Medicine, Department of
Obstetrics and Gynaecology, Department of
DSpace
UBCV
Sekhon, Prabhreet
Master of Science - MSc
University of British Columbia
Crohn’s disease (CD) is one form of inflammatory bowel disease (IBD) in which inflammation penetrates all gut layers, and occurs discontinuously anywhere from the lips to the anus. One in 3 people with CD will develop a life-threatening complication called fibrosis. Fibrosis is driven by excessive deposition of extracellular matrix components and thickening of the bowel wall due to fibroblast activation and smooth muscle cell hyperplasia, respectively. There are currently no therapies to directly treat intestinal fibrosis, and surgery is the only available intervention. The Sly laboratory has previously characterized the src homology 2 (SH2) domain-containing inositol polyphosphate 5′-phosphatase deficient (SHIP-/-) mouse model of CD-like intestinal inflammation and fibrosis. They have shown a critical role of phosphatidylinositol 3-kinase (PI3K) p110δ-mediated upregulation of arginase I (ArgI) in intestinal fibrosis. ArgI is an enzyme that uses L-arginine to produce L-ornithine, which ultimately gets broken down into precursors used for collagen biosynthesis and cell proliferation. Previous studies have implicated a pro-fibrotic role of interleukin (IL)-33 by promoting ArgI expression in the context of lung fibrosis. Based on this, I hypothesized that IL-33 promotes fibrosis in the SHIP-/- model of CD by inducing ArgI activity. I found that IL-33 concentrations and relative gene expression were higher in the fibrotic sections of SHIP-/- mice compared to SHIP+/+ controls, specifically the 30 kDa isoform. IL-33 may synergize with IL-13 to induce ArgI expression and activity in SHIP-/- bone marrow derived macrophages (BMDMs). IL-33 neutralization and genetic ablation in vivo reduced mean ArgI activity in SHIP-/- non-fibrotic and fibrotic sections, however, did not improve disease. Because high IL-1β concentrations persisted upon IL-33 neutralization and genetic ablation in SHIP deficiency, I speculate that targeting both the pro- and anti-inflammatory responses are required to treat CD-associated fibrosis. Overall, these findings implicate an association between IL-33 and fibrosis in SHIP-/- mice, however, targeting IL-33 was insufficient to ameliorate intestinal fibrosis.
Targeting IL-33 to treat Crohn’s Disease-associated fibrosis by Prabhreet Sekhon BSc., The University of British Columbia, 2020 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Women+ and Children’s Health Sciences) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2024 © Prabhreet Sekhon, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Targeting IL-33 to treat Crohn’s Disease-associated fibrosis submitted by Prabhreet Sekhon in partial fulfilment of the requirements for the degree of Master of Science in Women+ and Children’s Health Sciences Examining Committee: Dr. Laura Sly, Professor, Department of Pediatrics, UBC Supervisor Dr. Kelly Brown, Associate Professor, Department of Pediatrics, UBC Supervisory Committee Member Dr. Alice Mui, Associate Professor, Departments of Surgery and Biochemistry & Molecular Biology, UBC Additional Examiner Additional Supervisory Committee Members: Dr. Wilfred Jefferies, Professor, Department of Medical Genetics, UBC Supervisory Committee Member iii Abstract Crohn’s disease (CD) is one form of inflammatory bowel disease (IBD) in which inflammation penetrates all gut layers, and occurs discontinuously anywhere from the lips to the anus. One in 3 people with CD will develop a life-threatening complication called fibrosis. Fibrosis is driven by excessive deposition of extracellular matrix components and thickening of the bowel wall due to fibroblast activation and smooth muscle cell hyperplasia, respectively. There are currently no therapies to directly treat intestinal fibrosis, and surgery is the only available intervention. The Sly laboratory has previously characterized the src homology 2 (SH2) domain-containing inositol polyphosphate 5′-phosphatase deficient (SHIP-/-) mouse model of CD-like intestinal inflammation and fibrosis. They have shown a critical role of phosphatidylinositol 3-kinase (PI3K) p110δ-mediated upregulation of arginase I (ArgI) in intestinal fibrosis. ArgI is an enzyme that uses L-arginine to produce L-ornithine, which ultimately gets broken down into precursors used for collagen biosynthesis and cell proliferation. Previous studies have implicated a pro-fibrotic role of interleukin (IL)-33 by promoting ArgI expression in the context of lung fibrosis. Based on this, I hypothesized that IL-33 promotes fibrosis in the SHIP-/- model of CD by inducing ArgI activity. I found that IL-33 concentrations and relative gene expression were higher in the fibrotic sections of SHIP-/- mice compared to SHIP+/+ controls, specifically the 30 kDa isoform. IL-33 may synergize with IL-13 to induce ArgI expression and activity in SHIP-/- bone marrow derived macrophages (BMDMs). IL-33 neutralization and genetic ablation in vivo reduced mean ArgI activity in SHIP-/- non-fibrotic and fibrotic sections, however, did not improve disease. Because high IL-1β concentrations persisted upon IL-33 neutralization and genetic ablation in SHIP deficiency, I speculate that targeting both the pro- and anti- inflammatory responses are required to treat CD-associated fibrosis. Overall, these findings implicate an association between IL-33 and fibrosis in SHIP-/- mice, however, targeting IL-33 was insufficient to ameliorate intestinal fibrosis. iv Lay summary Crohn’s Disease (CD) is a life-long condition that affects the gastrointestinal tract. 1 in 3 people with CD develop a complication called fibrosis. Fibrosis happens when too much scar tissue builds up and causes a narrowing or complete blockage of the bowel. There are currently no therapies to directly treat intestinal fibrosis, and surgery remains the only option. To identify targets for therapy, our lab uses a CD mouse model. These mice lack a protein called SHIP which is also reduced in a subset of people with CD. Using this mouse model, I demonstrated that a protein called IL-33 has higher expression in fibrotic sections compared to sections from healthy mice. Although blocking or removing IL-33 in mice with CD reduced activation of a pathway involved in fibrosis, it did not improve disease. These results reveal IL-33 as an ineffective therapeutic target for treating CD-associated fibrosis, specifically in people that that exhibit low levels of SHIP. v Preface All the work presented in this thesis was performed at the BC Children’s Hospital Research Institute. All animal studies were performed in accordance with the guidelines from the Canadian Council on Animal Care (protocol numbers A21-0035 and A23-0073). The C57BL/6 IL33-/- mice were generously provided by Dr. Fumio Takei at the BC Cancer Terry Fox Laboratory (Vancouver, BC). All experiments, data acquisition and analyses presented in Chapters 3, 4, and 5, and writing were done by me, Prabhreet Sekhon, with the following exceptions: Susan Menzies bred and genotyped all the mice used in experiments, Maggie Ma and Yvonne Pang conducted histological damage scoring in Figures 4.3B and C, Chloé Bender-Thorsen and Yvonne Pang conducted fibrosis scoring in Figures 4.5B and C as well as in Figures 5.5B and C, Maggie Ma and Kwestan Safari conducted histological damage scoring in Figures 5.3B. Wax-it UBC provided histology services for sample processing and staining (H&E, Masson’s Trichrome, and Alcian Blue+PAS). Laura Sly, the principal investigator, provided guidance on concept formation, experimental design, and revised the thesis. My committee members, Dr. Kelly Brown, and Dr. Wilfred Jefferies also contributed to revisions of my thesis. Figure 1.1 was reproduced with permission from Neurath M, Nature Reviews Immunology 2014. Permission was granted through Springer Nature BV and Copyright Clearance Center (Licence ID: 1532859-1). Figures 1.2 and 1.3 were reproduced with permission from Dobranowski and Sly, Journal of Leukocyte Biology, 2018. Permission was granted by Oxford University Press through Copyright Clearance Center (License number: 5882111031089). Figure 1.4 was reproduced with permission from Dwyer et al, Annual Review of Immunology 2022. Permission was granted by Annual Reviews Inc through Copyright Clearance Center (License ID: 1515102-1). vi Table of contents Abstract ................................................................................................................................... iii Lay Summary .......................................................................................................................... iv Preface ....................................................................................................................................... v Table of contents ...................................................................................................................... vi List of tables ............................................................................................................................. ix List of figures ............................................................................................................................ x List of abbreviations ...............................................................................................................xii Acknowledgements ................................................................................................................ xvi Dedication.............................................................................................................................. xvii Chapter 1: Introduction ........................................................................................................... 1 1.1 Inflammatory bowel disease ............................................................................................. 1 1.1.1 Disease presentation and diagnosis .............................................................................. 1 1.1.2 Global incidence and emerging populations ................................................................ 3 1.1.3 Current strategies and therapies for disease management ............................................ 4 1.1.4 Etiology of disease ....................................................................................................... 6 1.1.4.1 Genetics ..................................................................................................................... 7 1.1.4.2 Environmental factors................................................................................................ 8 1.1.4.3 Microbial factors........................................................................................................ 9 1.1.4.4 Immune factors ........................................................................................................ 10 1.2 Crohn’s Disease-associated fibrosis ............................................................................... 13 1.2.1 Cellular contributors of intestinal fibrosis.................................................................. 13 1.2.1.1 Epithelial cells in intestinal fibrosis ......................................................................... 14 1.2.1.2 Macrophages in intestinal fibrosis ........................................................................... 15 1.2.2 Molecular mediators of fibrosis ................................................................................. 16 1.2.3 Therapeutic approaches.............................................................................................. 18 1.3 Murine models of intestinal fibrosis .............................................................................. 19 1.4 Src homology 2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP) . 22 1.4.1 Description .............................................................................................................. 22 vii 1.4.2 SHIP in people with IBD ......................................................................................... 24 1.4.3 The SHIP-deficient mouse model of Crohn’s disease-like inflammation and fibrosis .............................................................................................................. 25 1.4.4 SHIP-deficient macrophages ................................................................................... 26 1.5 IL-33 ................................................................................................................................. 28 1.5.1 IL-33 description ..................................................................................................... 28 1.5.2 Nuclear IL-33 .......................................................................................................... 29 1.5.3 IL-33 structure and release ...................................................................................... 29 1.5.4 Negative regulation of IL-33 signaling ................................................................... 30 1.5.5 IL-33 signaling in macrophages .............................................................................. 31 1.6 Thesis hypothesis and objectives .................................................................................... 32 1.6.1 Summary of rationale .............................................................................................. 32 1.6.2 Aims ........................................................................................................................ 33 1.6.3 Significance ............................................................................................................. 33 Chapter 2: Experimental Methods ....................................................................................... 35 2.1 Mice .................................................................................................................................. 35 2.2 Cytokine analyses ............................................................................................................ 35 2.3 Gene expression analyses ................................................................................................ 36 2.3.1 RNA extraction ........................................................................................................ 36 2.3.2 cDNA synthesis and quantitative PCR (qPCR) ....................................................... 37 2.3.3 cDNA synthesis and PCR for IL-33 splice variants ................................................ 37 2.4 Western blot analyses...................................................................................................... 38 2.4.1 Full-thickness ileal homogenates ................................................................................ 38 2.4.2 Serum .......................................................................................................................... 39 3.4.3 Cell lysates .................................................................................................................. 39 2.5 Histology ........................................................................................................................... 39 2.5.1 Hematoxylin and eosin (H&E), Masson’s trichrome and Alcian blue + Periodic Acid-Schiff (PAS) staining...................................................................................... 39 2.5.2 IL-33 Immunohistochemistry .................................................................................. 40 viii 2.6 Histological Analyses ....................................................................................................... 41 2.6.1 H&E staining ........................................................................................................... 41 2.6.2 Masson’s trichrome fibrosis score ........................................................................... 42 2.6.3 Masson’s trichrome image analysis ......................................................................... 43 2.7 Cell stimulations .............................................................................................................. 44 2.8 IL-33 neutralization experiments ................................................................................... 45 2.9 Arginase activity assays .................................................................................................. 45 2.10 Statistical analyses .......................................................................................................... 46 Chapter 3: Results .................................................................................................................. 48 3.1 Fibrotic sections in SHIP-/- mice have higher concentrations of the 30 kDa IL-33 isoform and relative gene expression compared to SHIP+/+ controls ........................ 48 3.2 IL-33 synergizes with IL-13 but not IL-4 to induce ArgI expression and activity in SHIP+/+ BMDMs ............................................................................................................ 57 3.3 IL-33 neutralizing antibodies are effective in vitro and in vivo ................................... 61 3.4 IL-33 neutralizing antibodies do not improve disease severity in SHIP-/- mice ........ 63 3.5 IL-33 neutralizing antibodies do not reduce fibrosis in SHIP-/- mice ........................ 68 3.6 Genetic ablation of IL-33 in SHIP deficiency does not ameliorate disease ................ 72 3.7 Genetic ablation of IL-33 in SHIP deficiency does not reduce fibrosis ...................... 78 3.8 Investigating role of nuclear IL-33 in inducing ArgI in BMDMs ............................... 81 Chapter 4: Discussion ............................................................................................................ 83 Chapter 5: Conclusion ........................................................................................................... 91 5.1 Limitations ........................................................................................................................ 92 5.2 Future Directions ............................................................................................................... 94 References ............................................................................................................................... 96 ix List of tables Table 2.1 Histological damage scoring ............................................................................. 41 Table 2.2 Fibrosis scoring ................................................................................................. 43 x List of figures Figure 1.1 Etiology of IBD .................................................................................................. 6 Figure 1.2 Function of SHIP .............................................................................................. 23 Figure 1.3 SHIP domains ................................................................................................... 24 Figure 1.4 IL-33 structure and release ............................................................................... 31 Figure 3.1 Fibrotic sections in SHIP-/- mice have higher IL-33 concentrations and relative gene expression compared to sections from SHIP+/+ mice ............... 49 Figure 3.2 Fibrotic tissue sections from SHIP-/- mice have higher expression of the 30 kDa IL-33 isoform ........................................................................................... 51 Figure 3.3 Expression of 20-22 kDa isoforms of IL-33 is higher in the sera of SHIP-/- mice .................................................................................................................. 53 Figure 3.4 IL-33 splice variants were not detected in full-thickness ileal tissue from SHIP+/+ or SHIP-/- (non-fibrotic and fibrotic sections) .................................. 55 Figure 3.5 Epithelial cells are one source of IL-33 in the ilea of SHIP deficient mice ...... 56 Figure 3.6 IL-33 may synergize with IL-13 to induce ArgI expression and activity in BMDMs ............................................................................................................ 59 Figure 3.7 IL-33 does not synergize with IL-4 to induce ArgI expression and activity in BMDMs ............................................................................................................ 60 Figure 3.8 IL-33 neutralizing antibodies are effective at blocking IL-33 in vitro and at lowering mean ArgI activity in SHIP-/- treated mice ...................................... 63 Figure 3.9 IL-33 blocking antibodies do not improve gross ileal pathology in SHIP-/- mice .................................................................................................................. 64 Figure 3.10 IL-33 blocking antibodies do not improve histological damage in SHIP deficient mice ................................................................................................... 67 Figure 3.11 IL-33 blocking antibodies do not reduce mucin accumulation in SHIP-/- mice .................................................................................................................. 68 Figure 3.12 IL-33 blocking antibodies did not reduce fibrosis in SHIP-/- distal ilea .......... 71 Figure 3.13 Anti-IL-33 blocking antibodies do not reduce IL-33 or IL-1β concentrations in SHIP-/- mice ..................................................................................................... 72 xi Figure 3.14 Genetic ablation of IL-33 reduces arginase activity in full-thickness ileal tissue homogenates from SHIP-/- mice ...................................................................... 73 Figure 3.15 Genetic ablation of IL-33 in SHIP deficient mice does not improve gross pathology .......................................................................................................... 74 Figure 3.16 No significant improvement in histological damage after ablation of IL-33 in SHIP-/- mice .................................................................................................... 76 Figure 3.17 Genetic ablation of IL-33 in SHIP deficient mice does not reduce mucin staining in ileal cross-sections .......................................................................... 77 Figure 3.18 Genetic ablation of IL-33 in SHIP deficiency did not reduce fibrosis ............. 80 Figure 3.19 Genetic ablation of IL-33 does not reduce IL-1β concentrations in full- thickness ileal tissues from SHIP-/- mice......................................................... 81 Figure 3.20 Nuclear IL-33 in BMDMs may play a role in repressing ArgI activity ........... 82 xii List of abbreviations αIL-33 Anti-IL-33 α-SMA Alpha smooth muscle actin ANCA Antineutrophil cytoplasmic antibodies APC Antigen presenting cell ArgI Arginase I ASCA anti-Saccharomyces cerevisiae ATG16L1 Autophagy-Related 16-like 1 BMDM Bone marrow derived macrophages BMe β-mercaptoethanol Ccl2 C-C motif chemokine ligand 2 CD Crohn’s disease c-IL-33 Cleaved IL-33 CRP C-reactive protein CSF1 Colony stimulating factor 1 Cxcl C-X-C motif chemokine ligand CTGF Connective tissue growth factor DC Dendritic cell DEFA5 Alpha defensin 5 DR3 Death receptor 3 DSS Dextran sodium sulfate ECM Extracellular matrix ELISA Enzyme-linked immunosorbent assay EMT Epithelial mesenchymal transition ERK Extracellular signal-regulated kinase FGF Fibroblast growth factor f-IL-33 Full-length IL-33 GATA3 GATA binding protein 3 GM-CSF Granulocyte macrophage colony-stimulating factor H&E Hematoxylin and eosin HLA Human leukocyte antigen xiii IBD Inflammatory bowel disease ICC Interstitial cells of Cajal IECs Intestinal epithelial cells IFN-β Interferon-β IFN‐γ Interferon-gamma Ig Immunoglobulin IL Interleukin IL-1Ra IL-1 receptor antagonist IL-1RAcP IL-1R accessory protein IL-23R IL-23 receptor IL-4R IL-4 receptor ILC2 Group 2 innate lymphoid cells IMDM Iscove’s modified dulbecco’s medium iNOS Inducible nitric oxide synthase iPSC Induced pluripotent stem cells ISPF α-isonitrosopropiophenone JAK Janus kinase JNK c-Jun N-terminal kinase LANA Latency-associated nuclear antigen LPS Lipopolysaccharide MAPK Mitogen-activated kinase MCP-1 Monocyte chemoattractant protein-1 MDP Muramyl dipeptide MET Mesenchymal epithelial transition MMP Matrix metalloproteinase MYD88 Myeloid differentiation primary response protein 88 NF-κB Nuclear factor kappa B NK Natural killer NO Nitric oxide NOD2 Nucleotide-binding oligomerization domain 2 PAS Periodic acid-schiff xiv PBMCs Peripheral blood mononuclear cells PDGF Platelet-derived growth factor PH-R Pleckstrin homology-related PI(3,4,5)P3 Phosphatidylinositol (3,4,5)-trisphosphate PI(3,4)P2 Phosphatidylinositol-3,4 bisphosphate PI(4,5)P2 Phosphatidylinositol (4,5)-bisphosphate PI3K Phosphatidylinositol 3-kinase PRR PTEN Pattern recognition receptor Phosphatase and tensin homologue deleted on chromosome 10 qPCR quantitative polymerase chain reaction Rag2 Recombination activating gene 2 RALDH2 Retinaldehyde dehydrogenase 2 rIL Rplp0 Recombinant interleukin Ribosomal protein lateral stalk subunit P0 SAMP SAMP1/YitFc SCFA Short chain fatty acid SEMF Sub-epithelial myofibroblast SH2 Src homology 2 SHIP Src homology 2 domain-containing inositol polyphosphate 5'- phosphatase SIGIRR Single Ig IL-1-related receptor SMAD Small mothers against decapentaplegic SMC Smooth muscle cell sSHIP Stem cell-restricted SHIP ST2 Suppression of tumorigenicity 2 sST2 Soluble suppression of tumorigenicity 2 STAT Signal transducer and activator of transcription TDM Therapeutic drug monitoring TGF-β Transforming growth factor-β Th T helper TIMP Tissue inhibitors of metalloproteinase xv TIR Toll/IL-1 receptor TL1A TNF-like cytokine 1A TLR Toll-like receptor TNBS 2,4,6-Trinitrobenzene sulfonic acid TNFα Tumor necrosis factor alpha Treg Regulatory T cell UC Ulcerative colitis VEO Very early onset xvi Acknowledgements This work was funded by the CIHR Canadian Graduate Scholarship-Master’s. I would like to thank my supervisor, Dr. Laura Sly, for the immense support and guidance that allowed me to grow as a scientist. I am very thankful, Laura, for providing me continuous feedback throughout my degree whether it was my project, writing or presentations. And for providing opportunities to attend conferences. I am grateful for the learning space in which I got the opportunity to ask questions and be excited about science while being supported throughout. I would also like to thank my committee members, Dr. Kelly Brown, Dr. Wilfred Jefferies and for your time and guidance on my project. Dr. Angela Devlin and Dr. James Lim for chairing my committee.Your questions and suggestions have been very valuable to my learning and my project. Thank you for your support in achieving my goals. Thank you to all the present and past members of the Sly lab: Susan Menzies, Yvonne Pang, Kwestan Safari, Chloé Bender-Thorsen, Maggie Ma, and Chanel Ghesquiere. I appreciate all your help and support throughout the years. As well as the members of the Animal Care Facilities at BCCHRI, Wax-It Histology at UBC, Sadarangani lab, Gibson lab, Steiner lab and Turvey lab for your support and equipment. I would also like to thank Dr. Fumio Takei and members of the lab for providing the C57/BL6 IL33-/- used in these experiments. Personally, I would like to thank my family and friends. My grandparents who have been a source of my happiness. A special shout out to Kwestan, Maggie, and Bruno (Levings lab) for your endless encouragement, guidance and support throughout my degree. You’ve all taught me something to influence my growth, both as a scientist and an individual. I am grateful for our friendship. My friends Pav and Al for encouraging me to follow my dreams especially during the pandemic, when it seemed impossible. xvii For mom and dad, I am forever in debt of the sacrifices that you made in order to provide me with the opportunities to pursue my dreams. This success is yours and was only made possible with your unconditional love. I will continue to try to make you proud. I love you. For my brother, Anmol, Thank you for being the selfless individual you are and supporting me in my goals. Although you are younger, I look up to you in many ways. I am extremely proud of you and wish you all the best in your future. For my uncle, Dr. Amandip Gill, Thank you for setting an example of what it means to dream big and work hard. You have been my role model from when I was a child, and I aspire to have the same dedication to my work as you do. 1 Chapter 1: Introduction 1.1 Inflammatory bowel disease 1.1.1 Disease presentation and diagnosis Inflammatory Bowel Disease (IBD) is characterized by chronic, relapsing and remitting, or progressive inflammation in the gastrointestinal tract. It encompasses two main subtypes, Crohn’s disease (CD) and ulcerative colitis (UC)1. CD and UC presents in both males and females2, however, they differ in disease location, penetrance, symptoms, complications, and disease management4. Diagnosis requires involvement of many medical professionals including gastroenterologists, pathologists, and radiologists4. It involves using a combination of clinical, endoscopic, radiologic testing, and histopathological assessments3. Despite medical intervention, CD and UC are life-long diseases, which can involve periods of active disease and remission5,9. Unfortunately, this has consequences on an individual’s quality of life6. CD presents with discontinuous inflammation, referred to as skip lesions, which can occur anywhere from the mouth to the anus. The inflammation occurs transmurally, penetrating all layers of the bowel. The most common site of inflammation in people with CD is the terminal ileum9. Clinically, people with CD exhibit heterogenous symptoms dependent on the location and severity of inflammation. Most common symptoms include diarrhea, which may be bloody, abdominal pain (most commonly in lower right quadrant), weight loss, and fatigue3, 10. As disease progresses, inflammatory regions can evolve into fibro-stenotic complications, which can lead to strictures3. Extraintestinal manifestations are also common in approximately 50% of people with CD. This can include complications involving the skin, joints, and eyes9, 11. UC differs from CD with respect to disease location, penetrance, symptoms, and complications. In contrast to CD, inflammation in UC usually begins in the rectum and progresses continuously to the colon. The inflammation is confined to the mucosal layer and does not penetrate the layers of the bowel3. A major symptom of UC is diarrhea along with 2 passage of mucus and blood, depending on severity of inflammation. Other symptoms include stomach cramping, abdominal pain, urgency, and tenesmus3,4. A serious complication of UC is colorectal cancer caused by the chronic inflammation in the colon13. In addition, people with UC can develop arthritis, which has been reported in 21% of people with disease9. The prolonged nature of UC can lead to life-threatening complications. CD and UC presents in both males and females2. There are no extreme sex-based differences in CD, however, some studies have reported a slight predominance in females that may be geography-dependent2,7. The age of onset for CD is usually 15 - 35 years of age or 55 - 70 years8. In contrast, UC has a slight male predominance after 45 years of age12 and disease onset usually occurs between 15 - 35 years of age8. To diagnose CD and UC, medical professionals commonly begin with clinical assessments followed by laboratory testing, imaging techniques, and histopathology. In the clinic, doctors will evaluate symptoms, seek family history and gain information on environmental factors such as diet. Blood tests will be conducted for measuring inflammatory markers, including C-reactive protein (CRP), which are helpful in assessing a possible diagnosis of IBD. However, CRP cannot differentiate between CD and UC3. Blood samples also can be used to detect antibodies including atypical anti-Saccharomyces cerevisiae (ASCA) and perinuclear antineutrophil cytoplasmic antibodies (ANCA) that tend to associate with CD and UC, respectively14. The gold standard for diagnosis is the combination of a colonoscopy and biopsies. Esophagogastroduodenoscopy may be used if the inflammation is suspected to be in the upper gastrointestinal tract3, 4. Other radiologic imaging techniques can also be used to confirm the diagnosis. This includes computed tomography and magnetic resonance imaging. These tools are especially useful in CD to assess thickening of the bowel wall. Biopsies are used for histopathological assessment4. In CD, biopsies are scored based on the presence of villous blunting, crypt distortion, and granulomas to evaluate disease14. Granulomas are comprised of lymphocytes as well as epithelioid histiocytes14. Deep biopsies can also be used to evaluate the presence of inflammation extending into the submucosa14. Histopathological diagnosis of UC involves a score-based criteria to evaluate disease based on the presence of crypt abscesses, crypt shortening, Paneth cell metaplasia, and an increased number of immune cells in the lamina propria14. If CD and UC cannot be differentiated using these diagnostic tools, then the patient is diagnosed with indeterminate colitis. However, 3 recent studies report the presence of human alpha defensin 5 (DEFA5) in the colon crypt mucosa as a marker for CD when diagnosis is not clear15. The tools discussed are not only used for diagnosis but can also be used to monitor disease as CD and UC are lifelong conditions3, 5, 9. 1.1.2 Global incidence and emerging populations More than 200 population-based studies have shown North America to have the highest prevalence of IBD in addition to Western Europe16, 17. In 2018, Crohn’s and Colitis Canada’s Impact of IBD report revealed Canada to have amongst the highest disease burdens in the world18. The rising incidence in Canada has been attributed to an increase in the number of pediatric-onset IBD cases whereas incidence remains stable in the adult and senior populations18,19. A report in 2019 estimated approximately 270,000 Canadians are living with IBD. Assuming trends remain the same, this number is estimated to increase to 402,000 in 203020. The increasing prevalence not only has major implications on an affected individual’s quality of life, but also adds burden on the healthcare system. For example, the direct cost associated with IBD care in Canada was > $1.28 billion in 2018. Prescription drugs contribute to majority of the costs, especially with the increasing use of biologics. In addition, surgical procedures, testing, emergency department visits and outpatient care all add to the direct costs. Indirect costs of IBD come from absenteeism of individuals from work, premature retirement as well as co-morbidities21. High prevalence of IBD in Canada and the continuing rise in cases is a concern for the individual’s quality of life as well as institutional economic burden associated with diagnosis and care. IBD was thought to be a disease of the western world, however, incidence has been rising in newly industrialized countries including the Middle East, Asia, and South America22. Overall, the number of cases globally have risen from 3.32 million in 1990 to 4.90 million in 2019. This corresponds to a 47.45% increase over 30 years22, 23. This global increase in incidence has been correlated with “westernization” including urbanization and dietary changes. For example, “westernization” includes consumption of a diet rich in fat, refined sugar, processed food, and low in fiber. These dietary changes have been associated 4 with increased risk of developing IBD24. However, it is important to note that increasing disease awareness and availability of diagnostic tools may also explain increases in newly industrialized countries16, 25. Although in the past, IBD was thought to be a disease impacting Caucasian people of European descent as well as Jewish people, the global rise in cases has reinforced that IBD is a disease impacting people of all ethnicities18. Studies have shown that migrating from a low incidence region to a region with high incidence of IBD increases risk of developing the disease. Additionally, this risk is higher when migration occurs at a younger age26. Rising incidence in newly industrialized countries highlight the role of environmental influences on increasing global incidence22. 1.1.3 Current strategies and therapies for disease management The goal of treating people with IBD is to improve quality of life by inducing and maintaining remission, and by preventing complications27. The introduction of biologics over the past two decades has shown to be effective in meeting these goals and has revolutionized treatment regimens28. In order to assess efficacy of treatment, a treat-to-target approach is employed in which a target is assessed for disease improvement. Targets to assess disease improvement have shifted from more subjective measures of clinical remission such as patient symptoms to more objective measures including mucosal healing. Clinicians use a combination of biomarkers, imaging, endoscopy, and histology evaluations to assess mucosal healing. Using mucosal healing as a target for disease improvement has shown to extend remission time and reduce the need for surgeries27, 29, 30. Mucosal healing leads to amelioration of disease and reduces risk of developing complications27. With respect to therapeutic intervention, a step-up approach is traditionally used. This approach involves use of less effective drugs that can cause lower number of side effects before moving onto more efficacious drugs that can potentially cause more side effects. Therapies include 5’- aminosalicylate/antibiotics then moving up towards corticosteroids, immunomodulators, and then biologics31. The step-up approach is common in individuals with mild to moderate disease who are at low risk for disease progression. The time to move onto more efficacious therapies in the step-up approach can be accelerated if therapeutic goals are not being met at defined time points. This is to induce remission faster and prevent complications from lingering disease. Alternatively, a top-down approach can 5 be used where more potent biologics and immunomodulators are started earlier in the disease course. This is mainly implemented for individuals with severe disease or if they have a high risk of complications27, 31. The past two decades have led to increased availability of therapies to treat disease. Examples of therapies include biologics that neutralize tumor necrosis factor alpha (TNFα) to suppress inflammation through TNFα neutralization. Anti-TNFα agents, such as Adalimumab and Infliximab, are used to treat moderate to severe CD and UC32. Other therapies include an anti-integrin biologic, namely Vedolizumab. Vedolizumab binds α4β7 integrin expressed on T lymphocytes to prevent their trafficking into the gut. However, studies have shown that it may act selectively to prevent trafficking of specific T cell subsets and have proposed alternative mechanisms of action. Vedolizumab is approved for use in moderate to severe CD and UC33. Anti-IL therapy includes Ustekinumab, an antibody binding to the p40 subunit of IL-12 and -23. It works to prevent IL-12/23 signaling in T and Natural Killer (NK) cells to reduce inflammation. It is used to treat moderate to severe CD and UC34. Lastly, Janus Kinase (JAK) inhibitors (Tofacitinib) are new class of small molecules used currently only to treat UC. Tofacitinib works by binding the active site of Jak3 to prevent downstream signal transducer and activator of transcription (STAT) signaling. Research on its use in CD is ongoing35. With the development of biologics and small molecule inhibitors, clinicians have treatment options, allowing for tailored treatment regimens towards an individual’s needs. Once treatment has begun, it is critical to monitor disease to ensure a state of remission. This strategy is referred to as Therapeutic Drug Monitoring (TDM)27, 36. Plasma can be assessed for drug concentrations to ensure optimal dosage, especially during active disease. Some individuals may also require higher than the recommended dosage, especially in cases of perianal fistulizing CD27. Moreover, it is important to monitor antibodies against the drug in the plasma as some individuals may become refractory over time. By switching to another drug, this can prevent the time the individual is left untreated, improving quality of life27. A trial by Assa et al., revealed that when pediatric patients with CD were given adalimumab, remission rates were higher in individuals who received TDM than those that did not37. Overall, TDM ensures clinical remission. 6 1.1.4 Etiology of disease Although the exact cause of IBD remains unknown, it is known to occur at the intersection of genetic, environmental, and immune factors. It is believed that genetically predisposed individuals mount aberrant immune responses to the gut microbiota38. Due to the complexity of disease, there have been many advances in trying to understand the contribution of each factor in disease. Figure 1.1 Etiology of IBD IBD is a multifactorial disease involving complex interplay between environmental and genetic factors that can compromise the integrity of the epithelial barrier. The impaired barrier allows for commensal microbes and their products to translocate into the bowel wall from the gut lumen, initiating innate and adaptive immune responses. Failure of anti- inflammatory responses to resolve inflammations leads to development of chronic 7 inflammation, which increases risk for developing complications. Reproduced with permission from Neurath M, Nature Reviews Immunology 2014. 1.1.4.1 Genetics A group of individuals that display very early-onset IBD (VEO-IBD) have a strong genetic component associated with disease. VEO-IBD encompasses individuals that develop disease before 6 years of age. These individuals display more severe disease and tend to be refractory to conventional therapies39. In contrast to older individuals that tend to exhibit polygenic variations, disease in VEO-IBD is often monogenic. The genetic variations are often related to primary immunodeficiencies and can fall into one of the 6 categories: (i) general immune dysregulation, (ii) defects in T and B cells, (iii) dysfunctional epithelial barrier, (iv) hyper- and auto- inflammatory conditions, (v) defects in phagocytosis, and (vi) other conditions39, 40. Specifically, several mutations within IL10, IL10Rα (IL-10 receptor α), and IL10Rβ have been associated with disease41, 42. Moreover, 19-41% of individuals with VEO-IBD show a strong family history of disease39. VEO-IBD highlights the contribution of genetics in disease. In addition to VEO-IBD, twin and familial studies reveal a strong genetic influence in IBD. Twin studies allow for better control of environmental variables assuming shared spaces, allowing for the understanding of genetic influences43. Studies involving twins from Sweden, Denmark, and the UK show that monozygotic twins have 50-55% concordance rates in CD and 15.0-18.8% concordance rates in UC43, 44, 45. Concordance rates in dizygotic twins are lower, 10% and 4% in CD and UC, respectively. Family history of IBD also influences risk of developing disease47. In a Swedish cohort, 1.5%-28.0% of CD cases and 1.5%-24.0% of UC cases have a family history of disease. This corresponds to an 8-fold and 4-fold higher risk of disease in first degree relatives of people with CD and UC, respectively46, implicating genetics in disease susceptibility. Genome-wide association studies have reported over 240 genes associated with susceptibility to developing IBD38, 50. In 2001, nucleotide-binding oligomerization domain 2 (NOD2) was the first gene associated with CD 48. It is expressed in monocytes, macrophages, intestinal epithelial cells (including Paneth cells), and lymphocytes. It is a cytosolic receptor 8 that binds to muramyl dipeptide (MDP), a component of the bacterial cell wall. Downstream signaling ultimately activates nuclear factor kappa B (NF-κB) leading to secretion of pro- inflammatory cytokines48, 51. NOD2 is also involved in autophagy, which is dependent on ATG16L1 (Autophagy-Related 16-like 1). Mutations in ATG16L1 are strongly associated with CD. People with CD carrying NOD2 or ATG16L1 tend to have localized inflammation to the small intestine that has been attributed to dysfunctional Paneth cells48, 49. Other examples of genes associated with both CD and UC include human leukocyte antigen (HLA). The exact mechanism how HLA variants contribute to disease is unknown. However, it is thought that molecular mimicry may play a role where foreign antigens may activate self-reactive T cells46. Polymorphisms in IL-23 receptor (IL-23R) also present in both CD and UC. IL-23 signaling is important in the maintenance of Th17 cells and loss of function of IL-23R has been shown to be protective in IBD52. GWAS has reported gene variants that are shared between CD and UC as well as some specific to each disease suggesting shared and unique mechanisms associated with each disease phenotype. 1.1.4.2 Environmental factors Environmental factors can influence susceptibility to disease very early in life. Breastfeeding has shown to be protective in reducing risk of developing IBD53. Breastmilk influences microbiome composition and promotes immune tolerance to dietary and microbial antigens53. Moreover, antibiotic use in the first year of life has shown to increase risk for developing IBD54. Growing up in a westernized setting has also been associated with disease susceptibility. For example, having less than 2 siblings, not sharing beds, no pets, or no exposure to farm animals increases risk of developing IBD55, 56. This relates to the hygiene hypothesis where increased sanitation with reduced exposure to microbes can increase risk of immune-mediated diseases56. In addition to living conditions, westernization involves dietary changes. In general, western diets tend to be calorie dense, high in sugar, animal proteins, carbohydrates, and processed foods. Studies on western diets revealed an enrichment of microbes in the gut responsible for metabolizing fat, animal proteins, and sugars as well as a decrease in fiber fermenting bacteria, and increased intestinal permeability58, 59. Also, high fat diets in mice induce endoplasmic reticulum stress in intestinal epithelial cells, compromising barrier integrity57. Thus, dietary interventions have been explored with the aim of improving 9 disease outcomes. An example includes the consumption of a Mediterranean diet, which is high in fruits, vegetables, nuts, whole grains, and fish, with olive oil being the primary fat source60. Short-term, Mediterranean diet consumption in people with mild to moderate IBD showed improvement in quality of life61, 62. Although promising, more research is needed to gain a deeper understanding of the mechanisms by which therapeutic diets may alleviate symptoms. In addition to macronutrients, micronutrients play a role in IBD. For example, people with IBD have increased incidence of deficiency in vitamin D. Vitamin D is critical for maintenance of epithelial integrity as it regulates the tight junction proteins63. It also modulates anti-inflammatory responses in immune cells63. Finally, smoking has been shown to worsen CD, possibly through impairment of autophagy, whereas it is protective in UC, as it can promote mucus production in the colon38. In conclusion, there are many environmental factors that can influence risk of developing IBD. 1.1.4.3 Microbial factors The healthy gut microbiome is comprised of 10-100 trillion microbes including bacteria, protozoa, fungi, and viruses70. Bacteria make up majority of the population with the dominating phyla being Firmicutes and Bacteroidetes65. The localization of bacteria is dependent on multiple factors, including, chemical, nutritional, and immunological gradients within the gut. The density and species of microbes is lower in the stomach and small intestine compared to the jejunum and colon66. The small intestine fosters an environment with high oxygen and acid levels and low transit time compared to the colon, which is mainly anaerobic. As such, fast growing, facultative aerobes that can adhere to the mucus are able to thrive in the small intestine compared to anaerobes that can digest complex carbohydrates, which thrive in the colon66. Commensal bacteria reside in a symbiotic relationship with the host and are essential for maintaining homeostasis. They prevent pathogen colonization by competing for space and nutrients, a mechanism called “colonization resistance”67. Moreover, they promote epithelial integrity and immune tolerance by producing key metabolites. For example, they 10 produce short chain fatty acids (SCFAs) from indigestible fiber, which is an energy source for colonic epithelial cells. Butyrate, a type of SCFA, has indirect anti-inflammatory properties by contributing to expansion of regulatory T cells (Tregs)67, 68, 69. To prevent commensals from triggering unwanted immune responses, the host has also evolved mechanisms to limit their contact with the epithelial layer. Examples include production of mucus, antimicrobial peptides, and immunoglobulin (Ig) A to segregate the microbes from the epithelium as well as control their density67, 68, 69. In summary, the gut microbiome is beneficial to the host and must be maintained to prevent aberrant immune responses. Perturbations in gut microbial composition have been associated with IBD. It has been reported that people with IBD exhibit an increased proportion of Proteobacteria and Bacteroidetes, and reduced proportion of Firmicutes67. Additionally, people with IBD have reduced bacterial diversity compared to healthy individuals64, 70. Specifically, a decrease in SCFA-producing bacteria, increase in mucus-degrading bacteria, and presence of pathogenic bacteria (i.e. adherent-invasive Escherichia coli, Clostridioides difficile) have been reported64, 70. Together, these changes can negatively influence epithelial cell growth, Treg populations, and mucus lining, all of which can increase intestinal barrier permeability and promote inflammation70. Although the causal role of the gut microbiota remains unclear, there is clear evidence for its contribution to disease. For example, murine colitis models show amelioration of inflammation in germ-free settings71. In addition, transfer of fecal matter from people of IBD into germ-free mice promotes colitis72. Moreover, antibiotics are used clinically to reduce intestinal inflammation and prevent reoccurrence after surgery71. Together, perturbations in microbial populations can lead to alterations in the composition of microbes and their metabolites and functions, which can contribute to IBD. 1.1.4.4 Immune factors IBD is an immune-mediated disease. The intestinal immune system is separated from luminal contents by a single epithelial layer protected by mucosal barriers73. The immune system has evolved mechanisms to maintain tolerance to microbial and food antigens but also protect the host from pathogens by mounting an inflammatory immune response. The immune response comprises both innate and adaptive immunity that when dysregulated, can 11 contribute to IBD73. Both, the innate and adaptive immune responses are discussed below in detail. The first line of defense includes innate immunity, which comprises of the physical barriers and immune cells that recognize antigens in a non-specific manner74. The mucus layer separates the gut luminal contents from the epithelium. Goblet cells in the epithelium secrete mucins that form the mucus layer. The mucus layer is comprised of an inner dense layer that is resistant to microbe penetration and a permeable outer layer, which houses commensals75. IBD pathogenesis has been associated with goblet cell loss or altered composition of the mucus in CD and UC, respectively76, 77. The next line of defense involves the epithelium. Specialized intestinal epithelial cells (IECs) are derived from self-renewing stem cells located at the base of the crypts. People with IBD exhibit defects in epithelial cell renewal, possibly due to the damage caused to stem cells during inflammation206. People with IBD also show reduced production of antimicrobial peptides by Paneth cells, more so in CD compared to UC74, 75. Immune cells that constitute innate immunity include antigen presenting cells (APCs). There are two main APCs, dendritic cells (DCs) and macrophages. DCs are found in the gut-associated lymphoid tissues, isolated lymphoid follicles and Peyer’s patches77. They can encounter antigens from the lumen that are transported by M cells. This is important in maintenance of tolerance to commensals and food antigens74, 78. In the intestine, CD103+ DCs produce Transforming growth factor-β (TGF-β) which induces Tregs. They also express retinaldehyde dehydrogenase 2 (RALDH2), which converts vitamin A to retinoic acid, further promoting Treg expansion and homing to the intestine79. In the context of both CD and UC, DCs exhibited higher expression of Toll-like receptor (TLR) 2 and 4 and produce more IL-12 and IL-6, contributing to disease74, 75. Macrophages are another APC, which, unlike DCs, do not migrate to the draining lymph nodes, but rather, act locally. They can exhibit inflammatory and anti-inflammatory properties depending on the environment. They can produce IL-1β which can induce Th17 cells. They can also produce IL-10 which can limit effector T cell functions while promoting Tregs80. In homeostasis, they are crucial for scavenging apoptotic cells and debris as well as maintaining epithelial integrity. In 12 disease, genetic variants in pathways involved in intracellular bacterial sensing and subsequent clearance have demonstrated that defects in macrophage functions can promote disease80. Moreover, an increase in pro-inflammatory cytokine (TNF, IL-1β, IL-6, and IL-23) producing macrophages has been confirmed in active disease. Interestingly, clinical remission following anti-TNF therapy has been associated with a reduction in pro- inflammatory macrophage activity80. Overall, DCs and macrophages exhibit a common role of maintaining immune tolerance. In disease, dysregulated immune functions are associated with disease promoting rather than tolerance inducing phenotypes. Adaptive immunity involves highly specific immune responses to an antigen. Key players in adaptive immunity involve T cells, which are activated by DCs in the lymph nodes into memory and effector T cells81. Dysregulated T cell responses can lead to excessive production of chemokines and cytokines that can promote disease82. Major T cell subsets include Th1, Th2, and Th17 cells. Th1 cells are mainly activated in response to intracellular pathogens and produce IFNγ, IL-12, and TNFα. Th2 cells are involved in eliminating extracellular microbes and helminths and produce the cytokines IL-4, IL-5, IL-13, IL-21, and IL-25. Th17 cells are responsible for protection against pathogens at mucosal surfaces and secrete IL-17 and IL-2274, 81. Originally, CD was associated with Th1 responses whereas UC was thought to involve Th2 cells. This paradigm has shifted over the years through the discovery of the involvement of other cell types (i.e. Th9, Th17, and Th22)81. Moreover, T cells have been shown to exhibit plasticity dependent on the cytokine environment83, 84. As such, this Th1/Th2 paradigm does not consider the altering cytokine profiles of T cells that occur during disease83. Moreover, two drugs, Anrukinzumab and Tralokinumab, that neutralize IL-13 failed to show clinical efficacy in improving ulcerative colitis85, 86. These trials provide evidence for rejecting the Th1/Th2 paradigm as it does not hold in human studies. In addition to T cells, B cells are also involved in adaptive immunity. In homeostasis, they secrete IgA, which gets transported to the gut lumen by epithelial cells76. However, in IBD, antibody responses get skewed towards IgG. Antibodies to microbes such as Saccharomyces cerevisiae and E. coli have been found in people with IBD. Moreover, antibodies against self-antigens have also been reported such as the colon epithelial antigen, 13 tropomyosin, and neutrophilic cytoplasmic antibodies75, 87. The immune system is a complex network of cells and further understanding about how these cells communicate to promote disease is required. 1.2 Crohn’s Disease-associated fibrosis More than one-third of people diagnosed with CD will develop a complication called fibrosis, which can be life-threatening88. Fibrosis is an aberrant wound healing response that may occur downstream of inflammatory tissue damage88. Fibrosis can occur transmurally, affecting the mucosa, submucosa, muscularis propria, and serosa layers of the bowel89. Key cell types involved are mesenchymal cells, which include fibroblasts and smooth muscle cells (SMCs). They contribute to fibrosis by depositing excessive amounts of extracellular matrix (ECM) components and thickening of the muscle layer88. Ultimately, the narrowing of the lumen can cause bowel obstruction and symptoms including abdominal pain, vomiting, cramping, and nausea. Early introduction of biologics has reduced the number of hospitalizations and surgeries in people with CD; however, incidence of fibrosis remains high91. Some researchers have proposed that fibrosis can progress independently in a self- perpetuating manner once established81, 92. There are currently no approved therapies to treat intestinal fibrosis91. As such, it is crucial to understand the mechanisms contributing to intestinal fibrosis to identify novel targets or strategies for therapy. 1.2.1 Cellular contributors of intestinal fibrosis Mesenchymal cells are non-hematopoietic cells that play a key role in contributing to intestinal fibrosis. They include fibroblasts, myofibroblasts, and SMCs. These cell types can be distinguished by their expression of vimentin, desmin, and alpha-smooth muscle actin (α- SMA)93. Fibroblasts are involved in maintaining tissue integrity and promoting inflammation resolution94. Activated fibroblasts in CD can lead to excessive deposition of extracellular matrix (ECM) components such as collagen subtypes I, III, and V88. They can also promote inflammation by releasing chemokines. Examples include C-X-C motif chemokine ligand (Cxcl) 12, C-C motif chemokine ligand 2 (Ccl2), colony stimulating factor 1 (CSF1), Cxcl1, and Cxcl8 to recruit lymphocytes, monocytes, macrophages, and neutrophils, respectively93. 14 McKaig et al. showed faster proliferation of fibroblasts that were isolated from the mucosa of people with IBD95. In the presence of IL-1β and TGF-β1, fibroblasts can differentiate into myofibroblasts94, 95. Myofibroblasts possess migratory capabilities through production of fibronectin, which provides them with an intermediate phenotype between fibroblasts and SMCs. Interestingly, it has been shown that myofibroblasts exposed to various microbial products selectively responded to flagellin, resulting in increased ECM production91. Myofibroblast differentiation could also be promoted through a positive feedback loop where ECM linking leads to tissue stiffness, triggering differentiation of fibroblasts into myofibroblasts. In CD, this positive feedback may aggravate progression of fibrosis88, 96. There are two types of myofibroblasts in the intestine, sub-epithelial myofibroblasts (SEMFs), which are located at the crypt base. These cells are thought to be involved in influencing epithelial cells to maintain a stem cell niche97 and are found to be dysregulated in colitis93. Interstitial cells of Cajal (ICC) are another type of myofibroblasts primarily found in the submucosa and muscularis propria and are involved in smooth muscle motility. SMCs are found in the muscularis mucosa and are responsible for producing collagen types I, III, and V. Proliferation of SMCs in CD can contribute to muscle thickening, contributing to narrowing of the lumen94. In summary, the three main cell types involved in intestinal fibrosis are fibroblasts, myofibroblasts, and SMCs. Further understanding of the cellular mechanisms involved in pathosis could allow identification of targets for therapy. 1.2.1.1 Epithelial cells in intestinal fibrosis The complex network between epithelial cells, immune cells, and fibroblasts contributes to the development of intestinal fibrosis. In fibrotic tissue, epithelial cells can serve as a source of fibroblasts, a process referred to as epithelial mesenchymal transition (EMT). During EMT, epithelial cells begin to lose polarity and epithelial markers and gain markers of fibroblasts as well as a migratory phenotype98. During the transition, the cells possess an intermediate phenotype, which maintain basement structures and gap junctions99. TGF-β1 is the key trigger of EMT. Specifically, TGF-β1 acts on epithelial cells to activate Small mothers against decapentaplegic (SMAD)2/3/4 complex, which upregulates transcription factors related to EMT (i.e. SNAIL, ZEB, and TWIST)98. Additionally, in vitro 15 studies have shown that TGF-β1 stimulation can induce IL-13 expression in epithelial cells that acts in an autocrine manner to promote EMT99, 100. Ultimately, the cells begin to express mesenchymal markers such as α-SMA and vimentin, and downregulate epithelial markers, including E-cadherin and claudins99. Using a 2,4,6-Trinitrobenzene sulfonic acid (TNBS) mouse model of CD with lineage tracing of epithelial cells, the Kalluri group have reported around 30% of fibroblasts were derived from epithelial cells98, 99. Presence of EMT has been confirmed in fibrotic tissue from people with CD using staining for transcription factors and cell markers101. It has been proposed that EMT may be a compensatory mechanism for the reduced migratory ability of myofibroblasts in IBD99. The reduced migration was shown in vitro by Leeb et al. using fibroblasts isolated from the colonic lamina propria of people with IBD102. To maintain homeostasis, EMT is balanced by mesenchymal epithelial transition (MET). However, in CD, stimuli for fibrosis alter the EMT/MET balance to favor EMT98. Inhibiting EMT by administering bone morphogenic protein 7 in TNBS mouse model of CD ameliorated fibrosis100. Targeting EMT is a potential therapeutic strategy to treat intestinal fibrosis. 1.2.1.2 Macrophages in intestinal fibrosis Macrophages are a heterogenous group of cells that exhibit a range of phenotypes depending on the cytokine environment. They can exhibit a pro-inflammatory phenotype upon tissue injury and express the markers CD64, F4/80, MHCII, CD206, and CD163103. Pro-inflammatory macrophages can be activated by microbial products (i.e. lipopolysaccharide (LPS)) and inflammatory cytokines (interferon-gamma (IFNγ)). They contribute to inflammation by producing reactive oxygen species, proinflammatory cytokines (i.e. TNF-α, IL-1β, IL-12, and IL-23), and by recruiting immune cells103, 104. During a type 2 immune response, Th2 cytokines such as IL-4 and IL-13 can activate macrophages to exhibit an anti-inflammatory phenotype. These macrophages express markers such as CD206, CD163, ArgI, Ym1, and IL-1 receptor antagonist (IL-1Ra). Key products secreted include TGF-β1 that activates fibroblasts and tissue inhibitors of metalloproteinase (TIMP)-1 that suppresses collagen degradation103. 16 In mice (not humans), ArgI upregulation in macrophages by IL-4 or IL-13 promotes collagen biosynthesis105. Mechanistically, ArgI competes with inducible nitric oxide synthase (iNOS) for their common substrate, L-arginine. ArgI will break down L-arginine into ornithine which ultimately gets broken down into precursors used for cell proliferation and collagen synthesis. By sequestering L-arginine from iNOS, nitric oxide (NO) production is prevented, reducing inflammation105, 106. In a CD-associated mouse model of fibrosis, the Sly laboratory has shown that blocking PI3K p110δ-mediated upregulation of ArgI to ameliorates intestinal fibrosis107. In people with CD, intestinal biopsies from fibrotic sections revealed an increase in similarly activated macrophages108. Understanding the role of macrophages in intestinal fibrosis may shed light onto pathways that can be targeted for anti- fibrotic therapy. 1.2.2 Molecular mediators of intestinal fibrosis In addition to the contribution of mesenchymal cells in ECM deposition, molecular mediators sustain the process of fibrosis through activation and proliferation of these cells109. These mediators, including multiple cytokines and growth factors, are discussed below. TGF-β1 is the most well described factor contributing to fibrosis in the gut as well as other organs, and is primarily produced by macrophages and fibroblasts. TGF-β1 is secreted as an inactive form, which gains activity upon proteolytic cleavage. Its function is mediated through binding to its receptors, TGFβR1 and 2, resulting in phosphorylation of SMAD2/3. Consecutively, a complex with SMAD4 is formed, which translocates to the nucleus to initiate transcription. SMAD7 is a negative regulator of the pathway94, 110. TGF-β1 can also signal in a SMAD independent manner involving ERK (extracellular signal-regulated kinase) 1/2, JNK (c-Jun N-terminal kinase), and p38 MAPK (mitogen-activated kinase)111. TGF-β1 signaling in fibroblasts results in activation of α-SMA allowing for differentiation of fibroblasts into myofibroblasts and increasing their migratory and collagen producing capabilities. Specifically, collagen types I and III are upregulated. In addition to inducing collagen in fibroblasts, TGF-β1 regulates the balance between matrix metalloproteinases (MMPs) and their inhibitors, TIMPs. MMPs are involved in degrading components of the 17 ECM such as collagen, fibronectin, and lamins while TIMPs work to inhibit MMP activity. TGF-β1 can promote TIMP production in fibroblasts while suppressing MMPs, thus contributing to a net positive accumulation of ECM94. Strictured areas of people with CD have high expression of TGF-β1 phosphorylated Smad2/3 and TIMP-1 while there is reduction in MMP-12, MMP-3, and SMAD7 expression compared to non-strictured areas112. In addition to TGF-β1, TNF-like cytokine 1A (TL1A) can contribute to fibrosis. It is a member of the TNF superfamily of proteins that binds to death receptor 3 (DR3). TL1A/DR3 signaling can promote collagen synthesis in myofibroblasts as well as enhance the IL-13 and IL-17 signaling pathways113. It may also lead to induction of IL-17. Both gain of function as well as loss of experiments in mouse models have shown TL1A to promote intestinal fibrosis. Additionally, TL1A and DR3 are both upregulated in people with CD113. It is generally believed that uncontrolled type 2 and type 17 responses can contribute to fibrosis. IL-13 is a type 2 cytokine that signals through IL-13Rα2 to regulate TGF-β1 expression. Although initially IL-13 was associated with UC, it has now been shown that fibrotic tissue sections from people with CD also have high expression of IL-13109, 114. IL-4 also has the capacity to promote fibrosis by inducing collagen in fibroblasts, however, its expression in the mucosa of people with CD is undetectable so the role of IL-4 in intestinal fibrosis remains controversial109. IL-17A, referred to as IL-17, is produced by Th17 cells that is also upregulated in fibrotic tissue sections of people with CD. Myofibroblasts stimulated with IL-17 upregulate MMP-3, MMP-12, collagen production, and downregulate TIMPs. IL- 17 also enhances EMT109. Despite the amelioration of intestinal fibrosis upon IL-17 blocking in mouse models113, clinical trials involving neutralizing IL-17A or its receptor worsened inflammation in people with CD109. Further research is needed to understand the mechanisms by which type 2 and 17 cytokines contribute to intestinal fibrosis. Growth factors such connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) produced by macrophages can contribute to fibrosis. CTGF is a downstream product of TGF-β194. It can stimulate proliferation of fibroblasts, collagen production, and trans-differentiation of epithelial cells into fibroblasts94, 18 149. In addition to promoting fibroblast proliferation and collagen synthesis, PDGF can also induce fibroblast migration94. FGF also acts on fibroblasts and increased serum FGF correlates with muscle thickening in people with CD94. Targeting growth factors responsible for fibroblast activation may be a potential strategy to combat intestinal fibrosis. 1.2.3 Therapeutic approaches Currently, there are no therapies to directly treat intestinal fibrosis. Rather, management involves the use of anti-inflammatory therapies, as well as endoscopic and surgical interventions. The goal of stricture management in CD is to alleviate symptoms115. If inflammation is detected in strictures using cross-sectional imaging techniques, then steroids may be used to reduce the swollen bowel and alleviate obstructive symptoms89. Early use of anti-TNF in people with CD appears to reduce stricture formation. Initially, there was concern surrounding the use of anti-TNF in CD due to the possibility of stricture formation caused by rapid healing of ulcers. However, it is now believed that anti-TNF does not promote strictures89. In addition to therapeutics, common management strategies involve endoscopic interventions. Endoscopic intervention includes endoscopic balloon dilation, a method which involves inflation of a balloon to dilate the narrow structure88, 116. This method is only employed if the instrument can reach the affected region, the stricture is less than 5 cm in length, and there are no other complications117. Other endoscopic options include intralesional injection of corticosteroid and infliximab. However, this strategy is not currently implemented due to limited research89, 117. Another strategy involving endoscopic stents did not have success clinically as the stents migrated117. Lastly, surgery is done if the stricture is greater than 5cm and/or is inaccessible in the ileocecal region. Strictureplasty involves surgically widening the stricture while preserving bowel length. This is preferred when there are concerns of short bowel syndrome, or when fibrosis has not penetrated all layers of the bowel. However, in the case of penetrating disease or malignancy, surgical resection is used. Despite surgery, strictures frequently recur highlighting the need for therapies117, 118. With the need to find an anti-fibrotic therapy comes the challenges associated with it. The first challenge is associated with targeting cytokines. Pleiotropic functions of cytokines 19 at different times during disease make it difficult to target fibrosis without causing negative side effects. An example includes metelimumab, a monoclonal antibody against TGF-β1, in the context of systemic sclerosis resulted in complications involving the skin, musculoskeletal pain, and even death119. Additionally, the nature of CD-associated fibrosis makes it difficult to carry out clinical trials and requires special considerations when designing drugs. For example, defining end points has been difficult due to the slow progression of disease, which makes clinical trials very long and costly119. Finally, with respect to drug design, the transmural nature of disease demands the drug penetrate all layers of the intestine118. Developing an anti-fibrotic therapy for CD is an important and active area of research. 1.3 Murine models of intestinal fibrosis Model systems are crucial systems to study intestinal fibrosis in ways that it cannot be studied in humans. Mouse models can be divided into categories based on the method for inducing fibrosis. This includes chemically-induced, genetically engineered, spontaneous models, bacteria-induced, and immune-mediated models of fibrosis that are discussed below120, 121. An example of a chemically-induced model is the dextran sodium sulfate (DSS) colitis model. In this model, DSS is administered in the drinking water of mice to induce UC- like pathology in the colon. DSS functions by damaging the epithelium to disrupt integrity, allowing for microbes to breach the barrier. Macrophages are thought to be involved in contributing to the inflammation by responding to the breaching microbes121, 122. Fibrosis is induced by chronic administration of DSS over multiple cycles. Although this model is convenient and yields reproducible results, its relevance to human disease has been questioned. Another model employs intrarectal administration of TNBS in ethanol to induce fibrosis in the distal colon. The ethanol breaks the gut barrier allowing for the TNBS to modify colonic proteins. The adaptive immune responses are then activated to initiate transmural disease. Caveats of this model include localization of disease to the distal colon 20 because of rectal administration as well as variability in mouse strains to develop colitis120, 121, 122. Genetically engineered models of intestinal fibrosis include mice deficient in IL-10, an anti-inflammatory cytokine. These mice develop enterocolitis in which inflammation is more severe in the colon compared to the small intestine123. CD4+ T cells are responsible for the phenotype as confirmed by injecting IL-10-/- naïve CD4+ T cells into recombination activating gene2 (Rag2) knockout mice, a strain deficient in mature T and B cells120. Disease onset in IL-10-/- mice is gradual, allowing for study of environmental factors and their contribution to disease progression121. These mice were also used to study post-operative fibrosis in which fibrosis developed at sites of resection120. In addition to IL-10-/- mice, TGF- β1 overexpressing mice also develop fibrosis. Specifically, an adenoviral vector containing bioactive TGF-β1 is administered rectally such that transfection occurs in colonic epithelium. This allows for mucosal and submucosal immune responses. Using this model, it was verified that TGF-β1 cannot be a target for therapy as TGF-β1 deficient mice developed multiorgan inflammation, highlighting its key role in maintaining homeostasis120, 122. Lastly, overexpression of monocyte chemoattractant protein-1 (MCP-1), a chemoattractant for monocytes, NK cells, and T cells, results in colonic fibrosis after intramural injection of the adenoviral vector. Inflammation is dependent on the presence of lymphocytes in this model120. Overall, genetic manipulation of IL-10, TGF-β1, and MCP-1 in mice can result in intestinal fibrosis. In addition to chemically-induced, genetically engineered and immune-mediated models, fibrosis can also be induced with bacterial infections. Specifically, giving Salmonella enterica serovar Typhimurium orally to mice after antibiotic treatment results in fibrosis in the cecum and colon after 3 weeks120, 122. However, clinically, intestinal fibrosis has not been linked to Salmonella enterica serovar Typhimurium, questioning its transability to human disease. Despite that, it offers a good tool to study the relationship between inflammation and fibrosis in a time dependent manner120. 21 So far, the models discussed above mainly develop fibrosis in the colon. This poses a limitation with respect to studying CD-associated fibrosis as the main site of disease and fibrosis is the terminal ileum. Although limited, there are spontaneous mouse models that can be used to study fibrosis in the terminal ileum. This includes a mouse model called SAMP1/YitFc (SAMP). These mice were generated by breeding AKR/J mice with an unknown strain, which were then subsequently bred together to create a senescence accelerated strain. By 30 weeks of age, these mice exhibit 100% penetrance of inflammation as well as thickened muscle layers and collagen accumulation. Disease is mainly localized to the terminal ileum However, they have a low breeding rate making it difficult to carry out large scale experiments120, 121. Another model in which fibrosis is confined to the terminal ileum is mice overexpressing TNF. These mice are heterozygous for a deletion of AU-rich elements in the 3’ untranslated region of the TNF mRNA yielding greater transcript stability, thus, more production of the protein. A limitation of this model is that the mice develop fibrotic pathology around 24 weeks and do not develop obstruction or fistulas124. Lastly, a mouse model that is deficient in SHIP spontaneously develop human CD-like intestinal inflammation and fibrosis125. This model is discussed in detail in section 1.4.1. Spontaneous mouse models that develop intestinal inflammation and fibrosis in the ileum can be beneficial in understanding CD specific mechanisms. Although mouse models are essential systems for understanding mechanisms and testing drugs in vivo, undeniably, there are differences in mice and humans that adds a layer of complexity when it comes to translation of results. As such, there has been an emergence in the use of intestinal organoids derived from humans. Intestinal organoids can be derived from induced pluripotent stem cells (iPSC), human embryonic stem cells or from stem cells taken from mucosal biopsies. Organoids from iPSCs contain mesenchymal cells in addition to the epithelium whereas organoids derived from biopsy stem cells contain only cells of the epithelium126, 127, 128, 129. Organoids can be grown into 3D structures that allows for cell-to- cell interactions that better mimic in vivo conditions129. With respect to specifically studying the role of epithelial cells in fibrosis, EMT can be induced in the organoids with TGF-β1130. However, the limitations of organoids involve understanding mechanisms restricted to 22 epithelial cells in the absence of the immune cell compartment and in some cases, the mesenchymal cells. When selecting the right model system to answer a question, some key considerations include the location of disease, involvement of the innate or adaptive immune system, cost (genetically engineered models can be more expensive than chemicals) as well as training for procedures such as intrarectal administration and access to facilities for experiments that require germ free conditions122. 1.4 Src homology 2 domain-containing inositol polyphosphate 5’-phosphatase (SHIP) 1.4.1 Description SHIP is a 145 kDa protein encoded by the gene Inpp5d. Its expression is restricted to hematopoietic cells including some parenchymal cells such as osteoclasts and endothelial cells131. It functions as a phosphatase to negatively regulate class I PI3K pathway, which is involved in cell proliferation, survival, and function. PI3Ks are lipid phosphatases located adjacent to the inner plasma membrane. They phosphorylate the 3’ hydroxyl group of the inositol ring found on phosphoinositides. There are three classes of PI3K which are defined by their substrate specificity. Specifically, class I PI3K phosphorylates phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) into phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3), which is a substrate for SHIP132, 133. Class IA is a subdivision of the class I PI3Ks that are activated upon engagement with TLRs, receptor tyrosine kinases, growth factors, and cytokines. They are comprised of one regulatory subunit (p85𝛼, p85𝛽, p55𝛼, p55𝛾, or p50𝛼) and one catalytic subunit (p110𝛼, 𝛽, or δ). Ultimately, PI(3,4,5)P3 activates AKT proteins to promote NF-κB mediated transcription. SHIP negatively regulates the PI3K pathway by dephosphorylating PI(3,4,5)P3 at the 5’ position to yield phosphatidylinositol-3,4 bisphosphate (PI(3,4)P2)106, 134. In summary, SHIP is a phosphatase that regulates PI(3,4,5)P3 levels. 23 Figure 1.2 Function of SHIP PI3Kp110 phosphorylates PI(4,5)P2 at the 3’ position to generate PI(3,4,5)P3, a critical second messenger that regulates multiple cellular processes including immune cell activation. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) removes the product of PI3Kp110 by de-phosphorylating the 3’ phosphate. SHIP mediates negative regulation of the PI3K pathway by dephosphorylating the 5’ phosphate to generate PI(3,4)P2. Reproduced with permission from Dobranowski and Sly, Journal of Leukocyte Biology, 2018. SHIP is comprised of multiple domains that have distinct functions. The src homology 2 (SH2) domain is located at the N-terminus. The SH2 domain allows for translocation of the protein to the plasma membrane. The cationic charges in the pY binding pocket of the SH2 domain can interact with membrane lipids as well as tyrosine phosphorylated proteins106. It has high binding affinity for phosphorylated tyrosine residues and can bind to intracellular receptors such as FcγRIIB, gp49B1, CD31, FcγRIIA, FcγRI- associated zeta-chain, TCR zeta chain, FcεRI, and BCR135, 136. The SH2 domain can also bind to cytoplasmic proteins (e.g. Shc1 and Dok-3), which may act as either adaptors to bridge SHIP to receptors or may compete with SHIP binding to prevent its activity. SHIP also contains a Pleckstrin homology-related (PH-R) domain that is located within the phosphatase domain. The PH-R domain has a high binding affinity for (PI(3,4,5)P3)137, 138. In addition, PH-R is required for the localization of SHIP to the phagocytic cup in macrophages137. The phosphatase activity of SHIP can be enhanced by phosphorylation of Ser440 within the phosphatase domain as well as binding of (PI(3,4)P2) by the C2 domain139. The C2 domain contains 2 NPXY sequences that when phosphorylated can interact with Shc1, Dok-1 and Dok-3. Lastly, the C terminus contains a proline rich domain, which can interact with SH3 binding motifs106,138. 24 Figure 1.3 SHIP domains SHIP consists of a SH2 domain at the N-terminus that allows for its translocation to the plasma membrane as well as interaction with some intracellular receptors. The phosphatase domain contains a PH-related domain that has high binding affinity for its substrate, PI(3,4,5)P3, and a C2 domain. The C-terminus proline rich domain can bind to SH3 binding motifs. Numbers represent amino acids for human SHIP. Reproduced with permission from Dobranowski and Sly, Journal of Leukocyte Biology, 2018. Alternative transcription, alternative splicing, post-translational modifications or protein degradation can give rise to multiple isoforms (145, 130, 125, and 110 kDa) of SHIP that retain phosphatase activity with differential protein-binding properties106, 140, 141, 142. Specifically, the 110 kDa isoform is referred to as stem cell-restricted SHIP (sSHIP). sSHIP is only expressed in embryonic and hematopoietic stem cells. It arises from a promoter with the intron between exons 5 and 6 and as a result, lacks the SH2 domain. As the hematopoietic progenitors differentiate, sSHIP gets replaced with full length SHIP135, 140. SHIP2 is another 150 kDa protein encoded on a separate gene, INPPL1. Compared to SHIP, SHIP2 is more ubiquitously expressed and is present in varying degrees in hematopoietic cells. It shares a similar structure to SHIP and possesses 5’ phosphatase activity. However, they differ in the C-terminal proline rich region135, 138, 143. 1.4.2 SHIP in people with IBD Reduced SHIP protein has been associated with IBD. Ngoh et al. showed lower SHIP protein and activity in ileal biopsies and peripheral blood mononuclear cells (PBMCs) from people with CD compared to people without disease144. Lower SHIP activity is inversely related to IL-1β production144. Fernandes et al. also have reported that 15% of people with IBD having severe reduction in SHIP protein in their blood as well as circulating CD4+ T cells145. Although the causal relationship is unknown, reduced SHIP did correlate with 25 disease severity143, 145. Moreover, people homozygous for ATG16L1 T300A-encoding risk allele in both tissue and PBMCs have reduced SHIP. Although SHIP deficiency is independent of ATG16L1 risk alleles, considering its proximity, a mechanism has been proposed in which autophagy upregulates SHIP146. The correlation of SHIP with IBD warrants further research to uncover mechanisms contributing to disease. Location of INPP5D in the chromosome may explain SHIP deficiency in people with IBD. Interestingly, INPP5D lies within 44kb of ATG16L1, the risk variant associated with IBD145. Although SHIP deficiency is independent of the ATG16L1 risk alleles, a novel fusion transcript was found in which exon 25 of SHIP was linked to exon 2 of ATG16L1. This fusion transcript could lead to reduced biologically active SHIP levels as the C terminal truncation of SHIP could lead to less stable protein that is rapidly degraded145. 1.4.3 The SHIP-deficient mouse model of Crohn’s disease-like inflammation and fibrosis In order to study SHIP deficiency, a germline SHIP-deficient mouse model was first created in 1998 by inserting a neomycin resistance gene within the first exon of Inpp5d147. These mice spontaneously develop human CD-like intestinal inflammation and fibrosis by 8 weeks of age. The Kerr and Sly groups characterized ileal pathology of these mice. By 8 weeks of age, these mice have visibly thicker areas of redness interspersed by non-inflamed areas (skip lesions)125, 131. The inflammation is restricted to the distal 10-15 cm of the ileum125. Based on histopathology, SHIP-/- mice have an increase in the number of immune cells, presence of granulomas, loss of crypt-villus architecture, and edema. Granulocytes/macrophages were present throughout the bowel wall whereas CD3 T cells were scarce131. Interestingly, adoptive transfer of SHIP-/- T cells and NK cells was unable to promote ileitis in wild type mice while reconstituting SHIP-/- bone marrow with bone marrow from wild type mice as able to alleviate disease pathology. Moreover, disease 26 pathology is similar to mice transplanted with bone marrow cells over expressing granulocyte-macrophage colony-stimulating factor135. As such, it is thought that disease phenotype is likely due to an excessive activation of myeloid cells in the absence of T cells131. The key mediator of inflammation in this model is IL-1β. Specifically, macrophages have upregulated PI3Kp110𝛼 mediated IL-1β production in the context of SHIP deficiency144. The role of IL-1β in promoting inflammation was demonstrated by treating SHIP-/- mice with IL-1 receptor antagonist and by depleting macrophages with clod- liposomes. Both experiments led to amelioration of intestinal inflammation which correlated with a reduction in IL-1β concentration144. SHIP-/- mice also display fibrotic pathology. Muscle layers are thicker due to proliferation of the smooth muscle cells and excessive collagen is deposited throughout the submucosa and muscle layers125. In addition to inflammation and fibrosis, these mice exhibit reduced body weight, splenomegaly, myeloproliferative disorder, infiltration of myeloid cells in the lungs, and osteoporosis135, 147. In summary, SHIP-/- mice develop spontaneous CD-like inflammation and fibrosis mediated by macrophage IL-1 production. 1.4.4 SHIP-deficient macrophages SHIP is a key regulator of macrophage differentiation and function. In the absence of SHIP, there is an increase in macrophage differentiation from myeloid progenitors150. With respect to macrophage function, SHIP is generally involved in repressing pro-inflammatory functions. SHIP represses production of IL-6, TNF, and interferon-β (IFN-β) upon activation of pattern recognition receptors (PRRs) in macrophages151. In the context of helminth infections, SHIP deficiency in macrophages results in higher production of IL-12/23p40 promoting Th1 immune responses152. Additionally, bone marrow derived SHIP-/- macrophages have higher IL-1β production when cultured in granulocyte macrophage colony-stimulating factor (GM-CSF)144. SHIP also limits pro-inflammatory cytokine production by promoting endotoxin tolerance. LPS is an endotoxin found on the cell wall of Gram-negative bacteria. It binds to TLR 4 to trigger pro-inflammatory cytokine production such as IL-1β and IL-6. However, the production of pro-inflammatory cytokines needs to be regulated to prevent endotoxin shock153, 154. Sly et al. have shown upregulation of SHIP in 27 macrophages upon LPS stimulation and subsequent reduction in pro-inflammatory cytokines when exposed to LPS a second time. The phenomenon is referred to as “endotoxin tolerance”. This tolerance is absent in SHIP-/- macrophages highlighting SHIP as a positive regulator of LPS induced tolerance155. Except for a few studies156, 157, there is substantial evidence implicating SHIP as a negative regulator of pro-inflammatory cytokine production. In addition to regulating pro-inflammatory cytokine production in macrophages, SHIP is also involved in the process of phagocytosis. SHIP is a negative regulator of FcR- mediated phagocytosis. Depending on context, SHIP can also be a positive regulator of phagocytosis. For example, SHIP-/- macrophages had impaired phagocytosis upon Pseudomonas aeruginosa exposure151. SHIP is also involved in phagosome maturation. Recruitment of SHIP on phagolysosomes can promote early production of reactive oxygen species158. Thus, SHIP is a regulator of microbial clearance in macrophages through phagocytosis. SHIP is also a negative regulator of the anti-inflammatory phenotype of macrophages. In the absence of SHIP, in vivo macrophages are skewed towards a reparative phenotype including production of ArgI159. SHIP-/- macrophages are hyperresponsive to IL-4. Upon binding of IL-4 to its receptor, IL-4Rα and the common γ chain, it initiates phosphorylation, dimerization and transcription by STAT6. IL-4 binding also activates PI3Kp110δ which is required for STAT6 driven transcription of ArgI160, 161. ArgI uses L-arginine to ultimately produce precursors used for collagen synthesis and cell proliferation162, 163. As SHIP is a negative regulator of the PI3K pathway, SHIP deficiency results in higher ArgI activity in ileal tissue125. To assess the role of ArgI in disease pathology, ArgI was directly targeted using an inhibitor125 as was upstream PI3Kp110δ, both of which resulted in alleviation of inflammation and fibrosis in SHIP deficient mice107. Overall, ArgI is involved in promoting fibrosis and macrophages exhibit higher ArgI expression in the absence of SHIP. 28 1.5 IL-33 1.5.1 IL-33 description IL-33, also referred to as nuclear factor from high endothelial venues, was first described in 2005 as belonging to the IL-1 family of cytokines164, 165, 166. IL-33 is located on human chromosome 9p24.1 and mouse chromosome 19qC1 and is comprised of 8 exons. Studies have identified alternative splice variants of IL-33 produced by specific cell types167. Alternative splice variants have been studied within the context of asthma. Specifically, transcripts missing exons 3 and 4 result in cytoplasmic localization whereas exon 5 was associated with cytokine activity168. The human gene consists of two promoters giving rise to transcripts Il33a and Il33b. These two transcripts encode for the same protein and differ by their unique 5’untranslated regions166. IL-33 protein consists of different domains which are discussed in sections 1.5.2 and 1.5.3. IL-33 is involved in immune cell activation. It is primarily produced by non- hematopoietic cells in homeostasis and inflammation. Cellular sources of IL-33 include endothelial cells, epithelial cells, fibroblasts, and myofibroblasts. In homeostasis, it is sequestered in the nuclei of cells and gets released upon cellular injury169. As such, it is thought to act as an alarmin, relaying information of tissue damage to immune cells179. It binds to its receptor, interleukin-1 receptor-like 1, also known as Suppression Of Tumorigenicity 2 (ST2)L. ST2L is a transmembrane receptor expressed mainly on immune cells. The main responders of IL-33 are tissue resident cells including group 2 innate lymphoid cells (ILC2s), mast cells, and Tregs. Other cell types expressing ST2L include macrophages, eosinophils, neutrophils, basophils, Th2 cells, dendritic cells, Th1 cells, CD8+ T cells, NK cells, and B cells169. ST2 is also present on non-immune cells such as epithelial cells. However, the role of IL-33 signaling in the epithelial cells is not well known. ST2 expression on a wide range of immune cells explains its role in both type 1 and type 2 immune responses169. In summary, IL-33 is a pleiotropic cytokine acting on multiple immune cells to relay signals of tissue damage. 29 1.5.2 Nuclear IL-33 In homeostasis, IL-33 is synthesized as a ~30 kDa isoform referred to as full-length IL-33 (f-IL-33). F-IL-33 is comprised of an N-terminal nuclear domain. The N-terminal nuclear domain contains a chromatin binding motif that attaches to the acidic pocket of histones, H2A/H2B169. Interestingly, the chromatin binding motif is homologous to the chromatin binding motif of the Kaposi sarcoma herpes virus LANA (latency-associated nuclear antigen). Similar to LANA, IL-33 is thought to be involved in chromatin compaction to repress transcription169, 170. Other studies have also implicated a role of IL-33 in regulating transcription. For example, Gatti et al. propose a mechanism where nuclear IL-33 restrains fibroblast activation in the context of unilateral ureteral obstruction171. Indirect effects of IL- 33 in regulating transcription have also been explored. IL-33 has been shown to bind to the p65 subunit of NF-κB to prevent its binding to DNA172. Moreover, transcriptomic analyses have led Park et al. to propose a mechanism where nuclear IL-33 can bind to RUNX2 to block SMAD6 expression. This interaction allows TGFβ induced epithelial proliferation162. However, the role of nuclear IL-33 in regulating transcription remains controversial. Jean- Philippe Girard used two independent RNA silencing strategies to knock down nuclear IL-33 and observed no differences in the endothelial proteome. As such, he proposes that nuclear IL-33 may be a sequestration mechanism to prevent IL-33 signaling in homeostasis169. In summary, the role of nuclear IL-33 remains controversial. 1.5.3 IL-33 structure and release In addition to the N-terminal domain, IL-33 contains a central protease-sensing domain, followed by the C-terminal cytokine domain. The C-terminal domain possesses the cytokine activity and is made up of 12 β-strands arranged in a β-trefoil fold similar to the other members of the IL-1 family cytokines169, 174. However, unlike some of the other members of the IL-1 family, IL-33 does not require cleavage for activation; it is gets transcribed in its full-length form, which is biologically active. In homeostasis, it is retained in the nuclei and upon cellular damage, it is passively released to act as an alarmin169. Upon release, the full-length form is active and signals through ST2L. Subsequently, the protease sensing domain of IL-33 can encounter proteases released by neutrophils (protease G and 30 elastase) and mast cells (chymase and tryptase) which can degrade IL-33 into a shorter isoforms (~18-20 kDa), which is 10-30 times more potent176. The potency arises from cleavage of the basic residues found in the N-terminus, allowing for the acidic residues in the C-terminus to make stronger interactions with ST2175. Upon binding of IL-33 to ST2L, it associates with IL-1R accessory protein (IL-1RAcP) to form a complex. This allows for juxtaposition of Toll/IL-1R (TIR) domains to recruit myeloid differentiation primary response protein 88 (MYD88), ultimately leading to downstream activation of NF-κB, c-JNK, p38, and ERK signaling pathways165. In summary, IL-33 contains 3 domains: N-terminal nuclear, central and C-terminal domain referred to as the full-length form. Post translational processing in the central domain can render a shorter, more potent forms of IL-33. Although the exact mechanism of its release is unknown, the most common method is thought to involve passive release upon mechanical stress or infection176. Other studies have shown the release of IL-33 from perforin-2 pores by conventional dendritic cells as well as translocation of nuclear IL-33 to the cytoplasm via Ca2+ dependent manner followed by Streptomyces hygroscopicus–derived potassium ionophore dependent gasdermin D release166. IL-33 release mechanisms warrant further investigation. 1.5.4 Negative regulation of IL-33 signaling In order to prevent excessive activation of the IL-33/ST2 signaling pathway, there are negative regulatory mechanisms. This includes binding of IL-33 to the alternative splice variant of ST2, referred to as soluble ST2 (sST2). sST2 is a decoy receptor that prevents binding of IL-33 to ST2L. Additionally, Single Ig IL-1-related receptor (SIGIRR) can bind ST2 to prevent signaling165, 176. Oxidation of the disulfide bridges in IL-33 is another regulatory mechanism that acts by shortening its half-life175. In addition, prior to its release during apoptosis, caspases 3 and 7 cleave the C-terminal domain to prevent IL-33 signaling176. In summary, IL-33 requires regulatory mechanisms to prevent excessive signaling as well as prevent signaling in the absence of tissue damage. 31 Figure 1.4 IL-33 structure and release Full-length IL-33 (f-IL-33) is comprised of a conserved nuclear domain with chromatin binding motifs and an IL-1-like cytokine domain that are joined by a linker region. F-IL-33 is released from the nuclei during cell damage (1), or it can be translocated to the cytoplasm via a calcium dependent mechanism and released through gasdermin-D pores (2). Alternatively, splice variants lacking the nuclear domain can be retained in the cytoplasm and released via perforin-mediated pores (3). Extracellular f-IL-33 can be processed by proteases to produce cleaved IL-33 (c-IL-33), both of which can bind to ST2 to initiate MyD88 dependent signaling. IL-33 signaling is negatively regulated by nuclear sequestration and oxidation of IL-33 as well as binding of IL-33 to the decoy receptor, sST2. Reproduced with permission from Dwyer et al, Annual Review of Immunology 2022. 1.5.5 IL-33 signaling in macrophages Macrophages are one cell type that are responsive to IL-33. The role of IL-33 and macrophages was studied in the context of Th1-mediated colitis including DSS and TNBS- induced models. In both models, administration of recombinant IL (rIL)-33 improves disease activity, restores goblet cell numbers and restores body weight. Interestingly, splenic populations of Treg cells were unaffected by rIL-33177. The authors attribute the presence of IL-23 to inhibit GATA binding protein 3 (GATA3) and ST2 in Treg, thereby limiting T cell responsiveness. The protective effects of IL-33 were due to the increased goblet cells and polarization of macrophages to the anti-inflammatory phenotype. Interestingly, they confirmed the protective effect of rIL-33 in the absence of MyD88 using a knockout mouse model177. However, it is important to note contradictory findings where IL-33 was also shown to exacerbate colitis in a DSS model210. Faas et al. (2021) studied the role of IL-33 in 32 macrophages to a deeper extent and demonstrate that MyD88 is crucial for polarization of IL-33 stimulated macrophages178. This finding implies a protective role of IL-33 in MyD88 deficiency demonstrated in the previous study177 may be due to alternative mechanisms. Interestingly, they show that compared to IL-4 stimulation, IL-33 induces a distinct gene expression profile involving upregulation of both inflammatory and anti-inflammatory markers. While pro-inflammatory cytokines are upregulated through NF-κB, they show an uncoupling of the mitochondrial respiratory chain in macrophages upon IL-33 stimulation, resulting in a shift in metabolites. This ultimately leads to upregulation of GATA3 and upregulation of markers involved in tissue repair178. The dual role of IL-33 in polarization of macrophages was also confirmed using human bone marrow derived macrophages. While macrophages that had not been previously skewed towards a particular phenotype were stimulated with IL-33, they upregulated pro-inflammatory markers. In contrast, macrophages that were already skewed towards pro- and anti-inflammatory phenotypes both enhanced anti-inflammatory markers upon IL-33 stimulation179. Another study involving airway inflammation confirmed a synergistic effect of IL-33 on macrophages stimulated with IL-13, a known inducer of the wound healing phenotype. However, they found this synergy to be dependent on IL-4Rα through which IL-13 binds to increase ST2L180. In summary, IL-33 can act on macrophages to polarize them into an inflammatory or anti-inflammatory phenotype depending on the environment. 1.6 Thesis objectives and hypothesis 1.6.1 Summary of rationale A subset of people with IBD exhibit low SHIP activity, which has been inversely correlated with disease severity144, 145. This phenotype has been replicated in a germ-line SHIP-/- mouse model that spontaneously develop CD-like intestinal inflammation and fibrosis by 8 weeks of age. Inflammation is characterized by immune cell infiltration, goblet cell hypertrophy and hyperplasia, and loss of crypt-villus architecture, while fibrosis involves the thickening of the muscle layer along with deposition of collagen125, 131. Macrophages are responsive to IL-4, a type 2 cytokine, which allows them to express markers related to an anti-inflammatory phenotype. In the context of SHIP deficiency, 33 macrophages exhibit enhanced a M(IL-4) phenotype160 mediated by the downstream kinase, PI3Kp110δ. Ultimately, SHIP-/- macrophages produce more ArgI, allowing for conversion of L-arginine into precursors used for cell proliferation and collagen biosynthesis. Our laboratory has shown that PI3Kp110δ mediated upregulation of ArgI is crucial to the development of intestinal inflammation and fibrosis by using inhibitors in vivo as well as employing genetic ablation methods107. IL-33 is a pleiotropic cytokine involved in both type 1 and 2 immune responses. IL- 33 has shown to be upregulated in biopsies of people with IBD suggesting a possible role in disease181. Additionally, studies have shown a role of IL-33 to act synergistically with other type 2 cytokines, IL-4 and IL-13, to enhance ArgI production in macrophages180. Many of the studies investigating the role of IL-33 have been conducted in colitis models182, 183, 184, 185. Thus, the role of IL-33 in intestinal fibrosis has been underexplored and warrants further investigation. 1.6.2 Aims Based on previous studies, I hypothesized that IL-33 plays a pathogenic role in intestinal fibrosis by enhancing ArgI expression in macrophages. Additionally, targeting IL- 33 will alleviate intestinal inflammation and fibrosis. In order to investigate IL-33 as a therapeutic target for intestinal fibrosis, I have three aims: • Aim 1. Characterize IL-33 expression, concentrations and localization in the fibrotic and non-fibrotic sections of SHIP-/- ileum compared to SHIP+/+ controls • Aim 2. Investigate the role of IL-33 in inducing ArgI in bone marrow derived macrophages from SHIP-/- and SHIP+/+ mice • Aim 3. Assess IL-33 as a target for therapy by utilizing IL-33 neutralizing antibodies in vivo and by genetically ablating IL-33 in SHIP-/- mice and SHIP+/+ controls 1.6.3 Significance By investigating the role of IL-33 in CD-associated intestinal fibrosis, these studies will provide a deeper understanding of the role of IL-33 in macrophage activation in vivo. In 34 addition to understanding mechanisms, this research will allow for testing of IL-33 as a target for anti-fibrotic therapy. The implications may be tremendous given that we currently have no therapies to directly treat intestinal fibrosis. 35 Chapter 2: Materials and Methods 2.1 Mice An F2 generation of C57BL/6  129Sv, which were heterozygous for SHIP expression, were bred to generate SHIP+/+ and SHIP-/- littermates. Mice heterozygous for SHIP were also bred with IL33-/- mice on a C57BL/6 genetic background to generate SHIP+/-IL33+/- mice. These mice were subsequently crossed to generate SHIP+/+IL33+/+, SHIP+/+IL33-/-, SHIP-/-IL33+/+, and SHIP-/-IL33-/-. C57BL/6 IL33-/- mice were generously provided by Dr. Fumio Takei at the BC Cancer Terry Fox Laboratory (Vancouver, BC). Mice were kept in specific pathogen-free conditions and co-housed in sterilized filter top cages. Autoclaved food and water were given to the mice ad libitum. The mice were maintained by the technicians in the Animal Care Facility at BC Children’s Hospital Research Institute (Vancouver, BC). Experiments were performed on 6- or 8-week old mice, males and females, and were performed in accordance with Canadian Council on Animal Care guidelines with approval from the UBC Animal Care Committee (Protocol A21-0035 and A23-0073). 2.2 Cytokine analyses Full-thickness ileal tissue (30-50 mg; healthy, non-fibrotic, and fibrotic sections) were homogenized in 1 mL ice-cold homogenization buffer (1PBS containing 10µg/ml of aprotinin and leupeptin). Samples were homogenized using the Kinematica Polytron MR2100 bench top homogenizer (Kinematica, New York, USA) and centrifuged at 10,000g for 5 min at 4°C. Clarified supernatants were aliquoted and stored at -80°C until assayed. Enzyme-linked immunosorbent assays (ELISAs) were performed on clarified supernatants for IL-33 (Cat. No. DY3626), IL-1β (Cat. No. DY401), and ST2 (Cat. No. DY1004) purchased from R&D (Minneapolis, MN, USA). 2 or 5-fold dilutions was used to assay IL-33 and ST2. For IL-1β, undiluted SHIP+/+ and SHIP-/- non-fibrotic samples and 10-20-fold diluted SHIP-/- fibrotic samples were assayed. ELISAs were performed according to the manufacturer’s instructions. 0.05% Tween-20 in PBS was used as the wash buffer, 36 TMB Substrate Solution (BD Biosciences, Cat. No. 555214), and 9.75% sulfuric acid solution in dH2O as the stop solution. Thermo Fisher Scientific Wellwash Microplate Washer (Thermo Fisher Scientific, Massachusetts, USA) was used to wash plates. The plates were read using the Thermo Fisher Scientific Varioskan LUX Multimode Microplate Reader (Software 7.0.2) at wavelengths of 450 and 570 nm. The cytokine concentrations were normalized to the weight of tissue homogenized. 2.3 Gene expression analyses 2.3.1 RNA extraction During harvesting, sections intended for RNA analyses were cleared of fecal matter and rinsed with ice-cold 1PBS. The sections were cut open longitudinally and further into smaller sections. The sections were then submerged in 1 mL of RNALater (Thermo Fisher Scientific, Cat. #AM7021) solution and incubated at 4°C overnight. The next day, sections were blotted dry on paper towel to remove excess RNALater and transferred to new Eppendorf tubes. Samples were stored at -80°C until ready to use. Samples were thawed on ice and 6-8 mg of tissue was added to an RNAse/DNAse free microcentrifuge tube. Samples were homogenized using a handheld homogenizer (Cole- Parmer, Cat. #UZ-44468-25) and RNA was extracted using the RNAeasy Plus Mini Kit (Qiagen, Cat. No. 74134) according to manufacturer’s instructions. Tissue sections were homogenized on ice in 350 µL of Buffer RLT and 3.5 µL β-mercaptoethanol (BMe). Tissue was further digested using a 20-guage needle and 1mL syringe to ensure maximum RNA extraction. The optional DNAse digestion step was included in the extraction using the RNase-Free DNase set (Qiagen, Cat. #79254) according to kit instructions. Additionally, an extra wash step was included after step 9 of the protocol by adding 500 µL of Buffer RPE and centrifuging at room temperature (RT) for 2 min at 8000g. RNA was eluted in 30 µL of RNAse free dH2O. Purity and concentration of RNA was assessed using a Nanodrop. For the first few extractions, the efficacy of this extraction protocol in yielding intact RNA was verified using Thermo Scientific™ High Range RNA Ladder (Fisher Scientific, Cat. No. 37 FERSM1821). 2 µL of 2 RNA loading dye and 2 µL of sample was mixed and prepared according to kit instructions. The samples were run on a 1.2% agarose gel. 2.3.2 cDNA synthesis and quantitative PCR (qPCR) RNA (500 mg) was reverse transcribed to cDNA using the iScript Reverse Transcription Supermix for RT-qPCR (BioRad, Cat. #1708841) according to the manufacturer’s protocol. cDNA samples were then diluted 1:10 in RNAse/DNAse free H2O and used in qPCR reactions with SsoAdvanced Universal SYBR Green Supermix (BioRad, Cat. #1725271). Master mixes were prepared for each gene and 8 µL of mastermix was added to each reaction with 2 µL of the 1:10 diluted cDNA. Mastermixes were prepared such that each reaction contained 1 µL of 10 µM forward and reverse ribosomal protein lateral stalk subunit P0 (Rplp0) primers (housekeeping gene). Primer sequences are as follows: Rplp0 forward 5'- TGACATCGTCTTTAAACCCCG-3', Rplp0 reverse 5'-TGTCTGCTCCCACAATGAAG-3'. Primers were purchased from Integrated DNA Technologies (Coralville, Iowa). 20 stock IL- 33 primers were purchased from BioRad (Assay ID qMmuCED0046391). 0.5 µL of 20 IL- 33 primer was used in each 10 µL reaction. BioRad CFX96 C100 was used to carry out the reactions with the following cycling conditions: polymerase activation: 95°C for 2 min, followed by 40 cycles of denaturation: 95°C for 5 sec and annealing/extension: 60°C for 30 sec, and melting curve analysis. With respect to quality control, standard curves were generated for each primer to ensure optimal primer efficiencies and qPCR products were run on an agarose gel to ensure specific amplification in the absence of primer dimers. 2.3.3 cDNA synthesis and PCR for IL-33 splice variants RNA (1 g) extracted from full-thickness ileal tissue was reverse transcribed into cDNA as described previously. Each 20 µL reaction contained 0.13 µL Taq DNA polymerase (FroggaBio, Cat. No. MB101-0500K), 2.5 µL of 10 PCR buffer (FroggaBio, Cat. No. MB101-0500K), 0.5 µL of 10 mM dNTP Mix (GeneDireX, Cat. No. DN0010), 1 µL or 0.5 µL each of 10 µM forward or reverse IL-33 or Rplp0 primers, respectively, and RNAse free dH2O. Primers sequences for detecting IL-33 splice variants are as follows: 38 mIL33_splice_forward 5'- CAACTCCAAGATTTCCCCGG-3', mIL33_splice_reverse 5'- TGCAGCTCTCATCTTTCTCCT-3'. Primers sequences for Rplp0 were the same as used previously. PCR reaction was carried out with the following cycling conditions: initial denaturation: 3 min at 94°C, denaturation for 20 sec at 95°C and primer annealing for 20 sec at 55°C, and extension 45 seconds at 72 °C (35 cycles), and final extension for 10 min at 72°C. PCR products were run on a 1.5% agarose gel and visualized using the iBright FL1500 imaging system (Thermo Fisher Scientific, Massachusetts, USA). 2.4 Western blot analyses 2.4.1 Full-thickness ileal homogenates Full-thickness ileal tissue was cut longitudinally and washed in a petri dish with ice cold 1PBS. It was noted not to vigorously wash intestines using a needle and syringe as it could initiate release of IL-33. After washing, sections were flash frozen in liquid nitrogen and stored at -80°C. To prepare samples for western blot analysis, ~30 mg of tissue was homogenized in 495 µL RIPA lysis buffer (Thermo Fischer Scientific, Cat. No. 89900) containing 5 µL of Halt™ Protease Inhibitor Cocktail (100X) (Thermo Fischer Scientific, 87786). Samples were homogenized using the Kinematica Polytron MR2100 bench top homogenizer (Kinematica, New York, USA). DNA was sheared after homogenizing using a 26 gauge needle and 1 mL syringe. Samples were centrifuged at 12,000g for 15 min at 4°C. Protein concentration was measured in the clarified supernatants using the Qubit™ Protein Broad Range (BR) Assay Kit (Thermo Fischer Scientific, Cat No. A50668). Protein (30 µg) was mixed with 2Laemmli buffer (4% SDS, 20% glycerol. 0.125M Tris-Cl, pH 6.8, bromophenol blue, and 10% BMe). Samples were boiled for 1 min at 100°C and were run on a 10% SDS-PAGE gel at 125 V. 6 µL of Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standards (BioRad, cat # 1610375). Proteins were transferred onto a nitrocellulose membrane using the Power Blotter Cassette XL (Invitrogen, Cat. No. PB0003). Blocking was done using 5% milk in PBS for 1 hour and primary antibody incubation was done overnight at 4°C. Antibodies used were: 1:250 polyclonal anti-mouse IL-33 raised in goat (R&D, Cat. No. AF3626) and 1:1000 monoclonal anti-mouse β-actin raised in rabbit (Cell Signaling Technology, Cat. No. 4970). Western blots were 39 visualized using the Licor Odyssey Imager (software version 3.0). Densitometry was performed using ImageJ (National Institute of Health, USA). 2.4.2 Sera Blood was collected from mice via cardiac puncture and clotted for 30 min at RT. Samples were spun at 15,000g for 10 min at 4°C. Protein concentrations were determined using the Qubit™ Protein Broad Range (BR) Assay Kit (Thermo Fischer Scientific, Cat No. A50668). Samples were diluted (10- or 100-fold) with Assay buffer from the kit and used to determine protein concentrations. Protein (50 µg) was loaded onto the 10% SDS-PAGE gel and the western blot was conducted as previously described. 2.4.3. Cell lysates Cells were stimulated and lysed as described under Section 2.7 Cell stimulations. Protein sample (10 g) was diluted in 4Laemmli’s buffer and boiled for 1 min at 100°C. Protein was loaded onto a 10% SDS-PAGE gel and western blot was conducted as previously described. Antibodies were used at the following dilutions: 1:2000 anti-mouse ArgI (Cell Signaling Technology, Cat No. 93668), and 1:1000 anti-mouse β-actin (Cell Signaling Technology, Cat. No. 4970). 2.5 Histology 2.5.1 Hematoxylin and eosin (H&E), Masson’s trichrome and Alcian blue + Periodic Acid – Schiff (PAS) staining Distal ileal tissue sections were harvested and fixed in 10% formalin (Formalde-Fresh Solution, Fisher Chemical SF934, Waltham, MA, USA) for 24 hours and stored in 70% ethanol. 5 µm sections were paraffin embedded and stained with H&E and Masson’s trichrome by Wax-It Histology services at the University of British Columbia. For Alcian Blue + PAS staining, ileal samples were fixed in Carnoy’s solution (60% of 100% ethanol, 40 30% chloroform, and 10% glacial acetic acid) for 24 hours and stored in 100% ethanol. Tissue processing and staining was also done by Wax-It Histology services at the University of British Columbia. Images were taken using the Olympus BX61 microscope with a 10 objective lens, DP71 camera, and cellSens Dimension software (Olympus, Tokyo, Japan). The tiling feature on the microscope was used to obtain an image of entire sections at 10. Scale bars were added using ImageJ software (National Institute of Health, USA). 2.5.2 IL-33 Immunohistochemistry Formalin fixed and paraffin embedded ileal sections (5 µm; processed by Wax-It Histology services at the University of British Columbia) were tightly sealed in coplin jars and heated in a water bath at 60°C for 30 min. This was done to ensure tissue adherence during staining. Sections were deparaffinized and rehydrated with xylene (3 x 3 min each), 100% ethanol (3 x 3 min each), 95% ethanol (2 x 3 min each), 80% ethanol (1 x 3 min), and TBST (5 min). Slides were then immersed in pre-heated sodium citrate buffer for antigen retrieval and incubated for 15 min in a steamer at the highest temperature setting. Slides were then allowed to cool to RT undisturbed for 1 hour. Peroxidase block was then performed with 3% H2O2 (Fischer Scientific, Cat. No. H324) for 15 min at RT. After washing the slides 2 x 5 min each with TBST, Avidin/Biotin Blocking Kit (Vector Laboratories, Cat. No. SP-2001) was used as per kit instructions to block endogenous avidin binding sites and biotin. After washing 5 min with TBST, the slides were blocked with 10% of 10 casein solution (Vector Laboratories, Cat. No. SP-5020-250), 10% of 10 PBS in dH2O for 30 min at RT. Anti- mouse IL-33 (R&D, Cat. No. AF3626) or goat IgG isotype control (Thermo Fischer Scientific, Cat. No. 02-6202) was diluted 1:600 in blocking buffer (5% rabbit serum, 3% BSA, and 0.3% TritonX-100 in 1X PBS) and incubated overnight at 4°C. The next day, slides were brought to RT and rinsed 3 x 5 min on a shaking platform. VECTASTAIN® ABC-HRP Kit, Peroxidase (Goat IgG) (Vector Laboratories, Cat. No. PK-4005) was used to add biotinylated secondary antibody conjugated to HRP enzyme as per kit instructions. Slides were developed using DAB substrate (DAKO, Cat. No. S0809) according to manufacturer’s instructions. After rinsing 3 x 5 min with TBST, slides were incubated with Harris Hematoxylin Solution, Modified (Millipore Sigma, Cat. No. HHS32) for 1 min and 41 gently rinsed in running tap water. Slides were briefly dipped twice in acid ethanol (1% HCl in 70% EtOH) and rinsed in tap water to remove excess stain. Slides were then incubated in Scott’s Tap water for 30 sec to blue the hematoxylin. After rinsing in tap water, the slides were dehydrated (the reverse of rehydration steps described above). Slides were mounted with nail polish and cured overnight. Slides were then visualized at 10 and 20 magnification using the EVOS M5000 Imaging System. 2.6 Histological Analyses 2.6.1 H&E staining H&E-stained ileal sections were scored on a 16-point scale107 according to the criteria in Table 2.1. Images were scored by three reviewers blinded to the experimental conditions. Median scores from the three analyses were plotted in Graphpad Prism (version 10.3.0; GraphPad Software, Inc.). Table 2.1 Histological damage scoring Damage Component Score Loss of architecture 0 = none 1 = <25% loss 2 = 25%-50% loss 3 = 50%-75% loss 4 = >75% loss Immune cell infiltration 0 = none 1 = occasional immune cell in lamina propria 2 = increased immune cells in lamina propria 3 = confluent immune cells in lamina propria and breaching mucosa 42 4 = immune cell infiltration throughout the section Goblet cell hyperplasia/hypertrophy 0 = none; 1 = <50% change 2 = >50% change Ulceration 0 = none 1 = intermediate ulceration 2 = substantial ulceration Edema 0 = none 1 = <50% of section 2 = >50% of section Muscle thickening 0 = none 1 = intermediate thickening 2 = substantial thickening 2.6.2 Masson’s trichrome fibrosis score Masson’s trichrome slides were scored for fibrosis based on 2 parameters listed in Table 2.2. Scoring criteria was adapted from Colgan lab124. Slides were scored by three people blinded to experimental condition. 43 Table 2.2 Fibrosis scoring Fibrosis Component Score Disorganization/Disarray of Collagen Fibers 0 = none, physiologically organized 1 = minimal disorganization, few small breaks 2 = moderately disorganized but structure still easy to see 3 = severely disorganized, no evidence of normal physiological pattern, collagen infiltrates muscular layers Fibromuscular Hyperplasia/Muscularization/Muscular Obliteration of the Submucosa 0 = none, normal muscle submucosa content 1 = mild increase in muscle content 2 = large amount of muscle in submucosa, affects the morphology but roughly normal structure 3 = muscle has obliterated the entire submucosal space 2.6.3 Masson’s trichrome image analysis Images of Masson’s trichrome stained slides were taken at 20 magnification using the EVOS M5000 Imaging System. 3 images were taken per section separated by > 50 µm. Images were analyzed in ImageJ2 (version 2.14.0/1.54f) using the colour deconvolution2 package186. Different thresholds were set for healthy, non-fibrotic, and fibrotic Masson’s trichrome stained sections in order to best eliminate capture of artifacts. After setting the threshold, an area was defined to measure collagen percentage and maintained for all sections. For each image, three measurements were averaged from different regions of the lamina propria to prevent selection bias. At the end, an average of the three images from one mouse was calculated as percent area positive for collagen. 44 2.7 Cell stimulations In order to generate bone marrow derived macrophages, the femurs and tibias of a euthanized 8-week-old mouse was harvested under sterile conditions. The bones were flushed with 50 mL of bone flush media (10% FBS in Iscove’s Modified Dulbecco’s Medium (IMDM)) using a 10 mL syringe and 26 gauge needle. The resuspended cells were plated in a T75 cm2 flask for 1 hour at 37°C and 5% CO2. After 1 hour, the monocytes remained in suspension and were spun at 300 g for 5 min at RT. The monocytes were resuspended in 30 mL of macrophage media (10% FBS, 10 ng/mL MCSF, 0.125% 1:100 monothioglycerol and 1% Pen/Strep in IMDM). Cells were plated in a tissue culture T75 cm2 flask and incubated at 37°C and 5% CO2 for 10 days. Media was changed on day 4 and day 7. This protocol has previously been established in lab and purity analysis by flow cytometry reveal that 95% cells are positive for Mac-1 and F4/80207. On day 10, cells were dissociated in 10 mL of Cell Dissociation Buffer, enzyme-free, Hanks' Balanced Salt Solution (Gibco, Cat No. 13150016). After incubating the flask for 10 min at 37°C, cells were scraped and transferred to a 50 mL canonical tube. The flask was rinsed 3 times with bone flush media and the cells were resuspended in a total volume of 50 mL. Cells were centrifuged at 300 g for 5 min at RT. The pellet was resuspended in macrophage media and cells were counted using a hemocytometer. Cells were plated at a density of 500,000 cells/well in a Corning 12-well plate in a total volume of 1.5 mL of macrophage media. The cells were incubated for 1 hour at 37°C to allow them to adhere. Cells were then incubated with the following cytokines prepared in 500 µL total volume of macrophage media: 10ng/mL rIL-13 (Biolegend, Cat. No. 575902), 10ng/mL rIL-10 (StemCell, Cat No. 78079), rIL-33 (R&D, Cat. No. 3626-ML), or rIL-4 (Biolegend, Cat. No. 574302). Cells stimulated with IL-13 were incubated for 48 hours whereas cells stimulated with IL-4 were incubated for 24 hours. Additionally, BMDMs derived from IL-33 genetically ablated mice were grown and stimulated as described. However, the cells were plated at a density of 250,000 cells/well in a 24-well plate in 500 µL of macrophage media. The stimulations were done in a total volume of 200 µL. After stimulation, the cells were placed on ice and rinsed 3 times with ice cold 1PBS. Cells were 45 lysed in 80 µL or 100 µL arginase cell lysis buffer for IL-13 stimulation experiments and IL-4 stimulations, respectively. BMDMs from genetically ablated mice were lysed with 45 µL lysis buffer. Arginase cell lysis buffer was comprised of 0.1% Triton X-100, 25 mM Tris pH 8, aprotinin (40 μg/mL), leupeptin (8 μg/mL), and PMSF (100 μM). After lysis, protein concentrations were measured using the Qubit™ Protein Broad Range (BR) Assay Kit (Thermo Fischer Scientific, Cat No. A50668). 2.8 IL-33 neutralization experiments SHIP+/+ and SHIP-/- mice (6-week-old) were intraperitoneally injected with sterile PBS (vehicle control), 5 mg/kg of anti-mouse IL-33 (Adipogen, Cat. No. AG-27B-0013PF), or 5 mg/kg of mouse IgG2b isotype control (Adipogen, Cat. No. AG-35B-0008). Anti-IL-33, a monoclonal antibody, is composed of single chain fragment variable generated using HEK 293 cells and fused to the mouse lgG2b Fc domain. In vitro experiments verified that the neutralizing antibody can bind to the 18 kDa isoform of IL-33 (R&D, Cat. No. 3626-ML-010/CF). Anti-IL-33 was aliquoted and stored at -20°C at 1 mg/mL and was diluted to 0.5 mg/mL on each injection day. Similarly, IgG2b isotype control was stored at 4°C at a concentration of 1 mg/mL and diluted to 0.5 mg/mL for injections. The volume to inject was calculated based on the weight of the mouse. Volume of PBS injected corresponded to the volume of 0.5 mg/mL treatment the mouse would receive at that weight. Treatments were administered every other day for two weeks. The mice were weighed and monitored daily for signs of post-injection distress and clinical health. Criteria that were assessed to evaluate post-injection distress included: loss of body weight, attitude/activity, appearance, posture/gate, hydration, and pain. Daily clinical health scores were based on attitude/activity, appearance, posture/gait, abdomen, weight loss, and pain. On day 14, the mice were euthanized by treating with isoflurane followed by asphyxiation by CO2. Lungs were harvested from mice first. As such, cervical dislocation was not used in the euthanization process. The distal 15 cm of intestine was also harvested for histology. Sections for cytokine analyses and arginase activity assays were flash frozen in liquid nitrogen and stored at -80°C. 46 2.9 Arginase activity assays Arginase activity assays were conducted on cell lysates and full-thickness ileal tissue homogenates. Cell lysates were prepared as previously described in “cell stimulations”. For preparing full-thickness ileal homogenates, 20-35 mg of distal ileal tissue was homogenized in 1 mL of arginase lysis buffer (0.1% Triton X-100, 25 mM Tris pH 8, aprotinin (40 μg/mL), leupeptin (8 μg/mL), and PMSF (100 μM)). Lysis buffer was prepared without PMSF, which was added right before homogenization of each sample to prevent its inactivation in aqueous solution. Homogenates were then centrifuged at 10,000 g for 5 min at 4°C. Protein concentrations were measured in clarified supernatants using the Qubit™ Protein Broad Range (BR) Assay Kit (Thermo Fischer Scientific, Cat No. A50668). To measure the concentration of urea produced by ArgI dependent hydrolysis of L- arginine, 5 µg of protein from cell lysates and 10 µg from full-thickness ileal homogenates was added to Eppendorf tubes topped up to 100 µL with arginase lysis buffer. 10 µL of manganese (II) chloride (MnCl2) was added to each sample and blank and incubated for 10 min at 55°C. 100 µL of 0.5 M L-arginine pH 9.7 was added to each sample. For cell lysates, the samples were incubated for 30 min at 37°C whereas ileal homogenates were incubated for 1 hour at 37°C. 800 µL of acid mixture (1:3:7 (v/v/v) H2SO4 (36 N):H3PO4 (44.6 N): dH2O) was added to stop the reaction. 40 µL of 9% (w/v) α-isonitrosopropiophenone (ISPF) (Sigma, Cat. No. 13502) in absolute ethanol was then added to the reactions and vortexed briefly. Standards were prepared by 2-fold serial dilutions of 50 mM urea prepared in dH2O along with a blank. To the standards, 400 µL of acid mixture and 25 µL of 9% (w/v) ISPF in absolute ethanol was added. Samples and standards were boiled at 100°C for 30 min, protected from light in locking Eppendorf tubes. Samples and standards were allowed to cool to RT (~10 min) and read at 550 nm within 30 min. The plate reader used was the Thermo Fischer Scientific Varioskan LUX Multimode Microplate Reader (Software 7.0.2). 2.10 Statistical analyses 47 All statistical analyses were performed using Graphpad Prism (version 10.3.0; GraphPad Software, Inc.). A single comparison between SHIP +/+ and SHIP-/- mice was assessed for statistical significance with an unpaired, two-tailed Student’s t-test assuming unequal variance between groups. Comparisons between non-fibrotic and fibrotic SHIP-/- sections were calculated using a paired two tailed Student’s t-test. When multiple t-tests were used to assess statistical significance for in vivo mouse experiments, an unpaired Welch’s t-test with Šídák-Bonferroni correction was used to correct for multiple comparisons. When performing multiple t-tests for cell stimulation experiments, a paired Student’s t-test with Šídák- Bonferroni correction was used to correct for multiple comparisons. P values were considered significant at p ≤ 0.05. 48 Chapter 3: Results 3.1 Fibrotic sections in SHIP-/- mice have higher concentrations of the 30 kDa IL-33 isoform and relative gene expression compared to SHIP+/+ controls IL-33 expression has been shown to be upregulated in SAMP mice, a model of CD- like intestinal inflammation and fibrosis181. Based on this, I asked whether IL-33 and ST2 concentrations and gene expression were also upregulated in the distal ileum of SHIP-/- mice compared to SHIP+/+ mice. ELISAs were conducted on full-thickness ileal tissue homogenates from 8-week-old SHIP+/+ and SHIP-/- (non-fibrotic and fibrotic) sections, and concentrations were normalized to weight of tissue (Figure 3.1A). SHIP+/+ mice had baseline concentration of IL-33 as expected as it is retained in the nuclei in homeoatasis169. In SHIP-/- ilea, mean IL-33 concentrations were higher in non-fibrotic and fibrotic sections compared to SHIP+/+ control; this difference was only significant in SHIP-/- fibrotic sections (p = 0.0420). In contrast to IL-33, there were no significant differences in ST2 concentrations between SHIP+/+ and SHIP-/- (non- fibrotic or fibrotic) sections (Figure 3.1B). In addition to protein concentration, I assessed IL-33 gene expression by RT-qPCR on full-thickness ileal tissues from 8-week-old SHIP+/+ and SHIP-/- mice. SHIP-/- non-fibrotic and fibrotic sections had higher IL-33 gene expression than sections from SHIP+/+ mice. This difference was statistically significant in SHIP-/- fibrotic sections compared to control (p = 0.0472) (Figure 3.1C). The higher relative IL-33 gene expression data aligns with higher concentrations in fibrotic sections of SHIP-/- mice compared to SHIP+/+ mice. Taken together, these results suggest that higher IL-33 concentrations and relative gene expression correlate with CD-like intestinal pathology in SHIP-/- mice. 49 Figure 3.1 Fibrotic sections in SHIP-/- mice have higher IL-33 concentrations and relative gene expression compared to sections from SHIP+/+ mice. Full-thickness ileal homogenates from 8-week-old SHIP+/+ and SHIP-/- mice (including non-fibrotic and fibrotic sections) were analyzed for (A) IL-33 concentrations and (B) ST2 concentrations by ELISA (N=15-17 for SHIP+/+, N=7 for SHIP-/- non-fibrotic, N=15 for SHIP-/- fibrotic). Concentrations were normalized to weight of tissue. (C) Relative IL-33 gene expression quantified from RT-qPCR (N=5 for SHIP+/+, N=7 for SHIP-/- non-fibrotic and fibrotic groups). The bars and whiskers depict the means and SEM, respectively. P values for comparisons between SHIP+/+ and SHIP-/-, either non-fibrotic or fibrotic sections, were calculated using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple comparisons. P values for comparisons between non-fibrotic and fibrotic SHIP-/- sections were calculated using a paired two-tailed Student’s t-test.050100150SHIP+/+ SHIP-/-IL-33 (ng/g of tissue)Ap = 0.0420p = 0.4931p = 0.1928IL-33relative gene expression012345p = 0.0472p = 0.0628p = 0.5794050010001500p = 0.3481p = 0.3353p = 0.5838ST2 (ng/g of tissue)FibrosisB CSHIP+/+ SHIP-/-FibrosisSHIP+/+ SHIP-/-Fibrosis50 30 kDa fIL-33 is released upon tissue damage and cleaved by neutrophils and mast cell proteases. The resulting shorter isoforms (~18-20 kDa) are known to be more potent ligands for ST2 signaling176. As such, I investigated whether the different isoforms can be detected in SHIP deficient mice. Full-thickness ileal homogenates from 8-week-old SHIP+/+ and SHIP-/- mice (non-fibrotic and fibrotic sections) were probed for IL-33 by western blotting (Figure 3.2A). β-actin was used as the loading control. SHIP-/- fibrotic sections had faint bands at ~25 kDa, correlating with cleavage products of caspase 3 and 7 under apoptotic conditions187. Additionally, there were two faint bands at ~18-20 kDa implying the presence of products processed by extracellular proteases. SHIP+/+ mice exhibited baseline expression of the 30 kDa isoform. In SHIP deficiency, expression of the 30 kDa isoform was higher in fibrotic sections compared to SHIP+/+ controls (Figure 3.2A). Densitometry was conducted on all biological replicates for the 30 kDa isoform (Figure 3.2B). Relative signal intensity (IL-33/β-actin) was quantified for each mouse and normalized to SHIP+/+ controls. The results confirmed a significantly higher relative signal intensity of the 30 kDa isoform in SHIP-/- fibrotic sections normalized to SHIP+/+ (p = 0.0466). SHIP-/- non-fibrotic sections had no significant difference in signal intensity of 30 kDa bands normalized to controls. In summary, SHIP-/- fibrotic sections had significantly higher expression of the 30 kDa isoform compared to SHIP+/+ sections. 51 Figure 3.2 Fibrotic tissue sections from SHIP-/- mice have higher expression of the 30 kDa IL-33 isoform. Full-thickness ileal tissue homogenates from 8-week-old SHIP+/+ and SHIP-/- mice (non- fibrotic and fibrotic sections) were analyzed for IL-33 isoforms by Western blot (A). β -actin was used as the loading control. Data is representative of 3 independent experiments using samples from 3 different mice with similar results. (B) Relative band intensity (IL-33/β-actin) of the 30 kDa isoform was quantified using densitometry and normalized to SHIP+/+ controls. Each data point represents one mouse, and bars represent means ± SEM. P values for comparisons between SHIP+/+ and SHIP-/- (either non-fibrotic or fibrotic sections) were calculated using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple comparisons. The P value between non-fibrotic and fibrotic sections of SHIP-/- mice was calculated using a paired two tailed Student’s t-test. 0.00.51.01.52.02.5SHIP+/+ SHIP-/-Fibrosisp = 0.0446p = 0.8993p = 0.101130 kDaRelative band intensity (IL-33/β-actin) normalized to SHIP+/+SHIP+/+ SHIP-/-Fibrosisβ-actin30 kDa25 kDaAB20 kDa52 Next, I investigated the presence and relative abundance of IL-33 isoforms in the sera of 8-week-old SHIP+/+ and SHIP-/- mice by western blotting (Figure 3.3A). There were faint bands present at 30 kDa in both SHIP+/+ and SHIP-/- sera, however, there were no visible differences. In contrast to tissue homogenates that revealed bands at ~18-20 kDa, sera of SHIP-/- mice had visibly higher expression of ~20-22 kDa isoforms. These results aligned with previous studies conducted in human sera of people with IBD compared to healthy individuals181. Whether ~20-22 kDa isoforms are more potent activators of ST2 remains controversial and dependent on the cleaving enzyme. Caspase-1 has shown to cleave 30 kDa IL-33 into 20 kDa, rendering it inactive208. Whereas mast cell proteases can generate ~18-21 kDa IL-33 isoforms that are 30-fold more potent activators of ILC2s209. These observational differences were confirmed using densitometry (Figure 3.3B). IL-33 signal intensities were calculated relative to β-actin and normalized the values to SHIP+/+ controls. Densitometry was conducted for both 30 kDa and ~20-22 kDa isoforms. Since sera from SHIP+/+ mice produced one cleavage product compared to two in SHIP-/- sera, I assessed total cleavage product by conducting densitometry in a range of ~20-22 kDa. The relative signal intensity of the ~20-22 kDa isoforms was significantly higher in sera of SHIP-/- mice compared to controls (p = 0.0250). There were no significant differences in relative signal intensity of the 30 kDa isoform in SHIP deficiency compared to controls. In conclusion, sera of SHIP-/- mice have higher expression of proteolytically cleaved isoforms of IL-33. 53 Figure 3.3 Expression of 20-22 kDa isoforms of IL-33 is higher in the sera of SHIP-/- mice. Sera samples from 8-week-old SHIP+/+ and SHIP-/- mice were assessed for IL-33 isoforms by (A) Western blot. β-actin was used as the loading control. Data are representative of 4 experiments with similar results. (B) Relative band intensity (IL-33/β- actin) of the ~20-22 kDa and 30 kDa isoforms were quantified using densitometry and normalized to SHIP+/+. Each data point represents a mouse, bars represent means ± SEM. P- values were calculated using an unpaired two tailed Student’s t-test. ABSHIP+/+ SHIP-/-051015Relative band intensity (IL-33/β-actin) normalized to SHIP+/+20-22 kDap = 0.025030 kDap = 0.1975012330 kDa20 kDaβ-actinSHIP+/+ SHIP-/-SHIP+/+ SHIP-/-Relative band intensity (IL-33/β-actin) normalized to SHIP+/+54 In addition to post-translational modifications, IL-33 can undergo splicing post- transcription. A study by Gorden et al. found a  exon 3, 4 splice variant in airway epithelial cells associated with type 2 inflammation in the context of asthma168. Thus, I next asked whether alternative splice variants can be detected in SHIP deficiency that may be associated with disease phenotype. Primers were designed within exon 2 and exon 8 using the mus musculus reference genome (Figure 3.4A). IL-33 and Rplp0 cDNA made from full-thickness ileal mRNA of SHIP+/+ and SHIP-/- (non-fibrotic and fibrotic) mice was amplified with PCR. PCR products were run on a 1% agarose gel (Figure 3.4B). There was a band around ~745 bp corresponding to f-IL-33 transcript in SHIP+/+ and SHIP-/- mice (non-fibrotic and fibrotic sections). Splicing events would have produced amplicons below the ~745 bp product. However, no bands present under the ~745 bp product were detected for either genotype. Exposure times on gel were increased to increase sensitivity for detection of low expressed transcripts; however, there were still no bands detected for alternative splice variants. 2 out of the 3 times the experiment was conducted, the non-fibrotic and fibrotic sections of SHIP-/- mice appeared to have lower fIL-33 transcript than SHIP+/+ sections as shown. However, the third time, the non-fibrotic and fibrotic sections had higher expression of fIL-33 compared to controls. Overall, it can be concluded that splice variants cannot be detected in full-thickness ileal tissue from SHIP+/+ and SHIP-/- (non-fibrotic and fibrotic) sections. 55 Figure 3.4 IL-33 splice variants were not detected in full-thickness ileal tissue from SHIP+/+ or SHIP-/- (non-fibrotic and fibrotic sections). A) Portion of chromosome 19 of the mus musculus (mm10) reference genome zoomed into the IL-33 locus and the two transcripts derived from alternative promoters (shown in blue). Primers were designed within exon 2 and exon 8 (highlighted by red boxes). Image was generated using Integrated Genome Browser134. (B) PCR products of IL-33 and Rplp0 (reference gene) amplification from mRNA extracted from full thickness ileal tissue from 8- week-old SHIP+/+ and SHIP-/- (non-fibrotic and fibrotic sections). Data is representative of N=3 independent experiments. SHIP-/-ABChromosome 19IL33IL33Rplp0IL-33850 bpSHIP+/+FibrosisSHIP-/-SHIP+/+hromosome 1929, 930 kb 29, 940 kb 29, 950 kb 29, 960 kbIL-3356 Since IL-33 is known to be expressed in epithelial cells in homeostasis169, I asked whether epithelial cells are the source of IL-33 in SHIP deficient mice. To assess localization of IL-33, I conducted IHC on distal ileum sections harvested from 8-week-old SHIP+/+ and SHIP-/- mice (including non-fibrotic and fibrotic sections) (Figure 3.5). Sections stained with pooled goat IgG isotype were included as negative controls. All SHIP+/+ and SHIP-/- (non- fibrotic and fibrotic) sections showed IL-33 staining in the nuclei of epithelial cells. Interestingly, in fibrotic sections of SHIP-/- mice, there were some crypt-villus structures that did not stain for IL-33 compared to SHIP+/+. Immune cells in the lamina propria also stained for IL-33 in SHIP+/+ and SHIP-/- (non-fibrotic and fibrotic) sections. Overall, this experiment investigating IL-33 localization in SHIP deficiency confirms epithelial cells as being one of the major producers. Figure 3.5 Epithelial cells are one source of IL-33 in the ilea of SHIP deficient mice. Ileal sections from 8-week-old SHIP+/+ and SHIP-/- (non-fibrotic and fibrotic sections) mice were stained for IL-33 by IHC. Positive signal for IL-33 is reflected by brown stain and negative controls were stained with pooled polyclonal goat IgG isotype. Images were captured at 10× and40× magnification with scale bars equal to 300 μm and 75 μm, respectively. Data are representative of sections from 6 mice per group. 10× 40×IL-33SHIP-/-non-fibroticSHIP+/+Isotype control IL-33 Isotype controlScale bars = 300 μm Scale bars = 75 μm SHIP-/-fibrotic57 3.2 IL-33 synergizes with IL-13 to induce ArgI expression and activity in SHIP+/+ BMDMs The Sly laboratory previously elucidated a mechanism by which PI3Kp110δ mediated ArgI upregulation mediates intestinal inflammation and fibrosis in SHIP deficiency107. ArgI is a marker of reparative macrophages and is upregulated in response to type 2 cytokines (IL-4 and IL-13)188. Since studies have implicating a role of IL-33 in macrophage activation188, I asked whether IL-33 enhances macrophage polarization to an M(IL-4) phenotype (characterized by ArgI upregulation). I conducted in vitro experiments, stimulating 10-day old bone marrow derived macrophages (BMDMs) with IL-13 ± IL-10 (positive control), and ± IL-33. After 48 hours, I harvested and resolved lysates from 500,000 cells/well for ArgI using western blot (Figure 3.6A). Densitometry analysis of the western blots was conducted by calculating the signal intensity of ArgI relative to β-actin and normalizing to the unstimulated controls for 3 independent experiments (graphs depicted at bottom of blots). Western blot analyses were further validated with arginase activity assays on the lysates from the stimulated SHIP+/+ and SHIP-/- BMDMs (Figure 3.6B). IL-13+IL-33 stimulated SHIP+/+ BMDMs produced significantly higher signal intensities of ArgI bands relative to β-actin compared to IL-13 alone (p = 0.0408). This synergy was consistent in the IL-13+IL-33 stimulated SHIP+/+ arginase activity assays compared to IL-13 alone (p = 0.0267). Unexpectedly, although there are trends implying synergy of IL-13+IL-10 stimulated samples compared to IL-13 alone, the results were not significant. Similarly, although SHIP-/- BMDMs displayed trends of synergy of IL-13 with IL-10 and IL-33, results from western blots and arginase activity assays were not significant. The lack of significance may be attributed to low sample size. The observed trends align with previous studies180 and imply a potential role of IL-33 in enhancing ArgI in the presence of IL-13 in SHIP+/+ and SHIP-/- BMDMs. 58 ABunstimulated IL-13 IL-10 IL-33 IL-13+10 IL-13+33 unstimulated IL-13 IL-10 IL-33 IL-13+10 IL-13+33β-actinβ-actinArgI ArgISHIP+/+ SHIP-/-p = 0.1648SHIP+/+IL-13IL-10IL-33SHIP-/-20406080100Arginase Activity(µg urea/µg protein/hr)0p = 0.0267p = 0.2231p = 0.16233020100IL-13IL-10IL-33302010IL-13IL-10IL-330Relative band intensity (ArgI/β-actin) normalized to unstimulated controlsRelative band intensity (ArgI/β-actin) normalized to unstimulated controlsp = 0.0408p = 0.0756p = 0.1403p = 0.134359 Figure 3.6 IL-33 may synergize with IL-13 to induce ArgI expression and activity in BMDMs. (A) Western blot of ArgI in SHIP+/+ and SHIP-/- BMDMs treated with IL-13 ± IL-10 (positive control for synergy) or ± IL-33. Left and right blots depict ArgI expression in SHIP+/+ and SHIP-/- stimulated macrophages, respectively. β-actin was used as the loading control. Graphs at bottom of blots represent relative band intensity (ArgI/β-actin) normalized to the unstimulated controls. Blots are representative of 3 independent experiments using BMDMs from different mice. (B) Arginase activity assays on the stimulated BMDMs. Individual experiments are shown as closed circles, bars show means, and whiskers show SD of 3 independent experiments. P values were calculated using a paired Student’s t-test for densitometry analysis and a paired Student’s t-test with Šídák-Bonferroni correction for arginase activity assay. I also assessed whether IL-33 can induce ArgI expression and activity in the presence of IL-4. Similar to the previous experiment, I incubated SHIP+/+ and SHIP-/- BMDMs with IL-4 ± IL-10 (positive control), and ± IL-33 for 24 hours. Western blots were conducted on the cell lysates for ArgI using β-actin as the loading control (Figure 3.7A). Signal intensity of bands corresponding to ArgI relative to β-actin were calculated and normalized to unstimulated controls for the two independent experiments (graphs depicted at bottom of blots). Arginase activity assays were also conducted on the stimulated lysates (Figure 3.7B). Arginase activity assays on SHIP+/+ BMDMs revealed a trend for synergy of IL-4+IL-33 compared to IL-4 alone. However, results were not significant, and one independent experiment produced low values. In order to exclude any outliers, more replicates would be required. Additionally, no significant findings or trends were observed in SHIP-/- arginase activity assays with respect to IL-4+IL-33 synergy. These experiments were not further pursued due to recent findings from Sly lab revealing IL-13 and not IL-4 as a mediator of intestinal fibrosis in the SHIP-/- mouse model (unpublished findings by Safari et al.). 60 Figure 3.7 Investigating the role of IL-33 in inducing ArgI expression and activity in BMDMs in the presence of IL-4. ABunstimulated IL-4 IL-10 IL-33 IL-4+10 IL-4+33 unstimulated IL-4 IL-10 IL-33 IL-4+10 IL-4+33β-actinβ-actinArgI ArgISHIP+/+ SHIP-/-SHIP+/+IL-4IL-10IL-33SHIP-/-204080Arginase Activity(µg urea/µg protein/hr)0p = 0.0414p = 0.5083p = 0.1222p = 0.26740246IL-4IL-10IL-33Relative band intensity (ArgI/β-actin) normalized to unstimulated controls051015Relative band intensity (ArgI/β-actin) normalized to unstimulated controlsIL-4IL-10IL-336061 (A) Western blot for ArgI on IL-4 ± 10 (positive control for synergy) or ± IL-33 stimulated SHIP+/+ and SHIP-/- BMDMs. β-actin was used as the loading control. Numbers at bottom of blots represent relative band intensity (ArgI/β-actin) normalized to the unstimulated controls. Blots are representative of 3 independent experiments. (B) Arginase activity assays were performed on stimulated SHIP+/+ and SHIP-/- BMDMs. Closed circles represent individual experiments. Bars represent means, whiskers show SD of 3 independent experiments. P values were calculated using a paired Student’s t-test for densitometry analysis and a paired Student’s t-test with Šídák-Bonferroni correction for arginase activity assay. 3.3 IL-33 neutralizing antibodies are effective in vitro and in vivo After the in vitro findings implicating a role for IL-33 in promoting ArgI expression in the presence of IL-13, I next assessed IL-33 as a therapeutic target. Antibodies to neutralize IL-33 (αIL-33) were intraperitoneally injected in 6-week-old SHIP+/+ (control) and SHIP-/- mice at 5 mg/kg. The dosage was selected based on previous studies189. Controls included PBS (vehicle control) and IgG2b (isotype control) injections. Experimental timeline is shown is Figure 4.1A. The efficacy of αIL-33 was first validated in vitro with an indirect ELISA (Figure 4.1B). Decreasing concentrations of αIL-33 or IgG2b (isotype control) were added with 3 µg/mL of ST2 to each well coated with rIL-33. Optical density (OD) corresponds to secondary antibody binding to ST2. As expected, OD values were consistent across all concentrations of IgG2b implying no specific binding to rIL-33. Also as expected, decreasing αIL-33 concentrations produced higher OD readings until the maximum was reached. These results confirmed that αIL-33 can effectively block IL-33 binding to ST2 in vitro. Next, the efficacy of αIL-33 to reduce ArgI expression in vivo was assessed by conducting arginase activity assays on full-thickness ileal homogenates from SHIP+/+ and SHIP-/- treated mice (Figure 4.1B). PBS treated SHIP-/- (non-fibrotic and fibrotic) sections did not display significant higher arginase activity compared to PBS treated SHIP+/+ controls possibly due to low sample size. However, IgG2b treated fibrotic sections of SHIP-/- mice had higher mean arginase activity compared to IgG2b treated SHIP+/+ controls. Compared to IgG2b treated, αIL-33 treated SHIP-/- (non-fibrotic and fibrotic) had lower mean arginase activity. Although there are trends in lower mean arginase activity upon αIL-62 33 treatment in SHIP deficiency, the results did not achieve statistical significance possibly due to low sample size and high variability in arginase activity in mice. The trends are consistent with a role for IL-33 in inducing ArgI expression in vivo. In summary, the IL-33 blocking antibodies are effective at neutralizing IL-33 in vitro. Additionally, the trends of lower mean arginase activity in SHIP-/- treated samples imply that αIL-33 may be neutralizing IL-33 in vivo at a dosage of 5 mg/kg. ABSHIP+/+SHIP-/-PBS (vehicle control)IgG2b (isotype control)αIL336 weeksof age8 weeksof ageDay 0 14Intraperitoneally inject 5 mg/kg every other day for 14 daysBlocking antibodyIgG2b isotype control0.00.20.40.60.81.0ODConcentration (µg/mL)0.0064CPBS IgG αIL33 PBSPBS IgG IgG αIL33αIL33FibrosisSHIP+/+ SHIP-/-0Arginase Activity(µg urea/µg protein/hr)5101520p = 0.6332 p = 0.9975 p = 0.4531 p = 0.5889 0.0320.160.84.02010063 Figure 3.8 IL-33 neutralizing antibodies are effective at blocking IL-33 in vitro and at lowering mean ArgI activity in SHIP-/- treated mice. (A) Experimental timeline of SHIP+/+ and SHIP-/- treated with PBS, 5 mg/kg IgG2b (isotype control), and αIL-33. (B) Indirect competitive ELISA measuring binding of ST2 to rIL-33 when incubated with different concentrations of IL-33 blocking antibody or IgG2b isotype control. Data shown are representative of 3 independent experiments. (C) Arginase activity in full- thickness ileal tissue of SHIP+/+ and SHIP-/- treated mice. Bars depict means and whiskers correspond to SEM. N=4 for PBS, N=8 for IgG and N=6 for αIL-33 treated SHIP+/+ mice. N=6 for all SHIP-/- groups including both non-fibrotic and fibrotic sections. P values were calculated using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple comparisons. 3.4 IL-33 neutralizing antibodies do not improve disease severity in SHIP-/- mice Next, I investigated whether αIL-33 treatment in SHIP-/- mice reduced features of intestinal inflammation. I examined gross pathology of SHIP+/+ and SHIP-/- treated mice (Figure 4.2A). As expected, PBS-treated SHIP+/+ distal ileum appeared thin, pink, and healthy. Additionally, there were no visible signs of disease observed in SHIP+/+ mice treated with IgG2b or αIL-33. This indicates that IgG2b and αIL-33 treatment did not have detrimental effects on healthy mice. In contrast, PBS-treated SHIP-/- mouse had visible signs of disease including areas of thickened, red patches followed by thinner, healthier looking sections in the distal ileum. Interestingly, IgG2b treatment in SHIP-/- mice appeared to reduce thickness of the red patches, implying some beneficial off target effects of the Fc portion of antibodies. However, αIL-33 treatment did not reduce presence of thickened red patches. As SHIP-/- mice have lower body weights compared to their SHIP+/+ counterparts (data not shown), I asked whether αIL-33 treatment could promote weight gain in SHIP deficiency. I also plotted mean percentage of initial weight of SHIP+/+ (left) and SHIP-/- (right) treated mice over 14 days (Figure 4.2B). Mice treated with αIL-33 showed no weight gain over the course of 14 days. Overall, these data suggest that αIL-33 treatment does not improve gross pathology or promote weight gain in SHIP-/- mice. 64 Figure 3.9 IL-33 blocking antibodies do not improve gross ileal pathology in SHIP-/- mice. (A) Distal ilea from SHIP+/+ and SHIP-/- mice treated with PBS (vehicle control), 5 mg/kg IgG2b (isotype control), or αIL33. (B) Percent initial weight of mice at each day during treatment. Data shown are representative (for (A)), or means ± SEM for N=4 for PBS, N=8 for IgG and N=6 for αIL-33 treated SHIP+/+ mice. N=6 for all treatment groups for SHIP-/- mice. In order to determine whether αIL-33 improved histological damage, I first examined H&E-stained cross sections of the distal ileum from treated mice (Figure 4.3A). Compared to PBS treated SHIP+/+ cross sections, SHIP-/- fibrotic cross sections were larger, displayed loss of crypt-villus architecture, edema, muscle thickening, immune cell infiltration, and goblet cell hyperplasia and hypertrophy. Although the non-fibrotic sections of SHIP-/- mice displayed characteristics of histological damage, the extent of damage appeared less compared to fibrotic sections. No obvious improvement was observed in SHIP-/- αIL-33 ABPBS(vehicle control)IgG2b(isotype control)αIL-33 SHIP+/+ SHIP-/-Day0 5 1095100105% of initial weightPBSIgGαIL-33PBSIgGαIL-3395100% of initial weight 105Day0 5 10SHIP+/+ SHIP-/-65 treated (non-fibrotic and fibrotic) sections compared to SHIP-/- IgG treated. In order to obtain a quantitative measure of histological damage, scores for histological damage were assigned to each mouse and plotted in Figure 4.3B. The stacked bars correspond to the criteria. Total scores were also plotted in Figure 4.3C. SHIP-/- non-fibrotic and fibrotic sections had higher median total damage scores compared to PBS treated SHIP+/+ sections. The fibrotic sections had statistically significant higher median scores (p = 0.0009). IgG treatment in the SHIP-/- non-fibrotic sections resulted in lower median histological damage scores compared to PBS treated SHIP-/- non-fibrotic controls. Specifically, there was an absence of edema, lower goblet cell hyperplasia and hypertrophy, immune cell infiltration, and improved architecture. However, there was no significant improvement in total histological damage score in SHIP-/- (non-fibrotic and fibrotic) sections upon IL-33 blocking compared to IgG-treated controls. IL-33 blocking antibodies did not reduce histological damage in SHIP-/- mice. 66 ABFibroticNon-fibroticSHIP+/+ SHIP-/-αIL-33 PBS(vehicle control)IgG2b(isotype control)Scale bars = 200 μm ArchitectureImmune cell infiltrationGoblet cell hyperplasia/hypertrophyUlcersEdemaMuscle thicknessp = 0.0009 p = 0.1619 p = 0.5342 p = 0.6948 05101520Total Damage ScorePBS IgG αIL33 PBSPBS IgG IgG αIL33αIL33FibrosisSHIP+/+ SHIP-/-PBS IgG αIL33 PBSPBS IgG IgG αIL33αIL33FibrosisSHIP+/+ SHIP-/-05101520Damage Score67 Figure 3.10 IL-33 blocking antibodies do not improve histological damage in SHIP deficient mice. (A) H&E images are representative cross-sections from SHIP+/+ and SHIP-/- mice treated with PBS (vehicle control), 5 mg/kg IgG2b (isotype control), and anti-IL-33. Images are taken at 10× magnification and scale bars are equal to 200 μm. (B) Histological damage scores were assigned by three people blinded to experimental condition. Stacked bars represent each criterion with median ± range. (C) Total damage scores were plotted for each individual mouse. Data shown are median ± range. For SHIP+/+ mice, N=4 for PBS, N=8 for IgG2b, and N=6 for αIL-33. For SHIP-/- mice, N=6 for all treatment groups. P values were calculated using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple comparisons. IL-33 is known to indirectly promote goblet cell differentiation via IL-13 produced by ILC2s190. I asked whether IL-33 neutralization reduced mucin production. Production of mucins in SHIP+/+ and SHIP-/- treated mice was visually assessed in Alcian Blue+PAS- stained distal ileal cross sections. Acidic mucins stain deep blue while neutral mucins stain magenta. In PBS treated SHIP-/- mice (non-fibrotic and fibrotic sections), there are more acidic mucins that span throughout the section compared to PBS treated SHIP+/+ sections. IL-33 blocking antibodies did not reduce mucin staining in SHIP deficient mouse (non-fibrotic and fibrotic) sections compared to IgG2b treated SHIP-/- controls. This data aligns with histological damage scoring where IL-33 neutralization did not reduce goblet cell hyperplasia/hypertrophy. Overall, IL-33 blocking antibodies did not reduce mucin production in goblet cells. 68 Figure 3.11 IL-33 blocking antibodies do not reduce mucin accumulation in SHIP-/- mice. Alcian Blue PAS staining of ileal sections from SHIP+/+ and SHIP-/- mice treated with PBS, IgG2b (isotype control), or anti-IL-33. Acidic mucins are stain deep blue while neutral mucins are stain magenta. Images are taken at 10× magnification and are a representative of 4 PBS, 8 IgG2b and 6 αIL-33 treated SHIP+/+ mice. Non-fibrotic and fibrotic SHIP-/- sections are representative of 6 mice for each treatment. Scale bars = 200 μm. 3.5 IL-33 neutralizing antibodies do not reduce fibrosis in SHIP-/- mice I next assessed whether IL-33 blocking antibody reduced fibrosis in SHIP deficiency. Masson’s Trichrome-stained cross sections from SHIP+/+ and SHIP-/- (non-fibrotic and fibrotic) treated mice were qualitatively assessed for collagen localization and muscle thickening (Figure 4.5A). Collagen is stained in blue and smooth muscle fibers in magenta. PBS-treated SHIP-/- (non-fibrotic and fibrotic) sections reveal dispersion of collagen fibers from the submucosal space into the lamina propria as well as localization in the muscle layer. This contrasts with PBS treated SHIP+/+ cross sections in which a dense collagen band is detected in the submucosal space only. Collagen in IgG or αIL-33 treated SHIP-/- cross sections was detected in all layers of the intestine implying no obvious improvement in fibrosis. FibroticNon-fibroticSHIP+/+ SHIP-/-Scale bars = 200 μm αIL-33 PBS(vehicle control)IgG2b(isotype control)69 In order to obtain a quantitative measure, fibrosis scores were assigned to the Masson’s Trichrome stained sections based on the criteria in Table 2.2 (Figure 4.5B). Stacked bars represent median for each criterion. Total scores were plotted for each mouse in all treatment groups (Figure 4.5C). As expected, fibrotic sections of PBS treated SHIP-/- mice had significantly higher median total score compared to SHIP+/+ mice (p = 0.0009). Interestingly, non-fibrotic sections of IgG treated mice had lower median total fibrosis score compared to PBS treated control. This implies alleviation of fibrosis by IgG2b to some extent, aligning with gross pathology results. There was no significant reduction in total fibrosis score in SHIP-/- αIL-33 treated (non-fibrotic and fibrotic) sections compared to IgG- treated controls. As an additional measure of fibrosis, percent collagen in a defined area in the lamina propria treated mice was quantified in ImageJ. There were no significant differences in percentage of collagen positive area in the lamina propria of αIL-33 treated SHIP-/- mice compared to IgG controls for either non-fibrotic or fibrotic sections. In summary, IgG2b isotype control may be alleviating fibrosis to a certain extent in SHIP-/- mice, however, αIL- 33 treatment did not reduce fibrosis. PBS IgG αIL33 PBSPBS IgG IgG αIL33αIL33FibrosisSHIP+/+ SHIP-/-2Fibrosis Score4680AFibroticNon-fibroticSHIP+/+ SHIP-/-αIL-33 PBS(vehicle control)IgG2b(isotype control)Scale bars = 200 μm BDisorganization/disarray of collagen fibersFibromuscular hyperplasia/muscularization/muscular obliteration of the submucosap = 0.0009 p = 0.1504 p = 0.9769 p = 0.9069 C2Total fibrosis Score4680PBS IgG αIL33 PBSPBS IgG IgG αIL33αIL33FibrosisSHIP+/+ SHIP-/-70 PBS IgG αIL33 PBSPBS IgG IgG αIL33αIL33FibrosisSHIP+/+ SHIP-/-2Fibrosis Score4680AFibroticNon-fibroticSHIP+/+ SHIP-/-αIL-33 PBS(vehicle control)IgG2b(isotype control)Scale bars = 200 μm BDisorganization/disarray of collagen fibersFibromuscular hyperplasia/muscularization/muscular obliteration of the submucosap = 0.0009 p = 0.1504 p = 0.9769 p = 0.9069 C2Total fibrosis Score4680PBS IgG αIL33 PBSPBS IgG IgG αIL33αIL33FibrosisSHIP+/+ SHIP-/-D0510152025PBS IgG αIL33Area positive for collagen (%)SHIP+/+ SHIP-/- Non-fibrotic SHIP-/- Fibroticp = 0.2099 p = 0.7455 p = 0.9925 0510152025Area positive for collagen (%)PBS IgG αIL330510152025Area positive for collagen (%)PBS IgG αIL3371 Figure 3.12 IL-33 blocking antibodies did not reduce fibrosis in SHIP-/- distal ilea. (A) Masson’s trichrome staining for collagen in ileal cross sections from SHIP+/+ and SHIP- /- mice (non-fibrotic and fibrotic sections) treated with PBS (vehicle control), IgG2b (isotype control), or anti-IL-33 blocking antibody. Collagen is stained in blue, and the smooth muscle fibers are stained in magenta. Images are taken at 10× and scale bars = 200 μm. (B) Fibrosis scores were assigned by three people blinded to experimental condition. Stacked bars represent median for each criterion and whiskers correspond to range. (C) Total damage scores were plotted for each mouse. Data shown are median ± range. P values were calculated using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple comparisons. (C) Image analysis for percent collagen present in a defined area of the lamina propria of treated SHIP+/+ and SHIP-/- mice. P values were calculated using an unpaired, two-tailed Welch’s t-test. N=4 for PBS, N=8 for IgG and 6 for αIL-33 treated SHIP+/+ mice. N=5 or 6 for all SHIP-/- treatment groups. I next examined cytokine responses to IL-33 neutralization. I conducted IL-33 ELISAs on full-thickness ileal tissue of treated SHIP+/+ and SHIP-/- mice in order to assess whether IL-33 signaling has positive feedback mechanisms to upregulate its expression (Figure 4.6A). PBS treated SHIP-/- fibrotic sections had higher mean IL-33 concentration compared to PBS treated SHIP+/+ sections. This result was close to achieving statistical significance using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple corrections (p = 0.0618). However, there was no statistical difference between αIL-33, and IgG treated SHIP-/- ileal sections (non-fibrotic and fibrotic). These results suggest IL-33 secretion may not be positively regulated by IL-33/ST2 signaling. Since IL-1β is the key driver of inflammation in the SHIP-/- model144 and IL-33 can have dual roles as a pro- inflammatory and anti-inflammatory cytokine, I asked whether blocking IL-33 influences IL- 1β production. I conducted ELISAs for IL-1β on full-thickness ileal treated samples from SHIP+/+ and SHIP-/- mice (Figure 4.6B). Mean IL-1β concentrations in PBS treated SHIP-/- fibrotic sections was higher compared to PBS treated SHIP+/+. However, this difference did not reach statistical significance (p = 0.1261). Interestingly, non-fibrotic SHIP-/- PBS treated sections had lower IL-1β concentrations compared to fibrotic sections implying an association between IL-1β and fibrosis. Additionally, fibrotic sections of IgG treated SHIP-/- mice had a lower mean IL-1 concentration compared to PBS control. This aligns with previous findings relating to ability of IgG2b to improve disease phenotype. However, αIL- 33 treatment did not significantly reduce IL-1β concentrations in SHIP deficiency compared 72 to IgG controls. Overall, IL-33 blocking does not lower IL-33 or IL-1β concentrations in SHIP deficiency. Figure 3.13 Anti-IL-33 blocking antibodies do not reduce IL-33 or IL-1β concentrations in SHIP-/- mice. Clarified full thickness ileal tissue homogenates from SHIP+/+ and SHIP-/- mice treated with PBS, IgG2b (isotype control), and anti-IL-13 were assayed by ELISA for IL-33 (A) or IL-1β (B). Concentrations were normalized to weight of tissue. Data shown are means ± SEMs. For SHIP+/+ mice, PBS (N=4), IgG isotype (N=8), and αIL-33 (N=6); for SHIP-/- mice, N=6 for all treatment groups. P values were calculated using an unpaired Welch’s t-test with Šídák- Bonferroni correction for multiple comparisons. 3.6 Genetic ablation of IL-33 in SHIP deficiency does not ameliorate disease Since initial characterization of IL-33 in SHIP deficiency revealed upregulation of the 30 kDa IL-33 isoform containing the chromatin binding motif (Figure 3.2A), I asked whether genetically ablating IL-33 in SHIP deficiency could improve disease outcome. I hypothesized that eliminating both secreted and nuclear isoforms of IL-33, disease outcomes would improve. As in IL-33 neutralizing experiments, I assessed whether arginase activity was reduced by genetic ablation of IL-33 in SHIP deficient mice. Arginase activity was significantly higher in non-fibrotic and fibrotic sections of SHIP-/-IL33+/+ sections compared A050100150200IL-33 (ng/g)0255075100125IL-1β (pg/mg)p = 0.0765p = 0.1261 p = 0.8351p = 0.9991p = 0.5458p = 0.0618 p = 0.9989p = 0.7448BPBS IgG αIL33 PBSPBS IgG IgG αIL33αIL33FibrosisSHIP+/+ SHIP-/-PBS IgG αIL33 PBSPBS IgG IgG αIL33αIL33FibrosisSHIP+/+ SHIP-/-73 to SHIP+/+IL33+/+ controls (p = 0.0040, 0.0012, respectively). Upon genetic ablation of IL- 33 in SHIP deficient mice, arginase activity was significantly lower in both non-fibrotic and fibrotic sections compared to SHIP-/-IL33+/+ controls. These data align with results of IL-33 blocking experiments where blocking antibodies reduced mean arginase activity in SHIP deficiency. In conclusion, this data strengthens the finding that IL-33 plays a role in up- regulating arginase activity in vivo. Figure 3.14 Genetic ablation of IL-33 reduces arginase activity in full-thickness ileal tissue homogenates from SHIP-/- mice. Arginase activity in full-thickness ileal tissue homogenates from 8-week-old SHIP+/+IL- 33+/+, SHIP+/+IL-33-/-, SHIP-/-IL-33+/+, and SHIP+/+IL-33-/- mice. Data are shown are means and whiskers depict SEM. N=7-11 mice per group. P values were calculated using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple comparisons. Next, to assess whether lower arginase activity in SHIP-/-Il33-/- correlated with improvement in disease, I examined gross pathology of SHIP+/+ (control) and SHIP-/- mice with or without genetic ablation of IL-33. Distal ilea from both SHIP+/+ ± IL33-/- appeared thin, pink, and healthy. This suggests that genetic ablation of IL-33 has no detrimental effects 51015200IL-33SHIP+/+ SHIP-/-FibrosisArginase Activity(µg urea/µg protein/hr)p = 0.0008p = 0.0012p = 0.0040 p = 0.015674 on gross pathology in SHIP+/+ mice. Ileum of SHIP-/-IL33+/+ had thickened areas of red patches found alongside thinner sections, as expected. With genetic ablation of IL-33, SHIP- /- mice retained features of disease including visible patchiness found along the distal ileum. However, visually, there appeared to be a reduction in thickened patches of genetically ablated SHIP-/- mice. Taken together, these results suggest that IL-33 may play a role in contributing to disease pathology to an extent, however, genetic ablation in SHIP deficiency does not completely improve gross pathology. Figure 3.15 Genetic ablation of IL-33 in SHIP deficient mice does not improve gross pathology. Images depict gross pathology of distal ilea from 8-week-old SHIP+/+IL-33+/+, SHIP+/+IL-33-/-, SHIP-/-IL-33+/+, and SHIP+/+IL-33-/- mice. Images are representative of N=7 or 8 mice per group. In order to determine any improvement in SHIP deficiency upon genetic ablation of IL-33, I assessed histological damage. I visually examined H&E-stained ileal sections from SHIP+/+ and SHIP-/- mice with or without genetic ablation of IL-33 (Figure 5.3A). There were no obvious differences in SHIP+/+ ileal sections upon genetic ablation compared to SHIP+/+ without ablation. This suggests no aberrant damage to the ileum upon IL-33 ablation in healthy mice. Fibrotic sections from SHIP-/-IL33+/+ mice displayed characteristics of histological damage including loss of crypt-villus architecture, immune cell infiltration, goblet cell hypertrophy/hyperplasia, edema, and muscle thickening. Non-fibrotic sections looked comparable to SHIP+/+IL33+/+ controls with some characteristics of damage SHIP+/+IL33+/+SHIP+/+IL33-/-SHIP-/-IL33+/+SHIP-/-IL33-/-75 including some presence of immune cells. Fibrotic sections of SHIP-/-IL33-/- mice appeared to have reduced immune cell infiltration. For quantitative analysis, median histological damage scores were plotted for each criterion according to Table 2.1. Whiskers represent range (Figure 5.3B). Fibrotic sections from genetically ablated SHIP-/- mice had slightly lower scores for immune cell infiltration and edema. Total scores were also plotted for each mouse in the groups (Figure 5.3C). As expected, fibrotic sections from SHIP-/-IL33+/+ mice had significantly higher total damage scores compared to SHIP+/+IL33+/+ controls (p = 0.0014). There was no significant improvement in total damage scores upon genetic ablation of IL-33 in SHIP deficiency (non-fibrotic and fibrotic sections) compared to SHIP-/-IL33+/+ controls. Significance was calculated using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple corrections. Overall, no significant improvement in histological damage upon IL-33 ablation was observed in SHIP deficient mice. 76 Figure 3.16 No significant improvement in histological damage after ablation of IL-33 in SHIP-/- mice. (A) H&E-stained cross sections of distal ilea from mice (genotypes as indicated). Images are representative of N=7-11 for each genotype. Images are taken at 10× magnification and scale bars = 200 μm. (B) Histological damage scores from the same mice. Stacked bars correspond to the criteria and represent the median ± range for each parameter. (B) Total damage scores for each mouse, bars represent median, and whiskers represent range. P values were calculated using the unpaired two tailed Student’s t-test. FibroticNon-fibroticSHIP+/+ SHIP-/-IL33+/+IL33-/-Scale bars = 200 μm AArchitectureImmune cell infiltrationGoblet cell hyperplasia/hypertrophyUlcersEdemaMuscle thickness0IL-33SHIP+/+ SHIP-/-Fibrosis5101520Damage Scorep = 0.0002p = 0.6545 p = 0.4908p = 0.7799Total Damage ScoreIL-33SHIP+/+ SHIP-/-FibrosisB C0510152077 Similar to IL-33 blocking experiments, I asked whether genetic ablation of IL-33 in SHIP deficiency reduced mucin production. Fibrotic sections of SHIP-/- IL33+/+ mice had more blue staining for acidic mucins compared to non-fibrotic and SHIP+/+IL33+/+ controls, as expected. Genetic ablation of IL-33 in SHIP deficiency did not reduce mucin staining compared to SHIP-/-IL33+/+ controls. This suggests that genetic ablation of IL-33 does not reduce mucin production in goblet cells in SHIP deficiency. Figure 3.17 Genetic ablation of IL-33 in SHIP deficient mice does not reduce mucin staining in ileal cross-sections. Alcian blue PAS staining of 8-week-old SHIP +/+IL33+/+, SHIP+/+IL33-/-, SHIP-/-IL33+/+, and SHIP-/-IL33-/- mice. Fibrotic and non-fibrotic sections are indicated. Acidic mucins stain deep blue and neutral mucins stain magenta. Images are representative of N=7-11 mice per group and are taken at 10× magnification. Scale bars = 200 μm. FibroticNon-fibroticSHIP+/+ SHIP-/-IL33+/+IL33-/-Scale bars = 200 μm 78 3.7 Genetic ablation of IL-33 in SHIP deficiency does not reduce fibrosis I next assessed parameters of fibrosis to determine whether genetic ablation of IL-33 in SHIP deficiency can ameliorate pathology. Masson’s Trichrome stained ileal sections were visualized to obtain a qualitative analysis of collagen localization and muscle thickening (Figure 5.5A). A dense collagen band stained in blue in SHIP+/+IL33+/+ appeared more dispersed in SHIP-/-IL33+/+ fibrotic sections. Additionally, collagen in the fibrotic sections of SHIP-/-IL33+/+ mice was localized in the lamina propria and muscle layer, as previously noted125. Whereas collagen structure in the non-fibrotic sections appeared more organized. Fibrotic sections of genetically ablated SHIP-/- mice appeared to have reduced muscle thickening compared to controls, however, collagen appeared dispersed throughout muscle layers. To further validate the results quantitatively, fibrosis scores were assigned to each Masson’s Trichrome stained section based upon the criteria listed in Table 2.2 (Figure 5.5B). Total fibrosis scores were plotted for each mouse (Figure 5.5C). Although not statistically significant, median total fibrosis score of sections from SHIP-/-IL33-/- mice were lower compared to fibrotic sections of SHIP-/-IL33+/+ mice. The lower fibrosis score was due to improvements in both parameters (muscle thickening and collagen disorganization) as shown in Figure 5.5B. Finally, I quantitated collagen as a percentage of a defined area in the lamina propria of Masson’s Trichrome stained sections (Figure 5.5D). Notably, there was no significant difference in percentage of collagen in the lamina propria of fibrotic sections from SHIP-/- IL33-/- mice compared to controls. In conclusion, these results do not show any significant improvement in fibrosis upon genetic ablation of IL-33 in SHIP deficient mice. 79 AFibroticNon-fibroticSHIP+/+ SHIP-/-IL33+/+IL33-/-Scale bars = 200 μm 2Fibrosis Score4680Total Fibrosis ScoreDisorganization/disarray of collagen fibersFibromuscular hyperplasia/muscularization/muscular obliteration of the submucosap = 0.4332p = 0.00002p = 0.2223 p = 0.9549IL-33SHIP+/+ SHIP-/-FibrosisC24680IL-33SHIP+/+ SHIP-/-FibrosisB80 Figure 3.18 Genetic ablation of IL-33 in SHIP deficiency did not reduce fibrosis. Ileal sections from 8-week-old SHIP +/+IL33+/+, SHIP+/+IL33-/-, SHIP-/-IL33+/+, and SHIP- /-IL33-/- mice stained for collagen using Masson’s Trichrome staining for fibrosis. Collagen is stained in blue, and the smooth muscle fibers are stained in magenta. 10× magnification was used to capture images and scale bars = 200 μm. Images are representative of 7-11 mice in each group. (B) Fibrosis scores were assigned by three people blinded to experimental condition. Stacked bars represent each criterion and correspond to median ± range. (C) Total damage scores were plotted for each mouse. Data shown are median ± range. P values were calculated using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple comparisons. (C) Image analysis for percent collagen present in a defined area of the lamina propria of treated SHIP+/+ and SHIP-/- mice. N=6-9 mice per group. P values were calculated using an unpaired, two-tailed Welch’s t-test. I also investigated whether IL-33 plays a role in inflammatory responses by examining IL-1β concentrations as it is the key driver of inflammation in SHIP deficient mice144. As expected, fibrotic sections of the SHIP-/-IL33+/+ sections had significantly higher concentrations of IL-1β compared to SHIP+/+IL33+/+ controls (p = 0.0223). However, genetic ablation of IL-33 in SHIP deficiency did not lower IL-1β concentrations in fibrotic sections compared to SHIP-/-IL33+/+ controls. Statistical significance was calculated using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple corrections. In summary, IL-1β is not reduced after genetic ablation of IL-33 in SHIP-/- mice. D051015IL-33SHIP+/+Area positive for collagen (%)2025SHIP-/- FibroticSHIP-/- Non-fibroticp = 0.1393 p = 0.0968 p = 0.8865IL-33 IL-33Area positive for collagen (%)05101520250510152025Area positive for collagen (%)81 Figure 3.19 Genetic ablation of IL-33 does not reduce IL-1β concentrations in full- thickness ileal tissues from SHIP-/- mice. IL-1β concentrations in full thickness ileal tissue homogenates from 8-week-old SHIP +/+IL33+/+, SHIP+/+IL33-/-, SHIP-/-IL33+/+, and SHIP-/-IL33-/- mice measured by ELISA. Concentrations were normalized to tissue weight. Closed circles represent individual mice, bars show means, and whiskers show ± SEMs. N=7-11 mice per group. P values were calculated using an unpaired Welch’s t-test with Šídák-Bonferroni correction for multiple comparisons. 3.8 Investigating a role of nuclear IL-33 in inducing ArgI in BMDMs Overall, the results from genetic ablation refuted the hypothesis that by eliminating both secreted and nuclear isoforms of IL-33, intestinal fibrosis would improve in SHIP deficient mice. However, whether nuclear IL-33 was playing a role in disease pathology warranted further investigation. I derived BMDMs from SHIP+/+IL33+/+, SHIP+/+IL33-/-, SHIP-/-IL33+/+, and SHIP- /-IL33-/- mice. 10-day old BMDMs were stimulated with IL-13, IL-33, or IL-13+IL-33 and assessed for arginase activity as previously described. In both SHIP+/+ and SHIP-/- 050100150200IL-1β (pg/mg)p = 0.8770p = 0.0223p >0.9999 p = 0.9875IL-33SHIP+/+ SHIP-/-Fibrosis82 stimulated with IL-13+IL-33, the synergy in arginase activity was higher in the genetically ablated BMDMs compared to IL33+/+ controls. This suggests that nuclear IL-33 may play a role in repressing arginase induction, however, further experiments are required to test this hypothesis. Figure 3.20 Nuclear IL-33 in BMDMs may play a role in repressing ArgI activity. Arginase activity was measured in (A) SHIP+/+ and (B) and SHIP-/- BMDMs with or without genetic ablation of IL-33. The cells were stimulated with IL-13 ± IL-33. Bars represent means ± SEM and N=2-3 for each stimulation and for all genotypes. IL33+/+0510152025IL33-/-IL-13IL-33Arginase Activityµg urea/µg protein/hrSHIP+/+ SHIP-/-AIL-13IL-33B0510152025Arginase Activityµg urea/µg protein/hr83 Chapter 4: Discussion IL-33 exhibits a complex duality as both a pro-inflammatory and anti-inflammatory cytokine, with its effects varying depending on the disease stage192. While studies have identified both pathogenic and protective roles of IL-33 in ulcerative colitis (UC), its function in Crohn's disease (CD) remains underexplored, particularly in relation to fibrosis192. Given that previous studies have implicated a potential pro-fibrotic role of IL-33 in the context of lung fibrosis193, it is a compelling cytokine to investigate as a target for CD-associated fibrosis. In this study, I utilized the SHIP-/- mouse model of CD-like intestinal inflammation and fibrosis to understand the mechanisms by which IL-33 can promote fibrosis as well as assess its role as a target for anti-fibrotic therapy. I demonstrated that IL-33 concentrations are higher in fibrotic sections of SHIP-/- mice, specifically the 30 kDa isoform. Additionally, IL-33 can possibly increase ArgI concentrations and activity in SHIP-/- macrophages in vitro, and ArgI protein and activity are reduced in vivo upon IL-33 neutralization or genetic ablation. Furthermore, IL-1β concentrations, a key driver of inflammation in this model, were not reduced during ArgI reduction. Ultimately, both prophylactic treatment with IL-33 neutralizing antibody and genetic ablation of IL-33 were not sufficient to ameliorate fibrosis in SHIP deficient mice. Taken together, these findings suggest that IL-33 can promote fibrosis by inducing ArgI activity, however, targeting IL-33 is not sufficient to ameliorate fibrosis. The data contradicts with the emerging model proposing that fibrosis can occur independently of inflammation192 and suggest amelioration of fibrosis may require downregulation of both the pro- and anti-inflammatory immune responses. The data presented in this thesis demonstrates an association between IL-33 expression and fibrosis. I show significant upregulation of IL-33 concentrations and gene expression in fibrotic sections of SHIP-/- mice. These results are consistent with findings in the SAMP mouse model of CD-like intestinal inflammation and fibrosis. Similar to this study, full- thickness ileal homogenates from SAMP mice were used to assess IL-33 gene expression over time. IL-33 expression was significantly higher in the SAMP mice compared to the AKR controls and gene expression correlated with disease severity181. In comparison 84 to human studies, although IL-33 concentrations and expression have shown to be dysregulated in people with IBD, generally the differences have been associated with UC, rather than CD191. The study by Pastorelli et al.181, investigated IL-33 expression in biopsies taken from inflamed and non-inflamed areas of the colon of people with UC and CD. They found higher IL-33 expression in the inflamed UC sections compared to healthy controls181. Although the mean IL-33 expression of inflamed CD sections was higher than the healthy controls, there was no statistically significant differences reported181. These results are consistent with the study of Seo et al., which confirmed significantly higher expression of IL-33 in colonic biopsies from people with UC compared to healthy controls, but not in CD177. Researchers have speculated that the association of IL-33 with UC, but not CD, support a paradigm of UC and CD involving type 1 and type 2 immune responses, respectively191. However, it is also important to consider the nature of disease and the sample taken for analyses in these studies. Inflammation in UC is confined to the mucosal layer whereas inflammation in CD may present transmurally, in all layers of the intestine. Accordingly, although the study by Seo et al. provided clinical characteristics of CD samples (i.e. stricturing and penetrating disease), data was not stratified to detect any disease phenotype specific differences. Two of the six samples they assessed had higher IL-33 gene expression. Additionally, since biopsy samples do not capture all layers of the intestine, then the probability of detecting significant differences may be higher in UC than CD. Hence, differences observed in mouse models of CD (SHIP-/- and SAMP) and in human CD samples could be due to sample collection methods of full-thickness ileal tissue versus biopsies, respectively. Higher IL-33 concentrations and relative gene expression in the SHIP-/- mice are not consistent with a paradigm in which CD is dominated by a type 1 immune response. Additionally, it highlights the need for further investigation of IL-33 in human CD samples, specifically in fibrotic tissues. Taken together, these results from the SHIP-/- model implicate an association of IL-33 with CD-like ileitis in mice that may be applicable to human CD. In addition, I found the 30 kDa IL-33 to be upregulated in fibrotic tissue sections and sera of SHIP-/- mice, respectively. The bands at 30 kDa align with the expected molecular weight of f-IL-33 which includes the nuclear binding motif as well as the cytokine and linker domains. Once released, f-IL-33 can be cleaved by proteases released by mast cells and 85 neutrophils. The processed isoforms are ~18-20 kDa and are referred to as c-IL-33195. The findings from this study that fibrotic sections in SHIP-/- mice have significantly higher concentrations of f-IL-33 as well as lower concentrations of c-IL-33 compared to SHIP+/+ mice are consistent with the study of Pastorelli et al.181. The authors showed upregulation of both the c-IL-33 and f-IL-33 isoforms in full-thickness ileal tissue from SAMP mice and CD biopsies181. C-IL-33 in the SAMP mice consisted of ~20 kDa isoform implying different proteases contributing to additional ~18 kDa isoform in the SHIP-/- mice. Additionally, in control biopsy samples, c-IL-33 was the predominant isoform181. In contrast, SHIP+/+ and AKR181 controls expressed only f-IL-33, which is expected as f-IL- 33 is known to be retained in cells during homeostasis169. Thus, the presence of c-IL-33 in control CD biopsies may result from release of IL-33 during sample processing. I tested whether harsher washing methods influenced IL-33 isoforms compared to more gentle processing methods, as IL-33 acts as an alarmin. I found that vigorous flushing of intestine with PBS using a needle and syringe resulted in low expression of f-IL-33. In contrast, utilization of less harsh washing methods involving cutting open the intestine longitudinally and swirling in a petri dish containing PBS preserved f-IL-33 (data not shown). Time between biopsy collection and freezing also may play a role, which may not be in the control of the researcher. In addition to intestinal tissue samples, IL-33 concentrations, particularly c-IL-33, were higher in the sera of people of CD and in the SAMP mice181. These results are consistent with the findings of this study and potentiate a role of IL-33 as a biomarker of disease. However, it is important to note contradictory results of the study by Seo et al.177 in which sera samples from people with CD had a lower mean concentration of IL-33177. Since anti-TNF has been shown to modulate circulating concentrations of IL-33 such that concentrations are lower during the maintenance phase181, these contradicting results may be due to differences in disease status and/or treatment regimes. If so, IL-33 may also be a useful biomarker to evaluate the efficacy of anti-inflammatory therapy during the maintenance phase of therapy, and this warrants further investigation. Taken together, the results from this study are consistent with studies demonstrating higher concentrations of f-IL-33 in mouse intestinal tissue and c-IL-33 in the sera. With regards to targeting the IL-33/ST2 pathway as a therapeutic target, previous studies by the Pizarro group have demonstrated a pathogenic role of IL-33 in the SAMP mice 86 using ST2 neutralization strategies195, 196. They administered anti-ST2 in 4- and 14-week-old mice to prevent and treat disease, respectively. A dose of 5 mg/kg was administered twice a week for 6 weeks. In both regimens, they observed a reduction in disease severity195, 196. The SAMP mouse model is characterized by early mucosal expression of IFNγ and TNF whereas late immune responses involve expression of IL-5 and IL-13197. In order to elucidate mechanisms by which IL-33 could be playing a pathogenic role in the SAMP mice, the Pizarro group investigated the role of ILC2s in pathosis. They found absolute numbers of ILCs were significantly higher in SAMP mice at 10 weeks of age, and ILC2s comprise the majority of the immune cell population195. Upon ST2 neutralization, ILC2 numbers were reduced, implying that IL-33 drives expansion of these cells in early disease. Consistently, in human intestinal biopsies, a slightly higher percentage of ILC2s have been reported195. Pathogenesis of IL-33 in the SAMP model has also been attributed to ILC2 driven IL-5 production which can promote eosinophilia. Interestingly, anti-ST2 reduced IL-4 and IL-5, but not IL-13 in the SAMP model. The authors suggest that CD4+ T cells may be an alternative source of IL-13 explaining why it was not downregulated by ST2 neutralization196. In addition to innate immunity, pathogenesis of disease in the SAMP mice also involves adaptive immunity as confirmed by backcrossing the mice onto a Rag2-/- background197. Specifically, the mLN of the SAMP were found to have more B cells and CD4+ T cells expressing the αEβ7 integrin198. These αEβ7 integrin expressing CD4+ T cells express IL-10 and exhibit a regulatory phenotype. However, they were unable to ameliorate disease in the T cell transfer model of colitis. αEβ7 expressing CD4+ T cells augmented B cell expansion that promoted colitis. B cells were also found to suppress the Treg phenotype of the αEβ7 expressing CD4+ T cells198. Since IL-33 can act on Tregs to promote expansion via ST2169, it is possible that the αEβ7 expressing CD4+ T cells proliferate upon IL-33 stimulation. Thus, by blocking IL-33 signaling, disease was improved. However, upon ST2 neutralization, it is critical to note that the authors reported a reduction in disease severity of 35% and 29% for the preventative and treatment regimens, respectively196. Taken together, IL-33 has been shown to play a pathogenic role in the SAMP mouse model of CD. In contrast, IL-33 neutralization in the SHIP-/- mouse did not improve disease severity as assessed by gross pathology, histological damage, and fibrosis parameters. In 87 order to understand these different outcomes in the SAMP and SHIP-/- model, it is important to compare the immune profiles. As opposed to the SAMP model, the SHIP-/- mice exhibit inflammation dependent on IL-1β and have high concentrations of IL-4, but not IL-5125, 144. Moreover, SHIP-/- mice exhibit overproduction of granulocytes/monocytes and the disease phenotype is dependent on the myeloid cell compartment135, 147. Since IL-5 is not upregulated in SHIP-/-mice compared to SAMP mice, it is probable that IL-33 does not contribute to ILC2 driven IL-5 production and eosinophilia. Moreover, there is a scarcity of T cells in the ileum of the SHIP-/- mouse131. Compared to the SAMP model, IL-33 most likely does not contribute to intestinal pathogenesis by promoting T cell expansion in SHIP deficient mice. Lastly, while IL-33 IHC staining revealed localization of IL-33 in the nuclei of epithelial cells, IL-33 was localized to the cytoplasm of epithelial cells in the SAMP mice perhaps suggesting that it is secreted181. It is thought that epithelial cells play a key role in the onset of disease in the SAMP mice as morphological and functional alterations of epithelial cells precede histological evidence of inflammation197. Given that epithelial cells are the key producers of IL-33 and that epithelial alterations are detected early in disease, it is possible that IL-33 release is triggered early in SAMP compared to SHIP-/- mice. Thus, IL-33 may be important in initiating disease in the SAMP. In addition to IL-33/ST2 neutralization, other targeting strategies in SAMP and SHIP-/- mice have also resulted in conflicting data. For example, IL-4 neutralization in the SAMP model reduced disease197. In vitro experiments elucidated a mechanism by which IL-4 could promote type 1 immune responses by synergizing with IFNγ197. Targeting IL-4 in the SHIP-/- mouse model, however, did not alleviate disease (unpublished data by Safari et al.). It is possible that IL-4 in the SHIP-/- model does not contribute to inflammation in the SHIP-/- mouse as it does in the SAMP because IFNγ is not upregulated. Moreover, IL-13 neutralization in SHIP-/- mice ameliorated disease (unpublished data by Safari et al.) whereas ST2 neutralization in SAMP ameliorated disease without reducing IL-13 concentrations196. These results suggest alternate immune-mediated mechanisms in the SHIP-/- and SAMP models that promote disease and require further exploration. Since IL-33 neutralization did not alleviate disease despite its role in promoting IL-13 production in ILC2s, it remains unknown whether ILC2s are a source of IL- 13 in SHIP deficiency and whether IL-13 production is being driven by other activators in ILC2s. Overall, these differences in immune mechanisms driving disease phenotype in 88 SAMP and SHIP-/- mice can contribute to the contradicting results not just with IL-33/ST2 neutralization but other targeting strategies as well. Since IL-33 can act as a cytokine and potentially a nuclear factor169, I investigated whether eliminating both functions of IL-33, a cytokine and nuclear factor, by genetic ablation can reduce disease. Interestingly, disease severity was reduced but not completely ameliorated as assessed by gross pathology. However, there were no significant reductions in histological damage scores. Although the median fibrosis score was lower in fibrotic sections of SHIP-/-IL33-/- compared to SHIP-/-IL33+/+ controls, the result was not significant. Reduction in disease severity observed in gross pathology could be due to the global knockdown of IL-33 as a cytokine which could imply that IL-33 neutralization at the dosage used was not enough. Another possibility is that nuclear IL-33 may play a role in promoting CD. However, it is also important to consider lung pathology of the SHIP-/- mice. In addition to intestinal fibrosis, SHIP-/- mice exhibit lung pathology characterized by myeloid cell infiltration. Compared to healthy pink lungs of SHIP+/+ mice, the lungs of SHIP-/- mice appear larger in size and have white patchiness through all the lobes147. Since concentrations of the c-IL-33 were higher in sera, I asked whether the lungs could also be a source of IL-33. Interestingly, the lungs from SHIP-/- mice had ~10 times higher concentrations of IL-33 than intestinal tissues (data not shown). Similar to the intestine, genetic ablation of IL-33 also improved gross pathology of the lungs although not completely (data not shown). It is plausible that cross-talk between the lungs and gut or systemic effects of lung inflammation could be promoting intestinal pathology. Allergic asthma models of lung inflammation have shown type 2 cytokines to play key roles199. Specifically, IL-33 mediated expansion of ILC2s can promote IL-13 secretion199 and our laboratory has shown that IL-13 is a critical cytokine driving fibrotic responses in the intestine (unpublished data by Safari et al.). Thus, considering the lungs had 10 times higher concentrations of IL-33 in SHIP deficient mice, the extent to which lung pathology promotes intestinal disease remains to be determined, as do the cell types promoting disease. Although IL-33 neutralization or genetic ablation were not able to ameliorate disease, they did reduce ArgI activity in full-thickness ileal tissue. IL-33 could possibly promote ArgI 89 expression in BMDMs and activity by synergizing with IL-33 as shown in results of the in vitro experiments. In order to consolidate these findings, more replicates will be required. However, the trends align with results from other studies that have shown synergistic effects of IL-33 with IL-13 in inducing ArgI in macrophages180. Specifically, IL-13 can upregulate expression of ST2L in BMDMs, allowing IL-33 to initiate signaling leading to upregulation of ArgI180, 188. Studies involving human macrophages have also demonstrated IL-33 being an enhancer of M(IL-4) activation when the macrophages are already polarized179. Since macrophages found within the peritoneal cavity of SHIP-/- mice are an M(IL-4) phenotype, it is likely that IL-33 may be promoting that phenotype in vivo in the presence of IL-13. Also, IL-33 alone did not induce ArgI, implying IL-13 to be critical in promoting IL-33 signaling in macrophages. Since IL-13 was found to be the critical driver of intestinal fibrosis in the SHIP-/- mouse model (unpublished data from Safari et al.), these results imply potential for IL-33 to be contributing to disease to an extent via macrophage activation. In addition to macrophages, ILC2s are another responder of IL-33 and a key source of ArgI. In the lungs, it has been shown that ArgI is expressed in bone marrow progenitors of ILC2s and maintain expression in mature cells. ArgI expression is critical to the proliferative capacity of ILC2s, but not their survival200. Thus, the possibility of ILC2s contributing to ArgI expression in SHIP-/- mice via IL-33 activation cannot be excluded. In summary, IL-33 has the potential to contribute to ArgI in vivo. ArgI downregulation upon IL-33 neutralization or genetic ablation did not improve histological damage or fibrosis. This result was unexpected as ArgI is involved in producing L-ornithine from L-arginine, which ultimately gets metabolized into precursors used for collagen biosynthesis105, 106. Additionally, the Sly lab has shown PI3Kp110δ-mediated ArgI to be critical in CD pathology107. However, targeting PI3Kp110δ not only reduced ArgI activity but also reduced IL-1β concentrations107. This finding contrasts with the results presented in this thesis where either IL-33 neutralization or genetic ablation reduced mean ArgI activity but not IL-1β concentrations. Moreover, non-fibrotic sections of SHIP-/- mice, either control or experimental mice, had almost no IL-1β compared to fibrotic sections. These results suggest that IL-1β may also play a role in promoting fibrotic pathology. 90 IL-1β is known to be a key pro-inflammatory cytokine that is tightly regulated within cells. Stimulation of PRRs initiates transcription of genes involved in formation of the NLRP3 inflammasome. Upon activation of the inflammasome, caspase-1 gets activated which processes the pro-form of IL-1β into the activated form201. There are additional caspase-1 independent mechanisms involving processing of pro-IL-1β into the active form by proteases in the extracellular space201. Macrophages are key producers of IL-1β in SHIP-/- mice144. IL-1 exerts its pro-inflammatory functions by the recruitment of immune cells201. However, IL-1β has shown to also exert anti-inflammatory effects, which could promote fibrosis. For example, IL-1β secreted from macrophages was shown to induce IL-6 in fibroblasts which in turn stimulated production of the pro-fibrotic factor osteopontin (OPN)202. Interestingly, other studies have reported a suppressive role of IL-1β on fibroblast production of collagen in the presence of TGFβ203. In addition to fibroblasts, in vitro studies have revealed a mechanism whereby IL-1β can enhance TGFβ mediated EMT204. The SHIP- /- mice have low concentrations of TGFβ and IL-6, therefore, mechanisms by which IL-1β influences fibrosis in SHIP deficiency requires further investigation. Taken together, these findings highlight that the IL-33 may play a role in fibrosis by promoting ArgI production, however, targeting IL-33 is insufficient to alleviate fibrosis in the SHIP-/- model of CD. I speculate IL-1β concentrations may be driving intestinal inflammation and fibrosis despite ArgI reduction upon IL-33 neutralization and blocking. 91 Chapter 5: Conclusion This study is the first to employ the SHIP-/- mouse model of CD-associated intestinal inflammation and fibrosis to evaluate IL-33 as an anti-fibrotic target for therapy. By separating analyses based on non-fibrotic and fibrotic sections of the mouse, I was able to infer its role in fibrosis. Additionally, I employed both IL-33 neutralization and genetic ablation strategies to definitively assesses IL-33 as a therapeutic target for treating intestinal fibrosis. In summary, IL-33 concentrations of the 30 kDa isoform were upregulated in full- thickness fibrotic tissue of SHIP-/- mice compared to SHIP+/+ controls. IL-33 neutralization and genetic ablation led to a reduction in ArgI activity in full-thickness ileal homogenates of SHIP-/- mice. In vitro studies reveal a mechanism by which IL-33 may act with IL-13 on BMDMs to synergistically enhance ArgI expression and activity. Ultimately, reductions in ArgI activity did not correlate with reduced disease severity. As such, IL-33 may play a role in promoting fibrosis in SHIP-/- mice, however, targeting both the secreted and nuclear forms is insufficient to abrogate disease pathology. Despite ArgI reduction upon IL-33 neutralization and genetic ablation in SHIP deficiency, I speculate that disease severity was not reduced due to the inability of these strategies to downregulate IL-1β concentrations. These findings challenge the notion of fibrosis propagating independently of inflammation once it is initiated205. However, the results and previous findings from the Sly lab suggest that amelioration of fibrosis may require downregulation of both pro- and anti-inflammatory responses in SHIP deficiency, highlighting the complex interplay between these two processes. With respect to clinical translatability, since IL-33 neutralization/genetic ablation was not successful in SHIP but ST2 neutralization therapies alleviated disease ~30% in the SAMP mice196, this work may suggest that IL-33 therapy may not be effective in the subset of people with CD who have low SHIP activity144 92 5.1 Limitations IL-33 isoforms Shorter forms of IL-33 are referred to as c-IL-33. However, in this study and compared to other studies, there is a range of molecular weights at which c-IL-33 can occur, some which have been associated with more potent forms as well as less active forms of IL-33. As such, in this study, I was not able to draw conclusions about the function of c-IL-33. IL-33 splice variants In this thesis, presence of splice variants was assessed in full-thickness ileal homogenates from SHIP+/+ and SHIP-/- mice (non-fibrotic and fibrotic sections). Although no splice variants were detected, it is possible that the transcripts may be expressed at low levels. To prevent the low expression of IL-33 splice variant transcripts from getting diluted in transcripts from full-thickness ileal homogenates, one cell type should be assessed. Specifically, epithelial cells should be interrogated as they are one of the key producers of IL-33, which was confirmed in SHIP deficient mice. Additionally, differences in IL-33 splice variants were found within airway epithelial cells in the study by Gorden et al. in the context of asthma reinforcing the idea of using epithelial cell monocultures for assessing splice variants. Since IL-33 is an alarmin, it is important to consider the protocol used for isolating epithelial cells. Ideally, a protocol that does not require harsh extracting conditions and maintains viable cells is ideal. Although not part of this thesis, epithelial cell isolation protocol was optimized for extraction from fibrotic tissues, however, it requires further optimization to improve cell viability. IL-33 neutralization experiments Although the IL-33 neutralization experiments did not alleviate disease, it is important to note that only a preventative regimen was tested. Studies in DSS-induced models of colitis have shown IL-33 administration early in disease has pathogenic effects whereas it promotes restitution when provided later in disease192. As such, a treatment 93 regimen could also be implemented in the SHIP-/- mice to confirm any time dependent roles for IL-33 in pathosis. Additionally, a dose titration experiment would have been ideal, however, it was not feasible at the moment because of the high cost of the IL-33 neutralizing antibody. Assessing improvement in gross pathology The IL-33 genetically ablated SHIP-/- mice did appear to have reduced number and thickness of inflamed regions as assessed by gross pathology. However, this improvement was not captured in quantitative measures such as histological damage scoring, which scores areas of active disease. It is plausible that evaluating non-fibrotic and fibrotic sections may not provide an overall picture of disease severity. Perhaps the use of Swiss Rolls in histology may help overcome this limitation by capturing the entire length of intestine. Assessing improvement in fibrosis One parameter used to assess fibrosis was the percentage of area positive for collagen using ImageJ. Masson’s Trichrome stained sections were used for the analysis. A limitation of the analysis done in this study is that staining was assessed in a defined region rather than the entire sample. Sections from SHIP-/- mice had more surface area compared to SHIP+/+ sections and difference in size of luminal space led to inaccuracy in results when normalized by surface area. As such, entire sections were not evaluated, but rather a defined region in the lamina propria. Additionally, threshold settings were set to avoid capture of debris or false positives. However, one set threshold value did not hold true for the different sections (SHIP+/+, SHIP-/- non-fibrotic and fibrotic). Perhaps differences in composition of sample with respect to collagen led to differences in capturing false positives. As such, different thresholds were set for each section meaning comparisons could not have been made across sections. Control and experimental samples from each section type were set at the same threshold. Overall, this tool is a good measure for the dispersion of collagen, however, does not correlate well with total collagen. Thus, ideally, total collagen should be measured or ELISAs for collagen subtypes should be implemented for better quantitative fibrosis measures. 94 Sample size Although trends were being observed in some data, the results were insignificant due to limited sample size. An example is data presented in Figure 4.1C where arginase activity was measured in tissue samples from treated SHIP+/+ and SHIP-/- mice. A trend of lower mean arginase activity upon IL-33 neutralization was observed in SHIP-/- mice compared to IgG controls. However, the results were not significant. In order to achieve an  = 0.05 with 80% power, 12 mice would be required in each group as determined by using a sample size calculator. Overall, increasing number of mice in each experiment could increase statistical power. 5.2 Future directions ILC2s are one cell type that responds to IL-33. ILC2s have been shown to promote type 2 inflammation in the lungs199 and they have been implicated in promoting disease in the SAMP model195. In SHIP-/- mice, both the lungs and fibrotic ileal sections exhibit higher concentrations of IL-33, which can be detected in the sera as well. Our laboratory has shown IL-13, but not IL-4, to be the critical driver of fibrotic pathology in SHIP-/- mice (unpublished data by Safari et al.). Thus, the question that remains unanswered is whether ILC2s are the source of IL-13 in SHIP deficiency. Visible improvement in the gross pathology after genetic ablation may suggest a potential role for IL-33 driven ILC2 expansion in disease. Experiments to test the contribution of ILC2s in SHIP-/- mice would be flow cytometry on lamina propria fractions of the distal ileum to determine total abundance of ILCs as well as the proportion of each cell type (ILC1, ILC2, ILC3). This flow cytometry experiment would be conducted at different ages including 4 weeks of age when disease is not established, 6 weeks of age during disease onset, and at 8 weeks of age when disease is established. These experiments would provide insight into differences in cell compositions at specific stages in disease as a result of cell expansion or cell plasticity. Additionally, rIL-33 administration into healthy SHIP+/+ mice and subsequent flow cytometry analysis for IL-13 positive ILC2s would confirm the role of IL-33 in ILC2 expansion and activation in vivo. Ex vivo analyses of ILC2s would also be conducted to confirm IL-13 production in SHIP deficiency. Specifically, the cells would be stimulated with or without a cocktail of Leukocyte Activation Cocktail in the presence of BD GolgiPlug Protein 112 Transport 95 Inhibitor (containing Brefeldin A) and analyzed for cytokine production by flow cytometry. Lastly, to validate if ILC2s promote CD pathology, IL7rCreRorαfl/fl could be bred with SHIP+/- mice to deplete ILC2s in SHIP deficient mice. In summary, the contribution of ILC2s to IL-13 production in the SHIP-/- mouse model warrants further investigation. The role of IL-33 as a nuclear factor remains controversial and requires further investigation. In this thesis, I presented a preliminary experiment in which I investigated the role of nuclear IL-33 in BMDMs. Based on the previous experiment where I showed that IL-33 synergizes with IL-13 to induce ArgI activity in BMDMs, I asked whether nuclear IL-33 plays a role in this synergy. I stimulated SHIP+/+IL33+/+, SHIP+/+IL33-/-, SHIP-/-IL33+/+, and SHIP-/-IL33-/- BMDMs with IL-13±IL-33 and assessed ArgI activity. Interestingly, in the absence of nuclear IL-33 in SHIP+/+ and SHIP-/- BMDMs, IL-13+IL-33 stimulation resulted in higher mean arginase activity compared to their IL-33+/+ counterparts. This implies that nuclear IL-33 may repress ArgI production upon IL-13+IL-33 stimulation. A similar result was shown in fibroblasts where nuclear IL-33 is proposed to repress fibroblast activation171. However, further work needs to be done to assess the role of nuclear IL-33 in BMDMs. First, the BMDMs would need to be stained for IL-33 using immunofluorescence microscopy in order to confirm nuclear localization in the controls. Additionally, bulk-RNA sequencing on stimulated BMDMs would provide a better overview of the phenotypic changes happening in the absence of nuclear IL-33. 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Thesis/Dissertation
eng
Women+ and Children’s Health Sciences
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Sly, Laura
Targeting IL-33 to treat Crohn’s Disease-associated fibrosis
Text