AUTOPHAGY, INFECTION,AND THE IMMUNE

RESPONSE

AUTOPHAGY, INFECTION,AND THE IMMUNE

RESPONSEEDITED BY

William T. Jackson and Michele S. Swanson

This edition first published 2015. © 2015 by John Wiley & Sons, Inc. All rights reserved

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Autophagy, infection, and the immune response / edited by William T. Jackson and Michele Swanson.

p. ; cm.

Includes bibliographical references and index.

ISBN 978-1-118-67764-3 (cloth)

I. Jackson, William T., 1971- , editor. II. Swanson, Michele, editor.

[DNLM: 1. Autophagy–immunology. 2. Immune System Processes. 3. Immunity, Innate.

4. Virus Physiological Processes. QU 375]

QR181.7

616.07′9–dc232014029016

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors xiiiPreface xviiAcknowledgments xix

1 AUTOPHAGY AND IMMUNITY 1Xu Liu and Daniel J. Klionsky1.1 Introduction 1

1.2 Autophagy 2

1.2.1 Types of autophagy 2

1.2.2 Morphology 3

1.2.3 Molecular machinery 3

1.2.4 Physiological roles 5

1.3 Autophagy and immunity 6

1.3.1 Xenophagy: autophagic clearance of intracellular

microorganisms 6

1.3.2 Autophagy and cryptides 9

1.3.3 Autophagy and pattern recognition receptors (PRRs) 9

1.3.4 Autophagy and MHC antigen presentation 10

1.3.5 Autophagy regulation by immune signaling molecules 11

1.3.6 Autophagy, inflammation, and autoimmunity 11

1.4 Conclusion 12

References 12

2 TECHNIQUES FOR STUDYING AUTOPHAGY 19Isei Tanida and Masato Koike2.1 Introduction 19

2.2 Reagents and tools for studying autophagy 21

2.2.1 Reagents to monitor the lysosomal flux of LC3-II 21

2.2.2 Reagents that induce autophagy 21

2.2.3 Reagents and recombinant tools that inhibit autophagy 22

2.3 Detection of LC3-I AND LC3-II by immunoblotting 22

2.4 Immunofluorescent analyses of endogenous LC3 23

2.5 Monitoring autophagy using fluorescent protein-tagged LC3 23

2.6 Morphological analyses of autophagosomes and autolysosomes

by TEM 24

2.6.1 Reagents or stock solutions 26

2.6.2 Resin embedding of cell pellets or microbes 26

v

vi CONTENTS

2.6.3 Resin flat embedding of cells grown on glass or plastic

coverslips 27

2.7 Techniques for immunoelectron microscopy 28

References 29

3 ROLE OF AUTOPHAGY IN DNA VIRUS INFECTIONS IN VIVO 33Xiaonan Dong and Beth Levine3.1 Introduction 33

3.2 In vivo interplay between autophagy and DNA viruses in plants and

invertebrates 34

3.3 In vivo interplay between autophagy and DNA viruses in vertebrates 35

3.3.1 Autophagy is an essential antiviral mechanism that protects

against HSV-1 in vivo 35

3.3.2 The autophagy-HBV interplay in vivo: a balance betweenviral exploitation and tumor suppression 40

3.3.3 Autophagy may suppress γ-herpesvirus persistent infection 42

3.4 Conclusion 43

Acknowledgments 44

References 44

4 STUDYING RNA VIRUSES AND AUTOPHAGY IN VIVO 49Mehrdad Alirezaei and J. Lindsay Whitton4.1 Introduction 49

4.2 In vivo interactions between autophagy and RNA viruses in plants

and invertebrates 50

4.2.1 Plants 50

4.2.2 Invertebrates 50

4.3 In vivo Interactions between autophagy and RNA viruses in

vertebrates 51

4.3.1 Togaviridae 51

4.3.2 Caliciviridae 51

4.3.3 Orthomyxoviridae 53

4.3.4 Flaviviridae 53

4.3.5 Picornaviridae 54

4.4 Conclusion 62

Acknowledgments 63

References 63

5 AUTOPHAGY AND PICORNAVIRUS INFECTION 67Tom Wileman, Zhigang Zhou, Matthew Whelband, Eleanor Cottam,Stephen Berryman, Terry Jackson and Rebecca Roberts5.1 Introduction 67

5.2 Selective autophagy involves autophagy receptors with

LC3-interacting domains 69

5.3 Autophagy is activated during virus infection 69

5.4 Picornaviruses and autophagy 69

5.4.1 Poliovirus 70

5.4.2 Coxsackievirus 72

CONTENTS vii

5.4.3 Human enterovirus 71 73

5.4.4 Encephalomyocarditis virus 73

5.4.5 Foot-and-mouth disease virus 74

5.4.6 Human rhinoviruses 75

5.5 Caution in interpretation of induction of LC3 puncta and

double-membraned vesicles in the context of autophagy 75

5.5.1 LC3 puncta 75

5.6 Conclusions and future research 77

References 78

6 FLAVIVIRUSES AND AUTOPHAGY 81Tristan X. Jordan and Glenn Randall6.1 Introduction 81

6.1.1 Autophagy 81

6.2 Flaviviruses 83

6.3 Dengue virus 83

6.3.1 Autophagosomes as a platform for replication? 85

6.3.2 Modulation of lipid metabolism 86

6.3.3 Potential role for the autophagy-related proteins USP10 and

USP13 in DENV virion maturation 87

6.3.4 Cytoprotective autophagy 88

6.3.5 The role of autophagy in an ADE model of monocyte infection 89

6.3.6 Autophagy in DENV infections in mice 89

6.4 Other Flaviviruses 90

6.4.1 Japanese encephalitis virus 90

6.4.2 Modoc virus 90

6.4.3 West Nile virus 90

6.5 Concluding remarks 92

Acknowlegments 92

References 93

7 AUTOPHAGY: A HOME REMODELER FOR HEPATITIS C VIRUS 101Marine L.B. Hillaire, Elodie Décembre, and Marlène Dreux7.1 Introduction 101

7.1.1 Autophagy 101

7.1.2 Hepatitis C virus (HCV) disease, genome and replication 103

7.2 HCV induces a proviral autophagy 111

7.3 How does HCV trigger autophagy vesicle accumulation? 111

7.4 Dynamic membrane remodeling by autophagy 113

7.5 Interlinkage of autophagy with the innate immune response 114

7.6 Autophagy and cell death 115

7.7 Removal of aberrant deposits and organelles by autophagy:

implications for liver injury associated with chronic hepatitis C 116

7.7.1 Autophagy and lipid metabolism 116

7.7.2 Mitophagy and HCV persistence 117

7.8 Conclusions and future directions 118

Acknowledgments 119

References 119

viii CONTENTS

8 MODULATING AUTOPHAGY TO CURE HUMANIMMUNODEFICIENCY VIRUS TYPE-1 127Stephen A. Spector and Grant R. Campbell8.1 Introduction 127

8.2 HIV subverts autophagy to promote its own replication 129

8.3 HIV infection inhibits autophagy during permissive infection while

induction of autophagy leads to inhibition of HIV 130

8.4 HIV-induced autophagy in bystander CD4+ T cells results in cell death 130

8.5 Modulation of autophagy as a mechanism for HIV-associated

neurocognitive impairment 132

8.6 How can autophagy be exploited to control and eradicate HIV? 134

Acknowledgments 137

References 138

9 AUTOPHAGY IN THE INFECTED CELL: INSIGHTS FROMPATHOGENIC BACTERIA 143Andrea Sirianni and Serge Mostowy9.1 Introduction 143

9.2 Autophagy–bacteria interactions 143

9.2.1 Salmonella typhimurium 144

9.2.2 Mycobacterium tuberculosis 145

9.2.3 Legionella pneumophila 146

9.2.4 Listeria monocytogenes 147

9.2.5 Shigella flexneri 149

9.2.6 Mycobacterium marinum 150

9.3 Conclusions 151

Acknowledgments 151

References 152

10 Rab PROTEINS IN AUTOPHAGY: STREPTOCOCCUS MODEL 159Takashi Nozawa and Ichiro Nakagawa10.1 Introduction 159

10.2 Rab GTPase 160

10.3 Rab GTPases in starvation-induced autophagy 160

10.4 Rab localization in autophagy during Streptococcus infection 161

10.5 Involvement of Rab7 in the initial formation of GcAV 163

10.6 Requirement of Rab23 for GcAV formation 163

10.7 Facilitation by Rab9A of GcAV enlargement and lysosomal fusion 164

10.8 Conclusion and perspective 165

References 167

11 HELICOBACTER PYLORI INFECTION CONTROL BY AUTOPHAGY 171Laura K. Greenfield, Frances Dang, and Nicola L. Jones11.1 Helicobacter pylori 171

11.2 H. pylori and evasion of host immune responses 176

11.3 Autophagy 178

11.4 Acute H. pylori infection: induction of autophagy in gastric

epithelial cells 180

CONTENTS ix

11.5 Chronic H. pylori infection: suppression of autophagy in gastric

epithelial cells 184

11.6 H. pylori induction of autophagy in immune cells 185

11.7 Host genetics affecting autophagic clearance of H. pylori 185

11.8 H. pylori disrupted autophagy and gastric cancer 186

11.9 H. pylori therapy: is autophagy a contender? 187

11.10 Concluding remarks 188

Acknowledgments 189

References 189

12 INTERACTIONS BETWEEN SALMONELLA AND THE AUTOPHAGYSYSTEM 201Teresa L.M. Thurston and David W. Holden12.1 Introduction 201

12.2 Salmonella’s life within the host 201

12.3 Salmonella’s survival in a harsh intracellular habitat 202

12.4 Models for studying Salmonella infection 203

12.5 Mechanisms of Salmonella autophagy 204

12.5.1 Salmonella is targeted for antibacterial autophagy 204

12.5.2 Antibacterial autophagy induction 205

12.5.3 Eat-me signals for antibacterial autophagy 206

12.5.4 Autophagy receptors provide cargo specificity 208

12.6 Autophagy of Salmonella in vivo 209

12.7 Bacterial countermeasures 210

12.7.1 Could Salmonella counteract autophagy? 210

12.7.2 Potential autophagy avoidance mechanisms 210

12.7.3 SseL deubiquitinates autophagy-targeted protein aggregates 210

12.7.4 Does Salmonella inhibit selective antibacterial autophagy? 211

12.8 Perspectives 211

References 213

13 HOST FACTORS THAT RECRUIT AUTOPHAGY AS DEFENSEAGAINST TOXOPLASMA GONDII 219Carlos S. Subauste13.1 Introduction 219

13.2 CD40, autophagy and lysosomal degradation of T. gondii 220

13.3 Events downstream of CD40 involved in the stimulation

of autophagy 222

13.4 Relevance of autophagy during in vivo infection with T. gondii 224

13.5 IFN-γ and ATG5 in T. gondii infection 224

13.6 T. gondii manipulates host cell signaling to inhibit targeting by LC3+

structures and to maintain the nonfusogenic nature of the

parasitophorous vacuole 227

13.7 Autophagy machinery within T. gondii 228

13.8 Conclusion 229

Acknowledgments 229

References 229

x CONTENTS

14 MYCOBACTERIUM TUBERCULOSIS AND THE AUTOPHAGICPATHWAY 233Gabriela María Recalde and María Isabel Colombo14.1 Mycobacterium tuberculosis, a pathogen that resides in a

self-tailored compartment to avoid killing by the host cell 233

14.2 The ESX-1 secretion system 235

14.3 Mycobacterium marinum, a close relative that escapes and forms

actin tails in the cytoplasm 235

14.4 Mycobacterium actively modulates autophagy 236

14.5 Mycobacterium tuberculosis, a pathogen also able to escape toward

the cytoplasm 239

14.6 Concluding remarks 240

References 241

15 AUTOPHAGY ENHANCES THE EFFICACY OF BCG VACCINE 245Arshad Khan, Christopher R. Singh, Emily Soudani, Pearl Bakhru,Sankaralingam Saikolappan, Jeffrey D. Cirillo, N. Tony Eissa,Subramanian Dhandayuthapani and Chinnaswamy Jagannath15.1 Introduction 246

15.2 Induction of autophagy through mTOR enhances antigen

presentation via the MHC-II pathway in macrophages

and dendritic cells 247

15.2.1 Rapamycin-induced autophagy enhances antigen

presentation in APCs 248

15.2.2 Rapamycin and Torin1-induced autophagy enhances

both antigen presentation and IL-1β secretion from BCG

infected APCs 248

15.3 Intracellular mechanisms of autophagic routing of particulate BCG

vaccine and secreted Ag85B into autophagosomes and enhanced

MHC-II mediated antigen presentation 251

15.3.1 Overexpression of secreted Ag85B in BCG vaccine leads to

aggresome formation in the cytosol of APCs 251

15.3.2 Overexpressed Ag85B from BCG vaccine forms

aggresomes, which enhance antigen presentation through

autophagy 251

15.3.3 Discussion: in vitro studies on autophagy and antigen

presentation 253

15.4 Rapamycin activation of dendritic cells enhances efficacy of

DC-BCG vaccine 255

15.4.1 Discussion 256

15.5 Rapamycin coadministration with BCG vaccine in mice enhances

CD4 and CD8 T cell mediated protection against tuberculosis 256

15.5.1 Discussion 262

15.6 Conclusions 262

Acknowledgments 263

References 263

CONTENTS xi

16 AUTOPHAGY’S CONTRIBUTION TO INNATE AND ADAPTIVEIMMUNITY: AN OVERVIEW 267Christina Bell, Michel Desjardins, Pierre Thibault and Kerstin Radtke16.1 Autophagy: different routes to the same goal? 267

16.2 Xenophagy: it is a dog-eat-dog world 269

16.3 Autophagy and Toll-like receptors: a mutual turn-on 269

16.4 Autophagy and antigen presentation: a cry for help to clear

pathogenic invaders 270

16.5 Autophagy and inflammasomes: Mutual regulation for an effective

immune response 273

16.6 Cross-talk between autophagy and cytokines 273

Acknowledgments 275

References 275

17 AUTOPHAGY IN IMMUNE RESPONSES TO VIRUSES 279Christophe Viret and Mathias Faure17.1 Innate immunity against viruses 279

17.2 Autophagy in antiviral innate immunity 281

17.2.1 Virus sensing for autophagy induction 281

17.2.2 Role of autophagy in xenophagy of viruses 282

17.2.3 Role of autophagy in antiviral innate immunity signaling 283

17.3 Autophagy manipulation by viruses to resist innate immunity 285

17.3.1 Autophagy manipulation by viruses to prevent IFN-I synthesis 285

17.3.2 Viruses subvert autophagy to interfere with inflammatory

responses 286

17.3.3 Autophagy and cell death during virus infection 287

17.4 Autophagy in antiviral adaptive immunity 287

17.4.1 Promotion of adaptive immune responses to viral infection

by autophagy 287

17.4.2 MHC class II-restricted presentation of viral epitopes 288

17.4.3 MHC class I-restricted presentation of viral epitopes 290

17.4.4 Autophagy and cross-presentation 292

17.5 Autophagy manipulation by viruses to escape adaptive immunity 294

17.5.1 MHC class II antigen presentation pathway 294

17.5.2 MHC class I antigen presentation pathway 295

17.5.3 Autophagy and antigen-presenting cell function 295

17.6 Concluding remarks 296

Acknowledgments 296

References 297

18 PROCESSING AND MHC PRESENTATION OF ANTIGENS AFTERAUTOPHAGY-ASSISTED ENDOCYTOSIS, EXOCYTOSIS, ANDCYTOPLASM DEGRADATION 303Christian Münz18.1 Introduction 303

18.2 Substrate recognition by macroautophagy 305

xii CONTENTS

18.3 Antigen processing for MHC class II presentation by macroautophagy 307

18.4 A role of macroautophagy in MHC class I antigen presentation 308

18.5 Antigen release by autophagy-assisted exocytosis 309

18.6 Autophagy-assisted phagocytosis 310

18.7 Conclusions and outlook 312

Acknowledgments 312

References 312

Index 317

CONTRIBUTORS

Mehrdad Alirezaei Department of Immunology and Microbial Science, The Scripps

Research Institute, La Jolla, California, USA

Pearl BakhruDepartment of Pathology and LaboratoryMedicine, University of Texas

Health Sciences Center, Houston, Texas, USA

Christina Bell Proteomics and Mass Spectrometry Research Unit, Institute for

Research in Immunology and Cancer, Département de Pathologie et Biologie

Cellulaire, Université de Montréal, Montreal, Quebec, Canada

Stephen Berryman The Pirbright Institute, Woking, Surrey, UK

Grant R. Campbell Department of Pediatrics, Division of Infectious Diseases, Uni-

versity of California, San Diego, La Jolla, California, USA

Jeffrey D. Cirillo Department of Microbial Pathogenesis and Immunology, Texas

A&M University Health Sciences Center—College of Medicine, College Station,

Texas, USA

María Isabel Colombo Laboratorio de Biología Celular y Molecular, Instituto de

Histología y Embriología (IHEM)-CONICET, Facultad de Ciencias Médicas, Uni-

versidad Nacional de Cuyo, Mendoza, Argentina

Eleanor Cottam The Pirbright Institute, Woking, Surrey, UK

Frances Dang Departments of Paediatrics and Physiology, University of Toronto,

Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto,

Ontario, Canada

Elodie Décembre CIRI, Université de Lyon, Lyon, France; Inserm, Lyon, France;

Ecole Normale Supérieure de Lyon, Lyon, France; Université Claude Bernard

Lyon 1, Lyon, France; CNRS, Lyon, France; LabEx Ecofect, Université de Lyon,

Lyon, France

Michel Desjardins Department of Pathology and Cell Biology, Université de

Montreal, Montreal, Quebec, Canada

Subramanian Dhandayuthapani Center of Excellence for Infectious Diseases,

Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas

Tech Health Sciences Center, El Paso, Texas, USA

Xiaonan Dong Center for Autophagy Research and Department of Internal Medicine,

University of Texas Southwestern Medical Center, Dallas, Texas, USA

Marlène Dreux CIRI, Université de Lyon, Lyon, France; Inserm, Lyon, France; Ecole

Normale Supérieure de Lyon, Lyon, France; Université Claude Bernard Lyon 1,

Lyon, France; CNRS, Lyon, France; LabEx Ecofect, Université de Lyon, Lyon,

France

xiii

xiv CONTRIBUTORS

N. Tony Eissa Department of Medicine, Baylor College of Medicine, Houston, Texas,

USA

Mathias Faure International Center for Infectiology Research (CIRI), CNRS, Uni-

versité Claude Bernard Lyon 1, Université de Lyon, Ecole Normale Supérieure de

Lyon, Lyon, France

Laura K. Greenfield Departments of Paediatrics and Physiology, University of

Toronto, Cell Biology Program, Research Institute, The Hospital for Sick Children,

Toronto, Ontario, Canada

Marine L.B. Hillaire CIRI, Université de Lyon, Lyon, France; Inserm, Lyon, France;

Ecole Normale Supérieure de Lyon, Lyon, France; Université Claude Bernard

Lyon 1, Lyon, France; CNRS, Lyon, France; LabEx Ecofect, Université de Lyon,

Lyon, France

David W. Holden MRC Centre for Molecular Bacteriology and Infection, Section of

Microbiology Imperial College London, London, UK

Terry Jackson The Pirbright Institute, Woking, Surrey, UK

Chinnaswamy Jagannath Department of Pathology and Laboratory Medicine, Uni-

versity of Texas Health Sciences Center, Houston, Texas, USA

Nicola L. Jones Departments of Paediatrics and Physiology, University of Toronto,

Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto,

Ontario, Canada

Tristan X. JordanDepartment of Microbiology, The University of Chicago, Chicago,

Illinois, USA

ArshadKhanDepartment of Pathology and LaboratoryMedicine, University of Texas

Health Sciences Center, Houston, Texas, USA

Daniel J. Klionsky Life Sciences Institute and Department of Molecular, Cellular and

Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA

Masato Koike Department of Cell Biology and Neuroscience, Juntendo University

School of Medicine, Tokyo, Japan

Beth Levine Center for Autophagy Research, Department of Microbiology, Depart-

ment of Internal Medicine and Howard Hughes Medical Institute, University of

Texas Southwestern Medical Center, Dallas, Texas, USA

Xu Liu Life Sciences Institute and Department of Molecular, Cellular and Develop-

mental Biology, University of Michigan, Ann Arbor, Michigan, USA

Serge Mostowy Section of Microbiology, MRC Centre for Molecular Bacteriology

and Infection, Imperial College London, London, UK

Christian Münz Viral Immunobiology, Institute of Experimental Immunology, Uni-

versity of Zürich, Zürich, Switzerland

Ichiro Nakagawa Department of Microbiology, Kyoto University Graduate School of

Medicine, Kyoto, Japan

Takashi Nozawa Department of Microbiology, Kyoto University Graduate School of

Medicine, Kyoto, Japan

Kerstin RadtkeDépartement de Pathologie et Biologie Cellulaire, Université deMon-

tréal, Montreal, Quebec, Canada

Glenn Randall Department of Microbiology, The University of Chicago, Chicago,

Illinois, USA

CONTRIBUTORS xv

Gabriela María Recalde Laboratorio de Biología Celular y Molecular, Instituto de

Histología y Embriología (IHEM)-CONICET, Facultad de Ciencias Médicas, Uni-

versidad Nacional de Cuyo, Mendoza, Argentina

Rebecca Roberts Department of Infection and Immunity, Sheffield University,

Sheffield, UK

Sankaralingam Saikolappan Center of Excellence for Infectious Diseases, Depart-

ment of Biomedical Sciences, Paul L. Foster School ofMedicine, Texas Tech Health

Sciences Center, El Paso, Texas, USA

Christopher R. SinghDepartment of Pathology and Laboratory Medicine, University

of Texas Health Sciences Center, Houston, Texas, USA

Andrea Sirianni Section of Microbiology, MRC Centre for Molecular Bacteriology

and Infection, Imperial College London, London, UK

Emily Soudani Department of Pathology and Laboratory Medicine, University of

Texas Health Sciences Center, Houston, Texas, USA

Stephen A. Spector Department of Pediatrics, Division of Infectious Diseases, Uni-

versity of California, San Diego, La Jolla and Rady Children’s Hospital, San Diego,

California, USA

Carlos S. Subauste Division of Infectious Diseases and HIV Medicine, Department

of Medicine, Department of Ophthalmology and Visual Sciences, Department of

Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio,

USA

Isei TanidaDepartment of Biochemistry and Cell Biology, National Institute of Infec-

tious Diseases, Tokyo, Japan

Pierre Thibault Proteomics and Mass Spectrometry, Research Unit, Institute for

Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec,

Canada

Teresa L. M. Thurston MRC Centre for Molecular Bacteriology and Infection,

Section of Microbiology, Imperial College London, London, UK

Christophe Viret International Center for Infectiology Research (CIRI), CNRS, Uni-versité Claude Bernard Lyon 1, Université de Lyon, Ecole Normale Supérieure de

Lyon, Lyon, France

Matthew Whelband Norwich Medical School, University of East Anglia, Norwich,

Norfolk, UK

J. Lindsay Whitton Department of Immunology and Microbial Science, The Scripps

Research Institute, La Jolla, California, USA

TomWilemanNorwichMedical School, University of East Anglia, Norwich, Norfolk,

UK

ZhigangZhouNorwichMedical School, University of East Anglia, Norwich, Norfolk,

UK

PREFACE

Since the discovery nearly 20 years ago that pathogenic bacteria and viruses intimately

associate with autophagosomal membranes, scientists have determined that autophagy is a

critical component of innate and acquired immunity. Of course, as with all aspects of the

host immune response, some pathogens have turned autophagy to their advantage. For this

volume, experts in the fields of bacteriology, virology, mycology, parasitology, immunol-

ogy, and cell biology describe the cellular mechanisms of autophagosome formation and

maturation, its contribution to host defenses, and themechanisms pathogenic microbes have

acquired to overcome and subvert this formidable barrier to infection. In addition, spe-

cialists discuss current efforts to exploit knowledge of the autophagy pathway to improve

vaccine design. Accordingly, this thorough examination of an extraordinary cellular bat-

tleground between host and pathogen can stimulate ongoing research to understand and to

manipulate autophagy to improve human health.

William T. Jackson

Michele S. Swanson

xvii

ACKNOWLEDGMENTS

The editors would like to thank the staff at Wiley-Blackwell, especially Mindy Okura-

Marszycki for her efforts in getting this project off the ground, and Stephanie Dollan,

Michelle Kwok, and Sangeetha Parthasarathy for helping us get it across the finish line.

We are, of course, especially indebted to all of the chapter authors for their tremendous

efforts throughout the process.

William T. Jackson

Michele S. Swanson

xix

1

AUTOPHAGY AND IMMUNITYXu Liu and Daniel J. Klionsky

Life Sciences Institute and Department of Molecular, Cellular and Developmental Biology,University of Michigan, Ann Arbor, Michigan, USA

1.1 INTRODUCTION

Autophagy is a highly controlled process in which cellular components are self-degraded

and subsequently recycled. This pathway in part plays a “house cleaning” role in the

cell, directing numerous cargoes to the lysosome (or the vacuole in yeast and plants) for

degradation. Depending on the specific conditions, the cargoes include random portions of

cytoplasm, protein aggregates, and damaged or superfluous organelles such as mitochon-

dria and peroxisomes. Dysfunction of autophagy is linkedwithmany pathologies, including

cancer, diabetes, myopathies, heart, liver and lung diseases, and certain types of neurode-

generative disease (Castets et al., 2013; Gonzalez et al., 2011; Klionsky and Codogno,

2013; Murrow and Debnath, 2013; Rubinsztein et al., 2012; Yang and Klionsky, 2010).

Emerging studies have revealed that autophagy plays important roles in immunity.

In 2004, independent studies demonstrated for the first time that invading pathogens can

be cargoes for autophagy (Gutierrez et al., 2004; Nakagawa et al., 2004). Today it is well

accepted that autophagy can directly eliminate intracellular pathogens, including bacteria,

fungal parasites, and viruses. Autophagy can also activate innate immune signaling cas-

cades such as Toll-like receptor (TLR) signaling to attack invading pathogens (Lee et al.,

2007; Xu et al., 2007). However, microbes constantly undergo strong selective pressure

to develop strategies to block host defense mechanisms. Indeed, studies indicate that

some adaptations that confer pathogenicity involve microbial inactivation or subversion of

Autophagy, Infection, and the Immune Response, First Edition.Edited by William T. Jackson and Michele S. Swanson.© 2015 John Wiley & Sons, Inc. Published 2015 by John Wiley & Sons, Inc.

1

2 AUTOPHAGY AND IMMUNITY

autophagy through distinct mechanisms (Deretic and Levine, 2009; Kuballa et al., 2012;

Levine et al., 2011; Yuk et al., 2012; Zhou and Zhang, 2012).

Autophagy’s role in immunity is not limited to controlling infection by direct elim-

ination of pathogens. For example, autophagy facilitates MHC (major histocompatibil-

ity complex) antigen presentation, indicating that autophagy is involved in adaptive as

well as innate immunity (English et al., 2009; Paludan et al., 2005). Moreover, defects in

autophagy are associated with autoimmune diseases such as Crohn disease (Levine et al.,

2011; Schroder and Tschopp, 2010; Shi et al., 2012). Thus, autophagy is an integral part of

our response to infection and plays a key role in immunity. A comprehensive understand-

ing of autophagy as it pertains to microbial infection and the molecular mechanisms that

underlie the interplay between autophagy and immune signaling pathways may enable us

to unravel the pathogenesis of many infectious and immune diseases, and develop more

effective therapeutic strategies for their treatment.

1.2 AUTOPHAGY

1.2.1 Types of autophagy

There are three main types of autophagy: chaperone-mediated autophagy (CMA), microau-

tophagy, and macroautophagy (Figure 1.1). CMA is a process where a cytosolic chaperone

protein, HSPA8/HSC70, specifically recognizes its cargo proteins through a KFERQ-like

Phagophore

Late endosome

Vesicle expansion

Hydrolases

Hydrolases

Lysosome

Amphisome

Autolysosome

Lysosome

Autophagosome

Vesiclematuration

Figure 1.1. Schematic model of mammalian autophagy. Cargoes including cytosolic proteins,

protein aggregates, and damaged organelles are sequestered by a phagophore, whichwill expand

and mature to form a complete autophagosome. The outer membrane of the autophagosome

fuses with either a late endosome (forming an amphisome, which then fuses with a lysosome) or

lysosome, forming an autolysosome. Finally, the cargoes together with the inner membrane are

degraded and the breakdown products are released back into the cytosol for reuse.

1.2 AUTOPHAGY 3

motif and facilitates their translocation directly across the lysosomal membrane for

degradation (Dice, 2007; Kaushik and Cuervo, 2012). Microautophagy involves the uptake

of portions of cytoplasm by the direct invagination or protrusion of the lysosomal or

vacuolar membrane (Mijaljica et al., 2011). The third process, macroautophagy, hereafter

referred to as autophagy, is the best characterized and will be the focus of this chapter.

1.2.2 Morphology

The morphological hallmark of autophagy involves the de novo formation of a double-

membrane organelle named the autophagosome; however, this structure is essentially an

end product of the sequestration process and as such is not really the primary functional

unit of autophagy. Rather, the precursor to the autophagosome, the phagophore, is the

dynamic membrane structure that is responsible for sequestering the cargos such as

damaged organelles and invading pathogens (Figure 1.1). The phagophore expands with

the addition of membrane, the sources of which are suggested to include almost every

intracellular organelle. Upon completion, the phagophore seals and becomes a completed

autophagosome. The autophagosome may fuse directly with a lysosome or, first, with a

late endosome to form an intermediate amphisome. The subsequent fusion of the outer

membrane of the autophagosome or the amphisome limiting membrane with a lysosome

generates an autolysosome and exposes the cargoes to the degradative lysosomal enzymes.

The degradation products, especially amino acids, are subsequently released back into the

cytosol and are used in generating energy or as substrates for biosynthetic pathways.

1.2.3 Molecular machinery

Even though autophagosomes have been observed by electron microscopy as early as the

1950s, the molecular mechanisms of autophagy have been poorly studied until the past

two decades (Stromhaug and Klionsky, 2001). The molecular machinery was first identi-

fied through studies in budding yeast, Saccharomyces cerevisiae, and to date more than 30

autophagy-related (ATG) genes have been identified as being involved in this process (Hard-ing et al., 1995; Klionsky et al., 2003; Thumm et al., 1994; Tsukada and Ohsumi, 1993).

Subsequent work with mammalian cells has revealed homologs of the core autophagy

machinery (Xie and Klionsky, 2007), supporting the notion that autophagy is evolution-

arily conserved. At the same time, there are also increasing numbers of ATG proteins being

identified in mammals and other model systems such as Caenorhabditis elegans that lackyeast homologs, suggesting an increased complexity and diversity of function in higher

eukaryotes (Klionsky and Codogno, 2013). For ease of discussion, the protein machinery

of autophagy is subdivided into four major complexes in the following sections, and we

focus on the mammalian autophagy machinery.

ULK1/ULK2 complex Autophagy occurs at a basal level in cells under normal condi-

tions. Upon stress or other stimuli, autophagy can be induced, and defects in regulation that

prevent proper induction can lead to aberrant cell physiology; however, toomuch autophagy

activity can also be detrimental to the cell. Thus, the level of autophagy must be tightly con-

trolled. Accordingly, there are various factors that regulate autophagy induction, and studies

have shown that the ULK1/ULK2 (unc-51 like autophagy activating kinase 1/2) complex

functions in part in an early stage of autophagy regulation.

ULK1 andULK2 are kinases and the other components of the complex include ATG13,

RB1CC1/FIP200 (RB1-inducible coiled-coil 1), and ATG101. ATG13 directly interacts

4 AUTOPHAGY AND IMMUNITY

with ULK1/ULK2 and RB1CC1 regardless of the nutrient availability (Hosokawa et al.,2009; Jung et al., 2009); however, the phosphorylation status of these proteins changesunder different conditions. In nutrient-rich conditions, a key upstream negative regulatorof autophagy, the mechanistic target of rapamycin complex 1 (MTORC1) interacts withthe complex and phosphorylates ULK1/ULK2 and ATG13, inhibiting ULK1/ULK2 kinaseactivity. Upon starvation,MTORC1 is released from the complex. ULK1/ULK2 andATG13are then partially dephosphorylated, leading to activation of ULK1/ULK2 kinase activ-ity, which in turn leads to phosphorylation of ATG13 (presumably on distinct sites fromthose used byMTORC1) and RB1CC1 to induce autophagy (Chan, 2009; Hara et al., 2008;Hosokawa et al., 2009). AMPK (AMP-activated protein kinase) also binds ULK1/ULK2and positively regulates autophagy through phosphorylation upon glucose starvation; asexpected, AMPK and MTORC1 phosphorylate ULK1 at different sites (Kim et al., 2011;Zhao and Klionsky, 2011).

Class III phosphatidylinositol 3-kinase complexes The class III phosphati-dylinositol 3-kinase (PtdIns3K) is generally thought to act downstream of the ULK1/ULK2complex, mediating formation of phosphatidylinositol-3-phosphate (PtdIns3P) on thephagophore membrane, an event essential for autophagy. PtdIns3P serves to recruitdownstream factors such as WIPI1 (WD repeat domain, phosphoinositide interacting 1)and WIPI2, which are involved in the trafficking of ATG9 and promote autophagosomematuration (Polson et al., 2010). In mammals, there are multiple class III PtdIns3K com-plexes with the core components being PIK3C3/VPS34 (phosphatidylinositol 3-kinase,catalytic subunit type 3), BECN1/Beclin 1 (beclin 1, autophagy related), and PIK3R4/VPS15/p150 (phosphoinositide-3-kinase, regulatory subunit 4). BECN1 can interact withseveral proteins, including AMBRA1 (autophagy/beclin-1 regulator 1), ATG14/ATG14L/Barkor, UVRAG (UV radiation resistance associated), KIAA0226/Rubicon and BCL2(B-cell CLL/lymphoma 2) to form distinct complexes (Furuya et al., 2005; Itakuraet al., 2008; Matsunaga et al., 2009; Petiot, 2000). BECN1 was first identified as aBCL2 binding protein. The interaction between BECN1 and BCL2 inhibits the bind-ing of the former with PIK3C3, thus inhibiting autophagy. The ATG14–BECN1–PIK3C3–PIK3R4–AMBRA1 complex is specific for autophagy; ATG14 may directthis complex to the phagophore to promote autophagosome biogenesis (Itakura et al.,2008; Matsunaga et al., 2009), whereas the SH3GLB1 (SH3-domain GRB2-likeendophilin B1)–UVRAG–BECN1–PIK3C3–PIK3R4 complex functions at a laterstep to promote autophagosome maturation (Itakura et al., 2008). In contrast, theKIAA0226–UVRAG–BECN1–PIK3C3–PIK3R4 complex localizes to late endosomesand negatively regulates autophagosome maturation (Matsunaga et al., 2009).

ATG9 trafficking system The Atg9 trafficking system is best characterized in yeast,although even in that model organism there are many questions that remain to be answered.The current model is that the transmembrane protein Atg9 cycles between the phagophoreassembly site (PAS) and peripheral (i.e., non-PAS) sites, and that this process is neededfor the proper delivery of membrane from various donor organelles to the expandingphagophore (Noda et al., 2000; Reggiori et al., 2005). Atg23 and Atg27 interact withAtg9 and facilitate its anterograde traffic from the peripheral sites to the PAS, whereasAtg2–Atg18 and the Atg1–Atg13 complex (yeast homolog of the ULK1/ULK2 complex)are required for its retrograde transport from the PAS back to the peripheral sites (Guanet al., 2001; Reggiori et al., 2004; Wang et al., 2001; Yen et al., 2007).

1.2 AUTOPHAGY 5

In mammals, ATG9 localizes to the trans-Golgi network and endosomes in nutrient-

rich conditions. A pool of ATG9 translocates toMAP1LC3 (microtubule-associated protein

1 light chain 3)/LC3-positive compartments upon starvation. This translocation is depen-

dent on ULK1 and PIK3C3 kinase activity (Young et al., 2006). The dynamic movement

between ATG9 and the phagophore membrane during autophagy suggests a conserved role

for ATG9 in membrane movement during phagophore expansion. Similar to yeast, ATG9

retrieval from the phagophoremembrane is dependent onWIPI2, a homolog of yeast Atg18,

but movement in this direction is ULK1 kinase independent (Orsi et al., 2012).

Ubiquitin-like conjugation systems There are two ubiquitin-like (Ubl) conjugation

systems, which involve the Ubl proteins ATG12 and LC3. These systems are quite

well-studied, playing important roles in phagophore expansion and maturation (Ichimura

et al., 2000; Mizushima et al., 1998, 2001). ATG12 is conjugated with ATG5 in a

manner that is similar to canonical ubiquitination (Mizushima et al., 1998). The E1-like

enzyme ATG7 activates ATG12 via a thioester bond (Tanida et al., 2001). ATG12 is then

transferred to an E2-like enzyme, ATG10, before it is finally conjugated to an internal

lysine of ATG5. ATG5 then noncovalently binds ATG16L1 (autophagy related 16-like

1 (S. cerevisiae)), which subsequently dimerizes. During autophagy, ATG5 directs the

ATG12—ATG5–ATG16L1 complex to the phagophore (Mizushima, 2003).

The different isoforms of LC3 (and the related GABARAP (GABA(A) receptor-

associated protein) subfamily proteins) are conjugated to the lipid phosphatidylethanol-

amine (PE), and this modification is required for association with the phagophore

membrane (Kabeya et al., 2004; Tanida et al., 2003). Initially, the cysteine protease

ATG4B removes the C-terminal amino acids of pro-LC3 to reveal a glycine residue,

generating a cytosolic form named LC3-I. LC3-I is then sequentially activated by ATG7

and conjugated via the E2-like enzyme ATG3, resulting in the membrane-associated form,

LC3-II (Tanida et al., 2001, 2002). The PE group can ultimately be cleaved by ATG4B

in a deconjugation step, which is important for maintaining the proper level of autophagy

activity (Tanida et al., 2006).

1.2.4 Physiological roles

Autophagy has many physiological roles. First, autophagy is a protective mechanism

against cellular stress (Kuma et al., 2004; Yang and Klionsky, 2010). For example,

autophagy’s role in supplying essential building blocks or metabolic substrates such

as amino acids under conditions of nutrient deprivation is critical for maintaining cell

viability under adverse conditions; autophagic degradation and recycling enable cells to

maintain the synthesis of essential proteins and to generate ATP.

Recent studies indicate that autophagy is also indispensible during development. One

example of such a role is seen after oocyte fertilization in C. elegans, where autophagy

is involved in the elimination of maternal mitochondria (Al Rawi et al., 2011; Sato and

Sato, 2011); however, this does not appear to be the case in mammals (Luo et al., 2013). In

addition, during embryonic development, clearance of apoptotic cells is achieved through

autophagy (Qu et al., 2007). Autophagy is also implicated in life span extension; induction

of autophagy increases longevity in several model organisms (Rubinsztein et al., 2011) and

its role in clearing aggregate-prone proteins and damaged mitochondria might be relevant

to its antiaging effects.

6 AUTOPHAGY AND IMMUNITY

As autophagy acts to eliminate many harmful components in a cell, malfunction of

autophagy has also been suggested to correlate with or be the cause of a variety of diseases,

such as cancer, neurodegeneration, cardiovascular myopathies, and lysosomal storage dis-

orders (Klionsky and Codogno, 2013). For example, the selective degradation of damaged

mitochondria is suggested to underlie the tumor suppressive effects of autophagy, possibly

through reducing oxidative stress and preventing DNA damage (Narendra et al., 2008). Sev-

eral lines of evidence suggest that the role of autophagy in clearing toxic aggregate-prone

proteins is critical to prevent certain types of neurodegeneration, including those associ-

ated with Huntington, Alzheimer, and Parkinson diseases (Bjørkøy et al., 2005; Ravikumar

et al., 2002).

1.3 AUTOPHAGY AND IMMUNITY

1.3.1 Xenophagy: autophagic clearance of intracellularmicroorganisms

For decades, scientists have explored how our body fights against invading pathogens. Even

though an understanding of our immune systems has steadily increased, a major problem,

how a cell breaks down an intracellular pathogen without harming itself, has been over-

looked or at least unanswered. Only recently have researchers realized that autophagy plays

a vital role in this process. This specific type of autophagy is termed “xenophagy.”

Autophagic degradation of bacteria and parasites Several independent stud-

ies have revealed that xenophagy acts to eliminate many different bacteria and other

microbes (Levine et al., 2011; Yuk et al., 2012). A good example of parasite clearance

is seen with Toxoplasma gondii (Andrade et al., 2006). This parasite is able to survive

within macrophages by residing in parasitophorous vacuoles that are modified to avoid

fusion with lysosomes. However, stimulation of T. gondii-infected macrophages with

CD40 (CD40 molecule, TNF receptor superfamily member 5), a member of the TNF

(tumor necrosis factor) receptor superfamily, causes colocalization of parasitophorous

vacuoles and LC3. Conversely, treatment of infected cells with the autophagy inhibitor

3-methyladenine (3-MA) or knockdown of BECN1 blocks the fusion of parasitophorous

vacuoles with lysosomal compartments (Andrade et al., 2006). Thus, these results suggest

that phagophores capture parasites that are residing within these vacuoles and direct them

to the lysosome for degradation.

As for bacterial clearance, evidence indicates that autophagosomes can sequester

both bacteria that reside within membranous compartments and those present free within

the cytosol, through mechanisms that are overlapping, but distinct (Figure 1.2) (Levine

and Deretic, 2007). The clearance of Mycobacterium tuberculosis is a good example of

engulfment of bacteria residing within phagosomes (Gutierrez et al., 2004). After entering

the cell through endocytosis, M. tuberculosis can actively survive in a host cell and evade

the host defense by inhibiting phagosomal maturation. However, if autophagy is induced

by either nitrogen starvation or rapamycin treatment, the inhibition of phagosomal matu-

ration by M. tuberculosis is suppressed and intracellular bacterial survival is significantly

decreased. Also, a substantial colocalization of M. tuberculosis-containing phagosomes

with autophagosomes is observed upon autophagy induction, supporting the idea that

phagophores capture bacteria residing within phagosomes and target them to lysosomal

compartments for degradation (Gutierrez et al., 2004).

1.3 AUTOPHAGY AND IMMUNITY 7

Phagosome

Phagophore

Autophagosome

Hydrolases

Lysosome

Autolysosome Autophagolysosome

Membrane

damage

ReceptorReceptor

UbLC3

Ub LC3

UbLC3

LC3

LC3

Figure 1.2. Models of autophagic elimination of invading pathogens. Intracellular virus pro-

teins are recognized by autophagy receptors and recruited to autophagosomes by interaction

between the receptors and LC3. Both bacteria within phagosomes and bacteria that have escaped

from phagosomes can be degraded through autophagy. Bacteria residing in a phagosome can be

engulfed by a phagophore; after completion of sequestration, the resulting autophagosome then

fuses with a lysosome forming an autophagolysome. (Note that we strongly recommend that this

term be reserved to describe the compartment that results from the fusion of lysosomes with

autophagosomes containing phagosomes, and not for the compartments that result from the

fusion of other autophagosomes with lysosomes, which are termed autolysosomes.) Some bacte-

ria are able to damage the phagosomal membrane and escape into the host cell cytoplasm. These

cytosolic bacteria are polyubiquitinated and recognized by autophagy receptor proteins, directing

their delivery to phagophores.

8 AUTOPHAGY AND IMMUNITY

With regard to cytosolic bacteria, a major problem/challenge is that these microbes

need to be specifically recognized and distinguished from other “self” endomem-

branes, including their endosymbiotic descendants, the mitochondria. Starvation-induced

autophagy is usually nonselective, but there are also selective types of autophagy. Recent

studies of selective autophagy reveal a common cargo–ligand–receptor–scaffold model

(Mijaljica et al., 2012). A receptor protein recognizes ligands on cargoes and at the same

time binds the scaffold protein of the autophagy machinery, selectively targeting cargoes

into the autophagy pathway. Specific receptors have been identified that recognize intracel-

lular bacteria during xenophagy, including SQSTM1/p62 (sequestome 1), NBR1 (neighbor

of BRCA1 gene 1), CALCOCO2/NDP52 (calcium binding and coiled-coil domain 2) and

OPTN (optineurin) (Kraft et al., 2010; Thurston et al., 2009; Wild et al., 2011; Zheng

et al., 2009). Usually, cytosolic intracellular bacteria are coated with polyubiquitin, and

these receptors are able to simultaneously bind the ubiquitinated bacteria and LC3, linking

the cargo with the autophagy machinery. In this way, intracellular bacteria are specifically

targeted for degradation.

Despite the utility of xenophagy in degrading intracellular bacteria, certain pathogens

have been successful in developing strategies for evading autophagy. One example of such

evasion is seen with Listeria monocytogenes (Birmingham et al., 2007). After infection

of its host macrophages, a population of L. monocytogenes damages phagosomes and is

released into the cytosol, where they will ultimately be recognized by autophagy. However,

the expression of the virulence factor ActA triggers host cell actin polymerization. This pro-

vides the bacteria with actin-based motility, which allows cell-to-cell spread and avoidance

of autophagic degradation.

Autophagic elimination of viruses The cargo of xenophagy is not restricted to pro-

tozoan parasites and bacteria; autophagy can also capture invading viruses. In general, the

mechanism involved in the recognition of viruses and their sequestration by phagophores

is conceptually similar to that of cytosolic bacteria (Figure 1.2). For example, after Sindbis

virus infects the mouse central nervous system, SQSTM1 interacts with Sindbis virus cap-

sid proteins, mediating their further degradation through autophagy (Orvedahl et al., 2010).

This action significantly reduces virally-induced cell death.

Similar to bacteria, many viruses also act to inhibit autophagy to confer virulence. First,

numerous viruses can either inhibit antiviral signaling pathways that induce autophagy or

they can activate an autophagy inhibitory pathway. EIF2AK2/PKR (eukaryotic translation

initiation factor 2-alpha kinase 2) is an interferon-inducible double-stranded RNA sensor

that mediates overall downregulation of translation in host cells via phosphorylation of

EIF2A (eukaryotic translation initiation factor 2A, 65 kDa). This signaling pathway also

positively regulates virus-induced autophagy (Levine and Deretic, 2007). Viruses develop

multiple strategies to block the EIF2AK2 pathway. For example, during infection herpes

simplex virus type 1 (HSV-1) expresses the US11 protein to antagonize EIF2AK2-mediated

phosphorylation of EIF2A by binding to the kinase, thus preventing autophagy induc-

tion (Lussignol et al., 2013). As discussed above, MTOR signaling is a negative regulator

of autophagy. Upon infecting dendritic cells, human immunodeficiency virus-1 (HIV-1)

downregulates autophagy by inducing MTOR and RPS6KB/p70 S6 kinase (ribosomal pro-

tein S6 kinase, 70 kDa) activation, thus promoting viral proliferation in host cells (Blanchet

et al., 2010).

In addition, a virulence factor may also directly target the autophagy machinery to neg-

atively regulate autophagy. For example, the HSV-1 protein ICP34.5 binds BECN1 to block

autophagy, possibly through inhibiting PIK3C3 kinase activity (Orvedahl et al., 2007).