Regulation of adaptive immune responses by the phagocyte-type NADPH oxidase in T cells and antigen presenting cells

Authors Shatynski, Kristen Elizabeth

Publication Date 2012

Abstract Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In contradiction to this innate immune dysfunction, CGD patients or animal mod...

Keywords adaptive immune system; T cells; Adaptive Immunity; Antigen- Presenting Cells; Granulomatous Disease, Chronic; NADPH Oxidases; Reactive Oxygen Species

Download date 06/10/2021 11:10:12

Link to Item http://hdl.handle.net/10713/2155 Name: Kristen Elizabeth Shatynski [email protected]

Degree and Date Conferred: Doctor of Philosophy, June 22, 2012

Collegiate Institutions Attended:

Ph.D., Molecular Microbiology and Immunology Dissertation defense date: June 22, 2012 Dissertation Topic: Regulation of adaptive immune responses by the phagocyte-type NADPH oxidase in T cells and antigen presenting cells Advisor: Mark S. Williams, Ph.D. University of Maryland School of Medicine, Baltimore, MD August 2006 – June 2012 GPA: 3.75

B.A., Biology (Magna Cum Laude Honors) La Salle University, Philadelphia, PA August 2002 - May 2006 GPA: 3.63

Peer-Reviewed Publications:

Shatynski KE , Chen H, and MS Williams. Decreased STAT5 phosphorylation and GATA-3 expression in NOX2 deficient T cells: Role in T helper differentiation. Submitted Manuscript – European Journal of Immunology 2012.

Lorenzo LPE, Shatynski KE , Clark S, Yarowsky PJ, and MS Williams. Defects in lymphoid progenitor development and mature T-cell function in a mouse model for Down syndrome. Submitted Manuscript - PLoS ONE 2012.

Lorenzo LPE, Chen H, Shatynski KE , Clark S, Rong Y, Harrison DE, Yarowsky PJ, and MS Williams. Defective hematopoietic stem cell and lymphoid progenitor development in the Ts65Dn mouse model of Down syndrome: potential role of oxidative stress. Antioxidants & Redox signaling 2011; 15 (8): 2083-2094.

Kwon J, Shatynski KE , Chen H, Morand S, de Deken X, Miot F, Leto TL, and MS Williams. The nonphagocytic NADPH oxidase Duox1 mediates a positive feedback loop during T cell receptor signaling. Science Signaling 2010; 3 (133): ra59.

Huard S, Chen M, Burdette KE , Fenyvuesvolgyi C, Yu M, Elder RT, and RY Zhao. HIV-1 Vpr-induced cell death in Schizosaccharomyces pombe is reminiscent of apoptosis. Cell Research. 2008; 18 (9): 961-973.

Published Abstracts and Symposia:

Shatynski KE , and MS Williams. T cell expression of NOX2 regulates T helper differentiation. Poster presentation – Crosstalk: Across Cells, Across Campuses, UMB-UMCP Annual Research Symposium; June 3, 2011; Baltimore, MD.

Shatynski KE , and MS Williams. T cell expression of NOX2 regulates T helper differentiation. Invited oral presentation for Block Symposium – American Association of Immunologists 98 th Annual Meeting; May 13-17, 2011; San Francisco, CA.

Shatynski KE , and MS Williams. T cell expression of NOX2 regulates T helper differentiation. Poster presentation – Maryland Branch ASM Dinner Meeting, University of Maryland Baltimore; May 11, 2011; Baltimore, MD.

Shatynski KE , and MS Williams. T cell expression of NOX2 regulates T helper differentiation. Oral presentation - 33 rd Annual Graduate Research Conference, University of Maryland Baltimore; April 7, 2011; Baltimore MD.

Shatynski KE , Chen H, and MS Williams. The phagocyte NADPH oxidase (NOX2) regulates adaptive immune response at the level of both T cells and APCs. Poster presentation –1st Annual Vascular Biology Training Program & Training Program in Cardiovascular Cell Biology Retreat; October 25, 2010; Baltimore MD.

Shatynski KE , Chen H, and MS Williams. The phagocyte NADPH oxidase (NOX2) regulates adaptive immune response at the level of both T cells and APCs. Poster presentation – American Association of Immunologists 97 th Annual Meeting; May 7-11, 2010; Baltimore, MD.

Kwon J, Burdette K , Miot F, and MS Williams. Expression and function of the non- phagocytic NADPH oxidase, Duox1 in T lymphocytes. Invited oral presentation for Block Symposium - T Cell Signaling, Experimental Biology 2008; April, 2008; San Diego, CA.

Blanton JL, Ogidan OS, and Burdette KE . Effect of the chemical warfare agent, sulfur mustard, on histone H2B serine 14 phosphorylation in primary blood lymphocytes and epithelial cell lines. Physiology and Immunology Branch, United States Army Medical Research Institute of Chemical Defense, US Army Medical Defense Bioscience Review; June 4-9, 2006; Hunt Valley, MD.

Professional Positions Held:

American Association for the Advancement of Science Congressional Science Fellow Sponsored by the American Society for Microbiology 1200 New York Avenue, NW Washington, DC 20005 Will be serving as a legislative fellow and science adviser to a member of Congress, and will be using scientific expertise, communication skills and science policy background to initiate programs and shape legislation that incorporates and promotes scientific excellence. September 2012 - August 2013

Graduate Research Assistant Department of Microbiology and Immunology Center for Vascular and Inflammatory Diseases University of Maryland Baltimore UMB Biopark-1, Room 315 800 West Baltimore Street Baltimore, MD 21201 Advisor: Mark S. Williams, Ph.D. Investigated the roles of phagocyte NADPH oxidase generated reactive oxygen species in shaping the adaptive and innate immune systems. Elucidated the biologic functions of the phagocyte oxidase in T cell signaling and development. June 2007 - August 2012

Oak Ridge Institute for Science & Education (ORISE ) Undergraduate Research Assistant Physiology & Immunology Branch United States Army Medical Research Institute of Chemical Defense 3100 Ricketts Point Road Aberdeen Proving Ground, MD 21010 Advisor: CPT Jason L. Blanton, Ph.D. Participated in the study and development of medical countermeasures to chemical warfare agents, by assisting in deciphering the molecular mechanisms through which chemical agents work. Summer 2004, 2005, and 2006

Membership in Professional Organizations:

Society for Free Radical Biology and Medicine, 2007-present The American Association of Immunologists, 2010-present The American Society for Microbiology, 2010-present American Association for the Advancement of Science, 2010-present United States Junior Chamber, 2010-present AcademyHealth, 2010-present Society for Leukocyte Biology, 2011-present

Awards:

- The University of Maryland Baltimore President’s Student Leadership Institute (PSLI), Platinum Honors, 2012 - 2011 AAI Abstract Trainee Award, American Association of Immunologists Annual Meeting, San Francisco, CA - NIH funded Vascular Biology T32 Training Grant, Center for Vascular and Inflammatory Diseases, 2009 - present - Graduate Program in Life Sciences, Molecular Microbiology and Immunology, Best Thesis Presentation Honorable Mention, 2008 - Graduate Program in Life Sciences, Molecular Microbiology and Immunology, Best Graduate Rotation Presentation, 2007 - Alpha Epsilon Delta pre-med honor society, La Salle University, 2004-2006 - The Honor Society of Alpha Epsilon, La Salle University, 2005 - 2006 - National Society of Collegiate Scholars, La Salle University, 2002 - 2006 - University Founders’ Scholarship, La Salle University, 2002 – 2006

Service:

- Coordinator, Graduate Student Presentations, University of Maryland Baltimore, Graduate Program in Molecular Microbiology and Immunology, 2011 - Individual Development Vice President, Towson Junior Chamber of Commerce – Chapter of the United States Junior Chamber, Towson, Maryland, 2010-present - Vice President, Students United for Policy, Education and Research – Student Chapter of AcademyHealth, University of Maryland Baltimore, 2010-present - Coordinator, Immunology Practice Comprehensive Examinations for Second-Year Students, University of Maryland Baltimore, Department of Microbiology and Immunology, 2009-2011 - Coordinator, Immunology Journal Club, University of Maryland Baltimore, Department of Microbiology and Immunology, 2008-2010 - Student Liaison, Graduate Student Applicant Day for the Graduate Program in Molecular Microbiology and Immunology, 2007- 2011 - Contributor, Microscoop Newsletter, University of Maryland Baltimore, Department of Microbiology and Immunology, 2007-2010 - President, Epsilon Zeta Chapter of Delta Phi Epsilon Sorority, La Salle University, 2005 - Resident Assistant, Community Development, La Salle University, 2003-2005

Abstract

Title of Dissertation: Regulation of adaptive immune responses by the phagocyte-type NADPH oxidase in T cells and antigen presenting cells

Kristen Elizabeth Shatynski, Doctor of Philosophy, 2012

Dissertation directed by: Mark S. Williams, Ph.D., Assistant Professor, Department of Microbiology and Immunology, Center for Vascular and Inflammatory Diseases

Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic

granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent

bacterial infections. In contradiction to this innate immune dysfunction, CGD patients or

animal models of the disease display improved response to infectious agents such as

Helicobacter pylori , influenza virus or Cryptococcus neoformans . These and other data

imply an altered adaptive immune response in CGD. In addition to antigen presenting

cells (APCs), NOX2 is expressed in T cells, and the goal was to determine if these

differences are T cell inherent, or if NOX2-deficient APCs promote T-helper

polarization. We hypothesize that NOX2 shapes both adaptive and innate immune

responses in a T cell and APC-dependent fashion.

In order to study T-helper polarization in vivo, wild type and NOX2-deficient

(NOX2 (-/-)) mice were immunized with OVA in CFA or Alum. Upon in vitro

(-/-) restimulation, lymph node cells from NOX2 mice had increased T H1 but decreased

(-/-) TH2 cytokine production. Adoptive transfer of OT-II T cells into wild type or NOX2

hosts followed by immunization also revealed increased IL-17 and IFN-γ, but decreased

IL-4 after restimulation in vitro. NOX2 (-/-) APCs (resident peritoneal cells and adherent splenocytes) displayed enhanced proinflammatory cytokine secretion in vitro, and

stimulation of OT-II T cells with antigen-pulsed NOX2 (-/-) APCs in vitro induced altered cytokine production, suggesting that NOX2 deficiency modifies APC function, resulting in T helper skewing.

In addition to APC-induced changes, T cells from NOX2 (-/-) mice were inherently

(-/-) skewed. Naïve T cells from NOX2 mice demonstrated T H1 skewed cytokine secretion as compared to their wild type counterparts, through increased IFN-γ, but decreased IL-4 in response to anti-CD3 and anti-CD28 stimulation. There were also selective decreases in GATA-3 and phosphorylated STAT5, with decreased Il4 gene expression, suggesting a mechanism for decreased T H2 responses. Finally, treatment with antioxidants recapitulated these selective changes in TCR-induced transcription factor activation.

These findings indicate that TCR-induced reactive oxygen species generation from

NOX2 activation selectively promotes STAT5 phosphorylation and downstream T H2 development in CD4 + T cells. Taken together, these findings suggest NOX2 affects cross talk between the innate and adaptive immune systems, resulting in changes in T helper differentiation.

Regulation of adaptive immune responses by the phagocyte-type NADPH oxidase in T cells and antigen presenting cells

by Kristen Elizabeth Shatynski

Dissertation submitted to the faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2012

©Copyright 2012 by Kristen Elizabeth Shatynski

All Rights Reserved

Table of Contents

Chapter I. General Introduction ...... 1

IA. Reactive Oxygen Species ...... 1

IB. NADPH oxidases ...... 4

IB1. The phagocyte NADPH oxidase ...... 4

IB2. Other members of the NOX family of NADPH oxidases ...... 7

IB3. Antioxidants and pharmacological inhibitors of NADPH oxidases ...... 11

IC. Regulation of Biologic Responses by ROS Generation ...... 16

ID. Helper T cell development ...... 20

ID1. The T H1/T H2 Paradigm ...... 20

ID2. Other T helper subsets ...... 25

ID3. T helper lineage plasticity ...... 27

IE. Chronic Granulomatous Disease (CGD) ...... 31

IE1. Disease Genetics ...... 31

IE2. CGD Pathophysiology ...... 36

IE3. Animal Models of CGD ...... 40

IF. Thesis Aims ...... 43

Chapter II. Materials and Methods ...... 48

IIA. Mice ……………………………………………………………………………...48

IIB. Antibodies ...... 48

IIB1. Antibodies used for negative selection ...... 48

IIB2. Antibodies used in cell culture ...... 48

IIB3. Antibodies used for flow cytometry ...... 49

iii

IIC. Reagents ...... 50

IIC1. Media ...... 50

IIC2. ELISA Buffers ...... 51

IID. Cell Isolation ...... 52

IID1. T cell negative selection ...... 52

IID2. Lavage of resident peritoneal macrophages ...... 54

IID3. Isolation of adherent splenocytes ...... 54

IIE. In vitro cell cultures ...... 57

IIE1. T cell cultures ...... 57

IIE2. Antigen presenting cell cultures ...... 58

IIE3. OT-II co-cultures ...... 58

IIF. In vivo experiments ...... 59

IIF1. Immunizations ...... 59

IIF2. Adoptive transfers ...... 59

IIF3. Tritiated thymidine proliferation assay ...... 60

IIG. Flow Cytometry ...... 60

IIH. CFSE cell proliferation analysis ...... 61

III. ELISA ...... 61

IIJ. Quantitative PCR ...... 62

IIK. Statistical Analysis ...... 63

Chapter III. APC expression of NOX2 regulates T helper differentiation through pro- inflammatory cytokine production...... 64

IIIA. Introduction ...... 64

iv

IIIB. Results ...... 68

IIIB1. Oxidase deficient mice mount a robust T H1 response during immunization ...68

IIIB2. Oxidase deficient APCs promote T H1 polarization in vivo ...... 70

IIIB3. CD11b + antigen presenting cells are most affected by oxidase deficiency .....72

IIIB4. Characterization of wild type and NOX2 deficient antigen presenting cells ..76

IIIB5. Oxidase deficiency results in changes in the cross-talk between T cells and

antigen presenting cells during antigenic stimulation ...... 83

IIIC. Discussion ...... 86

Chapter IV. T cell expression of NOX2 regulates T helper differentiation through

alterations in STAT5 and GATA-3 ...... 93

IVA. Introduction ...... 93

IVB. Results ...... 96

IVB1. NOX2 expression in T cell development ...... 96

IVB2. Oxidase deficient T cells are more T H1 skewed than their wild type

counterparts ...... 99

IVB3. Oxidase deficient T cells have decreased GATA-3 during primary activation 103

IVB4. Alterations in phosphorylated STAT proteins in oxidase deficient T cells ....106

IVB5. Decreased IL-4 and IL-4R α expression in NOX2 deficient T cells ...... 111

IVB6. Enhanced T H1 phenotype in BALB/c T cells treated with antioxidants ...... 115

IVC. Discussion ...... 119

Chapter V. Discussion ...... 123

v

List of Figures

Figure I.1. Reactive oxygen species ...... 3

Figure I.2. Activation of ROS generation by assembly of the phox regulatory proteins ... 5

Figure I.3. Structure of the NOX and DUOX enzymes ...... 8

Figure I.4. Mechanisms of NADPH oxidase inhibition ...... 13

Figure I.5. Cytokines and transcription factor networks regulating T helper cell

differentiation ...... 23

Figure I.6. Heterogeneity and plasticity of helper T cells ...... 29

Figure I.7. Distribution of Mutations in gp91 phox , p22 phox , p47 phox , and p67 phox ...... 33

(-/-) Figure III.1. Immunization of wild type and NOX2 mice using pro-TH1 and pro-TH2 adjuvants ...... 69

Figure III.2. Adoptive transfer of OT-IIs into wild type and NOX2 (-/-) mice ...... 71

Figure III.3. CD11b + resident peritoneal cells are the predominant producers of pro- inflammtory cytokines ...... 73

Figure III.4. CD11b + resident peritoneal cells and adherent splenocytes are the predominant producers of pro-inflammtory cytokines ...... 75

Figure III.5. Enhanced production of IL-6 and TNF-α from NOX2 (-/-) resident peritoneal

cells and adherent splenocytes after LPS challenge in vitro ...... 77

Figure III.6. Selective changes in cytokine production from wild type and NOX2(-/-)

APCs after in vitro LPS stimulation are dependent on tissue localization ...... 78

Figure III.7. Equivalent amounts of TGF-β from wild type and NOX2 (-/-) resident

peritoneal cells after LPS challenge in vitro ...... 79

vi

Figure III.8. Analysis of costimulatory molecule expression in peritoneal APCs from wild type and NOX2 (-/-) mice ...... 81

Figure III.9. Analysis of costimulatory molecule expression in adherent splenocyte APCs from wild type and NOX2 (-/-) mice ...... 82

Figure III.10. CFSE dilution of OT-II CD4+ T cells co-cultured with APCs from wild type and NOX2 (-/-) mice ...... 84

Figure III.11. Cytokine production from OT-II CD4+ T cells co-cultured with APCs from wild type and NOX2 (-/-) mice ...... 85

Figure IV.1. NOX2 gene expression in wild type mice ...... 97

Figure IV.2. Enhanced inflammatory cytokine and decreased T H2 cytokine production from oxidase deficient T cells ...... 100

Figure IV.3. Wild type and NOX2 (-/-) naïve CD4 + T cells have equal viability ...... 102

Figure IV.4. GATA-3 and T-bet levels in oxidase deficient CD4+ T cells ...... 104

Figure IV.5. The levels of phosphorylated STATs 1, 3, and 4 are unchanged between wild type and oxidase deficient CD4 + T cells ...... 107

Figure IV.6. Decreased phosphorylated STAT5 in NOX2-deficient CD4 + T cells ...... 109

Figure IV.7. Lack of changes in IL-2/IL-2R expression in NOX2-deficient CD4 + T cells

...... 110

Figure IV.8. Decreased IL-4 and IL-4R α expression in NOX2-deficient CD4 + T cells 112

Figure IV.9. NOX2 deficient T cells are able to undergo T H2 commitment ...... 114

Figure IV.10. Antioxidants inhibit T H2 development in BALB/c T cells ...... 116

Figure IV.11. Oxidative regulation of STAT5 phosphorylation ...... 118

vii

Abbreviations

AIR - auto-inhibitory region

AKT - protein kinase B

APC – antigen presenting cell

AR-CGD - autosomal recessive chronic granulomatous disease

BAX - Bcl-2–associated X protein

BCG - bacillus Calmette-Guérin vaccine

CFDASE - Carboxy-fluorescein diacetate succinimidyl ester

CD62L - CD62 ligand

CFA - complete Freund's adjuvant

CGD - chronic granulomatous disease c-MAF - musculoaponeurotic fibrosarcoma

COX - cyclooxygenase

DMNQ - 2,3-Dimethoxy-1,4-naphthoquinone

DPI - diphenyleneiodonium

DUOX - dual oxidase

EGF - epidermal growth factor

ERK - extracellular signal-regulated kinase

FAD - flavin adenine dinucleotide

Foxp3 - forkhead box P3

GATA-3 - “GATA” binding transcription factor

GDI - GDP-dissociation inhibitor

GM-CSF - granulocyte-macrophage colony-stimulating factor

viii

H2O2 - hydrogen peroxide

HPRT - hypoxanthine-guanine phosphoribosyl transferase

ICOS - inducible T cell costimulator

ICOSL - inducible costimulator ligand

IFN - interferon

ICAM-1 - intercellular adhesion molecule 1

Ig - immunoglobulin

IL - interleukin

IL-12Rβ2 - IL-12 receptor β2

IL-2R α - IL-2 receptor α

IL-4R α - IL-4 receptor α

JAK - janus kinase

JNK - c-Jun N-terminal kinase

Lck - lymphocyte-specific protein tyrosine kinase

LOX - lipoxygenase

LPO - lactoperoxidase

LPS - lipopolysaccharide

MCP-1 - monocyte chemotactic protein-1

MEK /ERK - mitogen/extracellular signal-regulated kinase

MIP-1 - macrophage inflammatory protein 1

MMR - macrophage mannose receptor

MPO - myeloperoxidase mTOR - mammalian target of rapamycin

ix

NAC - N-acetylcysteine

NADPH - nicotinamide adenine dinucleotide phosphate (reduced form)

NK cell - natural killer cell

NOS - nitric oxide synthase

NO - nitric oxide

NOX2 - NADPH Oxidase 2

NOX2 (-/-) - NOX2 deficient

NRAMP-1 - natural resistance-associated macrophage protein 1

•- O2 - superoxide

•OH - hydroxyl radical

OVA - ovalbumin

PAK - p21 activated kinase

PDGF - platelet-derived growth factor

PDK1 - 3-phosphoinositide dependent protein kinase-1

PGD2 - prostaglandin D2

Phox - phagocyte oxidase

PI3K - phosphatidylinositol 3-kinase

PKC - protein kinase C

PRR - proline-rich repeat

PTEN - phosphatase and tensin homolog

PTP - protein tyrosine phosphatase

PX - phox homology

ROR-γt - retinoid-related orphan receptor

x

ROS – reactive oxygen species

Runx3- runt-related transcription factor 3

SH2 - SRC homology 2 domain

SH3 - SRC homology 3 domain

SHP - SH2 domain-containing phosphatase

SOD - superoxide dismutase

SOCS - suppressor of cytokine signaling

STAT - signal transducer and activator of transcription

T-bet - T-box transcription factor expressed in T cells

TCR - T cell receptor

TFH - T follicular helper

TGF-β - transforming growth factor β

TH – T helper

TLR - Toll-like receptor

TNFR - tumor necrosis factor receptor

Treg – T regulatory

VEGF - vascular endothelial growth factor

WT – wild type

X91 0 – XL-CGD, NOX2 null

X91 - - XL-CGD, NOX2 diminished

X91 + - XL-CGD, NOX2 present

XL-CGD - X-linked chronic granulomatous disease

ZAP-70 - Zeta-chain-associated protein kinase 70

xi

Chapter I. General Introduction

IA. Reactive Oxygen Species

Reactive oxygen species (ROS) consist of a number of active and reactive partially reduced oxygen metabolites that form as the natural byproducts of many biological reactions, such as those in the mitochondria and peroxisomes (Figure I.1).

•- • Some ROS, such as superoxide (O 2 ) and hydroxyl radical ( OH) can be defined as true

free radicals and are reactive due to the presence of unpaired valence shell electrons 1. In

order to regain a more stable non-radical condition, they avidly interact with proteins,

lipids, carbohydrates, and nucleic acids in the cell, possibly irreversibly destroying or

altering the function of these target molecules. Other ROS, such as hydrogen peroxide

1 (H 2O2), are technically pro-oxidant non-radical agents .

Because of their ability to readily interact with biological macromolecules, ROS

have generally been identified as a major contributor of damage to cells. However,

contrary to their view as damaging agents, cells have evolved to utilize these potentially

toxic molecules and convert them into useful tools for the cell. ROS have been shown to

play important roles in signal transduction, gene expression and in responses to a variety

of inflammatory stimuli. Many enzyme systems are specifically designed to generate

ROS 2. These enzymes include cyclooxygenase (COX), which is responsible for

formation of biological mediators called prostanoids, including prostaglandins,

prostacyclin and thromboxane; nitric oxide synthase (NOS) which catalyzes the oxidation

of L-arginine to generate nitric oxide (NO); lipoxygenase (LOX), which gives rise to

H2O2 during the dioxygenation of polyunsaturated fatty acids in lipids; as well as

1

•- xanthine oxidase and NADPH oxidase which generate O 2 through the transfer of an electron from NADPH to molecular oxygen 1,3,4.

2

Figure I.1. Reactive oxygen species

Superoxide (O2 •-) is generated from various sources, which include the NADPH

•- oxidases. Two molecules of O2 can react to generate hydrogen peroxide (H 2O2) in a reaction known as dismutation, which is accelerated by the enzyme superoxide dismutase

(SOD). In the presence of iron, superoxide and H 2O2 react to generate hydroxyl radicals

• • ( OH). In addition to superoxide, H 2O2 and OH, other reactive oxygen species (ROS) occur in biological systems. In inflamed areas, these include hypochlorous acid (HOCl), formed in neutrophils from H 2O2 and chloride by the phagocyte enzyme myeloperoxidase

(MPO); singlet oxygen, which might be formed from oxygen in areas of inflammation through the action of NOX and MPO-catalysed oxidation of halide ions; and ozone (O 3), which can be generated from singlet oxygen by antibody molecules.

Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Immunology]

Lambeth JD. NOX Enzymes and the Biology of Reactive Oxygen , 4(3):181-189 copyright 2004

3

IB. NADPH oxidases

IB1. The phagocyte NADPH oxidase

The prototypic phagocyte NADPH oxidase (Figure I.2), belongs to the NOX

family of ROS-generating oxidases, all of which are multisubunit transmembrane

proteins that transport electrons across biological membranes to reduce oxygen to

superoxide 5. NOX2 was the first identified example of a system that intentionally generates ROS not as a byproduct, but as its primary function 5. This enzyme was first characterized in phagocytic cells such as macrophages and neutrophils. It is activated in these cells upon exposure to microorganisms or inflammatory mediators, catalyzing what is known as the respiratory burst. The respiratory burst is a large increase in oxygen consumption and reactive oxygen generation.

The NADPH oxidase consists of the catalytic heme-containing glycoprotein gp91 phox (referred to as NOX2) which associates in the membrane with p22 phox , together forming flavocytochrome b558 . The enzyme also consists of three cytosolic regulatory subunits: p40, p67 and p47 phox , as well as the small GTPase Rac. In unstimulated phagocytes, the enzyme complex is dissociated and inactive, with the membrane-bound

6,7 flavocytochrome b558 stored in intracellular granules . The other phox proteins (p40, p67 and p47 phox ) associate in a separate ternary cytosolic complex in a dephosphorylated state through interactions between their respective SH3 domains and proline-rich repeat (PRR) sequences. Rac is kept in a GDP-bound cytoplasmic complex dimerized with Rho-GDI 8.

4

Figure I.2. Activation of ROS generation by assembly of the phox regulatory proteins

Activation of NOX2 occurs through a complex series of protein-protein interactions.

Upon engulfment of a pathogen, the phosphorylation of p47 phox triggers the translocation of itself and the other cytosolic factors to the membrane to assemble with

phox flavocytochrome b558 . P22 functions as an adapter protein that provides a binding site

for the cytosolic regulatory subunits. The subunit p67 phox , contains an activation domain

that regulates the rate-limiting hydride transfer from NADPH to FAD in

flavocytochrome b558 , regulating overall activity of the enzyme. Once assembled, the

complex is active and transfers an electron from NADPH to molecular oxygen to

•- generate O 2 , which rapidly dismutates into H 2O2.

Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Immunology]

Lambeth JD. NOX Enzymes and the Biology of Reactive Oxygen , 4(3):181-189 copyright 2004

5

The p47 phox organizer subunit has two SH3 domains, which interact with its C- terminal auto-inhibitory region (AIR) to keep it in an auto-inhibited state 9. Activation

occurs through a complex series of protein-protein interactions, requiring the

phosphorylation of p47 phox by one or a combination of protein kinases, such as PKC-ζ,

PKC-δ, PAK, AKT, or ERK1/2, depending on the nature of stimulation 9. When a

macrophage or neutrophil engulfs a pathogen, the intracellular granules containing

flavocytochrome b558 fuse with newly forming phagosomes. The phosphorylation of the

p47 phox induces a conformational change in which the SH3 domains no longer interact

with the AIR, and convert to a p22 phox -polyproline rich-sequence 9. The conformational change also triggers the translocation of p47 phox with the other cytosolic factors to the

phox membrane to assemble with flavocytochrome b558 . P22 functions as an adapter protein and provides a binding site for the cytosolic subunits and allowing for assembly. This occurs through the interaction of p47 phox SH3 domains interacting with p22 phox , and through the p47 phox PX domain interacting with phospholipids in the membrane 7. The

activator subunit p67 phox contains an activation domain that regulates the rate-limiting hydride transfer from NADPH to FAD in flavocytochrome b558 , thereby regulating overall

activity of the enzyme. Once completely assembled, the complex is active and transfers

•- an electron from NADPH to molecular oxygen to generate O2 , which rapidly dismutates

into H 2O2. This can occur spontaneously, or by the enzyme superoxide dismutase (SOD).

Although H2O2 alone exerts modest antimicrobial action, it contributes to innate host

defense by functioning as a substrate for peroxidases such as myeloperoxidase in

neutrophils. Myeloperoxidase produces hypochlorous acid (HOCl) from H2O2 and

chloride anion (Cl -) during the neutrophil respiratory burst 7. This enzyme can also

6

oxidize tyrosines to tyrosyl radical using H2O2 as an oxidizing agent. Both HOCl and tyrosyl radical are cytotoxic, and are used by neutrophils to kill bacteria and other pathogens. The cascade of secondary ROS generation includes the formation of highly

•- reactive peroxynitrite, generated from O 2 and nitric oxide (NO), as well as hypochlorite, and hydroxyl radical (•OH) to name a few, all of which lead to destruction of the engulfed pathogen.

IB2. Other members of the NOX family of NADPH oxidases

After the identification of NOX2 (gp91 phox ), findings from several studies suggested the existence of other enzyme systems similar to the phagocyte oxidase that were expressed in non-phagocytic cells, such as neurons, hepatocytes, endothelial cells, hematopoietic stem cells, B cells, and T cells 5,10 . As a result of the availabilty of the human genome sequence, the first homolog of NOX2 was identified. This homolog was initally called mitogenic oxidase 1 (MOX1) or NADPH oxidase homolog 1 (NOH1), but is now termed NOX1 (Figure I.3) 11 . NOX1 is highly expressed in the colon as well as in the uterus, prostate and vascular smooth muscle cells. In endothelial cells, angiotensin II induced uncoupling of endothelial nitric oxide synthase (eNOS) requires the upstream

•- 12 activation of NOX1, inducing the production of O2 instead of NO . Like NOX2, NOX1 is composed of a catalytic transmembrane domain containing two hemes, which act as electron carriers capable of undergoing reduction and re-oxidation, and forms heterodimers with p22 phox in cell membranes.

7

Figure I.3. Structure of the NOX and DUOX enzymes

After the identification of NOX2, findings from several studies suggested the existence of other enzyme systems similar to the oxidase that were expressed in non-phagocytic cells.

As a result of the availabilty of the human genome sequence, the first homolog of NOX2 was identified, now termed NOX1. The discovery of NOX1 was followed by the identification of NOX3, NOX4, NOX5, and finally the dual oxidases DUOX1 and

DUOX2. These other six NOX family members are similar in overall structure to NOX2 but differ in some of their subunits and in their regulation.

Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Immunology]

Lambeth JD. NOX Enzymes and the Biology of Reactive Oxygen , 4(3):181-189 copyright 2004

8

•- phox phox Although NOX1 can generate O2 when co-expressed with p47 and p67 , it does not usually use them. NOX organizer 1 (NOXO1), is almost identical to p47 phox except that it lacks the autoinhibitory region, and NOX activator 1 (NOXA1) similarly shares the same domain organization as p67 phox but lacks one of the SH3 domains that are present in its NOX2 associated homologue 13 . Whereas inactive p47 phox is in the cytosol,

NOXO1 is pre-localized at membranes together with NOX1 14 . Neither translocation of

NOXO1 nor lipid kinases seem to be triggers of NOX1 activation, but there is some

evidence that activation occurs through the translocation of RAC1 15 .

NOX1 is functionally similar to NOX2 and can sometimes replace NOX2 in

•- 16 producing O2 , partially rescuing defective ROS production in CGD neutrophils . Like

NOX2, it has been shown to be involved in innate immunity. ROS generation by NOX1

was detected in guinea pig gastric pit cells primed with Helicobacter pylori

lipopolysaccharide (LPS), and in T84 cells primed with Salmonella enteritidis flagellin,

demonstrating that NOX1 can be activated by stimulation of TLR4 and TLR5,

respectively 17,18 .

Also structurally similar to NOX1 and 2 are NOXes 3 and 4 (Figure I.3). NOX3

expression has been found in several tissues, including kidney, liver, lung, and spleen. It

plays an important role in the inner ear. Head tilt ( het ) mice with mutations in NOX3 have impaired otoconial development and have vestibular defects 19,20 . NOX3 requires

p22 phox , but shows unusual flexibility in its ability to be activated by p47 phox /p67 phox or

NOXO1/ NOXA1 proteins 21 . Moreover, NOX3 can be activated by NOXO1 alone in the

absence of activator subunits.

9

NOX4 was originally described as an oxygen sensor and regulator of erythropoietin synthesis; and because of its renal abundance was termed “renox.” 22 It may have antimicrobial functions like NOXes 1 and 2, by releasing ROS into the glomerular filtrate 22 . Other possible functions include roles in oxidation or detoxification of urine wastes or in renal pH or electrolyte homeostasis, based on the proton-generating and electrogenic activities of all oxidases. NOX4 is also expressed in endothelial and vascular smooth muscle cells as well as in fibroblasts and adipocytes. Unlike renal

NOX4, which is constitutively active 23 , endothelial NOX4 can be stimulated by vasoactive agonists (angiotensin II), physical factors (shear stress and flow), and hypoxia, and promotes endothelial proliferation, migration, and tube formation 24,25 . NOX4's enothelial functions are mediated through the regulation of eNOS, which when

•- 26,27 uncoupled, generates O2 instead of NO .

NOX5 was the last of the conventional NOX enzymes to be identified, and has been found to be expressed in spermatocytes of testis, in B- and T-lymphocyte-rich areas of spleen and lymph nodes, and most recently in endothelial cells (Figure I.3) 19,28 .

Whereas the activity of NOXes 1–4 is tightly regulated by the coordinated assembly of their regulatory cytoplasmic proteins into a functional enzyme, the regulation of NOX5 activity is not dependent on the presence of the cytosolic factors 28 . P22 phox in particular, is required for NOXes 1–4 but dispensable for NOX5. NOX5 contains the same catalytic core as NOXes 1-4. However this core is regulated by an amino-terminal calmodulin- like domain containing four binding sites for calcium 5. Increases in calcium or binding of calcium-activated calmodulin induce a conformational change exposing hydrophobic residues that bind and subsequently activate the catalytic domain of the oxidase 29 . In

10

endothelial cells, in contrast to NOX1 and 4, NOX5 activates eNOS and promotes NO production by increasing the activity of Hsp90, a molecular chaperone that is important for overall eNOS enzymatic activity 30 .

The dual oxidase (DUOX1 and DUOX2) enzymes build on the NOX5 structure by the addition of an extra transmembrane α-helix and a peroxidase domain (Figure I.3) 5.

These enzymes are expressed in many tissues such as the colon, lungs, kidney, thyroid, and the pancreatic islets. In thyroid tissue, the DUOX enzymes are essential for thryoxine synthesis and are proposed to supply H 2O2 for thyroperoxidase-mediated iodination and cross-linking of thyroglobulin tyrosine residues 19,31 . They are also involved in mucosal immunity through the activity of lactoperoxidase (LPO), an enzyme with antimicrobial properties present in saliva, milk, tears, and airway secretions 24 .

DUOX generated H 2O2 has been shown to be used by LPO to oxidize substrates such as thiocyanate ions and halides, resulting in short lived oxidation products that are bacteriocidal 7. It has recently been demonstrated in respiratory tract epithelium that this activity can be upregulated in response to IFN-γ32 . Outside of innate immunity, DUOX expression in T cells is involved in TCR-induced signal transduction.

IB3. Antioxidants and pharmacological inhibitors of NADPH oxidases

Although antioxidants do not act by directly inhibiting NADPH oxidase activity, they are able to scavenge and neutralize their products (Figure I.4). Thus, they are able to inhibit the effects of NADPH activation. The use of antioxidants and pharmacologic inhibitors both in vitro and in vivo has been useful in elucidating NADPH oxidase function and the creation and discovery of new therapeutic compounds that can inhibit oxidase activity is an active area of investigation. The present study in particular will be

11

using apocynin (4-hydroxy-3-methoxy-acetophenone) and N-acetylcysteine (NAC) in proof of concept experiments as a way to consider the overall effect of redox signaling on

T helper differentiation.

The thiol antioxidant N-acetylcysteine is frequently used in studying ROS. It was developed in the 1960s and is the N-acetyl derivative of the naturally occurring amino acid L-cysteine 33 . NAC is used in the management of paracetamol (acetaminophen) overdose and as a mucolytic agent in chronic pulmonary disease 33,34 . It serves as a source of sulfhydryl groups in cells and ROS scavenger as it readily interacts with •OH and

H2O2. Other commonly used antioxidants include the superoxide dismutase mimetic tempol (tetramethylpiperidinol N-oxyl) and the selenium compound ebselen 35 . Both are potent scavengers of ROS. Ebselen in particular is being investigated as a possible treatment for reperfusion injury and stroke. Naturally occurring L-ascorbate and α- tocopherol (vitamins C and E) are widely administered antioxidants as well. Although they have provided disappointing results in the treatment of human disease, they are useful tools for studying redox balance in vitro.

12

Figure I.4. Mechanisms of NADPH oxidase inhibition

NADPH oxidases are multisubunit membrane proteins that are activated by translocation

of the cytosolic subunits p47 phox , p67 phox , and Rac to the gp91 phox /p22 phox complex. Most currently available specific inhibitors act by interfering with this translocation.

Nonspecific inhibitors target the flavin-containing subunit (DPI), or act to scavenge the reactive oxygen species produced by NADPH oxidases (antioxidants).

Adapted by permission from Wolters Kluwer Health: [Journal of Cardiovascular Pharmacology]

Williams HC, Griendling KK. NADPH Oxidase Inhibitors: New Antihypertensive Agents?,

50(1):9-16 copyright 2007

13

Chemical inhibitors of NADPH oxidases are more specific than antioxidants. The

two most widely used ones are diphenylene iodonium (DPI) and apocynin. DPI is a

commonly used potent inhibitor of NADPH oxidase activity. Originally identified as an

inhibitor of gluconeogenesis via inhibition of mitochondrial substrate oxidation, later

research identified a role for DPI as an NADPH oxidase inhibitor 35 . DPI is thought to

block NOX activity by removing an electron from the oxidase, causing adduct formation

•- with FAD and inhibiting O 2 formation (Figure I.4). However, DPI is toxic and very nonspecific because it inhibits all flavin-containing enzymes, such as NOS, xanthine oxidase and members of the cytochrome P450 family36-38 .

Apocynin, a constituent of the Himalayan medicinal herb Picrorhiza kurroa was

•- originally discovered as an inhibitor of O 2 in activated neutrophils. Apocynin is a pro- drug that is oxidized by peroxidases in the cell and converted into more active metabolites. The exact mechanism by which apocynin inhibits NADPH oxidase activity is unknown, but it is thought to prevent oxidase assembly by interfering with the binding of p47 phox to gp91 phox (Figure I.4). Apocynin has been shown to effectively reduce ROS production in animal models of arthritis, asthma and hypertension, but the effects are not entirely specific. Apocynin can influence other cellular events such as thromboxane A2 formation, AP-1 transcription factor induction, and arachidonic acid metabolism 39,40.

More concerning are findings from several studies that have shown that apocynin may increase oxidative stress under certain conditions 41,42 . Glutathione expression was found to be decreased in response to apocynin treatment in alveolar epithelial cells, and glial cells treated with it increased the expression of several markers of oxidative stress 35 .

14

Other small molecule inhibitors of oxidase activity are currently under

investigation as alternatives to DPI and apocynin. VAS2870 is a cell-permeable

thiotriazolopyrimidine compound designed by Vasopharm Biotech that has been

identified for the treatment of peripheral arterial occlusive diseases (PAOD) 35,43 . It is

reported to act as a rapid and reversible inhibitor of NADPH oxidase activity. VAS2870

has also been shown to efficiently abolish agonist-induced ROS generation and to offer

protection against oxidative stress 43 . Because VAS2870 inhibits only agonist-induced

ROS, leaving basal ROS production intact, it may have relative specificity within the

NOX family. However, comparing its effects on each of the family members has yet to

be performed.

Another molecule that shows some promise in treating oxidative stress in vascular

disease is the benzo(b)pyran-4-one derivative, S17834. S17834 has potent

antiatherosclerotic and anti-inflammatory properties, and was able to inhibit oxidase

activity in endothelial cell membranes 44 . Its major mechanism of action is believed to be

phox 38,44 preventing the binding of p47 to flavocytochrome b558 .

Peptide inhibitors that target sequences of NADPH oxidase components have the

potential to be even more specific than small molecule inhibitors. The naturally

occurring peptide PR-39 has been shown to regulate oxidase activity. This 39 amino acid

proline-arginine rich antibacterial peptide was originally isolated from swine intestine

and is present in macrophages and neutrophils 45 . PR-39 interferes with oxidase activity by binding to the SH3 domain of p47 phox and preventing oxidase assembly (Figure

I.4) 45,46 . However, PR-39 can be fairly nonspecific due to its ability to bind many proteins that have SH3 domains and interact with membrane lipids. One of the most

15

specific and efficacious inhibitors so far developed is gp91ds-tat, a chimeric 18 amino

acid peptide that also interferes with oxidase assembly 35,47 . The first nine of its amino acids mimic a region of gp91 phox that interacts with p47 phox . This sequence is able to bind

to p47 phox and inhibit subunit assembly, thus blocking activation (Figure I.4) 47 . The next

nine amino acids correspond to a specific sequence in the HIV viral coat, which allows

the peptide to be internalized by cells.

IC. Regulation of Biologic Responses by ROS Generation

The wide distribution and expression of the NOX family of enzymes in various tissues provides evidence for the intentional and tightly regulated generation of ROS involved in physiological functions outside of innate host defense. Various stimuli such as angiotensin II or PDGF can induce the intracellular production of ROS 48,49 . ROS function as requisite second messengers, necessary for ligand mediated regulation of protein kinase activation, gene expression and/or proliferation. NOX2 as well as its other isoforms have been found to regulate signaling though mechanisms which have evolved to harness ROS in specific ways. Oxidase activation in the respiratory burst of phagocytes leads to generation of high levels of ROS, producing a non-specific bactericidal effect within the phagosome. However, in non-phagocytic cells low levels of

ROS produced by oxidases are not cytotoxic, but rather regulate activation of signaling molecules, gene expression and/or cell proliferation.

The fact that ROS are very short lived coupled with the local availability of protein targets to react with ROS accounts for the specificity in redox-sensitive signal transduction. Redox signaling by H 2O2 typically results in the oxidation of thiol groups on cysteine residues. These reactive cysteine residues are easily oxidized to a sulfenic

16

form (RSOH), which is unstable and can undergo further oxidation via dismutation to a

50,51 sulfinic (RSO 2H) species . Under even greater oxidative stress, the sulphonic

(RSO 3H) species can be created. Other possibilities for post-translational cysteine modifications include nitrosylation (RSNO), glutathionylation (RSSG), or the formation of inter- or intramolecular disulfide bonds (RSSR). Oxidation of cysteines on proteins can alter their functions.

Multiple classes of proteins have been found to be reversibly oxidized by ROS and these oxidation events have downstream consequences. The best-characterized molecular targets of ROS are the protein tyrosine phosphatases (PTPs) because these important signaling enzymes require a conserved catalytic cysteine residue that exists as a thiolate anion, and has enhanced susceptibility to oxidation 52,53 . Oxidation of these cysteine residues modifies phosphatase activity. MAPK pathways can be activated by the direct inhibition of MAPK phosphatases by ROS 50,51 . ROS produced by NADPH oxidases have been shown to inhibit JNK-inactivating phosphatases through reversible oxidation of a catalytic-site cysteine to sulfenic acid, thus sustaining JNK activation.

ROS have also been shown to regulate dual-specific phosphatase 3 (DUSP3) activity, an example being that ROS generated by commensal bacteria inactivated DUSP3 by cysteine oxidation, resulting in ERK activation. PI3K and AKT activity have been shown to be reversibly regulated by ROS through the inactivation of PTEN 50,51 . PI3K activity phosphorylates and converts the lipid second messenger phosphatidylinositol

(4,5) bisphosphate (PIP 2) into phosphatidylinositol (3,4,5) triphosphate (PIP 3), which recruits and activates phosphatidylinositol-dependent kinase 1 (PDK1). PDK1 in turn phosphorylates and activates AKT, which promotes cell proliferation and survival. The

17

tumor-suppressor phosphatase with tensin homology (PTEN) negatively regulates PI3K signaling by dephosphorylating PIP 3, converting it back to PIP 2. H 2O2 generated by stimulation from growth factors such as insulin, EGF, or PDGF, has been shown to oxidize and inactivate human PTEN through disulfide bond formation between the catalytic domain Cys-124 and Cys-71 residues 50,51,54 . PTEN oxidation can be reversed by peroxiredoxin II, an antioxidant enzyme that that eliminates H 2O2 generated in response to growth factors and cytokines

More recently kinases have been show to undergo direct oxidative regulation as well 50,51 . The protein tyrosine kinase (PTK) Src as well as the fibroblast growth factor receptor type 1 (FGFR1) are fully active when reduced, and inactivated by oxidation; both of which are regulated by NOX1 55,56 . In addition to indirect regulation of the

MAPK pathway by ROS, mitogen-activated protein kinase kinase 6 (MKK6), a member of the MAP2K subfamily that specifically phosphorylates and activates the p38 MAPKs has been shown to be inactivated by oxidation due to the formation of a specific intramolecular disulfide bond between Cys109 and Cys196 in the kinase domain, which directly inhibits ATP binding 57 .

Though NOX2 is most highly expressed in phagocytes, its expression has also been detected in the CNS, endothelium, vascular smooth muscle cells, fibroblasts, cardiomyocytes, skeletal muscle, hepatocytes, and hematopoietic cells. NOX2 generated

ROS can be produced by receptor ligation and modulate downstream signaling events that alter cellular function. These ROS can in turn modulate downstream kinases and/or phosphatases regulating key signaling pathways, including those used by cells of the adaptive immune system. A recent study in fibroblasts found endosomal NOX2 to be

18

involved in TNF-α receptor signaling and downstream induction of the transcription factor NF-κB58 . The study proposes that after TNF α receptor ligation and endocytosis,

TRADD is recruited to the receptor, and NOX2 derived ROS promotes TRAF2 binding to the TNFR1/TRADD complex, activating I κB kinase (IKK) and promoting NF-κB

activation 59 . In addition to TNF-α, other cytokines, such as IL-2, IL-4, IL-6, IL-7 GM-

CSF, M-CSF, and TGF-β have been shown to induce the production of ROS, and can

have immunomodulatory effects on T cells and antigen presenting cells (APCs) 54,60-69 .

Within T cells themselves, T cell receptor (TCR) ligation induces the discrete generation

•- 70-73 of both O2 and H 2O2 which selectively regulate independent signaling pathways .

Both NOX2 and Duox1 are expressed in T cells and are responsible for regulating

specific signaling pathways. Upon TCR crosslinking, T cells activate the kinases ERK,

48,49,70,71 JNK, p38, and AKT, all of which are associated with the production of H 2O2 .

Duox1 has been shown to mediate the oxidation of SHP-2 and enhance proximal TCR

signaling through the activation of ZAP-70 and through the regulation of intracellular

calcium flux 73 . The role of NOX2 in T cell signaling is less understood, but NOX2

deficient T cells or wild type T cells treated with pharmacologic antioxidants have

demonstrated selectively augmented and sustained MEK-ERK activation 70,71 . Thus, ROS

in many types of nonphagocytic cells can mediate intracellular signaling events, which in

turn modulate various cellular responses, including those of the innate and adaptive

immune systems.

19

ID. Helper T cell development

ID1. The T H1/T H2 Paradigm

Upon antigen presentation to naive CD4 + T cells, the interaction of co-stimulatory molecules and T cell receptor (TCR) signal strength, together with the local cytokine environment and APC type, promote the differentiation of naive CD4+ T cells into various T helper subsets. In 1986, Coffman and Mosmann demonstrated the existence of

+ CD4 T helper 1 (T H1) and helper 2 (T H2) clones differing from each other in the

74 cytokines they preferentially produced . T H1 cells mainly produce IFN-γ, which is important for macrophage activation and clearance of intracellular pathogens, whereas

TH2 cells produce IL-4, IL-5, and IL-13, later shown to be critical for IgE production, eosinophil recruitment and clearance of extracellular parasites. Both subsets were initially thought to provide help to B cells, although each subset “instructed” the class- switching mechanism toward different classes of immunoglobulin (Ig). In addition to regulating their respective protective immune responses, T H1 and T H2 cells are also involved in many immmune-mediated diseases. T H1 cells have been suspected to be involved in many organ-specific autoimmune diseases, whereas T H2 cells are thought to be involved in asthma and other allergic reactions.

Several factors impact the development of T H1 and T H2 cells. These include antigenic type and load, the dominant cytokine environment, the composition of costimulatory molecules and balance of signaling cascades activated. All seem to be dependent, however, on a fundamental stepwise interaction between the APC and the naive T cell. The decision of the immune response to go in a certain direction is not

20

based on one signal alone, but rather on many different elements acting synergistically, antagonistically and through different feedback loops.

TH1 development is initiated by the signal transducer and activator of transcription (STAT) 1, which is activated in response to IFN-γ and IL-27 that are produced by natural killer (NK) cells and APCs, respectively 75 . Together with TCR- induced transcription factors, STAT1 induces the expression of the T H1-specific transcription factor T-box expressed in T cells (T-bet), which in turn induces the production of IFN-γ, the activation of the transcription factors H2.0-like homeobox

75-77 (HLX) and runt-related transcription factor 3 (Runx3) . Runx3 and T-bet block T H2 differentiation by opposing the inhibitory effects of GATA3 on T H1 differentiation, and by binding to and repressing the Il4 locus 78 . T-bet also induces the expression of the IL-

12 receptor-β2 (IL-12R β2), which pairs with IL-12R β1 to form a complete IL-12 receptor 78 . IL-12 receptor expression allows for APC-derived IL-12 to activate STAT4, which also induces the production of IFN-γ and further expression of T-bet 79 . Autocrine

IFN-γ signaling through the IFN-γR/STAT1 pathway further increases IFN-γ levels, forming a positive feedback loop (Figure I.5).

TH2-cell differentiation is initiated by the activation of STAT6 by IL-4, which, together with TCR-induced transcription factors, binds to and activates the T H2 master regulator GATA-3. GATA-3 is a member of the GATA family of zinc finger proteins, so-called because they bind to the DNA sequence GATA. GATA-3 induces the transcription factor c-MAF, which helps to activate Il4 , and together GATA-3 and c-

76,80,81 MAF activate the transcription of Il4 , Il5 and Il13 . T H2-lineage commitment is stabilized by the autoactivation of GATA-3, the autocrine and paracrine activation of

21

STAT6 by IL-4, as well as STAT6- and GATA3-dependent antagonism of IFN-γ expression 82 . The sources that produce the initial IL-4 that begin the STAT6-dependent positive-feedback loop during in vivo T H2 cell responses are still unresolved. Basophils,

NKT cells, as well as naïve T cells themselves are all possible early IL-4 producers 83-85 .

GATA-3 can be induced in a STAT6-independent manner through Notch signaling and also through the ligation of the IL-2 receptor and the TCR, which can induce both STAT5 phosphorylation and increase the rate of translation for GATA-3, respectively 86 . STAT5 can also be activated by TCR ligation, independent of IL-2, and the cooperation of

STAT5 and GATA-3 together induce early IL-4 production by naive CD4 + T cells and

87,88 initiate the early events of T H2 differentiation (Figure I.5) .

22

Figure I.5. Cytokines and transcription factor networks regulating T helper cell differentiation

Initial studies arising from in vitro cultured T H1 (A), T H2 (B), and T H17 (C) cells led to the idea that these subsets behaved like lineages, meaning that their phenotype (i.e., selective cytokine production) was heritable and inflexible. Accordingly, these subsets expressed lineage-restricted transcription factors that were sufficient to impart this selective cytokine production. As newer subsets of cytokine producing cells were identified, they too were viewed as stable lineages.

Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Immunology] Wilson

CB, Rowell E, Sekimata M. Epigenetic control of T-helper-cell differentiation , 9(2):91-105 copyright 2009

23

24

In addition to cytokine signaling, the strength of the TCR signal, in conjunction

with the nature of costimulatory molecules, influences the generation of T Hl and T H2 cells. In the absence of exogenous cytokines, the combination of a weak TCR signal and

89 strong costimulation through CD28 promotes the generation of T H2 cells . Enhanced

90,91 signaling though the inducible costimulator (ICOS) also promotes T H2 differentiation .

Signaling through CD28 and ICOS is also involved in T H1 development, but differentiation is much less dependent on costimulation and can be induced by a stronger

TCR signal. Strong TCR signaling is characterized by enhanced MEK/ERK signaling, and has been found to abrogate early TCR induced GATA-3 expression, IL-2 dependent phosphorylation of STAT5 and the early production of IL-485,89 . MEK inhibition has been shown to rescue early GATA-3 expression and responsiveness to IL-2, allowing for

85 early production of IL-4 and subsequent T H2 differentiation .

ID2. Other T helper subsets

While the T H1/T H2 paradigm gives valuable insight into the functions for helper T cells, it is now becoming clear that this model is too simplistic to define all of their roles.

Other subsets of helper T cells have been recently characterized and determined to be developmentally distinct from T H1 or T H2 cells.

TH17 cells produce IL-17a, IL-17f, IL-22 and IL-21. Upon initial characterization,

TH17 cells were broadly implicated in autoimmune disease, and auto-specific T H17 cells

92 were shown to be highly pathologic . The natural role of T H17 cells is to direct immune responses against extracellular bacteria and fungi. The main cytokines produced, IL-17a and IL-17f, are involved in the recruitment, activation and migration of neutrophils.

25

Transforming growth factor beta (TGF-β), IL-6, IL-21, IL-23, as well as IL-1β have been

implicated in their development 77 . The transcription factors STAT3 and the retinoic-

acid-receptor-related orphan receptor-γt (ROR γt) are critical for the differentiation of

93 TH17 cells , whereas the transcription factors that are involved in T H1-and T H2-cell

differentiation are not required for T H17 differentiation and have actually been shown to antagonize this process (Figure I.5)94 .

Opposite to T H17 cells are regulatory T cells (Tregs), which downregulate immune responses, resolve inflammation, maintain tolerance to self-antigens, and help in the prevention of autoimmune disease 95 . Besides thymic-derived natural Tregs (nTregs), it has been shown that naïve T cells in the periphery could acquire immunosuppressive properties and become inducible Tregs (iTregs), when treated with TCR stimulant and

IL-2, along with TGF-β or IL-10 96 . Both of these Treg subsets express the forkhead transcriptional repressor (Foxp3) and are highly dependent on STAT5 signaling, which upregulates Foxp3 expression 97 .

The induction of T H17 cells and Tregs is a unique balancing act that requires

TGF-β and is dependent on the presence of IL-6. TGF-β inhibits T H1-and T H2-cell

differentiation, and promotes Treg and T H17-cell lineage commitment by inducing the expression of Foxp3 and ROR-γt, respectively 93 . In the absence of IL-6, Foxp3 inhibits

RoR-γt, blocking T H17 development. However, in the presence of IL-6, STAT3 is activated and inhibits the expression of Foxp3, resulting in increased RoR-γt for T H17

76 differentiation . Once T H17 differentiation has been initiated, these cells produce IL-21, which also signals through STAT3 and induces the expression of IL-23R 98 . APC-derived

26

IL-23 can then bind to the IL-23R to further activate STAT3, dampening the production of IL-10 and driving the production of IL-22, thus stabilizing T H17 commitment.

The most recently characterized T helper subsets are the TH9 and T follicular helper (T FH ) lineages. T H9 cells are an IL-9 producing subset characterized to

77 be involved in allergic diseases and resistance against intestinal nematodes . T H9 cells do not express any well-defined transcription factors like T-bet, GATA-3, ROR-γt or

Foxp3, and differentiation of these cells oftentimes occurs downstream of T H2 differentiation by a combination of signals from TGF-β and IL-4. It is TGF-β that

77 reprograms T H2 cells to lose their characteristic profile and switch to IL-9 secretion .

The T follicular helper (T FH ) lineage is located in the follicular regions of lymph nodes and spleen, and can be identified by their constitutive expression of the B cell follicle homing receptor CXCR5, as well as expression of the transcription factor Bcl-

699,100 . Bcl-6, unlike Tbet and GATA-3 is actually a transcriptional repressor, suggesting that similar to T H9 cells, TFH cells may also subsequently arise from a separate helper subset 101 . These T cells provide help to B cells for high-affinity antibody responses and

B-cell memory. The costimulatory molecule ICOS has been shown to provide a critical

102 signal for T FH cells, as mice deficient in ICOS are unable to develop any of these cells .

ICOS has been shown to induce the secretion of IL-21 by activated T cells 103 , and IL-21

100 plays a crucial role in the development of the T FH subset .

ID3. T helper lineage plasticity

A differentiating T helper cell is able to assimilate multiple signals to acquire the appropriate effector phenotype. Within lineage subset variation, recent studies have demonstrated the "functional plasticity" of these subsets. Instead of lineage commitment

27

being something determined, it is more a process of specification; in that although

differentiation occurs, opportunities for alternative fates persist (Figure I.6) 104 . Cytokine

expression patterns are not as concrete as previously thought, and there is increasing

evidence that differentiated CD4 + T cell populations can alter the range of cytokines they

produce 101 . How frequently this occurs in vivo remains unclear, but "multi-functional" T

cells, producing multiple cytokine types have been isolated from hosts during certain

types of infection 105

IL-10 for example used to be considered a TH2 cytokine, but is now found to be

106 produced by several T cell subsets, including T H1, T H17 and Tregs . In addition to T H9

107 cells, IL-9 can be produced by T H17 cells as well . It is currently debated as to whether or not T H9 cells are technically even a "real" subset, as they do not have any particular lineage specifying transcription factors and they arise when T H2 cells are stimulated with

a combination of IL-4 and TGF-β. Other examples of plasticity describe T H17 cells that

produce IFN-γ with IL-17 simultaneously, and can even switch from producing IL-17 to

IFN-γ alone 101,106 .

28

Figure I.6. Heterogeneity and plasticity of helper T cells

Recent studies have revealed that helper T cells are more heterogenous and plastic than

originally thought, and there are now many examples of plasticity of T H cell phenotype.

CD4 + T cells can change their profile of cytokine production, and there are now circumstances in which the expression of master regulators is transient or instances where cells express more than one master regulator. Plasticity may be in part explained by the different combinations of expression of transcription factors in a single cell.

Copyright 2010 Wiley. Used with permission from Zhu J, Paul WE. Peripheral CD4 + T-cell differentiation regulated by networks of cytokines and transcription factors . Immunological

Reviews 238 (1):247–262, John Wiley and Sons

29

In addition to the simultaneous production of different cytokines, helper T cells

can also simultaneously express more than one type of lineage specifying transcription

factor. Cells expressing both Foxp3 and T-bet are present at sites of inflammation, and

paradoxically, sometimes Tregs can express Foxp3 and ROR-γt at the same time 101 .

Tregs overall have been found to be a relatively heterogeneous population and may or may not always express Foxp3, which itself only needs transient expression to allow for

Treg induction. Tregs can differentiate into TH2 cells as well as TFH cells in the Peyer's patches of the gut, implying that they acquire STAT6 or Bcl-6 expression,

101,108 99 respectively . In addition to Bcl-6 expression, TFH cells can express GATA-3 . A recent study comparing epigenetic changes in naïve, nTreg, iTreg, T H1, T H2, and T H17 cells found bivalent permissive and suppressive forms of histone modifications found at

Tbx21 (the gene locus encoding T-bet), in all non-TH1 cells, suggesting that they all may retain their ability to express T-bet 109 . This was also the case for the Gata3 locus in non-

TH2 cells. A study by Hegazy and others have shown that fully differentiated T H2 cells can be stimulated to express T-bet and produce IFN-γ110 . This change was completely dependent on the upregulation of T-bet. Indeed, recent evidence suggests that the ratio of

T-bet to GATA-3 expression is a more appropriate indicator to define the T helper

phenotype in immune cells, and that this balance between T-bet and GATA-3 expression

111,112 is representative of the balance between T H1 and T H2 cytokines . Confining T cells to a single phenotype could lead to inappropriate adaptive responses when encountering new pathogens; thus helper T cell plasticity provides a better evolutionary advantage to host survival 101 .

30

An additional factor that may influence T helper lineage fate decisions is the

involvement of redox signaling. Previous studies have demonstrated that altering the

redox balance in T cells can result in phenotypic changes in T helper function, although

the exact mechanisms are still being addressed. The antioxidant properties of the mineral

selenium as well as α-tocopherol (vitamin E) have been found to skew T cells toward a

113,114 TH1 response with increased IFN-γ and decreased IL-4 secretion . In a study

examining oxidative stress and its relationship to allergies and asthma, exposure of CD4 +

T cells to the intracellular pro-oxidant DMNQ (redox-cycling quinine which generates

•- O2 and H2O2) skewed them toward a T H2 phenotype, resulting in increased amounts of

IL-4, IL-5, and IL-13 115 . Multiple studies have demonstrated that T cells express a

functional NOX2, and its deficiency has been shown to alter the inherent function and

survival of these cells 116,117 . Our lab has also shown that T cells express the phagocyte-

type NADPH oxidase that is activated after TCR stimulation; and T cells deficient in the

oxidase have an altered cytokine profile 70,71 . T cell blasts from mice deficient in the

oxidase demonstrate T H1 skewed cytokine secretion as compared to their wild type

counterparts 71 . They demonstrated increased production of IFN-γ, TNF-α, and IL-2, with

decreased production of IL-471 . These data would suggest that the redox status within T cells plays an important role in lineage specification, and oxidase expression in T cells directly affects the T cell phenotype.

IE. Chronic Granulomatous Disease (CGD)

IE1. Disease Genetics

Mutations or deletions that affect the phox proteins of the phagocyte oxidase cause chronic granulomatous disease (CGD). Originally characterized in 1957 in four

31

boys from Minnesota with granuloma lesions in the lungs, and termed "a fatal

granulomatous disease of childhood"118,119 , CGD is a primary immunodeficiency

syndrome characterized by impaired ability of phagocytic cells (neutrophils, mononuclear

cells, macrophages, and eosinophils) to kill engulfed microorganisms, due to the inability

to generate the reactive oxygen species necessary for the respiratory burst 120 . Although

there are a number of different molecular defects that can cause CGD, they all affect one

component or another of the NADPH oxidase (Figure I.7). CGD can be relatively

heterogeneous in how severe it manifests itself and depends on which phox protein is

affected.

The most common form of the disease is due to an X-linked defect in gp91 phox .

X-linked CGD (XL-CGD) from mutations in the gp91 phox gene is responsible for 65-70% of the clinical cases in the United States. The gp91 phox gene is termed CYBB and spans a

30 kb region on the X chromosome at Xp21.1. Current studies describe 1156 unrelated families with 1259 patients, and a total of 621 different mutations, of which 368 mutations (59.3%) are unique for an individual kindred 121 . Deletions, frameshift, missense, nonsense, and splice site mutations have been described in this gene 122 . Protein

expression phenotypes of XL-CGD have been classified as X91 0, X91 - and X91 +;

32

Figure I.7. Distribution of Mutations in gp91 phox , p22 phox , p47 phox , and p67 phox

The top panel shows the positions of all identified mutations within the protein subunits of NADPH oxidase that were found in a cohort study by Kuhns and others. Changes in amino acids caused by missense mutations and 3-nucleotide in-frame deletions are indicated according to the mutation. The region of gp91 phox where missense mutations result in a loss of oxidase activity is highlighted in yellow. Missense mutations in the transmembrane regions (TM I, TM II, and TM IV) are on shown on the left side of the panel.

•- phox The lower panel shows the O2 production and expression of gp91 as a function of the nucleotide position of the mutation in patients with missense mutations in gp91 phox as compared with patients with all other mutations. In this cohort study, the level of expression of gp91 phox was scored on a scale from 0 (undetectable) to 3 (normal expression).

Domains of gp91 phox : transmembrane domains (TM I - TM VI), extracellular domains

(EC), intervening cytosolic domains, and flavin adenine dinucleotide (FAD) and NADPH binding domains.

Reproduced with permission from Kuhns DB, Alvord WG, Heller T, Feld JJ, Pike KM, Marciano

BE, Uzel G, DeRavin SS, Priel DAL, Soule BP and others. Residual NADPH Oxidase and

Survival in Chronic Granulomatous Disease . New England Journal of Medicine 363(27):2600-

2610 copyright 2010, Massachusetts Medical Society.

33

34

the superscript denoting whether gp91 phox protein expression is undetectable with oxidase activity completely abolished, diminished as a result of incomplete loss of protein or partial dysfunction, or normal, respectively.In patients with XL-CGD, gp91 phox protein levels were undetectable (X91 0) in 82%, diminished (X91 -) in 12%, and normal (X91 +) in

6% of cases 122 .

Autosomal recessive CGD (AR-CGD) seen in the remaining 35% cases, arise from mutations in genes for the other components of the oxidase: the CYBA gene

(16q24), the NCF1 gene (7q11) and the NCF2 gene (1q25) which encode p22 phox , p47 phox , p67 phox , respectively. Of these mutations, those in the p47 phox gene account for almost

25% of cases, followed by mutations in p67 phox (<5%) and p22 phox (<5%) 123 . Whereas there are a number of different mutations that lead to XL-CGD, most autosomal defects in the NCF1 gene for p47 phox are due to the same mechanism. In over 300 patients with p47 phox AR-CGD, the same deletion of two nucleotides was identified in a GTGT repeat, which corresponds to the acceptor site of the first intron-exon junction 124 . This common mutation may occur from recombination events between the NCF1 gene and an adjacent pseudogene, NCF1B , which has the GT deletion 124,125 . In general, patients with mutations in NCF1 have a more benign clinical course compared to other forms of the disease 123 , and overall more patients with AR-CGD (42%) have survived past their second decade of life than had patients with the X-linked form (22%) 126 .

Autosomal recessive mutations in the remaining subunits are less frequent.

Unlike NCF1 , CYBA encoding p22 phox , and NCF2 encoding p67 phox have been shown to have several heterogeneous mutations that are widely distributed throughout the genes.

These include missense and nonsense mutations, substitutions at splice sites, dinucleotide

35

insertions and various deletions. A single patient with a defect in NCF4 encoding p40 phox

has been reported, with mild disease limited to granulomatous colitis 127 . The patient's

•- neutrophils demonstrated defective intracellular, but not extracellular O2 production

during phagocytosis, which is different from other forms of CGD where both intracellular

and extracellular ROS production is affected 127 . To date, no patients with CGD have

been reported with defects in Rap1A, Rac1 or GDI components, but two reported cases of

dysfunctional Rac2 demonstrated a severe phagocyte immunodeficiency with clinical

features similar to CGD 128,129 .

IE2. CGD Pathophysiology

Classically, patients with CGD suffer from recurrent bacterial and fungal

infections. These infections oftentimes result in severe pneumonia; over 80% of CGD

patients have had pneumonia at least once. Abscesses and suppurative adenitis have also

occcured in more than 50% of the patients, with significant numbers also experiencing

osteomyelitis, sepsis, cellulitis, and meningitis 126,130 . Infections are limited to a specific

class of catalase positive microorganisms, such as Staphylococcus aureus and Aspergillus

fumigatus 130,131 . Additionally, recent evidence has shown a susceptibility to

mycobacterial species, such as Mycobacterium tuberculosis and even the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine 131-133 . Other unusual pathogens characteristic of CGD include Burkholderia cepacia , Chromobacterium violaceum,

Nocardia and invasive Serratia marcescens 8,131 . Additionally, infection-associated hemophagocytic syndrome triggered by Leishmania has been observed in CGD patients from Mediterranean regions 134 . CGD has about a 20% mortality rate, usually caused from complications caused by these infections 135 .

36

Prophylaxis for infections with trimethoprim-sulfamethoxazole is standard for the

management of patients with CGD. Trimethoprim-sulfamethoxazole treatment has been

shown to decrease the incidence of nonfungal infections without increasing the incidence

of fungal infections 136 . Additionally, IFN-γ is recommended as lifelong therapy for infection prophylaxis in CGD patients, although the molecular mechanisms associated with host defense improvement induced by this therapy are unknown 8,137 . IFN-γ is

believed to enhance the oxidase-independent antimicrobial pathways, possibly through

the upregualtion of iNOS 138 . It has also been shown to increase NADPH oxidase activity

in some rare variants of XL-CGD with neutrophils and monocytes characterized with

very low but detectable oxidase activity 125,139 . However, IFN-γ will not significantly

increase superoxide production in the X91 0 or autosomal recessive forms of the

disease 140 . Stem cell transplantation is a potentially curative option for CGD patients

when an HLA-matched donor is available, but graft-versus-host disease (GVHD) and

inflammation at infectious sites are the major risks associated with this treatment 141,142 .

In terms of future treatments, CGD is a promising candidate for gene therapy. Usually, X

linked female carriers with 10% - 20% normal phagocytes do not display disease

symptoms, suggesting that gene therapy is needed in only a small population of

phagocytes 123,125 . Gene therapy for CGD has been applied in cellular level studies,

murine CGD models 143 , and in some clinical trials. Oxidase activity could be restored by

gene transfer into EBV-transformed B cells and primary monocytes from patients with X-

linked or autosomal recessive forms in vitro 123 . Hopefully, the improvements in the

safety and efficacy of gene therapy could help achieve a clinically feasible cure for CGD

in the future.

37

Even when they do not have an infection, patients with CGD tend to have some

form of chronic inflammation. Characteristic granulomas, which are often times sterile,

develop in response to chronic inflammation 125,144 . Their development is not fully

understood, but it may be due to other indirect defects in the CGD innate immune system,

such as decreased degradation of phagocytosed material in phagocytes, or enhanced

production of proinflammatory cytokines. When stimulated with agonists that activate

the inflammasome, such as extracellular ATP, nigericin toxin, uric acid crystals or silica,

macrophages from CGD patients displayed enhanced caspase-1 activation, and IL-1β secretion 145 . Leukocytes from CGD patients have been found to produce enhanced IL-6

and TNF-α when stimulated with LPS or peptidoglycan 146 , and CGD neutrophils in particular, produce more IL-8 than normal neutrophils 147 , indicating an essential feedback loop in which oxidants curtail inflammation by limiting IL-8 production. These neutrophils also have delayed apoptosis, which normally serves to prevent tissue damage at sites of inflammation from necrotic lysis and release of proteases from dying neutrophils 147 . This survival defect may be due to decreased BAX, a pro-apoptotic Bcl-2 family protein that was found to be altered in CGD neutrophils. Additionally, microarray analysis of neutrophils from CGD patients revealed the upregulation of several proinflammatory genes 148 . The genes for CD11c, CD14 pattern recognition receptor,

ICAM-1, Fc γR1, Fc αR, TNF receptor, TLR5, IL-4R, CCR1, p47 phox, p40 phox, IL-8,

NRAMP1, and calgranulins A and B, were found to be constitutively upregulated in

unstimulated neutrophils from XL-CGD patients 148 . The combination of these factors

most likely contributes to the significant localized inflammation observed in the tissues of

patients.

38

Even less understood than the innate defects, are the alterations within the adaptive immune systems of CGD patients, who are prone to develop a variety of inflammatory or rheumatic diseases, such as inflammatory bowel disease and a lupus-like syndrome 149-151 . A variety of other disorders have been described in these patients for which no infectious etiology has been identified. In a report studying a national registry of 368 CGD patients, four were reported to have idiopathic/immune thrombocytopenia

(ITP), one had myasthenia gravis, and seven had chorioretinitis 126 . Obstructive lesions of the gastrointestinal and urinary tract as well as colitis/enteritis have been seen in a number of patients 149 . Many patients and their first-degree female relatives were reported to have systemic lupus erythematosus or discoid lupus erythematosus. Interestingly, lupus was significantly more common in the mothers, maternal grandmothers, and/or maternal aunts of patients with the X-linked form of the disease than it was in their counterparts in families of patients with the autosomal recessive forms 150 . These carriers generally have two populations for phagocytes, depending on which of their X chromosomes have been inactivated during lyonization 152 . Cells with inactivation of the

X chromosome carrying the defective gene will have a normal respiratory burst, whereas those with inactivation of the normal X will have an impaired respiratory burst. It is recognized increasingly that carrier status may be associated with a variety of manifestations of the disease 123,152 . To date, a convincing explanation for the autoimmunity seen in patients and carriers is lacking. However, it may have more to do with the absence of NOX2 intracellular signaling within immune cells than the absence of the respiratory burst. Studies examining lymphocytes from CGD patients have implicated possible alterations in survival. A study using 53 CGD patients and 42 age-

39

matched controls found that the patients with CGD had diminished T cell numbers 153 , and

other groups have shown specific decreases in pools of memory T and B cells 154,155 . In

addition, T cells derived from CGD patients also demonstrate decreased CD40L

156 157 expression but increased capacity to differentiate into T H17 cells , suggesting

functional changes in lymphocytes as well. Although the mechanisms are not yet

understood, it has been clearly established that oxidase deficiency in CGD patients results

in inherent changes in the innate and adaptive immune systems that are beyond the

respiratory burst.

IE3. Animal Models of CGD

The creation of animal models for CGD has helped in evaluating the roles of

NOX2-derived ROS during an immune response. The targeted disruption of the genes

for gp91 phox and p47 phox have generated oxidase deficient mice that have phenotypes

similar to those seen in X-linked CGD and the most common form of autosomal

recessive CGD, respectively 158,159 . Mice with these deletions develop lethal infections

and granulomatous inflammation similar to patients with the disease160-162 . Recently, the

characterizations of mice with deletions in p22 phox and p40 phox have also been described.

The p22 phox -deficient mouse strain, nmf333, has an immunodeficiency syndrome similar

to CGD, but also has a severe balance disorder 163 . This is most likely due to defective

NOX3 activity, which requires p22 phox . NOX3 is highly expressed in the inner ear,

including the cochlear and vestibular sensory epithelia and the spiral ganglion, and would

be rendered inactive without p22 phox 20 . However, it is still in question as to whether p22 phox is necessary for NOX3 function at least in humans, as no vestibular dysfunction

has been reported in p22 phox -deficient CGD patients 163 . There is also a mouse model for

40

p40 phox deficiency. P40 phox plays a role in NOX2 system activation, especially during Fc γ

receptor mediated phagocytosis, but has been shown to be dispensable for NOX2

activity 164 . Although human mutations in the p40 phox gene have only been described in

one case of mild colitis, neutrophils from mice with a lesion in the p40phox gene have

been shown to have some defects in NADPH oxidase activity 165 .

Gp91 phox and p47 phox knock out mice are the most commonly used murine CGD

models. Both models are characterized by an inability to eradicate infecting microbes as

well as by hyperinflammatory responses upon experimental challenge 161 . However, like

the human disease, in which differences in disease severity have been reported, these

models display slightly different inflammatory phenotypes for reasons not yet

understood 166 . In dextran sulphate sodium (DSS)-induced colitis, no significant

difference in inflammation between p47 phox deficient and wild type mice was reported,

but there was improved endothelium-dependent arteriolar dilation in gp91 phox deficient

mice, compared with that in wild type and p47 phox deficient mice 166 . Unlike gp91 phox

deficient mice, aging p47 phox deficient mice are more likely to develop proliferative

macrophage lesions and die of diffuse crystalline macrophage pneumonia. The secreted

crystalline material was found to be Ym1/Ym2 protein, which is secreted by alternatively

activated macrophages 160 . Generally, lung inflitrates in gp91 phox deficient mice have

phox large amounts of macrophages and T H1 cytokines, whereas the inflitrates of p47

deficient mice have copious amounts of T H1, T H2, and T H17 cytokines as well as

signature proteins for classical (IL-12) and alternatively (Ym1 and MMR) activated

macrophages 160 . Additionally, p47 phox deficient naïve T cells have displayed decreased survival and exhibited higher levels of intrinsic apoptosis than wild type cells in vitro 167 .

41

These cells could only be partially rescued by the addition of IL-7. However, in contrast,

gp91 phox deficient activated T cells have displayed improved survival in vitro in response to IL-2 removal 117 .

These differences may be attributed to subunit specificity. Gp91 phox is unique to the phagocyte-type oxidase, whereas the expression of p47 phox may be more promiscuous.

The subunits p47 phox and p67 phox are capable of activating NOX1 and NOX3 21,26 . In general, NOXO1 serves as the main organizer subunit for NOX1. However, in the vascular system, p47 phox may act as its replacement 168 . These findings suggest that p47 phox is not entirely specific for NOX2, and therefore p47 phox knock out mice have a more complicated disease phenotype due to possible alterations in other NOXes.

Because this study is focused on investigating the immunological contributions of the phagocyte-type oxidase, we will be using gp91 phox deficient mice.

Similar to human patients, there is also an association of CGD in animals with loss of tolerance and autoimmunity. These animals are more susceptible to develop severe arthritis and other autoimmune diseases 169,170 . Oxidase deficiency in mice breaks tolerance in models of collagen induced arthritis, resulting in an increased delayed-type hypersensitivity response and and serum levels of anti-collagen type II (CII) IgG antibodies, indicating enhanced activation of autoreactive T cells 171 . In other models of arthritis, and mice lacking p47 phox had a worsening of joint inflammation and granulomatous synovial lesions with extensive cartilage and bone erosion in response to the irritant zymosan or immune complexes 172 . Enhanced autoreactive T cells were also observed in p47 phox deficient mice with chronic experimental autoimmune encephalomyelitis (EAE) 169 . During allogeneic transplantation, oxidase deficient mice

42

exhibited exuberant migration of inflammatory cells consisting of activated T cells and

alveolar macrophages into the lungs 162 . These T cells, both resting naïve and activated

have been found to exhibit intrinsic survival defects. CGD animals also display enhanced

inflammation and an improved response to a certain selection of infectious agents. For

example, mice lacking a functional oxidase have been found to elicit a heightened

immune response against Cryptococcus neoformans and influenza infections, and are protected 173,174 . During Helicobacter pylori infection in mice, oxidase deficiency resulted in increased gastritis and spontaneous clearance of bacteria from gastric mucosa 175 . The

T cell response observed in CGD mice upon influenza or Cryptococcus infection was characterized by a heightened macrophage-driven T H1 response while in other systems

173,174,176 NOX deficiency correlated with a heightened T H17 response . These and other data imply an altered, perhaps augmented, adaptive immune response in chronic granulomatous disease.

IF. Thesis Aims

Absence of NOX2 activity in chronic granulomatous disease alters the functions of cells in both the innate and adaptive immune systems. The hyperinflammatory immune response seen in patients and animal models of the disease suggests that NOX2 plays a role in shaping the adaptive immune response. Specifically, altered T cell function in CGD would suggest that oxidase expression in T cells as well as antigen presenting cells affects the T cell phenotype. In addition to the obvious defect in the respiratory burst, oxidase deficiency in antigen presenting cells (APC) could alter their capability to activate T cells, instructing naive T cells down a certain differentiation pathway during priming. This could occur indirectly by altered cytokine production,

43

skewing T cells down a certain differentiation pathway. For example, Jendrysik et al

found that IFN-γ/LPS stimulated oxidase deficient dendritic cells (DC) secrete more IL-

12p70 than their wild type counterparts, and can subsequently induce ovalbumin-specific

OT-II lymphocytes to secrete more IFN-γ177 . Alternatively, changes in APC function could influence T cells directly during antigen presentation 178 . A group studying oxidase deficiency and arthritis susceptibility demonstrated that macrophages can mediate protection against arthritis by producing ROS that downregulates T cell activation 179,180 .

This suppression was mediated by ROS from the APC secreted directly upon the T cell 171 .

In contrast to this “APC instructive model”, the intrinsic expression of NOX2 in T cells themselves may also play a role in lineage fate decisions. The fact that adoptive transfer of CD4 + T cells from NOX2 deficient mice transferred their arthritogenic potential into recipient wild type mice would argue in favor of a direct role of NOX2 in T cells. T cells have been shown to express a functional phagocyte NADPH oxidase 116,117 , and previous studies have demonstrated that treating T cells with antioxidants promoted a

113,114 TH1 response with increased IFN-γ and decreased IL-4 while pro-oxidants biased

115 them toward a T H2 phenotype, resulting in increased amounts of IL-4, IL-5, and IL-13 .

Taken together, these findings suggest a redox dependent modulation of T helper

responses.

There are many studies that have attempted to elucidate the mechanisms

responsible for the changes observed in the CGD adaptive immune system. The

mechanism(s) by which oxidase/ROS deficiency leads to phenotypic changes in the

adaptive immune response remains unresolved. Therefore, the overall goal of this study

44

was to determine if differences in T cell responses that were previously observed are T cell inherent and can be attributed solely to the effect of oxidase deficiency in T cells, or if other oxidase deficient cells (i.e. antigen presenting cells) are creating an environment that promotes T helper skewing.

We hypothesize that chronic granulomatous disease is a disease that is both an innate and adaptive immune system disorder, in that absence of NOX2 activity alters

APC function resulting in the skewing T helper development. Furthermore, absence of

NOX2 activity within T cells alters intracellular signaling pathways that result in the skewing of their development. Thus, we predict that phagocyte type NADPH oxidase is involved in shaping both adaptive and innate immune responses in both a T cell and antigen presenting cell dependent fashion.

The first aim of this study was to determine whether or not oxidase deficient

APCs, specifically macrophages, create an environment that promotes skewing in T cells, both in vitro and in vivo. It was predicted that oxidase deficiency would alter T cell responses by changing the composition of pro-inflammatory cytokines produced by

APCs. Immunization of wild type and oxidase deficient mice with OVA in the presence of either CFA or Alum revealed altered cytokine profiles of total lymph node cells after restimulation in vitro, with oxidase deficient mice producing a more T H1-biased response, even when immunized with pro-TH2 adjuvants. Adoptive transfer of OT-II T cells into wild type or NOX2 (-/-) hosts followed by immunization also revealed altered cytokine profiles of T cells after restimulation in vitro. Analysis of APC localization demonstrated distinct differences in cytokine secretion and costimulatory molecule expression between resident peritoneal macrophages and adherent splenocytes. Upon maturation by LPS

45

stimulation, APCs from oxidase-deficient mice secreted significantly increased amounts

of pro-inflammatory cytokines such IL-6, GM-CSF and TNF-α but there were no

differences in other cytokines, including IL-10 and TGF-β, or in costimulatory molecule

expression as compared to wild type APCs. APC function was defined by the ability to

stimulate TCR-transgenic, ovalbumin-specific OT-II T cells in vitro. OT-II T cells

activated with oxidase deficient APC produced more IFN-γ in response to antigen but

secreted less IL-4 as compared to cells activated by APC from wild type mice. These

results suggest that the phagocyte NADPH oxidase is involved in the crosstalk between

the adaptive and innate immune systems by regulating cytokine production in APCs.

Previous data have demonstrated functional NOX2 expression in T cells, and the

second goal of this study was to determine if NOX2 deficient T cells are inherently

altered in their responses. We propose that NOX2 generated ROS are involved in

proximal signal transduction in T cells that is involved in their differentiation. Activation

of purified naive CD4 + T cells from NOX2-deficient mice led to augmented IFN-γ and

diminished IL-4 production and an increased ratio of expression of the T H1-specific

transcription factor T-bet versus the T H2-specfic transcription factor GATA-3, consistent with a T H1 skewing of naïve T cells. Selective inhibition of TCR-induced STAT5 phosphorylation was identified as a potential mechanism for skewed T helper differentiation. Exposure to anti-oxidants inhibited, while pro-oxidants augmented T H2 cytokine secretion and STAT5 phosphorylation, supporting the idea that these signaling changes are redox dependent. These data suggest that TCR-induced ROS generation from NOX2 activation can regulate the adaptive immune response in a T cell inherent fashion, and propose a possible role for redox signaling in T helper differentiation.

46

Taken together the findings of this study highlight the importance of crosstalk between the innate and adaptive immune systems and propose possible mechanisms for the hyperinflammatory phenotype observed in CGD patients and animal models of this disease. These results suggest that the redox status of a cell can determine the outcome of an immune response. Hopefully, this study and others will help open new avenues for an improved understanding of the role of ROS in the modulation of the immune response.

47

Chapter II. Materials and Methods

IIA. Mice

Female C57BL/6 (stock # 000664), NOX2 deficient B6.129S6-Cybb tm1Din (stock #

002365), Rag deficient B6.129S7-Rag1 tm1Mom (stock # 002216), OT-II TCR transgenic

C57BL/6-Tg (TcraTcrb)425Cbn (stock # 004194), and BALB/c (stock # 000651) mice

aged 4-8 weeks old were purchased from Jackson Laboratories. Animal care and all

experimental procedures were provided in accordance with Institutional Animal Care and

Use Committee procedures approved at the University of Maryland, Baltimore or at The

Jackson Laboratory.

IIB. Antibodies

IIB1. Antibodies used for negative selection Purified anti-CD8 (53-6.7; eBioscience), purified anti-CD16/32 (2.4G2; BD

Pharmingen), purified anti-CD44 (IM7; BD Pharmingen), purified anti-CD45R/B220

(RA3-6B2; eBioscience)

IIB2. Antibodies used in cell culture

Purified anti-CD3 (145-2C11; eBioscience), functional grade purified anti-CD28 (37.51;

eBioscience), no azide/low endotoxin (NA/LE) anti-IFN-γ (XMG1.2; BD Pharmingen),

anti-NA/LE IL-2 (S4B6; BD Pharmingen), anti-NA/LE IL-12 p40/p70 (C17.8 BD

Pharmingen)

48

IIB3. Antibodies used for flow cytometry

anti-CD4 FITC/APC (L3T4; eBioscience), anti-CD8a PE/APC (53-6.7; eBioscience),

anti-CD11b biotin/FITC/PerCP-Cy5.5 (M1/70; BD Pharmingen), anti-CD11c Alexa

Fluor 674/biotin/FITC (N418; eBioscience), purified anti-CD16/32 (2.4G2; BD

Pharmingen), anti-CD19 FITC/PerCP-Cy5.5 (1D3; BD Pharmingen), anti-CD25

APC/FITC (PC61.5, eBioscience), anti-CD28 PE (37.51; Biolegend), anti-CD44 PE

(IM7; eBioscience), anti-CD45R/B220 biotin/FITC (RA3-6B2; eBioscience), anti-

CD62L FITC (MEL-14; BD Pharmingen), anti-CD80 FITC/PE (16-10A1; Biolegend),

anti-CD86 PE/PerCP (GL-1; Biolegend), anti-CD274/PD-L1 PE (10F.9G2; Biolegend),

anti-CD275/ICOS-L PE (HK5.3; Biolegend), anti-CD278/ICOS PE (7E.17G9; BD

Pharmingen), F(ab')2 PerCP (donkey anti-mouse; Jackson ImmunoResearch), anti-FoxP3

PE-Cy7 (FJK-16s; eBioscience), anti-GATA-3 PE (TWAJ; eBioscience), anti-GM-CSF

FITC/PE (MP1-22E9; BD Pharmingen), anti-I-Ab Alexa Fluor 647 (AF6-120.1;

Biolegend), anti-IL-4R α PE (mIL4R-M1; BD Pharmingen), anti-IL-6 PE (MP5-20F3;

BD Pharmingen), anti-IL-10 APC/PerCP (JES5-16E3; BD Pharmingen), IL-12R β2

PerCP (305719; R&D Systems), anti-ROR-γt PE (AFKJS-9; eBioscience), anti-pY701-

STAT1 Alexa Fluor 488 (4a; BD Pharmingen), anti-pY705-STAT3 Alexa Fluor 488

(4/P-STAT3; BD Pharmingen), anti-pY693-STAT4 PE (38/p-Stat4; BD Pharmingen), purified anti-STAT5 (89/Stat5; BD Pharmingen), anti-pY694-STAT5 PE (47; BD

Pharmingen), anti-pY641-STAT6 PE (J71-773.58.11; BD Pharmingen), anti-T-bet

PerCP-Cy5.5 (4-B10; eBioscience), anti-TCR β FITC (H57-597; BD Pharmingen), anti-

TCR V α2 biotin (B20.1; eBioscience), anti-TCR V β5 FITC (MR9-4; BD Pharmingen), anti-TNF-α Alexa Fluor 647 (MP6-XT22; BD Pharmingen)

49

IIC. Reagents

IIC1. Media

Complete RPMI

RPMI 1640 (Gibco) 5% Heat Inactivated Fetal Bovine Serum (Atlanta Biologicals) 1 mM Sodium Pyruvate (BioWhittaker) 1 mM L-Glutamine (BioWhittaker) 1 mM Non-essential Amino Acids (BioWhittaker) 1% Penicillin/ Streptomycin (BioWhittaker) 5.5 mM β-mercaptoethanol

FACS Staining Buffer

100 ml 10X Ca- Mg- free Hank’s Balanced Salt Solution (HBSS, Gibco) 53.33 mM Potassium Chloride (KCl) 4.41 mM Potassium Phosphate monobasic (KH 2PO 4) 1379.31 mM Sodium Chloride (NaCl) 3.36 mM Sodium Phosphate dibasic (Na 2HPO 4-7H 2O) 55.56 mM D-Glucose 900 ml dH 2O 0.5% bovine serum albumin (BSA, Sigma) 0.1% sodium azide (NaN 3, Sigma) 1 mM ethylenediaminetetraacetic acid (EDTA)

Bead Separation Media

1X Hank’s Balanced Salt Solution (HBSS, Gibco) 1.26 mM Calcium Chloride (CaCl 2) anhydrous 0.493 mM Magnesium Chloride (MgCl 2-6H 2O) 0.407 mM Magnesium Sulfate (MgSO 4-7H 2O) 5.33 mM KCl 0.441 mM KH 2PO 4 4.17 mM Sodium Bicarbonate (NaHCO 3) 137.93 mM NaCl 0.338 mM Na 2HPO 4, anhydrous 5.56 mM D-Glucose 0.2% BSA (Sigma)

50

IIC2. ELISA Buffers

IL-4, IL-5 & IL-17 blocking buffer

1X Phosphate Buffered Saline (PBS) 1.06 mM KH 2PO 4 155.17 mM NaCl 2.97 mM Na 2HPO 4-7H 2O 1% BSA

IFN-γ blocking buffer

1X PBS 1% BSA 0.05% NaN 3

IL-4, IL-5 & IL-17 diluent

1X PBS 1% BSA

IFN-γ diluent

1X Tris Buffered Saline (TBS) 50 mM tris(hydroxymethyl)aminomethane (HOCH 2)3CNH 2) - Cl, pH 7.5 150 mM NaCl 0.1% BSA 0.05% Tween 20

Wash Buffer

1X PBS 0.05% Tween 20

Stop Solution

1N Sulfuric acid (H2SO 4)

51

IID. Cell Isolation

IID1. T cell negative selection

Naive CD4 + T lymphocytes were enriched by immunomagnetic depletion. Spleens and lymph nodes were harvested and gently minced to prepare single cell suspensions and red cells were removed by ACK lysis. After washing the cells were resuspended in bead separation media (HBSS + 0.2% BSA) at 20 × 10 6 cells/ml. The cells were incubated for

5 minutes on ice after the addition of 1 g/ml Fc block (2.4G2; BD Pharmingen).

Antibodies against CD8 and CD45R/B220 at 5 g/ml (53-6.7 and RA3-6B2;

eBioscience) and CD44 at 0.1 g/ml (IM7; BD Pharmingen) were added to the cells, followed by a 1 hour rotation at 4°C. After incubation, the cells were washed twice in bead separation media and added to BioMag goat anti-rat IgG conjugated magnetic beads

(Qiagen, 1 ml beads per 25 × 10 6 cells) at a concentration of 20 × 10 6 cell/ml. Following

1 hour incubation at 4°C with the magnetic beads, antibody-coated cells were removed by exposure to a magnetic field. The unbound naïve CD4 + T lymphocytes were pooled in a

50 ml conical tube and washed twice with cold complete media prior to counting and culture. Purity (> 98%) was assessed by flow cytometry (Figure II.1).

52

before negative selection after negative selection A 10 4 B 10 4

10 3 10 3 46.2 88.1

10 2 10 2

1 1 APC anti-TCR APC 10 anti-TCR APC 10

10 0 10 0 0 200 400 600 800 1000 0 200 400 600 800 1000 FSC forward scatter FSC forward scatter

C 10 4 D 10 4 6.96 8.18 0.18 0.46

10 3 10 3

10 2 10 2

PE anti-CD44 PE 10 1 anti-CD44 PE 10 1

3.2 81.7 5.26 94.1 10 0 10 0 0 1 2 3 4 10 0 10 1 10 2 10 3 10 4 10 10 10 10 10 FITC anti-CD62L FITC anti-CD62L

Figure II.1. Isolation of naïve CD4+ T cells from total spleen and lymph node

Spleens and lymph nodes were harvested and gently minced to prepare single cell suspensions and red cells were removed by ACK lysis. (A, C) CD44 and CD62L

expression were assessed upon gating on TCR+ cells in pooled spleen and lymph nodes

prior to negative selection. Naive CD4+ T lymphocytes were enriched by

immunomagnetic depletion using a cocktail of antibodies against CD8, B220, and CD44.

(B, D) Purity of isolated naïve CD4+ T cells (> 90%) was assessed by flow cytometry, by analysis of CD44 and CD62L in TCR + cells.

53

IID2. Lavage of resident peritoneal macrophages

Peritoneal cavities were washed with cold 1X PBS supplemented with 0.2% bovine

serum albumin (BSA). The collected resident peritoneal cells were pooled together and

washed two times with ice cold complete RPMI. The cells were counted and placed in

culture at 1 × 10 6 cells/ml in complete RPMI (Figure II.2 A-B).

IID3. Isolation of adherent splenocytes

Spleens were harvested and gently minced to prepare single cell suspensions and red cells

were removed by ACK lysis. After washing the splenocytes were counted and

resuspended at 15 × 10 6 cells/ml. The cells were cultured at 3 ml/well in six well plates for 4 hours at 37°C. After 4 hours, the splenocytes were washed to remove floating cells followed by overnight incubation at 37°C (Figure II.3 A-B).

54

A B WT NOX2 (-/-) 10 4 10 4 60 1.1 59 0.54

10 3 10 3

2 2 CD19 10 10

10 1 10 1

11.1 27.8 14.6 25.8 10 0 10 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CDllb CDllb CD11b

Figure II.2. Cell composition of wild type and NOX2 (-/-) peritoneal cells

Peritoneal cavities of wild type (A) and NOX2 (-/-) (B) mice were washed with cold 1X

PBS supplemented with 0.2% bovine serum albumin (BSA). Cell composition of the collected resident peritoneal cells was assessed by surface staining for CD19 and CD11b on gated live cells.

55

A B WT NOX2 (-/-) 10 4 10 4 16.2 0.73 13.2 0.78

10 3 10 3

2 2 CD19 10 10

10 1 10 1

9.12 73.9 7.08 78.9 10 0 10 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD11b CD11b

CD11b

Figure II.3. Cell composition of wild type and NOX2 (-/-) adherent splenocytes

Spleens were harvested and gently minced to prepare single cell suspensions and red cells were removed by ACK lysis. After washing the splenocytes were counted and resuspended at 15 × 10 6 cells/ml. The cells were cultured at 3 ml/well in six well plates for 4 hours at 37°C. After 4 hours, the splenocytes were washed to remove floating cells followed by overnight incubation at 37°C. Cell composition of the collected adherent splenocytes was assessed by surface staining for CD19 and CD11b on gated live cells.

56

IIE. In vitro cell cultures

IIE1. T cell cultures

Naive CD4 + T cells or total splenocytes were activated at 2 × 10 6 cells/ml in complete

RPMI on 0.5 g/ml immobilized anti-CD3 (145-2C11; eBioscience) with 1 g/ml anti-

CD28 (37.51; eBioscience) for various time points. In some scenarios, T cells were

cultured in the presence of .25 U/ml of recombinant human IL-2 (Boehringer

Mannheim/Roche). T cells cultured under T H2 skewing conditions were incubated with

50 ng/ml IL-4 (R&D Systems) as well as neutralizing antibodies to IL-12 and IFN-γ (10

g/ml from BD Pharmingen). Cells were also treated with antioxidants (10 mM N-

acetylcysteine [NAC] or 100 M apopcynin) or 1 M 2,3-Dimethoxy-1,4-

naphthoquinone (DMNQ) as a pro-oxidant during primary activation. After stimulation,

the supernatants from each culture were collected for ELISA (R&D Systems), and the

cells were harvested and extracted for mRNA isolation or analyzed by flow cytometry.

For T cell blasts, cells were initially activated at 2 × 106 cells/ml in complete RPMI on 5

g/ml immobilized anti-CD3 (145-2C11; eBioscience) with 1 g/ml anti-CD28 (37.51;

eBioscience) for 48 hours. After stimulation, the blasts were washed and grown for

approximately 3 days in complete RPMI supplemented with 50 U/ml of recombinant

human IL-2 (Boehringer Mannheim/Roche). After expansion in IL-2, the blasts were

washed and restimulated 1 × 10 6 cells/ml for 8 hours with 0.5 g/ml immobilized anti-

CD3. T cell blasts cultured in the presence of antioxidants were exposed throughout

activation and subsequent expansion in IL-2 prior to stimulation. After stimulation, the

supernatants from each culture were collected for analysis by ELISA.

57

IIE2. Antigen presenting cell cultures

Adherent splenocytes, and resident peritoneal cells were cultured for 4 or 24 hours in the presence or absence of 100 ng/ml LPS ( E. coli O111:B4; List Biological Laboratories,

concentration previously determined). After incubation, supernatants from each culture

were collected for analysis by multiplex bead array, and the cells were harvested for

analysis by flow cytometry.

IIE3. OT-II co-cultures

Adherent splenocyte APC were prepared by washing the cells from minced spleens and

treating with ACK lysing buffer. After RBC lysis, the splenocytes were washed,

resuspended at at 5 x 10 6 cells/ml and cultured in six well plates (3 ml per well) for four

hours. After incubation, the splenocytes were washed to remove any floating cells and

then incubated overnight. Peritoneal cells were cultured with 100 ng/ml LPS at 1 x 10 6

cells/ml in six well plates (2 ml per well) overnight prior to use as APC. OT-II CD4 + T

cells specific for chicken ovalbumin 323-339 (OVA 323-339 ) peptide presented on the MHC

class II molecule I-Ab from OT-II TCR transgenic C57BL/6-Tg (TcraTcrb)425Cbn mice

were isolated by negative selection from spleen and lymph nodes. The cells were cultured

with adherent splenocytes or LPS-primed resident peritoneal cells prepared the night

before from wild type C57BL/6 or NOX2 deficient B6.129S6-Cybb tm1Din mice. APC

were loaded with 1 g/ml OVA 323-339 peptide (New England Peptide) for 1 hour prior to

addition of T cells. Following peptide loading, OT-II T cells were added to APC at a 5 to

1 ratio of approximately 1 × 10 6 CD4 + T cells cells to 2 × 10 5 APC. After 48 hour

incubation, the supernatants from each culture were collected for analysis by ELISA.

58

IIF. In vivo experiments

IIF1. Immunizations

Three wild type C57BL/6 mice and three knock out B6.129S6-Cybb tm1Din mice were

immunized and euthanized after 10 days. For each group, one mouse was immunized

subcutaneously with 100 g OVA protein (albumin from chicken egg white, Sigma) in

CFA, one mouse intraperitoneally with 100 g OVA in Alum, and one mouse was left unimmunized. Lymph nodes were harvested from the mice after 10 days. For 48 hours, total lymph node cells were incubated at a concentration of 2 × 10 6 cells/ml in complete

RPMI with 25 g/ml OVA protein (as previously determined by titration). After incubation, the supernatants from each culture were collected for analysis by ELISA.

IIF2. Adoptive transfers

OT-II CD4 + T cells specific for chicken ovalbumin 323-339 peptide presented by the

MHC class II molecule I-Ab from OT-II TCR transgenic C57BL/6-Tg (TcraTcrb)425Cbn mice were isolated by negative selection from the spleen and lymph nodes. The cells were adoptively transferred by tail vein injection into three wild type C57BL/6 and three

NOX2 deficient B6.129S6-Cybb tm1Din mice. Approximately 2.5 × 10 6 cells were given to each animal. 24 hours after transfer the mice were immunized. For each group, one mouse was immunized subcutaneously with 100 g OVA in CFA, one mouse intraperitoneally with 100 g OVA in Alum, and one mouse was left unimmunized. After approximately 4 to 5 days, the mice were euthanized and the lymph nodes were harvested. Total lymph node cells were incubated at a concentration of 2 × 10 6 cells/ml

59

in complete RPMI with 100 g/ml OVA 323-339 peptide for 48 hours. After incubation, the supernatants from each culture were collected for analysis by ELISA.

IIF3. Tritiated thymidine proliferation assay

Total lymph node cells isolated from immunized animals were cultured at a concentration of 2 × 10 6 cells/ml in AIM V serum free medium in 96 well plates with and without 250

g/ml OVA protein or 100 g/ml OVA 323-339 peptide for general immunizations and

adoptive transfers, respectively. After 48 hours, the cells were pulsed with 1 Ci/well of

[3H] thymidine for 18 hours. After incubation the cells were harvested on a cell

harvester. Filter membranes were dried overnight and radioactivity was counted in a

Packard Matrix 9600 scintillation counter.

IIG. Flow Cytometry

Following harvest and washing in FACS buffer, cells were surface stained for 25 minutes

at 4°C with 5 g/ml Fc Block (2.4G2; BD Pharmingen) and surface staining antibodies

(approximately 1 l per million cells). After surface staining, the cells were washed and resuspended at 2 × 10 6 cells/ml in FACS buffer with 5 g/ml propidium iodide (PI) prior to flow cytometric analysis. Cells undergoing fixation were also surface stained with a live/dead fixable marker (LIVE/DEAD Fixable Dead Cell Stain Kit; Invitrogen). After incubation, the cells were washed twice and were fixed with 4% paraformaldehyde for 30 minutes at 4°C. For intracellular staining for transcription factors, following fixation, the cells were washed and then permeabilized in 80% methanol for 30 minutes at 4°C followed by incubation for 45 minutes in FACS buffer and antibody. For intracellular cytokine staining, following fixation, the cells were washed twice in FACS buffer

60

containing 0.5% Tween 20, followed by incubation for 45 minutes in FACS buffer +

0.5% Tween 20 and antibody. Positive staining and mean fluorescence intensity was

determined by flow cytometry gating on viable T cells and analysis using FlowJo

(Ashland, OR). Mean fluorescence intensity was calculated as the difference in

stimulated samples from unstimulated controls.

IIH. CFSE cell proliferation analysis

Prior to culture, purified T cells were washed in PBS and resuspended at a concentration

of 10 × 10 6 cells/ml in PBS. Carboxy-fluorescein diacetate succinimidyl ester (CFDASE,

Molecular Probes, Eugene OR) was added to the cell suspension at a final concentration of 0.25 M, and the cells were incubated at 37°C for 15 minutes. CFDASE is taken up

by the cells into the cytoplasm where it is deacetylated to carboxy-fluorescein

succinimidyl ester (CFSE). After incubation, the CFSE-labeled cells were washed two

times with cold complete RPMI to quench residual CFDASE followed by resuspension at

2 × 10 6 cells/ml for in vitro culture. The equal division of CFSE into daughter cells allows the determination of the degree of proliferation as well the proliferative cycle for each cell population. Proliferation was assessed after 48 and 72 hours by flow cytometry.

III. ELISA

Supernatants collected from cell cultures were analyzed in triplicates by ELISA using

Duosets for IFN-γ, IL-4, IL-5, and IL-17 from R&D Systems following the manufacturer’s instructions. Plates were developed using streptavadin-HRP and TMB substrate reagents (BD OptEIA), and read at 450 nm on a plate reader.

61

IIJ. Quantitative PCR

For analysis of NOX2 expression, CD4 + and CD8 + T cells were enriched by positive selection from spleen or lymph node by magnetic bead isolation (BioMag) using antibodies against CD4 and CD8 at 5 g/ml (53-6.7 and GK1.5; eBioscience), while for thymus, double negative thymocytes were isolated by negative selection using antibodies against CD4 and CD8 at 5 g/ml to remove mature thymocytes. Aortas were removed by micro-excision of the entire aorta from the heart to the iliac bifurcation. The aortas were then trimmed of adherent fat and were gently minced to prepare single cell suspensions.

Total RNA was isolated from cells or whole tissue using the NucleoSpin RNA II Total

RNA Isolation kit (Macherey-Nagel). Reverse transcription was carried out using the

AffinityScript RT-PCR kit (Agilent). mRNA levels in cultured cells were quantified by qRT-PCR (Agilent Brilliant SYBR Green-based qPCR) on a 7900HT real-time thermal cycler (ABI) using primers previously described 181-183 . The following primer sets were used in these studies: GATA-3, forward (5’-GAGGTGGTGTCTGCATTCCAA-3’) and reverse (5’-TTTCACAGCACTAGAGACCCTGTTA-3’); IL-2, forward (5'-

CCTGAGCAGGATGGAGAATTACA-3') and reverse (5'-

TCCAGAACATGCCGCAGAG-3'); IL-4, forward (5'-

GCATTTTGAACGAGGTCACAGG-3') and reverse (5'-

TATGCGAAGCACCTTGGAAGC-3'); IL-4R α, forward (5'-

ATGTTCTTCGAGTTCTCTGAAAACC-3') and reverse (5'-

TCTGATTGGACCGGCCTATT-3'); NOX2, forward (5'-

ACCTTACTGGCTGGGATGAA-3') and reverse (5'-TGCAATGGTCTTGAACTCGT-

3'); T-bet, forward (5‘-GTTCCCATTCCTGTCCTTC-3’) and reverse (5’-

62

CCTTGTTGTTGGTGAGCTT-3’). Relative mRNA expression levels were calculated based on the delta CT method.

IIK. Statistical Analysis

Statistical significance was analyzed using student’s T-test. Conditions were deemed significantly different if p< 0.05.

63

Chapter III. APC expression of NOX2 regulates T helper differentiation through pro-inflammatory cytokine production

IIIA. Introduction

Phagocytic cells, such as macrophages and neutrophils are the first line of defense against bacterial and fungal infections. In addition to their roles in innate immunity, certain phagocytic cells are necessary for the initiation of adaptive immune responses by serving as professional APCs. Chronic granulomatous disease is a rare clinical entity that affects phagocytes of the innate immune system and is characterized by a greatly increased susceptibility to severe infections from a relatively narrow spectrum of bacteria and fungi such as Staphylococcus aureus and Aspergillus fumigatus . It is also

characterized by a common set of inflammatory complications, most notably by the

formation of granulomas that develop in response to chronic inflammation. The disease

is a result of genetic mutations in the subunits of the phagocyte type NADPH oxidase, a

membrane-bound enzyme complex that generates oxygen free radicals used in the

destruction of invading pathogens.

In seeming contradiction to the dysfunction seen in the innate immune system,

CGD patients often display a hyperinflammatory phenotype in their adaptive immune

systems. They are known to present with autoimmune phenomena, such as Crohns-like

disease, juvenile rheumatoid arthritis and lupus-related syndromes 149 . In addition to

similar sensitivity to autoimmune disease 169 , animal models of CGD also display a

heightened response and enhanced clearance to certain other infectious agents including

Cryptococcus neoformans , influenza virus , and Helicobacter pylori 173-175 . Thus,

64

immunodeficiency in CGD is sometimes associated with enhanced protective immune

responses.

The local cytokine environment during T cell priming and the consequent

activation of lineage-specific transcription factors are two key elements that control

helper T cell differentiation. These factors can be determined by APC as well as other

cells present in the immune microenvironment. There are previous reports that oxidase

deficiency in antigen presenting cells may alter their capability to activate T cells 179 .

NOX2-derived ROS may indirectly regulate T cell differentiation by regulating the

composition of pro-inflammatory cytokines produced by the APC. ROS have been found

to play a role in the production of TNF-α, IL-1β, and IL-12p70 production, in that NOX2

deficient APC produced significantly increased levels of these cytokines in response to

stimulation with M. tuberculosis or S. aureus 177 . These APCs subsequently induced high levels of IFN-γ and IL-17 in T cells after activation.

It has also been suggested that NOX2 deficient APC may affect T cell differentiation directly during antigen presentation 184 . ROS produced by APC during antigen presentation has been found to determine T cell surface redox levels by altering thiols at the T cell surface 171 . These changes in surface redox levels are thought to

downregulate T cell activation 179 . T cells primed by oxidase deficient macrophages were

found to have enhanced autoreactivity resulting in increased production of IL-2 in vitro

and increased incidence in arthritis when transferred into mice 171 . With regard to other

APC types, NOX2 has been shown to regulate phagosomal pH during cross

presentation 178 , and CGD dendritic cells (DC) presenting OVA peptide induced higher

production of IFN-γ from TCR transgenic OT-II T cells in vitro than did wild type DC 177 .

65

The influence of B cell-generated ROS on T activity is still being identified, but many

•- studies in the past have demonstrated the generation of O 2 in Epstein-Barr virus (EBV)- transformed B cells after stimulation by TNF-α, IL-1β, and LPS; and more recent studies have demonstrated that H 2O2 can impair the capacity of B cells to process and present peptides to specific T cell clones 185,186 .

The discovery of NADPH oxidase expression in numerous types of non- phagocytic cells has demonstrated that ROS can function as signaling molecules with important regulatory functions controlling cell division, migration, apoptosis, and cytokine production. While oxidase activation in the respiratory burst leads to generation

•- of high levels of O 2 , low levels of ROS can act as second messengers that affect signaling molecules, gene expression and/or cell proliferation. Many reports have described oxidase generated ROS in response to receptor ligation by various stimuli 48,58,61,72,185,187 . These stimuli include angiotensin II, IL-1β, LPS, and EGF to name a few. Thus, NOX2-generated ROS could be involved in specific signal transduction pathways within APCs.

It is likely that oxidase deficiency results in cell intrinsic changes in APCs that subsequently affect their crosstalk with T cells, either directly or indirectly by changes in cytokine production, and thus skewing the adaptive immune system to produce a more inflammatory response. Therefore, the overall goal of this study was to understand how oxidase deficiency affects APC function and to identify the overall role APCs play in the inflammatory anomalies observed in CGD. Using wild type and oxidase deficient

(NOX2 (-/-)) mice, this study investigated the changes in APCs that could contribute to downstream changes in T helper modulation. By immunization of wild type and NOX2 (-

66

/-) mice, we were able to study the NOX2 deficient adaptive response in a biologically relevant immune microenvironment. Skewing the response with the use of adjuvants allowed for elucidation of the effects of oxidase deficiency on the redundancy of the cytokine network.

Immunization of NOX2 deficient mice resulted in increased T H1 cytokines but decreased T H2 cytokines upon in vitro restimulation of lymph node cells. Adoptive transfer of OT-II T cells into wild type or NOX2-deficient hosts followed by immunization with OVA in the presence of either CFA or Alum also revealed altered cytokine profiles from T cells after restimulation in vitro. Further analysis of APCs focused upon resident peritoneal cells and adherent splenocytes. CD11b + macrophages, were found to be the APC most affected by oxidase deficiency, and oxidase deficient resident peritoneal cells and adherent splenocytes showed enhanced secretion of IL-6 and

TNF-α after LPS stimulation in vitro. There were also selective increases in GM-CSF,

KC and MIP-1α production that were dependent on the localization and source of APC.

APC function was defined by the ability to stimulate TCR-transgenic, ovalbumin-specific

OT-II T cells in vitro. OT-II T cells incubated with oxidase deficient APC produced more IFN-γ and IL-17 upon antigen stimulation. These results suggest a role for the phagocyte NADPH oxidase in shaping adaptive and innate immune responses by changes in cytokine production that affect the local immune microenvironment.

67

IIIB. Results

IIIB1. Oxidase deficient mice mount a robust T H1 response during

immunization

CGD is characterized by an unusual predisposition to infection with bacteria and

fungi. However, CGD patients not only suffer from recurrent infections, but also present

with inflammatory, non-infectious conditions, which sometimes appear to be driven by a

heightened T H1 response. T H1 skewing of oxidase deficient T cells has been

demonstrated in vitro, but not yet in vivo. In vivo priming by immunization was

performed on wild type and NOX2 (-/-) mice in order to ascertain whether oxidase

deficiency skews the adaptive response in vivo toward a TH1 response regardless of adjuvant type. Wild type and NOX2 (-/-) mice were immunized with the protein antigen ovalbumin (OVA) in complete Freund's adjuvant (CFA), to induce a T H1 response, or

OVA in Alum, to induce a T H2 response. Ten days after immunization, whole lymph node single cell suspensions were restimulated in vitro with OVA protein and supernatants were collected for analysis by ELISA. Cells were also pulsed with 3H- thymidine, in order to evaluate the magnitude of the proliferative response after immunization. Total lymph node cells from NOX2 (-/-) mice immunized with OVA and

Alum had enhanced proliferative capacity as compared to wild type mice (Figure III.1

A). NOX2 (-/-) lymph node cells also produced significantly increased levels of IFN-γ upon in vitro restimulation, regardless of adjuvant type (Figure III.1 B).

68

A B 80000 30000 WT WT * NOX2 (-/-) * NOX2 (-/-) 60000 20000 * 40000 (pg/ml) γ 10000 *

20000 IFN- Proliferation(cpm)

0 0 unimmunized OVA OVA unimmunized OVA OVA CFA Alum CFA Alum C D 45 800 WT WT NOX2 (-/-) NOX2 (-/-) 600 30

400

15 IL-5(pg/ml) IL-17 (pg/ml) * * 200

0 0 unimmunized OVA OVA unimmunized OVA OVA CFA Alum CFA Alum

(-/-) Figure III.1. Immunization of wild type and NOX2 mice using pro-TH1 and pro- TH2 adjuvants

Wild type (hatched bars ) NOX2 (-/-) (closed bars ) mice were immunized either subcutaneously with 100 g OVA protein in CFA or intraperitoneally with 100 g OVA protein in Alum. After 10 days the mice were sacrificed and whole lymph node was incubated 48 hours with 25 g/ml OVA protein. At the end of 48 hours, supernatatants

were collected for analysis of IFN-γ (B) , IL-5 (C) , and IL-17 (D) by ELISA, and the cells

were pulsed with 3H-thymidine for 18 hours to assess proliferation (A) . (ELISA n = 4

separate experiments, 3H thymidine n = 3 triplicates, significantly different from wild

type * = p<0.05).

69

However, total lymph node cells from NOX2 (-/-) mice immunized with OVA in Alum or

OVA in CFA produced significantly decreased levels of IL-5 and IL-17, respectively, as compared to cells from wild type mice (Figure III.1 C, D). IL-4 levels were found to be below the limit of detection. In summary, oxidase deficient mice elicit a heightened T H1 response upon immunization with the pro-TH2 adjuvant Alum, as well as the pro-TH1 adjuvant CFA, as compared to wild type mice, suggesting that the oxidase deficient immune microenviornment is inherently more T H1 skewed.

IIIB2. Oxidase deficient APCs promote T H1 polarization in vivo

In order to address the question of whether oxidase deficient APC are creating an

environment that promotes T H1 polarization in vivo, TCR transgenic OT-II T cells were adoptively transferred into wild type and oxidase deficient mice. Following the transfer, the mice were immunized with the OVA in CFA or OVA in Alum and T cell responses were analyzed as before. Similar to total lymph node cells from oxidase deficient mice,

OT-IIs that were adoptively transferred into NOX2 (-/-) mice produced increased IFN-γ but

decreased IL-4 upon in vitro restimulation with with OVA 323-339 peptide (Figure III.2

B,C), and exhibited increased proliferation when taken from hosts immunized with OVA

in Alum (Figure III.2 A). This skewing occurred regardless of adjuvant type. However,

unlike the previous immunization experiments, OT-IIs that were adoptively transferred

into NOX2 (-/-) mice immunized with Alum had enhanced IL-17 secretion and no detectable levels of IL-5, bringing into question the altered status of NOX2 (-/-) T cells

(Figure III.2 D).

70

A B 60000 60000 WT WT * NOX2 (-/-) NOX2 (-/-) * * 40000 40000 (pg/ml) γ 20000 * 20000 IFN- Proliferation(cpm) * 0 0 unimmunized OVA OVA unimmunized OVA OVA CFA Alum CFA Alum C D 180 600 WT WT NOX2 (-/-) NOX2 (-/-) *

120 400

60 200 IL-4 (pg/ml) IL-4 * (pg/ml) IL-17 * 0 0 unimmunized OVA OVA unimmunized OVA OVA CFA Alum CFA Alum

Figure III.2. Adoptive transfer of OT-IIs into wild type and NOX2 (-/-) mice

OT-II T cells were adoptively transferred into wild type (hatched bars ) and NOX2 (-/-)

(closed bars ) mice. The mice were immunized 24 hours later either subcutaneously with

100 g OVA protein in CFA or intraperitoneally with 100 g OVA protein in Alum.

After 4 days the mice were sacrificed and whole lymph node cells incubated for 48 hours

with 100 g/ml OVA peptide. At the end of 48 hours, supernatatants were collected for

analysis of IFN-γ (B) , IL-4 (C) , and IL-17 (D) by ELISA, and the cells were pulsed with

3H-thymidine for 18 hours to assess proliferation (A) . (ELISA n = 4 separate

experiments, 3H thymidine n = 3 triplicates, significantly different from wild type * =

p<0.05).

71

Thus, NOX2 deficient APC promote T H1 differentiation of T cells, and under conditions of antigenic stimulation, these APC affect oxidase deficient T cells differently than wild type T cells.

IIIB3. CD11b + antigen presenting cells are most affected by oxidase deficiency

APC consist of dendritic cells, B cells and macrophages. Macrophages have the highest amount of NOX2 generated ROS and when compared to other APC such as dendritic cells, these cells have been implicated to be the most affected by oxidase deficiency. We began by studying resident peritoneal cells, since these cells best represented tissue resident macrophages. Adherent splenocytes were also examined because these cells are more likely to have the capacity to present antigen and activate

CD4 + T cells. However, because both cell types are mixed populations, intracellular cytokine staining was performed in order to confirm that the main APCs responsible for the changes in cytokine production were the CD11b + myeloid-derived cells, which are thought to be macrophages (Figure III.3 and Figure III.4). We did not see any major changes in cytokine production by CD19 +/CD11b - peritoneal cells (Figure III.3) or in

CD11b - adherent splenocytes (Figure III.4). As shown in Figure II.2 and Figure II.3 of the methods, surface staining of wild type and NOX2 (-/-) resident peritoneal cells and adherent splenoytes revealed that the cell composition of these populations were similar between the two types of mice. Taken together, these data suggest that overall macrophages appear to be more impacted by oxidase deficiency than B cells.

72

Figure III.3. CD11b + resident peritoneal cells are the predominant producers of pro-inflammtory cytokines

Peritoneal cells were cultured with 100 ng/ml LPS for 8, 16, or 24 hours in the presence of brefeldin-A (BFA). After incubation, the cells were harvested and underwent intracellular cytokine staining for TNF-α, and IL-6. Cells were analyzed by flow cytometry as described in the Methods, with gating based upon forward and side scatter profiles and surface staining for CD11b and CD19.

73

74

AB 100 100

80 80

60 60 peritoneal cells 40 40 % of Max % of Max % of

20 20

0 0 unstained 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD11b + PE anti-TNF-α PE anti-IL-6 CD11b - CD 100 100

80 80

60 60 adherent spleen 40 40 % of Max of % Max of %

20 20

0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 PE anti-TNF-α PE anti-IL-6

Figure III.4. CD11b + resident peritoneal cells and adherent splenocytes are the predominant producers of pro-inflammtory cytokines

Peritoneal cells (A-B) and adherent splenocytes (C-D) were cultured with 100 ng/ml LPS for 8 hours in the presence of brefeldin-A (BFA). After incubation, the cells were harvested and underwent intracellular cytokine staining for TNF-α (A, C) and IL-6 (B,

D). Cells were analyzed by flow cytometry as described in the Methods, with gating based upon forward and side scatter profiles and surface staining for CD11b on viable cells.

75

IIIB4. Characterization of wild type and NOX2 deficient antigen presenting cells

The pro-inflammatory T cell phenotype described in patients with CGD and

animal models of the disease could be attributed to changes in antigen presenting cell

function. This in turn could be skewing the adaptive immune system toward a T H1 response. Changes in APC function were initially observed by analysis of cytokine production. Resident peritoneal cells and adherent splenocytes isolated from wild type and oxidase deficient mice were incubated for 24 hours with or without the TLR4 agonist

LPS. Supernatants from these cells were then collected and analyzed by multiplex bead array, which screened for a variety of different cytokines and chemokines. After LPS challenge, oxidase deficient peritoneal cells and adherent splenocytes both demonstrated greatly enhanced secretion of IL-6 and TNF-α (Figure III.5). There were also selective

increases in GM-CSF production in NOX2 (-/-) peritoneal cells (Figure III.6 A), as well as increases in KC (murine IL-8) and MIP-1α production in NOX2 (-/-) adherent splenocytes

(Figure III.6 E, F).

Because of dramatic changes observed in the production of IL-6, it was possible that there would also be changes in TGF-β production. These cytokines together would

+ promote the differentiation of CD4 T cells into T H17 cells. Increased levels of IL-17 have been implicated to be heightened in CGD mice in other studies. However, as shown in Figure III.7, we did not see any detectable differences in the total levels of TGF-β

secreted from peritoneal cells from wild type and NOX2 (-/-) mice.

76

A B 600000 * 4000

400000 3000 (pg/ml)

α 2000

IL-6 (pg/ml) IL-6 200000 TNF- 1000

0 0 WT NOX2 (-/-) WT NOX2 (-/-)

C D 150 400 * * 300 100 (pg/ml) 200 α

IL-6 (pg/ml) IL-6 50 TNF- 100

0 0 (-/-) WT NOX2 (-/-) WT NOX2

Figure III.5. Enhanced production of IL-6 and TNF-α from NOX2 (-/-) resident peritoneal cells and adherent splenocytes after LPS challenge in vitro

Peritoneal cells (A-B) and adherent splenocytes (C-D) from wild type ( open bars ) and

NOX2 (-/-) (closed bars ) mice were cultured with 100 ng/ml LPS (as previously determined) for 24 hours. Supernatants were collected at 24 hours for for analysis of IL-6

(A, C) and TNF-α (B, D) by multiplex bead array (n = 6 replicates, significantly different from wild type * = p<0.05).

77

A B C 150 9000 3000 *

100 6000 2000 (pg/ml) α

50 KC(pg/ml) 3000 1000 MIP-1 GM-CSF (pg/ml) GM-CSF

0 0 0 WT NOX2 (-/-) WT NOX2 (-/-) WT NOX2 (-/-)

D E F 100 150 2400 * 75 * 100 1600 (pg/ml) 50 α

KC(pg/ml) 50 800 MIP-1

GM-CSF (pg/ml) GM-CSF 25

0 0 0 (-/-) (-/-) WT NOX2 WT NOX2 WT NOX2 (-/-)

Figure III.6. Selective changes in cytokine production from wild type and NOX2 (-/-) APCs after in vitro LPS stimulation are dependent on tissue localization

Peritoneal cells (A-C) and adherent splenocytes (D-F) from wild type ( open bars ) and

NOX2 (-/-) (closed bars ) mice were cultured with 100 ng/ml LPS for 24 hours.

Supernatants were collected at 24 hours for for analysis of GM-CSF (A, D) , KC (B, E)

and MIP-1α (C, F) by multiplex bead array (n = 6, significantly different from wild type

* = p<0.05).

78

160 WT NOX2 (-/-) 120

(pg/ml) 80 β TGF- 40

0 4 hrs overnight

Figure III.7. Equivalent amounts of TGF-β from wild type and NOX2 (-/-) resident peritoneal cells after LPS challenge in vitro

Peritoneal cells from wild type ( open bars ) and NOX2 (-/-) (closed bars ) mice were cultured with 100 ng/ml LPS for 24 hours. The supernatants were collected at 24 hours for for analysis of TGF-β production by ELISA (n = 3 duplicates from 3 separate experiments).

79

Aside from the local cytokine environment, the interaction of costimulatory

molecules between T cells and APCs is also an important contributing factor to T helper

lineage differentiation, and can shift the balance between T H1 and T H2. development.

Changes in expression of co-stimulatory molecules were examined in wild type and

oxidase deficient APC by surface staining after LPS challenge in vitro. As demonstrated

in Figure III.8 and Figure III.9, surface staining for class II MHC, CD80, CD86, PD-L1

and ICOS-L on resting and LPS stimulated resident peritoneal macrophages and adherent

splenocytes revealed that there were no detectable differences in the expression of class II

MHC, CD80, CD86, and PD-L1 between the wild type and knock out cells. However,

there was a significant decrease in the expression of ICOS-L in oxidase deficient

peritoneal cells, but not in adherent splenocytes (Figure III.8 D). Costimulation through

ICOS-L has been shown to augment the generation of T H2 responses, and ICOS-L knock

out mice produce lower levels of T H2 (IL-4 and IL-5) and anti-inflammatory (TGF-β and

IL-10) cytokines, but higher T H1-related (IFN-γ, IL-12p40, and IL-23) and

proinflammatory (IL-6 and TNF-α) cytokines 188 . Taken together these results suggest that NOX2 deficient APC primarily exert their immunomodulatory effects through altered cytokine production, but also through alterations in ICOS-L expression, and that these immunomodulatory effects are dependent on APC type and tissue localization.

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A B C 450 300 WT 1000 WT WT * NOX2 (-/-) NOX2 (-/-) NOX2 (-/-) 750 300 200

500

CD80 MFI CD80 150 MFI CD86 100 250 MFI II MHC

0 0 0 unstim 24hrs unstim 24hrs unstim 24hrs LPS LPS LPS D E 200 3000 WT WT NOX2 (-/-) NOX2 (-/-) 150 2000

100 * 1000 PD-L1 MFI PD-L1 ICOS-L MFI ICOS-L 50

0 0 unstim 24hrs unstim 24hrs LPS LPS

Figure III.8. Analysis of costimulatory molecule expression in peritoneal APCs from wild type and NOX2 (-/-) mice

Resident peritoneal cells from wild type ( open bars ) and NOX2 (-/-) (closed bars ) mice were cultured for 24 hours with or without 100 ng/ml LPS. Following incubation, the cells were harvested and surfaced stained for CD80, CD86, PDL-1, MHC class II, and

ICOS-L. Cells were gated based upon forward and side scatter profiles, live/dead discrimination with a live/dead fixable marker, and on CD11b + cells (n = 3 separate

experiments, significantly different from wild type * = p<0.05).

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A BC 300 1500 300 WT WT WT NOX2 (-/-) NOX2 (-/-) NOX2 (-/-)

200 1000 200

CD80MFI 100 CD86MFI 500 100 MHC II MFI MHCII

0 0 0 unstim 24hrs unstim 24hrs unstim 24hrs LPS LPS LPS DE 200 2000 WT WT NOX2 (-/-) NOX2 (-/-) 150 1500

100 1000 PD-L1 MFI PD-L1 ICOS-L MFI ICOS-L 50 500

0 0 unstim 24hrs unstim 24hrs LPS LPS

Figure III.9. Analysis of costimulatory molecule expression in adherent splenocyte APCs from wild type and NOX2 (-/-) mice

Adherent splenocytes from wild type ( open bars ) and NOX2 (-/-) (closed bars ) mice were cultured for 24 hours with or without 100 ng/ml LPS. Following incubation, the cells were harvested and surfaced stained for CD80, CD86, PDL-1, MHC class II, and ICOS-

L. Cells were gated based upon forward and side scatter profiles, live/dead discrimination with a live/dead fixable marker, and on CD11b+ cells (n = 3 separate experiments, significantly different from wild type * = p<0.05).

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IIIB5. Oxidase deficiency results in changes in the cross-talk between T cells

and antigen presenting cells during antigenic stimulation

ROS have been previously shown to exert effects on antigen processing and oxidation of target structures during the immunological synapse 171,184,189 . Recent data suggest that NOX2 deficient mice have accelerated antigen degradation due to an increased acidic environment within DC phagosomes 178 . We next examined whether oxidase deficiency in APC could influence T cells by changes in the APC during antigen presentation. APC function was defined by the ability to stimulate TCR-transgenic, ovalbumin-specific OT-II T cells in vitro. OT-II T cells incubated with oxidase deficient

APC had similar proliferative capacity as compared to OT-IIs incubated with wild type

APC (Figure III.10). However, these cells were found to produce more IFN-γ but less

IL-4 when stimulated with antigen presented on NOX2 deficient adherent splenocytes

(Figure III.11 A, C). Unexpectedly, in contrast to those activated with adherent

splenocytes, OT-IIs stimulated with antigen presented on NOX2 deficient peritoneal cells

produced significantly decreased IFN-γ, but increased IL-17 (Figure III.11 D, E). These

data suggest that during presentation of antigen, changes in NOX2 deficient APC affect

the naïve T cells they interact with. However, these changes vary depending on APC

type and tissue localization.

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peritoneal cells adherent spleen 100 100 unstim WT 80 80 NOX2 (-/-)

60 60 OVA 323-339 40 40

20 20

0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4

peritoneal cells adherent spleen 100 100 unstim WT 80 NOX2 (-/-) 80

60 60 anti-CD3 40 40

20 20

0 0 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4

OT-II CFSE Dilution

Figure III.10. CFSE dilution of OT-II CD4+ T cells co-cultured with APCs from wild type and NOX2 (-/-) mice

CFSE loaded OT-II CD4 + T cells isolated by negative selection were incubated with wild

(-/-) type or NOX2 APC with OVA 323-339 peptide or soluble anti-CD3 for 60 hours. After incubation, the proliferation was analyzed by flow cytometry as described in the

Methods.

84

A IL-4 B IL-17C IFN-γ 150 300 80000 * 60000 100 200

(pg/ml) 40000 * γ 50 100 IL-4 (pg/ml) IL-4 IL-17 (pg/ml) IL-17 IFN- 20000

0 0 0 WT NOX2 (-/-) WT NOX2 (-/-) WT NOX2 (-/-) D IL-17 E IFN-γ 1600 1500 *

1200 1000

800 (pg/ml)

γ * 500 IL-17 (pg/ml) IL-17 400 IFN-

0 0 WT NOX2 (-/-) WT NOX2 (-/-)

Figure III.11. Cytokine production from OT-II CD4+ T cells co-cultured with APCs from wild type and NOX2 (-/-) mice

OT-II CD4 + T cells isolated by negative selection were incubated with wild type (open bars ) or NOX2 (-/-) (closed bars ) adherent splenocytes (A-C) or peritoneal cells (D-E) with

OVA 323-339 peptide for 72 hours. After incubation, supernatants were collected and analyzed for IL-4 (A) , IL-17 (B, D) and IFN-γ (C, E) by ELISA (n = 6 separate experiments, significantly different from wild type * = p<0.05).

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IIIC. Discussion

CD4 + T cells cannot recognize, and therefore cannot react to free antigen. They

will only react to antigen that has been processed and presented on class II MHC by

antigen presenting cells. The interactions between T cells and APCs as well as the nature

of the local immune microenvironment drive T helper fate decisions. The results of this

report demonstrate that the loss of NOX2 generated ROS, alters functions in APCs,

+ possibly promoting the subsequent polarization of naïve CD4 T cells toward a T H1 or

TH17 phenotype during primary activation. These changes within APCs affect the cross talk during priming of T cells, and are associated with the enhanced production of pro- inflammatory cytokines, as well as changes in the expression of the costimulatory molecule ICOS-L.

The goal of this study was to determine whether or not oxidase deficient APCs create an environment that promotes skewing in T cells, both in vitro and in vivo.

Immunization of wild type and oxidase deficient mice with OVA protein in the presence of either CFA or Alum revealed altered cytokine profiles of total lymph node after restimulation in vitro, with oxidase deficient mice producing a more T H1-biased response,

even when immunized with the pro-TH2 adjuvant Alum. Adoptive transfer of OT-II T

cells into wild type or NOX2 (-/-) hosts followed by immunization also revealed altered

cytokine production from T cells after restimulation in vitro. The cytokine profiles of

these OT-II T cells were similar to those from total lymph node cells from wild type and

NOX2 (-/-) mice, implicating oxidase deficient APCs as drivers of T helper skewing.

There is a possibility that the in vitro cytokine production from the adoptive transfers was in part due to endogenous T cells of the host responding to immunization. However,

86

there are some factors which may rule them out and suggest that the response was

primarily specific to the adoptively transferred OT-IIs. First, cells were harvested for

restimulation 4 days after the transfer and immunization, as opposed to 10 days with the

normal immunization experiments, thus preventing optimal expansion of endogenous

host T cells. Second, in vitro restimulation was with OVA peptide specific to the OT-II T

cells. Finally, the cytokine profiles of cells from immunized wild type and NOX2 (-/-)

mice were not exactly the same as those from the adoptive transfer experiments, in that

OT-IIs from NOX2 (-/-) mice produced increased IL-17 in addition to IFN-γ, suggesting a

response that was independent of host endogeneous T cells.

NOX2's expression is highest in neutrophils and macrophages. Inflammatory

mediators that were found to be altered from oxidase deficiency were for the most part

cytokines produced by CD11b + cells. We believe these CD11b + cells to represent

macrophages. Dendritic cells can also express CD11b, but the proportion of dendritic

cells to macrophages in the peritoneal cavity is relatively low. B cells did not appear to

display any obvious changes in cytokine production, but we do not exclude the possibility

that B cells are affected, as NOX2 is also expressed in them, and the generation of ROS

has been observed upon stimulation with TNF-α, IL-1β, and LPS 185,186 . Future studies

may investigate them further.

Both NOX2 deficient peritoneal cells and adherent splenocytes produced

increased amounts of IL-6 and TNF-α, both of which are important mediators of fever and the acute phase response. TNF-α is a cytokine associated with T H1 polarization.

However, IL-6 is involved in T H17 polarization. It is generally accepted that the combination of IL-6 and TGF-β promote T H17 differentiation. However in humans,

87

CGD phagocytes have an impaired ability to produce anti-inflammatory mediators, such

190 as TGF-β and prostaglandin D2 (PGD2) . Both TGF-β and PGD2 can switch a response

191 from a T H1/T H17 towards a T H2/Treg immune profile . Although we saw no detectable differences in total TGF-β levels between wild type and NOX2 (-/-) peritoneal cells, the enhanced IL-6 alone could be enough to polarize a response toward T H17. Alternatively, the combination of IL-6 and TNF-α may be able to promote T H17 development.

Recently, TNF-α was shown in vitro to drive the production of IL-17 with the ability to

192 differentiate T cells towards a T H17 phenotype . In a psoriasis-like skin inflammation model, TNF-α enhanced the expression of T H17-related cytokine genes during priming but suppressed these cytokine transcripts when present during re-stimulation. In collagen-induced arthritis, TNF-α inhibitors reduced the number of T H17 cells in pathologic joints 193 . Taken together, these findings show that TNF-α and IL-6 can affect the expression of T H17-related cytokines, at least in animal models of autoimmune disease. The ability of TNF-α and IL-6 to promote a T H17 response most likely depends on antigen type as well as the site of inflammation. This was especially evident in this study where oxidase deficient peritoneal cells were found to induce OT-II T cells to produce slightly increased amounts of IL-17, but decreased amounts of IFN-γ. As mucosal sites such as peritoneal cavity are often exposed to exogenous pathogens, requiring rapid responses as a first line of defense, it may make sense for T cells activated by peritoneal APCs to produce a higher ratio of IL-17 to IFN-γ. Indeed, in animal models of CGD, improved clearance of Helicobacter pylori from the gastric mucosa correlated with increased IL-17 176 , whereas IFN-γ was primarily responsible for enhanced clearance of Cryptococcus neoformans from the lung 174 . Previous studies have

88

not been consistent in APC type under investigation, and many times NOX2 deficiency has been studied in the context of a disease model 116,169,172,194-196 . This may explain why some studies found an increased T H1 response, whereas other studies found an increased

TH17 response.

It may be that increased IL-6 production is the factor that links the APC inherent changes with T cell inherent changes in CGD. In the following chapter, we will show T cells to be inherently altered during oxidase deficiency, through selective decreases in the transcription factor STAT5. T cell inherent changes have been implied in this study through the observation that in vitro restimulated adoptively transferred OT-II T cells had a slightly different cytokine profile than total lymph node cells from NOX2 (-/-) mice. IL-6 primarily signals through the transcription factor STAT3, which can oppose signaling through STAT5, and the IL-6 induced upregulation of specific suppressors of cytokine signaling (SOCS) proteins have been demonstrated to inhibit the JAK2-dependent phosphorylation of STAT5 197 . Therefore, since oxidase deficient T cells already exhibit inherent defects in STAT5, changes in the dichotomy between STAT3 and STAT5 by increased IL-6 production from APCs would serve as a feed-forward mechanism to enhance T helper skewing.

MIP-1α, also known as CCL3, as well as KC (murine IL-8) were shown to be significantly increased in oxidase deficient adherent splenocytes. Both of these are pro- inflammatory chemokines. MIP-1α attracts granulocytes (neutrophils, eosinophils and basophils) which can lead to acute neutrophilic inflammation, and the primary function of

IL-8 being the induction of chemotaxis in neutrophils. These results are consistent with previous reports describing changes in chemokines during NOX2 deficiency. Neutrophil

89

migration and IL-8 production were found to be increased in CGD 148 , and increased expression of the CC chemokines MCP-1, MIP-1α, and MIP-1β was found in the bronchoalveolar lavage fluid of NOX2 deficient mice following allogeneic murine bone marrow transplantation 162 . Likewise, GM-CSF was selectively increased in oxidase deficient peritoneal cells, which is involved in the recruitment and maturation of macrophages. This influx of innate cells induces the synthesis and release of pro- inflammatory cytokines such as IL-1β, IL-6 and TNF-α. GM-CSF is also a pro-survival cytokine for neutrophils 198 , which have been shown to undergo delayed apoptosis in

CGD patients. GM-CSF has been shown to induce a rapid increase in ROS that is required for efficient signaling. This increase in ROS has been shown to be sensitive to antioxidant treatment 60 . Indeed, macrophages loaded with L-ascorbate (Vitamin C) demonstrated decreased GM-CSF induced JAK2 phosphorylation 60 , further suggesting the involvement of redox signaling.

Overall expression of costimulatory molecules were similar between wild type and NOX2 deficient cells, with the exception of ICOS-L, which was found to be decreased in oxidase deficient peritoneal cells. ICOS-L costimulation affects the production of cytokines in T cells, and has been demonstrated to endow maturing APCs with the ability to induce T cells to produce IL-10 188 . ICOS-L deficient mice produce lower levels of T H2 and anti-inflammatory (TGF-β and IL-10) cytokines, but higher T H1- related (IFN-γ and IL-12p40/IL-23) and proinflammatory (IL-6 and TNF-α) cytokines 188 .

These changes in proinflammatory cytokine composition, reflect changes observed in

NOX2 (-/-) mice, suggesting the possibility that changes in ICOS-L expression may be at least partially responsible for the increased inflammation observed in CGD. However,

90

the changes observed in ICOS-L in NOX2 deficient mice were not global, and were restricted to one type of APC, which brings in to play other factors affecting ICOS-L expression that are most likely dependent on APC tissue localization.

Taken together, the data suggest that NOX2 could be involved in intracellular signaling events that affect APC cytokine production. These events could be triggered by ligation of pattern recognition receptors (PRR) or cytokine receptors. Changes in cytokine production would affect downstream T cell function, which was demonstrated in the altered cytokine profiles of the OT-II T cells cultured with oxidase deficient APC.

The intracellular signaling events affected by NOX2 deficiency are not well understood.

Recent evidence has shown that certain cytokines and growth factors induce the generation of ROS in macrophages. These ROS serve as requisite chemical second messengers. GM-CSF signaling in particular has been shown to be sensitive to vitamin

C60 . Membrane signaling events initiated by GM-CSF induce the activation of JAK-2, which has recently been identified as having a redox-sensitive switch within its catalytic domain 60 . Alternatively, the affected signaling events may be associated with defective macrophage activation status. Kynurenine is produced from tryptophan by indoleamine-

•- 2,3-dioxidase (IDO), favoring T cell tolerance. O 2 is a required cofactor for tryptophan

•- oxidation by IDO. Thus the O 2 dependent step in tryptophan metabolism in APCs is blocked as a result of NOX2 deficiency. A recent study using CGD mice infected with

Aspergillus fumigatus demonstrated that oxidase deficiency altered the kynurenine pathway resulting in an expansion of γδ -T cells that produced enhanced amounts of IL-17 and were found to be autoreactive 199 . However, two independent studies recently showed that DCs generated from patients harbouring X-linked and autosomal recessive forms of

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CGD, and from healthy controls, produced similar amounts of kynurenine upon activation with LPS and IFN-γ200,201 . Thus, at least in humans, ROS are apparently dispensable for IDO activity and hyperinflammation in CGD patients cannot be attributed to disabled IDO activation.

In summary, the results of this study suggest that inflammation in CGD may be attributed to decreased intracellular ROS signaling in APCs, which alters their activity.

These changes subsequently affect cytokine production and the expression of costimulatory molecules on APCs, which in turn affect the cross talk between APCs and

T cells during antigenic stimulation. Thus, oxidase expression in APCs serves to regulate the nature and course of an adaptive immune response by modulating the inflammatory microenviornment during T cell priming.

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Chapter IV. T cell expression of NOX2 regulates T helper differentiation through alterations in STAT5 and GATA-3

IVA. Introduction

The NOX family of NADPH oxidases 5 is a group of transmembrane proteins that intentionally generate reactive oxygen species (ROS), and have been shown to play important roles in cell signaling and are involved in the cellular response to a variety of inflammatory stimuli. The phagocyte-type oxidase, of which NOX2 is the catalytic subunit, is the prototypic member of this family. Mutations or deletions that affect subunits of this complex cause chronic granulomatous disease, an immunodeficiency syndrome characterized by the impaired ability of phagocytic cells to kill engulfed microorganisms, primarily catalase positive organisms, such as Aspergillus and

Staphyloccal species 123,135 .

In contrast to the defects in innate immunity, CGD patients are prone to develop

autoimmune phenomena, such as Crohns-like disease, juvenile rheumatoid arthritis or

lupus-related syndromes 149,150 . In addition to similar sensitivity to autoimmune

phenomena, animal models of CGD also display a heightened response to certain

infectious agents including Cryptococcus neoformans , influenza virus, and Helicobacter

pylori 173-175 . Thus, immunodeficiency in CGD is sometimes associated with enhanced

inflammation and immune responses.

The T cell response observed in CGD mice upon influenza or Cryptococcus

174 infection was characterized by an augmented macrophage-driven T H1 response , while

196,202 in other systems NOX deficiency correlated with a heightened TH17 response .

93

Nevertheless, it is clear that NOX2 plays a role in shaping the adaptive immune response.

One model suggests that oxidase deficiency in APCs alters their capability to activate T

cells, instructing naive T cells down a certain differentiation pathway during priming. In

contrast to this “APC instructive model”, the intrinsic expression of NOX2 in T cells

themselves may also play a role in lineage fate decisions. Peripheral T cells have been

shown to express a functional phagocyte NADPH oxidase 70,71,116,117 . In support of an

effect of ROS on T cell differentiation, previous studies have demonstrated that treating T

cells with antioxidants promoted a T H1 response with increased IFN-γ and decreased IL-

113,114 4 while pro-oxidants biased them toward a T H2 phenotype, resulting in increased

amounts of IL-4, IL-5, and IL-13 115 . Taken together, these findings suggest a redox

dependent modulation of T helper responses. However, the mechanism(s) by which

oxidase/ROS deficiency leads to phenotypic changes in T cell activity and function

remains unresolved.

Several factors impact the decision of naive CD4 + T cells to develop down a particular differentiation pathway. Master regulator transcription factors, e.g., GATA-3

(T H2), T-bet (T H1) and ROR-γt (T H17) regulate key gene loci, in turn promoting or repressing expression of cytokines and receptors that determine the direction of differentiation 76,101 . Regulatory loops exist such that the cytokines can further augment

or inhibit expression of these master regulators, which then feedback and inhibit

expression of other master regulators. For example, the prototypical T H2 cytokine IL-4

can induce expression of GATA-3. GATA-3 binds and helps open the Il4 locus

(enhancing IL-4 production) and also inhibits expression of cytokines (IFN-γ) and

76 receptors (IL-12R β) that promote T H1 differentiation . The signal transducer and

94

activator of transcription (STAT) family of transcription factors regulates many of the

downstream signaling effects of cytokines in T helper development. The transcription

factors STAT4 and STAT1, activated by IL-12 and IFN-γ respectively, induce the

expression of T-bet, while IL-4 mediated STAT6 activation upregulates GATA-3

expression 76 . STAT5 is also an important player in T helper development. IL-2 mediated activation of STAT5 stimulates the proliferation and survival of T cells, but is

86 also needed for optimal induction of a T H2 response . Recent studies have identified IL-

2 dependent opening of the Il4 locus and upregulated expression of the IL-4R α on T cells

in a STAT5-dependent fashion after TCR ligation 88 .

The goal of this study was to determine whether and how the phagocyte oxidase

functions in naive T cells to modulate T helper differentiation. The results support

(-/-) + previous data, in that NOX2 , naive CD4 T cells are innately more T H1 skewed than

their wild type counterparts. These T cells secreted increased IFN-γ, but decreased IL-4

in response to anti-CD3 and anti-CD28 stimulation. There were also selective decreases

in GATA-3 and the phosphorylation of STAT5, with decreased Il4 gene expression

which suggested a mechanism for decreased T H2 conditions. Finally, treatment with

antioxidants or adding back ROS with pro-oxidants recapitulated these selective changes

in TCR-induced transcription factor activation. Taken together, these findings suggest

that during priming, TCR-induced ROS generation from NOX2 activation selectively

+ promotes STAT5 phosphorylation and downstream T H2 development in CD4 T cells.

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IVB. Results

IVB1. NOX2 expression in T cell development

The absence of NOX2 has been shown to affect T cell function both in vivo and

in vitro. However, it is unclear when in T cell development that NOX2 is expressed. As

shown in Figure IV.1 A, relatively low levels of NOX2 mRNA were found in immature

T cells from the thymus of C57BL/6 mice, but higher levels were found in resting CD4 + and CD8 + T cells of the spleen and lymph nodes, demonstrating that NOX2 is expressed in T cells as early as thymocyte development. However, the higher expression levels in T cells from the spleen and lymph nodes may indicate that NOX2 generated ROS play more of a role in mature T cells than in developing thymocytes.

In wild type C57BL/6 and oxidase deficient NOX2 knock out mice, flow cytometric analysis of the thymus (Figure IV.1 B, C) and spleen (Figure IV.1 B) demonstrated no detectable differences in lymphocyte composition of these organs. Total cellularity as well as cell viability of the spleen, thymus and lymph nodes were unchanged between wild type and oxidase deficient mice. There were, however, minor differences in the populations of naïve, effector and central memory T cells of the spleen

(Figure IV.1 D). In the oxidase deficient spleen, there were minor increases in naïve

(CD44 -,CD62L hi ) T cells, with slight decreases in effector (CD44 +CD62L lo ) and memory

(CD44 +CD62L hi ) T cells. This decrease in memory T cells is consistent with previous

reports of decreased levels of memory lymphocytes in CGD patients 154,155 .

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Figure IV.1. NOX2 gene expression in wild type mice

(A) - NOX2 expression was assessed in total thymus, double negative (DN) thymocytes,

CD4 + splenocytes, CD8 + splenocytes, total lymph node (LN), and aorta by qPCR in wild type mice. These data are expressed as the expression of the indicated mRNA relative to control HPRT mRNA. (n = 3).

(B-D) - Cell composition in wild type and NOX2 (-/-) mice: Cell populations from thymus

(B) and spleen (C) of NOX2 deficient mice ( closed bars ) or wild type controls ( open bars ) were assessed ex vivo by flow cytometry. (D) After gating on TCR + cells, splenocytes were further characterized into CD4 + or CD8 + T cells, as well as regulatory

(CD4 +, CD25 +), naïve (CD44 lo , CD62L hi ), effector (CD44 hi , CD62L lo ), and central memory (CD44 hi , CD62L hi ) T cells (n = 4 separate experiments).

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A B Thymus 0.75 100 WT NOX2 (-/-) 75 0.5

50

0.25 25 % Positive Cells %Positive

Relative NOX2 Expression NOX2 Relative 0 0 totalDN CD4 + CD8 + LN aorta DN CD4 + CD8 + DP thymus spleen T cells SP SP

C Spleen D Splenic TCR + Cells 100 75 WT WT NOX2 (-/-) NOX2 (-/-) 75 50

50

25 25 % Positive Cells %Positive Cells %Positive

0 0 TCR + CD19 + NK1.1 + Gr-1+ CD11b + CD4 + CD8 + CD4 + CD44 - CD44 + CD44 + CD25 + CD62L hi CD62L lo CD62L hi

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IVB2.Oxidase deficient T cells are more T H1 skewed than their wild type counterparts

Previous reports have suggested changes in cytokines from activated T cell blasts

from NOX2-deficient mice 71 . Therefore, the goal was to determine if there was intrinsic

TH1 skewing of the oxidase deficient adaptive immune system during primary activation.

Total resting splenocytes from wild type and oxidase deficient mice were stimulated for

72 hours on immobilized anti-CD3 and cytokine production was assessed by ELISA.

Cell supernatants from oxidase deficient splenocytes contained decreased amounts of IL-

4 but increased concentrations of both IL-17 and IFN-γ (Figure IV.2 A-C). Because total spleen includes both antigen presenting cells as well as memory T cells, oxidase deficient naïve CD4 + T cells were isolated to determine if they too demonstrated altered cytokine production during primary activation. Consistent with the phenotype observed in oxidase deficient splenocytes, naïve CD4 + T cells from NOX2 (-/-) mice produced significantly increased levels of IFN-γ and IL-17, but significantly decreased levels of IL-4 and IL-5

(Figure IV.2 D-F). After activation, no differences were observed in cell yield or cell viability (Figure IV.3). These findings suggest that oxidase deficient naive CD4 + T cells are themselves inherently more skewed away from a T H2 phenotype than their wild type counterparts, and this skewing may manifest itself as early as during primary activation.

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Figure IV.2. Enhanced inflammatory cytokine and decreased T H2 cytokine production from oxidase deficient T cells

Total splenocytes (A-C) and naïve CD4 + T cells (D-G) from wild type ( open bars ) and

NOX2 deficient ( closed bars ) mice were cultured for 48 to 72 hours on immobilized anti-

CD3 for total splenocytes, and immobilized anti-CD3 with soluble anti-CD28 for CD4 + T

cells. Supernatatants were collected for analysis by ELISA. (total spleen n = 7 separate

experiments, naïve CD4 + T cells n = 4 separate experiments, significantly different from wild type * = p<0.05).

100

101

unstimulated 12h αCD3 + αCD28 24h αCD3 + αCD28 48h αCD3 + αCD28 10 4 10 4 10 4 10 4 A 10 3 10 3 10 3 10 3

10 2 67.6 10 2 84.7 10 2 85.1 10 2 86.8

10 1 10 1 10 1 10 1 SSC side scatter side SSC 10 0 10 0 10 0 10 0 WT 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 10 4 10 4 10 4 10 4

10 3 10 3 10 3 10 3

10 2 10 2 10 2 10 2

10 1 10 1 10 1 10 1

FL4 Live Gate Live FL4 96.1 97.1 87.5 77.5 10 0 10 0 10 0 10 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 FSC forward scatter

unstimulated 12h αCD3 + αCD28 24h αCD3 + αCD28 48h αCD3 + αCD28 10 4 10 4 10 4 10 4 B 10 3 10 3 10 3 10 3

10 2 77.6 10 2 85.6 10 2 86.1 10 2 85.6

10 1 10 1 10 1 10 1 SSC side scatter side SSC 10 0 10 0 10 0 10 0 NOX2 (-/-) 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 10 4 10 4 10 4 10 4

10 3 10 3 10 3 10 3

10 2 10 2 10 2 10 2

10 1 10 1 10 1 10 1

FL4 Live Gate Live FL4 96.7 94.7 89.2 74.6 10 0 10 0 10 0 10 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 FSC forward scatter

Figure IV.3. Wild type and NOX2 (-/-) naïve CD4 + T cells have equal viability

Naïve CD4 + T cells from wild type (WT) (A) and NOX2 deficient (NOX2 (-/-)) (B) mice were cultured for 12, 24, or 48 hours on immobilized anti-CD3 with soluble anti-CD28.

Cells were harvested and viability was assessed by flow cytometric analysis of forward × side scatter profiles and by fluorescence of a fixable amine-reactive dye. Cells with low dye fluorescence gated on a high forward × side scatter profile were deemed viable.

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IVB3.Oxidase deficient T cells have decreased GATA-3 during primary activation

A hallmark feature of activated helper CD4 + T effectors is the expression of the lineage- specifying transcription factors T-bet, GATA-3, and ROR-γt for T H1, T H2, and T H17 cells, respectively. Control of these transcription factors, especially GATA-3 can be posttranscriptional in TCR stimulated T cells, 203 and oxidase deficient T cells had significantly decreased levels of GATA-3 protein by intracellular staining (Figure IV.4

A-B), whereas T-bet levels were significantly increased (Figure IV.4 D-E). When gene expression was examined, oxidase deficient T cells also had decreased mRNA expression of GATA-3 at 24 hours post stimulation (Figure IV.4 C), while T-bet expression was significantly increased at both 12 and 24 hours after activation (Figure IV.4 F). These data suggest that oxidase deficient T cells have altered expression of master regulator transcription factors during early activation through TCR stimulation.

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Figure IV.4. GATA-3 and T-bet levels in oxidase deficient CD4+ T cells

(A, D) Representative staining profiles for GATA-3 and T-bet. Naïve CD4 + T cells from wild type (WT) ( open bars ) and NOX2 deficient (NOX2 (-/-)) ( closed bars ) mice were cultured for 12 and 24 hours on immobilized anti-CD3 with soluble anti-CD28. Cells were harvested and underwent intracellular staining for GATA-3 (A-B) and T-bet (D-E) and were analyzed by flow cytometry as described in the Methods. The data are expressed as difference in mean fluorescence intensity after stimulation. (n = 5 separate experiments, * significantly different from wild type, p<0.05). GATA-3 (C) and T-bet

(F) mRNA expression was assessed in naïve CD4 + T cells by qPCR in wild type ( open bars ) and NOX2 deficient ( closed bars ) mice. These data are expressed as the expression of the indicated mRNA relative to control HPRT mRNA (triplicate wells from 2 separate experiments, * significantly different from wild type, p<0.05).

104

105

IVB4. Alterations in phosphorylated STAT proteins in oxidase deficient T cells

Signaling through cytokine receptors, activating JAK-STAT signaling pathways

are critical for control of T helper differentiation. Multiple STATs have been proposed to

regulate T H1 commitment, including STAT1 and STAT4, while TH2 commitment can be promoted by activation of STAT6, STAT5 or STAT3 76,204 . After primary activation, the levels of phosphorylated STAT1, STAT3, and STAT4 were found to be unchanged between wild type and oxidase deficient T cells (Figure IV.5). On the other hand, phosphorylation of STAT5 was found to be decreased nearly two fold in oxidase deficient T cells as compared to wild type controls (Figure IV.6 A-B). There were no differences in total STAT5 levels between wild type and oxidase deficient T cells, indicating that the defect is not due to decreased expression of STAT5. TCR stimulated

STAT5 phosphorylation and subsequent T H2 development has been proposed to be IL-2 dependent 86 , and stimulation in the presence of a neutralizing antibody to IL-2 led to strong decreases in STAT5 phosphorylation in both wild type and oxidase deficient cells

(Figure IV.6 C). Because of the dependence of STAT5 and GATA-3 on IL-2 in TCR stimulated cells, NOX2-deficient T cells were investigated for basic changes in the IL-

2/IL-2R signaling axis. Induction of IL-2R α (CD25) surface expression and IL-2 mRNA expression after TCR stimulation of naïve T cells were the same in wild type and NOX2- deficienct mice (Figure IV.7 A, B, & C).

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Figure IV.5. The levels of phosphorylated STATs 1, 3, and 4 are unchanged between wild type and oxidase deficient CD4 + T cells

Naïve CD4 + T cells from wild type (WT) ( open bars ) and NOX2-deficient (NOX2 (-/-))

(closed bars ) mice were cultured for 12, 24, and 48 hours on immobilized anti-CD3 with

soluble anti-CD28. Cells harvested at 12 and 24 hours underwent intracellular staining

for pSTAT3 (A, D). Cells harvested at 24 and 48 hours underwent intracellular staining

for pSTAT1 (B, E) and pSTAT4 (C, F). The data are expressed as the difference in

mean fluorescence intensity after stimulation (n = 3). (D-F) Representative staining profiles for STAT3, STAT1, and STAT4.

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108

A B 150 100 unstim WT WT NOX2 (-/-) 80 NOX2 (-/-) 100 60 *

40 % of Max %of 50

pSTAT5 MFI pSTAT5 * 20

0 0 10 0 10 1 10 2 10 3 10 4 12 hrs 24 hrs PE anti-pSTAT5 C D 150 WT 150 WT NOX2 (-/-) NOX2 (-/-)

100 * 100

50

pSTAT5 MFI pSTAT5 MFI pSTAT5 50 *

0 0 12 hrs 24 hrs 12 hrs 24 hrs + αIL-2 + αIL-2 + IL-2 + IL-2

Figure IV.6. Decreased phosphorylated STAT5 in NOX2-deficient CD4 + T cells

Naïve CD4 + T cells from wild type (WT) ( open bars ) and NOX2 deficient mice (NOX2 (-

/-)) ( closed bars ) were cultured for 12 and 24 hours on immobilized anti-CD3 with soluble anti-CD28 (A, B) , or were stimulated in the presence of 10 g/ml anti-IL-2 (C) or 0.25

U/ml IL-2 (D) . Cells were harvested and underwent intracellular staining for pSTAT5 and were analyzed by flow cytometry as described in the Methods. (A) Representative pSTAT5 staining profile. The data are expressed as the difference in mean fluorescence intensity after stimulation. (n = 5 separate experiments, * significantly different from wild type; p<0.05).

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A B C 1500 50 100 WT WT (-/-) NOX2 (-/-) 40 NOX2 80 1000 60 30

40 20 % of Max

CD25 MFI 500

20 10 ** Relative IL-2 Expression 0 0 0 10 0 10 1 10 2 10 3 10 4 12 hrs 24 hrs 12 hrs 24 hrs FITC anti-CD25

unstim WT NOX2 (-/-)

Figure IV.7. Lack of changes in IL-2/IL-2R expression in NOX2-deficient CD4 + T cells

(A-B) Naïve CD4 + T cells from wild type (WT) and NOX2 deficient (NOX2 (-/-)) mice

were cultured for 12 ( hatched bars ) and 24 hours ( solid bars ) on immobilized anti-CD3

with soluble anti-CD28. Cells were harvested and surface staining for CD25 was

analyzed by flow cytometry. (A) shows a representative staining profile and in (B) the

data are expressed as the difference in mean fluorescence intensity after stimulation (n =

5 separate experiments). (C) mRNA from wild type ( open bars ) and NOX2-deficient

(closed bars ) cells stimulated as in (B) was extracted for qPCR analysis for IL-2

expression. qPCR data are expressed as the expression of the indicated mRNA relative to

control HPRT mRNA (triplicate wells from 2 separate experiments).

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Furthermore, treatment of oxidase deficient T cells with exogenous IL-2 during activation

did not increase the levels of phosphorylated STAT5 (Figure IV.7 D) in oxidase deficient

T cells to those in wild type T cells, suggesting that these changes are not a result of

decreased IL-2 production or decreased sensitivity to IL-2. Taken together, it would

appear that oxidase deficient naive CD4 + T cells have a defect in TCR-stimulated STAT5 activation, which may subsequently affect downstream T H2 differentiation.

IVB5.Decreased IL-4 and IL-4R α expression in NOX2 deficient T cells

The model of STAT5 and GATA-3 regulation of T helper development suggests that these transcription factors facilitate the expression of both the Il4 and Il4r α genes.

Consistent with this, PCR analysis also found decreases in Il4 and Il4r α mRNA

expression in NOX2-deficient T cells (Figure IV.8 A, D). Surface expression of IL-4R α

was also downregulated in the absence of the oxidase (Figure IV.8 B, E). Modestly

decreased phosphorylated STAT6 levels were also observed in oxidase deficient T cells

(Figure IV.8 C, F), likely resulting as a consequence of reduced IL-4 production. In an

attempt to bypass T H1 skewing of NOX2-deficient T cells, naïve cells were activated

under T H2 skewing conditions. Consistent with normal T H2 lineage commitment, STAT6

phosphorylation and GATA3 expression (Figure IV.9 A-C) were greatly enhanced under

TH2 skewing conditions, but there were no detectable differences observed between wild

type and oxidase deficient cells. However, T H2 skewing of oxidase deficient T cells

during primary activation did not increase the expression of IL-4 to the levels seen in

wild type T cells (Figure IV.9 D) suggesting that defects in the opening of the Il4 locus

are still present in oxidase deficient T cells, likely due to alterations in STAT5 signaling.

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Figure IV.8. Decreased IL-4 and IL-4R α expression in NOX2-deficient CD4 + T cells

Naïve CD4 + T cells from wild type (WT) and NOX2 deficient (NOX2 (-/-)) mice were

cultured for 12 and 24 hours on immobilized anti-CD3 with soluble anti-CD28. mRNA

from wild type (open bars) and NOX2 deficient (closed bars) cells was extracted for

qPCR analysis for IL-4 (A) and IL-4R α (D) expression. qPCR data are expressed as the

expression of the indicated mRNA relative to control HPRT mRNA (triplicate wells from

3 separate experiments). (B, E) Naïve CD4 + T cells from wild type (open bars) and

NOX2 deficient mice (closed bars) were cultured for 12 and 24 hours on immobilized anti-CD3 with soluble anti-CD28. Following incubation, the cells were harvested and surface staining for IL-4R α expression was measured by flow cytometry. (C, F) Naïve

CD4 + T cells from wild type ( open bars ) and NOX2 deficient mice ( closed bars ) were cultured for 24 and 48 hours on immobilized anti-CD3 with soluble anti-CD28.

Following incubation, cells were harvested and intracellular staining for pSTAT6 was measured by flow cytometry. (B-C) The data are expressed as the difference in mean

fluorescence intensity after stimulation (IL-4R α n = 3 separate experiments, pSTAT6 n =

3 separate experiments, * significantly different from wild type; p<0.05).

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A TH2 skewing B TH2 skewing 45 100 WT unstim NOX2 (-/-) WT 80 NOX2 (-/-) 30 60

40

15 Max of % pSTAT6 MFI pSTAT6 20

0 0 12 hrs 24 hrs 10 0 10 1 10 2 10 3 10 4 PE anti-STAT6

C TH2 skewing D TH2 skewing 9 9 WT WT NOX2 (-/-) NOX2 (-/-)

6 6 *

3 3

Relative IL-4 Expression IL-4 Relative *

Relative GATA-3 Expression GATA-3 Relative 0 0 12 hrs 24 hrs 12 hrs 24 hrs

Figure IV.9. NOX2 deficient T cells are able to undergo T H2 commitment

Naïve CD4 + T cells from wild type (WT) ( open bars ) and NOX2-deficient (NOX2 (-/-))

(closed bars ) mice were cultured for 12 and 24 hours on immobilized anti-CD3 with soluble anti-CD28 under T H2 skewing conditions as described in the Methods. At 12 and

24 hours, the cells were harvested and underwent intracellular staining for pSTAT6 (A-

B) and were analyzed by flow cytometry. The data are expressed as the difference in mean fluorescence intensity after stimulation (n = 3 separate experiments). (C, D) mRNA was extracted from parallel samples for qPCR analysis of (C) GATA-3 or (D) IL-

4 expression. The data are expressed as the relative expression compared to HPRT expression (triplicate wells from 2 separate experiments).

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IVB6. Enhanced T H1 phenotype in BALB/c T cells treated with antioxidants

To consider the overall effect of redox signaling on T helper differentiation, CD4 +

T cell blasts from BALB/c mice were cultured in the presence or absence of the

antioxidant N-acetylcysteine (NAC). BALB/c T cells preferentially follow a T H2 differentiation pathway, and treatment of BALB/c CD4 + T cell blasts with 10 mM NAC

increased T H1 cytokine production and decreased T H2 cytokine production as compared

to untreated controls (Figure IV.10 A-C). BALB/c T cells treated with NAC also showed

decreased phosphorylated STAT5 (Figure IV.10 D), consistent with what was observed

in oxidase deficient T cells, and implicating oxidative signaling as a possible factor in T

helper differentiation. To support the possible role of NOX2 in TCR-stimulated T helper

determination, cells were also incubated with apocynin, an inhibitor of NADPH oxidase

activity. Similar to the effects of NAC, apocynin significantly inhibited STAT5

phosphorylation (Figure IV.10 D), implicating NOX2 as a major contributor to the

oxidative regulation of STAT5. Antioxidants were also shown to have similar effects on

STAT5 phosphorylation in T cells from C57BL/6 mice (Figure IV.11 A). Furthermore,

adding back ROS in the form of superoxide generated by treatment with DMNQ

enhanced STAT5 phosphorylation in NOX2-deficient cells close to that observed in T

cells from wild type mice (Figure IV.11 B).

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Figure IV.10. Antioxidants inhibit T H2 development in BALB/c T cells

(A-C) CD4 + T cell blasts from BALB/c mice were cultured and restimulated on immobilized anti-CD3 in the presence of 10 mM N-acetylcysteine (NAC) ( closed bars ).

Supernatants were collected after 8 hours of restimulation for ELISA (n = 4 separate experiments, * significantly different from wild type; p<0.05). (D) BALB/c naïve CD4 +

T cells were cultured on immobilized anti-CD3 with soluble anti-CD28 in the absence

(open bars ) or presence of NAC (10mM) ( hatched bars ) or the NADPH oxidase inhibitor

apocynin (100M) ( dotted bars ) for 12 hours. Following incubation the cells underwent

intracellular staining for pSTAT5 and were analyzed by flow cytometry. The data are

expressed as the difference in mean fluorescence intensity after stimulation (n = 4

separate experiments, * significantly different from wild type; p<0.05).

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A BALB/c IL-4B BALB/c IL-5 20 150

15 100

* 10

50 (pg/ml) IL-5 * IL-4 (pg/ml) IL-4 5

0 0 WT NAC WT NAC 10mM 10mM

C BALB/c IFN-γ D BALB/c pSTAT5 12000 60 * untreated NAC apocynin 8000 40 * (pg/ml) * γ

4000 20 IFN- pSTAT5 MFI pSTAT5

0 WT NAC 0 10mM

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A B 75 75 WT untreated WT untreated NOX2 (-/-) untreated NOX2 (-/-) untreated WT NAC NOX2 (-/-) DMNQ WT apocynin 50 50 * * * * 25 25 pSTAT5 MFI pSTAT5 pSTAT5 MFI pSTAT5

0 0

Figure IV.11. Oxidative regulation of STAT5 phosphorylation

(A) naïve CD4 + T cells from wild type (WT) mice were cultured in the absence ( open bars ) or presence of the antioxidant N-acetylcysteine (NAC) (10mM) ( hatched bars ) or the NADPH oxidase inhibitor apocynin (100 M) ( dotted bars ) for 12 hours, and compared to naïve CD4 + T cells from NOX2-deficient (NOX2 (-/-)) mice ( solid bars ). (B)

Naïve CD4 + T cells from NOX2-deficient mice were cultured in the absence ( closed bars ) or presence of 1 M of the pro-oxidant 2,3-Dimethoxy-1,4-naphthoquinone

(DMNQ) ( grey bars ) for 12 hours, and compared to naïve CD4 + T cells from wild type mice (open bars ). Following incubation the cells underwent intracellular staining for pSTAT5 and were analyzed by flow cytometry. The data are expressed as the difference in mean fluorescence intensity after stimulation (n = 4 separate experiments, * significantly different from untreated wild type; p<0.05).

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IVC. Discussion

The decision of naive T cells to differentiate into a particular helper subset is impacted by many different elements including signal strength, the local cytokine environment, the interaction of costimulatory molecules and the balance of downstream signaling cascades. The regulation of signaling in T helper development by changes in redox balance or oxidative stress in the adaptive immune system is not well understood.

The goal of this study was to help elucidate the involvement of ROS in the adaptive immune response. The results of this report demonstrate that the loss of NOX2 generated

ROS, induced by TCR stimulation, promotes the polarization of naïve CD4 + T cells toward a T H1 phenotype during primary activation. This polarization is associated with selective decreases in GATA-3 and phosphorylated STAT5, which is consistent with previous reports demonstrating the collaboration of STAT5 and GATA-3 in priming cells to acquire a T H2 phenotype.

Previous studies have demonstrated that altering the redox balance in T cells can result in phenotypic changes in T helper function, although the exact mechanisms were not addressed. The antioxidant properties of the mineral selenium as well as α- tocopherol (vitamin E) have been found to skew T cells toward a T H1 response with increased IFN-γ and decreased IL-4 secretion 113,114 . Conversely, in a study examining

oxidative stress and allergic asthma, exposure of CD4 + T cells to the pro-oxidant DMNQ biased them toward a T H2 phenotype, resulting in increased amounts of IL-4, IL-5, and

IL-13 115 . Multiple studies have demonstrated that T cells express a functional

NOX2 70,71,116,117 , and its deficiency has been shown to alter the inherent function and

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survival of these cells 117,167 . Although studies have indicated functional expression of

NOX2 in T cells, these reports have used T cell blasts or populations that included

memory T cells, allowing for the possibility that instruction of these T cells by previous

antigen experience or the presence of APCs could skew their subsequent response. Thus

in the current study, naïve T cells were activated through anti-CD3 and anti-CD28,

independent of APCs and therefore eliminating the possibility of any extrinsic

instruction.

In previous studies, low antigen concentration or signal strength during TCR

stimulation promoted T H2 development, and increasing the strength of TCR stimulation

inhibited the associated increases in GATA-3 and phospho-STAT5, consequently

85,89 promoting T H1 development . GATA-3 protein expression was found to be IL-2

independent, while increases in IL-4 mRNA were dependent on both GATA-3 and IL-

285 . These data suggested that TCR signals independently induced GATA-3 and STAT5 to open the Il4 locus and promote T H2 development. TCR signaling has been proposed to directly activate STAT5 phosphorylation 87 , and the current results suggest that NOX2

deficiency is not altering production of IL-2 or upregulation of the IL-2R α. Based on these observations, decreased STAT5 phosphorylation may be associated with lack of

TCR-induced generation of ROS, which affects proximal IL-2R signaling.

Under conditions of increased antigen stimulation, co-incubation with a MEK inhibitor reversed the pro- TH1 effects on T helper differentiation, allowing increases in

GATA-3 and phosphorylated STAT5 85 . These data suggest increased ERK activation would inhibit T H2 development. Previous data in NOX2 deficient T cells as well as those cells treated with antioxidants observed increased TCR-induced MEK/ERK

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activation 70,71 , suggesting that NOX2/ROS dependent alterations in MEK/ERK signaling

in T cells may inhibit T H2 development. Possible targets of NOX2 which may directly

regulate STAT5 and the ERK signaling pathway are the oxidation sensitive protein

tyrosine phosphatases (PTPs) SHP-1 and SHP-2 PTPs.

The data suggest that NOX2-derived ROS selectively regulate STAT5 activation

in TCR stimulated naïve T cells, which in turn affects expression of IL-4. This decreased

IL-4 production likely causes subsequent decreases in phosphorylated STAT6 in NOX2-

deficient cells. It was unexpected that phosphorylation of STAT1 and STAT4 were not

augmented in the T H1 skewed NOX2-deficient T cells, but the data only examined early

time points after activation and T-bet mRNA and protein expression were clearly

upregulated, suggesting that the T cells were pushed towards a T H1 phenotype.

Decreases in GATA-3 protein levels in NOX2-deficient T cells could also be contributing to the increased T-bet expression in addition to its effects on IL-4 expression. TCR signaling initially enhances GATA-3 protein levels by increasing its translation rate without increasing GATA-3 mRNA levels 203 . Since there was no significant difference in GATA-3 mRNA in wild type and NOX2-deficient T cells during the early signaling events at 12 hours, these data suggest that TCR signals may impact this early post- transcriptional control. However, IL-4 can strongly upregulate GATA-3 and the diminished IL-4 production from NOX2-deficient T cells may contribute to downregulation of GATA-3 levels early in T cell activation.

The decrease in phosphorylated STAT5 in NOX2 deficient T cells may contribute to phenotypic changes observed in immune cells from both CGD patients and animal models of this disease. The association of CGD with autoimmune/rheumatologic

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disorders and the development of autoimmune arthritis in animal models could be due to

an enhanced T H1 or T H17-mediated responses depending on the stimuli and immune microenvironment. Recent studies have shown that STAT3 and STAT5 competitively bind to multiple common sites across the Il17 locus 205 . It may be that decreased phospho-STAT5 levels allow for increased STAT3 binding to the Il17 locus, thus

increasing the expression of IL-17. Furthermore, reports suggest diminished induction of

T regulatory cells associated with CGD 199 and oxidase derived ROS has been shown to

be involved in direct Treg mediated suppression of effector T cells 206 . STAT5 can induce

the expression of the Treg transcription factor Foxp3 and decreased STAT5 could also

then inhibit Treg function or development. Additionally, decreased STAT5 activation

may play a role in defects in survival in oxidase deficient T cells. There is an association

of CGD with decreased memory T cells and B cells 154,155 and, in vitro, oxidase deficient

T cells demonstrate decreased survival that could only be partially rescued by IL-7167 .

Taken together, these observations and the results of this report suggest the importance of

NADPH oxidase mediated activation of STAT5 in T cells.

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Chapter V. Discussion

Since the first description of “a fatal granulomatous disease of childhood”, 118 much has been learned about chronic granulomatous disease, but the signaling pathways affected by oxidase deficiency have only partially been elucidated. Redox regulation of the adaptive immune system is still an emerging field, and the exact mechanisms behind the changes in the adaptive immune response observed in patients with CGD and animal models of the disease are still unresolved. Understanding the underlying mechanisms for the observed “hyperinflammation” is important in order to improve our understanding of the immune dysregulation seen in the disease, and allow for improved patient treatments.

The "dogma" in the field is that NOX2 functions primarily in myeloid cells and antigen presenting cells to “shape” adaptive immune responses. However, this APC instructive model does not fully account for all of the altered responses observed in CGD T cells.

Alternatively, the intrinsic expression of NOX2 in T cells themselves may also play a role in lineage fate decisions. In this scenario, oxidase expression in T cells directly affects their subsequent effector phenotype after activation. Differences in the animal models between studies have further complicated an understanding of what is going on, as using gp91 phox deficient animals versus p47 phox deficient animals versus the use of antioxidants and pharmacologic inhibitors have produced varying results 116,117,166,167,184,207 . However, there appears to be a trend in the field that the oxidase plays a role in shaping the adaptive immune response, and that decreased ROS inversely correlates with increased inflammation. The findings of this study suggest that NOX2 deficiency alters the adaptive immune system in ways beyond the respiratory burst, and

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demonstrate that CGD is a disease that is both an innate and adaptive immune system

disorder, in that absence of NOX2 activity alters APC function resulting in the skewing T

helper development. Furthermore, absence of NOX2 activity within T cells inherently

alters their intracellular signaling pathways that result in the polarization of their lineage

differentiation.

The first aim of this study was to determine whether oxidase deficient APCs,

specifically macrophages, create an environment that promotes skewing in T cells, both

in vitro and in vivo. Immunization of wild type and NOX2 (-/-) mice with OVA in the presence of either CFA or Alum revealed altered cytokine profiles of total lymph node cells after restimulation in vitro, with oxidase deficient mice producing a more T H1- biased response, even when immunized with the pro-TH2 adjuvant Alum. Adoptive transfer of OT-II T cells into wild type or NOX2 (-/-) hosts followed by immunization also revealed changes in cytokine production from T cells after restimulation in vitro.

Analysis of APC demonstrated that differences in their source and localization affected the composition of pro-inflammatory cytokines produced in response to LPS. Upon maturation by LPS stimulation, APCs from oxidase-deficient mice secreted significantly increased amounts of pro-inflammatory cytokines, such as IL-6 and TNF-α, which is consistent with previous reports of enhanced production of IL-6 and TNF-α from CGD leukocytes when stimulated with LPS or peptidoglycan 146 . However, oxidase deficient peritoneal cells did not act the same manner as oxidase deficient adherent splenocytes.

They did not produce the same composition of cytokines and they exhibited changes in the expression of ICOS-L that were not present in adherent splenocytes, suggesting the importance of the immune microenvironment in APC instruction. This may also explain

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varying responses observed in other studies using oxidase deficient APC. It appears that

the CGD phenotype is more complex than T cell versus APC, and displays a degree of

plasticity due to the nature of the stimulus and immune microenvironment. APC function was defined by the ability to stimulate TCR-transgenic, ovalbumin-specific OT-II T cells in vitro. OT-II T cells activated with oxidase deficient APC produced more IFN-γ in response to antigen but secreted less IL-4 as compared to cells activated by APC from wild type mice, suggesting that the phagocyte NADPH oxidase is involved in the crosstalk between the adaptive and innate immune systems by regulating cytokine production in APCs.

The second major aim of this study was to characterize the inherent changes in oxidase deficient T cells. Activation of purified naive CD4 + T cells from NOX2 (-/-) mice led to augmented IFN-γ and diminished IL-4 production and an increased ratio of expression of the T H1-specific transcription factor T-bet versus the T H2-specfic transcription factor GATA-3, consistent with a T H1 skewing of naïve T cells. The major

finding of specific inhibition of TCR-induced STAT5 phosphorylation could be a

potential mechanism for skewed T helper differentiation. Additionally, exposure to anti-

oxidants inhibited, while pro-oxidants augmented T H2 cytokine secretion and STAT5

phosphorylation, supporting the idea that these signaling changes are redox dependent.

Questions remain about the identity of signaling molecules in T cells and APCs

that are directly affected by NOX2-generated ROS. Our data show that in T cells,

STAT5 phosphorylation and GATA-3 expression are decreased during oxidase

deficiency. STAT5 phosphorylation may be oxidatively regulated by phosphatase

activity. The best-characterized molecular targets of ROS are the protein tyrosine

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phosphatases because these important signaling enzymes require a conserved catalytic cysteine residue that exists as a thiolate anion, and has enhanced susceptibility to oxidation 52,53 . Oxidation of these cysteine residues modifies phosphatase activity.

Oxidase generated ROS can transiently inactivate SHP-1 and stimulate downstream TNF-

α production 61 , and lung lesions in aging p47 phox(-/-) mice resemble those seen in SHP-1 null (motheaten) mice 160 , suggesting the possibility of a phospatase as the target of oxidative regulation. Previous studies have demonstrated that TCR-stimulated generation of ROS can selectively oxidize the protein tyrosine phosphatase SHP-2, but not SHP-1, despite its high homology to SHP-272,73 . Whether it be SHP-1 or SHP-2, by "inhibiting the inhibitor", the oxidation of a phosphatase negatively regulating STAT5 could allow for STAT5's subsequent phosphorylation and activation. Loss of NOX2 generated ROS could therefore prevent TCR induced STAT5 phosphorylation as well as GATA-3 translation because SHP-1/2 remains active, thus push naïve cells toward a more T H1 lineage.

MAPK pathways can be regulated by the direct ROS-mediated inhibition of

MAPK phosphatases as well 50,51 . ROS produced by NADPH oxidases have been shown to inhibit JNK-inactivating phosphatases through reversible oxidation of a catalytic-site cysteine to sulfenic acid, thus sustaining JNK activation. Downstream targets of TCR generated ROS are ERK and FasL, which were found to be regulated by TCR-induced

•- 70 H2O2 and O2 , respectively . Previous studies have found that oxidase deficient T cells have enhanced and sustained ERK activation 71 . Strong TCR signaling characterized by enhanced MEK/ERK signaling has been found to abrogate early TCR induced GATA-3 expression, IL-2 dependent phosphorylation of STAT5 and the early production of IL-

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485 . Conversely, MEK inhibition has been shown to rescue early GATA-3 expression

and responsiveness to IL-2, allowing for early production of IL-4 and subsequent T H2 differentiation 85 .

PI3K and AKT activity have been shown to be reversibly regulated by ROS through the inactivation of PTEN 50,51 . PI3K activity phosphorylates and converts the

lipid second messenger phosphatidylinositol (4,5) bisphosphate (PIP 2) into

phosphatidylinositol (3,4,5) triphosphate (PIP 3), which recruits and activates phosphatidylinositol-dependent kinase 1 (PDK1). PDK1 in turn phosphorylates and activates AKT, which promotes cell proliferation and survival. The tumor-suppressor phosphatase with tensin homology (PTEN) negatively regulates PI3K signaling by dephosphorylating PIP 3, converting it back to PIP 2. H 2O2 generated by stimulation from growth factors such as insulin, EGF, or PDGF, has been shown to oxidize and inactivate human PTEN through disulfide bond formation between the catalytic domain Cys-124 and Cys-71 residues 52,208 . In T-cell acute lymphoblastic leukemia cells, IL-7 induces

NOX-generated ROS by the PI3K/AKT/mTOR pathway 67 . Upon TCR ligation, the early increase in GATA-3 protein levels is a result of post-translational modifications which are mediated by PI3K 203 , suggesting the possibilty that the PI3K pathway is altered during oxidase deficiency. In this situation oxidase activation by TCR ligation induces

ROS production, which would in turn inactivate PTEN. Inactivation of PTEN would subsequently allow for PI3K-mediated increases in GATA-3, thus promoting T H2 differentiation.

Upon TCR crosslinking, T cells activate the kinases ERK, JNK, p38, and AKT, all of which are associated with the production of H 2O2. Certain kinases can undergo

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direct oxidative regulation. The kinase Src as well as the fibroblast growth factor receptor type 1 (FGFR1) are fully active when reduced, and inactivated by oxidation; both of which are regulated by NOX1 55,56 . JAKs, which propagate downstream signaling from the ligation of cytokine receptors, have been shown to be induced by oxidative stress 209-215 . JAKs induce tyrosine phosphorylation within receptor chains, which generate docking sites for STATs. Cytokine stimulation induces a conformational change in the homo- or hetero-dimers of STATs, which in turn bind to phosphorylated cytokine receptor chains via Src homology 2 domains. STAT dimers dissociate from cytokine receptors and translocate to the nucleus, where they act as transcription factors by binding to specific chromosomal DNA sequences. The JAK/STAT signal transduction pathway leads to a very rapid induction of gene activity. STAT5 signaling in macrophages can be mediated through JAK2 as well as TYK2, both of which have been shown to be oxidatively regulated 213,214 . Oxidative stress can also induce

JAK2/STAT3 signaling which mediates the production of cytokines, such as the

216 expression of IL-6 in adipocytes exposed to H 2O2 . A recent study demonstrated that oxidative stress in Mycoplasma pneumoniae -infected cells induced the expression of IL-8 through the activation of JAK2/STAT3 209 .

The regulation of the JAK/STAT pathway by NOX2 generated ROS would suggest oxidative regulation of cytokine signaling, which would in turn regulate lineage differentiation. NOX2 can be activated by ligation of cytokine receptors. In macrophages, GM-CSF signaling and STAT5 phosphorylation have been shown to be sensitive to the antioxidant L-ascorbate (vitamin C) 60 . Membrane signaling events initiated by GM-CSF include the activation of JAK-2, which has recently been identified

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as having a redox-sensitive switch within its catalytic domain215 . In T cells there is

extensive evidence for the crucial role of IL-2 receptor (IL-2R) signaling in T H2 and Treg

development 86 . The IL-2 signaling has been shown to be propagated by ROS

generation 54,62 . In human umbilical vein endothelial cells (HUVECs), IL-2 signaling promoted tube formation and angiogenesis, which was mediated through the production of ROS and phosphorylation of AKT 54 . STAT5 is not only activated by IL-2; all cytokine receptors that use the common gamma (γc) chain, such as the IL-4R, IL-7R, IL-

15R can activate STAT5 217 . A defect in γc signaling would correlate not only to

decreased T H2 differentiation, but also to the survival defects seen in oxidase deficient T

cells 117,167 , and to the decreased populations of memory cells observed in CGD

patients 154,155 . A recent report demonstrated that survival of CD8 + T cells cultured in IL-

15 correlated with increased accumulation of ROS 69 . IL-4 has been shown in numerous

studies to induce the production of ROS 63-65 . Myeloid cells from NOX2 deficient mice in

particular are found to have markedly attenuated IL-4-induced IL-6 and MCP-1

expression 64 . Tregs are another T helper subset that are highly dependent on IL-2 -

STAT5 signaling due to STAT5's ability to induce the expression of Foxp3 218 . There are reports that suggest diminished induction of T regulatory cells associated with

CGD 199,206,219 . NOX-derived ROS have also been shown to be involved in direct Treg mediated suppression of effector T cells 206 . Future studies will be addressing the correlation of oxidase deficiency with decreased Treg induction

In this study, oxidase deficient T cells were found to have an inherent defect in

STAT5 signaling. Common gamma chain signaling mediated by STAT5 in T cells is not mediated by JAK2, but by JAKs 1 and 3. However, if JAK2 can undergo redox

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regulation, the possibility exits that JAKs 1 and 3 can as well. Indeed, recent evidence

has shown, at least in cardiomyocytes, the oxidative regulation of JAK1 211 . However,

because treatment of NOX2 (-/-) cells with IL-2 did not fully restore phosphorylated

STAT5, the oxidative regulation of STAT5 signaling in T cells may be independent of the JAK-STAT pathway. STAT5 can directly associate with the TCR complex and be phosphorylated by the protein tyrosine kinase Lck, a key signaling protein in the TCR complex. This kinase has been demonstrated to activate DNA binding of transfected

STAT5a and STAT5b to specific STAT inducible elements, and activation of STAT5 by

TCR stimulation was found to be abolished in Lck deficient T cell lines 87 . In our lab, the phosphorylation of ZAP-70 and its association with Lck and the CD3 ζ chain of the TCR complex were found to be associated with DUOX1-mediated production of ROS 73 , implicating a strong link between TCR generated ROS, Lck, and STAT5 phosphorylation.

Because STAT5 has been shown in other studies to be oxidatively regulated in

APCs as well as T cells 60 , and it is tempting to speculate that oxidase deficient APCs and

T cells share a common defect in STAT5 signaling, causing the unique inflammatory phenotype observed in CGD. APC intrinsic changes in phosphorylated STAT5 will need to be investigated by future studies, but the increased IL-6 production from oxidase deficient APCs, suggests possible changes in the dichotomy between STAT3 and STAT5 signaling., which would be consistent with studies that found T cells from CDG patients

157 had an increased potential to differentiate into T H17 cells . IL-6 primarily signals through the transcription factor STAT3, which can oppose signaling through STAT5. In cardiomyocytes, the generation of ROS can block STAT3 signaling of IL-6-type

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cytokines by targeting JAK1 211 . STAT3 and STAT5 have opposing actions on the gene targets that they share, and they competitively bind to multiple common sites across the

Il17 locus 205,220 . Also, IL-6 induced upregulation of specific suppressors of cytokine signaling (SOCS) proteins have been demonstrated to inhibit the JAK2-dependent phosphorylation of STAT5 197 . In APCs, a reduction of STAT5 could thus result in increased STAT3 and subsequent increased production of IL-6. In T cells, decreased

STAT5 levels could allow for increased STAT3 binding to the Il17 locus 205 , thus increasing the expression of IL-17. The increased IL-6 production from APCs would serve as a feed-forward mechanism to further enhance T helper skewing. Additionally, increased IL-17 from oxidase deficient T cells can induce further production of IL-6 from

APCs, for the recruitment and activation of circulating neutrophils. This back talk from

T cells to APCs would further feed into the "vicious cycle" of decreased STAT5, increased IL-6 and increased inflammation observed in CGD. As mentioned before, reports suggest decreased Treg populations in CGD, and STAT5 induces the expression of the Treg transcription factor Foxp3. Therefore, decreases in STAT5, in addition to enhanced TNF-α and IL-6 from oxidase deficient APCs can inhibit Treg development, as

STAT3 signaling through IL-6 inhibits Foxp3 expression. Taken together, these changes in T cell and APC function could be responsible for the enhanced T H1 or T H17-mediated responses observed in animal models of CGD, as well as the predisposition towards autoimmunity in patients with this disease.

The results described here may help in the development and understanding of treatment options for patients with CGD. Adding back ROS to oxidase deficient T cells in the form of superoxide generated by treatment with DMNQ enhanced STAT5

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phosphorylation close to that observed in T cells from wild type mice. Pro-oxidants and

antioxidants are relatively nonspecific and thus can have adverse effects on cells. But

future studies may be able to find ways to specifically increase the generation of ROS in

cells from CGD patients. Phytol (3,7,11,15-tetramethyl-2-hexadecene-1-ol) is an acyclic

diterpene alcohol that can be used as a precursor for the manufacture of synthetic forms

of α-tocopherol (vitamin E) and phylloquinone (vitamin K 1), and has been shown to elevate levels of ROS. Treatment of NOX2 deficient rats with phytol increased oxidative burst in vivo. Importantly, phytol treatment also decreased autoimmunity and ameliorated both the acute and chronic phases of rheumatoid arthritis associated with

NOX2 deficiency 221 . Furthermore, when compared to standard therapies for rheumatoid arthritis such as anti-TNF-α and methotrexate, phytol showed equally good or better therapeutic properties, and mediated its effect within hours of administration 221 . Phytol treatment was believed to modulate T cell activation, as injection prevented the adoptive transfer of disease with arthritogenic T cells 221 . Perhaps, improvements in the safety and efficacy of pro-oxidants could help achieve a clinically feasible treatment option for CGD in the future. This study and others have also shown that NOX deficiency in mice leads to enhanced IFN-γ production. However, the standard treatment for CGD is IFN-γ treatment 137 . IFN-γ (Actimmune) has been shown to reduce infections and decrease their severity in CGD patients by nearly 70% 126 . The exact mechanism is still not entirely understood, but NOX2 gene expression is inducible in response IFN-γ139 . Many patients with CGD do not have complete deletion of NOX2. Therefore, in patients with certain variants of XL-CGD, IFN-γ treatment can slightly increase ROS production in their neutrophils and monocytes, increasing innate immunity 139 . However, IFN-γ will not

132

significantly increase superoxide production in the X91 0 or autosomal recessive forms of

the disease 140 . Because IFN-γ treatment has been shown to help almost all patients with

CGD, the more likely scenario is that IFN-γ upregulates oxidase-independent antimicrobial pathways, possibly through the upregualtion of iNOS resulting increased nitric oxide (NO) release from phagocytic cells. A recent study found that patients who received IFN-γ had significantly higher NO levels in their sera than the control group 138 .

One would think that Actimmune would not be necessary for a patient that has inherently

TH1 skewed IFN-γ producing T cells. However, the enhanced IFN-γ production observed

in this study and others was produced by the adaptive immune response, which takes

some time to develop. Actimmune may bypass the need for T cell help and provides

IFN-γ immediately to NOX deficient macrophages and neutrophils.

In addition to CGD, the results of this study open further dialogue about

the involvement of cellular redox status in many other diseases. Oxidative stress has

been shown to play critical roles in the pathogenesis of various diseases. In diabetes,

NADPH oxidase-dependent oxidative stress impairs glucose uptake in muscle and fat and

decreases insulin secretion from pancreatic β cells 222,223 . Increased oxidative stress also

underlies the pathophysiology of hypertension and atherosclerosis. Metabolic syndrome

(which includes hyperglycemia, dyslipidemia, and hypertension) is a growing medical

problem primarily caused by obesity 224 . Its causality to metabolic syndrome is not fully

understood, but it has been shown that elevated levels of fatty acids increase NADPH

oxidase activation leading to the dysregulated production of adipokines 224 . Asthma and chronic obstructive pulmonary disease (COPD) are also diseases characterized by oxidative stress 225 . Sources of oxidative stress arise from increased amounts of inhaled

133

oxidants, as well as elevated amounts of reactive oxygen species (ROS) released from

inflammatory cells 225 . In these situations, and in accordance with our model, treatment with apocynin or antioxidants may reduce oxidative stress and improve disease outcome.

Indeed, in an experimental model of allergic asthma, apocynin was able to attenuate

OVA-induced airway hyperresponsiveness and inflammation, characterized by decreases in macrophages and eosinophils as well as IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid 226 .

On the other hand, CGD patients who lack ROS often present with enhanced inflammation, and as demonstrated by this study, low levels of ROS function as signaling molecules that are important regulators of immune responses. Historically, ROS are suggested to increase rheumatoid arthritis (RA) severity. But recent studies have shown

NADPH oxidase generated ROS to be beneficial 180 . ROS can beneficial during antigen presentation and affect the response and reactivity of T cells 180,194 . Decreased oxidative

stress can also be detrimental in cancer, where ROS play an important role within cells to

act as secondary messengers in intracellular signaling cascades which induce cellular

senescence and apoptosis 227 . Therefore, hypothetically, target-specific administration of

ROS-inducing substances could be therapeutic in other certain diseases.

As mentioned before, all of these conditions suggest the possible use of pro- or

anti-oxidants as treatment options. Unfortunately though, clinical treatment with redox

modulating compounds has overall proved disappointing. Additionally, antioxidants are

double-edged swords. Most antioxidants have pro-oxidant activity under the right

conditions, especially when administered in high doses or in the presence of metal

ions 228 . Free radicals propagate chain reactions, and whether a compound acts as an

134

antioxidant depends on the fate of the secondary radical. Additionally, since ROS in many situations are beneficial, over-consumption of exogenous antioxidants may even lead to a state of “antioxidative” stress, where antioxidants might attenuate or block the adaptive stress responses 227 . Therefore the type, dosage and composition of redox modulating compounds are necessary determining factors that impact the balance between beneficial or deleterious effects in cells. All of these factors are still being tested both in vitro and in clinical settings.

In summary, the results of this study highlight the importance of crosstalk between cells of the innate and adaptive immune systems, demonstrating that the phagocyte NADPH oxidase acts in both a T cell and APC dependent manner to regulate the nature and course of adaptive immune responses. These findings propose potential mechanisms of how NOX2-derived ROS affect the adaptive immune system and suggest that the redox status of a cell can determine the outcome of an immune response. In the future this study and others may allow for an improved understanding of ROS in immune modulation and help in the discovery of new treatments for CGD, and other free radical- mediated diseases.

135

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