chronic granulomatous disease

IMMUNOLOGY AND IMMUNE SYSTEM DISORDERS

CHRONIC GRANULOMATOUS DISEASE GENETICS, BIOLOGY AND CLINICAL MANAGEMENT

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IMMUNOLOGY AND IMMUNE SYSTEM DISORDERS

CHRONIC GRANULOMATOUS DISEASE GENETICS, BIOLOGY AND CLINICAL MANAGEMENT Edited by

REINHARD A. SEGER DIRK ROOS BRAHM H. SEGAL AND

TACO W. KUIJPERS

Copyright © 2017 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. We have partnered with Copyright Clearance Center to make it easy for you to obtain permissions to reuse content from this publication. Simply navigate to this publication’s page on Nova’s website and locate the “Get Permission” button below the title description. This button is linked directly to the title’s permission page on copyright.com. Alternatively, you can visit copyright.com and search by title, ISBN, or ISSN. For further questions about using the service on copyright.com, please contact: Copyright Clearance Center Phone: +1-(978) 750-8400 Fax: +1-(978) 750-4470 E-mail: [email protected].

NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS.

Library of Congress Cataloging-in-Publication Data ISBN: 978-1-53612-498-9

Published by Nova Science Publishers, Inc. † New York

CONTENTS Introduction

vii

About the Authors and Contributors

ix

Section I Chapter 1 Chapter 2 Chapter 3 Section II Chapter 4

Biochemistry and Signaling Functions of NADPH Oxidase

1

Biochemistry of the Leukocyte NADPH Oxidase

3

NADPH Oxidase-Mediated Antimicrobial Activity: When Do We Need It?

35

Dysfunctional Processes Regulating IL-1β in Chronic Granulomatous Disease

69

Clinical Presentation and Diagnosis of CGD

89

Bacterial and Fungal Infections in Chronic Granulomatous Disease

91

vi Chapter 5

Contents Endemic Infections in Chronic Granulomatous Disease

125

Inflammatory Manifestations in Chronic Granulomatous Disease

163

Clinical Manifestations in X-Linked Carriers of Chronic Granulomatous Disease

203

Biochemical and Genetic Diagnosis of Chronic Granulomatous Disease

231

Section III

Management

301

Chapter 9

Checklists for Clinicians: Major Complications in CGD: How to Proceed

303

Allogeneic Hematopoietic Stem Cell Transplantation

311

Resources – Chronic Granulomatous Disease Websites

343

Section IV

Research

349

Chapter 12

Gene Therapy for CGD

351

Chapter 13

Chronic Granulomatous Disease – Quality of Life Studies

383

Future Directions in CGD Research

397

Chapter 6 Chapter 7 Chapter 8

Chapter 10 Chapter 11

Chapter 14 Index

403

INTRODUCTION This is the first e-book on Chronic Granulomatous Disease (CGD). This book is meant for everyone involved in the diagnosis, treatment or guidance of patients with this disease, as well as for the patients and their parents/caretakers, investigators and students learning from this disease. The authors hope that this book will help everybody involved to gain more knowledge about the cause of CGD, the symptoms and the treatment of the patients in various geographical regions to help to improve their lives. The authors have invited experts in the various aspects of CGD (fellow authors) to write chapters on their specialty, and have sent these chapters to other specialists (the contributors) for comments and additions. Nevertheless, if you think that this book contains errors or omissions, please do not hesitate to let us know. In future updates, the authors can improve the text if necessary. The editors invite comments and additions, and implore any readers with concerns to send them to one of them; their e-mail addresses can be found below.

viii

Reinhard A. Seger, Dirk Roos, Brahm H. Segal et al. Zürich, Amsterdam, Buffalo, July 2017 Reinhard A. Seger Dirk Roos Brahm H. Segal Taco W. Kuijpers

[email protected] [email protected] [email protected] [email protected]

Sponsored by Immunodeficiency Canada

ABOUT THE AUTHORS AND CONTRIBUTORS AUTHORS Alexandra Battersby, M.D., University of Newcastle and Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom. E-mail: [email protected] Martin de Boer, B.Sc., Dept. of Blood Cell Research, Sanquin Blood Supply Organization, Amsterdam, The Netherlands. E-mail: [email protected] Mary C. Dinauer, M.D., Ph.D., Washington University School of Medicine, St. Louis, MO 63110, U.S.A. E-mail: [email protected] Andrew R. Gennery, M.D., University of Newcastle Medical Society, Newcastle upon Tyne, United Kingdom. E-mail: [email protected]

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Reinhard A. Seger, Dirk Roos, Brahm H. Segal et al. David Goldblatt, M.D., Institute of Child Health, University College London and Great Ormond Street Hospital NHS Foundation Trust, London, United Kingdom. E-mail: [email protected] Tayfun Güngör, M.D., Division of Stem Cell Transplantation, University Children`s Hospital of Zürich, Zürich, Switzerland. E-mail: [email protected] Steven M. Holland, M.D., Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1684, U.S.A. Email: [email protected] Taco W. Kuijpers, M.D., Ph.D., Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, and Dept. of Blood Cell Research, Sanquin Blood Supply Organization, Amsterdam, The Netherlands. E-mail: [email protected] Yu-Lung Lau, M.D. Department of Paediatrics and Adolescent Medicine, Room 115, 1/F New Clinical Building, Queen Mary Hospital, Pokfulam, Hong Kong, and Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China. E-mail: [email protected] Pamela P.W. Lee, M.D. Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China. E-mail: [email protected]

About the Authors and Contributors

xi

Karin van Leeuwen, M.Sc., Dept. of Blood Cell Research, Sanquin Blood Supply Organization, Amsterdam, The Netherlands. E-mail: [email protected] Beatriz E. Marciano, M.D., Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1960, U.S.A. E-mail: [email protected] Janine Reichenbach, M.D., Division of Immunology, University Children’s Hospital Zurich, and Children’s Research Centre, Zurich, Switzerland. E-mail: [email protected] Dirk Roos, Ph.D., Dept. of Blood Cell Research, Sanquin Blood Supply Organization, and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. E-mail: [email protected] Brahm H. Segal, M.D., University at Buffalo Jacobs School of Medicine and Biomedical Sciences, Roswell Park Cancer Institute, Buffalo, NY 14263, U.S.A. E-mail [email protected] Reinhard A. Seger, M.D., Division of Immunology, University Children`s Hospital of Zürich, Zürich, Switzerland. E-mail: [email protected] Ulrich Siler M.D., Division of Immunology, University Children’s Hospital Zürich – Eleonoren Foundation, CH-8032 Zürich, Switzerland. E-mail: [email protected]

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Reinhard A. Seger, Dirk Roos, Brahm H. Segal et al. Adrian J. Thrasher, M.D., Institute of Child Health, University College London, WC1N 1EH, London, United Kingdom. E-mail: [email protected] Anton T.J. Tool, Ph.D., Dept. of Blood Cell Research, Sanquin Blood Supply Organization, Amsterdam, The Netherlands. E-mail: [email protected] Frank L. van de Veerdonk, M.D., Ph.D., Dept. of Pediatrics, Radboud University Medical Center, Nijmegen, The Netherlands. E-mail: [email protected]

CONTRIBUTORS Jacinta Bustamante, Laboratory of Human Genetics of Infectious Diseases, INSERM U550, and René Descartes University, Necker Medical School, Paris, France. E-mail: [email protected] Andrew Cant, Paediatric Immunology, Allergy and Infectious Diseases, Great North Children’s Hospital, Newcastle upon Tyne, United Kingdom. E-mail: [email protected] William M. Nauseef, M.D., Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Coralville, IA 52241, USA. E-mail: [email protected] Joachim Roesler, Dept. of Pediatrics, University Hospital Carl Gustav Carus, Dresden, Germany. E-mail [email protected]

SECTION I. BIOCHEMISTRY AND SIGNALING FUNCTIONS OF NADPH OXIDASE

Chapter 1

BIOCHEMISTRY OF THE LEUKOCYTE NADPH OXIDASE Mary C. Dinauer ABSTRACT The leukocyte NADPH oxidase catalyzes the transfer of electrons from NADPH to oxygen, thus generating superoxide (O2), which is the precursor to multiple reactive oxygen species (ROS) with microbicidal and immunoregulatory functions. Inactivating mutations in this enzyme result in the inherited immune deficiency chronic granulomatous disease (CGD), associated with defects in host defense and aberrant inflammation. The active enzyme complex includes flavocytochrome b, a heterodimer comprised of gp91phox (also known as NOX2 or CYBB) and p22phox (CYBA) and located in plasma and phagosome membranes, and four regulatory subunits that translocate from the cytosol to flavocytochome b upon cellular activation. The latter include p47phox (NCF1), p67phox (NCF2) and p40phox (NCF4), along with the GTP-bound form of Rac. Phosphorylation of p47phox and formation of Rac-GTP are two

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Mary C. Dinauer key events in assembling the active oxidase complex on the membrane. The p67phox subunit is a catalytic co-factor involved in the initial transfer of electrons from NADPH to flavocytochrome b, whereas the other subunits serve various roles in regulating assembly and activity of the NADPH oxidase complex. Additional events important for optimal oxidase activity include the production of phosphoinositides and the maintenance of charge compensation to counteract the electrogenic effects of electron transfer across the membrane.

INTRODUCTION The leukocyte NADPH oxidase is a multi-subunit phagosome and plasma membrane-associated enzyme (Figure 1) that generates large quantities of superoxide from molecular oxygen (O2) upon activation by particulate or soluble inflammatory stimuli [1-5]. The redox center of the enzyme is flavocytochrome b, found in plasma and phagosome membranes, which is activated upon binding of cytosolic regulatory subunits that in turn are regulated by protein phosphorylation and binding of phosphoinositides and other membrane-associated lipids (Figure 1). The enzyme is expressed in neutrophils, monocytes and macrophages, eosinophils, and dendritic cells, as well as B and perhaps T lymphocytes, although its function in lymphocytes is not well understood. Expression of NADPH oxidase subunits and enzyme activity is highest in neutrophils and in eosinophils. Autosomal or X-linked recessive defects in the NADPH oxidase complex result in chronic granulomatous disease (CGD), characterized by recurrent bacterial and fungal infections as well as aberrant inflammation that is not always associated with infection [1]. These symptoms reflect the importance of NADPH oxidase-derived ROS not only for killing

Biochemistry of the NADPH Oxidase

5

microbes but the emerging recognition of their influence on the digestion of protein antigens and on redox-regulated cellular processes that limit inflammation [2, 6]. This chapter describes the biochemistry of the leukocyte NADPH oxidase-mediated superoxide generation and the structure, function, and regulation of its constituent subunits. The activity of the NADPH oxidase and generation of microbicidal oxidants is also supported by ionic fluxes generated by membrane transporters.

BIOCHEMICAL PROPERTIES OF THE NADPH OXIDASE Quiescent neutrophils rely primarily on glycolysis and consume relatively little oxygen. However, following activation by chemoattractants or contact with microbes, O2 consumption increases as much as 100-fold [7]. This was originally believed to reflect increased mitochondrial respiration [8]. However, this phenomenon was subsequently recognized to reflect the consumption of O2 by the NADPH oxidase to produce O2-, and hence the enzyme is also referred to as the respiratory burst oxidase [9]. Notably, it was also discovered that this activity was absent in patients with CGD [10]. The reaction catalyzed by the NADPH oxidase involves the transfer of electrons from NADPH to molecular oxygen and is shown below: NADPH + 2O2

NADP+ + H+ + 2O2-

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Mary C. Dinauer

Figure 1. Leukocyte NADPH oxidase. The domain architecture of the NADPH oxidase subunits is shown as block diagrams. Blue dotted lines, interactions between phox subunits prior to activation. Red dotted lines, additional interactions that mediate NADPH oxidase assembly and activity. Membrane components include the gp91phox (NOX2) subunit of flavocytochrome b, containing an NADPH-binding site and FAD in its C-terminal flavoprotein domain and N-terminal membrane-spanning domains harboring a pair of heme groups. The p22phox subunit contains at least two membrane spanning domains and a hydrophilic C-terminus with a PxxP motif. Three soluble phox subunits, p47phox, p67phox, and p40phox, are linked in a trimeric complex in the cytosol prior to NADPH oxidase activation. p67phox is constitutively associated with p40phox via complementary PB1 (phox and Bem1) motifs. p67phox is also tethered to p47phox via a tail-to-tail interaction involving a SH3 domain and a proline-rich region (PRR) in the respective C-termini of these subunits. p67phox contains four tetratricopeptide (TPR) motifs targeted by Rac-GTP. p47phox contains tandem SH3 domains for binding to the p22phox PxxP motif as well as a regulatory PX domain that binds membrane phospholipids. The p47phox PX and SH3 domains are masked in the resting state by an adjacent polybasic auto-inhibitory domain. The PX domain in p40phox is highly specific for PI(3)P, whereas the p47phox PX domain can bind a broader range of phospholipids. GTP-bound Rac is an essential subunit of the NADPH oxidase. In unactivated cells, Rac-GDP resides in the cytoplasm in a complex with Rho-GDI. Following cellular activation, phosphorylation of the p47phox auto-inhibitory domain exposes its SH3 motifs, leading to translocation of the trimeric phox complex to flavocytochrome b558, where an activation domain (AD) in p67phox promotes electron transfer through gp91phox. Activation of guanine nucleotide exchange factors results in exchange of GTP for GDP on Rac as well as removal of Rho-GDI, allowing Rac-GTP to bind to the membrane via its isoprenyl group and adjacent polybasic domain, in order to activate p67 phox and gp91phox. PX domains in p47phox and p40phox additionally regulate the enzyme on plasma and phagosome membranes, respectively. Used with permission [2].


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NADPH is oxidized at the cytoplasmic surface and oxygen is reduced to form O2- on the outer surface of the plasma membrane (or interior surface of the phagosome membrane). Thus, oxidants are released at sites of microbial contact or within the phagocytic vacuole, where they can interact with contents of neutrophil granules or macrophage lysosomes to potentiate microbicidal activity. Superoxide is a relatively weak antimicrobial compound and also has a very short half-life. However, O2- serves as the precursor to potent reactive oxygen species (ROS) that are crucial for killing many species of bacteria and fungi [11]. Superoxide dismutates either spontaneously or via superoxide dismutase to form hydrogen peroxide (H2O2). Myeloperoxidase catalyzes the formation of hypochlorous acid (HOCl) from H2O2 [12]. Other ROS derivatives include the hydroxyl radical OH• whose formation is generated in a nonenzymatic reaction catalyzed by Fenton chemistry via either iron or copper ions [11]. H2O2 can diffuse from within phagosomes into the cytosol, and can oxidize glutathione as well as impact other redox-regulated protein modifications. In human neutrophils, NADPH is the physiologic substrate used by the oxidase, as the Km for NADPH is 33 µM, whereas it is 930 µM for NADP [13]. Continuous generation of NADPH is needed to provide a source of electrons for the NADPH oxidase. Replenishing NADPH from NADP+ occurs via utilization of glucose in the hexose monophosphate shunt, with glucose-6phosphate dehydrogenase as the first enzyme in this pathway [11].

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Mary C. Dinauer

COMPONENTS AND ASSEMBLY OF THE NADPH OXIDASE There are five subunits of the NADPH oxidase that have no known function outside of their role in the oxidase. These “dedicated” subunits are referred to by their molecular mass (kDa) and the designation “phox,” for phagocyte oxidase, or alternatively, named based on the corresponding gene. Studies of different genetic subgroups of CGD played a crucial role in the identification and molecular cloning of each of these subunits [1422]. The catalytic core and redox center of the NADPH oxidase is flavocytochrome b, a heterodimer of two integral membrane proteins, gp91phox (CYBB [cytochrome b beta subunit], also known as NOX2 [NADPH oxidase 2]) and p22phox (CYBA [cytochrome b alpha subunit]), affected in X-linked CGD and a form of autosomal recessive CGD, respectively. CYBB and CYBA refer to the large and small subunits of flavocytochrome b. There are three cytosolic subunits affected in other autosomal recessive forms of CGD, p47phox (NCF1), p67phox (NCF2), and p40phox (NCF4), where NCF refers to Neutrophil Cytosolic Factor. In addition, the GTPbound form of the Rac GTPase induces critical conformational changes in several phox subunits within the NADPH oxidase complex. The importance of Rac was discovered when identified as a protein factor, originally referred to as NCF3, that was required for high level activity in cell–free oxidase assays [23, 24]. With the exception of flavocytochrome b, most of the key functional domains have been crystallized, as summarized in several recent reviews that also include additional details on the properties of each subunit [3-5, 25].


9

Flavocytochrome b: gp91phox and p22phox

Flavocytochrome b is an unusual b-type cytochrome that has a spectral peak of light absorbance at 558 nm, and is often referred to as flavocytochrome b558. It also an extremely low midpoint potential of -245 mV and thus is also known as flavocytochrome b-245 [26]. While some flavocytochrome b is located in the plasma membrane, much is found in intracellular membrane compartments. In resting neutrophils, the majority of flavocytochrome b resides in specific granules, secretory vesicles, and gelatinase granules, whereas in resting monocytes, macrophages and dendritic cells, flavocytochrome b is found in endosomal compartments in addition to the plasma membrane [2729]. The gp91phox subunit is the electron transferase of the oxidase, and is a 570 amino acid glycoprotein encoded by the X-linked CYBB gene affected in the X-linked form of CGD (see also Chapter 8) [14, 16, 17, 30]. A topologic and functional model of gp91phox was deduced based on predicted features of its domains, epitope mapping and other studies [3, 4, 13, 31, 32] as well as insights from rare missense mutations in CYBB [33]. gp91phox harbors multiple hydrophobic regions in its N terminus that are likely membrane-spanning and contain a pair of heme moieties with different midpoint potentials (Figure 1) [3, 4, 13]. Its Cterminal flavoprotein domain is cytosolic, and homologous to ferredoxin-nicotinamide adenine dinucleotide phosphate phox reductases (FNR) [3, 13]. This gp91 domain contains a flavin adenine dinucleotide (FAD) moiety that accepts a pair of electrons from NADPH that are then shuttled as single electrons across the membrane via sequential transfer to the paired heme residues to reduce extracellular or phagosome-located O2, thereby generating superoxide.

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The p22phox subunit contains 195 amino acids and has Nterminal hydrophobic domains that are likely membrane-spanning and a hydrophilic C-terminus (Figure 1) [18, 32, 34]. This subunit has two major functions. First, p22phox plays a key role in assembly of the active enzyme complex. The C-terminus of p22phox contains a proline-rich PxxP motif that interacts with high affinity to tandem SH3 domains in p47phox [35-37]. This interaction mediates the docking of an otherwise cytosolic trimeric complex of p47phox, p67phox and p40phox [3, 4, 13]. Second, association of p22phox with gp91phox to form a heterodimer in the endoplasmic reticulum (ER) is important for the stability of each flavocytochrome b subunit, and also promotes heme incorporation into gp91phox and efficient transport from the ER to peripheral membrane compartments [13, 38, 39].

Regulatory Subunits: p47phox, p67phox and p40phox

The three soluble phox subunits, p47phox, p67phox, and p40phox are located in the cytosol prior to cellular activation (Figure 1) [3, 4, 13]. p67phox is constitutively associated with p40phox via complementary PB1 (phox and Bem1) motifs in their C-termini, and studies in both CGD patients and mice genetically deficient in one or the other subunit indicate this association is important for their mutual stability [1]. p67phox is also tethered to p47phox via a tail-totail interaction involving a SH3 domain and a proline-rich region (PRR) in the respective C-termini of these subunits. In resting cells, the abundance of the trimeric complex containing p47phox, p67phox, and p40phox varies, but is believed to increase upon cellular activation for translocation to flavocytochrome b [3, 40]. Whereas p67phox is an essential cofactor for activating electron transport, the


11

p47phox and p40phox subunits act as regulated adaptor proteins that optimize the assembly and activity of the enzyme complex. The p67phox subunit contains 526 amino acids in a series of domains that biophysical studies suggest are connected by flexible linkers [41]. The C-terminus of p67phox contains four tetratricopeptide (TPR) motifs that create a binding surface for Rac-GTP, and binding to Rac-GTP is necessary both to induce the active conformation of p67phox as well as to stabilize and retain p67phox in the active oxidase complex [3, 4, 13, 42, 43]. p67phox functions as a catalytic component of the NADPH oxidase and contains a discrete “activation domain” [44] that interacts with the flavoprotein domain of gp91phox (Figure 1), believed to promote NADPH binding and electron transfer to FAD. The p67phox subunit is also phosphorylated at sites within its N- and C-termini, which appears to modulate its activity [45]. p47phox is comprised of 390 amino acids and contains a pair of SH3 domains for binding to its target PxxP motif in the cytosolic Cterminus of p22phox as well as a PX (phox homology) domain that interacts with a variety of phospholipids (Figure 1) [3, 4, 13]. In resting cells, p47phox is in a “closed” conformation, with its PX and SH3 domains masked by an adjacent polybasic auto-inhibitory domain. Phosphorylation of serine residues within the p47phox autoinhibitory domain during oxidase activation releases this inhibitory interaction [37]. The major function of p47phox is to mediate translocation of the trimeric phox complex to membrane–bound flavocytochrome b. Neither p67phox nor p40phox are able to stably translocate to the phagosome membrane in CGD neutrophils lacking either p47phox, harboring a missense mutation in the p22phox PxxP motif, or lacking flavocytochrome b [3, 4, 46]. The p47phox subunit is also necessary for maximal translocation of p67 phox to the plasma membrane of activated neutrophils [47, 48]. However, a small amount of NADPH oxidase activity can still be detected in

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Mary C. Dinauer

p47phox–negative neutrophils [49], likely reflecting its function as an accessory protein rather than directly involved as a co-factor protein in electron transport. Allosteric effects of p47phox within the assembled complex also contribute to its role in the NADPH oxidase [3, 4, 13, 50, 51]. These include effects mediated by its regulatory PX domain that binds PI(3,4)P and, less strongly, other phosphoinositides and to phosphatidylserine and phosphatidic acid. One surface of the p47phox PX domain binds to cPLA2, which may generate arachidonic acid locally to augment oxidase activity [52]. The function of p40phox in the NADPH complex remained uncertain for many years, as it was is not needed for high level enzyme activity on the plasma membrane or in cell free systems [53]. It is now recognized that p40phox plays a specialized role in stimulating high levels of superoxide production on neutrophil phagosome membranes via a signal from the membrane lipid phosphatidylinositol 3-phosphate (PI(3)P) [54-58]. This role was first elucidated in studies using genetically engineered cells and mice [54-57, 59, 60]. PI(3)P binding to p40phox was recently shown to also positively regulate plasma membrane oxidase activity in macrophages [61]. The p40phox subunit contains 339 amino acids and harbors an N-terminal PX domain with high affinity and specificity for PI(3)P, which becomes unmasked during oxidase activation. Subsequently, a CGD patient with autosomal recessive mutations in NCF4 was discovered, including one allele with a missense mutation ablating PI(3)P binding [58]. Neutrophils from this patient had selective and profound defects in neutrophil phagosome but not plasma membrane oxidant production. How p40phox regulates phagosome oxidase activity in a PI(3)Pdependent manner is not fully understood. Most, but not all, of the requirement for PI(3)P appears to be mediated through its binding to the PX domain of p40phox [60]. In some settings, p40phox acts to assist p47phox in recruiting p67phox to phagosome flavocytochrome


13

b [43]. Other studies support a direct role for PI(3)P-bound p40phox in upregulating oxidase activity [56, 57]. Additional work suggests that p40phox-regulated translocation to phagosomes is of greater importance when p47phox is only partially phosphorylated [62]. Taken together, it appears that p40phox, similar to p47phox, contributes both to both assembly of the NADPH oxidase and to regulating its activity via conformational effects, with p40 phox specializing in the phagosome-localized enzyme via PI(3)P. PKC –mediated phosphorylation of p40phox on a threonine residue is also important for its optimal activity, but the underlying mechanism is uncertain [63]. Finally, there are poorly understood differences in mouse and human neutrophils in their requirement for p40phox. Phagocytosis-induced NADPH oxidase activity is much more profoundly reduced in human neutrophils deficient in p40phox PI(3)P-binding compared to similarly affected mouse neutrophils [43, 54, 58, 64]. In mouse phagocytes, oxidase activity elicited by IgG-opsonized particles, opsonized bacteria and TLR2 ligands are more dependent on PI(3)P binding to p40phox compared to fungal particles [54, 61].

Rac-GTP as Critical NADPH Oxidase Subunit

In addition to the oxidase-specific phox subunits, GTP-bound Rac interacts with both p67phox and gp91phox to induce conformational changes needed for high-level enzymatic activity [65]. The blood cell-specific Rac2 isoform is predominant in human neutrophils, whereas both Rac1 and Rac2 operate in macrophages. In unactivated cells, Rac is present in the GDPbound form and sequestered by Rho-GDI in the cytoplasm, which masks its isoprenylated and polybasic C-terminus that otherwise enables Rac binding to membranes. GTP loading of Rac results in

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conformational changes in its switch regions that allow Rac-GTP to bind to and activate downstream effectors. The main target of Rac-GTP in the NADPH oxidase is the p67phox subunit, which contains a Rac-binding domain created by four -helical tetratricopeptide repeat (TPR) motifs [42]. The GTP-bound form of Rac also appears to interact with the C-terminal flavoprotein domain of flavocytochrome b to regulate the stepwise electron transfer from NADPH to oxygen [66].

REGULATION OF NADPH OXIDASE ACTIVATION NADPH oxidase activity is tightly controlled, and triggered upon binding of particles, bacteria, fungi or soluble inflammatory mediators to specific receptors on the phagocyte cell surface that then activate a complex web of downstream signaling pathways [5, 67, 68]. These lead to activation of kinases and production of lipid mediators that induce assembly of the active membrane-bound enzyme complex from the otherwise spatially segregated cytosolic and membrane subunits. Translocation of the cytosolic phox complex may in part be facilitated by their association with the cytoskeleton, although definitive details remain uncertain. The focal point for oxidase assembly is flavocytochrome b. In response to soluble agonists, the oxidase complex is assembled on plasma membrane-associated flavocytochrome b (Figure 1, 2). During phagocytosis, additional flavocytochrome b for NADPH oxidase assembly is delivered to neutrophil phagosome by the membrane fusion of specific granules, or for macrophage phagosomes, via endosomes [2, 28, 29]. This delivery is in turn regulated by proteins involved in vesicular trafficking, including SNAP-23,


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Rab11, and Rab27 (reviewed in [2]). There is also some evidence for assembly of the NADPH oxidase on endosomes and, in neutrophils, specific granules prior to their fusion with phagosomes [25, 43, 69].

Figure 2. Regulation of phagosome NADPH oxidase activity. This schematic highlights features that contribute to phagosome NADPH oxidase and microbicidal activity. Binding of the membrane phosphoinositide PI(3)P to p40 phox strongly upregulates phagosome NADPH oxidase activity. Hv1 proton channels compensate NADPH oxidase-dependent charge build-up caused by its translocation of electrons into the phagosome lumen. Proton translocation is also mediated through V-ATPase, particularly in macrophages. Phagosomal ROS production and pH are closely linked, since superoxide reacts with and consumes luminal protons, producing H2O2. Clinflux into the phagosome via chloride transporters CFTR and ClC-3 support the synthesis of HOCl. Ca2+ efflux from phagosomes by STIM1-mediated SOC channel activation from tightly apposed ER may promote NADPH oxidase assembly and activity via calcium binding proteins S100A8/A9. Vesicular trafficking delivers additional flavocytochrome b into phagosomes, and regulators of this process include Rab11, Rab27 and/ or SNAP-23. Used with permission [2].

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Chief among events regulating oxidase assembly is phosphorylation of multiple serines in the auto-inhibitory region of p47phox by serine kinases, including various protein kinase C (PKC) isoforms, to render its SH3 and PX domains accessible to their targets (Figure 1) [3, 4, 70-72]. Release of auto-inhibition exposes tandem SH3 domains on p47phox that target the PxxP motif in p22phox, leading to translocation of the trimeric p47phox-p67phoxp40phox complex to the membrane flavocytochrome b. Partial phosphorylation of p47phox by agents such as TNF or endotoxin contributes to a more rapid and robust activation of the NADPH oxidase to subsequent stimulation, an effect often referred to as “priming” [73]. On phagosomes, the associations between phox proteins appear to be dynamic following their recruitment to flavocytochrome b. The tail-to-tail association between p47phox p67phox is lost [74] and PI(3)P binding is required to retain p40phox [57, 58], which implies that the PB1-PB1 domain interaction between p67phox and p40phox is also no longer operative. In addition to p47phox, phosphorylation of the other NADPH oxidase subunits is also observed, which likely positively regulates their function [3]. A second and independent critical event to turn on NADPH oxidase activity is the activation of Rac and its recruitment as part of the NADPH oxidase complex. As already mentioned, GTP loading of Rac results in conformational changes allowing its effector domains to bind to target motifs in both p67phox and gp91phox [3, 4, 66]. The formation of Rac-GTP first requires the release of Rac-GDP from Rho-GDI in the cytosol to expose its isoprenoid tail followed by membrane translocation and exchange of GDP to its active GTP-bound state. These events are controlled by phosphorylation of Rho-GDI [75] and activation of guanine nucleotide exchange factors (GEFs), respectively. In neutrophils activated by G protein coupled receptors for chemoattractants, P-


17

Rex1 is the major exchange factor that generates Rac-GTP [76, 77]. P-Rex1 is activated by Class I PI3 kinase-generated PIP (3,4,5) and G protein beta gamma subunits. The Vav family of GEFs is important for activating Rac in leukocytes stimulated via immunoreceptors. These immunoreceptors include Fc receptors for IgG, the dectin-1 receptor for -glucans and the 2 integrin CR3 (CD11b/ CD18) that binds to opsonic complement fragment iC3b, unopsonized bacteria and fungal -glucans. Upon receptor binding, signaling is initiated by Src family kinase–mediated phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) either directly on receptors or present on Dap12 and Fc adaptors [78-81]. This leads to recruitment of the Syk tyrosine kinase and activation of downstream effectors, including Vav1 and Vav3 that exchange GDP for GTP on Rac [82-86]. Membrane-associated lipids provide an additional level of NADPH oxidase regulation. These include the binding of phospholipids to PX domains in p47phox and p40phox [5]. PI(3)Pregulated oxidase activity on phagosomes is of particular importance, which strongly upregulates NADPH oxidase activity in this compartment in neutrophils and macrophages via the PX domain of p40phox [5, 61]. This phosphoinosotide is generated largely by the action of Class III PI3 kinase Vps34, which is present on intracellular membranes in a complex with p150 (Vps15) [87, 88]. Vps34 is active constitutively on Rab5-positive endosomes, and their rapid fusion with phagosomes shortly after ingestion leads to the synthesis of PI(3)P on the cytosolic leaflet of the phagosome membrane [57, 89-91]. In murine macrophages, PI(3,4)P2, generated by the inositol phosphatase SHIP-1 from PI(3,4,5)P, also contributes to oxidase activation elicited by IgGopsonized particles, possibly by enhancing activation of PKC , which can phosphorylate p47phox and p40phox [92]. The PX domain of p47phox instead plays a greater role in enhancing NADPH

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oxidase activity at the plasma membrane [51]. Interestingly, phorbol ester activation of the plasma membrane oxidase is insensitive to the PI3 kinase inhibitor wortmannin [51], suggesting its dependence on the p47phox PX domain may be mediated by PI(4,5)P2, an abundant plasma membrane phospholipid. Arachidonic acid and anionic membrane lipids are also implicated in inducing conformational changes in flavocytochrome b itself to enhance electron transfer [25, 68].

ACCOMPANYING IONIC FLUXES THAT SUPPORT NADPH OXIDASE AND OXIDATIVE MICROBICIDAL ACTIVITY The activity of the NADPH oxidase and generation of microbicidal oxidants is also supported by ionic fluxes generated by membrane transporters (Figure 2). The NADPH oxidase is electrogenic, since it transfers electrons across the membrane, and its sustained activity is dependent on mechanisms that mediate continuous charge compensation [93, 94]. Most evidence supports the voltage-gated proton channel [95] Hv1 encoded by the HVCN1 gene as the most important agent of charge compensation during NADPH oxidase activity [96-101]. The relationship between NADPH oxidase activity, proton ionic fluxes and pH within the phagosome is complex and varies between neutrophils, macrophages and dendritic cells, in large part related to differences in usage of vacuolar ATPases to regulate pH. Luminal protons are consumed by superoxide to produce H2O2, and thus pH is also influenced by the relative level of NADPH oxidase activity. These details are beyond the scope of this chapter, but are summarized in a recent review [2]. Influx of chloride (Cl-) into neutrophil phagosomes is needed to support the formation of hypochlorous


19

acid [11]. Chloride channels of importance for neutrophil phagosomes include CFTR (cystic fibrosis transmembrane conductance regulator) and the ClC-3 Cl-/H+ antiporter [102-105]. The Ca2+ content inside phagosomes decreases over time [106], and in neutrophils, this release involves activating phagosome store-operated Ca2+ channels by ER-bound STIM1 proteins that mediate juxtaposition to phagosome membranes [107]. STIM1 and its partner channel Orai1 promote neutrophil phagosome NADPH oxidase activity by stimulating the Ca 2+-dependent recruitment of S100A8/A9 proteins [108]. S100A8/A9 proteins are very abundant in neutrophil cytosol and act as Ca2+ sensors that can interact with flavocytochrome b and p67phox to enhance electron transfer [68, 109-111]. It is proposed that localized Ca2+ microdomain “hot spots” generated by Ca2+ release from phagosomes may directly modulate phagosome NADPH oxidase activity [2].

THE NOX FAMILY OF NADPH OXIDASES Although the gp91phox flavocytochrome was the first example recognized as an eukaryotic flavocytochrome, many homologs have since been discovered throughout the animal and plant kingdom. The most closely related are the NOX (NAD(P)H oxidase) proteins, of which there are 5 members, including gp91phox, which is designated as NOX2 in this classification [4, 13, 112, 113]. Other NOX proteins are expressed in various cell types, including lung and gut epithelium, smooth-muscle cells, and the endothelium, and generate superoxide, but generally at much lower levels than the leukocyte enzyme. At least some of the other NOX flavocytochromes appear to be regulated by homologs of p67phox and p47phox. The derivative oxidants are proposed to mediate signal transduction and, in epithelial cells, may also function in innate immunity.

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CONCLUSION The leukocyte NADPH oxidase plays important roles in both antimicrobial host defense and regulating processes of importance for innate and adaptive immunity. Activity of this enzyme is controlled by many layers of regulation that target assembly of the enzyme from cytosolic and membrane-bound components and fine-tune its output.

ACKNOWLEDGMENTS MCD is funded by the Children’s Discovery Institute at Washington University and St. Louis Children’s Hospital. I thank Tina McGrath for assistance with preparation of the manuscript. I apologize to colleagues whose work could not be cited due to space limitations.

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Mary C. Dinauer mediated phagosomal oxidative activity. J Immunol 2011; 186: 2182-2191. Brechard S, Tschirhart EJ. Regulation of superoxide production in neutrophils: role of calcium influx. J Leukoc Biol 2008; 84: 1223-1237. Berthier S, Nguyen MV, Baillet A, Hograindleur MA, Paclet MH, Polack B, et al. Molecular interface of S100A8 with cytochrome b558 and NADPH oxidase activation. PLoS One 2012; 7: e40277. Berthier S, Paclet MH, Lerouge S, et al. Changing the conformation state of cytochrome b558 initiates NADPH oxidase activation: MRP8/MRP14 regulation. J Biol Chem 2003; 278: 25499-25508. Al Ghouleh I, Khoo NK, Knaus UG, et al. Oxidases and peroxidases in cardiovascular and lung disease: new concepts in reactive oxygen species signaling. Free Radic Biol Med. 2011;51(7):1271-88. Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol. 2004; 4: 181-189.

Chapter 2

NADPH OXIDASE-MEDIATED ANTIMICROBIAL ACTIVITY: WHEN DO WE NEED IT? Taco W. Kuijpers With contributions by William M. Nauseef

ABSTRACT Neutrophils play a critical role in the prevention of invasive fungal infections. Mouse studies have demonstrated the role of various neutrophil pathogen recognition receptors (PRRs), signal transduction pathways and cytotoxicity in the murine antifungal immune response, but much less is known about the killing of fungi by human neutrophils. Most killing activity of phagocytes such as neutrophils is attributed to their capacity to produce large amounts of reactive oxygen species (ROS) by the NADPH oxidase complex. A defect in the phagocyte NADPH oxidase complex results in lack

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Taco W. Kuijpers of ROS production, a syndrome known as Chronic Granulomatous Disease (CGD). Studies with these human “knock-out” neutrophils, knock-out animal models and in-vitro cellular models have expanded our knowledge about the importance of and the interaction between ROS-mediated and ROS-independent killing mechanisms and the role of PRRs and signaling in human microbial killing, as will be discussed. From the studies with these patients it is becoming clear that neutrophils employ fundamentally distinct mechanisms to kill bacteria and fungi.

INTRODUCTION Chronic granulomatous disease (CGD) is a genetically heterogeneous disease characterized by recurrent life-threatening infections with bacteria and fungi, and dysregulated granuloma formation. CGD is caused by defects in NADPH oxidase, the enzyme complex responsible for the phagocyte respiratory burst that leads to the generation of superoxide and other reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) [1-3]. For details see Chapters 1 and 8. In the presence of myeloperoxidase (MPO) and chloride in the neutrophil phagosome, H2O2 is converted to hypochlorous acid (HOCl, bleach) with strong antimicrobial activity but with a short half life [4]. HOCl is subsequently converted to other microbicidal agents such as chloramides and aldehydes, with extended half lives [5]. The rapid consumption of oxygen for the production of superoxide and its metabolites is referred to as the so-called “respiratory burst.” In addition to ROS, neutrophils also use proteins and peptides stored in granules to kill microorganisms. Together with ROS, these products are released into the phagosome that contains ingested microbes. The best known of these proteins are serine proteases that degrade microbial constituents. Other cytotoxic

Antimicrobial Activity

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constituents sequester or target essential bacterial products and for that reason have microbicidal activity. Examples are the ironbinding protein lactoferrin and proteins that not only neutralize ‘endotoxin’ (i.e., lipopolysaccharide or LPS) in the membrane of Gram-negative bacteria but can also cause permeabilization (azurocidin and bactericidal/permeability-increasing protein [BPI]). Also small peptides with microbicidal activity (defensins) are released into this compartment. Although these mechanisms of ROS formation and the toxic capacity of granule proteins seem to act independently, it is already known for a long time that these different killing processes strongly synergize under many circumstances [6]. On the other hand, the killing mechanisms are not completely redundant, as is illustrated best by the clinical characterization of genetically proven phagocyte defects. It has become clear that different pathogens are killed by neutrophils with different mechanisms. This chapter describes for a number of the most frequently found bacteria and fungi in CGD patients how neutrophils and macrophages normally cope with these microbes. Several surface receptors and intracellular activation pathways for these killing reactions will be described. See also Tables 1 & 2. Many of the killing mechanisms are based on experimental models to support or understand their contribution to the pathophysiology observed in human disease. For that reason we will compare the molecular mechanisms in mice and men in this chapter and report on the similarities and differences in host defense between the two species.

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INFECTIOUS MANIFESTATIONS CGD patients with a defect in the NADPH oxidase activity are more susceptible to invasive bacterial and fungal infections, accompanied by increased rates of mortality. CGD can present any time from infancy to late adulthood, but the majority of patients are diagnosed during childhood. Lung, skin, lymph nodes, and liver are the sites of infection most frequently affected, followed by osteomyelitis, perianal abscesses, and gingivitis [7-11] (See Chapters 4 and 5). The overwhelming majority of infections in CGD consist of only a limited number of organisms: Staphylococcus aureus, Burkholderia (Pseudomonas) cepacia, Serratia marcescens, Nocardia and Aspergillus species, but also Klebsiella pneumoniae and Salmonella species [7, 8-10, 11]. With trimethoprim-sulfamethoxazole prophylaxis, the frequency of bacterial infections in general has diminished. Bacteremia is uncommon, and usually due to B. cepacia or S. marcescens. Also BCG complications following vaccination have been noted, ranging from none to self-limited localized or disseminated BCGitis. Increased prevalence of tuberculosis in CGD patients is found in areas where TB is endemic. See also Chapter 5. Fungal infections due to Aspergillus fumigatus are the leading cause of mortality in CGD [7, 8, 10, 11]. Itraconazole prophylaxis and the newer agents for treatment of filamentous fungal infections, such as voriconazole and posaconazole, have markedly reduced the frequency and mortality of fungal infections in CGD. Although less frequently isolated, Aspergillus nidulans is rather exclusive to CGD and goes along with an even higher rate of mortality than Aspergillus fumigatus [9].


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Table 1. Pattern recognition receptors (PRRs) involved in recognition by human host immune cells, with emphasis on phagocytes Name TLRs TLR2/1

TLR2/6 TLR3 TLR4

TLR5 TLR7 TLR8 TLR9 TLR10 CLRs Dectin-1 Dectin-2

Components involved

Microbial origin

Pam3CSK4

Gram-neg E coli-based lipopeptides Gram-pos Staph aureus Mycoplasma fermentans-derived Mycoplasma salivarium-based viruses Gram-neg bacteria Respiratory syncytial virus (RSV) Chlamydia pneumoniae Gram-neg bacteria viral & bacterial replication viral & bacterial replication bacterial DNA motifs

Lipoteichoic acid MALP-2 FSL-1 dsRNA Endotoxin (LPS) Glycoprotein F HSP60 Flagellin ssRNA ssRNA unmethylated CpG -β-1,3 glucan -mannan

Candida albicans, fungi Candida albicans; Schistosoma mansoni; Mycobacterium tuberculosis

Dectin-3 Mincle NLRs NOD1

-mannan --

diamino pimelic acid (DAP) NOD2 muramyl dipeptide (MDP) NLRC3 (NOD3) --

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Taco W. Kuijpers Table 1. (Continued)

Name

Components Microbial origin involved NLRC5 (NOD4) -NLRX1 (NOD5) -NLRC4 (IPAF) type III/IV secretion Gram-neg bacteria: system (cytosolic flagellin) Shigella flexneri, Salmonella spp, Legionella pneumophila, Pseudomonas aeruginosa NAIP (NLRB) cytosolic flagellin Salmonella typhimurium, Pseudomonas aeruginosa, Listeria monocytogenes NLRP1

NLRP3

NLRP6 NLRP12 ALRs AIM2

IFI16

MDP lethal toxin exotoxins

ESX1/ESAT6 (via RNA helicase Dhx15) -DNA

DNA

Bacillus anthracis, Toxoplasma gondii, Staph. aureus ( -hemolysin) Str. pyogenes (streptolysin O) Klebsiella pneumoniae Mycobacterium tuberculosis Norovirus, enterovirus Yersinia enterocolitica Vaccinia virus, CMV Francisella tularensis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Listeria monocytogenes Kaposi sarcoma-associated herpesvirus (KSHV, HHV8), HSV1, CMV, HIV


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Table 2. Ligands and corresponding microbial or endogenous PAMPs and DAMPs for the most commonly expressed Toll-Like Receptors TLR2 and TLR4 Ligand TLR2 Pam3CSK4 MALP-2

Origin

Known interactors

E. coli–based lipopeptides Mycoplasma fermentans– derived Mycoplasma salivarium–based Human cytomegalovirus Herpes simplex virus endogenous ligands?

TLR2/TLR1 TLR2/TLR6

TLR4 LPS

Gram-negative bacteria

TLR4, MD-2, CD14 (LBP)

Flagellin Fusion protein HSP60 Cleaved fibrinogen

Gram-negative bacteria RSV Chlamydia pneumoniae Aspergillus oryzae

Fibronectin

Endogenous, extracellular matrix

FSL-1 ? ?

(type III domain A) Biglycan Endogenous, extracellular matrix Hyaluronan Endogenous, extracellular matrix HSP60/70 Endogenous, cytoplasm HMGB1 Endogenous, cytoplasm/nuclear Beta-defensins Endogenous, mucosal Oxidized LDL Endogenous, plasma Surfactant protein Endogenous, lung A MRP8/14 Endogenous, cytoplasm, releasate Feutin A Endogenous, plasma

TLR2/TLR6 TLR2/TLR1 TLR2/TLR1/TLR6

TLR4, CD14 TLR4, MD-2 TLR4, proteinase, thrombin TLR4, MD-2

TLR4 (TLR2) TLR4 (TLR2) TLR4, MD-2 TLR4 TLR4 TLR4 (TLR6, CD36) TLR4 TLR4, MD-2, CD14 TLR4

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In the past the difference between catalase-negative and catalase-positive strains has been suggested to be the major discriminating factor to explain why certain strains are causing disease in CGD and others do not. Catalase-negative strains excrete H2O2, which might substitute for the lack of cellular H2O2 generated in CGD phagocytes and thus lead to killing of catalasenegative microbes even by CGD neutrophils. In contrast, catalasepositive strains, like S. aureus, degrade their own H2O2 and are therefore killed less efficiently by CGD neutrophils. This issue has been readdressed in a mouse model of CGD with clinical isolates of catalase-positive and -negative strains of S. aureus. The results show that these organisms were equally virulent and that the H2O2 they produced are insufficient for significant antimicrobial impact [12]. However, S. aureus is not a natural pathogen of mice, which is why murine “models” of human staphylococcal infection are contrived and frequently misleading. Nevertheless, since the large majority of all pathogens contains catalase, with the important exception of streptococci, the prevailing view now is that catalase is not a major pathogenicity factor in microbes infecting CGD patients.

DIFFERENT PERSPECTIVES Although the basis for the selectivity of pathogens in CGD is unknown, there may be some explanations possible that will be discussed in this chapter. Microbial killing depends on many factors, including factors strictly confined to the pathogens themselves, but also to the risk and extent of exposure, barrier functions of the epithelial layers of skin and mucosa, invasiveness and virulence factors, as well as various host defense mechanisms. The latter are related to how the microbes are being recognized


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and signal to oxidative and non-oxidative mechanism by phagocytes when they have infiltrated into the infected tissue. Apart from the above-mentioned factors and mechanisms that vary, the interaction between the phagocyte and microbial agents is not a static one. Starting with the premise that damage inflicted upon microbes in neutrophil phagosomes is the result of biochemical attack, it follows that variability of microbe composition would result in differential susceptibility to modification. For example, Grampositive bacteria such as Staphylococcus aureus provide an array of proteins, carbohydrates, and lipids for attack that differ in part from those presented by Gram-negative bacteria such as Escherichia coli or the mycolic acid-rich Mycobacterium tuberculosis. Not only do the potential substrates for biochemical attack differ among species but also between organisms of the same strain that are in different phases of growth. This is true for both bacteria and fungi, where the latter morphologically change from single-cell conidia into invasive hyphae. This adds an additional layer of antigenic variation. Moreover, the pathogen may be induced to adapt and change its composition because of the interaction with phagocytes.

RECEPTORS Both bacterial and fungal cell walls consist of specified determinants that are now being generally referred to as pathogenassociated molecular patterns (PAMPs), and multiple determinants have been identified that may be used for the relatively selective recognition by surface molecules. For binding fungal PAMPs human neutrophils express soluble (such as pentraxin-3 and

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ficolin), and membrane-bound pathogen recognition receptors (PRRs, including Toll-like receptors [TLRs], C-type lectin receptors [CLRs] and integrins) (Table 1). In particular the CD11b/CD18 heterodimer, the major beta-2 integrin on neutrophils, also known as the Complement Receptor-type 3 (CR3 or Mac1), plays an important role in this respect [13-16]. For S. aureus, and Gram-positive strains in general, the surface peptidoglycans and lipoteichoic acid (LTA) residues are recognized by TLR2, which is heterodimerized with either TLR1 or TLR6. TLR2 recognizes a broad repertoire of determinants, as has been supported by several experimental infection models [1718]. In case of fungi, both yeast and molds have a cell wall consisting of layers of β-glucans, mannans and chitin, that are recognized by immune cells as PAMPs [19]. These fungal constituents dramatically change in their expression and/or composition at the highly distinct and different stages of their growth. For instance, fungi reproduce by forming conidia and germinate into hyphae for dissemination. Upon germination, the composition and glycosidic linkages of the cell wall changes, e.g., the outer β-glucans are masked by mannans on the hyphal formations, and in this way escape host surveillance [20, 21]. Neutrophils express several TLRs, of which TLR2 (as dimers with either TLR1 or TLR6) and TLR4 are most prominently expressed; these TLRs can bind mannans among many other ligands (Table 2). The endosomal TLR9 binds microbial DNA motifs [19]. TLR3 and TLR7 are absent in human neutrophils [22]. Murine TLR2- and TLR4-deficient neutrophils show normal phagocytosis and killing of C. albicans, but are impaired in the uptake and killing of A. fumigatus [23, 24], whereas TLR9 was found to be dispensable in the neutrophil-mediated killing of bacterial strains, Aspergillus and Candida. Thus, murine studies show a role for TLR2 and TLR4 in the fungal host defense,


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whereas in humans genetic variation known as single nucleotide polymorphisms (SNPs) in TLR1 and TLR4 has been shown to predispose to fungal infections in immunocompromised patients [25-27]. Apart from microbial ligands or PAMPs, TLR2 and in particular TLR4 have been reported to also recognize a large number of endogenous ligands, known as damage-associated pattern molecules, (DAMPs, also known as ‘alarmins’), that alert the host defense system of injury, ischemia or inflammationrelated damage locally or in a systemic way. Hundreds of CLRs form a heterogeneous group that recognizes a wide range of microorganisms. All CLRs share a characteristic C-type lectin-like domain and can be either soluble or membrane bound. The human neutrophil-expressed CLRs dectin-1 (CLEC7A), dectin-2 (CLEC4A) and mincle (CLEC4D) bind fungal β-glucans and αmannose [28-30], but whether mouse dectin-1 has a broader pathogen-associated molecular recognition profile is uncertain. Dectin-1 knock-out mice are susceptible to candidiasis, dependent on the Candida strain and presence of chitin in the cell wall of the Candida [29, 31]. Murine neutrophils stimulated with Candida conidia depend on dectin-1 to activate via Vav signaling the integrin complement receptor-3 (CR3) for the cytotoxic response [32, 33]. Murine dectin-1-deficient granulocytes are also impaired in the uptake of A. fumigatus conidia [33, 34]. In humans, however, dectin-1 deficiency may perhaps contribute to chronic mucocutaneous candidiasis (CMC), but no invasive fungal infections have been reported to date [35]. Neutrophils derived from dectin-1-deficient individuals are completely normal in the binding and killing in vitro of both A. fumigatus and C. albicans [36, 37]. The data on dectin-1 demonstrate the different role of this CLR on granulocytes from mice and men. On the other hand, dectin-1 on human monocytes and dendritic cells is indeed required for the production of

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cytokines and for the polarization of T cells to control the adaptive IL17-dependent fungal immune response and to prevent CMC [38, 39]. Unlike dectin-1, both dectin-2 and mincle associate with the chain of Fc R, and its ITAM domain then activates signal transduction pathways [28, 30]. Both decrin-2 and mincle are expressed by human neutrophils. It has been suggested that Candida hyphae in particular are recognized by dectin-2 in the mouse [27, 28, 40]. Apart from the promotion of Th17-based antifungal immunity, dectin-2 is apparently also involved in NADPH oxidase activation, because murine dectin-2-deficient neutrophils produce lower amounts of ROS in response to C. albicans [41, 42]. HEK293 cells transfected with human dectin-2 do not bind Candida conidia or hyphae [36], indicating that the PAMPs for dectin-2 are still unknown. In contrast to the fact that mincle knock-out mice demonstrate increased susceptibility to systemic candidiasis, human neutrophils only show weak expression and a role in anti-fungal activity has yet to be shown. In summary, these CLRs are essential in the murine neutrophil cytotoxic response towards Candida, and dectin-1 and dectin-2 towards Aspergillus, but do not seem to be involved in the human neutrophil-mediated fungal killing. Likewise, the integrin CR3 contains a lectin-binding site [43]. CR3 is also able to bind iC3b-opsonized particles, which facilitates binding and killing of complement-opsonized microbes by neutrophils [44]. In serum, other opsonins, such as IgGs, are present. Antigen-specific antibodies often determine the response of human neutrophils. Whereas TLRs determine the cytokine response of neutrophils to both viable bacterial and fungal strains [45], the uptake of these strains and/or activation of the NADPH oxidase activity are not determined by TLRs. In fact, specific antibodies and complement are essential for in-vitro ROS


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induction and phagocytosis of extracellular bacteria, whereas capsular components, such as yeast-derived zymosan or the conidia of Candida yeast and molds such as Aspergillus, can be phagocytosed in the absence of antibodies or complement via the lectin-binding site of CR3 [36, 37]. On the other hand, neutrophilmediated killing of Aspergillus hyphae (but not Candida hyphae) strictly depends on opsonizing IgGs without the involvement of complement factors or CR3 [37]. For unopsonized conditions, see Tables 1 & 2, and schematic representation shown in Figure 1.

INTRACELLULAR SENSING AND INFLAMMASOME FORMATION Under certain conditions in either primary cells or upon overexpression in cellular systems, inflammasomes that bind and process pro-caspase-1 can be activated by eight members of NLRs (NLRP1, NLRP2, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4, and NAIP) and AIM2 (Table 1), while NLRP2 and NLRP4 act as negative regulators of the NF- B pathway. In human neutrophils, NLRP3 and NLRC4 are the main intracellular microbial sensors, whereas macrophages have a wider repertoire of NLRs. Caspase-1 processes the pro-inflammatory cytokine pro-IL-1 to generate mature IL-1 , which is presumably released by cell lysis during pyroptosis. Although IL-1 cleavage and release are tightly coupled events, it is still unclear whether caspase-1 plays a direct role in driving IL-1 release or whether release occurs as a result of compromised membrane integrity that precedes cell death. Caspase-1 also initiates pyroptosis by cleaving gasdermin D. Human caspase-4 and caspase-5 (murine caspase-11) oligomerizes upon binding with cytosolic LPS and becomes active.

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The finding of gasdermin D as the executioner of pyroptotic cell death argues against an alternative role for (multi)vesicular- and/or autophagy-dependent secretory pathways of the unconventional release of the leaderless peptide IL-1 [46, 47]. For further involvement in sensing microbial components during the whole process of killing of intracellular pathogens, see below.

Neutrophil killing of Aspergillus fumigatus Inhibition of A. fumigatus germination

Killing of A. fumigatus hyphae

CR3 IgG

Fc R SYK

kindlin3 PI3K

PI3K

Lactoferrin Fe

Non-oxidative killing mechanisms

H2O2 NADPH oxidase

Figure 1. Neutrophil-mediated killing of Aspergillus hyphae (but not Candida hyphae) strictly depends on opsonizing IgGs without the involvement of complement factors or the CD11b/CD18 integrin (ref 77).

INVOLVEMENT OF THE NADPH OXIDASE Activation of various signal transduction pathways triggers a number of cytotoxic effector mechanisms in neutrophils, including ROS production by the NADPH oxidase system and the release of


49

granule-derived proteins, including proteases and microbicidal peptides (Figure 1). This can be initiated through direct binding of the pathogen to surface molecules on the neutrophil or through soluble factors released by the pathogen. Human neutrophil killing of certain pathogens may depend completely on non-oxidative, proteolytic activity, as clearly indicated by the fact that human CARD9-deficient neutrophils produce normal amounts of ROS in response to certain fungal stimuli but show a defective Candida killing upon conidia germination [48]. On the other hand, CGD neutrophils are completely defective in killing serum-opsonized S. aureus and A. fumigatus hyphae, whereas these cells normally inhibit the germination of A. fumigatus conidia [37]. The latter suggests that once the Aspergillus conidia succeed in germinating into hyphae, CGD patients are at risk to develop aspergillosis, but not so upon minimal exposure to intact conidial membranes. This may explain why CGD patients do not suffer more often from fungal infections (even when not using antifungal prophylaxis). Finally, there are examples in which both killing mechanisms operate at the same time, possibly synergistically [5]. For instance, the killing of Gram-negative Escherichia coli depends on both ROS and the proteolytic activity of serine proteases such as elastase and cathepsin G, which represent some of the major constituents of the azurophil granule in neutrophils [49]. Inhibition of either mechanism leads to partial inhibition of E. coli killing. Using engineered S. typhimurium constitutively expressing flagellin or PrgJ, NLRC4 inflammasome activation was shown to inhibit systemic bacterial infection in mouse intraperitoneal infection. This effect is independent of IL-1 and IL-18, despite that many cytokines are upregulated upon sensing of flagellin and S. typhimurium infection [50]. Instead, restriction of S. typhimurium infection is critically mediated by caspase-1-

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mediated cell death (Table 3). Inflammasome activation induces in macrophages a rapid form of cell death known as pyroptosis, which can limit the replication of intracellular pathogens. Consistent with the absence of ASC in the ‘death NLRC4 inflammasome’, clearance of the flagellin-expressing S. typhimurium does not require ASC.

Table 3: Differences in killing mechanisms involved or used by human macrophages and neutrophils during host defense against bacterial pathogens Mechanism Macrophages Phagocytosis ++ ROS formation ++ Degranulation +/− Cytokine release +++ Autophagy-enhanced ++ phagocytosis Autophagy-induced ++ cell death Apoptosis ++ Necrosis ++ Pyroptosis ++++ NET formation −

Neutrophils ++++ ++++ ++++ +* ? ? ++ ++ − ++++

* IL-8 and IL-1 Receptor Antagonist (IL1RA), but not IL-6, IL-10, or TNF ,

It has been further suggested that caspase-1-induced pyroptosis releases the bacteria from infected macrophages, resulting in uptake by the neutrophil and consequently in killing of the bacteria by ROS. NLRC4 inflammasome activation can also clear


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Burkholderia or Legionella infection through a cytokineindependent mechanism [50]. Thus, neutrophil-mediated killing may apply generally to bacteria that are detected by NLRs. Nlrc4−/− mice succumb to infection with the flagellin-expressing S. typhimurium. This effect is also mediated primarily by pyroptosis, while IL-1 /18 only play a minor role. Neutrophils themselves respond to S. typhimurium both in vitro and during peritoneal infection [51]. The resulting NLRC4 inflammasome activation does not induce pyroptosis in neutrophils, but provides a major source of IL-1 in peritoneal S. typhimurium infection for NLRC4 inflammasome-mediated antibacterial defense involving murine neutrophils. To which extent these mechanisms of pyroptosis by macrophages and classical killing and IL-1 production by human neutrophils contribute to Salmonella clearance in humans is uncertain, but CGD patients are definitely more susceptible to Salmonella infections both by typhoid and non-typhoid strains. Although IL-1 can be produced in large amounts by human macrophages and these cells undergo pyroptosis, human neutrophils have not been shown to either undergo pyroptosis or produce large amounts of IL-1 - if any at all [45, 52]. Infections dominated by organisms of the Burkholderia cepacia complex (Bcc), a group of at least 17 closely-related species of Gram-negative bacteria, are particularly difficult to treat. Studies have shown remarkable differences in virulence between clinical isolates and environmental isolates of Bcc, and even clinical strains of the same species have been observed to have varying virulence in vitro and in mouse models [53]. Bcc produces at least four different exopolysaccharides. The predominant exopolysaccharide is the heptasaccharide cepacian, which is produced by most clinical isolates [54].

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Cepacian produced by B. cenocepacia has been shown to scavenge ROS and inhibit neutrophil chemotaxis in in vitro migration assays. Mutants that are unable to produce cepacian are less virulent than wild-type, cepacian-producing strains in a gp91phox−/− mouse model of CGD, which suggests that this exopolysaccharide is potentially an important bacterial factor for virulence in CGD [53]. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurs in p47phox−/− mice, whereas elastase x cathepsin G double knock-out mice, lacking most serine protease activity in their neutrophils, clear infection [52]. It may well be that the increased phagocytic uptake of Gram-negative Burkholderia strains results in an inadvertent survival advantage when ROS is not being generated for the usual killing of this commensal [53, 55].

GRANULAR PROTEINS FOR KILLING During granulopoiesis a variety of cytotoxic proteins and peptides are synthesized and stored in granules. These granular contents can be released by mature neutrophils upon activation, which is a highly effective cytotoxic mechanism [56]. MPO derived from the azurophilic granules converts H2O2 into the highly toxic antimicrobial HOCl [57]. Neutrophils from MPOdeficient patients are impaired in the intracellular killing of Candida conidia, which may contribute to their susceptibility to fungal infections [58]. The killing of Aspergillus hyphae by neutrophils that are completely MPO-deficient is also defective; however, the neutrophil-mediated conidia killing and inhibition of Aspergillus germination is normal [37]. Aspergillus infections have not been reported in MPO deficiency. This can either be explained by the normal capacity of MPO-deficient neutrophils to


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prevent Aspergillus germination, or by the fact that most MPOdeficient cases are not truly deficient and show residual MPO expression. In our experience over the last decades, truly complete MPO deficiency has a lower incidence than CGD (1:200,000), suggesting that the full clinical spectrum of MPO deficiency may be as yet not as clear as is generally believed. Elastase, a major neutrophil-derived serine protease stored in the azurophilic granules, has strong in vitro killing capacity against E. coli, by enzymatic attack of outer membrane protein A (OmpA) in the bacteria [59]. Also upon i.p. challenge with E. coli or K. pneumoniae, elastase-/- mice had poorer survival than w.t. mice [59, 60]. The bactericidal effect against E. coli and against S. aureus of the MPO system and of chymotrypsin-like cationic protein is synergistically enhanced by elastase, and this effect is independent of the catalytic activity of elastase [61]. The notion is that the cationic properties of elastase damage the bacterial cell wall. However, both elastase and cathepsin G are dispensable in the control of aspergillosis [60], although such double knock-out mice were previously suggested to be susceptible to systemic aspergillosis [62]. There are also patients that represent an intriguing human homologue of this multiple serine protease knock-out mouse model. Patients suffering from Papillon-Lefèvre syndrome (PLS) carry a mutation in the CTSC gene encoding cathepsin C. PLS is a rare autosomal recessively inherited disease that is characterized by palmoplantar keratosis and severe prepubertal periodontitis, and leads to premature loss of all teeth. CTSC mutations result in complete loss of activity and subsequent failure to activate immune response proteins. The severe periodontitis is thought to arise from failure to eliminate periodontal pathogens as a result of cathepsin C deficiency, but no invasive infections otherwise have been reported in these patients. Their neutrophils lack serine

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proteases (i.e., elastase, cathepsin G and proteinase 3) because of unsuccessful processing of these proteins during the early stage of myeloid development into neutrophils, whereas the small cationic antibiotic peptides known as the alpha-defensins as well as MPO are normally processed and stored [63-65]. Moreover, normal human neutrophils treated with serine protease inhibitors show a normal cytotoxic response against bacteria or fungi [36, 37], which may indicate incomplete blockade by the inhibitors even when used in combination, or activity of additional proteases or toxic agents in the killing process. In this respect, the direct role of defensins or cytotoxic proteins other than the proteolytically active enzymes such as the classical serine proteases elastase and cathepsin G may be more relevant than has generally been suggested [66]. In fact, azurophil granules, specialized lysosomes of the neutrophil, contain at least 10 proteins implicated in the killing of microorganisms. We have found that the neutrophil-mediated killing of Aspergillus hyphae strictly depends on the recognition of opsonizing IgGs by Fc Rs, which activates the NADPH oxidase activity in neutrophils, whereas neither complement nor CR3 are strictly involved. In contrast, Candida and Aspergillus conidia are recognized and killed independently of ROS [37]. For this killing activity, human neutrophils express various proteases and nonproteolytic antimicrobial peptides (AMPs), which most likely act together and have additive effects in the activity of human neutrophils against pathogens. First, apart from the membrane-bound PRRs, neutrophils also express several soluble PRRs, prestored in their specific granules, including ficolin-M and pentraxin-3 (PTX3), which may facilitate the cytotoxic response against A. fumigatus [46, 67] and Pseudomonas species in lung disease [68], and against E. coli in urinary tract infections [69].


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Secondly, several additional small antimicrobial peptides are known [66]. For instance, six small proteins called α-defensins from the granules of neutrophils (human neutrophil peptides [HNPs] 1-4) [46] and from Paneth cells (human defensins HD5 and HD6), as well as four human β-defensins derived from epithelial cells (HD1-4) have been identified. These HNPs represent more than 5% of the total protein of human neutrophils, but can also be derived from intestinal Paneth cells, mucosal epithelial cells and keratinocytes [66]. The human neutrophil defensins are synthesized as 94-amino-acid (aa) preproHNPs, which are converted to 75-aa proHNPs by cotranslational removal of a 19-aa endoplasmic reticulum signal peptide. At the promyelocytic stage of myelopoiesis, proHNPs are further proteolytically modified and accumulate in azurophil granules as 29-30-aa HNPs. Human neutrophils contain large amounts of three alpha-defensins (HNP-1-3), and smaller amounts of a fourth, HNP-4. Monocytes and macrophages generally lack defensins, but they release messengers that induce the synthesis of -defensins in epithelial cells. The α-defensins are released upon activation of the neutrophils. Thirdly, bactericidal/permeability-increasing protein (BPI) is another non-proteolytic AMP stored in leukocytes, with a high affinity for LPS of Gram-negative bacteria and is also active against viral capsids. The anti-bacterial properties of BPI include permeabilization of bacterial membranes, in addition to the neutralization of LPS. The structural arrangement of BPI includes two large, barrel-shaped domains connected by a central betasheet. Molecules containing this arrangement are known as BPI fold-containing (BPIF) proteins. Members of the BPIF superfamily can be separated into two clusters: one including the lipid-transfer proteins BPI, lipopolysaccharide-binding protein (LBP), cholesteryl-ester transfer protein (CETP), and phospholipid-

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transfer protein (PLTP), and another branch containing the Palate Lung and Nasal epithelium Clone (PLUNC) proteins, encoded by 12 genes and expressed almost exclusively in the respiratory tract [66]. Finally, lactoferrin is a multifunctional iron-binding glycoprotein belonging to the transferrin family. Lactoferrin is a glycoprotein located in the majority of exocrine secretions (e.g., milk, tears, nasal secretions) and in neutrophils, with a role in ROS-independent killing of Aspergillus conidia [37]. Although the working mechanism is not exactly known, bioactive peptides derived from lactoferrin can also exhibit strong antifungal activity, with some surpassing the potency of the whole protein. An immunodeficiency related to the function of the granules is familial hemophagocytic lymphohistiocytosis type 5 (FHL-5), characterized by a deficiency of the syntaxin-binding protein Munc18-2 (STXBP2) required for fusion of granules with the plasma or phagosomal membrane [70]. FHL-5 patients suffer from a complex syndrome, including defects in cytotoxic T and NK cell function, macrophage activation, hemophagocytosis and infections that may or may not trigger the recurrent inflammation and hemophagocytic events. Recently, we reported on the impaired phagolysosome formation by neutrophils from FHL-5 patients, resulting in a moderate killing defect of E. coli and Candida albicans. The ROS production in FHL-5 neutrophils is normal, and these cells effectively kill S. aureus as well as Aspergillus fumigatus hyphae [49; unpublished data]. Once again, these data indicate that the neutrophil is equipped with a ROS-independent killing mechanism, but the efficacy highly depends on the invading pathogen itself.


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NET FORMATION In the last decade it has been proposed that neutrophils possess an additional cytotoxic mechanism by the formation of neutrophil extracellular traps (NETs), weblike chromatin structures containing histones, granule-derived proteases and antimicrobial peptides [71]. Activation of the RAF-MEK-ERK pathway has been reported to result in MPO and elastase translocation to the nucleus and decondensation of the chromatin as a prerequisite for NET formation [72, 73]. This route of NET formation is dependent on NADPH oxidase activity and thus absent in CGD patients. However, bacteria, fungi and salival mucins can also induce NADPH oxidase-independent NET formation [74, 75], as supported by our findings with CGD neutrophils [37]. Whether NETs are able to directly kill microbes is under debate. A study on a single CGD patient with aspergillosis suggests that gene therapy restored NET formation, which then results in the resolution of the aspergillosis [76]. However, it may have been the recovery of the NADPH-oxidase activity itself, instead of the ability to form NETs, that in this CGD patient has resulted in the improved clinical condition. In fact, DNAse treatment of neutrophils incubated with Aspergillus hyphae prevents NET formation without any effect on the in-vitro killing [37]. The role of NETs in the microbicidal activity of neutrophils is doubtful, knowing that also the neutrophils from PLS patients are defective in NET formation, have a normal ROS production and lack part of the proteolytic capacity, but yet PLS patients live without clinical susceptibility to wide-spread infections [64, 65].

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CONCLUSION The identification and studies of novel primary immunodeficiencies in patients with a susceptibility to fungal infections have expanded our knowledge on PRRs and on signaling in neutrophil-mediated fungal killing mechanisms, and have in many cases provided an explanation for the observed clinical phenotype (Tables 1 & 2; Figure 1). It should be noted that the exact mechanisms of fungal killing in mice can be very different from those observed in humans. Human neutrophils use distinct killing mechanisms for bacteria, Candida and Aspergillus species, and with respect to the yeast and molds, their use in killing may also vary depending on the actual stage as conidia or hyphae [36, 37]. Which of the mechanisms are induced by the many signaling proteins and transcription factors for killing remains an interesting topic for future research. As importantly taught by the studies performed with CGD neutrophils and the help of our patients worldwide, the structure and assembly of the NADPH oxidase complex upon activation has been clarified to a very large extent. The production of ROS by the NADPH oxidase system and chlorination by MPO into toxic metabolites are involved in the killing of many serum-opsonized bacteria, and some of the stages of Candida (germination) and Aspergillus (hyphae). ROS production by the NADPH oxidase system and chlorination by MPO are not involved in the killing of Aspergillus conidia and inhibition of its germination by human neutrophils. Neutrophils may use lactoferrin to deplete the Aspergillus from iron, which the fungus needs for its growth, and possibly neutrophils use additional granule-derived products such as PTX3 [77]. The neutrophil-mediated killing of bacteria and Aspergillus hyphae strictly depends on IgG opsonization and recognition by Fc Rs. Neutrophils only recognize hyphae upon


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serum opsonization, which results in the activation of Syk and PKC signaling for the production of ROS by the NADPH oxidase system and chlorination by MPO, which is strictly required for the in-vitro hyphae killing – but not for the killing of various bacterial test strains [36, 37, 77]. The exact role of ROS and granulederived components has not yet been completely appreciated, and the neutrophil-mediated killing of microbes seems to proceed independent of NET formation.

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[34] Taylor PR, Tsoni SV, Willment JA, et al. Dectin-1 is required for beta-glucan recognition and control of fungal infection. Nat Immunol 2007; 8: 31-38. [35] Ferwerda B, Ferwerda G, Plantinga TS, et al. Human dectin1 deficiency and mucocutaneous fungal infections. N Engl J Med 2009; 361: 1760-1767. [36] Gazendam, RP, van Hamme JL, Tool ATJ, et al. Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects. Blood 2014; 124:590-597. [37] Gazendam RP, van Hamme JL, Tool AT, et al. Human neutrophils use different mechanisms to kill Aspergillus fumigatus conidia and hyphae: evidence from phagocyte defects. J Immunol 2016; 196: 1272-1283. [38] Ferwerda, G, Meyer-Wentrup, F, Kullberg, BJ, Netea, MG, Adema, GJ. Dectin-1 synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages. Cell Microbiol 2008; 10: 2058-2066. [39] Gringhuis SI, Kaptein TM, Wevers BA, et al. Dectin-1 is an extracellular pathogen sensor for the induction and processing of IL-1beta via a noncanonical caspase-8 inflammasome. Nat Immunol 2012; 13: 246-254. [40] Zhu, LL, Zhao, XQ, Jiang, C, et al. C-type lectin receptors Dectin-3 and Dectin-2 form a heterodimeric patternrecognition receptor for host defense against fungal infection. Immunity 2013; 39: 324-334. [41] Saijo S, Ikeda S, Yamabe K, et al. Dectin-2 recognition of alpha-mannans and induction of Th17 cell differentiation is essential for host defense against Candida albicans. Immunity 2010; 32: 681-691.

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Chapter 3

DYSFUNCTIONAL PROCESSES REGULATING IL-1Β IN CHRONIC GRANULOMATOUS DISEASE Frank L. van de Veerdonk and Mary C. Dinauer INTRODUCTION In addition to the well-known infectious complications, CGD can also present itself with abnormal inflammatory responses, which often result in the formation of granuloma in inflamed tissues [1]. Interleukin-1 is a very potent inflammatory cytokine that induces production of chemokines that can attract neutrophils and causes fever [2]. This cytokine has to be tightly controlled in order to prevent detrimental inflammation. In addition to transcriptional mechanisms regulating IL-1, inflammasome activation and autophagy are important to control the tight balance

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of IL-1 production [3]. Here we will focus on the mechanisms regulating IL-1β and review the role of NADPH-oxidase complex deficiency in these processes.

IL-1 BIOLOGY The cytokine interleukin-1 (IL-1) is associated with acute and chronic inflammation, and plays an essential role in the host response to infection. IL-1 consists of IL-1β and IL-1α, and defects in regulatory mechanisms that control these potent cytokines can result in inflammatory disorders [2]. One way to control IL-1-mediated inflammatory responses is the production of IL-1 receptor antagonist (IL-1Ra), which can bind the IL-1 receptor and block IL-1 signaling. A deficiency of IL-1 receptor antagonist (DIRA) causes one of the most severe autoinflammatory syndromes, due to the lack of inhibition of the biological effects of IL-1β and IL-1α [4], which highlights the importance of controlling excessive IL-1 to prevent detrimental inflammation. Moreover, the relevance of IL-1 in disease is highlighted by the effective therapeutic interventions targeting these molecules in disease [5]. IL-1β is a highly inflammatory cytokine that is primarily secreted by monocytes, macrophages and dendritic cells (DC). An important step in the activation of IL-1β is processing of its inactive precursor into the biologically active cytokine. This processing can be mediated by several mechanisms including activation of the intracellular cysteine protease caspase-1 by the inflammasome [6, 7], but also (and often neglected) by caspase-1independent cleavage mediated by neutrophil-derived serine proteases or proteases released by invading microorganisms [8, 9].

IL-1β Regulation in CGD

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The inflammasome is a multimeric protein complex that assembles in response to danger signals sensed by the immune cell and that results in the processing of inactive caspase-1 into its active form ready to cleave IL-1β. Sensing of danger signals is mediated by intracellular NOD-like receptors (NLR) that upon activation start the assembly of the inflammasome. Inflammasomes are named after their activating NLR, such as the Nlrp3 inflammasome, Nlrc4 inflammasome, and the AIM2 inflammasome [10]. The NLRP3 inflammasome is important to mention here. Activating mutations in NLRP3 (also called cryopyrin) leads to subsequent activation of IL-1β causing an autoinflammatory disease called the “familial cold auto-inflammatory syndrome” (FCAS) [11]. The potent inflammatory role of IL-1 becomes overt in diseases that are specifically associated with dysregulated IL-1 production, and which fall into the category of autoinflammatory diseases. As discussed below inflammatory disease in CGD might also contain an autoinflammatory component since several mechanisms regulating IL-1 seem to be dependent on NADPH-oxidase activity, and therefore inflammatory complications in CGD might be amendable to the drug anakinra (recombinant IL-Ra).

DYSREGULATED INFLAMMATORY PATHWAYS IN CGD In addition to the IL-1 pathway, various immunological pathways have been studied in CGD since the disease can present itself not only as an immunodeficiency but also as an autoimmune and inflammatory disease, including colitis mimicking Crohn’s disease [12]. These clinical characteristics highlight the concept that optimal NADPH-oxidase function is essential in preventing

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dysregulation of many immunomodulatory mechanisms. It is therefore not surprising that not one but many immunological pathways have been studied and were found to be affected in CGD [13-15] (see also Chapter 6 of this book). IL-1α is released upon tissue damage in sterile inflammation. In a sterile inflammation model of peritoneal injury, X-linked CGD mice had increased concentrations of IL-1α in response to tissue damage compared to wild-type mice, resulting in higher GCSF and subsequently increased peripheral neutrophilia [16]. Hematopoietic cells, most likely resident peritoneal macrophages, were the source of excessive IL-1α. These observations identify a role for the NAPDH-oxidase complex in controlling sterile inflammatory responses during tissue damage. The rationale to first study the IL-1β pathway and its regulatory mechanisms in CGD was the observation made by Dostert et al. [17] that NADPH-oxidase-dependent reactive oxygen species (ROS) in macrophages were the second signal needed to activate the Nlrp3 inflammasome in monocyte/macrophages. This observation drove the hypothesis that a deficient NADPH-oxidase complex would result in the absence of a second signal crucial to activate the Nlrp3 inflammasome and would subsequently lead to less IL-1β production. However, when this hypothesis was tested in peripheral blood mononuclear cells isolated from patients with CGD there was no defect in inflammasome activation [18]. By contrast, these cells produced increased concentrations of IL-1β in response to lipopolysaccharide (LPS) and other inflammatory stimuli [18]. Several other studies supported this observation and all together these studies suggested that the inflammasome and IL1β production was not defective in CGD but was rather increased; providing evidence that there was a defect in regulating the tight balance of IL-1β production [19, 20]. The knowledge from the


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field of autoinflammatory diseases and the findings of increased inflammasome activation and IL-1β production in CGD provided a rationale to explore treating inflammatory complications in CGD with anakinra, and the first case reports suggested that this might be beneficial in CGD in certain conditions [19, 21]. But the question remained how a deficiency in NADPHoxidase would lead to increased inflammasome activation. One possibility was that ROS would be able to inhibit caspase-1 directly and in this way maintain a balance of caspase-1 activation and thus IL-1β production. Indeed caspase-1 has redox-sensitive residues, namely Cys397 and Cys362, and deficiency of SOD1 results in increased ROS concentrations that can directly inhibit caspase-1 activity by oxidation of these residues [22]. However, whether NADPH-dependent ROS directly inhibit the inflammasome protein caspase-1 is not clear to date. Another possible explanation came from novel insights in the pathogenesis of Crohn’s disease, as discussed in the following section.

NON-CANONICAL AUTOPHAGY: LC3-ASSOCIATED PHAGOCYTOSIS Colitis in CGD can closely mimic Crohn’s disease, and therefore it was hypothesized that there might be overlapping pathological mechanisms at play between CGD and Crohn’s disease [23]. Large genome-wide association studies in Crohn’s disease have identified a process called autophagy to be involved in the pathogenesis of Crohn’s disease [24-26]. Autophagy, literally meaning “self-eating,” is a highly conserved mechanism in which a cell undergoes self-digestion and component recycling [27]. Under conditions of cellular stress, such as hypoxia or

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starvation, the autophagy machinery will be activated to maintain cell homeostasis. Autophagy involves the formation of doublemembrane vesicles that form autophagosomes, which upon fusion with lysosomes form autophagolysosomes (Figure 1). This fusion with the lysosome will result in the degradation of the captured cytoplasmic cargo. In addition to its distinct role in cell survival, autophagy also has multiple effects on the immune response, including the elimination of intracellular pathogens [28, 29].

Figure 1. LC3-associated phagocytosis (LAP). Left panel shows phagocytosis with a phagosome fusing with an endosome, LC3 recruitment, and eventually with a lysosome. Right panel shows canonical autophagy, with a autophagosome that is formed inside the cell, with a double membrane surrounding intracellular components and decorated with LC3 that is important for fusion with the lysosome.


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Several steps are needed in order to form the membrane of the autophagosome and to let it fuse with the lysosome; these steps are dependent on interactions between autophagy (ATG) protein complexes [28]. When autophagy is induced, for example by starvation, an initiation complex (ULK1 complex) consisting of the kinase ULK1/2 (Atg1 in yeast), FIP200 (Atg17 in yeast), and ATG13 is assembled. The next complex required for the early stages of the autophagy process is the Beclin 1 complex, consisting of Beclin 1 (Atg6 in yeast), the PI3K vacuolar protein sorting 34 and its subunit 15 (VPS34 and VPS15), and ATG14L. These two complexes further coordinate the recruitment of other ATG proteins, which will result in the elongation of the membrane of the autophagosome. An essential step in this elongation process is the lipidation of the protein microtubule-associated protein light chain 3 (LC3)-II. Two systems are required for this process. LC3 consists in the cytosol as pro-form and first needs to be cleaved by ATG4 and activated by ATG7 and ATG3. Activated LC3-I in its cytosolic form will next be conjugated to phosphatidylethanolamine (PE) by a multimeric complex formed by ATG5, ATG12 and ATG16L1. This lipidated form of LC3 inserts in the elongating membrane and remains there during formation and maturation of the autophagosome and eventually will also be responsible for optimal lysosomal fusion [30]. A large GWAS study in Crohn’s disease demonstrated that polymorphisms in ATG16L1 were associated with disease development [24, 25]. As mentioned above, a signal is needed to induce the complex machinery of autophagy, such as starvation. Interestingly, it had been described that starvation induces ROS, which in turn can induce autophagy and can regulate the function of ATG4 that is involved in the initial steps resulting in lipidated LC3 [31]. In the same GWAS studies, a polymorphism in NCF4, encoding a component of the NADPH-oxidase (p40phox) was also

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associated with disease development, and noteworthy, a mutation in NCF4 can also result in CGD [24, 32]. All together these observations provided a rationale to explore whether there was a linked between NADPH oxidase-generated ROS and autophagy. The first study that demonstrated a link between the NADPHoxidase complex and the autophagy machinery showed that LC3 recruitment to the phagosome upon activation of TLRs or Fcγ receptor was dependent on NOX2 (gp91phox), since murine NOX2deficient macrophages failed to optimally recruit LC3 to the phagosome [33]. This process, whereby the autophagy machinery is interacting with the process of phagocytosis, is a form of noncanonical autophagy, now termed LC3-associated phagocytosis (LAP) [34]. This process differs from the previously described autophagy since it does not involve double membrane formation and is not dependent on the ULK1 initiation complex.

INFLAMMATION AND DEFICIENT LAP The next question that needed to be addressed was whether the polymorphisms in the proteins found in the GWAS for Crohn’s disease, namely ATG16L1 and NCF4 (p40phox), would also be involved in this non-canonical from of autophagy. Indeed similar to NOX2, NCF4-deficient murine macrophages also failed to recruit LC3 to the phagosome in response to an E. coli strain associated with Crohn’s disease [35]. Furthermore, it has also been demonstrated that ATG16L1 is essential for optimal LAP [36]. ATG16L1 is not only important for this process but a deficiency in ATG16L1 has been associated with dysregulated inflammatory responses. ATG16L1-deficient mice have increased


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inflammasome activation in response to lipopolysaccharide (LPS) and subsequently produce much higher concentrations of IL-1β. ATG16L1-deficient macrophages also produced increased IL-1β in response to monosodium urate (MSU), a classical Nlrp3 inflammasome activator [37]. ATG16L1-deficient mice are more susceptible to DSS colitis than wild-type mice [37]. These observations highlight the importance of ATG16L1 in controlling intestinal inflammation, IL-1β production and the inflammasome. Although the ATG16L1 polymorphism in Crohn’s disease shows similar defects as murine ATG16L1-deficient cells, suggesting that this polymorphism is a loss-of-function (LOF) mutation [38], it has not been studied whether it is associated with a deficiency in LAP. Since NADPH-oxidase deficient cells have increased IL-1β production in response to LPS and MSU, have increased inflammasome activation, and CGD mice have a more severe colitis in response to DSS [18, 39], NADPH-oxidase deficiency shares many characteristic features of ATG16L1 deficiency. The link between dysfunctional autophagy machinery and the regulation of IL-1β is further underscored by the observation that blocking autophagy by 3MA in human peripheral blood mononuclear cells results in increased IL-1β production in response to LPS [18, 40]. In NADPH-oxidase deficient cells, LPS already induces high levels of IL-1β which cannot be increased by blocking autophagy [35]. All together these data provide evidence that the NADPH-oxidase complex is essential for LAP and that deficient LAP in CGD might underlie the increased inflammasome activation with subsequent higher IL-1β production in response to inflammatory stimuli, although the mechanisms are currently unclear.

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BLOCKING IL-1 IN CGD The hypothesis that IL-1β plays an essential role in CGD colitis was further explored in an experimental colitis model in CGD mice. Treatment with anakinra, a recombinant form of IL1Ra used for the treatment of autoinflammatory diseases, prevented severe colitis in CGD mice [35]. CGD mice treated with anakinra showed less cytokines in the colon, including IL-17, and had much less severe histology compared to CGD mice not treated with anakinra. These observations provided a rationale to treat several patients with CGD colitis with IL-1Ra. Two adult patients with CGD colitis were reported, who both responded to blocking IL-1 with anakinra for 3 months [35]. The dosage used was 300 mg intravenously for three days followed by 100 mg s.c. daily. One other pediatric patient was treated for eight days before conditioning for bone marrow transplantation, with a dose of 15 mg s.c daily [19]. Frequency of stools was not changed, but anakinra had an effect on abdominal pain, and the monocytes stimulated ex vivo showed decreased IL-1β production in response to LPS during anakinra treatment compared to the situation before the start of treatment [19]. However, another report of five patients treated with anakinra at the National Institute of Health showed variable outcomes [41]. Two out of five patients with mild colitis symptoms responded, but patients with severe colitis did not respond. Therefore, anakinra might be an option in certain settings for patients with CGD colitis, but its efficacy remains uncertain since anakinra is not consistent in reducing severe disease in all patients [41]. Importantly, during the treatment of anakinra none of the reported patients developed any infectious disease, although


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the observation period was relatively short. This is in contrast with anti-TNFα treatment. Although blocking TNFα was highly effective in refractory colitis in CGD in five patients treated with the TNFα inhibitor infliximab, all five developed infectious complications during treatment, with three having Burkholderia infections, one with fungal infection and one with staphylococcal infection [42]. These were all severe infections, and two of the five patients died [42]. Therefore, when anti-TNFα treatment is considered in CGD colitis, it should be performed with extreme caution, and close monitoring. In conclusion, initial observations indicate that use of anakinra in some patients with CGD colitis can have beneficial effects but cannot cure disease, also highlighting that processes other than altered LAP, inflammasome activation and IL-1β production are likely to play a role in the pathogenesis of CGD colitis. Since an excessive inflammatory response during infection can also be detrimental in CGD, the use of anakinra was further explored also in the setting of infection in a murine model of experimental pulmonary aspergillosis, since this is one of the most significant infections in patients with CGD. Indeed, anakinra treatment of CGD mice improved survival following pulmonary challenge with Aspergillus fumigatus [35]. Anakinra treatment reduced the presence of proinflammatory cytokines, such as IL-1α, Il-1β, and IL-17 at the site of infection [35]. Neutrophil influx was also reduced significantly. Although there is no direct evidence that LAP controls inflammation induced by Aspergillus, these studies show evidence that Il-1β controls inflammation in response to Aspergillus.

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Figure 2. IL-1 modulating processes in CGD and possible effects of anakinra in CGD. Stimuli that are phagocytosed, such as Aspergillus fumigatus (Af), will lead to the recruitment of LC3 to the phagosome to enhance lysosomal fusion and therefore more efficient degradation of its contents. Optimal NADPH-oxidase activity is needed for LC3-associated phagocytosis (LAP), and when NADPH-oxidase is deficient there is less LC3 recruited to the phagosome. LAP is a non-canonical form of autophagy, and similar to defects in canonical autophagy, defects in LAP can be associated with increased IL-1β production, although the mechanism is unknown. IL1Ra (anakinra) might be beneficial in the setting of deficient NADPH oxidase by restoring on the one hand the defective LAP and on the other hand dampening IL-1βdriven inflammatory responses, including inflammasome activation. Whether anakinra works by blocking the IL-1β receptor directly or whether it has direct effects on LAP remains to be elucidated.

More evidence has been reported on the concept that LAP plays a role in monocyte/macrophage clearing of Aspergillus. Knockdown of ATG5 in macrophages results in decreased control of Aspergillus germination from conidia (spores) [43]. Moreover, mice deficient in LAP due to deletion of autophagy-related proteins, such as Beclin-1, Rubicon and ATG7, display increased susceptibility to invasive pulmonary aspergillosis [36]. However, it must be noted that these mice were not neutropenic at the time of


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infection and therefore the dose of Aspergillus needed to induce infection was much higher than that needed to infect CGD mice. A striking observation was that the fungal burden in CGD mice infected with Aspergillus and treated with anakinra was also significantly reduced [35]. Anakinra restored defective LC3 recruitment to the phagosome in response to Aspergillus in murine CGD macrophages [35]. Moreover, defective LAP was also demonstrated in human CGD monocytes in response to Aspergillus and was restored in the presence of anakinra [35]. The mechanism via which recombinant IL-1Ra restores deficient LC3 recruitment to the phagosome in CGD is not known and is currently under investigation (Figure 2). The extreme susceptibility to Aspergillus infection in CGD might therefore be twofold. On the one hand CGD neutrophils are defective in controlling hyphal growth, and on the other hand the CGD monocyte/macrophages are deficient in controlling conidial killing, and improving the latter might be amendable to anakinra.

CONCLUSION The knowledge that NADPH-oxidase activity has a role in IL1β-regulating mechanisms, LAP and the inflammasome has contributed to the expanding understanding of the inflammatory conditions associated with CGD. However, several issues still need to be understood more completely. Is deficient LAP responsible for increased inflammasome activation? And if so, what is the mechanism? As mentioned, excessive inflammatory responses that are characteristic of CGD can also be detrimental to controlling infection. Can blocking of IL-1 be beneficial during infections in patients with CGD? However, the use of IL-1 blockade in this setting may need to be a balancing act given the

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importance of IL-1 in promoting recruitment of neutrophils and other inflammatory cells that contribute to non-oxidative control of pathogens (e.g., IL-1 is crucial for recruitment of the first immune response against invading Aspergillus in the lung [44, 45]). It must also be taken into account that many pathways other than IL-1β are also responsible for the inflammatory conditions seen in CGD. For example the microbiome plays an important role in the development of Crohn’s disease and has also been shown to play a role in an experimental murine model of colitis in CGD mice [46]. The microbiome is therefore likely to play a role in colitis development in CGD, and the search for a microbiome that would prevent colitis in CGD is under investigation. All together, data outlined here support the concept that in certain specific inflammatory conditions such as colitis as well as in infections, anakinra might be beneficial and provide the rationale to further explore anakinra as an important adjunctive treatment option CGD.

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[13] Holmdahl R, Sareila O, Olsson LM, Backdahl L, Wing K. Ncf1 polymorphism reveals oxidative regulation of autoimmune chronic inflammation. Immunol Rev 2016; 269: 228-247. [14] Rieber N, Hector A, Kuijpers T, Roos D, Hartl D. Current concepts of hyperinflammation in chronic granulomatous disease. Clin Dev Immunol 2012; 2012: 252460. [15] Segal BH, Grimm MJ, Khan AN, Han W, Blackwell TS. Regulation of innate immunity by NADPH oxidase. Free Radic Biol Med 2012; 53: 72-80. [16] Bagaitkar J, Pech NK, Ivanov S, Austin A, Zeng MY, Pallat S, Huang G, Randolph GJ, Dinauer MC. NADPH oxidase controls neutrophilic response to sterile inflammation in mice by regulating the IL-1alpha/G-CSF axis. Blood 2015; 126: 2724-2733. [17] Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 2008; 320: 674-677. [18] Van de Veerdonk FL, Smeekens SP, Joosten LA, et al. Reactive oxygen species-independent activation of the IL1beta inflammasome in cells from patients with chronic granulomatous disease. Proc Natl Acad Sci U S A 2010; 107: 3030-3033. [19] Meissner F, Seger RA, Moshous D, Fischer A, Reichenbach J, Zychlinsky A. Inflammasome activation in NADPH oxidase defective mononuclear phagocytes from patients with chronic granulomatous disease. Blood 2010; 116: 15701573. [20] Van Bruggen R, Koker MY, Jansen M, et al. Human NLRP3 inflammasome activation is Nox1-4 independent. Blood 2010; 115: 5398-5400.


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[21] Van de Veerdonk FL, Netea MG, Dinarello CA, van der Meer JW. Anakinra for the inflammatory complications of chronic granulomatous disease. Neth J Med 2011; 69: 95. [22] Meissner F, Molawi K, Zychlinsky A. Superoxide dismutase 1 regulates caspase-1 and endotoxic shock. Nat Immunol 2008; 9: 866-872. [23] Marks DJ, Miyagi K, Rahman FZ, Novelli M, Bloom SL, Segal AW. Inflammatory bowel disease in CGD reproduces the clinicopathological features of Crohn's disease. Am J Gastroenterol 2009; 104: 117-124. [24] Rioux JD, Xavier RJ, Taylor KD, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet 2007; 39: 596-604. [25] Hampe J, Franke A, Rosenstiel P, et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet 2007; 39: 207-211. [26] Cummings JR, Cooney R, Pathan S, et al. Confirmation of the role of ATG16L1 as a Crohn's disease susceptibility gene. Inflamm Bowel Dis 2007; 13: 941-946. [27] Yang Z, Klionsky DJ. Eaten alive: a history of macroautophagy. Nat Cell Biol 2010; 12: 814-822. [28] Deretic V, Saitoh T, Akira S. Autophagy in infection, inflammation and immunity. Nat Rev Immunol 2013; 13: 722-737. [29] Levine B. Eating oneself and uninvited guests: autophagyrelated pathways in cellular defense. Cell 2005; 120: 159162. [30] Lai SC, Devenish RJ. LC3-Associated phagocytosis (LAP): Connections with host autophagy. Cells 2012; 1: 396-408.

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[31] Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J 2007; 26: 1749-1760. [32] Matute JD, Arias AA, Wright NA, et al. A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity. Blood 2009; 114: 33093315. [33] Huang J, Canadien V, Lam GY, et al. Activation of antibacterial autophagy by NADPH oxidases. Proc Natl Acad Sci U S A 2009; 106: 6226-6231. [34] Romao S, Munz C. LC3-associated phagocytosis. Autophagy 2014; 10: 526-528. [35] de Luca A, Smeekens SP, Casagrande A,et al. IL-1 receptor blockade restores autophagy and reduces inflammation in chronic granulomatous disease in mice and in humans. Proc Natl Acad Sci U S A 2014; 111: 3526-3531. [36] Martinez J, Malireddi RK, Lu Q, et al. Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins. Nat Cell Biol 2015; 17: 893-906. [37] Saitoh T, Fujita N, Jang MH, et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature 2008; 456: 264-268. [38] Plantinga TS, Crisan TO, Oosting M, et al. Crohn's diseaseassociated ATG16L1 polymorphism modulates proinflammatory cytokine responses selectively upon activation of NOD2. Gut 2011; 60: 1229-1235.


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[39] Rodrigues-Sousa T, Ladeirinha AF, Santiago AR, et al. Deficient production of reactive oxygen species leads to severe chronic DSS-induced colitis in Ncf1/p47phox-mutant mice. PLoS One 2014; 9: e97532. [40] Crisan TO, Plantinga TS, van de Veerdonk FL,,et al. Inflammasome-independent modulation of cytokine response by autophagy in human cells. PLoS One 2011; 6: e18666. [41] Hahn KJ, Ho N, Yockey L, et al. Treatment With Anakinra, a Recombinant IL-1 Receptor Antagonist, Unlikely to Induce Lasting Remission in Patients With CGD Colitis. Am J Gastroenterol 2015; 110: 938-939. [42] Uzel G, Orange JS, Poliak N, Marciano BE, Heller T, Holland SM. Complications of tumor necrosis factor-alpha blockade in chronic granulomatous disease-related colitis. Clin Infect Dis 2010; 51: 1429-1434. [43] Kyrmizi I, Gresnigt MS, Akoumianaki T, et al. Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin1/Syk kinase signaling. J Immunol 2013; 191: 1287-1299. [44] Caffrey AK, Obar JJ. Alarmin(g) the innate immune system to invasive fungal infections. Curr Opin Microbiol 2016; 32: 135-143. [45] Gresnigt MS, Bozza S, Becker KL, et al. A polysaccharide virulence factor from Aspergillus fumigatus elicits antiinflammatory effects through induction of Interleukin-1 receptor antagonist. PLoS Pathog 2014; 10: e1003936. [46] Falcone EL, Abusleme L, Swamydas M, et al. Colitis susceptibility in p47(phox-/-) mice is mediated by the microbiome. Microbiome 2016; 4: 13.

SECTION II. CLINICAL PRESENTATION AND DIAGNOSIS OF CGD

Chapter 4

BACTERIAL AND FUNGAL INFECTIONS IN CHRONIC GRANULOMATOUS DISEASE Brahm H. Segal and Steven M. Holland ABSTRACT Chronic granulomatous disease (CGD) is an inherited disorder of the phagocyte NADPH oxidase in which phagocytes are defective in reactive oxidant generation. CGD patients suffer from recurrent and life-threatening bacterial and fungal infections as well as disorders of excessive inflammation, such as inflammatory bowel disease and obstructive granulomata of the genitourinary tract. The spectrum of pathogens afflicting CGD patients is distinct from other immunocompromised patients, such as those with neutropenia from anti-neoplastic chemotherapy. These clinical observations point to the critical role of the phagocyte NADPH oxidase in host defense against specific pathogens. Studies in mouse models further shed light on the pathogenesis of infections in CGD and the role of NADPH oxidase-dependent and –independent pathways in host defense. We review the major bacterial and fungal infections in

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Brahm H. Segal and Steven M. Holland CGD, including prevention approaches, diagnosis, and management. Future directions for research and development of new therapeutic approaches in CGD are discussed.

INTRODUCTION The phagocyte NADPH oxidase is a critical regulator of both antimicrobial host defense and inflammation. In the phagocyte, this oxidase is activated as an emergency response to microbial threat, generating superoxide anions and downstream reactive oxidant species (ROS) that target pathogens. However, it is also clear that it exists in many other cell types and may respond to other stimuli as well. The phagocyte NADPH oxidase is comprised of a cytochrome component consisting of gp91phox (phox, phagocyte oxidase), also called NOX2 (NADPH oxidase-2), and p22phox, which are embedded in membranes. Upon activation, the cytoplasmic subunits p47phox, p67phox, p40phox and rac translocate to the membrane-bound cytochrome. Phagocyte NADPH oxidase activation results in conversion of molecular oxygen to superoxide anion. Superoxide dismutase converts superoxide anion to hydrogen peroxide. In neutrophils, myeloperoxidase (MPO) converts hydrogen peroxide to hypohalous acids. The NADPH oxidase-MPO system generates microbicidal oxidants required for host defense. For more details see Chapter 1. Chronic granulomatous disease (CGD), an inherited disorder of the phagocyte NADPH oxidase, is a disease of both impaired host defense and dysregulated inflammation. CGD is estimated to affect approximately 1 in 200,000 to 250,000 live births [1]. This may be an underestimate since patients with less severe forms of CGD may go undiagnosed. X-linked CGD results from disabling mutations in the gp91phox gene (CYBB), and accounts for approximately two-thirds of cases of CGD. The remaining cases of

Bacterial and Fungal Infections

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CGD result from autosomal recessive defects, with mutations in the p47phox gene (NCF1) being the most common. Rarely, CGD is observed in a female carrier of X-linked CGD in whom the process of lyonization (X-chromosome inactivation) has led to a low proportion of circulating NADPH oxidase-competent neutrophils [2, 3]. For more details see Chapters 7 and 8. Patients with CGD suffer from recurrent and life-threatening bacterial and fungal infections [1, 4]. CGD patients are at increased risk for a distinct spectrum of bacterial infections. Infections by Aspergillus species and other filamentous fungi are the major causes of mortality in CGD [1, 4-6]. CGD is also characterized by disorders of dysregulated inflammation, including Crohn-like inflammatory bowel disease, obstructive inflammation of the genitourinary tract, and pneumonitis resembling sarcoidosis [7, 8]. These findings underscore the broad importance of the phagocyte NADPH oxidase in both host defense and in calibrating the inflammatory response. Patients with X-linked CGD typically have a greater risk for infectious complications than autosomal recessive forms of CGD [1]. This increased risk likely reflects the complete absence of NADPH oxidase activity with mutations that completely disable gp91phox function. Kuhns et al. [9] showed that residual NADPH oxidase activity in neutrophils from CGD patients was associated with less severe illness and a greater likelihood of long-term survival than for patients with little or no NADPH oxidase function. This effect on survival was observed in both X-linked and autosomal recessive forms of CGD, demonstrating that even low residual levels of ROS formation in neutrophils are protective, and supporting the notion that a small proportion of NADPH oxidase-competent neutrophils, such as that which may be achievable with gene therapy, will reduce infection risk in CGD patients.

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OVERVIEW OF INFECTIONS IN CGD FROM DATA REGISTRIES Given the rarity of CGD, the most reliable databases on infection risk are from multicenter data registries. These registries have limitations, including the potential of missing data and underreporting, and the fact that clinical practice varies among centers and changes over time. In a U.S. data registry of 368 patients with CGD, pneumonia was the most frequent infection, with Aspergillus species being the most common pathogens [1]. Other major infections included suppurative adenitis (Figure 1), soft tissue abscesses, and liver abscesses with Staphylococcus aureus being the most common cause. Serratia infection (commonly manifesting as osteomyelitis), Burkholderia cepacia (commonly manifesting as pneumonia and sepsis), and Salmonella sepsis were also common complications. Analysis of clinical data from 429 European patients with CGD showed that the most frequently cultured pathogens per episode were Staphylococcus aureus (30%) and Aspergillus spp. (26%); Aspergillus species (111 cases) was the most common cause of pneumonia [4]. Marciano et al. [10] evaluated the records of 268 patients followed at the National Institutes of Health (NIH; Bethesda, MD, USA) over a 40-year period. The incidence of infections was as follows: Aspergillus, 2.6 cases per 100 patient-years; Burkholderia, 1.06 per 100 patient-years; Nocardia, 0.81 per 100 patient-years; Serratia, 0.98 per 100 patient-years, and severe Staphylococcus infection, 1.44 per 100 patient-years. Pneumonia was the most common type of infection. The median age at death has increased from 15.53 years before 1990 to 28.12 years in the last decade. Fungal infection was the most common cause of mortality. Multiple recurring infections, rather than persistence of


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prior infections, are typical in CGD, emphasizing the need to establish the correct diagnosis [11].

CGD: Defect of Bacterial Killing Lymph node abscess

S. aureus killing by neutrophils

Control

0 min

60 min

120 min CGD

Figure 1. Lymph node abscess by S. aureus in a CGD patient. Neutrophil bactericidal assay showing that neutrophils from a CGD patient have defective killing of S. aureus.

These databases as well as data from large referral centers show that CGD patients are at high risk for a limited spectrum of pathogens [1, 4], highlighting the fact that different pathogens have varying sensitivity to NADPH oxidase-dependent host defense. CGD patients do not appear to be at increased risk for several pathogens that are commonly observed in the general population (e.g., streptococcal infections), or opportunistic pathogens that commonly affect other immunocompromised patients, such as Pseudomonas aeruginosa infections in patients with chemotherapy-induced neutropenia or Pneumocystis jirovecii in patients with impaired cellular immunity. Indeed, neutrophil bactericidal studies demonstrate ROS-independent killing of the pathogens that do not cause infections in CGD [12, 13]. These results show that the phagocyte NADPH oxidase has a required

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host defense function to protect against specific pathogens while NADPH oxidase-independent pathways are required to defend against others. For more details see Chapter 2. CGD patients are at increased risk for infections by specific catalase-producing bacteria. Catalase, which is found in mammalian cells and in a large group of bacteria, converts hydrogen peroxide to water and molecular oxygen, thereby protecting cells from oxidative damage. The classic view has been that bacterial catalase is a virulence factor in CGD patients because catalase can deplete very low levels of hydrogen peroxide produced by bacteria, thereby depleting the substrate for the MPOhalide system and enabling pathogen survival in CGD phagocytes. NADPH oxidase (if residual activity is present), and NADPH oxidase-independent pathways (e.g., xanthine oxidase, mitochondrial respiration) are additional sources of ROS. Messina et al. [14] showed that catalase-positive and catalase-negative strains of S. aureus had similar virulence in CGD mice, thereby showing that catalase was not required for pathogen virulence in CGD. Chang et al. [15] showed that catalase-deficient Aspergillus nidulans retained virulence in CGD mice [15]. Conceivably, other pathogen-generated ROS-scavenging systems can compensate for the loss of catalase. However, these studies showing a lack of effect of catalase in both bacterial and fungal virulence in CGD mice point to a more complex relationship of oxidant stress and detoxifying pathways in infection risk in CGD.

BACTERIAL INFECTIONS IN CGD Staphylococcus aureus

Staphylococcus aureus infections are among the most common in CGD, frequently manifesting during infancy or early childhood


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as recurrent soft tissue infections. More serious staphylococcal infections include pneumonia, osteomyelitis, and systemic infections. Staphylococcal hepatic abscesses are a recognized complication of CGD [1, 10, 16, 17] and rare in other patient populations. Treatment frequently requires percutaneous drainage in addition to prolonged antibiotics. Corticosteroid therapy has been used to control the aberrant inflammatory response in CGD patients with S. aureus liver abscesses refractory to antibiotics, and may cure the infection without surgery (Figure 2) [18, 19]. Hepatic disease and portal hypertension, which can result from infections and non-infectious causes, are associated with increased mortality in CGD (Figure 3) [20, 21]. In patients with CGD, prophylactic antibiotics with anti-staphylococcal activity are standard (see section on Antibiotic Prophylaxis).

CGD: Hepatic Abscess

Figure 2. CT (A) and MRI imaging of S. aureus liver abscesses in an X-linked CGD patient. The infection responded to intravenous antibiotics and systemic corticosteroids. From: Leiding JW et al. Corticosteroid therapy for liver abscess in chronic granulomatous disease. Clin Infect Dis. 2012; 54: 694-700.

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Since S. aureus infections are common in the general population, including in healthy children, when should S. aureus infection prompt an evaluation for an immunodeficiency, including CGD? Recurrent superficial S. aureus soft tissue infections that respond to incision and drainage are commonly observed in the general population, and do not per se warrant an evaluation for CGD. However, CGD should be considered when these infections are unusually frequent or severe, including requiring hospitalization. An evaluation for CGD is warranted in a patient with a staphylococcal visceral abscess. Likewise, S. aureus community-acquired pneumonia or osteomyelitis in the absence of predisposing factors should prompt an immunodeficiency evaluation.

Hepatic Pathology in CGD

Figure 3. Hepatic pathology in CGD can result from infection, granulomata, and drug toxicity. (A) Multiloculated abscesses are present within dense fibroinflammatory connective tissue. (B) Portal area showing obliteration of the normal portal vein. (C) A small central vein lumen is obliterated by loose connective tissue, with only the remnant of the collagenous vein wall present (arrow). (D) Nodular regenerative hyperplasia. Hepatic involvement and portal hypertension are associated with increased mortality in CGD. From: Hussain N et al. Hepatic abnormalities in patients with chronic granulomatous disease. Hepatology. 2007; 45: 675-683.


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Burkholderia cepacia

Burkholderia cepacia is a common opportunistic pathogen in two patient populations: cystic fibrosis and CGD. The most common manifestation in CGD is pneumonia and sepsis. Burkholderia gladioli and Burkholderia pseudomallei (causative agent of melioidosis) are also observed in CGD patients [22, 23]. Recurrent pneumonia with distinct Burkholderia strains is common in CGD [24]. Trimethoprim-sulfamethoxazole, the most commonly used prophylactic antibacterial agent in CGD, is active against Burkholderia strains. Bactericidal activity of human neutrophils against B. cepacia is NADPH oxidase-dependent, while neutrophil killing of P. aeruginosa, a genetically similar strain, occurs through ROSindependent pathways [13]. Gene therapy that restored NADPH oxidase function in a small proportion of neutrophils was protective against B. cepacia sepsis in CGD mice [23]. In addition, knock-in mice with NADPH oxidase functionally reconstituted in the monocyte/macrophage lineage were protected from B. cepacia sepsis [26]. These observations underscore the distinct role of ROS-dependent and -independent pathways in host defense against specific microbes and the sensitivity of B. cepacia to ROS.

Serratia Species

Serratia marcescens and other Serratia species are among the major pathogens in CGD [1, 10, 11]. Among the enterobacteriaceae, Serratia species are the most common infections in CGD. Serratia species can cause a number of infections, including suppurative adenitis, soft tissue abscesses, pneumonia, and bacteremia. Serratia species are the most common

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cause of osteomyelitis in CGD [1]. In a U.S. registry of infections in CGD patients, 29% of cases of osteomyelitis were caused by Serratia species, followed by Aspergillus species (22%). By contrast, Staphylococcus species, the most common cause of osteomyelitis in the general population, only accounted for 6% of cases in CGD. It remains unclear why CGD patients have such a high susceptibility to Serratia infections compared to other coliforms (e.g., Escherichia coli and Klebsiella species) or the cause of the tropism for Serratia for bone in CGD. Since several pathogens can cause osteomyelitis in CGD, when feasible, biopsy and cultures should be obtained prior to initiating treatment. Treatment for osteomyelitis requires prolonged antibiotics.

Nocardiosis

Outside of CGD, nocardiosis is principally an opportunistic infection in patients with severely impaired cellular immunity, such as patients receiving intensive and prolonged systemic corticosteroids. Nocardiosis is very rare among patients receiving cytotoxic antineoplastic regimens in the absence of concomitant corticosteroids or other agents that lead to prolonged suppression of cellular immunity. Nocardiosis typically manifests as pulmonary infection in CGD [10, 27]. In a review of the NIH experience of 28 episodes of nocardiosis in CGD, 25% had disseminated infection and one-third had concurrent fungal infections [27]. Given the propensity for Nocardia to disseminate to the brain, MRI imaging of the brain should be considered in cases of nocardiosis. The Nocardia isolates were variable, with N. asteroides and N. farcinica being the most common. The presence of co-infection with more than one pathogen is not unusual in CGD and highlights the need for a culture-based diagnosis. The


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majority of patients were successfully treated with a sulfonamidecontaining regimen, even though some patients had developed nocardiosis while receiving sulfonamide prophylaxis [27].

Salmonella Species

Although salmonellosis can occur in immunocompetent persons, it is most common and severe in patients with impaired cellular immunity. Salmonella species are an important cause of bloodstream infection in CGD. In a U.S. data registry, Salmonella species accounted for 18% of bloodstream infections in CGD [1], while in a European data registry, Salmonella species were the third most frequent cultured organism (following S. aureus and Aspergillus species) [4]. These results point to the need to educate CGD patients on the need for hand hygiene, avoidance of foodborne pathogens, including awareness of any foodborne outbreaks, and proper preparation of food. Salmonella species can be resistant to antimicrobial agents, and therapy must be tailored to susceptibility results.

Tuberculosis

Mycobacterium tuberculosis and other mycobacteria are longlived intracellular pathogens. Patients with impaired cellular immunity (e.g., advanced AIDS, use of corticosteroids or tumor necrosis factor-α inhibitors) are at highest risk for tuberculosis. Experiments in mouse models of CGD have shown variable results regarding the role of the phagocyte NADPH oxidase in defense against M. tuberculosis [28-30]. Deffert et al. [31] reported increased severity of Bacillus Calmette–Guérin (BCG; a live

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attenuated strain of bovine tuberculous bacillus) infection associated with impaired granuloma formation in CGD mice. In patients with CGD, case reports of disseminated infection following BCG vaccination point to an increased susceptibility to tuberculosis. In a study of 17 males in China (an area endemic for TB) with CGD, 7 patients had tuberculosis, 7 patients had prolonged scarring or abscess formation at the BCG injection site, and 1 had disseminated BCG infection [32]. See also Chapter 5. Bustamante et al. [33] showed that a variant of X-linked CGD in which NADPH oxidase activity was impaired in macrophages, but not in neutrophils or monocytes, predisposed to BCG and tuberculosis. Together, these results point to an increased susceptibility of CGD patients to tuberculosis that is most evident in TB endemic regions. BCG vaccination is contraindicated in CGD patients.

Additional Bacterial Pathogens in CGD

Case series point to a number of additional pathogens that are opportunistic in CGD. Reichenbach et al. [34] reported 10 cases of actinomycosis in CGD patients, with the most common manifestation being fever and elevated inflammatory markers (Figure 4). This observation is also notable since Actinomyces species are catalase-negative. In addition, CGD patients are at risk for bacterial pathogens that rarely affect other immunocompromised patients. Granulobacter bethesdensis is a recently recognized pathogen in patients with CGD that causes fever and necrotizing lymphadenitis (Figure 5) [35, 36]. In contrast to other bacterial infections in CGD, this organism can persist for months to years after apparent clinical cure [36]. This organism is intrinsically multidrug resistant, and surgery combined with


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prolonged antibiotics is advised [36]. Chromobacterium violaceum, a Gram-negative bacteria found in subtropical soil and water of the southeastern United States can cause lethal infection in CGD [37]. This organism was also highly virulent in NADPH oxidase-deficient mice [38]. Bacteria such as G. bethesdensis and C. violaceum with exquisite virulence in CGD can serve as model organisms for understanding the mechanisms underlying NADPH oxidase-dependent host defense.

Actinomycosis in CGD

C

Figure 4. Abdominal actinomycosis in an X-linked CGD patient. A) CT scan showed splenomegaly and thrombosis of the splenic vein, mesenteric adenopathy, gastric wall thickening, and a peripancreatic mass (white arrowhead). B) Liver histologic analysis showed extensive parenchymal necrosis and multiple abscesses with filamentous gram-positive bacteria within the granule surrounded by neutrophil “clubbing” cells typical for actinomycosis (Gram stain on the left and Grocott stain on the right). C) X-CGD patient with pulmonary actinomycosis. Positron emission tomographic-CT scan shows numerous foci of infection. From Reichenbach J et al. Actinomyces in chronic granulomatous disease: an emerging and unanticipated pathogen. Clin Infect Dis. 2009; 49: 1703-1710.

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Granulobacter bethesdensis lymphadenitis in CGD

Figure 5. Granulobacter bethesdensis lymphadenitis in a patient with X-linked CGD. A) MRI of the chest showing adenopathy in the precarinal, subcarinal, and hilar regions. Central areas of diminished enhancement suggest necrosis (arrow). B) Cervical lymph node showing necrotizing granulomata with abscess. (C) WarthinStarry stain of the cervical lymph node (magnification 600×) showing coccobacillary organisms. (D) Necrotizing granuloma containing neutrophils and cellular debris on the right, bounded by a poorly defined layer of palisaded epithelioid histiocytes. From: Greenberg DE et al. A novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease. PLoS Pathog. 2006; 2: e28.

Invasive Fungal Diseases in CGD

Aspergillosis and other filamentous fungi account for a substantial proportion of infection-related mortality in CGD. In a U.S. data registry, about one-third of the 368 patients had an episode of Aspergillus pneumonia [1]. In a French national database, the overall incidence of invasive fungal diseases was 0.04/patient-years, with aspergillosis accounting for 40% [39]. Among CGD patients treated at the NIH, fungal infections


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accounted for 55% of deaths, and were somewhat more common among patients with lower residual NADPH oxidase function [10]. Aspergillus fumigatus and Aspergillus nidulans are the most common Aspergillus species in CGD. With the exception of CGD patients, A. nidulans is a rare pathogen. We reviewed all cases in which A. nidulans was isolated from patients at the NIH between 1976 and 1997 [5]. A. nidulans infection occurred in 6 patients with CGD, but was not a pathogen in any other patient group. A. fumigatus was a more common pathogen in CGD (n = 17 cases), but A. nidulans was more virulent. A. nidulans was significantly more likely to result in death compared with A. fumigatus, to involve adjacent bone, and to cause disseminated disease (Figure 6). Patients with A. nidulans received longer courses of amphotericin B therapy than did patients with A. fumigatus, and were treated with surgery more often. In contrast to A. fumigatus, A. nidulans was generally refractory to intensive antifungal therapy, suggesting that early surgery may be important. However, the need for early resection of pulmonary lesions will need to be reevaluated with the availability of extended spectrum azoles. Kontoyiannis et al. [40] found that A. nidulans isolates were frequently resistant to amphotericin B, another potential cause of treatment failure in CGD patients. Aspergillus tanneri is a more recently described fungal infection in CGD, and characterized by resistance to amphotericin B and azoles [41]. CGD patients often do not have typical symptoms and signs of infection [42]. Fever and leukocytosis may be absent, and an elevated sedimentation rate or C-reactive protein may be the only abnormal laboratory test [42]. In a review of aspergillosis in CGD patients at the NIH, one third of patients were asymptomatic at diagnosis and only about 20% were febrile [5]. In many of these patients, a pulmonary infiltrate on routine screening chest X-ray or CT scan was the first indication of an infection. The white blood

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cell count was ≤10,000/µl in 13/23 cases and the sedimentation rate was ≤40 mm/hr in 9/20 cases.

Aspergillosis in CGD

Figure 6. Aspergillus nidulans infection in a patient with X-linked CGD. CT imaging showed a large left upper lung mass with chest wall and vertebral destruction. PostMortem examination showed extensive vertebral osteomyelitis. From: Segal BH at al. Aspergillus nidulans infection in chronic granulomatous disease. Medicine. 1998; 77: 345-354.

In contrast to patients with chemotherapy-induced neutropenia, hyphal angioinvasion is not a feature of invasive aspergillosis in CGD. In CGD mice, pulmonary aspergillosis is characterized by dense pyogranulomatous areas of consolidation in the absence of vascular hyphal invasion [15, 43]. These findings suggest that NADPH oxidase-independent pathways are sufficient to protect against hyphal angioinvasion. Serum galactomannan is not elevated in experimental pulmonary aspergillosis in CGD mice [43] and is an insensitive diagnostic marker of aspergillosis in CGD patients [44]. “Mulch pneumonitis” is a life-threatening fungal pneumonia in CGD patients characterized by an acute fulminant pneumonitis, which is treated with both antifungals and corticosteroids to


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dampen excessive inflammation (Figure 7) [45]. Mulch pneumonitis underscores the critical role for the NADPH oxidase in dampening inflammatory responses.

Fulminant Mulch Pneumonitis in CGD

Figure 7. Chest radiograph (A) and CT scan (B) of a patient with newly diagnosed p47phox-/- CGD with fulminant fungal pneumonitis. Thoracoscopic lung biopsy showed intense pyogranulomatous inflammation, with invasive hyphae, and cultures grew A. fumigatus and Rhizopus species. She was treated with broad spectrum antibacterial and antifungal therapy with corticosteroids to reduce inflammation. Imaging approximately 2 months later transfer (C and D) show resolution of infiltrates. From: Siddiqui S et al. Fulminant mulch pneumonitis: an emergency presentation of chronic granulomatous disease. Clin Infect Dis 2007; 45: 673-681.

CGD patients are at risk for a broad range of filamentous fungal infections. As an example of non-Aspergillus fungal pathogens, De Ravin et al. [46] described 7 cases of Geosmithia argillacea in CGD patients. In 5 cases, the fungus had been previously identified morphologically as Paecilomyces variotii.

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Pneumonia was the most common presentation, and extension to the chest wall and dissemination were observed. Response to antifungal treatment was poor, and 3 of the patients died. Examples of other rare fungal infections include zygomycosis (associated with iatrogenic immunosuppression) [47] trichosporonosis [48], Penicillium species [49], and Paecilomyces species [50]. Given the broad spectrum of opportunistic fungal pathogens in CGD, molecular-based identification of individual fungal pathogens is indicated when the diagnosis cannot be made based on morphologic criteria.

PROPHYLAXIS IN CGD Antibacterial Prophylaxis

Standard prophylaxis for CGD includes an antibacterial agent (generally trimethoprim-sulfamethoxazole), a mold-active antifungal agent (e.g., itraconazole), and recombinant interferon-γ. Trimethoprim-sulfamethoxazole has been used for decades in CGD. This agent has been proven to be safe and effective in reducing bacterial infections [51]. The protective benefit of trimethoprim-sulfamethoxazole may relate to its intracellular accumulation within neutrophils, which augments staphylococcal killing [52]. Trimethoprim-sulfamethoxazole is active against the majority of bacterial pathogens that cause infection in CGD, including most strains of S. aureus, including the majority of community-acquired oxacillin-resistant strains, Burkholderia species, and Nocardia species. In CGD patients who are allergic or intolerant of trimethoprim-sulfamethoxazole, alternative agents


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(e.g., cephalexin or doxycycline) with anti-staphylococcal activity should be used as prophylaxis.

Antifungal Prophylaxis

Prevention of invasive aspergillosis and other filamentous fungal diseases relies on avoiding environments where high levels of fungal spores are expected (e.g., gardening and building renovations) and mold-active antifungal prophylaxis. Itraconazole prophylaxis has been shown to be safe and effective in patients with CGD [6, 53]. In a French national database study of 29 episodes of invasive mold infections in CGD patients, the first proven fungal infection occurred later in the group that received itraconazole than in the group without (10 versus 4 years of age, respectively), with a higher proportion of A. nidulans and other opportunistic molds in itraconazole recipients [54]. The prognosis appeared to improve over time, possibly reflecting improvements in antifungal regimens [54]. Although not specifically evaluated as prophylaxis in CGD, other mold-active azoles (voriconazole or posaconazole), may be considered as antifungal prophylaxis.

Recombinant Interferon-γ

Recombinant interferon-γ has been widely used as prophylaxis in patients with CGD for approximately 25 years. In a randomized trial, prophylactic recombinant interferon-γ significantly reduced the incidence of serious infections, and was beneficial regardless of age, the use of prophylactic antibiotics, or the type of CGD (Xlinked or autosomal recessive) [55]. Although prior studies showed that interferon-γ could augment superoxide production in

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phagocytes from some CGD patients, there were no significant changes in the measures of superoxide production by phagocytes in the randomized trial. Thus, the benefit of prophylactic recombinant interferon-γ likely results from augmentation of oxidant-independent pathways. Long-term recombinant interferonγ has been generally well tolerated in CGD, with fever being the most frequent side effect [56]. In an Italian multi-center prospective analysis, CGD patients receiving recombinant interferon-γ had a mean rate of 0.03 total infections per patientyear as compared to 0.06 total infections per patient-year in controls (p = 0.07) [57]. All of these patients received trimethoprim-sulfamethoxazole and itraconazole as prophylaxis. The rates of overall and serious infections were lower than those reported in other studies, an effect that could be related to differences in antimicrobial prophylaxis, duration of follow-up, and how infections were monitored and reported. Limitations of this study include the small numbers of patients and lack of randomization. Our practice is to use recombinant interferon-γ as prophylaxis in CGD patients.

TREATMENT OF INFECTIONS IN CGD Treatment of bacterial and fungal infections in CGD relies on establishing the correct diagnosis. Chest CT imaging is more sensitive than X-radiography for detecting early infection, including invasive fungal disease. Since the differential diagnosis for pulmonary lesions is broad, a tissue-based diagnosis may be required. Invasive bacterial infections (e.g., pneumonia, osteomyelitis, and deep soft tissue infections) require prolonged antibiotic therapy. Bacterial infections involving bone or viscera frequently require surgery. Decisions about surgical intervention


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must be individualized based on the pathogen, location and extent of disease, and likelihood of response to antibacterial treatment alone. Invasive aspergillosis and other filamentous fungal infections are among the most challenging regarding therapy. Since CGD is a rare disease, there are no dedicated randomized trials of antifungal therapy for aspergillosis. Voriconazole was shown to be superior to conventional amphotericin B as primary therapy for aspergillosis [58]. Underlying conditions in enrolled patients were primarily hematologic malignancies and transplantation. Voriconazole has been evaluated in pediatric patients with invasive fungal infections, including CGD patients [59]. Based on this clinical database, the Infectious Diseases Society of America (IDSA) guidelines recommend voriconazole as primary therapy for invasive aspergillosis [60], a recommendation that can be reasonably applied to CGD patients. Lipid formulations of amphotericin B, posaconazole [61], isavuconazole, and echinocandins are additional options for therapy of invasive aspergillosis in patients who are intolerant to voriconazole or who have refractory disease. Similar to other immunocompromised patients with invasive aspergillosis, development of azole resistance is a serious complication for successful treatment in CGD patients [62, 63]. In addition to antifungal therapy, debridement or resection of infected tissue may be required. This is particularly the case for refractory aspergillosis or extension of fungal disease to vertebrae or chest wall.

Granulocyte Transfusions

Adjunctive granulocyte transfusions have been used for severe or refractory infections in CGD patients. The principle of

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granulocyte transfusions in CGD patients (who have normal circulating neutrophil numbers) is that a small proportion of normal neutrophils can augment host defense in CGD neutrophils by providing a source of diffusible ROS. Hydrogen peroxide generated by normal neutrophils can diffuse into CGD neutrophils and provide the necessary substrate to generate hypohalous acid and hydroxyl anion in vitro [64]. In addition, mixtures of small numbers of normal neutrophils with larger numbers of CGD neutrophils damaged A. fumigatus hyphae more efficiently than either population of cells alone [65]. These results support the notion of diffusible reactive oxidants generated by NADPH oxidase-competent neutrophils at least in part rescuing the impaired host defense in CGD neutrophils. Transfused granulocytes retain respiratory burst activity and appear to traffic normally, based on their recovery from sites of infection. However, a recent randomized trial of granulocyte transfusions in neutropenic patients without CGD who had severe bacterial or fungal infections failed to show benefit [66]. It is important to keep the potential complication of alloimmunization in mind when performing granulocyte transfusion, as this can be a major stumbling block to allogeneic stem cell transplantation [67]. Intralesional granulocytes have been used in a case of liver abscess with some success [68]. Overall, despite their theoretical attraction, the benefit of granulocyte transfusions in CGD remains unclear and is unlikely to ever be definitively evaluated.

Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation is usually curative in CGD, and is becoming accepted as a standard of care. The major risk is graft-versus-host disease [69, 70]. There are


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limited but growing reports of the use of allogeneic stem cell transplantation and gene therapy specifically as salvage treatments in CGD patients with refractory infections [54, 71, 72]. For more details see Chapters 10 and 12.

FUTURE DIRECTIONS FOR PREVENTING AND TREATING INFECTIONS IN CGD As a hematopoietic stem cell disorder, gene therapy in CGD has been evaluated for decades [73], and remains an active area of research; the major hurdle relates to maintaining a stable long-term population of gene-corrected cells. More details can be found in Chapter 12. While these approaches offer the potential for cure of CGD, knowledge gained about of the mechanisms by which NADPH oxidase mediates host defense and regulates inflammation also have the potential to lead to therapeutic advances that prevent or reduce infection-related morbidity in CGD. There is a number of examples that are relevant to aspergillosis in CGD. De Luca et al. [74] linked impaired antifungal host defense in CGD to defective autophagy in monocytes and macrophages. This defect in autophagy resulted in increased inflammasome activation and IL-1β release from macrophages, and IL-1 receptor blockade protected CGD mice from invasive aspergillosis and experimental colitis. See also Chapter 3. Long-lived macrophages are a promising target for therapeutic intervention in CGD, including modulation of autophagy and cytokine responses. Another therapeutic approach that merits evaluation in murine CGD is the use of agents that can stimulate NADPH oxidase-independent NET generation in neutrophils [75]. Finally, although CGD is a disorder of

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phagocytes, augmentation of cellular immunity can be of therapeutic value. As an example, a CD4+ T cell vaccine-based strategy led to enhanced cross-presentation of fungal antigens and resulted in long-term protection against aspergillosis in CGD mice [76]. Knowledge gained about the pathogenesis of infections in CGD is expected to lead to additional novel therapeutic approaches that augment host defense and limit excessive inflammatory responses.

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Brahm H. Segal and Steven M. Holland oral posaconazole and surgery. Med Mycol 2009; 47: 217220. Ohno Y, Gallin JI. Diffusion of extracellular hydrogen peroxide into intracellular compartments of human neutrophils. Studies utilizing the inactivation of myeloperoxidase by hydrogen peroxide and azide. J Biol Chem 1985; 260: 8438-8446. Rex JH, Bennett JE, Gallin JI, Malech HL, Melnick DA. Normal and deficient neutrophils can cooperate to damage Aspergillus fumigatus hyphae. J Infect Dis 1990; 162: 523528. Price TH, Boeckh M, Harrison RW, et al. Efficacy of transfusion with granulocytes from G-CSF/dexamethasonetreated donors in neutropenic patients with infection. Blood 2015; 126: 2153-2161. Heim KF, Fleisher TA, Stroncek DF, et al. The relationship between alloimmunization and posttransfusion granulocyte survival: experience in a chronic granulomatous disease cohort. Transfusion 2011; 51: 1154-1162. Lekstrom-Himes JA, Holland SM, DeCarlo ES, et al. Treatment with intralesional granulocyte instillations and interferon- gamma for a patient with chronic granulomatous disease and multiple hepatic abscesses. Clin Infect Dis 1994; 19: 770-773. Gungor T, Teira P, Slatter M, et al. Reduced-intensity conditioning and HLA-matched haemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicentre study. Lancet 2014; 383: 436-448. Martinez CA, Shah S, Shearer WT, et al. Excellent survival after sibling or unrelated donor stem cell transplantation for


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chronic granulomatous disease. J Allergy Clin Immunol 2012; 129: 176-183. Shigemura T, Nakazawa Y, Yoshikawa K, et al. Successful cord blood transplantation after repeated transfusions of unmobilized neutrophils in addition to antifungal treatment in an infant with chronic granulomatous disease complicated by invasive pulmonary aspergillosis. Transfusion. 2014; 54: 516-521. Siler U, Paruzynski A, Holtgreve-Grez H, et al. Successful combination of sequential gene therapy and rescue alloHSCT in two children with X-CGD - importance of timing. Curr Gene Therapy 2015; 15: 416-427. Malech HL, Maples PB, Whiting-Theobald N, et al. Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease. Proc Natl Acad Sci U S A 1997; 94: 12133-12138. De Luca A, Smeekens SP, Casagrande A, et al. IL-1 receptor blockade restores autophagy and reduces inflammation in chronic granulomatous disease in mice and in humans. Proc Natl Acad Sci U S A 2014; 111: 3526-3531. Douda DN, Khan MA, Grasemann H, Palaniyar N. SK3 channel and mitochondrial ROS mediate NADPH oxidaseindependent NETosis induced by calcium influx. Proc Natl Acad Sci U S A 2015; 112: 2817-2822. De Luca A, Iannitti RG, Bozza S, et al. CD4(+) T cell vaccination overcomes defective cross-presentation of fungal antigens in a mouse model of chronic granulomatous disease. J Clin Invest 2012; 122: 1816-1831.

Chapter 5

ENDEMIC INFECTIONS IN CHRONIC GRANULOMATOUS DISEASE Pamela Lee and Yu-Lung Lau With a contribution by Reinh ard Seger

ABSTRACT Patients with chronic granulomatous disease (CGD) are susceptible to a variety of bacterial and fungal pathogens. Staphylococcus aureus, Aspergillus, Burkholderia cepacia complex, Serratia marcescens and Nocardia are recognized as signature organisms for causing infections in CGD, but some other pathogens are clinically more important in different parts of the world. In this Chapter, we summarize the epidemiological pattern of salmonellosis, tuberculosis, BCG complications, melioidosis and Chromobacterium violaceum infection in CGD, based on published studies and data from the Asia Primary Immunodeficiency (APID) Network. For CGD patients, the exposure to M. tuberculosis and non-typhoidal Salmonella is determined by the prevalence of such

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Pamela Lee and Yu-Lung Lau diseases in the population, quality of healthcare service and socioeconomical factors, while climatic changes, habitats, human activities and personal behavior are important determinants of environmental exposure to B. pseudomallei and C. violaceum in the tropics and subtropics. ‘Red flags’ for CGD may be unique in different geographical regions, opening exciting opportunities to explore the biological and evolutionary roles of NADPH oxidase in protecting humans from environmental pathogens.

INTRODUCTION Patients with chronic granulomatous disease (CGD) suffer from a wide variety of bacterial and fungal infections. In North America, the most frequently encountered pathogens in CGD include Staphylococcus aureus, Aspergillus spp, Burkholderia cepacia complex, Serratia marcescens and Nocardia spp [1, 2]. In a large European CGD cohort, B. cepacia and Nocardia each accounted for < 1% of all isolated organisms; instead, Salmonella spp and Candida spp were amongst the ‘top five’ pathogens for CGD [3]. B. cepacia and Nocardia are also uncommon in CGD patients from Latin America; Mycobacterium bovis BCG was the most frequently isolated pathogen, followed by S. aureus, Aspergillus spp, Klebsiella, S. marcescens and Candida spp [4]. Such observations suggest that the types of infections seen in CGD vary widely in different geographical regions, and it is important for clinicians to recognize the spectrum of pathogens that commonly affect CGD patients in their locality in order to initiate appropriate investigations and provide targeted antimicrobial treatment. With improved awareness of primary immunodeficiencies (PID) in Asia, more patients with CGD have been diagnosed. The Asia Primary Immunodeficiency (APID) Network is a collaborative research network that has received more than 2,000

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patient referrals for genetic diagnosis from more than 50 centers from Asia. The APID Network has collected clinical and laboratory data from 91 patients with genetically proven CGD, and this multi-centered cohort allows us to analyze the trends and patterns of infections [5-7]. Burkholderia pseudomallei and Chromobacterium violaceum, which are bacterial pathogens indigenous to subtropical and tropical regions, have caused fatal illness in CGD patients and will be discussed in this Chapter. Although Salmonella and M. tuberculosis are not restricted to subtropical and tropical regions, disease burden is high in these areas due to environmental and socioeconomic factors, and their high prevalence in the community contributes to increased risk of exposure and disease in CGD patients.

SALMONELLA Salmonella enterica are gram-negative bacteria that cause various clinical syndromes in humans. S. typhi and S. paratyphi (A, B and C) cause enteric fever restricted to humans, and increased susceptibility or severity is not observed in primary or secondary immunodeficiencies. In contrast, non-typhoidal Salmonella (NTS) typically cause self-limiting diarrheal disease in humans, and have a broad vertebrate host range [8]. In most parts of the world, except Africa, NTS is rare in the community [9, 10]. NTS causes invasive infections mainly in immunocompromised hosts, such as HIV-infected persons, patients receiving immunosuppressive treatment and solid organ transplant recipients. CGD and Mendelian susceptibility to mycobacterial diseases (MSMD), particularly IL12RB1 and IL12B deficiencies, are major forms of PID with a high incidence of bacteremia caused by NTS [11-13].

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Pamela Lee and Yu-Lung Lau

The prevalence of enteric and invasive NTS disease varies substantially by geographic regions: while there is a higher population incidence of enteric NTS disease in Asia (3,280/100,000 in Asia / Oceania and 1,440/100,000 in Southeast Asia) compared with Europe (690/100,000) and USA (250/100,000), invasive NTS disease is less common in Southeast Asia (21/100,000) and Asia/Oceania (0.8/100,000) when compared with Europe (102/100,000) and USA (23/100,000) [9]. In contrast, NTS is a leading cause of invasive bacterial disease in sub-Saharan Africa. The magnitude of invasive NTS (227/100,000) is comparable to that of enteric NTS (320/100,000) [9], and it accounts for over 20% of community acquired bloodstream infections [14]. In Africa, invasive NTS is closely associated with malaria and malnutrition among infants and children, and with HIV infection among adults [14, 15]. Salmonella is the leading cause of bloodstream infection in CGD patients (Table 1). In two large CGD patient cohorts from Europe (n = 429) and US (n = 368), Salmonella accounted for 40.9% and 18.5% of isolated organisms from bloodstream infections, respectively [1, 2]. In the APID cohort, the frequency of bloodstream infections was 21.1%, which is comparable to that in the European and US CGD cohorts (19.8% and 17.7%). Salmonella accounted for 54.2% of isolated organisms from bloodstream infection. None of these patients are known to have HIV or malaria infection, which are risk factors for invasive nontyphoidal salmonellosis. In fact, an early cohort from Hong Kong during the 1980s revealed that Salmonella spp. accounted for 80% of septicemic episodes [16]. The predominance of Salmonella over other organisms in causing bacteremia in Asian patients with CGD may be related to increased exposure, as reflected by the high population incidence of enteric NTS disease [9]. The occurrence of invasive NTS disease probably has a high predictive value for PID

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such as CGD in Asia, especially in patients without secondary immunodeficiencies residing in regions with low prevalence of HIV and malaria. Table 1. Isolated organisms from bloodstream infections in patients with chronic granulomatous disease

No. of patients X-CGD AR-CGD Unknown % patients with bloodstream infection Isolated organisms from bloodstream infections Salmonella spp. Burkholderia pseudomallei Chromobacterium violaceum Burkholderia cepacia Staphylococcus spp.

91 75 (82.4%) 16 (17.6%)

European cohort 429 290 (67.6%) 139 (32.4%)

19 (20.9%)

85 (19.8%)

65 (17.7%)

54.2%

* 40.9%

18.5%

4.2%

* 3.4% * 12.5%

12.3% 9.2%

Streptococcus spp.

8.3%

* 10.2%

Candida spp. Pseudomonas spp. Klebsiella spp. Serratia spp. Acinetobacter spp.

4.2%

* 3.4% * 2.3% * 2.3% * 1.1%

10.7% 9.2% 4.6% 6.2% 4.6%

Aspergillus spp.

* 4.5%

E. coli spp.

* 2.3%

3.1% 1.5%

Asian cohort

USA cohort 368 259 (70.4%) 81 (22.0%) 28 (7.6%)

8.3% 8.3%

4.2% 4.2%

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Pamela Lee and Yu-Lung Lau Table 1. (Continued) Asian cohort

Others

Enterococcus spp. (4.2%)

European Cohort

USA Cohort

chronic granulomatous disease

chronic granulomatous disease

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