerogenic fashion. These cells are also important sentinels for clearance of invading micro-organisms. Despite the heterogeneity of macrophages there are obvious functional divisions between different subsets and it is useful to characterise macrophage subpopulations on the basis of location. Herein we briefly summarise the characteristics of macrophage subpopulations that are most frequently associated with parasitic infections. Although we omit discussion of macrophage subpopulations of the lung, brain and bone, macrophages at these locations have all been shown to harbour protozoan parasites and contribute to pathology in rare instances. For reviews on these cell types see references 44-47.

Kupffer Cells

Resident macrophages of the liver are termed Kupffer cells.48 The liver is an essential and active component of the innate immune response. Following infection at extrahepatic locations, local macrophages produce the cytokines IL-1, TNF-α and IL-6. Detection of IL-6 causes hepatocytes to produce a number of acute phase proteins that are responsible for the systemic effects of inflammation, and enhancing opsonic phagocytosis and complement activation. Extra-hepatic cytokines are detected by Kupffer cells; the cells become activated and have enhanced anti-microbial properties, although resident Kupffer cells have a less vigorous respiratory burst and are thus less efficient at killing certain pathogens than other types of macrophages.49 Kupffer cells express high levels of phagocytic receptors. These include Fc receptors by which they remove soluble IgG complexes and antibody coated particles or micro-organisms, complement receptors by which they remove complement coated bacteria and erythrocytes and scavenger and toll like receptors by which they remove bacteria and endotoxin from the circulation. Their avidity for clearing erythrocytes results in the characteristic accumulation of iron in these cells. Kupffer cells may be further subdivided on the basis of their location in the liver into cells of the periportal, midzonal and perivenous regions. Macrophages at different locations have different capacities for secretion of TNF-α, prostaglandin E, nitric oxide and IL-1. The Kupffer cells of the periportal region have the greatest phagocytic activity and highest lysosomal enzyme activity which is believed to be because this is the entry point for blood, and thus the first contact point for any blood borne pathogens. Kupffer cells are involved in both clearance and transmission of pathogens as they have been demonstrated to harbour a number of protozoan pathogens.50-52 For example, these cells may be especially important in the systemic spread of Plasmodium falciparum. Sporozoites move through the liver via the blood stream and are phagocytosed by Kupffer cells due to recognition of at least two proteins, circumsporozoite protein (CSP) and thrombospondin-related adhesive protein (TRAP).53,54 The sporozoites appear to be able to survive in the vacuoles of the macrophage and to exit the macrophage at a later time point.53,55,56 At this point the parasites invade neighbouring hepatocytes and cause their destruction, resulting in many of the symptoms of disease. The role of Kupffer cells in malaria is reviewed in reference 57 and the immunobiology of the liver is reviewed in reference 58.

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Splenic Macrophages The spleen is a unique lymphoid organ involved in clearance of pathogens and senescent erythrocytes from the blood as well as antigen presentation and activation of an adaptive immune response. Macrophages of the spleen are generally subdivided on the basis of location; however it is important to note that the architecture of mouse and human spleen is quite different and that the majority of our knowledge of splenic macrophages comes from murine studies.59 In the few comparative studies that have been performed it appears as though receptor distribution and function of different macrophage populations vary between humans and mice.60 Nevertheless the spleen functions as both a site of clearance of blood borne pathogens and of interactions between antigen presenting cells and B and T lymphocytes in both humans and mice. The summary below is based primarily on mouse studies. The white pulp is a specialised area of lymphocyte accumulation that contains B and T lymphocytes. The white pulp is separated from the red pulp, which is the major area of erythrocyte clearance, by the marginal zone. The marginal zone contains marginal zone B lymphocytes and dendritic cells as well as two types of macrophages, the marginal zone macrophages that are located adjacent to the red pulp, and the marginal zone metallophilic macrophages that are located adjacent to the white pulp. The macrophages of the marginal zone are involved in the clearance of apoptotic cells and micro-organisms as well as the maintenance of B lymphocytes. Like Kupffer cells, these macrophages are involved in turnover of erythrocytes and recycling of iron. The function of the marginal zone metallophilic macrophages is not entirely clear, although it is believed that they are involved in the response to viruses as they produce high levels of IFN-α and IFN-β. Splenic macrophages possess a variety of pattern recognition receptors60 that are of vital importance in clearance of blood-borne pathogens including Leishmania spp., and Plasmodium falciparum (reviewed in refs. 61,62). Certain pathogens that are easily cleared from circulation by the macrophages of the liver have virulence factors that prevent facile clearance from the spleen. For example, in experimental models of visceral leishmaniasis, the hepatic component of the infection is self-limiting (probably as a result of granuloma formation); however, amastigote growth in the spleen cannot be contained and results in tissue destruction. Although it is known that the marginal zone macrophages avidly phagocytose amastigotes it is not known whether their inability to clear the parasite is due to differences between hepatic and splenic macrophages such as differences in the mechanism of entry of the pathogen, differences in its ability to suppress cytokine production or some other unidentified mechanism. The importance of the spleen in the host’s response to infection is clear as splenectomised patients have a high risk of severe bacterial infections and must take prophylactic antibiotics.63 Patients who have undergone a splenectomy are also more likely to suffer from malaria and to have higher titres of parasites within their blood.64,65 Thus the macrophages of the spleen are important in host defence towards bacterial and parasitic infection.

Dendritic Cells The dendritic cell (DC) is the close cousin to the macrophage. Both macrophages and dendritic cells capture and present nonself antigens although the dendritic cell also presents self antigens and is involved in the induction of tolerance. The dendritic cell is referred to as an immature dendritic cell (iDC) before it encounters antigenic stimuli. These cells are found in nonlymphoid tissues and, like macrophages, dendritic cells are highly phagocytic, a function that is facilitated by the presence of motile, long dendrite-like processes that are able to sample antigen. In the absence of foreign or inflammatory stimuli immature dendritic cells may take up and process antigen, but do not interact with T cells because they do not express significant amounts of MHC class II or costimulatory molecules on their surface. Should the dendritic cell receive a “danger” signal (e.g., pathogen associated molecules or pro-inflammatory cytokines) it undergoes an activation process which increases expression of MHC class II, of costimulatory molecules (CD80 and CD86) and of selected chemokine receptors that allow it to migrate to

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the lymph node where antigen presentation occurs. These dendritic cells are referred to as mature dendritic cells (mDC). In contrast to macrophages, dendritic cells are able to present antigen to both naïve and activated T cells. Dendritic cells activate the adaptive immune response by both presenting antigen to T cells, but also by secreting a number of cytokines and chemokines. Often cytokine production by dendritic cells is far greater than that of macrophages, the result of which is greater recruitment and activation of T cells. Although it now appears that there may be as many as five classes of DCs we limit this discussion to the best characterised classes of dendritic cells that are most likely involved in the response to protozoan infections. Dendritic cell subtypes of mice and humans are reviewed in reference 66. The most recent class of circulating precursor cell to be identified is the plasmacytoid DC (pDCs) which is CD64-/CD16-. These cells comprise a very low percent of the total circulating population but despite those low numbers they are essential in the host’s response to viruses. Plasmacytoid cells can also be obtained from the spleen. They express TLR7, TLR9 and TLR11 (in mice) and are not responsive to TLR2 and TLR4 agonists such as LPS and peptidoglycan. Plasmacytoid DCs produce high amounts of IFN-α, but no or little IL-6 or TNF-α. Compared to other DC subsets, plasmacytoid DCs have limited phagocytic capacity, do not participate in ADCC and have very little interaction with either B or T lymphocytes. In general they are not believed to play a role in the host defence against protozoan pathogens, although it has been demonstrated that malaria blood stage schizonts can lead to increased expression of CD86 and stimulate production of IFN-α by pDCs in vitro.67 For a current review on the function of pDCs see reference 68. Myeloid DCs can also be detected in the circulation and are characterised by the expression of markers such as CD13, CD11c and CD33. Upon stimulation with pathogen associated microbial ligands via TLR1, TLR2, TLR5 and TLR8 these cells do not produce IFN-α or -β but rather the pro-inflammatory cytokines, IL-6 and TNF-α. Myeloid DCs produce high levels of IL-12 in response to protozoan pathogens in both toll like receptor-dependent69,70 and -independent fashion.71 In contrast to pDCs, these cells produce predominantly homeostatic chemokines72 and have a higher capacity to migrate towards chemokines such as MCP-1, RANTES and IP-10 produced during the course of protozoan infection.73 It is believed that under the correct conditions circulating myeloid DCs can migrate to the tissues where they differentiate into tissue DCs. With respect to parasitic infection the macrophages and dendritic cells of the skin and the gut are especially important. Because of the interplay between macrophages and dendritic cells at these sites in response to infection we summarise their properties on the basis of their location.

Macrophages/Dendritic Cells of the Skin The resident cells of the epidermis are crucial in the elimination of pathogens that are transmitted by insect bites or other breaches of the skin. There are two populations of dendritic cells in skin, the Langerhan’s cells which are characterised by the expression of CD207 (Langerin) and dermal dendritic cells which are characterised by expression of CD208 (DC-SIGN). Dermal DCs have been implicated in binding Leishmania amastigotes and Schistosoma mansoni egg antigens.74 Dermal dendritic cells are located at the capillaries and the reticular dermis whereas Langerhans cells are located at the basal and supra-basal layers of the epidermis.75 The long processes of these cells are uniquely adapted to capturing antigen, which is mediated by expression of C-type lectins and Fc receptors. These DCs present antigen in the context of both MHC class I and class II. Tissue macrophages and dendritic cells of the skin appear to have different abilities to phagocytose particulate matter and pathogens. Langerhans cells phagocytose 0.5 -1 μm beads whereas macrophages ingest larger particles (>3.5 μm). There are differences in the types of pathogens preferentially phagocytosed by different subsets of skin DCs and macrophages.76,77 The role of dendritic cells in the skin has been reviewed in reference 78.

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There is a complex interplay between tissue macrophages and dendritic cells during the course of parasitic infection. In cutaneous Leishmania infection the skin is inoculated with promastigotes that are ingested, and generally destroyed by resident macrophages via the production of reactive oxygen and nitrogen species. In vitro studies suggest that macrophages do not necessarily become activated or increase surface expression of various surface markers. Sequential activation of skin dendritic cells via ingestion of amastigotes is required to present antigen and to clear infection. CD4+ T lymphocytes must be involved in order to produce the Th1 promoting cytokines IL-12, IFN-γ and TNF-α.79 The pathology of Leishmania infection of the skin is reviewed in reference 80.

Macrophages and Dendritic Cells of the Gut/intestine The gut consists of many different immunological niches including Peyer’s patches, mucosal lymphoid follicles and the lamina propria. Antigen presenting cells can be found in all these areas. Macrophages of the mucosa and intestine are uniquely adapted to cope with the high antigenic and bacterial load of the gut. Although these cells are derived from CD14 expressing circulating monocytes they do not express CD14 or other bacterial recognition receptors and as such are essentially nonresponsive to stimulation with bacterial products. The inability of these macrophages to respond to bacterial stimuli by producing cytokines such as IL-1, IL-6, IL-10, IL-12, RANTES, TGF-β and TNF-α has lead to the suggestion that they as they develop from circulating monocytes that develop “inflammatory anergy”.13 It is believed that recruited CD14+ expressing monocytes develop this phenotype upon exposure to local cytokines such as TGF-β and that this is an essential adaptation to deal with the high load of predominantly commensal bacteria of the intestine. It should be noted that these macrophages are not defective in their ability to phagocytose or destroy phagocytosed bacteria. They do not express a number of other surface markers including CR3 and LFA-1 and receptors for IgA, IgG, but do express high levels of MHC class II and HLA-DR indicating that they have antigen presenting capacity. Macrophages may be found throughout the intestinal tract but appear to be most common in the lamina propria. The role of macrophages in the gut and intestine has been reviewed in references 81 and 82. Dendritic cells are also important antigen presenting cells in the gut and intestine. In addition to their antigen presenting functions these cells are particularly important in inducing the differentiation of regulatory T cells under steady state conditions. Immature dendritic cells are found in the Peyer’s patches and in the lamina propria. It is believed that pathogens and parasites are transported to the dendritic cells of the Peyer’s patches via M cells whereas dendritic cells of the lamina propria may sample pathogens using long dendrites that extend into the lumen of the gut between the tight junctions of the epithelial cells.83 Subsequent to infection the dendritic cells develop a mature phenotype (e.g., they express MHC class II, CD40, CD80, etc). It is believed that due to the capacity of these cells to migrate they are involved in dissemination of pathogens to distant sites throughout the body. The immunobiology of cells in the gut is described in reference 84. Many protozoan pathogens enter the host via ingestion. Giardia spp., Cryptosporidium parvum, Toxoplasma gondii and Entamoeba histolytica have all been demonstrated to multiply within the gut. Pathogens adhere to the epithelial layers of the intestine and in many cases are able to cross epithelial barriers at which point they may be detected by macrophages and dendritic cells of the intestine.85 A macrophage and dendritic cell mediated immune response is not mounted unless there is a breach of the integrity of the epithelial barrier or pro-inflammatory cytokines or chemokines are detected. Chemokine and cytokine production from epithelial cells and resident leukocytes is critical for both the mobilisation and activation of macrophages and dendritic cells.86,87 Once mobilised, macrophages, neutrophils and dendritic cells produce IL-12 and initiate a Th1 response. IL-12 production is essential for defence against protozoan pathogens in the gut because it stimulates the production of IFN-γ and activates macrophages.88 In fact, IFN-γ induced activation of macrophages is so critical for host defence that the ability to

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decrease or eliminate its production is an essential virulence determinent for protozoan pathogens.89,90 The mucosal immune response to parasites has been reviewed elsewhere.91,92

Recognition and Destruction by Macrophages and Subversion by Pathogens Despite the fact that the macrophages are exquisitely adapted to destroying intracellular bacterial and protozoan parasites, these infections occur at alarming rates, especially within the developing world. The World Health Organisation (WHO) lists malaria (Plasmodium spp), Chagas disease (Trypanosoma cruzi), leishmaniasis (Leishmania spp), and toxoplasmosis (Toxoplasma gondii) as major health risks in developing nations. It is believed that over 12 million people worldwide are affected by leishmaniasis,93 and there are 300 million cases of malaria.94 In order to understand what makes these pathogens so successful it is important to understand the mechanisms of recognition, uptake and destruction of pathogens by macrophages. The macrophage has a potent ability to recognise, phagocytose and destroy pathogens. The initial binding and recognition process that triggers phagocytosis varies with respect to the micro-organism. There are differences in the outcome of opsonin-dependent phagocytosis and -independent phagocytosis. The importance of opsonic phagocytosis is highlighted by the number of opsonins produced by the host, both constitutively and in response to infection. Opsonins such as C-reactive protein are important in enhancing phagocytosis of a number of intracellular parasites (e.g., Leishmania promastigotes). However, phagocytosis mediated by complement activation does not result in a strong oxidative burst from the macrophage and thus some pathogens exploit this mechanism of uptake. For example, Leishmania promotes complement-mediated uptake by expressing elongated lipidoglycans on its surface. These lipidoglycans do not prevent complement activation, but the parasite is not lysed because the activated complement is distant from the cell membrane. Furthermore opsonisation allows promastigotes to enter the macrophage through the complement pathway thus evading normal phagosome-lysosome fusion.95 Phagocytosis mediated through Fc receptors generally results in the maturation of the phagosome into an acidic, hydrolytically active compartment and destruction of the pathogen. Intracellular pathogens have a number of conserved strategies for subverting normal Fc receptor mediated uptake. The pathogens Toxoplasma, Plasmodium and Eimeria have a motile invasive stage, called zoites, in which they can use an actinomyosin-based motile system that mediates host cell invasion thus subverting both complement and Fc receptor mediated phagocytosis. Toxoplasma uses this system to create vacuoles that exist independently of the normal phagolysosomal pathway and is consequently not exposed to the destructive environment of the phagolysosome. Mechanisms of protozoan invasion of host cells are reviewed in reference 96. Upon Fc mediated phagocytosis the phagocytic vacuole undergoes numerous maturation steps that are accompanied by continuous remodelling of the phagosome membrane protein composition. Phagosomes sequentially fuse with the early endosomes, late endosomes and lysosomes and the maturation of the phagosome can be tracked by evaluating the accumulation of various surface markers. The pH drops slightly (pH 6.2) upon fusion with the early endosomes. This results in uncoupling of receptor/ligand pairs and receptor recycling mediated by the Rab proteins (Rab4 and Rab11). Upon fusion with the late endosomes the membrane of the phagolysosome accumulates acid resistant phospholipids and is characterised by the expression of Lamp1 and Lamp2. The resulting fusion with lysosomes results in a drop in pH (to 4.7 -5.2) that results in the activation of the proteolytic enzymes such as the cathepsins that are stored within. These enzymes are crucial not only for microbial degradation, but also to generate antigens for presentation by MHC molecules. Oxidative species such as O2- are rapidly produced upon phagocyte activation. The enzyme NADPH oxidase is essential for catalysis of various oxidative species including superoxide, hydrogen peroxide, and halogenated oxygen molecules in a process that is tightly coupled to cytoplasmic membrane and

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requires cytoskeletal elements and protein phosphorylation. Nitric oxide species are also involved in antimicrobial killing and are essential for the destruction of a number of intracellular parasites. NO production is catalyzed by nitric oxide synthase from L-arginine and molecular oxygen. Interactions of hydrogen peroxide with myeloperoxidase, reduced iron, or NO lead to formation of additional toxic intermediates such as hypochlorous anion, hydroxyl radicals, nitrogen dioxide and peroxynitrite. The acidification may also be required for the generation of the oxidative burst and subsequent cytokine production. Once the pathogen has been phagocytosed it has three options. It may either exist in the intralysosomal environment and develop mechanisms to deal with the acidic, hydrolytic environment therein or it may exist in the vacuole but prevent normal maturation from occurring thus remaining protected from the microbicidal properties of the macrophage. Some pathogens escape from the vacuole altogether and live in the more permissive environment of the cytosol. The majority of intracellular pathogens actively subvert phagolysome maturation. The pathogen may prevent acidification (e.g., Histoplasma capsulatum, Entamoeba histolytica), remodel the phagolysosome to a more permissive environment (e.g., Salmonella), or arrest the development of the phagosome at an earlier or less destructive stage (e.g., L. donovani, M. tuberculosis).97,98 Pathogens that have developed mechanisms for dealing with life in the lysosome include Leishmania and Coxiella. Leishmania resists hydrolysis by having a cell surface of resistant lipidoglycans and can resist antigen presentation by regulating the expression and accessibility of antigenic peptides.99,100 Escape from the phagocytic vacuole is a common theme amongst intracellular parasites including T. cruzi, Listeria, Shigella and Rickettsia. Pathogens have a number of mechanisms by which they escape the phagosomal membrane such as the production of pores (Listeria spp.), lysis (Shigella flexneri), and as of yet unidentified mechanisms (Rickettsia). Once the pathogens have escaped they are able to replicate in the more permissive environment of the cytosol. The macrophage has elaborate mechanisms to deprive the pathogen of essential components for survival such as iron and amino acids. In an unactivated state macrophages express the transferrin receptor by which they bind and internalise extracellular iron. Once they become activated by IFN-γ they down-regulate the transferrin receptor thus decreasing stores of intracellular iron. IFN-γ induced activation also activates the enzyme indoleamine 2, 3-dioxygease (IDO), which catalyses the degradation of L-tryptophan and thus limits the availability of this amino acid to intracellular organisms. Survival in the phagosome is the intracellular parasite’s most pressing issue, but once the immediate threat of degradation is dealt with the parasite must acquire scarce nutrients and avoid detection by the immune system. Activated macrophages prevent survival by sequestering free nutrients in the cytosol; however, Leishmania expresses nucleotidases on their surface in order to extract the purines from the host that they require for growth. The theme of extracting nutrients from a hostile environment is a common one used by C. burnetti, which has an active system for recruiting nutrients at an acidic but not neutral pH. The secretion and presence of cytokines have a number of indirect effects on macrophage killing. Besides being essential for macrophage activation, IFN-γ has a number of indirect effects that enhance anti-microbial activity. Exposure to IFN-γ induces the production of a number of chemokines such as IP-10/CXCL10 and CXCL11 which result in the recruitment of additional leukocytes with antimicrobial activity. Chemokines also contribute to the enhancement of antibacterial activity. RANTES, MIP-1α, MIP-1β increase the uptake and cause the intracellular destruction by macrophages of trypomastigotes and rickettsia by inducing NO production.101 Intracellular parasites also subvert host processes by inhibiting or promoting macrophage signalling. This results in disruption of normal host processes such as apoptosis (e.g., T. gondii)102 and pro-inflammatory cytokine production (e.g., T. gondii).103 Abrogation of pro-inflammatory cytokine production alone is not enough to ensure protection and some parasites alter cell

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signalling in such a way that the balance between Th1 and Th2 production is skewed, thus inhibiting the host’s normal anti-parasitic response.104 This may be done by altering signalling pathways through disruption or degradation of key signalling components105 or more directly by direct degradation of pivotal cytokines such as IL-12.106 For excellent reviews on the subversion of host cell defences by parasites see references 107-109.

Conclusion The macrophage is of crucial importance in host defence towards infectious disease. There is much work to be done on understanding of the subtleties of the macrophages response to infectious disease. First we must characterise macrophage heterogeneity and the intricacies of functional differences between subtypes and activation states and secondly we must investigate subtle differences in macrophage function and susceptibility between individuals. It is becoming apparent that differences at the genetic level, including subtle polymorphisms in genes encoding macrophage receptors, effector molecules and signalling pathways, may contribute to the host’s predisposition to infectious disease. This knowledge will be essential in order to translate in vitro observations to understanding of pathogenesis in vivo. Recent advances in the study of infection by protozoa have provided insight into how these pathogens subvert host defences and have illustrated that the macrophage is the essential target for eradication of these pathogens. Increased understanding of these mechanisms is required to develop novel macrophage-based therapies.

Acknowledgements The authors would like to acknowledge the contributors to their research. S. Gordon is supported by grants from the Wellcome Trust and the Medical Research Council. D.M.E. Bowdish is funded by a fellowship from the Canadian Institute for Health Research.

References 1. Janeway Jr CA, Medzhitov R. Introduction: The role of innate immunity in the adaptive immune response. Semin Immunol 1998; 10(5):349-350. 2. Taylor PR, Martinez-Pomares L, Stacey M et al. Macrophage receptors and immune recognition. Annu Rev Immunol 2005; 23:901-944. 3. Janeway Jr CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002; 20:197-216. 4. Medzhitov R, Janeway Jr C. Innate immune recognition: Mechanisms and pathways. Immunol Rev 2000; 173:89-97. 5. Medzhitov R, Janeway Jr CA. Innate immunity: Impact on the adaptive immune response. Curr Opin Immunol 1997; 9(1):4-9. 6. Santiago-Schwarz F. Positive and negative regulation of the myeloid dendritic cell lineage. J Leukoc Biol 1999; 66(2):209-216. 7. Clanchy FIL, Holloway AC, Lari R et al. Detection and properties of the human proliferative monocyte subpopulation. J Leukoc Biol 2006; 79(4):757-766. 8. Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 2003; 19(1):71-82. 9. Mizuno K, Toma T, Tsukiji H et al. Selective expansion of CD16highCCR2- subpopulation of circulating monocytes with preferential production of haem oxygenase (HO)-1 in response to acute inflammation. Clin Exp Immunol 2005; 142(3):461-470. 10. Cairns AP, Crockard AD, Bell AL. The CD14+ CD16+ monocyte subset in rheumatoid arthritis and systemic lupus erythematosus. Rheumatol Int 2002; 21(5):189-192. 11. Blaschke S, Koziolek M, Schwarz A et al. Proinflammatory role of fractalkine (CX3CL1) in rheumatoid arthritis. J Rheumatol 2003; 30(9):1918-1927. 12. Ancuta P, Rao R, Moses A et al. Fractalkine preferentially mediates arrest and migration of CD16+ monocytes. J Exp Med 2003; 197(12):1701-1707. 13. Smythies LE, Sellers M, Clements RH et al. Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity. J Clin Invest 2005; 115(1):66-75. 14. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol 2005; 5(12):953-964.

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15. Calder CJ, Nicholson LB, Dick AD. A selective role for the TNF p55 receptor in autocrine signaling following IFN-gamma stimulation in experimental autoimmune uveoretinitis. J Immunol 2005; 175(10):6286-6293. 16. Plata F, Garcia-Pons F, Wietzerbin J. Immune resistance to Trypanosoma cruzi: Synergy of specific antibodies and recombinant interferon gamma in vivo. Ann Inst Pasteur Immunol 1987; 138(3):397-415. 17. Baszler TV, Long MT, McElwain TF et al. Interferon-gamma and interleukin-12 mediate protection to acute Neospora caninum infection in BALB/c mice. Int J Parasitol 1999; 29(10):1635-1646. 18. Schofield L, Villaquiran J, Ferreira A et al. Gamma interferon, CD8+ T cells and antibodies required for immunity to malaria sporozoites. Nature 1987; 330(6149):664-666. 19. Skerrett SJ, Martin TR. Recombinant murine interferon-gamma reversibly activates rat alveolar macrophages to kill Legionella pneumophila. J Infect Dis 1992; 166(6):1354-1361. 20. Achbarou A, Ombrouck C, Gneragbe T et al. Experimental model for human intestinal microsporidiosis in interferon gamma receptor knockout mice infected by Encephalitozoon intestinalis. Parasite Immunol 1996; 18(8):387-392. 21. Swihart K, Fruth U, Messmer N et al. Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response. J Exp Med 1995; 181(3):961-971. 22. You X, Mead JR. Characterization of experimental Cryptosporidium parvum infection in IFN-gamma knockout mice. Parasitology 1998; 117(Pt 6):525-531. 23. Hertz CJ, Filutowicz H, Mansfield JM. Resistance to the African trypanosomes is IFN-gamma dependent. J Immunol 1998; 161(12):6775-6783. 24. Skurkovich B, Skurkovich S. Inhibition of IFN-gamma as a method of treatment of various autoimmune diseases, including skin diseases. Ernst Schering Res Found Workshop 2006; (56):1-27. 25. Skurkovich B, Skurkovich S. Anti-interferon-gamma antibodies in the treatment of autoimmune diseases. Curr Opin Mol Ther 2003; 5(1):52-57. 26. Daugherty A, Webb NR, Rateri DL et al. Thematic review series: The immune system and atherogenesis. Cytokine regulation of macrophage functions in atherogenesis. J Lipid Res 2005; 46(9):1812-1822. 27. Jozefowski S, Arredouani M, Sulahian T et al. Disparate regulation and function of the class a scavenger receptors SR-AI/II and MARCO. J Immunol 2005; 175(12):8032-8041. 28. Mukhopadhyay S, Chen Y, Sankala M et al. MARCO, an innate activation marker of macrophages, is a class A scavenger receptor for Neisseria meningitidis. Eur J Immunol 2006; 36(4):940-949. 29. Allen JE, Lawrence RA, Maizels RM. APC from mice harbouring the filarial nematode, Brugia malayi, prevent cellular proliferation but not cytokine production. Int Immunol 1996; 8(1):143-151. 30. Stein M, Keshav S, Harris N et al. Interleukin 4 potently enhances murine macrophage mannose receptor activity: A marker of alternative immunologic macrophage activation. J Exp Med 1992; 176(1):287-292. 31. Modolell M, Corraliza IM, Link F et al. Reciprocal regulation of the nitric oxide synthase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2 cytokines. Eur J Immunol 1995; 25(4):1101-1104. 32. Pearce EJ, MacDonald AS. The immunobiology of schistosomiasis. Nat Rev Immunol 2002; 2(7):499-511. 33. Scharton-Kersten T, Afonso LC, Wysocka M et al. IL-12 is required for natural killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis. J Immunol 1995; 154(10):5320-5330. 34. Namangala B, Noel W, De Baetselier P et al. Relative contribution of interferon-gamma and interleukin-10 to resistance to murine African trypanosomosis. J Infect Dis 2001; 183(12):1794-1800. 35. Iniesta V, Gomez-Nieto LC, Corraliza I. The inhibition of arginase by N(omega)-hydroxy-l-arginine controls the growth of Leishmania inside macrophages. J Exp Med 2001; 193(6):777-784. 36. Gratchev A, Guillot P, Hakiy N et al. Alternatively activated macrophages differentially express fibronectin and its splice variants and the extracellular matrix protein betaIG-H3. Scand J Immunol 2001; 53(4):386-392. 37. Schebesch C, Kodelja V, Muller C et al. Alternatively activated macrophages actively inhibit proliferation of peripheral blood lymphocytes and CD4+ T cells in vitro. Immunology 1997; 92(4):478-486. 38. Gordon S. Alternative activation of macrophages. Nat Rev Immunol 2003; 3(1):23-35. 39. Komohara Y, Hirahara J, Horikawa T et al. AM-3K, an Anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype. J Histochem Cytochem 2006.

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40. Ebert RH, Florey HW. The extravascular development of the monocyte observed in vivo. Brit J Exp Pathol 1939; 20:342-356. 41. Kennedy DW, Abkowitz JL. Kinetics of central nervous system microglial and macrophage engraftment: Analysis using a transgenic bone marrow transplantation model. Blood 1997; 90(3):986-993. 42. Kennedy DW, Abkowitz JL. Mature monocytic cells enter tissues and engraft. Proc Natl Acad Sci USA 1998; 95(25):14944-14949. 43. Merad M, Manz MG, Karsunky H et al. Langerhans cells renew in the skin throughout life under steady-state conditions. Nat Immunol 2002; 3(12):1135-1141. 44. Holt PG, Oliver J, Bilyk N et al. Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. J Exp Med 1993; 177(2):397-407. 45. Thepen T, McMenamin C, Oliver J et al. Regulation of immune response to inhaled antigen by alveolar macrophages: Differential effects of in vivo alveolar macrophage elimination on the induction of tolerance vs. immunity. Eur J Immunol 1991; 21(11):2845-2850. 46. Town T, Nikolic V, Tan J. The microglial “activation” continuum: From innate to adaptive responses. J Neuroinflammation 2005; 2:24. 47. Li Z, Kong K, Qi W. Osteoclast and its roles in calcium metabolism and bone development and remodeling. Biochem Biophys Res Commun 2006; 343(2):345-350. 48. Kupffer CV. Ueber Sternzellen der Leber. Arch mikr Anat 1876; 12:353-358. 49. Lepay DA, Nathan CF, Steinman RM et al. Murine Kupffer cells. Mononuclear phagocytes deficient in the generation of reactive oxygen intermediates. J Exp Med 1985; 161(5):1079-1096. 50. Shi M, Wei G, Pan W et al. Trypanosoma congolense infections: Antibody-mediated phagocytosis by Kupffer cells. J Leukoc Biol 2004; 76(2):399-405. 51. Albright JW, Long GW, Albright JF. The liver as a major site of immunological elimination of murine trypanosome infection, demonstrated with the liver perfusion model. Infect Immun 1990; 58(6):1965-1970. 52. Artan R, Yilmaz A, Akcam M et al. Liver biopsy in the diagnosis of visceral leishmaniasis. J Gastroenterol Hepatol 2006; 21(1 Pt 2):299-302. 53. Pradel G, Garapaty S, Frevert U. Proteoglycans mediate malaria sporozoite targeting to the liver. Mol Microbiol 2002; 45(3):637-651. 54. Meis JF, Verhave JP, Jap PH et al. An ultrastructural study on the role of Kupffer cells in the process of infection by Plasmodium berghei sporozoites in rats. Parasitology 1983; 86(Pt 2):231-242. 55. Pradel G, Frevert U. Malaria sporozoites actively enter and pass through rat Kupffer cells prior to hepatocyte invasion. Hepatology 2001; 33(5):1154-1165. 56. Meis JF, Verhave JP, Brouwer A et al. Electron microscopic studies on the interaction of rat Kupffer cells and Plasmodium berghei sporozoites. Z Parasitenkd 1985; 71(4):473-483. 57. Frevert U. Sneaking in through the back entrance: The biology of malaria liver stages. Trends Parasitol 2004; 20(9):417-424. 58. Parker GA, Picut CA. Liver immunobiology. Toxicol Pathol 2005; 33(1):52-62. 59. Mebius RE, Kraal G. Structure and function of the spleen. Nat Rev Immunol 2005; 5(8):606-616. 60. Martinez-Pomares L, Hanitsch LG, Stillion R et al. Expression of mannose receptor and ligands for its cysteine-rich domain in venous sinuses of human spleen. Lab Invest 2005; 85(10):1238-1249. 61. Engwerda CR, Ato M, Kaye PM. Macrophages, pathology and parasite persistence in experimental visceral leishmaniasis. Trends Parasitol 2004; 20(11):524-530. 62. Engwerda CR, Beattie L, Amante FH. The importance of the spleen in malaria. Trends Parasitol 2005; 21(2):75-80. 63. Kyaw MH, Holmes EM, Toolis F et al. Evaluation of severe infection and survival after splenectomy. Am J Med 2006; 119(3):276 e271-277. 64. Bach O, Baier M, Pullwitt A et al. Falciparum malaria after splenectomy: A prospective controlled study of 33 previously splenectomized Malawian adults. Trans R Soc Trop Med Hyg 2005; 99(11):861-867. 65. Chotivanich K, Udomsangpetch R, McGready R et al. Central role of the spleen in malaria parasite clearance. J Infect Dis 2002; 185(10):1538-1541. 66. Shortman K, Liu YJ. Mouse and human dendritic cell subtypes. Nat Rev Immunol 2002; 2(3):151-161. 67. Pichyangkul S, Yongvanitchit K, Kum-arb U et al. Malaria blood stage parasites activate human plasmacytoid dendritic cells and murine dendritic cells through a Toll-like receptor 9-dependent pathway. J Immunol 2004; 172(8):4926-4933. 68. Liu YJ. IPC: Professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol 2005; 23:275-306.

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69. Yarovinsky F, Sher A. Toll-like receptor recognition of Toxoplasma gondii. Int J Parasitol 2006; 36(3):255-259. 70. Scanga CA, Aliberti J, Jankovic D et al. Cutting edge: MyD88 is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells. J Immunol 2002; 168(12):5997-6001. 71. Kim L, Butcher BA, Lee CW et al. Toxoplasma gondii genotype determines MyD88-dependent signaling in infected macrophages. J Immunol 2006, (In press). 72. Penna G, Vulcano M, Roncari A et al. Cutting edge: Differential chemokine production by myeloid and plasmacytoid dendritic cells. J Immunol 2002; 169(12):6673-6676. 73. Penna G, Vulcano M, Sozzani S et al. Differential migration behavior and chemokine production by myeloid and plasmacytoid dendritic cells. Hum Immunol 2002; 63(12):1164-1171. 74. van Kooyk Y, Geijtenbeek TB. DC-SIGN: Escape mechanism for pathogens. Nat Rev Immunol 2003; 3(9):697-709. 75. Langerhans P. Ueber die Nerven der menschlichen Haut. Virchows Archiv 1868; 44(2-3):325-337. 76. Reis e Sousa C, Stahl PD, Austyn JM. Phagocytosis of antigens by Langerhans cells in vitro. J Exp Med 1993; 178(2):509-519. 77. Ritter U, Meissner A, Scheidig C et al. CD8 alpha- and Langerin-negative dendritic cells, but not Langerhans cells, act as principal antigen-presenting cells in leishmaniasis. Eur J Immunol 2004; 34(6):1542-1550. 78. Valladeau J, Saeland S. Cutaneous dendritic cells. Semin Immunol 2005; 17(4):273-283. 79. von Stebut E, Belkaid Y, Jakob T et al. Uptake of Leishmania major amastigotes results in activation and interleukin 12 release from murine skin-derived dendritic cells: implications for the initiation of anti-Leishmania immunity. J Exp Med 1998; 188(8):1547-1552. 80. Murray HW, Berman JD, Davies CR et al. Advances in leishmaniasis. Lancet 2005; 366(9496):1561-1577. 81. Smith PD, Ochsenbauer-Jambor C, Smythies LE. Intestinal macrophages: Unique effector cells of the innate immune system. Immunol Rev 2005; 206:149-159. 82. Makala LH, Nishikawa Y, Suzuki N et al. Immunology. Antigen-presenting cells in the gut. J Biomed Sci 2004; 11(2):130-141. 83. Rescigno M, Urbano M, Valzasina B et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2001; 2(4):361-367. 84. Kelsall BL, Leon F. Involvement of intestinal dendritic cells in oral tolerance, immunity to pathogens, and inflammatory bowel disease. Immunol Rev 2005; 206:132-148. 85. Barragan A, Sibley LD. Transepithelial migration of Toxoplasma gondii is linked to parasite motility and virulence. J Exp Med 2002; 195(12):1625-1633. 86. Lacroix-Lamande S, Mancassola R, Naciri M et al. Role of gamma interferon in chemokine expression in the ileum of mice and in a murine intestinal epithelial cell line after Cryptosporidium parvum infection. Infect Immun 2002; 70(4):2090-2099. 87. Mennechet FJ, Kasper LH, Rachinel N et al. Lamina propria CD4+ T lymphocytes synergize with murine intestinal epithelial cells to enhance proinflammatory response against an intracellular pathogen. J Immunol 2002; 168(6):2988-2996. 88. Gazzinelli RT, Wysocka M, Hayashi S et al. Parasite-induced IL-12 stimulates early IFN-gamma synthesis and resistance during acute infection with Toxoplasma gondii. J Immunol 1994; 153(6):2533-2543. 89. Wang W, Chadee K. Entamoeba histolytica suppresses gamma interferon-induced macrophage class II major histocompatibility complex Ia molecule and I-A beta mRNA expression by a prostaglandin E2-dependent mechanism. Infect Immun 1995; 63(3):1089-1094. 90. Salata RA, Pearson RD, Ravdin JI. Interaction of human leukocytes and Entamoeba histolytica. Killing of virulent amebae by the activated macrophage. J Clin Invest 1985; 76(2):491-499. 91. Buzoni-Gatel D, Schulthess J, Menard LC et al. Mucosal defences against orally acquired protozoan parasites, emphasis on Toxoplasma gondii infections. Cell Microbiol 2006; 8(4):535-544. 92. Kasper L, Courret N, Darche S et al. Toxoplasma gondii and mucosal immunity. Int J Parasitol 2004; 34(3):401-409. 93. Organization WH. The leishmaniases and Leishmania/HIV coinfections. Fact Sheets 2006, ([Website] http://www.who.int/mediacentre/factsheets/fs116/en/index.html). 94. Organization WH. Roll Back Malaria. What is malaria? 2006, (http://rollbackmalaria.org/ cmc_upload/0/000/015/372/RBMInfosheet_1.htm). 95. Joshi PB, Kelly BL, Kamhawi S et al. Targeted gene deletion in Leishmania major identifies leishmanolysin (GP63) as a virulence factor. Mol Biochem Parasitol 2002; 120(1):33-40. 96. Sibley LD. Intracellular parasite invasion strategies. Science 2004; 304(5668):248-253.

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97. Scianimanico S, Desrosiers M, Dermine JF. Impaired recruitment of the small GTPase rab7 correlates with the inhibition of phagosome maturation by Leishmania donovani promastigotes. Cell Microbiol 1999; 1(1):19-32. 98. Xu S, Cooper A, Sturgill-Koszycki S et al. Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages. J Immunol 1994; 153(6):2568-2578. 99. Lodge R, Descoteaux A. Modulation of phagolysosome biogenesis by the lipophosphoglycan of Leishmania. Clin Immunol 2005; 114(3):256-265. 100. Prickett S, Gray PM, Colpitts SL et al. In vivo recognition of ovalbumin expressed by transgenic Leishmania is determined by its subcellular localization. J Immunol 2006; 176(8):4826-4833. 101. Aliberti JC, Machado FS, Souto JT et al. beta-Chemokines enhance parasite uptake and promote nitric oxide-dependent microbiostatic activity in murine inflammatory macrophages infected with Trypanosoma cruzi. Infect Immun 1999; 67(9):4819-4826. 102. Sinai AP, Payne TM, Carmen JC et al. Mechanisms underlying the manipulation of host apoptotic pathways by Toxoplasma gondii. Int J Parasitol 2004; 34(3):381-391. 103. Butcher BA, Kim L, Johnson PF et al. Toxoplasma gondii tachyzoites inhibit proinflammatory cytokine induction in infected macrophages by preventing nuclear translocation of the transcription factor NF-kappa B. J Immunol 2001; 167(4):2193-2201. 104. Kim L, Del Rio L, Butcher BA et al. p38 MAPK autophosphorylation drives macrophage IL-12 production during intracellular infection. J Immunol 2005; 174(7):4178-4184. 105. Cameron P, McGachy A, Anderson M et al. Inhibition of lipopolysaccharide-induced macrophage IL-12 production by Leishmania mexicana amastigotes: The role of cysteine peptidases and the NF-kappaB signaling pathway. J Immunol 2004; 173(5):3297-3304. 106. Yun PL, Decarlo AA, Collyer C et al. Hydrolysis of interleukin-12 by Porphyromonas gingivalis major cysteine proteinases may affect local gamma interferon accumulation and the Th1 or Th2 T-cell phenotype in periodontitis. Infect Immun 2001; 69(9):5650-5660. 107. Gregory DJ, Olivier M. Subversion of host cell signalling by the protozoan parasite Leishmania. Parasitology 2005; 130(Suppl):S27-35. 108. Denkers EY, Butcher BA. Sabotage and exploitation in macrophages parasitized by intracellular protozoans. Trends Parasitol 2005; 21(1):35-41. 109. Denkers EY. From cells to signaling cascades: Manipulation of innate immunity by Toxoplasma gondii. FEMS Immunol Med Microbiol 2003; 39(3):193-203.

CHAPTER 1

Invasion and Intracellular Survival by Toxoplasma L. David Sibley,* Audra Charron, Sebastian Håkansson and Dana Mordue

Summary

T

oxoplasma gondii infects a wide range of warm-blooded vertebrates including humans and is one of the world’s most successful parasites. As a member of the phylum Apicomplexa, T. gondii is a model for understanding infection by a variety of related parasites such as Plasmodium and Cryptosporidium. Apicomplexans use a unique form of actin-based motility to directly penetrate their host cell, without the need for host uptake mechanisms. Invasion occurs more rapidly than phagocytic uptake and avoids triggering of the respiratory burst in macrophages. Within the host cell, the parasite resides in a modified vacuole that resists fusion with endosomes and lysosomes, while intimately associate with host cell ER and mitochondria. Active secretion of parasite proteins results in modification of the vacuole, rendering it permeable to small molecules. Within this porous vacuole the parasite acquires nutrients from the host cytosol, allowing rapid replication, and eventual consumption of the host cell prior to egress. Understanding the complex biology of intracellular survival by T. gondii has bearing on the mechanisms of host resistance during both acute and chronic infection.

Life Cycle and Basic Biology The phylum Apicomplexa contains some 5,000 members, most of which are parasitic, although only a few of these have been studied.1 Apicomplexans are unified by an apical complex consisting of a unique microtubule organizing center called the conoid, and a system of apical secretory organelles involved in cell invasion.2 A number of apicomplexans are important pathogens in humans (e.g., Plasmodium spp., Cryptosporidium spp., T. gondii) or in animals (e.g., Eimeria spp. Neospora caninum, Sarcocystis spp.). Apicomplexans are early branching eukaryotes and are most closely related to ciliates and dinoflagellates.3 Consequently, their basic cellular mechanisms are often unlike those of their host cells. Toxoplasma gondii is equipped with forward and reverse genetics, excellent animals models, and is amenable to cell biological and biochemical analyses,4 and thus it provides an ideal model organism for studying the unique biology of this phylum. Toxoplasma gondii is a generalist both in its host range and choice of cell type. Virtually all warm-blooded vertebrates are susceptible hosts5 and within its host, all nucleated cells are subject to infection. T. gondii is adept at entering and surviving in a variety of leukocytes and both monocytes and dendritic cells are highly permissive for replication.6 Early studies established that T. gondii enters macrophages without stimulating a respiratory burst.7 The parasite establishes a vacuole that resist fusion with endosomes and lysosomes8,9 and which maintain a *Corresponding Author: L. David Sibley—Department of Molecular Microbiology, Washington University, St. Louis, Missouri 63130, U.S.A. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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neutral pH.10 Avoidance of acidification and endosome fusion relies on active penetration of the cell and when parasites are opsonized by specific antibody and engulfed via Fc receptors they rapidly fuse and acidify.10,11 These observations led to the suggestion that parasite invasion was an active process and more recent molecular studies have validated this hypothesis (discussed further below). T. gondii has a complex life cycle consisting of haploid replicating stages that infect a variety of intermediate hosts and meiosis following infection of felines, which are the only known definite host.12 Transmission of T. gondii occurs by one of two routes: ingestion of oocysts that are shed in the feces from infected cats or ingestion of tissue cysts contained within undercooked meat. Direct infectivity of tissue cysts (containing bradyzoites) to other intermediate host is a unique feature in the life cycle of T. gondii, as all related parasites have a strictly obligatory two-host cycle. Following oral ingestion, sporozoites (contained within oocysts) or bradyzoites (contained within tissue cysts) emerge and penetrate epithelial cells of the small intestine. Herein they may develop, or may pass across the intestinal barrier to reach deeper tissues. When infection commences in the cat gut, haploid replication (schizogony) is followed by differentiation into gametocytes, which ultimately fuse to form a zygote and develop into an oocyst. When infection occurs in all other hosts, the parasite undergoes conversion to fast growing, lytic form called the tachyzoite, which is responsible for dissemination throughout the host. Replication of tachyzoites is ultimately curtailed by the innate and adaptive immune systems (dealt with elsewhere in this volume). Throughout these different developmental phases and within different tissues, T. gondii remains an obligate intracellular parasite.

Actin-Based Motility and Cell Invasion

Apicomplexan parasites are equipped with a unique form of motility termed gliding.13 Motility is strictly substrate dependent and occurs in the absence of cilia, flagella, or crawling behaviors exhibited by amoeboid cells. Instead, forward propulsion relies on a continuous conveyor belt of adhesive proteins attached the substrate.14 Rearward translocation of these adhesin-substrate complexes is governed by an actin-dependent myosin motor beneath the plasma membrane.14 Gliding propels the parasite forward at 1-2 microns per second15 and also powers cell invasion and enables migration across cellular barriers.16,17 Gliding motility is conserved in a variety of apicomplexan parasites including very early branching members such as Cryptosporidium spp.18 and Gregarines.19 Cryptosporidium occupies a vacuole that remains at the apical surface of enterocytes.20 While this compartment is modified by an underlying actin-rich pedestal that forms in the host cell cytosol, the initial mode of entry is based on actin-based motility by the parasite, similar to Toxoplasma.21 Treatment with cytochalasins, which disrupt actin filaments, impairs entry of T. gondii22 and P. falciparum23 into their respective host cells. Cytochalasins block parasite gliding and host phagocytic responses, thus confounding the interpretation of the observed inhibition of invasion. Conclusive demonstration that parasite actin filaments are essential for host cell invasion was provided by mutational analysis and molecular genetic studies in T. gondii.24 In contrast, there are no discernable changes in the host cytoskeleton during invasion25 and the host cytoskeletal system appears to be nonessential to the process of entry. This active mode of cell invasion is distinct from mechanism used by bacterial and viral pathogens and is likely conserved among other members of the Apicomplexa. Actin filaments are extremely dynamic in parasites and rapid turnover of filaments is likely important for regulating motility.26 Formation of new filaments appears to be rate limiting for motility.26 The motor force for motility is provided by a small myosin anchored as part of a complex in the inner membrane complex.27 Disruption of the gene encoding this myosin (TgMyoA) results in parasites that are nonmotile and unable to invade cells.28 The cytoplasmic tails of adhesins mediate connection between this motor complex and proteins that span the plasma membrane. The C terminal cytoplasmic domain of MIC2 contains a cluster of conserved acidic residues and a penultimate W residue, features that are shared by other microneme

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proteins in T. gondii and Plasmodium.29 Interaction between the C terminal domain of MIC2 and aldolase has been shown to bridge the interaction to the cytoskeleton.30 A similar interaction occurs between the Plasmodium adhesin known as TRAP, which is essential for gliding and invasion by sporozoites.31 Mutation of the key W residue disrupts aldolase binding and renders parasite nonmotile and noninvasive.30,32,33 The process of gliding motility requires the coordinated activities of protein secretion and translocation via the actin-based cytoskeleton. Adhesion to the cell substrate and host cell receptors is largely mediated by microneme proteins,34 which are discharged apically from small secretory vesicles. MIC secretion is a calcium regulated process that occurs constitutively and which is upregulated on contact with host cells.35 Apical discharge assures polarization and consistent with this gliding only occurs in a directional manner with the parasite moving forward along the substrate.15 Apically discharged adhesins form contacts with the substrate or host cell surface. These adhesin complexes are translocated rearward by the underlying actin-myosin cytoskeleton, thus propelling the parasite forward. The final step in the conveyor belt is the release of the adhesin at the posterior end of the cell, which occurs by intramembranous proteolysis mediated by a rhomboid-type protease.36 While this model seems inherently inefficient, it provides for several crucial features: (1) adhesins are sheltered from immune recognition until needed for motility (2) apical discharge of adhesins assures directional attachment to the host cell, (3) and rearward translocation of adhesins coupled to cell surface receptors provides for a directional process that drives cell penetration.

Host Cell Recognition and Entry Despite the fact that T. gondii is capable of invading nearly all types of nucleated cells form its vertebrate hosts, very little is known about specific host cell receptors that are utilized. Time-lapse video microscopy studies indicate that lateral binding and reorientation are not obligatory steps during entry of T. gondii.15,25 Rather invasion occurs when motile parasites contact the host cell with their apical end and this often occurs as a direct consequence of active gliding motility. The process is remarkably rapid and following initial contact entry is complete within 20-30 sec.15 Invasion occurs considerably faster than phagocytosis and is therefore capable of outmaneuvering a phagocytic cell attempting to engulf the parasite. During host cell invasion, T. gondii attaches to the host cell with the extreme apical end of the parasite (Figs. 1, 2). Invasion is accompanied by sequential discharged three sets of secretory organelles: micronemes, rhoptries, and dense granules.37 Micronemes are discharged first, on cell contact and they contain a family of proteins with adhesive domains including EGF, thrombospondin type I repeats, lectin-like, and integrin A/I domains.34 Most MIC adhesive proteins are only transiently associated with the cell surface before being translocated reward and shed. However, the microneme protein AMA-1 appears to be constitutively present on the parasite apical surface. AMA-1 plays an important role in mediating close apical attachment as invasion is severely impaired at this stage when AMA-1 expression is suppressed.38 A variety of MIC proteins have been shown to participate in cell attachment, although the host cell receptors recognized by these adhesins are less well defined. Interactions with glycosaminoglycans have been implicated in cell attachment.39,40 These interactions are generally low affinity, but multivalent, and thus they facilitate repeated rounds of attachment and release that would be expected to support gliding motility. During parasite invasion, the plasma membrane is stretched to form the parasitophorous vacuole (PV). The integrity of the membrane remains largely intact, and measurements of capacitance across the host cell membrane reveals that the majority of the membrane constituents come from invagination.41 This model is also supported by studies monitoring the redistribution of fluorescently labeled lipids incorporated into the host cell plasma.11,42 Most host cell surface proteins are excluded during entry, as they are apparently unable to pass through the tight junction that forms between the parasite and host cell plasma membranes.11,43 Exclusion of host cell surface proteins likely prevents activation of the endocytic fusion machinery as

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Figure 1. Invasion and intracellular niche of Toxoplasma gondii. A) Apical attachment of T. gondii establishes a tight junction between the host cell and parasite membranes. Micronemes (small oval secretory organelles) are clustered at the anterior end of the parasite where they discharge their contents. MIC proteins released at this apical site are involved in host cell adhesion. Reproduced with permission from reference 36, ©2005 Elsevier. Scale bar = 200 nm. B) Secretion of ROP protein contents into the host cell under cytochalasin D block. CytD treatment prevents cell invasion but not rhoptry secretion, hence large accumulations of ROP proteins form in the host cell (stained green here for ROP2). These small evacuoles (named so because they lack a parasite) traffic in the host cell in a similar manner to the parasitophorous vacuole. Evacuoles associate with host cell mitochondria (stained red here with mito-tracker) and ER, and avoid fusion with lysosomes and endosomes. Reproduced with permission from reference 56, ©2003 Blackwell Publishing. Scale bar = 5 microns. C) Transmission EM of intracellular T. gondii after one round of cell division. Two adjacent daughter cells show the elaborate cellular architecture of the parasite enveloped within the parasitophorous vacuole. Scale bar = 1 micron. Image courtesy of Wandy Beatty.

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Figure 2. Model depicting the intracellular fate of intracellular T. gondii, which occupies a nonfusigenic parasitophorous vacuole (left side). Invasion is accompanied by three successive waves of protein secretion from: (1) micronemes (adhesion), (2) rhoptries (vacuole formation) and (3) dense granules (modification of the intracellular compartment). The parasitophorous vacuole resists fusion with host cell endosomes and lysosomes and exocytic traffic from the Golgi has not been detected. Instead, the vacuole is drawn to host cell mitochondria and ER and migrates along microtubules to occupy a position near the host cell nucleus. In the presence of cytochalasin D, parasite invasion is blocked; however, apical attachment and secretion of rhoptry proteins occurs normally. Under these conditions, evacuoles form from the accumulations of ROP proteins injected into the host cell cytosol (right side). Evacuoles show very similar fate to the mature parasitophorous vacuole. Soluble proteins may be secreted by a similar process and act as effectors in modulate host cell responses. Model modified from studies originally reported by Håkansson et al.47

the newly formed PV lacks cytoplasmic domains from transmemebrane receptors that would otherwise recruit fusion machinery. Consistent with this, the PV membrane remains devoid of markers for endosomes (e.g., transferrin receptor, proton pump, LAMP1 ), or machinery involved in trafficking of endosome and lysosomes (e.g., rabs, NSF ), or antigen presentation (e.g., MHC class I and class II)11 (Fig. 2). Exclusion is evidently important for intracellular survival, as when parasites are opsonized with specific antibodies and engulfed by macrophages they enter into FcR-positive compartment that rapidly acquire markers in the endocytic fusion pathway.11 The mechanism of protein sorting during invasion has remained elusive. Initial models suggested that the large extracellular domains of cell surface proteins physically excluded them from entering the vacuole. However, a number of GPI-anchored proteins were found to readily enter the vacuole during invasion and this process was largely independent of their size.43 Comparison of cell surface ICAM-1 that was tethered in the plasma membrane by a conventional transmembrane domain versus a GPI anchor revealed that exclusion might be related to the partitioning of proteins within the membrane. However, further analysis of this model

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revealed a more complex sorting process that does not strictly depend on lipid partitioning within the membrane.42 Both raft and nonraft lipids and cytosolic leaflet proteins have access to the vacuole.42 Moreover, single transmembrane proteins may be excluded from the vacuole even when they are preferentially found in rafts.42 Collectively, these studies indicate that multiple mechanisms likely operate to exclude access to the vacuole including membrane fluidity, association with the cytoskeleton, and assembly into oligomeric complexes. Regardless of how this novel process is achieved, it is likely essential for intracellular survival as it assures the absence of signaling molecules that might otherwise drive endocytic fusion with the PV.

Vacuole Modification and Intracellular Survival The second round of parasite protein secretion that occurs during invasion is associated with discharge of the rhoptries (Fig. 1). These club-shaped organelles form a duct-like structure that connects to the apical end of the parasite. Rhoptries contain a family of proteins (ROPs) that is nonoverlapping with those founding micronemes or dense granules.44 Among the proteins found in the rhoptries are a subset that localize to the neck region of the rhoptry organelle, so called RONs. Recent studies reveal that RONs are localized specifically to the moving junction, a tight constriction that forms between the host and parasite membranes during invasion.45,46 RONs are thus candidates for controlling access of host cell surface components to the vacuole via a process of molecular sieving. Rhoptry discharge occurs rapidly at the time of entry37 and protein components are released not only into the forming PV but directly into the host cytoplasm47 (Figs. 1, 2). The discharge of rhoptries into the host cell can be accentuated by treating cells with cytochalasin, which blocks entry but not apical attachment.47 Following discharge into the cytosol, ROP proteins acquire a membranous appearance, perhaps by recruiting host membranes, or alternatively perhaps reflecting a contribution of secreted lipids that form membranes de novo. These ROP-rich vesicles are known as “evacuoles” due to their empty profile (Figs. 1, 2). Evacuoles behavior similarly to intact PV in that they avoid fusion with endosomes, recruit mitochondria and ER and traffic within the cell to occupy a paranuclear region.47 The similar properties of evacuoles and the mature PV suggest that ROP proteins mediate many of the key features of the PV. The recent identification of a family of ROP proteins44 may uncover specific functions for parasite proteins that act as effectors from within the host cell. A number of ROP family members contain degenerate kinase domains (unpublished), suggesting they may act to disrupt signaling networks within the host cell. Probable targets for intervention would be host signaling networks, gene transcription, and cytoskeletal turnover. Discharge of ROP proteins within the PV may also be a mechanism for insertion into the membrane, thus modifying its composition at the time of formation. ROP2 is one such protein that is targeted to the PV where it adopts a transmembrane orientation with the N terminus protruding into the cytosol of the host cell.48 ROP2 undergoes N-terminal processing to expose a sequence resembling a mitochondrial import peptide.49 Insertion of this sequence into the mitochondrial transporter appears to tether the host cell mitochondria to the vacuole.49 This finding explains the long appreciated phenomenon that the PV is tightly wrapped with host cell mitochondria, although it does not reveal why this association is important. The prevailing model is that host cell lipids may be acquired from closely opposed ER and mitochondria, and studies using a variety of lipid tracers have revealed uptake pathways that support this model.50 The final chapter in modification of the PV occurs with discharge of dense granules.37 While these organelles are capable of constitutive secretion, kinetic analysis of secretion following invasion reveals that discharge is greatly upregulated within the first 30 min after invasion. Dense granule proteins (referred to as GRA) contain a family of proteins that have little conservation to known proteins. GRA proteins occupy the lumen of the vacuole (GRA1) or are inserted into the PV membrane (GRAs 3 and 5) or are targeted to a membranous network that forms within the vacuole (GRAS 2,4,6).51 The specific functions of these modifications are poorly understood, but these adaptations may contribute to nutrient uptake.

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Enclosed with the PV, T. gondii remains sequestered from the host endocytic pathway and the extracellular environment (Figs. 1, 2). While this has obvious advantages in terms of avoiding immune detection, it also presents a challenge for nutrient acquisition. T. gondii may have solved this problem by insertion of protein complexes into the PV to render it porous to small molecules.52 These pores are predicted to allow passage of small metabolites such as amino acids, sugars, cofactors, nucleobases, etc., While the protein composition of these pores has not been defined, it seems likely that they are formed by insertion of either GRA or ROP proteins secreted into the PV. Consistent with this porous vacuole, the genome sequence of T. gondii reveals a large number of transporters that would be predicted to participate in uptake of small molecules from within PV lumen (http:///toxodb.org). Sequestered within the nonfusigenic vacuole, T. gondii is protected from access to both endogenous and exogenous antigen presentation pathways. Thus, it is somewhat surprising that both robust Class I and Class II MHC antigen presentation pathways are elicited during infection.53,54 One explanation for this would be the fact that not all parasites are able to enter cells successfully or avoid lysosomal fusion.8,9 Even a low level of parasite death and digestion might suffice to prime both pathways for presentation. A more intriguing possibility is that select antigens gain access to the cytosol to induce presentation by the class I pathway. The demonstration that many components of rhoptries are injected into the cell during invasion47 provides one possible route for this. A second mechanism has been suggested by recent studies demonstrating that model antigens (Ova) expressed in transgenic parasites can escape from the PV.55 Future studies aimed at identifying the major epitopes recognized during infection may help resolve the pathways by which these antigen reach the respective antigen processing pathways.

Concluding Remarks There are a number of remaining mysteries about the intracellular survival of T. gondii that have thus far remained elusive. Principle among these is the question of how antigens get processed in both the endogenous and exogenous antigen presentation pathways. Robust class I and class II-mediated responses occur during infection, yet the parasite antigens processed via these pathways remain largely undefined. Equally perplexing is the process by which parasite restricts access of host proteins within the PV during entry. It is unclear if this process occurs by coalescence of lipid rafts or by some other novel form of membrane partitioning. This ability is paramount to the success of T. gondii in entering host cells by creating a novel nonfusigenic compartment. On a broader scale, the ability of T. gondii to infect mononuclear phagocytes may be an important adaptation for dissemination to tissues within the body. Defining to what extent these cells are activated to migrate or to alter their maturation and signaling pathways may provide insight into basic cellular processes in macrophages and the pathogenesis of parasitic infections.

Acknowledgements I am grateful to members of my laboratory for many helpful discussions and for their contributions to the work cited here and to many colleagues who have provided critical advice and reagents. Supported by the National Institutes of Health.

References 1. Levine ND. The Protozoan Phylum Apicomplexa. Vols. 1,2. Boca Raton: CRC Press, 1988. 2. Morrissette NS, Sibley LD. Cytoskeleton of apicomplexan parasites. Microbiol Mol Biol Rev 2002; 66:21-38. 3. Baldauf SL, Roger AJ, Wenk-Siefert I et al. A kingdom-level phylogeny of eukaryotes based on combined protein data. Science 2000; 290:972-977. 4. Roos DS, Donald RGK, Morrissette NS et al. Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods Cell Biol 1994; 45:28-61. 5. Dubey JP, Beattie CP. Toxoplasmosis of animals and man. Boca Raton: CRC Press, 1988.

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6. Channon JY, Seguin RM, Kasper LH. Differential infectivity and division of Toxoplasma gondii in human peripheral blood leukocytes. Infect Immun 2000; 68:4822-4826. 7. Wilson CB, Tsai V, Remington JS. Failure to trigger the oxidative burst of normal macrophages. J Exp Med 1980; 151:328-346. 8. Jones TC, Hirsch JG. The interaction of Toxoplasma gondii and mammalian cells. II The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J Exp Med 1972; 136:1173-1194. 9. Jones TC, Yeh S, Hirsch JG. The interaction between Toxoplasma gondii and mammalian cells. I. Mechanism of entry and intracellular fate of the parasite. J Exp Med 1972; 136:1157-1172. 10. Sibley LD, Weidner E, Krahenbuhl JL. Phagosome acidification blocked by intracellular Toxoplasma gondii. Nature 1985; 315:416-419. 11. Mordue DG, Sibley LD. Intracellular fate of vacuoles containing Toxoplasma gondii is determined at the time of formation and depends on the mechanism of entry. J Immunol 1997; 159:4452-4459. 12. Petersen E, Dubey JP. Biology of toxoplasmosis. In: Joynson DH, Wreghitt TJ, eds. Toxoplasmosis: A comprehensive Clinical Guide. Cambridge: University Press, 2001:1-42. 13. King CA. Cell motility of sporozoan protozoa. Parasitol Today 1988; 11:315-318. 14. Sibley LD. Invasion strategies of intracellular parasites. Science 2004; 304:248-253. 15. Håkansson S, Morisaki H, Heuser JE et al. Time-lapse video microscopy of gliding motility in Toxoplasma gondii reveals a novel, biphasic mechanism of cell locomotion. Mol Biol Cell 1999; 10:3539-3547. 16. Barragan A, Sibley LD. Transepithelial migration of Toxoplasma gondii is linked to parasite motility and virulence. J Exp Med 2002; 195:1625-1633. 17. Barragan A, Sibley LD. Migration of Toxoplasma gondii across biological barriers. Trends Microbiol 2003; 11:426-430. 18. Arrowood MJ, Sterling CR, Healey MC. Immunofluorescent microscopical visualization of trails left by gliding Cryptosporidium parvum sporozoites. J Parasitol 1991; 77:315-317. 19. King CA. Cell surface interaction of the protozoan Gregarina with Concanavalin A beads - Implications for models of gregarine gliding. Cell Biol Intl Rep 1981; 5:297-305. 20. Clark DP, Sears CL. The pathogenesis of cryptosporidiosis. Parasitol Today 1996; 12:221-225. 21. Wetzel DM, Schmidt J, Kuhlenschmidt M et al. Gliding motility leads to active cellular invasion by Cryptosporidium parvum sporozoites. Infect Immun 2005; 73:5379-5387. 22. Ryning FW, Remington JS. Effect of cytochalasin D on Toxoplasma gondii cell entry. Infect Immun 1978; 20:739-743. 23. Miller LH, Aikawa M, Johnson JG et al. Interaction between cytochalasin B-treated malarial parasites and erythrocytes. J Exp Med 1979; 149:172-184. 24. Dobrowolski JM, Sibley LD. Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 1996; 84:933-939. 25. Morisaki JH, Heuser JE, Sibley LD. Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J Cell Sci 1995; 108:2457-2464. 26. Wetzel DM, Håkansson S, Hu K et al. Actin filament polymerization regulates gliding motility by apicomplexan parasites. Mol Biol Cell 2003; 14:396-406. 27. Gaskins E, Gilk S, DeVore N et al. Identification of the membrane receptor of a class XIV myosin Toxoplasma gondii. J Cell Biol 2004; 165:383-393. 28. Meissner M, Schluter D, Soldati D. Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 2002; 298:837-840. 29. Ménard R. Gliding motility and cell invasion by Apicomplexa: Insights from the Plasmodium sporozoite. Cell Micro 2001; 3:63-73. 30. Jewett TJ, Sibley LD. Aldolase forms a bridge between cell surface adhesins and the actin cytoskeleton in apicomplexan parasites. Molec Cell 2003; 11:885-894. 31. Buscaglia CA, Coppens I, Hol WGJ et al. Site of interaction between aldolase and thrombospondinrelated anonymous protein in Plasmodium. Mol Biol Cell 2003; 14:4947-4957. 32. Kappe S, Bruderer T, Gantt S et al. Conservation of a gliding motility and cell invasion machinery in apicomplexan parasites. J Cell Biol 1999; 147:937-943. 33. Jewett TJ, Sibley LD. The Toxoplasma proteins MIC2 and M2AP for a hexameric complex necessary for intracellular survival. J Biol Chem 2004; 275:9362-9369. 34. Soldati D, Dubremetz JF, Lebrun M. Microneme proteins: Structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii. Int J Parasitol 2001; 31:1293-1302. 35. Carruthers VB, Giddings OK, Sibley LD. Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cell Microbiol 1999; 1:225-236.

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36. Brossier F, Jewett TJ, Sibley LD et al. A spatially-localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma. Proc Natl Acad Sci USA 2005; 102:4146-4151. 37. Carruthers VB, Sibley LD. Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur J Cell Biol 1997; 73:114-123. 38. Mital J, Meissner M, Soldati D et al. Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion. Mol Biol Cell 2005; 16:4341-4349. 39. Carruthers VB, Håkansson S, Giddings OK et al. Toxoplasma gondii uses sulfated proteoglycans for substrate and host cell attachment. Infect Immun 2000; 68:4005-4011. 40. Ortega-Barria E, Boothroyd JC. A Toxoplasma lectin-like activity specific for sulfated polysaccharides is involved in host cell infection. J Biol Chem 1999; 274:1267-1276. 41. Suss-Toby E, Zimmerberg J, Ward GE. Toxoplasma invasion: The parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fusion pore. Proc Natl Acad Sci USA 1996; 93:8413-8418. 42. Charron AJ, Sibley LD. Molecular partitioning during host cell penetration by Toxoplasma gondii. Taffic 2004; 5:855-867. 43. Mordue DG, Desai N, Dustin M et al. Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring. J Exp Med 1999; 190:1783-1792. 44. Bradley PJ, Ward C, Cheng SJ et al. Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in T. gondii. J Biol Chem 2005; 280:34245-34258. 45. Alexander DL, Mital J, Ward GE et al. Identification of the moving junction complex of Toxoplasma gondii: A collaboration between distinct secretory organelles. PLos Path 2005; 1:137-149. 46. Lebrun M, Michelin A, El Hajj H et al. The rhoptry neck protein RON4 relocalizes at the moving junction during Toxoplasma gondii invasion. Cell Micro 2005; 7:1823-1833. 47. Håkansson S, Charron AJ, Sibley LD. Toxoplasma evacuoles: A two-step process of secretion and fusion forms the parasitophorous vacuole. EMBO J 2001; 20:3132-3144. 48. Beckers CJM, Dubremetz JF, Mercereau-Puijalon O et al. The Toxoplasma gondii rhoptry protein ROP2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. J Cell Biol 1994; 127:947-961. 49. Sinai AP, Joiner KA. The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane. J Cell Biol 2001; 154:95-108. 50. Charron AJ, Sibley LD. Host cells: Mobilizable lipid resources for the intracellular parasite Toxoplasma gondii. J Cell Sci 2002; 115:3049-3059. 51. Mercier C, Dubremetz JF, Rauscher B et al. Biogenesis of nanotubular network in Toxoplasma parasitophorous vacuole induced by parasite proteins. Molec Biol Cell 2002; 13:2397-2409. 52. Schwab JC, Beckers CJM, Joiner KA. The parasitophorous vacuole membrane surrounding intracellular Toxoplasma gondii functions as a molecular sieve. Proc Natl Acad Sci USA 1994; 91:509-513. 53. Hunter CA, Reichmann G. Immunology of toxoplasma infection. In: Joynson DH, Wreghitt TJ, eds. Toxoplasmosis: A Comprehensive Clinical Guide. Cambridge University Press, 2001:43-57. 54. Denkers EY, Gazzinelli RT. Regulation and function of T-cell mediated immunity during Toxoplasma gondii infection. Clin Micro Rev 1998; 11:569-588. 55. Gubbels MJ, Streipen B, Shastri N et al. Class I major histocompatibility complex presentation of antigens that escape from the parasitophorous vacuole of Toxoplasma gondii. Infect Immun 2005; 73:703-711. 56. Sibley LD. Toxoplasma gondii: Perfecting an intracellular life style. Traffic 2003; 4:581-586.

CHAPTER 2

Macrophages, Alternative Niches for Intracellular Growth of Trypanosoma cruzi Julio Scharfstein* and Marcos André Vannier dos Santos

Abstract

W

idely distributed in the peripheral and lymphoid tissues, macrophages are key effectors of cellular immunity during infection by Trypanosoma cruzi, the etiological agent of Chagas’ disease. At the onset of infection, insect-transmitted metacyclic trypomastigotes invade tissue-resident macrophages as well as a broad range of nonphagocytic cells. Metacyclic trypomastigotes are able to discriminate host cells through differential engagement of adhesive glycoproteins that convey either stimulatory or inhibitory signals for cellular invasion. After a few days of intracellular growth, the first cycle of infection terminates with host cell death and consequent release of parasites into interstitial spaces. Trypomastigotes invade and replicate safely in nonprofessional phagocytic cells, taking advantage of the low frequency of MHC-class I restricted CD8 effectors. In the meanwhile, extracellular amastigotes invade tissue-macrophages via the mannose scavenger receptor. As inflammation intensifies, macrophages are innately activated by microbial ligands for Toll-like receptors or by endogenously released danger signals. In spite of innate immunity, the parasites spread to lymphoid tissues, inducing polyclonal lymphocyte activation. Generation of apoptotic lymphocytes induces TGF-β/PGE2 production by macrophages, which then become permissive to T. cruzi growth. As the infection continues, tissue parasite load gradually subsides owing to the combined action of antibodies, type 1 cytokines and effector/memory CD8 T cells. Despite the vigor of anti-parasite immunity, a low-grade and insidious infection is established. Years later, immunoregulatory dysfunctions provoke a progressive form of chronic myocardiopathy in a significant proportion of chagasic patients. In this chapter, we will review information concerning the role of macrophages as “niches” for intracellular parasite growth during the course of Chagas’ disease.

Introduction Afflicting nearly 20 million people in South and Central America, Chagas’ disease is a chronic infection caused by the obligate intracellular protozoan Trypanosoma cruzi (phylum Sarcomastigophora, order Kinetoplastida). T. cruzi is a diploid organism that predominantly reproduces by binary fission. Remarkably, 50% of the T. cruzi genome is made of repetitive gene sequences, many of which code for large families of polymorphic surface antigens.1 T. cruzi is naturally transmitted to humans and wild mammals through blood-sucking insects of the family Reduviidae.2 As is true for the trypomastigotes that are generated within mammalian cells, the insect-borne metacyclic trypomastigotes (MT) invade a wide range of mammalian *Corresponding Author: Julio Scharfstein—Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, C.C.S., Ilha do Fundão, Rio de Janeiro, 21949-900, RJ, Brazil. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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host cells. The intracellular life cycle of T. cruzi involves the following events: first, the flagellates escape from the parasitophorous vacuole and lodge in the host cell cytoplasm. Once exposed to the cytoplasmic environment, the trypomastigotes transform into round-shaped amastigotes. After several cycles of binary division, the amastigotes transform into trypomastigote forms. Once released from disrupted host cells, T. cruzi propagates the infection systemically to multiple organs. Although the acute disease can be fatal, it is usually controlled by adaptive immunity; however, the parasites are not eradicated. After years of a silent infection, immunoregulatory dysfunctions cause a progressive form of chronic myocardiopathy in about 30% of the chagasic patients.3-6 Owing to progress in molecular genetics, the evolutionary history of the heterogeneous T. cruzi species is now well understood.2 T. cruzi isolates have arisen from three major ancestral lineages, designated as T. cruzi I, II, and III, each associated with a distinct ecosystem. The lineage designated as T. cruzi I, although less diversified, circulates mainly in a sylvatic ecosystem involving insect transmission to wild mammals while T. cruzi II, more diversified, predominates in “domestic” environment composed of home-dwelling hemiptera, domestic animals and humans. The T. cruzi III lineage, recently characterized, is thought to be involved in two genetic crosses with lineage T. cruzi II, producing evolutionarily viable hybrid progeny.7,8 In addition to phylogenetic lineage diversity, the clonal population structure of T. cruzi also accounts for biological variability. While not excluding the critical influence of host genetics,9 there is now compelling evidence that tissue tropism in laboratory animals is linked to phenotypic characteristics of T. cruzi clones.10,11 The analysis of T. cruzi clones isolated from a patient infected with HIV9 supports the concept that clonal heterogeneity influences parasite tissue tropism and possibly other variable aspects of Chagas’ disease.6 Viewed against this background, and due to the limited number of strains used in most laboratory studies, our knowledge of the intricate host-parasite interplay is still superficial. In spite of this, general principles can be drawn from the analysis of the literature on macrophage interactions with T. cruzi. For more detailed information on this subject, readers should refer to a recently published review (ref. 12). Here we aim to revisit the fundamental theme of cellular invasion mechanisms, highlighting the relationship between the dynamics of inflammation and macrophage susceptibility at early stages of infection.

Mechanisms of Cellular Invasion by Insect-Borne Metacyclic Trypomastigotes Knowledge of the molecular mechanisms underlying MT penetration comes mostly from culture studies performed with nonprofessional phagocytic target cells.13 Multiple interactions between stage-specific MT surface molecules with host components are required for establishment of a productive cycle of intracellular infection. During MT internalization, Ca2+ mobilization is induced both in the parasites and the target cells, owing to the triggering of signal transduction responses in both partners.14 For cell adhesion, MT engage surface glycoproteins, such as gp82,15 gp35/5016 and a recently characterized member of serine-, alanine-, and proline-rich protein family, termed as SAP,17,18 all of which are capable of eliciting Ca2+ signals in mammalian host cells. Analysis of signaling pathways activated in the protozoan cell revealed strain-dependent differences, i.e., while some MT invade epithelial cells through engagement of gp82, other stains depend on pg35/50 adhesive contacts.14,18,19 In the former subgroup of parasites, the triggering of gp82-signaling cascade leads to upstream activation of protein tyrosine kinase and of phospholipase C in the protozoan cell, and these events are coupled to Ca2+ release from IP3-sensitive stores.7,14 In contrast, in T. cruzi isolates that attach to target cells mainly through gp35/50, Ca2+ is released from acidocalciosomes.14 The signaling function of gp82 was confirmed by stably transfecting noninfective epimastigotes with a expression vector carrying the metacyclic stage gp82 cDNA.13,20 Although these gp82-epimastigote transfectants were capable of binding and triggering a Ca2+ response in HeLa cells, they were not internalized, indicating that

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epimastigotes lack other critical components of the MT invasion machinery.20 Analysis of the gp82-dependent signaling cascades in the epimastigote transfectants showed defective phosphorylation of intracellular parasite proteins,20 suggesting that target cell invasion via gp82 depends on the function of some of these downstream phosphorylated components. In another interesting finding, Yoshida and coworkers demonstrated that MTs are also equipped with cell-surface molecules that inhibit cellular invasion, e.g., gp90.21 These findings suggest that the insect-borne MT, once confronted with mammalian tissues, are able to scrutinize the potential target hosts cells by differentially engaging surface glycoproteins in adhesive contacts that either positively or negatively modulate cellular invasion. Studies of the impact of oral infection in mice showed that the gastric mucosa is particularly susceptible to infection by MT.15,22,23 For most parasite strains, gp82 is the most critical adhesive molecule involved in invasion of epithelial cells underlying the gastric mucosa.7,15,24 Interestingly, gp90 was degraded by pepsin-like gastric proteases, and for some of the parasite strains tested, loss of this negative modulator of cellular invasion correlated with increased parasite infectivity.24 Regarding MT interaction with macrophages, there is limited information available. Early studies with MT showed that parasites are opsonized, albeit to a moderate extent, by iC3b opsonins,25 i.e., the ligand for macrophage complement C3 receptor. Although MT may be also opsonized by natural antibodies present in human serum,26 the involvement of FcR was in this particular study explored with antiserum. As expected, the internalized parasites were killed in the inflammatory macrophages.27 The origin of the parasitophorous vacuole surrounding MT was investigated with human derived macrophages.25 This analysis revealed presence of β1-integrins as well as lysosomal membrane glycoproteins in such vacuoles, suggesting that plasma membrane fused with lysosomal vesicles during the penetration process.25 In the same study, it was demonstrated that FcR are not preferentially incorporated into parasitophorous vacuole membranes except when MT are deliberately opsonized with antibodies.25 Complement opsonins iC3b do not seem to play a major role in the determination of MT fate in human macrophages, since the membranes of parasitophorous vacuoles surrounding MT were free of CR3 whereas this receptor was conspicuously present in the vacuoles surrounding epimastigotes.25

Lessons from Cellular Invasion by Mammalian Stages of T. cruzi Research focusing on interactions of tissue culture derived trypomastigotes (TC) with nonphagocytic cells yielded a wealth of information about mechanisms underlying cellular invasion.28,29 Early studies suggested that TC adhesion to nonphagocytic host cells depends on trans-sialidase-dependent enzymatic transfer of sialic acid residues from host donor molecules onto mucin-like acceptors displayed at the cell surface of trypomastigotes.30 Subsequent studies revealed that Tc85, a member of the trans-sialidase superfamily of surface antigens of trypomastigotes, promotes parasite TC attachment to epithelial cells through the binding to laminin and cytokeratin 18.31,32 Independent studies indicated that fibronectin mediates T. cruzi attachment to fibroblasts33 and cardiomyocytes.34 In contrast to the classical phagocytic response, pseudopods were not commonly observed around the parasites internalized by nonphagocytic cells. In consonance with these observations, the penetration of the pathogen was not inhibited by cytochalasin D, a drug that blocks actin polymerization and phagocytosis.35 Early analysis of TC interaction with nonphagocytic cells revealed that parasite invasion depends on Ca2+-regulated lysosomal fusion to plasma membranes at the attachment sites.35-37 Recent studies showed that the fusion of lysosomes necessary for construction of the nascent parasitophorous vacuole depends on de novo microtubule polymerization at sites of parasite/ host cell “synapse” which then provides the infrastructure required for transport of lysosomes.38 More recently, it became apparent that lysosomal recruitment is not the sole mechanism by which T. cruzi penetrates nonphagocytic cells as at least half of the invading parasites bypass the lysosomal fusion via an actin-independent intimate association with host PtdInsP3/PtdIns

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(3,4)P2-enriched cell membrane regions.39 Rather than invading host cells through Ca2+-induced lysosomal fusion with plasma membranes, TC trigger formation of plasma membrane invaginations that result in the formation of nascent parasitophorous vacuoles.39 Subsequent studies revealed that actin remodelling via phosphatidylinositol-3 kinase (PI-3 kinase) is crucially required to retain the recently internalized parasites in the host cell, by promoting fusion of the nascent vacuole to lysosomes or to endosomes.40 A somewhat different picture was observed in studies of trypomastigote penetration in HeLa cells (Fig. 1 right panel) because in this particular case, pseudopodia and plasma-membrane internalization were observed.41 Of note, these pseudopodia display actin condensation associated with actin-binding proteins, indicating that nonprofessional phagocytes may indeed sometimes display macrophage-like pseudopodial emission.41 The dependence on actin skeleton formation was also observed in studies of the interaction of nonphagocytic cells with extracellular amastigotes, thus suggesting that pseudopodia formation is required for amastigote invasion of nonphagocytic cells. In the past decade, considerable effort was devoted at characterizing the signaling molecules that drive T. cruzi penetration in nonphagocytic cells.28,29,42-44 Early studies indicated a role for the TGF-β-dependent signalling pathway in the invasion of some types of nonphagocytic cells.45 Interest in the TGF-β signalling pathway resurged in view of recent data obtained with primary cardiomyocytes, indicating that trypomastigotes may convert inactive TGF-β into an active signal peptide through a pathway that depends on the proteolytic activity of serine and/ or cysteine proteases.46,47 Progress in the characterization of T. cruzi proteases implicated three different type of enzymes in cellular invasion mechanisms: oligopeptidase B,48 cruzipain44,49 and the prolyl oligopeptidase Tc80POP.50 Oligopeptidase B was characterized during attempts to identify the signalling molecules that trigger [Ca2+]i fluxes in nonphagocytic cells.35,42,48 Biochemical studies suggested that the putative agonist was generated by oligopeptidase B-dependent processing of a cytoplasmic precursor protein of T. cruzi.48,51 An important step forward was the finding that the Ca2+-stimulating molecule(s) promote parasite uptake by activating pertussis toxin-sensitive G-protein coupled receptors.52,53 Several years later, studies of TC interaction with primary endothelial cells and cardiomyocytes implicated the G-protein coupled kinin receptors in parasite invasion mechanisms.54,55 Analysis of the [Ca2+]i.-inducing responses evoked by TC implicated cruzipain, the major cysteine protease of T. cruzi54 as the kinin-releasing enzyme.44 Biochemical studies demonstrated that the substrate specificity of cruzipain resembles

Figure 1. Scanning electron microscopy showing tissue culture trypomastigotes binding to murine macrophages (left) and to HeLa cells (right). Note the sleeve-like pseudopodium attached to invading parasite. Figures were kindly provided by Drs. Tecia Carvalho and Wanderley de Souza (left) and Renato Mortara (right).

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that of tissue kallikrein, i.e., the parasite cysteine protease and the host serine protease share the ability to process kininogens, liberating the decapeptide hormone lysyl-bradykinin, from an internal segment of high molecular weight kininogens. 56 Noteworthy, analysis of the kinin-releasing reaction revealed that it is facilitated by cooperative interactions between heparan sulfate proteoglycans, cruzipain and high-molecular weight kininogen.57 Once excised from cell-bound kininogens, the short-lived “kinin” peptides then trigger robust [Ca2+]i responses in host cells (eg. endothelial cells and cardiomyocytes) that naturally overexpress kinin receptors, thereby stimulating the uptake of TC by these mammalian cells.44 Additional studies have confirmed that TC, but not epimastigotes, activate the kinin system in vivo58 through the activity of cruzipain (Monteiro et al, submitted). Of note, recent studies demonstrated that promastigotes of the L. donovani complex also activate the kinin system in vitro and in vivo by a cysteine protease dependent mechanism.59

Macrophage Signaling Pathways Involved in Uptake of Trypomastigotes Due to the wide range of T. cruzi strains and to the different models used in laboratory studies, the information on macrophage interaction with TC60 is still fragmented. In contrast to the unusual penetration mechanism observed in nonphagocytic cells, trypomastigotes trigger a typical phagocytic response in macrophages, i.e., the pathogen induce formation of profuse pseudopodia through the involvement of actin and actin-binding proteins, a process sensitive to low temperature and cytochalasin B. Prior to signaling, TC adhere to human or murine macrophages through fibronectin,61,62 most likely through the binding of β1 subunit of VLA integrins.63 Efforts to characterize the parasite surface molecules that promote macrophage invasion converged on gp83, another member of the trans-sialidase family. Human macrophages treated with gp83 were rendered more permissive to infection. The effect was associated with tyrosine kinase64 and protein kinase C65 activation. PD98059 or genistein abolished these effects, thus suggesting that MAP kinase-dependent tyrosine phosphorylation was required for productive infection.65 During infection of murine macrophages by TC, several proteins were phosphorylated at tyrosine residues, the response being linked to enhanced expression of src-like tyrosine kinases p53-56 lyn and fgr.66 Phosphotyrosine-presenting proteins accumulate at the site of parasite invasion in macrophages, colocalizing with a condensation of host F-actin.67 In another study, host PI 3-kinases were implicated in T. cruzi invasion.68 Pretreatment of macrophages with the PI 3-kinase inhibitor wortmannin markedly inhibited infection. It was further shown that T. cruzi markedly stimulates the formation of the lipid products generated by PI 3-kinases, PI 3-phosphate, PI 3,4-biphosphate, and PI 3,4,5-triphosphate, but not PI 4-phosphate or PI 4,5-biphosphate. This activation was inhibited by wortmannin.68 Infection with T. cruzi also stimulated a marked increase in the in vitro lipid kinase activities that are present in the immunoprecipitates of anti-p85 subunit of class I PI 3-kinase and anti-phosphotyrosine. In addition, T. cruzi invasion also activated lipid kinase activity found in immunoprecipitates of class II and class III PI 3-kinases. These data demonstrate that T. cruzi invasion into macrophages activates distinct PI 3-kinase isoforms and that inhibition of class I and class III PI 3-kinase activities abolishes parasite invasion. In summary, these studies suggest that once the surface components of TC are recognized by their macrophage receptors, there is activation of a tyrosine phosphorylation cascade leading to PI 3-kinase recruitment and assembly of actin filaments at the site of initial cell-to-cell contact, resembling the events described during phagocytosis. More recently, studies of the macrophage responses to a variety of pro-inflammatory mediators, such as β-chemokines, platelet-activating factor, leukotriene B459,69-71 and bradykinin,59 revealed that parasite phagocytic uptake by inflammatory macrophages is significantly enhanced due to the triggering of each of these GPCRs. It remains to be determined if adhesive contacts required for induction of pseudopodial emissions (Fig. 1, left panel) are somehow facilitated by the signaling responses relayed through these serpentine receptors.

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On the basis of studies of the phagocytic responses to various types of stimuli, it is known that actin polymerization and assembly is orchestrated by monomeric Ras GTPases, such as Rho, Rac and Cdc42.72 Indeed, studies with macrophage cultures suggested that they are indeed implicated in macrophage invasion by T. cruzi amastigotes 73 as well as by trypomastigotes.74,75 The Rho and ARF6 guanine nucleotide exchange factors (GEFs) may be regulated by tyrosine phosphorylation and binding to phosphoinositides via pleckstrin homology (PH) domains. PH domains also mediate the binding of myosin-X to PI-3 kinase products at the site of phagocytic cup formation.76 Interestingly the phagocytosis mediated by complement receptor and FcγRs, respectively trigger RhoA phosphorylation and ARF6 activation.77,78 Unlike the lysosomal exocytosis that is often observed during invasion of nonprofessional phagocytic cells by T. cruzi, the response of macrophages involves fusion of endoplasmic reticulum membranes and VAMP-3-presenting endosomal compartments with the plasma membrane.78 Besides regulating the actin cytoskeleton behavior, GTPases modulate signal transduction mechanisms, activating JNK and p38 MAP kinase pathway and therefore may be implicated in several downstream responses, including T. cruzi survival within macrophages.79 It was reported that T. cruzi infection down-modulates Rab 7 and 11 GTPases.80 Rac and Cdc42 can activate PI-3 kinase and phospholipase C and in addition can promote the generation of reactive oxygen species by activation of NADPH oxidase.72 Although not required for the phagocytic response, activation of Toll-like receptors (TLR) by their microbial ligands may modulate phagocytosis via Rac1 and PI-3 kinase.81 A limited number of studies have addressed the mechanisms of macrophage uptake of extracellular amastigotes.73,82,83 Early investigations implicated involvement of mannose scavenger receptors in amastigote adhesion to macrophages.84 Interestingly, the ligands for mannose receptors are developmentally regulated, i.e., they are displayed by extracellular amastigotes but are absent in the corresponding cell surface glycoproteins of trypomastigotes.84

Macrophages as Niches for T. cruzi Growth: Lessons from in Vivo Infection Models Except for sporadic cases of human infection by the oral route, which includes some fatal cases recently reported in Brazil,85 natural transmission of Chagas’ disease occurs when the blood-sucking triatomines releases feces containing metacyclic trypomastigotes on exposed wounded tissues or irrupted mucosae. After penetrating the exposed mucosa or skin-associated connective tissues, MT may penetrate macrophages or nonphagocytic host cells in the proximity of the entry site, or target macrophage populations residing in the draining lymph nodes. Interestingly, MT (unlike bloodstream trypomastigotes) do not elicit significant production of the IL-8 chemokine and nitric oxide by epithelial cells in vitro,86 suggesting that the insect-borne parasites have a seemingly “silent” phenotype. Consistent with this concept, MT are unable to evoke significant inflammatory edema in the mouse paw (J. Scharfstein, unpublished data) whereas TC, by contrast, induce prominent vascular responses.55 Although not proven, is conceivable that the low inflammatory activity of MT86 stems from low level expression levels of Toll-like receptor ligands.87,88 Indeed, biochemical studies revealed that MT do not express appreciable amounts of glycosylphosphatidyl-linked mucin anchor (tGPI-mucin), a potent ligand for Toll-like 2 receptors expressed by TC.87,89 It remains to be determined if MT may depend on gp90 negative signals to escape detection by tissue sentinel cells, such as DCs, mast cells, macrophages or by the recently described IFN-γ-producing killer dendritic cells.90 By the time that the first cycle of infection terminates, adaptive immunity (induced by cross-reactive MT antigens) is incipient, if present at all. As the host cells rupture, peripheral tissues are stormed with large numbers of trypomastigotes and/or incompletely differentiated amastigotes. While inflammation evolves, the extracellular trypomastigotes invade a broad range of nonphagocytic cells, such as cardiac muscle, fibroblasts, endothelial cells and adipocytes,91,92 taking advantage of the low frequency of CD8 effector cells generated at early stages of infection.

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Considering that host cell death is not a synchronic event in T. cruzi infection, the extracellular parasites are confronted with a rapidly changing environment. Depending on the time-frame of parasite discharge from the ruptured host cells, the adjacent microvasculature is involved, driving plasma leakage and/or neutrophil trans-endothelial migration. As inflammation evolves, the blood-borne monocytes recruited to injured tissues dynamically respond to changes in tissue homeostasis. Modifications of extracellular matrix components lead to changes in macrophage gene transcription and/or post-translational modifications, affecting cellular adhesion and migration properties. As discussed below, the phagocytes engulf (apoptotic or necrotic) through the engagement of scavenger receptors, eventually turning into safe shelters for T. cruzi growth. As mentioned, the extravasation of plasma proteins to peripheral tissues is one of the earliest manifestations of inflammation. Within minutes, the parasites retained in the peripheral tissues are exposed to a myriad of serum proteins, e.g., components of the complement and kallikrein-kinin systems. Early in infection, i.e., before serum IgG antibodies are present at significant levels, fibronectin seems to promote parasite invasion.62 C1q is another example of a plasma protein that binds to the TC cell surface thus increasing pathogen internalization by macrophages and fibroblasts.93 More recently, the nature of the C1q-docking molecule was characterized as a calreticulin-like protein (TcCRT).94 Interestingly, TcCRT is able to inhibit both the classical and the lectin-activation pathways by binding selectively to collagen-like recognition sites of both C1q and the mannan-binding lectin.94,95 In other studies, it was demonstrated that CD91 drives macropinocytosis and uptake of apoptotic bodies by macrophages through ligand interactions with C1q and the mannose binding lectin.96 The possibility that C1q-sensitized parasites are internalized by macrophages via CD91 remains to be assessed. This possibility is worth exploring in light of evidence that apoptotic cells fuel parasite growth in macrophages by stimulating vitronectin-receptor dependent release of TGF-β/PGE2, which then drives polyamine biosynthesis via the ornithinine decarboxylase pathway.97 It is unknown if these suppressive effects may occur when CD91-bearing macrophages simultaneously internalize apoptotic cells and C1q-decorated parasites. It is well-established that tissue sentinel cells, such as mast cells and dendritic cells (DCs) sense tissue injury and pathogen threat through alarm signals conveyed by endogenous signals and/or microbial signatures. After capturing antigen, DCs undergo a maturation program that enables them to prime virgin T cells in the draining lymph nodes. Recent studies in a subcutaneous model of T. cruzi infection revealed that Toll-like 2 receptors and bradykinin B2 receptors act cooperatively, driving type-1 immune responses (Monteiro et al, submitted). The dissection of the early stages of the inflammatory response evoked by TC revealed that initial triggering of Toll-like 2 receptors led to neutrophil-evoked plasma leakage, thus driving efflux of blood-borne kininogens into peripheral tissues. Within minutes, the inflammation is enhanced due to cruzipain-dependent proteolytic generation of bradykinin,55 recently characterized as a potent maturation signal for immature dendritic cells.98 Apart from accumulating kininogens, injured tissues are also permeated with α2-macroglobulin, a nonspecific protease inhibitor. It is well-established that molecular complexes formed between α2-macroglobulin and a variety of proteases are cleared from the blood or interstitial spaces by CD91, a macrophage multi-functional scavenger receptor (also known as α2-macroglobulin receptor or lipoprotein related receptor protein).99 Several studies have addressed the role of α2-macroglobulin and CD91 in cellular interactions between macrophages and T. cruzi. Initial observations documented that α2-macroglobulin binds to the cell surface of bloodstream trypomastigotes.100 Although the surface parasite molecule that acts as a docking site for α2-macroglobulin was not characterized, cruzipain is one of the possible candidates.101 Investigations addressing antigen-presentation mechanisms involved in human T cell response to cruzipain showed that the parasite cysteine protease is partially entrapped by α2-macroglobulin.102 Model studies carried out with human monocytes as APCs further revealed that α2-macroglobulin-cruzipain complexes are indeed cleared via binding to CD91.102 It was further shown that the CD91-driven uptake of the protease complexes resulted in enhanced presentation of cruzipain epitopes to Ag-specific human CD4 T cells.102,103

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Aside from influencing the antigen-presenting function of macrophages, CD91 and α2-macroglobulin may also contribute to maintenance of homeostasis in peripheral infection sites. This is because α2-macroglobulin binds to TGF-β, promoting CD91-dependent clearance of the complexes through CD91.99 Interestingly, infection of mice with targeted gene deletion of α2-macroglobulin increased tissue parasitism and fibrosis.104 The reasons for the increased morbidity observed in α2-macroglobulin deficient mice were not characterized, but may likely result from enhanced TGF-β activity in infected tissues, and/or to indirect tissue damage caused by unimpaired proteolytic activity of parasite proteases, such as cruzipain.57 TGF-β-dependent pathways of macrophage suppression may likely occur in lymphoid tissues because there is massive polyclonal activation at the early stage of infection.105 Studies in the mouse model showed that apoptotic lymphocytes trigger the TGF-β/PGE-dependent pathway of macrophage suppression, turning these phagocytes into permissive host cells.97 Other investigators suggested that parasite strains have differential abilities to induce host cell apoptosis while replicating in macrophages, fibroblast and cardiomyocytes.106 If confirmed, generation of “eat-me” tags in infected macrophages and cardiomyocytes may lead to their uptake by macrophages, perhaps rendering them susceptible to T. cruzi infection. Similarly, the parasites may take advantage of TGF-β dependent pathways to invade fibroblasts during tissue remodelling.45 Macrophages may also become permissive to T. cruzi growth due to direct suppressive effects of parasite molecules. For example, it was recently reported that T. cruzi expresses a GPI-linked mucin (Agc10) that suppresses human macrophage responsiveness to TNF-α. Using LPS as the activation stimulus, it was found that Agc10 down-modulate MAP-kinase activation through upstream inhibition of Jun kinase or extracellular regulated kinases.107 In other circumstances, IL-4/IL-13 may be produced endogenously at high levels by a given tissue, thus favoring activation of macrophages via the “nonclassical” mode.108 Since upregulated expression of the mannose scavenger receptor is one of the phenotypic changes displayed by the “alternatively” activated macrophages, these phagocytes can engage the mannose receptor in binding to amastigote-specific ligands (i.e., absent in TC).84 Model studies using murine macrophages revealed that enzymatically inactive forms of cruzipain, a mannose-rich glycoprotein, stimulate IL-10 and TGF-β production by macrophages, turning these cells permissive to parasite growth.109 In J774 cells, inactive cruzipain stimulated T. cruzi growth by activating the arginase pathway while at the same time down-modulating iNOS.109 Macrophage activation by T. cruzi can be also suppressed by mediators that stimulate high-level production of c-AMP via activation of receptors coupled to Gs, such as PGE2.110 As a consequence of parasite growth in lymphoid organs during acute infection, dendritic cells are infected, and their maturation program is impaired,111 possibly contributing to the aberrant immune responses observed in acutely infected mice. In some circumstances, the inflammatory state prevailing in peripheral tissues may be such that macrophages acquire full-fledged innate effector functions. As discussed elsewhere in this volume, macrophages can be innately activated by microbial stimuli, e.g., ligands for Toll-like receptors. Alternatively, endogenous pro-inflammatory signals, e.g., chemokines, 112 platelet-activating factor, leukortriene B469 and kinins58,59 may all converge to induce cellular release of TNF-α,113 which in turn triggers iNOS-dependent trypanocidal responses in the activated macrophages. Recently, several in vivo studies demonstrate the critical influence of chemokines in the control of T. cruzi infection.5,71,113-115 Studies performed with MIP-1α, MIP-1β, RANTES, and JE/MCP-1 show that they all increase T. cruzi uptake by inflammatory macrophages. Significantly, the GPCR-mediated responses elicited by these β-chemokines enhanced NO production, ultimately favoring control of parasite replication.70 Of note, JE potentiates parasite killing by macrophages incubated with low doses of IFN-γ, thus suggesting that GPCR triggering by murine β-chemokines may act cooperatively with IFN-γ. Another example of an endogenous mediator that activates macrophages came from analysis of the phenotype of mice with targeted gene deletion of PAF receptors.69 Macrophages from PAFR-/ mice exhibited impaired phagocytic responses along with reduced nitric oxide production

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when activated with PAF, leukotriene B4 or MCP-1. Collectively, these findings suggest that β-chemokines and endogenous downstream effectors, such as leukotriene B4 and PAF, control T. cruzi replication in wild-type mice through the facilitation of parasite uptake by inflammatory macrophages, a process that is normally linked to upregulated production of NO at early stages of infection.

Concluding Remarks Owing to the repertoire of stage-specific surface molecules displayed by MT, the insect forms invade tissues without evoking prominent inflammation. A different scenario can be envisaged when the first cycle of intracellular infection ends, several days later. As infected host cells rupture, large numbers of extracellular parasites are released into interstitial spaces. In this chapter, we have discussed how T. cruzi might exploit the complex dynamics of inflammation and tissue remodeling to invade a variety of host cells, outlining the strategies that convert macrophages into safe shelters for T. cruzi growth at early stages of infection.

Acknowledgements The authors are indebted to Claudio P. Figueira and Elisângela Sodré for help in the editorial process. We also wih to thank Drs. Tecia Carvalho, Wanderley de Souza, Emile Barrias and Renato Mortara for the electron microscopy pictures. Recent work by the authors was supported by CNPq, FAPERJ, CAPES and Wellcome Trust. The authors wish to dedicate this chapter to the memory of Nadia Nogueira, for her pioneer contributions to cellular immunity research in Chagas’ disease.

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69. Aliberti JC, Machado FS, Gazzinelli RT et al. Platelet-activating factor induces nitric oxide synthesis in Trypanosoma cruzi-infected macrophages and mediates resistance to parasite infection in mice. Infect Immun 1999; 67:2810-4. 70. Aliberti JC, Machado FS, Souto JT et al. beta-Chemokines enhance parasite uptake and promote nitric oxide-dependent microbiostatic activity in murine inflammatory macrophages infected with Trypanosoma cruzi. Infect Immun 1999; 67:4819-26. 71. Villalta F, Zhang Y, Bibb KE et al. The cysteine-cysteine family of chemokines RANTES, MIP-1alpha, and MIP-1beta induce trypanocidal activity in human macrophages via nitric oxide. Infect Immun 1998; 66:4690-5. 72. Jaffe AB, Hall A. RHO GTPases: Biochemistry and biology. Annu Rev Cell Dev Biol 2005. 73. Fernandes AB, Mortara RA. Invasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages. Microbes Infect 2004; 6:460-467. 74. Rosestolato CT, Dutra Jda M, De Souza W et al. Participation of host cell actin filaments during interaction of trypomastigote forms of Trypanosoma cruzi with host cells. Cell Struct Funct 2002; 27:91-98. 75. Ochatt CM, Mayorga LS, Isola EL et al. Inhibition of early endosome fusion by Trypanosoma cruzi-infected macrophage cytosol. J Eukaryot Microbiol 1997; 44:497-502. 76. Niedergang F, Chavrier P. Regulation of phagocytosis by Rho GTPases. Curr Top Microbiol Immunol 2005; 291:43-60. 77. Niedergang F, Chavrier P. Signaling and membrane dynamics during phagocytosis: Many roads lead to the phagos(R)ome. Curr Opin Cell Biol 2004; 16:422-428. 78. Jutras I, Desjardins M. Phagocytosis: At the crossroads of innate and adaptive immunity. Annu Rev Cell Dev Biol 2005. 79. Santiago HC, Feng CG, Bafica A et al. Mice deficient in LRG-47 display enhanced susceptibility to Trypanosoma cruzi infection associated with defective hemopoiesis and intracellular control of parasite growth. J Immunol 2005; 175:8165-72. 80. Batista DG, Silva CF, Mota RA et al. Trypanosoma cruzi modulates the expression of Rabs and alters the endocytosis in mouse cardiomyocytes in vitro. J Histochem Cytochem 2005. 81. Underhill DM, Gantner B. Integration of Toll-like receptor and phagocytic signaling for tailored immunity. Microbes Infect 2004; 6:1368-1373. 82. Procopio DO, da Silva S, Cunningham CC et al. Trypanosoma cruzi: Effect of protein kinase inhibitors and cytoskeletal protein organization and expression on host cell invasion by amastigotes and metacyclic trypomastigotes. Exp Parasitol 1998; 90:1-13. 83. Ley V, Andrews NW, Robbins ES et al. Amastigotes of Trypanosoma cruzi sustain an infective cycle in mammalian cells. J Exp Med 1988; 168:649-59. 84. Kahn S, Wleklinski M, Aruffo A et al. Trypanosoma cruzi amastigote adhesion to macrophages is facilitated by the mannose receptor. J Exp Med 1995; 182:1243-58. 85. Benchimol Barbosa PR. The oral transmission of Chagas’ disease: An acute form of infection responsible for regional outbreaks. Int J Cardiol 2006. 86. Eickhoff CS, Eckmann L, Hoft DF. Differential interleukin-8 and nitric oxide production in epithelial cells induced by mucosally invasive and noninvasive Trypanosoma cruzi trypomastigotes. Infect Immun 2003; 71:5394-7. 87. Campos MA, Almeida IC, Takeuchi O et al. Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite. J Immunol 2001; 167:416-423. 88. Oliveira AC, Peixoto JR, de Arruda LB et al. Expression of functional TLR4 confers proinflammatory responsiveness to Trypanosoma cruzi glycoinositolphospholipids and higher resistance to infection with T. cruzi. J Immunol 2004; 173:5688-96. 89. Almeida IC, Camargo MM, Procopio DO et al. Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. Embo J 2000; 19:1476-1485. 90. Chan CW, Crafton E, Fan HN et al. Interferon-producing killer dendritic cells provide a link between innate and adaptive immunity. Nat Med 2006; 12:207-213. 91. Mukherjee S, Huang H, Weiss LM et al. Role of vasoactive mediators in the pathogenesis of Chagas’ disease. Front Biosci 2003; 8:e410-9. 92. Combs TP, Nagajyothi, Mukherjee S et al. The adipocyte as an important target cell for Trypanosoma cruzi infection. J Biol Chem 2005; 280:24085-94. 93. Rimoldi MT, Tenner AJ, Bobak DA et al. Complement component C1q enhances invasion of human mononuclear phagocytes and fibroblasts by Trypanosoma cruzi trypomastigotes. J Clin Invest 1989; 84:1982-1989.

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94. Ferreira V, Valck C, Sanchez G et al. The classical activation pathway of the human complement system is specifically inhibited by calreticulin from Trypanosoma cruzi. J Immunol 2004; 172:3042-50. 95. Molina MC, Ferreira V, Valck C et al. An in vivo role for Trypanosoma cruzi calreticulin in antiangiogenesis. Mol Biochem Parasitol 2005; 140:133-140. 96. Ogden CA, deCathelineau A, Hoffmann PR et al. C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med 2001; 194:781-95. 97. Freire-de-Lima CG, Nascimento DO, Soares MB et al. Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages. Nature 2000; 403:199-203. 98. Aliberti J, Viola JP, Vieira-de-Abreu A et al. Cutting edge: Bradykinin induces IL-12 production by dendritic cells: A danger signal that drives Th1 polarization. J Immunol 2003; 170:5349-53. 99. Herz J, Strickland DK. LRP: A multifunctional scavenger and signaling receptor. J Clin Invest 2001; 108:779-84. 100. Coutinho CM, Cavalcanti GH, van Leuven F et al. Alpha-2-macroglobulin binds to the surface of Trypanosoma cruzi. Parasitol Res 1997; 83:144-150. 101. Murta AC, Persechini PM, Padron Tde S et al. Structural and functional identification of GP57/ 51 antigen of Trypanosoma cruzi as a cysteine proteinase. Mol Biochem Parasitol 1990; 43:27-38. 102. Morrot A, Strickland DK, Higuchi Mde L et al. Human T cell responses against the major cysteine proteinase (cruzipain) of Trypanosoma cruzi: Role of the multifunctional alpha 2-macroglobulin receptor in antigen presentation by monocytes. Int Immunol 1997; 9:825-34. 103. Arnholdt AC, Piuvezam MR, Russo DM et al. Analysis and partial epitope mapping of human T cell responses to Trypanosoma cruzi cysteinyl proteinase. J Immunol 1993; 151:3171-9. 104. Waghabi MC, Coutinho CM, Soeiro MN et al. Increased Trypanosoma cruzi invasion and heart fibrosis associated with high transforming growth factor beta levels in mice deficient in alpha(2)-macroglobulin. Infect Immun 2002; 70:5115-23. 105. DosReis GA, Freire-de-Lima CG, Nunes MP et al. The importance of aberrant T-cell responses in Chagas disease. Trends Parasitol 2005; 21:237-43. 106. de Souza EM, Araujo-Jorge TC, Bailly C et al. Host and parasite apoptosis following Trypanosoma cruzi infection in in vitro and in vivo models. Cell Tissue Res 2003; 314:223-35. 107. Alcaide P, Fresno M. AgC10, a mucin from Trypanosoma cruzi, destabilizes TNF and cyclooxygenase-2 mRNA by inhibiting mitogen-activated protein kinase p38. Eur J Immunol 2004; 34:1695-1704. 108. Gordon S. Alternative activation of macrophages. Nat Rev Immunol 2003; 3:23-35. 109. Stempin C, Giordanengo L, Gea S et al. Alternative activation and increase of Trypanosoma cruzi survival in murine macrophages stimulated by cruzipain, a parasite antigen. J Leukoc Biol 2002; 72:727-734. 110. Procopio DO, Teixeira MM, Camargo MM et al. Differential inhibitory mechanism of cyclic AMP on TNF-alpha and IL-12 synthesis by macrophages exposed to microbial stimuli. Br J Pharmacol 1999; 127:1195-205. 111. Chaussabel D, Pajak B, Vercruysse V et al. Alteration of migration and maturation of dendritic cells and T-cell depletion in the course of experimental Trypanosoma cruzi infection. Lab Invest 2003; 83:1373-1382. 112. Machado FS, Koyama NS, Carregaro V et al. CCR5 plays a critical role in the development of myocarditis and host protection in mice infected with Trypanosoma cruzi. J Infect Dis 2005; 191:627-36. 113. Teixeira MM, Gazzinelli RT, Silva JS. Chemokines, inflammation and Trypanosoma cruzi infection. Trends Parasitol 2002; 18:262-5. 114. Hardison JL, Wrightsman RA, Carpenter PM et al. The CC chemokine receptor 5 is important in control of parasite replication and acute cardiac inflammation following infection with Trypanosoma cruzi. Infect Immun 2006; 74:135-43. 115. Marino AP, da Silva A, dos Santo P et al. Regulated on activation, normal T cell expressed and secreted (RANTES) antagonist (Met-RANTES) controls the early phase of Trypanosoma cruzielicited myocarditis. Circulation 2004; 100:1443-1449.

CHAPTER 3

Macrophage – Leishmania Interactions: Complexities and Uncertainties from the Study of Leishmaniasis in Vivo Paul M. Kaye*

Abstract

L

eishmania parasites are intracellular pathogens residing predominantly within mononuclear phagocytes. Whilst valuable insights into the host-pathogen interaction have been obtained from the study of “typical” macrophage and dendritic cell populations in vitro, such studies may greatly underestimate the heterogeneity of mononuclear phagocytes encountered by Leishmania in vivo, and the microenvironmental constraints under which these cells function. In this review, some aspects of the in vivo interaction between Leishmania and the mononuclear phagocyte system are discussed.

Introduction A dozen or more species of Leishmania parasite infect man, giving rise to a complex spectrum of diseases known as the leishmaniases. At one end of this spectrum lies Old World cutaneous leishmaniasis, an ultimately self healing infection associated with the development of protective immunity and long lived memory to reinfection. At the other pole is visceral leishmaniasis, in most cases fatal if untreated. Lying in between are various less frequent manifestations including diffuse or disseminated leishmaniasis and metastatic mucocutaneous leishmaniasis.1,2 Leishmania parasites are vector borne, transmitted by female phelbotomine sandflies during the taking of a blood meal. Infection is initiated following regurgitation of metacyclic promastigote stage parasites into the pool of blood created by laceration of dermal capillaries.3 Parasite and host genetic diversity, environmental factors such as nutritional status and ultimately the nature of the host immune response all contribute to the diversity of clinical manifestations and the final outcome of infection. Epidemiological studies suggest most infections do not result in clinical disease. For visceral leishmaniasis caused by L. infantum in particular, subclinical infection with life long persistence of parasites may be the norm in a fully immunocompetent host. A number of factors contribute to the establishment of infection. Accompanying parasites during vector-borne transmission is an extracellular virulence factor, the promastigote secretory gel (PSG), secreted by promastigotes during their residence in the sandfly foregut. Sandfly salivary components also have potent anti-inflammatory properties, in addition to their role in anti-coagulation.3,4 Metacyclic promastigotes activate complement and thus rapidly become opsonised and targeted to phagocytes bearing complement receptors. In the immunologically naïve host, the extent of opsonisation by complement and natural antibodies, as well as *Paul M. Kaye—Immunology and Infection Unit, The Hull York Medical School, and Department of Biology, University of York, P.O. Box 373, York YO10 5YW, U.K. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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species-specific variation in expression of surface proteins and glycolipids recognized by alternate innate pathogen recognition receptors, determine whether metacyclic promastigotes are targeted for Type I or Type II phagocytosis.5,6 Infection of mononuclear phagocytes is one of the most common features associated with leishmaniasis, and the Leishmania life cycle in its mammalian host is often regarded as a relatively simple series of events: metacyclic promastigotes transform within the phagolysosome into replicative amastigotes; amastigotes survive basal levels of lysosomal activity and their multiplication eventually leads to host cell rupture; released amastigotes are phagocytosed by new macrophages and the cycle continues; finally, infected macrophages are taken up during the blood meal to infect further sandflies. At a time and place which remains to be precisely determined, induction of acquired immunity occurs. This sets in motion a complex immunoregulatory cascade, the nature of which will determine the subsequent course of infection. Amastigotes are eventually eliminated by a variety of well-understood effector mechanisms, generally regarded as involving the production of IFNγ by TH1 CD4+ T cells and activation of macrophage NOS2 gene expression.1 CD8+ T cells have a clear protective role in certain models.7-9 It is generally accepted that dendritic cells (DCs) play a significant role in the induction of immunity.10 Other chapters in this volume deal extensively with the ability of Leishmania parasites to subvert macrophage function, notably in terms of their interference with macrophage intracellular signaling pathways [see Mosser, this volume]. Largely, such studies have been performed in vitro using well characterized, readily obtainable mononuclear phagocyte populations. This chapter will briefly review the heterogeneity of mononuclear phagocytes encountered by Leishmania parasites in vivo. The importance of the tissue microenvironment for the initiation and maintenance of appropriate immune responses is highlighted, in relation to Leishmania-induced immunopathology and to infection-induced changes in the differentiation of monocytes, macrophages and DCs. Finally, the question of whether macrophages participate in the induction of T cell responses will be discussed, given that evasion of immune recognition by subversion of antigen presentation has been a dominant theme of in vitro studies. Only by understanding the breadth of function and diversity of mononuclear phagocytes are we likely to gain a full appreciation of the pathogenesis of the leishmaniases.

Diversity of Mononuclear Phagocytes The mononuclear phagocyte system (MPS) was proposed by van Furth in the 1970s to define cells originating as myeloid progenitors in the bone marrow and which through intermediary blood borne monocytes, become seeded in the tissues as mature resident macrophages.11 In subsequent years, the subdivision of the MPS has become more extensive with an ongoing tension between those wedded to a developmental continuum and those erring towards distinct precursor-product relationships to explain the origin of diverse tissue mononuclear phagocytes [ref. 12 and Gordon, this volume]. Although DCs were originally excluded from membership of the MPS, their close ontogenic relationship with other mononuclear phagocytes is now clear, and it is beyond question that they are mononuclear and phagocytic, notably in their immature form.10 Although lineage commitment can only strictly be studied by in vivo reconstitution assays, lineage assignment is, for all practical purposes during the study of infectious disease, reduced to an exercise in antibody-based phenotyping, occasionally complemented by analysis of transcription factor expression. Few single criteria provide a definitive assignment of lineage for the MPS, or even subdivide DCs from conventional macrophages. For example, the F4/80 (EMR-1) protein is expressed on most but not all macrophages—and rarely on DCs,12-15 whereas CD11c, often regarded as the cardinal DC marker, is abundant on alveolar macrophages.16 The transcription factor PU-1 and the CSF-1R (c-fms) are critical in the differentiation of all mononuclear phagocytes.12 JNK activation, as a consequence of c-fms engagement by CSF-1, appears essential for macrophage development, proliferation and survival. Hence inhibition of JNK leads to decreased c-fms expression and repressed differentiation in response to CSF-1, as

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well as disruption of PU1 binding to the c-fms promoter.17 C/EBPα is also required for myeloid cell fate determination,18 whereas Runx-1 KO mice develop neither macrophages nor granulocytes.19 Other factors e.g., hypoxia-induced factor-1 appear essential for acquisition of macrophage effector function but are dispensable for the differentiation of monocytes into macrophages.20 Inclusion of DCs as members of the MPS is compatible with a definition of Leishmania parasites as intracellular parasites of the MPS. But is this definition broad enough? Polymorphonuclear neutrophils (PMNs) have recently been shown to sustain short term infection by Leishmania, and to facilitate silent uptake into macrophages.21 PMNs express CSF-R1 (as determined in CSF-R1-EGFP mice) and F4/80 mRNA, supporting ancestry from a common myeloid progenitor cell.12 At least in vitro, some studies have observed apparent inter-conversion of PMNs to DCs.22 More controversially, some mononuclear phagocytes have the capacity to trans-differentiate into myofibroblasts, endothelial cells and even muscle23,24 and Leishmania amastigotes have been seen in a range of ostensibly nonhematopoietic cells.25,26 Perhaps further study of the host range of Leishmania using a broader range of criteria to define cell lineage will provide additional clues to the diversity of the MPS?

Host Cells for Leishmania In which mononuclear phagocytes do Leishmania parasites reside during the course of experimental infection in vivo? Two main experimental approaches to answering this question have been adopted, each bringing with it its own advantages and disadvantages. Immuno-histochemistry allows ready identification of cells containing parasites and/or their antigens and preserves the microenvironmental aspects of the infection. To unambiguously discriminate intracellular amastigotes from antigenic debris, the use of transgenic parasites that express fluorochromes such as DSRed27 or EGFP28 is slowly replacing immuno-labelling. An additional advantage of the histopathological approach is that host cell phenotype is captured at the time point of tissue isolation. Nevertheless, given the phenotypic complexity of mononuclear phagocytes, and a limitation of at best three-parameter analysis during immunohistology, subtle aspects of cell phenotyping may be difficult to resolve. Functional assessment of infected cells is also clearly problematic. However, the enthusiasm for employing multiple markers to provide a detailed phenotypic analysis of infected cells needs to be tempered by the knowledge that (i) heavily infected cells are fragile and may be lost and/or release amastigotes during the preparation of cell suspensions, (ii) that extended isolation procedures can dramatically alter cell surface phenotype and (iii) that all spatial and microenvironmental information is lost. Cells attributed as hosts for Leishmania are listed in Table 1. Some of the difficulties in identifying the true host cell for Leishmania become evident when considering what many regard as one of the key steps in pathogenesis—the initial fate of metacyclic promastigotes. Using high dose infection of L. major into the footpad, Langerhans cells were first attributed with the role of transport of parasites to the draining LN (dLN) and with the induction of CD4+ T cell responses.29 Subsequent analysis with more refined markers of DC subpopulations,30,31 as well as a greater understanding of the role of Langerhans cells derived from cell-specific depletion32 suggest this model is incorrect. Rather, parasite antigen, probably from dead parasites, is taken up over the first 16h post infection by CD11c+CD11b+CD8- DCs in the dLN to initiate a first wave of antigen presentation and T cell activation. Only rare parasites, apparently not within host cells, were seen at these early times. By 24-48h, however, CD11c+CD11b+CD8- CD205+ DCs containing EGFP+ parasites were detectable in the dLN.33 PMNs are also rapidly recruited into high dose lesions and acquire parasites readily.21 In contrast to these studies, building on the low dose L. major infection model established by Belkaid and colleagues,34 von Stebut has recently provided elegant evidence that dermal DCs largely ignore metacyclic promastigotes, this stage being avidly engulfed in an FcR/CR3 dependent manner by dermal macrophages.35 Strikingly, amastigotes of L. major only enter DCs when opsonised with immune IgG. Hence, DC infection is delayed

41

Macrophage – Leishmania Interactions

Table 1. Host cells for L. donovani and L. major in vivo Parasite

Tissue / Time p.i.

L. donovani

Spleen 5-24h Marginal zone

Spleen d14-56 Red pulp

PALS Marginal zone Bone marrow d28+

L. major

Dermis (5wk low dose)

Footpad lesion

dLN

Cell Phenotype

References

India ink+ER-TR9+ MOMA-1+ CD169hi CD68+

37 37 48

MOMA-2+FA/11+MHCII+ CD68+ ER-TR7+ MOMA-2+FA/11+MHCII+/-DEC-205CD169hiMOMA-2loMHCIIlo MOMA-2+MHCII+CD169lo CD169hiFA/11CD169-FA/11+ CD11c+CD205+MHCII+ F4/80+MHCIIlo/F4/80+GR-1+ F4/80- + Eosinophil morphology CD11c+CD11b-Gr1CD11c-CD11b+Gr1CD11c-CD11b-Gr1+ CD11c+ (inc. multinucleated cells) CD11clo CD11c+CD11b+F4/80+ MHCIIloCD86CD11c+CD11b+CD8- CD205+

38 26 26 38 38 38 74, 66 74 34 34 34 34 36 36 36 36 36 36 33

Data are mainly based on examination of tissues by immunochemistry or by cytospin of emigrating cells (for dermis).

until amastigotes become released from infected macrophages and serum IgG titres have increased appreciably - effectively 4-5 weeks after infection and corresponding to the end of the “silent” phase of infection.34 What remains to be determined is the cellular basis for this apparent DC-independent class-switched antibody response. Perhaps macrophages are the first cell to transport parasites to the LN after all?30 Host cell heterogeneity is also a complex issue later during infection. In probably one of the most comprehensive studies of its type in cutaneous leishmaniasis, Muraille and colleagues examined the distribution of L. major parasites in lesions and in the draining LN of mice infected for 4 weeks.36 Of those cells in the lesion that were positive for amastigotes by HE staining, approximately 20% were CD11b+CD11c-Gr1-, 10% were Gr1+CD11b-CD11cand less than 5% were Cd11c+CD11b-Gr1-. The phenotype of the remainder of infected cells was not reported. By contrast CD11c+ cells, referred to as DCs by the authors, contained the bulk of amastigotes in LN and CD11c+ cells contained on average 2 fold (in resistant B6) and 5-fold (in susceptible BALB/c) the number of amastigotes per cell as compared to CD11b+ cells in the same mice. In BALB/c mice, where amastigote load is considerably higher than in B6 mice at this time point, amastigotes were also identified in CD11cdimMHCIIdimCD86- cells in the subcapsular region and the cortical and paracortical sinus. CD11cdimMHCIIdimCD86- multinucleate cells were also seen in BALB/c mice and these contained very large numbers of amastigotes. Whilst the authors consider a number of possible origins for these CD11cdim cells, including that they are monocyte derived DCs,

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the possibility that these represent tissue macrophages that have upregulated CD11c was not discussed. Unfortunately the study also does not detail the absolute numbers of each cell type, so conclusions as to which cell type harbors the bulk of tissue amastigote burden is unclear. In the context of visceral leishmaniasis most studies have employed direct intravenous infection of amastigotes to initiate infection. Hence, little can be said about the tropism of visceralising species for skin or dLN macrophages and DCs. Nevertheless, their distribution in the spleen has been intensively studied. Within 1h of infection amastigotes were found distributed mainly in the marginal zone.37 At later time points, amastigotes were also found within MOMA-2+ Macrosialin+ MHC11+ macrophages in the red pulp and in some MOMA-2+ Macrosialin+ MHCII+ macrophages residing within the residual white pulp.38 Importantly, whereas infected PMNs were not found in situ, such cells could be readily observed following preparation of single cell suspensions of infected spleens. This result suggests that PMNs may acquire amastigotes rapidly during cell harvesting procedures. DEC-205+ splenic DCs were not found to be infected.38 Host cell heterogeneity is also observed in the liver, during the process of granulomatous inflammation (reviewed in refs. 39,40).

Remodeling the Environment In the steady state, blood borne monocytes undergo site specific differentiation to acquire the characteristics required of tissue macrophages.15 Hence monocytes may differentiate into Kupffer cells in the liver or alveolar macrophages in the lung. In secondary lymphoid tissues, tissue macrophages show extensive heterogeneity and local differentiation. Most striking in terms of organization are the macrophages of the rodent spleen, readily segregated anatomically, phenotypically, and functionally.41 Whilst sparse in the splenic white pulp, the red pulp contains abundant F4/80+ macrophages and the marginal zone plays host to two discrete populations of F4/80- macrophages. Marginal zone macrophages (MZMs) are usually defined by their avid uptake of injected carbon and by expression of SIGNR1. Marginal metallophilic macrophages (MMMs) express the as-yet uncharacterised MOMA-1 antigen as well as high levels of sialoadhesin (CD169).42,43 Whereas red pulp macrophages are largely involved in blood filtration and clearance of effete red cells, MZMs play a specialized role in the capture of polysaccharide antigens, form an essential component of innate defense against capsulated bacteria,44 and may assist in directing leucocyte traffic into the white pulp.45 MMMs are less phagocytic than MZMs (but can certainly acquire Leishmania amastigotes)37 but may acquire antigen via mannose receptor-mediated uptake and can be stimulated to migrate from the marginal zone into B cell follicles following LPS exposure.46,47 What governs this unique functional specialization of splenic macrophages and their equally remarkable spatial organization is largely not understood. Absence of specific populations of macrophages in the marginal zone is a feature of mice deficient in TNF superfamily cytokines and / or their receptors and is also a consequence of targeted deletion of various transcription factors.41 Nevertheless, how these factors actually regulate macrophage organization remains unclear. Similarly, although the chemokines CCL19 and CCL21 are involved in the positioning of MZMs at the outer rim of the marginal zone,48 it is not clear whether such positioning is necessary for MZMs to acquire other functional attributes or if this is cell-intrinsic. Intriguingly a role for MARCO, a scavenger receptor, in macrophage positioning has also been suggested.49 MARCO is expressed by MZMs in adult mice along with SIGNR1 and low level expression of CD169. However, during ontogeny, MARCO-expressing macrophages appear in the spleen before cells expressing SIGNR1, suggesting that interactions with endogenous MARCO ligands may guide macrophages to the marginal zone. L. donovani infection has a dramatic impact of splenic mononuclear phagocytes. MZMs are lost from the marginal zone,50 red pulp macrophages hypertrophy and heavily infected macrophages dominate involuted germinal centres and the residual white pulp region.51,52 Loss of MZMs can be partially attributed to the collapse of constitutive chemokine gradients,48 but

Macrophage – Leishmania Interactions

43

infected MZMs may eventually rupture and not be replenished or CTL-directed killing may occur, as in some viral infections.53,54 DCs in the chronically infected spleen also fail to migrate appropriately and become localized in the marginal zone and around infected CD11c- cells, a result of IL-10-mediated inhibition of CCR7 expression,55 and this may make some contribution to generalized immunosuppression seen during infection. Data on remodeling of the LN microenvironment during the progression of the various forms of cutaneous leishmaniasis is currently lacking. The recent use of Hox8 to immortalize myeloid progenitors for long term functional study of their progeny56 suggests exciting new approaches may soon be at hand to address the heterogeneity of macrophages in leishmaniasis. Studies in gene KO mice also suggest an intimate link between B cells and the structural integrity of the marginal zone and the development of MZMs. For example, B cell deficient mice lack MZMs, MMMs and the marginal sinus and transient B cell lymphopenia may trigger changes to marginal zone composition.57 Though B cells have generally been excluded as major players in anti-leishmanial immunity, altered B cell homeostasis may thus indirectly impact on macrophage behavior. Unfortunately, the development of immunopathology and tissue remodeling can not as yet be fully divorced from those mechanisms needed to control parasite multiplication. High levels of TNF appear critical for many of the structural alterations seen, whereas lower levels of TNF may be sufficient for appropriate host defence.58 TNF blockade has found a role in the treatment of chronic leishmaniasis,59 and the possibility that reconstitution of lymphoid tissue architecture resulted from this treatment and played a role in efficacy deserves further investigation.

Mononuclear Phagocyte Differentiation In the mouse, the spleen is a site of extramedullary hematopoiesis, reflecting both the presence of splenic hematopoietic stem / progenitor cells (HSC/HPCs) and stromal cells able to support their survival, differentiation and cell fate decisions. Recent work has demonstrated the capacity of stromal cells to direct DC differentiation, using both long term stromal cell cultures60 as well ex vivo stromal cell populations.26,61 Expression profiling has allowed partial characterization of the hematopoietic niche required for their development.62 Splenic stromal cells from naïve mice directed HPCs down the myeloid differentiation pathway, culminating in the development of CD11b+ CD11cmid cells bearing intracellular MHCII and producing IL-10 mRNA in high abundance.26 When exposed to TNF, these cells increased expression of CD11c and relocated MHCII to the plasma membrane—two characteristics of DC maturation. Strikingly, these CD11c+ cells suppressed primary mixed lymphocyte responses, induced IL-10 producing CD4+ T cells from naïve precursors in vitro, and when transferred in vivo, induced CD4+ T cells capable of transfer of tolerance to antigen. These experiments provided a plausible mechanism for the local steady state development of regulatory DCs in mouse spleen and hence the maintenance of local tolerance.63 More recently, splenic endothelium has also been shown to support HSC to DC differentiation, and though such DCs have regulatory function, this is largely mediated though NO production and they did not induce either regulatory T cells or T cell anergy.61 Endothelium also appears to induce a similar regulatory phenotype on mature DCs suggesting that endothelial interactions may also play a role in terminating immune responses.64 Like other chronic infectious diseases, infection with L. donovani65,66 and L. major67 results in an increase in hematopoietic activity. During L. donovani infection, increased hematopoiesis is associated with the onset of splenomegaly and loss of control over splenic amastigote replication.65 Ex vivo stromal cells (comprising ERTR7+ stromal fibroblasts and CD68+ stromal macrophages) from infected mice had greater capacity to generate regulatory DCs from HPCs.26 At least in vitro, infection with L. donovani amastigotes is sufficient to upregulate this supportive role of stromal cells (Svensson, unpublished). However, it remains circumstantial that this pathway of DC differentiation operates in vivo as a means of regulating anti-leishmanial immunity. As chronic infection progresses, increased numbers of CD11c+ cells accumulate in the

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spleen, most of which are MHCIIhigh. Nevertheless, functionally these cells are less effective in a bulk population at stimulating T cell responses, and on adoptive transfer to infected recipients they enhance disease. Other specialized mononuclear phagocytes in the spleen may also differentiate locally or undergo self-renewal, aided by alterations to distinct stromal cell niches, but quantitative data is unavailable in leishmaniasis.

T Cell Priming The ability to prime naive T lymphocytes is regarded as a hallmark of DCs and is taken to reflect a unique combination of cell biological attributes which allow i) efficient antigen/pathogen uptake ii) effective antigen processing to generate MHCI- and MHCII-binding peptides iii) highly regulated MHC expression iv) high levels of signal 2 (costimulation).10 Additionally, DCs have the capacity to produce cytokines ensuring appropriate T cell differentiation (signal 3).68 Evidence for this unique role of DCs stretches back to the mid 1980’s and is typified by the outstanding performance of isolated DCs as stimulators of the mixed lymphocyte response. The concept that DCs alone are capable of initiating T cell priming is important in leishmaniasis research for two reasons - first, it predicates that DCs induce MHCI and MHCII restricted T cell immunity, ergo that Leishmania-infected DCs or DCs that have captured leishmanial antigen take a preeminent place in the immunological hierarchy compared to other infected mononuclear phagocytes. Second, it questions the importance of some pathogen “immune evasion strategies” reported to be a feature of macrophage infection. Does the dogma therefore hold true in the complex setting of immune induction during Leishmania infection? Using the conventional footpad model of L. major infection, this question has been addressed in relation to MHCII restricted CD4+ T cell responses. By making use of mice in which MHCII was specifically targeted to CD11c+ DCs (including both the CD11b+ and CD8α+ subsets), Lemos et al69 demonstrated that cognate MHC dependent CD4+ T cell activation by “CD11c-” cells is neither essential for T cell priming nor for the expression of effector function, i.e., parasite killing and lesion resolution. Polyclonal CD4+ T cells transferred into TCRα-/- mice (lacking endogenous T cells but with WT MHCII expression) controlled L. major infection, whereas similar transfer into Aβb-/- mice (lacking all MHCII) failed to do so. Strikingly, transfer of these T cells into CD11c/Aβb mice (in which Aβ is reconstituted only in CD11c+ cells) resulted in mice fully able to resist lesion development and contain parasite numbers. Hence, restriction of MHCII to CD11c DCs is sufficient to promote all CD4+ T cell function associated with host resistance to L. major. Although this result is consistent with an exclusive role for DCs in CD4+ T cell priming, the data have wider implications. Most significantly, this result suggests that either macrophage effector function (i.e., NO-dependent parasite killing) requires DCs to participate in local reactivation of effector CD4+ T cell cytokine release or alternatively that CD4+ T cell effector function is essentially MHCII-independent, and does not require local antigen recognition during an effector cell-target cell synapse. On face value, both of these interpretations also suggest that attempts by amastigotes to inhibit MHCII processing and presentation in macrophages are futile and of no functional consequence! However, there are caveats to the study. Whilst MHCII expression on DCs is seen to be sufficient to promote resistance, these experiments do not address whether MHCII expression on macrophages contributes to resistance. Similarly, the authors did not formally rule out whether ectopic expression of CD11c on macrophages occurred during chronic L. major infection. It is certainly clear from studies both in visceral leishmaniasis and in other infectious and inflammatory conditions that CD11c expression can be observed in cells that would by other criteria be called tissue macrophages, e.g., foam cells in tuberculosis70 and granuloma macrophages in leishmaniasis (Beattie, unpublished). Ectopic CD11c expression may account for loss of classical marginal zone macrophage populations following administration of diphtheria toxin to CD11c-DT transgenic mice.71 Finally, these studies would be valuable to conduct using low dose infection models, to determine DC involvement in the early IgG response to L. major infection.35

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In addition to MHCII restricted CD4+ T cell responses, some models of Leishmania also show a role for MHCI-restricted CD8+ T cells, including the low dose L. major dermis model,7 and models of visceral leishmaniasis.9,72 Hence, the recent observation that peptide-pulsed macrophages can effectively stimulate murine CD8+ T cell responses after adoptive transfer into naïve mice and in the absence of cross-presentation by DCs is of potential significance. Notably, the migratory properties of macrophages reported in these transfer studies were very similar to those of DCs.73

Concluding Remarks Although many aspects mononuclear phagocyte biology are conserved, and may reflect a core environment for Leishmania survival, many distinctions in cell biology, receptor heterogeneity and cross-talk, cytokine and chemokine response and responsiveness, and effector capacity almost certainly exist and remain to be fully characterized. Over the next few years, advances in our understanding of the differentiation and microenvironmental control of mononuclear phagocytes, as well as new approaches to manipulate gene expression in a cell specific manner in vivo are likely to contribute greatly to expanding our appreciation of the complexities underlying Leishmania-macrophage interactions.

Acknowledgements I would like to thank the various members of my laboratory over the past years for their valuable input and discussion and Liz Hearn for help with preparation of this manuscript.

References 1. Davies CR, Kaye P, Croft SL et al. Leishmaniasis: New approaches to disease control. Bmj 2003; 326(7385):377-82. 2. Murray HW, Berman JD, Davies CR et al. Advances in leishmaniasis. Lancet 2005; 366(9496):1561-77. 3. Rogers ME, Ilg T, Nikolaev AV et al. Transmission of cutaneous leishmaniasis by sand flies is enhanced by regurgitation of fPPG. Nature 2004; 430(6998):463-67. 4. Bates PA, Rogers ME. New insights into the developmental biology and transmission mechanisms of Leishmania. Curr Mol Med 2004; 4(6):601-09. 5. Caron E, Hall A. Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases. Science 1998; 282(5394):1717-21. 6. Morehead J, Coppens I, Andrews NW. Opsonization modulates Rac-1 activation during cell entry by Leishmania amazonensis. Infect Immun 2002; 70(8):4571-80. 7. Belkaid Y, Von Stebut E, Mendez S et al. CD8+ T cells are required for primary immunity in C57BL/6 mice following low-dose, intradermal challenge with Leishmania major. J Immunol 2002; 168(8):3992-00. 8. Herath S, Kropf P, Muller I. Cross-talk between CD8(+) and CD4(+) T cells in experimental cutaneous leishmaniasis: CD8(+) T cells are required for optimal IFN-gamma production by CD4(+) T cells. Parasite Immunol 2003; 25(11-12):559-67. 9. Polley R, Stager S, Prickett S et al. Adoptive immunotherapy against experimental visceral leishmaniasis with CD8+ T cells requires the presence of cognate antigen. Infect Immun 2006; 74(1):773-76. 10. Mellman I, Steinman RM. Dendritic cells: Specialized and regulated antigen processing machines. Cell 2001; 106(3):255-58. 11. van Furth R, Cohn ZA, Hirsch JG et al. The mononuclear phagocyte system: A new classification of macrophages, monocytes, and their precursor cells. Bull World Health Organ 1972; 46(6):845-52. 12. Hume DA. The mononuclear phagocyte system. Curr Opin Immunol 2006; 18(1):49-53. 13. Austyn JM, Gordon S. F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur J Immunol 1981; 11(10):805-15. 14. Ezekowitz RA, Gordon S. Down-regulation of mannosyl receptor-mediated endocytosis and antigen F4/80 in bacillus Calmette-Guerin-activated mouse macrophages. Role of T lymphocytes and lymphokines. J Exp Med 1982; 155(6):1623-37. 15. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol 2005; 5(12):953-64. 16. Kirby AC, Raynes JG, Kaye PM. CD11b regulates recruitment of alveolar macrophages but not pulmonary dendritic cells after pneumococcal challenge. J Infect Dis 2006; 193(2):205-13.

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17. Himes SR, Sester DP, Ravasi T et al. The JNK are important for development and survival of macrophages. J Immunol 2006; 176(4):2219-28. 18. Suh HC, Gooya J, Renn K et al. C/EBP{alpha} determines hematopoietic cell fate in multipotential progenitor cells by inhibiting erythroid differentiation and inducing myeloid differentiation. Blood 2006. 19. Samokhvalov IM, Thomson AM, Lalancette C et al. Multifunctional reversible knockout/reporter system enabling fully functional reconstitution of the AML1/Runx1 locus and rescue of hematopoiesis. Genesis 2006; 44(3):115-21. 20. Oda T, Hirota K, Nishi K et al. Activation of hypoxia-inducible factor 1 during macrophage differentiation. Am J Physiol Cell Physiol 2006. 21. van Zandbergen G, Klinger M, Mueller A et al. Cutting edge: Neutrophil granulocyte serves as a vector for Leishmania entry into macrophages. J Immunol 2004; 173(11):6521-25. 22. Araki H, Katayama N, Yamashita Y et al. Reprogramming of human postmitotic neutrophils into macrophages by growth factors. Blood 2004; 103(8):2973-80. 23. Elsheikh E, Uzunel M, He Z et al. Only a specific subset of human peripheral-blood monocytes has endothelial-like functional capacity. Blood 2005; 106(7):2347-55. 24. Jabs A, Moncada GA, Nichols CE et al. Peripheral blood mononuclear cells acquire myofibroblast characteristics in granulation tissue. J Vasc Res 2005; 42(2):174-80. 25. Bogdan C, Donhauser N, Doring R et al. Fibroblasts as host cells in latent leishmaniosis. J Exp Med 2000; 191(12):2121-30. 26. Svensson M, Maroof A, Ato M et al. Stromal cells direct local differentiation of regulatory dendritic cells. Immunity 2004; 21(6):805-16. 27. Sorensen M, Lippuner C, Kaiser T et al. Rapidly maturing red fluorescent protein variants with strongly enhanced brightness in bacteria. FEBS Lett 2003; 552(2-3):110-14. 28. Misslitz A, Mottram JC, Overath P et al. Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes. Mol Biochem Parasitol 2000; 107(2):251-61. 29. Moll H, Fuchs H, Blank C et al. Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells. Eur J Immunol 1993; 23(7):1595-01. 30. Baldwin T, Henri S, Curtis J et al. Dendritic cell populations in Leishmania major-infected skin and draining lymph nodes. Infect Immun 2004; 72(4):1991-01. 31. Ritter U, Meissner A, Scheidig C et al. CD8 alpha- and Langerin-negative dendritic cells, but not Langerhans cells, act as principal antigen-presenting cells in leishmaniasis. Eur J Immunol 2004; 34(6):1542-50. 32. Bennett CL, van Rijn E, Jung S et al. Inducible ablation of mouse Langerhans cells diminishes but fails to abrogate contact hypersensitivity. J Cell Biol 2005; 169(4):569-76. 33. Misslitz AC, Bonhagen K, Harbecke D et al. Two waves of antigen-containing dendritic cells in vivo in experimental Leishmania major infection. Eur J Immunol 2004; 34(3):715-25. 34. Belkaid Y, Mendez S, Lira R et al. A natural model of Leishmania major infection reveals a prolonged “silent” phase of parasite amplification in the skin before the onset of lesion formation and immunity. J Immunol 2000; 165(2):969-77. 35. Woelbing F, Kostka SL, Moelle K et al. Uptake of Leishmania major by dendritic cells is mediated by Fcgamma receptors and facilitates acquisition of protective immunity. J Exp Med 2006; 203(1):177-88. 36. Muraille E, De Trez C, Pajak B et al. Amastigote load and cell surface phenotype of infected cells from lesions and lymph nodes of susceptible and resistant mice infected with Leishmania major. Infect Immun 2003; 71(5):2704-15. 37. Gorak PM, Engwerda CR, Kaye PM. Dendritic cells, but not macrophages, produce IL-12 immediately following Leishmania donovani infection. Eur J Immunol 1998; 28(2):687-95. 38. Lang T, Ave P, Huerre M et al. Macrophage subsets harbouring Leishmania donovani in spleens of infected BALB/c mice: Localization and characterization. Cell Microbiol 2000; 2(5):415-30. 39. Kaye PM, Svensson M, Ato M et al. The immunopathology of experimental visceral leishmaniasis. Immunol Rev 2004; 201:239-53. 40. Murray HW. Tissue granuloma structure-function in experimental visceral leishmaniasis. Int J Exp Pathol 2001; 82(5):249-67. 41. Mebius RE, Kraal G. Structure and function of the spleen. Nat Rev Immunol 2005; 5(8):606-16. 42. Kraal G. Cells in the marginal zone of the spleen. Int Rev Cytol 1992; 132:31-74. 43. Mebius RE, Nolte MA, Kraal G. Development and function of the splenic marginal zone. Crit Rev Immunol 2004; 24(6):449-64.

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44. Koppel EA, Wieland CW, van den Berg VC et al. Specific ICAM-3 grabbing nonintegrin-related 1 (SIGNR1) expressed by marginal zone macrophages is essential for defense against pulmonary Streptococcus pneumoniae infection. Eur J Immunol 2005; 35(10):2962-69. 45. Bruce Lyons A, Watkins M, Simpson CC et al. Modulation of lymphocyte migration to the murine spleen after marginal zone macrophage phagocytosis of blood-borne particulate material. Immunol Invest 2006; 35(1):75-92. 46. Martinez-Pomares L, Kosco-Vilbois M, Darley E et al. Fc chimeric protein containing the cysteine-rich domain of the murine mannose receptor binds to macrophages from splenic marginal zone and lymph node subcapsular sinus and to germinal centers. J Exp Med 1996; 184(5):1927-37. 47. Taylor PR, Martinez-Pomares L, Stacey M et al. Macrophage receptors and immune recognition. Annu Rev Immunol 2005; 23:901-44. 48. Ato M, Nakano H, Kakiuchi T et al. Localization of marginal zone macrophages is regulated by C-C chemokine ligands 21/19. J Immunol 2004; 173(8):4815-20. 49. Chen Y, Pikkarainen T, Elomaa O et al. Defective microarchitecture of the spleen marginal zone and impaired response to a thymus-independent type 2 antigen in mice lacking scavenger receptors MARCO and SR-A. J Immunol 2005; 175(12):8173-80. 50. Engwerda CR, Ato M, Cotterell SE et al. A role for tumor necrosis factor-alpha in remodeling the splenic marginal zone during Leishmania donovani infection. Am J Pathol 2002; 161(2):429-37. 51. Smelt SC, Engwerda CR, McCrossen M et al. Destruction of follicular dendritic cells during chronic visceral leishmaniasis. J Immunol 1997; 158(8):3813-21. 52. Melby PC, Tabares A, Restrepo BI et al. Leishmania donovani: Evolution and architecture of the splenic cellular immune response related to control of infection. Exp Parasitol 2001; 99(1):17-25. 53. Benedict CA, De Trez C, Schneider K et al. Specific remodeling of splenic architecture by cytomegalovirus. PLoS Pathog 2006; 2(3):e16. 54. Muller S, Hunziker L, Enzler S et al. Role of an intact splenic microarchitecture in early lymphocytic choriomeningitis virus production. J Virol 2002; 76(5):2375-83. 55. Ato M, Stager S, Engwerda CR et al. Defective CCR7 expression on dendritic cells contributes to the development of visceral leishmaniasis. Nat Immunol 2002; 3(12):1185-91. 56. Wang GG, Calvo KR, Pasillas MP et al. Quantitative production of macrophages or neutrophils ex vivo using conditional Hoxb8. Nat Methods 2006; 3(4):287-93. 57. Nolte MA, Arens R, Kraus M et al. B cells are crucial for both development and maintenance of the splenic marginal zone. J Immunol 2004; 172(6):3620-27. 58. Tumang MC, Keogh C, Moldawer LL et al. Role and effect of TNF-alpha in experimental visceral leishmaniasis. J Immunol 1994; 153(2):768-75. 59. Bafica A, Oliveira F, Freitas LA et al. American cutaneous leishmaniasis unresponsive to antimonial drugs: Successful treatment using combination of N-methilglucamine antimoniate plus pentoxifylline. Int J Dermatol 2003; 42(3):203-07. 60. O’Neill HC, Wilson HL, Quah B et al. Dendritic cell development in long-term spleen stromal cultures. Stem Cells 2004; 22(4):475-86. 61. Tang H, Guo Z, Zhang M et al. Endothelial stroma programs hematopoietic stem cells to differentiate into regulatory dendritic cells through IL- 10. Blood 2006. 62. Despars G, Ni K, Bouchard A et al. Molecular definition of an in vitro niche for dendritic cell development. Exp Hematol 2004; 32(12):1182-93. 63. Wakkach A, Fournier N, Brun V et al. Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity 2003; 18(5):605-17. 64. Zhang M, Tang H, Guo Z et al. Splenic stroma drives mature dendritic cells to differentiate into regulatory dendritic cells. Nat Immunol 2004; 5(11):1124-33. 65. Cotterell SE, Engwerda CR, Kaye PM. Enhanced hematopoietic activity accompanies parasite expansion in the spleen and bone marrow of mice infected with Leishmania donovani. Infect Immun 2000; 68(4):1840-48. 66. Cotterell SE, Engwerda CR, Kaye PM. Leishmania donovani infection of bone marrow stromal macrophages selectively enhances myelopoiesis, by a mechanism involving GM-CSF and TNF-alpha. Blood 2000; 95(5):1642-51. 67. Guilpin VO, Nosbisch L, Titus RG et al. Infection with Leishmania major stimulates haematopoiesis in susceptible BALB/c mice and suppresses haematopoiesis in resistant CBA mice. Parasitology 2003; 126(Pt 3):187-94. 68. Sher A, Pearce E, Kaye P. Shaping the immune response to parasites: Role of dendritic cells. Curr Opin Immunol 2003; 15(4):421-29. 69. Lemos MP, Esquivel F, Scott P et al. MHC class II expression restricted to CD8alpha+ and CD11b+ dendritic cells is sufficient for control of Leishmania major. J Exp Med 2004; 199(5):725-30.

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70. Ordway D, Henao-Tamayo M, Orme IM et al. Foamy macrophages within lung granulomas of mice infected with Mycobacterium tuberculosis express molecules characteristic of dendritic cells and antiapoptotic markers of the TNF receptor-associated factor family. J Immunol 2005; 175(6):3873-81. 71. Probst HC, Tschannen K, Odermatt B et al. Histological analysis of CD11c-DTR/GFP mice after in vivo depletion of dendritic cells. Clin Exp Immunol 2005; 141(3):398-404. 72. Tsagozis P, Karagouni E, Dotsika E. Function of CD8+ T lymphocytes in a self-curing mouse model of visceral leishmaniasis. Parasitol Int 2005; 54(2):139-46. 73. Pozzi LA, Maciaszek JW, Rock KL. Both dendritic cells and macrophages can stimulate naive CD8 T cells in vivo to proliferate, develop effector function, and differentiate into memory cells. J Immunol 2005; 175(4):2071-81. 74. Leclercq V, Lebastard M, Belkaid Y et al. The outcome of the parasitic process initiated by Leishmania infantum in laboratory mice: A tissue-dependent pattern controlled by the Lsh and MHC loci. J Immunol 1996; 157(10):4537-45

CHAPTER 4

Innate Recognition, Cell Signaling and Pro-Inflammatory Response during Infection with Trypanosoma cruzi Catherine Ropert and Ricardo T. Gazzinelli*

Abstract

I

nnate immunity has an important role in host resistance to early infection with the intracellular protozoan parasite, Trypanosoma cruzi. Here we review the studies that identified several parasite molecules which activate cells from innate immune system, such as macrophages and dendritic cells (DCs). We also cover the studies that demonstrate the involvement of different Toll-Like Receptors (TLRs) in T. cruzi recognition and discuss the potential role of these innate immune receptors in the pathogenesis of Chagas disease.

Introduction Protozoan parasites are highly versatile organisms; as such they have evolved in concert with their host and adapted to survive in the harsh environment encountered, which in many instances has developed to eliminate invasive organisms. Thus, in their different lifestyles, distinct protozoan parasites require the use of highly individualized strategies to deal with specific host defense mechanisms and to survive. The way of entry and the cellular compartment they live also define the needed survival strategy for the parasite. In this regard, a broad distinction can be made between microorganisms that lack a specialized mechanism for active invasion and depend on the phagocytic potential of host cells, and those parasites which developed specific organelles or molecular devices that enable them to play an active role in the invasion process. Leishmania spp belong to the first, whereas Trypanosoma cruzi, the ethiologic agent of Chagas´s disease, belongs to the second category of pathogens. Obviously, different strategies have been elaborated by these parasites. Leishmania lives exclusively within macrophages. Thus, Leishmania that replicates within the macrophage phagolysosomes, needs to shield itself against this hostile environment, and at the same time manipulate the host immune response to prevent activation of effector mechanisms that are displayed by macrophages. This includes protection against low pH and hydrolytic enzymes, as well as interference with cell signaling pathways to inhibit induction of reactive nitrogen intermediate (RNI) synthesis and cytokine production by macrophages. In contrast, T. cruzi has the capacity to infect and proliferate in any nucleated cell of the vertebrate host resulting in a highly efficient infection. Therefore, during T. cruzi infection, a *Corresponding Author: Ricardo T. Gazzinelli—Laboratory of Immunopathology Rene Rachou Research Center - FIOCRUZ, Av. Augusto de Lima 1715 30190-002 Belo Horizonte, MG, Brazil. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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strong activation of innate immunity and development of acquired cell-mediated acquired immune response are necessary to avoid uncontrolled parasite replication and host lethality. It appears from the different studies that a successful parasitism would emerge from pathogens that manage to balance their mechanisms of evasion and activation of the immune system, so they can establish parasitism, but at the same time limit their own replication to avoid excessive host tissue damage and lethality. In this context, host-parasite interactions have been extensively studied in many disciplines (including microbiology, immunology, biochemistry, proteomics) leading to a plethora of information. Currently, an important question addressed by many laboratories in the field, is regarding the nature of parasite molecules recognized by the host innate immune receptors, and responsible for initiation of host immune responses during the early stages of infection. Indeed, biochemical and immunological studies have revealed a very exciting role of molecules present on the surface of many parasites. Among these surface molecules the glycosylphosphatidylinositol (GPI) anchors are dominant glycolipids that cover the surface of most protozoan parasites. These molecules may be expressed in a free form as glycoinositolphospholipid (GIPL), or linked to proteins and glycoconjugates at the parasite surface.1,2 For instance, lipophosphoglycan (LPG), the most abundant cell-surface molecule of the promastigote stage of Leishmania, has been implicated in the subversion of host immune system.3-6 Other studies demonstrate that GPI-anchors present at the surface of other protozoa take a role in activation of different cells from host immune system.7-10 Accumulated evidence indicate that GPI anchors from Plasmodium falciparum contribute predominantly to the pathology of malaria.10 The deleterious effects of parasite GPI have been attributed to their ability to induce high levels of TNF-α and RNI during malaria.10,11 The discovery of a family of receptor that represents the primary line of defense against invading pathogens, Toll-Like receptors (TLRs),12-14 has emphasized the role of these parasite molecules in the interaction between parasites and cells from the host innate immune system.14 In fact, GPI anchors from different parasites have been identified as agonists of the same TLR, TLR2.15-19 While most of the small body of literature on innate immune activators from protozoan parasites has focused upon the GPI anchors, other molecules may also play an important role in the pro-inflammatory response. Similar to unmethylated bacterial CpG DNA motifs,20-22 DNA derived from various protozoan parasites such as T. cruzi, T. brucei and Babesia bovis are able to stimulate macrophages and DCs23 to produce pro-inflammatory cytokines. In this chapter we focus our interest on studies related to T. cruzi molecules that activate cells from the host innate immune system with emphasis on macrophages. Through different approaches, using various knockout (KO) mice and transfected cell lines, we discuss the importance of Toll-like receptors (TLRs) in activation of innate immunity during infection with T. cruzi. The potential role of TLRs in the pathogenesis of Chagas disease will be also discussed.

T. cruzi and Chagas Disease T. cruzi is the ethiologic agent of Chagas´ disease, also known as American trypanosomiasis.24 The natural transmission of T. cruzi parasites is widespread on the American continent, ranging from the southern area of USA to Argentina and Chile and is achieved by the hematophagous triatomine bugs. The T. cruzi life cycle is heteroxenic. Briefly, epimastigotes replicates extracellularly in the midgut of the invertebrate host and differentiate into the infective stage named metacyclic trypomastigotes that are eliminated in the feces, which then contaminate the wound caused by the bug biting the definitive host. To continue its life cycle, the trypomastigote metacyclics infect host cells and inside them differentiate into amastigotes that are responsible for parasitic multiplication in the vertebrate host. When the cells become packed, the amastigotes differentiate into blood trypomastigotes that are released in the extracellular environment.25 The trypomastigotes that reach the bloodstream will disseminate the infection to other tissues and organs of the vertebrate host. If uncontrolled by the immune system, as observed in immune deficient host, T. cruzi becomes even more virulent leading to generalized

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infection, which is often fatal in few days.26 During chronic infection, asymptomatic cycles of cell infection can occur for several years, and in some patients, pathology will emerge, characterized by localized lesions mainly in the heart and digestive system, associated with an increasing load of tissue parasitism. Approximately 18-20 millions individuals in Latin America are chronically infected with T. cruzi and 20-40% of them display the debilitating cardiac and/or digestive symptomatic forms of Chagas disease.26,27 However the cause and mechanism that make asymptomatic patients develop the chagasic cardiopathy are still in debate. The elimination of T. cruzi from infected patients might be a prerequisite to arrest the evolution of the disease and to avert its irreversible long-term consequences. Unfortunately, despite the advances in understanding different aspects of T. cruzi biology, the only drugs currently available for treating chagasic patients are those that were already registered 21 years ago. Nifurtimox and benznidazole (BZ) were developed empirically in the 1960s and 1970s and are active in curing approximately 70% of the treated acute cases of Chagas disease. However, these drugs present even more limited efficacy when used to treat chronically infected individuals.28 The existence of T. cruzi strains naturally resistant to both drugs is one factor that may contribute to the low rates of cure observed in treated chagasic patients.29 Further, studies performed in vivo, show that activation of host innate immune system is a fundamental aspect for the efficacy of treatment with BZ.30,31

Evidence for Activation of the Innate Immune System from Vertebrate Hosts Infected with T. cruzi As mentioned above, infection with T. cruzi is characterized by strong activation of the innate immune system. There are several means by which innate immunity translates its biological activity. The production of cytokines is an essential feature of host defense in the early stage of infection. Control of T. cruzi parasitism in the acute phase of infection is critically dependent on intracellular killing by macrophages activated with cytokines. In vitro and in vivo evidence indicates that infection with T. cruzi both in human and in murine models elicits monocytic lineage cells to produce high levels of pro-inflammatory cytokines.32-34 Most notably, macrophages exposed to these protozoan parasites produce IL-12 that is responsible for initiating interferon γ (IFN-γ) synthesis by natural killer cells.33,35 The strong and systemic activation of immune system culminates at the peak of parasitemia when the trypomastigote form is liberated from cells in the bloodstream. The importance of the host innate immune system was clearly demonstrated in mice lacking functional genes for IL-12, IFN-γ, IFN-γ receptor or inducible nitric oxide synthase (iNOS) genes, where experimental infection with T. cruzi is fatal.36-38 Thus far, the ability to survive infection is dependent upon IFN-γ, which is recognized as a major mediator of host resistance against T. cruzi.39 IFN-γ plays a major role in resistance through the activation of macrophages to produce high levels of RNI, which are very toxic to different protozoa.40,41 Initial experiments suggested that the release of RNI was the main mechanism involved in the microbicidal and/or microbiostatic effect against T. cruzi displayed by activated macrophages.42,43 More recently, a new mechanism involved in the control of T. cruzi in vivo has been identified. This mechanism involves a family of GTPases of 47 kDa (p47-GTPase) that are induced by IFN-γ.44 Mice deficient in Lrg47, a member of the p47-GTPase family, succumb to T. cruzi infection from unrestrained parasitism.45 Importantly, Lrg47 KO mice display unimpaired IL-12, IFN-γ and RNI production. In addition to involvement in the control of parasite replication, the early production of IL-12 and IFN-γ also appears to be critical in directing differentiation of Th precursor cells toward the Th1 phenotype.33,46 Th1 lymphocytes are the basis for a very effective immunity that controls the parasite dissemination during the chronic stages of infection. Th1 cells provide help for the development and activation of CD8+ T lymphocytes that are involved in the control of parasite replication by producing additional IFN-γ as well as displaying cytotoxic activity.47 In addition IFN-γ serves as an efficient coordinator of complementary activities, such as help for B lymphocytes to secrete high levels of parasite specific IgG2a antibodies,48,49

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that promote parasite killing by complement-mediated lysis or opsonization, facilitating parasite uptake by macrophage effector cells. It is clear that T. cruzi is highly effective in inducing IFN-γ and that this cytokine is a vital component in the immune response during infection. Indeed from the stand point of parasite survival in long term persistence it is advantageous for T. cruzi to elicit a protective immune response. Nevertheless, an immune response that is too vigorous can be equally disadvantageous for the host and consequently for the parasite. Consequently, a proper balance between pro- and anti-inflammatory mediators is necessary for regulating an adequate immune response toward a pathogen. There are two major anti-inflammatory cytokines, i.e., IL-10 and tumor growth factor β (TGF-β), which face the challenging task of limiting the induction of a vast variety of pro-inflammatory mediators. For instance, IL-10 KO mice succumb to normally nonlethal infection with T. cruzi and death is associated with decreased tissue parasitism and overproduction of IL-12, TNF-α and IFN-γ.50 These data clearly illustrate the necessity to control the pro-inflammatory reaction during acute infection with T. cruzi. In another way, the role of TGF-β may be to provide a mechanism of parasite escape.51 The production of TGF-β is associated with the removal of apoptotic cells during T. cruzi replication and a reduction of RNI production by infected macrophages that internalize apoptotic cells through a phagocytic process.41,52 This inhibition of RNI production may contribute to establishment of infection and parasite persistence in chronically infected host.

Pro-Inflammatory Activity of GPI Anchors from T. cruzi: Relationship of Structure and Function As pointed out above, high levels of cytokines in mouse sera are directly linked to the presence of the trypomastigote form of T. cruzi in the bloodstream. This constituted the first indication that the trypomastigote forms, and more specifically molecules from trypomastigotes are responsible for activation of immune system. In the last few years, the identification and characterization of protozoan parasite molecules that trigger cytokines and RNI production by cells of innate immune compartment has been the research aim of various groups. Different studies have documented the immunostimulatory and regulatory activities of protozoan derived GPI anchors that abound in the membrane of parasitic protozoans.7-10 GPI-linked proteins are also common on the surface membrane of eukaryotic cells. The most fundamental function of the GPI-anchor is to afford a stable association of proteins with the surface cell. Mammalian cells typically express in the order of ten thousand GPI anchors per cell, whereas a single parasitic protozoan expresses up to 10-20 106 copies of GPI-anchors.53,54 Obviously, it is assumed that this abundance has a physiologic significance in host-parasite interactions as in the case of P. falciparum, T. brucei and T. cruzi infection. Our in vitro studies have implicated GPI-anchored mucin-like glycoproteins (tGPI-mucin) from T. cruzi trypomastigotes as potent inducers of pro-inflammatory cytokines and effector functions by macrophages8,55-57 (Fig. 1). The same cytokines produced by macrophages exposed to T. cruzi were released by macrophages stimulated with tGPI-mucin. We proposed a possible role for tGPI-mucin as a parasite component capable of triggering various functions of innate immunity akin to the importance of lipopolysaccharide (LPS), a membrane component from Gram negative bacteria cellular wall.58 All GPI structures from T. cruzi contain the structural motif core which comprises the oligosaccharide sequence: Manα1-2Manα–Manα1–4GlcNα–myo-inositol-1-HPO4.59 Amongst the variations of GPI structure encountered are variations in carbohydrate branches, variation of the lipid inositol portion (glycerol vs ceramide), the number, length and degree of insaturation of the hydrocarbon chains. The importance of the fine chemical structure of GPI-anchor from T. cruzi in activation of macrophages was highlighted in studies employing tGPI-mucin and GPI-mucin from epimastigotes (eGPI-mucin) encountered in insect gut. We have shown that tGPI-mucin, but not eGPI-mucin potently elicits a pro-inflammatory response by IFN-γ primed murine macrophages.8 Comparing the structures of eGPI-mucin, and tGPI-mucin we note that

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Figure 1. Shematic representation of glycosylphosphatidylinositol (GPI) structures that cover the surface of T. cruzi membrane.57 The free GPI anchors, also named glycoinositolphospholipids (GIPL), from T. cruzi dominantly contain a ceramide as their lipid component. In contrast, the alkylacylglycerol is present in the GPI anchors from mucin like glycoproteins present in the surface of trypomastigote (tGPI-mucin) forms of T. cruzi. Depending on the developmental stage, the number of these molecules varies from 106 to 107 per cell, and they coat a significant amount (60-80%) of parasite plasma membrane. Reproduced with permission from reference 57.

the latter has a longer glycan core and a more fluid lipid moiety due to the presence of unsaturated fatty acid.8 These modifications may explain a better solubilization of tGPI-mucin in aqueous solutions, allowing a better interaction with its counterpart receptor at the macrophage surface. Interestingly, these results indicate that only molecules from T. cruzi stage, which is naturally encountered in vertebrate host, are capable of activating host macrophages. Additionally, other studies have demonstrated that tGPI-mucin and purified tGPI are indeed very potent activators of murine macrophages inducing RNI, IL-12, TNF-α in the 0.1-10 nM range.8,55 This suggests that the GPI anchor constitutes the active moiety of tGPI-mucin responsible for triggering macrophage functions. The bioactivity of these molecules was comparable to that obtained with intact parasite in the range of 0.1-10 parasite equivalents/macrophage. In order to establish the minimum structure required for bioactivity of the GPI anchor, chemical treatment has been performed. Chemical treatment of the GPI moiety with nitrous acid, periodate or methabolic ammonia lead to a diminution of GPI activity. We failed to dissociate the cytokine and or nitric oxide -inducing activity by breaking apart the GPI. Another important GPI structure that covers the T. cruzi surface is represented by the GIPLs (Fig. 1). These structures are not attached to proteins. GIPL from T. cruzi is characterized by the presence of ceramide instead of alkylacylglycerol present in tGPI-mucin structure.60 A role of the ceramide portion of GIPL from T. cruzi in the apoptosis of macrophage has been proposed.61 Another study has shown that GIPL purified from T. cruzi surface exerts a suppressive activity on LPS-stimulated human macrophages or DC, inhibiting the secretion of proinflammatory cytokines like TNF-α and IL-12.62 Importantly the ceramide portion was responsible for most of the activity exhibited by the whole molecule. Furthermore, GIPLs were also shown to exhibit either stimulatory or inhibitory effects on T and B lymphocyte

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functions.63-65 The complex activity of GIPL, exhibiting both inhibitory and stimulatory activities makes it difficult to predict the predominant effect in natural infection. Indeed, this may depend on parasite numbers, parasite strain or other yet-unknown factors.

Other Molecules from T. cruzi Involved in Activation of Cells from Innate Immune System Unmethylated CpG motifs within bacterial DNA and mimicking oligonucleotides (ODN) are able to directly activate murine and human B cells, macrophages and DCs. The reviews provided by Tokunaga66 and later by Krieg67 detailed how Bacillus Calmette-Guerin (BCG) and ODN, respectively, led to the discovery of immunostimulatory CpG-DNA motifs. The discrimination of pathogen from host DNA is attributed to the high frequency of unmethylated CpG dinucleotides in bacterial DNA and to the fact that CpG motifs are generally methylated in mammalian DNA. One potential strategy for escaping host innate immune surveillance would be for a pathogen to reduce the level of CpG motifs in its genome, and therefore decrease its immune stimulatory effects. Indeed, a possible correlation between the long term stable infection and the lowest CpG contents in pathogen DNA has been reported. As reported above, DNA derived from parasites such as T. cruzi, T. brucei and Babesia bovis were shown to stimulate macrophages and DCs.23 Another protein component of T. cruzi has been described as crucial for parasite survival and virulence. This molecule is a member of the thiol-disulfide oxidoreductase family and is named Tc52.68 The protein exerts immunoregulatory functions.69 In vitro, Tc52 modulates T cell proliferation,69 and activates human macrophages: it synergizes with IFN-γ to increase iNOS mRNA levels and RNI production, as well as induction of IL-1β, IL-12, and IL-10 mRNA expression by DCs.70 T. cruzi invades a large variety of mammalian cells and it was proposed that the T. cruzi trans-sialidase acts as a counter-receptor for trypomastigote binding to alpha 2,3-sialyl receptors on host cells as a prelude to T. cruzi invasion.71 Later, trans-sialidase was identified as a major inducer of IL-6 secretion upon T. cruzi infection in naive human intestinal microvascular endothelial cells or peripheral blood mononuclear cells, independently of immune cell activation.72 Such IL-6 secretion may underlie some features of Chagas´s disease, such as pyrexia, neuroprotection, and fibrosis, and lead to polyclonal activation of host lymphocytes, resulting in inefficient induction of T. cruzi specific acquired immunity.73

In Vitro and in Vivo Role of MyD88 and TLRs in Induction of Pro-Inflammatory Cytokines and Host Resistance to Infection with T. cruzi The challenge presented to the host by an infectious microorganism is to rapidly recognize the pathogen and mount an effective antimicrobial response. Following this concept, Janeway predicted that the innate immune system operates by means of receptors that have been selected over evolutionary time to recognize molecules that are both highly conserved and widely distributed. Janeway called these features ‘microbial patterns’ and coined the term of ‘pattern recognition receptors’(PRRs) for the receptors that recognize them.74 The paradigm for pathogen associated molecular pattern is LPS, a surface glycolipid widely distributed among Gram negative bacteria. An important observation that led to the discovery of the LPS receptor is the fact that while CD14, a glycosylphosphoinositol-anchored protein, was able to magnify the LPS signal, it was unable to signal by itself. It was believed that another receptor must exist, and that this would be a transmembrane receptor able to convey the signal across the host cell membrane. The discovery of the TLR family has modified the view of the detection of pathogen by cells from innate immunity. The history of these receptors is now well known. Briefly, Drosophila Toll regulates aspects of embryonic fly development, but deficiencies in Toll receptor also render the adult fly vulnerable

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to fungal infections.75 Based on the role of Toll in innate immunity of Drosophila, Janeway and colleagues identified functional homologs of Drosophila Toll in humans that were therefore named Toll-like receptors.76 In sequence, another group linked hyporesponsiveness to LPS from Gram-negative bacteria in C3H/HeJ mice to a natural mutation in a gene encoding the receptor of LPS, now named TLR4.77 This was the beginning of an explosion of studies related to TLR. Several mammalian TLRs have now been identified in humans and in mice.78-81 As sensors of microbial invasion, TLRs are expected to be found at sites of host-microbe interactions. The expression of TLRs on cells of the monocyte/macrophage lineage is consistent with the role of TLRs in eliciting inflammatory responses. Rapidly, the hypothesis emerged that each TLR senses a distinct repertoire of conserved microbial molecules, so that collectively, the TLRs can detect most, and perhaps all, microbes. While some of the TLRs reside at the cell surface (i.e., TLR1, 2, 4 and 6), some of the TLRs, specifically those involved in recognition of microbicidal nucleic acids (i.e., TLR3, 7, 8 and 9), are confined to the interior of the cells. A large number of studies have aimed to determine the TLR involved in the detection of molecules from viruses, bacteria or yeasts.82 Oligonucleotides containing unmethylated CpG motifs were identified as agonists for TLR9.83 A broad array of macromolecules have been reported to activate innate immunity through TLR2 including bacterial lipopeptides, peptidoglycan and zymosan.86-88 This “low specificity” compared to other TLR may be accounted for by the fact that TLR2 may dimerize with other TLRs to detect ligands.89 For instance, Akira and coworker have shown that there is an absolute need for both TLR2 and TLR6 in the recognition of MALP, a mycoplasma-derived diacylated lipoprotein. TLR1/TLR2 heterodimers recognize a variety of lipopeptides including the synthetic lipoprotein structure Pam(3)CSK(4).90,91 Regularly, new ligands of TLR have been proposed. More information can be found in several recent reviews.84,85 The intracellular signaling pathways activated by TLRs share much in common with IL-1R signaling owing to their conserved TIR (Toll/IL-R homology) domains. There are at least four TIR domain-containing adaptors (MyD88, TIRAP, TRIF and TRAM) that recently have been shown to play important roles in TLR signaling.92 These TIR domain-containing adaptors are associated with TLRs through homophilic interaction of TIR domains. Among these adaptors, MyD88 was first characterized.93 TLR mediated pro-inflammatory cytokine production in response to microbial recognition is critically dependent on MyD88 and its downstream mediators IRAK4, TRAF-6 that activate Mitogen-Activated Protein Kinases (MAPKs) and nuclear factor (NF)-κB.86,87,94,95 The MyD88-dependent pathway is critical to the production of inducible inflammatory cytokines. The importance of this pathway to host defense against a wide range of organisms was demonstrated when it was shown that MyD88 deficient macrophages are completely refractory to immunostimulatory components including LPS, lipoprotein, CpG DNA, showing an essential role in the response to all pathogen immunostimulatory molecules.96-98 More recently, a TLR3 and TLR4 mediated MyD88-independent pathway has been described.99-101 Further studies demonstrated that the MyD88-independent pathway is involved in IRF-3 activation, and induction of type I IFN and IFN inducible genes.102 Because the pattern of macrophage activation by protozoan tGPI-mucin is analogous to LPS, we speculated that tGPI-mucin may use the same family of receptor as LPS.103 To explore this hypothesis, we tried to induce cross-tolerance between GPI-mucin and LPS using macrophages from C57BL/6 mice. In fact, endotoxin or LPS tolerance is defined as a reduced capacity of the host (in vivo) or of cultured monocytes/macrophages (in vitro) to respond to LPS activation following a first exposure to this stimulus.104 Subsequently, studies were initiated to determine whether, in analogy to LPS tolerance, pretreatment with microbial nonLPS stimuli also induces hyporesponsiveness to subsequent restimulation.105-109 The experiments with bacterial DNA or lipopeptides indicate that down-regulation of macrophage responsiveness after stimulation is not restricted to LPS. In a similar way, the pretreatment of macrophages with tGPI-mucin induced a decrease of cytokine release in response to LPS.103 These data constituted evidence that LPS and tGPI-mucin shared a common signaling pathway. To further explore the capacity of tGPI-mucin to activate macrophage via

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TLRs, different strategies have been used. Employing Chinese hamster ovary cells transfected with TLR2 or TLR4 as well as the CD25 gene under a NF-κB dependent promoter as a reporter, we determined that tGPI-mucin or tGPI activate these cells in a TLR2 dependent manner.15 These findings were corroborated in experiments using macrophage from TLR2 KO mice, which did not produce cytokine in response to tGPI or tGPI-mucin. Finally, blocking of human monocyte TLR2 with antibody confirmed the need for TLR2 in the recognition of tGPI-mucin and consequently cytokine release. However, differences have emerged in the cytokine pattern expressed by macrophages from TLR2 KO mice exposed to tGPI-mucin or whole parasite. While macrophages from MyD88 deficient mice show no production of cytokines or RNI in vitro to either tGPI-mucin or live trypomastigotes the absence of TLR2 does not affect dramatically the production of TNF-α by macrophages when exposed to live T. cruzi trypomastigotes.16,110 To obtain additional insights into the involvement of other TLR in the inflammatory response induced upon infection with T. cruzi, we evaluated in vitro the production of TNF-α in response to tGPI-mucin or live trypomastigotes in cells from CD14-/-, TLR4-/-, and TLR6-/- mice. Compared with the wild type macrophages, no alteration of TNF-α or RNI was observed in TLR4-/- macrophages. In contrast, we observed an impaired response of TNF-α and RNI in macrophages from TLR6-/- mice stimulated with tGPI-mucin, and to a lesser extend upon in vitro exposure to live T. cruzi trypomastigotes. Using macrophages derived from CD14-/- mice, we showed a crucial role of CD14 in TNF-α release from tGPI-mucin stimulated cells. Importantly, the absence of CD14 did not affect the response to live trypomastigotes. According to these results, the complex TLR6/TLR2/CD14 appears essential for cytokine production in macrophage stimulated with tGPI-mucin, but not necessarily upon exposure to live parasites. We concluded that other T. cruzi components like GIPLs or DNA might be involved in the pro-inflammatory response elicited by trypomastigotes. In order tο determine the importance of TLR in Chagas disease, mice deficient in TLR or MyD88 were infected with T. cruzi. As expected MyD88-/- mice infected with T. cruzi showed an increase of parasite number in blood associated with an accelerated mortality. The surprise came from the mice deficient in TLR2. The absence of TLR2 did not affect the susceptibility of mice to infection with T. cruzi and more intriguing, an increase of cytokine production was observed when compared to wild type.16 Therefore, we proposed that TLR2 may have a regulatory role during T. cruzi infection in vivo. We hypothesize that after initial activation of TLR2, there probably occurs a down regulation of the common signaling pathways employed by most TLRs, and thus affecting the function of most TLRs. The identification of TLR4 as the receptor for GIPL-ceramide from T. cruzi reinforces the idea that the interactions of parasite with cells corresponds to the coordinated outputs of activation of multiple TLRs.111 The same group has proposed that TLR4 may be another receptor involved on interactions of T. cruzi and host cells and that this receptor may play a role in resistance to T. cruzi. In fact, they observed in C3H/HeJ mice that bear a functionally important mutation in Tlr4, a higher susceptibility to T. cruzi compared to C3H/HeN, which possess the functional TLR4. However, according to our data (unpublished results) the susceptibility to infection was not significantly altered in TLR4 KO mice in the C57BL/6 genetic background compared to the proper wild type animals. Importantly, C3H has been classified as a highly susceptible strain, whereas the C57BL/6 strain behaves as a resistant strain to T. cruzi infection. Thus, the lack of functional TLR4, may be more critical in a genetic background associated with susceptibility to infection, and this may explain the differences observed between the studies performed with the two mouse strains. With the exception of TLR3, the MyD88-dependent pathway is universally used by all TLRs in activating NF-κB. However, subsequent studies clearly demonstrated that there is a MyD88-independent pathway as well as a MyD88-dependent pathway in TLR4 signaling. In the MyD88-independent pathway, LPS stimulation leads to activation of the transcription factor IRF-3, and thereby induces IFN-β.99,112 IFN-β, in turn, activates Stat1, leading to the induction of several IFN-inducible genes. Interestingly, we found evidence for a MyD88-independent pathway during the T. cruzi infection leading to a small but significant

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production of cytokine like IL-12 and IFN-γ by spleen cells from T. cruzi infected MyD88 KO mice infected with T. cruzi.110 This hypothesis is also corroborated by the fact that MyD88 KO mice were not as susceptible as the IFN-γ KO mice infected with T. cruzi. One explanation for the difficulty in establishing a correlation between information obtained in vitro and in vivo using TLR deficient mice is provided by the fact that host defense to pathogens is orchestrated by multiple TLRs, that in addition to having a redundant role, may also present distinct output in different host cells. Indeed, a recent study shows that TLR2 and TLR9 synergize in mediating host resistance in mice experimentally infected with Mycobacterium tuberculosis.113 The hypothesis that different TLRs are simultaneously involved in parasite recognition during T. cruzi infection has led our group to evaluate the cooperation of different TLRs after infection with T. cruzi. We showed in vitro that tGPI-mucin and DNA from parasite cooperate to induce cytokine synthesis by macrophages and DCs. In addition, we showed that TLR2/9 double KO mice are more susceptible to acute infection with T. cruzi (Bafica et al submitted for publication). The loss in controlling parasite growth was found to correlate with a deficient Th1 like response in vivo after infection with T. cruzi. However, based on the survival curve, the TLR2/9 KO animals were not as susceptible to T. cruzi as MyD88 KO mice suggesting that additional TLR, possibly TLR4, is also contributing to early host resistance to infection. Nevertheless, this study clearly demonstrates the dominant role of TLR9 in induction of IL-12 and IFN-γ synthesis during T. cruzi infection. In Figure 2, we present a schematic representation of the interaction of TLRs interaction with different T. cruzi molecules within host macrophages.

Signalling Pathway and Tolerance Induced by tGPI-Mucins: An Alternative Way to Control Excessive Inflammatory Responses Generally, after ligand-mediated dimerization, TLR recruits an adaptor protein MyD88 as related above. MyD88 then assembles a signalsome containing IRAK, and TRAF6. Together, they mediate activation of NF-κΒ and MAPKs, such as ERK1/2, JNK and p38.102,114-116 The MAPK cascade is one of the most ancient and evolutionarily conserved signalling pathways, and is comprised of a group of serine/threonine protein kinases that are responsible for transmission of extracellular signals to the nucleus, where the transcription of specific genes is induced by phosphorylation and activation of transcriptions factors.117,118 The importance of MAPKs in the innate immune response was elegantly supported using mice deficient in TLRs or MyD88. Activation of NF-κB and MAPKs by mycoplasmal lipopeptide is completely abolished in TLR2or MyD88-deficient macrophages.97 The same observation was obtained in experiments involving tGPI-mucin from T. cruzi. The fundamental role of MAPKs in innate immunity is closely related with cytokine production by cells involved in innate immunity. The importance of these different signalling routes for tGPI-mucin -mediated cytokine production has been investigated using murine macrophages. tGPI-mucin or tGPI showed a pattern of activation of the three main MAPKs, ERK1/2, JNK and p38 similar to LPS in macrophages.103 Using specific inhibitors of these different pathways the results showed that ERK1/2 and p38 are involved in TNF-α and IL-12 synthesis (Fig. 3). Furthermore, inhibiting ERK1/2 we identified an unexpected stimulatory effect of IL-12 synthesis by macrophages exposed to tGPI-mucin. Other studies involving microbial products such as CpG DNA, and LPS have described the same negative regulation of IL-12 production by ERK1/2.6,119 It was proposed that some pathogens may suppress resistance to infection by switching on the ERK1/2 mediated negative regulation of IL-12 production.120,121 These data illustrate that these MAPKs are potential targets for the development of novel strategies to combat pathogens. The exact array of transcription factors activated by the MAPK pathways and involved in induction / regulation of gene expression remains to be defined. Nevertheless, CREB and NF-κB were identified as important transcription factors involved in TNF-α synthesis by macrophages stimulated with tGPI-mucin.103 As commented before, the immune system needs to constantly strike a balance between activation and inhibition to avoid detrimental and inappropriate inflammatory responses that is crucial for host survival and parasite persistance. This means that TLRs must be tightly regulated.

Figure 2. Schematic representation of molecular patterns and TLRs (TLR2, TLR4, TLR6 and TLR9), related coreceptor (CD14 and MD2) and signaling adaptor molecules (MyD88, Mal/TIRAP, and TRIF/TRAM) potentially involved in T. cruzi recognition and signaling by the vertebrate host innate immune system. GPI anchored mucin like glycoproteins from trypomastigotes (tGPI-mucin), glycoinositolphospholipids (GIPL) and DNA from T.cruzi interact simultaneously with TLR2, TLR4 and TLR9, respectively, in different cellular compartment, and play an important role in inducing the synthesis of cytokines by macrophages and DCs.

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Figure 3. A) Effects of PD98059 (40 μM), an inhibitor of ERK phosphorylation and/or SB203580 (10 μM), an inhibitor of p38 phosphorylation on tGPI-mucin induced phosphorylation of ERK1/ ERK2, SKK-1/MKK4 and SAPK-2/p38 and TNF-α synthesis by macrophages, after 15 minutes and 18 hr post-stimulation with tGPI-mucin (10 nM). B) Time course of IκB phosphorylation and degradation in inflammatory macrophages post-stimulation with 10 nM of tGPI-mucin. Macrophages were also cultured in the presence of different concentrations of SN50, an inhibitor of NF-κB translocation (closed symbols) or control peptide (open circle) and the levels of TNF-α measured in the cell supernatant at 18 h post-stimulation with 10 nM of tGPI-mucin. Reproduced with permission from reference 103.

We have previously commented that cytokine increase was observed in TLR2 KO mice infected with T. cruzi when compared to wild type mice. We have hypothesized that TLR2 may play a regulatory role during the infection with T. cruzi16,110 and we have suggested a role of tGPI-mucin as a parasite regulatory molecule, through the phenomenon of immune tolerance. In this context, we have investigated whether pretreatment of macrophages with tGPI-mucin could affect cytokine release in response to T. cruzi. Interestingly, tGPI-mucin pretreatment is associated with a decrease of cytokine and MAPK phosphorylation in macrophage infected with T. cruzi.16 We have confirmed that this phenomenon occurs in a TLR2-dependent way. The regulation of MAPK pathways involves the dynamic interplay between kinases and phosphatases.122,123 As MAPKs play a key role in the transcriptional regulation of the response to tGPI-mucin, we hypothesized that counterregulatory phosphatases that target MAPKs may play a role in the tolerance phenomenon. Analysis of the kinetics of phosphatase activity induced by tGPI-mucin or LPS revealed maximum levels between 12 and 24h that correlate with the macrophage hyporesponsiveness stage.124 Inhibition of protein phosphatase 2A (PP2A) by okadaic acid (OA) restores the cytokine production in macrophages tolerized by microbial stimuli suggesting the involvement of these phosphatases in the tolerance phenomenon. In

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Figure 4. Conceptual model for the role of TLR2 or TLR4-induced phosphatase in tolerance of macrophage pretreated with tGPI-mucin or LPS.124 A) Interactions of LPS or tGPI-mucin with TLR4 or TLR2 induce activation of IRAK-1, and downstream elements including MAPKs and I-κB (a) responsible for the production of TNF-α. p38 MAPK and NF-κB are also important for up-regulating phosphatase activity that will prevent IRAK-1, MAPKs and IκB phosphorylation upon a second stimulation with either TLR2 or TLR4 agonists (b). B) Hypothetical mechanism of tolerance reversion in macrophages treated with okadaic acid (OA). OA, a PP2A inhibitor, induces hyperphosphorylation of ERK1/2 (a) leading to blockage of p38 as well as IκB phosphorylation (a) and consequently down regulation of phosphatase activity (b) upon activation with either TLR2 or TLR4 agonists. The failure in inducing the phosphatase activity after the first stimulation favors the ability of macrophages to induce phosphorylation of IRAK-1, MAPKs and ΙκB in response to a second stimulation. Reproduced with permission from reference 124.

order to investigate mechanisms of regulation of phosphatase activity, macrophages were treated with inhibitors of MAPK phosphorylation or NF-κB translocation after LPS stimulation. Our data indicate the involvement of p38 and NF-κB induction of a phosphatase activity, after stimulation of macrophage via TLR. In Figure 4, we propose a model of tolerance involving p38 that controls ERK phosphorylation in a PP2A-dependent mechanism.124

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Concluding Remarks A considerable amount of information regarding the role of innate immunity during infection with T. cruzi in the vertebrate host is currently emerging from studies performed in different laboratories. The critical role of cells (i.e., macrophages, NK cells and neutrophils), cytokines (i.e., IL-12, TNF-α and IFN-γ), and IFN-γ inducible effector mechanisms (i.e., RNI and p47-GTPases) during infection with T. cruzi are now well established. The main focus of attention is currently the mapping of innate cognate receptors and signaling pathways involved in parasite recognition and activation of cells from innate immunity by T. cruzi parasites. Importantly, parasite molecules that activate TLR2, TLR4 and TLR9 have been defined, and the essential role of MyD88 for early cytokine synthesis and host resistance to infection demonstrated. New information indicates that different TLRs are simultaneously engaged and together mount the initial pro-inflammatory response during acute infection with T. cruzi. However, the complete array of TLRs triggered by T. cruzi components remains to be defined. Further, it is also clear that a yet undefined MyD88-independent pathway is also triggered by T. cruzi and is in part responsible for stimulation of innate immunity during initial stages of infection with this parasite. The new information regarding the role of innate immunity during experimental infection with T. cruzi also provides new insights for therapeutic and prophylactic interventions for Chagas disease, employing TLR agonists or compounds that interfere with signaling pathways triggered by these innate immune receptors.

References 1. Tachado SD, Mazhari-Tabrizi R, Schofield L. Specificity in signal transduction among glycosylphosphatidylinositols of Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. Parasite Immunol 1999; 21(12):609-617. 2. Ropert C, Gazzinelli RT. Signaling of immune system cells by glycosylphosphatidylinositol (GPI) anchor and related structures derived from parasitic protozoa. Curr Opin Microbiol 2000; 3(4):395-403. 3. Descoteaux A, Turco SJ, Sacks DL et al. Leishmania donovani lipophosphoglycan selectively inhibits signal transduction in macrophages. J Immunol 1991; 146(8):2747-2753. 4. Descoteaux A, Matlashewski G, Turco SJ. Inhibition of macrophage protein kinase C-mediated protein phosphorylation by Leishmania donovani lipophosphoglycan. J Immunol 1992; 149(9):3008-3015. 5. Desjardins M, Descoteaux A. Inhibition of phagolysosomal biogenesis by the Leishmania lipophosphoglycan. J Exp Med 1997; 185(12):2061-2068. 6. Feng GJ, Goodridge HS, Harnett MM et al. Extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leishmania phosphoglycans subvert macrophage IL-12 production by targeting ERK MAP kinase. J Immunol 1999; 163(12):6403-6412. 7. Camargo MM, Andrade AC, Almeida IC et al. Glycoconjugates isolated from Trypanosoma cruzi but not from Leishmania species membranes trigger nitric oxide synthesis as well as microbicidal activity in IFN-gamma-primed macrophages. J Immunol 1997; 159(12):6131-6139. 8. Almeida IC, Camargo MM, Procopio DO et al. Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. EMBO J 2000; 19(7):1476-1485. 9. Tachado SD, Schofield L. Glycosylphosphatidylinositol toxin of Trypanosoma brucei regulates IL-1 alpha and TNF-alpha expression in macrophages by protein tyrosine kinase mediated signal transduction. Biochem Biophys Res Commun 1994; 205(2):984-991. 10. Schofield L, Hackett F. Signal transduction in host cells by a glycosylphosphatidylinositol toxin of malaria parasites. J Exp Med 1993; 177(1):145-153. 11. Schofield L, Vivas L, Hackett F et al. Neutralizing monoclonal antibodies to glycosylphosphatidylinositol, the dominant TNF-alpha-inducing toxin of Plasmodium falciparum: Prospects for the immunotherapy of severe malaria. Ann Trop Med Parasitol 1993; 87(6):617-626. 12. Takeda K, Akira S. Toll-like receptors in innate immunity. Int Immunol 2005; 17(1):1-14. 13. Pasare C, Medzhitov R. Toll-like receptors: Linking innate and adaptive immunity. Adv Exp Med Biol 2005; 560:11-18. 14. Gazzinelli RT, Ropert C, Campos MA. Role of the Toll/interleukin-1 receptor signaling pathway in host resistance and pathogenesis during infection with protozoan parasites. Immunol Rev 2004; 201:9-25.

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15. Campos MA, Almeida IC, Takeuchi O et al. Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite. J Immunol 2001; 167(1):416-423. 16. Ropert C, Gazzinelli RT. Regulatory role of Toll-like receptor 2 during infection with Trypanosoma cruzi. J Endotoxin Res 2004; 10(6):425-430. 17. Becker I, Salaiza N, Aguirre M et al. Leishmania lipophosphoglycan (LPG) activates NK cells through toll-like receptor-2. Mol Biochem Parasitol 2003; 130(2):65-74. 18. Krishnegowda G, Hajjar AM, Zhu J et al. Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: Cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem 2005; 280(9):8606-8616. 19. de Veer MJ, Curtis JM, Baldwin TM et al. MyD88 is essential for clearance of Leishmania major: Possible role for lipophosphoglycan and Toll-like receptor 2 signaling. Eur J Immunol 2003; 33(10):2822-2831. 20. Gao JJ, Zuvanich EG, Xue Q et al. Cutting edge: Bacterial DNA and LPS act in synergy in inducing nitric oxide production in RAW 264.7 macrophages. J Immunol 1999; 163(8):4095-4099. 21. Sparwasser T, Miethke T, Lipford G et al. Macrophages sense pathogens via DNA motifs: Induction of tumor necrosis factor-alpha-mediated shock. Eur J Immunol 1997; 27(7):1671-1679. 22. Sparwasser T, Koch ES, Vabulas RM et al. Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol 1998; 28(6):2045-2054. 23. Shoda LK, Kegerreis KA, Suarez CE et al. DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi, and T. brucei is mitogenic for B lymphocytes and stimulates macrophage expression of interleukin-12, tumor necrosis factor alpha, and nitric oxide. Infect Immun 2001; 69(4):2162-2171. 24. Chagas C. Nova tripanozomiase humana. Estudo sobre a morfologia e o ciclo evolutivo do schyzotrypanum cruzi n.gen., n.sp., agente etiológico de nova entidade mórbida do homem. Mem Inst Oswaldo Cruz 1909; 1. 25. Brener Z. Biology of Trypanosoma cruzi. Annu Rev Microbiol 1973; 27:347-382. 26. Brener Z, Gazzinelli RT. Immunological control of Trypanosoma cruzi infection and pathogenesis of Chagas’ disease. Int Arch Allergy Immunol 1997; 114(2):103-110. 27. Dias JC. The indeterminate form of human chronic Chagas’ disease A clinical epidemiological review. Rev Soc Bras Med Trop 1989; 22(3):147-156. 28. Croft SL, Barrett MP, Urbina JA. Chemotherapy of trypanosomiases and leishmaniasis. Trends Parasitol 2005; 21(11):508-512. 29. Murta SM, Gazzinelli RT, Brener Z et al. Molecular characterization of susceptible and naturally resistant strains of Trypanosoma cruzi to benznidazole and nifurtimox. Mol Biochem Parasitol 1998; 93(2):203-214. 30. Romanha AJ, Alves RO, Murta SM et al. Experimental chemotherapy against Trypanosoma cruzi infection: Essential role of endogenous interferon-gamma in mediating parasitologic cure. J Infect Dis 2002; 186(6):823-828. 31. Michailowsky V, Murta SM, Carvalho-Oliveira L et al. Interleukin-12 enhances in vivo parasiticidal effect of benznidazole during acute experimental infection with a naturally drug-resistant strain of Trypanosoma cruzi. Antimicrob Agents Chemother 1998; 42(10):2549-2556. 32. Silva JS, Vespa GN, Cardoso MA et al. Tumor necrosis factor alpha mediates resistance to Trypanosoma cruzi infection in mice by inducing nitric oxide production in infected gamma interferon-activated macrophages. Infect Immun 1995; 63(12):4862-4867. 33. Aliberti JC, Cardoso MA, Martins GA et al. Interleukin-12 mediates resistance to Trypanosoma cruzi in mice and is produced by murine macrophages in response to live trypomastigotes. Infect Immun 1996; 64(6):1961-1967. 34. Van Voorhis WC. Coculture of human peripheral blood mononuclear cells with Trypanosoma cruzi leads to proliferation of lymphocytes and cytokine production. J Immunol 1992; 148(1):239-248. 35. Cardillo F, Voltarelli JC, Reed SG et al. Regulation of Trypanosoma cruzi infection in mice by gamma interferon and interleukin 10: Role of NK cells. Infect Immun 1996; 64(1):128-134. 36. Michailowsky V, Silva NM, Rocha CD et al. Pivotal role of interleukin-12 and interferon-gamma axis in controlling tissue parasitism and inflammation in the heart and central nervous system during Trypanosoma cruzi infection. Am J Pathol 2001; 159(5):1723-1733. 37. Holscher C, Kohler G, Muller U et al. Defective nitric oxide effector functions lead to extreme susceptibility of Trypanosoma cruzi-infected mice deficient in gamma interferon receptor or inducible nitric oxide synthase. Infect Immun 1998; 66(3):1208-1215. 38. Castanos-Velez E, Maerlan S, Osorio LM et al. Trypanosoma cruzi infection in tumor necrosis factor receptor p55-deficient mice. Infect Immun 1998; 66(6):2960-2968.

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39. Torrico F, Heremans H, Rivera MT et al. Endogenous IFN-gamma is required for resistance to acute Trypanosoma cruzi infection in mice. J Immunol 1991; 146(10):3626-3632. 40. Vespa GN, Cunha FQ, Silva JS. Nitric oxide is involved in control of Trypanosoma cruzi-induced parasitemia and directly kills the parasite in vitro. Infect Immun 1994; 62(11):5177-5182. 41. Gazzinelli RT, Oswald IP, Hieny S et al. The microbicidal activity of interferon-gamma-treated macrophages against Trypanosoma cruzi involves an L-arginine-dependent, nitrogen oxide-mediated mechanism inhibitable by interleukin-10 and transforming growth factor-beta. Eur J Immunol 1992; 22(10):2501-2506. 42. James SL, Kipnis TL, Sher A et al. Enhanced resistance to acute infection with Trypanosoma cruzi in mice treated with an interferon inducer. Infect Immun 1982; 35(2):588-593. 43. Nathan C, Nogueira N, Juangbhanich C et al. Activation of macrophages in vivo and in vitro. Correlation between hydrogen peroxide release and killing of Trypanosoma cruzi. J Exp Med 1979; 149(5):1056-1068. 44. Taylor GA, Feng CG, Sher A. p47 GTPases: Regulators of immunity to intracellular pathogens. Nat Rev Immunol 2004; 4(2):100-109. 45. Santiago HC, Feng CG, Bafica A et al. Mice deficient in LRG-47 display enhanced susceptibility to Trypanosoma cruzi infection associated with defective hemopoiesis and intracellular control of parasite growth. J Immunol 2005; 175(12):8165-8172. 46. Seder RA, Gazzinelli R, Sher A et al. Interleukin 12 acts directly on CD4+ T cells to enhance priming for interferon gamma production and diminishes interleukin 4 inhibition of such priming. Proc Natl Acad Sci USA 1993; 90(21):10188-10192. 47. Tarleton RL, Koller BH, Latour A et al. Susceptibility of beta 2-microglobulin-deficient mice to Trypanosoma cruzi infection. Nature 1992; 356(6367):338-340. 48. d’Imperio Lima MR, Eisen H, Minoprio P et al. Persistence of polyclonal B cell activation with undetectable parasitemia in late stages of experimental Chagas’ disease. J Immunol 1986; 137(1):353-356. 49. Brodskyn CI, da Silva AM, Takehara HA et al. Characterization of antibody isotype responsible for immune clearance in mice infected with Trypanosoma cruzi. Immunol Lett 1988; 18(4):255-258. 50. Hunter CA, Ellis-Neyes LA, Slifer T et al. IL-10 is required to prevent immune hyperactivity during infection with Trypanosoma cruzi. J Immunol 1997; 158(7):3311-3316. 51. Silva JS, Twardzik DR, Reed SG. Regulation of Trypanosoma cruzi infections in vitro and in vivo by transforming growth factor beta (TGF-beta). J Exp Med 1991; 174(3):539-545. 52. Freire-de-Lima CG, Nascimento DO, Soares MB et al. Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages. Nature 2000; 403(6766):199-203. 53. Ferguson MA, Brimacombe JS, Cottaz S et al. Glycosyl-phosphatidylinositol molecules of the parasite and the host. Parasitology 1994; 108(Suppl):S45-54. 54. Ferguson MA. The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research. J Cell Sci 1999; 112(Pt 17):2799-2809. 55. Camargo MM, Almeida IC, Pereira ME et al. Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes initiate the synthesis of proinflammatory cytokines by macrophages. J Immunol 1997; 158(12):5890-5901. 56. Ropert C, Ferreira LR, Campos MA et al. Macrophage signaling by glycosylphosphatidylinositol-anchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes. Microbes Infect 2002; 4(9):1015-1025. 57. Almeida IC, Gazzinelli RT. Proinflammatory activity of glycosylphosphatidylinositol anchors derived from Trypanosoma cruzi: Structural and functional analyses. J Leukoc Biol 2001; 70(4):467-477. 58. Beutler B. LPS in microbial pathogenesis: Promise and fulfilment. J Endotoxin Res 2002; 8(5):329-335. 59. Ferguson MA. The surface glycoconjugates of trypanosomatid parasites. Philos Trans R Soc Lond B Biol Sci 1997; 352(1359):1295-1302. 60. Bilate AM, Previato JO, Mendonca-Previato L et al. Glycoinositolphospholipids from Trypanosoma cruzi induce B cell hyper-responsiveness in vivo. Glycoconj J 2000; 17(10):727-734. 61. Freire-de-Lima CG, Nunes MP, Corte-Real S et al. Proapoptotic activity of a Trypanosoma cruzi ceramide-containing glycolipid turned on in host macrophages by IFN-gamma. J Immunol 1998; 161(9):4909-4916. 62. Brodskyn C, Patricio J, Oliveira R et al. Glycoinositolphospholipids from Trypanosoma cruzi interfere with macrophages and dendritic cell responses. Infect Immun 2002; 70(7):3736-3743. 63. Bellio M, Liveira AC, Mermelstein CS et al. Costimulatory action of glycoinositolphospholipids from Trypanosoma cruzi: Increased interleukin 2 secretion and induction of nuclear translocation of the nuclear factor of activated T cells 1. Faseb J 1999; 13(12):1627-1636.

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64. Bento CA, Melo MB, Previato JO et al. Glycoinositolphospholipids purified from Trypanosoma cruzi stimulate Ig production in vitro. J Immunol 1996; 157(11):4996-5001. 65. Gomes NA, Previato JO, Zingales B et al. Down-regulation of T lymphocyte activation in vitro and in vivo induced by glycoinositolphospholipids from Trypanosoma cruzi. Assignment of the T cell-suppressive determinant to the ceramide domain. J Immunol 1996; 156(2):628-635. 66. Tokunaga T, Yamamoto T, Yamamoto S. How BCG led to the discovery of immunostimulatory DNA. Jpn J Infect Dis 1999; 52(1):1-11. 67. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002; 20:709-760. 68. Schoneck R, Plumas-Marty B, Taibi A et al. Trypanosoma cruzi cDNA encodes a tandemly repeated domain structure characteristic of small stress proteins and glutathione S-transferases. Biol Cell 1994; 80(1):1-10. 69. Ouaissi A, Guevara-Espinoza A, Chabe F et al. A novel and basic mechanism of immunosuppression in Chagas’ disease: Trypanosoma cruzi releases in vitro and in vivo a protein which induces T cell unresponsiveness through specific interaction with cysteine and glutathione. Immunol Lett 1995; 48(3):221-224. 70. Fernandez-Gomez R, Esteban S, Gomez-Corvera R et al. Trypanosoma cruzi: Tc52 released protein-induced increased expression of nitric oxide synthase and nitric oxide production by macrophages. J Immunol 1998; 160(7):3471-3479. 71. Ming M, Chuenkova M, Ortega-Barria E et al. Mediation of Trypanosoma cruzi invasion by sialic acid on the host cell and trans-sialidase on the trypanosome. Mol Biochem Parasitol 1993; 59(2):243-252. 72. Saavedra E, Herrera M, Gao W et al. The Trypanosoma cruzi trans-sialidase, through its COOH-terminal tandem repeat, upregulates interleukin 6 secretion in normal human intestinal microvascular endothelial cells and peripheral blood mononuclear cells. J Exp Med 1999; 190(12):1825-1836. 73. Schenkman S, Eichinger D, Pereira ME et al. Structural and functional properties of Trypanosoma trans-sialidase. Annu Rev Microbiol 1994; 48:499-523. 74. Janeway Jr CA. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harb Symp Quant Biol 1989; 54(Pt 1):1-13. 75. Lemaitre B, Nicolas E, Michaut L et al. The dorsoventral regulatory gene cassette spatzle/Toll/ cactus controls the potent antifungal response in Drosophila adults. Cell 1996; 86(6):973-983. 76. Medzhitov R, Preston-Hurlburt P, Janeway Jr CA. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997; 388(6640):394-397. 77. Poltorak A, He X, Smirnova I et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene. Science 1998; 282(5396):2085-2088. 78. Takeuchi O, Kawai T, Sanjo H et al. TLR6: A novel member of an expanding toll-like receptor family. Gene 1999; 231(1-2):59-65. 79. Chuang TH, Ulevitch RJ. Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw 2000; 11(3):372-378. 80. Du X, Poltorak A, Wei Y et al. Three novel mammalian toll-like receptors: Gene structure, expression, and evolution. Eur Cytokine Netw 2000; 11(3):362-371. 81. Chuang T, Ulevitch RJ. Identification of hTLR10: A novel human Toll-like receptor preferentially expressed in immune cells. Biochim Biophys Acta 2001; 1518(1-2):157-161. 82. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 2003; 85(2):85-95. 83. Hemmi H, Takeuchi O, Kawai T et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408(6813):740-745. 84. Ishii KJ, Coban C, Akira S. Manifold mechanisms of toll-like receptor-ligand recognition. J Clin Immunol 2005; 25(6):511-521. 85. Kawai T, Akira S. TLR signaling. Cell Death Differ 2006. 86. Akira S, Takeda K, Kaisho T. Toll-like receptors: Critical proteins linking innate and acquired immunity. Nat Immunol 2001; 2(8):675-680. 87. Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000; 406(6797):782-787. 88. Dziarski R, Gupta D. Peptidoglycan recognition in innate immunity. J Endotoxin Res 2005; 11(5):304-310. 89. Ozinsky A, Underhill DM, Fontenot JD et al. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc Natl Acad Sci USA 2000; 97(25):13766-13771.

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90. Takeuchi O, Sato S, Horiuchi T et al. Cutting edge: Role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol 2002; 169(1):10-14. 91. Sandor F, Latz E, Re F et al. Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling. J Cell Biol 2003; 162(6):1099-1110. 92. Akira S. Toll-like receptor signaling. J Biol Chem 2003; 278(40):38105-38108. 93. Lord KA, Hoffman-Liebermann B, Liebermann DA. Nucleotide sequence and expression of a cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL6. Oncogene 1990; 5(7):1095-1097. 94. Medzhitov R, Janeway Jr C. The Toll receptor family and microbial recognition. Trends Microbiol 2000; 8(10):452-456. 95. Means TK, Golenbock DT, Fenton MJ. Structure and function of Toll-like receptor proteins. Life Sci 2000; 68(3):241-258. 96. Takeuchi O, Takeda K, Hoshino K et al. Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades. Int Immunol 2000; 12(1):113-117. 97. Takeuchi O, Kaufmann A, Grote K et al. Cutting edge: Preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2- and MyD88-dependent signaling pathway. J Immunol 2000; 164(2):554-557. 98. Hacker H, Vabulas RM, Takeuchi O et al. Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J Exp Med 2000; 192(4):595-600. 99. Kawai T, Takeuchi O, Fujita T et al. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 2001; 167(10):5887-5894. 100. Yamamoto M, Sato S, Hemmi H et al. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 2003; 301(5633):640-643. 101. Hoebe K, Du X, Georgel P et al. Identification of Lps2 as a key transducer of MyD88-independent TIR signalling. Nature 2003; 424(6950):743-748. 102. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004; 4(7):499-511. 103. Ropert C, Almeida IC, Closel M et al. Requirement of mitogen-activated protein kinases and I kappa B phosphorylation for induction of proinflammatory cytokines synthesis by macrophages indicates functional similarity of receptors triggered by glycosylphosphatidylinositol anchors from parasitic protozoa and bacterial lipopolysaccharide. J Immunol 2001; 166(5):3423-3431. 104. West MA, Heagy W. Endotoxin tolerance: A review. Crit Care Med 2002; 30(1 Suppl):S64-73. 105. Kreutz M, Ackermann U, Hauschildt S et al. A comparative analysis of cytokine production and tolerance induction by bacterial lipopeptides, lipopolysaccharides and Staphyloccous aureus in human monocytes. Immunology 1997; 92(3):396-401. 106. Sato S, Nomura F, Kawai T et al. Synergy and cross-tolerance between toll-like receptor (TLR) 2and TLR4-mediated signaling pathways. J Immunol 2000; 165(12):7096-7101. 107. Schwartz DA, Wohlford-Lenane CL, Quinn TJ et al. Bacterial DNA or oligonucleotides containing unmethylated CpG motifs can minimize lipopolysaccharide-induced inflammation in the lower respiratory tract through an IL-12-dependent pathway. J Immunol 1999; 163(1):224-231. 108. Lehner MD, Morath S, Michelsen KS et al. Induction of cross-tolerance by lipopolysaccharide and highly purified lipoteichoic acid via different Toll-like receptors independent of paracrine mediators. J Immunol 2001; 166(8):5161-5167. 109. Martin M, Katz J, Vogel SN et al. Differential induction of endotoxin tolerance by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli. J Immunol 2001; 167(9):5278-5285. 110. Campos MA, Closel M, Valente EP et al. Impaired production of proinflammatory cytokines and host resistance to acute infection with Trypanosoma cruzi in mice lacking functional myeloid differentiation factor 88. J Immunol 2004; 172(3):1711-1718. 111. Oliveira AC, Peixoto JR, de Arruda LB et al. Expression of functional TLR4 confers proinflammatory responsiveness to Trypanosoma cruzi glycoinositolphospholipids and higher resistance to infection with T. cruzi. J Immunol 2004; 173(9):5688-5696. 112. Toshchakov V, Jones BW, Perera PY et al. TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat Immunol 2002; 3(4):392-398. 113. Bafica A, Scanga CA, Feng CG et al. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. J Exp Med 2005; 202(12):1715-1724. 114. Adachi O, Kawai T, Takeda K et al. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 1998; 9(1):143-150. 115. Burns K, Martinon F, Esslinger C et al. MyD88, an adapter protein involved in interleukin-1 signaling. J Biol Chem 1998; 273(20):12203-12209.

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116. Muzio M, Ni J, Feng P et al. IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 1997; 278(5343):1612-1615. 117. Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature 2001; 410(6824):37-40. 118. Ropert C. Translating MAPKs inhibitors to anti-inflammatory compounds. Current Enzyme Inhibition 2005; 1:75-84. 119. Yi AK, Yoon JG, Yeo SJ et al. Role of mitogen-activated protein kinases in CpG DNA-mediated IL-10 and IL-12 production: Central role of extracellular signal-regulated kinase in the negative feedback loop of the CpG DNA-mediated Th1 response. J Immunol 2002; 168(9):4711-4720. 120. Tang N, Liu L, Kang K et al. Inhibition of monocytic interleukin-12 production by Candida albicans via selective activation of ERK mitogen-activated protein kinase. Infect Immun 2004; 72(5):2513-2520. 121. Matsunaga K, Yamaguchi H, Klein TW et al. Legionella pneumophila suppresses macrophage interleukin-12 production by activating the p42/44 mitogen-activated protein kinase cascade. Infect Immun 2003; 71(11):6672-6675. 122. Keyse SM. Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr Opin Cell Biol 2000; 12(2):186-192. 123. Camps M, Nichols A, Arkinstall S. Dual specificity phosphatases: A gene family for control of MAP kinase function. Faseb J 2000; 14(1):6-16. 124. Ropert C, Closel M, Chaves AC et al. Inhibition of a p38/stress-activated protein kinase-2-dependent phosphatase restores function of IL-1 receptor-associate kinase-1 and reverses Toll-like receptor 2and 4-dependent tolerance of macrophages. J Immunol 2003; 171(3):1456-1465.

CHAPTER 5

Modulation of Positive Signaling and Proinflammatory Responses by Hemozoin, a Plasmodium Metabolic Waste Martin Olivier* and Maritza Jaramillo

Abstract

D

uring its intraerythrocytic life cycle, Plasmodium digests up to 80% of host hemoglobin as its nutrient source. However, this process releases the monomer heme that is very toxic for the parasite. Therefore, heme is sequestered as an insoluble, crystalline, dark-brown pigment called hemozoin (HZ), which is formed by heme dimers that interact through hydrogen bonds. The presence of HZ inside erythrocytes and leukocytes has been used in malaria diagnosis and prognosis. Its synthesis is analogous to a process of “biocrystallization” and appears to be the target of potent antimalarial drugs; however, it is not completely understood whether it is autocatalytic or if it requires the participation of proteins and/or lipids. Even though HZ was considered as a nontoxic metabolic byproduct, different lines of evidence, including its rapid phagocytosis, its accumulation inside organs and its ability to modulate macrophage signaling and functions, suggest that HZ might contribute to malaria immunopathology.

Hemozoin and Macrophages Hemozoin (HZ) or malaria pigment was previously named “plasmodin” and also “hemo-melanin” until Sinton and Ghosh1 credited Sambon with the origin of the term “hemozoin” in 1934. This pigment was described in malaria patients long before the parasitic nature of this disease was clarified. In 1717, Lancisi was the first to note discoloration of the internal organs in malaria victims.2 In 1847, Meckel identified the characteristic pigment in the viscera and blood of people who died of “pernicious fever” and proposed that the pigment itself was the cause of malaria.3 In 1849, Virchow was the first to connect malaria infection with the presence of pigment in the blood4 and, in 1850 Hischl associated it with intermittent fevers.5 However, it was only thirty years later, when Laveran discovered the malaria parasite itself, that the source of HZ became clear.6 In his description of unstained smears from malaria patients, he noted that pigment granules were visible inside parasites. Confirming and extending these observations, Councilman and Abbot noted the blackish color of brain, lungs, liver *Corresponding Author: Martin Olivier—McGill University, Research Institute of the MUHC, Centre for the Study of Host Resistance, Duff Medical Building, Departments of Medicine, Microbiology and Immunology, 3775 University Street, Montréal, Québec, Canada H3A 2B4. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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and spleen of patients who died of malaria and associated it with the presence of HZ both inside and outside infected red blood cells (iRBC).7 Importantly, in 1897, when Ross detected the pigment inside bodies (parasites) contained in insects fed on malaria-infected blood, HZ helped to the identification of the Anopheles mosquito as the vector of malaria.8 Early this century, Brown identified the chromogenic species as hematin9 and suggested that HZ might be involved in the pathophysiology of malaria.10,11

Hemozoin Synthesis During the intraerythrocytic phase of its life cycle, the parasite Plasmodium matures within a cell in which hemoglobin (Hb) is the single major cytosolic protein. The malaria parasite has a limited capacity to synthesize amino acids de novo12 or to take them up exogenously.13 Therefore, while in the trophozoite stage, it avidly ingests and degrades up to 80% of host erythrocyte Hb14 into amino acids for use in protein synthesis15 and as an energy source.12 The parasite ingests Hb by means of a specialized mouth-like structure called the cytostome, which sucks RBC cytosol into the parasite by a process resembling pinocytosis.15 Hb-containing vesicles are pinched off from the cytostome and carry their contents to the digestive vacuole (DV), an acidic organelle (pH 5.0-5.5) where Hb is broken down.16 This process releases heme, which does not appear to be metabolized but rather accumulates within the DV as crystalline particles called HZ.17 In contrast to the trophozoite, during the ring stage, Hb digestion appears to be limited; however, HZ can still be observed,14 suggesting that the cellular machinery for ingestion and proteolysis is present. In fact, several studies have revealed that early in parasite development small portions of cytoplasm are taken up by micropinocytosis and, as the parasite matures, a larger volume of Hb is ingested via the cytostomal system.16,18 The process of HZ formation is of great interest to malariologists, not least because it is thought to be the target of some of the most effective antimalarial drugs.19 Even though the exact point in Hb digestion at which this process occurs has not been established,20 it is known that during this degradative pathway heme is released and immediately autoxidized to potentially toxic ferric heme. Free heme can damage cellular metabolism by inhibition of enzymes,21,22 peroxidation of membranes,23 and the production of oxidative free radicals in the acidic environment of the DV;24 therefore, its efficient disposal is crucial to parasite survival. In Plasmodium sp., rather than excreting or degrading heme, as occurs in mammalian cells,25 the parasite has evolved a novel pathway for its detoxification by incorporating it into an insoluble, crystalline dark-brown substance called HZ that is harmless to the parasite.26 Morphologically, the crystals exhibit a triclinic habit and are uniform in shape and size (see Fig. 1).27 These characteristics are

Figure 1. Scanning electron microscope images of Plasmodium falciparum hemozoin (left) and synthetic crystallized hemozoin (right). (Courtesy from Dr. Scott Bohle and Marie-Josée Bellemare, McGill University).

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typical of a process called “biologically controlled crystallization”—biocrystallization—in which an organism exerts a great deal of control over the nucleation and growth of the crystal.28,29 Even though it is widely recognized that a nucleation center or template is required to initiate the process,29 direct observation of HZ synthesis within the parasite is extremely challenging; thus, there is currently no consensus on the mechanisms of HZ formation in vivo.30

Hemozoin Accumulation and Disease Diagnostic-Prognosis HZ-containing parasites as well as free HZ are released as the iRBC bursts, and are avidly phagocytosed by circulating monocytes, neutrophils (Nφ) and tissue macrophages (Mφ).31-37 By reference to standard hematological measurements, it has been estimated that as much as 200 micromoles of HZ (~3 micromoles/Kg) are released into the circulation of a 70 kg Pf-infected human patient who has a 1% synchronized parasitemia.31 Since Laveran’s original description that pigment granules were visible not only in the blood but also in leukocytes,6 the presence of HZ in these cells has been a diagnostically useful marker for malaria.38-40 Later, intraleukocytic pigment was also associated with cellular activation and disease severity. In a study performed in adult patients with severe P. falciparum malaria, those who succumbed to infection had significantly higher proportions of HZ-containing Nφ and Mφ on admission than did survivors.34 Additionally, the count of peripheral HZ-containing Nφ was a better indicator of bad prognosis than the peripheral parasite count in infected patients.34,36 Evidence from autopsy and histological studies indicated that HZ accumulates in the reticuloendothelial system, turning the liver and spleen black in cases of chronic or repeated infection,41 where pigment persisted for 6 to 12 months after malaria parasite clearance.42 As patient conditions deteriorated and cerebral malaria (CM) occurred, brain capillaries were also filled with infected RBC and HZ-laden monocytes.43 Similarly, phagocytes located in the eyes, lungs, and kidneys also appear to be loaded with HZ.44,45 In line with these observations, HZ has been detected in liver and spleen of infected mice for at least 6-9 months after malaria parasitemia becomes undetectable,26 and its accumulation has been associated with increased parasite burden and disease chronicity.47 Moreover, histological analysis indicated that HZ accumulation occurred predominantly in the tissue Mφ of the infected animals.31,47,48 In a murine CM model, the amount of HZ in the brain was shown to be higher than in mice that did not develop this complication, indicating that the levels of HZ in this organ may be associated with the cerebral pathology.47 Altogether, these different lines of evidence suggested a clinical correlation between disease severity and HZ phagocytosis and tissue accumulation. However, it was not clear whether this association was only circumstantial or if it was due to a direct effect of HZ on the host immune system. The current available data indicate that HZ exerts immunomodulatory effects; however, the nature of the interaction between HZ and phagocytes, as well as its contribution to the pathogenesis of malaria, and particularly its severe complications, are still controversial. Indeed, both inhibitory48-58 and activating31,56,59-64 effects have been described in pigment-fed phagocytes.

Inhibition of Immune Cell Functions

Incubation of P. falciparum HZ with either murine Mφ31 or human monocytes37 led to rapid phagocytosis of the pigment. Although HZ-laden phagocytes retained their ability to degrade heme, HZ remained unmodified.37 Because HZ is a powerful generator of ROS49 and of highly potent products of lipoperoxidation,65 it has been postulated that the persistence of HZ in long-lived cells such as Mφ may thus represent a lasting source of noxious products.37 Although HZ was previously considered an inert storage form of toxic heme,66 several studies have shown that the malaria pigment can cause alterations of certain important functions of the phagocytes.49-58 Following phagocytosis of either HZ or P. falciparum-infected RBC, monocytes lost their microbicidal and tumoricidal capacities.67 Of interest, Schwarzer and colleagues49 demonstrated that human monocytes fed with trophozoite-parasitized RBC or with isolated HZ were unable

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to degrade the ingested pigment, to repeat the phagocytic cycle, and permanently failed to produce oxidative burst after phorbol ester (PMA) stimulation. In parallel, HZ-laden monocytes displayed a long-lasting oxidative burst and increased lipid peroxidation. In a separate study, comparison of the effects of HZ in monocytes and Nφ revealed that whereas PMA-induced ROS was reduced in HZ-laden monocytes, it remained unaffected in HZ-loaded Nφ,62 suggesting that the immunomodulatory properties of HZ are cell-type specific. Subsequently, Schwarzer et al52 proposed protein kinase C (PKC) as a potential target for HZ-inducible ROS and iron leading to phagocyte dysfunction. Consistent with this, membrane-associated PKC was shown to be irreversibly inactivated in HZ-fed human monocytes.52 A plausible explanation for these findings would include HZ-mediated oxidation of cysteine-rich regulatory domains on PKC.68 However, HZ-induced effects on phagocytes could not be solely ascribed to ROS-mediated damage to PKC because it was impossible to restore its activity and membrane translocation using radical scavengers.52 Given that PMA-mediated oxidative burst involves PKC-dependent phosphorylation of the cytosolic component of NADPH oxidase (NOX),68 it was next assessed whether inhibition of membrane-associated PKC by HZ was related to NOX inactivation.53 Even though HZ phagocytosis by human monocyte-derived Mφ resulted in NOX inhibition, its phosphorylation by PKC was impaired only after 24 h of HZ phagocytosis. Thus, only long-term inactivation of NOX by HZ seems to be dependent on PKC inhibition. Suppression of leukocyte-derived prostaglandin E2 (PGE2) has been associated with enhanced malaria pathogenesis in children.69 Consistent with this, elevated PGE2 levels in intervillous blood mononuclear cells (IVBMC) were associated with protection against placental malaria (PM) in infected pregnant women. Lower levels of phytohemagglutinin-promoted PGE2 were detected in IVBMC in association with increasing amounts of HZ acquired during natural infection.55 These data suggest that in vivo acquisition of HZ by placental mononuclear cells may lead to decreased synthesis of PGE2. Because PGE2 appears to have a protective role during both CM and PM,55,69 by reducing its production HZ could contribute to the pathology related to these clinical complications. In addition to its ability to impair monocyte phagocytic and microbicidal functions, a number of studies have reported the down-regulatory effects of HZ on monocyte/Mφ cytotoxic mediator production in response to proinflammatory stimuli.48,56,57,70 When murine peritoneal Mφ were loaded with P. vinkei HZ and further stimulated with IFN-γ and/or LPS, the production of IL-6 was drastically reduced.56 Similarly, in human endothelial cells, β-hematin inhibited the expression of IL-6 in response to LPS or TNF-α.58 Moreover, 24 h pretreatment with P. vinkei HZ caused a significant decrease of LPS+IFN-γ-mediated nitric oxide (NO) production and of zymosan-inducible ROS generation in murine peritoneal Mφ.48 Consistent with this, β-hematin or hematin led to a marked reduction on LPS-inducible NO and TNF-α production in murine peritoneal Mφ. In contrast, IL-1 production remained unchanged and that of IL-6 was impaired only at very high HZ concentrations, indicating a selective inhibition of Mφ functions. In contrast, the same treatment did not exert any effect in microglial cell lines.70 Accordingly, the down-regulatory effects of HZ seem to be due to its heme moiety and may be selective for specific phagocytes and cytotoxic mediators. Addition of antioxidant agents, such as reduced glutathione (GSH), during the HZ preincubation time partially counterbalanced the inhibitory actions of HZ, suggesting a role for HZ-inducible oxidative stress on peripheral Mφ unresponsiveness. In correlation with an earlier study describing the absence of inhibitory effects in microglial cell lines by β-hematin,70 a lower extent of induction of heme oxygenase (HO) and catalase as well as a lower accumulation of oxygen radicals was observed in β-hematin-fed microglial cells over peritoneal Mφ.57 It appears that according to their antioxidant defenses and/or their lipid membrane composition, phagocytes from different sources may differ in their susceptibility to the pro-oxidant activity of HZ. These findings may help to reconcile reports on both proinflammatory and suppressive activities of HZ in mononuclear phagocytes.57,71

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A major function of Mφ is to promote cell-mediated immune responses by presenting antigenic peptides via surface glycoproteins of the major histocompatibility complex (MHC) class II.72 In HZ-preconditioned human peripheral blood mononuclear cells (PBMC), the induction of MHC-II by IFN-γ stimulation was severely blunted both at the mRNA and protein levels.51 Mechanistically, the abrogation of IFN-γ responsiveness is unexplained. However, since PKC plays a role in IFN-γ-inducible MHC-II expression in Mφ73 and PKC inactivation appears to be due to 4-hydroxynonenol alipoperoxide (HNE) in HZ-loaded monocytes,65 the involvement of HZ-mediated PKC inhibition via HNE might be a potential pathway for MHC-II down-regulation by the malaria pigment.51 The constitutive expression of ICAM-1 and CD11c, were also decreased in PBMC upon P. falciparum HZ phagocytosis.51 ICAM-1 contributes to the capacity of APCs to adhere and stimulate T cell proliferation by reinforcing the signal from the T cell receptor,74 and CD11c has been identified as a ligand for ICAM-1.75 Therefore, down-regulation of their expression by HZ may help to explain the defective T cell response in malaria. In contrast, HZ ingestion had no effect in the constitutive expression of other receptors implicated in phagocytosis, including CR1, CR3, CD36, and CD64. Thus, only the expression of molecules that play essential roles in antigen presentation and T-cell response appears to be specifically hindered upon HZ phagocytosis.51 Thus supporting a role for HZ in cell-mediated immunity defects associated with malaria immunosuppression.76-78 In human malaria, immunosuppression appears late in the acute phase of the disease and can last a long time after the clearance of parasites from the circulation.76,79 Although the precise mechanisms are not clear, early investigations have suggested that defects in Mφ accessory cell function could account for the poor immune reactivity.80-82 In P. berghei-infected mice, HZ-laden liver and splenic Mφ displayed accessory cell dysfunction. In addition, either isolated P. falciparum HZ or P. berghei HZ from livers and spleens of infected animals markedly reduced splenic plaque-forming cell responses to sheep RBC.50 Further supporting a role for HZ in Mφ accessory cell dysfunction, either P. chabaudi HZ or β-hematin were able to mimic in vitro the immunosuppressive effects induced in peritoneal Mφ during in vivo infections with P. chabaudi.83 These studies suggest that the inhibition of Mφ accessory cell activity is due, at least in part, to the uptake and accumulation of HZ. However, whether or not similar responses to HZ occur in vivo and the exact cellular mechanisms involved remain to be elucidated.

Activation of Immune Responses

In 1994, Pichyangkul and colleagues59 first described HZ as a “potential virulence factor in the monokine-mediated induction of organ-specific and systemic pathology in P. falciparum malaria”. These conclusions were based on the observation that human PBMC cocultured with schizont-parasitized RBC or with isolated P. falciparum HZ rapidly released high levels of TNF-α and IL-1β upon treatment. However, protease treatment of the isolated pigment abolished PBMC cytokine production. In addition, β-hematin preparations induced only minimal production of TNF-α and IL-1β, suggesting that the active moiety of HZ was a protein or a protein-linked molecule associated with the malaria pigment. These data are in sharp contrast with those obtained in later investigations in which either β-hematin or native purified HZ was shown to induce phagocyte cytokine production.31,65 These discrepancies remain unclear and are probably related to differences in the preparation and characteristics of HZ/β-hematin samples used,84 as well as in the pigment concentrations used and the incubation times. Alternatively, the differences might be also associated with a variable susceptibility of the phagocytes to HZ treatment according to its antioxidant defenses and/or their lipid membrane content.57,71 Both purified P. falciparum HZ and chemically synthesized β-hematin potently induced the release of TNF-α, MIP-1α and MIP-1β, from murine Mφ and human PBMC.31 In contrast, phagocytosis of other well-characterized forms of heme, hemin and hematin as well as that of latex beads did not induce the production of any of these proinflammatory mediators. Additionally, incubation of human monocytes with P. falciparum HZ strongly stimulated TNF-α,

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IL-10 and IL-12 production62 suggesting that HZ might exert both pro- and anti-inflammatory effects in the infected host. In murine peritoneal Mφ, P. vinckei HZ enhanced TNF-α production after costimulation with LPS + IFN-γ,56 which might help to explain the some times very high TNF-α levels in peripheral circulation of patients with severe malaria and bacteriemia.85 Severe anemia is one of the main causes of morbidity and mortality related to P. falciparum malaria.86 Although the role of host-derived molecules (TNF-α, IFN-γ; IL-1β) as erythropoiesis inhibitors has been explored,87 only IL-12 appears to exert a protective effect.88 In a search for additional host factors contributing to malarial anemia, Martiney et al61 found that macrophage inhibitory factor (MIF) inhibited erythropoiesis in vitro in presence of erythropoietin. In the sera of P. chabaudi-infected mice, increased levels of MIF were detected, correlating with disease severity. Ingestion of P. chabaudi-iRBC or HZ induced MIF released from murine Mφ. In addition, MIF was elevated in the liver, spleen and bone marrow at peak levels of parasitemia, where high HZ and iRBC accumulation was observed. These data are in line with ultrastructural analysis of the bone marrow of severely anemic children showing Mφ containing iRBC and HZ,89 and with a previous report in which HZ-laden Mφ were seen adjacent to developing erythroblasts in the red pulp of the spleen in infected animals.87 Another characteristic feature of P. falciparum malaria is the cytoadherence of iRBC and leukocytes to vascular endothelium.90,91 Exposure of Nφ or Mφ to isolated P. falciparum HZ resulted in significant up-regulation of the adhesion molecule CD11b/CD18 and down-regulation of leukocyte adhesion molecule 1 (LAM-1). The involvement of CD11b/ CD18 and LAM-1 in leukocyte adhesion to endothelium during inflammatory responses is well documented,92 and their inverse expression has been detected in parallel upon leukocyte activation,93 including cellular responses to TNF-α, C5a and leukotriene B4 (LB4).94 Thus, it is likely that phagocytosis of HZ leads to release of several mediators, which in turn regulate the expression of these adhesion molecules. A causal relationship between HZ and proinflammatory mediator release in vivo remains elusive. However, a study using a rat model revealed that HZ has the ability to induce changes in body temperature31 via the induction of strong pyrogens (e.g., TNF-α, IL-1).90,95 In addition, colocalization of HZ and proinflammatory mediator production has been reported in both experimental and clinical malaria.96-98 Based on these different lines of evidence as well as on its effects in vitro, it is conceivable that HZ exerts immunomodulatory effects on the host.

Signaling Events Regulating HZ-Induced Inflammatory Response HZ and Nitric Oxide

In malaria, increased NO production has been reported in P. falciparum-infected patients99-103 and experimental models of infection.104,105 Both protective100,106-109 and detrimental109-111 roles have been attributed to this mediator in malaria. IFN-γ has been identified as an important Mφ activator during Plasmodium infection112 and thus, central in the regulation of NO synthesis.108 Recently we found that even though HZ itself did not induce Mφ NO production, it significantly enhanced it in response to IFN-γ.113 This synergistic effect was found to be dependent on the combined action of HZ and IFN-γ in inducible nitric oxide synthase (iNOS) expression. Analysis of the molecular events revealed the involvement of complementary intracellular signals and transcription factors. IFN-γ induced Jak2 phosphorylation and the subsequent activation of the transcription factor STAT1α (signal transducers and activators of transcription), two events that were essential in IFN-γ + HZ-mediated NO production. These observations are not surprising given that the Jak2/STAT1α pathway controls iNOS expression both directly, via STAT1α binding to the iNOS promoter.114 Despite the requirement of Jak2-dependent signals, HZ was found to exert its effect on iNOS expression through alternative pathways. In addition to its ability to activate ERK1/ 2-mediated binding of nuclear factor kappa B (NF-κB) to the murine iNOS promoter, HZ enhanced this event in response to IFN-γ (see Fig. 2). This is in line with a number of studies

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Figure 2. Signaling events involved in hemozoin plus IFN-γ-mediated iNOS expression in macrophages. Involvement of JAK2-STAT1 and MAP kinases-NF-κB pathways. (?) Other transcription factors such as CREB and AP-1 could play a role in this activation process. Whereas ERK1/2-NF-κB pathway was shown to be responsible for augmented NO generation when phagocytes were subjected to IFN-HZ stimulation, the JAK-STAT pathway was reported to be necessary for this activation but not modulated by HZ. It is clear that this response may vary from monocytes to tissue macrophages depending on their state of development and redox.

that have associated synergistic iNOS modulation with a dual up-regulation of NF-κB by IFN-γ in combination with other host cytokines,115 or microbial components.116 Of interest, the synergistic induction of inflammatory genes (e.g., iNOS, IP-10, ICAM-1) appears largely dependent on the cooperative action of NF-κB with STAT1α and/or IFN-γ responsive factor 1 (IRF-1).117-124 Whereas STAT1α and NF-κB, seem to bind independently to their respective sites on the promoter,117 the mechanism underlying the synergistic action of IRF-1 and NF-κB seem to necessitate their physical interaction.119,123,124 Thus, the possibility that NF-κB contributes to the HZ + IFN-γ-inducible iNOS promoter activation by acting in synergy with STAT1α and/or IRF-1 should not be ruled out. Because of the dual role proposed for NO in malaria, it is difficult to speculate at this point whether the contribution of HZ to increase its production is harmful or beneficial to the host. Before drawing any definitive conclusions, further investigations will need to be performed in vivo. Of interest, whereas contradictory results have been reported regarding a correlation between serum nitrate levels and human severe malaria,100,101 high levels of iNOS expression were detected in brain tissue from fatal cases of CM,102 underlining the importance of monitoring local iNOS and NO production during CM. Even though a role for NO has been proposed in human CM,99-102,125 and iNOS expression in circulating monocytes has been correlated with increased systemic NO production in malaria infected children,100 the ability

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of human Mφ to produce NO is still controversial.123-130 Therefore, whether HZ enhances human monocyte/Mφ NO generation in response to IFN-γ needs to be clarified.

Modulation of Chemokine Gene Expression by HZ High expression of chemokines and/or their receptors can create the potential for persistent or inappropriately exacerbated response (e.g., tissue damage, microvascular obstruction). In most cases, temporal and spatial patterns of chemokine/receptor expression are interwoven with ligand-receptor specificity to result in a multicomponent pathophysiological response.131 Therefore, over the last few years we have sought to identify the members of the chemokine system induced in response to HZ and to unravel the mechanisms controlling their expression in the Mφ, one of the major sources of inflammatory mediators in malaria.90 We found that HZ increases the expression of various Mφ chemokine transcripts (MIP-1α, MIP-1β, MIP-2 and MCP-1).132 Chemokine mRNA up-regulation was detected at early times of HZ stimulation and occurred in a concentration-dependent manner, suggesting that upon its release into the microvasculature, HZ might interact with resident Mφ and lead to rapid chemokine production. In this context, HZ could stimulate the immunological response in the sites of parasite sequestration by inducing potent chemoattractants and activators. However, at higher parasitemias and subsequent higher HZ concentrations, the malarial pigment might also contribute to an exacerbated proinflammatory response (e.g., leukocytosis, high reactive oxygen species (ROS) production) causing tissue damage and microvascular flow disturbance. The increase in HZ-inducible chemokine mRNA levels occurred through a mechanism dependent on ROS- and MEK1/2-ERK1/2-signals (see Fig. 3). The intracellular events

Figure 3. Hemozoin-induced macrophage chemokine genes expression regulated by oxidative stress-dependent and –independent/Erk1/2-dependent mechanisms involving NF-κB activation. (?) Other transcription factors such as CREB and AP-1 could play a role in this activation process. TLR9 has been reported to recognize hemozoin (HZ), however the identification of receptor(s) through which HZ triggers the signaling cascades conducting to chemokine expression needs to be clearly established. Crystal size and quality of synthetic hemozoin may influence host signaling in a different manner and therefore contribute to host cell activation.

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triggered by these second messengers resulted in the reduction of Mφ protein tyrosine phosphatases (PTP) activity, the nuclear translocation of NF-κB and its subsequent binding to the various chemokine promoters. Despite the activation of several transduction signals, this cellular regulation was specific, since other kinases—including p38 and JNK MAPK—shown to be involved in chemokine regulation were not activated in response to HZ.132 Our data indicating the requirement for MEK1/2-ERK1/2 and NF-κB activation are in agreement with several studies showing their involvement in chemokine induction by pathogens (e.g., H. pylori, Trichomonas vaginalis),133,134 cytokines (e.g., TNF-α)135-137 and host inflammatory particles (e.g., uric acid, monosodium urate (MSU) crystals).138-140 Of interest, in addition to ERK1/2 and NF-κB, the involvement of ROS was also reported,136-38 which might be related to their ability to act as a “danger signal” to the immune response.141 Because an important role of chemokines is to alert the immune system against pathogen invasion or tissue injury,131 it is plausible that immune cells have developed a rapid response to infection or inflammation through the activation of common intracellular messengers. Further supporting this idea and as discussed above, we found that the up-regulating effect of HZ on IFN-γ-mediated NO production also occurred via ERK1/2-dependent NF-κB activity. Thus, it is likely that by triggering the same intracellular signals, HZ modulates different Mφ functions, including chemokine expression and amplification of Mφ responses to IFN-γ, which would in turn contribute to enhance the inflammatory response during malaria infection. We found that HZ-inducible NF-κB activity was in part mediated by ERK1/2-dependent signals. Consistent with this, previous studies have shown that either specific inhibitors142-144 or constitutive mutants145 of the MEK1/2-ERK1/2 pathway abrogated NF-κB activation. Although the mechanism through which ERK1/2 enhances NF-κB translocation needs to be investigated, it is known that Ras/Raf/MEK1/2-ERK1/2-mediated signals can lead to NF-κB activation by phosphorylating and activating its positive regulators, IκB kinases (IKKs).146-148 Indeed, Ras/Raf-activated MEKK1 phosphorylates subunits of the IKK complex and modulates their activity.146 Even though MEKK1 can activate both IKKα and IKKβ,148 it preferentially phosphorylates IKKβ on Ser177 and Ser181.147 Whether MEKK1-mediated IKK phosphorylation constitutes a key step in HZ-inducible NF-κB nuclear translocation and chemokine transcription remains to be elucidated. In addition to the involvement of the MEK1/2-ERK1/2 pathway, we found that NF-κB nuclear translocation was partially dependent on ROS generation. These observations are consistent with our data demonstrating the activation of this transcription factor in response to oxidative stress.149 In this paper, we reported that H2O2 activated MEK1/2-ERK1/2-dependent signals were required for NF-κB activation and chemokine gene expression. From these data, it would have been logical to predict that in response to HZ-inducible ROS, ERK1/2 phosphorylation occurred and resulted in enhanced NF-κB activity. However, ERK1/2 phosphorylation by HZ appeared to be independent of ROS generation, since superoxide dismutase (SOD) pretreatment did not affect this event. These discrepancies might stem from the fact that the oxidative stress produced in response to HZ was much lower113 than that generated by the addition of H2O2 (500 μM), as we used previously.149 Thus, it is plausible that different levels of oxidative stress trigger different Mφ signaling pathways. Although the Mφ receptor(s) involved remain to be identified, several lines of evidence point to a role for members of the TLR family. Activation of TLRs (e.g., TLR-2, TLR-4) in response to parasitic infections, including Trypanosoma cruzi,150,151 Toxoplasma gondii152 and Leishmania major,153,154 appears to play an important role in host defense. Although this mechanism of immunity remains largely unexplored in malaria, some studies have reported TLR-mediated signaling in response to Plasmodium. In mice deficient in MyD88 P. berghei infection did not result in elevation of IL-12 levels and did not cause liver injury.155 Moreover, a recent study demonstrated that P. falciparum activates DC via TLR9-dependent signaling.156 Similarly, HZ has been reported to modulate immune cells through TLR9.157 These data suggest that the TLR system is also important in the inflammatory response during malaria infection.

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In Vivo Modulation of Proinflammatory Mediators by HZ

Despite the demonstration that HZ induces cytokine and chemokine expression in vitro,59,63 a causal relationship between HZ and proinflammatory mediator release in vivo was not clearly yet established. But over the last few years, we brought about clarification of this important issue. Initially we made use of the murine air pouch system, an experimental approach commonly used,158-160 because it provides a suitable closed environment to evaluate multiple inflammatory parameters in response to a given stimulus. HZ injection in the pouch resulted in significant leukocyte recruitment (mainly Nφ and monocytes), which was accompanied by increased levels of various chemokines (MIP-1α, MIP-1β, MIP-2 and MCP-1), chemokine receptors, (CCR1, CCR2, CCR5, CXCR2, CXCR4), proinflammatory cytokines (IL-1 and IL-6) and myeloid-related proteins (MRPs; S100A8, S100A9 and S100A8/A9).161 Based on these results, and in order to mimic HZ release into the circulation during malaria, HZ was next inoculated i.v.. The effects of HZ were monitored in the liver since its accumulation in this organ has been associated with infection chronicity in humans,41 and with cumulative parasite burden and duration of infection in experimental malaria.47 We found that HZ caused a concentration-dependent increase of various chemokine (MIP-1α, MIP-1β, MIP-2 and MCP-1) and cytokine (IL-β and IL-12) transcripts in the liver.161 Even though the cellular sources of these inflammatory mediators remain to be identified, several lines of evidence point to a role for Kupffer cells and monocytes via ROS generation. In a pilot study Sherry and colleagues detected HZ accumulation in Kupffer cells upon i.v. injection of the pigment.31 Moreover, liver chemokine induction31,162 as well as monocyte liver infiltration and increased O2- levels163 were reported in Plasmodium-infected mice. Additionally, it has been shown that the major sources of chemokines and ROS during liver disease are Kupffer cells, and the production of ROS by these cells enhances the secretion of cytokines and chemokines.164 Because HZ-inducible Mφ chemokine expression was in part ROS-dependent, and monocyte chemoattractants were detected in the liver of HZ-injected mice, it is possible that HZ triggers ROS-mediated cytokine and chemokine production by Kupffer cells, which would in turn contribute to monocyte infiltration and further oxygen radical generation and inflammatory mediator release. This inflammatory response could contribute to reduce parasite burden, but could also favor exacerbated phagocyte activation, resulting in hepatic damage, as observed in both human and murine malaria.165 Indeed, it has been proposed that ROS may play a role in the pathogenesis of severe malaria by causing endothelial damage,166 and their production has been associated with disease severity in adults and children.167-169 Thus, it will be important to assess the ability of HZ to induce ROS and chemokine production in Kupffer cells, and to define the contribution of ROS to the effects of HZ in vivo.

Concluding Remarks Collectively several findings have demonstrated that HZ is a potent proinflammatory agent in vitro and in vivo, and suggest that this molecule may play a role in mediating both beneficial and detrimental effects on the infected host. Because erythrocytic schizogeny and subsequent HZ release occur in the microvasculature of multiple organs where iRBC are sequestered, and both HZ and leukocyte accumulation have been reported in these locations, it is possible that the HZ-inducible immunological responses would be directed against the sites of maximal parasite sequestration. Therefore, by triggering specific intracellular signals leading to immune cell activation, HZ might function as a “danger signal” to alert the immune system against pathogen invasion. However, through an excessive inflammatory response, HZ is also likely to contribute to the pathogenesis of severe malaria. Although a causal link between HZ and the clinical manifestations of malaria remain to be established, our data—along with previous evidence from both human and experimental malaria—warrants further investigation of HZ as an immunomodulator. A better understanding of the antigenic properties of HZ as well as the analysis of (i) the local and temporal effects of HZ in vivo, (ii) the cellular sources and (iii) the signal transduction mechanisms through which this parasite metabolite controls the immune response, will be helpful in the design of future immune treatment against malaria.

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CHAPTER 6

Pro-Inflammatory Responses and Cell Signaling during Malaria Infection: The Parasite Glycosylphosphatidylinositol Ligand D. Channe Gowda*

Abstract

A

key feature of malaria infection is the production of high levels of TNF-α and other pro-inflammatory mediators that are thought to contribute to the systemic and organ-related malaria syndromes. In infection with Plasmodium falciparum, the parasite that causes fatal forms of malaria, the adherence and sequestration of infected erythrocytes in the microvascular beds of vital organs create pathogenic microenvironments and exacerbate the role of pro-inflammatory responses. Available information indicates that the glycosylphosphatidylinositols (GPIs) of P. falciparum are the specific and dominant parasite-associated molecular patterns recognized by the host innate immune system. The parasite GPIs appear to be mainly responsible for the ability of the parasite to induce potent pro-inflammatory responses in monocytes and macrophages, thereby playing an important role in malaria pathogenesis. The GPI-induced cellular activation is mediated mainly through the recognition of TLR2, involving MyD88-dependent signaling transduction and downstream initiation of ERK, p38 and JNK MAPK and NF-κB signaling pathways. The MAPK pathways and NF-κB family members differentially regulate the GPI-induced production of various pro-inflammatory cytokines and nitric oxide by macrophages. A comprehensive understanding of how the various signaling cascades of the cells of the host innate immune system induced by the malaria parasite factors regulate a wide range of cellular responses has important implications for the development of immunotherapeutics for severe malaria.

Introduction Malaria caused by the parasitic protozoa of the genus Plasmodium is a major public health crisis in the tropical and subtropical regions of the world.1-5 Nearly half of the global population lives in malaria endemic areas and is vulnerable to the disease. Afflicting about half a billion people, malaria causes 2-3 million deaths annually and ranks first among infectious diseases in rates of global morbidity and mortality. Additionally, since the disease is most prevalent in developing countries, its contribution to the socioeconomic burden of the people who are distressed by poverty, malnutrition and other infectious diseases is enormous.4 Furthermore, the emergence and high prevalence of AIDS in malaria endemic areas exacerbated the devastating effects of malaria.6,7 *D. Channe Gowda—Department of Biochemistry and Molecular Biology Pennsylvania State University College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033, U.S.A. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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During the past two decades, malaria has reemerged as a major threat because of the development of resistance of the mosquitoes to insecticides and the parasites to chloroquine and other commonly used drugs. The current rapid globalization and ever-increasing population mobility necessitate urgent efforts for novel treatments. In-depth understanding of the host-parasite interactions with regard to how the parasite exerts its deleterious effects on its human hosts, and how the host immune system recognizes the parasite and responds to infection would greatly facilitate efforts to develop therapeutics and/or a vaccine based on novel targets. Four species of Plasmodium, P. falciparum, P. vivax, P. malaraie, and P. ovale, infect humans.1,2 Of these, P. falciparum is the most deadly and accounts for more than 90% of deaths due to malaria. The mode of infection and developmental stages in the hosts are similar for all four human malaria parasites. The life cycle of Plasmodia is highly complex, involving asexual development in the animal host and sexual development in the mosquito vector to finally emerge as sporozoites to complete the life cycle.1,2 In both mosquito and human hosts, the parasites develop through several morphologically distinct stages. The parasites enter the human host in the form of sporozoites during a bite by infected mosquitoes and exclusively target the liver, invading hepatocytes. Infection at the liver stage is mostly asymptomatic. Over a period of 1-2 weeks, the parasites mature inside hepatocytes and differentiate into merozoites, with each infected liver cell releasing up to 30,000 merozoites into the bloodstream. These merozoites specifically invade red blood cells, and over the course of 48 h (72 h in the case of P. malariae), develop into trophozoites and finally to schizonts. Each schizont produce 8-24 merozoite progenies that go on to infect other erythrocytes.1,2 Multiplication of the parasites leads to massive destruction of red blood cells and stimulation of the host innate immune system to produce pro-inflammatory mediators. These and other events of infection collectively contribute to systemic and organ-related severe malaria, which are characterized by a wide range of clinical manifestations, including severe anemia, periodic fever and chills, shock, acute respiratory distress, pulmonary edema, cerebral complications with neurological problems such as seizure and coma, renal failure, and liver and other organ-related pathological conditions.1,2,5 Malaria has been the most extensively studied infectious disease in the past several decades with regard to the parasite biology, host immunity to infection, pathogenesis, and disease control-strategies. However, insight into the molecular basis of the host-parasite interactions that leads to the development of systemic and organ-related severe malaria is still limited. The available information indicates that severe malaria syndromes are complex and caused by the culmination of several pathogenic processes. Some of the well-recognized processes include the production of high levels of pro-inflammatory cytokines in response to rapidly expanding parasitemia, the rapid consumption of blood glucose by the parasite thus depriving vital organs of adequate levels of glucose needed for normal functions, and the destruction of infected and uninfected red blood cells leading to deprivation of oxygen for vital functions.2,5,8,9 Furthermore, P. falciparum, the parasite that causes the most severe malaria compared to the other three human parasites, has an important additional determinant that contributes to severe pathological conditions.5,8 The parasite expresses adherent protein(s) on the surface of the infected erythrocytes and sequesters in the microvascular beds of vital organs, causing capillary obstruction and creating pathogenic microenvironments. High parasite burden in localized areas and host responses to the parasite factors produce toxic levels of pro-inflammatory mediators in these microenvironments, damaging the endothelial cell lining. In addition, upregulation of endothelial cell adhesion molecules in response to the TNF-α that is produced augments the adherence of the infected erythrocytes, spiraling the destruction of the endothelial beds and causing organ-related pathological conditions.5,8-12 In this chapter, we discuss our current understanding of the pro-inflammatory responses that accompany malaria infection, key parasite factor and host receptors involved in eliciting the pro-inflammatory responses, and signaling pathways that govern these innate immune responses.

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Pro-Inflammatory Responses The pro-inflammatory cytokines elicited by the innate immune system in response to invading pathogenic organisms are critical for the host to control infections.13-17 In the absence of pro-inflammatory responses, pathogens will grow exponentially and can overwhelm the host with death to ensue. A large body of data accumulated over the past two decades indicates that during malaria infection the host immune system responds by producing high levels of pro-inflammatory mediators, which include TNF-α, IL-6, IL-12, IL-1, IFN-γ, and nitric oxide.18-21 This is evident from both population studies involving the analysis of sera samples from people in malaria endemic areas of different geographical settings as well as in experimental studies of human and rodent malaria.21-34 A number of studies using rodent malaria models have also amply demonstrated the beneficial function of pro-inflammatory cytokines and nitric oxide in reducing the parasite burden. Specifically, the cytokines such as TNF-α, IL-12, and IFN-γ are involved in the efficient inhibition of parasite growth.34-45 IFN-γ plays an important role in activating macrophages to induce the production of IL-6, IL-12, TNF-α, and reactive oxygen and nitrogen intermediates; of these, the latter mediators are directly cytotoxic to parasites.20,37 IL-12, a potent immunomodulatory cytokine, induces IFN-γ production by NK cells, potentiates cell-mediated immune responses, and modulates humoral responses by inducing antibody isotype-switching.46,47 Thus, pro-inflammatory responses elicited by the innate immune system are crucial for controlling parasite growth during the acute phase of malaria infection and in modulating parasite-specific adaptive immune responses. Pro-inflammatory responses that are produced in defense against the parasite can be harmful to the host if they are not properly regulated but instead are allowed to be produced in excess.18,20,48 Therefore, a balance between the control of parasitemia by pro-inflammatory mediators and the inhibition of these immune responses to avoid the deleterious effects of inflammatory responses on vital host functions is essential for preventing the development of pathogenic conditions. Normally, as the infection progresses, there will be a gradual transition from pro-inflammatory to anti-inflammatory response. First, the initial potent pro-inflammatory response effectively controls the acute phase of infection. Thereafter, as the adaptive immunity is developing to clear infection, the anti-inflammatory response progresses to inhibit the pro-inflammatory response from causing toxic effects. However, it appears that this delicate balance required for the resolution of infection without the development of severe illness is often not maintained in malaria-infected people. Depending on host genetic factors, such as the variation in the expression of receptor types for infected erythrocyte adherence, impaired innate immune responses due to defects in signaling cascades, and status of preexisting immunity, the infected individuals may develop a wide range of clinical conditions. High levels of pro-inflammatory mediators produced during malaria infection are associated with systemic effects, such as fever, chills, shock, and hypoglycemia, and organ-related pathological conditions, including cerebral syndromes.49-63 Excessive levels of TNF-α have been found in the plasma of patients with cerebral malaria and TNF-α level is much more elevated in the brains of patients who died of cerebral malaria.61-67 In rodent models as well, high levels of TNF-α and IFN-γ have been reported to be involved in cerebral syndromes.18,20,58,59,67 Anti-TNF-α and anti-IFN-γ antibodies have been shown to inhibit fever and promote faster recovery from clinical manifestations of severe malaria,59,68 although in one study a monoclonal antibody against TNF-α had no therapeutic benefits against cerebral malaria.69 The anti-inflammatory cytokines TGF-β, IL-10, and IL-4 have been shown to be crucial for protecting the host from the lethal effects of pro-inflammatory responses produced during infection.18,20,48 TGF-β plays a key role in the transition from pro-inflammatory response (Th1-type) to anti-inflammatory (Th2-type) response during the acute and resolving phases of malaria infection.70 The kinetics and level of TGF-β expression also appear to play an important role in malaria infection. Production of moderate levels of TGF-β early during infection leads to stimulation of monocytes/macrophages to kill parasites by phagocytosis.71 However,

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excessive production of TGF-β early in infection inhibits TNF-α and IFN-γ responses, thereby allowing the parasite to grow unchecked.72 Thus, unbalanced production of TGF-β during infection leads to the development of clinical conditions.73,74 IL-10 inhibits macrophage activation by suppressing the production of TNF-α, IL-6 and IFN-γ, thereby inhibiting the release of reactive oxygen and nitrogen species by macrophages.75 Therefore, early production of IL-10 potentially promotes parasite growth by prematurely inhibiting the pro-inflammatory responses necessary to effectively control parasitemia during the acute phase infection. High levels of IL-10 early on have been reported to be associated with acute malaria.76,77 However, efficient production of IL-10 after acute phase infection benefits the host by preventing the detrimental effects of pro-inflammatory responses.78-80 Thus, a complex interplay between pro-inflammatory anti-inflammatory responses occurs during malaria infection. These responses must be appropriately regulated for the proper outcome of infection. A comprehensive coverage of pro-inflammatory and anti-inflammatory responses in malaria infection is beyond the scope of this chapter. Several recent reviews covering these aspects are available.18,20,48 Compared to the large body of data available on pro-inflammatory responses during malaria infection and their roles in parasite growth control and immunopathology, relatively little is known about the parasite-host recognition mechanism involved in host cell activation and the accompanying signaling cascades. A characteristic feature of malaria infection is the occurrence of periodic fever, chills and shock that correspond to the period of synchronized burst of schizonts, producing peak levels of pro-inflammatory cytokines particularly TNF-α in response to the large amount of parasite mass released to the circulation.1,2 The components released from the schizonts include merozoites, parasite membrane fragments, digestive vacuoles containing the malaria pigment hemozoin, and parasite proteins expressed on the infected erythrocyte membrane. However, how these are recognized by the innate immune system and what is their relative contribution to overall pro-inflammatory responses remain largely unknown. Furthermore, studies from various laboratories demonstrated that circulatory monocytes, splenic macrophages, endothelial cells, dendritic cells, and NK cells are the major cells of the innate immune system that respond to malaria infection, triggering TNF-α, IL-12, IFNγ and other cytokine expression.18 However, to date, only limited information is available on the specific host receptors involved in the recognition of various parasite factors and cell signaling mechanisms.

Malaria Pro-Inflammatory Factor It has been known for a long time that severe malaria is caused by a toxin produced by the parasite.50 More than a century ago, the famous Italian Nobel laureate Camillo Golgi postulated that a parasite toxin released upon schizont rupture is responsible for the clinical manifestations of malaria.81 In 1921, Collier hypothesized that fever and other clinical symptoms of malaria were due to endogenous ‘auto-noxins’ produced by the host in response to the parasite-derived ‘auto-noxigens’.82 About 60 years later, it was proposed that TNF-α and other pro-inflammatory mediators released by monocytes and macrophages in response to parasite-derived endotoxin-like factor are responsible for malaria illness.83,84 During the past two decades, a number of studies have demonstrated the importance of pro-inflammatory responses in malaria pathogenesis. However, the nature of the parasite factor that exerts endotoxin-like activity remained unknown until 1993 when Schofield and Hackett found that the glycosylphosphatidylinositols (GPIs) purified from P. falciparum can induce TNF-α and IL-1 in macrophages and regulate glucose metabolism in adipocytes.85 The GPIs administered to animals could cause pyrexia, hypoglycemia and cachexia, the symptoms that are reminiscent of severe malaria illness. Later studies have shown that the parasite GPIs can induce the expression of inducible nitric oxide synthase, upregulate the expression of cell adhesion molecules, such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-selectin in endothelial cells, implicating these processes in malaria pathogenesis (discussed below). Thus, P. falciparum GPIs has been proposed as the dominant factor responsible for malaria pathogenesis.

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Studies from various laboratories during the past decade have demonstrated that GPIs are abundantly expressed in disease-causing protozoan parasites, including Plasmodia, Trypanosoma, Leishmania, and Toxoplasma.86-88 The GPIs of these parasites can activate host macrophages, initiating specific cell signaling pathways and triggering the production of pro-inflammatory cytokine responses similar to those elicited by bacterial LPS.86-90 From recent studies (Gowda and coworkers, unpublished data), it appears that, in malaria parasites, GPIs are the major molecular patterns recognized by the innate immune system, and therefore, they appear to play a prominent role in malaria pathogenesis.

P. falciparum GPI Structure The structures of P. falciparum GPIs were first studied by Schwarz and coworkers by analyzing the radiolabeled biosynthetic products and the GPI moieties of two of the major parasite surface molecules, merozoite surface protein-1 and -2.91,92 They found that the mature form of the parasite GPI (Man4-GPI) consists of the evolutionarily conserved glycan structure with an additional mannose residue attached to the third mannose residue via an α(1-2) glycosidic linkage and a phosphatidylinositol (PI) moiety comprised of acylated inositol and diacylglycerol. By mass spectrometry and by glc-ms analysis, Naik et al showed that the parasite Man4-GPI is very heterogeneous with regard to fatty acids at various positions and thus occurs as multiple species (Fig. 1).93 P. falciparum uses the Man4-GPI species for protein anchoring without discrimination toward fatty acid heterogeneity. The parasite contains considerable amount of Man4-GPIs in the free lipid form (not attached to protein) in its membranes, and the level of GPIs that are present in free form is about four- to five-fold higher than GPIs involved in anchoring parasite proteins.93 The structural features that distinguish P. falciparum GPIs from the GPIs of host are the uniform presence of a fourth mannose attached to the conserved glycan core, the absence of extra ethanolamine phosphate substituents on the first or second mannose, the exclusive presence of an unsaturated fatty acyl moiety at the sn-2 position, and extreme heterogeneity of the various fatty acid substituents.86,91-94

GPI Structure-Activity Relationship Early studies have reported that micromolar concentrations of P. falciparum GPIs are necessary for the efficient production of TNF-α and nitric oxide in macrophage cell lines.95,96 However, recent studies revealed that 5-20 nanomolar concentrations of P. falciparum GPIs are sufficient to induce high levels of TNF-α and nitric oxide in mouse macrophages and human monocytes primed with IFN-γ,97 indicating that the potency of malarial GPIs are physiologically relevant to their ability to contribute to malaria pathogenesis. Priming with IFN-γ was essential for macrophages and monocytes to produce IL-6, IL-12 and nitric oxide in response to GPIs.97 The structure-activity relationship studies in GPIs of P. falciparum, Trypanosoma and Leishmania by Tachado et al have shown that the conserved GPI structure containing three mannoses and the ethanolamine phosphate substituent at C6 of the third mannose is the minimal structure involved in the pro-inflammatory response-inducing activity.96 When the GPIs are cleaved into glycan and PI moieties, either one of those or a mixture of both was unable to induce pro-inflammatory responses, demonstrating that the intact structure is essential for the bioactivity of GPIs.96-99 Studies have also shown that dual interactions by the covalently bound glycan and lipid moieties of GPIs are critical for the bioactivity of GPIs.96-99 For the inflammatory activity of P. falciparum GPIs, the acyl substituent at the sn-2 position is not required, while the removal of fatty acids at both sn-1 and sn-2 positions abolishes the activity. This is in contrast to T. cruzi GPIs in which the removal of sn-2 fatty acid completely abolished the inflammatory activity.88,89 However, it should be noted that, unlike T. cruzi GPIs, P. falciparum GPIs have a fatty acyl substituent on the inositol residue. The presence of two fatty acids, one at the sn-1 position and the other at the C2 of inositol, are evidently sufficient for the activity of sn-2 lyso GPIs obtained from P. falciparum GPIs, whereas only one fatty acid at the sn-1

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Figure 1. The structures of P. falciparum GPIs. The heterogeneity in the fatty acid substituents at the sn-1, sn-2 and inositol C2 positions is indicated. The numeric values in parenthesis represent the percent mole proportions of the fatty acids in the parasite GPIs. Man, mannose; Ino, inositol, EtN, ethanolamine; P, phosphate.

position as in the case of T. cruzi sn-2 lyso GPIs, which lack inositol acylation is not adequate for the GPI activity.87-89,97-99 Furthermore, although a previous study had reported that the removal of the terminal fourth mannose residue from P. falciparum GPIs abolishes the pro-inflammatory activity,99 a recent study showed that the Man3-GPI obtained by the removal of the fourth mannose has 80% activity compared with that of the intact Man4-GPI.97 Thus, the Man3-GPI with the evolutionarily conserved glycan moiety (Man3-GlcN), as found earlier by Tachado et al is the minimal structural requirement for GPI activity, although the activity is lower than that of the GPIs with four mannoses (Man4-GPIs). The previously reported inactivity of Man3-GPIs was due to technical problems in assessing the activity because of the low solubility.97,99

Cell Signaling by P. falciparum GPIs Early Studies on Signaling Schofield et al have reported that P. falciparum GPIs can initiate protein tyrosine kinase (PTK) and protein kinase C (PKC) signaling pathways.87,95,96 These two pathways synergize to activate NF-κB/Rel family transcription factors, leading to the production of TNF-α, IL-1, IL-6, cell adhesion molecules, and nitric oxide in macrophages and endothelial cells. It was

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proposed that the parasite GPI could function as an agonist in signaling macrophages through distinct structural elements, the glycan and lipid moieties.87,96 The conserved glycan core was reported to activate PTK, while the diacylglycerol moiety was a second messenger for PKC activation.87,96 The glycan moiety could independently activate PTK, but this signaling alone was not sufficient to produce pro-inflammatory cytokines. The isolated PI moiety was also unable to activate PKC independently. However, in intact GPIs, the lipid could induce PKC activation. Thus, it was proposed that the PKC activation induced by the PI moiety and the simultaneous PTK activation by the glycan moiety together lead to the production of pro-inflammatory cytokines and nitric oxide in macrophages and the upregulation of cell adhesion molecules and inducible nitric oxide synthase in endothelial cells.87,95,96,100 The following hypotheses have been proposed to explain how the exogenous GPI agonist can initiate cellular activation and how this signal is transmitted across the cell membrane to the interior: (i) PTK is activated upon the binding of GPIs to a transmembrane receptor with a glycan-specific lectin domain in the microdomains of host cell membranes that are rich in GPI-anchored proteins or upon perturbation of the GPI-anchored protein assembly in the host cell membrane microdomains upon GPI insertion. (ii) The intact GPIs that are inserted into cell membranes may be translocated to the cell interior by the action of a “flippase”. (iii) The diacylglycerol, released by the hydrolysis of either membrane lipids or parasite GPIs at the cell membrane by membrane-associated GPI-specific phospholipase D, crosses from the lumen of the plasma membrane to enter the cytoplasmic side of the host cells. The PI moiety of the intact GPIs or the diacylglycerol in the cytoplasm then stimulates the PKC pathway.87,96 However, in view of the recent findings that GPIs of T. cruzi and P. falciparum activate macrophages through the recognition of TLR2, triggering various MAPK and NF-κB pathways (see below),89,97 it is important to reevaluate whether such an alternative pathway of GPI activation exist or those observations merely represent a part of the TLR2-mediated MAPK and NF-κB pathways. In this context, TLR2 may be viewed as the suggested transmembrane receptor with which GPIs specifically interact, activating a number of PTKs of the MAPK and NF-κB pathways. However, whether a PKC is involved in GPI-mediated signaling remains to be determined.

Host Receptor Specificity During the past few years, there have been a major advances in our understanding of the mechanism of pro-inflammatory responses to various microbial ligands, including GPIs. Particularly, it has been demonstrated that the members of the evolutionarily conserved Toll-like receptor (TLR) family of proteins function as receptors for a wide range of pathogen-associated molecular patterns to initiate pro-inflammatory responses by the innate immune system.101-105 Gowda and coworkers have recently studied in detail the signaling mechanisms in P. falciparum GPI-induced pro-inflammatory responses in macrophages.97,106 Assessment of the TNF-α response in primary bone marrow cells and macrophages from TLR gene-knockout mice has showed that, while P. falciparum GPI-induced pro-inflammatory response was only marginally affected in TLR4-deficient macrophages, the response was markedly decreased in TLR2-deficient macrophages (Fig. 2). The activity was almost completely absent in MyD88-deficient macrophages.97 The activation of human monocytes by the parasite GPIs also involves the recognition mainly by TLR2 since treatment of cells with anti-TLR2 or anti-TLR4 monoclonal antibody prior to stimulation with GPIs efficiently inhibited pro-inflammatory responses in dose-dependent manner.97 Thus, P. falciparum GPI-mediated cell signaling in monocytes/macrophages occurs predominantly by the TLR2 recognition, involving MyD88-dependent signal transduction and to a much lower level through TLR4 recognition. For ligand recognition by TLR2, dimerization of TLR2 with either TLR1 or TLR6 is essential.102 By a gene-transfection assay, it has been shown that P. falciparum GPIs are preferentially recognized by the TLR2-TLR1 heterodimer.97 The removal of the fatty acid substituent at the sn-2 position resulted in a switch in receptor specificity to the TLR2-TLR6 heterodimer. These observations were recently confirmed using TLR1- and TLR6-deficient macrophages obtained

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Figure 2. P. falciparum GPI-induced production of TNF-α by macrophages. Macrophages stimulated with the purified GPIs of P. falciparum produce TNF-α (shown here) and other pro-inflammatory mediators (not shown) in dose-dependent manner (see ref. 97). Closed circle, macrophages from wild type mice; open circles, TLR4-deficient (TLR2-/-) macrophages; closed triangle, TLR2-deficient (TLR2-/-) macrophages; open circle, MyD88-deficient (MyD88-/-) macrophages.

from gene-knockout mice (Gowda and coworkers, unpublished results). The pro-inflammatory activity of P. falciparum GPIs in TLR6-deficient macrophages was only slightly lower than that in the wild-type macrophages, but substantially lower in TLR1-deficient macrophages. In contrast, the activity of sn-2 lyso-GPIs in TLR1-deficient macrophages was marginally lower than that in the wild type cells, whereas the activity is considerably lower in TLR6-deficient macrophages. Thus, TLR2-TLR1 and TLR2-TLR6 heterodimers can discriminate GPIs with three and two fatty acids (intact P. falciparum GPIs and sn-2 lyso GPIs, respectively) in a manner similar to the known specificity of these heterodimers toward bacterial tri- and diacylated peptides, respectively. That is, TLR2-TLR1 heterodimer preferentially recognizes GPIs and lipoproteins with three fatty acyl substituents, whereas TLR2-TLR6 selectively recognizes those with two fatty acyl substituents.107,108

GPI-Induced Signaling Pathways The P. falciparum GPI-mediated TLR2- and MyD88-dependent signaling in macrophages leads to the downstream activation of ERK, JNK, p38, and NF-κB pathways, producing pro-inflammatory mediators (Fig. 3).97 This is evident by the observations that the parasite GPIs could efficiently phosphorylate ERK1/ERK2, p38, and JNK as well as cause the degradation of IκBα. The activation of these signaling pathways was markedly low in TLR2-deficient macrophages, but efficient in TLR4-deficient macrophages. Little or no activation was observed in MyD88-deficient and in macrophages deficient in both TLR2 and TLR4.97 Recent studies have also shown that the ERK, JNK, p38, and NF-κB signaling pathways are differentially involved in the parasite GPI-induced production of various pro-inflammatory mediators. Inhibition or lack of ERK activation leads to differential expression of pro-inflammatory mediators in macrophages.106 Inhibition of ERK1/ERK2 phosphorylation or a deficiency in ERK1/ERK2 activation caused marginal or no decrease in the levels of

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Figure 3. P. falciparum GPI-induced cell signaling. Schematic representation of P. falciparum -GPI-induced TLR2-MyD88-mediated cell signaling and production of pro-inflammatory mediators in macrophages primed with IFN-γ. The abbreviations for the signaling molecules of the indicated pathways are standard (see refs. 102,104,108).

TNF-α and nitric oxide production. However, upon inhibition of ERK1/ERK2 phosphorylation, the GPI-induced production of IL-6 and IL-12 increased by several-fold, indicating that lack of ERK phosphorylation dysregulates IL-6 and IL-12 production.106 The p38 MAPK pathway is crucial for the parasite GPI-induced production of IL-6 and IL-12, but is only marginally required or not essential for the production of TNF-α and nitric oxide.106 This conclusion is consistent with the finding that macrophages lacking p38 pathway activation produced TNF-α at levels similar to wild-type macrophages in response to LPS, while IL-12 levels were drastically reduced.109 The JNK pathway is also crucial and is differentially involved in the GPI-stimulated expression of various pro-inflammatory cytokines.106 Inhibition of JNK leads to the abolition of IL-12 production and substantial reduction in the levels of TNF-α and nitric oxide. The production of IL-6 was also significantly decreased, but the extent of reduction was lower compared to the three other inflammatory mediators. The JNK proteins comprise 10 isoforms formed by the alternative splicing of mRNA from three genes, jnk1, jnk2, and jnk3.110 Only jnk1 and jnk2 are expressed in monocytes and macrophages, while jnk3 is selectively expressed in nervous tissues.111 Studies using JNK1- and JNK2-deficient macrophages showed that the GPI-induced production of TNF-α, nitric oxide and IL-6 was normal in both macrophages compared to wild-type cells.106 IL-12 production was about 80% lower in JNK2-deficient macrophages while normal in JNK1-deficient cells. Therefore, the JNK1 and JNK2 are

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functionally redundant for TNF-α, IL-6, and nitric oxide production by macrophages stimulated with GPIs. However, JNK2, but not JNK1, is essential for the parasite GPI-induced IL-12 production.106 The P. falciparum GPI-elicited production of pro-inflammatory mediators, such as TNF-α, nitric oxide, IL-6 and IL-12, was markedly low in macrophages pretreated with the inhibitors of NF-κB activation or of translocation of the p50 component of the NF-κB complex.106 This is consistent with the concept that NF-κB family members are the key transcriptional factors that regulate the expression of cytokines, chemokines, and growth factors by cells of the innate immune system in response to microbial ligands.112,113 In efforts to determine whether the individual NF-κB family members are differentially involved in the parasite GPI-induced expression of pro-inflammatory mediators, Zhu et al have studied macrophages from NF-κB1-deficient mice.106 The production of TNFα and nitric oxide was normal in these macrophages, indicating that p50, which is derived from NF-κB1(p105), is not crucial for the GPI-induced expression of TNF-α and nitric oxide. In contrast, the expression of IL-6 and IL-12 was markedly reduced at both the transcriptional and protein levels in NF-κB1-deficient macrophages, demonstrating that p50 is critical for IL-6 and IL-12 production in macrophages stimulated with GPIs. These results are consistent with the recent finding that IκBζ, a newly identified transcriptional factor, is critical for TLR2 and TLR4 ligands-induced IL-6 and IL-12 expression.114,115 IκBζ requires p50 derived from NF-κB1 to exert its transcriptional activation function.

Regulation of GPI Activity by Cell Surface Phospholipases A previous study has shown that, upon incubation with macrophages, about 98% of P. falciparum GPIs remained in the culture medium, suggesting that very little GPI, if any, is bound to the cell surface or internalized by cells.99 Recently, Krishnegowda et al found that when GPIs were incubated with mouse macrophages or human monocytes, most of the GPIs were degraded by phospholipase A2 and GPI-specific phospholipase D present on cell surface.97 The major degradation products were those formed by the action of GPI-specific phospholipase D. Since the products of phospholipase D cannot elicit the production of pro-inflammatory responses, the results indicate that host macrophages regulate GPI activity at least partly by this mechanism. Thus, GPI-specific phospholipase D on monocyte/macrophage surface may play an important role in the control of malaria infection and pathogenesis.

Conclusions Pro-inflammatory responses that are triggered in response to Plasmodium infection contribute substantially to the severity of the systemic and organ-related malaria syndromes. The recognition that GPIs are the pro-inflammatory response-inducing factors of P. falciparum, and structural characterization of the parasite GPIs and their structure-activity relationship and cell signaling mechanism studies during the past decade have significantly broadened our understanding of how the malaria parasites are able to induce pro-inflammatory responses during infection. However, much remained to be learned: (i) How do the various signaling molecules of the downstream ERK, p38, JNK and NF-κB pathways activated by the parasite GPI-induced TLR2-MyD88-dependent signal transduction differentially regulate the expression of a wide range of pro-inflammatory and anti-inflammatory cytokines, chemokines and other mediators that are produced during malaria infection? (ii) How do the GPIs, which are in parasite membranes with their lipid tails inserted in the lipid bilayer, are presented to host cells for TLR2 recognition in vivo during infection? This is particularly important in view of the observation that parasite GPIs are not released as free lipids from parasite membranes to the medium during schizont burst (Gowda et al unpublished observations). (iii) How the cells of the innate immune system reprogram from eliciting pro-inflammatory responses during the acute phase infection to produce anti-inflammatory cytokines as the infection progresses? (iv) Are there receptors other than TLRs, such as other class of pattern recognition molecules or scavenger lectin-like receptors, in GPI recognition and activation of host cells? (v) Several

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studies have reported that hemozoin is able to induce pro-inflammatory and other cytokines and chemokines in macrophages.116-121 An important question that remains to be answered is whether the activity reported for hemozoin is due to the pure polymer per se or due to the polymer-associated parasite factors? A recent study implicates TLR9-mediated recognition in the hemozoin-induced innate immune responses by dendritic cells.122 However, it is hard to imagine how the heme crystalline polymer can function as the parasite-specific molecular pattern, precisely resembling CpG motifs of DNA, the known specific ligand for TLR9. Independent studies are needed to determine whether or not hemozoin is a TLR9 ligand. A full understanding of the various processes and signaling cascades that are activated during malaria infection and are involved in producing pro-inflammatory responses in the host will have important implications for the development of novel immunotherapeutics for malaria.

Acknowledgements I wish to thank Drs. Jianzhong Zhu and Gowdahalli Krishnegowda for their help in the preparation of figures, and the National Institute of Allergy and Infectious Diseases, National Institutes of Health for grant support AI41139.

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22. Jason J, Archibald LK, Nwanyanwu OC et al. Cytokines and malaria parasitemia. Clin Immunol 2001; 100:208-218. 23. Malaguarnera L, Imbesi RM, Pignatelli S et al. Increased levels of interleukin-12 in Plasmodium falciparum malaria: Correlation with the severity of disease. Parasite Immunol 2002; 24:387-389. 24. Lyke KE, Burges R, Cissoko Y et al. Serum levels of the proinflammatory cytokines interleukin-1β (IL-1β), IL-6, IL-8, IL-10, tumor necrosis factor alpha, and IL-12(p70) in Malian children with severe Plasmodium falciparum malaria and matched uncomplicated malaria or healthy controls. Infect Immun 2004; 72:5630-5637. 25. Bouyou-Akotet MK, Kombila M, Kremsner PG et al. Cytokine profiles in peripheral, placental and cord blood in pregnant women from an area endemic for Plasmodium falciparum. Eur Cytokine Netw 2004; 15:120-125. 26. Langhorne J, Quin SJ, Sanni LA. Mouse models of blood stage malaria infections: Immune responses and cytokines involved in protection and pathology. Chem Immunol 2002; 80:204-228. 27. Hensmann M, Kwiatkowski D. Cellular basis of early cytokine response to Plasmodium falciparum. Infect Immun 2001; 69:2364-2371. 28. Scragg IG, Hensmann M, Bate CA et al. Early cytokine induction by Plasmodium falciparum is not a classical endotoxin-like process. Eur J Immunol 1999; 29:2636-2644. 29. Cramer JP, Nussler AK, Ehrhardt S et al. Age-dependent effect of plasma nitric oxide on parasite density in Ghanaian children with severe malaria. Trop Med Intl Health 2005; 10:672-680. 30. Clark IA, Rockett KA, Burgner D. Genes, nitric oxide and malaria in African children. Trends Parasitol 2003; 19:335-337. 31. Maneerat Y, Viriyavejakul P, Punpoowong B et al. Inducible nitric oxide synthase expression is increased in the brain in fatal cerebral malaria. Histopathology 2000; 37:269-277. 32. Anstey NM, Granger DL, Hassanali MY et al. Nitric oxide, malaria, and anemia: Inverse relationship between nitric oxide production and hemoglobin concentration in asymptomatic, malaria-exposed children. Am J Trop Med Hyg 1999; 61:249-252. 33. Cot S, Ringwald P, Mulder B et al. Nitric oxide in cerebral malaria. J Infect Dis 1994; 169:1417-1418. 34. Nussler AK, Eling W, Kremsher PG. Patients with Plasmodium falciparum malaria and Plasmodium vivax malaria show increased nitrite and nitrate plasma levels. J Infect Dis 1994; 169:1418-1419. 35. Kwiatkowski D. Malarial toxins and the regulation of parasite density. Parasitol Today 1995; 11:206-212. 36. Bruce MC, Day KP. Cross-species regulation of Plasmodium parasitemia in semi-immune children from Papua New Guinea. Trends Parasitol 2003; 19:271-277. 37. Clark IA, al Yaman FM, Jacobson LS. The biological basis of malarial disease. Intl J Parasitol 1997; 27:1237-1249. 38. Sedegah M, Finkelman F, Hoffman SL. Interleukin 12 induction of interferon γ-dependent protection against malaria. Proc Natl Acad Sci USA 1994; 91:10700-10702. 39. Su Z, Stevenson MM. Central role of endogenous gamma interferon in protective immunity against blood-stage Plasmodium chabaudi AS infection. Infect Immun 2000; 68:4399-4406. 40. Su Z, Stevenson MM. IL-12 is required for antibody-mediated protective immunity against blood-stage Plasmodium chabaudi AS malaria infection in mice. J Immunol 2002; 168:1348-1355. 41. Balmer P, Phillips HM, Maestre AE et al. The effect of nitric oxide on the growth of P. falciparum, P. chabaudi and P. berghei in vitro. Parasite Immunol 2000; 22:97-106. 42. van der Heyde HC, Pepper B, Batchelder J et al. The time course of selected malarial infections in cytokine-deficient mice. Exp Parasitol 1997; 85:206-213. 43. Clark IA, Hunt NH, Butcher GA et al. Inhibition of murine malaria (Plasmodium chabaudi) in vivo by recombinant interferon-γ or tumor necrosis factor, and its enhancement by butylated hydroxyanisole. J Immunol 1987; 139:3493-3496. 44. De Souza JB, Williamson KH, Otani T et al. Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria. Infect Immun 1997; 65:1593-1598. 45. Muniz-Junqueira MI, dos Santos-Neto LL, Tosta CE. Influence of tumor necrosis factor-α on the ability of monocytes and lymphocytes to destroy intraerythrocytic plasmodium falciparum in vitro. Cellular Immunol 2001; 208:73-79. 46. Trinchieri G. Interleukin-12: A proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 1995; 13:251-276. 47. Trinchieri G. The two faces of interleukin 12: A pro-inflammatory cytokine and a key immunoregulatory molecule produced by antigen-presenting cells. Ciba Found Symp 1995; 195:203-220.

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48. Artavanis-Tsakonas K, Tongren JE, Riley EM. The war between the malaria parasite and the immune system: Immunity, immunoregulation and immunopathology. Clin Exp Immunol 2003; 133:145-152. 49. Odeh M. The role of tumor necrosis factor-α in the pathogenesis of complicated falciparum malaria. Cytokine 2001; 14:11-18. 50. Playfair JHL, Taverne J, Bate CAW et al. The malaria vaccine: Anti-parasite or anti-disease? Immunol Today 1990; 11:25-27. 51. Clark IA, Chaudri G. Tumor necrosis factor may contribute to the anemia of malaria by causing dyserythropoiesis and erythrophagocytosis. Br J Haemotol 1988; 70:99-103. 52. Clark IA, Chaudri G, Cowden WB. Roles of tumor necrosis factor in the illness and pathology of malaria. Trans Royal Soc Trop Med Hyg 1989; 83:436-440. 53. White NJ, Ho M. The pathophysiology of malaria. Adv Parasitol 1992; 31:83-127. 54. Bate CAW, Taverne J, Playfair JHL. Malarial parasites induce TNF production by macrophages. Immunology 1988; 64:227-231. 55. Bate CAW, Taverne J, Playfair JHL. Soluble malarial antigens are toxic and induce the production of tumor necrosis factor in vivo. Immunology 1989; 66:600-605. 56. Kwiatkowski D, Cannon J, Manogue K et al. TNF production in falciparum malaria and its association with schizont rupture. Clin Exp Immunol 1989; 77:361-366. 57. Clark IA, Cowden WB, Butcher GA et al. Possible role of tumor necrosis factor in the pathology of malaria. Am J Pathol 1987; 129:192-199. 58. Grau GE, Fajardo LF, Piguet PF et al. Tumor necrosis factor (cachectin) is an essential mediator in murine cerebral malaria. Science 1987; 237:1210-1212. 59. Grau GE, Heremans H, Piguet PF et al. Monoclonal antibody against interferon-γ can prevent experimental cerebral malaria and its overproduction of tumor necrosis factor. Proc Natl Acad Sci USA 1989; 86:5572-5574. 60. Curfs JH, Schetters TP, Hermsen CC et al. Immunological aspects of cerebral lesions in murine malaria. Clin Exp Immunol 1989; 75:136-140. 61. Kwiatkowski D, Hill A, Sambou I et al. TNF concentration in fatal cerebral, nonfatal cerebral, and uncomplicated Plasmodium falciparum malaria. Lancet 1990; 336:1201-1204. 62. Kwiatowski D. Tumor necrosis factor, fever and fatality in falciparum malaria. Immunol Lett 1990; 25:213-216. 63. Grau GE, Taylor TE, Molyneux ME et al. Tumor necrosis factor and disease severity in children with falciparum malaria. N Engl J Med 1989; 320:1586-1591. 64. Brown H, Turner G, Rogerson S. Cytokine expression in the brain human cerebral malaria. J Infect Dis 1999; 180:1742-1746. 65. Porta J, Carota A, Pizzolato GP et al. Immunopathological changes in human cerebral malaria. Clin Neuropathol 1993; 12:142-146. 66. Udomsanpetch R, Chivapat S, Viriyavejakul P et al. Involvement of cytokines in the histopathology of cerebral malaria. Am J Trop Med Hyg 1997; 57:501-506. 67. Grau GE, Piguet PF, Vassali P et al. Tumor necrosis factor and other cytokines in cerebral malaria: Experimental and clinical data. Immunol Rev 1989; 112:49-70. 68. Kwiatkowski D, Molyneux ME, Stephens S. Anti-TNF therapy inhibits fever in cerebral malaria. Q J Med 1993; 86:91-98. 69. van Hensbroek MB, Palmer A, Onyiorah E et al. The effect of a monoclonal antibody to tumor necrosis factor on survival from childhood cerebral malaria. J Infect Dis 1996; 174:1091-1097. 70. Tsunawaki S, Sporn M, Ding A et al. Deactivation of macrophages. Nature 1988; 334:260-262. 71. Ferrante A, Kumaratilake L, Rzepczyk CM et al. Killing of Plasmodium falciparum by cytokine activated effector cells (neutrophils and macrophages). Immunol Lett 1990; 25:179-187. 72. Omer FM, Kurtzhals JA, Riley EM. Maintaining the immunological balance in parasitic infections: A role for TGF-β? Parasitol Today 2000; 16:18-23. 73. Wenisch C, Parschalk B, Burgmann H et al. Decreased serum levels of TGF-β in patients with acute Plasmodium falciparum malaria. J Clin Immunol 1995; 15:69-73. 74. Omer FM, de Souza JB, Riley EM. Differential induction of TGF-β regulates proinflammatory cytokine production and determines the outcome of lethal and nonlethal Plasmodium yoelii infections. J Immunol 2003; 171:5430-5436. 75. Akdis CA, Blaser K. IL-10-induced anergy in peripheral T cell and reactivation by microenvironmental cytokines: Two key steps in specific immunotherapy. FASEB J 1999; 19:603-609. 76. Hugosson E, Montgomery SM, Premji Z et al. Higher IL-10 levels are associated with less effective clearance of Plasmodium falciparum parasites. Parasite Immunol 2004; 26:111-117. 77. Wilson JN, Rockett K, Jallow M et al. Analysis of IL10 haplotypic associations with severe malaria. Genes Immun 2005; 6:462-466.

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78. May J, Lell B, Luty AJ et al. Plasma interleukin-10: Tumor necrosis factor (TNF)-α ratio is associated with TNF promoter variants and predicts malarial complications. J Infect Dis 2000; 182:1570-1573. 79. Othoro C, Lal AA, Nahlen B et al. A low interleukin-10 tumor necrosis factor-α ratio is associated with malaria anemia in children residing in a holoendemic malaria region in western Kenya. J Infect Dis 1999; 179:279-282. 80. Kossodo S, Monso C, Juillard P et al. Interleukin-10 modulates susceptibility in experimental cerebral malaria. Immunology 1997; 91:536-540. 81. Goligi C. On the cycle of development of malarial parasites in tertian fever: Differential diagnosis between the intracellular malarial parasites in tertian and quartan fever (in Intalian). Extracts reprinted. In: Kean BH, Mott KE, Russell AJ, eds. Vol. 1. Ithaca: Cornell University Press, 1978, (Vol 1. Arch Sci Med (Torino) 1889; 13:173-196). 82. Collier WA. Ueber auto-noxine. Berlin klin Wichenschr 1921; 58:478-479. 83. Clark IA. Does endotoxin cause both the disease and parasite death in acute malaria and babesiosis? Lancet 1978; 2:75-77. 84. Clark IA, Virelizier JL, Carswell EA et al. Possible importance of macrophage-derived mediators in acute malaria. Infect Immun 1978; 32:1058-1066. 85. Schofield L, Hackett F. Signal transduction in host cells by a glycosylphosphatidylinositol toxin of malaria parasites. J Exp Med 1993; 177:145-153. 86. Gowda DC. Structure and activity of glycosylphosphatidylinositol anchors of Plasmodium falciparum. Microbes Infect 2002; 4:983-990. 87. Nebl T, De Veer MJ, Schofield L. Stimulation of innate immune responses by malarial glycosylphosphatidylinositol via pattern recognition receptors. Parasitology 2005; 130:S45-S62. 88. Almeida IC, Gazzinelli RT. Proinflammatory activity of glycosylphosphatidylinositol anchors derived from Trypanosoma cruzi: Structural and functional analyses. J Leukoc Biol 2001; 70:467-477. 89. Ropert C, Ferreira LR, Campos MA et al. Macrophage signaling by glycosylphosphatidylinositolanchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes. Microbes Infect 2002; 4:1015-1025. 90. Ropert C, Gazzinelli RT. Signaling of immune system cells by glycosylphosphatidylinositol (GPI) anchor and related structures derived from parasitic protozoa. Curr Opin Microbiol 2000; 3:395-403. 91. Gerold P, Dieckmann-Schuppert A, Schwarz RT. Glycosylphosphatidylinositols synthesized by asexual erythrocytic stages of the malarial parasite, Plasmodium falciparum. Candidates for plasmodial glycosylphosphatidylinositol membrane anchor precursors and pathogenicity factors. J Biol Chem 1994; 269:2597-2606. 92. Gerold P, Schofield L, Blackman MJ et al. Structural analysis of the glycosylphosphatidylinositol membrane anchors of the merozoite surface proteins-1 and -2 of Plasmodium falciparum. Mol Biochem Parasitol 1996; 75:131-143. 93. Naik RS, Branch OH, Woods AS et al. Glycosylphosphatidylinositol anchors of Plasmodium falciparum: Molecular characterization and naturally elicited antibody response that may provide immunity to malaria pathogenesis. J Exp Med 2000; 192:1563-1575. 94. Ferguson MAJ, Brimacombe JS, Brown JR et al. The GPI biosynthetic pathway as a therapeutic target for African sleeping sickness. Biochim Biophys Acta 1999; 1455:327-340. 95. Tachado SD, Novakovic S, Gerold P et al. Glycosylphosphatidylinositol toxin of Plasmodium induces nitric oxide synthase expression in macrophages and vascular endothelial cells by a protein tyrosine kinase-dependent and protein kinase C-dependent signaling pathway. J Immunol 1996; 156:1897-1907. 96. Tachado SD, Gerold P, Schwarz R et al. Signal transduction in macrophages by glycosylphosphatidylinositols of Plasmodium, Trypanosoma, and Leishmania: Activation of protein tyrosine kinases and protein kinase C by inositolglycan and diacylglycerol moieties. Proc Natl Acad Sci USA 1997; 94:4022-4027. 97. Krishnegowda G, Hajjar AM, Zhu J et al. Induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols of Plasmodium falciparum: Cell signaling receptors, glycosylphosphatidylinositol (GPI) structural requirement, and regulation of GPI activity. J Biol Chem 2005; 280:8606-8616. 98. Almeida IC, Camargo MM, Procopio DO et al. Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. EMBO J 2000; 19:1476-1485. 99. Vijaykumar M, Naik RS, Gowda DC. Plasmodium falciparum glycosylphosphatidylinositol-induced TNF-α secretion by macrophages is mediated without membrane insertion or endocytosis. J Biol Chem 2001; 276:6909-6912.

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100. Schofield L, Novakovic S, Gerold P et al. Glycosylphosphatidylinositol toxin of Plasmodium upregulates ICAM-1, VCAM-1, and E-selectin expression in vascular endothelial cells and increases leukocyte and parasite cytoadherence via protein tyrosine kinase-dependent signal transduction. J Immunol 1996; 156:1886-1896. 101. Janeway Jr CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002; 20:197-216. 102. Takeda K, Akira S. Toll-like receptors and pathogen resistance. Cellular Biol 2003; 5:143-153. 103. Kopp E, Medzhitov R. Recognition of microbial infection by Toll-like receptors. Curr Opin Immunol 2003; 15:396-401. 104. Takeda K, Akira S. Microbial recognition by Toll-like receptors. J Dermatol Sci 2004; 34:73-82. 105. Pasare C, Medzhitov R. Toll-like receptors: Linking innate and adaptive immunity. Adv Exp Med Biol 2005; 560:11-18. 106. Zhu J, Krishnegowda G, Gowda DC. Induction of proinflammatory responses in macrophages by the GPIs of Plasmodium falciparum: The requirement of extracellular signal-regulated kinase, p38, c-Jun N-terminal kinase and NF-κB pathways for the expression of proinflammatory cytokines and nitric oxide. J Biol Chem 2005; 280:8617-8627. 107. Takeda K, Takeuchi O, Akira S. Recognition of lipopeptides by Toll-like receptors. J Endotox Res 2002; 8:459-463. 108. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 2003; 85:85-95. 109. Lu HT, Yang DD, Wysk M et al. Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (Mkk3)-deficient mice. EMBO J 1999; 18:1845-1857. 110. Manning AM, Davis RJ. Targeting JNK for therapeutic benefit: From junk to gold? Nat Rev Drug Discov 2003; 2:554-565. 111. Yang DD, Kuan CY, Whitmarsh AJ et al. Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature 1997; 289:865-870. 112. Bonizzi G, Karin M. The two NF-κB activation pathways and their role in innate and adaptive immunity. Trends Immunol 2004; 25:280-288. 113. Hayden MS, Ghosh S. Signaling to NF-κB. Genes Dev 2004; 18:2195-2224. 114. Haruta H, Kato A, Todokoro K. Isolation of a novel interleukin-1-inducible nuclear protein bearing ankyrin-repeat motifs. J Biol Chem 2001; 276:12485-8. 115. Yamamoto M, Yamazaki S, Uematsu S et al. Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IkBζ. Nature 2004; 430:218-222. 116. Pichyangkul S, Saengkrai P, Webster YD. Plasmodium falciparum pigment induces monocytes to release high levels of TNF-α and IL-1β. Am J Trop Med Hyg 1994; 51:430-435. 117. Sherry BA, Alava G, Tracey KJ et al. Malaria-specific metabolite hemozoin mediates the release of several potent endogenous pyrogens (TNF-α, MIP-1, and MIP-1β) in vitro, and altered thermoregulation in vivo. J Inflamm 1995; 45:85-96. 118. Taramelli D, Basilico N, De Palma AM et al. The effect of synthetic malaria pigment (β-hematin) on adhesion molecule expression and interleukin-6 production by human endothelial cells. Trans R Soc Trop Med Hyg 1998; 92:57-62. 119. Jaramillo M, Gowda DC, Radzioch D et al. Hemozoin increases IFN-γ-inducible macrophage nitric oxide generation through extracellular signal-regulated kinase- and NF-κB-dependent pathways. J Immunol 2003; 171:4243-4253. 120. Jaramillo M, Plante I, Ouellet N et al. Hemozoin-inducible proinflammatory events in vivo: Potential role in malaria infection. J Immunol 2004; 172:3101-3110. 121. Jaramillo M, Godbout M, Olivier M. Hemozoin induces macrophage chemokine expression through oxidative stress-dependent and -independent mechanisms. J Immunol 2005; 174:475-484. 122. Coban C, Ishii KJ, Kawai T et al. Toll-like receptor 9 mediates innate immune activation by the malaria pigment hemozoin. J Exp Med 2004; 201:19-25.

CHAPTER 7

Pro-Inflammatory Responses in Macrophages during Toxoplasma gondii Infection Katherine S. Masek and Christopher A. Hunter*

Abstract

T

oxoplasma gondii is an obligate intracellular parasite that causes an asymptomatic infection in a significant percentage of the world’s population. Healthy hosts mount a robust innate response mediated by IL-12, which influences the development of protective cell-mediated immunity by stimulating NK and T cells to produce IFN-γ. Although macrophages can be infected by T. gondii, they are able to limit parasite replication and produce cytokines that contribute to resistance, making them important regulatory and effector cells during toxoplasmosis. A large body of work has focused on the interactions between parasites and these host cells, and contributed to our understanding of the pro-inflammatory activities of macrophages during T. gondii infection.

Introduction Toxoplasma gondii is an intracellular parasite that causes a persistent infection in 10-80% of the world’s population, depending on geographic location.1 Infection is normally asymptomatic in healthy individuals because control of this pathogen results from the host’s ability to mount a robust cell-mediated immune response which is dominated by production of Interferon-γ (IFN-γ) by NK cells during the earliest stages of infection, and by parasite specific CD4+ and CD8+ T cells thereafter.2-5 The clinical significance of these events is illustrated by patients with primary or acquired defects in T cell function, in whom a failure to control parasite replication results in overt disease. Thus, in AIDS patients, the decline in T cell numbers correlates with reactivation of latent infection and the development of Toxoplasmic Encephalitis (TE).6,7 Likewise, patients undergoing immunosuppressive therapy or affected by cancers that lead to defects in T cell function are also susceptible to TE.8 Consequently, studies on immunity to T. gondii have focused on the role of T cells in resistance to this organism. However, it is now recognized that Toxoplasma induces a strong innate immune response that provides a mechanism of resistance during acute infection and influences subsequent adaptive responses. Historically, macrophages have been considered important effectors of resistance during toxoplasmosis in large part because of early work that established their ability to kill parasites9-11 and produce chemokines and cytokines like IL-12 and TNF-α, which are critical for the production of IFN-γ.12-14 In the last decade, studies using macrophages and dendritic cells (DC) have significantly advanced our understanding of the parasite-derived factors and host signaling pathways that are involved in the innate recognition of Toxoplasma. Thus, this chapter will review the pro-inflammatory signaling and cytokine responses that are initiated by macrophages in response to T. gondii. *Corresponding Author: Dr. Christopher A. Hunter—Department of Pathobiology, University of Pennsylvania, 3800 Spruce St., Philadelphia, Pennsylvania 19104, U.S.A. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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Macrophage Effector Functions In macrophages, the fate of intracellular T. gondii depends on the mechanism of entry into the cell. Dead or opsonized parasites that are phagocytosed are targeted to lysosomal compartments for degradation15 whereas live parasites that actively invade the host cell become established in a protective nonfusogenic parasitophorous vacuole (PV).16 Macrophages that have been preactivated by IFN-γ are able to limit replication of these intra-vacuolar parasites, and work by several laboratories has focused on the mechanisms by which IFN-γ priming directs this response (Fig. 1). One of its anti-microbial activities in murine cells is to stimulate production of NO17 through upregulation of inducible nitric oxide synthase (iNOS). This occurs in combination with TNF-α, which provides a necessary “second signal” that triggers iNOS-dependent parasite control.18 The significance of NO production during Toxoplasma infection was evaluated using iNOS-/- mice, which survived acute parasite challenge but were highly susceptible to TE.19,20 This finding established a central role for NO in the control of toxoplasmosis in the CNS, but it also indicated that there were NO-independent mechanisms that conferred early resistance. In this regard, human macrophages have been shown to use reactive oxygen species to inhibit parasite replication.10,11 In addition to iNOS, IFN-γ stimulation induces expression of six 47-48 kDa GTPase proteins (p47 GTPases) in mouse macrophages, of which IGTP, LRG47 and IRG47 are essential for acute (IGTP and LRG47) or chronic (IRG47) resistance.21,22 Moreover, IFN-γ stimulated macrophages deficient in IGTP or LRG47 have been shown to be attenuated in their ability to limit parasite growth.23 The mechanisms by which these proteins effect anti-microbial activity are yet to be defined, however recent evidence suggests they may localize to and disrupt the PV.24 Notably, human homologues of mouse p47 GTPases have been identified through genome screening but have yet to be cloned.

Figure 1. Overview of pro-inflammatory responses generated by macrophages during toxoplasmosis. Exposure to T. gondii initiates MAPK and NF-κB signaling, leading to synthesis of cytokines, including IL-12, that stimulate production of IFN-γ from NK and T cells. Macrophages become activated by a combination of IFN-γ and TNF-α, leading to upregulation of iNOS and the p47 GTPases, which limits parasite replication. Interactions with activated T cells through CD40/ CD40L ligation also contributes to parasite control. In addition, activated macrophages express MHC class I and II, and can present parasite-derived antigen to activated CD4+ and CD8+ T cells.

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Although there is broad consensus that IFN-γ is the major mediator of resistance to T. gondii, there is evidence of IFN-γ independent anti-microbial activity in macrophages. This is based in part upon reports of patients with partial defects in IFN-γR1 signaling who exhibit serological evidence of Toxoplasma infection, but are clinically asymptomatic.25 One mechanism of resistance that has been suggested in these patients involves TNF-α, in combination with the CD40/CD40L interaction, which triggers parasite killing independently of IFN-γ or NO.26-28 The significance of this pathway in vivo is illustrated in CD40L-/- mice29 and in human patients with a primary defect in the CD40/CD40L interaction,30 which are susceptible to TE. In addition, a recent report using a forward genetic approach has identified a novel locus in rats, Toxo1, which confers resistance to Toxoplasma infection, apparently through the ability of macrophages to limit parasite replication.31 The function of this locus has yet to be described, but appears to be unique from other resistance mechanisms.

Production of IL-12

The discovery of IL-12 as a major mediator of acute resistance to T. gondii12,14,32 prompted investigation into its sources, and initial studies using inflammatory macrophages indicated that they could respond to T. gondii and make IL-12.12 However, it was also recognized that resting macrophages are poor sources of IL-12 when stimulated with live T. gondii, and host survival depends upon its production during the first three days of infection, before extensive macrophage activation has occurred.32 Thus, it was likely that there were other cell types that could produce this cytokine in response to infection without prior priming. Subsequent work identified that conventional DCs exposed to soluble toxoplasma antigens (STAg) could also make IL-12,33 and that neutrophils contained preformed stores that were released following parasite challenge.34 Consistent with this observation, depletion of neutrophils with anti-GR1 antibody antagonized development of protective T cell responses and had a profound effect on infection-induced activation of DC.35 This finding implicated neutrophils as important sources of IL-12 during acute toxoplasmosis, however it is now appreciated that anti-GR1 not only depletes granulocytes, but also affects other GR-1+ cell populations including certain monocytes, CD8+ T cells and the plasmacytoid subset of DC (pDC). Indeed, recent studies have shown that GR-1+ monocytes are the principal cell type recruited to the site of infection, and these cells not only generate NO and kill parasites directly without prior activation, but also can act as a source of IL-12.36,37 Thus, it appears that there are multiple cell types that can produce IL-12 during acute toxoplasmosis, and future work will likely define the extent to which each of these contributes to the development of protective immunity in infected hosts.

Innate Recognition of T. gondii Macrophages are a prominent source of proinflammatory cytokines, in particular IL-12, during toxoplasmosis, and the last decade has seen considerable progress in defining the mechanisms by which these cells recognize and respond to live parasites or their products. The Toll-like family of pattern recognition receptors (TLR) plays a critical role in the innate immune response to multiple types of microbial stimuli,38 and several studies have implicated their role in toxoplasmosis. Signaling downstream from TLRs is dependent on the adaptor molecules MyD88 and TRAF6, and studies conducted with knockout mice have associated these factors with resistance to T. gondii. MyD88-/- mice exhibit profound defects in IL-12 production and are highly susceptible to acute infection,39 and TRAF6 deficient macrophages are also deficient in their IL-12 response to T. gondii.40 Subsequent work has attempted to identify the TLRs that may be involved in parasite recognition, with conflicting results. No defect in IL-12 production was noted following infection of DC from either TLR2-/- or TLR4-/- mice,39 and it has been reported that mice lacking TLR1,2,4,6, or 9 are not acutely susceptible to Toxoplasma infection.41 Moreover, the recently identified TLR1142 has been implicated in parasite recognition, however, despite having a major defect in IL-12 production following parasite challenge, TLR11-/- mice are not susceptible to acute infection.43 In contrast to these findings,

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one study has reported that TLR2-/- mice are acutely susceptible to i.p. challenge, and peritoneal macrophages from these mice make reduced levels of IL-12.44 However these results are complicated by the high dose of parasites that was required to see an effect. In addition, a TLR-independent signaling pathway has been implicated in IL-12 production during toxoplasmosis. In studies using MyD88-/- mice, it was observed that there was a residual IL-12 response in vivo, and from accessory cells in vitro, which was sensitive to treatment with pertussis toxin, an uncoupler of G-protein signaling.39 This result suggested a MyD88-independent, G-protein mediated pathway to IL-12 production, and subsequent work identified a secreted parasite product, C-18, that induced IL-12 synthesis from DC through the chemokine receptor CCR5.45,46 Consistent with this observation, CCR5-/- mice were more susceptible to T. gondii, and produced lower levels of IL-12.47 Despite its prominent function in DC, the role of CCR5 in parasite recognition by other innate immune cells, including macrophages, is at present undefined.

Pro-Inflammatory Signaling Events in Macrophages Significant progress has been made in understanding the proximal events that facilitate parasite recognition, and several studies have begun to elucidate the downstream signaling pathways that effect the pro-inflammatory response (Fig. 2). In macrophages, parasite challenge initiates at least two prominent innate signaling cascades, Mitogen-activated protein kinases (MAPK) and NF-κB. Macrophages stimulated with STAg activate rapid, TRAF6-dependent phosphorylation of the MAPK family members p38 and ERK1/2.40 Using kinase-specific inhibitors, it has been shown that p38 acts as a positive regulator of IL-12p40 production, whereas activation of ERK1/2 is antagonistic.40,48 Moreover, the MAPK-activated transcription factors c-jun, ATF-2, and MAPKAPK2 are reported to translocate to the nucleus in infected cells,48 however the mechanisms by which MAPK direct transcription of IL-12p40 are unknown, and their role in production of IL-12p35, and hence bioactive IL-12p70, has yet to be investigated. Recent work has started to elucidate the T. gondii-induced mechanisms of MAPK activation, and it appears that the upstream pathways leading to ERK1/2 and p38 activation are divergent. Phosphorylation of p38 occurs by TAB1-dependent autophosphorylation,48 and is dependent on MyD88.49 In contrast, ERK1/2 activation by the upstream kinase MEK1/2 is MyD88 independent, and is sensitive to pertussis toxin and wortmannin, implicating activation by a G-protein coupled pathway and PI3-kinase.49 Notably, CCR5 is a G-protein coupled receptor involved in IL-12 production by DC, however it is dispensable for activation of p38 and ERK1/2 in macrophages,49 which suggests that there are other G-protein coupled receptor pathways that are activated in response to T. gondii. In addition to MAPK, Toxoplasma initiates rapid activation of the NF-κB pathway in macrophages. This evolutionarily conserved family of transcription factors is involved in many aspects of innate immunity and is ubiquitously expressed in mammalian cells.50 In the inactive state, they are maintained in the cytoplasm through association with an inhibitor protein, IκB, which becomes phosphorylated and then degraded following cellular stimulation. NF-κB homoand hetero-dimers are then free to translocate to the nucleus, where they bind DNA and initiate transcription of pro-inflammatory and anti-apoptotic genes. The upstream signaling events that facilitate NF-κB activation following stimulation with various TLR ligands or TNF-α are well-defined, and involve activation of the IKK complex as a proximal step to IκB phosphorylation and degradation. In macrophages, as well as numerous other cell types, acute infection with Toxoplasma induces robust IKK activation51,52 and phosphorylation and subsequent degradation of IκB.51-55 However, there is conflicting evidence with regard to the mechanism by which Toxoplasma initiates IKK activity in infected cells. A parasite-derived kinase activity that can phosphorylate IκB has been reported at the PV membrane.52 However, work from this laboratory demonstrates that infection of IKK knockout cells does not result in degradation of IκB, implicating only the host’s kinase activity in this response.51 Moreover, despite robust IKK activation and IκB degradation, whether NF-κB dimers access the nucleus in infected

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Figure 2. Signaling events induced by parasites and their products. Macrophages contacted by T. gondii or parasite-derived products initiate phosphorylation of the MAPK family members p38, ERK1/2 and JNK, which is associated with nuclear translocation of MAPK-dependent transcription factors MAPKAP2, ATF-2 and c-jun, and production of IL-12. Infection with virulent strains of T. gondii also induces robust IKK activity and subsequent degradation of IκB, however nuclear translocation of NF-κB dimers is blocked. A detailed description of the mechanisms that underlie these events is found in the text.

cells and initiate transcription is also controversial. Several groups have determined that virulent strains of T. gondii block nuclear translocation and DNA binding in infected cells,51,53,54,56,57 whereas another group reports that NF-κB accesses the nucleus and induces transcription of anti-apoptotic genes.55,58 In addition, recent studies indicate that while virulent strains of parasites induce low levels of IL-12 from infected cells and do not activate NF-κB, infection with avirulent, Type II strains of T. gondii leads to low level NF-κB activation, which correlates with increased production of IL-12,57 and survival of challenged mice.59,60 The contribution of NF-κB dependent signaling in the macrophage’s pro-inflammatory response to T. gondii, and to the outcome of disease, remains to be precisely defined, and may significantly depend on variations in parasite strain. It is well recognized that STAg and live tachyzoites induce pro-inflammatory signaling in macrophages, however there are differences in the pathways that become activated by these stimuli. For example, STAg does not induce IκB degradation in macrophages or NF-κB

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binding to DNA.40 In addition, whereas STAg activates p38 and ERK1/2,40 live infection also initiates phosphorylation of JNK,48,53,61 which may be involved in parasite-induced IL-12 production.48 Moreover, STAg induces significantly more IL-12 from macrophages and DC than live infection, which may be attributed to differences in pro-inflammatory signaling, as well as the ability of live parasites to actively suppress IL-12 production,53,62 through a recently described STAT3-dependent mechanism.63 Despite characterization of the pro-inflammatory signaling events that occur in infected macrophages, there are many questions regarding the downstream factors that effect transcription. The NF-κB family member c-rel is required for IL-12 production in response to multiple microbial stimuli, including LPS and CpG, but it appears to be dispensable in the Toxoplasma system since knockout macrophages treated with STAg make normal levels of IL-12p40 and in vivo cytokine levels are similar to WT following parasite challenge.64 The Interferon Regulatory Factors (IRF) comprise another group of transcription factors that is implicated in induction of IL-12 synthesis by microbial stimuli like LPS. Studies using IRF-8/ICSBP knockout mice have demonstrated that these animals are acutely susceptible to Toxoplasma infection, and this is associated with decreased IL-12p40 production in vivo.65 However, it is now recognized that ICSBP-/- mice have defective maturation and trafficking of CD8α+ DCs,66,67 which may contribute to enhanced susceptibility to acute toxoplasmosis.33 Nevertheless, peritoneal macrophages from ICSBP knockout mice produce low levels of IL-12p40 following challenge with live parasites or STAg in vitro, implicating a role for this transcription factor in the cell’s pro-inflammatory response.65 Notably, these studies have begun to shed light on the transcriptional elements involved in production of IL-12p40, however little is known about T. gondii-induced regulation of p35.

Conclusions Macrophages residing in tissues are among the first defenses against invading microbes, and there is a growing appreciation of their role in determining the outcome of the host-pathogen interaction. This is not only because they act as effectors to kill invading microbes, but also because they can bridge the gap between innate and adaptive immunity by acting as professional APCs, and through production of chemokines and cytokines. With the discovery of the TLR family of pattern recognition receptors, there has been significant progress in our understanding of the molecular mechanisms by which these cells sense bacterial, viral, parasitic, and fungal organisms. It is now clear that macrophages, together with a few other elite cell types such as DC, are crucial to the innate immune response through their unique ability to distinguish and respond to prokaryotic and eukaryotic infections. Microarray analysis, coupled with in vivo and in vitro investigation, has determined that these other accessory cell types share many functions with macrophages, however there are also distinct differences between them. Thus a future challenge will be to define the unique contributions of these cell types that help coordinate a tailored and targeted immune response to distinct classes of pathogens.

Acknowledgements This work was supported by NIH grants AI46288, T32 AI055400, NIGMS T32-07229 and the State of Pennsylvania.

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29. Reichmann G, Walker W, Villegas EN et al. The CD40/CD40 ligand interaction is required for resistance to toxoplasmic encephalitis. Infect Immun 2000; 68(3):1312-1318. 30. Levy J, Espanol-Boren T, Thomas C et al. Clinical spectrum of X-linked hyper-IgM syndrome. J Pediatr 1997; 131(1 Pt 1):47-54. 31. Cavailles P, Sergent V, Bisanz C et al. The rat Toxo1 locus directs toxoplasmosis outcome and controls parasite proliferation and spreading by macrophage-dependent mechanisms. Proc Natl Acad Sci USA 2006; 103(3):744-749. 32. Khan IA, Matsuura T, Kasper LH. Interleukin-12 enhances murine survival against acute toxoplasmosis. Infect Immun 1994; 62(5):1639-1642. 33. Reis e Sousa C, Hieny S, Scharton-Kersten T et al. In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas. J Exp Med 1997; 186(11):1819-1829. 34. Bliss SK, Butcher BA, Denkers EY. Rapid recruitment of neutrophils containing prestored IL-12 during microbial infection. J Immunol 2000; 165(8):4515-4521. 35. Bliss SK, Gavrilescu LC, Alcaraz A et al. Neutrophil depletion during Toxoplasma gondii infection leads to impaired immunity and lethal systemic pathology. Infect Immun 2001; 69(8):4898-4905. 36. Robben PM, LaRegina M, Kuziel WA et al. Recruitment of Gr-1+ monocytes is essential for control of acute toxoplasmosis. J Exp Med 2005; 201(11):1761-1769. 37. Mordue DG, Sibley LD. A novel population of Gr-1+-activated macrophages induced during acute toxoplasmosis. J Leukoc Biol 2003; 74(6):1015-1025. 38. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol 2001; 1(2):135-145. 39. Scanga CA, Aliberti J, Jankovic D et al. Cutting edge: MyD88 is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells. J Immunol 2002; 168(12):5997-6001. 40. Mason NJ, Fiore J, Kobayashi T et al. TRAF6-dependent mitogen-activated protein kinase activation differentially regulates the production of interleukin-12 by macrophages in response to Toxoplasma gondii. Infect Immun 2004; 72(10):5662-5667. 41. Hitziger N, Dellacasa I, Albiger B et al. Dissemination of Toxoplasma gondii to immunoprivileged organs and role of Toll/interleukin-1 receptor signalling for host resistance assessed by in vivo bioluminescence imaging. Cell Microbiol 2005; 7(6):837-848. 42. Zhang D, Zhang G, Hayden MS et al. A toll-like receptor that prevents infection by uropathogenic bacteria. Science 2004; 303(5663):1522-1526. 43. Yarovinsky F, Zhang D, Andersen JF et al. TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 2005; 308(5728):1626-1629. 44. Mun HS, Aosai F, Norose K et al. TLR2 as an essential molecule for protective immunity against Toxoplasma gondii infection. Int Immunol 2003; 15(9):1081-1087. 45. Aliberti J, Valenzuela JG, Carruthers VB et al. Molecular mimicry of a CCR5 binding-domain in the microbial activation of dendritic cells. Nat Immunol 2003; 4(5):485-490. 46. Aliberti J, Reis e Sousa C, Schito M et al. CCR5 provides a signal for microbial induced production of IL-12 by CD8 alpha+ dendritic cells. Nat Immunol 2000; 1(1):83-87. 47. Aliberti J, Sher A. Role of G-protein-coupled signaling in the induction and regulation of dendritic cell function by Toxoplasma gondii. Microbes Infect 2002; 4(9):991-997. 48. Kim L, Del Rio L, Butcher BA et al. p38 MAPK autophosphorylation drives macrophage IL-12 production during intracellular infection. J Immunol 2005; 174(7):4178-4184. 49. Kim L, Denkers EY. Toxoplasma gondii triggers Gi-dependent phosphatidylinositol 3-kinase signaling required for inhibition of host cell apoptosis. J Cell Sci 2006, (in press). 50. Caamano J, Hunter CA. NF-kappaB family of transcription factors: Central regulators of innate and adaptive immune functions. Clin Microbiol Rev 2002; 15(3):414-429. 51. Shapira S, Harb OS, Margarit J et al. Initiation and termination of NF-kappaB signaling by the intracellular protozoan parasite Toxoplasma gondii. J Cell Sci 2005; 118(Pt 15):3501-3508. 52. Molestina RE, Sinai AP. Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaB-alpha. Cell Microbiol 2005; 7(3):351-362. 53. Butcher BA, Kim L, Johnson PF et al. Toxoplasma gondii tachyzoites inhibit proinflammatory cytokine induction in infected macrophages by preventing nuclear translocation of the transcription factor NF-kappa B. J Immunol 2001; 167(4):2193-2201. 54. Shapira S, Speirs K, Gerstein A et al. Suppression of NF-kappaB activation by infection with Toxoplasma gondii. J Infect Dis 2002; 185(Suppl. 1):S66-72. 55. Molestina RE, Payne TM, Coppens I et al. Activation of NF-kappaB by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated IkappaB to the parasitophorous vacuole membrane. J Cell Sci 2003; 116(Pt 21):4359-4371.

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56. Goebel S, Gross U, Luder CG. Inhibition of host cell apoptosis by Toxoplasma gondii is accompanied by reduced activation of the caspase cascade and alterations of poly(ADP-ribose) polymerase expression. J Cell Sci 2001; 114(Pt 19):3495-3505. 57. Robben PM, Mordue DG, Truscott SM et al. Production of IL-12 by macrophages infected with Toxoplasma gondii depends on the parasite genotype. J Immunol 2004; 172(6):3686-3694. 58. Payne TM, Molestina RE, Sinai AP. Inhibition of caspase activation and a requirement for NF-kappaB function in the Toxoplasma gondii-mediated blockade of host apoptosis. J Cell Sci 2003; 116(Pt 21):4345-4358. 59. Gavrilescu LC, Denkers EY. IFN-gamma overproduction and high level apoptosis are associated with high but not low virulence Toxoplasma gondii infection. J Immunol 2001; 167(2):902-909. 60. Mordue DG, Monroy F, La Regina M et al. Acute toxoplasmosis leads to lethal overproduction of Th1 cytokines. J Immunol 2001; 167(8):4574-4584. 61. Kim L, Butcher BA, Denkers EY. Toxoplasma gondii interferes with lipopolysaccharide-induced mitogen-activated protein kinase activation by mechanisms distinct from endotoxin tolerance. J Immunol 2004; 172(5):3003-3010. 62. Butcher BA, Denkers EY. Mechanism of entry determines the ability of Toxoplasma gondii to inhibit macrophage proinflammatory cytokine production. Infect Immun 2002; 70(9):5216-5224. 63. Butcher BA, Kim L, Panopoulos AD et al. IL-10-independent STAT3 activation by Toxoplasma gondii mediates suppression of IL-12 and TNF-alpha in host macrophages. J Immunol 2005; 174(6):3148-3152. 64. Mason N, Aliberti J, Caamano JC et al. Cutting edge: Identification of c-Rel-dependent and -independent pathways of IL-12 production during infectious and inflammatory stimuli. J Immunol 2002; 168(6):2590-2594. 65. Scharton-Kersten T, Contursi C, Masumi A et al. Interferon consensus sequence binding protein-deficient mice display impaired resistance to intracellular infection due to a primary defect in interleukin 12 p40 induction. J Exp Med 1997; 186(9):1523-1534. 66. Aliberti J, Schulz O, Pennington DJ et al. Essential role for ICSBP in the in vivo development of murine CD8alpha + dendritic cells. Blood 2003; 101(1):305-310. 67. Tsujimura H, Tamura T, Gongora C et al. ICSBP/IRF-8 retrovirus transduction rescues dendritic cell development in vitro. Blood 2003; 101(3):961-969.

CHAPTER 8

Down-Modulation of Proinflammatory Signal Transduction in Toxoplasma gondii-Infected Macrophages Barbara A. Butcher,* Leesun Kim, Chiang W. Lee and Eric Y. Denkers

Abstract

T

oxoplasma gondii is an opportunistic apicomplexan parasite that displays a broad host range and high prevalence among humans and animals worldwide. Successful parasitism requires Toxoplasma to ensure its quiet existence within the host, at least until further transmission. Thus, the parasite treads the border of coexistence that separates immunopathology due to an overwhelming proinflammatory crisis and rampant pathogen proliferation resulting from an inadequate immune response. These disparate responses yield a similar and unproductive outcome-death of the host and thus, the parasite. Recent work highlighting the interplay between Toxoplasma’s survival strategies and the host’s immune response is discussed in this chapter.

Introduction Toxoplasma gondii is an opportunistic apicomplexan parasite that infects 30–80% of the human population worldwide. Felids are the definitive host of the parasite and shed oocysts in the feces.1 Infection occurs either by ingestion of food-or waterborne-oocysts or, more commonly, undercooked infected meat.2-4 Normally, infection is asymptomatic and progresses from an initial acute phase, during which tachyzoites rapidly replicate to a chronic stage characterized by the presence cyst-contained bradyzoites within tissues of the skeletal and central nervous systems.2 However, reactivation of the infection can be fatal in cases of immune suppression, such as in victims of HIV-AIDS. In addition, when the parasite is transmitted to the fetus of a previously uninfected mother, neurologic complications or even spontaneous abortion can occur. Toxoplasma infection stimulates a Th1 type cellular immune response that is dependent upon dendritic cell, neutrophil and macrophage production of IL-12 and T-cell and natural killer cell-derived IFN−γ.5-10 Mice lacking either of these cytokines succumb to infection during the acute phase.11,12 Despite a requirement for early IL-12 and IFN-γ production, recent evidence has demonstrated that Toxoplasma gondii wields a surprising array of tools to outmaneuver the infected macrophage’s early proinflammatory response. In this chapter, we focus on three pathways that Toxoplasma gondii coopts to its advantage in the host macrophage. These are the nuclear factor *Corresponding Author: Barbara Butcher—Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, U.S.A. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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κB (NFκB), mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription (STAT)3 pathways.

Transcriptional Profiling in the Toxoplasma Infected Cell Although Toxoplasma triggers an IL-12 dependent Th1 response in infected mice, several recent studies have shown that the parasite actively down-regulates proinflammatory signaling in infected innate immune cells13 (Fig. 1). Specifically, the parasite blocks lipopolysaccharide-(LPS) induced production of IL-12, TNF-α and nitric oxide (NO) in bone marrow-derived macrophages and dendritic cells (DC). Whereas IL-12 production eventually occurs, infected macrophages display long-term suppression of TNF-α. 14,15 Other studies demonstrated that IFN-γ-initiated upregulation of MHC class II is suppressed by T. gondii infection.16-18 An increasingly useful way to identify pathogen signatures on host cell responses is the use of microarray technology. This strategy affords the ability to examine transcriptional changes

Figure 1. MAPK/NFκB pathways in the LPS-treated macrophage. Ligation of the TLR4 receptor by LPS leads to MyD88 dependent activation of TAK1, a MAPK kinase kinase (MKKK) through intermediate signaling complexes. TAK1 can activate IκB kinase, which leads to phosphorylation and subsequent degradation of IκB. Thus, the nuclear localization sequence of NFκB is exposed and the transcription factor translocated to the nucleus. LPS-induced nuclear translocation of NFκB is blocked by parasite preinfection for at least 6-8 hours. TAK1 can also activate MAPK kinases that phosphorylate ERK1/2, p38, and SAPK/JNK MAPKs that go on to activate transcription factors. While Toxoplasma itself transiently activates MAPK, preinfection blocks LPS-triggered rephosphorylation. Although not shown above, Toxoplasma also activates STAT3, abrogating LPS-induced IL-12 and TNF-α production. It is not known at present if STAT3 activation interferes with MAPK or NFκB pathways.

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in clusters of genes related by function (immune response, metabolism), signaling pathways (i.e., MAPK, NFκB) or other criteria. The global effect of Toxoplasma infection on host cell transcription has been reported in several studies. Chaussabel and coworkers found that T. gondii upregulated interferon-inducible genes in human peripheral blood derived dendritic cells but not monocytes from the same donor.19 In addition, while transcription of several genes of the NFκB family was increased, the genes under NFκB transcriptional control were not induced. The transcriptional response of T. gondii infected human foreskin fibroblasts revealed similar changes in NFκB family members as well as genes involved in metabolism.20 To specifically examine down-regulation of LPS-responsive genes by Toxoplasma, Lee, et al recently published a microarray study that focused on the effect of preinfection with RH strain tachyzoites on LPS signaling in macrophages.21 While LPS induced a 2-fold or more induction of 77 genes, preinfection with Toxoplasma resulted in parasite-induced transcriptional repression of over two thirds of these genes. The repressed genes encode cytokines, chemokines, surface receptors, and proteins involved in antigen uptake, antigen presentation and signal transduction, indicating that the parasite reformats the LPS response early in infection. Specific examples of genes whose LPS-induced induction was down regulated by parasite infection include TNF-α and IL-12(p40), confirming previous results that assessed these cytokines by ELISA and RNase protection assay.14,22 In contrast, transcription of a small subset of LPS-induced genes was further increased by Toxoplasma infection. Most notably, the anti-inflammatory cytokine IL-10 was synergistically up-regulated by preinfection. Furthermore, another subset of LPS-responsive genes displayed little or no change upon RH infection. These included the genes for CD86, Toll-like receptor (TLR)2 and NFκB family member RelB. Together, these findings show selectivity of the LPS-inducible genes that are down-regulated by Toxoplasma.

Interference with NFκB Signaling An emerging survival strategy for Toxoplasma is interference with transcriptional regulators of the proinflammatory response.13,23-26 Several of the inflammatory mediators produced in acute situations (such as during endotoxin exposure) rely on transcription factor NFκB for their synthesis.24,27,28 Indeed, TNF-α, IL-12 and inducible NO synthase are all dependent to some extent on activation, nuclear translocation, and DNA-binding activity of NFκB.24,27,28 The NFκB pathway is a canonical signal transduction cascade, the activation of which is critical to many physiological pathways including proliferation, apoptosis, and the immune response to infection.23,29-32 Indeed, the importance of the NFκB pathway is illustrated by the varied mechanisms employed by numerous pathogens to thwart its function.33-36 The Rel/NFκB family comprises a diverse set of proteins that include RelA/p65, RelB, c-Rel, NFκB1 (p50) and NFκB2 (p52).32 These proteins can homo- or heterodimerize via their Rel homology domains. While both c-Rel and RelA bear strong transactivating domains, c-Rel appears to be the predominant NFκB species involved in LPS-induced transcription of IL-12(p40).37 NFκB dimer is sequestered in the cytosol by its inhibitory partner IκB. When the appropriate signal is delivered to the cell surface, the canonical NFκB pathway is initiated by activation of upstream IκB kinases (IKK). Phosphorylation of IκB serves as a signal for its ubiquitination-dependent degradation upon which the NFκB nuclear localization sequence is revealed. After nuclear translocation, NFκB initiates transcription of several proinflammatory genes encoding cytokines, chemokines and immune recognition receptors. The products of several genes whose transcription relies on NFκB, including TNF-α and IL-1β, can also directly activate this pathway, creating a positive feedback loop.38 Although Toxoplasma generates a strong Th1 response in vivo, with the appearance of IL-12 and TNF-α in the serum of infected mice,39-41 in vitro experiments suggest that infected macrophages may not be the early source of these cytokines. This lack of early cytokine production has been linked to the apparent inability of infected macrophages to accumulate nuclear NFκB.14,22,24,42 Indeed, tachyzoite infection alone does not appear to stimulate nuclear

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translocation of this transcription factor and the parasite actively inhibits endotoxin induced nuclear accumulation and cytokine production. Although NFκB fails to accumulate in the nucleus of infected cells, all upstream activation steps of the NFκB pathway-IκB phosphorylation, ubiquitination and degradation-occur in parasite-infected cells with or without subsequent endotoxin stimulation.14,15,43,44 When macrophages are subjected to longer infections (6-18 hours) there is still no apparent accumulation of NFκB in the nuclei of infected macrophages. However, when infected cells are stimulated with endotoxin at the later time points, the inhibition is lifted and NFκB undergoes translocation. Interestingly, Shapira and colleagues recently reported that NFκB is translocated to the nuclei of infected cells, but that it undergoes rapid export.44 The overall effect of displacing the localization equilibrium to the cytoplasm is likely to be absence of IL-12 and TNF-α production in parasite-infected and LPS-triggered macrophages. A second, noncanonical NFκB pathway is initiated by proteolytic generation of p50 from NFκB2 precursors.45,46 Homodimers (p50/p50) have been implicated in endotoxin tolerance and inhibition of TNF-α expression.47-49 It is unknown if the parasite affects the activation of the noncanonical NFκB pathway. There is some controversy about translocation of NFκB in Toxoplasma infected cells. By comparing the response of macrophages to infection by Type 1 (virulent) RH strain and the Type 2 (less virulent) PTG strains, Robben and colleagues50 demonstrated that parasite genotype is an important factor in both NFκB translocation and IL-12 production. The Type 2 strain induced NFκB translocation after 24 h and generated significantly more IL-12 at this time point than the more virulent Type 1 strain. The authors propose that this strong cytokine response contains the Type 2 parasite, thus making it less virulent. Molestina and colleagues find NFκB activation and translocation during RH infection of fibroblasts and attribute IκB phosphorylation to a parasite-derived kinase that localizes to the parasitophorous vacuole.43,51,52 It is likely that parasite strain and cell type are significant sources of discrepancy in these studies.

Effect of Toxoplasma Infection on MAPK Signaling The MAPK family comprises three members: the stress-activated and Jun N-terminal kinases (SAPK/JNK), the extracellular signal-regulated kinases (ERK) 1 and 2, and p38 MAPK.53 Activation of MAPK begins at the plasma membrane when cells are stimulated by ligation of several classes of receptors, including those for inflammatory cytokines and Toll-like receptors (TLR). For most TLR, signaling requires adaptor protein MyD88 and subsequent recruitment of TNF receptor-associated-associated factor (TRAF)6 and IL-1 receptor-associated kinases (IRAK) 1 and 4.54 This is followed by assembly of a complex containing transforming growth factor-β-activated kinase (TAK)1, and TAK1-binding proteins TAB1 and TAB2. Subsequently, TAK1, a MAPK kinase kinase (M3K) is activated and the signaling cascade bifurcates at this point: TAK1 initiates the MAPK cascade and phosphorylates IκB, leading to the activation of NFκB (Fig. 1). The three MAPK signaling modules are activated within minutes when macrophages are subjected to Toxoplasma infection (Fig. 1).55,56 Activation is transient, however, and within two hours phosphorylation returns to control levels. Kim and colleagues56 showed that MAPK rephosphorylation is defective when infected macrophages are subsequently stimulated with lipopolysaccharide (Fig. 1). This phenomenon is mechanistically different from endotoxin tolerance57,58 in several important ways. For example, LPS pretriggering followed by subsequent LPS exposure fails to rephosphorylate MKK3 and 6, major kinases that activate p38 MAPK. In contrast, Toxoplasma induces sustained phosphorylation of MKK3 while failing to activate MKK6.56 Moreover, LPS-induces down-regulation of TLR4, providing another proposed mechanism of LPS tolerance.58,59 However, Toxoplasma actually upregulates TLR4 during infection, another indication that the parasite effect is fundamentally different.56 Phosphorylation of p38 MAPK is essential for IL-12 production in both LPS-treated and parasite-infected cells.60-62 To delineate this pathway in parasite-infected cells, Kim and colleagues63 used active site-directed inhibitors and macrophages genetically deficient in upstream kinases. This series of experiments demonstrated that Toxoplasma infection activates p38 MAPK by stimulating autophosphorylation, rather than through activation of upstream p38-directed

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MKK. Autophosphorylation of p38 is a recently recognized mechanism for activation of this particular MAPK.64-66

Induction of STAT3 Signaling by Toxoplasma Inhibition of proinflammatory responses seems a perfect strategy for T. gondii to gain purchase within the macrophage. It is clear that Toxoplasma does not invade silently, but activates host MAPK pathways and sets into motion, perhaps unproductively, the NFκB pathway. The parasite takes over the macrophage’s defense pathways and directs them toward different ends. Recently it has come to light that Toxoplasma not only inhibits proinflammatory pathways, but also exploits the IL-10 anti-inflammatory pathway to accomplish down-regulation of cytokine signaling. IL-10 is an anti-inflammatory modulator that suppresses many proinflammatory cytokines, including IL-12 and TNF-α. To accomplish this, IL-10 requires the intracellular signaling molecule STAT3.67,68 While the mechanisms linking down-modulatory effects of IL-10 and STAT3 remain elusive, it is clear that deletion of either the IL-10 or STAT3 gene sets up an uncontrolled Th1 response. Mice that have tissue-targeted deletion of STAT3 succumb to overwhelming enterocolitis associated with a dramatic hyperinflammatory response, similar to IL-10 deficient animals.67 When IL-10 binds its receptor, the Janus kinase (JAK)/STAT signaling pathway is initiated through phosphorylation of the receptor and subsequent transphosphorylation of the JAK1/ Tyrosine kinase (Tyk2) molecules.69,70 STAT3 monomers are then recruited to the receptor/ JAK complex via its SH2 domain, whereupon JAKs phosphorylate STAT3. Phosphorylated STAT3 (p-STAT3) dimerizes and translocates to the nucleus where the p-STAT3 dimer is phosphorylated at a serine residue. The dually phosphorylated dimer then binds to target DNA to regulate STAT3-sensitive gene transcription.70 When macrophages are infected with Toxoplasma, STAT3 is rapidly phosphorylated and this activation state is maintained for at least 23 hours.71 The significance of Toxoplasma-induced STAT3 activation is revealed when STAT3 gene-deleted macrophages are infected. The ability of parasites to mediate inhibition of LPS-induced IL-12 and TNF-α is severely compromised when STAT3 deficient macrophages are infected, suggesting that the parasite exploits an established host cell immunosuppressive activity. It is not clear at present how STAT3 is activated by the parasite. STAT3 phosphorylation appears to be Pertussis toxin insensitive, indicating that activation is not G-protein linked (Butcher and Denkers, unpublished observations). Moreover, experiments in MyD88-deficient macrophages show that this common adaptor of TLR signaling is not involved in T. gondii-induced STAT3 activation (Butcher and Denkers, unpublished observations). The identity of host JAK molecules activated by the parasite is unknown. Indeed, it seems possible that the parasite itself may directly activate STAT3 during its interaction with the host cell.

Kinetics of Toxoplasma Invasion and Disruption of Host Signaling Pathways The concept of ongoing communication between Toxoplasma gondii and the macrophage raises the possibility of parasite-derived molecules directly interfering with host cell signaling pathways (Fig. 2). From the point of attachment through invasion and modification of the parasitophorous vacuole and on to parasite multiplication and egress from the cell, there are several opportunities for involvement of parasite proteins (Fig. 2). After orienting its apical end toward the macrophage, the parasite secretes proteins from the micronemes (Fig. 2, Step 1) These proteins do not associate with the parasitophorous vacuole and are thought to be involved only in the earliest phases of host cell contact and invasion.72-79 Because microneme proteins are not disgorged into membrane-bound vacuoles, these proteins are freely accessible to the plasma membrane and thus potentially able to participate in signaling events initiated at the cell surface. The release of microneme contents and parasite invasion occur with similar kinetics to Toxoplasma-induced MAPK and STAT3 activation (Fig. 2), begging the intriguing question of whether the parasite directly phosphorylates host cell signaling molecules during early invasion.

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Figure 2. Kinetics of parasite invasion and interference with signaling pathways. The ordered secretion of proteins contained in the micronemes, rhoptries, and dense granules during parasite attachment (1), invasion (2) and vacuole modification (3) should provide ample opportunity for exposure of the host cell to active parasite molecules (dard gray circles denote released microneme proteins; grey circles denote rhoptry derived evacuoles). The MAPK, STAT3 and NFκB pathways are all initiated within minutes of the parasite’s initial contact with the macrophage. Indeed, considering the rapid phosphorylation kinetics of STAT3 and the MAPKs, it is conceivable Toxoplasma either directly activates upstream kinases or directly phosphorylates these proteins. Although MAPK activation is short-lived, STAT3 phosphorylation is apparent for at least 24 hours. It is unknown at present if this sustained phosphorylation is due to continued activation from within the vacuole or parasite-mediated deactivation of phosphatases. Inhibition of LPS-induced NFκB translocation is lifted at 6-8 hours, however Toxoplasma still blocks TNF-α synthesis, indicating that there is still down-regulation of some LPS responses. The release of parasite-derived proteins (black triangles) from the vacuole and decoration of the vacuole with parasite molecules (black circles) may influence sequestration from the lysosomal pathway.

Vacuole modification, which begins with exclusion of most host cell proteins from the membrane, continues for the tenure of the vacuole. Modification of the vacuole by decoration with rhoptry and dense granule proteins provides means for direct communication with the host in terms of recruitment of host cell proteins and activation/deactivation of signaling pathways.80 Rhoptry proteins are released into the developing vacuole membrane and lumen only during invasion (Fig. 2 Step 2). Rhoptry proteins are also present in evacuoles, small vesicle-like compartments that are released into the host cytosol and are trafficked to the parasitophorous

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vacuole along microtubules.81 Dense granule proteins are discharged constitutively into the vacuole and parasite proteins may exit the vacuole to influence host cell function.82-85 Other modifications include creation of a “molecular sieve”, formed from insertion of rhoptry and dense granule proteins into the vacuolar membrane, that allows for acquisition of nutrients86 (Fig. 2, Steps 2 and 3). Rhoptry proteins have been implicated in the association of both the parasitophorous vacuole and evacuole with host mitochondria and endoplasmic reticulum.81,87 The ability of Toxoplasma to coordinate docking of its vacuole with host organelles raises the intriguing possibility that parasite proteins may influence other aspects of membrane acquisition and docking. For example, phagosome maturation relies upon a continuum of membrane interactions that are mediated in part via phosphatidylinositol kinases and small GTPases.88-90 The kinetics of rhoptry discharge and insertion of parasite proteins that remain with the vacuole would permit interference with these interactions. Thus, Toxoplasma may maintain its specialized habitat and sequestration from the endocytic pathway with its own enzymes, for example by parasite-mediated phosphorylation or dephosphorylation of vacuolar phosphatidylinositol phosphates. Testing this hypothesis awaits further identification of vacuole membrane constituents and their possible enzymatic activities.

Conclusions and Future Perspectives Toxoplasma gondii is an enormously successful parasite. Success, in this context, depends upon the relatively peaceful coexistence of both Toxoplasma and host, at least until further transmission. This coexistence relies on a carefully modulated immune equilibrium that is readily achieved in the healthy host. Tipping the balance either way, toward a hyperinflammatory response or an immunosuppressive event, will result in immune pathology or uncontrolled parasite replication, respectively. In light of this, it appears that T. gondii, relies upon immunosuppression of the infected cell to survive. Although we have identified several proinflammatory pathways that the parasite usurps for its survival, very little is known about the precise mechanisms or the parasite molecules that are involved. Information gained from both the developing field of parasite proteomics and the completion of the Toxoplasma genome will permit rapid advances in the understanding of this elegant parasite, especially at the cell biology and immunobiology interface.

Acknowledgements The authors appreciate the contributions of present and former laboratory members. This work is supported by the National Institutes of Health (AI50617).

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CHAPTER 9

Avoidance of Innate Immune Mechanisms by the Protozoan Parasite, Leishmania spp. David M. Mosser* and Suzanne A. Miles

Abstract

I

n this chapter, we will examine the mechanisms by which Leishmania parasites interact with host cells. We will try to develop the hypothesis that the success or failure of Leishmania infections can be traced to the initial mechanism(s) of parasite entry into mononuclear phagocytes. We will try to make the following points about the activation and modulation of innate immunity by Leishmania: First, promastigotes enter macrophages by a quiescent mechanism that fails to induce innate immune responses, and this may result in a delayed induction of an adaptive immune response. This delay in the development of adaptive immunity may provide the parasite with time to replicate within macrophages. Second, parasite replication disrupts macrophage responsiveness to the immune signals that are eventually generated. Third, the mechanism of amastigote entry into macrophages may also be the harbinger for successful parasitism. Amastigotes coat themselves in host IgG which ligates macrophage FcγR, resulting in the hyperproduction of IL-10 from infected macrophages. This IL-10 can prevent macrophage responses to IFN-γ allowing the parasites to survive even in the immunologically intact host.

Life Cycle The promastigote form resides in the midgut of infected sandflies (Fig. 1). A small pool of blood is formed when a female fly takes a blood meal. Infectious promastigotes (metacyclics) are regurgitated into this pool of blood, where they enter the human host.1 Phagocytic cells recruited to the site of infection rapidly internalize the promastigotes. The parasites not only survive but replicate within the acidic phagolysosome. Replication continues until the host cell is lysed, releasing parasites, to infect neighboring cells.

Receptor-Mediated Phagocytosis The uptake of Leishmania promastigotes by macrophages is a receptor-mediated process that involves the expenditure of energy by the macrophage, but not by the parasite.2 Due to the obligate intracellular nature of the pathogen, this organism expresses several different ligands on their surface that can interact with a variety of different macrophage receptors, to ensure their uptake by phagocytic cells.2 These include the receptors for complement,3,4 fibronectin,5 and sugars, such as the mannose-fucose receptor6 and others.7 These receptors bind parasites with different avidity. In some instances low affinity receptors can make a substantial contribution to parasite internalization without making an obvious contribution to parasite adhesion. A clear example of this is the fibronectin receptor (FnR), which binds to the parasite surface molecule *Corresponding Author: David M. Mosser—Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, U.S.A. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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Figure 1. The life cycle of Leishmania spp. The promastigote stage resides in the midgut of sandflies, and is transferred to the human host during a bloodmeal. Promastigotes are taken up by resident macrophages and cells recruited to the site of the bite. Inside macrophages they differentiate into the amastigote form. The cycle continues when a sandfly bites an infected host, ingesting macrophages infected with amastigotes. In the sandfly midgut, amastigotes differentiate back into the promastigote form.

gp63 with low affinity. During in vitro phagocytosis assays, this receptor appears to play little or no role in parasite adhesion. However cells lacking FnR, or parasites with a mutation in the fibronectin recognition domain on gp63, exhibit significant delays in parasite uptake, relative to wild-type cells,6 indicating that this receptor plays an important role in uptake. The ligands on promastigotes that have been implicated in parasite uptake include lipophosphoglycan (LPG) and gp63, as well as other phosphoglycan species in the parasite glycocalxy.8-10 However the most important ligands for parasite uptake are not parasite-encoded at all. Promastigotes rely heavily on host-derived opsonins to achieve maximal uptake by phagocytis cells. In addition to fibronectin, mentioned above, the complement system represents an important mediator of promastigote adhesions to phagocytic cells.11 The third component of complement (C3) and the complement receptor Type 3, (CR3, Mac-1, CD11b/CD18) is probably the most important of the macrophage complement receptors for parasite phagocytosis. This is due to the abundance of CR3 expression on macrophages and to the very transient nature of the C3b molecule, whose half-life on opsonized particles is measured in minutes.12 Promastigotes enter into macrophage phagosomes, which acidify and fuse with lysosomes. It is in these acidified phagolysosomes that the organisms replicate. The evidence to indicate that these phagolysosomes are fully competent, is the fact that debris and dead organisms are degraded in the same phagolysosomes that house viable amastigotes.13 There is now strong

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evidence that there is a delay in the maturation of promastigote phagosomes. This “pregnant pause”14 may give the promastigote the time needed to transform into amastigotes and upregulate the genes necessary for intracellular survival.15 Lipophosphoglycan from the surface of the promastigote may contribute to this delay in maturation. The current thought is that alterations in the lipid content of cellular organelles can influence the fusagenic competence of these vesicles with each other.16 The intercalation of LPG into the membrane of the phagosome may adversely influence this fusion process, delaying maturation long enough for parasite survival.17 This delay may not be exclusively mediated by LPG, since some LPG-deficient species, such as L. mexicana, survive quite well in macrophages.18 Amastigotes are also taken up by receptor-mediated phagocytosis and this process is remarkably efficient. A resting macrophage can internalize a dozen or more amastigotes within 30 minutes. While this developmental form may use some of the same receptors as the promastigote form, there are clearly some important differences with regard to amastigote uptake. Amastigotes appear to have a heparin binding activity, which allows them to adhere to cellular proteoglycans. This adhesion increases the efficiency of receptor-mediated phagocytosis. Mannose receptors and complement receptors have been implicated in amastigote phagocytosis,5 and the amastigote ligands include phosphoglycans19 and glycoinositol-phospholipids (GIPL).20 Interestingly, a study has shown that amastigotes may mimic apoptotic cells and bind to phosphatidylserine receptors on macrophages.21-23 This would be consistent with the failure of these organisms to activate inflammatory cytokine production from macrophages. Similar to promastigotes, a host opsonin plays an important role in amastigote uptake. The opsonin in this case is IgG, rather than complement. Amastigotes isolated from lesions of experimentally infected animals are coated with host IgG and they bind avidly to macrophage Fcγ receptors (FcγR).5 It is important to note that like the promastigote, there are multiple, redundant ways for amastigotes to enter macrophages. Consequently, neither the lack of IgG on amastigotes, nor the lack of FcγR on macrophages, prevents amastigote phagocytosis. The role of IgG on amastigotes will be discussed in detail in a subsequent section. Amastigote entry into macrophages does not result in a delay in phagolysosomal (P/L) fusion.24 It would be interesting to know whether signals transduced through the FcγR are responsible for the efficient uptake of amastigotes leading to P/L fusion. It should also be noted that dendritic cells express FcγR and are highly phagocytic for both developmental forms of Leishmania. There is solid evidence that signaling through FcγR leads to DC maturation25-27 and enhanced APC function. The role of FcγR-mediated phagocytosis of Leishmania by DCs may represent an important mechanism for the induction of a protective immune response.28

Leishmania and Innate Immunity There is a growing body of evidence to suggest that the activation of the innate immune response occurs during experimental infection with Leishmania, and that this activation can impact the development and the characteristics of the adaptive immune response. Mice on a resistant background, but deficient in the toll-like receptor (TLR) adaptor MyD88, developed progressive lesions and failed to mount protective Th1 immune responses.29,30 Similarly, genetically resistant mice, which carried a null mutation in tlr4, developed non healing lesions, when infected with L. major.31,32 There is also evidence that parasite derived molecules can activate TLRs. The most thoroughly studied of these is LPG, which has been shown to activate TLR 2.30 There is evidence from other protozoa that glycosyl-phosphatidylinositol (GPI) anchored structures can activate TLRs,33 although this may not be true for at least some GPI-linked structures from Leishmania.34 Despite these in vivo studies, which suggest a role for innate immunity, the results from in vitro infections point to fundamental differences in the innate response of macrophages and dendritic cells to this organism, relative to bacteria. The phagocytosis of even small numbers of bacteria is invariably associated with the rapid translocation of NF-κB,35 and the secretion of a large array of cytokines, most of them inflammatory in character, such as TNF, IL-1, and IL-12.

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Figure 2. Immunofluorescence microscopy to detect NF-κB (p65) translocation in macrophages. Untreated macrophages show cytoplasmic staining for NF-κB (p65) and dark unstained nuclei. LPS-treated macrophages show brightly stained nuclei indicating the translocation of NF-κB into nuclei. Macrophages infected with the bacterium Rhodococcus equi also show nuclear localization of NF-κB (arrows). Macrophages heavily infected with L. amazonensis fail to translocate NF-κB. Note the dark unstained nuclei.

This rapid activation of innate immune responses does not appear to occur following Leishmania infection. Even the phagocytosis of large numbers of promastigotes results in little, to no, detectible NF-κB activation (Fig. 2). Consequently, promastigote phagocytosis induces minimal cytokine secretion by infected macrophages (Fig. 3). This appears to be a characteristic of all Leishmania species tested, regardless of whether they are alive or heat-killed, provided that the organisms are grown in media that is free of contaminating LPS. Thus, the promastigote form of the parasite enters macrophages and dendritic cells by a quiescent mechanism that does not elicit substantial cytokine production. Importantly, the simultaneous addition of LPS and parasites to macrophages results in the secretion of a large number of LPS-induced cytokines. The only exception of which is IL-12 which will be discussed below. The logical interpretation of this latter observation is that Leishmania promastigotes do not actively prevent cytokine production by macrophages, but rather they fail to induce it. The easiest way to reconcile the in vivo observations suggesting that TLRs are an important component of early immunity to Leishmania, and the in vitro evidence that parasites induce minimal TLR activation, is to hypothesize that during infection, the indirect activation of endogenous TLR ligands by Leishmania, such as those associated with the inflammatory extracellular matrix, are responsible for the induction of innate immunity. This indirect activation of innate immunity may account for the slow induction of immune responses that occurs when mice are infected with low numbers of parasites in the ear.36

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Figure 3. TNF production by infected macrophages. Unprimed (black bars) or IFN-γ-primed (gray bars) macrophages were infected with increasing ratios of live or heat-killed promastigotes of L. amazonensis. Twenty-four hours later, TNF levels in supernatants were quantitated by ELISA. For a positive control, parallel monolayers were infected with increasing ratios of live R. equi.

Suppression of IL-12 and the Th1 Response The failure of Leishmania promastigotes to induce the secretion of macrophage cytokines extends to IL-12.37,38 However, in the case of IL-12, promastigotes not only fail to induce IL-12 synthesis, but they appear to actively downregulate its production (Fig. 4).39 This is different from other inflammatory cytokines, which are not actively suppressed by Leishmania infection. The in vitro infection of macrophages, with either developmental form of the parasite, results in a general suppression of IL-12 biosynthesis, even in response to exogenous stimuli, such as LPS. This observation is quite unexpected, given the importance of IL-12 in directing the development of a protective Th1 immune response.40 The downregulation of IL-12 leads to a failure in macrophage activation, and ultimately a failure in the intracellular killing of Leishmania. This inhibition may be a general response to infection, or it may be a result of the increased levels of intracellular calcium that seem to accumulate in infected macrophages (see below).41-43 Alternatively, it has been suggested that the specific suppression of IL-12 may relate to the transcription factor IFN-γ consensus sequence binding protein (ICSBP). Activation of the IL-12(p40) promoter requires ICSBP, which is regulated by signal transducer and activator of transcription 1 (STAT-1).44 Thus STAT-1 independent pathways may allow other cytokines to be produced, and remain unaffected by the presence of Leishmania. Similar to intact parasites, the stimulation of macrophages with purified LPG also diminishes the production of IL-12 by macrophages. It is likely, however, that this response is not specific to LPG, because the ligation of a large number of different receptors on macrophages has been

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Figure 4. IL-12 production by infected macrophages. IL-12 production by macrophages was measured by ELISA 24 hrs after exposure of cells to 10 μg/ml LPS alone or in combination with a 10:1 ratio of L. amazonensis promastigotes.

associated with a decrease in IL-12 biosynthesis.45-47 Regardless of the mechanism, the dramatic inhibition of IL-12 following Leishmania infection points to other nonmacrophage sources of IL-12 as being responsible for the initiation of the protective Th1 biasing that occurs during a protective immune response to this parasite.

Alterations in Signal Transduction following Leishmania Infection In addition to failing to efficiently activate innate immunity in parasitized cells, it also appears as though promastigote infection of macrophages is associated with alterations in cellular signal transduction mechanisms, leading to defective immune responses of infected macrophages. Thus, the parasite may take an active role in suppressing immune responses. The experimental model used for most of these studies is to infect macrophages with Leishmania, and then look for alterations in signal transduction by infected cells. Obviously in a model such as this, the multiplicity of infection is quite important, and to our knowledge, a careful study to correlate signaling alterations with MOI values has not been done. Nevertheless, several observations have begun to emerge about specific alterations in signaling in parasitized macrophages. One of the early observations, which has been confirmed by several groups, is that the infection of macrophages with Leishmania is accompanied by an increase in intracellular calcium levels.41 This may have several consequences on parasite survival. Alterations in the activation of various isoforms of protein kinase C (PKC)48,49 have been reported in infected macrophages, and these alterations may be causally related to the changes in intracellular calcium concentrations.

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The experimental inhibition of PKC activation in macrophages leads to increased numbers of parasites within these macrophages.49 Additionally, it has been shown that purified LPG can block PKC activity, and that promastigotes which do not express LPG on their surfaces exhibit decreased intracellular survival.50 These observations suggest that parasite induced impairment of PKC activation may be important mechanism of establishing an infection. Altered levels of intracellular calcium may also be involved in the reported alterations in mitogen-activated protein kinase (MAPK) activation that accompany Leishmania infection.51,52 MAPKs play an important role in promoting the transcription of many inflammatory cytokines,53,54 and the differential activation of MAPKs has been linked to the production of different cytokines during infection. Recent evidence suggests that activation of different MAPKs can influence parasite survival.55 The suppression of IL-12 production during infection may be a result of the preferential activation of extracellular signal related kinase (ERK) 1/2, by Leishmania.56 The strongest evidence for alterations in signal transduction pertain to the inhibition of protein tyrosine kinase (PTK) activity in infected macrophages. This observation has been made by several groups.57-59 The reduction of PTK activity in infected macrophages leads to reduced signaling through Janus kinases (JAKs) and STAT-1.60 The major result of this alteration is defective responses of infected macrophages to the activating effects of IFN-γ.61 Infected macrophages display reduced levels of total and phosphorylated JAK1 and JAK2.62 Additionally, STAT-1 plays a critical role in the signaling of IFN-γ. The STAT-1 mediated pathway has been shown to be necessary for a protective immune response against L. major.63 L. donovani has been shown to attenuate IFN-γ induced STAT-1 phosphorylation in macrophages, aiding the parasite in escaping host immunity.60 The mechanism whereby Leishmania modulate cellular PTK activity appears to involve the activation of cellular phosphatases.64 Cellular SHP-1 activity is increased following infection, and subsequent stimulation of macrophages with phorbol esters to stimulate protein tyrosine phosphorylation results in diminished levels of tryosine phosphorylation in infected cells.65 The mechanism of SHP-1 activation by Leishmania has not been defined, but given the sensitivity of these molecules to alterations in lipid composition, one cannot rule out a role for the inositol-linked phospholipids from the Leishmania surface, which appear to intercalate into the plasma membrane of infected macrophages.66

IL-10 Induction by IgG-Opsonized Amastigotes It is well-established that the infection of BALB/c mice with L. major results in the production of IL-4 from T cells, which ultimately results in progressive disease. However, there is increasing evidence that IL-4 may not be sufficient for susceptibility of BALB/c mice to cutaneous leishmaniasis.67,68 Early studies in human visceral leishmaniasis correlated IL-10 levels with disease severity,69 and several groups have subsequently examined the role of this cytokine in murine models of disease. The role of IL-10 in animal models of leishmaniasis has been demonstrated for both cutaneous and visceral forms of the disease.38,67,70 This is important, because IL-10 can suppress Th1 responses, and inhibit macrophage activation.71 It has been previously demonstrated that macrophages activated in the presence of immune complexes shut off their production of IL-12,45 and produce high levels of IL-10.72 This alteration in cytokine production occurs when immune complexes ligate the FcγRs on macrophages. The mechanisms involved in this cytokine switch are not fully understood, however it has been shown that ERK activation is required for the super-induction of IL-10 caused by immune complexes.73 FcγR ligation leads to the activation of the ERK, which leads to remodeling of the il-10 promoter, making it more accessible to transcription factors that bind there.73 The high level of IL-10 production from macrophages has two important effects. First, IL-10 makes macrophages refractory to the activating effects of IFN-γ.38 Second, when macrophages are used as antigen presenting cells, the production of IL-10 inhibits the development of Th1 cells and induces a Th2-like immune response.74 These basic observations predicted that the presence of immune complexes could adversely influence the development of cell mediated immunity to Leishmania spp. Two other previous

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observations reinforced this prediction. The first was the observation that the levels of immune complexes are quite high in patients with visceral leishmaniasis.75,76 The second was the observation that amastigotes isolated from the lesions of infected mice were opsonized with host IgG.38,77 Thus, immune complexes formed during Leishmania infections could interfere with protective immune responses to this organism. A series of studies was undertaken to determine whether lesion-derived amastigotes could induce IL-10 production from macrophages in an FcγR-dependent manner. In both murine and human systems this is precisely what we observed. Lesion derived amastigotes from BALB/ c mice induced high levels of IL-10 production from inflammatory macrophages expressing FcγR.38 Similarly, peripheral blood mononuclear cells from the blood of normal human donors produced high levels of IL-10 when infected with L. chagasi amastigotes that were opsonized with serum from a patient with visceral leishmaniasis (VL).78 In vivo studies in JH mice were consistent with these in vitro observations. JH mice have a targeted deletion of the immunoglobulin heavy chain J locus, and therefore, make no antibody.79 JH mice were more resistant to infections with L. major than were normal BALB/c mice. When these mice were reconstituted with sera from chronically infected BALB/c mice, they exhibited larger lesions, with higher numbers of parasites residing in those lesions. Treating the mice with a mAb to the IL-10 receptor prevented the IgG-mediated enhancement in lesion development.78 These studies demonstrate that host IgG can cause a novel form of immune enhancement, due to its ability to induce IL-10 production from macrophages. These studies also provided evidence to link the high titers of parasite-specific IgG in human VL with defective DTH responses and disease progression. These observations point to several interesting parallels between L. major infection of BALB/ c mice, and human VL. BALB/c mice develop high antibody titers, with frequent metastasis of parasites to the bone marrow, liver, and spleen.80 Additionally, the delayed-type hypersensitivity (DTH) response in BALB/c mice is smaller and more transient than in resistant C56BL/6 mice.81 Interestingly, 20 years ago it was demonstrated that the suppression of DTH responses in BALB/c mice required the presence of B cells.82 Taken together these observations suggest that parasite-specific antibody can play a role in disease by contributing to the formation of immune complexes, which in turn induce the production of IL-10 from host macrophages. These conclusions are consistent with previous reports by others,5,78,83,84 which demonstrated that B cell deficient mice, on the susceptible BALB/c background, were more resistant to disease, and developed smaller lesions. It is important to note that in addition to macrophages,85 Th2 and T regulatory cells (Tregs) have also been shown to be important producers of IL-10. The role of Tregs was specifically examined in mice infected with low doses of L. major.86 It was shown that IL-10 derived from Tregs made an important contribution to the persistence of parasites after clinical cure. Paradoxically, the sterile cure observed in mice deficient in IL-10 was actually detrimental to host immunity, because these mice were no longer immune to subsequent reinfection following clearance.87 This work indicated that the maintainance of memory T cells is dependant upon parasite persistence. The mechanism of Treg induction by these parasites is not known. However, there appear to be multiple ways that this organism induces an IL-10 response from the host that is conducive to parasite growth and/or persistence. The IL-10 produced by macrophages and Tregs appears to cause a localized and transient immunosuppression that allows the parasite to survive in an immunocompetent host.

Conclusion Parasites in the genus Leishmania have evolved a variety of intricate ways to modulate the host immune response. The promastigote relies on a stealth mechanism of entry to parasitize macrophages without inducing the panoply of cytokines that designates “danger” to the host. Furthermore, the intracellular residence of this parasite in phagocytes confuses the signaling pathways, making these cells less efficient at responding to immune activating signals. Finally, the amastigote form of the parasite takes advantage of what should normally be a protective

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immune response, and coats itself in host IgG, in order to induce the production of the immunosuppressive cytokine, IL-10.

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73. Lucas M, Zhang X, Prasanna V et al. ERK activation following macrophage FcgammaR ligation leads to chromatin modifications at the IL-10 locus. J Immunol 2005; 175:469-477. 74. Anderson CF, Mosser DM. Cutting edge: Biasing immune responses by directing antigen to macrophage Fc gamma receptors. J Immunol 2002; 168:3697-3701. 75. Carvalho EM, Andrews BS, Martinelli R et al. Circulating immune complexes and rheumatoid factor in schistosomiasis and visceral leishmaniasis. Am J Trop Med Hyg 1983; 32:61-68. 76. Pearson RD, de Alencar JE, Romito R et al. Circulating immune complexes and rheumatoid factors in visceral leishmaniasis. J Infect Dis 1983; 147:1102. 77. Guy RA, Belosevic M. Comparison of receptors required for entry of Leishmania major amastigotes into macrophages. Infect Immun 1993; 61:1553-1558. 78. Miles SA, Conrad SM, Alves RG et al. A role for IgG immune complexes during infection with the intracellular pathogen Leishmania. J Exp Med 2005; 201:747-754. 79. Chen J, Trounstine M, Alt FW et al. Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus. Int Immunol 1993; 5:647-656. 80. Howard JG, Hale C, Chan-Liew WL. Immunological regulation of experimental cutaneous leishmaniasis. 1. Immunogenetic aspects of susceptibility to Leishmania tropica in mice. Parasite Immunol 1980; 2:303-314. 81. Andrade ZA, Reed SG, Roters SB et al. Immunopathology of experimental cutaneous leishmaniasis. Am J Pathol 1984; 114:137-148. 82. Sacks DL, Scott PA, Asofsky R et al. Cutaneous leishmaniasis in anti-IgM-treated mice: Enhanced resistance due to functional depletion of a B cell-dependent T cell involved in the suppressor pathway. J Immunol 1984; 132:2072-2077. 83. Kima PE, Constant SL, Hannum L et al. Internalization of Leishmania mexicana complex amastigotes via the Fc receptor is required to sustain infection in murine cutaneous leishmaniasis. J Exp Med 2000; 191:1063-1068. 84. Colmenares M, Constant SL, Kima PE et al. Leishmania pifanoi pathogenesis: Selective lack of a local cutaneous response in the absence of circulating antibody. Infect Immun 2002; 70:6597-6605. 85. Lang R, Rutschman RL, Greaves DR et al. Autocrine deactivation of macrophages in transgenic mice constitutively overexpressing IL-10 under control of the human CD68 promoter. J Immunol 2002; 168:3402-3411. 86. Belkaid Y. The role of CD4(+)CD25(+) regulatory T cells in Leishmania infection. Expert Opin Biol Ther 2003; 3:875-885. 87. Belkaid Y, Hoffmann KF, Mendez S et al. The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure. J Exp Med 2001; 194:1497-1506.

CHAPTER 10

Survival Strategies of Toxoplasma gondii: Interference with Regulatory and Effector Functions of Macrophages Carsten G.K. Lüder*

Abstract

T

oxoplasma gondii establishes long-lasting asymptomatic infections in immunocompetent hosts including humans, but is an important opportunistic pathogen in immunocompromised patients or after transplacental transmission to fetuses. The ability to survive within hosts with an intact immune system relies on the parasite-mediated inhibition of regulatory functions of macrophages, e.g., the downregulation of the MHC class II expression and the presentation of antigens to CD4+ T lymphocytes. It is also facilitated by the downregulation of toxoplasmacidal effector functions of macrophages, e.g., the production of nitric oxide by the inducible nitric oxide synthase or the blockade of host cell apoptosis in parasite-infected cells. Progress that has been made in the characterization of the underlying molecular and cellular mechanisms reveals distinct interference by T. gondii with signal transduction cascades or other regulatory components of the host cell. These interactions provide fascinating examples of the elaborate mechanisms by which an intracellular parasite reprograms macrophages in order to establish chronic infection.

Introduction The apicomplexan Toxoplasma gondii is probably the most abundant protozoan parasite throughout the world. Its success may be related to the unique ability to infect and survive in a broad range of warm-blooded hosts including humans. Due to the parasite-driven active invasion of its host cells (see chapter 1) T. gondii is also able to infect and replicate within any nucleated mammalian cell. Despite the promiscuity of T. gondii in choosing a host cell, macrophages are nevertheless crucial for the course of toxoplasmosis. Channon et al1 established the fact that monocytes are more readily infected by T. gondii and are more permissive for efficient parasite replication than other human leucocytes. Monocytes/macrophages may thus represent an important host cell type for local parasite replication and for hematogeneous dissemination to other organs. During the development of anti-parasitic immunity and after activation with IFN-γ and other proinflammatory cytokines, macrophages fulfill anti-parasitic effector mechanisms thereby restricting replication or even killing T. gondii.2,3 Furthermore, macrophages are critical for the regulation of the immune response during toxoplasmosis due to their essential functions as phagocytes, professional antigen-presenting cells (APC) and producers of various immunomodulatory cytokines. T. gondii would, however, not be able to become one *Carsten G.K. Lüder—Institute for Medical Microbiology, Georg-August-University, Kreuzbergring 57, 37075 Göttingen, Germany. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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of the most successful parasites without evolving a variety of strategies by which regulatory and effector functions of macrophages are counterbalanced. Such mechanisms may not only facilitate the parasite’s own survival within macrophages but may also restrict the anti-parasitic immune response in order to establish and maintain persistent infections. While these parasite interferences are—as far as we know—not specific for macrophages, they may, nevertheless, be particularly relevant for this interaction because of the importance of macrophages as host cells and as regulator and effector cells of anti-Toxoplasma immunity.

Inhibition of MHC Class II Expression and Antigen Presentation Expansion of antigen-specific CD8+ or CD4+ T lymphocytes and microbicidal activity exerted by these cells require presentation of antigenic peptides in the context of major histocompatibility complex (MHC) class I and class II molecules, respectively.4 According to the classical paradigm in immunology, endogenous, i.e., cytosolic, peptides are bound to MHC class I molecules and presented to CD8+ T lymphocytes, whereas exogenous, i.e., extracellular-derived peptides are bound to MHC class II molecules and presented to CD4+ T cells. In macrophages, MHC class I molecules are constitutively expressed and MHC class II molecules are efficiently induced by activation with IFN-γ which also further upregulates MHC class I.5 Since efficient control of T. gondii both in humans6 and after experimental infection of mice7,8 relies on T cell-mediated immunity, presentation of parasitic peptides is particularly important for the outcome of infection. Lüder et al,9,10 however, have provided convincing evidence that T. gondii inhibits the expression of MHC class II molecules after activation of murine macrophages with IFN-γ. In addition, IFN-γ-induced upregulation of MHC class I molecules is also inhibited in parasite-infected macrophages while the constitutive expression of class I moleclules is not diminished.9 Importantly, the parasite-imposed blockade in the expression of MHC class II molecules leads to a dramatic impairment of T. gondii-infected macrophages to present antigens to CD4+ T lymphocytes indicating that this interference is of functional significance.10 This may at least partially explain longstanding observations that CD4+ T cells are less efficiently expanded during toxoplasmosis as compared to CD8+ T cells.11,12 Furthermore, it may relate to the fact that following infection of mice with T. gondii, CD4+ T lymphocytes are less effective in restricting parasite propagation than CD8+ T cells.7 Since T. gondii also blocks MHC class II-restricted antigen presentation of heterologuous antigens,10 resistance of mice to concomitant infections with intracellular bacteria appears also to be decreased during acute toxoplasmosis.13 The inhibition of MHC class II expression by T. gondii, thus, not only represents an important immune evasion strategy but may also affect the outcome of coinfections. How is the MHC class II expression blocked after T. gondii infection? IFN-γ triggers— via the activation of Janus kinases (JAK’s)—the phosphorylation and homodimerization of signal transducer and activator of transcription (STAT)-1 thereby leading to its import into the nucleus and binding to promoters containing a TTNCNNNAA consensus sequence, i.e., the gamma-activated site (GAS; Fig. 1).14 Together with a second IFN-γ-regulated transcription factor, i.e., IRF-1, and the ubiquitous upstream stimulatory factor 1 (USF-1), binding of STAT-1 activates trancription of the class II transactivator (CIITA) gene.15 CIITA in turn activates the promoters of MHC class II and related genes and represents the master regulator of MHC class II expression.16 In T. gondii-infected macrophages, this signaling pathway is disturbed at the level of gene transcription of CIITA and IRF-1 consequently leading to defective MHC class II expression (Fig. 1).10 In contrast, tyrosine phosphorylation of STAT-110 as well as nuclear import of activated STAT-1 (C Lang and CGK Lüder, unpublished) is not inhibited by the parasite. This indicates that T. gondii might alter the binding of STAT-1 to its consensus sequence and/or interferes with the assembly and activity of the basal transcriptional machinery that is required for polymerase II-driven mRNA synthesis.

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Figure 1. Interference of T. gondii with IFN-γ-mediated MHC class II and iNOS expression in infected macrophages. Binding of IFN-γ and assembly of a functional IFN-γ receptor (IFN-γR) leads to tyrosine phosphorylation of the receptor and of STAT-1. Phosphorylated STAT-1 dimerizes and is then translocated into the nucleus where it binds to the GAS consensus sequence in promoters of IFN-γ-responsive genes and activates transcription. T. gondii interferes with the transcription of CIITA, IRF-1 and iNOS by altering the binding of STAT1 or the activity of the transcriptional mashinery. The reduced expression of IRF-1 in T. gondii-infected macrophages further inhibits CIITA and iNOS expression due to IRF-1-binding sites in the promoters of these genes. Together, this results in an inability of infected macrophages to express MHC II molecules and to produce NO (shaded area). See text for further details.

Interference with iNOS Expression and NO-Mediated Anti-Toxoplasma Activity Beside the presentation of antigens to T lymphocytes, macrophages also function as important effector cells that potentially exert toxoplasmastatic or even toxoplasmacidal activity. Depending on the host species, effector mechanisms include nitric oxide (NO) production by the inducible NO synthase (iNOS),17 production of reactive oxygen species (ROS),18 tryptophan starvation19 and disruption of T. gondii-containing parasitophorous vacuoles as well as possibly other mechanisms exerted by distinct members of the p47 GTPases family.20,21 IFN-γ and other proinflammatory cytokines, e.g., TNF-α are required to activate macrophages for such anti-parasitic activity.2,3 Despite the production of both cytokines during infection,22 the host is nevertheless not able to clear the infection. The molecular basis of this discrepancy may relate to recent findings that infection with T. gondii significantly inhibits activation-induced NO production by activated murine macrophages.23,24 As expected, this is achieved by parasitic interference with the transcription of the iNOS gene induced by activation with either IFN-γ, lipopolysaccharide or both (Fig. 1).24 Most

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importantly, intracellular parasite replication in moderately activated macrophages, i.e., those treated with IFN-γ alone or LPS alone clearly depends on the parasite’s ability to partially inhibit iNOS expression and NO production.24 In contrast, when macrophages are synergistically activated by a combination of IFN-γ plus LPS, T. gondii also partially inhibits iNOS and NO production, however, the level of such evasion does not suffice to allow parasite replication.24 These results suggest a scenario of a balanced interaction between T. gondii and macrophages during toxoplasmosis: the parasite-driven partial downregulation of NO production allows replication of T. gondii in moderately activated macrophages and this may facilitate parasite dissemination and establishment of infection. During prolonged acute infection and with the development of cell-mediated adaptive immunity, however, more efficiently activated macrophages acquire toxoplasmastatic activity thereby avoiding life-threatening parasite tissue burdens. Partial inhibition of iNOS expression as well as downregulation of the secretion of proinflammatory cytokines (chapter 8) may, nevertheless, still allow parasite survival and eventually differentiation to the latent and potentially persisting bradyzoite stage.25 Such a hypothesis is consistent with findings that iNOS expression is not essential for host resistance during acute infection by mice with T. gondii.26,27 While partial inhibition of iNOS facilitates parasite survival and establishment of infection, it may at the same time also avoid overwhelming immunopathology as indicated by oral infection of iNOS-deficient mice.28 Interference of T. gondii with iNOS expression thus appears beneficial for both parasite and its host and establishes such immune evasion as a crucial mechanism for the host-parasite equilibrium. The murine iNOS promoter contains binding sites for STAT-1, IRF-1 and NF-κB29-31 and transcription relies on activation of the JAK/STAT and/or the NF-κB signal transduction pathways. Although it has not been explicitly elucidated, it therefore appears most likely that T. gondii inhibits IFN-γ-induced iNOS expression by a mechanism similar to that described above for the interference with MHC class II expression (Fig. 1). Furthermore, LPS-triggered iNOS expression either alone or in synergy with IFN-γ may be inhibited by parasite-driven avoidance of the nuclear translocation of NF-κB32,33 and/or hijacking of STAT3 activation34 (see chapter 8). Interestingly, inhibition of the IFN-γ-induced gene expression has recently also been described for p47 GTPases21 thus suggesting that T. gondii is able to broadly inhibit the transcription of genes coding for toxoplasmastatic effector molecules in murine macrophages.

Modulation of Macrophage Apoptosis Apoptosis, i.e., programmed cell death plays essential roles in the regulation of immune responses and as innate as well as adaptive effector mechanism against intracellular pathogens. It occurs continuously during the physiological turnover of phagocytes, e.g., neutrophils and macrophages and regulates the adaptive immune response by the elimination of autoreactive T cells and by the termination of T cell responses to foreign antigens. After intraperitoneal infection of mice, T. gondii leads to a sustained viability of phagocytes, including macrophages, thereby triggering the inflammatory response to the parasite.35-37 Interestingly, apoptosis of peritoneal macrophages is particularly inhibited after infection with a T. gondii strain of low virulence and correlates with the ability of the infected mice to control the infection.35 In contrast, infection with highly virulent parasite strains leads to macrophage apoptosis, the inability to control the parasites and the hosts’ death.35 These results indicate that the T. gondii-mediated inhibition of macrophage (and granulocyte) apoptosis is an important determinant for the pathogenesis of disease. In order to kill intracellular pathogens, apoptosis can also act as an antimicrobial effector mechanism induced by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells via the release of perforin and granzymes38 or the engagement of death receptors, e.g., Fas/CD95 on the surface of target cells.39 Upon induction of cellular stress including microbial infection, cells can also altruistically commit ‘suicide’ and, thereby, combat the intracellular pathogen.40 During apoptosis, cells undergo biochemically and morphologically defined changes

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that ultimately lead to disintegration of the cell into membrane-bound apoptotic bodies which are removed without inducing an inflammatory response.41 Obviously, the death of the host cell would interrupt further development of T. gondii and may even lead to parasite killing, since parasite-containing vesicles are readily engulfed by phagocytes. This has put considerable selective pressure on the parasite to invent mechanisms which counteract apoptosis. T. gondii indeed blocks apoptosis that has been induced in different cell types including monocyte-like cell lines by a variety of proapoptotic stimuli.42-45 Such inhibition requires the presence of viable intracellular parasites42,43 which, however, do not necessarily have to replicate.43 Infection of the host cell with a single viable parasite thus suffices to inhibit host cell apoptosis indicating a direct and highly efficient mechanism to interfere with apoptosis-regulating cascades. Importantly, such evasion of the host cell’s protective suicide response appears also to occur in vivo, since after infection of mice, T. gondii-infected peritoneal macrophages are considerably protected from apoptosis.37 In addition, the number of parasites are substantially lower in the apoptotic population than in the nonapoptotic.37 Although definitive proof is still lacking, the above experiments indicate that upon infection, T. gondii counteracts apoptosis that would normally ensue from the cellular stress imposed by parasitic host cell infection. Furthermore, the parasite may thereby also evade cell death of infected target cells induced by CTL’s or NK cells via the induction of apoptosis. Consistent with this, Denkers et al46 showed that granule-mediated cytotoxicity plays only a minor role in resistance of mice against T. gondii. Direct and indirect mechanisms contribute to the parasitic inhibition of host cell apoptosis (Fig. 2). The diminished rate of cell death in inflammatory macrophages at least partially depends on the increased expression of the heat shock protein (HSP) 6535 and the anti-apoptotic Bcl-2 family member A1.37 HSP 65 and A1 are upregulated in both parasite-positive and parasite-negative macrophages indicating that this effect does not rely on a direct interaction of intracellular parasites with their host macrophages. It rather depends on the secretion of proinflammatory cytokines, e.g., IFN-γ and TNF-α35 as well as growth factors including granulocyte monocyte colony-stimulating factor (GM-CSF)36 by activated bystander cells. This may then lead to the activation of survival-promoting signaling pathways including the NF-κB,47,48 the Akt/PKB,49,50 the MAPK50,51 and the JAK/STAT50,52 pathways (Fig. 2). In addition to these indirect mechanisms which particularly function in the inhibition of apoptosis in uninfected inflammatory bystander cells, T. gondii also evolved mechanisms to interfere with apoptosis-regulating cascades in a more direct manner. Such a blockade of apoptosis is confined to parasite-positive cells and results from a T. gondii-mediated reduced activation of caspases, i.e., distinct cysteine proteinases (Fig. 2).44,45 Caspases constitute central elements of apoptotic pathways and integrate several upstream signals to a common executioner program. They are activated during apoptosis in a cascade-like fashion, cleave structural and regulatory target proteins and lead to the death and disintegration of the cell.41 As expected, the cleavage of prototypic target proteins of caspases and the fragmentation of DNA as observed during apoptosis are clearly inhibited after infection with T. gondii.44 In addition to the reduced cleavage, Goebel et al44 also reported a decrease in the total protein level of the poly(ADP-ribose) polymerase (PARP) in parasite-infected cells. Since PARP possesses proapoptotic properties under conditions of excessive activation,53 its downregulation after parasitic infection might contribute to the blockade of host cell apoptosis. However, further experiments are clearly required to confirm such a scenario and to elucidate the underlying mechanisms. Whereas the role of altered PARP expression awaits clarification, there is good evidence that the T. gondii-mediated inhibition of caspase activation involves interference with the apoptogenic function of mitochondria. Both intrinsic, e.g., cellular stress and - at least under certain conditions also - extrinsic proapoptotic signals, e.g., engagement of CD95/Fas or TNF-R154 leads to the activation of Bax-like members of the Bcl-2 family, thereby, disturbing the integrity of the outer mitochondrial membrane and leading to the release of cytochrome c into the cytosol.55 Cytosolic cytochrome c then catalyzes the formation of the apoptosome

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Figure 2. Direct and indirect mechanisms T. gondii for the inhibition of apoptosis in macrophages that harbour intracellular parasites or in uninfected inflammatory bystander cells. Infection of a host with T. gondii leads to the production of proinflammatory cytokines, e.g., IFN-γ and TNF-α as well as growth factors, e.g., GM-CSF and M-CSF. This activates signaling cascades, i.e., NF-κB, Akt/PKB, ERK’s and STAT’s leading to the transcription of genes encoding anti-apoptotic and pro-survival molecules. In contrast, intracellular infection with T. gondii may require additional direct mechanisms to inhibit host cell apoptosis. Such cell death may be induced by cellular stress, e.g., intracellular infection via a mitochondrial pathway, or by cytotoxic lymphocytes via the release of granzymes or activation of a Fas-mediated pathway. T. gondii broadly inhibits these pathways by the upregulation of anti-apoptotic molecules, e.g., Mcl-1, A1 and IAP’s, by blocking the release of cytochrome c from mitochondria and possibly other mechanisms. Consequently, caspase-mediated cleavage of target proteins and DNA fragmentation is inhibited in infected cells (shaded area).

which results in initiation of the caspase casade.55 In T. gondii-infected cells, however, the integrity of the host cell mitochondria and the mitochondrial cytochrome c localization after treament of cells with proapoptotic signals is preserved and this correlates with the protection of the host cell from apoptosis (Fig. 2).44 Parasite-induced upregulation of certain antiapoptotic Bcl-2 proteins, i.e., Mcl-144 and Bfl-1/A156 which are known to inhibit the activation of Bax-like proteins may contribute to the blockade of apoptosis in T. gondii-infected cells. Other mechanisms, however, probably participate in the inhibition of cytochrome c release after parasitic infection and await future clarification. Furthermore, direct inhibition of caspases by the parasite or by parasite-induced host cell caspase inhibitors, e.g., inhibitors of apoptosis (IAP)56 might also be involved in the broad antiapoptotic activity of T. gondii.

Concluding Remarks and Future Directions During recent years, considerable progress has been made in understanding the elaborate mechanisms by which T. gondii manipulates its host cells to establish long-lasting infections in

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the host. Importantly, the examples discussed above or in other chapters of this book provide evidence that T. gondii does not only interfere indirectly with macrophage functions, e.g., by the modulation of cytokine secretion by infected or uninfected bystander cells but rather directly exploits cellular signaling pathways and other regulatory components in order to make its host cell a safe niche. The future molecular elucidation of these fascinating interactions will thus also unravel hitherto unknown regulatory mechanisms of macrophage signaling pathways. More importantly, however, such direct parasite-host cell interactions might open new avenues for the therapy of toxoplasmosis. In contrast to the progress being made in characterising underlying mechanisms of these interactions on the host cell side, we still have very limited information on the parasite molecules which interfere with regulatory components of the host cell. Distinct excretory-secretory proteins of T. gondii are inserted into the membrane of the parasitophorous vacuole and have access to the host cell cytosol57 or are even translocated into the host cell cytosol.58 Although such molecules are prime candidates to interfere with host cell signaling cascades their exact functions are still unknown. Future work—possibly with the help of recent improvements in the analyses of excretory-secretory proteins from T. gondii59—has to address this largely unresolved problem in order to obtain a complete picture of these crucial parasite-macrophage interactions.

Acknowledgements The author gratefully acknowledges the financial support of his investigations by the Deutsche Forschungsgemeinschaft (LU 777/2-1, 2-2).

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39. Dockrell DH. The multiple roles of Fas ligand in the pathogenesis of infectious diseases. Clin Microbiol Infect 2003; 9(8):766-779. 40. Williams GT. Programmed cell death: A fundamental protective response to pathogens. Trends Microbiol 1994; 2(12):463-464. 41. Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407:770-776. 42. Nash PB, Purner MB, Leon RP et al. Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis. J Immunol 1998; 160:1824-1830. 43. Goebel S, Lüder CGK, Gross U. Invasion by Toxoplasma gondii protects human-derived HL-60 cells from actinomycin D-induced apoptosis. Med Microbiol Immunol 1999; 187:221-226. 44. Goebel S, Gross U, Lüder CGK. Inhibition of host cell apoptosis by Toxoplasma gondii is accompanied by reduced activation of the caspase cascade and alterations of poly(ADP-ribose) polymerase expression. J Cell Sci 2001; 114:3495-3505. 45. Payne TM, Molestina RE, Sinai AP. Inhibition of caspase activation and a requirement for NF-κB function in the Toxoplasma gondii-mediated blockade of host apoptosis. J Cell Sci 2003; 116:4345-4358. 46. Denkers EY, Yap G, Scharton-Kersten T et al. Perforin-mediated cytolysis plays a limited role in host resistance to Toxoplasma gondii. J Immunol 1997; 159:1903-1908. 47. Zhong WX, Edelstein LC, Chen C et al. The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-κB that blocks TNFα-induced apoptosis. Genes Develop 1999; 13:382-387. 48. Shapira S, Speirs K, Gerstein A et al. Suppression of NF-κB activation by infection with Toxoplasma gondii. J Infect Dis 2002; 185(Suppl 1):S66-S72. 49. Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med 2005; 9(1):59-71. 50. Channon JY, Miselis KA, Minns LA et al. Toxoplasma gondii induces granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor secretion by human fibroblasts: Implications for neutrophil apoptosis. Infect Immun 2002; 70(11):6048-6057. 51. Anderson P. Kinase cascades regulating entry into apoptosis. Microbiol Mol Biol Rev 1997; 61(1):33-46. 52. Bowman T, Garcia R, Turkson J et al. STATs in oncogenesis. Oncogene 2000; 19:2474-2488. 53. Tanaka Y, Yoshihara K, Tohno Y et al. Inhibition and downregulation of poly(ADP-ribose) polymerase results in a marked resistance of HL-60 cells to various apoptosis-inducers. Cell Mol Biol 1995; 41:771-781. 54. Scaffidi C, Schmitz I, Zha J et al. Differential modulation of apoptosis sensitivity in CD95 type I and type II cells. J Biol Chem 1999; 274(32):22532-22538. 55. Jiang X, Wang X. Cytochrome c-mediated apoptosis. Ann Rev Biochem 2004; 73:87-106. 56. Molestina RE, Payne M, Coppens I. Activation of NF-κB by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated IκB to the parasitophorous vacuole membrane. J Cell Sci 2003; 116:4359-4371. 57. Beckers CJ, Dubremetz JF, Mercereau-Puijalon O et al. The Toxoplasma gondii rhoptry protein ROP2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm. J Cell Biol 1994; 127:947-961. 58. Gubbels MJ, Striepen B, Shastri N et al. Class I major histocompatibility complex presentation of antigens that escape from the parasitophorous vacuole of Toxoplasma gondii. Infect Immun 2005; 73(2):703-711. 59. Bradley PJ, Ward C, Cheng SJ et al. Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii. J Biol Chem 2005; 280(40):34245-34258.

CHAPTER 11

Targeting SHP-1 to Prevent Macrophage Activation Promotes Leishmania Pathogenesis Devki Nandan* and Neil E. Reiner

Abstract

P

rotozoa of the genus Leishmania have evolved mechanisms to sabotage host-cell signaling pathways to enhance their intracellular survival and perpetuate infection. Recent findings have shown that the Src-homology-2 domain containing tyrosine phosphatase-1 (SHP-1) is targeted in Leishmania-infected cells. Activation of SHP-1 contributes to macrophage deactivation and to disease pathogenesis. Interference with macrophage cell regulation is a dynamic process and is mediated by Leishmania effector proteins and other molecules that target critical pathways. This chapter highlights the remarkable strategies employed by Leishmania to attenuate host-cell signaling and identifies some of the key molecules involved. Leishmania spp. are obligate intracellular protozoa that reside within mononuclear phagocytes of their mammalian hosts. They have two distinct developmental stages. These include nonmotile amastigotes that reside intracellularly within mammalian macrophages and extracellular motile promastigotes that multiply within the alimentary tract of their sandfly vectors. The human Leishmaniases span a diverse set of tropical and subtropical diseases. The majority of deaths result from the visceral form of Leishmaniasis caused by L. donovani or L. chagasi. Infections with Leishmania represent a major health problem of global importance. According to the latest WHO report, twelve million people are infected with various Leishmania species have clinical evidence of Leishmaniasis worldwide, and two million new cases occur each year (Leishmaniases Control, home page www.who.int/health-topics/Leishmaniasis.htm, updated 2000). Moreover, the incidence of Leishmaniasis has been on the rise because of the AIDS epidemic, increased international travel, lack of effective vaccines, difficulty in controlling vectors, and the development of resistance to chemotherapy.

Mechanism of Infection Humans become infected when a female sandfly deposits promastigotes into the dermal structures while taking a blood meal. These parasites are phagocytized by tissue macrophages and by other phagocytes recruited to the site of inoculation. Recently, it has been shown that saliva from the sandfly plays an important role in the initial stages of infection.1 Parasite surface macromolecules are also important in promoting adhesion to macrophages.2-5 Following phagocytosis and over a period of time, the promastigote-containing vacuole is converted into a phagolysosome, and the organism differentiates into a nonmotile amastigote.6 Initially, promastigotes are susceptible to lysis by the acidic and proteolytic environment of the *Corresponding Author: Devki Nandan—Department of Medicine, Division of Infectious Diseases, University of British Columbia, Rm 452D, 2733 Heather St., Vancouver, BC. Canada, V5Z 3J5. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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phagolysosome. In contrast, in the hostile environment of the phagolysosome, amastigotes grow and multiply, eventually lysing the macrophage thereby releasing themselves to infect other mononuclear phagocytes. Of interest, it has been shown that promastigotes delay phagosome maturation by retarding phagosome-lysosome fusion,7 providing a window of opportunity during which time they are able to transform into the relatively resistant amastigotes.

Mononuclear Phagocytes Phagocytes are important effectors and regulators of both the innate and acquired immune systems. In particular, they are programmed to respond rapidly as part of the innate immune response and can function without delay against invading microorganisms once the epithelial surface has been breached. Below the epithelial barrier are found resident tissue macrophages and in addition rapid recruitment of mononuclear phagocytes from the blood occurs as soon as the inflammatory response is initiated. The biochemical basis for the microbicidal activity of macrophages is complex and has conventionally been divided into two general categories: (i) oxygen dependent killing mechanisms mainly involving the generation of reactive oxygen intermediates or reactive nitrogen intermediates, and (ii) oxygen-independent killing mechanisms. The latter include reduced pH, limiting the availability of nutrients in the phagolysosome, neutral proteases and lysosomal hydrolases and the production of antimicrobial peptides.8,9 In effect, the environment within a mononuclear phagocyte is typically a hostile one where microbial replication and survival is curtailed.

Microbial Strategy in Relation to Phagocytes The central importance of phagocytes in host defense against microbial invasion was clearly recognized by Elie Metchnikoff over 100 years ago. Indeed, most microbes that encounter phagocytes are ingested and killed in short order. In contrast, a diverse group of successful pathogens have evolved strategies to either evade or subvert the effector functions of these cells. Thus, some pathogens have evolved the capacity to inhibit phagolysosome fusion.10-12 Other organisms may escape from the vacuole,13 divert the vacuole to a specialized compartment,14 or otherwise demonstrate resistance to killing by either oxygen-dependent or independent mechanisms or both. Interestingly, Leishmania spp survive and multiply inside macrophages through the adoption of several evasion strategies.

Host Defense against Leishmania As discussed above, in spite of the hostile environment of phagolysosomes, there are numerous microbial pathogens including Leishmania spp that are able to survive and replicate inside this compartment.15-17,18-25 This is a curious paradox of biology which is not fully understood. Most of what we know about host defense against Leishmania has come from studies of Leishmaniasis in mouse models. It has been established that NO and ROI (reactive oxygen intermediates) in macrophages play a critical role in parasite clearance.26,27 Mice lacking inducible nitric oxide synthase are unable to control infection,28 however, mice deficient for the generation of ROI do ultimately control infection, after an initial period of increased susceptibility. This has led to the suggestion that ROI play a limited role in parasite clearance in the mouse. The most important conclusion from murine studies is that mice that mount a T-helper type 1 adaptive immune response leading to macrophage activation show acquired resistance to Leishmania infection.29 In this context, interferon-γ (IFN-γ) plays a major role in Leishmania clearance. In contrast, the phenotype of mice mounting a T-helper type 2 response where IL-4 is the major cytokine produced is one of susceptibility to chronic infection with Leishmania.30

Leishmania and Macrophage Deactivation Leishmania have evolved mechanisms to inhibit numerous macrophage functions and to prevent cell activation to ensure their survival inside these cells. Below, we review examples of macrophage activities and properties that are attenuated by Leishmania.

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Suppression of Microbicidal NO and Superoxide Release The synthesis of NO by the inducible isoform of NO synthase (iNOS) is the most important mechanism known to be involved in leading to the resolution of Leishmania sp in in vitro and in vivo murine models. On the other hand, macrophages infected with Leishmania have limited ability to induce NO production in response to IFN-γ and lipopolysaccharides.31,32 This inhibition seems to be dependent on parasite-derived products. It has also been shown that ROI may be involved in mediating host resistance to Leishmania infection,26 and here again Leishmania have been observed to interfere with either the production or action of macrophage-derived ROI.33,34

Dynamics of Cytokine Production Influence the Outcome of Leishmania Infection The activation state of macrophages depends on ambient concentrations of both stimulatory and inhibitory cytokines, the production of which can be influenced by Leishmania. As mentioned above, a Th1 immune response is protective against Leishmania infection. Conversely, immunosuppressive cytokines such as IL-10, IL-4 and TGF-β inhibit Th1 protective immune response. Moreover, these cytokines are associated with macrophage deactivation.35,36 For example, inhibition of NO production has been associated with both IL-10 and TGF-β and it has been shown that increased amounts of these cytokines are produced by macrophages infected with Leishmania. For example, IL-10 is significantly upregulated in macrophages infected with Leishmania37-39 and IL-10 transgenic mice fail to control parasite loads following footpad administration of L. major as compared to control mice.40 Production of TGF-β in cells infected with Leishmania has also been shown to be upregulated in vitro and in vivo41,42 and Leishmania-infected mice treated with exogenous TGF-β showed increased numbers of parasites in their footpads.43 Evolution of a Th1 immune response is favored by the production of proinflammatory cytokines such as IL-12, IL-18, IL-1, tumor necrosis factor alpha (TNF-α) and IL-6. In addition to inducing the production of immunosuppressive cytokines, Leishmania has developed the capacity to infect macrophages without inducing the production of these proinflammatory cytokines. Furthermore, once infected, these cells show defective responses to various agents known to induce these cytokines. For example, IL-12 plays a critical role in positive regulation of Th1 responses and is also one of the most potent inducers of IFN-γ leading to macrophage activation and subsequent enhanced microbicidal activity. It is not surprising, therefore, that Leishmania have evolved mechanisms to inhibit IL-12 production.44-46

Modulation of Antigen Presentation In addition to providing a first line of defense against infection during the innate immune response, macrophages also play an important role in acquired immunity as antigen presenting cells. Activation of naive T cells requires recognition of foreign peptide in the context of appropriate MHC molecules. Peptide antigens derived from pathogens multiplying in intracellular vesicles are carried to the surface by MHC class II molecule and presented to CD4+ T cells that can differentiate into effector T cells. MHC class II molecules are expressed exclusively on the surface of professional antigen presenting cells—including macrophages, dendric cells, and B cells—where they activate CD4+ T cells, thereby initiating the release of cytokines that regulate immune responses. In addition to MHC class II molecules, antigen presenting cells also must express costimulatory signals for T cell activation to occur. The best characterized costimulatory molecules on antigen-presenting cells are the structurally related glycoproteins B7.1 and B7.2. A series of studies over the past decade or more have identified several mechanisms used by Leishmania to interfere with macrophage antigen presentation and activation of T cells. For example, early studies showed that upregulation of MHC class II molecule expression is attenuated in cells infected with Leishmania.47-50 Interference with antigen presentation has also been linked to sequestration of Leishmania antigens in an endocytic compartment and

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defective transport of MHC peptide complexes to the cell surface.51 Leishmania infection has also been associated with reduced expression of the costimulatory receptor B7-1, important for biasing Th1 immune cell activation.52

Disruption of Host Cell Signaling Pathways Cells constantly receive and interpret signals from their environment. In many cases, these signals continue to propagate within the cell, ultimately reaching the nucleus where they induce changes in gene expression. Signaling is initiated when a ligand or pathogen surface molecule interacts with a cell surface receptor leading either to phosphorylation of the receptor or a conformational change in the receptor, or both. This in turn leads to signal propagation distally which frequently involves activation of one or more serine/threonine, tyrosine or lipid kinases. Activation events involving phosphorylation often result in stimulation of an opposing phosphatase leading to signal attenuation and ultimately signal termination. Recent evidence suggests that diverse intracellular microbes including Leishmania,15-17,53 Yersinia,24 Mycobacteria,18-21,54 and the human immunodeficiency virus55 have evolved mechanisms to impair macrophage activation through effects on cell signaling. In what appears to be an effort to shut down macrophage microbicidal activities, Leishmania have been shown to inhibit the activation of several kinases. Some of the earliest studies in this field demonstrated that Leishmania avoided triggering the oxidative burst by inhibiting protein kinase C (PKC) activity. 34 In fact, the Leishmania surface macromolecule lipophosphoglycan (LPG) is a potent inhibitor of PKC. Glycosylinositolphospholipids,56 which represent the most abundant glycoconjugates of the amastigote stage, also show inhibitory activity towards PKC. Although in vivo mechanisms of inhibition of PKC are not fully elucidated, this attenuation may contribute to inhibition of macrophage microbicidal activity. Leishmania have been shown to affect other signaling pathways as well. Many important activation related functions of macrophages such as NO production and upregulation of MHC class II expression are IFN-γ inducible. Responses to IFN-γ require the Jak-Stat1 signaling pathway, making this a logical focus for targeting by Leishmania. In fact, it has been clearly demonstrated that macrophages infected with Leishmania show defective activation of Jak1, Jak2 and Stat1 in response to IFN-γ.57-59 Activation of members of the family of MAP kinases is also impaired in Leishmania infected macrophages. ERK1/2, p38 and JNK are elements in all three MAP kinase subfamilies and Leishmania appear to target all of these kinases activities.60-62 While there are a number of potential mechanisms that could explain these findings, activation of a phosphatase in infected cells is a particularly attractive one, given that it could explain targeting of multiple kinases simultaneously.

Leishmania-Induced Negative Signaling In principle, Leishmania could manipulate a variety of host regulatory nodes that inhibit intracellular signaling. An attractive negative regulatory molecule is the Src homology domain containing protein tyrosine phosphatase (PTP) SHP-1. This PTP is predominantly expressed in cells of hematopoietic origin but is also found in smooth muscle and epithelial cells. SHP-1 has tandem SH2 domains and a phosphatase domain. The SH2 domains recognize and bind to other signaling molecules which contain specific phosphotyroine residues. SHP-1 is involved in the negative regulation of many signaling pathways in most hematopoietic cell types.63-66 The majority of the effects of SHP-1 are indirect and are brought about by dephosphorylation of various protein tyrosine kinases involved in cell signaling pathways. SHP-1 is the major negative regulator of the Jak-Stat pathways including the IFN-γ associated pathway. Evidence accumulated over a decade or more has firmly established that macrophage SHP-1 plays an important role in the pathogenesis of infection with Leishmania. The finding of defective activation and tyrosine phosphorylation of Jaks and Stat1 in response to IFN-γ and impaired activation of MAP kinases in Leishmania-infected macrophages led to investigation of the role of SHP-1 in the pathogenesis of Leishmania infection. To begin with, these studies

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showed that the specific activity of SHP-1 was increased in Leishmania infected cells.67 Moreover, attenuation of MAP kinase activation and defective induction of iNOS expression in response to IFN-γ were both reversed by incubating cells with sodium orthovanadate.67 a PTP inhibitor, prior to infection. Consistent with these in vitro findings, administration of bis-peroxovanadium (also a PTP inhibitor) to mice infected with Leishmania reduced the burden of infection.68 These findings provided clear evidence that Leishmania attenuates host cell signaling by activating a cellular PTP. That activation of host SHP-1 is involved in the pathogenesis of Leishmania infection is also supported by two additional independent findings. First, SHP-1-deficient, viable motheaten mice showed a phenotype of increased resistance to Leishmania infection.69 Consistent with this, histochemical and in situ hybridization analyses of infected footpads from motheaten mice showed increased expression of iNOS as compared to infected wild type controls. Second, the anti-Leishmanial drug sodium stibogluconate which is used clinically, was found to be an inhibitor of SHP-1.70 A direct role for SHP-1 in negative signaling induced by Leishmania infection is also supported by the observation that infected macrophages showed markedly enhanced association of Jak2 with SHP-1.58 Taken together, these findings provide clear evidence that Leishmania attenuates host cell signaling by activating host SHP-1.

Mechanisms of SHP-1 Activation by Leishmania As discussed above, activation of macrophage SHP-1 plays an important role in the pathogenesis of Leishmania infection and may be responsible for the deactivated phenotype of Leishmania-infected macrophages. To search for an activator of SHP-1 from Leishmania, an affinity column of SHP-1 was used to identify interacting protein(s) from Leishmania lysates. This led to the identification of elongation factor 1α (EF-1α) as a candidate SHP-1 activator.71 This finding was clearly surprising given that EF-1α is a highly conserved, ubiquitously expressed protein in all eukaryotic organisms. EF-1α plays an essential role in eukaryotic protein biosynthesis.72,73 It is involved in polypeptide chain elongation and has GTPase activity. It binds aminoacyl-t-RNAs in a GTP dependent manner and then delivers these complexes to the A-site of the ribosome. GTP is hydrolyzed upon cognate codon–anticodon interaction between a tRNA and mRNA in the ribosome, after which an inactive EF-1α·GDP disassociates from ribosome. To initiate the next round of the elongation step, EF-1α must bind to a GDP/GTP exchange factor to recharge it with GTP. EF-1α usually is found in a heterotetrameric complex with other subunits involved in translation including βγδ. As EF-1α is more abundant than the other members of this complex, it has been inferred that EF-1α has other functions in addition to its role in translation. For instance, EF-1α is an actin binding protein. It has been suggested that EF-1α affects polymerization and stability of actin filaments and may regulate the length and stability of microtubules.73-76 EF-1α may also help integrate signal transduction with the cytoskeleton by being an activator of phosphatidylinositol-4 kinase.76 EF-1α from Trypanosoma brucei and rabbit reticulocytes has been shown to bind calmodulin and may play a role in Ca2+ signaling.74

Leishmania EF-1α Targets and Activates Macrophage SHP-1 Leading to Macrophage Deactivation The fact that EF-1α is both highly conserved and ubiquitously expressed raised obvious concerns about the specificity of the interaction of Leishmania EF-1α with SHP-1 as compared to macrophage EF-1α. This concern was addressed by examining the interactions of purified Leishmania and human EF-1α’s in vitro with SHP-1. Leishmania EF-1α was found to bind directly and selectively to host SHP-1 whereas human EF-1α did not. Subsequent experiments showed that Leishmania EF-1α activated host SHP-1 in vitro and in vivo, but the mammalian orthologue did not.71 Selective modulation of host SHP-1 by Leishmania EF-1α suggested that this Leishmania protein might be involved in deactivating infected host cells. To examine this directly, purified, native Leishmania EF-1α was introduced into macrophages after which

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Figure 1. Model of Leishmania EF-1α as an activator of SHP-1 and inducer of macrophage deactivation. Leishmania EF-1α accesses the cytosol of infected macrophages leading to targeting and activation of host protein tyrosine phosphatase SHP-1. This phosphatase activation negatively affects Jaks and MAP kinases, thus causing attenuation of IFN-γ-inducible macrophage functions like iNOS and c-FOS.

the cells were incubated with IFN-γ. This resulted in significant attenuation of iNOS induction, compared with cells that had been exposed to purified mammalian EF-1α.71 This finding demonstrated that Leishmania EF-1α is capable of recapitulating the deactivated phenotype of Leishmania infected macrophages (Fig. 1).

Leishmania EF-1α Accesses the Cytosol of Infected Cells SHP-1 is mainly a cytosolic protein, whereas Leishmania lives inside phagolysosomes. This raised an important question about accessibility of SHP-1 to Leishmania EF-1α. Scanning confocal immunofluorescence microscopy using antibodies specific to Leishmania EF-1α localized this protein to the cytosol of infected macrophages.71 Moreover, the finding of Leishmania EF-1α in the cytosol of infected cells was independently confirmed by coimmunoprecipitating Leishmania EF-1α with host SHP-1 from Leishmania infected cells.

Leishmania and Mammalian EF-1α’s Are Structurally Distinct Comparison of EF-1α of Leishmania with EF-1α of human or murine origin revealed several distinguishing features for the pathogen protein including: (i) a distinct sub-cellular distribution of Leishmania EF-1α in comparison to host EF-1α (ii) unique behavior on 2D Gel electrophoresis, (iii) the presence of antibodies in the sera of Leishmania infected humans that recognize pathogen, but not host EF-1α, and (iv) Leishmania EF-1α has a higher molecular mass than its mammalian counterpart, despite the fact that its amino acid sequence is significantly shorter.77 Protein structure modeling of Leishmania EF-1α based upon the crystal structure of EF-1α for Saccharomyces cerevisiae revealed important and previously unappreciated

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structural differences between host and Leishmania EF-1α. A hairpin loop of 12 amino acids was found that was unique to human and other mammalian EF-1α’s and deleted from the Leishmania orthologue.77 An important aspect of the missing hairpin loop in Leishmania EF-1α is the opportunity to design novel, small molecule inhibitors that bind specifically to the exposed region of the Leishmania protein otherwise shielded by the hairpin loop in the human protein. Given the essential role played by EF-1α in protein translation, this unique structural property of the Leishmania protein makes it a potentially highly attractive candidate for targeted drug development.

Summary and Conclusions In many ways Leishmaniasis provides an excellent example of immune evasion. Leishmania are efficiently internalized by phagocytic cells and delivered into phagolysosomes ostensibly to be killed. In spite of the hostile environment of phagolysosome, the pathogen replicates inside macrophages leading to chronic disease. Over the years, we have learned that Leishmania has evolved multiple evasion mechanisms for intracellular survival within macrophages. These strategies to exploit the host immune system are not unique to Leishmania. It is becoming increasingly clear that other intracellular pathogens like Yersinia, M. tuberculosis,19-21 human immunodeficiency virus and others have developed strategies similar to Leishmania to avoid killing by macrophages. One emerging common theme is that many intracellular pathogens including Leishmania have evolved to exploit the host cell signaling network. Since tyrosine phosphorylation is a critical regulatory mechanism in many cytokine signaling pathways, targeting protein tyrosine phosphatase like SHP-1 is an excellent choice by Leishmania to control critical regulatory proteins. The molecular and functional characterization of microbial virulence factors and examination of their effects on cell signaling components of the host is likely to provide new insights into pathogenesis. Moreover, it should provide novel targets for development of novel therapeutics.

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39. Belkaid Y, Hoffmann KF, Mendez S et al. The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure. J Exp Med 2001; 194(10):1497-1506. 40. Spath GF, Epstein L, Leader B et al. Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major. Proc Natl Acad Sci USA 2000; 97(16):9258-9263. 41. Barral-Netto M, Barral A, Brownell CE et al. Transforming growth factor-β in Leishmanial infection: A parasite escape mechanism. Science 1992; 257:545-548. 42. Wilson ME, Young BM, Davidson BL et al. The importance of TGF-β in murine visceral Leishmaniasis. J Immunol 1998; 161(11):6148-6155. 43. Li J, Hunter CA, Farrell JP. Anti-TGF-β treatment promotes rapid healing of Leishmania major infection in mice by enhancing in vivo nitric oxide production. J Immunol 1999; 162(2):974-979. 44. Carrera L, Gazzinelli RT, Badolato R et al. Leishmania promastigotes selectively inhibit interleukin 12 induction in bone marrow-derived macrophages from susceptible and resistant mice. J Exp Med 1996; 183:515-526. 45. Weinheber N, Wolfram M, Harbecke D et al. Phagocytosis of Leishmania mexicana amastigotes by macrophages leads to a sustained suppression of IL-12 production. Eur J Immunol 1998; 28(8):2467-2477. 46. Piedrafita D, Proudfoot L, Nikolaev AV et al. Regulation of macrophage IL-12 synthesis by Leishmania phosphoglycans. Eur J Immunol 1999; 29(1):235-244. 47. Reiner NE, Ng W, McMaster WR. Parasite-accessory cell interactions in murine Leishmaniasis. II. Leishmania donovani suppresses macrophage expression of class I and class II major histocompatibility gene products. J Immunol 1987; 138:1926-1932. 48. Antoine JC, Jouanne C, Lang T et al. Localization of major histocompatibility complex class II molecules in phagolysosomes of murine macrophages infected with Leishmania amazonensis. Infect Immun 1991; 59:764-769. 49. Lang T, De Chastellier C, Frehel C et al. Distribution of MHC class I and of MHC class II molecules in macrophages infected with Leishmania amazonensis. J Cell Sci 1994; 107:69-82. 50. Lang T, Hellio R, Kaye PM et al. Leishmania donovani-infected macrophages: Characterization of the parasitophorous vacuole and potential role of this organelle in antigen presentation. J Cell Sci 1994; 107(Pt 8):2137-2150. 51. Antoine JC, Prina E, Lang T et al. The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages. Trends Microbiol 1998; 6(10):392-401. 52. Probst P, Skeiky YA, Steeves M et al. A Leishmania protein that modulates interleukin (IL)-12, IL-10 and tumor necrosis factor-alpha production and expression of B7-1 in human monocyte-derived antigen-presenting cells. Eur J Immunol 1997; 27(10):2634-2642. 53. Nandan D, Knutson KL, Lo R et al. Exploitation of host cell signaling machinery: Activation of macrophage phosphotyrosine phosphatases as a novel mechanism of molecular microbial pathogenesis. J Leukocyte Biology 2000; 67(4):464-470. 54. Campbell K, Diao H, Ji J et al. DNA immunization with the gene encoding P4 nuclease of Leishmania amazonensis protects mice against cutaneous Leishmaniasis. Infect Immun 2003; 71(11):6270-6278. 55. Baldwin GC, Fleischmann J, Chung Y et al. Human immunodeficiency virus causes mononuclear phagocyte dysfunction. Proc Natl Acad Sci USA 1990; 87:3933-3937. 56. Turco SJ, Descoteaux A. The lipophosphoglycan of Leishmania parasites. Annu Rev Microbiol 1992; 46:65-94. 57. Nandan D, Reiner NE. Attenuation of γ-interferon-induced tyrosine phosphorylation in mononuclear phagocytes infected with Leishmania donovani: Selective inhibition of signaling through Janus kinases and Stat1. Infect Immun 1995; 63:4495-4500. 58. Blanchette J, Racette N, Faure R et al. Leishmania-induced increases in activation of macrophage SHP-1 tyrosine phosphatase are associated with impaired IFN-gamma-triggered JAK2 activation. Eur J Immunol 1999; 29(11):3737-3744. 59. Ray M, Gam AA, Boykins RA et al. Inhibition of interferon-gamma signaling by Leishmania donovani. J Infect Dis 2000; 181(3):1121-1128. 60. Prive C, Descoteaux A. Leishmania donovani promastigotes evade the activation of mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase-1/2 during infection of naive macrophages. Eur J Immunol 2000; 30(8):2235-2244. 61. Junghae M, Raynes JG. Activation of p38 mitogen-activated protein kinase attenuates Leishmania donovani infection in macrophages. Infect Immun 2002; 70(9):5026-5035.

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62. Awasthi A, Mathur R, Khan A et al. CD40 signaling is impaired in L. major-infected macrophages and is rescued by a p38MAPK activator establishing a host-protective memory T cell response. J Exp Med 2003; 197(8):1037-1043. 63. Plutzky J, Neel BG, Rosenberg RD. Isolation of a Src homology 2-containing tyrosine phosphatase. Proc Nat Acad Sci 1992; 89:1123-1127. 64. Yi T, Cleveland JL, Ihle JN. Protein tyrosine phosphatase containing SH2 domains: Characterization, preferential expression in haemopoietic cells, and localization to human chromosome 12p12p-p13. Mol Cell Biol 1992; 12:836-846. 65. Matthews RJ, Bowne DB, Flores E et al. Characterization of hematopoietic intracellular protein tyrosine phosphatases: Description of a phosphatase containing an SH2 domain and another enriched in proline-, glutamic acid-, serine-, and threonine-rich sequences. Mol Cell Biol 1992; 12:2396-2405. 66. Perkins LA, Larsen I, Perrimon N. Corkscrew encodes a putative protein tyrosine phosphatase that functions to transduce the terminal signal from the receptor tyrosine kinase torso. Cell 1992; 70:225-236. 67. Nandan D, Lo R, Reiner NE. Activation of phosphotyrosine phosphatase activity attenuates mitogen-activated protein kinase signaling and inhibits c-FOS and nitric oxide synthase expression in macrophages infected with Leishmania donovani. Infection and Immunity 1999; 67(8):4055-4063. 68. Olivier M, Romero-Gallo BJ, Matte C et al. Modulation of interferon-γ-induced macrophage activation by phosphotyrosine phosphatases inhibition - Effect on murine Leishmaniasis progression. J Biol Chem 1998; 273(22):13944-13949. 69. Forget G, Siminovitch KA, Brochu SRS et al. Role of host phosphotyrosine phosphatase SHP-1 in the development of murine Leishmaniasis. Eur J Immunol 2001; 31(11):3185-3196. 70. Pathak MK, Yi T. Sodium stibogluconate is a potent inhibitor of protein tyrosine phosphatases and augments cytokine responses in hemopoietic cell lines. J Immunol 2001; 167(6):3391-3397. 71. Nandan D, Yi T, Lopez M et al. Leishmania EF-1alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation. J Biol Chem 2002; 277(51):50190-50197. 72. Ryazanov AG, Rudkin BB, Spirin AS. Regulation of protein synthesis at the elongation stage: New insights into the control of gene expression in eukaryotes. FEBS Lett 1991; 285:170-175. 73. Condeelis J. Elongation factor 1 alpha, translation and the cytoskeleton. Trends Biochem Sci 1995; 20(5):169-170. 74. Kaur KJ, Ruben L. Protein translation elongation factor-1 alpha from Trypanosoma brucei binds calmodulin. J Biol Chem 1994; 269(37):23045-23050. 75. Murray JW, Edmonds BT, Liu G et al. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends. J Cell Biol 1996; 135(5):1309-1321. 76. Yang W, Boss WF. Regulation of phosphatidylinositol 4-kinase by the protein activator PIK-A49. Activation requires phosphorylation of PIK-A49. J Biol Chem 1994; 269(5):3852-3857. 77. Nandan D, Cherkasov A, Sabouti R et al. Molecular cloning, biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: Comparison with the mammalian homologue. Biochem Biophys Res Commun 2003; 302(4):646-652.

CHAPTER 12

Negative Signaling and Modulation of Macrophage Function in Trypanosoma cruzi Infection Flávia L. Ribeiro-Gomes, Marcela F. Lopes and George A. DosReis*

Abstract

M

acrophages serve either as host and primary effector cells against Trypanosoma cruzi, the protozoan parasite responsible for Chagas disease. Although the parasite mobilizes innate and adaptive immune responses that induce macrophage activation and keep infection under control, T. cruzi persists in the host for life. Parasite persistence is associated with inflammatory destruction of skeletal and cardiac muscle. Here, the roles of T. cruzi molecules and immune mechanisms involved in parasite evasion of macrophage defenses are discussed. Targeting these molecules and mechanisms will be essential for attaining successful therapies and vaccination.

Introduction Macrophages play important roles in Trypanosoma cruzi infection by serving as host and effector cells against the parasite. Macrophages are among the first cells to be parasitized by T. cruzi.1 Early studies demonstrated that T. cruzi multiplies in resident macrophages, leading to rupture of the host cell, and to the release of infective trypomastigote forms.2,3 However, it was soon apparent that macrophages could be instructed by the host immune response to become effector cells against the parasite. One central observation was that previous activation of macrophages by products released from activated lymphocytes - later identified as proinflammatory cytokines, including IFN-γ-induced microbicidal activity, conferring the ability to control infection.2,3 Macrophages play a protective role in T. cruzi infection in vivo. Macrophage depletion by treatment with silica increases parasitemia and rates of mortality in infected mice.4 However, macrophages could play a deleterious role, as well. Depletion of macrophages by treatment with clodronate liposomes resulted in decreased parasitism and damage to the central nervous system in suckling rats,5 suggesting that macrophages might serve to disseminate infection. These contrasting roles of macrophages could be explained by induction of functionally diverse pathways of macrophage activation.

Distinct Programs of Macrophage Activation Microbial products acting in concert with cytokines secreted by T-cell subsets influence the functional outcome of effector macrophage differentiation. At least three distinct phenotypes *Corresponding Author: George A. DosReis—Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ 21949-900, Brazil, and Institute for Investigation in Immunology (iii), Millenium Institutes, Brazil. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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have been identified. The best characterized cells are the classically activated macrophages (caMϕ), which are the effector cells of Th1 immune responses.6 caMϕ differentiate in the presence of two signals. One is IFN-γ, a major cytokine secreted by Th1 CD4+ T cells and CD8+ T cells. The second signal is TNF-α, which can be induced by microbial products acting as ligands for Toll-like receptors (TLRs). caMϕ produce nitric oxide (NO), increase expression of class II MHC and costimulatory CD86 molecules, and have increased antigen presenting capacity. caMϕ play an essential protective role against intracellular pathogens due to combined NO release and increased oxidative burst, as well as secretion of proinflammatory cytokines TNF-α, IL-1 and IL-6.7 Macrophages activated in a Th2 environment express different gene products, and were termed alternatively activated macrophages (aaMϕ). In contrast to caMϕ, aaMϕ fail to generate NO from L-arginine. Macrophages upregulate arginase activity following interaction with Th2 cytokines IL-4 and IL-10,8 or with the immuno-suppressive cytokine TGF-β.9 Arginase and the downstream enzyme ornithine decarboxylase (ODC) shift L-arginine metabolism towards production of polyamines, such as putrescine.10 Arginase activity is a marker of aaMϕ.7 aaMϕ also express increased class II MHC and CD86 expression, and efficiently present antigen to T cells.11 However, aaMϕ secrete antiinflammatory cytokines, such as IL-10 and TGF-β, and actively participate in reactions involving tissue remodelling, angiogenesis and wound repair during the healing phase of acute and chronic inflammatory diseases.7 Recently, it has been demonstrated that differentiation to caMϕ requires the enzyme src homology 2-containing inositol-5' phosphatase (SHIP), a potent inhibitor of phosphatidylinositol 3-kinase (PI3-kinase).12 Macrophages from SHIP-deficient mice differentiate into an aaMϕ phenotype in vivo, which requires the action of active TGF-β present in the serum.12 These studies will help understanding signaling pathways involved in distinct macrophage differentiation programs. Macrophages activated by immune complexes through ligation of FcγRs in the presence of a microbial TLR ligand such as bacterial lipopolysaccharide (LPS), downregulate IL-12 secretion and produce increased amounts of IL-10.6 These cells preferentially induce Th2 responses characterized by IL-4 secretion and antibodies of the IgG1 isotype, and were called Type 2-activated Mϕ because they are biased to induce a Th2 response.6 Type 2-activated Mϕ comprise a distinct subset sharing some properties with both caMϕ and aaMϕ. Type 2-activated Mϕ secrete TNF-α, IL-1 and IL-6, like caMϕ. However, they do not secrete IL-12, they do secrete IL-10, and drive a Th2 response, like aaMϕ. In contrast to aaMϕ, type 2-activated Mϕ do not express arginase activity.7 At present, it is unclear whether discrete activation states exist in vivo, or whether macrophages instead show a wide range of phenotypes, depending on multiple inflammatory stimuli.13 Monocyte and macrophage heterogeneity has been investigated by selective expression of cell surface markers, and this topic has been recently reviewed.13

Protective Mechanisms against T. cruzi Induced by IFN-γ The ability of caMϕ to mediate microbicidal activity against intracellular forms of T. cruzi depends on priming with IFN-γ and requires production of NO.14,15 Administration of IFN-γ reduced parasitemia and increased survival of mice infected with T. cruzi.16 Furthermore, mice receiving neutralizing antibody against IFN-γ, or deficient in IFN-γ Receptor had high parasitemias and succumbed from infection.17,18 These results indicate a central role for IFN-γ in resistance against infection. IFN-γ is produced by effector Th1 CD4+ T cells and by CD8+ T cells upon antigen recognition, and activates transcription of a large number of genes in macrophages. Ligation of IFN-γ Receptor by IFN-γ induces nuclear translocation of STAT1 to stimulate the transcription of IFN-regulated genes. Several products of IFN-regulated genes inhibit pathogen survival.19 Indoleamine 2,3-dioxygenase (IDO), nitric oxide synthase 2 (NOS2), and phagocyte oxidase (Phox) inhibit the survival of intracellular bacteria and protozoa. NOS2 is located in the cytosol, where it catalyses the production of NO, which rapidly diffuses to the phagosome to effect pathogen killing. Phox is found in plasma membrane,

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cytosol and intracellular granules, but translocates to the phagosome, where it catalyses the conversion of O2 to superoxide, which gives rise to molecules that are toxic to phagosome-bound pathogens. It has been suggested that NO combines with superoxide to generate peroxynitrite, which is more toxic to T. cruzi than NO.10 Several studies indicate that NO production by caMϕ is a major mechanism of defense against T. cruzi. However, one recent study argued that NO-independent mechanisms triggered by IFN-γ are important for resistance against T. cruzi, specially in infections mediated by less virulent parasite isolates.20 In this regard, IFN-γ induces expression of several GTP-binding proteins. A family of 47 kDa GTPases induced by IFN-γ has been implicated in intracellular defense against pathogens independently of NO.19 Although their mechanism of action is incompletely understood, p47 GTPases associate with lipid membranes and promote fusion of phagosomes harboring microbial pathogens with lysosomes.19 In addition, p47 GTPases regulate survival and function of memory-activated CD4+ T cells.19 Interestingly, IFN-γ induces autophagy, and the p47 GTPase LRG-47 is involved in autophagic killing of Mycobacteria by macrophages.21 p47 GTPases are important for resistance against intracellular parasites residing in phagosomes, such as Toxoplasma, Leishmania and Mycobacteria.19 Surprisingly, mice deficient in LRG-47 also express enhanced susceptibility to infection by T. cruzi,22 a pathogen that rapidly escapes the phagosome and enters the cytosol.1 In this model, LRG-47 appears to be required for proper control of host lymphocyte numbers, and for NO-independent killing of T. cruzi induced in macrophages by IFN-γ.22 The mechanism of parasite killing promoted by LRG-47 remains to be identified. Other potential mechanisms of protection mediated by IFN-γ could involve antigen processing and presentation, and lymphocyte migration to infection sites.20 These important issues also remain to be investigated.

Evasion of Innate Macrophage Defenses Microscopic examination of T. cruzi replication in resident and inflammatory macrophages illustrates how permissive these cells are for the parasite. Studies performed with nonprofessional phagocytes indicate that T. cruzi invades host cells by a mechanism resembling membrane wound repair. This mechanism involves sustained increases in cytosolic Ca2+ levels, leading to recruitment of lysosomes to the site of parasite attachment.23 The ability of parasites to trigger intracellular free Ca2+ transients in host cells is associated with the activity of a T. cruzi serine hydrolase, oligopeptidase B. Deletion of the gene encoding this enzyme results in a marked defect in host cell invasion and in the ability to infect mice.24 Endocytosis of the parasite is achieved through fusion of lysosomes with the plasma membrane, followed by membrane recycling. Recent studies suggest the existence of a second route of parasite invasion independent of targeted lysosome exocytosis, provided by tightly associated plasma membrane derived vacuoles.25 On the other hand, it has been suggested that T. cruzi invades professional phagocytes by a mechanism dependent on PI3-kinase and actin filament assembly, that resembles phagocytosis.26 Interestingly, recent studies have suggested that the lysosome dependent pathway of invasion of nonphagocytic cells also requires PI3-kinase and host cell actin polymerization.25,27 Both the parasite and the host cell rapidly initiate signaling pathways, allowing T. cruzi entry and intracellular survival. Similar to host cell signaling, penetration of T. cruzi into mammalian cells depends on the activation of the parasite protein tyrosine kinase activity and Ca2+ mobilization.28 Later on, the parasitophorous vacuole is lysed under the action of a T. cruzi protein named TcTox, that resembles C9 complement component, and the parasite is released into the host cell cytoplasm.29 Therefore, the parasite rapidly evades the hostile environment of the phagosome.30 Compared with other pathogenic parasites, T. cruzi invades host cells silently, eliciting few changes in host cell transcription during the first hours of infection.31 Once in the cytoplasm, the parasite differentiates into an amastigotes, replicates, and differentiates back to a trypomastigote. T. cruzi induces NF-kB activation in a number of cells which are relatively resistant to infection; but fails to do so in muscle cells, which are susceptible to invasion.32 It has been suggested that this cell type

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specific activation of NF-kB could explain the tissue tropism of T. cruzi.32 In addition, T. cruzi inhibits programmed cell death in certain mammalian cells, such as fibroblasts. The parasite posttranscriptionally upregulates expression of cellular FLICE inhibitory protein (c-FLIP); the only known mammalian inhibitor specific for death receptor signaling.33 In this way, the parasite prolongs host cell survival, allowing more time for completing its replicative cycle. Following rupture of the host cell, trypomastigotes are released to perpetuate the infective cycle. This infectious mechanism is facilitated by T. cruzi trans-sialidase,34 and requires TGF-β signaling,35 possibly to prevent harmful reactions of the host cell, and to provide essential cofactors and nutrients to the parasite. The exact nature of ligands and receptors critically involved in parasite invasion is unclear. However, invasion of host cells by T. cruzi appears to involve a complex array of host and parasite surface molecules. The extracellular matrix protein Fibronectin binds to T. cruzi trypomastigotes,36 and facilitates infection of fibroblasts and macrophages.36,37 In agreement, β1 integrins with avidity for fibronectin are involved in infection.38 Complement component C1q enhances invasion of phagocytes and fibroblasts by T. cruzi trypomastigotes opsonized wtih human serum.39 Interestingly, C1q also bridges apoptotic cells to calreticulin-CD91 complexes on phagocytes, promoting engulfment of apoptotic cells.40 Since engulfment of apoptotic cells inhibits proinflammatory properties of macrophages,41 it will be important to investigate whether trypomastigotes also engage calreticulin-CD91 complexes at the host cell. This strategy has been called “apoptotic mimicry” to indicate that parasites expose ligands similar to apoptotic cells in order to inactivate host macrophages and facilitate infection.42 The role of NO production at this early stage favors parasite infection. T. cruzi induces a modest and IFN-γ independent increase in NOS2 expression.43 Together with parasite NOS,10 parasitized cells produce low amounts of NO, which facilitates proliferation of amastigotes.43

Evasion of Activated Macrophages Once a specific immune response has developed, caMϕ which have differentiated through the IL-12/IFN-γ axis play a central role in control of parasitemia.44 In fact, caMϕ are not permissive to T. cruzi infection,2 and parasite killing can be triggered in these cells by autocrine TNF-α secretion.14,15 Microbicidal activity relies on large levels of NO production by highly expressed NOS2. Furthermore, in the presence of opsonizing antibodies, phagocytosis of T. cruzi engages FcγRs to stimulate peroxide production by Phox.10,45 Peroxide reacts with NO to generate peroxynitrite in vitro.10 It has been suggested that the combined activation of NOS2 and Phox could lead to peroxynitrite formation, accounting for more efficient microbicidal activity of caMϕ.10 Since infection by T. cruzi cannot be spontaneously cured, even in genetically resistant hosts, the parasite must be able to evade responses mounted by caMϕ. Similar to other parasites that replicate in macrophages, T. cruzi has evolved molecular mechanisms to escape the microbicidal arsenal of the activated phagocyte,30 or even to prevent phagocyte activation.46 In fact, macrophages infected with T. cruzi have been observed in inflammatory lesions of chronically infected mice.47 Some mechanisms of evasion could be intrinsic to the biology of the parasite. For example, the ability of T. cruzi to induce capping and shedding of antigen-antibody complexes from its surface,48 must be relevant to avoid FcγR engagement and peroxide formation upon invasion of caMϕ. In addition, some studies have suggested that macrophages infected with T. cruzi are defective in antigen presentation to CD4+ T cells. In one case, a deficit in protein catabolism and processing was reported.49 Another study did not find any defects in antigen processing or class II MHC expression, although physical interactions between macrophages and T cells were reduced.50 A deficit in antigen presentation could reduce IFN-γ secretion by interacting Th1 T cells, and failure to generate a caMϕ phenotype. However, it is unclear whether these alterations occur in vivo. Molecules produced by T. cruzi transduce signals to cells of the host immune system.51 Some of these molecules alert the organism of the presence of the parasite. Glycosylphospha tidylinositol

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(GPI)-anchored mucins from T. cruzi activate cytokine release in caMϕ by interacting with TLR2.52 A T. cruzi released protein related to oxidoreductase family, called Tc52, also activates macrophages and induces dendritic cell maturation via TLR2.53 On the other hand, other parasite molecules appear to have an immunoregulatory role that helps parasite evasion. AgC10 is a GPI-anchored mucin that inhibits macrophage secretion of TNF by inhibiting the activity of p38 MAPK.54 The cysteine proteinase cruzipain induces aaMϕ differentiation through TGF-β and IL-10 release.55 Glycoinositolphospholipid (GIPL) from T. cruzi exerts immunosuppressive effects and counteracts macrophage activation through its ceramide domain.56,57 Interestingly, pretreatment of macrophages with GPI-anchored T. cruzi mucins reduces subsequent cytokine responses to whole T. cruzi parasites. Furthermore, TLR2-deficient mice produce enhanced levels of cytokines following challenge with live parasites.58 These results suggest that GPI-anchored mucins, which bind to TLR2, 52 might have an immunoregulatory role by reducing proinflammatory reactions to the parasite. The immunoregulatory role of GPI-anchored mucins could be related to another phenomenon, known as LPS desensitization, which involves downregulation of TLR4 signaling.59 A recent study suggested that previous LPS desensitization also promotes increased replication of T. cruzi in peritoneal macrophages,60 suggesting that interference with TLR signaling compromises macrophage defenses against the parasite. Parasite molecules that subvert immune responses constitute an important obstacle for the development of effective vaccines. Table 1 lists T. cruzi molecules and mechanisms implicated in evasion of macrophage defenses. Mechanisms of evasion that modify macrophage activation have been investigated by immunological approaches. The antiinflammatory cytokines IL-10 and TGF-β could be important for evading caMϕ. Both IL-10 and TGF-β have deleterious effects that exacerbate infection in vivo.61,62 In addition, both IL-10 and TGF-β inhibit NO-dependent trypanocidal activity of macrophages primed by IFN-γ.15,62 TGF-β has potent deleterious effects in vivo, and increases parasitemia, even in animals that have received IFN-γ in vivo.62 Both IL-10 and TGFβ are secreted by aaMϕ,6,7 and can induce the differentiation of additional aaMϕ.8,9,12 Therefore, it is likely that T. cruzi evasion relies on generation of aaMϕ, which in turn could function as permissive host cells for the parasite. One mechanism for induction of aaMϕ is through secretion of IL-4 and IL-10 by Th2 T cells.8 In this regard, Th2-biased mice show exacerbated parasite loads and tissue inflammation, compared to control mice.63 Furthermore, even a residual Th2 response is necessary for both parasite persistence and tissue inflammation in T. cruzi infection.63 Humoral immunity dependent on Th2 T cells is necessary for protection against infection.64 Therefore, generation of aaMϕ could be an unavoidable by-product of the host humoral immune response. A mixed Th1/Th2 response arises during T. cruzi infection,63 but it is not clear how aaMϕ and Th2 T cells are generated. Certain molecules produced by the parasite could play an important role. Cruzipain, a cysteine proteinase produced by T. cruzi, induces a Th2 response in BALB mice.65 More importantly, cruzipain induces an alternatively activated phenotype in macrophages even in the absence of T cells, due to increased secretion of TGF-β and IL-10 by macrophages.55 Once differentiated, these aaMϕ supported increased intracellular replication of T. cruzi,55 indicating that aaMϕ are permissive hosts for the parasite. Cruzipain increases arginase activity and the subsequent T. cruzi growth by activation of tyrosine kinase, protein kinase A (PKA), and p38 mitogen activated protein kinase (MAPK) activities in macrophages.66 A second mechanism for induction of aaMϕ is through secretion of the antiinflammatory cytokine TGF-β.9,12 Secretion of TGFβ has been associated with resolution of inflammation. In fact, induction of aaMϕ is regarded as an intrinsic immunoregulatory response of the host, that counteracts potentially harmful Th1 immune responses.7 Acute infection with T. cruzi induces an early and potent Th1 response, which is drastically reduced once parasitemia is resolved.67 Usually, downregulation of Th1 responses results from apoptosis of effector Th1 T cells mediated by Fas/Fas ligand (FasL) interactions.68 Apoptosis of CD4+ T cells has been described during the acute phase of T. cruzi infection,69 and Fas/FasL interactions

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Table 1. Parasite molecules and immune mechanisms involved in evasion of macrophage defenses by T. cruzi Molecule / Mechanism in: Resident Mϕ

caMϕ

TcTox Trans-sialidase ? ? Ag/Ab capping and shedding GPI-mucins AgC10 (GPI-mucin) Cruzipain GIPLs

Th2 cells/IL-4/IL-10 Polyclonal lymphocyte activation; apoptosis; clearance of apoptotic cells

Proposed Mechanism

Refs.

Escape from phagosome Escape from phagosome TGF-β signaling Limited NO production by host cell and parasite Avoidance of FcγR engagement and oxidant generation TLR2 ligands; downregulation of proinflammatory response Downregulation of TNF production Arginase activity; TGF-β and IL-10; induction of aaMϕ Downregulation of TNF and costimulatory molecules through ceramide moiety Arginase activity; induction of aaMϕ Prostaglandin, TGF-β production; induction of aaMϕ

29 34 35 10,37 48 58 54 55,65,66 56,57

8,63 46,71,72

Note: Mechanisms that operate in resident Mϕ are likely to contribute to escape from activated Mϕ, as well, except for NO production.

are involved in death of T cells from infected hosts.70 Furthermore, removal of apoptotic cells by macrophages is antiinflammatory, leading to arrest of TNF-α production, and to TGF-β secretion.41 Therefore, it is possible that phagocytic burial of apoptotic lymphocytes induces aaMϕ differentiation, and helps survival and replication of parasites inside TGF-β producing macrophages. To test this hypothesis, macrophages infected with T. cruzi were cocultured with CD4+ T cells from infected mice. When T cells were killed by agents that increase FasL expression, or by an agonist anti-Fas antibody, parasite replication inside macrophages was exacerbated.71 This deleterious effect required physical interaction between CD4+ T cells and infected macrophages,71 suggesting that phagocytosis of dead lymphocytes drives T. cruzi replication inside macrophages. In fact, apoptotic but not necrotic lymphocytes exacerbated T. cruzi replication in macrophages.72 The integrin αVβ3 (vitronectin receptor) plays a critical role in phagocytosis of apoptotic cells.73 In agreement, both binding of apoptotic lymphocytes, and increased T. cruzi replication could be blocked by an antagonist anti-αV Fab fragment, and parasite growth could be exacerbated by intact anti-αV antibodies in the absence of apoptotic cells.72 Parasite replication driven by apoptotic cells or by αVβ3 engagement were dependent on PGE2 and TGF-β production.72 Furthermore, PGE2 and TGF-β upregulated ODC activity and polyamine production. Increased parasite growth relied on putrescine production.72 Induction of protracted ODC activity, an enzyme placed downstream to arginase, strongly suggests that apoptotic cell ingestion triggers aaMϕ differentiation, and that this was accomplished through delayed effects of TGF-β.72 Furthermore, addition of apoptotic cells reverted the trypanocidal effect of Mϕ treated with LPS plus IFN-γ, resulting in increased parasite replication.72 Therefore, interaction with apoptotic

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Figure 1. Clearance of apoptotic cells shifts arginine metabolism and drives parasite growth in macrophages. Left, in the absence of apoptotic cells, infected macrophages interact with parasite-specific Th1 T cells, leading to IFN-γ production, and induction of NOS2 activity. NOS2 produces NO from L-arginine, mediating parasite killing. Right, in the presence of apoptotic cells, apoptotic bodies engage phagocytic receptors that trigger TGF-β release and disable signals from IFN-γ. TGF-β induces arginase and ODC activities. Arginase produces ornithine from L-arginine and downregulates NO production. ODC activity leads to putrescine production, which fuels intracellular parasite replication. Infected macrophages release infective trypomastigote forms.

cells is an efficient way to prevent caMϕ differentiation. In agreement, injection of apoptotic cells increased and accelerated parasitemia in infected mice.72 During the acute phase of infection, the parasite induces intense polyclonal lymphocyte activation that resolves by lymphocyte apoptosis. T. cruzi exploits apoptotic cell clearance and the associated shift in macrophage L-arginine metabolism to replicate inside permissive macrophages.74 Figure 1 shows the opposing outcomes of T. cruzi replication in macrophages, during infection in the absence or in the presence of apoptotic cells.

Prospects for the Future The biochemical pathway that links apoptotic cell removal to intracellular parasite replication provides new targets for therapeutic intervention. For example, treatment of infected mice with blockers of cyclooxygenase,72 or with zVAD-fmk, a pan-caspase inhibitor that functions as a general blocker of apoptosis (Lopes MF and DosReis GA, unpublished results), markedly reduces parasitemia in infected mice. In addition, polyamine production is another potential target for chemotherapic intervention against protozoal parasites.75 T. cruzi is unable to synthesize putrescine and is dependent on uptake of exogenous polyamines by high affinity transporters.76 Polyamines are essential for T. cruzi survival and division.

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Polyamine analogs block T. cruzi intracellular replication in vitro.77 Furthermore, T. cruzi conjugates glutathione with the polyamine spermidine to synthesize trypanothione, a critical antioxidant molecule and a potential target for chemotherapy.76 Finally, clearance of apoptotic cells could be involved in tissue fibrosis in situations of exagerated or sustained apoptosis. It has been reported that TGF-β shifts L-arginine metabolism, leading to both polyamine and collagen synthesis.78 Together, the results indicate that Th2 cytokines and the clearance of apoptotic cells induce aaMϕ, which serve as safe host cells for parasite replication and spread. Since aaMϕ upregulate the arginase/ODC/polyamine axis, this biochemical cascade is a promising target for therapies aimed at reducing parasite replication, and fibrogenesis secondary to inflammation.

Acknowledgements Authors’ cited work was financed by Brazilian National Research Council (CNPq), Rio de Janeiro State Science Foundation (FAPERJ), and Howard Hughes Medical Institute (HHMI). FLRG is a post-doctoral fellow from FAPERJ. MFL and GADR are investigators from CNPq. GADR is a Howard Hughes International Research Scholar.

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18. Holscher C, Kohler G, Muller U et al. Defective nitric oxide effector functions lead to extreme susceptibility of Trypanosoma cruzi-infected mice deficient in gamma interferon receptor or inducible nitric oxide synthase. Infect Immun 1998; 66:1208-1215. 19. Taylor GA, Feng CG, Sher A. p47 GTPases: Regulators of immunity to intracellular pathogens. Nat Rev Immunol 2004; 4:100-109. 20. Cummings KL, Tarleton RL. Inducible nitric oxide synthase is not essential for control of Trypanosoma cruzi infection in mice. Infect Immun 2004; 72:4081-4089. 21. Gutierrez MG, Master SS, Singh SB et al. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 2004; 119:753-766. 22. Santiago HC, Feng CG, Bafica A et al. Mice deficient in LRG-47 display enhanced susceptibility to Trypanosoma cruzi infection associated with defective hemopoiesis and intracellular control of parasite growth. J Immunol 2005; 175:8165-8172. 23. Andrews NW. Lysosomes and the plasma membrane: Trypanosomes reveal a secret relationship. J Cell Biol 2002; 158:389-394. 24. Caler EV, Vaena de Avalos S, Haynes PA et al. Oligopeptidase B-dependent signaling mediates host cell invasion by Trypanosoma cruzi. EMBO J 1998; 17:4975-4986. 25. Woolsey AM, Sunwoo L, Petersen CA et al. Novel PI3-kinase-dependent mechanisms of trypanosome invasion and vacuole maturation. J Cell Sci 2003; 116:3611-3622. 26. Vieira M, Dutra JM, Carvalho TM et al. Cellular signaling during the macrophage invasion by Trypanosoma cruzi. Histochem Cell Biol 2002; 118:491-500. 27. Woolsey AM, Burleigh BA. Host cell actin polymerization is required for cellular retention of Trypanosoma cruzi and early association with endosomal/lysosomal compartments. Cell Microbiol 2004; 6:829-838. 28. Yoshida N, Favoreto Jr S, Ferreira AT et al. Signal transduction induced in Trypanosoma cruzi metacyclic trypomastigotes during the invasion of mammalian cells. Braz J Med Biol Res 2000; 33:269-278. 29. Andrews NW, Abrams CK, Slatin SL et al. A T. cruzi-secreted protein immunologically related to the complement component C9: Evidence for membrane pore-forming activity at low pH. Cell 1990; 61:1277-1287. 30. Denkers EY, Butcher BA. Sabotage and exploitation in macrophages parasitized by intracellular protozoans. Trends Parasitol 2005; 21:35-41. 31. Vaena de Avalos S, Blader IJ, Fisher M et al. Immediate/early response to Trypanosoma cruzi infection involves minimal modulation of host cell transcription. J Biol Chem 2002; 277:639-644. 32. Hall BS, Tam W, Sen R et al. Cell-specific activation of nuclear factor-kappaB by the parasite Trypanosoma cruzi promotes resistance to intracellular infection. Mol Biol Cell 2000; 11:153-160. 33. Hashimoto M, Nakajima-Shimada J, Aoki T. Trypanosoma cruzi posttranscriptionally upregulates and exploits cellular FLIP for inhibition of death-inducing signal. Mol Biol Cell 2005; 16:3521-3528. 34. Hall BF, Webster P, Ma AK, et al. Desialylation of lysosomal membrane glycoproteins by Trypanosoma cruzi: A role for the surface neuraminidase in facilitating parasite entry into the host cell cytoplasm. J Exp Med 1992; 176:313-325. 35. Ming M, Ewen ME, Pereira ME. Trypanosome invasion of mammalian cells requires activation of the TGF beta signaling pathway. Cell 1995; 82:287-296. 36. Ouaissi MA, Afchain D, Capron A et al. Fibronectin receptors on Trypanosoma cruzi trypomastigotes and their biological function. Nature 1984; 308:380-382. 37. Wirth JJ, Kierszenbaum F. Fibronectin enhances macrophage association with invasive forms of Trypanosoma cruzi. J Immunol 1984; 133:460-464. 38. Fernandez MA, Munoz-Fernandez MA, Fresno M. Involvement of beta 1 integrins in the binding and entry of Trypanosoma cruzi into human macrophages. Eur J Immunol 1993; 23:552-557. 39. Rimoldi MT, Tenner AJ, Bobak DA et al. Complement component C1q enhances invasion of human mononuclear phagocytes and fibroblasts by Trypanosoma cruzi trypomastigotes. J Clin Invest 1989; 84:1982-1989. 40. Ogden CA, deCathelineau A, Hoffmann PR et al. C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells. J Exp Med 2001; 194:781-795. 41. Fadok VA, Bratton DL, Konowal A et al. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE-2 and PAF. J Clin Invest 1998; 101:890-898. 42. de Freitas Balanco JM, Moreira ME, Bonomo A et al. Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity. Curr Biol 2001; 11:1870-1873.

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43. Rottenberg ME, Castanos-Velez E, de Mesquita R et al. Intracellular colocalization of Trypanosoma cruzi and inducible nitric oxide synthase (iNOS): Evidence for dual pathway of iNOS induction. Eur J Immunol 1996; 26:3203-3213. 44. Michailowsky V, Silva NM, Rocha CD et al. Pivotal role of interleukin-12 and interferon-gamma axis in controlling tissue parasitism and inflammation in the heart and central nervous system during Trypanosoma cruzi infection. Am J Pathol 2001; 159:1723-1733. 45. Cardoni RL, Antunez MI, Morales C et al. Release of reactive oxygen species by phagocytic cells in response to live parasites in mice infected with Trypanosoma cruzi. Am J Trop Med Hyg 1997; 56:329-334. 46. Lopes MF, Freire-de-Lima CG, DosReis GA. The macrophage haunted by cell ghosts: A pathogen grows. Immunol Today 2000; 21:489-494. 47. Andrade SG, Grimaud JA. Chronic murine myocarditis due to Trypanosoma cruzi; an ultrastructural study and immunochemical characterization of cardiac interstitial matrix. Mem Inst Oswaldo Cruz 1986; 81:29-41. 48. Schmunis GA, Szarfman A, Langembach T et al. Induction of capping in blood-stage trypomastigotes of Trypanosoma cruzi by human anti-Trypanosoma cruzi antibodies. Infect Immun 1978; 20:567-569. 49. Plasman N, Guillet JG, Vray B. Impaired protein catabolism in Trypanosoma cruzi-infected macrophages: Possible involvement in antigen presentation. Immunology 1995; 86:636-645. 50. La Flamme AC, Kahn SJ, Rudensky AY et al. Trypanosoma cruzi-infected macrophages are defective in major histocompatibility complex class II antigen presentation. Eur J Immunol 1997; 27:3085-3094. 51. DosReis GA, Freire-de-Lima CG, Nunes MP et al. The importance of aberrant T-cell responses in Chagas disease. Trends Parasitol 2005; 21:237-243. 52. Campos MA, Closel M, Valente EP et al. Impaired production of proinflammatory cytokines and host resistance to acute infection with Trypanosoma cruzi in mice lacking functional myeloid differentiation factor 88. J Immunol 2004; 172:1711-1718. 53. Ouaissi A, Guilvard E, Delneste Y et al. The Trypanosoma cruzi Tc52-released protein induces human dendritic cell maturation, signals via Toll-like receptor 2, and confers protection against lethal infection. J Immunol 2002; 168:6366-6374. 54. Alcaide P, Fresno M. AgC10, a mucin from Trypanosoma cruzi, destabilizes TNF and cyclooxygenase-2 mRNA by inhibiting mitogen-activated protein kinase p38. Eur J Immunol 2004; 34:1695-1704. 55. Stempin C, Giordanengo L, Gea S et al. Alternative activation and increase of Trypanosoma cruzi survival in murine macrophages stimulated by cruzipain, a parasite antigen. J Leukoc Biol 2002; 72:727-734. 56. DosReis GA, Pecanha LM, Bellio M et al. Glycoinositol phospholipids from Trypanosoma cruzi transmit signals to the cells of the host immune system through both ceramide and glycan chains. Microbes Infect 2002; 4:1007-1013. 57. Brodskyn C, Patricio J, Oliveira R et al. Glycoinositolphospholipids from Trypanosoma cruzi interfere with macrophages and dendritic cell responses. Infect Immun 2002; 70:3736-3743. 58. Ropert C, Gazzinelli RT. Regulatory role of Toll-like receptor 2 during infection with Trypanosoma cruzi. J Endotoxin Res 2004; 10:425-430. 59. Nomura F, Akashi S, Sakao Y et al. Cutting edge: Endotoxin tolerance in mouse peritoneal macrophages correlates with downregulation of surface toll-like receptor 4 expression. J Immunol 2000; 164:3476-3479. 60. Perez AR, Tamae-Kakazu M, Pascutti MF et al. Deficient control of Trypanosoma cruzi infection in C57BL/6 mice is related to a delayed specific IgG response and increased macrophage production of pro-inflammatory cytokines. Life Sci 2005; 77:1945-1959. 61. Reed SG, Brownell CE, Russo DM et al. IL-10 mediates susceptibility to Trypanosoma cruzi infection. J Immunol 1994; 153:3135-3140. 62. Silva JS, Twardzik DR, Reed SG. Regulation of Trypanosoma cruzi infections in vitro and in vivo by transforming growth factor beta (TGF-beta). J Exp Med 1991; 174:539-545. 63. Tarleton RL, Grusby MJ, Zhang L. Increased susceptibility of Stat4-deficient and enhanced resistance in Stat6-deficient mice to infection with Trypanosoma cruzi. J Immunol 2000; 165:1520-1525. 64. Kumar S, Tarleton RL. The relative contribution of antibody production and CD8+ T cell function to immune control of Trypanosoma cruzi. Parasite Immunol 1998; 20:207-216. 65. Guinazu N, Pellegrini A, Giordanengo L et al. Immune response to a major Trypanosoma cruzi antigen, cruzipain, is differentially modulated in C57BL/6 and BALB/c mice. Microbes Infect 2004; 6:1250-1258.

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66. Stempin CC, Tanos TB, Coso OA et al. Arginase induction promotes Trypanosoma cruzi intracellular replication in cruzipain-treated J774 cells through the activation of multiple signaling pathways. Eur J Immunol 2004; 34:200-209. 67. Zhang L, Tarleton RL. Characterization of cytokine production in murine Trypanosoma cruzi infection by in situ immunocytochemistry: Lack of association btween susceptibility and type 2 cytokine production. Eur J Immunol 1996; 26:102-109. 68. Krammer PH. CD95’s deadly mission in the immune system. Nature 2000; 407:789-795. 69. Lopes MF, da Veiga VF, Santos AR et al. Activation-induced CD4+ T cell death by apoptosis in experimental Chagas’ disease. J Immunol 1995; 154:744-752. 70. Lopes MF, Nunes MP, Henriques-Pons A et al. Increased susceptibility of Fas ligand-deficient gld mice to Trypanosoma cruzi infection due to a Th2-biased host immune response. Eur J Immunol 1999; 29:81-89. 71. Nunes MP, Andrade RM, Lopes MF et al. Activation-induced T cell death exacerbates Trypanosoma cruzi replication in macrophages cocultured with CD4+ T lymphocytes from infected hosts. J Immunol 1998; 160:1313-1319. 72. Freire-de-Lima CG, Nascimento DO, Soares MB et al. Uptake of apoptotic cells drives the growth of a pathogenic trypanosome in macrophages. Nature 2000; 403:199-203. 73. Savill J, Dransfield I, Hogg N et al. Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature 1990; 343:170-173. 74. DosReis GA, Barcinski MA. Apoptosis and parasitism: From the parasite to the host immune response. Adv Parasitol 2001; 49:133-161. 75. Muller S, Coombs GH, Walter RD. Targeting polyamines of parasitic protozoa in chemotherapy. Trends Parasitol 2001; 17:242-249. 76. Ariyanayagam MR, Oza SL, Mehlert A et al. Bis(glutathionyl)spermine and other novel trypanothione analogues in Trypanosoma cruzi. J Biol Chem 2003; 278:27612-27619. 77. Majumder S, Kierszenbaum F. Inhibition of host cell invasion and intracellular replication of Trypanosoma cruzi by N,N’-bis(benzyl)-substituted polyamine analogs. Antimicrob Agents Chemother 1993; 37:2235-2238. 78. Durante W, Liao L, Reyna SV et al. Transforming growth factor-beta 1 stimulates L-arginine transport and metabolism in vascular smooth muscle cells: Role in polyamine and collagen synthesis. Circulation 2001; 103:1121-1127.

CHAPTER 13

Effector Functions of Macrophages in Plasmodium Parasite Infections Mariela Segura, Rebecca Ing, Zhong Su, Neeta Thawani and Mary M. Stevenson*

Abstract

M

alaria, due to infection with protozoan parasites of the genus Plasmodium, is a major cause of high morbidity and mortality worldwide, especially in sub-Saharan Africa. The multiplication of Plasmodium parasites in host red blood cells (RBC) during the asexual blood stage results in the severe symptoms associated with malaria, including anemia, lactic acidosis and cerebral malaria. Immunity to blood stage malaria is complex and must be finely balanced to achieve protection of the host but prevent the development of pathology that is immunologically mediated. Innate and adaptive immune mechanisms are involved in protective immunity to Plasmodium parasites, and a network of proinflammatory cytokines, especially interferon (IFN)-γ, and a variety of cell types including dendritic cells (DCs), natural killer (NK) cells, CD4+ T cells and B cells as well as macrophages play important roles. These cell types act in concert to generate a type 1 antibody-dependent immune response, which mediates control of parasite replication and clearance. The focus of this review is how Plasmodium-infected RBC interact with macrophages and other antigen presenting cells (APCs), such as DCs, and how such interactions affect the host immune response to Plasmodium infection and influence the outcome of malaria. Data are summarized from studies in humans and mice and evidence from both in vitro and in vivo studies are discussed. Information derived from studies on the interaction between Plasmodium parasites and macrophages will likely contribute to the development of an effective malaria vaccine and improved immunotherapies.

Introduction Malaria, caused by protozoan parasites of the genus Plasmodium (Table 1), is a major infectious disease that results in more than 300 million infections and 1-3 million deaths per year. The life cycle of Plasmodium parasites is complex (reviewed in ref. 1). Transmitted during a bite of an infected female Anopheles mosquito, Plasmodium sporozoites are deposited in the skin of the host and migrate to the liver where they infect and develop in hepatocytes into merozoites (Fig. 1). The liver stage, also called the exo-erythocytic (or preerythrocytic) stage, is typically asymptomatic. During the blood stage, merozoites infect red blood cells (RBC) wherein they develop into ring-shaped trophozoites, late-stage schizonts, and new merozoites which infect other RBC, thus propagating the infection. With each replication cycle, the destruction of *Corresponding Author: Mary M. Stevenson—Centre for the Study of Host Resistance, Research Institute of the McGill University Health Centre, Montreal General Hospital, Room L11-409, 1650 Cedar Avenue, Montreal, Quebec, Canada H3G 1A4. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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Table 1. Description of Plasmodium species in humans and mouse models Plasmodium Species In humans: Plasmodium falciparum

Plasmodium vivax

Plasmodium malariae

Plasmodium ovale

In mouse: Plasmodium chabaudi

Plasmodium berghei

Plasmodium yoelii

Plasmodium vinckei

Comments*

Causes the most severe form of malaria and can be fatal. Can cause chronic infections (up to 2-3 years), but does not form hypnozoites (dormant stages that persist in hepatocytes) and does not relapse. A common cause of severe, acute febrile illness, but is rarely fatal. Distribution is restricted by the absence of Duffy antigen (which determines entry into red blood cells) in African populations. This parasite forms hypnozoites and might relapse many years after apparent cure. Infrequent cause of clinical malaria, especially in Africa. Induces minor disease, although untreated infections can persist as low-grade parasitaemia for several decades. Infrequent cause of mild-moderate clinical malaria, but might be found in mixed infections with other species. Forms hypnozoites and might relapse. Both P. chabaudi chabaudi AS and P. chabaudi adami are commonly used to study immune mechanisms and immunoregulation by cytokines, to identify susceptibility loci, and to study the immunological basis of pathology. P. chabaudi chabaudi AS causes nonlethal infection in resistant mouse strains and lethal infection in susceptible mouse strains. P. chabaudi adami causes a mild, nonlethal infection. P. berghei ANKA and P. berghei K173 are widely used to study pathogenesis. P. berghei ANKA serves as a model of experimental cerebral malaria (ECM); the development of ECM varies among inbred mouse strains and this genetic variation correlates with the production of pro-inflammatory cytokines. P. yoelii 17XL, P. yoelii 17XNL and P. yoelii YM are used to study immune mechanisms and pathogenesis, including ECM. As recombinant merozoite surface protein 1 (MSP1) is available, P. yoelii 17XL is often used to elucidate vaccine-induced immune responses. P. vinckei vinckei, which causes a lethal infection, is used to study pathogenesis and for chemotherapy studies; P. vinckei petteri, which causes a nonlethal infection, is used to study immune mechanisms.

*Adapted from reference 2.

infected RBC (iRBC) and release of parasite waste products result in the episodic chills, headaches and fever that characterize the disease. Malaria parasites can multiply eight-fold every two days, resulting in infection of a high proportion of RBC within a short time. As yet unknown factors trigger a subset of merozoites to differentiate into gametocytes, which are taken up by a feeding mosquito. In the mosquito mid-gut, the gametocytes mature and fuse to form gametes which develop into sporozoites that can be transmitted during the next blood meal. Innate immune responses are important for the control of parasitemia in infected humans and experimental mice. Cells that provide the first line of host defense, including macrophages, play key roles in immune protection against both initial infection and reinfection with different parasite variants. Indeed, the generation and maintenance of clinically protective immune responses requires repeated infections over the lifetime of the individual.2-4 During the blood stage, highly variable parasite-derived proteins are expressed on the surface of iRBC. These

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Figure 1. The life cycle of Plasmodium falciparum in the human host and mosquito vector. Sporozoites injected during a mosquito bite are carried via the blood to the liver, where they invade hepatocytes and undergo a process of asexual replication to give rise to exoerythrocytic schizonts. After about seven days, the liver schizonts rupture to release many thousands of merozoites into the blood. Merozoites invade red blood cells (RBC) and develop into ring-formed trophozoites, which divide mitotically into erythrocytic schizonts that may contain up to 20 daughter merozoites. These merozoites can reinfect new RBC, resulting in cyclical blood-stage infection with a periodicity of 48-72 h, depending on the Plasmodium species. As yet unknown factors trigger a subset of developing merozoites to differentiate into male and female gametocytes, which are taken up by a feeding mosquito and therein develop into extracellular gametes. In the mosquito mid-gut, the gametes fuse to form a motile zygote (ookinete), which penetrates the mid-gut wall and forms an oocyst that develop into sporozoites. Reprinted with permission from Stevenson MM, Riley EM. Nat Rev Immunol 2004; 4:169-180.2

proteins continually undergo antigenic variation to evade detection by the host’s immune system, mediate sequestration of parasites, and can activate and modulate immune responses. Here, we discuss how iRBC interact with macrophages and other APCs, such as dendritic cells (DCs), and how such interactions affect innate and adaptive immune responses to Plasmodium infection.

The Role of the Spleen in Host Defense against Malaria The spleen plays a crucial role in mediating protection against blood-borne pathogens. In malaria infection, the spleen is a key site for removal of damaged RBC or iRBC, generation of protective immune responses, and in mice is also involved in production of new RBC.5 Immune mechanisms during acute infection are necessary to control and prevent exponential increases in parasitemia. Studies in humans and various mouse models have demonstrated that interactions between circulating parasites or iRBC and key innate immune cells, such as macrophages, DCs and natural killer (NK) cells, can limit parasitemia, preventing severe pathology and death.2 Furthermore, splenic phagocytes can extract parasites from iRBC, a mechanism known as pitting, and the intact RBC subsequently return to the circulation.6,7

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In the mouse spleen, two subtypes of macrophages are found abundantly in the marginal zone.8 Marginal-zone macrophages lie in the outer ring adjacent to the red pulp and express the C-type lectin SIGNR1, a mouse homologue of human DC-SIGN (CD209), and the type I scavenger receptor MARCO.9-11 These receptors recognize polysaccharide antigens of pathogens, facilitating their clearance.10,12 Marginal-zone metallophilic macrophages, forming an inner ring of macrophages, express the adhesion molecule SIGLEC1, which recognizes sialic-acid residues of blood-borne pathogens.13 In humans, splenic macrophages are found in the perifollicular zone surrounding the marginal zone and also express high levels of SIGLEC molecules. When activated by pathogens or their products, macrophages can interact with DCs, T cells and B cells in the marginal zone, although only marginal-zone DCs and B cells are known to migrate further into the white pulp to activate naïve T cells.8 The majority of red pulp macrophages are located in the splenic cords and function to remove aged or damaged RBC, thereby contributing to the turnover of erythrocytes and recycling of iron.8 Splenomegaly is a common feature of malaria infection in both humans and experimental animals. Increased spleen size is a result of white and red pulp hyperplasia due to expansion of lymphoid, myeloid and erythroid components.14 In Plasmodium chabaudi AS-infected mice, spleen size is indicative of resistance to malaria. C57BL/6 mice, which survive and resolve the infection within 4 weeks, develop marked splenomegaly, whereas A/J mice, which succumb to the infection, exhibit minimal splenomegaly. Immunochemical analysis of infected spleens showed that marginal-zone macrophages are depleted during malaria infection.14 In P. chabaudi adami-infected mice, iRBC are not phagocytosed in the marginal zone but are removed by macrophages in the red pulp.15 However, recent histological studies of spleens from patients dying from Plasmodium falciparum infection did not confirm the changes in splenic macrophage populations observed in malaria-infected mice.16 Nonetheless, an important role for the spleen in parasite clearance is clearly demonstrated by the responses of splenectomized humans and mice to malaria infection. Splenectomized patients infected with P. falciparum display a considerable delay in parasite clearance,17 and splenectomized mice infected with Plasmodium yoelii show higher parasitemia and delayed parasite clearance.18 Thus, macrophages in the spleen perform important functions of phagocytosis and destruction of parasites, contributing to the removal of damaged RBC or iRBC (see “Functional Properties of Macrophages during Blood-Stage Malaria Infection” section below).

Functional Properties of Macrophages during Blood-Stage Malaria Infection Circulating monocytes and tissue resident macrophages, particularly in the spleen and liver, facilitate the control and resolution of infection via clearance of parasites and activation of immune responses. The molecular mechanisms by which these cells recognize iRBC and initiate innate immunity are not fully understood. To date, most studies have focused on monocyte/macrophage phagocytosis of immune-opsonized iRBC.19,20 However, during acute infection, nonimmune individuals, those most at risk for severe malaria, have yet to develop antigen-specific T cell and antibody responses and must rely on innate immune mechanisms to recognize and clear iRBC. In the “Macrophage Interactions with Plasmodium Blood-Stage Parasites in Humans” section, we will discuss these interactions in the context of human infections and provide further insights provided by studies in mouse models of malaria in “Macrophage Interactions with Plasmodium Blood-Stage Parasites in Mouse Models” section below.

Macrophage Interactions with Plasmodium Blood-Stage Parasites in Humans Four Plasmodium species infect humans, causing different outcomes of varying pathology and severity (Table 1). However, the vast majority of severe malaria cases and deaths are caused by P. falciparum, which is endemic in sub-Saharan Africa and many other tropical countries. Severe disease includes anemia, metabolic acidosis, cerebral malaria, hypoxia, hypoglycemia

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and lactic acidosis.2-4 Much of our understanding of macrophage function in human malaria is based on studies on interactions of RBC infected with P. falciparum and parasite-derived products with human monocytes/macrophages.

Opsonin-Independent Adhesion and Phagocytosis A key feature of P. falciparum-infected RBC is the ability to adhere to the linings of small blood vessels. Immune responses to P. falciparum-infected RBC are linked to the parasite replication cycle, as shown by the ability of RBC infected with mature parasite stages to bind to a variety of host receptors, including CD36, intercellular adhesion molecule-1 (ICAM-1), thrombospondin, hyaluronic acid, and others.3,21,22 iRBC can also rosette uninfected RBC and clump platelets. Collectively, these adhesive properties are responsible for sequestration of iRBC in the microvasculature, which has been associated with the development of severe clinical disease.3,23 Cytoadherence is also thought to protect the parasite from immune recognition and destruction, as nonadherent iRBC are rapidly cleared in the spleen. The adhesion phenotype is not homogenous; different parasites can bind to variable numbers and combinations of host receptors, affecting their tissue distribution and pathogenesis.24 The surface of RBC infected with P. falciparum trophozoites or schizonts is covered with knob-like excrescences that are the contact points with host cells.23 These structures are composed of several polypeptides, including PfEMP-1 (P. falciparum membrane protein-1), a highly variable antigen with multiple adhesion domains, that bind to many different host receptors such as CD36 and ICAM-1. PfEMP-1 variants permit not only heterogeneous adhesive phenotypes but also efficient evasion of protective humoral immune responses.21,23 In addition to PfEMP-1, phosphatidylserine (PS) also contributes to the adherence of P. falciparum-parasitized RBC to CD36 and thrombospondin.25 Other surface oligopeptides, such as the rosettins/rifins and sequestrins, may also participate in binding, but their role in sequestration of iRBC is unclear.3 Cytoadherence may also be involved in interaction of iRBC with cells of the innate immune system such as professional phagocytes as well as DCs (see “Role of Dendritic Cells as Phagocytic Cells during Malaria Infection” section). Although macrophages are critical for parasite clearance, especially in the spleen,26 their function can be altered and downregulated during malaria infection.27 Early studies showed that monocyte chemotactic responsiveness was suppressed in patients with acute primary attacks of P. falciparum, P. vivax or P. ovale malaria.28 Malaria infection also induces a decrease in predominantly monocyte-derived macrophage subpopulations that mediate high levels of phagocytosis.29 Furthermore, ingestion of a few iRBC stimulates phagocytosis but large amounts or longer exposures eventually paralyze the entire phagocytic system.27,30 Given that macrophages contribute to host defense as phagocytes and as APCs, the selective suppression of macrophage activity by malaria parasites is detrimental to the ability of the host to mount both nonspecific and specific immune responses.27 Monocytes can bind cytoadherent iRBC in the absence of antibody and ingest iRBC even when complement and Fc receptor pathways are blocked.19,31,32 Early studies showed that RBC infected with P. falciparum trophozoites or schizonts bind to CD36 expressed on human monocytes,33-35 but are not phagocytosed through this receptor.36 More recent studies, however, showed that RBC infected with mature parasite stages and expressing the appropriate PfEMP-1 variant are phagocytosed by purified human monocytes and culture-derived macrophages via CD36.37,38 This is in agreement with the finding that nonopsonic phagocytosis of iRBC is dependent on the parasite stage.19 In addition, phagocytosis of iRBC via CD36 does not prime an elevated proinflammatory response (see “Cytokine Production by Activated Monocytes/Macrophages” section below), and thus constitutes an alternate mechanism for modulation of macrophage function.38 Phagocytosis of iRBC is accompanied by CD36 clustering and is dependent on extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways.37 Moreover, proliferation-activated receptor γ-retinoic acid X receptor agonists and 9-cis-retinoic acid, a metabolite of vitamin A, increase the levels of

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CD36 expression on macrophages and phagocytosis of iRBC while reducing the secretion of proinflammatory cytokines.39,40 Together, these findings suggest a protective role for CD36 expression by monocytes/macrophages via parasite clearance with minimal pathology for the host. Studies with CD36-deficient or transgenically-rescued rodents have supported a role for CD36 in macrophage phagocytosis of iRBC.41 Furthermore, it has been demonstrated that CD36 also partially mediates uptake of RBC infected with P. falciparum gametocytes.42 Although not directly beneficial to the host, this mechanism may be a novel target for decreasing parasite transmission. In addition to PfEMP-1, iRBC uptake is also partially dependent on the expression of PS.19 Although PS is known to bind to CD36, CD36-mediated uptake of iRBC does not involve PS and is similar to the phagocytosis of apoptotic cells.39 Thus, the specific receptor(s) for PS-mediated uptake of iRBC have yet to be defined. Hemozoin, a metabolic product of blood-stage Plasmodium parasites and also known as malaria pigment, has been shown to modulate macrophage function.43 Large proportions of tissue macrophages and circulating monocytes are loaded with hemozoin during malaria infection; once loaded, hemozoin may persist unchanged for months and accumulate to high levels that can suppress macrophage activity.30,44 Indeed, ingestion of iRBC containing hemozoin inhibits further phagocytosis of infected cells or pathogenic bacteria or fungi.45 As discussed in “Macrophage-Derived Mediators of Cytotoxicity” section, other anti-tumor and anti-microbial activities are also perturbed in hemozoin-laden macrophages. However, the ingestion of newly infected RBC, which have less accumulated hemozoin, does not lead to impaired macrophage function.30,45,46 Based on these findings, it is not clear if the poor disease outcome associated with hemozoin-loaded phagocytes is due to prolonged high levels of malaria parasites or to more general cellular dysfunction.21 In infected individuals, free pigment is also internalized by both circulating and resident phagocytes, subsequently modulating their functions. Phagocytosis of free native or synthetic hemozoin was shown to be comparable to internalization of asbestos minerals and other crystals by both human and mouse phagocytes.47 By contrast, P. falciparum intraerythrocytic parasites are phagocytosed via the classical endocytic pathway. These results suggest differences between the ingestion of free particles and that of whole parasites in terms of modulation of phagocyte function. Nevertheless, the production of hemozoin appears to benefit parasite survival by suppressing some of the immune responses mediated by macrophages as effector cells.

Opsonin-Dependent Parasite Clearance by Human Monocytes Antibodies that recognize specific antigens expressed by iRBC mediate Fc receptor (FcR)-dependent uptake of blood-stage parasites by phagocytes. In addition, antibodies that mediate phagocytosis of free merozoites are associated with protective immunity.20 The importance of antibodies in enhancing clearance of blood-stage parasites has been long documented.48 Serum from malaria-immune individuals or purified IgG enhances the ingestion of P. falciparum-infected RBC by human monocytes.49 Opsonization of iRBC and subsequent phagocytosis vary with the developmental stage of the parasite; late-stage intraerythrocytic parasites are preferentially phagocytosed compared to ring forms.49 While phagocytosis of iRBC harboring ring forms depends mainly on deposition of IgG and complement, a process similar to that described for senescent or oxidatively damaged RBC, recognition signals for phagocytosis of iRBC with trophozoites/schizonts depend, as mentioned above, on nonimmune mechanisms as well as immune opsonization.19,50 Opsonic phagocytosis increases not only iRBC uptake but also their intracellular destruction.37 Antibody-dependent killing of P. falciparum merozoites by monocytes has also been reported.51-54 Induction of high levels of protective antibodies is undoubtedly a major challenge in modern anti-malarial vaccine strategies given that several exposures are required to induce large titers of these antibodies in naturally infected individuals. Furthermore, immune evasion by the parasite through antigenic variation and gene polymorphism results in prolonged infection, thereby ensuring parasite survival for continued transmission.3

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In contrast to the large body of research on antibodies, the roles of complement and other serum proteins in opsonization and phagocytosis of iRBC have been poorly addressed. Complement has been shown to promote killing of P. falciparum blood stages by the differentiated human myelomonocytic THP-1Mo cell line,55 but other studies reported that heat-inactivated immune or normal serum has no effect on the opsonic activity of freshly isolated human monocytes against iRBC.56 Conversely, more recent studies showed that P. falciparum ring-stage iRBC induce binding of autologous antibodies and activated C3 to the RBC membrane, a mechanism by which senescent and damaged RBC are removed by phagocytosis via the complement receptor CR1.57 Enhanced CR1-dependent phagocytosis of ring-parasitized RBC has also been proposed to explain protection against falciparum malaria in individuals with genetic disorders such as the sickle cell trait, homozygous hemoglobin C, and glucose-6-phosphate dehydrogenase deficiency.58 Thus, the role of complement in human malaria infection remains to be further elucidated. Serum proteins, such as fibrinogen and albumin, are involved in parasite rosetting, although their role in opsonic phagocytosis is inconclusive.3 In addition, serum mannose-binding lectin (MBL), which binds to the surface of RBC infected with mature parasite forms, might also function as an opsonin. Individuals with variant alleles in the MBL gene causing low levels of functional MBL develop higher parasitemia, but the role of MBL in phagocytosis was not addressed in this study.59 Other soluble immune mediators, such as cytokines, have also been shown to contribute to increased opsonin-dependent phagocytosis. For instance, tumor necrosis factor (TNF)-α enhances phagocytic activity by increasing the expression of FcR on monocytes or alternatively by modulating FcR-specific signaling pathways.32,60

Cytokine Production by Activated Monocytes/Macrophages In both infected humans and mouse models, innate immune responses are essential during acute infection to limit the initial phase of parasite replication before the host develops antigen-specific adaptive responses that can clear the parasites and resolve the infection. However, the inflammatory response needed to control parasite growth can lead to considerable tissue damage. Activation of phagocytes to kill intracellular or extracellular parasites requires the production of inflammatory cytokines, which may contribute to clinical disease such as severe anemia and cerebral malaria. Thus, the outcome of infection depends on a balance between proinflammatory and anti-inflammatory responses (for reviews see refs. 2-4). Cytoadherence of P. falciparum-infected RBC and release of products, such as glycosylphosphatidylinositol (GPI)-anchored proteins and hemozoin, by late-stage parasites may modulate the function of monocytes/macrophages and damage host tissues directly or through overstimulation of cytokine production. Human peripheral blood mononuclear cells (PBMC) release abundant levels of TNF-α and other proinflammatory cytokines when incubated with intact or lysed late-stage iRBC or hemozoin in vitro.61-63 Although TNF-α has anti-parasitic functions, it has also been implicated as a mediator of fever and other severe complications. Native hemozoin contains ferriprotoporphyrin IX and several lipids as well as proteins of both host and parasitic origin.64 Thus, the ingestion and destruction of intraerythrocytic parasites and release of their products provide an enormous stimulus for the macrophage population. High levels of TNF-α activity are consistently detected in monocytes cocultured with P. falciparum schizont-infected RBC that subsequently rupture. Pigment recovered from ruptured schizonts induces monocytes to produce high levels of TNF-α and interleukin (IL)-1β, while particulate-free culture supernatant induces relatively low levels of cytokine release. Treatment of isolated hemozoin with protease abolishes the cytokine-inducing activity.65 In addition to TNF-α, both native and synthetic hemozoin potently induce the release of several other pyrogenic cytokines, including MIP-1α and MIP-1β, from murine macrophages and human monocytes in vitro.66,67 Trophozoite-infected RBC or hemozoin-fed human monocytes also display increased activity of matrix metalloproteinase-9, which not

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only degrades matrix proteins but also activates TNF-α from circulating precursors, resulting in disruption of the basal lamina of endothelia and activation of a positive feedback loop for TNF-α production.68 In addition, phagocytosis of hemozoin derived from ruptured schizonts upregulates the expression of CD11b/CD18 and other adhesion molecules by both neutrophils and monocytes, thereby enhancing their cytoadherence to capillaries and resulting in local tissue edema, vascular clogging and severe tissue damage.69 The molecular mechanisms underlying the stimulatory effects of hemozoin were recently elucidated. P. falciparum hemozoin functions as a nonDNA ligand for Toll-like receptor (TLR) 9 on human DCs.70 TLR9-mediated activation of innate immune responses by hemozoin results in the production of cytokines, chemokines, and upregulation of costimulatory molecules in a myeloid differentiation factor 88 (MyD88)-dependent manner. Synthetic hemozoin also activates innate immune responses in vivo via a TLR9-dependent pathway,70 although it is unclear whether TLR9 is also the major receptor for recognition of hemozoin by monocytes in vivo. In addition to TLR9-mediated signals, both purified and synthetic P. falciparum hemozoin induces macrophage chemokine expression through oxidative stress-dependent and -independent mechanisms involving ERK1/2 phosphorylation and NF-κB activation.71 In contrast to its stimulatory activity, the accumulation of hemozoin inside macrophages is associated with suppression of both antigen processing and immune activation by macrophages. Higher percentages of heavily hemozoin-laden leukocytes and macrophages correlate with severe disease.72 Hemozoin-laden monocytes have defects in the induction of MHC class II responses to interferon (IFN)-γ stimulation.73 In addition, expression of CD54, CD62L (L-selectin) and CD11c as well as protein kinase C (PKC) signaling pathways are decreased, suggesting that hemozoin loading may contribute to the impairment of the immune response and antigen presentation by macrophages during malaria infection.69,73,74 IL-10, produced by hemozoin-loaded monocytes/macrophages, also plays a role in the suppression of proliferation and cytokine responses of mitogen-stimulated human PBMC.75 Thus, it remains unresolved whether hemozoin exerts stimulatory or suppressive effects on macrophage function. The immune function of monocytes/macrophages may also be modulated after interaction of iRBC with CD36.37,38 Phagocytosis of iRBC via CD36 decreases LPS-induced TNF-α production by monocytes,37,38,40 an outcome that has been linked to both CD36-mediated phagocytosis of iRBC and soluble factors released by iRBC.76 The anti-inflammatory effects resulting from adhesion of iRBC to CD36 may affect the survival of both parasite and host, thereby influencing the outcome of infection. GPI, a toxin produced by P. falciparum, is thought to contribute to the pathogenesis of severe malaria in part by inducing the secretion of proinflammatory cytokines by macrophages (for review see refs. 77,78). This activity involves protein tyrosine kinase (PTK)- and PKC-mediated cell signaling pathways, which cooperate in regulating the downstream NF-κB/ rel-dependent gene expression of IL-1α, TNF-α, and other proinflammatory mediators. Treatment of parasite extracts with monoclonal antibodies to GPI blocks the induction of TNF-α, indicating that GPI stimulates the parasite-induced TNF-α response.79-82 The receptors for GPI-specific recognition and signaling were recently identified: GPI signaling is mediated mainly via TLR2 and, to a lesser extent, TLR4.83,84 GPIs containing different numbers of fatty acids vary considerably in the requirement of auxiliary receptors; GPIs with three fatty acids are preferentially recognized via TLR2/TLR1, and GPIs with two fatty acids via TLR2/TLR6. However, all GPI types engage similar signaling pathways involving MyD88-dependent activation of MAPKs, including ERK, c-Jun N-terminal kinase (JNK), and/or p38 MAPK, and NF-κB up-regulation. The differential activation of these signaling molecules by GPIs leads to very distinct patterns of cytokine production.83,84 Malarial GPIs are therefore important determinants of parasite virulence and disease severity. In response to these parasite toxins, macrophages express surface phospholipases, which can degrade GPIs into inactive species, suggesting that regulation of GPI activity by host macrophages may determine in part the outcome of infection.83

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Macrophage-Derived Mediators of Cytotoxicity Activated macrophages rely on a complex array of oxygen-dependent antimicrobial molecules to inhibit or kill pathogens. The production of reactive oxygen species (ROS), such as superoxide (O2-) and hydrogen peroxide (H2O2), by phagocytes requires NADPH oxidase activity and coincides with a period of oxygen consumption called the respiratory or oxidative burst. This burst is followed by a prolonged nitrosative phase during which nitric oxide synthases (NOS) catalyze the oxidation of guanidino nitrogen of L-arginine to nitric oxide (NO). The NO radical and its oxidized derivatives, nitrite and nitrate, are collectively termed reactive nitrogen intermediates (RNI). Of the multiple NOS isoforms that catalyze NO synthesis, inducible NOS (iNOS) is most commonly associated with antimicrobial activity. Host expression of iNOS is regulated primarily at the transcriptional level and can be induced after interaction with microbial products or in response to cytokines such as IL-1, TNF-α and IFN-γ. The temporal progression from the predominant production of ROS to the production of NO results in a reduced microbial burden, but these reactive species can also target host tissues, thus contributing to the development of clinical disease (for reviews see refs. 85,86). Given that hemoglobin has a high affinity for NO and RBC act as permanent sinks for this molecule, it is controversial as to whether NO plays a role in immune protection against intraerythrocytic parasites. Nonetheless, there is evidence that NO and, to a lesser extent, ROS may protect against blood-stage malaria. NO activity and parasitemia are inversely correlated in infected individuals.87-91 Furthermore, in vitro studies have demonstrated that monocytes matured or activated with IFN-γ can inhibit P. falciparum parasite growth via release of RNI or ROS following interaction with merozoites or iRBC.92-94 P. falciparum is indeed killed in vitro by ROS.95 Intraerythrocytic parasites are also sensitive to NO in vitro under low oxygen tension, a condition typically observed during parasite sequestration in deep-tissue capillaries.96-98 Importantly, recent studies show that hemoglobin releases NO under low oxygen conditions, enabling NO and its associated RNI to be available for anti-parasitic effector mechanisms.96-98 Despite its protective properties, an excess of NO or ROS has been associated with severe anemia and cerebral malaria,90,99,100 and parasite-derived molecules may induce the overproduction of these reactive species. Purified P. falciparum GPIs upregulate iNOS expression and NO production as well as synergize with IFN-γ to induce NO production by mouse macrophages via a pathway requiring PTK-, PKC- and NF-κB/rel-dependent signaling.80,101 The recent discovery of GPI interaction with TLRs has provided novel insights into the signaling mechanisms, including the relative contributions of JNK pathways, that control NO production.83,84 In contrast to the anti-inflammatory effects attributed to CD36, binding of P. falciparum-infected RBC to CD36 induces oxidative burst activity via a pathway that involves PKC activation in the absence of phagocytosis of iRBC.35,38 Hemozoin is also postulated to induce NO and ROS overproduction but this remains controversial. Neither native nor synthetic hemozoin by themselves induce NO production by murine macrophages, although these molecules significantly increase IFN-γ-mediated iNOS expression and NO production via ERK- and NF-κB-dependent pathways.102 Experiments using PBMC from noninfected donors confirmed that P. falciparum hemozoin alone has no effect but augments iNOS transcription and NO production in synergy with LPS and IFN-γ.103 Hemozoin also modulates macrophage oxidative burst; P. falciparum-derived or synthetic hemozoin stimulates ROS production by murine macrophages with consequent activation of chemokine gene transcription.71 In agreement with these findings, human monocytes fed with purified P. falciparum hemozoin show a long-lasting period of oxidative burst although further phorbol myristate acetate (PMA)-induced oxidative burst is suppressed.30,44 Similarly, human monocytes that have ingested trophozoite-infected RBC show a reduced capability to produce superoxide following stimulation with PMA.45 This inhibitory effect of hemozoin on nonparasite activation of the oxidative burst might be partially explained by the finding that hemozoin inhibits PKC activation and consequently NADPH oxidase activity.46,74

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Long chain polyunsaturated fatty acids (LCPUFAs), such as arachidonic acid (AA), have been shown to be directly toxic against P. falciparum and to enhance respiratory burst and free radical generation by macrophages. Thus, a deficiency of LCPUFAs may render an individual more susceptible to malaria.104 Alternatively, LCPUFAs have been proposed to be a potential mechanism of hemozoin-mediated inhibition of macrophage function through the peroxidation of AA, leading to the generation of biologically active lipoperoxides. Lipoperoxides isolated from hemozoin and/or hemozoin-fed macrophages have been shown to inhibit macrophage function in vitro, such as PMA-induced oxidative burst and PKC activation.105-107

Macrophage Interactions with Plasmodium Blood-Stage Parasites in Mouse Models Infections of mice with rodent strains of Plasmodium species (Table 1) have been used extensively to elucidate the complex interactions between Plasmodium parasites and the host immune system. Although no single mouse model fully replicates human malaria in terms of either pathology or immune responses, infections of mice with P. chabaudi, P. yoelii, P. berghei, or P. vinckei have been useful and convenient tools for elucidating mechanisms of immunity and pathogenesis, identifying genes that regulate host susceptibility to malaria, and revealing important targets for development of vaccines and chemotherapies.2-4

Opsonin-Independent Adhesion and Phagocytosis Depending on the parasite species and the genetic background of the host, different mouse strains show divergent outcomes of infection with blood-stage parasites. Resistant strains are able to control acute parasitemia and resolve the infection, whereas susceptible strains show high, fulminating parasitemia and succumb to the infection. Activation of nonspecific immune responses by Th1-associated cytokines is critical for efficient control of acute malaria and host survival (see “Cytokine Production by Activated Macrophages” section below). Early studies provided evidence that phagocytosis of iRBC or merozoites is an important effector mechanism for controlling acute blood-stage malaria. Mice pretreated with immunomodulating agents such as Mycobacterium bovis (BCG), muramyl dipeptide-liposomes or heat-killed Corynebacterium parvum, which activate nonspecific immune responses including macrophage phagocytosis, show reduced acute parasitemia and improved survival.108-111 Depletion of mononuclear phagocytes by silica treatment results in increased parasitemia and mortality in resistant P. chabaudi AS-infected C57BL/6 mice.111 Studies in resistant versus susceptible mice also show that macrophage phagocytic activity correlates with the ability to control acute parasitemia; specifically, peritoneal macrophages from P. chabaudi AS-infected resistant C57BL/6 mice show higher nonopsonic phagocytosis of iRBC and free merozoites than susceptible A/J mice and IFN-γ- or IL-12p40-deficient mice.112 Macrophages and endothelial cells express scavenger receptors that recognize ligands expressed on iRBC or merozoites and mediate opsonin-independent phagocytosis. Mice deficient in type I and II class A scavenger receptors (SR-AI/II knockout mice) develop similar levels of parasitemia as wild-type control mice following blood-stage P. chabaudi or P. berghei infection.112,113 However, in vitro blocking of scavenger receptors on macrophages with polyinosinic acid, a synthetic ligand for these receptors, inhibited phagocytosis of P. chabaudi free merozoites and, to a lesser extent, iRBC.112 Furthermore, treatment of P. chabaudi-infected mice with polyinosinic acid in vivo results in increased levels of parasitemia. These observations suggest that members of the scavenger receptor family other than SR-AI/II may mediate the phagocytosis of iRBC or merozoites. As discussed above, although macrophages from rodents deficient in the scavenger receptor CD36 show diminished ability to phagocytose P. falciparum-infected RBC, the role of this receptor in interactions between macrophages and rodent Plasmodium species has not been addressed.41

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Opsonin-Dependent Parasite Clearance by Mouse Macrophages Although nonopsonic phagocytosis limits malaria parasite replication, it is not sufficient for parasite clearance and resolution of the infection. The latter critically require antibody-mediated adaptive immunity, as demonstrated in B cell-deficient mice which can control acute malaria but are unable to resolve a chronic infection.2-4 As discussed in “Opsonin-Dependent Parasite Clearance by Human Monocytes” section, malaria-specific antibodies play an important role in immune protection by neutralizing merozoites or opsonizing iRBC for FcR-mediated phagocytosis. Similarly, sera obtained from P. chabaudi AS-infected mice during acute infection or from mice rendered hyperimmune by repeated infections contain antibodies that are able to recognize and bind malarial antigens expressed on the surface of iRBC. Antibodies in immune or hyperimmune sera can opsonize iRBC and significantly enhance phagocytosis by macrophages in vitro.114,115 Using fractionated immune serum, it was further demonstrated that only IgG antibodies are able to opsonize iRBC and promote phagocytosis. 114 Whether antibody-mediated phagocytosis contributes in vivo to immune protection against blood stage malaria is still controversial. Polyclonal immune sera from mice immunized with merozoite surface protein-1 (MSP-1) of P. yoelii 17XL or infected with P. yoelii 17XL confer immune protection to both wild-type BALB/c recipients and FcR γ chain-deficient mice which are unable to mediate phagocytosis through FcγRI-III receptors.116 Similarly, passive transfer of monoclonal IgG3 specific for MSP-1 protects against P. yoelii YM infection in FcR α chain-deficient mice that lack high affinity receptor FcγRI.117 It is possible that antibodies against MSP-1 may directly kill P. yoelii 17XL merozoites, such that phagocytosis is not involved in IgG3-mediated protection. However, macrophages from wild-type controls show enhanced phagocytosis of iRBC opsonized by immune serum from P. berghei XAT-infected mice, but this activity is absent in macrophages from FcR γ chain-deficient mice. Importantly, transfer of anti-P. berghei XAT polyclonal immune serum to FcR γ chain-deficient mice fails to confer immune protection against live P. berghei XAT infection.118 Together, these observations demonstrate the complexity of host immune mechanisms against malaria involving multiple antibody isotypes and immune effector cells, and different mechanisms may occur with various Plasmodium species, target antigens and developmental stages of the malaria parasite. As discussed in “Opsonin-Dependent Parasite Clearance by Human Monocytes” section, the role of complement in enhanced opsonin-dependent phagocytosis is unclear. Mice deficient in classical and alternative complement pathways are able to control and resolve a primary P. chabaudi infection, indicating that complement fixation by antibody is not essential for protective immunity.119 However, mice lacking the classical complement pathway are more susceptible to challenge infection than wild-type mice,119 suggesting a role for complement in the development of memory immune responses.

Cytokine Production by Activated Macrophages Mouse models of malaria have yielded important insights into immune mechanisms mediated by cytokines and effector cells against blood-stage parasites.2,120-122 In P. chabaudi-infected mice, IL-12 and IFN-γ are critical cytokines for the development of Th1-type immunity.123,124 Depending on the genetic background, the ability of mice to control infection with nonlethal Plasmodium strains is associated with high levels of IL-12 and IFN-γ production.124-126 Following P. chabaudi AS infection, splenic macrophages from resistant C57BL/6 mice produce higher levels of biologically active IL-12p70 than macrophages from susceptible A/J mice.126 Moreover, abundant IL-12p70 production correlates with enhanced IFN-γ synthesis by iRBC-stimulated spleen cells.126 On the other hand, splenic macrophages from infected C57BL/ 6 or A/J mice secrete equivalent levels of TNF-α in response to iRBC or soluble P. chabaudi AS antigens.127 Importantly, IFN-γ-deficient mice also show higher parasitemia, reduced LPS-stimulated IL-12 and NO production by splenic macrophages, and severe morality compared to wild-type mice.123 These results suggest the presence of a positive feedback loop for

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optimal production of IL-12 and IFN-γ, both of which are required for Th1-dependent protective immunity to blood-stage malaria. As discussed in “Macrophage-Derived Mediators of Cytotoxicity” section below, IFN-γ also plays an important role in activating macrophages for increased phagocytosis of iRBC and release of reactive species.123,125 Peritoneal macrophages stimulated by P. chabaudi-infected RBC or synthetic hemozoin release macrophage migration inhibitory factor (MIF), a cytokine that has been linked to the pathogenesis of malarial anemia.128 Mice injected with high doses of synthetic hemozoin show strong inflammatory responses, including the recruitment of leukocytes, which in excess may contribute to the development of malarial disease.129 Similarly, the amount of intraleukocytic pigment correlates with prognosis of severe malaria in humans,130 and peritoneal macrophages loaded with P. vinckei hemozoin have increased TNF-α production after LPS or IFN-γ stimulation, although IL-6 production is reduced.131 As observed in human studies, evidence for the ability of hemozoin to elicit cytokine responses from murine macrophages is conflicting; crude P. chabaudi hemozoin suppresses macrophage antigen presenting function, while synthetic hemozoin either increases or suppresses cytokine production.71,132-135

Macrophage-Derived Mediators of Cytotoxicity The role of NO or RNI in immunity against murine malaria is also controversial. No increases in iNOS activity or NO metabolism are observed in mice infected with P. berghei ANKA or P. vinckei petteri.136 In contrast, upregulated iNOS or NO activity has been reported in mice infected with various strains of P. chabaudi,120,124,137-139 P. vinckei,139 or P. berghei XAT.140 Although the level of NO in blood is unchanged following infection with P. berghei N/13/1A/ 4/203, the levels in the liver and spleen, along with RNI concentration in plasma, are increased.141 These results show that different levels of NO and RNI production are induced during blood-stage malaria, depending on the tissue, the Plasmodium species, and the method of measuring NO biosynthesis.140,141 Likewise, there are conflicting data on the functional role of NO in protection against blood-stage malaria. Mice deficient in iNOS or treated with pharamacological agents blocking NO production show either higher or unaltered parasitemia levels, which may vary with the Plasmodium species and strains, the genetic background of the host, and the extent to which alternative effector mechanisms compensate for the absence of NO production.124,137-139,142-147 In P. chabaudi AS malaria, the ability of mice to control infection is associated with Th1-dependent induction of NO production by splenic macrophages.121,124,143 Resistant C57BL/6 mice but not susceptible A/J mice show upregulated expression of iNOS in the spleen and increased levels of RNI in serum.143 In vivo administration of IL-12 protects susceptible A/J mice against a primary P. chabaudi infection in a mechanism that partially requires NO.124 Interestingly, NO-mediated resistance to P. chabaudi infection involves pathways that do not affect parasitemia levels.124,143 Based on these findings, it was proposed that NO plays a role in host protection via direct parasite killing and/or regulation of the immune response. In support of the latter contention, macrophage-derived NO has been shown to suppress spleen cell proliferation and production of IL-12, IFN-γ and TNF-α, thus limiting Th1-associated immune responses to maintain a balance between the protective and pathologic effects of proinflammatory cytokines.123,138,139,142,148,149 In addition to NO, there is also evidence that ROS produced by activated macrophages can kill parasites, although the efficiency of killing varies among different murine hosts as well as distinct rodent Plasmodium strains.98,142,150-152 Accordingly, the capacity of splenic macrophages to generate ROS in vitro is higher in response to nonlethal P. yoelii or P. chabaudi than to lethal P. berghei or P. yoelii 17XL.125,152,153 While some studies show no correlation between ROS production by macrophages and protective immunity to blood-stage malaria,154,155 oxygen metabolism is higher among macrophage populations from resistant C57BL/6 mice than from susceptible A/J mice after P. chabaudi AS infection.127

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As described for human malaria in “Macrophage-Derived Mediators of Cytotoxicity” section, the induction of NO and other reactive species during murine malaria is thought to be mediated by the parasites or their secreted products in synergy with cytokines such as TNF-α and IFN-γ.122,123,156 For instance, phagocytosis of P. berghei- or P. yoelii-infected RBC activates mouse peritoneal macrophages to release ROS in vitro and this occurs to a greater extent with a lysed iRBC preparation.125,153,157 Cytokines, such as IFN-γ, and immune serum enhance the ROS response to iRBC, confirming the activating role of cytokines and antibody-mediated phagocytosis in macrophage production of cytotoxic mediators.125,150,157-159 As described for P. falciparum, hemozoin derived from rodent Plasmodium species have divergent capacities to induce production of NO or ROS. P. chabaudi hemozoin activates NO production by murine macrophages, P. vinckei hemozoin suppresses LPS or IFN-γ-mediated induction of NO and ROS by peritoneal exudate macrophages, and synthetic hemozoin suppresses NO-inducible activity of LPS but increases the oxidative stress and lipid peroxidation in macrophages.132-134,160,161

Functional Properties of Macrophages during Liver-Stage Malaria Although blood-stage parasites are cleared primarily in the spleen, the liver is a major site of removal of exo-erythrocytic or liver stages. Kupffer cells, the largest population of mammalian tissue macrophages, are pivotally positioned in the sinusoidal lumen of the liver for removal of cellular debris or foreign substances from the blood. Kupffer cells can disrupt the development of exo-erythrocytic forms possibly through the production of IL-6.162 In vivo depletion of Kupffer cells prior to infection with P. berghei sporozoites results in increased parasitemia.162,163 However, a conclusive role for Kupffer cells in immunity against liver-stage parasites is unclear. Earlier studies suggested that Kupffer cells phagocytose sporozoites although the parasites retain their integrity inside endocytotic vacuoles and no signs of lysosomal digestion are observed.164 Furthermore, recent studies suggest that Kupffer cells are targets for sporozoite entry into the liver.165,166 Surface receptors on Kupffer cells bind circumsporozoite protein (CSP) and thrombospondin-related adhesive protein expressed by sporozoites. Following adhesion, sporozoites actively invade and traverse Kupffer cells while contained inside a vacuole, which does not fuse with lysosomes and thus allows the parasites to exit in the macrophage unharmed.165,167-169 Moreover, during parasite gliding and invasion, the release of CSP, which binds to RNA-associated sites on ribosomes and blocks translation, may result in the destruction of Kupffer cells.170 Kupffer cells are also fully mature APCs that may play a central role in portal vein tolerance.169 Tolerance to sporozoite antigens and continuous elimination of Plasmodium-specific CD8+ T cells in the liver may help explain the poor, short-lived protective immune response against liver-stage parasites. An effective vaccine against the liver stage would require a switch of the immune response from tolerance to immunity.171,172 Indeed, it has been shown that immunization with radiation-attenuated P. berghei sporozoites can reverse the tolerogenic milieu in the liver and facilitate the induction of protective immunity.173

Role of Dendritic Cells as Phagocytic Cells during Malaria Infection Monocytes give rise not only to tissue macrophages but also develop into DCs following exposure to the appropriate cytokine microenvironment. DCs are highly specialized APCs that control immune responses to pathogens. During steady state, low numbers of immature DCs circulate in the periphery and continually sample the local environment for signs of infection or tissue injury. DCs use several mechanisms to recognize and capture antigens.174 First, DCs take up particles, microbes and infected or damaged cells via actin-dependent phagocytosis. They also form large pinocytic vesicles to sample extracellular fluid and solutes, a process called macropinocytosis. Third, DCs perform absorptive endocytosis using receptors that include the family of C-type lectin receptors, such as DC-SIGN and DEC-205 (CD205), as well as Fc, complement and scavenger receptors.174 During antigen uptake, DCs receive information about

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the nature of the infection through various TLRs and other pattern recognition receptors (PRRs) capable of recognizing a diverse but overlapping repertoire of conserved microbial structures.175 The information conveyed by TLRs, PRRs, and other receptors also allows DCs to distinguish between apoptotic cells and those that die by externally activated necrosis.176 Macrophages are less efficient than DCs at priming T cells because they possess fewer MHC class II molecules and most of the antigens internalized in their lysosomes are fully digested into amino acids.177 Therefore, DCs are regarded as classical APCs that initiate and regulate host immunity rather than as professional phagocytes. DCs may play an important role in the induction of protective immunity to blood-stage malaria.178 To initiate anti-malarial immunity, DCs selectively recognize and capture iRBC circulating in the blood or deposited in the spleen for removal, and subsequently process and present the iRBC-derived antigens to other immune cells. Early studies showed that DCs can internalize RBC infected with P. falciparum or P. yoelii.179,180 More recently, our laboratory demonstrated that DCs internalize P. chabaudi-infected RBC via actin-dependent phagocytosis181 (Fig. 2). The uptake of iRBC by splenic DCs is selectively enhanced after P. chabaudi AS infection in vivo (Fig. 2A) and is associated with phenotypic and functional maturation, including upregulated costimulatory molecule expression, production of Th1-polarizing cytokines, and stimulation of CD4+ T cell proliferation and IFN-γ production.181 In contrast, DCs take up noninfected RBC at significantly lower levels (Fig. 2A) and this does not induce DC maturation or DC-primed T cell activation (Fig. 2D,E). Thus, DCs in the spleen are pivotally positioned to detect and capture erythrocytic parasites during acute malaria infection. Phagocytic uptake of iRBC is a critical event in the early interactions between DCs and malaria parasites, leading to DC activation and initiation of DC-mediated innate and adaptive immune responses.178,181

Concluding Remarks Studies conducted in humans as well as in mice provide evidence of an important role for monocytes/macrophages as effector cells during malaria. Following interactions with Plasmodium-infected RBC mediated via PRRs, such as TLRs, myeloid lineage cells including DCs may phagocytose iRBC by either opsonin-independent or -dependent mechanisms (Fig. 3). In addition to interactions with iRBC, monocytes and macrophages also encounter parasite-derived antigens and products such as hemozoin or GPI. The consequences of these interactions between macrophages and the malaria parasite may lead to one of two possible scenarios. On one hand, down-modulation of macrophage functions, for example phagocytosis and antigen presentation together with decreased production of proinflammatory cytokines such as TNF-α and IL-12 and increased production of immunomodulatory cytokines such as IL-10, may occur and lead to impaired immune responses with uncontrolled parasite replication and possibly severe disease and death of the host. Alternatively, activation of macrophages may occur with increased cytokine and chemokine production, increased expression of adhesion molecules, and increased activities of key enzymes involved in the generation of reactive oxygen and nitrogen metabolites important in the microbicidal activity of this cell population; these enhanced responses are important for control and clearance of the parasite. However, over-abundant activation of monocytes/macrophages may lead to excessive inflammation resulting in immune-mediated pathology and severe disease with a fatal outcome. Whether or not the functions of a particular macrophage population are suppressed or enhanced following encounter with Plasmodium parasites likely depends upon a number of critical factors, such as the heterogeneity among cells of the macrophage lineage as well as their tissue location and microenvironment.182 To date, most studies have focused on the roles of monocytes/macrophages in malaria as nonspecific effector cells. Emerging studies, however, suggest that macrophages interact with innate immune cells, especially NK cells, early during immune responses in clinical disease settings, including P. falciparum infection, and provide a critical source of stimulatory cytokines

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Figure 2. Splenic CD11c+ DCs preferentially take up parasitized RBC (pRBC) versus normal RBC (nRBC) at increasing levels in the early days following P. chabaudi infection in vivo and is associated with T cell priming. A) Splenic DCs enriched from naïve (left panels) or day 5 infected (right panels) C57BL/6 mice were incubated with CFSE-labeled pRBC or nRBC and the level of uptake by nongated cells (dot plots) or gated CD11c+ cells (histograms) was determined by CFSE fluorescence as detected by flow cytometry. B) Kinetics of pRBC or nRBC uptake by splenic DCs enriched from mice before and after P. chabaudi infection, and course of parasitemia in mice infected with P. chabaudi during the same experimental period. C) Splenic DCs preferentially take up pRBC vs. nRBC at all ratios of DCs incubated with RBC and uptake is inhibited by treatment with cytochalasin D (CyD, dotted lines). D) DCs pulsed with pRBC at ratios of 1:10 or 1:20 stimulated significantly higher levels of CD4+ T cell proliferation than nonpulsed DCs or DCs pulsed with nRBC. E) DCs pulsed with pRBC stimulated significantly higher levels of IFN-γ production by CD4+ T cells than nonpulsed DCs or DCs pulsed with nRBC. Data are presented as mean ± SEM of 3-4 mice per group and are representative of three independent experiments. Statistical analyses in A-C compared pRBC vs. nRBC and in D-E compared DC + pRBC (1:20) vs. DC + nRBC (1:20) at each DC:T cell ratio (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Reprinted with permission from Ing R, Segura M, Thawani N et al. J Immunol 2006; 177:441-450.181

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Figure 3. Overview of nonopsonic interactions between Plasmodium-infected RBC (iRBC) and macrophages. Macrophages recognize host- or parasite-derived molecules expressed on the surface of iRBC. For instance, P. falciparum membrane protein-1 (PfEMP-1) is recognized via CD36 receptor, while the receptor(s) for phosphatidylserine (PS) and other surface components remain to be elucidated. Members of the scavenger receptor family (SRs), other than CD36, may also be involved in macrophage phagocytosis of iRBC. Ligation of CD36 by iRBC leads to activation of the mitogen-activated protein kinase (MAPK) signaling pathway with consequent phagocytosis of iRBC, a process that is accompanied by down-modulation of macrophage pro-inflammatory activity. In addition, phagocytosis of RBC infected with mature parasite stages containing high levels of the malarial pigment hemozoin (HZ) or alternatively phagocytosis of free HZ also contributes to down-modulation of macrophage functions, such as reduced expression of MHC class II and adhesion molecules and decreased protein kinase C (PKC) signaling and oxidative burst. In addition, interleukin-10 (IL-10) produced by HZ-loaded macrophages and lipoperoxide formation contribute to the impairment of the phagocytic and antigen presentation functions of macrophages during malaria infection. On the other hand, under conditions that remain controversial, ligation of Toll-like receptor (TLR)-9 by HZ leads to activation of macrophage effector functions rather than immune suppression in a mechanism that depends on MyD88 and MAPK signaling pathways. Lastly, glycosylphosphatidylinositol (GPI)-anchored proteins contribute to the pathogenesis of severe malaria by inducing the secretion of proinflammatory cytokines and nitric oxide by macrophages via TLR2 and, to a lesser extent, TLR4 signaling pathways, involving MyD88-dependent activation of MAPKs. In contrast to the anti-inflammatory effects attributed to CD36, binding of P. falciparum-infected RBC to CD36 also induces oxidative burst activity via a pathway that involves PKC activation in the absence of phagocytosis of iRBC. Activated macrophages can interact with innate immune cells, especially NK cells, and provide a critical source of stimulatory cytokines to induce IFN-γ production and promote a protective type 1 adaptive immune response against malaria.

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to induce IFN-γ production and promote a protective type 1 adaptive, antibody-dependent immune response against malaria.183-185 Additional studies are clearly warranted to delineate the pivotal role of macrophages in innate and adaptive immunity to malaria. Given the urgent need for efficacious eradication strategies, information derived from these studies will likely contribute to the development of an effective malaria vaccine and improved immunotherapies.

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97. Taylor-Robinson AW. The sequestration hypothesis: An explanation for the sensitivity of malaria parasites to nitric oxide-mediated immune effector function in vivo. Med Hypotheses 2000; 54:638-641. 98. Rockett KA, Awburn MM, Cowden WB et al. Killing of Plasmodium falciparum in vitro by nitric oxide derivatives. Infect Immun 1991; 59:3280-3283. 99. Gyan B, Kurtzhals JA, Akanmori BD et al. Elevated levels of nitric oxide and low levels of haptoglobin are associated with severe malarial anaemia in African children. Acta Trop 2002; 83:133-140. 100. Clark IA, Rockett KA, Cowden WB. Proposed link between cytokines, nitric oxide, and human cerebral malaria. Parasitol Today 1991; 7:205-207. 101. Tachado SD, Gerold P, McConville MJ et al. Glycosylphosphatidylinositol toxin of Plasmodium induces nitric oxide synthase expression in macrophages and vascular endothelial cells by a protein tyrosine kinase-dependent and protein kinase C-dependent signaling pathway. J Immunol 1996; 156:1897-1907. 102. Jaramillo M, Gowda DC, Radzioch D et al. Hemozoin increases IFN-gamma-inducible macrophage nitric oxide generation through extracellular signal-regulated kinase- and NF-kappa B-dependent pathways. J Immunol 2003; 171:4243-4253. 103. Keller CC, Kremsner PG, Hittner JB et al. Elevated nitric oxide production in children with malarial anemia: Hemozoin-induced nitric oxide synthase type 2 transcripts and nitric oxide in blood mononuclear cells. Infect Immun 2004; 72:4868-4873. 104. Arun Kumar C, Das UN. Lipid peroxides, nitric oxide and essential fatty acids in patients with Plasmodium falciparum malaria. Prostaglandins Leukot Essent Fatty Acids 1999; 61:255-258. 105. Schwarzer E, Muller O, Arese P et al. Increased levels of 4-hydroxynonenal in human monocytes fed with malarial pigment hemozoin. A possible clue for hemozoin toxicity. FEBS Lett 1996; 388:119-122. 106. Schwarzer E, Ludwig P, Valente E et al. 15(S) hydroxyeicosatetraenoic acid (15-HETE), a product of arachidonic acid peroxidation, is an active component of hemozoin toxicity to monocytes. Parasitologia 1999; 41:199-202. 107. Schwarzer E, Kuhn H, Valente E et al. Malaria-parasitized erythrocytes and hemozoin nonenzymatically generate large amounts of hydroxy fatty acids that inhibit monocyte functions. Blood 2003; 101:722-728. 108. Nussenzweig RS. Increased nonspecific resistance to malaria produced by administration of killed Corynebacterium parvum. Exp Parasitol 1967; 21:224-231. 109. Clark IA, Allison AC, Cox FR. Protection of mice against Babesia and Plasmodium with BCG. Nature 1976; 259:309-311. 110. Cottrell BJ, Playfair JHL, De Sousa B. Plasmodium yoelii and Plasmodium vinckei: The effects of nonspecific immunostimulation on murine malaria. Exp Parasitol 1977; 43:45-53. 111. Stevenson MM, Ghadirian E, Phillips NC et al. Role of mononuclear phagocytes in elimination of Plasmodium chabaudi AS infection. Parasite Immunol 1989; 11:529-544. 112. Su Z, Fortin A, Gros P et al. Opsonin-independent phagocytosis: An effector mechanism against acute blood-stage Plasmodium chabaudi AS infection. J Infect Dis 2002; 186:1321-1329. 113. Nogami S, Watanabe J, Nakagaki K et al. Short report: Involvement of macrophage scavenger receptors in protection against murine malaria. Am J Trop Med Hyg 1998; 59:843-845. 114. Mota MM, Brown KN, Holder AA et al. Acute Plasmodium chabaudi chabaudi malaria infection induces antibodies which bind to the surface of parasitized erythrocytes and promote their phagocytosis by macrophages in vitro. Infect Immun 1998; 66:4080-4086. 115. Mota MM, Brown KN, Do Rosario VE et al. Antibody recognition of rodent malaria parasite antigens exposed at the infected erythrocyte surface: Specificity of immunity generated in hyperimmune mice. Infect Immun 2001; 69:2535-2541. 116. Rotman HL, Daly TM, Clynes R et al. Fc receptors are not required for antibody-mediated protection against lethal malaria challenge in a mouse model. J Immunol 1998; 161:1908-1912. 117. Vukovic P, Hogarth PM, Barnes N et al. Immunoglobulin G3 antibodies specific for the 19-kilodalton carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein 1 transfer protection to mice deficient in Fc-gammaRI receptors. Infect Immun 2000; 68:3019-3022. 118. Yoneto T, Waki S, Takai T et al. A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT infection. J Immunol 2001; 166:6236-6241. 119. Taylor PR, Seixas E, Walport MJ et al. Complement contributes to protective immunity against reinfection by Plasmodium chabaudi chabaudi parasites. Infect Immun 2001; 69:3853-3859. 120. Phillips RS, Mathers KE, Taylor-Robinson AW. T cells in immunity to Plasmodium chabaudi chabaudi: Operation and regulation of different pathways of protection. Res Immunol 1994; 145:406-412.

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121. Bastos KR, Barboza R, Elias RM et al. Impaired macrophage responses may contribute to exacerbation of blood-stage Plasmodium chabaudi chabaudi malaria in interleukin-12-deficient mice. J Interferon Cytokine Res 2002; 22:1191-1199. 122. Favre N, Ryffel B, Bordmann G et al. The course of Plasmodium chabaudi chabaudi infections in interferon-gamma receptor deficient mice. Parasite Immunol 1997; 19:375-383. 123. Su Z, Stevenson MM. Central role of endogenous gamma interferon in protective immunity against blood-stage Plasmodium chabaudi AS infection. Infect Immun 2000; 68:4399-4406. 124. Stevenson MM, Tam MF, Wolf SF et al. IL-12-induced protection against blood stage Plasmodium chabaudi AS requires IFN-γ and TNF-α and occurs via a nitric oxide-dependent mechanism. J Immunol 1995; 155:2545-2556. 125. Shear HL, Srinivasan R, Nolan T et al. Role of IFN-gamma in lethal and nonlethal malaria in susceptible and resistant murine hosts. J Immunol 1989; 143:2038-2044. 126. Sam H, Stevenson MM. Early IL-12 p70, but not p40, production by splenic macrophages correlates with host resistance to blood-stage Plasmodium chabaudi AS malaria. Clin Exp Immunol 1999; 117:343-349. 127. Stevenson MM, Huang DY, Podoba JE et al. Macrophage activation during Plasmodium chabaudi AS infection in resistant C57BL/6 and susceptible A/J mice. Infect Immun 1992; 60:1193-1201. 128. Martiney JA, Sherry BA, Metz CN et al. Macrophage migration inhibitory factor release by macrophages after ingestion of Plasmodium chabaudi-infected erythrocytes: Possible role in the pathogenesis of malarial anemia. Infect Immun 2000; 68:2259-2267. 129. Jaramillo M, Plante I, Ouellet N et al. Hemozoin-inducible proinflammatory events in vivo: Potential role in malaia infection. J Immunol 2004; 172:3101-3110. 130. Nguyen PH, Day N, Pram TD et al. Intraleucocytic malaria pigment and prognosis in severe malaria. Trans R Soc Trop Med Hyg 1995; 89:200-204. 131. Prada J, Malinowski J, Muller S et al. Hemozoin differentially modulates the production of interleukin-6 and tumor necrosis factor in murine malaria. Eur Cytokine Netw 1995; 6:109-112. 132. Scorza T, Magez S, Brys L et al. Hemozoin is a key factor in the induction of malaria-associated immunosuppression. Parasite Immunol 1999; 21:545-554. 133. Taramelli D, Recalcati S, Basilico N et al. Macrophage preconditioning with synthetic malaria pigment reduces cytokine production via heme iron-dependent oxidative stress. Lab Invest 2000; 80:1781-1788. 134. Taramelli D, Basilico N, Pagani E et al. The heme moiety of malaria pigment (beta-hematin) mediates the inhibition of nitric oxide and tumor necrosis factor-alpha production by lipopolysaccharide-stimulated macrophages. Exp Parasitol 1995; 81:501-511. 135. Huy NT, Trang DTX, Kariu T et al. Leukocyte activation by malarial pigment. Parasitol Int 2006; 55:75-81. 136. Jones IW, Thomsen LL, Knowles R et al. Nitric oxide synthase activity in malaria-infected mice. Parasite Immunol 1996; 18:535-538. 137. Taylor-Robinson AW, Phillips RS, Severn A et al. The role of Th1 and Th2 cells in a rodent malaria infection. Science 1993; 260:1931-1934. 138. Taylor-Robinson AW, Severn A, Phillips RS. Kinetics of nitric oxide production during infection and reinfection of mice with Plasmodium chabaudi. Parasite Immunol 1996; 18:425-430. 139. Rockett KA, Awburn MM, Rockett EJ et al. Possible role of nitric oxide in malarial immunosuppression. Parasite Immunol 1994; 16:243-249. 140. Yoneto T, Yoshimoto T, Wang CR et al. Gamma interferon production is critical for protective immunity to infection with blood-stage Plasmodium berghei XAT but neither NO production nor NK cell activation is critical. Infect Immun 1999; 67:2349-2356. 141. Nahrevanian H, Dascombe MJ. Nitric oxide and reactive nitrogen intermediates during lethal and nonlethal strains of murine malaria. Parasite Immunol 2001; 23:491-501. 142. Balmer P, Phillips HM, Maestre AE et al. The effect of nitric oxide on the growth of Plasmodium falciparum, P. chabaudi and P. berghei in vitro. Parasite Immunol 2000; 22:97-106. 143. Jacobs P, Radzioch D, Stevenson MM. Nitric oxide expression in the spleen, but not in the liver, correlates with resistance to blood-stage malaria in mice. J Immunol 1995; 155:5306-5313. 144. Favre N, Ryffel B, Rudin W. Parasite killing in murine malaria does not require nitric oxide production. Parasitology 1999; 118:139-143. 145. van der Heyde HC, Gu Y, Zhang Q et al. Nitric oxide is neither necessary nor sufficient for resolution of Plasmodium chabaudi malaria in mice. J Immunol 2000; 165:3317-3323. 146. Basir R, Dascombe MJ. Nitric oxide involvement in rodent malaria. Asia Pacif J Pharmacol 2000; 14:S72-S73. 147. Dascombe MJ, Nahrevanian H. Pharmacological assessment of the role of nitric oxide in mice infected with lethal and nonlethal species of malaria. Parasite Immunol 2003; 25:149-159.

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148. Taylor-Robinson AW, Smith ED. A dichotomous role for nitric oxide in protection against blood stage malaria infection. Immunol Lett 1999; 67:1-9. 149. Ahvazi BC, Jacobs P, Stevenson MM. Role of macrophage-derived nitric oxide in suppression of lymphocyte proliferation during blood-stage malaria. J Leukoc Biol 1995; 58:23-31. 150. Ockenhouse CF, Shear HL. Oxidative killing of the intraerythrocytic malaria parasite Plasmodium yoelii by activated macrophages. J Immunol 1984; 132:424-431. 151. Clark IA, Hunt NH. Evidence for reactive oxygen intermediates causing hemolysis and parasite death in malaria. Infect Immun 1983; 39:1-6. 152. Dockrell HM, Playfair JHL. Killing of blood-stage murine malaria parasites by hydrogen peroxide. Infect Immun 1983; 39:456-459. 153. Brinkmann V, Kaufmann SH, Simon MM et al. Role of macrophages in malaria: O2 metabolite production and phagocytosis by splenic macrophages during lethal Plasmodium berghei and self-limiting Plasmodium yoelii infection in mice. Infect Immun 1984; 44:743-746. 154. Cavacini LA, Guidotti M, Parke LA et al. Reassessment of the role of splenic leukocyte oxidative activity and macrophage activation in expression of immunity to malaria. Infect Immun 1989; 57:3677-3682. 155. Potter SM, Mitchell AJ, Cowden WB et al. Phagocyte-derived reactive oxygen species do not influence the progression of murine blood-stage malaria infections. Infect Immun 2005; 73:4941-4947. 156. Jacobs P, Radzioch D, Stevenson MM. In vivo regulation of nitric oxide production by tumor necrosis factor alpha and gamma interferon, but not by interleukin-4, during blood stage malaria in mice. Infect Immun 1996; 64:44-49. 157. Makimura S, Brinkmann V, Mossmann H et al. Chemiluminescence response of peritoneal macrophages to parasitized erythrocytes and lysed erythrocytes from Plasmodium berghei-infected mice. Infect Immun 1982; 37:800-804. 158. Li M, Li YJ. The production of reactive oxygen species in mice infected or immunized with Plasmodium berghei. Parasite Immunol 1987; 9:293-304. 159. Wozencraft AO, Croft SL, Sayers G. Oxygen radical release by adherent cell populations during the initial stages of a lethal rodent malarial infection. Immunology 1985; 56:523-531. 160. Omodeo-Sale F, Basilico N, Folini M et al. Macrophage populations of different origins have distinct susceptibilities to lipid peroxidation induced by beta-haematin (malaria pigment). FEBS Lett 1998; 433:215-218. 161. Prada J, Malinowski J, Muller S et al. Effects of Plasmodium vinckei hemozoin on the production of oxygen radicals and nitrogen oxides in murine macrophages. Am J Trop Med Hyg 1996; 54:620-624. 162. Vreden SG, van den Broek MF, Oettinger MC et al. Cytokines inhibit the development of liver schizonts of the malaria parasite Plasmodium berghei in vivo. Eur J Immunol 1992; 22:2271-2275. 163. Vreden SG, Sauerwein RW, Verhave JP et al. Kupffer cell elimination enhances development of liver schizonts of Plasmodium berghei in rats. Infect Immun 1993; 1936-1939. 164. Meis JF, Verhave JP, Jap PH et al. An ultrastructural study on the role of Kupffer cells in the process of infection by Plasmodium berghei sporozoites in rats. Parasitology 1983; 86:231-242. 165. Pradel G, Frevert U. Plasmodium sporozoites actively enter and passage through Kupffer cells prior to hepatocyte invasion. Hepatology 2001; 33:1154-1165. 166. Barnwell JW. Hepatic Kupffer cells: The portal that permits infection of hepatocytes by malarial sporozoites? Hepatology 2001; 33:1331-1333. 167. Pradel G, Garapaty S, Frevert U. Proteoglycans mediate malaria sporozoite targeting to the liver. Mol Microbiol 2002; 45:637-651. 168. Meis JF, Verhave JP, Brouwer A et al. Electron microscopic studies on the interaction of rat Kupffer cells and Plasmodium berghei sporozoites. Z Parasitenkd 1985; 71:473-483. 169. Frevert U. Sneaking in through the back entrance: The biology of malaria liver stages. Trends Parasitol 2004; 20:417-424. 170. Frevert U, Galinski MR, Hugel FU et al. Malaria circumsporozoite protein inhibits protein synthesis in mammalian cells. EMBO J 1998; 17:3816-3826. 171. Rajan TV. Why does Plasmodium have a preerythrocytic cycle? Parasitol Today 1997; 13:284-287. 172. Krzych U, Schwenk R, Guebre-Xabier M et al. The role of intrahepatic lymphocytes in mediating protective immunity induced by attenuated Plasmodium berghei sporozoites. Immunol Rev 2000; 174:1-12. 173. Steers N, Schwenk R, Bacon DJ et al. The immune status of Kupffer cells profoundly influences their responses to infectious Plasmodium berghei sporozoites. Eur J Immunol 2005; 35:2335-2346. 174. Guermonprez P, Valladeau J, Zitvogel L et al. Antigen presentation and T cell stimulation by dendritic cells. Ann Rev Immunol 2002; 20:621-667. 175. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol 2001; 1:135-145.

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176. Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: Endogenous activators of dendritic cells. Nat Med 1999; 5:1249-1255. 177. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392:245-252. 178. Stevenson MM, Urban BC. Antigen presentation and dendritic cell biology in malaria. Parasite Immunol 2006; 28:5-14. 179. Urban BC, Ferguson DJP, Pain A et al. Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 1999; 400:73-77. 180. Ocana-Morgner C, Mota MM, Rodriguez A. Malaria blood stage suppression of liver stage immunity by dendritic cells. J Exp Med 2003; 197:143-151. 181. Ing R, Segura M, Thawani N et al. Interaction of mouse dendritic cells and malaria-infected erythrocytes: Uptake, maturation and antigen presentation. J Immunol 2006; 177:441-450. 182. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nature Rev Immunol 2005; 5:953-964. 183. Artavanis-Tsakonas K, Eleme K, McQueen KL et al. Activation of a subset of human NK cells upon contact with Plasmodium falciparum-infected erythrocytes. J Immunol 2003; 171:5396-5405. 184. Dalbeth N, Gundle R, Davies RJO et al. CD56bright NK cells are enriched at inflammatory sites and can engage with monocytes in a reciprocal program of activation. J Immunol 2004; 173:6418-6426. 185. Baratin M, Roetynck S, Lépolard C et al. Natural killer cell and macrophage cooperation in MyD88-dependent innate responses to Plasmodium falciparum. Proc Natl Acad Sci USA 2005; 102:14747-14752.

CHAPTER 14

Innate Control of Toxoplasma gondii through Macrophage-Based Effector Mechanisms Gregory A. Taylor*

Abstract

M

acrophages and other host cells possess an array of effector mechanisms that restrict intracellular replication of Toxoplasma gondii in a cell autonomous manner. These effectors are diverse and include proteins such as phagocyte oxidase that catalyze the production of substances toxic to T. gondii, and proteins such as indoleamine 2,3-dioxygenase that catabolize the breakdown of nutrients essential for parasite growth. In contrast, a new family of interferon-γ-regulated effector proteins—the p47 GTPases—has recently been identified that employ a much different mechanism to suppress T. gondii growth in activated macrophages. Current theories suggest that the p47 GTPases traffick from intracellular membrane compartments to the T. gondii vacuole, where they modulate processing of the vacuole, eventually undermining the integrity of the vacuole and survival of the parasite. The essential role of p47 GTPases in controlling T. gondii is illustrated by mice that lack the proteins, and consequently, are profoundly susceptible to acute infection. In this chapter, evidence is reviewed supporting the functions of p47 GTPases and other effector proteins in macrophage-based control of T. gondii.

Introduction T. gondii is an intracellular protozoan parasite that resides in a highly modified vacuole in macrophages and other cells.1 In immunocompetent hosts, T. gondii infection is often asymptomatic, but in situations in which the immune response is compromised, such as in the cases of those with AIDS or the developing fetus, infection can result in serious disease culminating in loss of vision, neurologic complications, or death.2 Infection proceeds in two phases: an acute phase in which the rapidly replicating form of the parasite, the tachyzoite, disseminates throughout the host infecting most cell types, and a chronic phase in which the slowly dividing bradyzoite primarily inhabits brain and muscle cells. The acute phase must be met with a potent immune response to insure host survival and drive the parasite into latency. Central to a successful acute response is cell-mediated immunity in which the macrophage plays a critical role.3 Macrophages initiate the response to acute infection by secreting several cytokines including IL-12 and TNF-α, which in turn, trigger production of IFN-γ, *Gregory A. Taylor—Departments of Medicine, Immunology, and Molecular Genetics and Microbiology; Division of Geriatrics; Center for the Study of Aging and Human Development, Duke University Medical Center; and GRECC, VA Medical Center, Durham, North Carolina, U.S.A. Email: gregory.taylor @ duke.edu

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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predominantly from natural killer cells. The IFN-γ then activates effector mechanisms in macrophages, other hematopoietic cells, and nonhematopoietic cells, which enable these diverse cells to promote killing of intracellular parasites. The importance of the cytokine cascade in initiating the immune response to T. gondii is well-established: For instance, mice that lack production of IL-12, IFN-γ, or IFN-γ receptors have profoundly elevated susceptibility to the parasite.4-6 What is less clear is the identity of the IFN-γ-induced effectors and the mechanisms through which they limit survival of T. gondii in host cells. This chapter will review current knowledge of anti-toxoplasma effector mechanisms with emphasis on those that are active in the macrophage. In particular, the focus will be on a newly recognized family of IFN-γ-induced proteins, the p47 GTPases, which play a pivotal role in T. gondii control.

Reactive Oxygen Intermediates Phagocytosis of pathogens, in general, is accompanied by increases in oxygen consumption and concomitant production of reactive oxygen intermediates (ROI). These substances are highly toxic to pathogens, and consequently, they are a major weapon that aid macrophages and neutrophils as they fight intracellular protozoa and bacteria.7 Formation of ROI is stimulated by a complex of at least five phagocyte oxidase (phox) proteins, including the plasma membrane proteins gp91phox and p22phox, and cytosolic proteins p40phox, p47phox, p67phox. These proteins complex on the plasma membrane following an appropriate stimulation,8 and then catalyze the production of superoxide from molecular oxygen, using NADPH as a source of reducing potential. Subsequently, superoxide reacts spontaneously to produce ROI, including hydrogen peroxide, hydroxyl radical, and hypochlorous acid. Phagocyte oxidase activity is activated by IFN-γ, which promotes transcription of certain phox components including gp47 and gp91.9,10 ROI constitute a major mechanism that prevents growth of T. gondii in macrophages. For instance, they are necessary for the ability of mouse peritoneal macrophages to both inhibit T. gondii growth, and to kill the parasite in vitro.11 Similarly, ROI are required for the activity of human monocyte-derived macrophages against T. gondii,12 particularly activity stimulated by IFN-γ.13 Despite these findings, ROI do not seem to be required for T. gondii killing or growth restriction in all types of cells, or in response to all activating stimuli. For instance, ROI are not required for T. gondii inhibition in endothelial cells,14 nor are they required for IFN-γ-induced inhibition in astrocytes.15 This illustrates an important generalization concerning T. gondii effector mechanisms, that while multiple effectors exist, their involvement in parasite restriction occurs in a cell-specific manner, and only in response to certain activators.

Nitric Oxide It is well-established that generation of the free radical nitric oxide (NO) by macrophages increases cytotoxicity against tumor cells and pathogens.16 Nitric oxide is produced by the intracellular enzyme, nitric oxide synthase, which exists in constitutively-expressed and inducible isotypes, the principal form being inducible nitric oxide synthase (iNOS) in activated leukocytes. iNOS uses L-arginine as a nitrogenous donor to produce NO, which is, in itself, microbicidal, but also gives rise to byproducts including nitrate and nitrite that are microbicidal as well. Production of iNOS is stimulated at the transcriptional level by microbial products and cytokines, among them being IFN-γ.17 Several lines of evidence suggest that NO is an important effector of IFN-γ-mediated protection against T. gondii. These data result from largely in vitro studies that make use of the iNOS inhibitor L-NMMA to demonstrate that iNOS activity is required for cytokine-induced anti-toxoplasma activity in peritoneal macrophages, bone marrow macrophages, and microglial cells.18-20 As persuasive as these studies are, however, other evidence suggests that NO is not critical for T. gondii killing in vivo, at least during the acute phase of the infection.21 iNOS-deficient mice survive acute infection and have parasite burdens comparable to those of wild-type animals. In contrast, an important role for NO is suggested during chronic infection, as iNOS-deficient mice also demonstrate decreased survival and increased parasite burden in

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the chronic phase of infection. These data imply that much like ROI, NO is important for T. gondii control, but only in certain cell types and/ or under certain physiological conditions. Thus, the exact role of NO in temporal control of T. gondii growth in specific types of cells including the macrophage, remains to be determined.

Iron Deprivation Pathogens have an obligate requirement to obtain iron to support their growth; consequently, they have developed sophisticated mechanisms to obtain iron from their environment within the host.22 The hosts, in turn, have developed means of withholding iron from pathogens, these pathways serving, in effect, as innate immune mechanisms that prevent pathogen growth. Several iron binding proteins are present in mammalian hosts that maintain iron homoeostasis; these include ferritin that maintains intracellular iron stores, transferrin which is a serum protein involved in iron transport through the organism and into cells, and lactoferrin that is released from neutrophil granules at sites of infection.8 By modulating levels of these proteins, the host can effectively reduce iron stores on which the pathogen is dependent. For example, activated macrophages are able to increase production of tranferrin which binds intracellular iron and prevents its access to intracellular pathogens.23 Furthermore, activated macrophages have decreased transferrin receptors on their cell surface, which decreases influx of iron and additionally limits access of iron to pathogens.24 Regulation of iron stores has been implicated as a host response that may control growth of T. gondii in some settings. The evidence is, perhaps, most persuasive in rat enterocytes, in which iron and iron chelators have been used to demonstrate that IFN-γ-induced inhibition of T. gondii replication is iron-dependent.25 In contrast, it has also been shown that modulation of iron stores is not involved in T. gondii inhibition in either IFN-γ-activated human mononuclear phagocytes26 or IFN-γ-activated mouse astrocytes.15 More recent studies have shown that transferrin receptors are downregulated in mouse macrophages at 18 hours after tachyzoite infection;27 yet, it has not been demonstrated that this response assists the macrophage in restricting the parasite. Therefore, based on current data, there is no strong evidence that iron deprivation plays a role in controlling T. gondii growth in macrophages, though it may well be the case in other cells such as enterocytes.

Tryptophan Degradation Tryptophan is an essential amino acid that is required for growth of pathogens including T. gondii. Much in the same way that host cells deprive pathogens of iron as a means of control, host cells also deprive them of tryptophan. This occurs mainly through actions of the enzyme indoleamine 2,3-dioxygenase (IDO) which catabolizes tryptophan.28 In addition, metabolic products of tryptophan degradation, such as hydroxyanthranilic acid, are also toxic to pathogens. Expression of IDO is very strongly induced by IFN-γ in macrophages, while LPS, TNF-α and IFN-α/β also induce production, albeit not as strongly.8 Following T. gondii infection in vivo, expression of IDO is highly increased in lung and brain, while tryptophan concentrations in lung are greatly decreased. These effects are mediated by IFN-γ, as evidenced by the fact that IDO fluctuations are not noted in IFN-γ-deficient mice.29 As was true for the effector mechanisms previously, IDO appears to be critical for the restriction of T. gondii growth in some types of host cells but not others. For instance in one study examining human fibroblasts, the anti-T. gondii activity that was induced by IFN-γ was highly dependent on the tryptophan concentration in the cell culture medium, implicating the importance of tryptophan starvation in mediating the anti-T. gondii activity.30 In another report, IFN-γ clearly induced IDO production in human macrophages and a glioblastoma cell line; however, an IDO inhibitor was used to show that only in the glioblastoma line, and not in the macrophage line, was IDO actually involved in restriction of T. gondii growth.31 In yet a third study, human macrophages displayed IFN-γ-induced IDO induction that contributed to T. gondii growth inhibition, as determined by the reversal of growth inhibition following addition

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of tryptophan to the cell culture medium.32 In mouse macrophages, conversely, IFN-γ-induced IDO induction was not observed. Thus to reiterate, IDO plays a role in the restriction of T. gondii growth, particularly that triggered by IFN-γ, but only in certain cell types.

p47 GTPases To this point, the four effector mechanisms that have been mentioned function either by creating toxins that are microbicidal for T. gondii, or by depleting stores of key nutrients that are required for T. gondii growth. A fifth group of effector proteins, the p47 GTPases, are likely to act through a much different mechanism. Although these proteins appear to have multiple functions, current theories suggest that p47 GTPases regulate T. gondii growth largely by modifying processing of the toxoplasma vacuole, thus undermining the intracellular niche that the parasite establishes and allowing eradication from the host cell. The evidence supporting a role for these proteins as host resistance effectors will be presented following a discussion of their molecular properties. The p47 GTPases are family of 47-48 kDa proteins that exist in mice, rats, dogs, and humans;33 however, to date their genes have only been extensively studied in mice, with the focus being on IGTP,34 LRG-47,35 GTPI,36 IRG-47,37 IIGP,36 and TGTP.38,39 The expression of these proteins is strongly induced by interferons at the transcriptional level following parasitic, bacterial, or viral infection, with induction being seen in nearly all tissues. IFN-γ is the most potent inducer of transcription, acting through a STAT1-dependent pathway.40-42 Expression is also triggered by type I interferons, albeit to a lesser extent, while other cytokine have little affect on expression. IFN-induced expression seems to occur in nearly all cells types; thus, the proteins are present in both nonhematopoietic and hematopoietic cells, including macrophages. Each p47 GTPase contains consensus guanosine triphosphate (GTP)-binding motifs, much like those found in other GTPases such as Ras.43 GTPases, in general, catalyze the hydrolysis of GTP to either GDP or GMP. The guanosine moiety functions as a molecular switch that converts the protein from an active state, when GTP bound, to an inactive state, when GDP bound. Once the GTPase reaches the inactive conformation, exchange factors that exist in the cell are able to displace GDP/ GMP with GTP to reactivate the GTPase. Concerning the p47 GTPases, biochemical characterization of IGTP44 and IIGP45 has shown that the GTP-binding motifs are indeed functional, in that the proteins can bind and hydrolyze GTP. In vivo, IGTP is predominantly GTP-bound, and hence in an active state, though the proportion of protein that is GTP-bound does not seem to be affected by IFN-γ stimulation.44 Thus, this cytokine regulates p47 GTPase production at the level of transcription, but does not affect biochemical activity. Within macrophages and other cells, p47 GTPases are bound to different lipid membranes to varying degrees (see Table 1). IGTP, for instance, is >90% membrane bound, predominantly to the endoplasmic reticulum (ER),44 while LRG-47 is also >90% bound to membranes, but to the Golgi.46 In comparison, IIGP is only 60-70% membrane bound, which in this case is the ER and Golgi;46,47 furthermore, IRG-47 is <10% membrane bound, to a currently unidentified compartment.46 These data in combination with others allows division of the protein family into two subfamilies, with one branch including IGTP, LRG-47, and GTPI that tightly bind intracellular membranes, and the second branch including IIGP, IRG-47, and TGTP that have a greater fraction of soluble protein within the cell.46 Interestingly, the presence of a GKS or a GMS amino acid motif within the first GTP-binding motif of the proteins correlates with the division into the first and second subfamilies, respectively. Finally, localization to intracellular lipid membranes is mediated by different elements within the p47 GTPases. IIGP, for instance, localizes to the ER via a N-terminal myristoyl modification while in contrast, LRG-47 has no apparent post-translational lipid modifications, but localizes to the Golgi via an amphipathic helix near the C-terminus.46 Currently the picture of p47 GTPase function at the molecular level is incomplete, but a few studies have revealed facets of their activities within pathogen-infected cells. First, it has been demonstrated that p47 GTPases traffick from their intracellular compartments to

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Table 1. p47 GTPase membrane binding properties Localization to Pathogen-Containing Vacuoles

Basal Localization

Compartment

Percent Membrane Bound

Mechanism

Documented Vacuole Contents

Mechanism

LRG-47

Golgi, ER

>90%

C-terminal amphipathic helix

Latex beads; M. tuberculosis

Nucleotide dependent

IGTP

ER

> 90%

nd*

Latex beads; T. gondii

nd

IIGP1

ER, Golgi

60-70%

N-terminal myristoylation; G domain signal

Latex beads; T. gondii

Nucleotide dependent

TGTP

nd

20-30%

nd

T. gondii

nd

IRG-47

nd

<10%

nd

T. gondii

nd

* nd indicates not determined. Adapted from reference 46.

pathogen-containing phagosomes or vacuoles. This includes trafficking of LRG-47 to Mycobacterium tuberculosis-containing vacuoles in macrophages,40 trafficking of LRG-47 and IGTP to latex bead-containing vacuoles in macrophages,46,48 and trafficking of IIGP1, GTPI, IRG-47, and IGTP to T. gondii vacuoles in astrocytes.49 Note that latex beads are often used in the study vacuole maturation; the beads are particles that are actively phagocytosed by macrophages, resulting in bead-containing vacuoles that are efficiently processed by the host cell without the subversive influences of pathogens. Trafficking of LRG-47 and IGTP to latex bead vacuoles is rapid;48 in fact, LRG-47 has been shown to accumulate at the phagocytic cup as the bead enters the cells.46 Furthermore, both proteins remain concentrated on the vacuoles at high levels that vary only slightly for the first few hours after vacuole formation.48 The functional consequence of the presence of p47 GTPases in pathogen-containing vacuoles is thought to be modulation of vacuole processing. This is supported by studies using LRG-47-deficient macrophages, in which acidification and lysosomal fusion of the M. tuberculosis vacuole is reduced, paralleling a reduction in IFN-γ-induced killing of the bacteria.40 Additionally, trafficking of late endosomal and lysosomal markers to the bacterial vacuole is attenuated in LRG-47-deficient macrophages, while trafficking of early endosomal markers is unaltered. These results suggest that LRG-47, and perhaps other p47 GTPases, traffick from their intracellular membrane compartments to pathogen-containing vacuoles and drive late events in vacuole processing. However, the mechanism through which this occurs is unknown. Gene targeting studies in mice have established that p47 GTPases are critical requirements for IFN-γ-induced resistance to intracellular pathogens50 (Table 2). Importantly, different GTPases seem to be required for resistance to specific pathogens. For instance, IGTP is required for resistance to some protozoa, such as T. gondii,51 but not intracellular bacteria; in contrast, LRG-47 is required for resistance to a broader range of protozoa including T. gondii,52 and to many intracellular bacteria, including L. monocytogenes,52 M. tuberculosis,40 M. avium,53 and S. typhimurium.50 The molecular properties that underlie the differential roles of IGTP and LRG-47 in host resistance have not been determined, though it may be speculated that steady-state localization to different lipid membrane compartments could underlie their differential functions at some level.

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Table 2. Increased susceptibility to intracellular pathogens in mice lacking p47 GTPases Wild-Type

IFN-γ KO

LRG-47 KO

IGTP KO

IRG-47 KO

Intracellular Protozoa Toxoplasma gondii Leishmania major Trypanosoma cruzi

R* R R

S (acute) S S

S (acute) S S

S (acute) S R

S (chronic) R nt

Intracellular Bacteria Listeria monocytogenes Mycobacterium avium Mycobacterium tuberculosis Salmonella typhimurium

R R R R

S S S S

S S S S

R R R R

R nt R R

Virus MCMV

R

S

R

R

R

* The responses of mice with targeted deletions in the indicated proteins to experimental pathogens are scored as normal resistance (R) or increased susceptibility. nt indicates not tested.

Specifically concerning T. gondii, the role of IGTP in host resistance has been the most extensively characterized. Mice that lack IGTP succumb rapidly to intraperitoneal infection of an avirulent T. gondii strain.51 The loss of resistance is profound, as IGTP-deficient mice are unable to restrict spread of the parasite, leading to lethality within the same timeframe as that seen in IFN-γ-deficient mice. Similar losses in resistance are seen in LRG-47-deficent mice, indicating that these GTPases have essential, yet nonredundant roles in resistance.52 Further studies have shown that IGTP is required for both the acute and chronic phases of infection, though its role during the chronic phase does not seem to be as critical, in that resistance is only slightly attenuated in its absence.42 In contrast to these results, IRG-47-deficient mice have normal acute resistance to T. gondii, and only modestly impaired chronic resistance.52 Thus, current data suggest that the subfamily of p47 GTPases that contains IGTP and LRG-47 may have a more dominant role in resistance to T. gondii. What is the mechanism through which p47 GTPases regulate resistance to T. gondii? Conclusive data are yet to be presented, but the preponderance of current studies suggest that IGTP and LRG-47 are able to promote intracellular killing of T. gondii, particularly that induced by IFN-γ. In primary macrophage cultures, IGTP or LRG-47-deficiency, but not IRG-47 deficiency, leads to impaired IFN-γ-induced killing of T. gondii.48 Further, the role of IGTP in regulating T. gondii killing is not limited to macrophages: Reciprocal bone marrow chimera studies show that IGTP is required in both hematopoietic and nonhematopoietic compartments for acute T. gondii resistance,42 and studies of astrocytes cultures indicate that IGTP is required for IFN-γ-induced T. gondii killing in these cells.54 Absence of IGTP and LRG-47 does not affect other IFN-γ-induced effector mechanisms, including NO and IDO; thus, the p47 GTPases represent truly novel anti-toxoplasma activities in macrophages. The current challenge is to determine the mechanism through which the p47 GTPases regulate intracellular killing of T. gondii. It was mentioned above that LRG-47 drives processing of M. tuberculosis vacuoles, and this line of thinking could be extended to suggest that LRG-47 and IGTP drive processing of T. gondii vacuoles. Nevertheless, it is not clear how the GTPases might do this. One possibility is that they promote membrane fusion events between nascent vacuoles and late endosomal/ lysosomal compartments, in order to facilitate

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movement of the vacuole along the maturation pathway. Certainly, there are precedents for this type of activity, which would include the rab proteins, a group of GTPases that mediate early and late endosomal fusions with pathogen-containing vacuoles.55,56 This remains a viable theory, but at least in one respect, the p47 GTPase do not fit this model, as they are acquired by nascent vacuoles very rapidly and remain at consistently high levels. This is in contrast to classical endosomal and lysosomal markers known to influence maturation, such as the rabs, which show more dynamic and transient localization to maturing vacuoles. An alternative model is that the p47 GTPases impact formation or stability of the T. gondii vacuole. This theory is supported by a recent study focusing on IIGP1 which showed that overexpression of this protein resulted in vesiculation of the vacuolar membrane, eventually leading to complete disruption of the vacuole and elimination of the parasite.49 The mechanism through which IIGP1 and other p47 GTPases might regulate vacuole stability has not been identified; yet one possibility would be that the proteins modify interaction of the vacuole with the cytoskeleton of the host cell, which would in turn affect movement and stability of the vacuole. In fact, it has been shown that IIGP1 binds to the cellular protein hook3, a microtubule binding protein,57 thus supporting such a model. While future work will undoubtedly address in more detail the means through which the p47 GTPases regulate vacuole maturation and vacuole stability, currently we are left with the provocative findings that the p47 GTPases are critical factors that are essential for IFN-γ-induced clearance of T. gondii from macrophages and other cells.

References 1. Sibley LD. Toxoplasma gondii: Perfecting an intracellular life style. Traffic 2003; 4(9):581-6. 2. Luft BJ, Remington JS. Toxoplasmic encephalitis in AIDS. Clin Infect Dis 1992; 15(2):211-22. 3. Robben PM, LaRegina M, Kuziel WA et al. Recruitment of Gr-1+ monocytes is essential for control of acute toxoplasmosis. J Exp Med 2005; 201(11):1761-9. 4. Deckert-Schluter M, Rang A, Weiner D et al. Interferon-gamma receptor-deficiency renders mice highly susceptible to toxoplasmosis by decreased macrophage activation. Lab Invest 1996; 75(6):827-41. 5. Scharton-Kersten TM, Wynn TA, Denkers EY et al. In the absence of endogenous IFN-gamma, mice develop unimpaired IL-12 responses to Toxoplasma gondii while failing to control acute infection. J Immunol 1996; 157(9):4045-54. 6. Suzuki Y, Orellana MA, Schreiber RD, Remington JS. Interferon-gamma: The major mediator of resistance against Toxoplasma gondii. Science 1988; 240(4851):516-8. 7. Iles KE, Forman HJ. Macrophage signaling and respiratory burst. Immunol Res 2002; 26(1-3):95-105. 8. Stafford JL, Neumann NF, Belosevic M. Macrophage-mediated innate host defense against protozoan parasites. Crit Rev Microbiol 2002; 28(3):187-248. 9. Cassatella MA, Bazzoni F, Flynn RM et al. Molecular basis of interferon-gamma and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components. J Biol Chem 1990; 265(33):20241-6. 10. Nathan CF, Murray HW, Wiebe ME, Rubin BY. Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J Exp Med 1983; 158(3):670-89. 11. Murray HW, Juangbhanich CW, Nathan CF, Cohn ZA. Macrophage oxygen-dependent antimicrobial activity. II. The role of oxygen intermediates. J Exp Med 1979; 150(4):950-64. 12. Robertson AK, Cross AR, Jones OT, Andrew PW. The use of diphenylene iodonium, an inhibitor of NADPH oxidase, to investigate the antimicrobial action of human monocyte derived macrophages. J Immunol Methods 1990; 133(2):175-9. 13. Murray HW, Rubin BY, Carriero SM et al. Human mononuclear phagocyte antiprotozoal mechanisms: Oxygen-dependent vs oxygen-independent activity against intracellular Toxoplasma gondii. J Immunol 1985; 134(3):1982-8. 14. Woodman JP, Dimier IH, Bout DT. Human endothelial cells are activated by IFN-gamma to inhibit Toxoplasma gondii replication. Inhibition is due to a different mechanism from that existing in mouse macrophages and human fibroblasts. J Immunol 1991; 147(6):2019-23. 15. Halonen SK, Weiss LM. Investigation into the mechanism of gamma interferon-mediated inhibition of Toxoplasma gondii in murine astrocytes. Infect Immun 2000; 68(6):3426-30.

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16. MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol 1997; 15:323-50. 17. Xie QW, Whisnant R, Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. J Exp Med 1993; 177(6):1779-84. 18. Adams LB, Hibbs JB Jr, Taintor RR, Krahenbuhl JL. Microbiostatic effect of murine-activated macrophages for Toxoplasma gondii. Role for synthesis of inorganic nitrogen oxides from L-arginine. J Immunol 1990; 144(7):2725-9. 19. Chao CC, Anderson WR, Hu S et al. Activated microglia inhibit multiplication of Toxoplasma gondii via a nitric oxide mechanism. Clin Immunol Immunopathol 1993; 67(2):178-83. 20. Bohne W, Heesemann J, Gross U. Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: A possible role for nitric oxide in triggering stage conversion. Infect Immun 1994; 62(5):1761-7. 21. Scharton-Kersten TM, Yap G, Magram J, Sher A. Inducible nitric oxide is essential for host control of persistent but not acute infection with the intracellular pathogen Toxoplasma gondii. J Exp Med 1997; 185(7):1261-73. 22. Jurado RL. Iron, infections, and anemia of inflammation. Clin Infect Dis 1997; 25(4):888-95. 23. Byrd TF, Horwitz MA. Lactoferrin inhibits or promotes Legionella pneumophila intracellular multiplication in nonactivated and interferon gamma-activated human monocytes depending upon its degree of iron saturation. Iron-lactoferrin and nonphysiologic iron chelates reverse monocyte activation against Legionella pneumophila. J Clin Invest 1991; 88(4):1103-12. 24. Byrd TF, Horwitz MA. Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. J Clin Invest 1989; 83(5):1457-65. 25. Dimier IH, Bout DT. Interferon-gamma-activated primary enterocytes inhibit Toxoplasma gondii replication: A role for intracellular iron. Immunology 1998; 94(4):488-95. 26. Murray HW, Granger AM, Teitelbaum RF. Gamma interferon-activated human macrophages and Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani: Antimicrobial role of limiting intracellular iron. Infect Immun 1991; 59(12):4684-6. 27. Dziadek B, Dytnerska-Dzitko K, Dlugonska H. The modulation of transferrin receptors level on mouse macrophages and fibroblasts by Toxoplasma gondii. Pol J Microbiol 2004; 53(Suppl):75-80. 28. Taylor MW, Feng GS. Relationship between interferon-gamma, indoleamine 2,3-dioxygenase, and tryptophan catabolism. Faseb J 1991; 5(11):2516-22. 29. Fujigaki S, Takemura M, Hamakawa H et al. The mechanism of interferon-gamma induced anti Toxoplasma gondii by indoleamine 2,3-dioxygenase and/or inducible nitric oxide synthase vary among tissues. Adv Exp Med Biol 2003; 527:97-103. 30. Pfefferkorn ER. Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan. Proc Natl Acad Sci USA 1984; 81(3):908-12. 31. MacKenzie CR, Langen R, Takikawa O, Daubener W. Inhibition of indoleamine 2,3-dioxygenase in human macrophages inhibits interferon-gamma-induced bacteriostasis but does not abrogate toxoplasmastasis. Eur J Immunol 1999; 29(10):3254-61. 32. Murray HW, Szuro-Sudol A, Wellner D et al. Role of tryptophan degradation in respiratory burst-independent antimicrobial activity of gamma interferon-stimulated human macrophages. Infect Immun 1989; 57(3):845-9. 33. Bekpen C, Hunn JP, Rohde C et al. The interferon-inducible p47 (IRG) GTPases in vertebrates: Loss of the cell autonomous resistance mechanism in the human lineage. Genome Biol 2005; 6(11):R92. 34. Taylor GA, Jeffers M, Largaespada DA et al. Identification of a novel GTPase, the inducibly expressed GTPase, that accumulates in response to interferon gamma. J Biol Chem 1996; 271(34):20399-405. 35. Sorace JM, Johnson RJ, Howard DL, Drysdale BE. Identification of an endotoxin and IFN-inducible cDNA: Possible identification of a novel protein family. J Leukoc Biol 1995; 58(4):477-84. 36. Boehm U, Guethlein L, Klamp T et al. Two families of GTPases dominate the complex cellular response to IFN-gamma. J Immunol 1998; 161(12):6715-23. 37. Gilly M, Wall R. The IRG-47 gene is IFN-gamma induced in B cells and encodes a protein with GTP-binding motifs. J Immunol 1992; 148(10):3275-81. 38. Carlow DA, Marth J, Clark-Lewis I, Teh HS. Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif. J Immunol 1995; 154(4):1724-34.

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39. Lafuse WP, Brown D, Castle L, Zwilling BS. Cloning and characterization of a novel cDNA that is IFN-gamma-induced in mouse peritoneal macrophages and encodes a putative GTP-binding protein. J Leukoc Biol 1995; 57(3):477-83. 40. MacMicking JD, Taylor GA, McKinney JD. Immune control of tuberculosis by IFN-gammainducible LRG-47. Science 2003; 302(5645):654-9. 41. Gavrilescu LC, Butcher BA, Del Rio L et al. STAT1 is essential for antimicrobial effector function but dispensable for gamma interferon production during Toxoplasma gondii infection. Infect Immun 2004; 72(3):1257-64. 42. Collazo CM, Yap GS, Hieny S et al. The function of gamma interferon-inducible GTP-binding protein IGTP in host resistance to Toxoplasma gondii is Stat1 dependent and requires expression in both hematopoietic and nonhematopoietic cellular compartments. Infect Immun 2002; 70(12):6933-9. 43. Wennerberg K, Rossman KL, Der CJ. The Ras superfamily at a glance. J Cell Sci 2005; 118(Pt 5):843-6. 44. Taylor GA, Stauber R, Rulong S et al The inducibly expressed GTPase localizes to the endoplasmic reticulum, independently of GTP binding. J Biol Chem 1997; 272(16):10639-45. 45. Uthaiah RC, Praefcke GJ, Howard JC, Herrmann C. IIGP1, an interferon-gamma-inducible 47-kDa GTPase of the mouse, showing cooperative enzymatic activity and GTP-dependent multimerization. J Biol Chem 2003; 278(31):29336-43. 46. Martens S, Sabel K, Lange R et al. Mechanisms regulating the positioning of mouse p47 resistance GTPases LRG-47 and IIGP1 on cellular membranes: Retargeting to plasma membrane induced by phagocytosis. J Immunol 2004; 173(4):2594-606. 47. Zerrahn J, Schaible UE, Brinkmann V et al. The IFN-inducible Golgi- and endoplasmic reticulum- associated 47-kDa GTPase IIGP is transiently expressed during listeriosis. J Immunol 2002; 168(7):3428-36. 48. Butcher BA, Greene RI, Henry SC et al. p47 GTPases regulate Toxoplasma gondii survival in activated macrophages. Infect Immun 2005; 73(6):3278-86. 49. Martens S, Parvanova I, Zerrahn J et al. Disruption of Toxoplasma gondii parasitophorous vacuoles by the mouse p47-resistance GTPases. PLoS Pathogens 2005; 1(3):e24. 50. Taylor GA, Feng CG, Sher A. p47 GTPases: Regulators of immunity to intracellular pathogens. Nat Rev Immunol 2004; 4(2):100-9. 51. Taylor GA, Collazo CM, Yap GS et al. Pathogen-specific loss of host resistance in mice lacking the IFN-gamma-inducible gene IGTP. Proc Natl Acad Sci USA 2000; 97(2):751-5. 52. Collazo CM, Yap GS, Sempowski GD et al. Inactivation of LRG-47 and IRG-47 reveals a family of interferon gamma-inducible genes with essential, pathogen-specific roles in resistance to infection. J Exp Med 2001; 194(2):181-8. 53. Feng CG, Collazo-Custodio CM, Eckhaus M et al. Mice deficient in LRG-47 display increased susceptibility to mycobacterial infection associated with the induction of lymphopenia. J Immunol 2004; 172(2):1163-8. 54. Halonen SK, Taylor GA, Weiss LM. Gamma interferon-induced inhibition of Toxoplasma gondii in astrocytes is mediated by IGTP. Infect Immun 2001; 69(9):5573-6. 55. Pfeffer S, Aivazian D. Targeting Rab GTPases to distinct membrane compartments. Nat Rev Mol Cell Biol 2004; 5(11):886-96. 56. Vieira OV, Botelho RJ, Grinstein S. Phagosome maturation: Aging gracefully. Biochem J 2002; 366(Pt 3):689-704. 57. Kaiser F, Kaufmann SH, Zerrahn J. IIGP, a member of the IFN inducible and microbial defense mediating 47 kDa GTPase family, interacts with the microtubule binding protein hook3. J Cell Sci 2004; 117(Pt 9):1747-56.

CHAPTER 15

Phagocyte Effector Functions against Leishmania Parasites Christian Bogdan*

Abstract

L

eishmania parasites are sandfly-transmitted protozan pathogens that cause a spectrum of important diseases in humans, but have also served as important model organisms for the characterization of antimicrobial effector mechanisms. In both mice and humans NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) seem to be most relevant for the control and killing of intracellular Leishmania, but oxygen-independent pathways might also exist. This chapter will review the in vitro and in vivo evidence for the antileishmanial function of phox and iNOS.

Introduction Leishmania parasites are flagellated protozoan pathogens that are transmitted between mammalian organisms by blood-sucking sandflies of the genus Phlebotomus or Lutzomyia. Inoculation of immunocompetent humans can lead to inapparent infections or, depending on the parasite species and strain, to various forms of clinical disease. These include localized, chronic, but ultimately self-healing skin lesions (cutaneous leishmaniosis, e.g., caused by L. major, L. tropica and L. infantum in the Old World and by L. braziliensis, L. mexicana, L. amazonensis, and L. guyanensis in the New World); the progressive destruction of mucosal tissues (mucocutaneous leishmaniosis, e.g., after an infection with L. braziliensis); and the infiltration of spleen, liver and bone marrow (visceral leishmaniosis, e.g., after infection with L. donovani, L. infantum or L. chagasi), which is fatal if untreated.1,2 Leishmaniosis is prevalent on all five continents and belongs to the ten most common parasitic diseases in the world.3,4 In addition to many endemic foci several dramatic outbreaks of cutaneous or visceral disease have been reported in recent years, most notably in Afghanistan, Pakistan, and Sudan.4,5 Whereas Leishmania exist in an extracellular, motile, promastigote form (carrying a flagellum) in the sandfly vectors, they rapidly transform into amastigotes when taken up by phagocytic cells of the mammalian host. Although Langerhans cells, granulocytes, dendritic cells and fibroblasts can endocytose Leishmania parasites in vitro and contribute to the initial survival, degradation, processing and presentation, and/or late phase evasion of the pathogen in vivo,6-9 macrophages are thought to be the key effector cells that account for the massive decrease in the parasite burden during the clinical resolution of the disease and for the containment of residual parasites during the subsequent phase of lifelong persistence. During the past 30 years detailed mouse studies and analyses of infected humans have identified CD4+ type 1 T helper *Christian Bogdan—Abteilung Mikrobiologie und Hygiene-Institut für Medizinische Mikrobiologie und Hygiene, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Straße 11, D-79104 Freiburg, Germany. Email: [email protected]

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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cells, CD8+ T cells, NK cells and NKT cells as sources of interferon (IFN)-γ, which was shown to be indispensable for macrophage control of all Leishmania species investigated to date10-12 (Fig. 1). Other signals that stimulate macrophages for killing of intracellular Leishmania amastigotes may include (a) the interaction of CD40 ligand, tumor necrosis factor (TNF), or intercellular adhesion molecule (ICAM-1, CD54) expressed on the T cell membrane with CD40, TNF receptor or leukocyte-functional-antigen-1 (LFA-1, CD11a/CD18) on the surface of macrophages;13-16 (b) the generation of type I interferons (IFN-α/β);17,18 and (c) the ligation of macrophage Fc receptors.19 A number of oxygen-dependent and -independent effector mechanisms have been invoked to operate against Leishmania, including the NADPH phagocyte oxidase (phox) and the inducible nitric oxide synthase (iNOS) pathways, 20 the IFN-γ-inducible indolamine-2,3-dioxygenase pathway,21 as well as various antimicrobial peptides and proteins including the saposin-like proteins,22 the dermaseptins23 and the temporins.24 This chapter presents the well-studied oxygen-dependent antileishmanial effector mechanisms that operate in macrophages and other myeloid cells.

Figure 1. Some of the components of the immune system required for the control of Leishmania major during the early (innate), acute and chronic phase of infection. During the innate phase of infection NK cells are activated by IL-12, IL-18 and type I interferons (IFN-α/β), presumably released by macrophages (MΦ) and dendritic cells (DC), and possibly also by other cells (?), in response to Leishmania infection. Inducible NO synthase (iNOS) is initially induced via IFN-α/β in the infected skin; subsequently, IFN-γ (released by NK cells, Th1 cells, CD8+ T cells and possibly also by NKT cells) becomes the dominant inducer of iNOS (with TNF synthesized by macrophages being an important coinducer). During the acute and chronic phase of infection iNOS is prominently expressed in the skin lesion and the draining lymph node. NADPH oxidase (or, in the absence of arginine, also uncoupled iNOS) synthesize superoxide (O2-). Both NO and O2- (as well as subsequent reactive nitrogen and oxygen intermediates) contribute to the control of Leishmania parasites in the tissue. After the clinical resolution of the disease Leishmania parasites persist in lymphoid tissue, with macrophages, DCs and fibroblasts as host cells. In the case of L. major the persisting parasites are under the control of iNOS-derived NO. For references see text.

Phagocyte Effector Functions against Leishmania Parasites

195

NADPH Oxidase Background The key enzyme for the generation of reactive oxygen intermediates (ROI) is the phagocyte NADPH oxidase (phox), a NADPH:O2-oxidoreductase, which assembles as a multimer enzyme complex on the plasma membrane, but also on intracellular membrane compartments such as the specific granules of neutrophils. During phagocytosis the phox is internalized to the membrane of phagocytic vacuoles. In resident phagocytes the catalytic core unit of the phox, the flavocytochrome b558, is constitutively expressed as an heterodimeric integral membrane protein that consists of a glycosylated 91 kDa protein (gp91phox, a member of the Nox/Duox family) and a nonglycosylated 22 kDa subunit (p22phox). When phagocytes encounter microbial pathogens, bacterial peptides (e.g., N-formyl-methionyl-leucyl-phenylalanine), fungal cell wall components (e.g., zymosan), immune complexes, chemoattractants (IL-8, leukotriene, C5a, platelet-activating factor) or various experimental compounds (e.g., phorbol esters that activate protein kinase C), at least four cytosolic proteins (p40phox, p47phox, p67phox, small GTPase Rac) are translocated to the membrane, where they associate with the flavocytochrome to form the active phox. The process of translocation requires specific phosphorylation events (e.g., of p47phox). The active phox enzyme transfers electrons from NADPH to molecular oxygen and generates superoxide anions (O2-) in granulocytes, monocytes and macrophages, a process which is termed “oxidative burst”. O2- can dismutate into molecular oxygen (partly in the form of highly reactive singlet oxygen) and hydrogen peroxide (H2O2). This reaction occurs spontaneously at acidic pH (e.g., in phagolysosomes) or is catalyzed by superoxide dismutases (SOD) found in the cytosol or mitochondria.25-27 In granulocytes and monocytes myeloperoxidase (MPO) converts H2O2 in the presence of halide anions (e.g., Cl-) to a variety of toxic products including hypohalous acids (e.g., HOCl), hydroxyl radicals (OH-), and singlet oxygen (1O2).27

Evidence for Antileishmanial Activity of Reactive Oxygen Intermediates in Vitro In vitro studies showed that extracellular L. donovani, L. chagasi or L. tropica promastigotes or amastigotes are killed by H2O2, which possibly is transformed into highly toxic hydroxyl radicals (OH-) via a parasite iron-catalyzed Fenton reaction.28-31 The activity of H2O2 was greatly enhanced in the presence of peroxidase and halide.29 The use of cell-free enzyme systems that generated several different ROI species (H2O2, O2-, OH., 1O2) and of various ROI scavengers and quenchers revealed that H2O2 was necessary and sufficient to mediate killing of Leishmania promastigotes.28 L. tropica, L. guyanensis, L. donovani, or L. chagasi promastigotes stimulated resting or lymphokine-activated mouse or human macrophages for the release of H2O2 and O2-, which was paralleled by a rapid destruction of the promastigotes.28,29,32-35 Conversely, resting macrophage cell-lines (J774G8, IC-21) that were unable to generate ROI upon infection with L. donovani or L. tropica promastigotes failed to exhibit antileishmanial activity.36,37 Addition of catalase (but not of SOD or other ROI scavengers or quenchers) to nonactivated or lymphokine-activated mouse macrophages infected with L. donovani or L. tropica promastigotes resulted in a 2- to 5-fold increase of the number of intracellular parasites at 4 and 18 hours of infection.28,36,38 In contrast, SOD was reported to abrogate the elevated antileishmanial activity of L. donovani-infected mouse peritoneal macrophages after stimulation with muramyl dipeptide at 20 hours of infection, whereas catalase and other ROI scavengers were inactive.39 Apocynin, an inhibitor of the assembly of the functional phox complex, completed suppressed the release of ROI by mouse peritoneal macrophages in response to L. guyanensis promastigotes and prevented the subsequent elimination of L. guyanensis amastigotes by otherwise nonstimulated macrophages.35 In the case of human macrophages the addition of the O2- scavenger TEMPOL increased the

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number of intracellular parasites in nonactivated cells after 24 hours of infection with L. chagasi promastigotes, but did not revert the parasite killing observed in IFN-γ stimulated macrophages.34 Compared to promastigote Leishmania, amastigotes elicited only a weak H2O2 and O2release by resting or cytokine-pretreated mouse macrophages.29,38,40 However, cytokine activation of primary mouse macrophages infected with L. donovani amastigotes killed more than 90% of the intracellular parasites after 72 or 96 hours of infection, which was reversed by catalase, glucose deprivation or prior treatment with phorbol myristate acetate (which depletes the cells of phox activity).29,38 All these findings support the idea that ROI (notably H2O2, but also O2-) released by macrophages in response to Leishmania promastigotes and/or cytokines contribute to the control of the parasites. However, a number of studies published during the past 20 years yielded results that clearly argue against an essential and sufficient role of ROI for the killing of intracellular Leishmania parasites. First, Leishmania promastigotes of the highly infective, stationary growth phase as well as Leishmania amastigotes were much less susceptible to killing by H2O2 than promastigotes of the hypoinfective logarithmic growth phase.30,38 At the same time amastigotes contained 3- or 14-fold higher concentrations of catalase and glutathione peroxidase than promastigotes, although these levels were still much lower than those found in lysates of the parasite Toxoplasma gondii that was entirely resistant to H2O2.38 Second, a macrophage cell line (IC-21) that failed to generate ROI after L. donovani infection and lymphokine activation still efficiently killed intracellular L. donovani amastigotes.37 Third, mouse peritoneal macrophages activated with lipopolysaccharide showed a strong respiratory burst, but were unable to kill intracellular L. enriettii amastigotes unless they were additionally stimulated with recombinant IFN-γ.41 Fourth, after infection with pro- or amastigotes the fate of intracellular parasites was comparable in unstimulated peritoneal or bone marrow-derived macrophages from wildtype and gp91phox gene-deleted mice; upon activation with IFN-γ and TNF the capacity of gp91phox-/- macrophages to kill intracellular L. major was at least as prominent as of wildtype macrophages.42 These findings on the killing of Leishmania by gp91phox-/- macrophages clearly contrast with the earlier studies that used O2- and H2O2 quenchers or scavengers (see above). However, several observations help to explain the discrepant findings and the unaltered antileishmanial activity of phagocytes in the absence of phox: (1) Various species and isolates of Leishmania promastigotes and amastigotes were reported to be resistant to O2- due to the expression of parasite superoxide dismutases.28,43-46 Therefore, gp91phox-/- macrophages infected with O2—resistant Leishmania can show the same antileishmanial activity as wildtype macrophages. Furthermore, the lipophosphoglycan on the surface of Leishmania promastigotes and the proteophosphoglycans of pro- and amastigotes confer resistance to various species of ROI.47-49 (2) Intracellular promastigotes and amastigotes of several species and strains of Leishmania were found to actively suppress the oxidative burst of neutrophils, monocytes and macrophages.50-53 Possible underlying mechanisms include the inhibition of protein kinase C by the promastigote lipophosphoglycan,54 the blocking of phagosomal maturation (and of the functional assembly of phox at the phagosome membrane) via accumulation of periphagosomal F-actin,55,56 and the degradation of heme via amastigote-induced induction of heme oxygenase-1, which disrupts the maturation of gp91phox.57 (3) O2- or H2O2 can be generated in a phox-independent manner (e.g., via xanthine oxidase or the mitochondrial electron transport chain).27,58 (4) The applied quenchers or scavengers of ROI might lack specificity or contain previously not recognized contaminants. For example, commercial preparations of catalase were shown to inhibit the synthesis of nitric oxide (NO) by cytokine-activated macrophages as a result of the presence of arginase (which deprived the culture medium of arginine, the substrate of NO synthase)59 or an activity that depleted tetrahydrobiopterin (THB), an essential cofactor of the NO synthases.60

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Evidence for Antileishmanial Activity of Reactive Oxygen Intermediates in Vivo The role of ROI for the control of Leishmania parasites in vivo and the clinical resolution of the disease was studied in mice deficient for the flavocytochrome b unit of the phox (gp91phox-/ ). In experimental murine visceral leishmaniasis caused by L. donovani, the absence of gp91phox caused a transiently enhanced parasite burden in the liver, but did not alter the overall course of infection; the effect on the parasite load in the spleen was not investigated.61 In an independent second study using a different strain of L. donovani, gp91phox-/- mice did not exhibit significantly increased parasite loads in the liver or spleen compared to the wildtype controls.62 In experimental cutaneous leishmaniasis caused by L. major, gp91phox-deficiency did not lead to more severe dermal lesions42,62 and only to a very limited increase of the number of parasites in the skin and draining lymph nodes during the acute phase of infection.42 In contrast, phox-deficiency caused parasite persistence in the spleen at high levels and splenomegaly.42 After resolution of the primary skin lesions, phox-deficiency was associated with the recurrence of skin lesions which spontaneously occurred in ca. 60% of the mice after an infection period of 200 to 400 days.42 Unlike the L. major model, infections of mice with L. braziliensis did not require phox activity for parasite control in the spleen (C. Bogdan, unpublished observations). To date there is only circumstantial and indirect evidence for an antileishmanial function of phox in humans. In a Spanish patient with chronic granulomatous disease due to a mutation of p47phox visceral leishmaniasis was diagnosed along with several severe bacterial infections.63 In Indian patients with visceral leishmaniasis (kala-azar) the levels of phox and myeloperoxidase activity in peripheral blood monocytes were lower compared to healthy controls.64

Inducible Nitric Oxide Synthase Background A seminal finding in macrophage research was the discovery that mouse macrophages activated by IFN-γ (± TNF) and/or microbial products (e.g., LPS) require the amino acid L-arginine for the killing of certain tumors, bacteria, protozoa, fungi or parasites.65-73 Subsequent research revealed that activated macrophages express an enzyme termed inducible (or type 2) NO synthase (iNOS, NOS2), which converts the amino acid L-arginine and molecular oxygen into Nω-hydroxy-L-arginine and further into citrulline and the cytotoxic, gaseous molecule nitric oxide (NO)74 (Fig. 2). NO can interact with oxygen and ROI to form further reactive nitrogen intermediates (RNI) with antiviral or antimicrobial potential such as nitrogen dioxide (.NO2) or peroxynitrite.20 Once induced iNOS forms an active dimer that utilizes multiple cofactors and cosubstrates (THB, FAD, FMN, NADPH, calmodulin, heme, thiol) for the complex oxidoreduction of arginine.75 In macrophages iNOS is positively and negatively regulated by cytokines and microbial products at the level of transcription, mRNA stability, translation, and protein stability.76 More recently, it was found that the strongly suppressed production of NO at low concentrations of arginine does not only reflect the lack of iNOS substrate, but is also the consequence of a strikingly reduced synthesis of iNOS protein.77 Arginine depletion can result from the activity of arginase I, which converts arginine into urea and ornithine and is readily induced in macrophages by cytokines (IL-4, IL-13, TGF-β)78,79 (Fig. 2). IL-4, IL-13 and TGF-β are potent suppressors of iNOS expression,76 and at least in the case of IL-4 and IL-13 part of their iNOS suppressive activity is due to the upregulation of arginase I in macrophages.77,80 Arginases are also expressed by microbial pathogens,81,82 which due to the inhibition of NO production can facilitate their survival in the host.81 In addition to direct static or cidal effects on infectious pathogens, NO and other RNI can also exert indirect antimicrobial effects due to their numerous immunomodulatory functions, which include the regulation of host and immune cell adhesion and migration, T cell differentiation, cytokine production, and NK cell cytotoxicity.76,83-85

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Figure 2. Inducible NO synthase (NO) and arginase as the key enzymatic pathways of macrophages metabolizing L-arginine. CAT, cationic aminoacid transporter. Arg, L-arginine.

Evidence for the Antileishmanial Activity of Reactive Nitrogen Intermediates in Vitro Early during NO-Leishmania research extracellular pro- or amastigotes of different Leishmania species were shown to be killed by NO gas as well as by a variety of chemical NO donors, e.g., acidic sodium nitrite, S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO) and diethylenetriamine-NO-adducts (DETA-NO).86 Some NO donors were also reported to reach intracellular Leishmania residing in macrophages, but damage to the host cells has been a matter of concern and NO donors which selectively target intraphagosomal Leishmania are currently designed.87 Strains of Leishmania parasites with lower in vitro susceptibility to NO have been described.88 In activated mouse macrophages the endogenous L-arginine-iNOS-NO pathway was convincingly shown to exert killing activity against all tested Leishmania species by four different experimental approaches, i.e., the removal of arginine from the culture medium (e.g., by arginase),71 the inhibition of iNOS activity by L-arginine substrate analogues,71 the downregulation of iNOS mRNA, protein or activity by suppressive cytokines,77,89-94 and the use of iNOS gene-deleted macrophages.17,42 In all cases iNOS-dependent generation of NO from L-arginine turned out to be essential for the elimination of intracellular Leishmania amastigotes by macrophages. Activation of various populations of mouse macrophages for the induction of iNOS and the killing of Leishmania was achieved by IFN-γ plus TNF,71,72,95-97 IFN-γ plus IL-4,98 IFN-γ plus LPS,99-103 IFN-α/β plus Leishmania,17,18 chemokines (MCP-1, MIP-1α),104 CD40/ CD40L and LFA-1/ICAM-1,16 and by Toll-like receptor (TLR)-4 (LPS) and TLR-9 ligands (CpG oligodesoxynucleotides).105 Despite claims to the contrary there are unequivocal data that human macrophages can also be activated for the expression of iNOS (reviewed in refs. 76,83,106). Although the optimal stimuli for the induction of iNOS are distinct from the mouse system, NO-dependent antileishmanial activity of human macrophages has been repeatedly reported.34,86,107,108

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The mechanism of action of NO against Leishmania parasites is poorly understood to date. Published data support the concept that NO leads to multiple effects within the parasite, including the loss of iron from iron-sulfur prosthetic groups (in particular cis-aconitase),99,109 the inactivation of parasite cysteine proteinase (and other proteins) by S-transnitrosylation,110,111 and the proteasome-dependent, oligonucleosomal fragmentation of genomic DNA.112 Alternatively, it has been proposed that the antileishmanial activity of iNOS is related to the metabolic intermediate of the L-arginine-iNOS-NO reaction, Nω-hydroxy-L-arginine (Fig. 2), a known inhibitor of arginases,113 which was found to be cytotoxic to intracellular L. major or L. infantum parasites in otherwise untreated (i.e., no cytokine stimulation) macrophages.114 A subsequent study showed that Nω-hydroxy-L-arginine partially inhibited the arginase activity of extracellular L. major promastigotes, but did not affect the viability of the parasites.115 In macrophages that had been activated for the expression of arginase activity by IL-4, parasite growth was impeded by the addition of Nω-hydroxy-L-arginine. In this case, exogenously added Nω-hydroxy-L-arginine blocked the arginase activity of the host cell, i.e., the synthesis of ornithine and of polyamines, which led to reduced growth of the intracellular parasite.115 However, these results did not provide evidence that the shortlived intermediate Nω-hydroxy-L-arginine endogenously synthesized after induction of iNOS accounts for the antileishmanial effect of the L-arginine-iNOS-NO pathway.

Evidence for the Antileishmanial Activity of Reactive Nitrogen Intermediates in Mice L. major was the first infectious pathogen for which a L-arginine- and NO-dependent control was documented in vivo.116 Studies with iNOS k.o. mice revealed that iNOS-derived NO was essential for the control of different species of Leishmania such as L. major,17 L. donovani61 and L. mexicana.117 Infection of iNOS k.o. mice with L. major led to nonhealing cutaneous lesions and strikingly increased parasite numbers in the skin and draining lymph nodes, whereas the parasite burden in the spleen of iNOS k.o. mice was barely higher than in wildtype controls. This clearly indicates an organ-specific control of L. major by iNOS (skin and lymph node) and phox (spleen) (see also NADPH oxidase above)42 (Fig. 3). In a different strain of iNOS-/- mice118 L. major-induced skin lesions were later reported to heal,119,120 but this knockout mouse strain expressed an alternative iNOS mRNA transcript and the macrophages exhibited residual iNOS activity.118 In the case of L. donovani infection the original findings on unrestrained parasite growth in the liver of iNOS-/- mice61 were not confirmed with another iNOS-/ mouse strain.62 The discrepancy between both studies might be due to the fact that iNOS-/and wildtype mice were genetically heterogenous with respect to the two existing forms of the natural resistance-associated macrophage protein-1 gene (NRAMP-1, also termed solute carrier family 11a member 1 [Slc11a1]), which is known to prominently influence the course of L. donovani infections in mice. In addition to iNOS k.o. mice, the protective effect of iNOS-derived NO against various species of Leishmania has been strongly suggested by a number of other experimental approaches and observations, i.e., the exacerbation of clinical disease by the application of iNOS inhibitors,116,121 the inverse correlation between the extent of iNOS expression in the tissue and the parasite load,122 the therapeutic effect of immunomodulators that led to enhanced expression of iNOS,123-129 and the reactivation of parasite replication and recrudescence of disease after inhibition of iNOS.130 Despite this overwhelming evidence that iNOS-derived NO is associated with a curative course of Leishmania infection and long-term suppression of persisting parasites, there are a number of in vivo observations that either document iNOS-independent control of Leishmania or demonstrate that iNOS expression alone is not sufficient to confer parasite containment and protection. For example, failure of parasite clearance, noncuring disease and/or death have been described in L. major-infected mice deficient for TNF,131 Fas119 or FasL,132 despite high level expression of iNOS in the tissues of these mice. Nonhealing infections with L. major have

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Figure 3. Organ-specific and stage-dependent control of Leishmania major infection by inducible nitric oxide synthase (iNOS) and phagocyte NADPH oxidase.42

also been observed, when regulatory T cells and IL-10 counteracted a prominent Th1 response with high expression of iNOS in the lesion.133 Whereas in L. major-infected mice functional inhibition of IL-10 led to sterile cure of cutaneous leishmaniasis,134,135 in L. mexicana-infected mice deletion of IL-10 was insufficient to induce resolution of the skin lesions, despite the presence of iNOS-expressing cells.136 Finally, in L. donovani-infected mice that had been treated with amphotericin B to kill 90-95% of the parasites, relapse of parasite replication and disease occurred in IFN-γ-/- mice or in mice depleted of both CD4+ and CD8+ T cells, but not in mice deficient for either IL-12p40 or iNOS or phox.137 Similarly, visceral infection with L. donovani was controlled and reactivation did not occur in iNOS/phox-double deficient mice after treatment with antimony.138 Thus, there are definitely IFN-γ-dependent, but iNOS- and phox-independent leishmanicidal pathways.

Evidence for the Antileishmanial Activity of Reactive Nitrogen Intermediates in Humans To date, no polymorphism of the human iNOS promotor or mutation of the iNOS gene has been found that is associated with enhanced susceptibility to Leishmania infection or noncuring or progressive cutaneous or visceral disease in humans. Several studies showed that iNOS mRNA or protein is expressed in leishmanial skin lesions.139-141 Evidence that iNOS might be associated with a less severe course of Leishmania infections mainly originates from comparative immunhistological analyses of biopsies from patients with self-healing, localized skin lesions or with nonhealing, disseminated cutaneous disease. Two different studies on patients infected with L. mexicana139 or L. pifanoi142 came to the conclusion that in lesions of patients with localized cutaneous leishmaniasis the number of iNOS protein-positive cells is significantly higher than in the dermis of patients with diffuse cutaneous leishmaniasis. The curative effect of the NO-donor S-nitroso-N-acetylpenicillamine in a small uncontrolled study on patients with L. braziliensis-induced skin lesions from Ecuador further supports the concept that NO can kill Leishmania in vivo and promotes the resolution of cutaneous leishmaniasis.143

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Conclusions Depending on the parasite species, strain and developmental stage as well as on the species, tissue origin, and activation state of the phagocytes, ROI (generated by the NADPH oxidase or other mechanisms) contribute to the control of Leishmania in vitro to a varying degree. In L. major-infected mice NADPH oxidase was essential for the control of L. major in the spleen, but dispensable for the killing of L. braziliensis in the spleen and of L. donovani in the liver. The antileishmanial activity of iNOS and RNI was most striking in the skin and draining lymph node of mice with cutaneous leishmaniasis (e.g., L. major, L. mexicana). In visceral organs (spleen, liver) the contribution of iNOS to parasite control was either very weak (L. major) or dependent on the genetic background of the mice (L. donovani). Based on a so far limited set of in vitro and in vivo studies on mice and humans compounds releasing ROI or RNI upon activation by infected host cells are future candidates for the treatment of leishmaniasis.

Acknowledgments The preparation of this review and conduct of some of the studies reviewed was supported by grants to C.B. by the Deutsche Forschungsgemeinschaft (Bo996/3-1, 3-2, and 3-3; SFB620 A9).

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71. Green SJ, Meltzer MS, Hibbs Jr JB et al. Activated macrophages destroy intracellular Leishmania major amastigotes by an L-arginine-dependent killing mechanism. J Immunol 1990; 144:278-283. 72. Liew FY, Li Y, Millott S. Tumor necrosis factor-α synergizes with IFN-γ in mediating killing of Leishmania major through the induction of nitric oxide. J Immunol 1990; 145:4306-4310. 73. Adams LB, Franzblau SG, Vavrin Z et al. L-arginine-dependent macrophage effector functions inhibit metabolic activity of Mycobacterium leprae. J Immunol 1991; 147:1642-1646. 74. Stuehr DJ, Griffith OW. Mammalian nitric oxide synthases. Adv Enz Rel Areas Mol Biol 1992; 65:287-346. 75. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992; 6:3051-3064. 76. Bogdan C. Nitric oxide and the immune response. Nature Immunol 2001; 2:907-916. 77. El-Gayar S, Thüring-Nahler H, Pfeilschifter J et al. Translational control of inducible nitric oxide synthase by IL-13 and arginine availability in inflammatory macrophages. J Immunol 2003; 171:4561-4568. 78. Boutard V, Havouis R, Fouqueray B et al. Transforming growth factor-β stimulates arginase activity in macrophages. Implications for the regulation of macrophage cytotoxicity. J Immunol 1995; 155:2077-2084. 79. Munder M, Eichmann M, Moran JM et al. Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells. J Immunol 1999; 163:3771-3777. 80. Rutschman R, Lang R, Hesse M et al. Stat6-dependent substrate depletion regulates nitric oxide production. J Immunol 2001; 166:2173-2177. 81. Gobert AP, McGee DJ, Akhtar M et al. Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: A strategy for bacterial survival. Proc Natl Acad Sci USA 2001; 98:13844-13849. 82. Roberts SC, Tancer MJ, Polinsky MR et al. Arginase plays a pivotal role in polyamine precursor metabolism in Leishmania. J Biol Chem 2004; 279:23668-23678. 83. Bogdan C. The function of nitric oxide in the immune system. In: Mayer B, ed. Handbook of Experimental Pharmacology. Volume: Nitric Oxide. Heidelberg: Springer, 2000:443-492. 84. Bogdan C. Nitric oxide and the regulation of gene expression. Trends in Cell Biol 2001; 11:66-75. 85. Nathan C. Specificity of a third kind: Reactive oxygen and nitrogen intermediates in cell signaling. J Clin Invest 2003; 111:769-778. 86. Bogdan C, Röllinghoff M, Diefenbach A. Nitric oxide in leishmaniasis: From antimicrobial activity to immunoregulation. In: Fang F, ed. Nitric oxide and infection. New York: Kluwer Academic/ Plenum Publishers, 1999:361-377. 87. Keefer LK, Davies KM, Saavedra JE, et al. Targeting nitric oxide to macrophages with prodrugs of the O2-glycosylated diazeniumdiolate family. Nitric Oxide 2006; 14:A50 (P103). 88. Holzmuller P, Sereno D, Lemesre JL. Lower nitric oxide susceptibility of trivalent antimony-resistant amastigotes of Leishmania infantum Antimicrob. Agents Chemother 2005; 49:4406-4409. 89. Liew FY, Li Y, Severn A et al. A possible novel pathway of regulation by murine T helper type-2 (Th2) cells of a Th1 cell activity via the modulation of the induction of nitric oxide synthase in macrophages. Eur J Immunol 1991; 21:2489-2494. 90. Bogdan C, Vodovotz Y, Paik J et al. Mechanism of suppression of nitric oxide synthase expression by interleukin-4 in primary mouse macrophages. J Leukoc Biol 1994; 55:227-233. 91. Vieth M, Will A, Schröppel K et al. Interleukin-10 inhibits antimicrobial activity against Leishmania major in murine macrophages. Scand J Immunol 1994; 40:403-409. 92. Bogdan C, Thüring H, Dlaska M et al. Mechanism of suppression of macrophage nitric oxide release by IL-13. J Immunol 1997; 159:4506-4513. 93. Nelson BJ, Ralph P, Green SJ et al. Differential susceptibility of activated macrophage cytotoxic reactions to the suppressive effects of transforming growth factor-β1. J Immunol 1991; 146:1849-1857. 94. Vodovotz Y, Bogdan C, Paik J et al. Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor-β. J Exp Med 1993; 178:605-613. 95. Bogdan C, Moll H, Solbach W et al. Tumor necrosis factor-α in combination with interferon-γ, but not with interleukin 4 activates murine macrophages for elimination of Leishmania major amastigotes. Eur J Immunol 1990; 20:1131-1135. 96. Liew FY, Li Y, Millott S. Tumor necrosis factor (TNF-α) in leishmaniasis. II. TNF-α induced macrophage leishmanicidal activity is mediated by nitric oxide from L-arginine. Immunology 1990; 71:556-559. 97. Betz-Corradin S, Fasel N, Buchmüller-Rouiller Y et al. Induction of macrophage nitric oxide production by interferon-γ and tumor necrosis factor-α is enhanced by interleukin-10. Eur J Immunol 1993; 23:2045-2048.

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98. Stenger S, Solbach W, Röllinghoff M et al. Cytokine interactions in experimental cutaneous leishmaniasis. II. Endogenous tumor necrosis factor-α production by macrophages is induced by the synergistic action of interferon (IFN)-γ and interleukin (IL) 4 and accounts for the antiparasitic effect mediated by IFN-γ and IL-4. Eur J Immunol 1991; 21:1669-1675. 99. Mauël J, Ransijn A, Buchmüller-Rouiller Y. Killing of Leishmania parasites in activated murine macrophages is based on an L-arginine-dependent process that produces nitrogen derivatives. J Leukocyte Biol 1991; 49:73-82. 100. Betz-Corradin S, Mauël J. Phagocytosis of Leishmania enhances macrophage activation by IFN-γ and lipopolysaccharide. J Immunol 1991; 146:279-285. 101. Liew FY, Li Y, Moss D et al. Resistance to Leishmania major infection correlates with the induction of nitric oxide synthase in murine macrophages. Eur J Immunol 1991; 21:3009-3014. 102. Roach TIA, Kiderlen AF, Blackwell JM. Role of inorganic nitrogen oxides and tumor necrosis factor alpha in killing Leishmania donovani amastigotes in gamma interferon/ lipopolysaccharide-activated macrophages from Lshs and Lshr congenic mouse strains. Infect Immun 1991; 59:3935-3944. 103. Assreuy J, Cunha FQ, Epperlein M et al. Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major. Eur J Immunol 1994; 24:672-676. 104. Dey R, Sarkar A, Majumder N et al. Regulation of impaired protein kinase C signaling by chemokines in murine macrophages during visceral leishmaniasis. Infect Immun 2005; 73:8334-8344. 105. von Stebut E, Belkaid Y, Nguyen B et al. Skin-derived macrophages from Leishmania major-susceptible mice exhibit interleukin-12- and interferon-gamma-independent nitric oxide production and parasite killing after treatment with immunostimulatory DNA. J Invest Dermatol 2002; 119:621-628. 106. Weinberg JB. Nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: A review. Mol Med 1998; 4:557-591. 107. Vouldoukis I, Bécherel PA, Riveros-Moreno V et al. Interleukin-10 and interleukin-4 inhibit intracellular killing of Leishmania infantum and Leishmania major by human macrophages by decreasing nitric oxide generation. Eur J Immunol 1997; 27:860-865. 108. Brandonisio O, Panaro MA, Fumarola I et al. Macrophage chemotactic protein-1 and macrophage inflammatory protein-1 alpha induce nitric oxide release and enhance parasite killing in Leishmania infantum-infected human macrophages. Clin Exp Med 2002; 2:125-129. 109. Lemesre JL, Sereno D, Daulouède S et al. Leishmania spp.: Nitric oxide-mediated inhibition of promastigote and axenically grown amastigote forms. Exp Parasitol 1997; 86:58-68. 110. Bocedi A, Gradoni L, Menegatti E et al. Kinetics of parasite cysteine proteinase inactivation by NO donors. Biochem Biophys Res Commun 2004; 315:710-718. 111. de Souza GF, Yokoyama-Yasunaka JK, Seabra AB et al. Leishmanicidal activity of primary S-nitrosothiols against Leishmania major and Leishmania amazonensis: Implications for the treatment of cutaneous leishmaniasis. Nitric Oxide 2006; 14, (Epub ahead of print). 112. Holzmuller P, Sereno D, Cavaleyra M et al. Nitric oxide-mediated proteasome-dependent oligonucleosomal DNA fragmentation in Leishmania amazonensis amastigotes. Infect. Immun 2002; 70:3727-3735. 113. Boucher JL, Moali C, Tenu JP. Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for L-arginine utilization. Cell Mol Life Sci 1999; 55:1015-1028. 114. Iniesta V, Gomez-Nieto LC, Corraliza I. The inhibition of arginase by Nw-hydroxy-L-arginine controls the growth of Leishmania inside macrophages. J Exp Med 2001; 193:777-783. 115. Kropf P, Fuentes JM, Fahnrich E et al. Arginase and polyamine synthesis are key factors in the regulation of experimental leishmaniasis in vivo. FASEB J 2005; 19:1000-1002. 116. Liew FY, Millott S, Parkinson C et al. Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from L-arginine. J Immunol 1990; 144:4794-4797. 117. Buxbaum LU, Uzonna JE, Goldschmidt MH et al. Control of New World cutaneous leishmaniasis is IL-12-independent, but STAT4 dependent. Eur J Immunol 2002; 32:3206-3215. 118. Wei Xq, Charles IG, Smith A et al. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 1995; 375:408-411. 119. Huang FP, Xu D, Esfandiari EO et al. Mice defective in Fas are highly susceptible to Leishmania major infection despite elevated IL-12 synthesis, strong Th1 responses, and enhanced nitric oxide production. J Immunol 1998; 160:4143-4147. 120. Niedbala W, Wei XQ, Piedrafita D et al. Effects of nitric oxide on the induction and differentiation of Th1 cells. Eur J Immunol 1999; 29:2498-2505. 121. Evans TG, Thai L, Granger DL et al. Effect of in vivo inhibition of nitric oxide production in murine leishmaniasis. J Immunol 1993; 151:907-915.

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122. Stenger S, Thüring H, Röllinghoff M et al. Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major. J Exp Med 1994; 180:783-793. 123. Li J, Hunter CA, Farrell JP. Anti-TGF-β treatment promotes rapid healing of Leishmania major infection in mice by enhancing in vivo nitric oxide production. J Immunol 1999; 162:974-979. 124. Murphy ML, Wille U, Villegas EN et al. IL-10 mediates susceptibility to Leishmania donovani infection. Eur J Immunol 2001; 31:2848-2856. 125. Das L, Datta N, Bandyopadhyay S et al. Successful therapy of lethal murine visceral leishmaniasis with cystatin involves up-regulation of nitric oxide and a favorable T cell response. J Immunol 2001; 166:4020-4028. 126. Datta N, Mukherjee S, Das L et al. Targeting of immunostimulatory DNA cures experimental visceral leishmaniasis through nitric oxide up-regulation and T cell activation. Eur J Immunol 2003; 33:1508-1518. 127. Ukil A, Biswas A, Das T et al. 18β-glycyrrhetinic acid triggers curative Th1 response and nitric oxide up-regulation in experimental visceral leishmaniasis associated with the activation of NF-κB. J Immunol 2005; 175:1161-1169. 128. Arendse B, van Snick J, Brombacher F. IL-9 is a susceptibility factor in Leishmania major infection by promoting detrimental Th2/Type 2 responses. J Immunol 2005; 174:2205-2211. 129. Buxbaum LU, Scott P. Interleukin-10- and Fcγ receptor-deficient mice resolve Leishmania mexicana lesions. Infect Immun 2005; 73:2101-2108. 130. Stenger S, Donhauser N, Thüring H et al. Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase. J Exp Med 1996; 183:1501-1514. 131. Wilhelm P, Ritter U, Labbow S et al. Rapidly fatal leishmaniasis in resistant C57BL/6 mice lacking tumor necrosis factor. J Immunol 2001; 166:4012-4019. 132. Chakour R, Guler R, Bugnon M et al. Both the Fas Ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major, whereas the contribution of tumor necrosis factor is minimal. Infect Immun 2003; 71:5287-5295. 133. Anderson CF, Mendez S, Sacks DL. Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice. J Immunol 2005; 174:2934-2941. 134. Belkaid Y, Hoffmann KF, Mendez S et al. The role of interleukin-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure. J Exp Med 2001; 194:1497-1506. 135. Belkaid Y, Piccirillo CA, Mendez S et al. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. Nature 2002; 420:502-507. 136. Jones DE, Ackermann MR, Wille U et al. Early enhanced Th1 response after Leishmania amazonensis infection of C57BL/6 interleukin-10-deficient mice does not lead to resolution of infection. Infect Immun 2002; 70:2151-2158. 137. Murray HW. Prevention of relapse after chemotherapy in a chronic intracellular infection: Mechanisms in experimental visceral leishmaniasis. J Immunol 2005; 174:4916-4923. 138. Murray HW. Responses to Leishmania donovani in mice deficient in both phagocyte oxidase and inducible nitric oxide synthase. Am J Trop Med Hyg 2006; 74:1013-1015. 139. Qadoumi M, Becker I, Donhauser N et al. Expression of inducible nitric oxide synthase in skin lesions of patients with American cutaneous leishmaniosis. Infect Immun 2002; 70:4638-4642. 140. Arevalo J, Ward B, Matlashewski G. Detection of iNOS gene expression in cutaneous leishmaniasis biopsy tissue. Mol Biochem Parasitol 2002; 121:145-147. 141. Serarslan G, Atik E. Expression of inducible nitric oxide synthase in human cutaneous leishmaniasis. Mol Cell Biochem 2005; 280:147-149. 142. Diaz NL, Arvelaez FA, Zerpa O et al. Inducible nitric oxide synthase and cytokine pattern in lesions of patients with American cutaneous leishmaniasis. Clin Exp Dermatol 2005; 31:114-117. 143. Lopez-Jaramillo P, Ruano C, Rivera J et al. Treatment of cutaneous leishmaniasis with a nitric oxide donor. Lancet 1998; 351:1176-1177.

CHAPTER 16

Effector Mechanisms of Macrophages Infected with Trypanosoma cruzi Fredy R.S. Gutierrez, Flavia S. Mariano, Isabel K.F. Miranda-Santos and João S Silva*

Abstract

T

he main effector mechanisms that control infection by T. cruzi depend upon activation of macrophages. These cells are activated soon after infection by mechanisms that are dependent on production of several cytokines and chemokines. Once activated, macrophages, as well other cells of the innate immune system, including cardiomyocytes produce several oxidative molecules, such as nitric oxide. These free radicals kill the intracellular parasites by chemically modifying the structural properties of their proteins and inactivating catalytic sites of their enzymes. Some of these molecules present systemic effects and activate other cells of the innate and adaptive immune responses, recruiting them to the inflammatory site, hence improving the host’s immune response to infection. These energy-consuming responses must be controlled in order to avoid damage to host tissues and macrophages also participate in this aspect of homeostasis. Here we discuss the mechanisms that lead to activation of macrophages, killing of parasites and migration of cells, as well as the consequences of the inflammatory reaction caused by infection with T. cruzi.

The Clinical Outcome of Infection with Trypanosoma cruzi and its Preference for an Intracellular Habitat Trypanosoma cruzi, the causative agent of Chagas’ disease, is classified as an intracellular parasite, in reference to its preferential location for development within the mammalian host. The term “intracellular” also refers to the escape strategies that it have evolved in order to deal with the effector or killing mechanisms of the host’s phagocytes, the cell that the amastigote form of this parasite is very adept at living in. In fact, parasite escape mechanisms underscore the relevant host defenses that are important for maintaining pathogen numbers in check. These effector mechanisms and T. cruzi’s preference for the very cell that is supposed to destroy it must be reconciled with the pathology and clinical presentations of infections caused by this parasite. Most patients infected with T. cruzi survive the acute infection and progress to the so-called indeterminate phase. Of these, less than 30% develop the clinical complications of chronic disease such as carditis and organomegally. The different outcomes of the initial infection reflect the stochastic nature of the interplay between parasite and host, which is affected by their genetic makeup. *Corresponding Author: João S. Silva—Depto of Biochemistry and Imunology, School of Medicine-USP, Av. Bandeirantes, 3900, CEP 14049-900; Ribeirão Preto, SP, Brazil, Email: [email protected].

Protozoans in Macrophages, edited by Eric Denkers and Ricardo Gazzinelli. ©2007 Landes Bioscience.

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The parasite’s molecular environment in both the insect vector and the mammalian host trigger the biochemical changes that result in the different developmental forms of its life cycle. In the vector, a hematophagous triatomid bug, noninfective epimastigote forms proliferate in the gut, where they differentiate into metacyclic trypomastigotes, which are infective for vertebrates. After inoculation into the host’s conjunctival mucosa or the dermal layers of skin that is injured at the site of the blood meal, a variety of innate immune system cells, mainly fibroblasts, endothelial cells, cardiomyocytes,1 NK cells2 and macrophages3 participate in mounting an initial acute inflammatory response. This response is clinically translated into variable degrees of nonspecific symptoms, and while the adaptive response is mounted, high parasitemia and tissue parasitism are observed.

Triggering the Macrophage to Kill Trypanosoma cruzi A network of molecular signals orchestrates the triggering of the cellular immune mechanisms that effect killing of T. cruzi.4 After cells of the innate immune system sense the parasite and respond to it, a wide response occurs mediated by a potent and systemic synthesis of pro-inflammatory factors, that is, cytokines, adhesion molecules and chemokines. The receptors and signaling pathways that control these responses are dealt with in other chapters of this book. These signaling pathways lead to the synthesis of IL-12, and TNF-α by the infected macrophages and dendritic cells, which in turn activate NK cells to produce IFN-γ and stimulate the development of a strong adaptive immune effector response (see Fig. 4). This response relies on the support of Th1-biased CD4+ cells and deploys activated macrophages.5-8 This potent and systemic inflammatory environment is responsible for the clinical manifestations seen in the acute phase of disease. Infection with T. cruzi also increases the expression of chemoattractants like β-chemokines CCL2, CCL3, CCL4, and CCL5 in macrophages, as well as their receptor CCR5 in heart tissue. This mechanism potentiates parasite uptake and macrophage microbicidal mechanisms, and improves the Th1-biased response although CCR5-/mice have increased susceptibility to infection.9,10 The cytokine TNF-α has a crucial role in the response against T cruzi by indirectly inducing transcription of the enzyme inducible nitric oxide synthase (iNOS),8 the product of which is one of the most important effector molecules for killing the parasite and which will be described in further detail below. In fact, macrophages and monocytes are the main sources of TNF-α in vivo.11,12 Accordingly, a significant reduction in the synthesis of NO and a concomitant increase in parasitemia and mortality was observed in experiments using anti-TNF-α antibodies both in vivo and in vitro.8 Therefore, TNF-α is essential triggering activation of macrophages for killing of T. cruzi. It acts in an autocrine and paracrine fashion and potentiates IFN-γ-mediated effects13 and its own production as well as that of other cytokines.14 Stimulation of the macrophage with IL-12 and IFN-gamma activates the iNOS biochemical system within this cell, leading to the generation of killing mechanisms (see Figs. 1 and 2). In addition, lipid mediators of inflammation like platelet-activation factor (PAF) and leukotriene B4 enhance the IFN-γ-induced production of NO by macrophages during infection and thus also determine the control of parasite proliferation.15,16 However, in vitro experiments showed that regardless of its activation by IFN-gamma, the stage of maturation of the T. cruzi-infected macrophage is critical for its ability to produce NO and control infection. The probability of the parasite encountering a suficient number of mature macrophages may explain in part the diverse outcomes of infection with T. cruzi. The factors that promote and enhance encounters between mature macrophages and T. cruzi during natural infections remain to be established. Coexisting microbes and pathogens may affect the cytokine profile and thus the outcome of macrophage activation. Indeed, in gnotobiotic mice components of indigenous microbiota can skew the immune response towards production of inflammatory cytokines during experimental infection with T. cruzi. A direct correlation is observed between a higher survival rate and increased production of IFN-γ and TNF-α in mice concomintantly infected with Enterococcus

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Figure 1. Murine macrophages infected with T. cruzi. Mouse peritoneal macrophages were harvested 3 days after the injection of a 3% solution of thioglycollate in PBS. The cells were cultured in cover slips for 12 h, infected with trypomastigostes of T. cruzi, and washed with PBS. The cultures were incubated for 48 h without (A) or with (B) 10 U/ml of rMuIFN-γ. Arrowheads show amastigote forms inside the macrophages. Note the vacuolization (asterisks) and absence of viable parasites in the macrophages activated with IFN-γ.

faecalis, Bacteroides vulgatus and Peptostreptococcus.17 It is well established that HIV infection induces cellular immunosuppression, mainly through depletion of T helper cells, leading to development of several secondary infections with opportunistic microorganisms, including protozoa. However, the defect does not occur at the level of the macrophage because HIV-infected

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macrophages are able to adequately respond in vitro to stimulation by IFN-γ and kill T. cruzi and T. gondii.18

Killing of Trypanosoma cruzi by the Lysosome Regardless of a pathogen’s preference for an intracellular habitat, the adaptive effector immune response begins within the phagocyte, which possesses two main functions: to digest the pathogen’s components and to present its antigens to parasite-specific T cells (see Fig. 2) in order to enlist their helper functions for activation of effector cells and molecules such as, respectively, activated macrophages and antibodies. T. cruzi gains access to this cell by means of two processes. One entails phagocytosis, which involves actin-dependent invagination of the plasma membrane followed by intracellular fusion of the phagosome with lysosomes. This leads to the formation of the lysosome-like parasitophorous vacuole. This structure has lysosomal properties and is the pivotal feature for preventing trypomastigotes from completing the intracellular life cycle and exit host cells. In the second process, and distinct from most other intracellular prokaryote pathogens, T. cruzi can directly invade host cells independently of the polymerization of actin. This process, however, also involves recruitment and fusion of host lysosomes at the site of parasite entry,19-22 and retaining of these highly motile parasites inside cells.23,24 Recent studies highlight a role for host cell phosphatidylinositol 3-kinases (PI3Ks) in the invasion process by T. cruzi.25-27 Invasion of host cells by T. cruzi is reviewed further in another chapter of this book.

Figure 2. Macrophages and dendritic cells trigger innate immune mechanisms to kill T cruzi. Infection with trypomastigotes results in the production of TNF-α, IL-12, and IL-1-β, IL-10 and TGF-β. IL-12 and TNF-α, and IL-1β induce activation of NK cells, resulting in production of IFN-γ, which induces activation of the macrophage, triggering its effector functions and improving killing of parasites. Production of IL-10 and TGF-β modulates macrophage activation in order to avoid self-destruction and tissue damage.

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Impact of Humoral Effector Mechanisms on Killing Functions of the Macrophage Once the trypomastigote gains access to macrophages and subject to escape from the lysosomal killing mechanisms, it undergoes transformation into the amastigote developmental form, which follows two possible fates: it can escape from additional intracellular resistance mechanisms and proliferate or it can be killed. After the intracellular proliferation of amastigotes, they transform into new, infective trypomastigote forms that then disrupt the cell. These gain access to the bloodstream and then infect other cells, including non phagocytic cells (mainly cardiomyocytes, skeletal muscle cells and astrocytes). In the extracellular milieu, during their transit through the bloodstream from ruptured phagocytes to intact cells, the trypomastigote forms must deal with host humoral effector mechanisms such as complement and antibodies. While these mechanisms per se are not the focus of this chapter, they have important and well known roles in regulating the effector mechanisms of the macrophage. These are the generation of macrophage-activating opsonins derived from components of complement activated by immune complexes and the activation or the inhibition of phagocytes and induction of chemokines through the crosslinking by antibodies of the several types of receptors for antibody Fc (FcR). The parasitic escape mechanisms that subvert the function of complement, for example, T cruzi’s decay accelerating factor simile28 and antibodies (its receptor for mammalian IgG Fc29 and the inhibition of B cells30) indicate the great importance of these host defence molecules. Antibodies modulate inflammatory responses and several lines of evidence show that they may protect the host against the damage that results from infection (by inducing IL-10, as discussed below, and by promoting clearance of antigens) or, conversely, they may amplify inflammation.31 These divergent outcomes are a function of the isotype as well as the levels of antibody. Indeed, isotypes and functional properties of anti-T. cruzi antibodies correlate with the different clinical forms of infections with the parasite.32 Antigen-antibody complexes enhance expression of inducible nitric oxide synthase,33 an enzyme which participates in one of the mechanisms for killing T. cruzi described below. The role of antibodies in the shift of cytokine profiles produced in Chagas’ disease and in the modulation of macrophage function is a line open for investigation.

Killing of Trypanosoma cruzi by Free Radicals: Reactive Oxygen Species, Nitric Oxide and Peroxynitrite Parasites that survive lysosomal hydrolases and develop into amastigotes must next confront the biochemical weapons of the macrophage that rely on short-lived free radical structures such as nitric oxide (NO), reactive oxygen species (ROS) and peroxynitrite. In regard to trypanosomes, T. brucei- and T. cruzi-infected macrophages produce high levels of NO, which has antiparasitic effects in vitro and in vivo.34-37 Additionally, the NADPH oxidase system is also activated by trypanosomes to produce ROS such as the superoxide radical and hydrogen peroxide.38-40 In addition to macrophages, other cells of the immune system, including Kupffer cells, and neutrophils, produce and release superoxide anion and NO as part of their microbicidal effector molecules, and the adequate combination of all of them is responsible for control of infection. Superoxide anion is a normal product of the aerobic cellular metabolism, being mainly produced by xanthine oxidase, by leakage of electrons from the respiratory chain, and by autoxidation of cellular reductants such as ascorbate, NADPH, and glutathione (GSH). In addition to the production of ROS by mitochondria, a membrane-bound NADPH-oxidase is present in cells of the immune system. This latter system participates in the so-called the respiratory burst and is dependent on antibody-mediated phagocytosis. As it enters into phagocytes, T. cruzi tries to avoid triggering the respiratory burst41 and the consequent production of toxic oxygen radicals by sorting host cellular receptors and thus avoiding the molecular interactions among them.42 Superoxide can be also formed by the parasite itself (e.g., during the generation

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of the irontyrosyl radical center in the small subunit of ribonucleotide reductase,43 by mitochondrial respiration,44 or by redox cycling of antichagasic drugs).45 Once formed, O2- can directly attack biomolecular targets of the parasite and impair their function or it can dismutate spontaneusly or enzymatically to H2O2. The extracellular liberation of H2O2 by peritoneal macrophages is associated with killing of parasites.46 The reactivity of O2- is low compared to that of other free radicals and the toxicity of this intermediate is mostly mediated by OH produced in the Haber-Weiss mechanism. Besides damaging molecular targets, ROS affect several biological processes, because they are also able to activate a variety of transcription factors and signal proteins, including , AP-1, c-fos, tumor suppressor protein p53, and protein kinase C (PKC).47-50 On the other hand, an excessive production of ROS may cause lipid peroxidation, with membrane injury, or even DNA structural modifications (because of which they are considered to be carcinogenic).51 NO participates in the other killing mechanism that relies on the oxidation of molecular targets in T. cruzi. It is best known for its function in host defense and in cyto-protective activities and these roles have been observed in bacterial, fungal, and parasitic infections. In vitro and in vivo studies with murine and human macrophages52-54 have demonstrated that NO is involved in both intracellular and extracellular killing of various intracellular pathogens (see Fig. 3). NO was first described as a gaseous molecule with signaling functions in several physiological phenomena, including relaxation of smooth muscle, neurotransmission, tissue homeostasis, memory and learning processes, and inhibition of platelet aggregation. It is now well known, however, that a large amount of NO is produced during infections caused by almost all pathogens including bacteria, viruses, parasites, and fungi. NO is also involved in the pathogenesis of several inflammatory autoaggresive conditions. The metabolism of L-arginine is the crucial pathway used by phagocytes and nonphagocytic cells for generating this oxidant. It is synthesized by means of an enzymatic reaction catalyzed by NO synthases and involves a two-step oxidation of the terminal guanidine nitrogen of L-arginine, the result being formation of NO and L-citrulline. NO synthases (NOSs) are expressed as constitutive enzymes primarily in endothelial cells (eNOS) and neuronal cells (nNOS) and in this situation they

Figure 3. Nitric oxide mediates intracellular killing of T. cruzi. In Figure 3A peritoneal macrophages were infected with T. cruzi and incubated with medium only (), L-NMMA (▲), IFN-γ (Ξ), or IFN-γ plus L-NMMA (•). The parasites released after these treatments were counted in a hemocytometer. The macrophages are able to kill T. cruzi after activation by IFN-γ, but only in absence of L-NMMA, which inhibits iNOS and, therefore, production of NO.71 Figure 3B shows that in the absence of iNOS or IFN-γ the animals are highly susceptible to the infection. Under this condition, the parasite is able to proliferate because without this mediator its killing by host cells is impaired. The effect of lack of iNOS is comparable to that of lack of IFN-γ and in this situation all infected mice die by the second week of infection.70

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produce nanomolar quantities of NO. The inducible isoform (iNOS) is expressed in various cells including macrophages, neutrophils, epithelial cells, and hepatocytes, and catalyzes production of micromolar amounts of NO during infections and inflammation. The two pathways for generating toxic oxidant molecules can converge and form an additional oxidant. A diffusion-controlled reaction between NO and O2- results in the formation of the peroxynitrite anion,55 a very strong oxidant that inhibits proliferation of T. cruzi epimastigotes in a dose-dependant fashion in vitro.56,57 In spite of some reports showing that iNOS-deficient mice present no difference in mortality or parasitemia when compared to wild-type mice,58 it is well accepted that synthesis of NO by macrophages and other host cells is responsible for the killing of Trypanosoma, since iNOS inhibitors impaired the control of parasite proliferation both in vivo and in vitro.34-37 In fact, a cysteine proteinase from T. cruzi, called cruzipain (CP), is an important molecular target of NO. The parasite employs CP in an escape mechanism, which will be described below. NO directly inhibits the action of CP by nitrosylating the Cys catalytic residue, in a dose-dependent fashion.59 NO-releasing compounds can also directly inhibit cysteine proteases (CP) of P. falciparum and L. infantum in a dose-dependent manner.60 Comparative analysis of 3-D amino acid sequence models of CP from a broad range of living organisms, from viruses to mammals, suggests that the Sy atom of the Cys catalytic residue undergoes NO-dependent chemical modification (S-nytrosilation and disulfide bridge formation), with the concomitant loss of enzyme activity. The NO-donor S-nitroso-N-acetilpenicillamine (SNAP) kills epimastigotes of T. cruzi and promastigotes of L. infantum in culture, while a combination of nitrite plus acid organic salts was highly effective against amastigotes of L. major infecting mouse macrophages. A parasitostatic effect (with inhibition of both encystation and excystation) of S-nitrosoglutathione and spermine-NONOate was documented in cultures of Giardia duodenalis trophozoites. Recently, a novel formulation of metronidazole bearing a NO-releasing group was found to enhance significantly the killing of E. histolytica trophozoites in vitro, as compared to unmodified metronidazole. Despite the evidence for NO’s role as a natural anti-protozoal weapon, few efforts have been made to develop and test NO-based drugs in human medicine. This state of affairs is probably due to the difficulty in designing suitable chemical carriers that are able to release the right amount of NO, in the right place and in the right time in order to avoid toxic effects against nontarget host cells. The production of NO, apparently the main effector molecule produced by the macrophage against T. cruzi, can be regulated at any level during production production of protein (transcriptional, post-transcriptional and post-translational).61 The cytokines IFN-γ and TNF-α can up regulate production of NO.61 Among the chemokines, JE/MCP-1, RANTES, MIP-1α, MIP-1β, MIP-2 and CRG-2 are also positive inducers of NO synthesis.62,63 An opposite role is performed by IL-10, IL-4, IL-13 and TGF-β. NO also modulates proliferation of cytotoxic T lymphocytes in response to IL-2, as demonstrated by the diminished uptake of 3H-thymidine by these cells in response to various doses of IL-2 when an inhibitor of NO synthase was added to the culture.64 It is noteworthy that cytotoxic T cells are another important effector mechanism against T. cruzi.65 NO also influences leukocyte migration, by modulating the production of chemokines, by inhibiting activity of chemokines, as well as by acting as an intracellular signaling molecule in chemokine pathways. For a full review of the effects of NO on the immune system, please refer to the review by Bogdan (61).

Quantitative Effects of NO upon the Growth of Trypanosoma cruzi and in the Pathology of Chagas’ Disease NO can play two opposite roles, either favoring or impairing host defense and either killing or promoting the growth of the pathogen.66 NO-mediated killing is carried out only when it is present at micromolar quantities. However, in vitro, when unprimed, non-IFN-γ-activated macrophages are invaded by T. cruzi through an IgG-independent pathway, a smaller, nanomolar quantity of NO is produced. This favors not killing, but proliferation of the parasite, possibly

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by the action of signalling through cGMP.67 NO is able to react with the redox forms of oxygen, thiols, amines and transition metals, as well as participate in the nitrosilation of proteins.68 This may negatively affect the physiology of uninfected, bystander host cells. The protection mediated by this oxidant may come, therefore, at a price: damage of host tissues by NO has been documented in several diseases including arthritis, encephalitis, ulcerative colitis, and viral infections.69 Protective and toxic effects of NO are frequently seen in parallel during infection with T. cruzi67,70-72 and a role for NO in the pathogenesis of heart disease has been reported.73 The expression of cardiac iNOS has also been associated with myocardial dysfunction.74 An excessive activation of NO systems can cause adverse effects to the host, as suggested by the inflammatory lesions seen in the central nervous system of mice chronically infected with T. brucei brucei and in the myocardium of acute chagasic rats. In the latter infection, animals express type II nitric-oxide synthase and show protein 3-nitrotyrosine immunoreactivity, which is ascribed to formation of peroxynitrite and/or nitrogen dioxide (NO2).75-77 A very tight control of expression of inducible NO synthase is therefore required in order to prevent auto-injury. Polymorphisms for the promoter of the NOS-II gene in Africans are associated with protection from severe malaria or susceptibility to this complication; indeed, a pathogenic role for endogenous NO has been documented in cerebral malaria. However, such polymorphisms were not associated with determination of susceptibility nor resiatance to T cruzi infection.78 In summary, NO can be involved in host effector and immunomodulatory functions and can kill T. cruzi or favor its growth.

Killing of Trypanosma cruzi by Depletion of Tryptophan In addition to the effector mechanisms described above, IFN-γ-induced depletion of the essential amino acid L-tryptophan in the macrophage’s intracellular milieu also appears to contribute in the killing of some intracellular pathogens.79,80 This is achieved by the enzyme indolamine 2,3-dioxygenase (IDO), which catabolizes tryptophan into kinurenins.81 However, whilst the expression of IDO is induced by IFN-γ,82 its products appear to have immunoregulatory properties,83 and induce a TH2-biased immune profile,84 constituting a feedback mechanism and perhaps favoring the parasite in spite of the depletion of a component essential for its growth. In all, it is not known if T. cruzi has evolved an escape mechanism that exploits the IDO pathway, but it was demonstrated that depletion of L-tryptophan does not participate in killing of this parasite.85 The main source of IDO is the eosinophil,84 which in its activated state is a significant component of the infiltrate in cardiac lesions of Chagas’ disease patients.86 However, a role for this cell in the pathology and in the effector and regulatory mechanisms that operate during infections with T. cruzi has not been well established.

Control of Macrophage Effector Responses by Trypanosoma cruzi and by the Host The host inflammatory response against T. cruzi can be controlled by several means. Besides the parasite escape mechanisms mentioned above, T. cruzi also releases factors that induce apoptosis of host cells,87 a phenomenon that is essential for determining the level of infection and of pathology caused by the host’s response to the parasite. The phagocytosis of apoptotic, infected cells by macrophages favors parasite growth by enhancing secretion of TGF-β, which deactivates the macrophage killing mechanisms.88 The increase in production of this cytokine shifts the balance between the two opposing pathways of L-arginine metabolism from the production of nitric oxide catalyzed by the enzyme nitric oxide synthase (NOS)89 to the production of polyamines catalyzed by arginase and ornithine decarboxylase (ODC). Whereas nitric oxide can kill T. cruzi, polyamines are growth factors for the parasite and also precursors for the synthesis of trypanothione which protects it against chemical and oxidative stresses.89-91 Besides apoptosis, activation of arginase and shifting to the polyamine pathway is also achieved

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by T. cruzi’s cysteine protease, cruzipain.92 In addition, NO itself can also induce apoptosis of macrophages,93 constituting a regulatory mechanism. This phenomenon appears to be exclusive of macrophage because it was not observed in neutrophils.94 Modulation of the macrophage is also achieved by countering the effects of IFN-γ with TGF-β and IL-10, which are also produced during the infection (see Fig. 4). These cytokines down regulate production of NO, leading to impaired microbicidal activity and increased susceptibility to infection. 95,96 It is noteworthy that T. cruzi induces expression of a TGF-β-responsive reporter gene and gains access to host cells by means of receptors for TGF-β, which signals through pathways that deactivate the effector functions triggered by IFN-γ,97 facilitating both entry and survival of the parasite. The release of TGF-β seems to be concomitant with the production of NO: uninfected macrophages can produce high levels of NO under synergistic stimulation with IFN-γ and TNF-α, however, infected phagocytes produce less NO under the same stimulus and present reduced trypanicidal activity.98 TGF-β also acts as a suppressor of macrophage activation. TGF- β suppresses expression of iNOS by three distinct mechanisms: decreased stability and translation of its mRNA, and increased degradation of iNOS protein.98 Furthermore, when the transcription of TGF-β is manipulated to down-regulate the production of its protein, increased concentrations of IFN-γ are detected in supernatants of phagocytes in culture.99 Natural killer T cells can positively or negatively regulate the inflammatory response during infections with T. cruzi. The response outcome depends on the presence of variant or invariant T cell receptor chains. In response to the same infection, NKT cells expressing variant chains promote an inflammatory response and produce more gamma interferon, TNF-γ, and NO. On the other hand, NKT cells expressing invariant chains dampen this response, possibly by down regulating the proinflammatory NKT cells. Strikingly, Jalpha18-/- mice, which lack NKT

Figure 4. Macrophages trigger inflammatory responses during infection with T. cruzi. After infection with trypomastigote forms of T. cruzi, macrophages produce several cytokines (mainly TNF-α, IL-12 and IL-1α), inflammatory mediators such as NO, as well as several chemokines. These mediators are able to induce migration of inflammatory cells to the site of infection, which include macrophages, NK cells and T lymphocytes. Chemokines and IL-12 induce NK cells and T lymphocytes to produce IFN-γ, resulting in activation of macrophages. Other cytokines such as IL-10 and TGF–β are produced and may control cellular activation and the local inflammatory response.

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cells expressing invariant chains, die. This outcome may be a consequence of the excessive activation of inflammatory responses and damage to host cells by NO.100 T. cruzi also modulates expression of several molecules in host monocytes. For example, monocytes from Chagas’ disease patients presented with decreased expression of CD11b and HLA-DR compared to those from uninfected individuals. Additionally, monocytes from indeterminate-disease patients are committed to expressing IL-10, while monocytes from patients with clinically active Chagas’ disease tend to produce higher levels of TNF-α. This finding prompts the suggestion that cells of the innate immune system obtained from indeterminate-disease patients can be used in the clinical therapy of Chagas disease, as they could improve adaptive immunity and probably modulate myocarditis and other ominous forms of the disease. However, purification and isolation of such cells has been made difficult by the absence of specific markers.101

Conclusions The distinct clinical outcomes of infections with Trypanosoma cruzi reflect the immunochemical, immunopathological and genetic balance that individual hosts achieve after killing mechanisms exert their effects upon the parasite and, inevitably, upon their infected cells or uninfected, bystander cells. Oxidant radicals, while efficient effector molecules for killing this parasite, when in excess exact a toll from the host in the form of nerve damage and cardiomyophathy. When parasite escape mechanisms gain a leeway or host immunomodulatory reactions are excessively up regulated, they dampen the triggers for host effector mechanisms, resulting in less parasites being killed and less damage to the host. The extent of microbial killing and of the accompanying pathological insults, the characteristics of pathogens that coexist with T. cruzi, as well as the overall physiological environment of infection will determine the final outcome of the effector functions of the macrophage against T. cruzi.

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Index A Adhesion 18, 21, 26, 27, 30, 31, 72, 85, 87, 89, 90, 118-120, 139, 163, 164, 167, 169, 172, 173, 175, 179, 194, 197, 208 Amastigotes 6-8, 25, 26, 28, 30, 39-44, 50, 118-120, 124, 125, 139, 140, 151, 152, 211, 213 Antibodies 20, 25, 27, 31, 51, 86, 144, 150, 152, 154, 165-167, 170, 208, 210, 211 Antigen presentation 3, 6, 7, 10, 20, 22, 39, 40, 71, 110, 131, 141, 152, 167, 173, 175 Antigen-presenting cells (APC) 130, 141 Apoptosis 10, 32, 53, 110, 130, 133-135, 153-156, 214 Atively activated macrophages 3, 4, 150

C Calreticulin-like protein 31 Caspase 134, 135, 155 Cell signaling 49, 84, 87-90, 93, 112, 136, 139, 142, 143, 145, 151, 167 Chagas disease 9, 149, 216 Chemokines 1, 2, 7, 8, 10, 29, 32, 33, 42, 74-76, 93, 94, 99, 104, 110, 167, 198, 207, 208, 211, 213, 215 Classically activated macrophage 3, 4, 150 Complement 2, 5, 9, 27, 30, 31, 38, 39, 51, 52, 72, 118-120, 151, 164-166, 170, 173, 211 CR3 8, 27, 71, 119 Cruzipain 28, 29, 31, 32, 153, 154, 213, 214 Cytokine 1-8, 10, 11, 25, 42, 44, 45, 49-59, 61, 71, 73, 75, 76, 84-88, 90, 92-94, 99-101, 104, 108, 110-112, 120-122, 124-126, 130, 132-134, 136, 140, 141, 145, 149, 150, 153, 156, 160, 164-173, 175, 176, 184-187, 196-199, 207, 208, 211, 213-215

D Dendritic cells (DC) 2, 6-8, 16, 30-32, 38, 39-41, 43, 44, 49, 53, 75, 87, 94, 99, 101, 102, 104, 109, 110, 120, 121, 153, 160, 162, 163, 172-174, 193, 194, 208, 210

Differentiation 8, 17, 39, 40, 42-45, 51, 133, 149, 150, 153-155, 167, 197

E Effector immune response 210

F Fas ligand (FasL) 153, 154, 199

G Glycosylphosphatidylinositol (GPI) 20, 32, 50, 52, 53, 55, 58, 84, 87-93, 120, 153, 154, 166-168, 173, 175 GPI anchors 50, 52, 53 GTPase 51, 100, 143, 151, 187, 189, 190, 195

H Hematopoiesis 43 Hemozoin 67-69, 73, 74, 87, 94, 165-169, 171-173, 175 Host receptor 85, 87, 90, 164

I IFN 3, 4, 6-8, 10, 30, 32, 51, 52, 54, 55, 56, 57, 61, 70-75, 86-88, 93, 99-101, 108, 109, 118, 122-124, 130-134, 140-144, 149-155, 160, 167-176, 184-190, 194, 196-198, 200, 208-210, 212-215 IFN-γ 3, 4, 8, 10, 30, 32, 51, 52, 54, 57, 61, 70-75, 86-88, 93, 99-101, 108, 109, 118, 122-124, 130-134, 140-144, 149-155, 168-176, 184-190, 194, 196-198, 200, 208-210, 212-215 IL-10 2, 4, 8, 32, 43, 52, 54, 72, 86, 87, 110, 112, 118, 124-126, 141, 150, 153, 154, 167, 173, 175, 200, 210, 211, 213, 215, 216 IL-12 3, 4, 7, 8, 11, 51-54, 57, 61, 72, 75, 76, 86-88, 92, 93, 99-104, 108-112, 120-124, 141, 150, 152, 170, 171, 173, 184, 185, 194, 208, 210, 215

222 Immune evasion 44, 131, 133, 145, 165 Immunity 25, 26, 30, 33, 38, 39, 43, 44, 49-52, 54, 55, 57, 61, 71, 75, 85, 86, 99, 101, 102, 104, 118, 120, 121, 123-125, 130, 131, 133, 141, 153, 160, 163, 165, 169, 170-173, 176, 184, 216 Inducible nitric oxide synthase 51, 72, 87, 90, 100, 130, 140, 185, 193, 194, 197, 200, 208, 211 Infection and pathogenesis 93 Inflammation 1, 2, 5, 25, 26, 30, 31, 33, 42, 75, 153, 156, 173, 208, 211, 213 Innate immune response 1, 5, 57, 99, 101, 104, 120, 140, 141 Innate immunity 25, 49, 50-52, 54, 55, 57, 61, 102, 118, 120, 121, 123, 163 iNOS 3, 32, 51, 54, 72, 73, 100, 132, 133, 141, 143, 144, 168, 171, 185, 193, 194, 197-201, 208, 212-215 Interferon-γ 3, 99, 140, 184 Interferon (IFN) 3, 4, 6-8, 10, 30, 32, 51, 52, 54, 55, 56, 57, 61, 70-75, 86-88, 93, 99-101, 108, 109, 118, 122-124, 130-134, 140-144, 149-155, 160, 167-176, 184-190, 194, 196-198, 200, 208-210, 212-215 Invasion 1, 9, 16-18, 20, 21, 25-31, 49, 54, 55, 75, 76, 112, 113, 130, 140, 151, 152, 172, 210

K Kinase 21, 26, 28-30, 32, 70, 89, 102, 108, 109, 111, 112, 123, 124, 142, 143, 150, 151, 153, 164, 167, 175, 195, 196, 212

L Leishmania 4, 6-10, 38-40, 42, 44, 45, 49, 50, 75, 88, 118, 120-125, 139, 140, 141, 142-145, 151, 188, 193-201 Lipid 21, 22, 29, 52, 53, 70, 71, 88, 90, 93, 120, 124, 142, 151, 172, 187, 188, 208, 212 Lipopolysaccharide (LPS) 3, 4, 7, 32, 42, 52-57, 59, 60, 70, 72, 88, 92, 104, 108-112, 120-122, 132, 133, 150, 153, 154, 167, 168, 170-172, 186, 196-198 Lymphocyte 5, 6, 25, 43, 44, 53, 151, 154, 155

Protozoans in Macrophages

M Macrophages 1-11, 16, 20, 22, 25-33, 38-45, 49-61, 67, 69, 72-74, 84, 86-94, 99-104, 108-112, 118-125, 130-134, 136, 139-145, 149-155, 160-173, 175, 176, 184-190, 193-199, 207-216 Macrophage inflammatory protein-1α (MIP-1α) 10, 32, 71, 74, 76, 166, 198, 213 Major histocompatibility complex (MHC) 71, 131 Malaria 5-7, 9, 50, 67-76, 84-88, 93, 94, 160-173, 175, 176, 214 MAPK and NF-κB signaling pathways 84 Marginal zone 6, 40, 42-44, 163 MCP-1 7, 32, 33, 74, 76, 198, 213 Membrane fusion 189 Metacyclic trypomastigotes (MT) 25-27, 30, 33, 50, 208 Mitochondria 16, 18, 21, 114, 134, 135, 195, 196, 211, 212 Mitogen-activated protein kinase (MAP-kinase) 32, 55, 57, 59, 60, 75, 84, 90, 92, 100, 102, 103, 108-112, 124, 153, 164, 167, 175 Monocytes 1, 2, 4, 5, 8, 16, 31, 39, 40, 42, 55, 69-71, 73, 76, 84, 86-88, 90, 92, 93, 101, 110, 130, 163-168, 170, 172, 173, 195-197, 208, 216 Mononuclear 22, 38-40, 42-45, 54, 70, 71, 118, 125, 139, 140, 166, 169, 186 Mononuclear phagocytes 22, 38-40, 42, 44, 45, 70, 118, 139, 140, 169, 186 Motility 16-18 Mouse models 140, 160, 162, 163, 166, 169, 170 Moving junction 21

N NF-κB 56, 57, 59, 60, 72-75, 84, 89-91, 93, 100-104, 108-112, 120, 121, 133, 134, 151, 152, 167, 168, 212 Nitric oxide (NO) 30, 32, 33, 43, 44, 51, 53, 70, 72-75, 84, 86-90, 92, 93, 100, 101, 109, 110, 130, 132, 133, 140-142, 150-155, 168, 170-172, 175, 185, 186, 189, 193, 194, 196-200, 207, 208, 211-215 Nucleotide exchange factors 30

Index

O

223

Ornithine decarboxylase (ODC) 150, 154-156, 214

Spleen 6, 7, 40, 42-44, 57, 68, 69, 72, 125, 162-164, 170-173, 193, 197, 199, 201 Stromal cell 43, 44 Superoxide dismutase (SOD) 75, 195

P

T

Parasites 2, 5, 6, 8-11, 16-18, 20, 22, 25-28, 30-33, 38-41, 49-51, 54, 56, 61, 67-69, 71, 85, 86, 88, 93, 99-104, 112, 118, 119, 121, 122, 124, 125, 131, 133, 134, 139, 141, 151-155, 160-166, 168-173, 185, 193-200, 207, 209-212, 216 Phagocyte NADPH oxidase (phox) 150, 152, 185, 193-197, 199, 200 Phagocytosis 18, 27, 29, 30, 39, 67, 69-72, 86, 118-121, 139, 151, 152, 154, 163-173, 175, 185, 210, 211, 214 Phosphatidylinositol-3 kinase (PI-3 kinase) 28, 30 Plasmodium 5, 6, 9, 16, 18, 50, 67, 68, 72, 75, 76, 84, 85, 93, 160-163, 165, 169, 170-173, 175 Plasmodium falciparum 5, 6, 50, 68, 84, 160, 163 Pleckstrin homology (PH) 30 Pro-inflammatory responses 84-87, 88, 90, 93, 94, 99, 100 Protein secretion 18, 21

T cells 3, 4, 6-8, 25, 31, 39, 40, 43-45, 54, 71, 99-101, 124, 125, 131, 133, 141, 150-155, 160, 163, 172-174, 194, 197, 200, 210, 213, 215 T. cruzi calreticulin-like protein (TcCRT) 31 Tetrahydrobiopterin 196 TGF-β 4, 8, 25, 28, 31, 32, 52, 86, 87, 141, 150, 152-156, 197, 210, 213-215 Th1/Th2 153 THB 196, 197 Tissue culture derived trypomastigote (TC) 27-32 Tolerance 6, 43, 55, 57, 59, 60, 111, 172 Toll-like receptor (TLR) 1, 3, 5, 7, 25, 30, 32, 49, 50, 54-58, 60, 61, 75, 90, 93, 101, 102, 104, 110-112, 120, 121, 150, 153, 167, 168, 173, 175, 198 Toxoplasma gondii 8, 9, 16, 18, 99, 108, 112, 114, 130, 184, 188, 196 Trypanosoma cruzi (T. cruzi ) 9, 25-33, 49-59, 61, 75, 88-90, 149-156, 188, 207, 208, 210-216 Tumor necrosis factor (TNF) 3, 5, 7, 8, 32, 42, 43, 50, 52, 53, 56, 57, 59-61, 70-72, 75, 84-90, 92, 93, 99-102, 108-112, 120, 123, 132, 134, 141, 150, 152-154, 166-168, 170-173, 184, 186, 194, 196-199, 208, 210, 213, 215, 216

R Reactive nitrogen intermediates (RNI) 49, 50-54, 56, 61, 168, 171, 197, 201 Reactive oxygen intermediates (ROI) 49, 140, 141, 168, 185, 186, 195-197, 199-201 Receptors 1, 2, 3, 5-11, 17, 18, 20, 25, 27-32, 38, 39, 42, 45, 49-51, 54-56, 61, 71, 74-76, 85-87, 90, 93, 101, 102, 104, 108, 110-112, 118-120, 122, 125, 133, 142, 150, 152, 154, 155, 163-165, 167, 169, 170, 172, 173, 175, 185, 186, 194, 198, 208, 211, 215 Respiratory burst 3, 4, 5, 16, 169, 196, 211

S Signal transduction 26, 30, 76, 90, 93, 108, 110, 123, 124, 130, 133, 143 Signaling 21, 22, 26-29, 39, 49, 55, 56, 58, 61, 67, 72-75, 84-91, 93, 94, 99-104, 108-113, 120, 123-125, 131, 134, 136, 139, 142, 143, 145, 149-154, 164, 166-168, 175, 208, 212, 213

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Denkers • Gazzinelli

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Protozoans in Macrophages

Denkers ISBN 978-1-58706-150-9

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Eric Y. Denkers and Ricardo T. Gazzinelli

Protozoans in Macrophages