The Role of Sigirr in Innate and Adaptive Immunity

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Muhammet Fatih Gulen Cleveland State University

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This Dissertation is brought to you for free and open access by EngagedScholarship@CSU. It has been accepted for inclusion in ETD Archive by an authorized administrator of EngagedScholarship@CSU. For more information, please contact [email protected]. THE ROLE OF SIGIRR IN INNATE AND ADAPTIVE IMMUNITY

MUHAMMET FATIH GULEN

Bachelor of Science in Molecular Biology and Genetics

Middle East Technical University, TURKEY

June, 2004

Submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY IN REGULATORY BIOLOGY

at the

CLEVELAND STATE UNIVERSITY

February, 2010

©Copyright by Muahmmet Fatih Gulen 2010

This thesis/dissertation has been approved for doctoral degree for the Department of

Biological, Geological, and Environmental Sciences and for the College of Graduate

Studies of Cleveland State University by

______Date:______Xiaoxia Li, PhD, CCF-LRI Major Advisor

______Date:______Crystal M Weyman, PhD, CSU-BGES Advisory Committee Member

______Date:______Christine S Moravec, PhD, CCF-LRI Advisory Committee Member

______Date:______Clemencia Colmenares, PhD, CCF-LRI Advisory Committee Member

______Date:______Vincent K Tuohy, PhD, CCF-LRI Internal Examiner

______Date:______Lina Lu, MD, CCF-LRI External Examiner

DEDICATION

To my uncle for his great guidance and my parents for their steady love and support during all my life. ACKNOWLEDGEMENTS

Many thanks to my advisor Dr. Xiaoxia Li for her great support in every ways and understanding during my Ph.D. education. I thank my advisory committee Dr. Crystal M

Weyman, Dr. Clemencia Colmenares and Dr. Christina S Moravec for their valuable suggestions and for their time.

To the Molecular Medicine CSU-CCF Program for the financial support for this project.

To all my colleagues in LI LAB for their friendship and moral support.

THE ROLE OF SIGIRR IN INNATE AND ADAPTIVE IMMUNITY

MUHAMMET FATIH GULEN

ABSTRACT

Single immunoglobulin IL-1R-related receptor (SIGIRR) is a transmembrane

protein, which belongs to interleukin 1 receptor (IL-1R)/Toll like receptor (TLR) superfamily (Toll-IL-1R superfamily). SIGIRR has been identified as a negative modulator for IL-1R/TLR signaling. To asses the physiological function of SIGIRR, the

expression of SIGIRR mRNA in different mouse tissues was analyzed. Although SIGIRR is expressed in many tissues, its expression in different cell types was more restricted, with extremely high expression in primary kidney, colon epithelial cells and T cells, and

moderate expression in dendritic cells (DCs). This expression pattern shows a possible

regulatory role of SIGIRR in innate and adaptive immune systems.

In chapter II, we present in vivo and ex vivo study results showing that SIGIRR in

colon epithelial cells plays an important role in the colonic epithelial homeostasis and

inflammation by maintaining the microbial tolerance. The colonic epithelium of Sigirr-/- mice has increased response to microflora which leads to altered colonic epithelial homeostasis. Moreover, Sigirr-/- mice showed hypersensitivity to DSS-induced colitis due to uncontrolled inflammation in the colon, and Sigirr-/- mice developed increased tumors

in the colon in response to AOM induced tumorigenesis. Interestingly, overexpression of

vi SIGIRR in colon epithelium of Sigirr-/- mice normalize some of the defects seen in Sigirr-

/- mice colonic homeostasis, and reduced the hypersensitivity of Sigirr-/- mice to DSS induced colitis and AOM induced tumorigenesis.

In chapter III, we also investigated the function of SIGIRR in Th17 cells. We presented data showing that Sigirr-/- mice have increased Experimental Autoimmune

Encephalomyelitis (EAE) phenotype due to increased MOG 35-55-specific Th17 cell

polarization in response to MOG35-55 immunization. This increased polarization has been

shown to be dependent on negative regulatory function of SIGIRR in Th17 cells. SIGIRR

in Th17 cells limits the IL-1R dependent Th17 cell expansion by suppressing IL-1R

signaling. More importantly, our data indicated that mTOR mediated cell proliferation by

IL-1R signaling in Th17 cells is the key component which leads to increased Th17 cell

proliferation in SIGIRR-deficient Th17 cells. Thus, our studies have a great significance

showing the important regulatory role of SIGIRR in innate and adaptive immune

systems.

vii TABLE OF CONTENTS

ABSTRACT ...... VI

ABBREVIATIONS USED ...... XI

LIST OF FIGURES ...... XVI

CHAPTER I. INTRODUCTION...... 1

1. The interleukin 1 receptor/Toll like receptor superfamily……………………………1

1.1. The Immunoglobulin (Ig) Domain Subgroup…………………………………..3

1.2. The Leucine Rich Repeat (LRR) Subgroup……………………………………4

1.3. IL-1R/TLR Signaling Pathways………………………………………………..5

2. Negative Regulation of Toll-IL-1R Superfamily……………………………………10

2.1. Single Immunoglobulin Relate Receptor (SIGIRR)…………………………..12

3. Commensal-host interaction in gut and the pathogenesis of Inflammatory

Bowel Diseases…………………………………………………………………...... 15

3.1. Innate Immune System Components…………………………………………16

3.2. Adaptive Immune System Components……………………………………...18

3.3. DSS induced colitis/ AOM induced tumorigenesis models……………...... 19

4. Experimental Autoimmune Encephalomyelitis (EAE)………………………...... 21

4.1. Effector T helper (Th) cells……………………………………………...... 24

4.1.1. Th1/Th2 cells……………………………………………………….24

4.1.2. Th17 cells in mouse....……………………………………………...27

viii CHAPTER II. THE TOLL–INTERLEUKIN-1 RECEPTOR MEMBER SIGIRR

REGULATES COLONIC EPITHELIAL HOMEOSTASIS, INFLAMMATION

AND TUMORIGENESIS…………………………………………………………..30

ABSTRACT………………………………………………………………...... 31

INTRODUCTION………………………………………………………………31

RESULTS…………………………………………………………………...... 35

The colon epithelium of SIGIRR-deficient mice exhibits altered homeostasis……………………………………………………………………...... 35

Constitutive signaling in SIGIRR-deficient colon epithelium is dependent on commensal bacteria………………………………………………….37

The impact of SIGIRR deficiency on intestinal inflammation……...... 43

The impact of SIGIRR deficiency on colitis-associated cancer….……..51

Hyperactivation of NF-κB and STAT3 contribute to increased tumorigenesis in SIGIRR-deficient mice…………………………………………...55

The impact of gut-epithelial cell specific expression of SIGIRR

on the homeostasis of colon epithelium, intestinal inflammation and tumorigenesis……………………………………………………………………….59

The role of epithelial-derived SIGIRR in the regulation of chemokine gene expression…………………………………………………………66

DISCUSSION…………………...... 69

MATERIAL and METHODS…………………………………………………..74

REFERENCES…………………………………………………………….……79

ix CHAPTER III. THE RECEPTOR SIGIRR SUPPRESSES TH17 CELL

PROLIFERATION VIA INHIBITION OF THE INTERLEUKIN-1 RECEPTOR

PATHWAY AND MTOR KINASE ACTIVATION……………...... 82

ABSTRACT………………………………………………………………...... 83

INTRODUCTION………………………………………………………….....83

RESULTS…………………………………………………………………...... 87

SIGIRR expression is induced during Th17 cell differentiation………87

SIGIRR deficiency leads to increased susceptibility to Th17 cell-dependent EAE……………………………………………………………...... 89

SIGIRR directly regulates Th17 but not Th1 cell development…….....92

SIGIRR suppresses Th17 cell differentiation and proliferation through IL-1R signaling……………………………………………………………96

SIGIRR deficiency leads to increased IL-1-induced JNK and

mTOR phosphorylation……………………………………………………………99

SIGIRR-modulated IL-1-activated mTOR pathway signals through the IRAK proteins…………………………………………………………………104

DISCUSSION……………………………………………………………...... 107

MATERIAL and METHODS……………………………………………...... 112

REFERENCES……………………………………………………………….116

CHAPTER IV. GENERAL DISCUSSION AND FUTURE WORK…………….122

REFERENCES [For Chapter I and Chapter IV]………………….……………….127

x ABBREVIATIONS

AOM – azoymethane

AP-1 – activator protein 1

APC – antigen presenting cell

ATF – activating transcription factor

ATG16L1 – autophagy-related 16-like 1 gene

BBB – blood brain barrier

BrDU – bromodeoxyuridine

BSA – bovine serum albumin

CD – Crohn’s Disease

CIA – collagen induced arthritis

CNS – central nervous system

CREB – cAMP response element binding cDNA – complementary DNA

DC – dendritic cell

DEPC – diethylene pyrocarbonate

DMEM – Dulbecco’s modified Eagle’s medium

DNA – deoxyribonucleic acid

DNTPs – deoxynucleotide triphosphates

DSS – dextran sulphate sodium

EAE – experimental autoimmune encephalomyelitis

ELISA – enzyme-linked immuno sorbent assay

ER – endoplasmic reticulum

xi ERK – extracellular-signal regulated kinase

EST – Expressed Sequence Tags Database

FCS – fetal calf serum

Fabp1 – fatty acid binding protein 1

FITC – fluorescein isothiocyanate

GFP – green fluorescence protein

GMCSF – granulocyte-macrophage colony-stimulating factor

GWAS – genome-wide association studies

HEK – human epithelial kidney cells hGH – human growth hormone

HMGBP1 – high mobility group box protein 1

IBD – inflammatory bowel disease

IC – immune complex

IEC – Intestinal epithelial cells

IFN – interferon

Ig – immunoglobulin

IKK – IκB Kinase

IL-1R – interleukin 1 receptor

IP-10 – interferon-gamma-induced protein

IRAK – interleukin-1 receptor associated kinase

IRF – interferon regulatory factor

IRGM – immunity-related GTPase family M

ITS – insulin/transferrine/selenium

xii JNK – C-jun N-terminal protein kinase

KC – keratinocyte-derived chemokine

LB – luria-bertani broth

LBP – LPS binding protein

LPS – lipopolysacharide

LRR-leucine rich repeat

MCP-1 (CCL2) – monocyte chemoattractant protein-1

MDP – muramyl dipeptide

MEKK – mitogen-activated protein kinase kinase kinase

MHC – major histocompatibility complex

MIP-2 (CXCL2) – macrpphage inflammatory protein 2

MMP – matrix metalloproteases

MOG – myelin oligodendrocyte glycoprotein

MS – multiple sclerosis mTOR – mammalian target of rapamycin

MyD88 – myeloid differentiation protein 88

NF-κB – nuclear factor kB

NK – natural killer cells

NLR – Nod like receptor

NLS – nuclear localization sequence

NOD – nucleotide-binding oligomerization domain

PAMPs – pathogen-associated molecular patterns

PBS – phosphate-buffered saline

xiii PCR – polimerase chain reaction

PE – phycoerythrin

PI3K – phosphoinositide 3-kinase

PRR – pattern recognition receptors

PS – penicillin/streptomycin

Rag1 – recombination activating protein

RIG – retinoic acid-inducible gen 1

RNA – ribonucleic acid

RORγt – retinoic acid-related orphan receptor

ROS – reactive oxygen species

RT – reverse transcriptase

RV – retrovirus

SARM – sterile alpha and HEAT/armadillo motif protein

SIGIRR – single immunoglobulin IL-1-related receptor

SLE – systemic lupus erythematosus

SNP – single nucleotide polymorphism snRNP – small nuclear ribonucleoproteins

SOCS-1 – suppressor of cytokine signaling 1 ss/ds – single-, double-stranded

STAT – signal transducer and activator of transcription

TAK1 – transforming growth factor-β-activated kinase 1

TBS – tris buffered saline

TCF4 – transcription factor 4

xiv TCR – T cell receptor

TGFβ – transforming growth facor β

Th – T helper cells

TIR8 – toll interleukin-1 receptor 8

TIZ – TRAF6-inhibitory zinc finger protein

TLR – toll-like receptor

TNF – tumor necrosis factor

TRAF – TNF receptor associated factor

TRAM – TRIF-related adaptor molecule

TRIF – TNF receptor-inhibitory factor

TSC1/2 – tunerous sclerosis protein 1/2

TUNEL – terminal dUTP nick-end labeling

U – unit

VNMA – vancomycin, neomycin, metronidazole, and ampicillin

xv LIST of FIGURES

Figure 1.1. The known members of Toll-IL-1R superfamily and their lingands……….2

Figure 1.2. IL-1R/TLR signaling pathways……………………………………………..7

Figure 1.3. Inhibitory molecules in IL-1/TLR signaling………………………………11

Figure 1.4. Development of EAE……………………………………………………...22

Figure 1.5. Identified effector T helper cell lineages…………………………………..26

Figure 2.1. Cell Type-Specific Expression of SIGIRR………………………………..36

Figure 2.2. Increased cell proliferation and survival in Sigirr-/- mice colon……….…..38

Figure 2.3. Commensal Bacterial Counts in Antibiotic-Treated Mice…………….…..42

Figure 2.4. Constitutive upregulation of proinflammatory cytokines and chemokines in colon of Sigirr-/- mice…………………………………………………………….….44

Figure 2.5. Sigirr-/- mice are highly susceptible to develop DSS-induced colitis….….46

Figure 2.6. Sigirr-/- Mice Develop More Severe Colitis than Wild-Type Mice……....48

Figure 2.7. Sigirr-/- Colon Showed Increased Inflammatory Cell Infiltration after DSS Treatment………………………………………………………..…….……49

Figure 2.8. Expression of Cytokines in Colon Tissues from Wild-Type and

Sigirr-/- Mice……………………………………………………………………….….50

Figure 2.9. SIGIRR deficiency enhances tumorigenesis in mouse colon……………..53

Figure 2.10. Macroscopic View of Tumors…………………………………………...54

Figure 2.11. Increased cell proliferation and enhanced inflammatory response in

Sigirr-/- mice during early stage of tumorigenesis induced by AOM+DSS treatment...56

Figure 2.12. DSS-Induced Colitis in Bone Marrow-Transplanted Mice…………...…60

xvi Figure 2.13. Construction of Gut-Specific SIGIRR Transgenic Mice………………..61

Figure 2.14. Gut-epithelial specific expression of SIGIRR-transgene rescued the

Sigirr-/- mice from severe DSS-induced colitis………………………………………..63

Figure 2.15. Chemokine production was highly induced in Sigirr-/- colon tissue

and crypt cells after DSS treatment……………………………………………………67

Figure 2.16. Expression of Chemokines in Colon Tissue from Wild-Type and

Sigirr-/- Mice……………………………………………………………………..…...68

Figure 3.1. SIGIRR expression is induced during Th17 cell differentiation………....88

Figure 3.2. SIGIRR deficiency leads to increased susceptibility of Th17-dependent

EAE and hyper activation of MOG-specific Th17 cells………………….………..….90

Figure 3.3. T-cell-derived SIGIRR regulates Th17 cell development in vivo…….…93

Figure 3.4. SIGIRR suppresses Th17 differentiation and expansion through

IL-1R signaling………………………………………………………………….……97

Figure 3.5. SIGIRR suppresses Th17 expansion through IL-1-induced mTOR-mediated cell proliferation……………………………………………….….100

Figure 3.6. IL-1 activates mTOR pathway through IRAK proteins………………...105

The experiments done by Muhammet Fatih Gulen in Chapter II and Chapter III (based on Figures) :

Chapter II: Figure 2.2.-Figure 2.7., Figure 2.14. (A, C, D, E), Figure 2.15 (A and B)

Chapter III: All the experiments related the figures in this chapter were done by Muhammet Fatih Gulen.

xvii

CHAPTER I

INTRODUCTION

1. THE INTERLEUKIN 1 RECEPTOR/TOLL LIKE RECEPTOR

SUPERFAMILY

The Toll-Interleukin 1 Receptor superfamily is composed of a large number of type I transmembrane receptors, which play crucial role in host defense by initiating inflammation and shaping adaptive immune responses. While all the members within this family contain an intracellular Toll-IL-1 receptor (TIR) domain, which mediate a highly conserved signaling network, whereby promotes proinflammatory responses, they can be further divided into two subfamilies based on their distinct extracelluar domains. Toll- like receptor subfamily receptors possess leucin-rich repeats (LRR) in their extracellular domains, which can directly recognize the so-called Pathogen-Associated Molecular

Patterns (PAMPs) carried by invading pathogens. On the other hand, IgG-domain containing subfamily members bind to endogenous cytokines, regulating both innate and adaptive immunity.

Figure 1.1: The known members of Toll-IL-1R superfamily and their lingands

2 1.1. The Immunoglobulin (Ig) Domain Subgroup

The immunoglobulin (Ig) domain subgroup includes IL-1RI (the first identified

member of the family), interleukin accessory protein (IL-1RAcP), IL-1RII, IL-1Rrp2, IL-

18R (IL-1Rrp), IL-18RAcP (also termed AcPL), ST-2 and the orphan receptors IL-1 receptor accessory protein-like (IL-1RAPL) and SIGIRR. Except SIGIRR, all IL-1R family members contain three Ig domains in their extracellular domains, and other than

IL-1RII, all receptors have intracellular TIR domain which recruits TIR-containing adaptors to initiate a proinflammatory signaling network through homotypic protein- protein interaction.

IL-1RI and IL-1R AcP are the bona-fide receptors for IL-1, and all the physiological functions of IL-1 and IL-1 are mediated by the IL-1RI/IL-1R AcP heterodimers. Like IL-1RI, IL-1RII can also bind to IL-1, but fail to initiate signaling events due to its shortened intracellular domain. Interestingly, IL-1RII seems to function as a negative regulator for IL-1 signaling by competing with IL-1RI for IL-1.

IL-18R is very similar to IL-1RI complex. Like in IL-1R signaling, IL-18/IL-

18R/IL-18RAcP heterotrimeric complex formation occurs in response to IL-18 stimulation. However IL-18R expression is more restricted and signaling intensity is modulated differently. IL-18R is expressed on leukocytes such as naïve T cells, mature

Th1 cells, NK cells, macrophages, B cells, neutrophils, basophils, mast cells, as well as endothelial cells, smooth muscle cells, synovial fibroblasts, chondrocytes, and epithelial cells (Yoshimoto et al., 1998; Nakamura et al., 2000; Gerdes et al., 2002; Sims, 2002). In

3 these cells IL-18R signaling is modulated by the regulation of IL-18RAcP expression in

synergism different cytokines such as IL-12 and IL-21 (Strengell et al., 2003; Neumann

and Martin, 2001). IL-18 is known as the interferon-γ (IFNγ) inducer. IL-18 stimulation

of naïve T cells promotes development and differentiation of Th1 cells. Cytolytic activity of NK cells and cytotoxic lymphocytes is upregulated by IL-18 during the inflammation.

While IL-18R signaling is one of the main players in Th1 response, ST2 had been

shown to play a critical role in Th2 cell activation. ST2 is highly expressed on Th2

cells(Lohning et al., 1998). In response to IL-33 which is an IL-1 like cytokine, ST2 forms heterodimeric receptor complex with IL-1RAcP and induce signaling through NF- kB pathway and MAPKs (Schmitz et al., 2005; Chackerian et al., 2007). When we consider the effect of IL-1 itself on another T helper cell lineage, Th17 cell, expansion

(Chung et al., 2009), IL-1R family seems to have a key regulatory role in T helper cell activation and function.

1.2. The Leucine Rich Repeat (LRR) Subgroup

The LRR subgroup consists of at least twelve Toll-like receptors (TLRs) (Chung et al., 2009; Medzhitov et al., 1997; Rock et al., 1998; Takeuchi et al., 1999b; Chuang and Ulevitch, 2000; Hemmi et al., 2000; Zhang et al., 2004). These receptors have

received intense attention because different TLRs were found to be activated by specific

pathogen products. TLRs share conserved intracellular TIR domain with IL-1R family in

their C-terminal. However, N-terminal domain of TLRs consists of 16 to 28 leucin-rich

repeats with a conserved motif “LxxLxLxxN” which is responsible for the pathogen

4 recognition (Medzhitov, 2007; Beutler, 2009). While TLR4 has been genetically identified as a signaling molecule essential for the responses to LPS, a component of

gram negative bacteria(Poltorak et al., 1998), TLR2 responds to mycobacteria, yeast, and

gram-positive bacteria (Takeuchi et al., 1999a; Takeuchi et al., 2000; Underhill et al.,

1999a; Underhill et al., 1999b). TLR2 associates with TLR6 and recognizes lipoproteins

from microplasma, or TLR2 may also act together with TLR1 in recognition of bacterial

lipoproteins (bLP). While TLR5 mediates the induction of the immune response by

bacterial flagellins (Hayashi et al., 2001), TLR9 has been shown to recognize bacterial

DNA(Hemmi et al., 2000), and TLR3 recognizes dsRNA (Alexopoulou et al., 2001).

While a synthetic compound (imidazoquinoline compound R848) with antiviral activity

has been described as a ligand for TLR7 and TLR8(Jurk et al., 2002), recent studies

showed that ssRNA is the natural ligand for TLR7/8(Diebold et al., 2004; Heil et al.,

2004). Natural ligand for TLR11 has been identified as the protozoan derived profiling-

like protein, which shows that eukaryotic microorganisms can be recognized by TLRs as

well(Yarovinsky et al., 2005). Although it has been shown that TLR10 is selectively

expressed and functional in human B cells, the natural ligand for TLR10 is still not

known (Hasan et al., 2005; Zhang et al., 2004) [Figure 1.1]. Upon ligand stimulation,

signaling cascades differentiate and leads to phosphorylation of different kinases and

activation of several transcription factors.

1.3. IL-1R/TLR Signaling Pathways

In response to IL-1, IL-1R associates with IL-1RAcp and recruits

MyD88(Wesche et al., 1997). MyD88 acts as an adapter, recruiting two Ser/Thr kinases,

5 IRAK4 and IRAK1, to the receptor complex, resulting the phosphorylation of IRAK1

(Cao et al., 1996; Li et al., 2002; Li et al., 1999; Suzuki et al., 2002) [Figure 1.2]. The phosphorylated IRAK1 then recruits TRAF6 to the receptor complex and dissociate from

MyD88. Dissociated IRAK-TAF6 complex then recruit and activate transforming growth

factor-β-activated kinase 1 (TAK1) or mitogen-activated protein kinase kinase kinase 3

(MEKK3) which subsequently phosphorylates IKK complex which includes IκB Kinase

α (IKKα), IKKβ and IKKγ. While the kinase activity of IRAK is not necessary for its function, loss of entire IRAK protein in the cells cause impaired IL-1R signaling (Yao et al., 2007). On the other hand, the kinase activity of IRAK4 is required for TAK1 dependent Nuclear Factor-κB (NFκB) activation (Fraczek et al., 2008). Activated TAK1 phosphorylates IKKα/β, resulting in phosphorylation and degradation of IκBα, releasing

NFκB to be translocated to nucleus. When activated through IL-1R signaling, MEKK3 can phosphorylate IKKγ and activate IKKα, resulting in IκBα phosphorylation and subsequent dissociation from NFkB without degradation. Both TAK1 and MEKK3 pathways activate NFkB, their relative contributions in Toll-IL-R-induced physiological functions remain to be uncovered.

Upon activated, TLRs can activate NFkB or/and IRF3/7, leading to the induction of interferons, cytokines and chemokines. . TLR3, TLR7, TLR8 and TLR9 are expressed on endosome or endoplasmic reticulum (ER) (Ewald et al., 2008; Tabeta et al., 2004), sensing the nucleic acids released from pathogen degraded by lysosome. TLR2 and

TLR5 are present on the cell surface, and mainly activate NFkB, but not IRF3/7.

Interestingly, TLR4 has been shown to initiate signaling both on cell membrane and

Figure 1.2. IL-1R/TLR signaling pathways

7 endosome compartment (Kagan et al., 2008). On the cell surface, activated TLR4 recruits

two different TIR domain containing adapter proteins, MyD88 and Mal, to mediate early phase NFkB activation. Activated TLR4 then traffics to endosome where it recruits another TIR domain containing adapter protein, TRIF, through TRAM. Trif-mediated signaling leads to the activation of IRF3, which induce type I interferon production, as well as the late phase NFkB activation.

TLR5 are present on the cell surface, and mainly activate NFkB, but not IRF3/7.

Interestingly, TLR4 has been shown to initiate signaling both on cell membrane and endosome compartment (Kagan et al., 2008). On the cell surface, activated TLR4 recruits two different TIR domain containing adapter proteins, MyD88 and Mal, to mediate early phase NFkB activation. Activated TLR4 then traffics to endosome where it recruits another TIR domain containing adapter protein, TRIF, through TRAM. Trif-mediated signaling leads to the activation of IRF3, which induce type I interferon production, as well as the late phase NFkB activation.

In addition to NFκB activation, IL-1R-mediated signaling also activate MAPKs, such as extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinases (JNKs), p38 MAPK, which activates transcription factors ATF2, AP-1 or CREB to regulate gene

8 expression(O'Neill, 2008). Together with NFκB and IRF3/7, activation of these kinases

mediate expression of genes involved in the regulation of inflammation, host defense, cell

proliferation and apoptosis (Kumar et al., 2009). These genes not only define the fate of

the stimulated cell, but also secreted cytokines and chemokines regulate the innate and

adaptive immune responses against pathogens.

While proinflammatory cytokines, chemokines and interferons are directly

involved in inflammation and host defense mechanisms, TLRs induced genes are also associated with tissue remodeling and regulation of adaptive immunity. Upon infecton,

TLRs induce the expression of major histocompatibility complex class II (MHC II) and

co-stimulatory molecules on antigen presenting cells (APCs), as well as the secretion of

proinflammatory cytokines, IL-6, IL-12, TNFa to activate T cell and B cell-mediated

immune responses

The impact of Toll-IL-1R signaling on the development stage and effector stage

of T cells is crucial. Defects in the Toll-IL-1R signaling in mice may cause susceptibility

to infections. However, enhanced signaling may also cause other complications such as

changes in tissue homeostasis and increased autoimmune responses. To avoid these

complications, Toll-IL-1R signaling is tightly controlled.

9 2. NEGATIVE REGULATION of TOLL-IL-1R SUPERFAMILY

Uncontrolled inflammatory responses can cause autoimmune diseases, organ failure and death. While the role of Toll-IL-1R signaling in innate and adaptive immune responses has been well studied, recent studies have linked dysregulated Toll-IL-1R signaling to autoimmune diseases and cancer, highlighting the important role of a variety of negative regulators. A large number of negative regulators have been demonstrated to regulate Toll-IL-1R signaling via a variety of mechanisms. While inhibitory orphan receptor molecules, decoy receptors and nonfunctional mutated ligands, regulate signaling at receptor level, several inhibitory molecules have been reported to block intracellular signaling pathways of Toll-IL-1R signaling. Interestingly, the expression of the negative regulators may be regulated differently in specific tissues, suggesting that these molecules may be involved in controlling Toll-IL-1R signaling in a cell type specific manner.

Among the inhibitory molecules which negatively regulate the intracellular signaling components, IRAKM, a member of IRAK family proteins, is implicated in the negative regulation of TLR signaling specifically in monocyte/macrophages (Kobayashi et al., 2002) [Figure 1.3]. Although the detailed mechanism by which IRAKM negatively regulates Toll-IL-1R-mediated signaling is still unclear, it has been shown that in the presence of IRAKM, dissociation of IRAK1 from receptor complex and the subsequent formation of IRAK1-TRAF6 complex is inhibited. Toll-IL-1 stimulation and bacterial challenge cause increased cytokine production in IRAKM deficient cells as compared to

Figure 1.3. A) Inhibitory molecules in IL-1/TLR signaling (labeled in red color) B)

Structure function analysis of SIGIRR protein: Both extracellular and intracellular domains of SIGIRR are necessary to inhibit IL-1R signaling (left). Intracellular TIR domain alone can inhibit TLR4 signaling (right).

11 wild type cells. Splicing variants of IRAK2, IRAK2c and IRAK2d are shown to be negative regulators for Toll-IL-1R superfamily (Hardy and O'Neill, 2004). Like splicing variant MyD88s, an non-functional version of MyD88, IRAK2 splicing variants prevents recruitment of IRAK4 to the receptor (Janssens et al., 2002; Burns et al., 2003). On the other hand, SARM, a newly identified TIR domain containing adaptor protein, negatively regulates TRIF dependent TLR pathway (O'Neill and Bowie, 2007). In the downstream complexes, interaction of TRAF6 with a newly identified zinc finger domain containing protein members such as A20, Cezanne, TRAF6-inhibitory zinc finger protein (TIZ),

FLN29 and ZCCHC11 cause suppression of TLR dependent NFkB activation (Mashima et al., 2005; Minoda et al., 2006; Shin et al., 2002). Furthermore, a Ring finger protein,

Triad3A, which acts as an E3 ubiquitin-protein ligase, regulates the intensity and duration of TLR signaling (Chuang and Ulevitch, 2004) by targeting TLRs for their ubiquitination and degradation.

2.1. Single Immunoglobulin Relate Receptor (SIGIRR)

Screening of EST database for TIR domain containing proteins revealed SIGIRR as a TIR domain containing receptor which has only one Ig domain in its extracellular site (Thomassen et al., 1999). The open reading frame of the transcript encodes a protein in size of 410 amino acids in humans and 409 amino acids in mice. However, the actual size of the protein is much larger and varies among different tissues due to its differential glycosylation. The first 118 amino acids in N-terminal cover a very short extracellular Ig domain with 4 to 5 glycosylation sites. Possibly, the small size of this extracellular domain do not allow protein to form a ligand binding site (Barclay, 2003). A very long

12 TIR domain containing intracellular site (268 amino acids) is connected to a transmembrane domain. The TIR domain of SIGIRR displays two critical amino acid substitutions (Ser447 and Tyr536), which may contribute to its distinct structure and incapable of initiating signaling (Garlanda et al., 2009). Although SIGIRR is member of

IL-1R family, chromosomal location of SIGIRR is totally different from the other IL-1R family members. Except SIGIRR gene, which is located on chromosome 11 at 11p15 locus (Sims et al., 1995; Thomassen et al., 1999), the rest of IL-1R family gene members all reside on chromosome 2. The expression of Sigirr is highly cell type specific..

SIGIRR mRNA is abundantly present in the epithelium of kidney, gastrointestinal tract and lung, moderately expressed in splenocytes, and low or undetectable in fibroblast and endothelial cell lines (Garlanda et al., 2004; Wald et al., 2003). While SIGIRR is expressed in spleen T cells and dendritic cells (DCs), no expression is detected in B cells

(Wald et al., 2003). Interestingly, the expression level of SIGIRR in macrophages is downregulated in response to LPS under in vivo and in vitro conditions (Garlanda et al.,

2004; Polentarutti et al., 2003).

A number of studies have identified SIGIRR as a negative regulator for IL-1 and

TLR signaling. It has been shown that SIGIRR inhibits IL-1, IL-18, ST-2, TLR-4 and

TLR-9 receptor signaling (Bulek et al., 2009; Wald et al., 2003). In response to ligand stimulation, SIGIRR binds and form heterodimer with the receptor. While SIGIRR interacts with TLRs through its TIR domain, both extracellular domain of SIGIRR and

TIR domain are required for SIGIRR to bind to IL-1R. Through its binding to ligand bound receptors, SIGIRR blocks the recruitment of TIR domain containing adaptor

13 proteins to the receptor and inhibits downstream signaling. The SIGIRR deficient cells

showed increased cytokine/chemokine secretion in response to LPS, CpG and IL-1

stimulation. In consistent with this data, overexpression of SIGIRR in Jurkat cells inhibits

IL-1R and IL-18R signaling (Wald et al., 2003).

Further studies showed that in response to Ischemia-reperfusion, SIGIRR

deficient mice displayed increased postischemic acute renal failure, probably due to more lymphocyte infiltration to kidney and higher cytokine/chemokine production from renal myeloid cells in SIGIRR deficient mice as compared to wild type mice (Lech et al.,

2009). SIGIRR deficient mice have been crossed with lupus mice to produce SIGIRR- deficient-lpr/lpr mice, and SIGIRR deficiency on lpr background caused severe autoimmune lung disease and lupus nephritis due to increased active T cells and autoantibody production against nuclear lupus autoantigens (Lech et al., 2008). The lupus phenotype has been associated with the function of SIGIRR in DCs.

Moreover, SIGIRR-deficient mice showed increased susceptibility to LPS induced septic shock and dextran sulphate sodium (DSS)-induced chronic colitis

(Garlanda et al., 2004; Wald et al., 2003). However, the underlying mechanisms and the

cell types involved in hyper inflammation due to DSS treatment in SIGIRR deficient

mice have not been studied. Our recent understanding shows that in addition to

leukocytes, other cell types such as epithelial cells and endothelial cells may contribute to initiation and severity of inflammation in gut. To investigate the role of SIGIRR in innate immune and adaptive immune regulation, we have examined the function of SIGIRR in

14 colon epithelial cells (innate immune response) and T cells (adaptive immune response) in the pathogenesis of autoimmune diseases, such as colitis and EAE.

3. COMMENSAL-HOST INTERACTION IN GUT AND THE PATHOGENESIS

OF INFLAMMATORY BOWEL DISEASES

Human colon is heavily colonized by a large number and diverse groups of microbes. Under physiological conditions, microflora can supply nutrients and energy to host through their metabolism, modulate the development of host immune system and limit pathogen infection, while avoiding triggering harmful inflammatory responses.

(Duerkop et al., 2009). Molecular mechanisms attribute to Microbial-tolerance have begun to unveil. However, when “Microbila-tolerance” is disrupted, microflora also plays a causative role in the pathogenesis of Inflammatory Bowel Diseases. While mice deficient in IL-10, TGFb or IL-2 develop spontaneous colitis in pathogen-free facility, they are completely disease free under germ-free conditions (Davidson et al., 2000;

Hahm et al., 2001; Hoentjen et al., 2005; Mombaerts et al., 1993; Rakoff-Nahoum et al.,

2006).

Intestinal epithelial cells (IECs) express pattern recognition receptors (PRRs) such as TLRs and NOD-like receptors (NLRs). Expression of TLR2, TLR3, TLR4, TLR5 and

TLR9 is detected in the colon epithelium. The recognition of commensal flora by TLRs and activation of NFκB have been shown to play critical role in the maintenance of colon homeostasis (Rakoff-Nahoum et al., 2004). However, TLR-induced NFkB activation has

15 to be elegantly controlled, and inappropriate activation of NFkB can lead to chronic

inflammation and even cancer development.

For example, epithelial cell specific deletion of A20, suppressor partner of

TRAF6 which is a signaling component in TLR signaling [Figure 1.1], leads to

spontaneous colitis and premature death in A20 deficient mice (Turer et al., 2008).

The gut epithelium layer constitute the physical barrier essential to segregate

microflora from a large number of immune cells reside in lamina propria. Tight junctions

formed between epithelial cells contribute to the integrity of epithelium layer, and are

crucial to maintain microbial tolerance and prevent pathogen invasion as well. As

epithelium layer is under constant renewal, signaling pathways controlling cell

proliferation, differentiation and apoptosis have also been implicated in regulating

microbial tolerance and inflammatory bowel diseases.

3.1. Innate Immune System Components

Interestingly, the first genetic risk factor identified for Crohn’s disease (CD) (a subtype of IBD) was NOD2, an intracellular receptor for bacteria component

MDP(Hugot et al., 2001). NOD2 is expressed in dendritic cells, macrophages and intestinal epithelial cells. Upon activated by bacterial peptidoglycan, Muramyl dipeptide

(MDP), NOD2 induced NFkB activation, and the induction of antibacterial defensins.

Consistent with this finding, the distribution of the antibacterial defensins were shown to

be altered in IBD patients (Shi, 2007; Wehkamp et al., 2007).

16 NALP3, another member of NLR controls the activation of inflammasome, which

is essential for maturation and secretion of IL-1β, has also been linked to Crohn’s disease

by Genome Wide Association Study. Moreover, monocytes of the patients displayed

reduced NALP3 expression and compromised IL-1β secretion in both basal level and

stimulated cells (Villani et al., 2009).

In addition to NLRs, genetic variation of TLR receptor family members has been

associated with IBD patients. TLR4 and its adaptor molecule TIRAP have been linked to

elevated risk of IBD on a pooled analysis of 4805 cases (De Jager et al., 2007).

TLRs are expressed in many cells, but their expression in colon is specifically studied enable to understand the relation of microflora and epithelium. The defect in TLR signaling leads to disruption of homeostasis in the colon epithelium and this disrupted homeostasis make TLR deficient mice more susceptible to chemical induced colitis models (Rakoff-Nahoum et al., 2006).

Another very important gene identified related to CD susceptibility is autophagy-

related 16-like 1 gene (ATG16L1)(Zhang et al., 2009a). The ATG16L1 has a function in

autophagy pathway which is an intracellular response to lowlevel invasive bacteria. This

pathway is necessary for the destruction of bacterial pathogens and the cytotoxic effect of

bacterial toxins. ATG16L1 is broadly expressed in the intestinal epithelium, APCs, T

cells and memory B cells. ATG16L1-deficient mice die shortly after birth and primary cells obtained from these animals showed autophagy defect. When hematopoietic cells of

17 ATG16L1-deficient mice transferred to wild type mice, chimeric mice showed increased

levels of the pro-inflammatory cytokines IL-1β and IL-18. Also, these mice had severe

susceptibility to DSS induced colitis (Saitoh et al., 2008). Recent publications implicated

another autophagy gene, IRGM in CD risk. It was initially found that IRGM has an

important role in protection against M. tuberculosis infection. This shows a possible

analogous role of IRGM in granulomatous response which is observed in CD pathogenesis. Genetic and functional studies, after the implication of ATG16LI and

IRGM in CD, show the key role of autophagy pathway in the pathogenesis of CD.

Common genetic variations in ATG16LI and IRGM and other autophagy genes seem to have a major impact on the innate immune response to intestinal flora and cause susceptibility to IBD.

3.2. Adaptive Immune System Components

Innate immune dysregulaion has been extensively studied in IBD pathogenesis.

Another factor affecting this pathogenesis is excessive activation of adaptive immunity which is more proximate driver in tissue damage in IBD patients. Adaptive immunity in

GI tract involves resident and recruited cell populations including mucosal B cells producing secretary IgA and IgG, and a mixture of T cell populations (Th1, Th2, Th17 and regulatory T/B cells). Th1 or Th2-type inflammation is observed in different subtype of IBD patients (Xavier and Podolsky, 2007). However, like in many autoimmune diseases Th17 response play a critical role in all IBD pathogenesis.

18 For a long time the involvement of adaptive immunity in IBD has been suspected

but until first GWAS study there was no evidence. First strong evidence showing the role

of adaptive immunity in IBD comes from the first GWAS with the discovery of involvement of IL-23R in IBD (Duerr et al., 2006). Recent studies showed that there are multiple variants in the region coding IL-23R independently associated with risk of IBD.

IL-23R is part of the heterodimeric membrane receptor for cytokine IL-23. The cytokine itself also is a heterodimeric protein which shares a common subunit with IL-12 (p40 (IL-

12B)). Not only the receptor, but also the IL-12B gene encoding this common subunit has

been recently associated with IBD (Barrett et al., 2008). IL-23R signaling is widely

studied with its function on Th17 cells. Although the real mechanism how IL-23

maintains Th17 responses in vivo is not known, recent studies have shown that IL-23 is

critical in the expansion or maintenance of Th17 cell population. This was the first and

strongest evidence combines IBD susceptibility and Th17 response. IL-23R signaling is

associated with other autoimmune diseases such as ankylosing spondylitis and psoriasis

as well.

3.3. DSS induced colitis/ AOM induced tumorigenesis models

While there are multiple spontaneous colitis animal models such as A20-/-, Muc2-/-

, IL-10-/- and IL-10R-/-, some transgenic animals need to be manipulated in order to observe their sensitivity to colitis. DSS (Dextran Sulfate Sodium) induced colitis is one of

the animal models trigger acute and chronic colitis in rodents. Treating animals with

DSS, which is a detergent, damage the mucosal layer of the intestine and brings the mucosal flora in contact with the apical layer of intestinal epithelial cells. This causes

19 either acute (during DSS administration) or chronic (little time after DSS administration) inflammation in intestine. As a result, shortening of intestine, ulceration and infiltration of lymphocytes into intestinal epithelium is observed with DSS treated animals. Acute colitis is considered as a result of innate immune response, whereas infiltrated lymphocytes are the main cause of chronic colitis (Hibi et al., 2002).

Human IBD patients suffering chronic intestinal inflammation are shown to have a very high risk to develop colorectal cancer. The chronic inflammation in intestine promotes the secretion of cytokines, chemokines and cell growth factors that stimulate surrounding resident cells. These stimuli can either damage the resident cell DNA through the production of reactive oxygen species (ROS), or provide active signaling for the growth and survival of cancer cells. Specifically, IL-6, IL-23 and NFκB activation are shown to contribute the tumor development. To investigate the function of NFκB in the tumor development, AOM (Azoxymethane) induced tumorigenesis model has been used on epithelial specific IKKβ (one of the key kinase in NFκB pathway) deficient mice.

AOM induced tumorigenesis model is used to mimic the pathogenesis of inflammation induced colorectal cancer patients. Injection of AOM, which is highly potent carcinogen, and promoting chronic inflammation in the intestine by DSS treatment develop tumors in the gut. Although epithelial specific IKKβ deficient mice developed reduced number of tumors in the colon, the size and proliferation index of tumor cells were comparable to the tumor in wild type mice. And when the tumor incidences were studied in myeloid cell specific IKKβ deficient mice, tumor sizes reduced greatly. Collectively these data shows that while NFκB pathway in epithelial cells is necessary for tumor initiation, this pathway

20 in myeloid cells leads to expression of cytokines and growth factors which maintain the

progression of tumors. These animal models are very beneficial to study the impact of

different cell populations on inflammation and inflammation induced tumor formation.

4. EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE)

EAE is a Th17 driven autoimmune animal model which mimics Multiple

Sclerosis (MS) pathogenesis. Immunization of animals with peptides derived from

myelin of neural cells lead to activation of peptide specific T cells, which pass through

Brain Blood Barrier (BBB), attack neurons and cause paralysis in animals. Although it has been shown that adoptive transfer pathogenic Th1 cells can induce EAE in mice,

pathogenic function of Th17 cells in active immunization and human patients is more accepted [Figure 1.4].

EAE can reproduce many of the clinical, neuropathological aspects of multiple sclerosis and is therefore widely used to dissect molecular mechanisms of MS and to develop new therapeutic strategies. The concept that MS is a T-cell-mediated autoimmune disease mainly stems from studies with EAE. Studies of the EAE model have helped define the sequence of immunopathogenesis events involved in the development of autoimmune CNS-directed inflammatory diseases. The development of

EAE can be subdivided into initiation stage (activation and expansion of neuroantigen- reactive CD4 T lymphocytes outside of CNS) and effector stage (recruitment and reactivation of neuroantigen-reactive CD4 T lymphocytes and subsequent inflammatory response within CNS).

Figure 1.4. Development of EAE

22 During the initiation stage, protein antigens (myelin components) and peptides are

presented by antigen presenting cells (APCs) within secondary lymphoid organs to the

neuroantigen-reactive T cells leading to their activation and expansion(Steinman, 2001).

During the initiation stage of EAE, in addition to T cell activation and expansion,

the APCs produce cytokines to regulate the differentiation of effector CD4 T cells,

including the classical Th1 (producing IFNγ and TNFα) and Th2 (producing IL-4, IL-5,

and IL-10) subsets of T cells and newly identified Th17 T cell lineage. While Th1 cells

were shown to play a critical role in the initiation of inflammatory responses in

CNS(Agrawal et al., 2006; Yang et al., 2009), Th2 cells were considered as counter-

inflammatory(Kleinschek et al., 2007; Ramirez and Mason, 2000). This Th1/Th2 paradigm is challenged by the fact that lack of IFNγ actually leads to increased EAE

phenotype and mice lacking the Th1-inducing cytokine IL-12 or IFNγ are also hyper-

susceptible to EAE(Cua et al., 2003). Importantly, recent data demonstrate that both Th1

and Th17 cells can independently induce EAE possibly through different

mechanisms(Kroenke et al., 2008; Stromnes et al., 2008). EAE is markedly suppressed in

mice lacking IL-17 or IL-17 receptor and IL-17-specific inhibition attenuates

inflammation, indicating that IL-17-mediated signaling plays a critical role in the effector

stage of EAE(Gonzalez-Garcia et al., 2009; Komiyama et al., 2006). However, the

precise mechanism by which IL-17 participates in EAE development and pathogenesis

remains unclear.

23 4.1. Effector T helper (Th) cells

T lymphocytes are adaptive immune components, derive from hematopoietic stem cells. Their commitment takes place in thymus where CD4+ cells undergo “thymic education” to select the mature T cells which have T cell receptors (TCRs) with a low affinity to self major histocompatibility (MHC) molecules (Fietta, 2007). Matured T cells leave thymus, migrate into bloodstream and reach the peripheral lymphoid organs where their extrathymic maturation occurs. T helper cells develop from naïve CD4+ cells by their interaction with antigen presenting cells in lymphoid organs. Antigen presentations by MHC class II molecule carrying APCs activate naïve T cells and differentiate them into T helper cell lineages. However final commitment of antigen presented naïve T cells depends on complex variables, including antigen type, antigen load, costimulatory signals and cytokines in the microenvironment (Bettelli et al., 2007). Multiple signals induced by these variables may result in differentiation of Th1, Th2 and Th17 cells with distinct cytokine production and immune functions [Figure 1.5].

4.1.1. Th1/Th2 cells

When APCs are exposed to intracellular pathogens, they secrete IL-18 and IL-12 which are the main Th1-polarizing cytokines. In addition to TCR-mediated intracellular signals, Th1- polarizing cytokines stimuli the expression and activation of transcription factors, signal transducer and activator of transcription (STAT) 4 and T-box expressed in

T cells (T-bet), which specifically regulate Th1 cell generation and inhibit Th2 differentiation (Szabo et al., 2000). Differentiated Th1 cells selectively produce IL-2, IL-

3, tumor necrosis factor β (TNF-β) and IFN-γ which strengthen Th1 phenotype in an

24 autocrine loop. Th1 cells peculiarly target intracellular pathogens and malignant cells by supporting cell mediated immunity. IFN-γ secretion activates phagocytosis activity of macrophage and stimulate production of opsonizing, complement fixing immunoglobulin

(Ig)G2a and production of antibodies by B cells. Moreover, this prototypic Th1 cytokine activates endothelial cell expression of adhesion molecules and chemokines for the attraction of mononuclear cells to the inflammation site.

Th2 cells function in the elimination of extracellular pathogens. While IL-4 is considered as the main Th2-polarizing cytokine, IL-13 (through ST2) and IL-25 can also mediate Th2 differentiation. In early studies, STAT6 and GATA-binding protein 3

(GATA-3) were identified as two major transcription factors regulating Th2 differentiation. Recently, another transcription factor, Ikaros, was shown as a key protein in Th2 differentiation by regulating chromatin accessibility (Quirion et al., 2009). Active

Th2 cells secrete cytokines such as IL-4, IL-5, IL-6, IL-10, IL-13 and IL-25 which enhance humoral immune system. Specifically, these cytokines promote B cell differentiation in plasma cells and antibody production. While IL-4 alone activates mast cell/basophil sensitization and enhances Th2 phenotype in an autocrine loop, together with IL-13, it stimulates plasma cell generation and IgG1 and IgE production. IL-5, another important cytokine produced by Th2 cells, induces eosinophil and mast-cell proliferation as well as their IgE receptor expression. In parasitic infections, upregulation of adhesion molecules in eosinophil is enhanced by IL-5 signaling to draw eosinophils to the infection site. While Th2 cytokines IL-4, IL-5 and IL-10 suppress Th1 differentiation,

IFN-γ and IL-10 can inhibit Th2 polarization (Bettelli et al., 2007).

Figure 1.5: Identified effector T helper cell lineages.

26 Recently, a third distinct T helper cell lineage has been identified. This third

lineage has been characterized first for their interleukin17 (IL-17) secretion and so called as Th17 cells. Because of the pathogenic function of Th17 cells in autoimmune diseases, these cells are being widely studied.

4.1.2. Th17 cells in mouse

Th17 cells are proposed as a distinct third T helper lineage with an important role

in host defense and in the development of human and animal autoimmune diseases

(Bettelli et al., 2006; Kolls and Linden, 2004). Multiple bacterial and fungi-like pathogen

infection, such as Citrobacter rodentium, Klebsiella pneumoniae, Mycobacterium

tuberculosis and Candida albicans, develop Th17 response in the host (Huang et al.,

2004; Khader et al., 2007; Korn et al., 2009). Th17 mediated response leads to expression

of antimicrobials as well as expression of chemokine receptors and cytokines by

epithelial cells which leads to clearance of these pathogens. In human, deficiency of Th17

response against pathogens ends with hyper-IgE syndrome, which patients lack the ability to fight against C. albicans and Staphylococcus aureus in their skin and lungs (Milner et al., 2008). While deficiency in Th17 development causes severe infections, uncontrolled

Th17 response is associated with multiple autoimmune diseases. Th17 cells are shown to involve in the pathogenesis of rheumatoid arthritis, psoriasis, asthma, inflammatory bowel diseases and multiple sclerosis (Miossec et al., 2009).

Transcription factors RORγt and RORα synergistically function in the development of Th17 cells. Th17 differentiation was completely attenuated in the mice

27 which both transcription factors were deleted (Yang et al., 2008). STAT3, which is another transcription factor, is also shown to play an important role in Th17 development.

RORγt is one of the target molecule expressed by STAT3 activation (Dong, 2009).

Moreover, STAT3 directly binds to IL-17 promoter and leads to IL-17 production (Chen et al., 2006). In mouse T cells, combination of IL-6/TGF-β/ IL-23 activates these transcription factors and induces Th17 differentiation (Veldhoen et al., 2006). The differentiated cells preferentially secrete IL-17A, IL-17F, IL-21 and IL-22, which target multiple cells including epithelial cells and fibroblasts.

IL-17A and IL-17F are effector cytokines with proinflammatory properties. These cytokines induce expression of cytokines (TNF, IL-6, IL-1β, and GMCSF), chemokines

(CXCL1, CXCL8 and CXCL10) and metalloproteinases in a broad range of cell types

(Fossiez et al., 1996; Laan et al., 1999; Yao et al., 1995). Expression of these cytokines and chemokines by IL-17A and IL-17F leads to activation and recruitment of neutrophils to the inflammation site (Moseley et al., 2003). When the IL-17 receptor is deleted in mouse, the mice suffer from several pathogenic infections such as Klebsiella (Ye et al.,

2001) and Candida(Huang et al., 2004) because of the deficiency in neutrophil trafficking. Another Th17 producing cytokine, IL-22 also acts against pathogens by inducing antimicrobial agents in keratinocytes (Liang et al., 2006). However, IL-22 has been more associated with dermal inflammation and IBD and it has been used on mice to induce psorasis like animal model (Zheng et al., 2007). While IL-17A, IL-17F and IL-22 have proinflammatory and antimicrobial functions, IL-21 acts as an autocrine growth factor for Th17 cells (Zhou et al., 2007). Although it is less potent than IL-6, with TGFβ,

28 IL-21 can also promote Th17 differentiation by inducing RORγt expression. IL-21 itself is also sufficient to induce IL-23R expression in Th17 cells.

IL-2 is the key cytokine for the expansion of Th1 and Th2 cells. Instead, IL-23, which is a member of IL-12 family of cytokines, function in survival and expansion of

Th17 cells (Cua et al., 2003). Moreover, it has been showed that IL-23 can expand pathogenic Th17 cells which are capable of inducing Experimental Autoimmune

Encephalomyelitis (EAE) in mice, whereas without IL-23 stimulation, this pathogenic function of Th17 cells can be inhibited by IL-10 which is produced by Th17 cells themselves (Langrish et al., 2005; McGeachy et al., 2007).

CHAPTER II

THE TOLL–INTERLEUKIN-1 RECEPTOR MEMBER SIGIRR REGULATES

COLONIC EPITHELIAL HOMEOSTASIS, INFLAMMATION AND

TUMORIGENESIS

Hui Xiao1*, Muhammet F. Gulen*1,5, Jinzhong Qin*1, 5, Jianhong Yao1, Katarzyna

Bulek,1 Danielle Kish1, Cengiz Z. Altuntas1, Dave Walde1, Caixia Ma2, Hang Zhou3,

Vincent K. Tuohy1, Robert L. Fairchild1, Carol de la Motte1, Daniel J. Cua4, Bruce A.

Vallance2, and Xiaoxia Li1

1Department of Immunology, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland 2Division of Gastroenterology, University of British Columbia and BC Children’s Hospital, Vancouver, B.C. Canada 3Department of Epidemiology and Biostatistics, Case Western Reserve University 4DNAX Research Inc., 901 California Avenue, Palo Alto, CA 94304-1104 5Department of Biology, Cleveland State University, Celeveland, USA * Equal contributors.

Appeared as Immunity 26(4): 461-75, April 2007

30 ABSTRACT

Despite constant contact with the large population of commensal bacteria, the

colonic mucosa is normally hyporesponsive to these potentially proinflammatory signals.

Here we report that the single immunoglobulin IL-1 receptor related molecule (SIGIRR),

a negative regulator for Toll-IL-1R signaling, plays a critical role in gut homeostasis,

intestinal inflammation and colitis-associated tumorigenesis by maintaining the microbial

tolerance of the colonic epithelium. SIGIRR-deficient (Sigirr-/-) colonic epithelial cells

displayed commensal bacteria-dependent homeostatic defects, as shown by constitutive upregulation of inflammatory genes, increased inflammatory responses to dextran sulfate

sodium (DSS) challenge and increased Azoxymethane (AOM)+DSS-induced colitis-

associated tumorigenesis. Gut epithelium-specific expression of the SIGIRR-transgene in

the SIGIRR-deficient background reduced the cell survival of the SIGIRR-deficient colon epithelium, abrogated the hypersensitivity of the Sigirr-/- mice to DSS-induced colitis and

reduced AOM+DSS-induced tumorigenesis. Taken together, our results indicate that

epithelium-derived SIGIRR is critical in controlling the homeostasis and innate immune

responses of the colon to enteric microflora.

INTRODUCTION

Up to 100 trillion commensal bacteria, which are highly diverse in strains, are

present in the colon providing the host with important functions including the metabolism

of nutrients and organic substrates (Berg, 1996; Hooper and Gordon, 2001; Hooper et al.,

2001; Sansonetti, 2004). The beneficial effects of the commensal bacteria have evolved

in parallel with the so called “microbial tolerance” of the intestinal epithelial layer, in

31 which the lack of responsiveness to this bacterial population, results in the co-existence of the host and the bacteria. The intestinal epithelial layer functions not only as a physical barrier but also an innate and adaptive immune barrier against commensal bacteria. Inappropriate activation of the immune system by commensal bacteria is thought to lead to the pathogenesis of human inflammatory bowel diseases (IBD), including Crohn’s disease and ulcerative colitis (Podolsky, 2002).

Ulcerative colitis is associated with an elevated risk for colorectal cancer(Clevers,

2004). It is now commonly believed that the chronic inflammation process is responsible for the neoplastic transformation of the intestinal epithelium(Balkwill and Mantovani,

2001; Clevers, 2004). The proinflammatory cytokines and chemokines, such as TNF,

IL-1, IL-6, and IL-8, as well as matrix-degrading enzymes, growth factors and reactive oxygen species (ROS), create a microenvironment that enhances cell proliferation, cell survival, cell migration, and angiogenesis, thereby promoting tumorigenesis (Coussens et al., 2000; Coussens and Werb, 2002; Dvorak, 1986). The common view is that the association of ulcerative colitis with cancer involves the inflammation of the submucosa, induced by direct contact with the intestinal microflora, promoting tumor outgrowth in the overlaying epithelium. Therefore, the “microbial tolerance” of the intestinal epithelial layer is not only important for controlled state of inflammation but also for preventing tumorigenesis in the colon.

This “microbial tolerance” of the intestinal epithelial layer is a consequence of multiple factors involved in the interaction between the host and bacteria. The Toll-like

32 receptors (TLRs), is a family of pattern-recognition receptors that detects conserved

molecular patterns of microorganisms, thereby playing a critical role in the interaction

between host and bacteria (Chuang and Ulevitch, 2000; Hemmi et al., 2000; Medzhitov et

al., 1997; Rock et al., 1998; Takeuchi et al., 1999; Zhang et al., 2004). TLR4 (a receptor for LPS, lipopolysaccharide), TLR2 (a receptor for LTA, lipoteichoic acid) and TLR9 (a

receptor for CpG DNA) are sensors for bacterial infection and are critical for the

initiation of inflammatory and immune defense responses. One confounding issue with

this hypothesis is that TLR ligands are present on both commensal bacteria and

pathogens. As such, it remains unclear how the host remains tolerant to commensal

bacteria yet can initiate an effective inflammatory and immune responses against

pathogens when they appear. It has been shown that the spatial and temporal expression

of TLRs in the mucosal surfaces of the intestinal tract contributes to the ability of the host

to discriminate between pathogen and resident microflora (Melmed et al., 2003; Ortega-

Cava et al., 2003). TLRs are expressed in innate immune cells, including macrophages

and dendritic cells (Akira et al., 2001). The lack of pathogenic factors in commensal

bacteria prevents them from crossing the physical barrier of the gut epithelium, reducing

and/or excluding the contact between commensal bacteria and innate immune cells in the

gut mucosa. However, recent studies suggest that the intestinal epithelium is not totally

“blind” to TLR ligands. Experiments with TLR2-, TLR4-, and MyD88 (adaptor for

TLRs)-deficient mice suggest that TLR signaling is required for the homeostasis of the

intestinal epithelium and protects gut epithelium from injury (Rakoff-Nahoum et al.,

2004). One of the major TLR signaling pathways is the activation of the IKKβ-NFκB

signaling cascade, which provides the survival signal for epithelial cells (Greten et al.,

33 2004; Karin and Greten, 2005). Deletion of IKKβ in the epithelial cells reduced the

colitis-associated tumor incidence in a mouse model, indicting the direct contribution of

the epithelial cells in colitis-associated tumorigenesis (Greten et al., 2004; Karin and

Greten, 2005).

SIGIRR (also named as TIR8) represents a unique subgroup of the Toll-IL-1R

superfamily, with a single immunoglobulin extracellular domain and a TIR (Toll/IL-1R)

intracellular domain (Garlanda et al., 2004; Wald et al., 2003). We previously showed

that SIGIRR functions as a negative regulator for IL-1 and LPS signaling, through its

interaction with the TLR4 and IL-1R complex (Qin et al., 2005). In this manuscript, we

report that SIGIRR-deficient (Sigirr-/-) mice exhibit dysregulated signaling in the gut

epithelium in response to commensal bacteria, which disrupts the homeostasis of the

colon epithelium and leads to hyper susceptibility of these mice to dextran sulfate sodium

(DSS)-induced colitis and increased colitis-associated tumor incidence and tumor growth.

The important role of SIGIRR in gut epithelium is demonstrated by the fact that gut-

epithelial specific expression of SIGIRR was able to restore the homeostasis of the colon

epithelium, attenuated the severity of DSS-induced colitis and reduced colitis-associated

tumor incidence and tumor growth in the Sigirr-/- mice. Our results show that SIGIRR is an important modulator of intestinal epithelial homeostasis and a key regulator of

mucosal immunity, maintaining “microbial tolerance” of the intestinal epithelial layer.

34 RESULTS

The colon epithelium of SIGIRR-deficient mice exhibits altered homeostasis.

Previous studies have shown that recognition of commensal bacteria by TLR2 and

4 is required for the homeostasis of intestinal epithelium (Rakoff-Nahoum et al., 2004),

which is controlled by the balance of proliferation and differentiation along the crypt

axis. Because SIGIRR is a negative regulator for IL-1R- and TLR4-mediated pathways

and highly expressed in colonic epithelial cells (Fig. 2.1), we hypothesized that Sigirr-/- epithelial cells might be more responsive to commensal bacteria in the colon. To test this hypothesis, we examined whether the gut epithelium in the Sigirr-/- mice might suffer

intrinsic homeostatic defects. The proliferation state of the colon crypts of the Sigirr-/- mice was compared with the wild-type mice by labeling the proliferating cells with BrDU administrated by intraperitoneal injection. Colon sections were harvested 2 and 24 hours after BrDU injection and stained for BrDU positive cells (Fig. 2.2A-B and data not shown). There was an increased number of proliferating epithelial cells in the crypts of the Sigirr-/- colon as compared to that in the wild-type mice. Furthermore, although the

BrDU positive cells were only in the stem cell zone localized at the bottom of the crypts

in the wild-type colon, the proliferating epithelial cells were also found in the middle and

upper regions of the crypts in the Sigirr-/- colon, where cells normally are differentiated

and non-proliferating. Taken together, these results indicate that the epithelial cells in the

Sigirr-/- colon exhibit a dysregulated state in their basal proliferation.

To further compare the homeostasis of the colon epithelium between Sigirr-/- mice

and wild-type mice, we examined epithelial cell survival and apoptosis in the crypts. In

Figure 2.1. Cell Type-Specific Expression of SIGIRR. Primary cells were isolated from mice and total RNA was prepared using Trizol from Sigma. Northern blot was performed to examine SIGIRR expression in various cell types.

situ Tunnel assay was performed on colon sections to examine the DNA fragmentation

caused by apoptosis. As shown in Fig. 2.2C, Sigirr-/- colon epithelium had fewer

apoptotic cells at the top of the crypts as compared to the wild-type colon, which provides

further evidence for imbalanced homeostasis in the Sigirr-/- colon epithelium.

To address the impact of this imbalance over an extended period, we examined

aged Sigirr-/- and wild-type mice kept under identical conditions. When 9 months old

mice were examined, we noted a striking elongation in Sigirr-/- colon crypts, at more than

twice the length of wild-type mice. In addition, goblet cells increased in both size and

number in aged Sigirr-/- mice as compared to litter mate wild-type control mice (Fig.

2.2D). The elongation of colon crypts was observed in as early as 5 months old Sigirr-/- mice as compared to wild-type mice (data not shown). These phenotypic changes in the

Sigirr-/- colon crypts are consistent with the increased proliferation and cell survival of

the Sigirr-/- epithelial cells.

Constitutive signaling in SIGIRR-deficient colon epithelium is dependent on

commensal bacteria

To investigate the molecular mechanism for the altered homeostasis of the Sigirr-/- colon epithelium, we examined signaling in these epithelial cells. Interestingly, we found that Sigirr-/- colon epithelial cells revealed constitutive NFκ-B, IκB and JNK phosphorylation, indicating constitutive NFκ-B and JNK activation (Fig. 2.2E). Upon

37 Figure 2.2. Increased cell proliferation and survival in Sigirr-/- mice colon. (A and B)

38 Bromodeoxyuridine (BrDU, 1mg/ml) was administrated to age-and sex-matched wild-

type and SIGIRR-deficient mice by I.P. injection. Twenty-four hours after BrDU

injection, the colon sections were stained for BrDU positive cells (stained brown).

Hemotoxylin (blue) was used as counter staining to visualize the whole tissue. Well-

oriented crypts (at least 30 crypts) were counted on slides (at least two slides) from each

mouse and 3 pairs of mice were analyzed. Error bars represent ±SEM, and student’s T

test was used. ** = p<0.01. (C) DNA fragmentation in wild-type and Sigirr-/- mice colon

was detected by in situ TUNEL assay. Apoptotic cells were stained as green and nuclei

were stained as blue by DAPI. Well-oriented crypts (at least 30 crypts) were counted on

slides (at least two slides) from each mouse and 3 pairs of mice were analyzed. Error

bars represent ±SEM, and student’s T test was used. * = p<0.05. (D) Aged wild-type and

Sigirr-/- mice (9 months old) were sacrificed and colon was excised. Following PBS

wash, the colon was cut open longitudinal and Swiss-roll was made. Sections of Swiss-

roll were stained by Hematoxylin and eosin and crypt length was measured. Well-

oriented crypts (at least 30 crypts) were measured on slides (at least two slides) from each mouse and 3 pairs of mice were analyzed. Error bars represent ±SEM, and student’s T test was used. ** = p<0.01. Scale bar=100M. (E-G) Colon epithelial cells from Sigirr-/-

mice are constitutively activated and hyper-responsive to IL-1 and LPS stimulation.

Colon epithelial cells prepared from wild type or Sigirr-/-were untreated or stimulated

with IL-1 (10 ng/ml), LPS (10 g/ml) or TNF (10 ng/ml) for 15 or 30 minutes. Cells

were then lysed and cell lysates were resolved by SDS-PAGE. Immunoblotting was

performed to detect TLR and IL-1R signaling components with anti-phospho-NFB (p-

p65), anti-phospho-JNK, anti-IB and anti-phosphor- IB (E-F) or proliferation and

39 apoptosis markers with anti-Cyclin D1 and anti-Bcl-xL (G). (H-I) Depletion of commensal bacteria abolished dysregulated cell proliferation in colon of Sigirr-/- mice.

Wild-type and Sigirr-/- mice were treated with four different antibiotics in drinking water

(1 g/l ampicillin, 500mg/l vancomycin, 1 g/l neomycin sulfate, and 1 g/l metronidazole) for 4 weeks. Bromodeoxyuridine (BrDU, 1mg/ml) was administrated to antibiotic-treated mice via I.P. injection 24 hours prior to sacrifice. Colon sections from 4 pairs of mice were examined for BrDU positive cells (brown staining).

40 IL-1 or LPS, but not TNFα stimulation, the Sigirr-/- colon epithelial cells showed even higher activation, compared to the control cells (Fig. 2.1E-F). Such constitutive signaling and hyper activation probably underlie the increased proliferation and survival of the

Sigirr-/- colon epithelial cells. Furthermore, Sigirr-/- colon epithelial cells had upregulated

expression of Cyclin D1 and Bcl-xL compared to that in wild-type cells. IL-1 stimulation

further induced the expression of Cyclin D1 in the Sigirr-/- colon epithelial cells,

compared to the control cells (Fig. 2.2G). These results indicate that Cyclin D1 and Bcl- xL are probably part of the effector molecules responsible for the increased cell

proliferation and reduced apoptosis in the colon epithelium of the Sigirr-/- mice.

To test whether the constitutive signaling in Sigirr-/- colon epithelium is

dependent on commensal bacteria-derived ligands, we removed commensal bacteria from

Sigirr-/- and wild-type mice through oral administered antibiotics, including vancomycin,

neomycin, metronidazole, and ampicillin (VNMA). The depletion of the commensal

bacteria was confirmed by counting the bacteria in the stool of untreated and antibiotic-

treated mice (Fig. 2.3 and data not shown). To compare the proliferation of the epithelial

cells in theSigirr-/- mice with that in wild-type mice after the removal of commensal

bacteria, we injected BrDU into the antibiotic-treated Sigirr-/- and wild-type mice

intraperitoneally.

Colon sections were harvested 24 hours after BrDU injection and stained for

BrDU positive cells. As shown in Fig. 2.2H-I, the number and pattern of BrDU positive

Figure 2.3. Commensal Bacterial Counts in Antibiotic-Treated Mice. Stool of the antibiotics untreated or treated mice as described in Fig. 1H and I was diluted, grinded in PBS and fixed with formalin. This dilution was filtered through 0.2 μm Whatman Anodisc filter (VWR). Attached bacteria were stained in SYBR green solution (Invitrogen) and examined under fluorescence microscope. At least six representative pictures were counted from each mouse and data from 3 pairs of mice were subjected to statistical analysis. Error bars represent ±SEM, and student’s T test was used. ** = p<0.01.

42 cells (proliferating epithelial cells) in the crypts were similar between the antibiotic-

treated Sigirr-/- colon and that in wild-type mice. These results clearly showed that the

removal of commensal bacteria abolished that hyper-proliferative state of the Sigirr-/- colon epithelium, indicating that the increased proliferation of the colon epithelial cells in

Sigirr-/- mice (Fig. 2.2A-B) depends on the presence of commensal bacteria in the colon.

Furthermore, colonic epithelial cells from antibiotic-treated Sigirr-/- and wild-type mice

were also examined for signaling. Similar amounts of NF-κB and JNK phosphorylation

were observed in colon epithelium cells of the Sigirr-/- and wild-type mice after the

removal of commensal bacteria, indicating that the constitutive signaling in Sigirr-/- colonic epithelial cells is indeed dependent on commensal bacteria (data not shown).

The impact of SIGIRR deficiency on intestinal inflammation

Based on these homeostatic changes, we wondered whether the mircoflora- mediated constitutive activation of Sigirr-/- colon epithelium may ultimately lead to

spontaneous colitis. Comparison of the colon sections between wild-type and Sigirr-/- mice (2-12 months old) did not reveal significant infiltration of inflammatory cells and did not detect obvious tissue damage, although the crypts were dramatically elongated in

5-9-month old Sigirr-/- distal colon as compared to that in wild-type mice (Fig. 2.2D).

However, by ELISA, we did find that the Sigirr-/- colonic mucosa showed significantly

higher expression of cytokines (TNF-α, IL-6 and IFN-γ) and chemokines (MIP-2, MCP-

1, and KC) as compared to wild-type colon (Fig. 2.4). These results suggest that

although the Sigirr-/- mice do not develop overt spontaneous colitis, the physiologic inflammation typically found in the colonic mucosa is exaggerated in Sigirr-/- mice. This

Figure 2.4. Constitutive upregulation of proinflammatory cytokines and chemokines

in colon of Sigirr-/- mice. Same amount of colon tissue from Wild type and Sigirr-/- mice

(ranging from 200-300mg) was cut into small pieces and incubated in serum-free RPMI medium for 24 hours and secreted cytokines (IL-6, TNF and IFN) and chemokines

(KC, MIP-2 and MCP-1) in the medium were measured by ELISA. Error bars represent

±SEM, and student’s T test was conducted. * = p<0.05.

44 may make them far more susceptible to colitis when the colon is challenged by noxious stimuli.

Sigirr-/- mice has been reported to be more sensitive to DSS-induced colitis

(Garlanda et al., 2004). We also employed the DSS-induced colitis model to investigate

the mechanism by which SIGIRR regulates intestinal inflammation. Both Sigirr-/- and

wild-type mice were continuously treated with 3% DSS in drinking water. Nine days after DSS treatment, the Sigirr-/- mice began to die and at the end of day 13 all of the

mice (n=15) had died. In contrast, the entire cohort of wild-type mice was still alive even

at day 16 (Fig. 2.5A). These results clearly indicate that the Sigirr-/- mice were much

more susceptible to DSS treatment as compared to wild-type mice. The DSS-induced

colitis phenotype in Sigirr-/- mice appeared to be much stronger than that described by

Garlanda et al for the Sigirr-/- mice, which could be due to strain difference or difference

of the microflora spectrum in the colon.

The higher mortality of the Sigirr-/- mice was associated with increased gut injury caused by DSS treatment. Histology analysis of colon sections showed increased damage

in Sigirr-/- epithelial layer as compared to that in wild-type mice after the DSS treatment

(Fig. 2.5B and Fig. 2.6). By immunofluorescent staining for infiltrating leukocytes, we

found that colon sections of DSS-treated Sigirr-/- mice had highly increased numbers of infiltrated inflammatory cells as compared to those in wild-type mice (Fig. 2.7).

Furthermore, inflammatory cytokine gene and protein expression were also clearly induced to much higher amounts in DSS-treated Sigirr-/- colon than the wild-type mice,

including IL-12 p40, IFN-γ, IL-17, IL-6, and IL-1β (Fig. 2.5C and Fig. 2.8). Taken

Figure 2.5. Sigirr-/- mice are highly susceptible to develop DSS-induced colitis.

SIGIRR-deficient and wild-type control mice were treated continuously with 3% DSS in drinking water. (A) Kaplan-Meier plot of survival of wild type and Sigirr-/- mice (n=15)

46 following DSS treatment for 16 days. (B) Hematoxylin and eosin staining of colonic cross sections of mice treated with 3% DSS for various days (original magnification=100). (C) Increased cytokine production in colon of Sigirr-/- mice after

DSS treatment. 200 mg of colon tissue was removed from Sigirr-/- and wild-type mice.

Whole colon culture was collected on various days post-treatment with 3% DSS and

cytokine production was examined by ELISA. Error bars represent ±SEM, and student’s

T test was conducted. * = p<0.05, **=p<0.01. (D) Removal of commensal bacteria

abolished the hyper sensitivity of Sigirr-/- colon to DSS treatment. Wild-type and Sigirr-/-

mice were treated with four different antibiotics as described in Experimental Procedure

for 4 weeks and followed by DSS (3%) treatment for 3-7 days. H&E staining was

conducted to examine the histology of cross-sections from Sigirr-/- and WT mice.

Magnification=50×.

Figure 2.6. Sigirr-/- Mice Develop More Severe Colitis than Wild-Type Mice. (A)

Epithelial injury and (B) Infiltration of leukocytes in WT and Sigirr-/- mice after DSS treatment. H&E stained cross-sections from at least 3 mice at each time point was scored as described in Experimental Procedures. Data shown is the mean ±SEM, p<0.05 using student’s T test.

Figure 2.7. Sigirr-/- Colon Showed Increased Inflammatory Cell Infiltration after

DSS

Treatment. Colonic cross-sections of wild type or Sigirr-/- mice treated with DSS for 6 days were subjected to immunofluorescence staining for the cell markers GR-1

(neutrophil, A), F4/80 (macrophage, B), CD-4 (T-cells, C) or iNOS (D), respectively.

Figure 2.8. Expression of Cytokines in Colon Tissues from Wild-Type and Sigirr-/-

Mice. Expression with or without 3% DSS treatment was quantified by measuring mRNA in total colon tissue using RT-PCR.

50 together, the above results indicate that severe damage of colonic epithelium in DSS- treated Sigirr-/- mice is due to increased inflammation.

We then examined whether hypersensitivity of the Sigirr-/- mice to DSS challenge is dependent on the presence of commensal bacteria. Antibiotic-treated Sigirr-/- and wild- type mice were treated with 3% DSS and followed by histology analyses. As shown in

Fig. 2.5D, antibiotic treatment reduced the sensitivity of the Sigirr-/- mice to DSS treatment. The antibiotic-treated Sigirr-/- mice showed similar inflammatory response to

DSS treatment as the antibiotic-treated wild-type mice. These results demonstrate that the regulatory role of SIGIRR in DSS-induced colitis is commensal bacteria-dependent, probably through its negative regulation on TLR-IL-1R-mediated-signaling induced directly or indirectly by commensal bacteria.

The impact of SIGIRR deficiency on colitis-associated cancer

We next examined the impact of SIGIRR deficiency on colitis-associated cancer.

The link between chronic inflammation and tumorigenesis has been well established for colorectal cancer (Balkwill and Mantovani, 2001; Clevers, 2004). Because SIGIRR plays an important role in maintaining colonic epithelial homeostasis and controlling intestinal inflammation, we predict that SIGIRR should have an impact on inflammation-induced cancer in the colon tissues. To test this hypothesis, we employed a mouse model of colitis-associated cancer (CAC), injecting mice with procarcinogen AOM

(Azoxymethane) followed by three cycles of oral administration of DSS (Okayasu et al.,

1996). Although AOM treatment introduces genetic instability and mutation of oncogenes in epithelia, mice developed chronic colitis after repeated treatment with DSS,

51 accelerating tumor promotion and progression in colon. Sigirr-/- and wild-type mice were

subjected to this AOM+DSS-induced colitis-associated cancer model. Whereas 63% of

the wild type mice developed macroscopic polyps, all the Sigirr-/- mice developed colon

tumors (Fig. 2.9A). The tumors detected in both wild-type and Sigirr-/- mice were

exclusively located in distal colon. Some of Sigirr-/- mice showed severe rectal bleeding,

diarrhea or loss of weight towards the end of treatment. Furthermore, the average tumor

number per mouse (17±4.03) in Sigirr-/- mice was twice more than that in wild type mice

(7±3.3) (Fig. 2.9B). These results showed that tumor incidence was increased in Sigirr-/- mice, indicating that SIGIRR deficiency enhances colitis-associated tumor promotion.

SIGIRR deficiency also had impact on the size of the tumors. About 60% polyps developed in wild-type mice were small adenomas (<2mm diameter), whereas the size of the other 40% polyps were 2-5mm in diameter (Fig. 2.9C and Fig. 2.10). Remarkably about 70% of the polyps formed in Sigirr-/- mice were big tumors (>2mm diameter), and

each mouse developed one or two large tumor (>5mm diameter) with histology

characteristics of adenocarcinoma (Fig. 2.9C-D). These results indicate that SIGIRR

deficiency not only enhances tumor promotion but also increases tumor progression.

Histology analyses showed that all the tumors formed in wild-type mice were tubular

adenomas, with no sign of invasion to mucosa or muscular layer (Fig. 2.9D). However,

the large tumors formed in Sigirr-/- mice showed high-grade dysplasia, less differentiation

and obvious infiltration of inflammatory cells (Fig. 2.9D). We frequently observed crypts

penetrating into the mucosa or muscular mucosa (Fig. 2.9D).

Figure 2.9. SIGIRR deficiency enhances tumorigenesis in mouse colon. (A) Tumor

incidence induced by AOM plus DSS regime in Sigirr-/- and WT mice. Wild type n=13;

Sigirr-/- n=14. (B) Total colonic polyps formed in colorectal of Sigirr-/- and WT mice. (C)

Size distribution of colonic tumors formed in Sigirr-/- and WT mice. The data shown is

the mean ± SD (WT n=13, KO n=14), and p<0.0001 using ANOVA analysis. (D)

Histology of tumors formed in Sigirr-/- and WT mice. Photomicrograph of hematoxylin

and eosin stained sections of Sigirr-/- and WT mice are shown. Magnification = 16 or

100. Arrows point to crypts found in the mucosa and muscular mucosa.

Figure 2.10. Macroscopic View of Tumors. Tumors were formed in Sigirr-/-and WT mice following AOM+DSS. Scale bar=5mm.

54 Hyperactivation of NF-κB and STAT3 contribute to increased tumorigenesis in

SIGIRR-deficient mice

To examine how SIGIRR deficiency leads to increased tumor promotion and

progression, we first compared the proliferation status of colon epithelium of the Sigirr-/- mice with that of wild-type control mice at the early stage of tumorigenesis upon AOM and DSS treatment. After AOM injection and the first cycle of DSS treatment, we detected increased Ki-67-positive cells in the colon epithelium of Sigirr-/- mice as compared to that in wild-type control mice, indicating that cell proliferation is increased in Sigirr-/- epithelium (Fig. 2.11A). Previous studies have shown that NF-κB activation

in colon epithelial cells has important tumor-promoting function(Karin and Greten,

2005). The NF-κB pathway was indeed activated at a higher amount in the colon tissue

of Sigirr-/- mice (with more phosphorylation of IKK, IκB and NFκB) after AOM+DSS treatment as compared to that in wild-type control mice (Fig. 2.11B). NF-κB nuclear accumulation was increased in epithelium of Sigirr-/- mice, confirming the activation of

NF-κB (data not shown). Consequently, expression of NF-κB target genes, Bcl-xL and

Cyclin D1 that are important for cell survival and proliferation, was highly induced in colon tissues of Sigirr-/- mice (Fig. 2.11B-C). In addition to increased cell proliferation,

inflammation and damage of crypt cells were much more evident in Sigirr-/- mice at the

early stage of tumorigenesis after the initial AOM and DSS treatment. We detected much

stronger induction of inflammatory gene COX2 (Fig. 11B) and cytokines (IL-17, TNF-α

and IL-6) in colon tissues of Sigirr-/- mice as compared to that in wild-type mice (Fig.

2.11D). In particular, IL-6 has been shown to promote tumor progression in

Figure 2.11. Increased cell proliferation and enhanced inflammatory response in

Sigirr-/- mice during early stage of tumorigenesis induced by AOM+DSS treatment

(A) Increased cell proliferation of colon epithelia of Sigirr-/- mice. Immunohistochemical

56 staining was performed to examine Ki-67 positive cells on cross-sections of colons from

Sigirr-/- and WT mice on day 15 under AOM plus DSS regime (magnification 100). Ki-

67 positive cells were quantitated from 6 cross-sections of two pairs of mice. Error bars

represent ±SEM, * = p<0.05 using student’s T test. (B) NFκB pathway is highly activated

in Sigirr-/- mice. Protein lysates were prepared from whole colon tissue from two pairs of

Sigirr-/- and WT mice on day 15 of AOM plus DSS regime. Western blotting was

performed using antibodies against p-IKK/β, p-p65, COX2 and Actin. 1# and 2# represent samples from two individual mice. (C) Immunochemical staining was performed to examine the nuclear accumulation of cyclin D1 on colonic cross-sections from Sigirr-/- and WT mice on day 15 under AOM plus DSS regime. (D) Cytokine

production in Sigirr-/- and WT mice. On day 15 under the AOM plus DSS regime, 200

mg of colon tissue was removed from Sigirr-/- and wild-type mice. Whole colon cultures

were subsequently prepared as described in Experimental Methods. ELISA was

conducted to measure cytokine production. Error bars represent ±SEM, and student’s T test was conducted. * = p<0.05. (E) Immunohistochemical staining of p-STAT3 on colonic cross-sections of Sigirr-/- and WT mice on day 15 of AOM plus DSS regime. (F)

Nuclear β-Catenin staining in tumors from WT and Sigirr-/- mice after the completion of

the AOM plus DSS regime. Note the line separate tumor from adjacent normal tissue.

Magnification: 100 or 400.

57 inflammation-associated cancer models through the activation of oncogene

STAT3(Becker et al., 2004).

Consistent with this, nuclear accumulation of phospho-STAT3 was indeed

increased in Sigirr-/- epithelium as compared to that in wild-type mice (Fig. 2.11E), which

probably contributed to the increased cell proliferation observed in the Sigirr-/- epithelium. Taken together, the above results showed that NF-κB and STAT3 were highly activated during the early stage of tumorigenesis in Sigirr-/- colon epithelium,

which in turn promote tumor formation through the expression of their target genes

(including Cyclin D1 and Bcl-xL) important for cell survival and proliferation.

Human colon cancers often have mutation and/or loss of heterozygosity in the key components of APC (adenomatous polyposis coli)- β-catenin pathway, indicating an

essential role for β-catenin in carcinogenesis of the colon (Gregorieff and Clevers, 2005;

Radtke and Clevers, 2005). It has been reported that β-catenin, rather than APC, is often

mutated in AOM-induced murine colon tumors (Greten et al., 2004; Guda et al., 2004).

β-catenin nuclear accumulation was indeed detected by immunohistochemistry showing a

shift of β-catenin from the membrane toward a cytoplasmic and nuclear localization in

the tumor tissues from both wild-type and Sigirr-/- mice (Fig. 2.11F), indicating that β- catenin pathway was activated in this tumor model. β-catenin nuclear accumulation probably resulted from AOM-induced mutations in components of the β-catenin pathway in both wild-type and Sigirr-/-mice. The hyperactivation of NF-κB and STAT3 in Sigirr-/- colon epithelium after AOM+DSS treatment are probably responsible for the

58 increasedtumor incidence and tumor growth in Sigirr-/- mice by promoting tumor formation of the transformed cells (with activated β-catenin) mutated by AOM.

The impact of gut-epithelial cell specific expression of SIGIRR on the homeostasis of colon epithelium, intestinal inflammation and tumorigenesis

One important question is what cell type is mainly responsible for the regulatory role of SIGRR in gut mucosal immunity. Although SIGIRR is highly expressed in colon epithelial cells, substantial amounts of SIGIRR expression were also detected in T cells

(Fig. 2.1). Therefore it is important to determine the relative contribution of bone marrow-derived versus non-bone marrow-derived cells for SIGIRR’s action in the gut.

Interestingly, we found that the Sigirr-/- mice received bone marrow from either wild-type

or Sigirr-/- mice died much faster than wild-type mice transplanted with wild-type or

Sigirr-/- bone marrow upon DSS treatment, indicating the main contribution of the non-

bone marrow-derived tissue cells to SIGIRR’s function in intestinal inflammation (Fig.

2.12). The fact that wild-type or Sigirr-/- mice transplanted with bone marrow from

Sigirr-/- mice had somewhat stronger phenotype as compared to those transplanted with

wild-type bone marrow also implicated some degree of contribution of bone marrow-

derived cells to SIGIRR’s function in regulating DSS-induced colitis.

To examine the function of SIGIRR in gut epithelial cells, we generated a gut-

epithelial specific SIGIRR-transgenic mouse by constitutively expressing SIGIRR in the

distal small intestinal and colonic epithelium. We chose to express flag-tagged SIGIRR

under the control of transcriptional regulatory elements derived from a fatty acid-binding

Figure 2.12. DSS-Induced Colitis in Bone Marrow-Transplanted Mice. (A) Kaplan- Meier plot of survival of bone-marrow transplanted mice following DSS treatment. Bone- marrow cells (1×107) isolated from wild type or Sigirr-/- mice were injected into lethalirradiated wild type or Sigirr-/- recipients (5 males and 5 females for each group). Six weeks after bone-marrow transplant, 3% of DSS was administrated in drinking water for 15 days. Black line: wild type donor cells to wild type recipients; Red line: Sigirr-/- donor cells to wild type recipients; Green line: wild type donor cells to Sigirr-/- recipients; Blue line: Sigirr-/- donor cells to Sigirr-/- recipients. P=0.0027, wild type donor cells to wild type recipients versus wild type donor cells to Sigirr-/- recipients; P=0.0002, Sigirr-/- donor cells to wild type recipients versus Sigirr-/- donor cells to Sigirr-/- recipients; P<0.0001, wild type donor cells to wild type recipients versus Sigirr- /- donor cells to Sigirr-/- recipients. Statistical analyses were performed by Kaplan-Meier method, and all tests were two-sided and p-values less than 0.05 were considered to be statistically significant. (B) Flow cytometry analysis of the lymph nod T cells from sentinel chimeras (described in Experimental Procedures). B6 Thy1.1 bone- marrow cells were transplanted into lethal-irradiated B6 Thy 1.2 wild type and Sigirr-/- mice, or vice versa. In six weeks, lymph nodes were removed and single cell suspensions were stained with APCconjugated anti-CD4 and anti-CD8, FITC-conjugated anti-Thy 1.2 and PE- conjugated anti-Thy 1.1. Data were analyzed on a FACSCaliber and gated around CD4 and CD8 positive T cells.

Figure 2.13. Construction of Gut-Specific SIGIRR Transgenic Mice. (A) Diagram for the Fabpl4x at –132/Sigirr construct. The details are described in text and material and methods. (B) Southern analysis for the identification of the founders carrying the SIGRR transgene. Genomic DNA was prepared from the mouse tails followed by genomic

Southern analysis using hGH reporter cDNA as a probe. (C) Tissue specific expression of

SIGIRR-transgene. Protein lysates were prepared from various tissues of the SIGIRR- transgenic mice and subjected to immunoprecipitation with anti-Flag M2 (Sigma), followed by western analysis with anti-SIGIRR (R&D).

61 protein gene (Fig. 2.13A-13B), a gut-epithelial specific gene (Saam and Gordon, 1999).

The expression of SIGIRR-transgene was examined by immunoprecipitation with Flag antibody followed by immunoblot analysis with anti-SIGIRR. The SIGIRR transgene was specifically expressed in intestine and colon but not in other tissues.

To address the function of SIGIRR in gut epithelial cells, the gut epithelial specific SIGIRR-transgenic mouse was bred to SIGIRR-deficient mice to create a mouse

in which SIGIRR is only expressed in gut epithelial cells but not in other tissues or cell

types. We named this mouse SIGIRR-TG–KO. The expression of SIGIRR-transgene in

the colon of SIGIRR-TG–KO mice was comparable to that in wild-type mice (Fig.

2.14A). Because SIGIRR deficiency leads to increased cell proliferation and survival in

the colon epithelium, we examined the impact of gut-epithelial expression of SIGRR-

transgene on homeostasis of colon epithelium. We compared cell survival and apoptosis

of the colon epithelium between SIGIRR-TG-KO and Sigirr-/- mice by in situ Tunnel

assay. Interestingly, we found that more apoptotic cells were detected in SIGIRR-TG-

KO colon epithelium as compared to Sigirr-/- mice (KO) (Fig. 2.14B), suggesting that

gut-epithelial cell specific expression of SIGIRR-transgene reduced cell survival.

To examine the effect of gut-epithelial cell specific expression of SIGIRR on

intestinal inflammation, the SIGIRR-TG-KO mice were compared with Sigirr-/- mice for their susceptibility to DSS-induced colitis. Both SIGIRR-TG-KO and Sigirr-/- mice were

continuously treated with 3% DSS in drinking water. Although all of the Sigirr-/- mice

died 14 days after DSS treatment, only 45% of the SIGIRR-TG-KO mice died at day 16

Figure 2.14. Gut-epithelial specific expression of SIGIRR-transgene rescued the

Sigirr-/- mice from severe DSS-induced colitis. (A) Protein lysates were prepared from

colon crypts of the Sigirr-/- , SIGIRR-TG-KO and wild-type mice and followed by

western analysis with anti-SIGIRR and anti-Actin (R&D). (B) DNA fragmentation was

detected on frozen colon sections by in situ TUNNEL assay. At least thirty intact crypts

were counted on each slide of three pairs of mice. The data shown is the mean ±SEM.

63 *p<0.05 compared to Sigirr-/- mice, student’s T test. (C) Kaplan-Meier plot of survival

study of SIGIRR-TG-KO and Sigirr-/- mice under 3% DSS treatment (p<0.0001 and

n=14). (D) Hematoxylin and eosin staining of colonic cross sections of untreated or

DSS-treated mice (magnification 200). (E) Whole colon cultures were prepared from

colons of SIGIRR-TG-KO or Sigirr-/- mice under DSS treatment for indicated days. The same segments of colon with the weight of 250 mg were cut into small pieces and incubated with 1 ml of serum-free RPMI medium for 24 hours and the secreted cytokines and chemokines in the medium were then measured by ELISA. Data shown is the mean

±SEM from three experiments. Student’s T test was conducted. * = p<0.05, **=p<0.01.

(F-H) Intestine-specific transgene of SIGIRR suppress colonic carcinogenesis in Sigirr-/- mice. The data shown is the mean ± SD (Sigirr-/- n=13, SIGIRR-TG-KO n=11), p<0.0001

using ANOVA analysis.

64 after DSS treatment (Fig. 2.14C). These results clearly showed that the SIGIRR-TG-KO

mice are more resistant to DSS treatment as compared to Sigirr-/- mice.

In supporting of the lethality curve, histology analysis of colon sections detected

much reduced inflammation and less tissue damage in epithelial layer of the SIGIRR-TG-

KO mice as compared to that in Sigirr-/- mice (Fig. 2.14D). Consistent with the histology

data, the inflammatory gene expression was induced at much lower amounts in DSS-

treated SIGIRR-TG-KO colon as compared to that in Sigirr-/- colon, including IL-6, TNF-

α, and IFN-γ (Fig. 2.14E). Taken together, the above results indicate that the specific

expression of SIGIRR in gut epithelial cells can rescue the Sigirr-/- mice from developing severe DSS-induced colitis.

Lastly, we examined the effect of gut-epithelial cell specific expression of

SIGIRR on colitis-associated cancer. We first injected the SIGIRR TG-KO mice with

AOM followed by three cycles of oral administration of DSS. In comparison with Sigirr-

/- mice, re-expression of SIGIRR exclusively in intestinal epithelia reduced the tumor

incidence to 68% (Fig. 2.14F), which is close to that of wild-type mice. Furthermore, the average total and big tumors (>2mm) per mouse was greatly reduced in SIGGIR TG-KO mice (Fig. 2.14G and H), indicating that SIGIRR’s expression in epithelial cells has the ability to suppress colitis-associated tumorigenesis.

65 The role of epithelial-derived SIGIRR in the regulation of chemokine gene

expression

The above results showed that epithelial-derived SIGIRR can specifically rescue

DSS-induced colitis and suppress colitis-associated tumorigenesis. The question was

then how SIGIRR modulates intestinal inflammation and tumorigenesis through its

function in epithelial cells in response to intestinal microflora. Considering that the

epithelium can play an important role in attracting inflammatory and immune cells, we

examined the expression of several chemokine genes within the inflamed colon. By RT-

PCR and ELISA, it was found that the DSS-treated and AOM+DSS-treated Sigirr-/- colon had much greater expression of chemokine genes that play critical role in the recruitment of T cells (IP-10 and MIG), neutrophils (KC and MIP-2) and macrophages (Rantes and

MCP-1) as compared to that in wild-type mice (Fig. 2.15A and Fig. 2.16). To address the specific role of the colonic epithelium in the DSS-induced inflammatory response, we isolated crypt epithelial cells from wild-type and Sigirr-/- colon untreated or treated with

DSS. Interestingly, the Sigirr-/- crypt epithelial cells produced much higher amounts of chemokines after DSS treatment as compared to wild-type crypt cells, including MCP-1,

MIP-2 and KC (Fig. 2.15B). These results indicate that much of the increased chemokine expression found in the DSS-treated Sigirr-/- mice was mediated by epithelial cells, and

the epithelial response probably plays a critical role in DSS-induced colitis.

We then further addressed the role of epithelial-derived SIGIRR in the regulation

of chemokine gene expression by using the SIGIRR-TG-KO mice. Interestingly, the

colon tissues from SIGIRR-TG-KO had reduced basal amounts of chemokines and also

Figure 2.15 Chemokine production was highly induced in Sigirr-/- colon tissue and

crypt cells after DSS treatment. Chemokine production from whole colon tissue culture

(A) or protein lysate of isolated crypt cells (B) from Sigirr-/- and WT mice was measured

by ELISA. Chemokine production from whole colon tissue culture (C) or protein lysate

of isolated crypt cells (D) from Sigirr-/- and SIGIRR-TG-KO mice was measured by

ELISA. 200 mg of colon tissue was used for whole colon tissue culture or isolation of crypt cells as described in Experimental Procedure. Error bars represent ±SEM, and student’s T test was conducted. * = p<0.05, **=p<0.01.

Figure 2.16. Expression of Chemokines in Colon Tissue from Wild-Type and Sigirr-

/- Mice. (A) Expression of chemokines in colon tissues from wild type and Sigirr-/- mice with or without 3% DSS treatment was quantified by measuring mRNA levels in total colon tissue using RT-PCR. (B) Chemokine production in Sigirr-/- and WT mice. On day

15 under the AOM plus DSS regime, 200 mg of colon tissue was removed from Sigirr-/- and wild-type mice. Whole colon cultures were subsequently prepared as described in

Experimental Methods. ELISA was conducted to measure chemokine production. Error bars represent ±SEM, p<0.05 using student’s T test.

68 reduced induction of chemokines after DSS treatment as compared to Sigirr-/- mice (Fig.

2.15C). These results suggest that the expression of SIGIRR-transgene in gut-epithelial cells suppressed the constitutive activation of chemokine genes and also DSS-induced chemokine gene expression. Furthermore, the crypt cells from DSS-treated SIGIRR-TG-

KO mice produced much lower amounts of chemokines as compared to the crypt cells from DSS-treated Sigirr-/- mice (Fig. 2.15D). These results indicate that the expression of

SIGIRR-transgene in gut epithelial cells suppressed the production of chemokines, which

is likely to the main mechanism for how the SIGIRR-transgene rescued the Sigirr-/- mice from developing severe DSS-induced colitis, thereby suppressing colitis-associated cancer. Taken together, our results clearly indicate that the impact of SIGIRR on the intestinal inflammation and tumorigenesis is mainly through its function in gut-epithelial cells.

DISCUSSION

The results presented here reveal an important role of SIGIRR in regulating the interaction between commensal microflora and colonic epithelium. The recent study by

Rakoff-Nahoum et al suggests that activation of TLR signaling by commensal microflora is required for the homeostasis of the gut epithelium (Rakoff-Nahoum et al., 2004). Mice deficient in TLR signaling have dysregulated intestinal homeostasis causing them to be more sensitive to DSS-induced injury. The results in this manuscript indicate that excessive commensal bacteria-induced TLR-IL-1R-mediated signaling in the colon is also detrimental. This suggests that there is a fine balance of pro- and anti-inflammatory signals in the colonic epithelium, and that this balance critically involves SIGIRR.

69 Deletion of SIGIRR, the negative regulator of TLR-IL-1R signaling, leads to exaggerated

commensal bacteria-induced TLR-IL-1R signaling disrupting the homeostatic regulation

of the proliferative and inflammatory responses of the colonic epithelium to commensal

bacteria, resulting in enhanced colitis-associated tumorigenesis.

The fact that the there were more proliferating cells in the Sigirr-/- colon crypts

than that in control mice indicates an intrinsic proliferative dysregulation in SIGIRR-

deficient colonic epithelial cells. Importantly, dysregulated proliferation in the Sigirr-/- colonic epithelial cells was no longer detected after removal of commensal bacteria from

SIGIRR-deficient mice. These results support the hypothesis that the TLR ligands (such as LPS) carried by commensal bacteria are recognized by Toll-like receptors in the colon epithelial layer, mediating the proliferation and survival of the epithelial cells to maintain the homeostasis of the epithelium. SIGIRR probably modulates the levels of TLR signaling in the epithelial cells either directly through its interaction with the Toll-like receptors that are activated by commensal bacteria or indirectly through its impact on pathways regulated by TLR signaling. .

It has been reported that mice deficient in TLR signaling were more sensitive to

DSS-induced injury (Rakoff-Nahoum et al., 2004). It was clearly shown that the colon damage and associated mortality in TLR-deficient mice after DSS treatment was not due to overt inflammatory cell infiltration. However, our results showed that inflammation was the primary cause for the increased tissue damage and high mortality in Sigirr-/- mice

after DSS treatment. Importantly, gut-epithelial specific expression of SIGIRR-transgene

70 rescued the Sigirr-/- mice from developing severe DSS-induced colitis, indicating that epithelial-derived SIGIRR has a major impact on intestinal inflammation.

Previous studies showed that chronic inflammation and cancer are closely associated in the intestine (Balkwill and Mantovani, 2001; Clevers, 2004). Indeed, we found that Sigirr-/- mice were much more susceptible to develop colitis-associate cancer induced by carcinogen AOM plus DSS as compared with wild-type mice, indicating that the Sigirr-/- mice are an excellent model to study the link between inflammation and tumorigenesis . Tumorigenesis can be divided into three mechanistic stages, initiation

(genomic alteration), promotion (proliferation of genetically altered cells) and progression (tumor growth) (Karin and Greten, 2005). Inflammatory cells and the innate immune system are shown to be important mediators of tumor promotion and progression, but not for tumor initiation. In our colitis-associated cancer model, carcinogen AOM introduces genomic instability and mutation of oncogenes in the epithelia, whereas repeated DSS treatment creates a microenvironment of chronic inflammation in the colon. Mutations in the APC or β-catenin gene that lead to stabilization and nuclear accumulation of β-catenin and transcriptional activation with

TCF-4 play a critical role in the initiation of colorectal tumorigenesis. The activation of

β-catenin pathway (β-catenin nuclear accumulation) was detected in the tumor tissues from both wild-type and Sigirr-/- mice, implicating that AOM probably introduced mutations to the key components of the β-catenin pathway in these mice. When the

Sigirr-/- mice were subjected to AOM injection or repeated DSS treatment alone, colon

71 tumors were not developed (data not shown), indicating that both AOM-induced tumor

initiation and DSS-induced chronic inflammation are necessary for this tumor model.

NF-κB links inflammation and immunity to cancer development and progression

(Karin and Greten, 2005). As discussed above, the constitutive activation of TLR- signaling (activation of NFκB and JNK) in the Sigirr-/- epithelial cells led to upregulation

of genes for cell survival and proliferation (Cyclin D1 and Bcl-xL) resulting in increased

cell survival and cell proliferation. Enterocyte-specific ablation of IKKβ was shown to

decrease tumor incidence markedly, indicating that IKKβ-dependent NFκB-activation in

intestinal epithelial cells operates during early tumor promotion (Greten et al., 2004).

Therefore, the constitutive NFκB activation in Sigirr-/- colon epithelium is likely to

contribute to the increased tumor incidence in the Sigirr-/- mice.

In addition to increased NFκB activation, we also detected hyperactivation of

STAT3 in colon epithelium of Sigirr-/- mice upon AOM+DSS treatment. IL-6, which has

been shown to promote cancer growth in inflammation-associated cancer models, was

highly induced in Sigirr-/- mice treated with AOM+DSS. The elevated IL-6 production is

probably responsible for the increased nuclear phospho-STAT3 detected in Sigirr-/- epithelium. Hyperactivation of STAT3 has been shown to promote tumor progression in gastric and colonic cancers (Becker et al., 2004). Colorectal tumors result from accumulation of multiple changes that lead to activation of oncogenes combined with the inactivation of tumor suppressor genes (Radtke and Clevers, 2005). Although deletion of

SIGIRR probably does not directly introduce mutations to the colon epithelium, it does

72 lead to the activation of two important transcription factors NF-κB and STAT3 during

early stage of colitis-associated tumorigenesis, which in turn promotes cell proliferation

and cell survival through the up-regulation of their target genes (including Cyclin D1 and

Bcl-xL) in Sigirr-/- epithelium, leading to tumor promotion and progression.

Our studies also implicate that SIGIRR-regulated chemokine gene expression

plays a critical role for epithelial-derived SIGIRR to modulate intestinal inflammation

and colitis-associated cancer. Importantly, epithelial specific expression of SIGIRR-

transgene reduced the expression levels of chemokines before or after DSS stimulation, confirming the critical role of epithelial-derived SIGIRR in the regulation chemokine gene expression. We hypothesize that the DSS-induced chemokines produced by the

Sigirr-/- epithelial cells play critical roles in the recruitment of the inflammatory cells to the colon mucosa, leading to severe inflammation, which in turn promotes tumorigenesis in the AOM+DSS model. While our studies demonstrate a critical role for SIGIRR expression by the colonic epithelium, it is also clear that we can not rule out modest regulatory roles of SIGIRR in other cell types. In addition to its high expression colon epithelial cells, SIGIRR is also expressed in T cells and has low level of expression in dendritic cells. Future experiments are required to dissect the specific function of

SIGIRR in each cell type.

Despite the density of commensal bacteria and their products, the colonic mucosa maintains a controlled state of inflammation (physiologic inflammation). What is the role of SIGIRR in the microbial tolerance of the colonic epithelial layer? Based the

73 findings in this manuscript, we propose that in addition to its function as a physical

barrier, the colon epithelial layer also functions as an active innate immune barrier.

SIGIRR is an important modulator to regulate the interaction between commensal

bacteria and gut epithelium to maintain the innate immune tolerance of the colon

epithelial layer. SIGIRR plays a critical role in preventing over-reaction of the colon

epithelial layer to commensal bacteria, contributing to the microbial tolerance of the

colon. While loss of SIGIRR was insufficient on its own to cause spontaneous colitis in

these mice, our findings suggest that SIGIRR is a strong candidate for regulating

inflammatory responses against real or perceived luminal threats, with its dysregulation

possibly predisposing to exaggerated or even chronic forms of infectious, idiopathic

colitis or colitis-associated cancer.

MATERIAL and METHODS

Construction of SIGIRR-transgenic mouse

To generate the Fabpl4x at –132/Sigirr construct, DNA encoding SIGIRR was placed

under the control of transcriptional regulatory elements derived from a fatty acid-binding protein gene (Saam and Gordon, 1999) followed by the human growth hormone reporter gene (hGH). Fabpl4x at –132 include elements consist of nucleotides –596 to +21 of rat

Fabpl with 4 additional copies of a 35-bp sequence [spanning nucleotides –177 to –133,

that has been inserted at nucleotide –132]. Earlier light and EM immunohistochemical studies of transgenic mice demonstrated that Fabpl4x at –132 can direct the expression of a

human growth hormone (hGH) reporter throughout the epithelium of crypts in the distal

small intestine, cecum, and colon of adult mice (Saam and Gordon, 1999). A Flag tag

was included at the N-terminus of SIGIRR to distinguish the transgene from the

74 endogenous gene. Fabpl4x at –132/Sigirr was sent to the Transgenic Mouse Service in the

University of Cincinnati and injected into the pronucleus of the fertilized eggs, followed by implantation into the oviduct of a 0.5-day p.c. pseudopregnant female mouse. The founders that carry the Sigirr transgene were identified by genomic Southern analysis using hGH reporter cDNA and Sigirr cDNA as probes. The Sigirr transgenic founder lines were bred to generate F1. Sigirr transgenic mice were bred to Sigirr-/- mice to generate mice only express SIGIRR in gut-epithelial cells. SIGIRR-TG-KO mice were maintained on C57BL/6×129/SvJ background. All of the mice utilized in this manuscript were housed in animal facility (with SPF condition) at the Cleveland Clinic Foundation in compliance with the guidelines set by Institutional Animal Care and Use Committee.

DSS-induced colitis

Experimental colitis was induced by giving 3% (w/v) DSS (M.W. 40,000 kDa;

MP Biomedicals Inc., Solon, Ohio) in drinking water ad libitum. Mice (6-8 weeks) were treated for 16 days for survival studies. For histological, gene expression and cytokine production studies, mice were sacrificed following DSS treatment for indicated days.

TUNNEL assay

Frozen sections obtained from untreated mice were fixed with 4% paraformadehyde and permeabilized by 0.1% Triton X-100. TUNEL assay kit (Roche) was used to detect apoptotic cells and DAPI was used to stain the nuclei.

BrDU staining

1mg/ml of BrDU in PBS was injected to mice via I.P. Mice were sacrificed in 24 hours following BrDU injection. The same segment of distal colon was fixed in 10%

75 neutral formalin and paraffin embedded. Proliferating cells were detected by using BrDU detection kit (BD Bioscience). Tissues were counterstained with hematoxylin. The number of BrDU positive cells was quantified by number of cells in intact, well orientated crypts.

Whole colon culture

200-300mg of colon tissue was washed in cold PBS supplemented with penicillin and streptomycin. These segments were cut into small pieces and cultured in 12-well flat bottom culture plates (Falcon) in serum-free RPMI medium. High concentration of penicillin and streptomycin was supplemented to prevent bacteria growth. After incubation at 37C for 24 hours, medium was collected and stored at -80C until use.

Colon crypt isolation and treatment

Mice colon was washed with cold PBS and cut longitudinally. Following incubation with 0.04% sodium hypochlorite (Sigma) for 30 minutes, colon was cut into small pieces and shaken continuously in PBS buffer with 1mM EGTA and 1mM EDTA at room temperature for 30 min. Crypts in the supernatant were collected. Collected crypt cells were lysed directly (for ELISA) or following treatment with 10 ng/ml IL-1

(National Cancer Institute), 10 g/ml LPS (Sigma) or 10 ng/ml TNFα (R&D System) (for western blotting).

ELISA

Whole colon culture or crypt lysate from DSS-treated mice was examined for cytokine and chemokine production using ELISA kits obtained from R&D Systems, following manufacture’s instruction. Cytokine and chemokine production is normalized

76 by total colon tissue weight (whole colon culture) or total protein amount (crypt protein lysate) measured by BCA analysis (Pierce). 3-4 pairs of mice were used for each time point.

Western blot

Crypt protein lysates were resolved by SDS-PAGE and transferred to PVDF membrane. Blots were probed with phospho-IB, phospho-p65, phospho-JNK (Cell

Signaling), anti-IB, Cyclin-D1, Bcl-xL, and β-Actin (Santa Cruz Biotechnology, CA).

Following incubation with HRP-conjugated secondary antibody, ECL (Amersham,

Arlington Heights, IL) was used to develop.

Commensal depletion

6-8 weeks mice were treated with ampicillin (A, 1 g/l, Sigma), vancomycine (V,

500 mg/l), neomycin sulfate (N, 1 g/l) and metronidazole (M, 1g/l) in drinking water for

4 weeks. Stool collected from antibiotic treated and untreated mice was diluted and grinded in 1.5 ml PBS. The grinded stool was diluted 10 times with PBS and fixed with formalin. 2ul of fixed bacteria was diluted in 1ml PBS. This dilution was filtered through

0.2 m Whatman Anodisc 25 filter (VWR) with the help of low vacuum. Attached bacteria were incubated in 100 l of SYBR green solution (SYBR Green 1 nucleic acid gel stain, Invitrogen). The stained filters were dried and covered with mounting medium on a slide. Bacteria stained on the filter were counted under fluorescence microscope.

Following commensal depletion, mice were then either sacrificed for BrDU staining, or

77 treated with 3% DSS in drinking water for indicated days and then sacrificed for

histological study.

Tumorigenesis procedure

8-weeks old mice (Sigirr-/- and WT littermates on C57BL/6 background, and

Sigirr-/- mice and SIGIRR-TG-KO littermates on mixed C57BL/6129/SvJ background)

were injected with AOM (Sigma) dissolved in 0.9% NaCl intraperitoneally at a dose of

12.5mg/kg body weight. Five days following injection, mice were treated with 2.5% DSS in drinking water, then followed by regular water for 16 days. This cycle was repeated twice (at the third cycle, mice were treated with 2.0 % DSS for 4 days)(Greten et al.,

2004). Two weeks after DSS treatment, mice were sacrificed and murine colon was removed and flushed carefully with PBS buffer. Colon was then cut longitudinally and fixed flat in 10% neutral buffered formalin overnight. All the colon tumors were counted and measured under a stereomicroscope. Representative tumors were paraffin-embedded and sectioned at 5m. Histology analysis was carried out on H&E stained tumor sections.

Immunohistochemistry

Formalin-fixed and paraffin-embedded colon sections or tumor samples were deparaffined, rehydrated and pretreated with 3% hydrogen peroxidase in PBS buffer for

20 minutes. Antigen retrival in DAKO’s antigen retrival buffer was conducted in a steam cooker for 20 minutes at 96C, followed by slowly cooling down at room temperature.

After blocking with DAKO’s block buffer, avidin/biotin block, sections were incubated with anti-Ki67 (1:150, Dako, USA), anti-β-Catenin (1:1000, BD pharmingen), anti-

78 CyclinD1 (1:50, Santa Cruz), anti-p65 (1:1000, Abcam) or anti-p-STAT3 (1:50, Abcam) for 1 hour at room temperature. Followed incubation with biotin-conjugated secondary antibody and streptavidin-HRP, positive signals were visualized by DAB kit (BD pharmingen), and counterstained with Harris hematoxylin (Fisher Scientific).

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CHAPTER III

THE RECEPTOR SIGIRR SUPPRESSES TH17 CELL PROLIFERATION VIA

INHIBITION OF THE INTERLEUKIN-1 RECEPTOR PATHWAY AND MTOR

KINASE ACTIVATION

Muhammet F Gulen1,2, Zizhen Kang1, Katarzyana Bulek1, Wan Youzhong1, Tae Whan

Kim1 , Yi Chen3, Cengiz Z. Altuntas1, Kristian Bak-Jensen3, Mandy J. McGeachy3,

Jeong-Su Do1, Hui Xiao 1, Greg M. Delgoffe4, Booki Min1, Jonathan D. Powell4,

Vincent K. Tuohy1, Daniel J. Cua3*, Xiaoxia Li X1,2*

1Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation

2Department of Biological, Geological, and Environmental Sciences, Cleveland State

University

3Schering-Plough Biopharma), 901 California Ave. Palo Alto, CA 94304, USA

4Sidney-Kimmel Comprehensive Cancer Center, Johns Hopkins University School of

Medicine, Baltimore, MD 20205 USA

*Co-corresponding author

Accepted for publication by Immunity, November 2009

82 ABSTRACT

Interleukin-1 (IL-1)-mediated signaling in T cells is essential for T helper 17

(Th17) cell differentiation. We showed here that SIGIRR, a negative regulator of IL-1

receptor and Toll-like receptor signaling, was induced during Th17 cell lineage

commitment and governed Th17 cell differentiation and expansion through its inhibitory

effects on IL-1 signaling. The absence of SIGIRR in T cells resulted in increased Th17

cell polarization in vivo upon myelin oligodendrocyte glycoprotein (MOG35-55) peptide immunization. Recombinant IL-1 promoted a marked increase in the proliferation of

SIGIRR-deficient T cells under an in vitro Th17 cell-polarization condition. Importantly, we detected increased IL-1-induced phosphorylation of JNK and mTOR kinase in

SIGIRR-deficient Th17 cells compared to wild-type Th17 cells. IL-1-induced proliferation was abolished in mTOR-deficient Th17 cells, indicating the essential role of mTOR activation. Our results demonstrate an important mechanism by which SIGIRR controls Th17 cell expansion and effector function through the IL-1-induced mTOR signaling pathway.

INTRODUCTION

Interleukin-17 (IL-17) is a proinflammatory cytokine that plays an important role in host defense against infections and is involved in the pathogenesis of multiple human and animal autoimmune disease as well as allergen-specific immune responses (Bettelli et al., 2006; Dong, 2008; Harrington et al., 2005; Iwakura and Ishigame, 2006; Kolls and

Linden, 2004; Mangan et al., 2006; McGeachy and Cua, 2008; Nakae et al., 2002; Park et al., 2005; Veldhoen et al., 2006). IL-17 concentrations are elevated in patients with

83 rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD),

and asthma (Matusevicius et al., 1999; Teunissen et al., 1998; Tzartos et al., 2008). Anti-

IL-17 inhibition can lead to substantial reduction of chemokine production in the central

nervous system (CNS), markedly reduce experimental autoimmune encephalomyelitis

(EAE) severity and reverse the progression of active EAE (Kroenke et al., 2008). The

pathogenic role of IL-17 has also been demonstrated in experiments with IL-17- and IL-

17R-deficient mice, in which various autoimmune disorders, including EAE and

collagen-induced arthritis (CIA), were suppressed (Gonzalez-Garcia et al., 2009;

Hofstetter et al., 2005; Taylor, 2003). The main function of IL-17 is to coordinate local

tissue inflammation via the interaction with IL-17 receptor, inducing the expression of

pro-inflammatory and neutrophil-mobilizing mediators (including IL-6, G-CSF, TNFα

and IL-1, CXCL1, CCL2, CXCL2, CCL7, and CCL20), as well as matrix

metalloproteases (MMPs) that allow activated T cells to penetrate extracellular matrix

(Andoh et al., 2005; Bamba et al., 2003; Chabaud et al., 2000; Fossiez et al., 1996;

Gaffen, 2008; Kao et al., 2005; Laan et al., 1999; Qian et al., 2007; Yao et al., 1995).

CD4+ T helper (Th) lymphocytes are essential in regulating immune responses

and autoimmune and inflammatory diseases. Upon activation, naïve CD4+ T helper (Th)

cells differentiate into three effector subsets: Th1 cells are characterized by production of

IFN-γ which mediates cellular immunity; and Th2 cells, which synthesize IL-4, IL-5 and

IL-13 and are important in humoral immunity and allergic responses (McGuirk and Mills,

2002; Murphy and Reiner, 2002). Th17 cells belong to a third lineage of CD4+ Th cells, which produce IL-17, IL-17F, IL-21 and IL-22 (Bettelli et al., 2006; Harrington et al.,

84 2005; Iwakura and Ishigame, 2006; Kolls and Linden, 2004; Langrish et al., 2005;

Mangan et al., 2006; Nakae et al., 2002; Park et al., 2005; Veldhoen et al., 2006). Several

cytokines including TGF-β and IL-6 are required for Th17 cell differentiation upon T cell

receptor (TCR) activation. Transcription factors including STAT3 together with RORγ

and RORγt determine Th17 cell lineage specific differentiation program through

induction of a set of signature cytokines and cytokine receptors, including IL-23R and IL-

1R (Ivanov et al., 2006; Korn et al., 2007; McGeachy et al., 2007; Wei et al., 2007; Yang

et al., 2008). Although the detailed molecular mechanism is still unclear, IL-1 and IL-23

have major influences on Th17 cell differentiation, possibly through cell proliferation,

and survival to maintain the differentiated state of Th17 cells (Korn et al., 2009).

Importantly, mice lacking either IL-1 or IL-23 are resistant to disease induction in Th17

cell-dependent CIA, EAE, and IBD diseases models (Ben Sasson et al., 2009; Brereton et

al., 2009; Chung et al., 2009; Langrish et al., 2005; Murphy et al., 2003; Sutton et al.,

2006; Thakker et al., 2007). While much effort has been focusing on how cytokines

promote Th17 cell differentiation, it is equally critical to investigate how Th cell effector

functions are regulated. Multiple cytokines including IFN , IL-4 (Harrington et al.,

2005; Park et al., 2005), IL-2 and IL-27 (Laurence et al., 2007; Stumhofer et al., 2006)

have been shown to down-regulate Th17 cell differentiation through direct and/or indirect

impact on the transcriptional program mediated by STAT3, RORγt and RORα. Here, we

demonstrate that SIGIRR, a negative regulator of IL-1 receptor and Toll-like receptor, suppressed Th17 cell expansion and IL-17-dependent disease.

85 SIGIRR (also referred as TIR8) is a negative regulator for IL-1R, TLR4 and

TLR9 signaling. SIGIRR contains a single immunoglobulin (Ig) extracellular domain

and a TIR (Toll-IL-1R) intracellular domain (Garlanda et al., 2004; Wald et al., 2003),

inhibiting IL-1 and lipopolysaccharide (LPS) signaling through its interaction with the

TLR4 and IL-1R complexes (Qin et al., 2005). In addition to the role of SIGIRR in

mucosal immunity (Garlanda et al., 2004; Xiao et al., 2007), recent studies have shown

the critical role of SIGIRR in modulating autoimmunity and inflammatory responses

associated with infections (Bozza et al., 2008; Garlanda et al., 2007). SIGIRR is highly

expressed in intestinal epithelial cells, contributing to immune tolerance to commensal

bacteria. Importantly, SIGIRR is also expressed in T cells and DCs, exerting the fine-

tuning modulation in inflammatory responses, manifested by the hypersusceptibility of

SIGIRR-deficient mice to pathogen infection or autoimmune lupus (Lech et al., 2008).

Here, we report that whereas SIGIRR expression was induced in differentiated

Th17 cells, SIGIRR-deficient mice were more susceptible to experimental autoimmune

encephalomyelitis (EAE) due to hyper activation of Th17 cells upon immunization with

MOG (myelin oligodendrocyte glycoprotein) peptide. IL-1 treatment resulted in

increased TGFβ and IL-6-mediated Th17 cell differentiation in SIGIRR-deficient T cells compared to that in wild-type T cells, suggesting the impact of SIGIRR on Th17 cells is probably through its modulation on IL-1 signaling during Th17 differentiation. We also examined IL-1 signaling mechanism in Th17 cells and detected increased IL-1-induced phosphorylation of JNK and mTOR kinase in SIGIRR-deficient Th17 cells compared to wild-type Th17 cells. Rapamycin, an inhibitor of mTOR, specifically inhibited SIGIRR-

86 regulated IL-1-induced Th17 cell proliferation and IL-17 production. Taken together, our

study suggests that SIGIRR plays an important regulatory role in Th17 differentiation and

proliferation and IL-17-dependent EAE pathogenesis, probably through its modulation on

IL-1 signaling in Th17 cells.

RESULTS

SIGIRR expression is induced during Th17 cell differentiation

Although SIGIRR is expressed in splenic T cells, its physiological function in T cell is unclear. SIGIRR-deficient mice are highly susceptible to DSS-induced colitis and

DSS-treated SIGIRR-deficient colon tissues produce high amounts of IL-17 (Xiao et al.,

2007), implicating a possible regulatory role of SIGIRR in Th17 cell function. Therefore,

we sought to determine the potential role of SIGIRR in modulation of Th17 cell function.

Interestingly, we found that SIGIRR and the known Th17 cell-associated molecules (IL-

17, IL-1R1 and IL-23R) were expressed at a much higher amount in Th17 cells compared

to IFNγ-producing Th1 or naïve CD4+ T cells, indicating a possible functional role of

SIGIRR in Th17 cell subset (Fig. 3.1A-C). A time-course was performed to examine the

kinetics of SIGIRR and IL-1R1 expression by real-time PCR during Th17 cell

differentiation. Although IL-1R1 expression was highly induced on day 1 of polarization, SIGIRR, IL-17 and IL-23R expression was induced more gradually during polarization, suggesting a possible feedback regulatory role of SIGIRR for Th17 cell

function (Fig. 3.1D).

Figure 3.1. SIGIRR expression is induced during Th17 cell differentiation. (A-C)

Naïve CD4+ T cells (CD4+CD44lo) were sorted by flow cytometry and polarized under

Th1 (IL-12 and anti-IL-4) and Th17 (TGF-β, IL-6, anti-IFN-γ and anti-IL-4) cell- inducing conditions (on anti-CD3 and anti-CD28-coated plates) for 3 days, followed by

(A) immunoblot analysis for the expression of SIGIRR, (B) real-time PCR analysis for the expression of IL-1R1 and IL-23R, and (C) ELISA assay for the production of IL-17 and IFN-γ. (D) Real-time PCR analysis for the expression of IL-17, SIGIRR, IL-1R1 and

IL-23R over the time course of Th17 cell polarization. The presented data represent three independent experiments. Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test).

88 SIGIRR deficiency leads to increased susceptibility to Th17 cell-dependent EAE

IL-1 signaling in T cells is essential in Th17 cell responses in vivo (Ben Sasson et

al., 2009; Sutton et al., 2006). To determine the regulatory role of SIGIRR in IL-1-

mediated signaling in Th17 cells, we first examined the impact of SIGIRR deficiency on

Th17 cell effector function in vivo. Compared to WT mice, Sigirr-/- mice showed an

earlier onset of neurologic impairment and markedly increased disease severity compared

to WT littermates (Fig. 3.2A). These data show that SIGIRR is critical for the

modulation of MOG35-55-induced EAE in mice. Consistent with the clinical signs,

mononuclear cell infiltrates were more prominent in the white matter of spinal cords of

-/- MOG35-55 immunized Sigirr mice compared to that in WT mice (Fig. 3.2B-C).

Compared with the brain tissues from WT mice, Sigirr-/- mice showed substantially more

infiltrating CD45+, CD4+, CD11b+ and Gr-1+ cells. In addition, the amount of TNF, IL-

17 and IL-6 was increased in the spinal cords of Sigirr-/- mice compared to that in control

-/- mice (Fig. 3.2D). Thus, the increased susceptibility of Sigirr mice to MOG35-55-induced

EAE is associated with increased mononuclear cell infiltration in the CNS during EAE induction and elevated inflammatory cytokines in the spinal cords.

Figure 3.2. SIGIRR deficiency leads to increased susceptibility of Th17-dependent

EAE and hyper activation of MOG-specific Th17 cells. (A) EAE was induced by

MOG35-55 immunization. Mean clinical scores were calculated each day for WT (n = 13) and Sigirr-/- mice (n = 10). (B) Hematoxylin and eosin and anti-CD3 staining of spinal

-/- cord of WT and Sigirr mice 15 days after immunization with MOG35-55. (C) Immune

-/- cell infiltration in the brain of MOG35-55 immunized WT and Sigirr mice (n=3, 7 days

after disease onset) was analyzed by flow cytometry. (D) Real-time PCR analysis of

relative expression of IFN-γ, IL-17, TNF-α and IL-6 in spinal cords of MOG35-55

immunized WT and Sigirr-/- mice (n=3, 7 days after disease onset) as compared to CFA

treated WT control mice. Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test). Data

are representative of three independent experiments.

Figure 3.2(continue). SIGIRR deficiency leads to increased susceptibility of Th17- dependent EAE and hyper activation of MOG-specific Th17 cells. (E-F) Draining lymph node cells from wild-type mice and Sigirr-/- mice were collected 10 days after

immunization with either MOG35-55 emulsified in complete Freund's adjuvant or complete

Freund's adjuvant alone and were re-stimulated with MOG35-55 in vitro for 4 days,

followed by ELISPOT analysis (E) and ELISA (F) of IL-17 and IFN-γ. Error bars, s.d.; n = 10 mice per group. p<0.05; **.p<0.01 (two tailed t-test). (G) Primed MOG35-55

specific T cells (10 days) were re-stimulated with MOG35-55 in vitro in the presence of

recombinant IL-23 for 4 days, and then transferred to naïve wild-type and Sigirr-/- mice.

Graph represents the average clinical score after T-cell transfer. n=5. *, p<0.05;(ANOVA). Data are representative of three independent experiments.

91 To determine the effect of SIGIRR deficiency on the activation of MOG35-55- specific T cells in EAE, we examined responses of Sigirr-/- and WT lymph node cells to

MOG35-55 in vitro for 10 days after immunization of mice with MOG35-55. Lymph node

cells from Sigirr-/- mice showed higher frequencies of T cells secreting IL-17, but similar

numbers of IFNγ producing cells, compared to WT mice (Fig. 3.2E). In the recall

-/- responses to MOG35-55, supernatants from 10-day primed lymph node cells from Sigirr

mice showed elevated production of IL-17, but not IFNγ, compared to that in WT mice

(Fig. 3.2F). These results suggest that SIGIRR deficiency enhances Th17 polarization in vivo. The functionality of MOG-specific Th17 from the WT and Sigirr-/- mice was

further compared by adoptive transfer to WT and Sigirr-/- recipient mice to induce EAE.

-/- Adoptive transfer of MOG35-55-specific Sigirr Th17 cells induced more severe EAE in

either WT or Sigirr-/- recipient mice compared to WT Th17 cells (Fig. 3.2G). Thus,

SIGIRR deficiency enhances Th17 polarization in vivo, leading to increased severity of

IL-17-dependent EAE.

SIGIRR directly regulates Th17 but not Th1 cell development

To test the intrinsic role of SIGIRR in T cells, we sorted naïve T cells

(CD4+CD44lo) from WT and Sigirr-/- mice and transferred them into Rag1-/- mice. Upon

-/- immunization with MOG35-55, mice with Sigirr T cells developed more severe EAE than

the mice with WT T cells (Fig 3.3A). Consistent with the clinical scores, inflammatory

cell infiltration and secretion of inflammatory cytokines were significantly increased in

the spinal cord of the Rag1-/- recipient mice transferred with Sigirr-/- T cells compared to

that in the mice transferred with WT T cells (Fig 3.3B-C). Elevated IL-17 mRNA

Figure 3.3: T-cell-derived SIGIRR regulates Th17 cell development in vivo. Wild- type and Sigirr-/- naïve T cells were intravenousely transferred into Rag1-/- mice and EAE

was induced by MOG35-55 immunization. (A) Mean clinical scores were calculated each day for Rag1-/- mice transferred with wild-type and Sigirr-/- T cells. n=5 *, p<0.05;

(ANOVA). (B) Hematoxylin and eosin staining of spinal cord of Rag1-/- mice transferred

-/- with wild-type and Sigirr T cells 15 days after immunization with MOG35-55. (C) Real-

time PCR analysis of relative expression of IFN-γ, IL-17, TNF-α and IL-6 in spinal cords

-/- -/- of MOG35-55 immunized Rag1 mice transferred with wild-type and Sigirr T cells (n=3,

10 days after disease onset). (D-E) Draining lymph node cells from SIGIRR-TG-KO

(TG-KO) and littermate Sigirr-/- mice were collected 10 days after immunization with

MOG35-55 emulsified in complete Freund's adjuvant and were re-stimulated with MOG35-

55 in vitro for 4 days, followed by ELISA (D) and ELISPOT analysis (E) (See also Figure

S1), Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test). Data are representative of

three independent experiments.

Figure 3.3(continue): T-cell-derived SIGIRR regulates Th17 cell development in vivo.

(F-H) Naïve T cells (CD4+CD44low) from wild-type and Sigirr-/- mice were co-cultured

with wild-type splenic antigen-presenting cells (APCs) in the presence of anti-CD3 and polarized them under Th1 (IL-12 and anti-IL-4), or Th17 (TGF-β, IL-6, anti-IFN-γ and anti-IL-4) conditions, followed by intracellular cytokine staining for IL-17 and IFN-γ (F) and ELISA for IL-17 (H). (G) Wild-type and Sigirr-/- mice Th1 cells were treated with

5ng/ml IL-18 for 48 h, followed by ELISA for IFN-γ. (I) Real-time PCR analysis of

relative expression of IL-17, IL-22, IL-23R, ROR-γt and IL-10 in wild-type and Sigirr-/-

Th17 cells as compared to naïve T cells. Data are representative of at least three (A-C) independent experiments. Error bars (A-C), s.d. *, p<0.05; **.p<0.01 (two tailed t-test).

94 expression in the spinal cord of mice with Sigirr-/- T cells supports the regulatory role of

SIGIRR on Th17 cell differentiation and expansion (Fig. 3.3C). To confirm T cell-

derived SIGIRR has an intrinsic impact on Th17 cell development, we examined whether

T cell-specific expression of SIGIRR is able to rescue the hyper Th17 cell activation in

Sigirr-/- mice. We generated T cell-specific SIGIRR-transgenic mice using the CD2

promoter to drive the over-expression of SIGIRR, and crossed CD2-SIGIRR onto Sigirr-/-

mice to generate SIGIRR-TG-KO mice (Lang et al., 1988). In the recall responses to

MOG35-55, supernatants from 10-day primed lymph node cells from SIGIRR-TG-KO

mice showed reduced production of IL-17 compared to Sigirr-/- mice (Fig 3.3D).

Consistent with ELISA data, the number of MOG35-55 specific IL-17 producing cells, but

not IFNγ producing cells, was decreased in SIGIRR-TG-KO mice as compared to that in

Sigirr-/- mice (Fig 3.3E). Thus, T cell-specific expression of SIGIRR is able to rescue the

hyper Th17 cell activation in Sigirr-/- mice.

To further determine whether SIGIRR has direct impact on Th17 cells, we co-

cultured naïve T cells from WT and Sigirr-/- mice with WT splenic DCs and differentiated

them into Th1 and Th17 cell lineages. The frequency of IFNγ-positive cells as well as amount of secreted IFNγ from Th1 cell differentiated cultures was indistinguishable between WT and Sigirr-/- cells (Fig. 3.3F), suggesting that SIGIRR does not have a

substantial impact on Th1 cell differentiation. Furthermore, similar amounts of IFN-γ

were detected in WT and Sigirr-/- Th1 cells in response to IL-18 stimulation (Fig. 3.3G).

In contrast, the frequency of IL-17-positive cells and total IL-17 production were

significantly increased in Sigirr-/- Th17 cells compared to WT (Fig. 3.3F and 3.3H). In

95 addition to IL-17, the expression of other Th17-associated molecules (including IL-17,

IL-22, and IL-23R) was also higher in Sigirr-/- Th17 cells than that in WT cells (Fig.

3.3I). Thus, SIGIRR directly regulates Th17 but not Th1 cell development.

SIGIRR suppresses Th17 cell differentiation and proliferation through IL-1R

signaling

IL-1 signaling in T cells synergizes with IL-6 to regulate TH17 cell differentiation

and maintain cytokine production in effector Th17 cells (Chung et al., 2009). To confirm

the role of IL-1 signaling in Th17 cell differentiation and maintenance, we purified naïve

T cells from WT and Il1r1-/- mice and differentiated them into Th17 cells (TGFβ+IL-6) with or without IL-1β. IL-1β indeed enhanced Th17 cell differentiation, which was abolished in the absence of IL-1R (Fig. 3.4A). SIGIRR inhibits IL-1R-TLR signaling through its interaction with the receptor complexes. We hypothesize that the impact of

SIGIRR on Th17 cell differentiation and proliferation is due to enhanced IL-1R signaling in these cells. To test this hypothesis, WT and Sigirr-/- naïve T cells were differentiated

into Th17 cells with or without IL-1β stimulation. Similar numbers of IL-17-positive

cells ( 8-10%) were detected in WT and Sigirr-/- T cells under TGFβ+IL-6-mediated differentiation condition (in the absence of IL-1β stimulation) (Fig. 3.4B). Importantly, although IL-1β promoted Th17 cell differentiation in both WT and Sigirr-/- T cells,

addition of IL-1β resulted in a significantly higher IL-17-positive population in Sigirr-/- T cells than that in WT T cells (Fig. 3.4B). Consistent with flow cytometry analysis, the expression of other Th17 cell-associated molecules (including IL-17, IL-21,

97 Figure 3.4. SIGIRR suppresses Th17 differentiation and expansion through IL-1R signaling (A) Naïve wild-type and Il1r1-/- CD4+ T cells (CD4+CD44low) were polarized to

Th17 cells (TGF +IL-6 on anti-CD3 and anti-CD28 coated plates) in the presence and absence of IL-1β, followed by ELISA for IL-17 and IL-22. (B) Naïve wild-type and

Sigirr-/- CD4+ T cells (CD4+CD44low) were polarized to Th17 cells (TGF +IL-6 on anti-

CD3 and anti-CD28 coated plates) in the presence and absence of IL-1β, followed by intracellular cytokine staining for IL-17 and IFN-γ. While IL-1β promoted Th17 cell differentiation in both WT and Sigirr-/- T cells, addition of IL-1β resulted in a significantly higher IL-17-positive population in Sigirr-/- T cells than that in WT T cells.

γt, IL-10 and IRF4 in wild-type and Sigirr-/- Th17 cells as compared to the naïve T cells.

Data are representative of at least three (A-C) separate experiments. Error bars (A-C), s.e.m. *, p<0.05; **.p<0.01 (two tailed t-test).

98 IL-22, and IL-23R) was also much higher in Sigirr-/- T cells than that in WT cells in the

presence of IL-1β stimulation (Fig. 3.4C). In addition, consistent with IL-1 induction of

transcription factor IRF4 and RORγt expression, SIGIRR deficiency significantly enhanced the expression of IRF4 and RORγt during Th17 cell differentiation (Fig. 3.4C).

Thus, SIGIRR negatively regulates Th17 cell differentiation and proliferation by suppressing IL-1R signaling during differentiation of Th17 cells.

SIGIRR deficiency leads to increased IL-1-induced JNK and mTOR phosphorylation

To understand the molecular mechanism by which SIGIRR regulates IL-1- dependent Th17 cell differentiation and proliferation, we examined IL-1 signaling in WT and Sigirr-/- Th17 cells. Although IL-1 stimulation induced p38 and IκBα

phosphorylation in both WT and Sigirr-/- Th17 cells, IL-1 treatment leads to increased

JNK and mTOR phosphorylation in Sigirr-/- Th17 cells compared to that in WT cells (Fig.

3.5A). Consistent with this, IL-1-dependent phosphorylation of 4E-BP1 (eukaryotic

initiation factor 4E-binding protein, an mTOR substrate) and S6 (a downstream

component of mTOR signaling) were also increased in Sigirr-/- Th17 cells compared to

that in WT cells. Thus, SIGIRR deficiency markedly enhances at least some of the IL-1-

mediated signaling cascades in Th17 cells.

To determine the impact of SIGIRR-modulated IL-1-induced signaling events on

Th17 effector function, we used specific inhibitors to block these IL-1-induced signaling

Figure 3.5. SIGIRR suppresses Th17 expansion through IL-1-induced mTOR- mediated cell proliferation. (A) Cell lysates from wild-type and Sigirr-/- Th17 cells

untreated or treated with IL-1 (10ng/ml) for different time points were analyzed by

western blot analysis using antibodies as indicated.

100

Figure 3.5(continue). SIGIRR suppresses Th17 expansion through IL-1-induced mTOR-mediated cell proliferation. (B) Wild-type and Sigirr-/- Th17 cells were rested for two days, followed by incubation with different doses of IL-1 as indicated in the presence or absence of 50nM rapamycin, 2μM SB203580 or 10μM SP600125 for three days. The treated cells were analyzed for production of IL-17 by ELISA. Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test). Data are representative of three independent experiments.

101

Figure 3.5(continue). SIGIRR suppresses Th17 expansion through IL-1-induced mTOR-mediated cell proliferation. (C) Cell lysates from Sigirr-/- Th17 cells pre-treated

with rapamycin for 2h and then treated IL-1 (10ng/ml) treatment for 15, 30 and 60 min

were analyzed by western blot analysis using antibodies as indicated. (D) Wild-type and

Sigirr-/- Th17 cells were rested for two days, followed by incubation with different doses of IL-1. The treated cells were incubated one additional day with 3H for thymidine incorporation experiment. (E) Frap1fl/fl naïve CD4+ T cells were differentiated into

Th17 cells and infected with retrovirus expressing GFP or Cre/GFP. Infected CD4+GFP+ cells were isolated, analyzed for mTOR expression (left), and cultured in the presence and absence of IL-1. GFP+ cells were analyzed by thymidine incorporation assay for

proliferation (right). Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test). Data are

representative of three independent experiments.

102 -/- cascades in WT and Sigirr TH17 cells, including inhibitors for mTOR (rapamycin), p38

(SB203580) and JNK (SP600125). All three inhibitors significantly reduced IL-1-

induced IL-17 production in both WT and Sigirr-/- Th17 cells, indicating the importance

of mTOR, JNK and p38 activation for IL-1-induced Th17 effector function (Fig. 3.5B).

Although the inhibitors of p38 and JNK decreased IL-1-induced IL-17 production in both

WT and Sigirr-/- Th17 cells, Sigirr-/- Th17 cells still produced more IL-17 in the presence of either inhibitor compared to WT cells. In contrast, the impact of SIGIRR deficiency on Th17 cell effector function was abolished after rapamycin treatment (Fig. 3.5B).

Similar residual amounts of IL-17 production were detected in IL-1-stimulated WT and

Sigirr-/- Th17 cells after rapamycin treatment, suggesting that the mTOR-mediated signaling is critical for SGIRR to modulate IL-1-dependent Th17 cell effector function.

The inhibitory effect of rapamycin is specific for the mTOR pathway because rapamycin

treatment inhibited the IL-1-induced 4E-BP1 and S6 but not JNK phosphorylation (Fig.

3.5C).

Because mTOR signaling is known to have a major impact on cell growth and

-/- proliferation, we examined WT and Sigirr TH17 cell proliferation in response to IL-1

stimulation. Whereas IL-1 induced proliferation in both WT and Sigirr-/- Th17 cells, the

IL-1-induced cell proliferation was much higher in Sigirr-/- Th17 cells than that in WT

cells (Fig. 3.5D). These results strongly suggest that SIGIRR deficiency has a substantial

impact on Th17 cell proliferation, which leads to increased production of cytokines

associated with Th17 cells. Importantly, rapamycin abolished IL-1-induced cell

proliferation in both WT and Sigirr-/- Th17 cells. Thus, one important mechanism for

103 SIGIRR to suppress Th17 proliferation and effector function is through IL-1-induced

mTOR-mediated cell proliferation. To exclude the possible non-specific effect of rapamycin, we examined the responsiveness of WT and mTOR-deficient Th17 cells to

IL-1 stimulation. Because Frap1-/- T cells are unable to differentiate into Th17 cells, we

first differentiated Frap1fl/fl (wild-type) naïve T cells into Th17 cells. The differentiated

Th17 cells (Frap1fl/fl) were infected with hCre-GFP-RV to delete mTOR (Delgoffe et al.,

2009; Zhu et al., 2004) (Fig. 3.5E). Whereas IL-1 stimulation increased the proliferation

of WT Th17 cells, the proliferation of mTOR-deficient cells was abolished in response to

IL-1 treatment (Fig 3.5E). Thus, IL-1-induced Th17 expansion is mTOR-dependent.

SIGIRR-modulated IL-1-activated mTOR pathway signals through the IRAK

proteins

We next investigate how SIGIRR modulates IL-1-mediated mTOR activation.

Upon IL-1 stimulation, adaptor MyD88 is recruited to the IL-1 receptor, followed by the

recruitment of serine and threonine kinases IRAK4 and IRAK1 (interleukin-1 receptor-

associated kinases) (Yao et al., 2007). At the receptor complex, IRAK4 mediates the

phosphorylation of IRAK1, followed by the ubiquitination and degradation of IRAK1,

which are crucial for the activation of downstream signaling cascades (Yao et al., 2007).

We have previously shown that SIGIRR negatively regulates IL-1 signaling through its

interaction with the receptor complex, attenuating the recruitment of these receptor

proximal signaling components to the receptor (including MyD88, IRAK4 and IRAK1)

(Qin et al., 2005). Consistent with this, we found that IRAK1 degradation was

104

Figure 3.6. IL-1 activates mTOR pathway through IRAK proteins. (A) Cell lysates from

wild-type, Irak4-/- and Irak1-/- Th17 cells untreated or treated with IL-1 (10ng/ml) for different

time points were analyzed by western blot analysis using antibodies as indicated. (B) Naïve

wild-type, Irak4-/- and Irak1-/- CD4+ T cells (CD4+CD44low) were polarized to Th17 cells

(TGF +IL-6 on anti-CD3 and anti-CD28 coated plates) in the presence and absence of IL-

1β, followed by intracellular cytokine staining for IL-17 and IFN-γ, Error bars, s.d.; *, p<0.05; **.p<0.01 (two tailed t-test). (C) 293 cells transfected with IL-1R (293-IL-1R) cells,

IRAK1-deficient cells and 293-IL-1R transfected with SIGIRR were treated or untreated with

IL-1 for 15 min. TSC1/TSC2 protein complex was immunoprecipitated and analyzed by western blot analysis using antibodies as indicated. Data are representative of at least three independent experiments.

105 We and others have recently reported that IRAK4 and IRAK1 are required for

Th17 cell polarization and development of EAE (Staschke et al., 2009). Therefore, we wondered whether SIGIRR modulates IL-1-mediated mTOR activation and the IL-1- dependent Th17 cell proliferation through the IRAK proteins. To test this hypothesis, we examined the impact of IRAK4 and IRAK1 deficiency on IL-1-induced mTOR signaling and IL-1-dependent Th17 cell development. IL-1-induced phosphorylation of mTOR,

4EBP, and S6 were all abolished in IRAK4-deficient Th17 cell and greatly reduced in

IRAK1-defcient Th17 cells (Fig. 3.6A), indicating that IRAK4 and IRAK1 are indeed

required for IL-1-induced activation of mTOR and its downstream signaling components.

Consistent with this, IL-1-induced Th17 cell proliferation was significantly reduced in

IRAK4- and IRAK1-deficient Th17 cells, implicating the importance of IRAK4 and

IRAK1-mediated mTOR activation in IL-1-induced Th17 cell proliferation (Fig. 3.6B).

Taken together, these results suggest that SIGIRR modulates Th17 cell expansion

through the IRAK4-IRAK1-mTOR pathway. Interestingly, IRAK4 and IRAK1 were

required for IL-1-induced mTOR and JNK but not p38 activation (Fig. 3.6A). Consistent

with this, SIGIRR preferentially affected mTOR and JNK activation, but not p38 in

response to IL-1 stimulation, which further supports the notion that SIGIRR modulates

IL-1-mediated downstream signaling through its impact on the activation of IRAK4 and

IRAK1 (Fig. 3.6A)

The tuberous sclerosis 1 (TSC1)-TSC2 tumor suppressor complex serves as a

repressor of the mTOR pathway, and the disruption of TSC1-TSC2 complex leads to

mTOR activation (Lee et al., 2007). We found that IL-1 stimulation can also lead to

106 disruption of TSC1-TSC2 complex in human 293 cells transfected with IL-1R (293-IL-

1R). Importantly, IL-1-mediated disruption of TSC1-TSC2 complex was greatly reduced

in IRAK1-deficient cells derived from 293-IL-1R cells (Li et al., 1999), suggesting that

IRAK1-dependent mTOR activation is through the disruption of TSC1-TSC2 complex

(Fig. 3.6C). Overexpression of SIGIRR suppressed the IL-1-induced disruption of TSC1-

TSC2 complex, which is likely due to the inhibitory effect of SIGIRR on IRAK1

activation (as evident by reduced IRAK1 modification upon SIGIRR overexpression)

(Fig. 3.6C). Taken together, these results suggest that SIGIRR modulates IL-1-induced

mTOR activation through its impact on IRAK1 activation and consequent effect on

IRAK1-mediated disruption of TSC1-TSC2 complex.

DISCUSSION

We report here a mechanism for the regulation of Th17 cell differentiation and

expansion. IL-1-mediated signaling in T cells is essential for in vivo Th17 cell

differentiation and IL-17-dependent autoimmune diseases. We showed here that SIGIRR

is induced during Th17 cell differentiation and it suppresses Th17 cell differentiation and

proliferation through direct inhibition of multiple IL-1-dependent signaling pathways.

IL-1-induced phosphorylation of JNK and mTOR is enhanced in Sigirr-/- Th17 cells.

Rapamycin, an inhibitor of mTOR specifically blocked SIGIRR-regulated IL-1-induced

Th17 cell proliferation and cytokine production. Importantly, IL-1-dependent proliferation was abolished in mTOR-deficient Th17 cells, confirming the essential role of mTOR activation in IL-1-dependent Th17 cell proliferation. We also showed that

SIGIRR is critical for the control of Th17 cell-dependent development of CNS

107 autoimmune inflammation. In the absence of this IL-1 regulatory mechanism, Th17 cells

infiltrated the CNS in greater numbers and showed enhanced pathogenic functions. Thus,

SIGIRR belongs to a class of immune modulators, including IL-2, IL-4, IL-25, IL-27,

IFN , IFN , and ATAR, capable of suppressing the development and function of IL-17-

producing T cells.

Both IL-1R1 and IL-23R are induced by IL-6-dependent RORγt expression

(Chung et al., 2009). Although stimulation with TGF-β and IL-6 is sufficient to drive the initial development of IL-17-producing T cells in vitro, Il1r1-/- T cells can not

differentiate into inflammatory Th17 cells in vivo. Accordingly, IL-1 signaling in T

cells is absolutely essential for the induction of autoimmune encephalomyelitis (Chung et al., 2009; Sutton et al., 2006). In support of this, IRAK4 (a key kinase in IL-1 signaling)

kinase-inactive (IRAK4 KI) Th17 cells fail to induce EAE in either WT or IRAK4 KI

recipient mice (Staschke et al., 2009). In contrast, WT Th17 cells are able to induce EAE in both WT and IRAK4 KI recipient mice, indicating that IL-1 signaling in the CNS

resident cells is not essential for IL-1-dependent induction of EAE disease. Further

evidence supporting a direct effect of IL-1 on T cells came from adoptive transfer of

OVA-TCR transgenic T cells into Il1r1-/- recipients, which demonstrate that T cells are a

major target of IL-1 in vivo (Ben Sasson et al., 2009). In addition, deficiency of IL-1R1

on T cells but not on DCs blocked Th17 cell polarization (Chung et al., 2009).

Consistent with these previous results, we found that the lack of SIGIRR on T cells leads

to increased autoantigen-induced Th17 cell polarization in vivo. Importantly, Th17 cell

differentiation and cytokine production were significantly increased in Sigirr-/- T cells

108 after they were polarized in the presence of WT DCs, indicating that deficiency of

SIGIRR on T cells—not DCs—results in increased Th17 cell development. It is also noteworthy that WT Th17 cells were able to induce similar EAE severity in both WT and

Sigirr-/- recipients, demonstrating that SIGIRR deficiency does not have a major impact

on the responses of APCs and CNS resident cells to autoantigen-specific Th17 cells.

Recent studies have shown that IL-1 stimulation enhances the expression of

transcription factors IRF4 and RORγt, indicating that IL-1 is capable of directly

influencing Th17 cell differentiation (Chung et al., 2009). It is thought that naïve T cell-

derived TGF-β in the presence of exogenous IL-6 is sufficient to initiate the RORγt-

dependent gene expression program of Th17 cells. However, further stabilization of the

Th17 cell program is required for their in vivo function. We and others have

demonstrated that two additional signaling pathways are essential for this stabilizing

effect; these include the IL-1R and IL-23R pathways (Chung et al., 2009; McGeachy and

Cua, 2007; McGeachy and Cua, 2008). Consistent with the concept that SIGIRR has a

central role in Th17 cell differentation through the regulation of IL-1 signaling, SIGIRR

deficiency did not impact Th17 cell differentiation induced by TGFβ+IL-6 in vitro. It is

only in the presence of exogenous IL-1 (TGFβ+IL-6+IL-1) that we observed enhanced

Th17 cell proliferation and cytokine production in Sigirr-/- T cells. Our findings also

suggest that SIGIRR negatively regulates Th17 cel differentiation and proliferation by

suppressing IL-1 signaling during initial differentiation of Th17 cells as well as in

differentiated effector Th17 cells. Furthermore, the absence of SIGIRR regulation enabled the overexpression of IRF4 and RORγt that is mediated by IL-1 in the presence

109 of TGFβ and IL-6, which might account for the increased expression of IL-17 and other

Th17 cell -associated cytokines in Sigirr-/- T cells. Hence our findings are consistent with

the known effects of IL-1 on Th17 cell transcriptional regulation, where SIGIRR is a key

regulator to prevent over-activation of Th17-mediated pathogenic effects.

What specific IL-1-mediated signaling events are critical for Th17 differentiation and effector function and how they are modulated by SIGIRR? Although IL-1 induced

similar amounts of p38 phosphorylation in WT and Sigirr-/- Th17 cells, SIGIRR

deficiency allowed greater IL-1-induced phosphorylation of JNK, mTOR and 4E-BP1 (a

substrate of mTOR). We showed that the inhibitors of p38, JNK and mTOR all

decreased IL-1-induced IL-17 production in both WT and Sigirr-/- Th17 cells. However,

only rapamycin abolished the impact of SIGIRR deficiency on Th17 cell effector

function, indicating the critical role of mTOR pathway for SIGIRR-mediated modulation on Th17 cell function. In addition, the mTOR activated pathways have been linked to cell cycle progression and indeed, IL-1 induced more Th17 cell proliferation in Sigirr-/-

Th17 cells than that in WT cells. Consistent with our findings, a recent study also

reported that IL-1 signaling maintains Th17 cells in the absence of TCR stimuli (Chung

et al., 2009). Thus, whereas IL-1 signaling contributes to tissue immunity mediated by

Th17 cells by promoting Th17 cell differentiation and maintenance, SIGIRR-modulated

IL-1-induced mTOR pathway is essential to ensure a stringent control for the

proliferation of potentially pathogenic Th17 cells. The fact that p38 and JNK inhibitors

reduced IL-1-induced IL-17 production in WT and Sigirr-/- Th17 cells indicates that these

MAPKs are also required for IL-1-induced Th17 effector function. Future studies are

110 required to identify the specific substrates of p38 and JNK that are critical for Th17 cell

function.

We found that IL-1-mediated disruption of TSC1-TSC2 complex was greatly

reduced in IRAK1-deficient cells derived from 293-IL-1R cells, indicating that IL-1-

induced IRAK1-dependent mTOR activation is also through the disruption of TSC1-

TSC2 complex. Consistent with the negative regulation of IRAK1 activation by SIGIRR,

SIGIRR overexpression suppressed IL-1-induced IRAK1 modification and as well as the disruption of TSC1-TSC2. IKKβ interacts with and phosphorylates TSC1, resulting in suppression of TSC1, which plays a critical role in IL-1-mediated mTOR activation (Lee et al., 2007). Because IRAK4 and IRAK are known to mediate the IL-1-induced IKKβ activation, it is possible that IRAK4 and IRAK1 mediate mTOR activation through the activation of IKKβ. Future studies are required to elucidate the detailed signaling cascade for IL-1-induced IRAK4 and IRAK1-mediated mTOR activation.

We have defined the molecular mechanisms for Th17 cell survival and competition for resources in vivo, which explains why Th17 cells are completely absent in IL-1R- deficient mice (Sutton et al., 2006). Compared to in vitro cultures, activating CD4+ T

helper cells in lymph nodes are exposed to relatively low nutrient and antigen load. In this restrictive in vivo environment, IL-1 activation of the mTOR (nutrient sensor and cell cycle regulator), p38, and JNK pathways enable Th17 cells to divide faster and dominate an immune response compared to Th1 cells that might have the same antigenic specificity. Hence, presence of IL-1 during an inflammatory response will give Th17

cells important survival and functional advantages over other T cell subsets. In this

111 study, we also identified SIGIRR as the key regulator of IL-1 function in Th17 cells.

Our earlier studies have shown that SIGIRR can regulate innate immunity; however, as demonstrated in the present study, SIGIRR is expressed on Th17 cells and directly regulates adaptive immunity. In support of this, recent studies have demonstrated critical roles of SIGIRR in modulating lupus autoantigen priming andantigen-specific antimicrobial responses (Bozza et al., 2008; Lech et al., 2008). Based on these observations, we propose that SIGIRR is an important negative “feedback switch” that prevents detrimental autoimmune and chronic inflammatory responses. Given the potency of rapamycin for blockade of SIGIRR-IL-1-regulated TH17 cell proliferation and cytokine production, it may be feasible to consider this well-developed drug and related compounds for treatment of autoimmune and chronic inflammatory diseases.

MATERIAL and METHODS

Mice

Il1r1-/-, and Rag1-/- mice were purchased from Jackson Laboratory. Sigirr-/- mice were generated as described (Wald et al., 2003). The CD2-SIGIRR construct contains an

EcoRI-BamHI cDNA fragment, coding for the SIGIRR protein, inserted between 5 kilobase (kb) upstream and 5.5 kb downstream flanking sequences of the human CD2 gene (Lang et al., 1988). A Flag tag was included at the N-terminus of SIGIRR to distinguish the transgene from the endogenous gene. The founders that carry the SIGIRR transgene were identified by genomic Southern analysis with SIGIRR cDNA as probes.

SIGIRR transgenic mice (CD2-SIGIRR) were bred to Sigirr-/- mice to generate SIGIRR-

112 TG-KO mice. Mice were housed in animal facility (SPF condition) at the Cleveland

Clinic Foundation in compliance with the guidelines set by Institutional Animal Care and

Use Committee.

T cell differentiation

Naïve CD4+CD44lo T cells from Sigirr-/-, C57BL/6 and Il1r1-/-mice were sorted

by flow cytometry and activated with 1 mg/ml anti-CD3 and splenic APCs in the

presence of 5 ng/ml TGF-β and 10 ng/ml IL-6 (Peprotech). For anti-CD3 and anti-CD28-

stimulated differentiation, naïve sorted CD4+CD44lo T cells were activated with plate-

bound 1 mg/ml anti-CD3 and 1 mg/ml anti-CD28 and in the presence of 5 ng/ml TGF-

(Peprotech), 20 ng/ml IL-6 (Peprotech), anti-IL-4 (11B11), 5 mg/ml anti-IFN-γ (XMG

1.2), 10 ng/ml IL-1β (National Cancer Institute) or combination of these stimuli. Three days after activation, cytokines in the supernatant were measured by ELISA. For intracellular staining, cells were stimulated with PMA and ionomycin in the presence of

Golgi-stop for 4 hr, after which IL-17- and IFN-γ-producing cells were analyzed with intracellular staining.

Transfection, coimmunoprecipitations and Western analysis

Procedures for transfection, coimmunoprecipitations and western analysis using

293 cells were previously described (Yao et al., 2007). Antibodies used in this study include primary anti-mouse SIGIRR antibody (R&D systems), β-actin, IRAK1 (Santa

Cruz), p-4EBP1, 4EBP1, p-IκBα, p-JNK, p-p38, p-mTOR, mTOR, p-S6, TSC1, and

TSC2 (Cell Signaling) and GAPDH (Ambion).

113

EAE Induction and Adoptive transfer experiment

Procedures for EAE induction and adoptive transfer experiment were previously

described (Qian et al., 2007). For EAE induction of Rag1-/- mice, CD4+ T cells from wild

type and Sigirr-/-mice were sorted by AutoMacs and intravenousely and transferred into

Rag1-/- mice (7X106 cells/transfer). One day later, the recipient mice were immunized

with MOG(35-55) as described above.

Quantitative Real-Time RT-PCR

Expression of the genes encoding IL-17A, IL-23R, IL-22, IL-21, IL-10 and IRF-4

were quantified with the SYBER Green PCR Master Mix kit (Applied Biosystems) using

primer pairs selected for amplification of each individual cytokine: Il17a: 5’-

CTCCACCGCAATGAAGAC-3’ and 5’- CTTTCCCTCCGCATTGAC-3’: Il23r: 5’-

TTTTGTCGGGAATGGTCTTC-3’ and 5’- GCCACTTTGGGATCATCAGT-3’; Il22:

5’- TCATCGGGGAGAAACTGTTC-3’ and 5’- CATGTAGGGCTGGAACCTGT-3’;

Il10: 5’-TGAATTCCCTGGGTGAGAAG-3’ and 5’-TTCATGGCCTTGTAGACACCT-

3’; Il21: 5’-CGCCTCCTGATTAGACTTCG-3’ and 5’-

TTGAGTTTGGCCTTCTGAAAA-3’; β-actin: 5’- GGTCATCACTATTGGCAACG-3’

and 5’- ACGGATGTCAACGTCACACT-3’; Irf4: 5’-

TCCTCTGGATGGCTCCAGATGG-3’ , and, 5’-CACCAAAGCA CAGAGTCACCTG-

3’. Relative gene expression was determined as the ratio of cytokine to β-actin gene-

expression levels for each sample.

114 Proliferation Assay

Th17 cells were cultured in 96-well flat-bottom microtiter Falcon plates (BD

Labware) at 20x103 cells/well in DMEM (Mediatech CellGro) supplemented with 10%

FBS (HyClone), 5% HEPES buffer, 2% L-glutamine, and 1% penicillin/streptomycin

(Invitrogen Life Technologies). IL-1β was added in serial dilutions to triplicate wells

with negative control wells (without IL-1β). Cells were cultured at a final volume of 200

µl/well. In some wells, rapmaycin (50nM) (Sigma) was added. After incubation for 96 h,

wells were pulsed with [methyl-3H]thymidine (1.0 µCi/well, specific activity 6.7 Ci/mM;

New England Nuclear) and harvested 16 h later by aspiration onto glass fiber filters. For proliferation assay, levels of incorporated radioactivity were determined by scintillation

spectrometry. Results are expressed as mean cpm of experimental cultures with IL-1β

divided by mean cpm of cultures without IL-1β (stimulation index).

Retroviral infection

Frap1fl/fl naïve T cells were sorted and differentiated into Th17 cells on anti-CD3

and anti-28 coated plate as described above. After differentiation, Th17 cells were rested

for two days and re activated with 0.2 mg/ml anti-CD3 and splenic APCs in the presence

of 5ng/ml TGF-β (Peprotech) and 10 ng/ml IL-6 (Peprotech). Twenty-four hours after

activation, the cells were infected by retroviruses expressing either Cre–GFP (a gift from

Dr. William Paul at National Institute of Health), or control empty vector (containing

only IRES-GFP). Four days after infection, the cells were washed; CD4+GFP+ cells were sorted and cultured in the presence and absence of IL-1 for three days. At the end of three days GFP+ cells were analyzed for cell proliferation by thymidine incorporation assay.

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121

CHAPTER IV

GENERAL DISCUSSION AND FUTURE WORK

The expression pattern of SIGIRR in different tissues shows its possible function

and possible target receptors for its inhibitory function. In colon epithelium, the necessity of TLR2, TLR4 and TLR9 signaling in the maintenance of colonic homeostasis has been shown (Lee et al., 2006; Rakoff-Nahoum et al., 2004). The interaction between epithelial cells and commensal microflora regulates the homeostasis in colon epithelium through

TLR signaling. Interestingly, our results showed that not only the absence of TLR signaling but also the excessive signaling leads to dysregulation of intestinal homeostasis.

In the absence of SIGIRR, the changes in the cell cycle and survival as well as increased cytokine and chemokine secretion can be explained by increased TLR signaling in Sigirr-

/- mice. However the physiological inflammatory condition in Sigirr-/- mice did not cause

a severe colitis phenotype like in A20-/- mice (Turer et al., 2008). Although it is not

tested, it is possible that downstream inhibitory molecules in TLR signaling compensate

the absence of SIGIRR at some level.

122 The increased TLR signaling in Sigirr-/- mice colon epithelium may be directly

affecting the epithelial cells or it is possible that highly activated epithelial cells are

triggering secondary intraepithelial cells such as DCs which stimulate the changes in the homeostasis. The interaction between DCs and epithelial cells are well studied. It has been shown that thymic stromal lymphopoietin (TSLP) secreted by epithelial cells stimulate DCs in the intestinal mucosa which cause the secretion of IL-4 and IL-10 by

DCs to establish a Th2 like environment in the intestinal mucosa against pathogens

(Rimoldi et al., 2005). Moreover, while DCs in the intestinal mucosa continuously

sample luminal antigens and initiate T cell and B cell responses, in the absence of TLR

signaling in epithelial cells, the sampling process in DCs is dramatically diminished

(Chieppa et al., 2006). These findings not only suggest that the DCs in the Sigirr-/- mice

intestinal epithelium may contribute to the phenotype, but also it shows a possible change in intraepithelial cell population, regulated by the signals come from epithelial cells, and a possible change in the population of commensal microflora, regulated by both epithelial cells and intraepithelial cells.

We have shown the increased TLR signaling in Sigirr-/- mice colon epithelial cell

in the homeostasis conditions by showing increased NFκB activation in the epithelial cells in response to IL-1 and LPS treatment. However, our second study related to IL-1 signaling in Th17 cells showed that in addition to NFκB and MAP kinases, IL-1/TLR signaling can also activate mTOR pathway which is known to be important in cell proliferation and cell survival. It is highly possible that some of the phenotype in colonic homeostasis we have observed in Sigirr-/- mice is related to increased mTOR signaling as

123 well. Studying the activation of mTOR pathway in wild type and Sigirr-/- mice, and the

effect of differential inhibition of NFκB and mTOR pathway in the colon epithelium of these mice will show the impact of both pathways on the homeostasis of colonic

epithelium.

The impact of mTOR is not limited on the homeostatic conditions of colon

epithelium. mTOR activation was also linked to the hyper proliferation of colon

epithelium in inflammation-induced tumorigenesis model (Deng et al., 2009). Moreover,

inhibition of mTOR pathway in human colon cancer patients resulted in decrease in

tumor growth (Zhang et al., 2009b). These recent findings indicate that in addition to

NFκB pathway, increased mTOR activation also affect tumor growth in Sigirr-/- mice.

Although the relation between NFκB/mTOR and STAT3 in some cancer cells is shown

(Ma et al., 2010; Yu et al., 2009), the mechanism how increased NFκB and/or mTOR

pathway leads to increased β-catenin accumulation in Sigirr-/- mice colon epithelium is

not clear.

In our study, we have used inflammation-induced tumorigenesis model, because

the link between NFκB dependent inflammation and tumor development has been shown

(Karin and Greten, 2005). However, our recent understanding suggest that not only

inflammation but also mTOR dependent activation of STAT3 can lead to development of

cells which have tumorigenic potential (Ma et al., 2010). To further analysis of the tumor

development in Sigirr-/- mice, a genetic approach such as breeding colon specific Sigirr-/-

mice to Apcmin mice, which develop spontaneous tumors, will reveal the importance of

SIGIRR not only in the progression but also in the initiation of the tumors.

124 Although our study showed the importance of SIGIRR function in colon

epithelial cells in the development of colonic inflammation and tumor, the effect of

SIGIRR in Th17 cells and DCs to the phenotype has not been studied. Interestingly,

different studies showed that Th17 cells are protective against tumors (Martin-Orozco et

al., 2009; Benatar et al., 2009). Based on our EAE study, it is expected to observe

increased Th17 cell population in the Sigirr-/- mice colon after DSS treatment. Increase tumor number and enlarged tumor in Sigirr-/- mice colon suggest that either Th17 cell

population in Sigirr-/- mice colon does not change or SIGIRR deficiency in colon

epithelial cells has more decisive effect on the phenotype of tumorigenesis.

Importance of Th17 cells in tumor immunity and in autoimmune diseases is well

accepted. Although Th17 mediated immune response is considered protective against

tumors, in autoimmune diseases such as IBD autoimmune Th17 cells are pathogenic.

Thus, in inflammation induced tumor formation, suppressing Th17 cells and NFκB

pathway in early inflammation conditions has protective impact on tumor formation

(Yamamoto and Gaynor, 2001). We showed in our studies that IRAK4 is a common kinase in both NFκB and mTOR pathways. Inhibition of IRAK4 in IBD patients not only block NFκB pathway in epithelial cells, but also it inhibits the expansion of Th17 cells.

IRAK4 kinase activity is shown to be the one of necessary event in receptor complex for

mTOR activation. However, the other proteins in IL-1R/TLR signaling which activates

mTOR are unrevealed.

125 The regulation of Toll-IL-1R signaling through different inhibitory molecules is necessary for the maintenance of homeostasis and regulation of inflammation. In colon, inhibition of TLR signaling at receptor level by constitutively expressed SIGIRR seems to be important for colon epithelium function. On the other hand, in Th17 cells SIGIRR expression is observed at later stages of differentiation, showing that inhibition of Th17 cells expansion is maintained by SIGIRR to prevent exaggerated Th17 response. Thus, our studies showed the function of SIGIRR in two distinct cell types differentially.

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