STAT3 Regulation of Mucosal Inflammation in Pediatric Crohn's

STAT3 Regulation of Mucosal Inflammation in Disease and Murine Colitis

A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment for the requirements for the degree of

DOCTOR OF PHILOSOPHY (Ph.D)

In the Department of Cancer and Cell Biology of the College of Medicine February 2012 by Tara A. Willson

B.S. 2002, University of Kentucky, Kentucky Committee Chairman: Lee A. Denson, MD Abstract

Significance: Signal Transducer and Activator of Transcription 3 (STAT3) is crucial for both

innate and adaptive mucosal immune responses in human inflammatory bowel disease (IBD) and

murine models of colitis. Background: Our lab has reported up-regulation of IL-6:STAT3 ¡

dependent biological networks in colonic mucosal biopsies from IBD patients. We identified

increased frequency of STAT3 activated lamina propria and epithelial cells that correlated with

histologic disease severity and epithelial injury. Recent genome-wide association studies

(GWAS) have associated genetic variants in STAT3 with risk for IBD. Goals: Goals that are met

in this dissertation are as follows: (1) Determine the functional effect of rs744166 STAT3 IBD

£ ¤ ¥ ¦ § ¨ © ¦ ¢ £ ¤ ¥ £ £ ¢ £ ¢ ¤ £ ¤ ¤ ¢ leukocyte recruitment

to the gut, peripheral T lymphocyte and granulocyte STAT3 activation, and STAT3

signaling. (2) As no suitable mode for the study of in-vivo epithelial STAT3 specific affects

exits, create and characterize an epithelial Stat3 deficient murine model (3) and determine if the

loss of STAT3 in the epithelial compartment would promote the development of chronic colitis

¢ £ ¤ ¥ £ ¤ following acute dextran sodium sulfate (DSS) injury. Results: (1)The ¦

associated with colonic up-regulation of chemokines IL-8, CXCL1 , and CXCL3 , which promote

CXCR2+ neutrophil recruitment to the gut, and the neutrophil activation products, calprotectin

(S100A8/S100A9) and S100A12 . The frequency of neutrophils expressing CXCR2 was increased,

and the frequency of pSTAT3+ or CXCR2+ neutrophils correlated with histologic severity in

colonic biopsies from patients carrying the risk allele. Peripheral blood frequency of basal CD4 +

lymphocytes and basal and IL-6/IL-6R stimulated granulocytes expressing pSTAT3 was

¢ £ ¦ ¢ £ ¤ ¥ ! "

increased in patients ca ¢ -transformed lymphocytes from

£ ¤ ¢ ¢ £ ¦ ¢ £ ¤ ¥ # £ $ £ £ ¢ ¤ $ ¤ % &

-6 stimulated STAT3

£ ' £ ¨ £ £ £ ( ¢ £ ¢ ¢ £ ¤ £ ¤ £ £ ) ) ¢ $ ¤ ¦

risk allele genotype although the above-mentioned phenotypic differences seem to define a

newly described sub-population of pediatric CD patients. (2-3) In our murine model of intestinal

+ , - epithelial cell Stat3 deletion (Stat3 * ) chronic inflammation at day 28 was more severe

following 7 days of acute injury with DSS which was not accounted for by a sustained defect in

epithelial proliferation or apoptosis as compared to littermate control (Stat3 Flx/Flx ) mice. Colonic

lamina propria frequency of total pSTAT3+ cells was increased and correlated with histologic

injury and F480+pSTAT3+ macrophages, and CD3+pSTAT3+ T-lymphocytes were increased in

+ , - . / 0 1 2

Stat3 * mice compared to littermate controls. Colonic expression of Stat3 target genes

/ 0 1 3 * + , -

and . that mediate epithelial restitution were significantly decreased in Stat3

4 5

mice. However colonic expression of Il-17a, I 3 , Cxcl2 , Cxcl10, Ccl2, and Ccl4 were

significantly increased and Il-17a expression correlated with the increased lamina propria

+ , -

frequency of CD3+pSTAT3+ T-lymphocytes in Stat3 * mice. Conclusion: The rs744166

¢ £ ¤ ¥ £ ¤ ¤ ¤ £ 6 £ £ ¢ ¤ 7 ¢ ¦ £ ' £ 7 ¦ -regulation of

pathways which promote recruitment of CXCR2+ neutrophils to the gut. The loss of STAT3

activation in the epithelial compartment ultimately leads to a more severe chronic inflammation,

reduced epithelial restitution gene expression and expansion of pSTAT3+ lymphocytes and Il-

17a expression.

Chapter 2 contains published orginal research: citation:

Willson, TA et al., STAT3 Genotypic Variation and Cellular STAT3 Activation and Colon

7 ¥ 9 ¢ 7 £ ( £ ¨ £ ¢ £ ¢ ¤ : £ ¤ ¤ ; ¨ < § = > ? ? &

Doi: 10.1097/MPG.0b013e318246be78

PMID: 22197944

8 Acknowledgments

I would first like to acknowledge my graduate mentor Dr. Lee A. Denson. His guidance

and mentoring has developed and matured my scientific career. I have really enjoyed my time

training in a cross functional environment with both translation and basic science research. I also

thank Dr. Denson for serving as Chair of my thesis committee. I would like to thank my thesis

committee Dr. David Hildeman, Dr. Nelson Horseman, Dr. Marshall (Chip) Montrose, and Dr.

Yi Zheng.

I would like to thank all of the past and present members of the Denson Laboratory.

! @ ( £ A 7 ¢ ¤ ¦ ( ¤ ¢ £ B ¦ ¢ : B C £ D ¦ £

Gilbert. A special thanks to Ingrid Jurickova, who has been instrumental in helping me see the

light at the end of the tunnel, thank you for being so wonderful! Another special thanks to Erin

Bonkowski, for keeping the lab running, we would be lost without you! Thanks to Kris

Steinbrecher and Eleana Harmel-Laws for experimental support, scientific insight, and helping

me remember to have fun and enjoy every day. Special thanks to Sandy Geideman for her kind

words and helping me administratively. All of these people have not only been instrumental in

my progression and development as a scientist, but also have become my friends.

Lastly and most importantly, I would like to thank my family and friends for their never-

ending support, encouragement, and understanding especially my mother Carol Willson, my

brother Kyle Willson, and my grandfather Glenn Chipps. I am so blessed to have your constant

love and support. Thanks Mom for helping me believe in myself and giving me courage, I am so

proud to be your daughter. I would also like to thank my grandmother, Mary Chipps, who is no

longer with us for the ambition that she inspired in me. Finally, I would like to express my 8 utmost thankfulness for my best friend Brian Pan whose unwavering love, support, encouragement, and patience have given me the confidence to rise above and move forward.

8 Preface

This thesis dissertation is manuscript-based consisting of two independent research hypotheses, which resulted in one first author publication and one publication under review. There are four chapters herein:

1- Introduction and literature review

2- Manuscript one: preface, abstract, introduction, materials and methods, results,

discussion, and bibliography

3- Manuscript two: preface, abstract, introduction, materials and methods, results,

discussion, and bibliography

4- Overall Discussion, Alternative hypothesis and Future Directions

Table of Contents

$ ¤ ¢ E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E £ £

......

£ E E E E E E E E E E E E E E E E E E E E E E E E E E E E £ '

¥ 6 ( ¤ E E E E E E E E E E E E E E E E E E E E E E E E E E E

...... v

) E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E

Pr .. .. .vii

$ ) ¤ E E E E E E E E E E E E E E E E E E E E E E E E E E E E

...... viii

£ ¤ ) F £ 7 ¢ ¤ E E E E E E E E E E E E E E E E E E E E E E E E E E E

& ...... xiv

£ ¤ ) $ ¤ E E E E E E E E E E E E E E E E E E E E E E E E E E & ...... xvii

Chapter 1- Literature Review

¢ 7 £ E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E % . .1-2

Epidemiology of IBD...... 1-4

Incidence and Prevalence...... 1-4

Age...... 1-5

Ethnic Differences and Geographic Location...... 1-5

) £ £ ¢ ¤ : £ ¤ ¤ : ...... 1-7

Epidemiology...... 1-7

Location and Intestinal Manifestation...... 1-8

Disease Progression...... 1-10

Surgery...... 1-13

Mortality...... 1-14

Defining Ulcerative Colitis...... 1-15

Epidemiology...... 1-15

8 Location and Intestinal Manifestation...... 1-15

Disease Progression...... 1-16

Surgery...... 1-17

Mortality...... 1-18

Extraintestinal Features...... 1-18

Diagnosis...... 1-20

Hematologic Tests...... 1-20

Fecal Markers...... 1-21

Serologic Markers...... 1-21

Genetic Markers...... 1-23

Endoscopic Evaluation...... 1-25

Radiologic Evaluation...... 1-26

Clinical Disease Activity...... 1-27

Clinical Histology Disease / Mucosal Healing...... 1-31

Treatment...... 1-33

5-ASA...... 1-34

Corticosteroid...... 1-35

Immunosuppressant...... 1-37

Biologic Therapy...... 1-39

Antibiotic...... 1-41

Biologic Treatment options...... 1-42

The fundamental basis of inflammatory bowel disease...... 1-45

Hypothesis regarding Pathogenesis...... 1-46

G Hygiene Hypothesis...... 1-47

Environmental Risk Factors...... 1-48

Intestinal microbiota...... 1-48

Smoking...... 1-49

NSAID Exposure...... 1-49

Genetic Susceptibility with a focus on STAT3...... 1-49

Signal Transducer and Activator of Transcription (STAT)-3 in IBD...... 1-52

B ) £ E E E E

¦ ...... 1-52

E E E E E E E The IL-6:STAT3 Signaling Pathway ...... E . . 1-54

Negative Regulators of the IL-6 signal transducing subunit GP130...... 1-56

IL-10:STAT3 Signaling Pathway...... 1-56

£ D 7 ( % ! : E

STAT ...... 1-60

! : £ ¤ 6 £ 7 £ £ ¤ H E E E E E E E % ...... 1-62

STAT3 in Innate and Adaptive Immunity, One Cell at a Time...... 1-64

Dendritic Cells...... 1-65

Macrophages...... 1-66

Neutrophils...... 1-67

Neutrophils hold the key in Transition from Acute to Chronic inflammation...1-71

T ¡ Lymphocytes...... 1-73

Intestinal Epithelial Cells...... 1-76

7 ( ( ¢ E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E ? ¦ -78

References...... 1-80

G Chapter 2 - Willson et al. STAT3 Genotypic Variation and Cellular STAT3 Activation and Colon Leukocyte Recruitment in Pediatric Inflammatory Bowel Disease.

Preface...... 2-2

Abbreviations...... 2-2

Author Contributions...... 2-3

Abstract and Key Words...... 2-4

Introductions...... 2-5

Materials and Methods...... 2-7

Results...... 2-11

Discussion...... 2-16

Acknowledgments...... 2-21

Figures and Tables...... 2-22

References...... 2-39

Chapter 3 - Deletion of Intestinal Epithelial Cell STAT3 Promotes T Lymphocyte STAT3

Activation and Chronic Colitis Following Acute Dextran Sodium Sulfate Injury in Mice

Preface...... 3-2

Abbreviations...... 3-2

Author Contributions...... 3-2

Abstract and Key words...... 3-3

Introduction...... 3-4

Material and Methods...... 3-6

Results...... 3-11

G Discussion...... 3-17

Acknowledgments...... 3-21

Figures and Tables...... 3-22

References...... 3-45

Chapter 4- Overall Discussion, Alternative hypothesis and Future Directions

Introduction...... 4-2

Pediatric versus Adult IBD...... 4-4

Biochemical interrogation of the IL-6:STAT3 signaling pathway...... 4-5

Alternative Hypothesis and Future Directions ...... 4-7

Membrane localization of IL-6R/GP130/JAK2...... 4-7

¦ I

The STAT3 splice variant ¡ ...... 4-7

£ J 7 ( ¤ ) ¦ 9 £ ¤ ¥ P patients...... 4-9

Alternative Hypothesis and Future Directions...... 4-10

IL-8 as a marker for colonic disease...... 4-10

Il-6 classical or trans-signaling?...... 4-11

The JAK2/STAT3 axis as a biomarker for disease...... 4-14

Therapeutic Targeting of STAT3...... 4-15

STAT3 ¡ A Double Edged Sword...... 4-16

Selecting the appropriate animal model of IBD...... 4-17

Location, Location, Location...... 4-22

Interpretation of immunofluorescence and gene expression data...... 4-23

Alternative Hypothesis ...... 4-25

G Loss of IEC STAT3 results in enhanced epithelial bacterial sensing...... 4-25

SHP2 is protective in mice devoid of IEC STAT3...... 4-25

Future Directions...... 4-26

A need to define the STAT3/Th17/IL22 axis...... 4-26

IEC STAT3 in the chronic phase...... 4-27

Defining the Cellular Source ...... 4-28

Summary...... 4-30

References...... 4-32

G List of Figures

Chapter 1- Literature Review

E E E E E E E E E E E E E E E

Figure 1- Factors involved in IBD pathogenesis E ...... 1-2

E E E E E E E E E E E E E E Figure 2- Global map of inflammatory bowel disease E .. . ..1-4

Figure 3- Temporal trends of incidence rates for Crohn's disease and ulcerative colitis in studies reporting incidence at multiple time points...... 1-6

Figure 4- Distribution (%) of affected sites initially and maximally among Olmsted County,

Minnesota, residents diagnosed with CD, 1943-1982...... 1-11

Figure 5- 5-ASA therapy and recommendations for IBD...... 1-35

Figure 6- Corticosteroid therapy and recommendations for IBD...... 1-36

Figure 7- Immunosuppressant therapies and recommendations for ulcerative colitis...... 1-38

¢ £ ¤ ¢ ( ( £ ¤ ) ¢ ¢ ¤ : £ ¤ ¤ Figure 8- Immunosuppressant ...... 1-38

Figure 9- Biologic therapy and recommendations for ulcerative colitis...... 1-39

¢ ¢ ( ( £ ¤ ) ¢ ¢ ¤ : £ ¤ ¤ Figure 10- Biologic ...... 1-41

Figure 11- A model for IBD pathways based on GWAS...... 1-51

Figure 12 -The structural domains of the STAT3 protein...... 1-53

Figure 13- Regulation of intracellular STAT3 signaling...... 1-55

Figure 14- Negative Regulatory Mechanisms in IL-6-type Cytokine Signaling...... 1-57

Figure 15- Strength and duration of STAT3 activation determines cellular responses...... 1-59

Figure 16-Schematic model of IL-6 trans-signaling in IBD...... 1-72

Figure 17 - Contribution of different STATs in T-helper Cell differentiation...... 1-74

G 8

Chapter 2 K Willson et al. STAT3 Genotypic Variation and Cellular STAT3 Activation

M N M O P Q R S M T U V Q W Q T X R Y V Z Q O V Y O [ Q \ Y ] V X Y T L X M ^ O _ ` a Y ` Q ] ` Q and L . J Pediatr Gastroenterol

and Nutr, Dec 2011.

Figure 1 - Colon Expression of the STAT3 Target Gene SOCS3 and Chemokines Expressed on

? ¢ % ¢ ¤ £ : £ ¤ ¢ ¢ £ ¦ 9 £ ¤ ¥ E E E E E 4q12-b ...... 2-22

Figure 2 - The Frequency of CXCR2+Neutrophils is Increased in Colon Biopsies from CD

¨ £ ¤ ¢ ¢ £ ¦ 9 £ ¤ ¥ F ¢ b 7 ) ¦ c C 9 = c

E E E E E E E E E E E E E E Neutrophils Correlates with Histological Severity. E ...... 2-24

Figure 3 - Peripheral Blood Lymphocyte and Granulocyte STAT3 Tyrosine Phosphorylation is

¢ ¤ £ : ¨ £ ¤ ¢ ¢ £ ¦ 9 £ ¤ ¥ E E E E E E E E E E E % .. ..2-26

Figure 4 - Cytosolic protein abundance of STAT3, STAT1, IL-6 receptor, JAK2, GP130, nor

J ¦ ' ¢ £ $ ¦ ¢ £ ¤ ¥ E E E E E E E E E E E E E E E E E E ¦ ...... 2-28

Figure 5 - IL-6 Stimulated STAT3 Activation and Expression of STAT3 and SOCS3 are

¢ ¢ £ ¦ 9 £ ¤ ¥ Increased in Immortalized B-cell lines from IBD Patients C

...... 2-30

Figure 6 - Membrane Localization of the IL-6 receptor, GP130, and JAK2 are Increased in

£ ¤ ) ¢ ( % ! : ¨ £ ¤ ¢ ¢ £ ¦ 9 £ ¤ ¥ E E E immortalized B-c . ...2-32

Figure 7 - IL-6 Stimulated Nuclear Localization of STAT3 is Increased in EBL Carrying the

9 £ ¤ ¥ E E E E E E E E E E E E E E E E E E E E E E E E E E ¦ ...... 2-34

G 8 Chapter 3 K Willson et al. Deletion of Intestinal Epithelial Cell STAT3 Promotes T

Lymphocyte STAT3 Activation and Chronic Colitis Following Acute Dextran Sodium

Sulfate Injury in Mice.

IEC

Figure 1 - Figure 1. Stat3 * mice exhibit more severe acute and chronic colitis after DSS administration...... 3-22

IEC

Figure 2 - Figure 2. All histologic sub-scores are increased in Stat3 * mice compared to

Stat3 Flx/Flx littermate controls at day 28...... 3-24

IEC

Figure 3 - Figure 3. IEC proliferation and apoptosis do not differ at day 28 between Stat3 * mice and littermate controls...... 3-26

IEC

Figure 4 - Figure 4. Chronic colitis in Stat3 * mice involves Stat3 activation in the non- epithelial compartment...... 3-28

Figure 5 - Figure 5. Colonic expression of Stat3 target genes that mediate epithelial restitution is

IEC * decreased while SOCS3 , IFN-3 , and IL-17A expression are increased in Stat3 mice at Day

28...... 3-30

Figure 6 - Figure 6. Colonic expression of CXCL2 , CXCL10 , CCL2 , and CCL4 is significantly

IEC increased in Stat3 * mice at Day 28...... 3-32

Figure 7 - Colonic expression of IL-6, IL-10 , IFN-y and IL-17A correlates with histologic severity and IL-17A expression correlates with the frequency of colon pSTAT3 +CD3 + T-

IEC lymphocytes in Stat3 * mice at day 28...... 3-34

Supplemental Figure 1 - Supplemental Figure 1. Characterization of Stat3 Intestinal Epithelial

IEC Flx/Flx cell deficient mice (Stat3 * ) compared to littermate controls (Stat3 )...... 3-37

G 8

7 ( F £ 7 ¢ = £ ' £ ) § F d ! ¦ ? ¦ e Supplemental Figure 2 - ¦

plausible compensatory mechanisms for protection of IECs during colitis in a STAT3 deficient

IEC Flx/Flx

epithelium, did not vary qualitatively in Stat3 * mice compared to Stat3 littermate

controls...... 3-39

Supplemental Figure 3 - Supplemental Figure 3. Neutrophil mediated responses during chronic

Flx/Flx IEC

colitis in Stat3 littermate controls and Stat3 * mice...... 3-41

Supplemental Figure 4 - Supplemental Figure 4. Colonic expression of IL-10, Th2 cytokine IL-

§ F g % & ? I C & ? & e & = > - expression do not vary between genotypes at Day 0

or Day 28...... 3-43

Chapter 4 K Overall Discussion, Alternative Hypothesis and Future Directions

Figure 1 ¡ Inflammatory bowel disease susceptibility loci...... 4-3

Figure 2 ¡ Circulating levels of Inflammatory cytokines...... 4-12

Figure 3 ¡ TNBS colitis model in IEC STAT3 deficient mice...... 4-19

* + , - Figure 4 ¡ STAT3 mice are not susceptible to piroxicam induced colitis...... 4-20

G 8 List of Tables

Chapter 1 - Literature Review

¢ ¤ : £ ¤ ¤ Table 1 -Montreal classification of ...... 1-10

Table 2- Montreal classification for Ulcerative Colitis...... 1-16

¢ ¤ : £ ¤ ¤ £ ' £ % #

Table 3- (CDAI) ...... 1-27

£ ¢ £ ¢ ¤ : £ ¤ ¤ £ ' £ % # Table 4- ¨ (PCDAI)...... 1-29

Table 5- Ulcerative Colitis Disease Activity Index(UCDAI)...... 1-30

Table 6- Pediatric Ulcerative Colitis Disease Activity Index (PUCDAI)...... 1-31

¢ ¤ : £ ¤ ¤ D £ ¤ £ % # ) ¦ ' ¢ £ © : D % ¦ Table 7- ...... 1-32

Table 8 - Ulcerative Colitis Histology Index of Severity (UCHIS)...... 1-33

Chapter 2 K Willson et al. STAT3 Genotypic Variation and Cellular STAT3 Activation

Q \ Y ] V X Y T L X M ^ O _ ` a Y ` Q ] ` Q and Colon Leukocyte Recruitment in [ . J Pediatr Gastroenterol and Nutr, Dec 2011.

Table 1 ¡ Clinical and Demographic Characteristics...... 2-36

Table 2 ¡ Colonic Expression of JAK/STAT Signaling and Leukocyte Recruitment Genes

E E E E E E E E E E E E E E E E E E E E E

Stratified by STAT3 Genotype E ...... 2-37

E E E E E E Table 3 - EBV Cell line Patient Demographics and Medication Exposures E .2-38

Chapter 3 K Willson et al. Deletion of Intestinal Epithelial Cell STAT3 Promotes T

Lymphocyte STAT3 Activation and Chronic Colitis Following Acute Dextran Sodium

Sulfate Injury in Mice.

Table 1 - Real-time quantitative reverse transcription- PCR (RT-PCR) Primers...... 3-36

G 8

Chapter 1

Literature Review

Introduction

Autoimmune diseases affect 50 million Americans, this in direct comparison to 81 million affected by heart disease and 11 million by cancer 1. Autoimmune diseases are the second highest cause of chronic illness in the United States and Inflammatory Bowel Disease

(IBD) ranks 3 rd in prevalence among the autoimmune diseases 2 . IBD, a chronic relapsing inflammation of gastrointestinal tract can arise in an otherwise healthy individual resulting in increased morbidity and decreased quality of life. Approximately four million people worldwide

currently suffer from IBD frequently

experiencing diarrhea, abdominal

pain, fever, chronic fatigue, and

weight loss . The clinical

presentation, inflammatory location

and the type of inflammation, is

phenotypically characterized as

k l m n o p q r s q t u q t v k r w x y z { t l u | s } t

Colitis (UC), or IBD unspecified

(IBD-U). While the precise etiology Figure 1. Factors involved in IBD Pathogenesis include genetics, environmental triggers , the immune response, is unknown, IBD is thought to be the and luminal bacterial antigens. consequence of environmental factors triggering an inappropriate and continuing mucosal inflammatory immune responses to enteric bacteria in genetically susceptible individuals (Figure 1) 3.

IBD is chronic, with relatively low mortality. Thus, medical management focuses on

maintaining remission but refractory disease is highly likely and this creates high caseloads for

h i j current gastroenterologists. The estimated annual disease-attributable direct cost of IBD is $6.3 billion (Crohn's disease accounting for $3.6 billion of that figure) and the cost is significantly higher for children with IBD compared with adults 4. Moreover, the yearly inpatient burden of pediatric IBD is immense, $152.4 million (2010 US$) with 64,985 days spent in the hospital, evidence that improved therapeutics are needed to reduce the significant inpatient burden5.

Since 1972, hundreds of research grants have been funded for IBD research in areas

expanding infectious pathogens, immunology, therapeutics, genetics and intestinal bacterial

l s } u | t o s o q m l { t q q { n u q | n t k l m n o p q u o k m z s | s q m o u | s m o m ¡ t l s { u

microflora.

(CCFA) and the Eli and Edythe Broad Foundation have provided millions for IBD research

| q p o s o m l within the United States. The National Institutes of Health (NIH) has increased s

IBD research each year during a period when overall funding for the NIH remained flat, this in part due to the quality of the applications, the interest of people at the NIH in IBD, and congressional recommendations encouraging the agency to develop its IBD research portfolio 6.

These efforts have translated into approximately 688 current IBD focused research projects sponsored by the NIH. As there is still no cure for IBD, clinicians and researchers have focused new technologic and scientific approaches to develop therapeutics and further our understanding of IBD etiology.

h i Epidemiology of IBD

Incidence and Prevalence . Population-based studies reveal the uneven distribution of

IBD throughout the world. The highest disease rates occur in western or industrialized countries with globalization inducing increased incidence in previously low incidence areas, such as

Southern Europe and Asia (Figure 2)7. The annual incidence of UC was highest in Europe (0.6 to 24.3 per 100,000 person-years) then North America (0 to 19.2 per 100,000 person-years) and finally Asia and the Middle East (0.1 to 6.3 per 100,000 person-years)8. The annual incidence of

CD was highest in North America (0 to 20.2 per 100,000 person-years) followed by Europe (0.3 to 12.7 per 100,000 person-years) and then Asia and the Middle East (0.04 to 5.0 person-years)8.

Molodecky et al. also reported that IBD has the highest reported prevalence in Europe (UC: 505

per 100,000 persons; CD: 322 per 100,000 persons) and North America (UC: 249 per 100,000

h i ¢ persons; CD: 319 per 100,000 persons) 8. Overall, it is estimated that 2 million and 1.4 million individuals in Europe and the United States are suffering with IBD, respectfully 9.

Age. The descriptive epidemiology of IBD differs when comparing an adult population to a pediatric population. Recent reports suggest that pediatric-onset IBD is a sub population of

IBD due to the varying phenotypic and natural history differences in young patients 10 .

Childhood IBD incidence are on the rise as the number of younger patients (younger than 18 years) has increased significantly 11-13 . IBD is diagnosed 25% of time during childhood and estimates suggest 100,000 children in North America currently suffer from IBD with three CD cases for every new UC case 14,15 . A recent comprehensive review of worldwide trends in epidemiology for pediatric-onset IBD suggested that the incidence of IBD is rising and the incidence of CD has risen significantly in several countries, while most studies have reported stable incidence of pediatric-onset UC 16 (Figure 3).

Sex. Adult- onset IBD shows equal ratio of male to female disease with perhaps slightly more women having the disease 15 . However, the incidence of UC verse CD in males and females varies among high and low-incidence rate areas. In high-incidence areas males are more affected by UC whereas CD is more frequent in females 17-19 but incidence of CD is men has increased and is becoming equivalent to that of women 20-23 . However, in pediatric-onset IBD,

CD has a male predominance whereas UC has a female predominance and there has been no molecular elucidation for this difference in adult versus pediatric CD:UC gender ratio 15,24 .

Interestingly, the male to female ratio normalizes between 14 and 17 years 25 .

Ethnic Differences and Geographic Location. Studies describing IBD characteristics in racial/ethnic groups are beginning to surface as most of the current studies have been conducted

in primarily Caucasian populations. In the studies conducted it appears that Caucasian are at the

h i £

¦ § ¨ © ª « ¬ ® ¬ ¯ ° ¬ ¯

½ ¾ ¿ À Á Â Ã

± ² ³ ´ µ ¶ · ¸ ¹ ´ º » ² ¸ · ¸ ¼

½ ¾ ¿ À Á Â Ã

Figure 3. Temporal trends of incidence rates for (A) Crohn's disease and (B) ulcerative colitis in studies reporting incidence at multiple time points. Where a year range is reported, incidence rate is reported for the final year in the range (e.g., if in cidence is

reported for 1990 ¥ 1999, rate is plotted as incidence for 1999 ). Reprinted from Inflamm Bowel Disease, 17, Benchimol et al., Epidemiology of pediatric inflammatory bowel disease: a systematic review of international trends. 423-439, Copyright (2011 ), with permission form John Wiley and Sons.

highest risk for IBD and in a systematic review African American incidence come close to that

h i ¤ of Caucasian with Asian Americans and Hispanics incidence lower but on the rise 26,27 . The

assessment of ethnic/racial variation of indigenous prevalence and incidence rates compared to

that of the immigrated prevalence and incidence rates will be essential to identify the

environmental factors that control IBD development in genetically susceptible individuals. The

genes that incur disease susceptibility also seem to be ethnic-specific 28,29 . In regards to

geographical location, the further distance from the equator the higher the incidence of CD in

both the Western Hemisphere and Western Europe 27 . This trend is also evident within the same

country as seen in the studies from the United States and in the south of France 30,31 . Recently

low sunlight exposure was associated with increased incidence of CD in France and may explain

the reduced incidence with low degrees of latitude from the equator 32 . Vitamin D is a fat soluble

vitamin that is produced in the skin following UV exposure and can also be absorbed through the

diet 33 . Vitamin D status is low in patients with IBD and Genome Wide Association Studies

(GWAS) show polymorphisms in the VDR gene are associated with increased susceptibility to

CD and UC 34,35,36 . However defining if vitamin D status or VDR genotype cause or effect the

disease still remains to be elucidated.

Æ Ç È É È É Ê Ë Ì Í Î É Ï Ð Å È Ð Æ Ñ Ð Æ

m | n Ó t s u | l s { u o u z | m o q t | Ô Õ r x k l m n o p q s q t u q t s q z t q q

Epidemiology. Considering Ò

l m n o p q s q t u q t common than ulcerative colitis. k is most commonly diagnosed between 15 and

30 years of age 37 . CD has a particularly high incidence in Ashkenazi Jews reported to be

associated with the IBD5 locus in a pediatric-onset population however other studies have failed

to define this genetic susceptibility and hypothesize the existence of novel, yet unidentified,

¡ t l m Ó t m Ó z t Ö s | n k l m n o p q s q t u q t n u q 38,39 o

genetic variants unique to this population . The Ò

been steadily increasing, particularly among children and in the non-Western world 16 . In the

h i Ä Olmsted County, Minnesota cohort, since 1991 the prevalence of CD has increased about 31%

and in direct comparison UC has decreased 7% 21 .

q Ù s Ó z t q s m o Ú

Location and Intestinal Manifestation. CD is commonly referred to as a Ø where inflammation is discontinuous or patchy and can affect any part of the gastrointestinal tract. The immune response can affect all layers of the gastrointestinal tract, (transmural), can extend to the serosa (penetrating disease) and can progress to fistulae, an abnormal connection

between different parts of the intestine, and stricturing. Expected histologic findings include

Û t l p q Ó u | { n v u Ó n | n m s z { t l w u o m { u z { n l m o s { small superficial ulcerations over a Pt inflammation extending to the submucosa, sometimes accompanied by noncaseating granuloma formation. Generally, CD mostly affects the distal ileum and colon. In pediatric patients the terminal ileum is the most common site of CD, with 60% having ileo-colonic disease and 20% to

30% having colon only disease 24 . Unfortunately CD presentation can be highly variable leading to a delayed diagnosis with symptoms depending on location and extent and severity of involvement 40 . Gastrointestinal symptoms depend on location and become symptomatic when lesions are extensive or distal, accompanied by systemic inflammatory reaction, or when stricture or abscesses and fistulas, are involved 40,41 . No correlation exists with symptoms and progression of anatomical injury as stricture and fistulas can develop for years without any symptoms but ileitis alone can be associated with refractory abdominal pain and fatigue 41 .

About 1 in 4 patients present with abdominal pain, weight loss and diarrhea 42 , evaluated individually 70 to 90% of patients present with diarrhea, 45 to 66% abdominal pain and/or 65 to

70% weight loss 43 . In pediatric IBD, growth failure is a critical concern and may be the only initial indicator of CD even before gastrointestinal symptoms present 44 . Clinical disease activity

involves relapsing and remitting inflammation (flares) with varying duration of remission as 10-

h i × 15% of patients experience chronic continuous disease course 45,46 . The major complications associated with CD are bowel obstruction resulting from inflammation induced increased thickness of the intestinal wall and often results in surgery to remove the diseased portion of the bowel. Ulcers or open sores can arise from chronic inflammation anywhere in the digestive tract and the worst circumstances extend completely through the intestinal wall to create fistulas.

Fistulas can occur between the intestine and skin, or between the intestine and another organ, such as the bladder or vagina. Internal fistulas may result in food bypassing areas of the bowel that are necessary for absorption and ultimately cause retardation in growth and malnutrition.

External fistula can cause continuous drainage of bowel contents to the skin and may become infected forming an abscess, which can be life threatening if left untreated. Perianal fistulas are the most common kind of fistula and referred to as an anal fissure or crack/cleft in the anus or in the skin around the anus where infections can occur and is often associated with painful bowel movements. The pathogenesis of fistulae formation is still unknown.

CD, unlike UC, has a wide spectrum of chronic intestinal inflammatory phenotypes categorized by the Montreal Classification developed in 2005 to sub-divide IBD patients by age of diagnosis, location of disease, and disease behavior (Table 1) 47 . The dynamic nature of pediatric CD disease phenotype was not sufficiently captured by the Montreal Classification particularity in disease behavior over time, disease location, and growth failure. Therefore this system was recently redefined for pediatric IBD in 2009 and is known as the Paris Classification

(Table 1) 48,49 . This accurate phenotypic classification system was set up to be used in conjunction with genotypic data to better predict clinical course and streamline interpretation of genetic analysis.

h i Ü

Age at Diagnosis Location Behavior Growth A1 = < 16 years L1 = terminal ileal + limited B1 = non -stricturing non -penetrating N/A cecal disease A2 = 17 - 40 years L2 = colonic B2 = stricturing A3 = > 40 years L3 = ileo -colonic B3 = penetrating *L4 = isolated upper GI *P = perianal disease disease * is a modifier that can be * is a modifier that can be added to B1, added to L1 , L2 or L3 B2 or B3 Paris (Pediatric) Modification Age at Diagnosis Location Behavior Growth A1a = 0 -10 years L1 = distal ileal 1/3 ileum + B1 = nonstricturing/nonpenetratring G0: No evidence limited cecal disease of growth delay

A1b = 10 -17 years L2 = colonic B2 = stricturing G1: Growth delay A2 = 17 -40 years L3 = ileo -colonic B3 = penetrating L4a = upper disease B2B3 = both penetrating and stricturing proximal to ligament of disease, either at the same or different Treitz.* times L4b = upper disease distal *P = perianal disease to ligament of Treitz and proximal to distal 1/3 ileum.* * is a modifier that can be added to B1, B2 or B3 *In both the Montreal and Paris Classification systems L4 and L4a/L4b may coexist with L1, L2, L3, respectively. B1 - Nonstricturing, nonpenetrating disease: uncomplicated inflammatory disease without evidence of stricturing or penetratin g disease. B2 - Stricturing disease: the occurrence of constant luminal narrowing demonstrated by radiologic, endoscopic, or surgical examina tion combined with prestenotic dilation and/or obstructive signs or symptoms but without evidence of penetrating di sease. B3 - Penetrating disease: the occurrence of bowel perforation, intraabdominal fistulas, inflammatory masses and/or abscesses at any time in th e course of

the disease, and not secondary postoperative intra -abdominal complication (excludes isolated perianal or rectovaginal fistulae). B2B3 Þ Stricturing and penetrating disease: the presence of both B2 and B3 phenotypes in the same patient, either at the same moment in time, or separately over a period of time. Adapted from Inflamm Bowel Dis 17, Levine et al., Pediatric modification of the Montreal classification for inflammatory bowel disease: the Paris classifica tion. 1314-1321, Copyright (2011), with permission from John Wiley and Sons

Disease Progression. Disease progression (remission, relapse, and surgery rate) is rather challenging to assess within a single cohort therefore interpreting results from multiple studies is even more difficult as quantification of these parameters vary from study to study as do definitions of remission, relapse, and surgery. Thus many studies involving referral centers and population based cohorts have tried to extrapolate this data and the most reputable studies are prospective population based studies. These studies follow a cohort of individuals diagnosed

within a population with a known incidence and prevalence, having proper exclusion and

h i h Ý inclusion criteria (migration), routine follow up in clinical course, and document surgical and or

drug therapy and mortality. As difficult as these studies are, several have been completed for CD

worldwide. In the Olmstead Country, Minnesota population based study (residents who were

diagnosed with CD from 1970 to 2004) at diagnosis, 35% had pure ileitis (L1), 36% had colitis

alone (L2), and 29% had ileocolitis (L3) and under a median follow-up of 8 to 9 years, 60% of

patients had progressed to more extensive disease 46 . Within this same cohort 13% had

unremitting disease, 75% experienced a chronic intermittent course, 2% could not be adequately

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{ 46 Ø Ø intestinal resection) .

In a retrospective study ileal only disease progressed to include the colon in less than 20% of

patients 50 and colon

only disease

extended to the

small bowel in less

than 20% of

patients 51 (Figure 4).

About 3-5% of

patients in L1, L2 or Figure 4. Distribution (%) of affected sites initially (left panel) and L3 classifications also maximally (right panel) among Olmsted County, Minnesota, residents diag nosed with CD, 1943-1982. Reproduced from Gut, 29, Gollop et al., have associated upper The Epidemiologic aspects of Crohn's disease: a population based study in Olmsted County, Minnesota, 1943 -1982, 49-56, Copyright (1988 ), with GI lesions 41 . The permission from BMJ Publishing Group Ltd.

Inflammatory Bowel

South-Eastern Norway (IBSEN) population-based study reported that CD patients at diagnosis

had 27% pure ileitis (L1), 45% colitis alone (L2), 26% ileocolitis (L3), and 4% upper

h i h h gastrointestinal disease (L4). After 5 years of follow-up, the resultant figures were 23%, 37%,

38%, and 6%, respectively and overall changes in disease location observed in 14% of patients at

5 years 52 . A European population-based study involving 358 patients with Crohn's disease showed 15% had ileitis, 42% had colitis, 37% had ileocolitis, and 6% had upper gastrointestinal disease at the time of diagnosis 53 . Overall, these studies suggest an equal ratio of disease classification for L1, L2, or L3 upon diagnosis.

What has troubled gastroenterologists is how to accurately predict clinical disease course and severity based on the initial presentation and evolution of disease behavior. Recent reports involving the Olmsted Country and IBSEN population-based cohorts have elucidated changes in disease behavior over time. In the IBSEN study 200 Norwegian patients with Crohn's disease were compared at diagnosis and in a 5 year follow up where 61% had nonstricturing nonpenetrating disease compared to 47% (at 5 years), 28% had stricturing phenotype compared to 33% (at 5 years), and 12% had penetrating disease compared to 21% (at 5 years) 52 . The

Olmsted County, Minnesota cohort had almost 18.6% of the patients with evidence of a stricturing or penetrating intestinal complication at diagnosis or within 90 days of diagnosis, 50% experienced intestinal complications 20 years after diagnosis, and found that factors associated with development of CD complications were the presence of ileal involvement and perianal disease 54,55 .

Perianal (anal tags, perirectal abscesses, nonhealing deep fissures, and fistula) and perirectal disease are complications of CD not found in UC. In a retrospective study on

Stockholm County, Sweden patients with colonic or rectal Crohn's disease, perianal or rectal fistulas occurred in 37% of those followed between 1955 and 1989 56 . In the Olmsted County

cohort, the cumulative risk for developing a perianal fistula was 21% and 26% after 10 and 20

h i h j years, respectively with 75% of patients undergoing surgery and 24% managed medically 57 . In pediatric-onset CD approximately 33% of patients will develop a perianal fistula or abscesses in their lifetime 15 .

Medical management is focused on mucosal healing and clinical remission commonly referred to as less need for steroid therapy, and reduced rates of hospitalization and surgical resection 58,41 . Clinical remission (including surgical resection if needed to achieve remission) and relapse rates in two Scandinavian population-based cohorts reported that the probability of a continuously active course without clinical remissions was 4% and 1% after 5 and 10 years45,59 .

However, the probability of a relapse-free course decreased rapidly with time, from 22% after 5 years to 12% after 10 years, respectively 45 59 .

Surgery. The removal of a diseased portion of the bowel is reserved for complications of disease such as obstructions, fistulas and/or abscesses, or if the disease does not respond to medical therapy. Surgery may not be curative as inflammation can reside at the site of the resection or can appear in other locations. Approximately 70%-80% of patients with CD will require intestinal surgery 20 years post diagnosis 60 . Cumulative incidence of surgery at 10, 15, and 20 years was found to be 61%, 70%, and 82% 61 . Within the first year of diagnosis the cumulative risks for bowel surgery, non-bowel surgery, and all CD-related surgeries was 3.4%,

1.4%, and 4.8%, and at 5 years was 13.8%, 4.5%, and 17.7% 62 . Increased age at diagnosis, greater disease severity, and stricturing or penetrating disease increased the risk of bowel surgery

62 . Schaefer et al. also reported that other factors such as initiation of immunomodulator therapy within 30 days of diagnosis, sex, race, and family history of inflammatory bowel disease did not influence the risk of bowel surgery. In a recent detailed analysis of a large multicenter cohort of

pediatric CD patients, the incidence of surgery was 5.7% at 1year, 17% at 5 years, and 28.4% at

h i h 10 years from the time of diagnosis 63 . In this cohort, female gender, poor growth at presentation, initial diagnosis of UC, and development of an abscess, fistula, or stricture was associated with an increased risk of surgery 63 .

An important clinical question is how medication affects the risk of surgery, which is difficult to interpret as the relationship may reflect a patient p s clinical disease severity or the efficacy of the drug(s). A recent publication by Bouguen and Peyrin-Biroulet evaluated all know data sets for randomized trial, referral centers and population based studies to compare surgical rates before and after the era of biologics. They were able to show in population based study that before the era of biologics the rate of surgical requirements ranged from 27% to 61% at 5 years and in the era of biologics the rate of surgery ranged from 25% to 33% within 5 years 64 . They also showed that in a randomized control trial, 1 year of treatment to evaluate the efficacy of adalimumab (TNF Inhibitor), the rate of surgery was nine times greater in the placebo group than in the adalimumab group. In pediatric CD, treatment with infliximab or 5-aminosalicylic acid were associated with decreased risk for surgery 63 .

Current studies dichotomize patients by medication exposure (exposed or not exposed).

However, prospective investigations that utilize defined disease severity scores as clinical endpoints and collect uniform data for medications variables such as start, stop, and duration of exposure will clarify associations between medication use and surgical risk. These studies will ultimately lead to risk stratification during the course of CD and help to guide therapy that may improve disease course and decrease the need for surgery.

Mortality. A meta-analysis on mortality in CD, was conducted in population-based studies and revealed a slight but significantly increased mortality in patients, especially women,

with CD compared with the background population 65 . The study identified an increased

h i h ¢ mortality from pulmonary cancer, malignant melanoma, and potentially from intestinal cancer combined, but not from colorectal cancer. The pooled standardized mortality ratio (SMR) was

1.39 reduced from a previous meta-analysis (SMR 1.52) which included both population-based and referral center studies 66 .

Defining Ulcerative Colitis

Epidemiology. Long-term follow up studies can provide valuable contributions, however limitations exist when comparing across cohorts because information is not systematic or consistently reported in the same way. The most reliable data comes from population based studies, more specifically the IBSEN and Olmsted County, Minnesota cohorts. The yearly incidence of UC reported in the IBSEN (Norway) cohort was 12.8/100,000 in 1997 67 . This is higher than the Olmsted County cohort incidence between 1940 and 1993 which was

7.0/100,000 person-years . The Olmstead cohort incidence increased in 1990 ã 2000 to

8.8/100,000 person-years, however these increases were shown to be seen after 1940 and it is

believed that UC incidence has remained stable over the past 30 years21 . When calculated for ã 10-year age groups, the highest incidence was among the 20 ã 29 and 30 39 year age groups

(13.9/100,000 and 12.0/100,000 person-years, respectively) and the lowest incidence was found

21 in the 0 ã 19 year age group (2.4/100,000 person-years) . Since 1991 the prevalence of UC has decreased by 7%, while the prevalence of CD increased about 31% 21 .

Location and Intestinal Manifestation. UC involves the rectum in 95% of patients with variable proximal extension 40 . Inflammatory lesions are generally limited to the mucosa and are continuous with variable severity of ulceration, edema, and hemorrhage. Inflammatory lesions

do not progress to strictures or fistulae. Expected histologic finding are acute and chronic

h i h £ mucosal inflammation with infiltration of polymorphonuclear leukocytes and mononuclear cells, crypt abscesses, distortion of the mucosal gland, and goblet cell depletion. At the time of presentation ulcerative colitis is equally distributed to either the rectum, the colorectum distal to the splenic flexure or proximal to the splenic flexure 41 . UC has a wide spectrum of chronic intestinal inflammatory phenotypes categorized by the Montreal Classification developed in

2005 to sub-divide IBD patients by location of disease, and disease behavior (Table 2) 47 . As the features in pediatric disease phenotype are not sufficiently captured by the Montreal

Classification, this system was recently redefined for pediatric IBD in 2009 and is known as the

Paris Classification (Table 2) 48,49 .

Table 2. Montreal Classification for Ulcerative Colitis Extent Severity E1 = ulcerative proctitis SO= Clinical remission E2 = Left sided UC (distal to splenic flexure) S1 = Mild disease E3 = extensive (proximal to splenic flexure) S2 = moderate UC S3= Severe UC

Paris (Pediatric) Modification Extent Severity E1 = Ulcerative proctitis SO = never severe * E2 = Left sided UC (distal to splenic flexure) S1 = ever severe * E3 = Extensive (hepatic flexure distally) E4 = Pancolitis (proximal to hepatic flexure)

* Severe defined by Pediatric Ulcerative Colitis Activity Index (PUCAI) 65 Adapted from Inflamm Bowel Dis 17, Levine et al., Pediatric modification of the Montreal classification for inflammatory bowel disease: the Paris classifica tion. 1314-1321, Copyright (2011 ), with permission൒ from John Wiley and Sons. Disease progression . A small fraction of UC patients have unremitting activity, while most bear relapsing inflammation with periods of remission 41 . Flare severity and response to treatment is unpredictable and ranges from minor to life-threatening fulminant colitis that is non responsive to treatment and requires colectomy 41 . A recent report by Magro et al. included twenty-one studies from seven large cohorts. This summarized data revealed that overtime

disease extends from proctitis to pancolitis with approximately 30% of patients having proctitis

h i h ¤ upon diagnosis. A 5 and 10 year follow up revealed extension from initial location varied 10%ã

19% and 11%ã 28%, respectively. Preventing relapse is the most important goal for UC medical management however it appears that most patients will have a relapse in the first year of disease

69 with cumulative relapse rates between 67% ã 83% after 10 years of diagnosis . In the IBSEN cohort within 5 years of diagnosis 78% of UC patients had at least one relapse and having the first relapse in the first year of UC was associated the fact that more relapses were imminent 70 .

Surgery. Surgical procedures in UC patients are categorized as either emergency or elective. Emergency operations are due to perforation of the bowel and massive haemorrhage usually indicated by failed medical therapy and toxic megacolon 71 . Elective surgery indications included chronic continuous symptoms, corticosteroid dependence, partial response to medical therapy and the presence of malignancy/dysplasia 71 . In a comprehensive review of large cohorts,

69 from 1962 ã 2004 there was a decreasing trend in the proportion of colectomies . The major reasons for surgery were acute serious flares or chronic relapsing disease 72 . The Olmsted County

cohort (1949 ã 2001), had a cumulative probability of proctocolectomy of 9.0% after 5 years,

18.7% after 15 years, and 20.8% after 25 years of disease . The IBSEN cohort from (1990ã

1993), had an excessive risk of surgery in patients with pancolitis at diagnosis compared to those patients with proctitis and left-sided UC at diagnosis 67 . At 10-year follow up, the majority of colectomies were performed during the first 2 years of disease and in patients with extensive colitis, this was also similar in the European Collaborative Study IBD cohort 67,72 . These patterns were also similar in the Danish cohort (1962-1987) where colectomy was generally higher in the first 2 years of disease and among patients with pancolitis at diagnosis. 10 years after diagnosis, patients older than 50 had a 70% reduced risk of colectomy than those less than 30 years of age

in the IBSEN cohort. 72 .

h i h Ä Mortality. Risk factors associated with increased UC mortality are having a short time interval from UC onset and diagnosis within the first 5 years of disease, having systemic symptoms of weight loss and fever and having pancolitis at diagnosis 74 . Langholz et al also reported that the median age at death for UC-related causes was 58 years for men and 76 years for women, and for other causes was 70 years for men and 75.5 years for women 74 . In the

IBSEN cohort at 1 year of disease, 0.2% and at 5 years of disease 6% of patient deaths were

IBD-related (spontaneous colon perforation, cholangiocarcinoma and septic shock)67,70 . At 10 years of disease, the 6% of deaths also resulted from complications associated with UC that included colorectal cancer 75 . In the Olmsted Country cohort the standardized mortality ratio

(SMR) from 1940-1959 was 0.8 and from 1990-2001 was 0.5, while having pancolitis was associated with a higher risk of mortality 76 . The overall survival was 81% at 20 years and 69% at 30 years of follow-up 68 . In the 2004 update of the Olmsted County cohort, (median follow-up of 15 years) 19% of deaths related to UC were due to complications of severe UC (perforation, intestinal hemorrhage, and postoperative acute myocardial infarction or sepsis, primary sclerosing cholangitis, and metastatic colorectal cancer) and the median age at death was 81 years for men and 71 years for women 76 .

Extraintestinal Features

Given the systemic and immune mediated nature of IBD, extraintestinal manifestations

(EIMs) are commonly experienced and may often precede the development of gastrointestinal symptoms, delaying diagnosis (especially in CD) and contributing to the morbidity and mortality of IBD patients 40 . Approximately 25% to 40% of patients will exhibit some EIM during their

lifetime 77,78 . Fever, usually low grade and chronic, malnutrition (due to reduced caloric intake in

h i h × response to abdominal pain), anemia, and fatigue are common non-specific symptoms. Weight loss can be seen in both adult and pediatric onset IBD but in pediatric onset failure to maintain a normal growth velocity is the most common systemic feature of IBD and is more frequent in CD than UC 40 .

Growth delay (60-80% in CD; 6 -12% in UC) and delayed onset of puberty may be the initial presentation of CD in children 44 and patients may experience delayed skeletal maturation 40 . Growth delay in children with IBD may be the result of a number of factors, including nutritional status, activity of inflammation, disease severity, genotype (IBD5 -

OCTN1/OCTN2 79 and Card15 in a neutralizing GM-CSF antibody background 80 ) all of which may lead to a growth hormone resistant state 80,81 . Growth failure is present at diagnosis in up to

80% of children with Crohn's disease (CD), and persists into adulthood in 30% 82 . Growth failure

is defined as either reduced growth velocity over a period of 3-4 months, in centimeters per year,

| n t { n s z p q Ó l t } s m q z t } t z for age or a fall in height percentile fro¡ along with delayed skeletal maturation and delayed onset of puberty 40,83 . Osteoporosis affects approximately 40% of adult

IBD patients and therefore decreased bone mass density is extremely important EIM in the pediatric population 15 .

Other EIMs that are slightly less common but parallel disease activity are joint pain or swelling 84 , clubbing of the distal phalanges which is rare but most frequent in children with

CD 40 , oral aphthous stomatitis (seen in 20-30 % of CD and 5-10% UC)85 , and characteristic skin lesions (described in 10-15% of IBD patients) 77 such as erythema nodosum or pyoderma gangrenosum. Other less frequent EIMs are ocular manifestations (episcleritis and uveitis) and the hepatobiliary complication sclerosing cholangitis 15 .

h i h Ü Diagnosis

Diagnosis of IBD is established on the basis of clinical, laboratory, bowel imaging, endoscopic assessment, and histologic findings. A full medical history should include onset of symptoms, recent travel, family history of IBD, drug history including antibiotics and nonsteroidal anti-inflammatory drugs (both thought to be environmental triggers of IBD), prior appendectomy (appendicitis can mimic CD) and smoking. A proper physical exam including abdominal signs of tenderness and or palpable mass (abscess or inflammatory masses) should be carefully examined. Localized pain to the right lower quadrant is indicative of CD and diffuse tenderness associates with small bowel involvement and overall pain depends on extent of colonic involvement 15,40 . UC patients may also have bloody and mucus mixed stool, along with lower abdominal cramping 71 . Rectal bleeding without diarrhea can be due to fissure, polyp, rectal ulcer syndrome, or Meckel diverticulum 15 . Perianal disease as seen in CD, presents with skin tags, fissures, fistulae and abscess. A thorough examination should be conducted for enteric pathogens often encountered during travel which may mimic IBD and should be ruled out before making a diagnosis 40 . Laboratory testing is usually performed to identify patients at high risk, for whom definitive diagnostic procedures such as endoscopy and colonoscopy are warranted.

Hematologic Tests. Initial tests should include full blood count, renal function, albumin and inflammatory markers (C-reactive protein (CRP) or erythrocyte sedimentation ratio (ESR)), and metabolic profile that includes liver enzymes 40,86 . C-reactive protein (CRP) is an acute phase protein that is increased in nearly 100% of patients with CD and approximately 50% with

UC 87 . However CD patients with established active disease do not have increased levels of

CRP 88 . Elevated liver enzymes levels should prompt an evaluation for associated liver

disease 40 .

h i j Ý Fecal Markers. The two most commonly utilized fecal markers with the most reliable read out for IBD are calprotectin and lactoferrin. The concentration of calprotectin

(S100A8/S100A) in feces is an indirect measure of neutrophil infiltrate in the bowel mucosa and has been linked to disease activity and severity 89 . At 50ug/g the estimated sensitivity and specificity for identification of patients with IBD from those without were 89% and 81%, respectively 90 . Lactoferrin is an iron-binding protein found in neutrophil granules and serum and is secreted by mucosal membranes. Gisbert et al reported that the lactoferrin test identified

IBD patients with a mean sensitivity of 80% and specificity of 82%91 . Lastly, these tests can help differentiate between IBD and irritable bowel syndrome (IBS) but can p t distinguish IBD from various other types of intestinal inflammation 92,93 . S100A12 is a S100 protein and is a marker for neutrophil activation that correlates with disease activity and may be more specific to

IBD. In a pediatric cohort, fecal levels of S100A12 greater than 10 mg/kg identified IBD with a sensitivity of 96% and a specificity 92% 94 . In adults, Kaiser et al reported that S100A12 distinguished patients with IBD from those with IBS and viral enteritis with high sensitivity and specificity, and correlated better with intestinal inflammation than fecal calprotectin or other biomarkers. They also concluded that S100A12 reflects inflammatory activity of chronic IBD 95 .

Serologic Markers. Anti-Saccharomyces cerevisiae antibodies (ASCA) and perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) are serologic markers that can be used in combination with other diagnostics for di agnosis of IBD 96 . Increased titers of ASCA was found

to identify patients with CD by high specificity (96% ã 100%) but low sensitivity (approximately

50%) while increased levels of pANCA was more common in patients with UC or those with CD who had UC-like pancolitis 96 . In a meta-analysis comprising 3,841 UC and 4,019 CD patients

the ASCA with pANCA ä test offered the best sensitivity for CD ((54.6%) with 92.8%

h i j h specificity), pANCA+ sensitivity and specificity for UC were 55.3% and 88.5%, respectively and in a pediatric subgroup sensitivity and specificity were improved to 70.3% and 93.4% when

97 combined with an ASCA ä . However diagnostic precision decreased for ASCA discrimination between CD and UC in isolated colonic CD 97 . Newer additions to serological evaluations include antibodies to Escherichia coli outer membrane porin (OmpC), Pseudomonas fluorescens -associated sequence (I2), and flagellin (CBir1) however the sensitivity of these individual markers for identifying patients with IBD is low, ranging from 11% to 83% and thus only offer modest contributions 98 . Individual assays are merged into panels to maximize the sensitivity and specificity of these serological tests and the IBD Serology 7 (IBD7) panel

(Prometheus Laboratories, San Diego, CA), is the most widely used in the United States and has been available since 2006 99 . However in a head to head competition, in a population of children referred for initial evaluation of suspected IBD, the predictive value of routine blood tests

(aneamia, thrombocytosis, and increased erythrocyte sedimentation rate (ESR)) had better sensitivity and specificity than the Prometheus IBD7 panel 99 .

Other serologic markers in the early stages of investigation for utility in IBD

phenotyping, clinical course and need for surgery are neutralizing intrinsically produced

o Ô æ antibodies against specific cytokines. TGF- u -10 are two cytokines important in peripheral tolerance and development of T-regulatory cells. Ebert el al. reported that anti- TGF-å antibodies were significantly higher in UC patients and anti-IL-10 antibodies significantly

increased in CD patients compared to controls100 . They also noted that anti- IL-2, IL-6, IL-8, IL-

u o Ô è ç 12, IL-18, IFN- -é antibodies were not different between UC and CD patients compared to controls 100 . Our lab has reported that granulocyte-macrophage colony-stimulating factor

(GM-CSF) autoantibodies, which decrease neutrophil antimicrobial function, are increased in the

h i j j serum of pediatric and adult CD patients 101 . The anti-GMCSF antibody levels are associated

with ileal involvement (P < .001), ileal location, duration of disease, and stricturing/penetrating

behavior (odds ratio, 2.2; P = .018) 101 . These studies suggest that a cytokine deficient state may

promote disease by inhibiting the cytokine action. This brings forth the idea that rather than

cytokine production, cytokine action would be a true measure of its deficiency or excess in IBD.

Auto-antibody studies are still in their infancy and larger prospective studies are needed to

elucidate the association with disease phenotype, clinical course and therapy.

Genetic Markers. Genome-wide association studies (GWAS) have identified 100

genetic risk loci of which 28 associate with both UC and CD. These loci reside in genes

involved in innate and adaptive immunity, autophagy, and intestinal homeostasis. Researchers

are avidly seeking to define how these loci affect disease phenotypic expression, clinical course,

and treatment modalities. Experts predict that genetic testing will have a very limited role in

diagnosing patients. However, recent studies from our lab and others justify the importance of

genetic markers in an integrated prognostic approach. Prometheus has just released the first

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ê ë ì í î ï í î genetic diagnostic for IBD -

generation IBD diagnostic test, the first and only test to combine serologic, genetic, and

s o z u ¡ ¡ u | s m o ¡ u l Ù t l q x v q s Ú w

Ø , in the proprietary Smart Diagnostic Algorithm for added

diagnostic clarity. This assay includes evaluation for single nucleotide polymorphisms

associated with disease risk in ATG16L1(Autophagy), STAT3(IL-6/IL-10/IL-23/IL-17 signaling

pathway), NKX2-3(intestinal differentiation), and ECM1(involved in angiogenesis, and tumor

biology).

Recent studies have tried to associate genetic variation with disease phenotype. Giving

only a modest capacity to predict clinical outcomes (AUC .59 to .76) NOD2, JAK2 and IL23R

h i j along with smoking habit and age at onset were associated with disease phenotype in CD patients with 10 years of follow-up 102 . A large cohort of CD patients from Belgium were genotyped for 50 SNPs (including the Wellcome Case Control Consortium GWAS SNPs and 13

SNPs from confirmed susceptibility loci [IL23R, ATG16L1, STAT3, TNFSF15, IBD5 (5q31 and

10q21]) and homozygosity at the minor allele of the rs1363670, in close proximity to IL12B gene, was found to be associated with stricturing disease 103 . In that same cohort any IBD-related surgery was associated with homozygosity of the minor allele of rs1363670 and carriage of a

NOD2 variant 103 . In abstract form at Digestive Disease Week 2011, Dubinsky et al. presented a predictive model of surgery by extrapolating genetic markers (the IL12B locus and Chr. 4q31), small bowel disease location, age at onset, and a serologic antibody sum in a cohort of 1115 CD patients with an AUC of .77 104 . In a cohort of 385 CD patients Lichtenstein et al utilized

105 serologic and NOD2 genetic markers to achieved an AUC of 0.80 (95% CI, 0.757 ã 0.846) .

These studies are the future of IBD research and illustrate the need for researchers and clinicians to work cohesively to aggregate larger patient cohorts complete with full genotypic profiles for multivariate analysis of genotype-phenotype (disease location, progression, behavior, treatment response) associations in the context of environmental factors (smoking, geographic location, ethnicity, diet, appendectomy, intestinal microbiota). The Immunochip project of the

International IBD Genetics consortium has utilized a custom-designed chip to provide genotypic data of 200,000 markers (IBD loci and additional candidate genes based on functional and genomic positional data) in a large cohort (15,000 CD, 12,000 UC, 21,000 healthy controls).

The first preliminary results were presented in October 2011 at the United European

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Gastroenterology week and Ò

meeting this December 2011. These studies promise great insight into the complexity of IBD

h i j ¢ pathogenesis. Lastly, newer models of IBD pathogenesis are even moving beyond genetic advances toward RNA biology and protein-protein interactions. Furthermore, the utility of biomarkers in defining disease activity, mucosal healing, predicting disease course and relapse, and response the therapy are of great interest for the clinician in defining disease management.

The current status of applying biomarkers beyond clinical diagnosis of IBD and distinguishing

CD vs UC can be reviewed elsewhere 89 .

Endoscopic Evaluation. Endoscopic examination with biopsies establishes the diagnosis of IBD. Endoscopy and histologic assessment of biopsies is the most perceptive and specific evaluation to aid in assessing the extent and severity of disease and differentiate a diagnosis of

UC or CD 40 . Therefore, Ileo-colonscopy with multiple biopsies taken from all segments of the

GI tract is the first-line procedure. A newer recognized endoscopic technique for complete small bowel interrogation is wireless capsule endoscopy (WCE). The capsule is the size of a pill and contains a tiny camera to allow excellent small-bowel visualization. Published studies have demonstrated that this technique is superior to standard endoscopic and radiographic modalities in diagnosing small bowel CD, is a first-line imaging modality for suspected CD in adults, and approved for use in children aged 2 years and older 106-109 . WCE has also been indicated for reclassification of IBD-U patients and in previously diagnosed CD patients allows greater detection of the overall burden of small bowel disease resulting in more targeted and effective therapy 110 . WCE has been reported to be non-specific (10% of healthy individuals have mucosal breaks and erosions in the small bowel) and contraindications include intestinal obstruction, stenosis or strictures, and pacemakers 43 . However, capsule retention occurs with a frequency of

1-2% and is a major complication 111 .

h i j £ Radiologic Evaluation. Radiologic/Enterography allows the clinician to see beyond the mucosa to understand the full transmural view and assess for extra-enteric complications such as

fistulas or abscesses and obstruction. Radiographic evidence is usually needed for patients with

t m z ð | u o u l Ú õ CD to assess extent, location, and severity of disease and as of 2002, th Ø was small bowel follow-through. However, newer technologies such as magnetic resonance imaging

(MRI) computed tomography scans (CT), and ultrasound (US) are the most successful widely used to date 112 . CT and MR enterography involve oral luminal contrast and have a high diagnostic sensitivity in detecting small bowel involvement and extra luminal complication of

CD, including fistula and abscess 43 . Plain abdominal X-rays can be non-specific, however if toxic megacolon is suspected or obstruction plain films should be used before any contrast 15 .

Since most children with IBD will receive multiple radiologic exams, their total radiation exposure is of concern and MRI, the modality with the least ionizing radiation, is the imaging

modality of choice in children 112 . Ultrasound is also a non-ionizing imaging modality to evaluate

q m l ¡ u | s m o s o k l m n o p q s q t u q t ó the site, extent, activity, and complications such as absce q It is widely available and inexpensive but operator dependent and has wide inter-observer

variability 43 .

t Ó u | s t o | p q 113 n

A white blood cell scan can help in distinguishing CD and UC . î leukocytes are isolated and labeled with a radioactive substance, then infused back into the patient and watched for uptake in areas of infection or inflammation within the body. Positron emission tomography (PET) for IBD patients involves administering of radio-actively labeled glucose (18 F-FDG), that is more readily taken up by cells that are inflamed or cancerous, allowing clinicians to see areas of active inflammation. PET has not been shown to be a better

diagnostic than CT or MR enterography in the detection and anatomic characterization of

h i j ¤ intestinal disease but is more useful in accurate and non-invasive quantification of disease

activity by assessing the presence and degree of inflammation 114 . PET can also be combined

Ö s | n k Ù o m Ö o u q l t s q | l u | s m o Ú Ø î co- of CT however a recent study in children with IBD found

that the co-registration of CT had no additional benefit in diagnosis 115 .

Clinical Disease Activity. Clinical disease activity indices should include grading and

weighting of specific symptoms and intestinal manifestations, with defined cutoff scores

corresponding to clinically important disease states from remission to severe disease activity that

is then clinically validated for reliability, responsiveness (improvement or worsening) and

feasibility 116,117 . Inflammatory bowel disease indices include composite evaluation of symptom

severity, quality of life, laboratory tests and endoscopic findings.

l m n o p q s q t u q t The k activity index (CDAI) was developed in 1976 and evaluates eight

disease variables including liquid stools, abdominal pain, general well-being, extra intestinal

Variable Description Multiplier Number of liquid stools Sum of 7 days x 2 Abdominal pain Sum of 7 days rating x 5 (graded from 0 -3 on severity) General well -being, subjectively Sum of 7 days x 7

assessed (graded as 0 = well to 4 = terrible)

l | n l s | s q ö u l | n l u z s u x s l s | s q ö } t s | s q x Extra intestinal complications x 20 erythema nodosum, pyoderma

gangrenosum, aphtous stomatitis, anal

s q q l t ö s q | z u ö u q { t q q x t } t l ÷ ø ù ó ú û k

Ò Antidiarrheal drugs ( 0 = no, 1 = yes) Use in the previous 7 days x 30 Abdominal mass (0 = none, 2 = questionable, 5 = definite) x 10 Hematocrit Males:<0.47 x 6 Women<0.42 Body weight, % deviation from [1 - (ideal/observed)] x100 x 1 standard weight

Sum of CDAI (0 -600)

ü ý þ ÿ ¡ ¢ ý £ ¤ ÿ ¥ ¦ § ¨ © ¢ ÿ ¥ ý ¡ ¢ ý ÿ ¥ ¥ ý © ¢ ý ¢ ¨ ý ý ¥ þ ¦ ý ¡ ¢ ¥ ¤ ¨ ÿ ¥ ¡ © © ý ¨ © ý ¨ ¢ ¢ ¡ £ ý ¨ ¢ ¥ ¡ ¨

¥ ¥ þ ý ÿ ¨ ¢ ý ÿ ¥ ¡ © © ý ¨ © ý

tudy. 439 -444. Copyright (1976 ), with permission from Elsevier.

h i j Ä complications, antidiarrheal drugs, abdominal mass, hematocrit, and body weight (Table 3) 118 .

These variables are weighted by using an individual multiplier for each score and complied to

give the CDAI score that can range from 0 to approximately 600. Investigators have arbitrarily ã labeled CDAI scores of 150 ã 219 as mildly active disease and scores of 220 450 as moderately

active disease however clinical studies continually determine cutoffs for a defined population or

specific clinical endpoint 119 . The Harvery-Bradshaw (HBI) is a simplified version of CDAI and

the correlation between CDAI and HBI scores are high however the CDAI is the most widely

t t s u | l s { k l m n o p q s q t u q t u { | s } s | Û 118,120 n

used in clinical trials for CD . T index (PCDAI) was

modified from the CDAI and is the preferred method for assessing disease activity in clinical

l s u z q m Ó t s u | l s { k l m n o p q s q t u q t | (Table 4) 121 . PCDAI modifications disregarded the use of

antidiarrheal agents, decreased weighting of subjectively reported abdominal pain and general

well-being, however assess limitations to participate in normal activities and laboratory

parameters of linear growth, serum albumin, and erythrocyte sedimentation rate (ESR) 121 .

Therefore, this index functions as a noninvasiveness, valid, response index used as a primary

outcome measure to reflect disease activity (remission, severe disease, moderate disease) for

prognosis and treatment decisions 122 . Another measurement of clinical disease activity is the

k l m n o p q s q t u q t t o m q { m Ó s { s o t m q t } t l s | Û v k r Ô ð w x t } t z m Ó t s o à ú x s q q t | m u q q t q q | n t

severity of mucosal inflammation or mucosal healing 123 . Parameters for the CDEIS include

healed ulceration, erythema, mucosal oedema, aphthoid ulcerations, superficial and deep

ulcerations and stenosis. A simplified endoscopic severity index for CD (SES-CD) was

developed in 2004 to evaluate the size and penetration of ulceration as a reflection of disease

severity in CD 124 .

h i j ×

Ñ Æ ! " Æ # È Ñ $ Ì È % Ë Ì Í Î É Ï Ð Å È Ð Æ Ñ Ð Æ & % $ È ' È $ ( ) É # Æ * + " Ë Å & ) , Variable Description Score No pain 0 Abdominal pain Pain can be ignored 5 Moderate/Severe: daily, nocturnal 10

0-1 liquid, no blood 0

. / 0 1 2 Stools / day - -formed +small blood or 2 -5 liquid 5

with or without blood

4 5 2 6 7 2 8 / 9 : : 5 / ; < = : / / > 5 : : 8 ; : = ? : @ 9 7 = ? A 5 3 10 diarrhea

Patient Functioning B General Well - No limitations of activities 0 being Occasional difficulties in maintaining 5 appropriate activities Frequenty Limitation of activity 10 Weight Weight Gain or stable 0 Weight Loss < 10% 5 Weight Loss > 10% 10 Height at diagnosis < 1 channel decreased from * 0

* is previous percentile 1 to 2 < channel decrease from * 5

. @ C A ? ? 0 5 8 0 @ = 0 A / 0 D = : 1 E 3 10 - -1 standard * 0 Height Velocity -1 to < -2 standard * 5 * is deviation from normal 3 -2 standard * 10 Abdomen No tenderness or mass 0 Tenderness or mass without tenderness 5 Tenderness and definite mass 10 Peri -rectal disease None, asymptomatic tags 0 1-2 indolent fistula, drainage, non -tender 5 Active fistula, drainage, tenderness, or 10 abscess Extra -intestinal Manifestations None 0

1 Manifestation 5

F A ? 2 D 0 / 9 A 9 2 : ? /

3 10

0 A = / J 3 K K

H I

M/F 6 - G

0 A = / J 3 K M

L I

M 11 - G 0

0 A = / J 3 K

N I L

F 11 - G

0 A = / J 3 K O

N I M 15 - G M/F 6 -10 years:28 -32 M 11 -14 years: 30-34 2.5 Hematocrit (%) M=Male and F 11 -19 years: 29-33 F=Female M 15 -19 years: 32 -36 M/F 6 -10 years: <28 M 11-14 years: < 30 F 11 -19 years: < 29 5 M 15 -19 years: < 32 < 20 0 ESR mm/hr 20 -50 2.5

>50 5

K M 3 0

Albumin (g/L) 31 -34 5

- K

H 10

Sum PCDAI

A 1 / 0 9 A 5 Q R 0 S 0 5 : T 1 0 ? 9 A ? 8 U A 5 2 8 A 9 2 : ? : D A V 0 8 2 A 9 = 2 @ W = : C ? X / R 2 / 0 A / 0 Y @ 9 2 S 2 9 ? 8 0 [ Q \

I I Z Pediatr Gastroenterol Nutr 12, 439-447. Copyright (1991 ) with permission from Wolters Kluwer

Health

h i j Ü Several disease scores for measuring disease activity in UC patients have been developed but still there is no clinically defined gold standard 125,126 . The two methods most readily used in clinical trials are the Ulcerative colitis disease activity index (UCDAI) and the Mayo Disease

Activity Index (MDAI) which are a composite of clinical and endoscopic disease activity (Table

127,128 5, UCDAI) . Table 5. Ulcerative colitis disease activity index (UCDAI) Variable Description Score

In children, the Stool frequency 1] 2 Stools /day>normal 1

3] 4 Stools /day>normal 2 Pediatric Ulcerative >4 Stools /day>normal 3 Rectal Bleeding None 0 Colitis Activity Streaks of blood 1 Obvious blood 2 Mostly blood 3 Index (PUCAI) is Mucosal appearance Normal 0 Mild friability 1 a noninvasive Moderate friability 2

Exudation, spontaneous bleeding 3

_ ` a b c b d e f a g d h b e i j k l b a m d a m d c h b n b h ` ^ Normal 0 evaluation of UC * Includes endoscopic assessment Mild 1 Moderate 2 disease activity that Severe 3

Sum UCDAI

¥ ¥

has been shown as Each variable is scored from 0 ¥ 3 so that the total index score ranges from 0 12; 0 2: ¥ remission; 3 ¥ 6: mild; 7 10: moderate; >10: severe UC. Adapted from N Engl J Med. 317, Schroeder et al. Coated oral 5 -aminosalicylic acid therapy for mildly to moderately active a valid, highly ulcerative colitis. A randomized study. 1625 -1629. 1987. reliable, and a responsive index with which to assess disease activity(Table 6)129,130 . Turner et al demonstrated a very strong correlation of the PUCAI with macroscopic mucosal inflammation supporting the use of this score as a measure of disease activity in children without endoscopic assessment. The development of and endoscopic assessment of has recently been reported for

UC by Travis et al. They report that the Ulcerative Colitis Endoscopic Index of Severity

(UCEIS) accurately predicts overall assessment of endoscopic severity of UC however they the validity and responsiveness need further testing before it can be applied as an outcome measure

in clinical trials or clinical practice 131 .

h i Ý Table 6. Pediatric Ulcerativ e Colitis Activity Index (PUCDAI) Variable Description Score No pain 0 Abdominal pain Pain can be ignored 5 Pain cannot be ignored 10 None 0 Small amount only, in less than 10 Rectal bleeding 50% of stools Small amount with most stools 20 Large amount (>50% of the 30 stool content) Stool consistency of most Formed 0 stools Partially formed 5

Completely unformed 10

o p

á 0

o q

Number of stools per 24 ø 5

o ú hours r 10 >8 15 Nocturnal stools (any No 0 episode causing wakening) Yes 10 No limitation of activity 0 Activity level Occasional limitation of activity 5

Severe restricted activity 10

s t u

Ç " v Ë & ) + w x y z , Í Reprinted from Gastroenterology 133, Turner et al. Development, validation, and evaluation of a pediatric ulcerative colitis activity index: a prospective multicenter study, 423 -432, Copyright (20 07 ), with permission from Elsevier.

Histologic Disease Activity / Mucosal Healing

The histopathologic features of IBD are variable and depend on the type of disease (CD or UC), the duration of disease and the severity of bowel inflammation. In newly diagnosed CD patients, the earliest lesions are apthous ulcers132 . The apthous ulcers occur in the small and large bowel, and are associated with submucosal lymphoid aggregates covered by specialized antigen-transporting cells called membranous (M) cells. CD histology reveals neutrophilic infiltration of surface epithelial cells and crypts leading to crypt abscesses, crypt destruction and

mucosal ulceration. A hallmark of CD is the presence of noncaseating granuloma which can be

h i h identified in 15% of endoscopic biopsies and up to 70% of operative specimens 133 . UC is histopathologic features include a granular mucosa with areas of superficial ulceration,

neutrophilic infiltration of crypts and the development of crypt abscesses. Contrary to CD,

s s

Ñ Æ { ! Ë Ì Í Î É Ï Ð Å È Ð Æ Ñ Ð Æ | È Ð $ Í Í Ê È % ) É # Æ * Í Ç Æ ' Æ Ì È $ ( + Ë Å | ) , inflammation is Variable Description Score largely mucosal Normal 0 Epithelial Damage Focal Pathology 1 and depletion of Extensive Pathology 2 Normal 0 goblet cells is more Architectural Changes Moderately disturbed (<50%) 1 Severely disturbed (>50%) 2 prominent. In the Normal 0 Infiltration of mononuclear cells chronic phase in the lamina propria Moderate increase 1 histologic features Severe increase 2 Infiltration of Normal 0 of IBD included polymorphonuclear Moderate increase 1 cells in the lamina propria Severe increase 2 distorted crypt In surface epithelium 1 Polymorphonuclear cells in Cryptitis 2 architecture, crypt epithelium Crypt abscess 3 atrophy, increased Presence of erosion and/or ulcers No 0 Yes 1 numbers of round Presence of granuloma No 0 Yes 1

cells and None (0 of 6) 0

ø ø â v à m l p m r w No. of biopsy specimens affected } 1 neutrophils in the

33%ã 66% (3 or 4 of 6) 2

r r â v q m l r m r w ~ 3 lamina propria, Sum CDHIS Each topic is scored independently. Moderate increase, up to twice the number of

giant cells and cells that can normally be expected; severe increase, more than twice the normal

basal lymphoid of recur ileum, 262 -267, Copyright (1998), with permission from Elsevier. aggregates 133 .

Mucosal healing or endoscopic remission as a therapeutic endpoint in research trials and clinical

practice is effective in optimizing long-term outcomes 134 . Histopathologic analysis of CD and

h i j UC as it relates to biologic disease activity and predictions of future outcomes remains largely unknown 135 . The current method for assessment of histologic outcomes is a reduction in the CD or UC Endoscopic Index of Severity (CDEIS/UCEIS) and the CD or UC Histologic Index of

Severity (CDHIS/UCHIS) (Table 7 and 8)136,137 .

Table 8. Ulcerative Colitis Histologic Index of Severity (UCHIS) Variable Description Score Normal 0 Chronic inflammation only 1 Mild active cryptitis (but no 2 abscesses) Polymorphonuclear cells in epithelium Moderate Active (few crypt 3 abscess) Severe active inflammation 4 (numerous crypt abscesses)

None (0 of 6) 0

ø ø â v à m l p m r w No. of biopsy specimens affected } 1

33%ã 66% (3 or 4 of 6) 2

r r â v q m l r m r w ~ 3 Sum UCHIS Each topic is scored independently. Moderate increase, up to twice the number of cells that can normally be expected; severe increase, more than twice the normal number of cells. Reprinted from Gastroenterology, 126, Rutter et al. Severity of inflammation is a risk factor for colorectal neoplasia in ulcerative colitis, , 451-459, Copyright (2004), with permission from Elsevier.

Treatment

Medical therapy relies on classic anti-inflammatory and immunosuppressant drugs: corticosteroids, mesalamine compounds, corticosteroids, azathioprine and the like. Biologic drugs such as TNF-ç , have added greatly to the ability to control IBD while newly described mechanisms of controlling the gut microbiome through probiotics, antibiotics and fecal transplant are on the horizon. In April 2011 the American College of Gastroenterology IBD

Task Force, consisting of selected experts in epidemiology and therapy of IBD, released an

evidence-based systematic review on medical therapies for IBD in the American Journal of

h i Gastroenterology. They developed a protocol for systematically reviewing the data on currently available therapies for UC and CD pertaining to both inducing remission and in preventing relapse of the disease. The group reported evidence-based statements with strength of recommendation graded according to standard criteria. The following pages are summarized drug description and listed recommendations from this report.

5-ASA Therapy. 5-ASA was the first drug used for UC and combines the antibacterial properties of sulfonamides with the anti-inflammatory effects of salicylates. Also known as mesalamine (free, unconjugated 5-ASA) and mesalamine prodrugs (azo-bonded 5-ASA) have similar modes of action. Sulfasalazine, the standard azo-bonded 5-ASA designer drug, is engineered to release free 5-ASA in the colon, protecting it from proximal absorption 138 . Newer topical and oral 5-ASA agents are delivered to different anatomic sites, corresponding to the distribution of disease thus having minimal systemic side effects 139 . The precise mode of action is unclear, but the effectiveness of the compound is related to its mucosal concentration and it is absorbed by colonic epithelial cells 140,141 . 5-ASA is believed to be anti-inflammatory by

modulating inflammatory cytokine production along with nuclear factor kappa B RelA/p65

t Ù m | l s t o t p q phosphorylation and inhibiting the biosynthesis of prostaglandins and z 142 . 5-ASA is also thought to be an antioxidant that traps free radicals, which are potentially damaging byproducts of metabolism 143 . Although very rare, serious adverse effects of 5-ASA are interstitial nephritis, pancreatitis, pneumonitis, pericarditis, and hepatitis 144 . Recommendations from the American College of Gastroenterology IBD Task Force (Talley et al., 2011) for treatment of IBD with 5-ASA are as follows: 5-ASA therapies are effective at inducing remission in mild-to moderately-active UC with a strong recommendation and moderate quality

of evidence. In preventing relapse of quiescent UC 5-ASA is very effective with strong

h i ¢ recommendation and high quality of evidence. 5-ASA therapies are not recommended for inducing remission in active CD with a weak recommendation and low quality of evidence. In preventing relapse of quiescent CD 5-ASA therapies are not recommended with a weak recommendation and low quality of evidence.

5-ASA

Remission Relpase of Remission Relpase of of UC UC of CD CD

strong rec./ strong rec. / not rec. / low moderate high QOE QOE QOE

Figure 5 . 5 -ASA treatment recommendations for IBD . ). QOE, Quality of evidence . Reprinted by permission with Macmillan Publishers Ltd: Talley, N.J., et al. An evidence- based systematic review on medical therapies for inflammatory bowel disease. Am J Gastroenterol 106 Suppl 1, S2-25; (2011)

Corticosteroid Therapy. The first Randomized Controlled Trial of cortisone in UC was

1954145 . Since then corticosteroids have been widely used for UC and CD but in only acute exacerbations due to the short-term adverse effects (risk of infection and psychiatric disorders) and long term adverse effects (loss of bone mineral density and diabetes mellitus)146 147-150 .

Therefore budesonide was synthetically derived to achieve enhanced corticosteroid concentration to the distal ileum and proximal colon while limiting systemic bioavailability to minimize systemic adverse effects 151,152 . Corticosteroids inhibit the immune system through modulation of glucocorticoid receptor interaction in the nucleus, trafficking of inflammatory cells to the

intestine, inducing apoptosis of activated lymphocytes, and decreasing inflammatory cytokine

h i £ expression 153-156 . Corticosteroids were reviewed only for treatment of acute CD and UC and maintenance therapy of CD with budesonide was evaluated since the systemic adverse effects are lower with this preparation 27,156 . Recommendations from the American College of

Gastroenterology IBD Task Force (Talley et al., 2011) for treatment of IBD with corticosteroids are as follows: Corticosteroid therapies are effective at inducing remission in active UC and CD with a strong recommendation and low quality of evidence. Budesonide therapy is effective at inducing remission in mild-to moderately-active CD with a strong recommendation and loquacity of evidence. Standard corticosteroids are more effective than budesonide at inducing remission in mild-to-moderately-active CD with a weak recommendation and low quality of evidence. Budesonide is not recommended for preventing relapse in quiescent CD with weak recommendation and low quality of evidence.

Corticosteriods

Remissin Relapse Relapse of Remission of CD UC for UC CD

strong rec/ low QOE corsticosteriods better Budesonide strong rec./ than Budesonide not evaluated not rec./ low low QOE Budesonide in QOE moderately active CD is strongly rec. / low QOE

Figure 6. Corticosteroid therapy in IBD and recommendations . QOE, quality of evidence . Reprinted by permission from Macmillan Publishers Ltd: Talley, N.J., et al. An evidence-based systematic review on medical therapies for inflammatory bowel disease. Am J Gastroenterol 106 Suppl 1, S2 -25; (2011).

h i ¤ Imunosuppressant Therapy. Thiopurine analogs (6-MP and its pro-drug azathioprine), methotrexate, cyclosporin and more recently tacrolimus are immunosuppresants. Azathioprine and 6-MP are purine anti-metabolites which impede DNA synthesis and inhibit the proliferation of rapidly dividing cells such as lymphocytes 157 . These drugs are associated with nausea, allergic reactions, acute pancreatitis, hepatitis, increased risk of infection, malignancy, and bone marrow suppression 158 . Methotrexate acts by inhibiting the metabolism of folic acid and decreasing the availability of folate needed to synthesize DNA in rapidly dividing lymphocytes.

The adverse effects of methotrexate include hepatotoxicity, pneumonitis, increased risk of infection, malignancy, alopecia, stomatitis, and myelosuppression. Cyclosporin and tacrolimus both inhibit calcineurin which impedes T-lymphocyte signal transduction and IL-2 transcription leading to reduced effector T-cell responses. The main adverse effect of these drugs is renal toxicity and drug levels should be monitored 158 . Other side effects include hypertension, hirsuitism, headache, opportunistic infections, seizures, and paresthesia158 . Recommendations from the American College of Gastroenterology IBD Task Force (Talley et al., 2011) for treatment of IBD with Immunosuppressant are as follows: Azathioprine and 6-MP are not recommended for inducing remission in active UC but are effective at preventing relapse in quiescent UC with weak recommendation and low quality of evidence. Methotrexate is not recommended for inducing remission in active UC or for preventing relapse in quiescent UC with a weak recommendation and low quality of evidence. IV cyclosporin is effective at improving symptoms in hospitalized patients with severely active UC not responding to corticosteroids. Tacrolimus is not recommended in mild-to-moderately active UC with weak recommendation and very low quality of evidence. Azathioprine and 6-MP are not recommended

for inducing remission in active CD but are effective at preventing relapse in quiescent CD with

h i Ä Immunosuppresant

Remission UC Relapse UC

Azathiopurine IV Cyclosporin Tacrolimus Azathioprine & Methotrexate effective severe methotrexate & 6-MP not rec. mild-to 6-MP effective, not rec. / very UC not not rec. / low not rec. / very moderately UC weak rec. / low low QOE responding to QOE low QOE Corticosteriods / very low QOE QOE

Figure 7. Immunosuppressant Therapies and recommendations for UC. QOE, quality of evidence. Reprinted with permission from Macmillan Publishers Ltd. Talley, N.J., et al. An evidence -based systematic review on medical therapies for inflammatory bowel disease. Am J Gastroenterol 106 Suppl 1, S2-25; (2011). a weak recommendation and low quality of evidence. Intramuscular methotrexate is effective at inducing remission in active CD with weak recommendation and low quality of evidence.

Cyclosporin is not recommended for inducing remission in active CD or for preventing relapse in quiescent CD with weak recommendation and very low quality of evidence. Methotrexate is effective at preventing relapse in quiescent CD with weak recommendation and low quality of evidence.

Immunosuppresant

Remission CD Relapse CD

Intramuscular Azathioprine Azathioprine Methotrexate methotrexate Cyclosporin & 6-MP Cyclosporin & 6-MP effective, effective, not rec. / very effective not rec. / low weak rec. / not rec. / low weak rec. / low QOE weak rec. / QOE low QOE QOE low QOE low QOE

Figure 8. Immunosuppressant Thera pies and recommendations for CD. QOE, quality of evidence . Reprinted with permission from Macmillan Publishers Ltd: Talley, N.J., et al. An evidence-based systematic

review on medical therapies for inflammatory bowel disease. Am J Gastroenterol 106 Suppl 1, S2 -25; (2011).

h i × Biologic Therapy. Treatment of IBD started to include biologics in 1998 with current therapy guidelines devised from composite evidence of clinical trials, clinical series, and expert opinions 27 . Thus the IBD Task Force conducted a systematic review and meta-analysis of randomized control trials (RCTs) to estimate the effectiveness and safety of these drugs in

IBD 159 . Overall there were no statistically significant differences between adverse events in patients randomized to biological therapy compared with placebo but the risk of infection increases if these agents are used in combination and concerns that the biological therapies may

increase the risk of lymphoma have been evaluated 27 . Most biologics target tumor necrosis w factor alpha (TNF-ç a pleiotropic inflammatory cytokine shown to be linked to many of the

Biologics

Remission UC Relapse UC

Infliximab effective ambulatory Infliximab effective (moderate to severe) improved symptoms in No trials strong rec. / moderate hospitalized patients QOE

Figure 9. Biological therapies and recommendations in UC. QOE, quality of evidence. Reprinte d with permission from Macmillan Publishers Ltd Talley, N.J., et al. An evidence -based systematic review on medical therapies for inflammatory bowel disease. Am J Gastroenterol 106 Suppl 1, S2-25; (2011). clinical problems associated with autoimmune disorders such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, hidradenitis suppurativa and refractory asthma. Therapies

used in CD inhibit TNF using a monoclonal antibody such as infliximab (Remicade), ô adalimumab (Humira), certolizumab pegol (Cimzia). Another biologic used for IBD is anti-ç

integrin antibodies (natalizumab) which works by blockade of leucocyte adhesion to

h i Ü endothelium, inhibiting extravasation and the inflammatory process. Recommendations from the

American College of Gastroenterology IBD Task Force (Talley et al., 2011) for treatment of IBD with biologics are as follows: Infliximab is effective at inducing remission in ambulatory patients with moderate-to-severely-active UC with a strong recommendation and moderate quality of evidence. Infliximab is effective at improving symptoms in hospitalized patients with severely active UC with a weak recommendation and very low quality of evidence. Anti-

TNF antibody therapies (infliximab, adalimumab, and certolizumab pegol) are effective at inducing remission in ambulatory patients with moderate-to-severely-active CD with a strong recommendation and moderate quality of evidence. Anti-ç 4 integrin antibodies (natalizumab) are effective at inducing remission in ambulatory patients with moderately-to-severely-active CD with a weak recommendation and moderate quality of evidence. Anti-TNF ç antibodies

(infliximab, adalimumab, certolizumab pegol) are effective in cessation of fistula drainage in CD with a strong recommendation and lwo quality of evidence. Anti-TNF ç antibodies (infliximab, adalimumab, certolizumab pegol) are effective at preventing relapse in quiescent CD with a strong recommendation and high quality of evidence. Anti-ç 4 integrin antibodies (natalizumab) are effective in prevention of relapse in quiescent CD with a weak recommendation an low quality of evidence. Infliximab is effective in prevention of relapse in healed fistulizing CD with a weak recommendation and low quality of evidence.

h i ¢ Ý Biologics

Remission CD Relapse CD

Anti-TNF

(Infliximab, Anti- L integrin Anti-TNF Anti-TNF

Infliximab

(Infliximab, Anti- L integrin adalimumab/ (natalizumab) (Infliximab, effective relapse adalimumab, (natalizumab) certolizumab effecitve adalimumab, of healed certolizumab effective pegol) ambulatory certolizumab fistulizing CD. ambulatory moderate to pegol) effective pergol) moderate to severe fistula drainage weak rec. / low severe weak rec. / low weak rec. / strong rec. / strong rec. / QOE QOE strong rec. / moderate QOE low QOE high QOE moderate QOE

Figure 10. Biologic therapies and recommendations in CD. Reprinted with permission from Macmillan Publishers Ltd Talley, N .J., et al. An evidence-based systematic review on medical therapies for inflammatory bowel disease. Am J Gastroenterol 106 Suppl 1, S2-25; (2011). QOE, quality of evidence

Antibiotics. Modulating the gut flora using probiotics, transplanting the entire intestinal micro biota, or using antibiotics to inhibit harmful bacterial luminal antigens are currently being evaluated as treatment modalities for IBD. Since the substantial clinical efficacy of probiotics in

IBD is less evident and fecal transplant is in its infancy, these studies were not considered.

Antibiotics were evaluated showing promise but had no recommendation for a particular class of drug. Recommendations from the American College of Gastroenterology IBD Task Force

(Talley et al., 2011) for treatment of IBD are as follows: Antibiotic therapies have a statistically significant effect at inducing remission in UC and CD but are not recommended as no particular class of drug could be recommended in clinical practice with a weak recommendation very low quality of evidence. Antibiotic therapies are effective at reducing fistula drainage in CD with a weak recommendation and low quality of evidence. Antibiotic therapies shows a statistically

significant effect at preventing relapse in quiescent CD but these are not recommended as no

h i ¢ h particular class of drug can be recommended in clinical practice with a weak recommendation

and very low quality of evidence.

Biologic Treatment options. Other potential biologic treatment options under active

investigation involve inhibition of inflammatory cell recruitment and adhesion, recombinant

anti-inflammatory cytokines and cytokine inhibition, inhibitory T-cell antibodies, growth factors,

and hormones160,161 . Biologics with clinical efficacy in IBD are reviewed in the proceeding

paragraphs.

The development of anti-adhesion drugs that can selectively inhibit recruitment and

inappropriate retention of leukocytes has shown promise for the treatment of chronic

¡ m o m { z m o u z u o | s m Û s l t { | t u u s o q | | n t ô ç

inflammation. Ò -integrin, Natalizumab, binds both

å å

ô à u o ô ù s o | t l s o q | m z m { Ù z Û ¡ Ó n m { Û | t u n t q s m o u o s q u Ó Ó l m } t m l q t s o k r ç ç 162

- - Ò .

y o m l | o u | t z Û ô à s o | t l u { | q Ö s | n } u q { z u l { t z z z u l u n t q s m o ¡ m z t { z t ç - -1 (VCAM-1), which is

present on every vascular bed in the body and this immunosuppression can reactivate the latent

JC virus inducing a progressive multifocal leukoencephalopathy (PML), that could be fatal 163 .

ô ù The monoclonal antibody directed against ç , Vedolizumab (MLN-02), selectively inhibits

Madcam-1-mediated leukocyte gut mucosal adhesion and was found to be effective in a dose

dependent manner for both CD and UC164,165 . Vedolizumab alters gut inflammation without

ô ù

inducing systemic immunosuppression by exclusively binding to ç integrin and inhibiting

ô ù

adhesion of ç -expressing cells to both Madcam-1 and fibronectin but not Vacam-1166 . q Vedolizumab p binding capacity was the highest for subset of human peripheral blood memory

CD4(+) T lymphocytes (25%) that included gut-homing interleukin 17 T-helper lymphocytes

with no binding in the majority of memory CD4(+) T lymphocytes (60%), neutrophils, and most

h i ¢ j monocytes 166 . Vedolizumab is currently in phase III clinical trials (GEMINI) for both UC and

CD 167-169 .

Another anti-adhesion drug in phase III clinical trials is an orally bioavailable small

molecule CCX282-B (GSK-1605786, Traficet-ENTM or anti-CCR9) that selectively blocks the

human CCR9 receptor, a small bowel specific chemokine receptor found on the majority of

lamina prorpia T-cells that binds CCL25 170 . The Phase II (PROTECT-1) and Phase III(

SHIELD-3) clinical trials showed promising end point reductions in CDAI and maintained

remission rates however the results are still are not fully published. GlaxoSmithKline has

reformulated CCX-282 for bioavailability and GSK1605786A is currently recruiting for Phase

III clinical trials to investigate the efficacy and safety in patients with moderate to severe

l m n o p q s q t u q t k with completion expected in 2014 171 .

Blockade of lymphocyte-vascular gut endothelium interactions with monoclonal

antibodies seem to show promise in patients with UC. Visilizumab, an IgG2 monoclonal anti-

CD3 antibody, was shown to be clinically beneficial in UC patients not responding to iv

corticosteroids and decreased circulating CD4(+) T-cell in all patients 172 . Basiliximab, an IgG1

monoclonal anti-CD25(IL-2 receptor) antibody, promoted prolonged remission after a single

treatment, in moderate steroid-resistant UC 173 .

Cytokine inhibition of IL-6 and CD T-helper1 polarizing cyotkines IL-12, IL-18, and

Ô è n u } t u z q m t t o t q s

é 174

Ò ned and tested for efficacy . The humanized anti-IL-6 receptor

monoclonal antibody, Tocilizumab, blocks both the membrane-bound and the soluble form of

IL-6R and has been studied for potential use in the treatment of CD. A pilot study showed

promising results with 80% of patients given a biweekly infusion for 12 weeks showing a

significantly higher clinical response rate then the placebo group, however only two patients

h i ¢

receiving tocilizumab achieved remission and endoscopic and histologic examination showed no

Ô è u o | s m Û

175,176 é difference between tocilizumab and placebo groups. . A humanized anti- Ò , fontolizumab, showed a biologic effect in a recent randomized clinical trial by reducing CRP levels in all fontolizumab groups compared to placebo 177 . The anti-IL-12 antibody, ustekimumab, targeted against the p40 subunit of two important cytokines in CD pathophysiology, IL-12 and IL-23 has shown effects in CD patients. Treatment of either moderate-to-severe CD disease, CD patients previously given infliximab, and CD non- responders to infliximab with ustekinumab improved clinical response compared to placebo and reduced serum CRP levels 178,179 . The anti-inflammatory cytokine, recombinant human IL-10 was not successful at inducing remission in patients with active CD showing no benefit for treatment of active disease 180 .

The immunostimulator and growth factor, sargramostim (recominant human granulocyte- macrophage colony-stimulating factor) and filgrastin (granulocyte-colony stimulating factor) have shown promising results for inducing CD remission and reducing clinical disease activity but there are some inconsistencies across studies and these drugs need further investigation 181-

183 . The growth factor, growth hormone (GH), has been shown to modulate intestinal inflammation and mucosal repair in murine models of colitis 184,185 186 . In clinical trials GH has

improved linear growth, remission rates, and clinical disease activity in pediatric CD patients but q larger clinical trials are needed to investigate GH p potential in mucosal healing and repair in human colitis 187 188,189 .

h i ¢ ¢ The Fundamental Basis of Inflammatory bowel disease

The pathogenesis of IBD is complex and still not completely understood. Basic concepts are conserved in human and murine colitis and in spite of different genetic predispositions and environmental triggers, the common mediator of disease is still an altered immune response

190,191 . Under normal homeostatic conditions, the intestine has an immunologic tone of tolerance

toward commensal flora and dietary antigens that is referred to as a state of Ø nonpathogenic inflammation Ú that keeps the immune response conditioned and has the ability to induce an immune response to pathogens 192 . This also establishes a hypo-responsive phenotype to pathogenic organisms upon infection that can be quickly controlled and is effective. IBD constitutes a loss of tolerance toward commensal flora, dietary antigens, and components of the actual immune response as evidenced by serological antibodies to a patient p s own neutrophils, colonizing yeast, E. coli, flagella, and cytokines (autoantibodies) that are routinely found in the serum of IBD patients 193,194 . Regulatory immune cells mediate tolerance and in IBD these cells are inadequately stimulated or maintained allowing an inflammatory response to either commensal or pathogenic to be over stimulated or persist and develop into a destructive inflammatory reaction 195,196 . The characterization of this immune dysfunction has been associated with T-cell activation and for over two decades the pattern of helper T-cell dysfunction has been traditionally distinguished as Th1 and Th2. It is clear that CD and UC are immunologically distinct as CD patients overproduce cytokines within the lamina propria associated with a T helper (Th) 1 response, such as IL-12 and interferon-é 197 . Conversely, UC patients overproduce cytokines associated with the Th2 response, such as IL-5 and IL-13 198 .

Murine models of innate and adaptive mucosal inflammation also follow this Th1/Th2 paradigm

and advance our understanding of CD4 T-cell biology. In 2006, T-cell mediated mucosal

h i ¢ £ immunology became more complex with the discovery that a subset of T cells, Th17 cells, induce the inflammatory pathology originally believed to be mediated by Th1 cells and IL-12 that require the IL-12 family member IL-23 as a growth factor 199 . Furthermore, new advances in uncovering this complex mucosal immunology has revealed that in various settings of mucosal inflammation, Th1, Th2, and Th17 cells have pro- or anti-inflammatory properties 200,201 . Recent data also suggests that neutrophils thought to be the final effector cell in acute inflammatory response also have the ability to crosstalk in chronic mucosal inflammation 202 . Thus altered immune responses in both innate and adaptive immunity are considered to be key elements related to the pathogenesis of IBD.

Hypothesized Pathogenesis in IBD

IBD pathogenesis studies involve multiple basic scientific disciplines, population and social studies, psychology, genetics, microbiology, immunology, biochemistry, cellular and molecular biology, drug discovery, and DNA engineering. What we currently understand is that both CD and UC are the result of the combined effects of four basic components: environmental factors, the input of multiple genetic variations, alterations in the intestinal microbiota, and aberrations of innate and adaptive immune responses. It has become clear that none of these four components can, by itself, trigger or maintain intestinal inflammation but that the combination of these various factors is needed to bring about CD or UC in individual patients. Unfortunately there is no smoking gun in IBD pathogenesis and of substantial proof is that the concordance rate in monozygotic twins is 10-15% in UC and 30-35% in CD 203 . Moreover genetic factors shown to have high penetrance in IBD including NOD2, IL-23R and ATG16L1 do not account for the striking rise in the incidence of CD and UC seen in the 20th century implicating the strong

influence of environmental factors 204 . Because patients have various combinations of these

h i ¢ ¤ disease susceptibility factors it creates diverse mechanisms of pathogenesis, clinical course, and treatment response. As the awareness of individual variability increases, so does the observation that we face the perplexity of elucidating these already complex mechanisms involved in chronic intestinal inflammation within the setting of each patient's individual(environmental, genetic, inflammatory and gut microbial) variability. So as we move forward clinicians and researchers have to reformulate study designs that incorporate and control for these complex factors with the hope of a more tailored approach for medical therapy. There are several hypotheses for the pathogenesis of IBD which are still evolving.

Hygiene Hypothesis. The increased incidence of IBD and other autoimmune diseases parallels globalization and is thought to be the result of better hygiene. Improved hygienic conditions deplete organisms and their antigens that are crucial in developing normal levels of

immunoregulation. The first to recognize this environmental effect was Charles Harrison

o | n t à ú ù á p q Blackely s when he noticed that aristocrats and city-dwellers were more likely to get hay fever than were farmers 205 . Microorganisms are thought to be necessary for programming the immune system particularly in the gut and damping future inflammatory responses by

helping to establish the immunological balance between pro-inflammatory T-cell subsets and

s q s q l t t l l t | m u q | n t n Û s t o t n Û Ó m | n t q s q Ú u o s q

206 n Ø tolerance-inducing regulatory T cells . î centered on the belief that a child overprotected from exposure to common environmental infectious agents then becomes infected with the pathogenic agents later in life (delayed exposure) and induces an inappropriate immunologic response leading to the development of an abnormal or ineffective inflammatory process 207,208 . A literature review conducted by Koloski et al. found that helminthic infection, Helicobacter pylori exposure, antibiotic use, breastfeeding

and sibship represented factors potentially associated with development of IBD supporting the

h i ¢ Ä hygiene hypothesis in IBD 209 . Furthermore, helminth Trichuris suis ova therapy in patients with

active CD and active UC has shown improved patient response and remission rates with no

adverse side effects in just 12 weeks of therapy 210,211 .

Environmental Risk Factors . Environmental risk factors are highlighted by the fact that

twins studies do not show a 100% concordance rate and that increased IBD prevalence in

immigrant populations from low IBD risk areas have a similar or higher risk of developing IBD

when compared to the native population 212-214 . Recent review of studies investigating the impact

of environmental factors in familial IBD reported that the increased frequency of smoking habit,

appendectomy, fecal-oral transmitted infections, eating habits, and pets were associated with the

development of IBD 215 . The most well characterized environmental risk factors involve the

u o s ¡ u z u o { t t | Ö t t o n t u z | n Û Ú u o

Ò Ø

breakdown of intestinal barrier function, dysbiosis ( Ò

o n t u z | n Û Ú u { | t l s u w u o m l | n t Ò Ø presence of a pathogenic agent.

The intestinal Microbiota. The intestinal Microbiota in patients with IBD reveals a

dysbiosis selection toward mucosal associated bacteria and reduced commensal species diversity

compared to healthy controls 216-219,220 . Reduced bacteria with anti-inflammatory properties

and/or more bacteria with proinflammatory properties are found in IBD patients compared to

healthy controls 216 . Experimental models of colitis have shown that the intestinal microbiota is a

major contributor to chronic inflammation and antibiotic treatment or housing in germ free

conditions suppresses the colitis phenotype. In humans, fecal stream diversion resolves

downstream inflammation in CD but reappears in postoperative exposure 136,221 . Several studies

have demonstrated increased risk of developing IBD following gastroenteritis 222,223 . In a Danish,

population-based cohort, long-term follow-up study showed that gastroenteritis caused by

nontyphoid Salmonella or thermophilic Campylobacter increased the risk of developing IBD 222 .

h i ¢ × q In CD patient p colonization with a pathogenic adherent-invasive Escherichia coli (AIEC) is

increased compared to controls and AIEC adheres to the brush border of primary ileal

enterocytes isolated from CD patients but not from individuals without IBD 224-226 . AIEC

s o t { | t ¡ u { l m Ó n u t q l t z t u q t z u l t u ¡ m o | q m è ç

î and CD patient AIEC infected

macrophages have been shown to induce granulomas in vitro that can recruit lymphocytes 227,228 .

Many other infectious organisms have been suggest to be involved in IBD pathogenesis such as

mycobacteria paratuberculosis, Listeria, Pseudomonas fluorescens, Bacteroides vulgatus but

warrant further investigation.

Smoking. Smoking is an indisputable environmental factor in IBD. Smoking contributes

to initiation of CD but has a protective effect in the development of UC and passive smoking has

also been shown to be a novel risk factor in both CD and UC 229 . In addition to the impact on

disease susceptibility, smoking habit also modifies the disease clinical course increasing the risk

of experiencing a relapse and the need for surgery 230-232 . Smokers have luminal microbiota that

consist of significantly higher bacteroides and investigation of whether this is a mechanism for

the negative effects of smoking on CD is necessary 233 .

Nonsteroidal anti-inflammatory drugs (NSAID)s . NSAIDs are analgesic, anti-

inflammatory drugs readily available over the counter. NSAID acute and chronic ingestion

promotes altered intestinal barrier dysfunction by inhibition of protective prostaglandins 234 . The

link between NSAIDs use and IBD activity is strong, however confounding factors make it

difficult to establish cause and effect 235 .

Genetic Risk Factors with a focus on STAT3 . Genome-wide association studies

examine common genetic variants in different individuals to see if any variant is associated with

a trait. These variants are single-nucleotide polymorphisms (SNPs) and studies compare

h i ¢ Ü millions of genetic variants using SNP arrays to DNA from patients with disease (cases) and similar people without the disease (controls). If one type of the variant (one allele) is more frequent in people with the disease, the SNP is thus "associated" with the disease and considered to mark a region of the human genome that influences the risk of disease. Results are presented as allele frequency with the fundamental unit for reporting effect sizes being the odds ratio 236 .

The odds ratio is the proportion of individuals in the case group having a specific allele, and the proportions of individuals in the control group having the same allele. When the allele frequency in the case group is much higher than in the control group, the odds ratio will be higher than 1 236 .

GWAS has identified 99 non-overlapping genetic risk loci, including 28 that are shared by CD and UC 237 . Loci implicated in IBD reveal many pathways important in intestinal homeostasis and barrier function, epithelial restitution, autophagy, innate and adaptive immune regulation, reactive oxygen species (ROS) generation, and endoplasmic reticulum (ER) stress(Figure 11) 237 . Interestingly, loci in multiple genes that involve STAT3 signaling are associated with susceptibility to CD as well as UC. These include TYK2, JAK2, STAT3, interleukin-10(IL-10), interleukin-23(IL-23), interleukin-23 receptor(IL-23R), interleukin-12 B precursor(IL-12B), and interleukin 12 receptor (IL-12R)238-244 . The tyrosine kinases TYK2 and

JAK2 directly phosphorylate STAT3 in response to both the IL-6 and IL-10 family of cytokines.

IL-10 is an anti-inflammatory cytokine that directly activated STAT3 and is involved in regulatory T cell generation and maintenance. IL-23 and the IL23R induce STAT3 activation and are involved in Th17 cell generation whereas IL-12B and the IL-12R help mediate Th1 cell lineage commitment. The association of these loci with IBD implicate that STAT3 plays a central role in the pathogenesis of IBD and functions as a key conductor of innate and adaptive

inflammatory responses.

h i £ Ý Figure 11. A model for IBD Pathways based on GWAS. Intestinal homeostasis involves the coordinated actions of epithelial, innate and adaptive immune cells. Innate immu ne system and the epithelial barrier orchestrate appropriate tolerogenic, inflammatory and restitutive responses. SNPs within STAT3 itself, IL -10, and the IL/23/Th17 pathway all utilize STAT3 as a major effector molecule and are associated with increased risk for IBD (Red Box). Genes in linkage disequilibrium (r2 > 0.8) with IBD -associated single nucleotide polymorphisms (SNPs) were manually curated and

classified according to their function(s) in the context of intestinal homeostasis and immunity. Text color indicates

ý ¢ ý ÿ ¢ ý ý ¡ ý © ¨ ÿ ý ¡ ¡ ý £ ¢ ¥ ÿ © ¡ ¥ ¨ © © ¥ ¨ ¢ ý £ ¢ ÿ ¥ ¡ © £ © ý ¨ © ý ¢ £ ¤ ¨ ¡ ¥ ¦ ý ÿ ¨ ¢ ý ¥ ¢ © ¢ § £ ¤ ¦ ý ¥ ¥ ÿ ¤ ¥ ¢

(red). Asterisk denotes corresponding coding mutations; cis -eQTL effects are underlined. G, goblet cell; P, Paneth cell. Reprinted with permission from Macmillan Publishers Ltd: Nature. Vol 474, Kohr et al. Genetics and pathogenesis of inflammatory bowel disease. 307-317, Copyright (2011).

Functional studies to determine causation have focused on coding variants however, non- coding SNPs can also modify gene expression through alternative splicing, transcription factor binding, locus accessibility, translational efficiency, and trans -regulators such as non-coding

RNAs and micro RNAs 237 . The rare IL10RA polymorphisms were identified by fine mapping

and are associated with the loss of IL-10 signaling and development of early-onset IBD 245 .

h i £ h Recent data has shown that the IL23R R381Q gene variant protects against immune-mediated

diseases by impairing IL-23-induced Th17 effector response 246 . Additionally, the functional

Ú l s q Ù u z z t z t s q

consequence of the non-coding STAT3 rs744166 Ø investigated in a cohort of pediatric CD patients and EBV-transformed lymphocytes in Chapter 2.

Signal Transducer Activator of Transcription-3 (STAT3) in IBD

Cytokine responses are involved in the pathogenesis more specifically the initiation, dysregulation, perpetuation and resolution of the immune response in IBD 191,247 . Current research has focused on the role of STAT mediated cytokine response in IBD. Among STATs, the role of STAT3 is the most well documented in human IBD studies and experimental IBD models 248-257 . Once in the nucleus, STAT3 induces gene expression of molecules with diverse biological functions including cell growth, survival and apoptosis, cell motility, inflammation, and negative feedback 254,257-261 . Therefore, STAT3 activation is highly context-dependent with

distinct roles in innate and acquired immune cells that differ in acute and chronic inflammation q

and ultimately dependent on STAT3 p mode of action.

¨ © ª © « ¬

Mode of Action. The JAK ã STAT paradigm is employed by most cytokine receptors to transmit chemical signals outside the cell, through the cell membrane, and into gene promoters on the DNA to induce rapid gene expression 262 . The mammalian Janus Kinase family, are intracellular, non-receptor tyrosine kinases, that transduce cytokine-mediated signals and have four members: JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2). There are seven mammalian STAT family members identified: STAT1, STAT2, STAT3, STAT4, STAT5

(STAT5A and STAT5B), and STAT6 that function as latent cytoplasmic transcription factors

and mediate cytokine and growth factor signaling pathways 263 . The STAT3 protein similar to

h i £ j other STATs has a conserved amino-terminus for tetramerization, a DNA binding domain with a sequence specificity for a palindromic IFN-é -activated sequence (GAS) element analogous to

STAT1, an SH2 domain involved in receptor recruitment along with STAT protein dimerization, and a carboxy-terminal trans-activation domain (Figure 12) 264,265 . Cytoplasmic membrane receptor tails not only serves as a docking sites for JAKs but certain residues have been identified to be crucial for JAK activation 266 . Ligand binding at the cell surface induces receptor chain dimerization activating receptor associated JAKs that in turn phosphorylate STATs at a single conserved carboxy-terminally located tyrosine residue 267 . This induces reciprocal phospho-tyrosine SH2 domain interactions allowing STATs to form stable homo and heterodimers that traverse the nuclear pore and bind GAS elements in conjunction with other

Figure 12. The structural domains of the STAT3 protein . The STAT protein contains five structural domains. The amino -terminal domain (N-term) of STATs mediates the formation of higher-order structures when two STAT dimers bind neigh boring STAT-binding sites on the DNA. The coiled-coil domain is involved in the receptor binding but also the nuclear translocation of STATs. The DNA -binding domain with the

consensus core sequence TT(N4 ¥ 6)AA enable binding to STAT response elements promot er region of STAT target genes. The SRC -homology 2 (SH2) domain enables docking of STATs to the receptor or the formation of dimers via binding to phosphorylated tyrosine residues (pY). The transactivation domain is involved in recruitment and binding of transcription coregulators, such as histone acetyltransferases. Some STATs contain serine phosphorylation (pS) residue in the carboxy -terminal domain (C-term) that enhances transcriptional activity. Reprinted from Inflamm Bowel Dis. Li et al. New Insights into the role of STAT3 in IBD,1-7, Copyright (2011) with permission from John Wiley and Sons. cofactors to induce transcriptional regulation of target genes 268 . The most prominent mechanism utilized to negatively regulative JAK/STAT signaling is the suppressor of cytokine signaling

(SOCS) proteins. The SOCS family members modulate signaling by several mechanisms shown to include binding and inactivation of JAKs, blocking access of STATs to receptor binding sites, and ubiquitination of signaling proteins and their subsequent targeting to the proteasome 269 .

Different STAT proteins show preferred specificity for individual cytokine families.

® ¯ ° ± STAT3 is the most pleiotropic member activated by a variety of cytokines and certain growth factors, including the interleukin-6 (IL-6) and Interleukin-10 (IL-10) cytokine family, and epithelial growth factor (EGF) 270 . In human and murine intestinal inflammation the most important STAT3 regulators are IL-6 considered a pro-inflammatory cytokine and IL-10 an anti- inflammatory cytokine that both activate STAT3 by different mechanisms with different gene expression profiles 271 .

The IL-6-STAT3 Signaling Pathway. The IL-6 family of cytokines (IL-6, IL-11, IL-27,

IL-31, leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic factor (CNTF), and

ø á l t { t Ó | m l CT-1 are characterized by the use of the IL-6 signal transducing subunit, gp à

(GP130). IL-6 and IL-11 are restricted to GP130 homodimerisation whereas IL-27, IL-31, LIF,

OncostatinM, CNTF, and CT-1 induce heterodimeric GP130 receptor complexes 272 . IL-6

q o s | ç

binding the IL-6 receptor- Ò (CD126) triggers engagement and dimerization of GP130

n t l t { t Ó | m l u q q m { s u | t ³ u Ù p q 273 ó that activates receptor associated JAK1, JAK2 and Tyk2 î mediate subsequent tyrosine phosphorylation of the GP130 receptor allowing docking sites for

STAT3 SH2 domains to create the close proximity needed for JAK1/2 to phosphorylate STAT3

274-276 . STAT3 activation seems to be induced by JAK2 and to a lesser extent JAK1 in response to IL-6 and in human colorectal cancer cell lines JAK2 and to a lesser extent JAK1 but not

JAK3 induced STAT3 phosphorylation and subsequent activation involved in survival, cell cycle, and tumor cell invasion 277,278 . JAK mediated tyrosine phosphorylation at a single site

(Y705) in the carboxy-terminus of STAT3 is required for dimerization, nuclear translocation, and DNA binding 279 . STAT3 can also be activated by serine phosphorylation at a site within the transactivation domain (S727) thought to be involved in maximal transcriptional activity by

enhanced recruitment of necessary transcriptional cofactors 280 . Cancer associated cytosolic and

® ¯ ° ² Figure 13. Regulation of intracellular STAT3 signaling Stat3 signaling is induced by various kinases (green) in a phosphorylation dependent manner (green arrows), and counteracted by several regulatory proteins (red).

Stat3 activation occurs in response to gp130 hetero or homodimerization following binding IL 6 type cytokines

¤ ¦ ý ¥ ¢ ¥ ¢ ý ÿ © þ ý ¤ ¢ ÿ ¨ ¡ © ¦ ý ¦ ¤ ÿ ¨ ¡ ý ÿ ý ý þ ¢ ¥ ÿ ´ ¢ -subunits (white). Phosphorylation of four membrane distal tyrosine (Y, black) residues by constitutively associated JAK -family tyrosine kinases (TK) is sufficient to enable src -homology domain (SH)-2 mediated binding of STAT3 and, to a lesser extent, of STAT1. Once tyrosine phosphorylated, STAT3/1 form homo - and heterodimers, which translocate and transactivate target genes, including the negative regulator SOCS3. STAT3 can also be phosphorylated by certain cytosolic TKs and receptor TK. The serine threonine kinases (STK) mediate serine -phosphorylation that maximizes the transcriptional activity. Gp130 also engages the Ras/ERK pathway through binding of the tyrosine phosphatase SHP2 to the m embrane proximal phospho-Y (Y757 in mouse and Y759 in humans). This phosphor-tyrosine also provides the binding site for SOCS3 to mediate proteasomal degradation of ligand -occupied receptor complexes. Gp130 signaling is also attenuated by the Y -phosphatase activity of SHP2, while cytoplasmic PIAS3 protein

sequesters Y -phosphorylated STAT3 from homodimerization, nuclear translocation and target gene activation.

¥ ÿ ý ¢ ¨ ¡ ¨ µ ¦ ¢ ¶ ¤ ý ý ¡ ý Adapted from Jarnicki and Ernst. Stat3: linking inflammation to epithelial cancer - ¦ Division 2010 5:14, Copyright (2010) with permission from BioMed Central Ltd. receptor tyrosine kinases can also activate STAT3 281 . Elegant genetically engineered murine models have shown that the cytoplasmic tail of GP130 contains four tyrosine residues that are required and sufficient for activation of STAT3 and STAT1, with STAT3 having the predominant role in vitro and in vivo 273 . Additionally in the cytoplasmic tail of GP130 another

tyrosine residue (Y757 in mouse, Y759 in humans) is responsible for engagement of the

® ¯ ° ° cytoplasmic Src homology tyrosine phosphatase (SHP2) and subsequent activation of the Ras-

ERK pathway to transduce activities regulating mitogenesis (Figure 13)273 .

Negative Regulators of the IL-6 signal transducing subunit GP130. The Suppressor of cytokine signaling-3 (SOCS3), is a negative regulator of GP130 induced STAT3/STAT1 activation and functions as a classic negative feedback loop via direct transcription by STAT3 activation 282 . SOCS3 binds to the same phopho-tyrosine that enables the activation of SHP2

(Y757 in mouse and Y759 in humans), and inhibits further binding of STAT3 SH2 domains to the GP130 receptor and binds the receptor associated JAKs to restrain their activity 283,284 .

Binding of SOCS3 to activated GP130 has been shown to induce SOCS3 mediated JAK associated proteosomal degradation maintaining the transient nature of STAT3 285 . The tyrosine phophotase activity of SHP2 can also inhibit GP130 signaling and cytoplasmic protein inhibitor of activated STAT, 3 (PIAS) can sequester tyrosine-phosphorylated STAT3 from homodimerization, nuclear translocation and target gene activation 283,286 .

The IL-10-STAT3 Signaling Pathway. The IL-10 family of cytokines IL-19, IL-20, IL-

¹ º » º ¼ ½ ¾

22, IL-24, IL-26, IL-28 and IL-29 all share the common IL-10 receptor-¸ . In the case of

À Á Â the IL-10R, the IL- ¿ -chain has two defined docking sites for STAT3, whereas GP130

provides four docking sites and STAT3 activation via IL-10 mediates an anti-inflammatory

Ç ½ ¼

¾ 287,288

À Ã Ä Å Ä Æ Ã Â Å È É response (AIR) in cells expressing the IL- ¿ . . Interleukin-10 is a pleiotropic cytokine, whose main function is the limitation and ultimately termination of immune responses in IBD. The primary role of IL-10 shown in vitro and in vivo is to inhibit the powerful stimulatory effects of TLR agonists such as LPS that act on macrophages and dendritic cells 289 .

IL-10 acts as a key mediator for maintaining gut homeostasis and mice lacking IL-10 are

characterized by excessive cytokine release and development of enterocolitis 290 . The IL-10

® ¯ ° · Figure 14 Negative Regulatory Mechanisms in IL -6-type Cytokine Signaling. SHP2 is a ubiquitously expressed cytoplasmic PTP, all tyrosine -phosphorylated signaling components are potential substrates for the SHP2 phosphatase. PIAS3 specifically inhibits STAT3 -mediated gene expression (after IL-6 stimulation). SOCS3 is functional ly most related and inhibits IL-6-type cytokine signaling by acting on JAKs and thereby inhibiting the phosphorylation of gp130, STATs and the JAKs themselves. The balance of protein synthesis and degradation also affects IL -6- type cytokine signal transduction and the half-lives of the various players involved in IL -6 signaling differ substantially. The feedback inhibitor SOCS3 is very short-lived, STAT3 and SHP2 have slow turnover rates while Janus kinases JAK1, JAK2, TYK2 and gp130 show intermediate ha lf-lives1. (Representation of the negative regulators and their sites of action (red arrows). Reprinted with permission from Biochem. J. 374, Heinrich et al. Principles of interleukin (IL)-6-type cytokine signalling and its regulation, 1 -20, Copyright (2003) the Biochemical Society. family receptor subunits lack SOCS3 binding sites, thus IL-10 receptor engagement results in sustained STAT3 activation due to the loss of SOCS3 inhibition, this mediates the difference in effector properties of IL-6 versus IL-10 induced STAT3 activation (Figure 15)271 . Importantly, heterologous cytokine receptor systems that activate STAT3 but are naturally refractory such as the IL-22R, or an engineered IL-6R to be refractory to SOCS3 mediated inhibition activate an

AIR indistinguishable from IL-10 291 . Thus transient STAT3 activation by IL-6 in wild-type

® ¯ ° Ê macrophages promotes inflammatory responses. However, macrophages lacking the SOCS3 gene or carrying a mutation in the SOCS3-binding site of GP130 have both IL-10 and IL-6 suppression of lipopolysaccharide-induced tumor necrosis factor (TNF) and IL-12 292 . In vitro,

SOCS3-deficient CD4+ T cells produced more transforming growth factor (TGF)-beta1 and interleukin (IL)-10 and chromatin immunoprecipitation assay revealed that more STAT3 was recruited to the TGF-beta1 promoter in SOCS3-deficient T cells than in control T cells 293 .

SOCS3 also has a significant role in constraining the generation of Th17 cells by attenuating the phosphorylation of STAT3 and IL-23 induced Stat3 phosphorylation is enhanced in the absence of SOSC3294 . However the AIR response induced by IL-10-STAT3 is still not completely

understood. The AIR is selective, targeting subsets of inflammatory genes accounting for 10 Ì

of the genes induced by TLR signaling after LPS stimulation. IL-10 mediated STAT3׽ 15%

activation induces the transcriptional repressor, ETV3 and a transcriptional corepressor, Î Strawberry notch homologue 2 (SBNO2) which has strong repressive activity for NF-Í 295 .

Therefore, perhaps unrecognized or undetectable additional signals other that the IL-10R can potentially act in conjunction with an IL-10-STAT3 response in an anti-inflammatory manner to

regulate inflammation. This could explain divergent effects of STAT3 activation in effector T- ¸ cell function as STAT3 via IL-6 and IL-23, in combination with factors like IL-1¸ , TGF- , and

296

RoR Ï t, induce T helper 17 (Th17) formation . Whereas IL-2-induced STAT5 activation is required for the development of FOXP3 + regulatory T cells (Tregs) and STAT3 activation by IL-

10 is important for the function of Tregs such as their ability to suppress pathogenic Th17 responses 297,298 .

® ¯ ° Ë Figure 15. Strength and duration of STAT3 activation determines cellular responses. STAT3 activation patterns and associated outcomes differ between IL6 and IL10 family cytokine -induced signaling. In response to binding of ligand (blue), signaling from gp130 is transient, because the phosphorylated, membrane proximal tyrosine residue (Y, red) provides a binding site for the negative regulator SOCS3. Although SOCS3 is also induced in response to activation of the IL10 receptor family, STAT3 signaling remains sustained, because the IL10 receptor chains lack the corresponding YxxV motif. Similarly, signaling from the mutant gp130 Y757F receptor is sustained due to phenylalanine (F) substitution of the Y757 residue (Y759 in human GP130), resulting in exaggerated activation of its down -stream signaling molecules. The range of target genes activated differs between acute and sustained STAT3 activation, most evident in macrophages where the former promotes

and the latter suppresses in flammatory response. Adapted from Jarnicki and Ernst. Stat3: linking inflammation

¦ ¥ ÿ ý ¢ ¨ ¡ ¨ µ ¦ ¢ ¶ ¤ ý ý ¡ ý © ¥ ¡ Ñ Ò Ó Ô Ò Õ ¥ þ ÿ ¢ ¢ Ñ Ò ¥ ¢ þ ý ÿ ¦ © © ¥ ¡ ¤ ÿ ¥ ¦ to epithelial cancer -

BioMed Central Ltd.

¯ ° Ð ® STAT3 in Human IBD

Many studies have shown that compared to other STAT proteins, STAT3 is heavily involved in the pathogenesis of IBD. In IBD there have been no known activating mutations in the STAT3 gene but it is well known that the pro-inflammatory overproduction of IL-6 is the main mediator of constitutive STAT3 activation while the regulatory and anti-inflammatory action of IL-10 induced STAT3 activation is lost 271 . IL-6 is believed to regulate mucosal inflammation in large part via tyrosine phosphorylation of STAT3 in effector lymphocytes and granulocytes. Some of the first studies to better highlight STAT3 activation in mucosal inflammation were by Suzuki et al. who detected phosphorylation of STAT3 but not STAT1,

STAT5, or STAT6 in whole colonic tissue extracts from DSS treated mice and mucosal biopsies

of UC and CD patients 253 . These studies also found STAT3 activation in all colitis models

¼ Ç Ç º ¾

¼ +

× Ä Ä Ø Å Ø examined including DSS treatment, TCR-Â , CD4 T cell transfer, IL-10 knockout, macrophage- and neutrophil specific STAT3 conditional knockout as well as TNBS treated 253 . However, these studies were done from whole colonic extracts whereas Lovato et al. reported constitutive activation of STAT3 and SOCS3 in colonic mucosal CD3 + and CD4 + T cells of patients with CD but not healthy controls 248 . One criticism of these experiments was the use of intestinal and peripheral T-cell lines grown with high doses of IL-2 before assessing

STAT3 activation. Musso et al. went on to demonstrate that activated STAT3 was detected by immunobloting of freshly isolated lamina propria mononuclear cells from inflamed IBD tissues, but not in peripheral blood mononuclear cells from control subjects or IBD patients 249 . They also demonstrated that activation of STAT3 is confined to actively inflamed areas of the mucosa in IBD, where phosphorylated STAT3 is found exclusively in infiltrating CD3 + T lymphocytes

and CD68 + macrophages but not neutrophils 249 . However more recent studies have suggested

® ¯ · Ö that STAT3 is also activated in peripheral and lamina propria neutrophils 299 . Mudter et al. reported an increased amount of total STAT3 protein in both UC and CD compared to non- inflammatory control cells, and total STAT3 correlated with increased STAT3 activation in tissue sections from both UC and CD 250 . These studies also revealed that the increased frequency of pSTAT3+ cells directly correlated with the histological degree of inflammation and that activated T lymphocytes and innate colonic epithelial cells demonstrated STAT3 activation 250 .

Neurath et al. evaluated cytokine production of stimulated T cell and macrophage enriched lamina propria cells in patients with CD compared to control. Using ELISA they described an upregulation of IL-6 compared to IL-4, IL-5, IL-10 and IL-11 251 . Furthermore, lamina propria T cells in CD patients activated by IL-6 induced STAT3 dependent induction of anti-apoptotic genes such as bcl-2 and bcl-xl 251 . Mitsuyama et al. established that colonic mucosa from patients with active disease contains larger amounts of IL-6 than colonic mucosa from patients with

inactive disease or normal controls 300 . RT-PCR analysis of mucosal specimens showed that of

¸ Ù Ú Û Ü ¼ Ý

Â É the inflammatory cytokines IL- ¿ IL-6, IL-6 mRNA levels were the highest in active

IBD patients 301 . Furthermore, spontaneous production of IL-6 by peripheral blood and lamina propria mononuclear cells was increased in patients with IBD 302-304 . These were the first studies to propose that IL-6 mediated STAT3 could foster T-cell survival and aggravate chronic colitis.

These above mentioned studies have all involved adult patients receiving immunomodulatory therapies with longstanding chronic disease. Brown et al. revealed that in intestinal biopsies from newly diagnosed untreated pediatric IBD patients the level of IL-6 secretion significantly correlated with the graded severity of histological inflammation 305 . Carey et al. was the first to identify a pro-inflammatory IL-6:STAT3 biological network that drives

mucosal inflammation in pediatric onset CD both at diagnosis and during therapy 252 . Serum IL-6

® ¯ · ® was significantly increased in CD and UC patients at diagnosis and treated CD patients with active disease. In the peripheral blood, CD3+CD4+ lymphocytes were hyper-responsiveness to

IL-6 stimulation and the basal frequency of pSTAT3-positive granulocytes was elevated in IBD patients compared to healthy controls 252 . In colonic biopsies, the frequency of total pSTAT3- positive cells (lamina propria and epithelial) correlated with overall mucosal inflammation and increased epithelial injury and the frequency of pSTAT3+ peripheral blood granulocytes correlated with the severity of mucosal inflammation 252 . In addition, Carey et al. identified biologically relevant networks present where IL-6 and STAT3 functioned as central regulatory nodes with genes involved in immune and inflammatory mediators or chemokines, cytokines, and growth factors. Recent studies indicate that serum IL-6 has excellent diagnostic accuracy for detecting patients that have active disease as evaluated by CDEIS 306 . These studies have been seminal in developing a role for STAT3 activation more specifically IL-6 induced STAT3 activation within the intestinal mucosa of pediatric-onset and adult-onset IBD. Importantly, this activation is observed in both acquired and innate immune cells such as T-lymphocytes and the intestinal epithelial cells. Additionally, in a novel murine model of CD, the SAMP/YIT mouse,

IL-6 induced STAT3 activation contributes to the development of this spontaneous chronic ileitis and colitis model 307,308 . These data implicate that IL-6:STAT3 signaling mediates pro- inflammatory gene expression within the mucosa of IBD patients.

IBD Patients with True Acute Colitis? To understand the function of STAT3 during intestinal inflammation, it is helpful to divide inflammation into three distinct stages (1) acute involving the innate immune response, (2) chronic, involving the adaptive immune response and

(3) recovery or wound healing and resolution of the adaptive immune response also referred to as

clinical remission in human subjects. Sophisticated tissue specific conditional transgenic and

® ¯ · Þ gene deletion murine models have allowed the investigation of STAT3 in innate and adaptive colitis. However, murine models are often difficult to interpret in comparison to human studies especially when a patient diagnosed with IBD may have the disease for several months and is ultimately in the chronic phase. Therefore in human studies the acute phase and timing of inflammation is hard to decipher and study design may involve dichotomized patient populations of newly diagnosed untreated versus treated and patients in remission. The only true acute IBD lesion is the postoperative recurrence model of CD where an ileocolic resection with anastomosis to remove diseased bowel induces an immediate disease remission. However even with diseased bowel removed the endoscopic recurrence of disease arises in the neoterminal ileum in 30% of

patients after 3 months and in up to 80% of patients after 1 year 309 . In these patients that

¼ Ý Ú Û Ü ¹ ¸ Ù ß à

É Â â Ä ã Ä â subsequently developed recurrence, ileal mucosal IL- ¿ - at the time of

remission postoperatively were significantly higher than in the group who remained in remission

Ù Ú Û Ü Ù Ç ¼ º ¼ ½ ½ ¾ ¼ ¹ ½ ¹ ¸ Ù ß à

á 310

Æ ä À å À À è ç Â Æ ä À å À è é ã É Ã É Ä É É â ê Æ ä À å À æ ç at 1 year (IL- ¿ - . In the same study a multivariate analysis revealed that only elevated mucosal IL-6 levels were shown to be an independent risk factor for postoperative disease recurrence. In this same model, patients with low IL-10 mRNA levels in ileal biopsies preoperatively were found at 3 months to have higher rates of endoscopic recurrence than those with high IL-10 mRNA levels (80% vs 40%, p=0.02) 311 . These data highlight the pro-inflammatory role of mucosal IL-6 along with a deficiency in mucosal IL-10 in disease initiation or acute colitis. Another important factor shown to be involved in disease initiation is the bacterial flora and/or other components of the faecal stream. Interestingly, postoperative recurrence patients with diversion of faecal stream proximal to an ileocolic anastomosis had no endoscopic evidence of recurrence in the

neoterminal ileum 6 months after surgery however, after restoring bowel continuity, essentially

® ¯ · ± all patients had rapid endoscopic recurrence in the neoterminal ileum 221 . The aforementioned studies point out the importance of mucosal IL-6 and IL-10 as well as the bacterial flora and/or other components of the fecal stream in initiation of IBD. Moreover, the postoperative recurrence model is the closest human model to deduce initiating events in IBD.

STAT3 in Innate and Adaptive Immunity, One Cell at a Time

The innate immune response is the first line of defense against invading pathogens. The innate immune system consists of physical and chemical barriers, cells and molecules that recognize pathogen-associated molecular patterns (PAMPs), and effector systems that facilitate destruction of pathogens. PAMPs are recognized by pattern-recognition receptors, such as Toll- like receptors (TLRs) on the cell surface and intracellular Nod-like receptors (NOD) of innate immune cells 312 . Dysfunction in the innate immune system and/or inappropriate adaptive immune response can mediate chronic intestinal inflammation 313 . IBD is more associated with a defective adaptive immune system as inflamed mucosa from CD and UC patients consistently shows the presence of infiltrating activated CD4+ T lymphocytes. Abnormalities in regulatory pathways ultimately control both the innate and the adaptive response whereas dendritic cells

(DCs) and various cytokines, important in the innate immune system determine the type of adaptive T cell response that predominates during the inflammatory process 314 .

The gut associated lymphoid tissue (GALT), is the largest reservoir of lymphoid tissue in the body separated by a single layer of epithelial cells and mucus from an immense amount of luminal bacterial (more than 1000 species) and dietary antigens 315 . Luminal bacteria aid in digestion of food, protection against pathogens, and help maintain immune system conditioning.

The immunologic tone within the GALT is regulated by unique mechanisms that involve

® ¯ · ² development and maintenance of Preyer ë s patches, isolated lymphoid follicles and connection to

the mesenteric lymph node and spleen. A key cell type within the GALT that regulates tolerance

and antigen presentation is the dendritic cell (DC).

Dendritic Cells. DCs have a unique function in sensing and then reacting to the gut

microenvironment. DCs shape the immune response by responding with tolerance to normal

commensal microbiota and luminal antigens or generate protective immune responses against

pathogens without causing pathology 316 . DCs directly sense the luminal content, extending

dendrites between epithelial cells, and recognize luminal antigens in the lamina propria through

apoptosis of epithelial cells or transported by specialized epithelial cells present in the follicles of

¹ ¾ ¹ Ù

ì 317

ê Ä Ã ë Æ É Å È Ä Ä microfold (M) cells . DCs migrate from the lamina propria to the draining

mesenteric lymph nodes (MLNs) constitutively delivering antigens to present to B and T cells

initiating either a tolerogenic or an inflammatory response 318 319 . In the periphery regulatory T

cells (Tregs) induced by DCs can suppress the function of effector T cells diverting naïve T cells

that are reactive to inoffensive(self) antigen 316 . DCs also mediate the protective immune é response involved in effector cell induction and determine whether a T helper (Th) 1 (IFN-Ï ,

Th2(IL-4), or Th17(IL-17) response will prevail while the inflammatory milieu, containing

cytokines such as IFN-Is, TNF, IL-6, IL-12, and IL-23, may also regulate DC activity and direct

effector T cell differentiation 320 . Murine models of colitis have shown accumulation of activated

DCs throughout the lamina propria and MLNs 247 . Targeted deletion of the Stat3 gene with a

DC-specific CD11c-cre (Stat3 CKO) impairs weight gain and mice develop ileocolitis that is

associated with impaired mucosal tolerance 321 . Moreover, Stat3 CKO DCs develop normally,

but are resistant to IL-10 suppression and exhibit both an enhanced ability to secrete

inflammatory cytokines and stimulate T cells. These experiments emphasize an important role

® ¯ · ° for Stat3 in antagonizing DC activity and directly link DC hyperactivity with inflammatory

disease 321 .

Macrophages. Another innate immune cell type in the intestine is the resident

macrophage. Studies in human and animal models have shown a role for abnormal macrophage

immune responses to the intestinal microbiota in the pathogenesis of IBD 322 . Resident

macrophages vary greatly compared to newly recruited inflammatory monocytes /macrophages

during an infection. Resident macrophages reside underneath the epithelium in the lamina

propria, waiting to phagocytose luminal microbes that breach the epithelial barrier 323 . These

macrophages are efficient at eliminating bacteria within the lamina propria but are not

professional antigen-presenting cells and have low expression of surface costimulatory

molecules, and do not initiate and immune response 323,324 . Resident macrophages maintain

homeostasis in the intestine by clearing apoptotic cells and debris, promoting epithelial repair

and producing IL ̻10 that is shown to maintain expression of FOXP3 in colonic Tregs 325 .

Resident macrophage function is determined by cytokines in the microenvironment such as TGF-

Ù ß à ¸ -10, and IL-6 and they mediate tolerance by inducing differentiation or inhibition of T cells

and influence the adaptive immune response 326 . In the midst of tissue injury or infection newly

recruited macrophages exhibit an inflammatory phenotype and secrete pro-inflammatory

Ú Û Ü

mediators such as Â , nitric oxide (NO), and IL-1 that are involved in the activation of

various microbial defense mechanisms 327 . In patients with IBD, macrophages are increased in

the inflamed mucosa and are functionally different than resident macrophages in that they can

mount a robust immune response to luminal microbes and express T costimulatory molecules

(CD40, CD80, and CD86), PAMP receptors (TLR2, TLR4, CD89, TREM1, and CD14), and

í Ú Û Ü Ç 328,329

become an important source Â . Deletion of STAT3 in the myeloid cell linage

® ¯ · · induces spontaneous colitis due to impaired IL-10 signaling in macrophages and neutrophils 259 .

Similarly, mice lacking expression of IL-10 spontaneously develop colitis, which is a consequence of macrophage differentiation into pro-inflammatory subsets that produce large amounts of IL-12 and IL-23 and depletion of macrophages in IL-10 KO mice prevents development of colitis 322 . STAT3-deficient macrophages also demonstrated enhanced CD8 T cell cross presentation with antigen 330 . Thus IL-10 induced STAT3 activation in the myeloid cell linage helps suppress a pro-inflammatory phenotype and maintains gut homeostasis.

Neutrophils. Neutrophils were regarded as short-lived innate immune effector cells that engulf and kill invading pathogens however new evidence suggests that neutrophils also play a part in effector and regulatory circuits in both the innate and adaptive immune systems 331 .

Neutrophils express a vast array of pattern recognition receptors (PPRs) including TLRs and

NLRs (Nod-like-receptors) and are characterized as phagocytic cells that release noxious enzymes from their granules and produce reactive oxygen intermediates (ROI) with antimicrobial potential 332 . Neutrophils also either spontaneously or following appropriate stimulation secrete multiple cytokines and chemokines that enhance phagocytosis, activate complement and regulate inflammation 333 . Other non-classical forms of neutrophil effector function include release of extracellular fibrillary networks termed neutrophil extracellular traps

(NETs) that snare microorganisms and soluble pattern recognition molecules (PRMs) with antibody-like properties 334 .

The production of neutrophils is extensive in steady state and the major activity in hematopoiesis is devoted to the production of monocytes and granulocytes (myelopoiesis).

Granulocyte colony-stimulating factor (G-CSF), promotes myeloid progenitor cell proliferation,

neutrophil differentiation, and neutrophil mobilization from the bone marrow into the circulating

® ¯ · Ê blood 335 . G-CSF binds the homodimeric G-CSF receptor (G-CSFR), which activates associated

Jak1 and Jak2 protein tyrosine kinases whereby experiments in vitro demonstrate that STAT3 is

strongly activated and to a lesser extent so are STAT1 and STAT5 336 . The STAT3 receptor

recruitment site within the G-CSF receptor is critical for controlling G-CSF-responsive

neutrophil production/mobilization and SOCS3 has been shown to be important in terminating

signals from the G-CSF receptor 336,337 . STAT3 controls macrophage inflammatory protein-2

MIP-2(CXCL2) responsive neutrophil mobilization from the bone marrow and STAT3-deficient

neutrophils have defective migration in response to CXCR2 ligands, the main receptor for ¸

338 ß

ï î Á æ ð â ñ Ã é

CXCL2 . G-CSF-stimulated î transcription occurs by direct binding of STAT3 to

ß ¸

¾ lo

Ä â ñ Ã È promoter in immature neutrophils (Gr-1 ) that enhances their responsiveness to

CXCL2(MIP-2) 338 . Furthermore, in mice deletion of STAT3 in the bone marrow causes death

¹ í ¾ » ½ ¾ ½ ¾ Ç ¼ ¹ Ý ½ ¹ ¹

á ò ò

Ä Ä Ø É Ä Ã Ã È È î Ã È ë Ä É Ä within 4 Ì -like pathogenesis in both the small and large

intestine inducing inflammatory cell infiltration, ulceration, bowel wall thickening, and

granuloma formation 339 . Interestingly these STAT3 deficient mice have neutrophilia with a

½ ¹ Ç í Ú Û Ü ¼ Ý ß Ü Û

× È â Ä ã Ä â Â É Ï

pseudoactivated innate immune responses with È (Th1-type

¼ Ý ½ ¼ ¹ Ý Û Ü ¾ ½ ½ ¾ í ¾ à

ì ó

Í Î

É Å Ã Ä É Ä Å ã ê É Ä Ã response), inhibition of the NAPDH oxidase activity, É

stimulation 339 . Therefore STAT3 has critical roles in the development, regulation and tolerance

of innate immunity, and deletion of STAT3 during hematopoiesis results in abnormalities in

º ¹ ¹ Ç ¼ ¹ Ý ½ ¹ ¹

É Ä î Ã È ë Ä É Ä myeloid cells that Å -like pathogenesis.

Once neutrophils leave the bone marrow they are recruited to sites of infection through

the help of endothelial cell adhesion molecules. Removal of circulating neutrophils from the

blood ameliorates inflammation in IBD patients suggesting that they play a major role in the

course of inflammation 340 . Microbes and resident macrophages generate signals as sites of

® ¯ · Ë infection that activate local endothelial cells to provide signals for neutrophil to gain access to tissues. Bypassing neutrophils are captured and guided across the endothelial cell lining where chemokines, cytokines and products generated by live microorganisms guide neutrophils toward the microbes 332 . Neutrophil cell surface expression of PSG-1(CD162), L-selectin(CD62 ligand),

ESL-1, and CD44 engages endothelial cell P-selectin/E-selectin to mediate rolling and activation

of the neutrophil integrins that interact with ICAM-1 and VCAM-1 expressed on endothelial ¸

341-343 à

Â æ é

cells . LFA-1ð mediates stop of rolling and firm adhesion together with Mac-

¸ í ¾

ò 344,345

ô æ é É Ä Ã

1(Â hich paracellular or transcellular migration occurs . Concurrently

¸ ½ ¹ ¾ ½ ¾ Ý » Ú Û Ü Ù ß à

â É â Å Ä â â É Å ã É Ä ê Â endothÄ -1 , and IL-17 produce IL-8 and MIP-2 that activates neutrophils 332 . IL-8 and the murine homologues KC(CXCL1) and MIP-2(CXCL2), are neutrophil chemoattractants that orchestrate neutrophil activation and recruitment from the blood into sites of infection, inflammation and injury by promoting endothelial adhesion and transmigration 346 . Their functional effects are mediated by binding CXCR1 and CXCR2 347 .

CXCR2 has proved to be a potent mediator of neutrophil recruitment in murine models of autoimmune diseases including IBD and increased mucosal expression of these chemokine receptors and their ligands in IBD may explain the massive influx of leucocytes in active disease 348-350 . Acute induction of human IL-8 production by the intestinal epithelium triggered neutrophil infiltration and in a model of dextran sulphate sodium (DSS) induced acute colitis there is a marked increase in KC(CXCL1) and MIP-2(CXCL3) within the colon 351-353 .

Furthermore, the up-regulation of IL-8 in the colonic mucosa of IBD patients correlates with the histological degree of inflammation and chemokine mRNA expression 354-357 . Interestingly, calprotectin (S100A8/S100A9) a neutrophilic cytosolic protein can be detected in stool and is

thought to be a valid biomarker for intestinal inflammation358 . In pediatric IBD, faecal

® ¯ · Ð calprotectin was a direct measure of intestinal inflammation and a good marker of the risk of histological relapse 359 . Therefore, targeting neutrophil influx is a potential therapeutic strategy

for IBD. Additionally, Chapter 2 of this PhD thesis reveals, that pediatric CD patients carrying

½ ¹

ö ÷ Ã Ø É â â Ä â Ä the STAT3 rs744166 õ have increased frequency of peripheral blood pSTAT3 positive leukocytes, increased STAT3 acitvation, increased colonic expression of IL-8, CXCL1,

CXCL3, calprotectin (S100A8/S100A9), S100A12, and increased frequency of colonic CXCR2 positive neutrophils 299 . Therefore, the rs744166 SNP may be a biomarker for a new cohort of pediatric CD patients in which blockade of CXCR2 would potentially be therapeutically relevant.

Chemokine-directed leukocyte trafficking can also be directed by IL-6. IL-6 stimulates myelopoiesis and causes accompanying changes in the numbers of peripheral neutrophils due to increased demargination of intravascular neutrophils 360 . IL-6 participates in neutrophil migration by inducing production of chemokines such as IL-8 and CCL2(MCP-1/Monocyte chemoattractant protein-1) and by inducing expression of adhesion molecules such as ICAM-1 on endothelial cells 361-363 . Moreover, increased mRNA levels in colonic mucosal biopsies and enhanced lamina propria mononuclear cell IL-6 production has been shown in both CD and

UC 304,364 . In CD patients circulating IL-6 was considered to correlate with the inflammatory activity and intestinal IL-6 production was highly correlated with the severity of endoscopic and histopathological signs of inflammation 304,365 . Thus, elevated serum IL-6 levels in CD patients with steroid induced remission were highly predictive for a following relapse 366,367 . Studies show that lamina propria T cells, macrophages (CD68+), and epithelial cells are the main cell types responsible for elevated intestinal IL-6 concentration 251,365 . Increased circulating levels

can be detected in both pediatric(early-onset) and adult IBD patients 252,300,305,368 . Studies show

® ¯ Ê Ö that the transition from acute to sustained or chronic response involves enhanced leukocyte activation, differentiation of T-cell subsets and T-cell proliferation mediated by STAT3 and controlled by a mechanism known as IL-6 trans-signaling 251,369 .

Neutrophils hold the key in transition from acute to chronic Inflammation. In the lamina propria, classical IL-6 signaling is restricted to neutrophils, monocytes/macrophages and

epithelial cells since they express the IL-6R Â (Rose-John et al, 2006). IL-6 trans-signaling is initiated by shedding of the IL-6R from the cell surface of the initial neutrophil infiltrate, resulting in soluble IL-6R (sIL-6R) (Figure 16). This sIL-6R has been shown to induce CXC and CC chemokine expression of IL-8(CXCL8), CCL2(MCP-1), CCL20(MCP-3) and in acute inflammation inhibit neutrophil recruitment while promoting the attraction of monocytes and lymphocytes 370 . This induces IL-6-s-IL-6R complexes that can signal via the GP130 receptor.

Gp130 is ubiquitously expressed on all cell types whereas the expression of the IL-6R is restricted to some lymphoid cells including monocytes, macrophages, neutrophils, B-cells, subpopulations of T-cells and non-lymphoid cells such as hepatocytes 371-373 . Atreya et al.

demonstrated that the IL-6Ì s-IL-6R system modulates T cell resistance against apoptosis and that

a pathway of T-cell activation driven by IL-6Ì sIL-6R strongly contributes to the perpetuation of chronic intestinal inflammation 251 . In CD patients, isolated lamina propria mononuclear cells and T-cells secreted elevated levels of IL-6 and sIL-6R that then form IL-6-sIL-6R complexes 251 .

These complexes then stimulate gp130 on the surface of lamina propria T cells in the gut, causing a STAT3-dependent up regulation of intestinal T-cell resistance to apoptosis through induction of the anti-apoptotic genes Bcl-2 and Bcl-xl whereas the expression of the STAT3

independent pro-apoptotic gene Bax is unaffected 251,374 . The increased Bcl-xl/Bax ratio has been

® ¯ Ê ® Figure 16 Schematic model of IL -6 trans -signaling in inflammatory bowel disease. There is increased production of IL -6 by macrophages, lymphocytes and intestinal epithelial cells in response to continuous stimuli by intestinal microflora. The s oluble IL-6 receptor (sIL-6R) is generated through shedding of the membrane-bound receptor from the surface of macrophages or neutrophils by proteolytic cleavage. This process can occur under stimulation by the acute -phase protein CRP in macrophages and by microbial metalloproteases in human monocytes. The increased formation of IL -6/IL-6R complexes that interact with gp130 on the membrane of T cells lacking membrane -bound IL-6R through trans-signaling leads to an increased expression and nuclear translocat ion of STAT3. This causes the induction of anti-apoptotic genes, such as Bcl-xl and Bcl-2 and resulting in an augmented resistance of T cells to apoptosis. The ensuing T -cell expansion contributes to the perpetuation of intestinal inflammation. Reprinted from Cytokine and Growth Factor Reviews, 17, Mitsuyama et al. Interleukin -6 trans-signaling in Inflammatory Bowel Disease. 251-261. Copyright (2006) with permission from Elsevier. shown to mediate resistance against intestinal T-cell apoptosis in experimental models of colitis

as in IBD patients 251,375,376 .

® ¯ Ê Þ T-Lymphocytes . CD4+ T cells play a major role in initiating and shaping the chronic immunopathologic process of IBD. The inflamed mucosa of IBD patients is characterized by the heavy infiltration of activated CD4+ T lymphocytes and blockade of the activation or activity of these CD4+ T cells can reduce the ongoing mucosal inflammation in both murine models of colitis as well as in patients with IBD. It is commonly accepted that pathogenic T cells constantly stimulated by commensal enteric bacterial antigens cause chronic intestinal injury 377 .

IL-6:STAT3 signaling is an important inflammatory pathway that promotes T-cell survival, proliferation and effector function. Both CD and UC are characterized as an exaggerated immune response in genetically susceptible individuals. However, they differ by the nature of the inflammation and subsets of T helper cells that mediate the perpetual recruitment and/or commitment of naïve T cells and granulocytes. Most experimental models of chronic intestinal inflammation are dependent on lamina propria T-cells even if there is a primary epithelial defect, solidifying the importance of mucosal T-cells during chronic inflammation 378 . T-cell effector lineage is influenced by the cues derived from the microenvironment, particularly cytokines.

CD4+T cells can diverge into four main subgroups, three sets of helper T cells Th1, Th2, Th17 and the regulatory Treg cells that all secrete characteristic types of cytokines. Tregs suppress excessive immune responses and this regulation is lost in CD and UC patients. CD is driven by a

Th1/Th17 response in which IL-12(p34/p40) and IL-23(p19/p40) play key roles whereas the

Th2-like response in which NKT cells producing IL-13 and IL-5 is the major response in UC

(Figure 17) 379 . However, recent murine and human studies from several labs also implicate a role for the IL-17 producing Th17 lineage in UC 380,381 . Thus, the unique adaptive immune

response differentiates CD from UC.

® ¯ Ê ± Figure 17 . Contribution of different STATs in Th Cell differentiation. Naive CD4+ T cells can differentiate to Th1 cells in the presence of IL -12, and require activation of the master regulator transcription factor, T -bet, through STAT1. IL-4-induced STAT6 mediates Th2 differentiation in combination with GATA3 signals. Th17 cells require a combination of TGF -b and proinflammatory cytokines (IL-1, IL-6, IL-21, IL-23) with the activation of STAT3 and RoRct. IL -2-induced activation of STAT5 is needed for the induction of FOXP3þ Tregs, but in these cells STAT3 is also needed for their regulatory function. . Reprinted from Inflamm Bowel Dis. Li et al. New Insights into the role of STAT3 in IBD,1-7, Copyright (2011) with permission from John Wiley and Sons.

STAT3 plays a critical role in T-cell apoptosis, proliferation and differentiation however, these STAT3 functions ultimately depends on the microenvironment and the activation status of the T-cell. In the initial studies regarding T-cell STAT3 deficiency, IL-6-induced proliferation was severely impaired and did not prevent apoptosis as compared to wild type STAT3 T-cells, which was independent of the anti-apoptotic protein Bcl-2261 . Teague et al. found a difference in the response of T cells to IL-6, depending on the activation status of the cells whereby IL-6 inhibited the death of naive T cells but had no effect on the death of either superantigen-activated

T cells, or T cells bearing memory markers 382 . Activated T cells had a lower level of surface IL-6

® ¯ Ê ² receptors but there was a similar level of STAT3 phosphorylation (Y705/S727) in both naïve and activated T-cells treated with IL-6382 . Interestingly, they did however observe a profound

inhibition of IL-6Ì induced Stat1 phosphorylation in activated T cells compared with resting T cells 382 . In this model system IL-6 was not sufficient to induce T-cell survival in activated and memory T-cells. In the aforementioned studies STAT3 induced by IL-6 trans-signaling enhanced mucosal T-cell survival in murine models of colitis and human IBD and blockage of this STAT3 induced signal with an antibody against the IL-6R enhanced T-cell apoptosis and had the downstream effect of inhibiting mucosal inflammation 251 . In a T cell-transfer colitis model of lymphopenic Rag2-/- mice reconstituted with naive T cells from control (Stat3flx/flx) or Stat3-/-

(CD4-Cre;Stat3flx/flx) mice resulted in protection of CD4+T cell STAT3 deficient mice 383 .

Further experiments revealed that STAT3 in CD4+ cells was essential for differentiation of Th17 cells and in its absence T cells were nonpathogenic and tended to divert toward a Treg cell fate

383 . Chip-Seq in T-cells from these mice identified that almost all the genes known to regulate

Th17 cell differentiation as well as genes that promote survival and proliferation were direct targets of STAT3 383 . In the opposing T cell lineage Chaundhry et al. utilized Foxp3-

Cre:Stat3flx/flx mice to delete STAT3 in Treg cells and demonstrated a selective dysregulation of TH17 responses upon Treg-specific ablation of Stat3 and identify Treg dependent control of

TH17 responses as an essential component of immune homeostasis 384 . Finally, certain Th17 cytokines such as IL22 induce epithelial barrier protective properties such as increasing defensin secretion by paneth cells, enhancing mucin production by goblet cells, and promoting IEC migration, thereby limiting inflammation. These studies in entirety demonstrate that action of

STAT3 in T lymphocytes is highly context-dependent and may mediate anti or pro-inflammatory

effector function.

® ¯ Ê ° Intestinal Epithelial Cells. The intestinal epithelium is the primary barrier between large quantities of commensal and potentially pathogenic bacterial species from the underlying gut associated lymphoid tissue (GALT). Thus intestinal epithelial cells in addition to digestion and absorption of nutrients also perform indispensable barrier functions that mediate homeostasis and inhibits host immune-microbial interactions. The intestinal epithelial cells (IECs) maintain this important homeostatic barrier through intracellular tight junctions that maintain a selective and polarized epithelium. This physical barrier is reinforced by mucin and antimicrobial peptides secreted to the apical surface by goblet and paneth cells 378,385 . Epithelial cells themselves can also secrete antimicrobial peptides including defensins, cathelicidins, and calprotectin 386,387 .

IECs also express TLRs and NLRs, at low levels to help combat an innate or adaptive immune

response 388,389 . Tonic signaling through PRRs on IEC seems to be tolergenic as IEC-specific

½ ¼ ½ » ½ ¾ Ç Ç í Û Ü ß ½ ¼ ¹ Ù Ù Ç

á ø ù

Í Î Í Î

ð È Ã ð Ø É Ä é Æ Á Ä â ö Ã deletion of components in the IKK complex é murine transgenic expression of the human NOD2 gene increase susceptibility to intestinal inflammation in multiple disease models 390-392 .

Murine models and human genetic and environmental linkage studies in IBD reveal that increased epithelial permeability and bacterial immune activation as the major contributor in the development of acute and chronic intestinal inflammation 377,393 . Almost all experimental mouse models of inflammatory bowel disease can be inhibited in mice devoid of intestinal flora 394-396 .

A recent report revealed that overexpression of CD97, an adhesion G-protein-coupled receptor in intestinal epithelial cells of transgenic mice attenuates colitis by strengthening adherens junctions

397 . The epithelial barrier is an important consideration in all phases of murine colitis and human

IBD.

® ¯ Ê · Another mechanism that mediates IEC homeostasis is efficient epithelial wound healing and restoration of the epithelial barrier in response to acute mucosal insults. Non-steroidal anti- inflammatory drugs (NSAIDs), antibiotics, enteric or other systemic infections, and stress are all reported potential triggers of IBD and are plausible inducers of gut injury 235 . As the actual primary cause of IBD remains unclear it is suspected to be the result of either an initial defect in the intestinal barrier or a breakdown of immune tolerance 191 . In dextran sodium sulfate (DSS) induced murine colitis, IEC STAT3 activation has a crucial role in the maintenance of the intestinal epithelial barrier and homeostasis via proliferation, survival, and wound healing 254,257,398 .

DSS exerts direct toxic effects on epithelial cells independently of the underlying gut immune system and its transient administration provides an experimental system to assess the

critical first phase of wound healing 393 . Murine models in which gp-130-STAT1/3 signaling is

û ü ý ü abolished (gp130 ú ) or in IL-6 deficient mice (IL-6-/-), there is exaggerated acute intestinal colitis in response to DSS as compared to wild type 256 . This was due to impaired epithelial wound healing with consistently more severe epithelial erosion 256 . This suggests that IL-6 induced STAT3 activation promotes epithelial restitution and wound repair important during the innate immune response. However, these models evaluated STAT3 contribution to innate immune response by inactivating STAT3 in multiple cells types. Bollrath et al. and Grivennikov et al. went on to show that ablation of STAT3 signaling in IEC resulted in greater susceptibility to DSS induced epithelial damage and mucosal inflammation 254,257 . However, this protected animals from the colitis associated cancer tumorigenesis, whereby hyper-activation of GP130 mediated STAT3 promoted tumor incidence and growth. Gene chip analysis of IECs isolated

from DSS treated IEC STAT3 deficient mice compared to controls demonstrated a reduction in

® ¯ Ê Ê the expression of genes involved in cellular stress response, apoptosis, and pathways associated with IEC wound healing specifically, Reg3 family members 398 . Additionally, IL-22 knockout animals were also more susceptible to DSS induced colitis and showed reduced in-vivo wound closure rate compared to IL-6KO and control 398 . Thus epithelial cell STAT3 seems to regulate survival, proliferation, and wound healing events during acute colitis and colitis-associated cancer. However, these studies have investigated the contribution of IEC STAT3 in acute injury or during tumorigenic chronic colitis conditions. Thus loss of STAT3 within the epithelial compartment would likely increase risk for the development of chronic inflammation following an acute self-limited gut injury, which has not been formally tested. A model of acute injury and either resolution or development of chronic colitis in a genetically susceptible host would be expected to be more applicable to the current concept of IBD pathogenesis in humans.

Therefore, chapter 3 of this PhD thesis investigates the functional consequences of IEC STAT3 deletion in a model of chronic colitis in which mice are exposed to DSS for seven days, and then assessed during recovery over the next twenty-one days. These studies reveal that deletion of

IEC STAT3 promotes T- lymphocyte STAT3 activation and chronic colitis.

Summary

STAT3 is a master regulator of mucosal inflammation with activation occurring in multiple cell types in a highly context dependent manner. Transient STAT3 activation is essential for gut homeostasis while constitutive activation leads to chronic inflammation. During the innate immune response, IL-6 induced STAT3 activation results in effector function of DCs, macrophages, and polymorphonuclear cells to mediate robust clearance of infection while IL-10

induced STAT3 activation leads to inhibition of these cell types. The function of regulatory T

® ¯ Ê Ë cells mediated by IL-10 induced STAT3 activation and Th17 cells mediated by IL-6 induced

STAT3 activation allows the mucosal immune system to fight infection and return to steady

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ë Ã × É Å Ä ÿ Ä â Å â state. î -6 induced STAT3 activation mediates proliferation and survival while IL-22 induced STAT3 activation mediates wound healing responses. Importantly, the loss of STAT3 within the epithelial compartment following an acute self-limited gut injury, has not been formally investigated during chronic phases of colitis.

In IBD there is loss of IL-10 induced regulatory pathways and the pro-inflammatory overproduction of IL-6 drives STAT3 activation and continued mucosal inflammation. Thus, with STAT3 being such an integral part of the mucosal immune response it is not surprising that recent GWAS studies have associated the genetic variant rs744166 in STAT3 with risk for IBD.

Chapter 2 of this PhD thesis builds upon the understanding that newly diagnosed pediatric CD

patients have up-regulation of mucosal IL-6:STAT3 dependent biologic networks that drive

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disease and stratifies these patients by Å These

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Ä Ã ¿ ö è ÷ ö ÷ Ã Ø É â â Ä â Ä É Å É Ä È Å Ã Ä É Ä Å Ä â â â É Ã ö è studies show that t È activation and up-regulation of pathways which promote recruitment of CXCR2+ neutrophils to the gut 299 . Additionally, Chapter 3 reveals that the loss of Stat3 activation in the epithelial compartment ultimately leads to more severe chronic colitis with reduced epithelial restitution gene expression and expansion of pSTAT3+ lymphocytes and Il-17a expression. These studies as a whole reveal the kaleidoscopic nature of STAT3 activation in the context of IBD and suggest that downstream mediators of disease may be more relevant in therapeutic intervention.

® ¯ Ê Ð References

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® ¯ Ð Ê Chapter 2

STAT3 Genotypic Variation and Cellular STAT3

Activation and Colon Leukocyte Recruitment in

Tara A. Willson BS 1,4,^ , Benjamin R. Kuhn DO1,^ , Ingrid Jurickova MD 1, Shaina Gerad BS 1, David Moon BS 1,

Erin Bonkowski BS 1, Rebecca Carey MD 1, Margaret Collins MD 2, Huan Xu MS 3, Anil G. Jegga DVM 3,

Stephen L. Guthery MD, MS 5, and Lee A. Denson MD 1,4*. STAT3 Genotypic Variation and Cellular STAT3

! " # " " $ J Pediatr Gastroenterol and Nutr.

Dec 22; 2011

Doi: 10.1097/MPG.0b013e318246be78

PMID: 22197944

License # 2847790442225

Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Preface

^ denotes both authors contributed equally to this paper

Abstract: 250 words

Text: 4164 words (excludes abstract, keywords, references, and figure legends)

Figures: 7

Tables: 4

! " # " " % " & ' ( ) * + # % ' * , Abbreviations: , Epstein Barr Virus transformed lymphocytes (EBL), Granulocyte-Macrophage Stimulating Factor auto-antibodies (GM-CSF Ab),

Inflammatory Bowel Disease (IBD), Interleukin 6 (IL-6), IL-6 receptor (IL6R), Janus-associated Kinase

! " # " " ' ( + # ' , - 2 (JAK2), Single Nucleotide Polymorphism (SNP),

Signal Transducer and Activator of Transcription 3 (STAT3), Ulcerative Colitis (UC).

Transcript Profiling: NCBI gene Expression Omnibus ( http://www.ncbi.nlm.nih.gov/geo ) accession number GSE9686.

Writing assistance: not applicable

Competing Interest: None to declare

Grant Support: This work was supported by the Bioinformatics, Gene Expression, Integrative

Morphology and Flow Cytometry cores of the National Institutes of Health (NIH)-supported Cincinnati

! " % " . " / # & " % + Childre 1P30DK078392-01), and NIH grants

R01 DK078683 (LAD), R01 DK068164 (LAD), T32 DK007727 (BK & RC), and DK069513 and the

! " 0 Foundation (SLG). This investigation was supported by Public Health Service research grant UL1-RR025764 and CO6-RR11234 from the National Center for Research Resources.

2-2

Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Author Contributions:

Study concept and design: TW, BK, SG, LD

Acquisition of data: TW, BK, IJ, SG, EB, RC, MC

Analysis and interpretation of data: TW, BK, HX, AJ, SG, TD

Drafting of the manuscript: TW, BK, LD

Critical revision of the manuscript for important intellectual content: TW, SG, LD

Statistical analysis: TW, BK, SG, LD

Obtained funding: SG, LD

Administrative, technical, or material support: IJ, SG, EB, RC, HX

Study supervision: SG, LD

2-3

Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Abstract & Key Words

Objectives: Genotypic variation in STAT3 increases risk for IBD, and STAT3 dependent inflammatory

networks are induced in the colon in these patients. We hypothesized that STAT3 1 risk allele carriage would be associated with increased cellular STAT3 activation and colon leukocyte recruitment.

Methods: Colonic expression of genes regulating STAT3 signaling and leukocyte recruitment and function was measured in pediatric CD patients stratified by STAT3 genotype. The frequency of colonic

pSTAT3+ and CXCR2+ neutrophils was determined using immunohistochemistry. STAT3 tyrosine phosphorylation (pSTAT3) was measured in circulating leukocytes by flow cytometry, and mechanisms regulating STAT3 activation were tested in IBD EBV-transformed lymphocytes (EBL). Results:

Colonic expression of IL-6, the STAT3 target gene SOCS3 , the neutrophil chemo-attractants IL-8,

CXCL1 , and CXCL3, and the neutrophil products S100A8, S100A9 and S100A12 were increased in

patients carrying the STAT3 1 risk allele. The frequency of neutrophils expressing the cognate receptor for IL-8, CXCR2, was increased in colonic biopsies from patients carrying the risk allele, and the frequency of pSTAT3+ or CXCR2+ neutrophils correlated with histologic severity. The frequency

of CD4 + lymphocytes and granulocytes expressing pSTAT3 was increased in patients carrying the

2 2 ! " ) . " & 2 2 4 STAT3 risk allele. 3 STAT3 risk allele exhibited increased basal and IL-6 stimulated STAT3 tyrosine phosphorylation, increased transcription of STAT3 and SOCS3 after

IL-6 stimulation, and increased membrane localization of the IL-6 receptor, GP130, and JAK2.

Conclusions: The STAT3 1 risk allele is associated with increased cellular STAT3 activation and up- regulation of pathways which promote recruitment of CXCR2+ neutrophils to the gut.

! " " "

Key words 5 ; Signal Transducer and Activator of Transcription 3 (STAT3); neutrophil recruitment; CXCR2

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Introduction

! " " " (CD) and ulcerative colitis (UC) are chronic relapsing and remitting inflammatory disorders of the gastrointestinal tract commonly referred to as the inflammatory bowel diseases (IBDs). IBD pathogenesis is complex involving the gut flora, epithelial barrier function, and innate and adaptive immunity. The precise etiology remains unclear but evidence suggests that it involves dysregulation of the host immune response to luminal flora. Genome wide association studies

(GWASs) have now identified multiple genetic factors that confer IBD susceptibility, with some unique to CD or UC, and some shared.[1-3]. Genotypic variation in STAT3 has been linked to risk for both CD and UC [1]. Recently, newly described permutations to build protein-protein interaction (PPI) networks from GWAS identified risk associated genetic loci revealed that in CD the core candidate network involved JAK2 and STAT3 [4].

In IBD STAT3 activation has been well documented with several potential roles. In animal models, Stat3 activation in intestinal epithelial cells is required for acute wound healing responses, but also promotes development of colitis-associated cancer during chronic inflammation [5, 6]. Stat3 activation in myeloid cells mediates anti-inflammatory effects of IL-10; targeted deletion of Stat3 in this cell type leads to severe spontaneous entero-colitis [7]. Conversely, Stat3 activation in CD4+ T cells is required for differentiation of Th17 effector lymphocytes, and blockade of IL-6:Stat3 signaling ameliorates both ileitis and colitis in animal models [8, 9]. Recent work by Nguyen et al. has shown that

Stat3 is essential for the neutrophil migratory response to CXCR2 ligands such as CXCL2 via activation of G-CSF induced CXCR2 expression and modulation of CXCR2 signal transduction [10]. We have

previously shown that STAT3 activation was increased in PB granulocytes, IL-6-stimulated CD3 +/CD4 + lymphocytes, and affected colon biopsies of pediatric IBD patients at diagnosis and during therapy [11].

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Furthermore, we identified an IL-6:STAT3 biological network that drives leukocyte recruitment and thereby mucosal inflammation in this setting.

We sought to define the functional consequences of the recently identified G>A intronic SNP

(rs744166) within the STAT3 gene in pediatric CD patients. We hypothesized that the STAT3 risk allele 2

1 A would be associated with increased cellular STAT3 activation, and differences in colonic

2 expression of chemokines driving leukocyte recruitment. We found that carriage of the STAT3 2 risk allele is associated with increased cellular STAT3 activation and up-regulation of chemokines expressed on 4q12-13 which promote CXCR2+ neutrophil recruitment to the gut.

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Materials and Methods

Materials. Human IL-6 and soluble IL-6 receptor (sIL-6R) were from R&D Systems (Minneapolis,

MN). Tyrosine phosphorylation state specific STAT3 (pSTAT3) (SC-7993, Santa Cruz Biotechnology,

Santa Cruz, CA), CXCR2 (CD182, BD Biosciences) and secondary antibodies (Vector Laboratories,

West Grove, PA) were used for immunohistochemical (IHC) analysis. pSTAT3 antibodies and antibodies for CD3, CD4, IL6R, and GP130 for flow cytometry and ImageStream x (amnis® Seattle,

WA) analysis were from BD Biosciences (San Jose, CA). Western blot antibodies to pSTAT3 (SC-

7993), STAT3, pSTAT1 (SC-135648), STAT1, GP130, IL-6 receptor alpha, JAK2, and SOCS3 were from Santa Cruz Biotechnology (Santa Cruz, CA).

Study Subjects for microarray and interrogation of colonic biopsies. Colon biopsies were obtained from an area of active disease in the ascending colon in pediatric CD patients, and from the same

segment of normal colon in healthy controls. The diagnosis of CD was made using established clinical,

& - " & $ 6 ! " # " " ' ( + # ' , 7 " "

! " # " " % " & ' ( ) * + # % ' * , 7 " " to measure clinical severity, and the Croh to measure mucosal severity [12, 13]. The Montreal system was used to classify disease location and

behavior [14]. The mean age (range) for the healthy controls for the colon biopsy studies was 10 (6-18) years; 57% were male.

Genotyping. Genomic DNA was extracted from whole blood using the PureGene Kit (Gentra System,

Minneapolis, MN). Patients were genotyped for the STAT3 G>A (rs744166) single nucleotide

polymorphism (SNP) using the TaqMan system [1].

8 Gene Array Analysis. Two colon biopsies from the ascending colon were placed in RN

(Qiagen, Valencia, CA) at 4°C. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen,

Valencia, CA). Samples were submitted to the CCHMC Digestive Health Center Microarray Core

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011. where the quality and concentration of RNA was measured and the global pattern of gene expression was determined using Affymetrix GeneChip Human Genome HG-U133 Plus 2.0 arrays as previously reported [11]. These data were previously published in a study which examined overall biologic networks induced in the colon in pediatric IBD, without stratification by STAT3 genotype [11]. Data

were normalized to allow for array-to-array comparisons, and differences between groups were detected

in Genespring with a significance at the 0.05 level and mean fold change relative to healthy control

samples. The complete dataset is available at the NCBI gene Expression Omnibus

(http://www.ncbi.nlm.nih.gov/geo ) accession number GSE9686.

Gene expression by real-time quantitative reverse transcription- PCR (RT-PCR). Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) or an RNeasy Plus Kit (Qiagen, Valencia, CA) and reverse transcribed using Accuscript High Fidelity First-Strand Synthesis System (Stratagene, Cedar

Creek, TX). Brillant SYBR Green based detection (Stratgene) utilizing the Stratagene Mx3000P PCR machine was used to determine gene expression. The mRNA levels of the gene of interest and that of the internal standard, hypoxanthine phosphoribosyltransferase ( HPRT ) or glyceraldehyde phosphate

dehydrogenase ( GAPDH ), were measured and expressed as a ratio to HPRT or GAPDH 9 Primer

! ; !

sequences are as follows: STAT3 forward : -ATG GAA GAA TCC AAC AAC GGC AGC- ; STAT3

" ! ; ! < = ; ) 7 ! :

: -AGG TCA ATC TTG AGG CCT TGG TGA- -AGC ACC AAC TGA

; ! < = ; " ! ; ! < '

CAG GAG AGA AGT - : - AGT CCT TTC CAG CTG TCC CTA GAA- -8

) 7 ! ; ! < ' ! :

: -AGA AAC CAC CGG AAG GAA CCA TCT - -8 reverse - AGA GCT GCA GAA

; ! < * > * ; ) 7 ! ; ! <

ATC AGG AAG GCT- : - ATT CGC CTT AAA TGC TCC CTG TCC-

* > * ; " ! 6 ? ? 6 6 6 6 ? ? ? 6 ? ; ! $

: -

Colon Histology and Immunohistochemistry (IHC). Paraffin-embedded hematoxylin-stained colon

biopsies were scored in a blinded manner by a pediatric pathologist (M.C.) using the CDHIS [13]. For

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

IHC, paraffin-embedded slides were deparaffinized and antigen unmasking was done by boiling for 10 minutes 10mM sodium citrate (pH 6) for CXCR2 and 1mM EDTA (pH 8) for pSTAT3. Endogenous

peroxide was quenched with 3% hydrogen peroxide for 15min at RT and tissue was permeabilized with

.3% Triton X-100 for 15 min at RT. Slides were subsequently blocked with 3% serum and then incubated overnight at 4ºC with primary antibodies. Detection and visualization of stained cells was achieved using the R.T.U kit (Vector Laboratories, West Grove, PA) with DAB (diaminobenzidine) as the chromogen.

Enzyme-Linked Immunosorbent Assay (ELISA) for measurement of serum IL-6. IBD patient sera were tested using sandwich ELISAs for the presence of human IL-6 as previously reported [11, 15].

Media from EBL cultures was analyzed for IL-6 concentration by ELISA at baseline prior to stimulation.

Flow Cytometry. Whole blood was collected in sodium heparin tubes at the time of colonoscopy and

placed directly on ice. Samples were stimulated with IL-6/sIL-6R and surface and intracellular staining was performed as described [11].

EBV-transformed lymphoblastoid cell lines (EBLs) culture and stimulation. Peripheral blood

samples were obtained from 18 IBD patients, and cells were isolated by gradient centrifugation. These

cells were then transfected with Epstein-Barr Virus (EBV) to create immortalized EBLs. EBLs were

cultured overnight in serum-free media with 1x10 6 cells/mL and then stimulated with 100 ng/mL IL-6 and 50ng/mL IL-6 receptor for 10 minutes prior to protein isolation and for 3 hours prior to RNA isolation.

Preparation of EBL Cytosolic, Membrane, and Nuclear Proteins. Nuclear and Cytosolic protein

fractions were obtained using NE-PER Nuclear and Cytoplasmic Extraction Kit according to

) ! " " (Thermo Scientific ® Waltham, MA). Membrane protein fraction was

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

obtained using MEM-PER Eukaryotic Membrane Protein Extraction Reagent Kit according to

) ! " " (Thermo Scientific ® Waltham, MA).

Immunoblot analysis. 40 µg cytoplasmic protein was separated by NuPAGE® Novex® 4-12% Bis-

Tris gel electrophoresis and transferred onto nitrocellulose membranes. Membranes were independently stained for rabbit-anti-human antibodies against STAT3, STAT1, IL-6 receptor, GP130, JAK2, SOCS3,

and @ -Actin loading control. 20 µg of nuclear protein was separated by gel electrophoresis in a similar

; - . * 6 6 A - 6 / ' ' &

fashion and stained for rabbit-anti-human antibodies against pSTAT @

control. 40 µg membrane protein was separated by gel electrophoresis in a similar fashion and stained

B . - ? A ; C - D E F -

for rabbit-anti-human antibodies against IL- @ -Tubulin loading control.

Protein bands were quantified by normalized chemoluminescent arbitrary units (AU) via LAS Image

Reader and MultiGauge Software (Fujifilm®).

ImageStream x analysis. EBLs were stimulated as described above and then stained with antibodies for

pSTAT3, IL-6 receptor, and DRAQ5 nuclear marker. 5x10 4 cells were analyzed by IDEAS application

Statistical analysis. Statistical analyses were performed using GraphPad PRISM© Version 4.01.

G ! "

Continuous variables were analyzed using unpaired t test, two sample t test 7 , or

" 7 # ! "

Kruskal-G est for multiple comparisons. Discrete variables were analyzed using

" ! " ( " / . A p-value <0.05 was considered significant.

Ethical Considerations. The patient-based studies were approved by the CCHMC and University of

Utah Institutional Review Boards, and consent was obtained from parents and assent from subjects age

11 and above.

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Results

Clinical and Demographic Characteristics. The clinical and demographic data for the CD patient

cohort stratified by STAT3 1 risk allele utilized for the FACS and colon biopsy studies are provided in

Table 1. There were no differences for age, gender, disease location, medication exposure, or clinical or

2 histologic disease activity in patients stratified by the STAT3 2 risk allele.

Colon Expression of Genes Regulating Leukocyte Recruitment and Function is Up-regulated in

I J 1 CD Patients Carrying the STAT3 H Risk Allele. We first asked if the STAT3 risk allele would

be associated with differences in colonic expression of genes we had previously reported to be up-

regulated in active colonic IBD and involved in leukocyte recruitment. We stratified gene expression

2 "

determined by microarray as a function of STAT3 2 (Table 2). We found that patients

2 carrying the STAT3 2 risk allele exhibited a significant increase in colonic expression of IL-6, the IL-

6:STAT3 target gene SOCS3 , leukocyte recruitment genes expressed on 4q12-q13, and S100A8, S100A9 and S100A12 . Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated in children with active IBD, have enhanced expression in pathological conditions of chronic inflammation, and are involved in phagocyte chemotaxis and function [16]. By comparison, expression of CXCL9-11 or CCL11 did not differ between the groups. Importantly differences in colon gene expression were not accounted for by differences in overall histologic severity, epithelial injury or lamina propria cellularity (mononuclear or polymorphonuclear cells), as measured by the CDHIS sub- scores (Table 1).

Since SOCS3 expression is an indicator of STAT3 activity and IL-8 and CXCL3 play a critical

role in neutrophil chemotaxis we elected to confirm their up-regulation by quantitative real time PCR.

SOCS3 expression was induced five-fold in CD patients carrying the STAT3 1 risk allele (p = 0.03,

Figure 1A). IL-8 expression trended towards a three-fold higher value in CD patients carrying the

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

STAT3 2 risk allele (p = 0.07, Figure 1B). CXCL3 mRNA expression was induced twelve-fold in CD

patients carrying the STAT3 2 risk allele (p = 0.01, Figure 1C). Collectively, these results

demonstrated that the STAT3 1 risk allele is associated with increased expression of genes mediating leukocyte recruitment located on 4q12-q13 and phagocyte S100 products.

The Frequency of Neutrophils Expressing CXCR2 is Increased in the Colon of CD Patients

I J Carrying the STAT3 J Risk Allele. We then asked if there would be an increase in the frequency of

neutrophils expressing activated STAT3 (pSTAT3) or the cognate receptor for CXCL3 and IL-8,

2 "

CXCR2, a STAT3 target gene, within colon samples stratified by STAT3 1 . In disease

controls lacking the risk allele the frequency of pSTAT3+ neutrophils was equal to 1.8 + 0.5 per hpf,

compared to 3.6 + 0.8 per hpf in CD patients carrying the STAT3 1 risk allele (p=0.09, Figure 2A and

2B). The frequency of pSTAT3-positive neutrophils per hpf was positively associated with the overall

2 histologic index of severity (r=0.65, p= 0.02; Figure 2C). CD patients carrying the STAT3 2 risk allele exhibited an increased frequency of neutrophils expressing CXCR2 in the colon compared to disease controls (p=0.03; Figure 2D & 2E). The frequency of CXCR2+ cells per hpf was highly related to the overall histologic index of severity within the colon biopsy (r=0.68, p=0.01; Figure 2F).

Moreover, the frequency of total neutrophils in non-risk allele patients was 5.5+1.3 per hpf compared to

2 " 7 7 " " & ) ) )

7.5+1.9 per hpf in CD patients carrying the STAT3 1 however both were significantly increased compared to 2.1+.3 neutrophils per hpf in controls (data not shown).

Peripheral Blood Lymphocyte and Granulocyte STAT3 Tyrosine Phosphorylation are increased

I J in CD Patients Carrying the STAT3 J Risk Allele.

We then asked whether differences in the frequency of pSTAT3 neutrophils observed in the mucosa would be reflected in differences in circulating granulocytes. We have previously shown that

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

STAT3 activation was increased in PB granulocytes, IL-6-stimulated CD3 +/CD4 + lymphocytes, and

affected colon biopsies of pediatric IBD patients [11]. We therefore asked whether carriage of the

STAT3 1 risk allele would be associated with increased cellular STAT3 tyrosine phosphorylation. We measured intracellular CD3+/CD4 + lymphocyte and granulocyte STAT3 tyrosine phosphorylation

(pSTAT3) before and after stimulation with IL-6/IL-6R by flow cytometry [17]. The cell surface

markers CD3 and CD4 were used to identify the lymphocyte population and granulocytes were

identified based upon scatter properties (Figure 3A). Patients carrying the STAT3 1 risk allele exhibited a significantly higher basal frequency of pSTAT3 +CD3 +/CD4 + lymphocytes (p=0.01, Figure

3B) and pSTAT3 +granulocytes (p=0.0004, Figure 3C) compared to non-risk allele patients. Moreover,

" & * 6 6 ; 2 " + p 1 also exhibited a significantly higher frequency of pSTAT3 granulocytes after IL-6/IL-6R stimulation (p=0.001, Figure 3C) compared to non-risk allele patients.

Comparison of un-stimulated and stimulated cells within the same genotype revealed that only samples from patients carrying the risk allele exhibited a significantly higher frequency of pSTAT3+ lymphocytes (p=.003, Figure 3B) or granulocytes (p=.02, Figure 3C) following IL-6/IL-6R stimulation.

This was specific as the frequency of pSTAT5 +CD3 +/CD4 + lymphocytes was not different in STAT3

2 " . " A ; K L + M F A , . N $ O K L + M , :

1 -risk allele patients. Similarly,

" 7 " ) ) * 6 6 ; 2 " . " O L K ; O + & the frequency of pSTAT5 1

(n=21) compared to 36+22 (n=5) in non-risk allele patients. We also measured the circulating

concentration of the STAT3 activating cytokine IL-6 and found that there was significantly less in

. " & * 6 6 ; 2 "

1 at 34+5pg/ml (n=22) compared to non-risk allele patients

86+25pg/ml (n=3, p<0.05). Collectively, these data demonstrated that the STAT3 1 risk allele is associated with increased STAT3 activation in primary peripheral blood leukocytes.

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

IL-6 stimulated STAT3 activation and membrane localization of the IL-6 Signaling Complex are

increased in Immortalized B-cell lines from IBD patients carrying the STAT3 risk allele. To

investigate the mechanism by which the STAT3 1 risk allele could be mediating differences in cellular

STAT3 activation we utilized EBV transformed B-cell lines (EBL) from IBD patients genotyped for the

STAT3 risk allele. Table 3 provides the clinical and demographic data for the EBL patient cohort

2 " &

stratified by STAT3 1 , while Table 4 provides B cell phenotyping stratified by the

2 STAT3 2 risk allele. There were no differences by genotype for age at diagnosis, gender, IBD

phenotype, or medication exposures at the time of sample collection. Moreover, the B cell phenotype

2 "

did not vary by IBD phenotype, so results were stratified by STAT3 1 within the entire IBD

cohort.

2 We first tested whether the STAT3 2 risk allele would be associated with differences in

cytosolic abundance of the IL-6:STAT3 signaling complex, or STAT3 itself. Neither cytosolic protein

2 " abundance of STAT3, STAT1, IL-6 receptor, JAK2, GP130, nor SOCS3 varied by STAT3 2

allele (Figure 4). However, nuclear protein abundance of tyrosine phosphorylated STAT3 was

2 " . " O P A

significantly increased at baseline in EBLs from STAT3 1 A normalized chemoluminescent arbitrary units (AU), compared to 1±0.4 AU in non-risk allele patients (p=0.04,

Figure 5A and 5B). Following IL-6 stimulation, the nuclear protein abundance of phosphorylated 2

STAT3 was also significantly increased to 32±4 AU in EBLs from STAT3 1 A risk allele patients compared to 19±4 AU in non-risk allele patients (p=0.04). Conversely, analysis of the alternative IL-

6:STAT1 pathway determined that the nuclear protein abundance of phosphorylated STAT1 was 2

significantly decreased following IL-6 stimulation in EBLs from STAT3 1 A risk allele patients compared to non-risk allele patients (p=0.001, Figure 5C and 5D). Since nuclear STAT3 accumulation was increased we then asked if transcription of STAT3 target genes including STAT3 and SOCS3 would

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011. also be increased. Neither STAT3 nor SOCS3 basal mRNA expression differed by STAT3 risk allele

carriage; however, following IL-6 stimulation, STAT3 and SOCS3 mRNA expression were significantly 2

increased in EBLs from STAT3 1 A risk allele patients compared to non-risk allele patients (p=0.003 and p=0.04, Figure 5E and 5F).

Membrane protein abundance of the IL-6 receptor under basal conditions was increased two-fold in 2

EBLs from STAT3 1 A risk allele patients compared to non-risk allele patients (p=0.04, Figure 6A and 2

6B). The membrane protein abundance of GP130 was increased two-fold in EBLs from STAT3 1 A risk

allele patients compared to non-risk allele patients (p=0.003, Figure 6C and 6D). The membrane 2

protein abundance of JAK2 was increased three-fold in EBLs from STAT3 1 A risk allele patients compared to non-risk allele patients (p=0.008, Figure 6E and 6F). Importantly, IL-6 did not appear to be

acting in an autocrine fashion to regulate the IL-6:STAT3 signaling complex as EBL supernatants 2

measured by ELISA demonstrated no difference in IL-6 concentration between STAT3 1 A risk allele

patients at 22pg/ml compared to non-risk allele patients at 20 pg/ml. Additionally, analysis of EBLs by

ImageStream x (Amnis®) demonstrated increased nuclear co-localization of pSTAT3 following IL-6

" * 6 6 ; 2 " + A C $ L

3 4 1 fold increase) compared to the non-risk allele EBL

(Figure 7). Collectively, these data demonstrated that targeting of the IL-6:STAT3 signaling complex to

the membrane, and IL-6:STAT3 signaling was enhanced in EBLs from patients carrying the STAT3 1 risk allele.

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Discussion

Recently identified IBD risk loci encode candidate genes involved in maintenance of the epithelial barrier, innate responses to microbial products, and differentiation and function of effector and regulatory lymphocytes. STAT3 activation has been well documented in these processes in both human and murine colitis where transient activation induces protective mechanisms but persistent activation furthers disease progression and ultimately malignant transformation. The aim of the current study was

to delineate how the recently identified intronic G>A STAT3 SNP (rs744166) is associated with specific

pathways involved in the pathogenesis of CD. We found that the STAT3 1 risk allele is associated with increased cellular STAT3 activation, and induction of pathways regulating leukocyte recruitment and function in the affected colon in the patient sub-group with this genotype.

The mechanism of enhanced cellular STAT3 responsiveness was not known and to dissect the

biochemical pathway we utilized EBLs created from IBD patients. We demonstrated increased IL-6

2 " . " .

dependent STAT3 tyrosine phosphorylation in EBLs from STAT3 1

non-risk allele patients. This mirrored our findings regarding increased peripheral blood leukocyte

2 " $ G ) "

3 4

STAT3 tyrosine phosphorylation in patients carrying the STAT3 1

2 " . " " " " " Q . Q ) '

carrying the STAT3 1 -6:STAT3 receptor complex (IL-6 receptor, GP130, and JAK2), in the absence of a difference in autocrine IL-6 exposure. The enhanced membrane accumulation of the IL6R signaling complex likely accounts for the

increased cellular responses to IL-6 via STAT3 activation. We feel this is specific to the STAT3

. 7 7 ) " * 6 6 A " & * 6 6 ; 2 " $

4 1 3

These differences in cell signaling may drive disease in the sub-group of patients who carry the STAT3 risk allele via STAT3 dependent effects upon T-lymphocyte and granulocyte differentiation, activation and survival.

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

The STAT3 risk SNP (rs744166) is located within the intron between exon 1 and exon 2 and

might be predicted to regulate gene expression; however, we did not observe association between the

6 6 ; 2 2 " R ( . " " * . Future work will ultimately require sequencing of the entire gene and its surrounding genomic sequence to delineate the genetic basis for differences in cellular STAT3 responses. The IL-6:STAT3 biologic network was the focus for our work and we realize our limitation in that we did not interpret responses to other STAT3 IBD effector cytokines such as IL-10, IL-11, IL-17, IL-22, IL-23, and IL-27 or the expression level of their cognate receptors. We utilized the EBV-transformed B cell lines as a model system for testing the effect of STAT3 genotype upon JAK:STAT signaling. While the B cell signaling responses may have been influenced by the

EBV-transformation, we observed a similar enhancement in lymphocyte STAT3 activation in primary cells from IBD patients. Future studies will be required to characterize the mechanistic basis for differences in cell signaling in IL-6 stimulated T-cells and granulocytes from the peripheral blood of

IBD patients.

Patients carrying the STAT3 1 risk allele exhibited increased colonic expression of chemokines located on 4q12-q13 (IL-8, CXCL2 , and CXCL3) and S100A8, S100A9 and S100A12 .

Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, known as damage associated molecular patterns, are found at high concentrations in inflamed tissue and have been shown to be involved in neutrophil chemotaxis [19]. Increased mucosal release correlates with fecal markers of

IBD disease activity, and in myeloid progenitor cells up-regulation of S100A8 and S100A9 was shown

by direct binding of STAT3 to the gene promoter via chromatin immunoprecipitation [18] [20]. Here

we demonstrate that S100A8, S100A9 and S100A12 are up-regulated in CD patients carrying the

2 STAT3 2 risk allele, in the absence of an overall difference in clinical or mucosal disease activity.

Furthermore, we did not find differences in the expression of genes classified in other pathways such as

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

immune and inflammatory mediators, cancer and cell proliferation, ECM tissue remodeling, or

2 " & Q &

metabolism. This suggests that patients carrying the STAT3 1 that involves increased neutrophil chemotaxis and activation. However, future studies which directly measure neutrophil chemotaxis will be required to test this.

We did investigate pathways which are involved in neutrophil mobilization since they are closely associated with the outcome of inflammation.[21, 22]. It has been shown that STAT3 regulates CXCR2 expression during mobilization responses and CXCR2 binds IL-8 and CXCL6 to promote neutrophil migration while CXCL1, -2, -3, and -5 enhance neutrophil chemoattractant activity.[10, 23, 24]. In murine models of chemically induced colitis small molecule antagonism of the CXCR2 receptor or genetic deletion reduces MPO (neutrophil) activity, colonic damage and clinical symptoms [25, 26].

Thus we evaluated the frequency of neutrophils expressing pSTAT3, and the cognate receptor for IL-8,

2 CXCR2+, and found them to be increased in colonic biopsies from CD patients carrying the STAT3 2 risk allele. Consistent with the murine studies, we found that the frequency of pSTAT3+ or CXCR2+ neutrophils was highly correlated with histologic severity. Importantly, overall lamina propria cellularity measured within the same biopsies from which we scored the above parameters did not vary for mononuclear or polymorphonuclear sub-scores. These data demonstrate that current clinical scoring systems are not able to distinguish between these differential pathways driving disease and also suggest the utility of fecal calprotectin as a plausible biomarker for this sub-population of CD. We did not observe an association between STAT3 risk allele carriage and disease location or severity within out

cohort. However, recent work from Ferguson et. al confirmed increased risk for CD associated with the

* 6 6 ; 2 "

1 and demonstrated associations with clinical phenotypes{Ferguson, 2010 #1236}.

6 ; ? ? 2 S & " & 7 " T

In that study the frequency of CD patients with ST 1

12.3%. They reported a significantly increased frequency of extra-intestinal manifestations,

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

inflammatory disease behavior, and colonic involvement in individuals who have the STAT3 1 risk

allele. This is consistent with our studies regarding mechanisms of colonic disease in patients carrying

* 6 6 ; 2 " - & 2 1 1 patients homozygous for G allele were at very low numbers in our patient population, ultimately limited the power of our analyses. Future studies with greater power to detect clinical associations will be needed to elucidate associations with disease behavior, response to therapy, and rates of colorectal cancer and surgery.

Lastly, data from human and murine models of colitis indicate that STAT3 may be an important target for the treatment of IBD. Recent clinical trials reported in abstract form have shown that the oral

Janus Kinase inhibitor, CP-690,550 (CP), is effective in moderate-to-severe UC patients in a dose- dependent manner with improvements in clinical response and remission rates[28]. However CP was not effective in CD[29]. The specificity of CP is for JAK1 and JAK3 over JAK2, and JAK3 is restricted to hematopoietic cells whereas JAK1 and JAK2 are ubiquitously expressed [30, 31]. The divergent result in CD versus UC may reflect differences in the underlying pathogenesis and supports further study of specific JAK:STAT signaling pathways in these disorders.

It is likely that there are several immunogenetic forms of IBD, with CD and UC representing the

broadest clinical classifications. While therapeutic options have increased over the past decade, our ability to target newer biologic therapies to specific subgroups of patients has lagged behind. Our data

suggests that inhibition of JAK:STAT3 signaling warrants further clinical investigation, and that

2 "

stratification of CD patients by the STAT3 1 define patient populations that have varying clinical efficacy to investigational agents including the oral Janus Kinase inhibitor, CP-690,550.

Furthermore, activation of STAT3 occurs during innate and acquired immune responses in multiple cell types having both pro and anti-inflammatory functions [7, 32-35]. Thus STAT3 activation has been referred to as a double-edged sword and investigating factors which mediate inflammation downstream

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

of STAT3 may lead to more targeted approaches. Collectively, our studies demonstrate that the STAT3

IBD 1 risk allele (rs744166) is associated with increased cellular STAT3 activation and up-regulation of chemokines which promote CXCR2+ neutrophil recruitment to the gut in a newly described sub- population of CD patients.

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Acknowledgment

This work was supported by the Bioinformatics, Gene Expression, Integrative Morphology and Flow

. . ! " % " . Cytometry cores of the National Institutes of Health (NIH)-"

Research Foundation Digestive Health Center (1P30DK078392-01), and NIH grants R01 DK078683

(LAD), R01 DK068164 (LAD), T32 DK007727 (BK & RC), and DK069513 and the Primary

! " 0 Foundation (SLG). This investigation was supported by Public Health

Service research grant UL1-RR025764 and CO6-RR11234 from the National Center for Research

Resources. Ramona Bezold, Kathleen Lake, and Ann Rutherford provided outstanding support with subject recruitment.

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figures and Tables

Figure 1

2-22 Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figure 1. Colon Expression of the STAT3 Target Gene SOCS3 and Chemokines Expressed on

I J 4q12-q13 are Increased in CD patients Carrying the STAT3 H Risk Allele. Colon mRNA expression was measured by quantitative real time PCR for (A) SOCS3, (B) IL-8, and (C) CXCL3 in

biopsies taken from active areas of disease. Data are expressed relative to hypoxanthine

phosphoribosyltransferase (HPRT) expression as the mean + SEM. GG (n=4) and AG/AA (n= 10):

! " # " " & " " 2 " $

null, heterozygous or homozygous for the STAT3 1

" 7 G ! " $ Differences were tested by unpaired t-

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figure 2

2-24 Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figure 2. The Frequency of CXCR2+Neutrophils is Increased in Colon Biopsies from CD Patients

I J Carrying the STAT3 J Risk Allele and the Frequency of pSTAT3+ and CXCR2+ Neutrophils

Correlates with Histological Severity. Immunohistochemistry for (A) pSTAT3 was performed in colon biopsies taken at diagnosis from active areas of disease and (B) the frequency of pSTAT3+ neutrophils in 10 random high-powered fields (HPF; 400x magnification) was determined. CTR:

healthy control (n= 6); GG (n=3), AG/AA (n=12): CD at diagnosis null, heterozygous or homozygous

2 " $ " 7 G ! "

for the STAT3 1 Differences were tested by unpaired t- correction. (C)

" ! " & " " " " 7 " " test for an association between the frequency of pSTAT3+ neutrophils per HPF and histological severity measured by the CDHIS (r = .65, p<0.02). Line arrows depict pSTAT3 positive neutrophils; block arrows depict non stained neutrophils.

Immunohistochemistry for (D) CXCR2 was performed in colon biopsies taken at diagnosis from active

areas of disease and the frequency of CXCR2+ (E) neutrophils in 10 random high-powered fields (HPF;

! "

400x magnification) was determined. CTR: healthy control (n=6); GG (n=3), AG/AA (n=14): C

2 " $

Disease at diagnosis null, heterozygous or homozygous for the STAT3 1 Differences were

" 7 G ! " " ! " tested by unpaired t- . (F) regression analysis was used to test for an association between the frequency of CXCR2+ neutrophils per HPF and histological severity measured by the CDHIS (r = 0.68, p<0.01). Line arrows depict CXCR2 positive neutrophils; block arrows depict non stained neutrophils.

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Figure 3

2-26 Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figure 3. Peripheral Blood Lymphocyte and Granulocyte STAT3 Tyrosine Phosphorylation is

I J Increased in CD Patients Carrying the STAT3 J Risk Allele. Intracellular STAT3 tyrosine phosphorylation (pSTAT3) was measured by flow cytometry in washed peripheral blood leukocytes

before and after stimulation with IL-6/IL-6R. (PBLs). T helper lymphocytes were identified as the

CD3 +CD4 + population and granulocytes were identified based upon forward and side scatter properties.

(A) Representative scatter grams for the frequency of CD3 +CD4 + lymphocytes or granulocytes expressing pSTAT3 is shown, with and without IL-6/IL-6R stimulation. Frequency of (B) CD3+CD4 +

lymphocytes and (C) granulocytes expressing pSTAT3 are shown as the mean + SEM. GG (n=5),

! " 2

AG/AA (n=20-22): C Disease at diagnosis null, heterozygous or homozygous for the STAT3 1

" 7 G ! " risk allele. Differences were tested by unpaired t- ..

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Figure 4

2-282-28 Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figure 4. Cytosolic protein abundance of STAT3, STAT1, IL-6 receptor, JAK2, GP130, nor

I J U V W X Y Z Z [ Z [ \ SOCS3 varied by STAT J EBL cytosolic protein abundance of (A) STAT3, (B) STAT1,

. " " " S " Q " + ] , &

are ex @ -actin via LAS

Image Reader and MultiGauge Software (Fujifilm®). Representative immunoblot for each are shown.

2 " $ # ) ) " 7

GG (n=6) and AA (n=9) : EBLs null or homozygous for the STAT3 1 tested by unpaired t-test.

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figure 5

2-30 Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figure 5. IL-6 Stimulated STAT3 Activation and Expression of STAT3 and SOCS3 are Increased

I J in Immortalized B-cell lines from IBD Patients Carrying the STAT3 J Risk Allele. EBL nuclear

protein abundance of (A) tyrosine phosphorylated STAT3 and (C) tyrosine phosphorylated

STAT1before and after IL-6/IL-6R stimulation was measured by immunoblot analysis. Data are

+ ] , & 6 / ' ' *

expressed as normalized chemoluminescent arbitrary units @

Image Reader and MultiGauge Software (Fujifilm®). Representative immunoblot for (B) pSTAT3 and

(D) pSTAT1. EBL mRNA expression was measured by quantitative real time PCR for (E) STAT3 and

(F) SOCS3 before and after stimulation with IL-6/ IL-6R. Data are expressed relative to glyceraldehyde

phosphate dehydrogenase (GAPDH) as mean + SEM. GG (n=6) and AA (n=6-9) : EBLs null or

2 " $ # ) ) " 7 " Q - - , . 3

homozygous for STAT3 1 -test with

! " / , . G -test.

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figure 6

2-32 Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figure 6. Membrane Localization of the IL-6 receptor, GP130, and JAK2 are Increased in

I J Immortalized B-cell lines from IBD Patients Carrying the STAT3 J Risk Allele. EBL Membrane

protein abundance of (A) IL-6 receptor (C) GP130 and (E) JAK2 were measured by immunoblot

" " $ # ( . " " " S " Q " + ] , &

@ -

Tubulin via LAS Image Reader and MultiGauge Software (Fujifilm®). Representative immunoblot for

(B) IL-6 receptor, (D) GP130, and (F) JAK2. GG (n=7) and AA (n=10) : EBLs null or homozygous

2 " $ # ) ) " 7 " Q .

for the STAT3 1 -test.

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Figure 7

2-34 Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Figure 7. IL-6 Stimulated Nuclear Localization of STAT3 is Increased in EBL Carrying the

2 " " " "

I J 1

STAT3 J Risk Allele. EBLs from IBD patients carrying the STAT3

& ? 2

1 -risk allele were examined under basal conditions and following IL-6 stimulation.

ImageStream x (Amnis®) analysis demonstrated increased nuclear co-localization of pSTAT3 following

B " * 6 6 ; 2 " + A C $ L ) " , .

3 4

IL- 1 -change in the

" ? 2 $

3 4 non- 1

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Willson et al. J Pediatr Gastroenterol and Nutr, 2011.

Tables

Table 1. Clinical and Demographic Characteristics STAT3 Age Male Colon-only STER 6-MP 5ASA PCDAI CDHIS CDHIS Sub-Scores Genotype Gender Location Epithelial Mono Poly

GG (n=8) 12(3) 88% 38% 38% 25% 25% 36(16) 5(4) 4.5 (2) 2.3(.5) 4.3(1)

AA or AG 13(3) 75% 40% 39% 25% 25% 36(17) 7(4) 4.5 (3) 2.8(2) 4.8(3)

(n=29)

_ ` a b c ^ d ` e f d ^ g h i j k l m n o j h p ` ^ g q ` e k q b r a s c l ` c h ^ c h m e g ` t ` g f n a p h u i k l v n w o k q b r a s c STER: corticosteroid, 6-MP: 6-mercaptopurine, 5ASA: 5-^ Disease Histological Index of Severity at the time of sample collection for FACS or microarray. Data are shown as the mean (SD) or frequency. CDHIS Sub -Scores (Epithelial, score for epithelial damage, architectural distortion, and erosion/ulcers; Mono, mononuclear cell sub-score; Poly, polymorphonuclear cell sub-score). Data are shown as the mean (SEM) or frequency. Data are shown as the mean (SEM).

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Table 2. Colonic Expression of JAK/STAT Signaling and Leukocyte Recruitment Genes Stratified by STAT3 Genotype Gene Symbol Gene Name Cytoband FOLD CHANGE STAT3 GG AG/AA Genotype: JAK/STAT Signaling IL-6 Interleukin 6 7p21 1.4 2.8* IL-6R Interleukin 6 Receptor 1q21 .83 .84 IL-6ST Interleukin 6 Signal Transducer 5q11 1.3 1.4 JAK2 Janus Kinase 2 9p24 1.3 1.9 STAT1 Signal Transducer and Activator of Transcription 1 2q32.2-q32.3 1.4 1.6 STAT3 Signal Transducer and Activator of Transcription 3 17q21 1.1 1.2 STAT5A Signal Transducer and Activator of Transcription 5A 17q11.2 1.3 1.3 STAT5B Signal Transducer and Activator of Transcription 5B 17q11.2 1.1 1.1 SOCS2 Suppressor of Cytokine Signaling 2 12q 1.0 .90 SOCS3 Suppressor of Cytokine Signaling 3 17q25.3 1.0 2.0**

Chemokines / Cytokines CCL4 Chemokine Ligand 4 (CC Motif) 17q12 1.6 2.4 CCL5 Chemokine Ligand 5 (CC Motif) 17q11.2-q12 1 .83 CCL11 Chemokine Ligand 11 (CC Motif) 17q21.1-q21.2 3.7 3.5 CCL17 Chemokine Ligand 17 (CC Motif) 16q13 0.83 .92 CCL25 Chemokine Ligand 25 (CC Motif) 19p13.2 0.73 .70 CCR9 Chemokine Receptor 9 (CC Motif) 3p21.3 1.2 1.0 CSF1R Colony Stimulating Factor 1 Receptor 5q33.2-q33.3 1.8 1.6 CSF2RA Colony Stimulating Factor 2 Receptor Alpha Xp22.32 1.6 1.6 CXCR3 Chemokine Receptor 3 (CXC Motif) Xq13 1.0 1.0 CXCL1 Chemokine Ligand 1 (CXC Motif) 4q12-q13 3.3 8.0* CXCL2 Chemokine Ligand 2 (CXC Motif) 4q12-q13 2 4.8 CXCL3 Chemokine Ligand 3 (CXC Motif) 4q12-q13 2.1 5.5* CXCL5 Chemokine Ligand 5 (CXC Motif) 4q12-q13 4.3 12.3 CXCL6 Chemokine Ligand 6 (CXC Motif) 4q12-q13 2.4 6.3 IL-8 Interleukin 8 4q12-q13 1.7 7.4* CXCL9 Chemokine Ligand 9 (CXC Motif) 4q21 3.6 5.1 CXCL10 Chemokine Ligand 10 (CXC Motif) 4q21 3.6 6.3 CXCL11 Chemokine Ligand 11 (CXC Motif) 4q21.2 7.7 17.5 CXCL12 Chemokine Ligand 12 (CXC Motif) 10q11.1 1.5 1.6 IL-8RA Interleukin 8 Receptor Alpha 2q35 1 1.1 IL-8RB Interleukin 8 Receptor Beta 2q35 1.2 5.0

S100 Family Members S100A8 S100 Calcium Binding Protein A8 1q21 1.6 5.6* S100A9 S100 Calcium Binding Protein A9 1q21 1.3 3.1* S100A12 S100 Calcium Binding Protein A12 1q21 1.1 5.8*

y z { | } ~

GG, AG/AA: CD null, heterozygous or homozygous for the STAT3 x -change is relative to healthy control colon, *p<0.05 or **p<.01 versus

| { } | } { { |

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Table 3. EBV Cell line Patient Demographics and Medication Exposures STAT3 Age Male CD UC 5-ASA Azathioprine STER Infliximab Methotrexate Genotype Gender GG 16(8-35) 25% 25% 75% 88% 38% 63% 13% 0% (n=8) AA 15(9-19) 50% 20% 80% 80% 50% 90% 20% 11% (n=10) 5-ASA: 5-aminosalicylate, STER: prednisone, Data are shown as the mean(range) or frequency.

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2.Imielinski M, Baldassano RN, Griffiths A, et al. Common variants at five new loci associated with early-onset inflammatory bowel disease. Nat Genet. 2009;41:1335-40.

3.Mathew CG. New links to the pathogenesis of Crohn disease provided by genome-wide association scans. Nat Rev Genet. 2008;9:9-14.

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5.Grivennikov S, Karin E, Terzic J, et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell. 2009;15:103-13.

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7.Takeda K, Clausen BE, Kaisho T, et al. Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity. 1999;10:39-49.

8.Atreya R, Mudter J, Finotto S, et al. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo. Nat Med. 2000;6:583-8.

9.Chaudhry A, Rudra D, Treuting P, et al. CD4+ regulatory T cells control TH17 responses in a Stat3- dependent manner. Science. 2009;326:986-91.

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10.Nguyen-Jackson H, Panopoulos AD, Zhang H, et al. STAT3 controls the neutrophil migratory response to CXCR2 ligands by direct activation of G-CSF-induced CXCR2 expression and via modulation of CXCR2 signal transduction. Blood. 2010;115:3354-63.

11.Carey R, Jurickova I, Ballard E, et al. Activation of an IL-6:STAT3-dependent transcriptome in

pediatric-onset inflammatory bowel disease. Inflamm Bowel Dis. 2008;14:446-57.

12.Hyams JS, Ferry GD, Mandel FS, et al. Development and validation of a pediatric Crohn's disease activity index. J Pediatr Gastroenterol Nutr. 1991;12:439-47.

13.D'Haens GR, Geboes K, Peeters M, et al. Early lesions of recurrent Crohn's disease caused by infusion of intestinal contents in excluded ileum. Gastroenterology. 1998;114:262-7.

14.Satsangi J, Silverberg MS, Vermeire S, et al. The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications. Gut. 2006;55:749-53.

15.Han X, Uchida K, Jurickova I, et al. Granulocyte-macrophage colony-stimulating factor autoantibodies in murine ileitis and progressive ileal Crohn's disease. Gastroenterology. 2009;136:1261-

71, e1-3.

16.Leach ST, Yang Z, Messina I, et al. Serum and mucosal S100 proteins, calprotectin

(S100A8/S100A9) and S100A12, are elevated at diagnosis in children with inflammatory bowel disease.

Scand J Gastroenterol. 2007;42:1321-31.

17.Krutzik PO, Clutter MR, Nolan GP. Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry. J Immunol. 2005;175:2357-65.

18.Cheng PY, Corzo CA, Luetteke N, et al. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein. Journal of Experimental

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20.Foell D, Wittkowski H, Ren Z, et al. Phagocyte-specific S100 proteins are released from affected mucosa and promote immune responses during inflammatory bowel disease. J Pathol. 2008;216:183-92.

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CXCR2, in inflamed gut tissue. J Pathol. 2000;192:533-9.

25.Bento AF, Leite DF, Claudino RF, et al. The selective nonpeptide CXCR2 antagonist SB225002 ameliorates acute experimental colitis in mice. J Leukoc Biol. 2008;84:1213-21.

26.Farooq SM, Stillie R, Svensson M, et al. Therapeutic effect of blocking CXCR2 on neutrophil recruitment and dextran sodium sulfate-induced colitis. J Pharmacol Exp Ther. 2009;329:123-9.

27.Ferguson LR, Han DY, Fraser AG, et al. Genetic factors in chronic inflammation: single nucleotide polymorphisms in the STAT-JAK pathway, susceptibility to DNA damage and Crohn's disease in a New

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28.Sandborn WJ, Ghosh S, Panes J, et al. Phase 2 Study of CP-690,550, an Oral Janus Kinase Inhibitor, in Active Ulcerative Colitis. Gastroenterology. 2011;140:S-110.

29.Sandborn WJ, Ghosh S, Panes J, et al. Phase 2 Randomized Study of CP-690,550, an Oral Janus

Kinase Inhibitor, in Active Crohn's Disease. Gastroenterology. 2011;140:S-124.

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35.Alonzi T, Newton IP, Bryce PJ, et al. Induced somatic inactivation of STAT3 in mice triggers the development of a fulminant form of enterocolitis. Cytokine. 2004;26:45-56.

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Chapter 3

Deletion of Intestinal Epithelial Cell STAT3

Promotes T Lymphocyte STAT3 Activation and

Chronic Colitis Following Acute

Dextran Sodium Sulfate Injury in Mice

Tara A. Willson BS 1,4, Ingrid Jurickova MD 1, Margaret Collins MD 2, and Lee A. Denson MD 1,4*

Willson et al.

Preface

Abstract: 251 words

Text: 5,763 words

Abbreviations: Dextran Sodium Sulfate (DSS) Chronic Colitis, Signal Transducer and Activator of Transcription 3 (STAT3), Interleukin-17 (IL-17), Azoxymethane (AOM), macrophage ( )

Financial disclosures: The authors have no financial arrangement(s) with a company whose product figures prominently in the submitted manuscript or with a company making a competing product.

Writing assistance: not applicable

Competing Interest: None to declare

¡ ¢ £ ¤ £ ¢ ¢ ¥ ¦ § ¨ ¥ © ¡ ª ¡ ¡ © ¡ « ¦ ¥ ¬ ¢ ¢ ¡ « ® ¥ ¢ ¥ ª ¯ ¦ ¡

Digestive Health Center (1P30DK078392-01), and NIH grants R01 DK078683 and DK068164.

Author Contributions:

Study concept and design: TW, LD Acquisition of data: TW Analysis and interpretation of data: TW, MC, LD Drafting of the manuscript: TW, LD Critical revision of the manuscript for important intellectual content: TW, LD Statistical analysis: TW Obtained funding: LD Administrative, technical, or material support: IJ Study supervision: LD

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Willson et al.

Abstract and key words

Background : Intestinal epithelial cell (IEC) Stat3 is required for wound healing following acute

Dextran Sodium Sulfate (DSS) injury, and we have reported that the IBD JAK2 risk polymorphism is associated with reduced cellular STAT3 activation. We hypothesized that loss of IEC STAT3 would

promote the development of chronic colitis following acute DSS injury. Methods : Colitis was induced

Flx/Flx

± ² ³ in IEC-specific Stat3 deficient mice (Stat3 ° ) and littermate controls (Stat3 ) with 4%DSS for 7 days, followed by water consumption for 21 days. Epithelial and immune mediators and severity of

colitis were determined. Results : Survival, colon length, and histologic injury were significantly worse

± ² ³ at day 28 in Stat3 ° mice. IEC proliferation and apoptosis did not vary by genotype at day 28. The

colonic lamina propria frequency of pSTAT3+ cells was increased at day 28 and correlated with

± ² ³ histologic injury in Stat3 ° mice. The frequency of colonic F480+pSTAT3+ macrophages and

Flx/Flx

± ² ³

CD3+pSTAT3+ T-lymphocytes were increased in Stat3 ° mice as compared to Stat3 controls. In

¡ ª ¥ µ ª « ¡ ª ¥ ¶ ¥ ¢ ¢ ¡ · ¸ ¹ ¥ ¹ ¥ ¥ ¢ ® ¥ ¹ ¸ º ¦ ® ¥ ¹ ¸ » £ ¡ ª ¥ ¦ ¡ ¥ ¥ ¡ ¥ « ¡

´ ´

± ² ³

Stat3 ° al

¼ ¯ ½ » µ © ¾ © ¿ À µ © ¾ © ¿ Á Â µ © © ¿ À µ restitution were significantly decreased, while expression of IL-17aµ and CCL4 were significantly increased and correlated with the increase in histologic severity at Day

28(p<.05). IL-17a expression also correlated with the increased lamina propria frequency of

CD3+pSTAT3+ T-lymphocytes. Conclusions: Loss of intestinal epithelial Stat3 leads to more severe chronic inflammation following acute injury which is not accounted for by a sustained defect in epithelial proliferation or apoptosis but rather expansion of pSTAT3+ lymphocytes and IL-17a expression.

Key words : Dextran Sodium Sulfate (DSS), Chronic Colitis, Signal Transducer and Activator of

Transcription 3 (STAT3), and Interleukin 17A

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Willson et al.

Introduction

¬ ¢ ¦ ¡ ¢ ¥ ¢ ¥ Ã © Ä Å © and ulcerative colitis (UC) are chronic relapsing and remitting inflammatory disorders of the gastrointestinal tract commonly referred to as the inflammatory bowel diseases (IBDs). The precise etiology remains unclear but evidence suggests that it involves the deregulation of the host immune response to luminal flora. Large scale genome wide association studies

(GWASs) have now identified multiple genetic factors that confer IBD susceptibility. 1-3 Genotypic variation in STAT3 and JAK2 has been linked to IBD risk. 1 We have recently reported that carriage of

the STAT3 risk allele is associated with increased cellular STAT3 activation and up-regulation of

chemokines expressed on 4q12-13 which promote neutrophil recruitment to the gut 4. Conversely, we

have found that patients carrying the JAK2 risk allele have decreased STAT3 activation in IL-6-

stimulated PB CD3+/CD4+ lymphocytes and reduced colonic expression of the STAT3 target gene

REG3Æ suggesting that reduced STAT3 activation may also be associated with increased risk for IBD 5.

Signal transducers and activators of transcription (STATs) mediate cytokine signaling.

Constitutive activation of STATs, especially STAT3, has been reported in several inflammatory and malignant disorders. STAT3 is a pleiotropic transcription factor that displays tissue specific differences in function. STAT3 is activated by multiple cytokines and growth factors and roles for IL-6, IL-11, IL-

17, IL-22, IL-27, growth hormone and leptin in experimental colitis have been elucidated. 6-15 In animal models, Stat3 activation in intestinal epithelial cells is required for acute wound healing responses via induction of Reg3 family members, but also promotes development of colitis-associated cancer during chronic inflammation. 16, 17 Stat3 activation in myeloid cells mediates anti-inflammatory effects of IL-

10; targeted deletion of Stat3 in this cell type leads to severe entero-colitis. 18 Conversely, Stat3 activation in CD4+ T cells is required for differentiation of Th17 effector lymphocytes, and blockade of

IL-6:Stat3 signaling ameliorates both ileitis and colitis in animal models. 6, 9 Tyrosine phosphorylation

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Willson et al. of STAT3 induced by IL-6 has also been linked to effector lymphocyte and granulocyte activation in

IBD. 19 We have previously shown that STAT3 activation was increased in PB granulocytes, IL-6- stimulated CD3 +/CD4 + lymphocytes, and affected colon biopsies of pediatric IBD patients at diagnosis

and during therapy. 20 We identified an IL-6:STAT3 biological network that drives leukocyte

recruitment and thereby mucosal inflammation in this setting.

Previous studies in IEC Stat3 deficient mice have examined the effects of either acute DSS induced colon injury, or multiple rounds of DSS administration together with injection of the carcinogen

Azoxymethane (AOM) to induce colitis associated cancer. These studies have shown that while Stat3 activation promotes survival and proliferation of IECs in response to acute injury, during chronic colitis under tumorigenic conditions epithelial Stat3 drives the development of colitis associated cancer 16 , 17 .

While these prior studies suggested that loss of Stat3 within the epithelial compartment would likely increase risk for the development of chronic inflammation following an acute self-limited gut injury, this had not been formally tested. This model of acute injury and either resolution or development of chronic colitis in a genetically susceptible host would be expected to be more applicable to the current concept of IBD pathogenesis in humans. We therefore sought to define the functional consequences of intestinal epithelial specific deletion of Stat3 in a model of chronic colitis in which mice are exposed to

DSS for seven days, and then assessed during recovery over the next twenty-one days. We hypothesized that loss of IEC Stat3 would promote the development of chronic colitis following acute DSS injury.

We found that loss of intestinal epithelial Stat3 leads to more severe chronic inflammation following acute injury which is not accounted for by a sustained defect in epithelial proliferation or apoptosis but rather expansion of pSTAT3+ lymphocytes and IL-17A expression.

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Willson et al.

Material and Methods

Animals

To delete Stat3 in intestinal epithelial cells (IEC), we crossed Stat3 lox/lox mice (termed Stat3 Flx/Flx ) 21 obtained with permission from Takeda via Dr. Jeffrey Whitsett at CCHMC and backcrossed for five generations on a C57BL/6 background before intercrossing to villin-Cre (B6.SJL-Tg(Vil-

Cre)997Gum/J, Jackson Laboratories, Bar Harbor, Maine) to generate compound mutant villin-

lox/lox

± ² ³ Cre/Stat3 mice (termed Stat3 ° ). Cre-negative littermates were used as controls (termed

Stat3 Flx/Flx ). We confirmed deletion of intestinal epithelial STAT3 using an in-vivo model of LPS

± ² ³ exposure and PCR for total STAT3 in isolated enterocytes (Supplemental Figure 1). Stat3 ° mice were born in mendelian ratios, developed normally and showed no obvious spontaneous phenotype as has been previously published 10, 17 . All mice were maintained in conventional housing at CCHMC and all experiments were performed in accordance with CCHMC and Institutional Animal Care and Use

Committees of CCHMC.

DSS Colitis

Intestinal inflammation was induced by 4% DSS water (m.w. 36,000-50,000):MP Biomedical, LLC,

Solon, OH) for 7 days in acute studies designated (day 7) or 7 days followed by 21 days of water designated (day 28) to study progression from acute phase to chronic inflammation and recovery.

Healthy control animals received water only designated (day 0). The dosing of DSS was established by initial experiments within our colony to induce moderate to severe colitis while minimizing mortality.

Body weight change during DSS protocol was calculated by dividing body weight on the specified day

by body weight at day 0 (starting body weight) and expressed in percentage and recorded on Day 0, 3, 5,

7, 10, 12, 18, 20, 22, 26, and 28. In all experiments, littermate controls were used to assure comparison of mice on the same genetic background. DSS injury occurs throughout the colon at varying levels and

3-6

Willson et al. the area involved increases from the proximal to the distal colon22 . Therefore we utilized the anal verge as the most distal reference point from which to perform histopathological analysis.

Tissue sampling

Mice were sacrificed and the colon from the ileocecal valve to the anal verge was removed. Colon length was determined from the cecocolic junction to the anal verge. The cecum was removed at the cecocolic junction and the whole colon flushed with ice-cold PBS. The colon was then cut into two equal segments one labeled as distal and the other as proximal. The distal and proximal segments were fileted open and cut into two equal portions the length of the segment. One portion of distal and

proximal colon was laid flat and fixed in 10% neutral buffered formalin overnight, transferred to 70% ethanol, processed and embedded, sectioned at 5-µm, and utilized for immunohistochemistry. The remaining distal and proximal portions were Swiss-rolled at the base of a vinyl cryomold that was subsequently filled with O.C.T compound (Tissue Tek®, Sakura Finetek USA, Inc) frozen on dry ice, and stored at -80°C for later use in immunohistochemical analyses and RNA extraction 23 . All sections from paraffin embedded and Swiss-rolled OCT fixed colons were processed so that the two segments had continual villus crypt architecture from the anal verge to the cecocolic junction for representation of the whole colon.

Histo-pathological assessment of DSS-induced colitis.

After staining with hematoxylin-eosin colon sections were scored as previously described with the following alternations 24 . It is known that C57BL/6J mice upon DSS administration develop distal and middle colonic disease with little disease in the most proximal portion 22 . To orient a starting point for analysis we developed a scoring system to account for total colonic disease by using the anal verge to mark the most distal starting point for scoring and the following four consecutive longitudinal 5x High

Powered Fields (HPF) scored as the distal colon and the following four, 5x HPF scored as the proximal

3-7

Willson et al. colon. With the observer blinded to genotype, a disease score was determined separately for the distal and proximal portions by adding the scores for area involved, erosion/ulceration, crypt loss, edema, and infiltration of immune cells into the mucosa. The total disease score was calculated as the sum of distal and proximal disease score divided by two.

Immunohistochemical Analyses (IHC)

For IHC, paraffin-embedded slides were deparaffinized and antigen unmasking was done by boiling for

10 minutes in 10mM sodium citrate (pH 6) for cleaved caspase-3 (Cell Signaling, Danvers, MA), and

1mM EDTA (pH 8) for tyrosine phosphorylation state specific (Tyr705) STAT3 (pSTAT3-XP) (Cell

Signaling, Danvers, MA). Endogenous peroxide was quenched with 3% hydrogen peroxide for 15min at RT and tissue was permeabilized with .3% Triton X-100 for 15 min at RT. Slides were subsequently

blocked with 3% serum and then incubated overnight at 4ºC with primary antibodies. Biotinylated secondary anti-rabbit antibodies (Vector Laboratories, Burlingame, CA) were added and incubated at room temperature for 1hr. Detection and visualization of stained cells was achieved using the R.T.U kit

(Vector Laboratories, West Grove, PA) with DAB (diaminobenzidine) as the chromogen.

Quantification of cleaved caspase-3 staining in epithelial cells of the distal and proximal colon was

performed, with observer blinded to genotype, by counting positive cells in three 400x high-powered fields (HPFs) in the mucosa that bordered ulcers of the distal and proximal colon or in the corresponding area of the colon in untreated controls. BrdU staining was performed as suggested by the manufacturer

(BrdU staining kit; Invitrogen, Carlsbad, CA). Mice were injected with BrdU labeling reagent

(Zymed®, Invitrogen, Carlsbad, CA) per manufacturer instructions 2hr before sacrifice and processed for immunohistochemistry as above. Quantification of pSTAT3 or BrDU positive epithelial cells from properly oriented crypts defined as those with clear cell column lining both sides of an open luminal area, were counted within .5cm (or one 50x HPF) of the mucosa that bordered ulcers in the distal and

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proximal colon or in the corresponding segment of the colon in untreated controls. The number of

pSTAT3 or BrdU-positive cells per crypt divided by the total number of cells per crypt was determined with the observer blinded to genotype and data are expressed as a labeling index. Histological analysis and microphotographs were attained on an Olympus BX51 microscope utilizing the Olympus DP71 digital camera system and analyzed using DP-BSW ver .03.02 software.

Immuno-fluorescence

5-µm-thick colonic sections were fixed with 3.7% formaldehyde in phosphate-buffered saline for 15 min and washed with phosphate-buffered saline once followed by permeabilization with ice-cold methanol at

-20 for 10 min. The cells were then blocked with 5% bovine serum albumin for 60 min. To double label, sections were incubated with rabbit anti-mouse pSTAT3-XP antibody overnight at 4°c. The following day sections were incubated with Rhodamine (TRITC) conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and then sequentially incubated

with either rat anti-mouse F4/80 (eBioscience, San Diego, CA ) to stain macrophages, or goat anti- Ã mouse CD3-Ç Santa Cruz Biotechnology, Santa Cruz, CA.) to stain T lymphocytes for 1 hour at room

temperature and finally incubated with DyLight È 488-conjugated anti-goat or Fluorescein (FITC)- conjugated anti-Rat secondary antibodies (Jackson ImmunoResearch Laboratories Inc.). Cell nuclei were labeled with DAPI diluted in the aqueous mounting media, Fluoromount-G (Southern Biotech), to

prevent fluorochrome quenching during analysis and for a semi-permanent seal. Images were captured using Olympus BX51 microscope utilizing the Olympus DP71 digital camera system and analyzed using

DP-BSW ver .03.02 software.

Real-time RT-PCR

Swiss Rolled colonic tissues embedded in O.C.T compound were cross sectioned into four to five 30-

µm-thick sections, giving approximately 20mg of total colonic tissue. The sections were rinsed in

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Willson et al.

RNA later ® RNA stabilization reagent (Ambion, Austin, TX) to remove O.C.T medium and RNA was extracted using RNeasy Plus Kit (Qiagen, Valencia, CA). RNA integrity was validated on a denaturing gel with proper 2:1 ratio (28S:18S) and quantified using NanoDrop® 1000 Spectrophotometer

(ThermoScientific, Wilmington, DE). After DNase I treatment (Invitrogen, Carlsbad, CA), RNA was reverse transcribed with Oligo-dT using SuperScript III 1 st Strand cDNA Synthesis kit. SYBR Green

based detection (Stratgene) utilizing the Stratagene Mx3000P PCR machine was used to determine gene

É ¥ ¦ º expression. Expression data were normal ¡ -actin mRNA expression and presented as relative message level. Primer sequences are listed in Table 1.

Statistical analysis

All values are presented as mean + SEM. Total histology scores, colon length, and histology sub-scores

· ¥ ¡ ¬

were compared using an analysis of variance (ANOVA) followed by Ê s test for multiple comparisons. %Body Weight was compared using a 2 way Analysis of Variance (ANOVA).

Flx/FLx

± ² ³ Comparisons were made between Stat3 littermate controls and Stat3 ° mice at day 0, day 7 or day

28 using un-paired Student t test. Association between two variables was measured by Pearson's correlation coefficient. Statistical analyses were performed using GraphPad PRISM© Version 4.01. A p- value <0.05 was considered statistically significant.

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Results

Ì Í Î Stat3 Ë mice exhibit more severe acute and chronic colitis following DSS administration.

Flx/Flx IEC

Stat3 littermate controls and Stat3 ° mice were administered 4%DSS for 7 days to induce acute colitis (day 7) followed by water consumption alone for 21 days (day 28) with untreated mice used as

IEC controls (day 0) (Figure 1A). Colitis was not apparent at day 0 in either genotype. Stat3 ° mice at day

7 exhibited a more severe total histology score of 12.6 + .55 compared to 9.1+1.5 for Stat3 Flx/Flx

littermate controls (p<.0001) (Figure 1B). Day 28 analysis revealed that both genotypes had sustained

± ² ³ chronic colitis however the Stat3 ° mice had more total histology scores of 8.6+.44 compared to

5.3+.38 for Stat3 Flx/Flx littermate controls (p<.0001) (Figure. 1B). Kaplan-Meyer analysis demonstrated

± ² ³ increased mortality in Stat3 ° mice which became apparent by day 12 (Figure. 1C). Both genotypes

began to lose weight after the 7th day of DSS administration and by day 10 the body weight of the

Flx/Flx

± ² ³ Stat3 ° mice was reduced by 10.6% compared with 4.3% in Stat3 littermate controls (p<.001)

(Figure 1D). Colon length was shortened to a similar extent in both genotypes at day 7, but the length of

± ² ³ the colon was significantly reduced at day 28 to 8 +.2cm in Stat3 ° mice compared to 9+.2cm in

Stat3 Flx/Flx littermate controls (p<.005) (Figure 1E).

The histologic sub-scores for overall area involved at day 7 and day 28 were significantly worse

IEC Flx/Flx in Stat3 ° mice at 3.4+.2 and 2.8+.2 compared to 2.6+.4 and 2.0+.2 in Stat3 littermate controls

(p<.001) (Figure 2B). There was substantial specific injury to the epithelial cell layer as measured by

IEC IEC ° ulceration and crypt loss that persisted within the Stat3 ° deficient animals. At day 7 Stat3 mice had almost twice the amount of ulceration with a score of 1.7+.2 compared to .8+.3 in Stat3 Flx/Flx

IEC littermate controls (p<.0001) (Figure 2C). The ulceration persisted in the Stat3 ° mice at day 28 and was almost four times more severe in at .73+.1 compared to .15+.1 in the Stat3 Flx/Flx littermate controls

(p<.0001) (Figure 2C). Crypt loss at day 7 was not significantly different between genotypes but at day

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Willson et al.

IEC

28 Stat3 ° mice had almost double the amount of crypt loss at 2.46+.2 compared to 1.7+.13 in the

Flx/Flx IEC

Stat3 littermate controls (p<.05)(Figure 2D). The immune cell sub-score at day 7 in Stat3 ° mice was also significantly higher at 2.4+.1 compared to 1.5+.3 in Stat3 Flx/Flx littermate controls (p<-001)

IEC

(Figure 2E). At day 28 both genotypes still had infiltration of immune cells but in Stat3 ° mice it was more significant at 2.1+.1 compared to 1.3+.1 in Stat3 Flx/Flx littermate controls (p<.001) (Figure 2E).

Ì Í Î IEC proliferation and survival does not differ from controls at Day 28 in Stat3 Ë mice.

IEC Stat3 deficiency in DSS induced acute colitis has shown that Stat3 functions to induce intestinal wound healing responses, proliferation, and protection from apoptosis during acute injury 10 ,17 ,

16 . We confirmed Stat3 activation in response to DSS in colonic enterocytes at day 7 in the littermate controls (Figure 3A). The frequency of nuclear pSTAT3 positive IEC per crypt was equal to 4.8+1.6

percent at day 0, which was significantly increased to 15.2+6 percent at day 7, and returned to basal

± ² ³ levels of 1.6+.77 percent at day 28 (p<0.05) (Figure 3B). Stat3 ° mice as expected had no nuclear IEC

pSTAT3 staining. To examine proliferation of IECs we injected untreated control, acute and chronic phase mice with BrdU, and sacrificed the animals 2 hours later. At Day 7 we found a significant reduction in basal crypt proliferation rates as the percentage of BrdU positive cells per crypt was 3.8+.6

IEC Flx/Flx in Stat3 ° mice compared to 7.3+.6 in Stat3 littermate controls (p<.005) (Figure 3D). There was no difference at day 28 between genotypes. To examine whether IEC-specific deletion of Stat3 renders enterocytes more susceptible to apoptosis at baseline and during acute and chronic phases of colitis we

quantified immunohistochemical staining for cleaved caspase-3 positive cells per HPF at day 0, day 7

± ² ³ and day 28. We found that at day 7 Stat3 ° mice had a trend for increased caspase-3 positive IECs per

HPF but this comparison was not significant and there was no difference between genotypes at day 0 or day 28 (Figure 3F).

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Willson et al.

We therefore asked if other signaling pathways that control the expression of anti-apoptotic, pro-

proliferative and immune response genes known to overlap and have redundant functions to Stat3, such

¢ ¥ ½ ¯ Ï

Ê signaling pathway, might mediate wound healing, proliferation and enhanced survival in the IEC Stat3 deficient animals 25, 26 27, 28, 29, 30, 31, 32,33 . We stained for nuclear phosphorylated

Rela(p65) but did not observe a difference in the percentage of nuclear RelA(p65) positive IEC per crypt at day 0, day 7 or day 28 between the genotypes (Suppl. Figure 2B). As expected, the frequency of

RelA(p65) positive IEC per crypt was increased in both genotypes at day 7. We also did not observe a

¡ ¥ ¥ ¶ ¥ ¢ ¢ ¡ · ¼ Ï Ð difference Ê , which is direct downstream target of activated RelA(p65), between the genotypes at day 28 (Suppl. Figure 2C)34,35,36. STAT1 or STAT5 activation might also promote a

survival signal which could compensate for the loss of STAT3 37, 38, 39 . Immuno-histochemical analysis

of consecutive colonic sections for pSTAT1 and pSTAT3 showed no activation of pSTAT1 in the

epithelial compartment of either genotype at day 0, day 7 (data not shown) or day 28 (Suppl. Figure 2D).

Similarly, we observed no nuclear IEC staining for pSTAT5 in either genotype at day 28 (Suppl. Figure

± ² ³ 2E). Collectively, these data suggested that the increased severity of colitis at day 28 in Stat3 ° mice was not accounted for by differences in IEC survival or proliferation, or induction of compensatory IEC signaling pathways.

Development of chronic colitis involves Stat3 activation in the non-epithelial compartment in

Ì Í Î Stat3 Ë mice.

In order to identify the lamina propria cellular populations involved during DSS induced chronic colitis, colonic sections of control (day 0) and DSS-induced chronic colitis (day 28) mice were examined

for the frequency of pSTAT3 positive enterocytes and lamina propria cells, and double positive

¦ pSTAT3 +/F4/80 + M pSTAT3 +/CD3 + T-lymphocytes per high powered field (HPF). In Stat3 Flx/Flx

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Willson et al.

littermate controls the number of IEC that were positive for nuclear pSTAT3 staining at day 28 was

± ² ³ equal to 4.5+2 cells per HPF with no IEC nuclear pSTAT3 detectable in the Stat3 ° mice (Figure 4A).

IEC +

In the lamina propria, the Stat3 ° mice exhibited a significant increase in pSTAT3 cells per HPF at

23.5+3, compared to 9.2+2 cells per HPF in the Stat3 Flx/Flx littermate controls (p<.001) (Figure 4B). The frequency of cells in which surface staining for F4/80 co-localized with nuclear pSTAT3 was significantly increased as well from 2+.3 cells per HPF in Stat3 Flx/Flx mice to 8.7+1 cell per HPF in

IEC +

Stat3 ° mice (p<.001) (Figure 4C). The frequency of total F4/80 M per HPF was also significantly

Flx/Flx

± ² ³ increased in Stat3 ° compared to Stat3 littermate controls (data not shown). The frequency of cells in which surface staining for CD3 Ç co-localized with nuclear pSTAT3 was also significantly

IEC Flx/Flx increased in Stat3 ° mice at 1+.3 cells per HPF compared to .1+.02 cells per HPF in Stat3 mice

± ² ³ (p<.05). The frequency of total CD3 cells per HPF was also significantly increased in Stat3 ° compared to Stat3 Flx/Flx littermate controls (data not shown). Neutrophils did not seem to be playing a

major role in the chronic colitis at day 28. We assessed mucosal S100 proteins, S100A8/S100A9,

± ² ³ involved in phagocyte chemotaxis and function and found only S100A9 increased in STAT3 ° mice compared to STAT3 Flx/Flx littermate controls (Suppl. Figure 3A&B) 40, 41 . However, when we stained for neutrophil elastase, secreted by neutrophils during inflammation, the frequency of lamina propria

Flx/Flx

± ² ³ neutrophil elastase positive cells per HPF was decreased in Stat3 ° mice compared to Stat3 littermate controls at day 28 (Suppl. Figure 3C). We also examined the colonic expression of CXCR2, a

receptor known to mediates neutrophil migration to sites of inflammation, in Stat3 Flx/Flx littermate

± ² ³ controls and Stat3 ° mice at day 0 and day 28 and found no differences (Suppl. Figure 3E). Finally, the overall frequency of lamina propria cells expressing nuclear pSTAT3 was significantly associated

IEC with histology scores at day 28 in the Stat3 ° mice, but not in littermate controls (r=.77, p =.03)

(Figure 4E).

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Willson et al.

Colonic expression of Stat3 target genes that mediate epithelial restitution is significantly

Ë Ì Í Î decreased while SOCS3 , IL-6, IFN- Ñ and IL-17A expression are increased in Stat3 mice at day

28. Since mice deficient in IEC Stat3 exhibited a more significant lamina propria infiltrate at day 28 we next investigated colonic expression of genes involved in epithelial restitution and mucosal inflammation. We performed the expression analysis from the same colonic sections for which we determined the composition of cellular infiltrate, which allowed us to correlate colonic gene expression

with immune cell population frequencies and histology scores. Using quantitative RT-PCR we found

Ó Ô Õ Ö Ò Ó Ô Õ × that mRNA expression for the Stat3 dependent epithelial restitution genes Ò and was

IEC Flx/Flx significantly decreased at both day 0 and day 28 in Stat3 ° mice compared to Stat3 littermate controls (p<.005) (Figure 5A&5B). Conversely, colonic expression of Socs3 , a direct downstream target

IEC of Stat3 activation, was significantly increased in Stat3 ° mice at day 28, likely reflecting the increased

lamina propria Stat3 activation observed at this time point (p<.04) (Figure 5C). Expression of IL-6 was

Ù Ú × not different between genotypes at day 0 or day 28 (Figure 5D). Ø expression was significantly

Flx/Flx

± ² ³ increased at day 0 and day 28 in Stat3 ° mice compared to Stat3 littermate controls (p<.01)

IEC

(Figure 5E), while IL-17A expression was also increased at day 28 with Stat3 ° mice expressing

163.6+67 relative units (RU) compared to 33.9+7 RU in Stat3 Flx/Flx littermate controls (p<.02) (Figure

5F). By comparison, we did not observe a significant difference in colonic expression of IL-10, IL-4,

½ ¯ µ ¼ ¿ Á º

Ð - between genotypes at day 28 (Suppl. Figure 4A,B,C&D), suggesting that the differences

Ù Ú ×

in Ø and IL-17A expression were specific.

Ì Í Î Colonic expression of chemokines involved in leukocyte recruitment is increased in Stat3 Ë mice at Day 28. We have found that the frequency of colonic pSTAT3 +/F4/80 +M , pSTAT3 +/CD3 + T-

IEC

Ù Ú × ° lymphocytes, and colonic expression of Ø and IL-17A were increased at day 28 in Stat3 mice compared to littermate controls. We therefore next asked whether there would be differences in

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Willson et al. expression of chemokines which mediate trafficking of these cells. Colonic expression of CXCL5 was increased in both genotypes at day 28 (Figure 6A). By comparison, day 28 CXCL2 expression was

IEC Flx/Flx increased significantly more in Stat3 ° mice at 54.3+21 RU compared to 11.6+2 RU in Stat3 littermate controls (p<.03) (Figure 6B). The day 28 expression of CXCL10 was significantly increased

IEC Flx/Flx in Stat3 ° mice at 77+21 RU compared to 20+3 RU in Stat3 littermate controls (p<.001) (Figure

IEC

6C). CCL2 expression was also significantly increased at day 28 in Stat3 ° mice at 3.4+.6 RU compared to 2.1+.63 RU in Stat3 Flx/Flx littermate controls (p<.05) (Figure 6D). Lastly, the expression of

IEC

CCL4 was significantly increased at day 28 in Stat3 ° at 2.5+.1 RU compared to 1.1+.1 RU in

Stat3 Flx/Flx littermate controls (p<.05) (Figure 6E). By comparison, we did not observe differences

between genotypes at day 28 for colonic expression of CXCL1 , CCL5, or CCL20 , supporting the specificity of the differences observed (Suppl. Figure 4E, F&G).

Colonic expression of IL-6, IL-10 , IFN g and IL-17A correlates with histologic severity and colonic

+ +

Ì Í Î expression of IL-17A correlates with the frequency of pSTAT3 CD3 T-lymphocytes in Stat3 Ë

mice at day 28. We then used linear regression analysis to determine whether the colonic expression of

Ù Ú ×

IL-6, IL-10 , Ø , and/or IL-17A was associated with the histology scores. We found that colonic

± ² ³

expression of IL-6 was associated with increased histologic score in Stat3 ° mice (r=.76, p=.03) as was

Ù Ú × IL-10 (r=.77, p=.02), Ø (r=.82, p=.01), and IL-17A (r=.77, p=.03). However there was no significant correlation in the Stat Flx/Flx littermate controls for any of these cytokines. We next asked whether the

frequency of CD3 +/pSTAT3 + T-lymphocytes per HPF correlated with IL-17A colonic mRNA

± ² ³ expression, and found that this was the case in the Stat3 ° mice (r=.90, p=.002) but not in the

Stat3 Flx/Flx littermate controls.

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Willson et al.

Discussion

In the present study we have demonstrated that the loss of IEC Stat3 promotes the development

of chronic colitis after administration of DSS, in terms of increased degree of histologic severity,

± ² ³ reduced survival, and reduced colon length. Body weight was significantly reduced in Stat3 ° mice as compared to Stat3 Flx/Flx littermate controls after removal of DSS at day 10 which was followed by a rapid recovery by day 12 in both genotypes. This is not surprising in that C57BL/6J mouse strain has been shown to develop chronic colitis while returning to their original weight 3 to 4 weeks after removal of

± ² ³ DSS . Since some of the Stat3 ° mice died around day 12 it is likely that those animals would have had a significant negative impact on the body weight, colon length, and histological score parameters,

EC ± however we nevertheless observed significant differences at day 28 in the Stat3 ° cohort.

IEC Stat3 deficient mice exhibited more severe histologic injury at day 7, recapitulating what has

previously been published, and here we show that they continue to exhibit more severe disease through three weeks of water recovery at day 28. The severity was worse in all areas scored including area involved, ulceration, crypt loss, and inflammatory infiltrate. Over the course of the 21 day water

Flx/Flx

± ² ³ consumption Stat3 littermate controls and Stat3 ° mice were able to significantly reduce ulceration and crypt loss when comparing day7 to day 28 within genotypes. This is thought to be mediated by intestinal epithelial cell proliferation, survival and wound healing responses. However, the

immune cell score remained high in both genotypes from day 7 to day 28. This suggests that those

± ² ³ Stat3 ° mice that survived were able to induced regenerative responses to the initial DSS induced injury regardless of a loss in Stat3 activation within the epithelium but however these mice still had more severe colonic injury compared to Stat3 Flx/Flx littermate controls at day 28.

Upon evaluation of pSTAT3 within the epithelial crypts we observed that at day 7 more Stat3 activation was induced than at day 28 in the Stat3 Flx/Flx littermate controls. We did not evaluate this at

3-17

Willson et al. the biochemical level, as epithelial preps from ulcerated and colitic tissue can be contaminated by other cell populations. Secondly, we elected to stain for the activated phosphorylation site of Stat3 within the

IEC cellular niche. This allowed us to gain better insight into the relative area in which Stat3 was induced and how it relates to active or disease free areas of the colon and the patchy nature of the DSS colitis model. Thus we concentrated our efforts within 0.5cm from the ulcer border or similar areas of the colon if no ulcer was present. Our studies at day 7 revealed, as previously published, that the

Stat3 Flx/Flx mice had a greater epithelial proliferative response however at day 28 there was no difference

± ² ³ between genotypes. Epithelial cell survival was not reduced in Stat3 ° mice at day 7 however it has

been suggested that the most significant portion of apoptosis occurs within the first 2 or 3 days of DSS administration and therefore we may have missed this difference between genotypes at day 7 43 .

Furthermore, there was also no difference for IEC apoptosis at day 28 between genotypes. Both

genotypes exhibited an improvement in crypt loss and ulceration between day 7 and 28, although these

± ² ³ remained more severe in the Stat3 ° mice. We did not observe a compensatory increase in the

frequency of crypt epithelial cells expressing the activated forms of p65, pSTAT1, or pSTAT5 at day 7

± ² ³

or day 28 in the Stat3 ° mice. However, we did observe a persistent defect in expression of the Stat3

Ó Ô Õ Ö Ò Ó Ô Õ × ° ± ² ³ dependent epithelial restitution genes Ò and in the Stat3 mice, which likely contributed to

the relative defect in wound healing 10 . This may also explain why patients carrying the JAK2 risk allele

¸ Ð have reduced colonic expression of R Û and why they may be at greater risk for development of

IBD 5. Furthermore, recent data in UC patients associated the level of SOCS3 activation within the

epithelium with a shorter time to relapse and more severe inflammation during relapse hypothesizing

that the increased SOCS3 may make the epithelial cells more susceptible in various conditions. 44

Chemokines are small chemotactic cytokines that regulate the trafficking and migration of

leukocytes 45 . Previous reports in C57BL/6J mice have determined that expression of CXCL1 , CXCL2 ,

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Willson et al.

CXCL10 , CCL2 , CCL3 and CCL4 remain elevated during the chronic phase of DSS induced colitis 46 .

Our results were consistent with these prior reports in that we observed increased expression of CXCL2 ,

± ² ³ CXCL10 , CCL2 and CCL4 in Stat3 ° mice in the setting of more severe chronic colitis which is likely contributing to the differences in the inflammatory infiltrate observed at day 28. It will be important in future studies to determine whether the Stat3 deficient epithelium is the source of these chemokines, and

IEC if so, whether Stat3 directly regulates their expression. The cellular infiltrate in Stat3 ° mice

remarkable showed an increased frequency within the lamina propria of pSTAT3 + cells including

pSTAT3 + -lymphocytes along with increased frequency of total M and T-lymphocytes (data not shown) compared to Stat3 Flx/Flx littermate controls. The frequency of pSTAT3 + cells within the lamina propria was positively correlated with a more severe histologic score suggesting that these cells are either induced in response to the healing process or are directly mediating worse disease.

SOCS3 , a direct downstream target of activated Stat3, was increased in expression in the

± ² ³

Stat3 ° mice which we believe is consistent with the pronounced pSTAT3 infiltrate within the lamina

± ² ³ propria. Colonic IFN expression was increased at both day 0 and day 28 in the Stat3 ° mice. We did

not observe qualitative differences in pSTAT1 activation in the epithelial cell compartment at these time

¯ ½ » ¡ ¢ ¦ ª ¥ ¦ § ¨ Á ª ¥ « « ¨ ¥ ¢ points. However, as ¼ , and IL-4 expression did not differ between

± ² ³ genotypes, this likely represents a basal Th1 skewing in the Stat3 ° mice . Colonic IL-17A

IEC expression was increased only at day 28 in Stat3 ° mice, in the setting of chronic colitis. IL-17A is released by Th17 inflammatory T-cells, shown to involve direct binding of Stat3 to the IL-17A

promoter, and known to be involved in many chronic autoimmune disorders 48-50 . We did not detect a

Ö Ý Ú Ù

difference between genotypes for expression of IL-10 , IL-6, IL- Ü or , suggesting that there was

± ² ³ not a global difference in activation of mucosal inflammatory pathways in the Stat3 ° mice.

Consistent with an effector function, we observed a strong correlation between the frequency of

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Willson et al.

pSTAT3+ T lymphocytes and IL-17A expression, and between IL-17A expression and histologic injury.

Future studies employing Th17 blockade will be required to determine whether this will ameliorate chronic colitis in this setting. In summary, we have shown that loss of IEC Stat3 leads to more severe chronic inflammation following acute injury which is not accounted for by a sustained defect in epithelial proliferation or apoptosis, but rather expansion of pSTAT3+ lymphocytes and up-regulation of

IL-17A expression.

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Willson et al.

Acknowledgements

We would like to thank Eleana Laws, MS and Erin Molden, MS for excellent technical assistance. This

(1P30DK078392-01), and NIH grants R01 DK078683 and DK068164 (LAD).

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Willson et al.

Figures and Tables

Figure 1

3-223-22 Willson et al.

Ì Í Î Figure 1. Stat3 Ë mice exhibit more severe acute and chronic colitis after DSS administration.

(A) Schematic representation of the DSS-induced colitis model. Mice lacking intestinal epithelial Stat3

± ² ³ (Stat3 ° ) and littermate controls were given 4% DSS for 7 days followed by consumption of water alone for 21 days. (B) Colon histologic injury was determined as shown. (C) Survival (n>21 mice per genotype) and (D) weight loss were determined and are shown. (E) Colon length was measured as shown with (F) representative images of the gross appearance. Data are shown as mean + SEM.

Differences between genotypes were analyzed by (B and E) one-way ANOVA or (D) two-way

ANOVA, with Bonnferroni post-test (*, p < .05; ** p < .005; *** p < .0001).

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Willson et al.

Figure 2

3-24 Willson et al.

Flx/Flx

Ì Í Î Figure 2. All histologic sub-scores are increased in Stat3 Ë mice compared to Stat3 littermate controls at day 28. (A) Representative images from the same segment of the colon at day 0 and day 28 utilizing the anal verge as an anatomical landmark. Original magnification was 100x. (B)

Area involved, (C) epithelial monolayer ulceration (D) crypt loss and (E) extent of immune cell infiltration are shown at day 7 and day 28 (n>5 mice per group, per genotype). Data are shown as mean

+ SEM. Differences within (dotted line) and between (solid line) genotypes were analyzed by one-way

ANOVA with Bonnferroni post-test (* p < .05; ** p < .005; *** p < .0001).

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Willson et al.

Figure 3

3-263-26 Willson et al.

Ì Í Î Figure 3. IEC proliferation and apoptosis do not differ at day 28 between Stat3 Ë mice and littermate controls. Imuno-histochemical analysis of (A) phophos-STAT3 quantified as (B) IECs positive per crypt and proliferation measured by BrDU incorporation shown in (C) expressed as a

proliferation index (D) (average of 12 properly oriented crypts/mouse n>5 mice per genotype) and apoptosis measured by (E) cleaved caspase-3 quantified as (F) positive cells per HPF (n>5 mice per

Flx/Flx

± ² ³ genotype and three fields counted per mouse) in colons from Stat3 mice and STAT3 ° mice is shown. Analysis was performed in .5 cm area of mucosa that bordered ulcers in the distal and middle colon or in corresponding areas of the colon if no ulcer was found. Data are shown as mean + SEM.

Differences within and between genotypes were compared by un-paired t-test (*p < .05 and ** p <

.005).

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Willson et al.

Figure 4

3-283-28 Willson et al.

Ì Í Î Figure 4. Chronic colitis in Stat3 Ë mice involves Stat3 activation in the non-epithelial compartment. Colon sections were quantified by fluorescent microscopy for hosphor-Stat3 and the

frequency of (A) enterocyte pSTAT3 positive cells and (B) lamina propria pSTAT3 positive cells in 10

random high powered fields (HPF; 400x magnification) was determined in Stat3 Flx/Flx littermate controls

± ² ³ and Stat3 ° mice at day 28 (n > 6 mice per group). Immunofluorescent labeling of hosphor-Stat3 and (C) (F4/80 +) macrophages and (D) (CD3 +) T-lymphocytes was used to determine the frequency of

double positive cells in 10 random high powered fields (HPF; 400x magnification) of Stat3 Flx/Flx

± ² ³

littermate controls and Stat3 ° mice at day 28 (n > 6 mice per group, positive cells are marked by

¥ ¢ ¬ ¢ ¥ ¹ ¥ ¢ ¢ ¡ « ¨ ¢ ¡ ¢ £ ¢ ¢ ¥ ¦ ¥ ¢ · ¢ ¢ ª ¡ ¡ § ¥ £ ¥ ¥

arrowhead). (E) ß the frequency of lamina propria pSTAT3+ cells per HPF and histological severity measured by the histology score at

Flx/Flx

± ² ³ day 28 in Stat3 littermate controls (r = .-17, p = .67) and Stat3 ° mice (r = .77, p = .03). Data are shown as mean + SEM. Differences between genotypes were compared by un-paired t-test (*, p < .05;

** p < .005; *** p < .0001).

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Willson et al.

Figure 5

3-30 Willson et al.

Figure 5. Colonic expression of Stat3 target genes that mediate epithelial restitution is decreased

Ë Ì Í Î

while SOCS3 , IFN- Ñ , and IL-17A expression are increased in Stat3 mice at Day 28. Real-time

Ó Ô Õ Ö Ò Ó Ô Õ × RT-PCR was performed on mRNA isolated from whole colonic serial sections for (A) Ò (B)

Flx/Flx

° ± ² ³ (C) SOCS3 (D) IL-6 (E) IFN-× and (F) IL-17A expression in Stat3 littermate controls and Stat3 mice at day 0 and day 28 (n>7 mice per genotype). Data are normalized to b-actin mRNA expression and shown as mean + SEM. Differences between genotypes at day 0 and day 28 were compared by unpaired t-test (*, p < .05; ** p < .005; *** p < .0001).

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Figure 6

3-32 Willson et al.

Figure 6. Colonic expression of CXCL2 , CXCL10 , CCL2 , and CCL4 is significantly increased in

Ì Í Î Stat3 Ë mice at Day 28. Colon mRNA expression was measured by quantitative real time PCR for

(A) CXCL5 (B) CXCL2 (C) CXCL10 (D) CCL2 and (E) CCL4 in Stat3 Flx/Flx littermate controls and

± ² ³ Stat3 ° mice at day 0 and day 28 of the colitis model (n>7 mice per genotype). Data were normalized to b-actin mRNA expression and are shown as the mean + SEM (*, p < .05; ** p < .005; *** p < .0001

by unpaired t-test).

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Figure 7

3-34 Willson et al.

Figure 7. Colonic expression of IL-6, IL-10 , IFN-y and IL-17A correlates with histologic severity

and IL-17A expression correlates with the frequency of colon pSTAT3 +CD3 + T-lymphocytes in

Ì Í Î

Stat3 Ë mice at day 28. Pearson regression analysis was used to test for an association between

Ù Ú × normalized colonic expression of (A) IL-6 (B) IL-10 (C) Ø and (D) IL-17A and histologic severity score. E) Pearson regression analysis was used to test for an association between IL-17A colonic expression and the frequency of CD3 +/pSTAT3 + T-lymphocytes per 400x high-powered field (r=.90,

p<.002) (n>7 mice per genotype). Data are shown as mean + SEM (*, p < .05; ** p < .005; *** p <

.0001 by unpaired t-test).

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Tables

Table 1. Real-time quantitative reverse transcription- PCR (RT-PCR) Primers Gene Forward Reverse

STAT3 Signaling

â â

á á à á á

IL-6 à -CAAAGCCAGAGTCCTTCAGAGAGATAC- -ATTGGATGGTCTTGGTCCTTAGC-

â â

á á à á á

IL-10 à -GGT TGCCAAGCCTTATCGGA- -ACCTGCTCCACTGCCTTATCGGA-

â â

á á à á á

SOCS3 à -GCTCCAAAAGCGAGTACCAGC- -AGTAGAATCCGCTCTCCTGCAG-

â â

à á á à á á

RegIII ã -TCCCAGGCTTATGGCTCC TA- -GCAGGCCAGTTCTGC ATC A-

â â

ä å æ ç ç ç è

á á à á á à -TTCCTGTCCTCCATGATCAAAA- -CATCCACCTCTGTTGGGTTCA-

p65 Signaling

â â

ç é ê ë

á á à á á à -TGGCGAGGTCTGACTGTTGTGG- -GCTCATCAAAAAGTTCCCTGTGC-

Cytokines

â â

à á à ì á

IL-4 -TGTACCAGGAGCCATATCCAC- á -GTTCTTCGTTGCTGTGAGGAC-

â â

í ã à á à á á

IL- -CAGAGTTCCCCAACTGGTACAT- á -AAGGAGGAAACACAGGCTCTCT-

â â

ç î ï è

á á à á á

à -CATTCATGAGTATTGCCAAGTTTG- -GCTGGATTCCGGCAACAG-

â â

ð ï î ë

á á à á á

à -AGGCAGGTTCTGTCCCTTTCA- -AGATAGCAAATCGGCTGACGG-

â â

á á à á á IL-17A à - GCTCCAGAAGGCCCTCAGA- -AGCTTTCCCTCCGCATTGA-

Chemokines

â â

á á á à á

CXCL1/KC à -CCCAAACCGAAGTCATAGCCACAC- -TTGTCAGAAGCCAGCGTTCACCAG-

â â

á á à á á

CXCL2 / MIP-2 à -GGCTGTTGTGGCCAGTGAA- -TGTTCAGTATCTTTTGGATGATTTTCTG-

â â

á á à á á

CXCL5 / Lix à -AGCTGCGTTGTGTTTGCTTAACCG- -TTGAACACTGGCCGTTCTTTCCAC-

â â

á á à á á

CXCL10 à -GCCGTCATTTTCTGCCTCAT- -GCTTCCCTATGGCCCTCATT-

â â

á á à á á

CCL2/MCP-1 à -CACCCCATTACATCTCTTCCCC- -TGTTTCCCTCTCACTTCACTCTGT-

â â

ã à á á à á á

CCL4/MIP- í -GCCCTCTCTCTCCTCTTGCT- -GAGGGTCAGAGCCCATTG-

â â

á á á à á

CCL5 à -TTGCCTACCTCTCCCTCGCGC- -GGTTCCTTCGAGTGACAAACA-

â â

á á á à á CCL20 à -GGTGGCAAGCGTCTGCTC- -GCCTGGCTGCAGAGGTGA-

S100 products

á á à á á

S100A8 à - AGTGTCCTCAGTTTGTGCAG- -ATCCCTTGTGGCTGTCTTTG-3

â â

á á à á á S100A9 à -GTTGATCTTTGCCTGTCATGAG- -AGCCATTCCCTTTAGACTTGG-

Receptors

â â

á á à á á CXCR2 à -AGCAAACACCTCTACTACCCTCTA- -GGGCTGCATCAATTCAAATACCA-

3-36

Willson et al.

Supplemental Figures

Supplemental Figure 1

3-37 Willson et al.

Supplemental Figure 1. Characterization of Stat3 Intestinal Epithelial cell deficient mice

Flx/Flx

Ì Í Î (Stat3 Ë ) compared to littermate controls (Stat3 ). (A) Representative immunofluorescence images of phospho-STAT3 (Tyr705) staining in the colon of mice injected for 4hrs with 200mg/kg

endotoxin showing robust epithelial STAT3 activation in Stat3 Flx/Flx littermate controls and only STAT3

± ² ³ activation is the lamina propria of Stat3 ° mice. (B) Western blot analysis of phophos-STAT3

(Tyr705) in isolated colonic enterocytes 4hrs after injection of 200mg/kg endotoxin showing enhanced

Flx/Flx

± ² ³ Stat3 activation in Stat3 littermate controls and loss of Stat3 activation in Stat3 ° mice. (C) IECs

Flx/Flx

± ² ³

from the ileum and colon of Stat3 and Stat3 ° mice were isolated and assessed for expression of

ß -actin shown as a reaction control.

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Willson et al.

Supplemental Figure 2

3-393-39 Willson et al.

ñ ò ó ó ô õ ö õ ÷ ø ù ô ú û ü ò ý õ þ ÿ ¡ ø û ¢ ù ø û £ ÷ £ ¤ ¥ ú ¦ § ¨ ñ © © ù ÷ ñ © © ¨ ó ô ù ò û ô õ ¡ £ ö ó õ ÷ ù ø £ ý mechanisms for protection of IECs during colitis in a STAT3 deficient epithelium, did not vary

Flx/Flx

Ì Í Î qualitatively in Stat3 Ë mice compared to Stat3 littermate controls. Imuno-histochemical analysis of (A) phophos-p65 (Ser276) quantified as (B) IECs positive per crypt within .5cm of disease area expressed as a labeling index (average of 12 properly oriented crypts/mouse n>5 mice per

genotype). (C) Real-time RT-PCR was performed on mRNA isolated from whole colonic serial sections

¼ Ï µ ¦ ¡ ¥ ª ¦ £ ¢ ¥ ¹ ¥ · ª ¡ Þ ¥ ¦ ® ¥ « µ ¦ ¥ ¥ ¶ ¥ ¢ ¢ ¡ ¡ ¸

´ Flx/Flx Ð

for Ê littermate

¡ ª ¥ ¦ ¨ Â ¦ ¦ ¨ À Ã ¡ ª ¥ ¥ ¹ ¥ ¨ ¥ Å Ä ¥ « ¡ É ¥ ¦ º

´ ´ ´

± ² ³ controls and Stat3 ° -actin mRNA expression. (D)Representative Imuno-histochemical images of consecutive fields in colonic tissue stained for phospho-STAT1 and phospho-STAT3 at day 28 showing little to no STAT1 activation in the epithelium or lamina propria in both genotypes. (E) Representative image of colonic tissue

Flx/Flx

± ² ³ stained for phosphor-STAT5 (Tyr694) at day 28 in Stat3 littermate controls and STAT3 ° mice.

Analysis was performed in .5 cm area of mucosa that bordered ulcers in the distal and middle colon or in corresponding areas of the colon if no ulcer was found. Data are shown as mean + SEM. Differences within and between genotypes were compared by un-paired t-test (*p < .05 and ** p < .005).

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Willson et al.

Supplemental Figure 3

3-413-41 Willson et al.

Supplemental Figure 3. Neutrophil mediated responses during chronic colitis in Stat3 Flx/Flx

Ì Í Î littermate controls and Stat3 Ë mice. (A)Real-time RT-PCR was performed on mRNA isolated from whole colonic serial sections for phagocytic products (A) S100A8 and (B) S100A9, known to be

Flx/Flx

± ² ³ involved in neutrophil chemotaxis and function, in Stat3 littermate controls and Stat3 ° mice at day 0 and day 28 (n>7 mice per genotype). Colon sections were quantified by fluorescent microscopy of neutrophil elastase which is secreted by neutrophils during inflammation. The frequency of (C) lamina propria neutrophil elastase positive cells in 10 random high powered fields (HPF; 400x

Flx/Flx

± ² ³ magnification) was determined in Stat3 littermate controls and Stat3 ° mice at day 28 (n > 6 mice

per group). (D) Representative image showing neutrophil elastase positive cells marked by arrow head.

(E) Real-time RT-PCR was performed on mRNA isolated from whole colonic serial sections for

CXCR2, a receptor that mediates neutrophil migration to sites of inflammation, in Stat3 Flx/Flx littermate

± ² ³

controls and Stat3 ° mice at day 0 and day 28 (n>7 mice per genotype). All Real-time PCR data are

« ¡ É ¥ ¦ º ´ -actin mRNA expression. Data are shown as mean + SEM. Differences between genotypes were compared by un-paired t-test (*, p < .05).

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Willson et al.

Supplemental Figure 4

3-433-43 Willson et al.

CCL5 and CCL20 expression do not vary between genotypes at Day 0 or Day 28. Real-time RT-

PCR was performed on mRNA isolated from whole colonic serial sections for (A) IL-10 (B) IL-4 (C)

Æ Flx/Flx

Ú Ù Ü Ö

Ý (D) IL- (E) CXCL1 (F) CCL5 and (G) CCL20 expression in Stat3 littermate controls and

± ² ³ Stat3 ° mice at day 0 and day 28 (n>7 mice per genotype). Data are normalized to b-actin mRNA expression and shown as mean + SEM. Differences between genotypes at day 0 and day 28 were compared by unpaired t-test (*, p < .05).

3-44

Willson et al.

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3-50

Chapter 4

Overall Discussion, Alternative Hypotheses and Future Directions

Introduction Estimates suggest that 1.4 million individuals in the United States are suffering from

IBD. IBD pathogenesis is multifactorial involving the gut flora, epithelial barrier function, and innate and adaptive immunity. The precise etiology remains unclear, but evidence suggests that it involves dysregulation of the host immune response to luminal flora. Augmented STAT3 activation has been well documented in both human and murine colitis. Transient activation induces protective mechanisms but persistent activation furthers disease progression and ultimately malignant transformation. Therefore, deciphering the cellular, temporal and transcriptional context of STAT3 activation as it relates to mucosal inflammation is of great interest. In murine models, Stat3 activation in intestinal epithelial cells is required for acute wound healing responses via induction of Reg3 family members, but also promotes development of colitis-associated cancer during chronic inflammation 1,2. Stat3 activation in myeloid cells mediates anti-inflammatory effects of IL-10; targeted deletion of Stat3 in this cell type leads to severe entero-colitis 3. Conversely, Stat3 activation in CD4+ T cells is required for differentiation of Th17 effector lymphocytes, and blockade of IL-6:Stat3 signaling ameliorates both ileitis and colitis in animal models 4,5. Tyrosine phosphorylation of Stat3 induced by IL-6 has also been linked to effector lymphocyte and granulocyte activation in IBD. We had previously shown that STAT3 activation was increased in PB granulocytes, IL-6-stimulated

CD3+/CD4+ lymphocytes, and affected colon biopsies of pediatric IBD patients at diagnosis and during therapy 4,5. We were the first to identify an IL-6:STAT3 biological network that drives leukocyte recruitment and thereby mucosal inflammation in this setting.

4-2

Figure 1. Inflammatory bowel disease susceptibility loci. The loci (depicted by lead gene name) attaining genome-wide significance are shown for CD (red), UC (blue) and IBD (black where p<5x10 -8 in CD and UC; red where p<5x10 -8 in CD and 5x10 -4 in CD). Reproduced from Gut, Lee et al., 60, New IBD genetics: common pathways with other disease, 1739-1753, Copyright (2011) with permission from BMJ Publishing Group Ltd. Large-scale genome-wide association studies (GWAS) have determined that IBD risk loci encode candidate genes involved in maintenance of the epithelial barrier, innate responses to microbial products, and differentiation and function of effector and regulatory lymphocytes.

Genotypic variation in STAT3 is linked to risk for both CD and UC and there are now 99 genetic factors that confer IBD susceptibility, with 30% shared between CD and UC (Figure 1) 6-9.

Several of the other shared susceptibility loci(IL-10, JAK2, TYK2, and IL-23) also utilize

STAT3 as the downstream signaling effector. Newly described permutations to build protein- protein interaction (PPI) networks from GWAS identified risk-associated loci, revealed that in

CD the core candidate network involved Janus-associated kinase 2 (JAK2) and STAT3 10 .

4-3

Waterman et al. evaluated the overlap between CD and UC genetic loci stratified by pathogenic pathways and by disease location and found that gene variants associated with immune response amplification and perpetuation such as IL12B (p40), IL-23R, and STAT3 associated with Th17 differentiation, and genes involved in barrier function, were similar in their prevalence between

UC and CD 11 . This data supports the hypothesis that host immune response to luminal flora seem to differentiate CD and UC and highlight the pivotal role STAT3 has in the overall course of intestinal inflammation and mucosal pathology. Evidence suggests that IBD disease course that starts earlier in life may have a stronger genetic influence however, 11 GWAS have been performed in adult IBD, compared to only two GWAS performed exclusively in early-onset disease 12-15 . These studies however have revealed that early-onset and adult disease share the majority of the identified risk loci and that the STAT3 rs744166 is associated with risk in both early-onset and adult IBD.

Pediatric versus Adult Onset IBD

IBD is diagnosed 25% of the time during childhood and estimates suggest 100,000 children in North America currently suffer from IBD with three CD cases for every new UC

16,17 case . Childhood IBD incidence ranges from 0.3 10.9/100,000/year and recent reports suggest that pediatric-onset IBD is a sub population of IBD due to the varying phenotypic and natural history differences in young patients 18,19 . Childhood IBD incidence is on the rise as the number of younger patients (younger than 18 years) has increased significantly 20-22 . Adult- onset

IBD shows equal ratio of male to female disease with perhaps more women having the disease 17 .

However, in pediatric-onset IBD, CD has a male predominance whereas UC has a female predominance and there has been no molecular elucidation for this difference in adult versus

4-4 pediatric CD:UC gender ratio 17,23 . In early-onset disease, presentation has a more extensive anatomical involvement compared with adult disease with major lesions scattered throughout the gastrointestinal tract in CD and a pancolitis in UC 16,23 . Disease behavior is similar to adult disease, with progression from inflammatory disease to stricturing/penetrating complications however surgical need is earlier in early-onset UC but later for CD 23-25 . Amre et al. recently reported in a Canadian pediatric population that the STAT3 rs744166 SNP was significantly associated with overall susceptibility for CD and ileal disease with or without colonic involvement at diagnosis 26 . Ultimately we did not find differences for age, sex, disease location,

medication exposure, or clinical or histologic disease activity at diagnosis in patients stratified by

! " # $ % $ & ' ' % ( ( ) * + , - . . " . " in our pediatric cohort. We ultimately wanted to investigate the underlying functional consequences that could potentially infer risk for development of IBD in association with the STAT3 rs744166.

Biochemical interrogation of the IL-6:STAT3 signaling pathway

/ 0 1 2 3 ! - ! " # $ % $ & ' ' % ( ( ) * + , - . . " . " * + - + + 0 4 * - " 3 5 * ! * 2 4 ) " - + " 3 W" cellular STAT3 activation in peripheral blood leukocytes. To dissect the mechanism of enhanced cellular

STAT3 responsiveness we utilized EBLs (Epstein Barr virus (EBV) transformed lymphoblastoid cell lines) created from patients with IBD. These cell lines are a readily available and renewable resource for functional genotype-phenotype studies in humans.

However, aberrant gene expression induced by EBV transformation leads to skepticism for the usefulness of EBLs as an alternate model to primary tissues.

Caliskan et al. compared global gene expression and methylation profiles in primary B cells and transformed EBLs and revealed that most of the differences in gene expression levels

4-5

are of small magnitude, and that EBLs recapitulate the naturally occurring gene expression

variation in primary B cells 27 . This suggests that EBLs are a sufficient model for interrogating

genetic architecture that underlies regulatory variation 27 . They did find inter-individual variation

in EBV copy number that was significantly greater than the intra-individual variation, as

suggested by previous studies showing that differences in EBV copy numbers may significantly

contribute to regulatory variation across cell lines 28 . However when Caliskan et al. investigated

gene expression data they identified only 160 genes whose expression levels were significantly

associated with EBV copy numbers and found that most of these genes were involved in the anti-

apoptotic role of NFkB signaling.

Furthermore, only 33 genes were differentially expressed between primary B cells and

EBLs with a fold change greater than 1.5. Therefore, EBLs are an important in-vitro tool to

assess the biochemical differences of IL-6 induced STAT3 activation in patients carrying the

STAT3 risk allele. Furthermore, we demonstrated increased IL-6 dependent STAT3 tyrosine

! 0 + ! 0 ) 7 . - * 0 2 * 2 8 9 : + / ) 0 ; # $ % $ & ' ' % ( ( ) * + , - . . " . " - * " 2 + 4 0 ; - ) " 3 0 2 0 2

6 6 6 6 -risk allele

patients. This mirrored our findings regarding increased peripheral blood leukocyte STAT3

! " # $ % $ & ' ' % ( ( ) * + , - . . " . " < = " / 0 1 2 3 ! - 8 9 : +

tyrosine phosphorylation in patients carrying

4 - ) ) 7 * 2 > ! " # $ % $ & ' ' % ( ( ) * + , - . . " . " 0 + + " + + * 2 4 ) " - + " 3 ; " ; ? ) - 2 " ) 0 " * 2 - ? 1 2 3 - 2 4 " 0 / ! " @ : 6 6 -

6:STAT3 receptor complex (IL-6 receptor, GP130, and JAK2), in the absence of a difference in

autocrine IL-6 exposure. The enhanced membrane accumulation of the IL6R signaling complex

likely accounts for the increased cellular responses to IL-6 via STAT3 activation. We feel this is

specific to the STAT3 pathway in that we found a decrease in STAT1 activation in EBLs

* 2 > ! " # $ % $ & ' ' % ( ( ) * + , - . . " . " < $ ! " + " 3 * / / " ) " 2 4 " + * 2 4 " . . + * > 2 - . * 2 > ; - 7 3 ) * A " 3 * + " - + " * 2 ! " carr 7

4-6

subgroup of patients who carry the STAT3 risk allele via STAT3-dependent effects upon T-

lymphocyte and granulocyte differentiation, activation, and survival.

Alternative hypothesis and Future Directions

Membrane localization of IL-6R/GP130/JAK2. The increased membrane localization

+ * ) " . - " + 0 ! " # $ % $ & B % C ) * + , - . . " . " + 1 > > " + * 2 4 ) " - + " 3 . " A " . + 0 / of the Il-6R and GP130 -

synthesis and targeting to the membrane. The receptor complex via endocytosis is removed from

the membrane after IL-6 induced signaling therefore, experiments to investigate rates of receptor

internalization, degradation or retargeting to the membrane can be interpreted with pulse chase

methodology in conjunction with cycloheximide to prevent de novo protein synthesis.

Alternatively, JAK2 has been shown to mediate increased stabilization and surface expression of

E F IFN-D in Th17 cells as well as the erythropoietin receptor through favoring Golgi processing

and cell surface expression 29,30 . We could investigate this mechanism of JAK2 mediated

receptor stabilization and surface expression in EBLs utilizing SiRNA technology to reduce

JAK2 expression and then quantify the abundance of membrane IL-6R/GP130 receptor in

patients with and without the STAT3 risk allele. Furthermore, if the STAT3 risk allele is

associated with JAK2 mediated IL-6/GP130 receptor stability then heterozygous patients should

have intermediate levels of membrane receptor compared to non-risk allele and homozygous risk

allele patients.

H I H J K The STAT3 splice variant - G . Functional studies to determine causation have

focused on coding variants, however the STAT3 risk SNP (rs744166) is located within the intron

between exon 1 and exon 2 and may be predicted to regulate gene expression. We did not

0 ? + " ) A " - + + 0 4 * - * 0 2 ? " 5 " " 2 ! " # $ % $ & ' ' % ( ( ) * + , - . . " . " - 2 3 ; % " M ) " + + * 0 2

6 L E in colonic

biopsies but did find an increase in unstimulated EBLs that could not be explained by IL-6

4-7

$ % $ & N - 2 3 # $ % $ & O <

induced autocrine activation. STAT3 has two distinct isoforms, # The

. * 4 * 2 > A - ) * - 2 # $ % $ & O 1 + " + - 2 - . " ) 2 - * A " - 4 4 " 0 ) + * " 5 * ! * 2 " M 0 2 & ! - . " - 3 + 0 - ) 1 2 4 - " 3

6 6 s F

isoform lacking the C-terminal transactivation domain that acts as a dominant negative inhibiting

0 " 2 * - . 0 / # $ % $ & O 5 - + * 2 A " + * > - " 3 - 2 3

STAT3 activation. Recently the antitumorigenic 6

3 0 > " 2 0 1 + # $ % $ & O . " - 3 0 - 0 0 + * + - 2 3 4 " . . 6 induction of en 6 -cycle arrest in cell lines with

persistent STAT3 tyrosine phosphorylation 31 . This group also found down-regulation of

$ % $ P O # the proliferative antagonist isoform of STAT1 which is thought to primarily promotes

growth arrest, apoptosis, and anti-tumor immunity downstream of type I and II interferons 32 31 .

$ % $ & N - 2 3 O * + 0 / 0 ) ; + 4 0 1 . 3 ! - A "

This not only infers that a differences in the ratio of #

" # $ % $ & O * + 0 / 0 ) ; 4 0 1 . 3 ; " 3 * - "

profound effects on gene expression but also indicates that t !

. " A " . + 0 / # $ % $ P N ? 7 4 0 2 ) 0 . . * 2 > # $ % $ P O " M ) " + + * 0 2 < 6 This could have profound effects on T

lymphocyte and granulocyte activation and gene expression. Primers utilized for EBL

experiments and the Affymetrix probe set for STAT3 in the Human Gene U1333 Plus 2.0 array

for colonic biopsy mRNA expression do not distinguish between the different isoforms for

! - ! " # $ % $ & B % C ) * + , - . . " . " 4 0 1 . 3 ? " - + + 0 4 * - " 3 5 * ! -

STAT3. Therefore, it is plausibl "

$ % $ & N - 2 3 # $ % $ & O

difference in the ratio of the # the dominant negative form that would

* 2 ! * ? * # $ % $ & - 4 * A - * 0 2 ? 7 ? * 2 3 * 2 > ! " - A - * . - ? . " # $ % $ & N * + 0 / 0 ) ; - / " ) + * ; 1 . - * 0 2 < $ ! * + 4 0 1 . 3

also explain why patient EBLs null for the STAT3 risk allele have higher levels of pSTAT1

2 ! - ! " # $ % $ & O * + 0 / 0 ) ; 3 0 5 2 ) " > 1 . - " + ! " # $ % $ P O * + 0 / 0 ) ;

activation after IL-6 stimulation *

. . 0 5 * 2 > * 2 4 ) " - + " 3 # $ % $ P N + * > 2 - . * 2 > - . The Utilization of RNA-Seq with Next generation

sequencing technology from both EBLs and colonic biopsies would allow the assessment of total

# $ % $ & N R # $ % $ & O 0 ) - 2 1 2 , 2 0 5 2 ; 1 - 2 S

STAT3 transcripts Q between STAT3 risk allele and

non-risk allele patients. Moreover there are no known constitutively activation mutations in

4-8

STAT3, and future work will ultimately require sequencing of the entire gene and its surrounding genomic sequence to delineate the genetic basis for differences in cellular STAT3 responses.

Phenotypic outcomes in STAT3 risk allele patients

B % C ) * + , - . . " . " " M ! * ? * " 3 * 2 4 ) " - + " 3 4 0 . 0 2 * 4 " M ) " + + * 0 2 Patients carrying the STAT3 6 of chemokines located on 4q12-q13 (IL-8, CXCL2 , and CXCL3) and S100A8, S100A9 and

S100A12 . Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, known as damage associated molecular patterns, are found at high concentrations in inflamed tissue and have been shown to be involved in neutrophil chemotaxis 33 . Increased mucosal release correlates with fecal markers of IBD disease activity, and in myeloid progenitor cells up- regulation of S100A8 and S100A9 was shown by direct binding of STAT3 to the gene promoter

via chromatin immunoprecipitation 33,34 . Here we demonstrate that S100A8, S100A9 and

% C ) * + , - . . " . " R * 2 ! " - ? + " 2 4 " 0 / - 2 S100A12 are up-regulated in CD patients carrying the STAT3 C overall difference in clinical or mucosal disease activity. Furthermore, we did not find differences in the expression of genes classified in other pathways such as immune and

inflammatory mediators, cancer and cell proliferation, ECM tissue remodeling, or metabolism.

% C ) * + , - . . " . " ; - 7 ! - A " 1 2 3 " ) . 7 * 2 > ? * 0 . 0 > 7 ! - This suggests that patients carrying the STAT3 B leads to increased neutrophil chemotaxis and activation. However, future studies which directly measure neutrophil chemotaxis will be required to test this.

We did investigate pathways which are involved in neutrophil mobilization since they are closely associated with the outcome of inflammation. 35,36 . It has been shown that STAT3 regulates CXCR2 expression during mobilization responses and CXCR2 binds IL-8 and CXCL6

4-9 to promote neutrophil migration while CXCL1, -2, -3, and -5 enhance neutrophil chemoattractant activity. 37-39 . In murine models of chemically induced colitis small molecule antagonism of the CXCR2 receptor or genetic deletion reduces MPO (neutrophil) activity, colonic damage and clinical symptoms 40,41 . Thus we evaluated the frequency of neutrophils

expressing pSTAT3, and the cognate receptor for IL-8, CXCR2+, and found them to be

% C ) * + , - . . " . " < T 0 2 + * + " 2 increased in colonic biopsies from CD patients carrying the STAT3 C with the murine studies, we found that the frequency of pSTAT3+ or CXCR2+ neutrophils was highly correlated with histologic severity. Importantly, overall lamina propria cellularity measured within the same biopsies from which we scored the above parameters did not vary for mononuclear or polymorphonuclear sub-scores. These data demonstrate that current clinical scoring systems are not able to distinguish between these differential pathways driving disease and also suggest the utility of fecal calprotectin as a plausible biomarker for this sub-population of CD.

Alternative Hypothesis and Future Directions

IL-8 as a marker of colonic disease. In UC a colon only disease, IL-8 serum concentration has been related to endoscopic and histological severity and IL-8 seems to be a reliable biomarker closely related to disease activity. CD patient neutrophil migration to the gut lumen has been shown to be a feature of colonic disease irrespective of associated ileal lesions 42 .

Utilizing whole gut lavage, twenty-seven of 36 patients with isolated colonic Crohn's disease had detectable neutrophil elastase, a neutrophil-bound enzyme, whereas 3 of 15 with small-bowel involvement alone had detectable neutrophil elastase 42 . This supports our results in that IL-8 seems to drive neutrophil mediated migration in colonic mucosal inflammation of which we interrogated in colonic biopsies from our pediatric CD patient cohort. Our patient population

4-10 had approximately 40% colon only disease in both the non-risk allele and risk allele genotypes.

Therefore differences we see in colonic expression of IL-8, CXCL2, and CXCL3 and frequency of neutrophils expressing pSTAT3, and the cognate receptor for IL-8, CXCR2+ may be even more enhanced in CD with colon only or severe colonic involvement at diagnosis. We did not

investigate UC patients but it would be predicted that UC has an even greater difference in gene

B % C ) * + , - . . " . " < expression for these neutrophil mediated pathways with carriage of the STAT &

IL-6 classical or trans-signaling? One of the most intriguing and unexplainable

findings was that the serum level of IL-6 was significantly reduced in patients carrying the

% C

STAT3 C risk allele (Figure 2). Compared to other circulating cytokines known to be

S R 1 ; 0 ) 2 " 4 ) 0 + * + / - 4 0 ) - . ! - $ N

F E L U upregulated in IBD including soluble IL-2R (sIL- Q ) and interleukin-5(IL-5), IL-6 was the only one that was increased in our CD patient cohort at diagnosis. This supports our previous publication showing that in newly diagnosed CD patients, a pro-inflammatory IL-6:STAT3 induced biologic network drives mucosal disease. One

explanation could be that we only evaluated circulating IL-6 in a small number of patients or that

% C patients carrying the STAT3 B risk allele have increased STAT3 activation that enhances negative feedback mechanisms. However the former conclusion it is hard to connect with overall reduced circulating IL-6. Even more interesting is that with suppressed circulating IL-6

levels we still found an increased frequency of peripheral blood leukocytes with basal STAT3

* 2 - * " 2 + 4 - ) ) 7 * 2 > ! " # $ % $ & B % C ) * + , - . . " . " < activation 6 An alternative hypothesis would be that classical and trans-signaling mechanisms of IL-6 induced STAT3 activation vary with carriage of the STAT3 risk allele. Thus patients with the STAT3 risk allele may have increased

IL-6/sIL-6R(soluble IL-6) complexes that also induce STAT3 activation but have bound the free

IL-6 making it

4-11

200 2500

2000 150

(pg/mL) 1500 a 100 1000 50 500 Serum TNF Serum

0 (pg/mL) sIL2R Serum CTL GG AG_AA 0 CTL GG AG_AA

10 Serum IL-5 (pg/mL) IL-5 Serum

0 CTL GG AG_AA

Figure 2. Circulating levels of Inflammatory cytokines. Serum cytokine levels were measured by ELISA and shown as the mean + SEM. CTL, healthy control; GG, AG_AA, CD at diagnosis null, heterozygous and homozygous for the STAT3 risk allele, (n = 3 - 17), *p<.05; **p<.01; ***p<.001 by t-test.

undetectable by the IL-6 ELISA. Therefore suggesting the STAT3 risk allele patients have increased basal levels of trans-signaling.

Not all cell types express the IL-6R, however GP130 is expressed ubiquitously in the body. In chronic intestinal inflammation the IL-6R is cleaved through ectodomain shedding from the cell surface of innate immune cells and is thus free to bind free IL-6 and induces IL-6- sIL-6R trans-signaling in cells devoid of the IL-6R 43 . This mechanism has been shown to enhance lymphocytes proliferation and survival 44 . We stimulated our cells with IL-6 and the sIL-6R to make sure we induced similar levels of STAT3 activation regardless of the cellular receptor expression. We also investigated the frequency of CD4+ T-lymphocytes and

4-12 granulocytes expressing the IL-6(CD126) and GP130(CD130) receptor by flow cytometric analysis and found no difference with carriage of the STAT3 risk allele (data not shown).

However, this method may not be sensitive enough to detect differences of the receptor at the protein level. Therefore, quantification by western blot analysis of the receptor complex in the membrane and cytosolic fractions along with the comparison of STAT3 activation in response to

IL-6 alone or in combination with the IL-6R would help decipher classical or trans-signaling mechanisms in peripheral blood leukocytes. Trans-signaling is also thought to be highly

regulated by sGP130 (soluble gp130), that exist in high concentrations within the blood 45 . sGP130 binds to the IL-6 sIL-6R complex and thereby inhibits the binding of the IL-6 sIL-6R complex to the membrane bound GP130 45,46 . Interestingly there are no reports on circulating levels of this sGP130 in human IBD. Administration of a synthetic soluble GP130 (gp130-Fc) inhibits Stat3 phosphorylation and ameliorates colitis in the SAMP1/Yit murine model 47 . The

SAMP1/Yit murine model is the most closely related model of human CD as these mice spontaneously develop ileal-colonic disease very similar to CD. IL-6 trans-signaling has been shown to mediate chronic murine colitis and development of colonic cancer 4,43,48,49 . However much less is known about this mechanism in human IBD. It has been shown that sIL-6R levels are significantly elevated in patients with active IBD compared to inactive IBD and increased IL-

6 and sIL-6R production was also observed in the lamina propria during active IBD 4,50 . Thus, sIL-6R molecules are able to bind free IL-6 and interpreting circulating free IL-6, sIL-6 and sGP130 in IBD patients is a necessity. A very recent publication has elegantly shown by in-vitro culture systems and mice that depending on the molar excess of IL-6 over sIL-6R, sgp130Fc plays a role in blocking trans-signaling because free IL-6 will not or will only partially be trapped in IL-6·sIL-6R·sgp130Fc complexes 51 . We currently do not yet have a clear

4-13 understanding of these concepts in IBD patients and further studies are warranted. This may be why the humanized anti-IL-6 receptor monoclonal antibody, Tocilizumab that blocks both the membrane-bound and the soluble form of IL-6R has marginal clinical efficacy in CD patients 52,53 . We ultimately did find increased circulating IL-6 in both STAT3 risk allele and non-risk allele genotypes compared to control. However, since we encountered such dramatic

differences as a function of gneotype it may be very interesting to investigate the level of free IL-

- 2 3 + V W P & X * 2 - * " 2 + + ) - * / * " 3 ? 7 ! " # $ % $ & B % C ) * + , - . . " . " < 6, free sIL-R, IL-6-sIL-6R 6

The JAK2/STAT3 axis as a biomarker for disease. Researchers are avidly trying to define how these loci affect disease phenotypic expression, clinical course, and treatment modalities. The increase in relative risk conferred by individual risk alleles may only be moderate, however the combined associated risk may be substantial. Therefore, studies in patients carrying multiple risk alleles assessed and related to disease location, disease behavior, medical therapy, and remission will ultimately delineate a more personalized medical approach.

Combined genetic variants in genes that utilize STAT3 as a downstream effector include IL-6,

JAK2, IL-10, IL-23, and IL-27. Associations with clinical disease phenotype and gene expression profiles of biopsies from inflamed and non- inflamed tissue for the same location as it relates to CD or UC at diagnosis and during treatment would allow interpretation of this genetic

JAK2/STAT3 axis in disease behavior. We have interrogated only one of the these SNPs,

STAT3 rs744166. The complexity is immense, exemplified by our studies that show individual

JAK2 or STAT3 alleles can mediate signaling and gene expression outcomes in combined

JAK2/STAT3 analysis (data not shown). This indicates a matrix of possibilities in interpreting multiple SNPs and warrants that future studies assessing risk loci as biomarkers be as coherent as

4-14

possible in defining known IBD risk loci in patient demographics for enhanced interpretation of

the combined associated risk.

Therapeutic targeting of STAT3. While therapeutic options have increased over the past

decade, our ability to target newer biologic therapies to specific subgroups of patients has lagged

behind. Data from human and murine models of colitis indicate that STAT3 may be an important

target for the treatment of IBD. However, considering that STAT3 mediates both IL-10 induced

anti-inflammatory and IL-6 induced pro-inflammatory pathways at various times within the

disease course of both murine and human IBD it seems unlikely that an oral inhibitor of STAT3

would be feasible 3,58-61 . Recent clinical trials reported in abstract form have shown that the oral

Janus Kinase inhibitor, CP-690,550 (CP), is effective in moderate-to-severe UC patients in a

dose-dependent manner with improvements in clinical response and remission rates 54 . However

CP was not effective in CD 55 . The specificity of CP is for JAK1 and JAK3 over JAK2, and

JAK3 is restricted to hematopoietic cells whereas JAK1 and JAK2 are ubiquitously expressed

56,57 . The divergent result in CD versus UC may reflect differences in the underlying

pathogenesis and supports further study of specific JAK:STAT signaling pathways in these

disorders. Our data suggests that inhibition of JAK:STAT3 signaling warrants further clinical

* 2 A " + * > - * 0 2 R - 2 3 ! - + ) - * / * 4 - * 0 2 0 / T Y - * " 2 + ? 7 ! " # $ % $ & B % C ) * + , - . . " . " ; - 7 3 " / * 2 " 6

patient populations that have varying clinical efficacy to investigational agents including the oral

Janus Kinase inhibitor, CP-690,550. Thus STAT3 activation has been referred to as a double-

edged sword and investigating factors which mediate inflammation downstream of STAT3 may

9 Y B % C lead to more targeted approaches. Collectively, our studies demonstrate that the STAT3 @

risk allele (rs744166) is associated with increased cellular STAT3 activation and up-regulation of

4-15 chemokines which promote CXCR2+ neutrophil recruitment to the gut in a newly described sub- population of CD patients.

STAT3 a double-edged sword

As mentioned above STAT3 activation is a double-edged sword. Studies suggest that

IEC STAT3 activation has a crucial role in the maintenance of intestinal epithelial barrier and homeostasis. In Chapter III we examined this dichotomy as loss of IEC Stat3 promoted the development of chronic colitis after administration of DSS. IEC Stat3 deficient mice had increased degree of histologic severity, reduced survival, and reduced colon length. Body weight

Flx/Flx

[ \ ] was significantly reduced in Stat3 Z mice as compared to Stat3 littermate controls after

removal of DSS at day 10 which was followed by a rapid recovery by day 12 in both genotypes.

^ _ ` _ Murine models in which gp-130-STAT1/3 signaling is abolished (gp130 Z ) or in IL-6 deficient mice (IL-6-/-), there is exaggerated acute intestinal colitis in response to DSS as compared to wild type 58 . This was due to impaired epithelial wound healing with consistently more severe epithelial erosion 58 . This suggests that IL-6 induced STAT3 activation promotes epithelial restitution and wound repair important during the innate immune response. However, these models evaluated STAT3 contribution to innate immune response by inactivating STAT3 in multiple cells types. Thus no suitable model for the study of in-vivo epithelial STAT3 specific affects in colitis existed. During creation and characterization of our epithelial Stat3 deficient murine model Bollrath et al. and Grivennikov et al. published that IEC ablation of

STAT3 signaling resulted in greater susceptibility to DSS induced epithelial damage and mucosal inflammation but protected animals from the colitis associated cancer tumorigenesis.

Moreover, hyperactivation of GP130 mediated STAT3 promoted tumor incidence and growth 1,2.

Gene chip analysis of IECs isolated from DSS treated IEC STAT3 deficient mice compared to

4-16 controls demonstrated a reduction in the expression of genes involved in cellular stress response, apoptosis, and pathways associated with IEC wound healing specifically, Reg3 family members 59 . Additionaly recent publications have shown that loss of IL-22 mediated STAT3 activation in IL-22 knockout animals induces greater susceptible to DSS induced colitis and showed reduced in-vivo wound closure rate compared to IL-6KO and control 59 . Thus epithelial cell STAT3 seems to regulate survival, proliferation, and wound healing events during acute colitis and colitis-associated cancer. However, these studies have investigated the contribution of IEC STAT3 in acute injury or during tumorigenic chronic colitis conditions. Thus loss of

STAT3 within the epithelial compartment would likely increase risk for the development of chronic inflammation following an acute self-limited gut injury, which has not been formally tested. A model of acute injury and either resolution or development of chronic colitis in a genetically susceptible host would be expected to be more applicable to the current concept of

IBD pathogenesis in humans. Therefore, we defined the functional consequences of IEC STAT3 deletion in a model of chronic colitis in which mice are exposed to DSS for seven days, and then assessed during recovery over the next twenty-one days.

Selecting the appropriate animal model of IBD

Alternatively, we could have utilized other chemically induced murine models of IBD.

Trinitro Benzene Sulfonic acid (TNBS) dissolved in ethanol and given intra-rectally induces an

IL-12 mediated immune response immunologically accepted as a Th1 model with increased

$ N R @ : P a R - 2 3 @

U U L D levels of L -6, IL-12, IL- . Ethanol, required to break the mucosal barrier, and TNBS haptenize colonic autologous or microbiota proteins rendering them immunogenic to the host immune system 60 . The conversion rate for colitis in a C57BL/6 background, of which

4-17 are mice are on, is only about 15% and large numbers of mice are needed to assure statistical significance due to variability. Pilot experiments inducing TNBS colitis assessed 3 and 7 days after administration did not show differences in weight, histologic score, or proliferation in

Flx/Flx

[ \ ] Stat3 Z compared to Stat3 littermate controls (Figure 4). We also tested Piroxicam induced ileitis that mimics an NSAID induced environmental trigger reported in human CD.

This model has been shown to induce focal transmural ileitis in CARD15-/- mice with GM-CSF

Ab injection that ultimately induces defects in innate immunity and leads to development of ileitis 61 . We found no differences in weight after 2 weeks of Piroxicam exposure and did not

Flx/Flx

[ \ ] find disease in the ileum or colon of Stat3 or Stat3 Z mice (Figure 5). These studies suggested that we utilize an approach more effective in mediating epithelial responses.

4-18

d b c

4-194- 19

g h i

Figure 4. STAT3 f mice are not susceptible to Piroxicam induced colitis .

k l m Mice lacking intestinal epithelial Stat3 (Stat3 j ) and littermate controls were given 200ppm piroxicam containing chow . %Weight gain did not differ after two week exposure.

Dextran Sodium Sulfate (DSS) exerts direct toxic effects on epithelial cells independent of the underlying gut immune system and its transient administration provides an experimental system to assess the critical first phase of wound healing 62 . In murine models DSS induced colitis follows a predictable time course, is very reproducible, and has a similar pathogenesis and therapeutic response to human IBD 63,64 . Initially, DSS impacts the epithelial cell with loss of the basal one-third of the crypt by day 3 62 . This imparts a breakdown in the epithelial barrier causing normal mucosal microflora substances to be exposed to the underlying lamina propria innate immune cell populations, which in turn provokes and adaptive immune response 62,64 .

Severe combined immunodeficient mice also develop severe intestinal inflammation and suggests that the adaptive immune system does not play a major part in the acute phase of this model 65 .

DSS induced acute colitis is a 5-7 day treatment that results in inflammation driven by polymorphonuclear cells and macrophages, resulting in bloody diarrhea, ulceration, and infiltration with granulocytes 66 . Importantly this model has a 95% conversion rate and in pilot

Flx/Flx

[ \ ] experiments, we were able to induce a consistent acute colitis in both Stat3 and Stat3 Z

4-20 mice. This model induces initial Th1 response however in the chronic phase it is mixed Th1/Th2 and also has inflammatory features of both UC and CD pathology. In the C57BL/6 background it has been shown that one initial dose of DSS for 5-7 days induces a chronic colitis characterized with a significant inflammatory response, progressive production of IL-1, active

IL-12, and IL-17 and increased production of chemokines CXCL1, CXCL2/3, CXCL10, CCL2,

CCL3, CCL4 67,68 . In C57BL/6 mice DSS-induced colitis treated with current clinical therapeutic agents, cyclosporine A, methotrexate, IL-12p40 and agonistic anti-CD3 antibody have given sufficient evidence for the models use in translation of mouse and human disease 63 . Ultimately, there are many other murine models of IBD however in the C57BL/6 DSS was the perfect model to test epithelial dependent loss of Stat3 function in acute injury and evolution into chronic colitis.

Additionally, murine models of intestinal inflammation are greatly affected by the resident enteric bacteria. The gut microbiota is a key component in intestinal homeostasis and in humans a dysbiosis or imbalance of healthy bacteria has been linked to the development of

IBD 69 . Almost all experimental mouse models of inflammatory bowel disease can be inhibited in mice devoid of intestinal flora 62,70,71 . The development of spontaneous colitis and immune system activation in IL-10-deficient mice is lost if animals are housed in germ-free conditions thus resident enteric bacteria are necessary for development of spontaneous colitis in these mice 72 . Conventional housing unlike specific pathogen free barrier facilities increase the incidence and severity of the colitis phenotype and is more indicative of human disease 73,74 .

Moreover, variability in animal responses, particularly to immune-related stimuli, when assessed in the context of conventional versus barrier conditions deserves consideration. The murine studies in chapter 3 include animals housed in conventional facilities and this is likely to enhance

4-21 differences in immune phenotype and pathology. Thus, phenotypes may be more elusive in barrier facilities. Furthermore, understanding the role enteric bacteria may play in acute and chronic colitis as a function of epithelial dependent loss of Stat3 was outside the scope of this thesis but warrants further investigation.

Location, Location, Location

[ \ ] In our murine model of intestinal epithelial cell Stat3 deletion (Stat3 Z ) chronic inflammation at day 28 was more severe following 7 days of acute injury with DSS which was not accounted for by a sustained defect in epithelial proliferation or apoptosis as compared to littermate control (Stat3 Flx/Flx ) mice. We concentrated our efforts within 0.5cm from the ulcer border or similar areas of the colon if no ulcer was present. Analysis from areas that were

Flx/Flx

[ \ ] unaffected at day 7 and day 28 in both Stat3 and Stat3 Z mice were very similar for proliferation and apoptosis (data not shown). Even more striking were the differences between distal and proximal disease between genotypes (data not shown). Distal disease was very similar between genotypes however proximal disease varied and thus we had to be very consistent with our approach for harvesting the colon and the overall analysis of disease histology, proliferation, apoptosis, and immunohistochemistry for activated p65/RelA, pSTAT1, pSTAT5. To orient a starting point for analysis we developed a scoring system to account for total colonic disease by using the anal verge to mark the most distal starting point for scoring and the following four consecutive longitudinal 5x High Powered Fields (HPF) scored as the distal colon and the following four, 5x HPF scored as the middle colon. We did not observe in any of the animals, regardless of genotype, inflammation past the middle/proximal portion that was scored in our developed system and thus we feel we were able to score total colonic disease. We did not find

4-22 patchy disease we had very consistent distal and middle disease, with the most proximal middle disease having greater variation between genotypes. We reported our data as a total colonic score, but observed very different levels of disease in the middle colon suggesting differential regulation of STAT3 depending on colonic location. This may facilitate differences in investigator interpretation of disease severity depending on how stringent one is in evaluation of total colonic disease. Therefore we feel we have devised a very stringent and important scoring system to evaluate total histologic disease severity.

Interpretation of immunofluorescence and gene expression data

In the same mice that we interrogated proliferation and apoptosis, we had residual

Swiss Rolled colonic tissues embedded in O.C.T compound. We cross sectioned these tissues into four to five 30-µm-thick sections, giving approximately 20mg of total colonic tissue that we could extract RNA from using RNA later to wash away the O.C.T compound. This was helpful as we could also asses the frequency of different cell populations from subsequent portions of the same tissue while also interpreting gene expression. We ultimately found that colonic lamina propria frequency of total pSTAT3+ cells was increased and correlated with histologic injury and

[ \ ]

F480+pSTAT3+ macrophages, and CD3+pSTAT3+ T-lymphocytes were increased in Stat3 Z

o p q r n o p q s

mice compared to littermate controls. Colonic expression of Stat3 target genes n and

[ \ ]

that mediate epithelial restitution were significantly decreased in Stat3 Z mice. However

u v s colonic expression of Il-17a, t , Cxcl2 , Cxcl10 , Ccl2 , and Ccl4 were significantly increased

and Il-17a expression correlated with the increased lamina propria frequency of CD3+pSTAT3+

[ \ ]

T-lymphocytes in Stat3 Z mice. Our results were consistent with prior reports in that we

[ \ ] observed increased expression of CXCL2 , CXCL10 , CCL2 and CCL4 in Stat3 Z mice in the

4-23 setting of more severe chronic colitis which is likely contributing to the differences in the

inflammatory infiltrate observed at day 28. SOCS3 , a direct downstream target of activated Stat3,

[ \ ] was increased in expression in the Stat3 Z mice which we believe is consistent with the pronounced pSTAT3+ infiltrate within the lamina propria. Moreover, this infiltrate most likely

was responding to IL-6/GP130 receptor activation as SOCS3 is not a direct target of IL-10

T 0 . 0 2 * 4 @ " M ) " + + * 0 2 5 - + * 2 4 ) " - + " 3 - ? 0 ! 3 - 7 X - 2 3 3 - 7 * 2

L D F w

induced STAT3 activation. U

[ \ ]

the Stat3 Z mice. We did not observe qualitative differences in pSTAT1 activation in the

" * ; " 0 * 2 + < x 0 5 " A " ) R - + @ * + ) 0 3 1 4 " 3 ? 7 $ ! P 4 " . .

6 6

L D epithelial cell compartment at thes U types, and IL-4 expression did not differ between genotypes, this likely represents a basal Th1

[ \ ]

skewing in the Stat3 Z mice . Colonic IL-17A expression was increased only at day 28 in

[ \ ] Stat3 Z mice, in the setting of chronic colitis. IL-17A is released by Th17 inflammatory T- cells, shown to involve direct binding of Stat3 to the IL-17A promoter, and known to be involved

in many chronic autoimmune disorders 76-78 . Although there was a trend, we did not detect a

r z v u {

difference between genotypes for expression of IL-10 , IL-6, IL- y or , suggesting that there

[ \ ] was not a global difference in activation of mucosal inflammatory pathways in the Stat3 Z mice. Consistent with an effector function, we observed a strong correlation between the frequency of pSTAT3+ T lymphocytes and IL-17A expression, and between IL-17A expression and histologic injury. Future studies employing Th17 blockade will be required to determine whether this will ameliorate chronic colitis in this setting. In summary, we have shown that loss of IEC Stat3 leads to more severe chronic inflammation following acute injury which is not accounted for by a sustained defect in epithelial proliferation or apoptosis, but rather expansion of pSTAT3+ lymphocytes and up-regulation of IL-17A expression.

4-24

Alternative Hypothesis

Loss of IEC STAT3 results in enhanced bacterial sensing . Is it possible that the loss of

STAT3 in the epithelial compartment enhances microbial sensing by intestinal epithelial cells after activation or inflammation. The intestinal epithelium provides a critical interface between luminal bacteria and the mucosal immune system. Whereas normal commensal flora do not trigger acute inflammation, pathogenic bacteria trigger a potent inflammatory response. IFN-D has been shown to positively regulated MD-2 promoter activity in IEC and co-expression of

D 79

u v s SOCS3 blocked IFN- -mediated MD-2 promoter activation . In our model t expression is

IEC Flx/Flx increased at day 0 and day 28 in Stat3 Z mice compared to Stat3 littermate controls.

IEC

Therefore in the epithelial compartment of Stat3 Z mice there would be reduced transcription of

; " 3 * - " 3 ? 7 @

L D SOCS3 allowing for enhanced expression U of the critical TLR4 co-receptor

MD-2 which could alter toll-like receptor expression and bacterial reactivity.

SHP2 is protective in mice devoid of IEC STAT3. Recent data in abstract form suggests that knockdown of the Src homology tyrosine phosphatase (SHP2) in the human colonic cell line

Caco-2 inhibited the ability of the cells to migrate and adhere compared to control cells.

Additionally upon IL-6 induced GP130 activation SHP2 mediated activation induces Ras-ERK pathway seeming to transduce activities regulating mitogenesis 80 . The SHP-2 inducing site in

IEC the cytoplasmic tail of GP130 is the binding site for SOCS3 inhibition. In our Stat3 Z mice reduced levels of SOCS3 would therefore allow enhanced SHP2/Ras/ERK activation. This maybe an important mechanism in wound healing that is mediated at the early stages of acute colitis in response to IL-6. Thus may also be the alternative protective mechanism used to

IEC promote the wound healing and proliferation that allowed Stat3 Z mice to recover after day 7 compared to littermate controls and may be involved in epithelial proliferation from day 7 to day

4-25

28. We did not see other compensatory mechanisms including pSTAT1, pSTAT5, or p65

upregulated in the epithelium at day 7, or day 28. Therefore an increase in SHP2 activity may be

extremely protective in the model and evaluating downstream effectors important in proliferation

and survival induced by Ras and ERK activation would be important.

Future Directions

A need to define the STAT3/Th17/IL22 axis. The cytokine IL-23 is one of the key

components involved in the induction and maintenance of Th17 cells through STAT3 activation.

Recent data has shown that the IL23R R381Q gene variant protects against immune-mediated

diseases by impairing IL-23-induced Th17 effector response 81 . Th17 cells secrete IL-22 shown

to induce STAT3-dependent IEC survival, regeneration, and the production of antimicrobial

compounds that directly limit the bacterial and/or the inflammatory T-cell component of the

inflammatory response following inflammatory insult specifically directed by Th17 cells 82 .

However, IL-22-induced STAT3 activation can also induce the production and release of IL-8

- 2 3 $ N U L , further enhancing IL-17A-driven inflammation 83 . Specifically IL-6, IL21, and IL23

are the cytokines known to drive the differentiation and survival of these cells. We did not find

an increase in IL-6 expression but we did not investigate the level of expression for IL-21 , IL-23,

O S " M ) " + + * 0 2 ! - + ? " " 2 3 " ; 0 2 + ) - " 3 or IL-22 . Transforming growth factor beta (TGF- 6

throughout wound healing and shown to regulate many processes involved in tissue repair,

including production of ECM, migration, chemotaxis, and proliferation of macrophages,

0 ) " 0 A " ) $ V O

84 U

fibroblasts of the granulation tissue, epithelial and capillary endothelial cells . |

along with IL-10 are involved in mediating regulatory T-cell expansion as IL- 10-induced

STAT3 is important for the regulatory function of Tregs on Th17 cells 85 . We did not find an

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! " . " A " . 0 / $ V O ; - 7 increase in IL-10 expression between our genotypes at day 28 however, U give insight into the overall regenerative capacity of the colon between genotypes. Th17 mediated IL-22 dependent STAT3 expression of membrane-bound mucin referred to as Muc1, functions as a lubricant and a physiological barrier between luminal contents and mucosal surface. Muc1 deficient mice develop more severe forms of Th1- and Th2-induced colitis than controls with increased colonic permeability and Th17-cells. This data suggests that IL-22 induced Muc1 expression functions as a negative feedback pathway that prevents an excessive

Th17-cell response in inflamed colons of mice 86 . In our model we did not evaluate Muc1 but at

IEC day 28 STAT3 induced Muc1 expression is probably substantially lower in the Stat3 Z mice compared to controls. We did find a increase in IL17A is probably mediated by increased

IEC number of Th17 cells that are also secreting IL-22 to which Stat3 Z mice cannot respond

reducing the regenerative capacity and barrier function that is defective at day 28. Ultimately,

& R $ V 9 O - 2 3 1 4 P * 2 0 1 ) F we would want to define the level of expression of IL-21, IL-22, IL- | murine model at day 0 and day 28.

IEC STAT3 in the chronic phase . These studies highlight the protective and important role of STAT3 in maintaining barrier function and terminating inflammatory reaction during the chronic phase of colitis. This has only recently been appreciated as loss of STAT3 in the chronic phase has only been associated with a reduced colonic cancer phenotype. This data supports our hypothesis that loss of STAT3 in the epithelial compartment mediates more severe colitis. The specific mu-opioid receptor agonist DALDA induces STAT3 activation-dependent proliferation and migration of IEC, and data from DSS induced colitis models suggest that targeting epithelial

STAT3 activation with this compound could be a potential therapeutic 87 . This also supports recent data from humans showing that high expression of SOCS3 could conceivably interfere

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with regenerative responses and epithelial cell homoeostasis enhancing the sensitivity of SOCS3

expressing epithelial cells to inflammatory damage. The high SOCS3 expression in IEC in UC

remission is associated with a shorter time till relapse and an increased severity of inflammation

during relapse 88 . This supports other preliminary data from our lab in that patients carrying the

JAK2 risk allele have decreased STAT3 activation reduced colonic expression of the STAT3

} ~ q { target gene n suggesting that reduced STAT3 activation may also be associated with

increased risk for IBD 89 . The JAK2 risk allele has also been associated with increased intestinal

permeability and we would suggest that this is probably mediated by reduced epithelial STAT3

activation 90 . These data and our own suggest that epithelial STAT3 activation is needed for

protection from acute colitis through IL-6 induced proliferation and survival and that during the

chronic phase STAT3 helps maintain mucosal homeostasis by inducing greater barrier function

and wound healing/regeneration responses. This could be tested utilizing an IEC STAT3

deficient mouse with the tissue-specific and inducible Cre-mediated (vil-Cre-ER T2 ) line 91 . Thus,

we could mediate loss of STAT3 in a time and phase dependent to more elegantly explain how

STAT3 deficiency in the epithelial compartment impacts colitis.

Defining the Cellular Source. Ultimately, defining the cellular source of IL-17A and

@ - 2 3 ! " 0 - . / ) " 1 " 2 4 7 0 / ! " + " 4 " . . +

L D U within the lamina propria using flow cytometric

analysis would be informative. However, one could also quantify these cells within the lamina

propria by immunofluorescence double staining for the surface marker CD3/IL-17A and

CD3/IFN. Interestingly, gamma delta T-cells have also been shown to be an important source of

IL-17 92 . Therefore comparison of double staining for the gamma delta TCR or CD4 with IL-17A

would help further interpret which T-cells are producing the IL-17A. Additionally, the

significant increase in F4/80+/pSTAT3+ macrophages should be characterize as an M1 or M2

4-28 phenotype. M1 is classically activated, generally inflammatory and mediates protection against invading pathogens with activation of STAT3 and STAT1 and induction of SOCS3 for release of

IL23 and I L12. Alternatively, M2 an anti-inflammatory phenotype known to regulate wound healing, activates STAT6 and induces SOCS1 expression 93 . Based on our gene expression and

immunofluorescence data we assume that these macrophages are M1 inducing pro-inflammatory

o p q r n o p q s r

antimicrobial functions. Epithelial expression of Muc1 , n , , and -defensin-2

t u v s (epithelial restitution/barrier function genes ) Il-6, Il- y , Cxcl2, Cxcl5, Cxcl10, ccl2, ccl4, ccl20 could be quantified using laser capture micro-dissection of epithelial cells within colonic

Flx/Flx

[ \ ] areas of active disease and inactive disease in both Stat3 and Stat3 Z mice. The ability to define gene expression within the same animal in regards to epithelial crypts undergoing wound healing compared to adjacent non disease epithelial crypts would be extremely interesting in and of itself. We observed in histologic assessment differences in BrdU and caspase-3 in cells closest to the ulcer verses adjacent healthy tissue. This suggests that there are completely different gene expression profiles depending on colonic location and disease involvement.

Therefore, it would be interesting to decipher epithelial genes important in wound healing and extracellular matrix interactions that are STAT3 dependent utilizing laser capture micro- dissection of epithelial cells form the colonic niche in regards to diseased and healthy tissue from

Flx/Flx

[ \ ] Stat3 Z mice compared to Stat3 littermate controls. Lastly, the lamina propria could also be interrogated utilizing laser capture micro-dissection protocol to help define the gene expression coming from the lamina propria infiltrate verse the epithelial barrier.

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Summary

The STAT3 transcription factor is important in almost all cell types in the intestine. In

the epithelial compartment STAT3 mediates proliferation and survival, wound healing, and

regeneration. In the innate immune system, STAT3 induces proper protective and highly

regulated immunity and in the adaptive immune system STAT3 mediates the highly aggressive

pro-inflammatory Th17 cell type and the opposing regulatory T cell phenotype. Moreover,

inflammation per se is both protective yet can be detrimental in a chronic setting to the host.

Therefore, as STAT3 is involved in protection, perpetuation, and ultimately resolution and

regeneration, it is a master regulator of mucosal inflammation. The studies herein have shown

that loss of IEC Stat3 leads to more severe chronic inflammation following acute injury which is

not accounted for by a sustained defect in epithelial proliferation or apoptosis, but rather

expansion of pSTAT3+ lymphocytes and up-regulation of IL-17A expression. Further studies

employing Th17 blockade will be required to determine whether this will ameliorate chronic

colitis in this setting.

Lastly, in trying to define how the STAT3 rs744166 is associated with risk for IBD we

9 Y B % C ) * + , - . . " . " * + - + + 0 4 * - " 3 5 * ! * 2 4 ) " - + " 3 4 " . . 1 . - ) # $ % $ & revealed that the STAT3 @

activation and up-regulation of chemokines which promote CXCR2+ neutrophil recruitment to

the gut in a newly described sub-population of CD patients. It is likely that there are several

immunogenetic forms of IBD, with CD and UC representing the broadest clinical classifications,

our studies implicate the potential of defining subpopulations in regards to improved therapeutic

efficacy. Even though we did not observe differences in overall clinical demographics our

- * " 2 + ! 0 ; 0 7 > 0 1 + / 0 ) ! " B V C - . . " . " 5 " ) " - A " ) 7 . 0 5 2 1 ; ? " ) + * 2 6 our patient population

which ultimately limited the power of our analyses. Therefore, future studies with greater power

4-30 to detect clinical associations will be needed to elucidate associations with disease behavior, response to therapy, and rates of colorectal cancer and surgery.

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