The Differential Roles of pH Sensing G Protein Coupled Receptors in Inflammation and Cancer by Mona Abdou Ahmed Marie December 2022 Director of Dissertation: Li V. Yang, PhD Department: Internal Medicine Intestinal inflammation is a burdening disease that may occur due to an aberrative immune response to certain drugs, infections, genetics, or unknown environmental factors. Inflammatory bowel disease (IBD) is of special concern as long-standing chronic intestinal inflammation may increase the risk for colitis-associated colorectal cancer (CAC) development due to cellular transformation to neoplastic lesions. Both the inflamed and tumor microenvironments are complex in nature and are characterized by an acidic environment. Bacterial byproducts, leukocytes respiratory bursts, and tissue ischemia and glycolysis are the main sources of protons produced in the inflamed environment, which cause local tissue acidosis. Tissue acidosis may alter both the immune and vasculature responses and subsequent cytokines and chemokines production. The pH sensing G-protein coupled receptors (GPCRs), GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A) have emerged as a new class of proton sensing receptors that are expressed by immune and non-immune cells. GPR65 is mainly expressed in immune cells and is functionally critical for intestinal homeostasis, as identified by Genome-Wide Association Study (GWAS), for being a genetic risk factor in patients with IBD. We and others observed an anti-inflammatory role for GPR65 in pre-clinical mouse models, possibly through modulating the innate immune response towards a less inflammatory phenotype. GPR4, on the other hand, is mainly expressed in endothelial cells and confers a proinflammatory role that our group had previously uncovered the mechanism for. Upon acidic activation of GPR4, a proinflammatory program in the endothelium is activated, upregulating cytokines, chemokines, adhesion molecules, and ER stress responses. This activation is particularly crucial in the process of immune cell extravasation to the site of inflammation which is a critical step in IBD pH homeostasis and chronic inflammation. We and others observed reduced inflammatory response in IBD preclinical mouse models using both genetic and pharmacological inhibition for GPR4. Additionally, a biological, as well as a pathological proangiogenic role for GPR4 has been previously described. Therefore, based on the differential roles observed for GPR65 and GPR4 in inhibiting and mediating intestinal inflammation, respectively, we sought to investigate their functional roles in CAC development. To this end, we utilized the well-established azoxymethane/dextran sodium sulfate (AOM/DSS) CAC mouse model, using GPR65-null, and GPR4-null mice, to study their functional roles in CAC. Our observations indicate an anti-inflammatory role during chronic intestinal inflammation and an anti-tumoral role of GPR65, leading to less tumor development in CAC mice. Thus, we propose that the use of GPR65 agonist will be of therapeutic benefit in IBD treatment and CAC prophylaxis. Conversely, our results indicate a proinflammatory role for GPR4 during intestinal inflammation in addition to protumorigenic and proangiogenic roles, contributing to CAC development. Hence, we propose the use of GPR4 antagonism as a strategy for IBD and CAC treatment. Finally, we observed that abolishing GPR4 alleviates another type of colitis, immune checkpoint inhibitors-mediated colitis (IMC). This type of colitis occurs as a side effect for the use of immunotherapy treatment in cancer patients. Using an IMC mouse model, our results indicate that abolishing GPR4 reduces disease activity, macrophage clusters, and fibrosis, suggesting that inhibiting GPR4 may provide an effective treatment for IMC. Collectively, this dissertation work provides new insights into the roles of GPR4 and GPR65 in intestinal inflammation and cancer development. The Differential Roles of pH Sensing G Protein Coupled Receptors in Inflammation and Cancer A Dissertation Presented to the Faculty of the Department of Internal Medicine East Carolina University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Interdisciplinary Biological Science Mona Abdou Ahmed Marie December, 2022 DIRECTOR OF DISSERTATION: Li V. Yang, PhD COMMITTEE MEMBER: Yan-Hua Chen, PhD COMMITTEE MEMBER: Isabelle M. Lemasson, PhD COMMITTEE MEMBER: James A. McCubrey, PhD COMMITTEE MEMBER: Akshaya K. Meher, PhD © Mona Abdou Ahmed Marie, 2022 Dedication To my beloved family, who have been my rock throughout this journey. My unique and progressive mother, Dr. Sabah El-Beer, and my kind-hearted brother, Khaled Marie. To my dad’s soul, may you rest in peace, Abdou Marie. To my soul mate and husband, Dr. Tamer El-Shamy, who has endured a long- distance marriage and has been nothing short of supportive, loving, caring, and always wise. I am blessed to have such a family, and I can never do you justice. Acknowledgments I would like to acknowledge my dissertation advisor, Dr. Li Yang, for giving me the opportunity to grow under his mentorship. I would like to express my gratitude for the time and effort he put towards reinforcing my critical thinking and approach towards science. I would also like to thank my committee members for their support and encouragement, Drs. Yan-Hua Chen, James A. McCubrey, Akshaya K. Meher, and Isabelle M. Lemasson. You all have been very supportive, and I am indebted to you forever. Dr. Lemasson, I cannot express my gratitude for your support to me during my search for my next role. I would also like to acknowledge Yang lab’s past members who paved the way for our current research to take place through their hard work and amazing scientific discoveries. I acknowledge Dr. Elizabeth Krewson, and Dr. Edward Sanderlin, in addition to Ms. Sarah Hammon and the future Dr. Druid Atwell, whom I collaborated with for the first two years in the Yang lab. I would like to acknowledge Kylie and Fatema, the current Yang lab members, for always providing a fun and friendly environment in the lab. I also acknowledge my dear friends, Drs. Eman Soliman, Ahmed El-Hasssanny, Hosni Hussein, and Tracey Woodlief who have always been great advisors and colleagues. They are truly a family away from family. I acknowledge the amazing ladies who tirelessly provided emotional and administrative support Ms. Delores Bowling, Ms. Glenda Daniels, and Ms. Lorelei Senna. Delores and Glenda, we will always be family. I also acknowledge Dr. Karen Oppelt, Mr. Jerry Register, Ms. Cindy Kukoly and Ms. Joani Zari Oswald for being wonderful technical advisors, experts in their fields, and an amazing support system for me during this journey. Also, the amazing Anagha Malur and Nan Leffler for being generous and always supportive neighbors. I also acknowledge the amazing friends and future Drs. Justin, Xander, Cassie, Aaron, and Ashley, thank you for your hard work and great spirit in contributing to my projects. Table of Contents List of Figures ......................................................................................................................................... viii List of Abbreviations ............................................................................................................................... ix Chapter I: Introduction ............................................................................................................................ 1 A. Inflammatory response..................................................................................................................... 1 B. Inflammatory bowel disease (IBD) and immune-checkpoint inhibitors mediated colitis (IMC) .... 3 C. Acidic microenvironment and the immune response in IBD and tumorigenesis ............................ 7 D. Carcinogenesis-promoting-inflammation: Colitis Associated Colorectal Cancer (CAC): .............. 9 (Inflammation-carcinoma axis) ............................................................................................................... 9 E. IBD therapeutic landscape ............................................................................................................. 12 F. pH-sensing G- protein-coupled receptors ...................................................................................... 13 G. Rational for evaluating the role of pH sensing GPR65 and GPR4 in inflammation and CAC ..... 20 Chapter II: Materials and Methods ...................................................................................................... 26 A. Summary ........................................................................................................................................ 26 B. Materials and Methods used for the investigation of CAC development and IMC....................... 27 B.1. AOM and DSS-induced colitis associated colorectal cancer mouse model ........................... 27 B.2. Immune checkpoint inhbitors-mediated colitis (IMC) mouse model .................................... 27 B.3. Clinical phenotype scoring ..................................................................................................... 28 B.4. Tissue collection, evaluation, and processing ........................................................................ 28 B.5. Histopathological analysis...................................................................................................... 29 B.6. Immunohistochemistry ........................................................................................................... 29 B.7. Leukocyte and myofibroblast quantification .......................................................................... 30 B.8. Tumor necrotic area quantification ........................................................................................ 30 B.9. Microvessel density quantification ......................................................................................... 31 B.10. Tumor proliferation quantification ....................................................................................... 31 B.11. Statistical analysis ................................................................................................................ 31 Chapter III: The Role of GPR65 in Colitis-Associated Colorectal cancer (CAC) ............................ 32 A. Summary ........................................................................................................................................ 32 B. Introduction .................................................................................................................................... 33 C. Results ............................................................................................................................................ 35 C.1. GPR65 KO mice show aggravated intestinal inflammation in the CAC mouse model ......... 35 C.2. Genetic knockout of GPR65 increases intestinal tumorigenesis in the AOM/DSS-CAC model ..................................................................................................................................................... 44 D. Discussion ....................................................................................................................................... 48 Chapter IV: The Role of GPR4 in colitis-associated colorectal cancer (CAC) ................................. 53 A. Summary ........................................................................................................................................ 53 B. Introduction .................................................................................................................................... 54 C. Results ............................................................................................................................................ 55 C.1. Genetic knockout of GPR4 reduces intestinal inflammation in the AOM/DSS- CAC mouse model ..................................................................................................................................................... 55 C.2. GPR4 deficiency reduces tumor burden in the CAC mouse model ....................................... 59 C.3. GPR4 is highly expressed in the tumor blood vessels of AOM/DSS mice............................ 62 C.4. GPR4 genetic knockout decreases angiogenic blood vessel formation in the tumors of AOM/DSS mice ............................ ……………………………………………………………………65 C.5. GPR4 KO increases necrosis and cell death in the tumors of AOM/DSS mice ……………68 D. Discussion ...................................................................................................................................... 71 Chapter V: The Role of GPR4 in Immune-checkpoint Inhibitors Mediated Colitis (IMC) ............ 76 A. Summary ........................................................................................................................................ 76 B. Introduction .................................................................................................................................... 77 C. Results ............................................................................................................................................ 80 C.1. GPR4 genetic knockout decreases disease severity index in the IMC mouse model ............ 80 C.2. Lack of GPR4 expression reduces Immune checkpoint inhibitors mediated colitis histopathological inflammation ............................................................................................................. 85 D. Discussion ...................................................................................................................................... 99 Chapter VI: General Discussion .......................................................................................................... 105 References .............................................................................................................................................. 117 Appendix A: Animal use protocol ....................................................................................................... 143 Appendix B: Elsevier license to publish .............................................................................................. 144 Appendix C: BioRender license to publish ......................................................................................... 145 Appendix D: BioRender license to publish ......................................................................................... 146 List of Figures Figure 1.1. Roles of proton sensing GPCRs in different systems…………………………………….…22 Figure 1.2. A graphical illustration of GPR4- mediated leukocytes extravasation………..………….....24 Figure 3.1. Macroscopic parameters of colitis in WT and GPR65 KO AOM/DSS mice…………….....37 Figure 3.2. Colon shortening, fibrosis, and myofibroblast expansion in AOM/DSS mice…...…………40 Figure 3.3. Leukocyte infiltration in colons of AOM/DSS mice……………………………………......42 Figure 3.4. Effects of GPR65 on tumorigenesis in the CAC mouse model……………………………..45 Figure 4.1. Clinical indicators of intestinal inflammation in the WT and GPR4 KO CAC mice……….56 Figure 4.2. Effects of GPR4 on tumorigenesis in the CAC mouse model………………………….…...60 Figure 4.3. GFP signal and double labeling with CD31 in the CAC mouse model……………………..63 Figure 4.4. Micro-vessel density and VEGFR2 protein expression assessment in the blood vessels of the CAC mice………………………………………………………………………………………...…66 Figure 4.5. Tumor necrosis and cell death in the tumor tissues of the CAC mouse model…………......69 Figure 5.1. Clinical indicators of intestinal inflammation in the IMC mouse model…………………...82 Figure 5.2. Histopathological analysis of IMC mice colons……………………………………………87 Figure 5.3. Pathological histiocytic (macrophage) clusters analysis in IMC mice colons……………..91 Figure 5.4. Pathological fibrosis analysis of IMC mice colons………………………………………...95 List of Abbreviations 5-ASA 5-aminosalicylic acid ACTA2 Actin alpha-2 AOM Azoxymethane APC Antigen-presenting Cell ASIC Acid sensing ion channels ATF-3,4,6 Activating transcription factor-3,4,6 BMDM Bone marrow derived macrophage CAC Colitis Associated Colorectal Cancer cAMP Cyclic adenosine monophosphate CCL-2,3,5,12,20 C-C Motif Chemokine Ligand- 2,3,5,12,20 CD Crohn’s disease CD-4,8,28,40,45 Cluster of differentiation-4,8,28,40,45 CDH1 Calcium-Dependent Adhesion Protein, Epithelial/ E-cadherin CHOP C/EBP homologous protein CLR C-type lectin receptors COL1A1 Collagen type I alpha 1 chain COL3A1 Collagen Type III Alpha 1 Chain COPD Chronic obstructive pulmonary disease COVID-19 Corona Virus Disease 2019 COX-2 Cyclooxygenase-2 CSF2 Colony stimulating factor-2 CTCAE National Cancer Institute’s Common Terminology Criteria for Adverse Events CTLA-4 Cytotoxic T-lymphocyte antigen-4 CX3CL-1,7,12 C-X3-C motif chemokine ligand-1,7,12 CXCR-4,7 C-X-C chemokine receptor type-4,7 DAMP Damage-associated molecular patterns DNA Deoxyribonucleic acid DSS Dextran sodium sulfate EC Endothelial cell eIF2? Eukaryotic initiation factor-2? ER Endoplasmic reticulum GFP Green fluorescent protein GM-CSF Granulocyte-macrophage colony stimulating factor GPR 4,65,86,132 G-protein coupled receptor 4,65,86,132 GWAS Genome-Wide Association Studies H&E Hematoxylin and eosin HLA Human leukocyte antigen HMVEC Human microvascular endothelial cells HMVEC-L Human microvascular endothelial cells from lung HPAEC Human pulmonary artery endothelial cells HUVEC Human umbilical vein endothelial cells IBD Inflammatory bowel disease ICAM-1 Intercellular adhesion molecule-1 ICI Immune checkpoint inhibitors IEC Intestinal epithelial cells IFN? Interferon gamma ? IgA Immunoglobulin A IHC Immunohistochemistry IL-1,2,4,5,6,8,10,12 Interleukin-1,2,4,5,6,8,10,12 IL23R Interleukin receptor 23 ILC Innate lymphoid cells IMC Immune checkpoint inhbitors mediated colitis irAEs Immune-related adverse events IRE1 Inositol-requiring enzyme type-1 JAK2 Janus kinases KO Knock out LRR Leucine-rich repeat receptors M1 Classically activated macrophage M2 Alternatively activated macrophage MAdCAM-1 Mucosal addressin cell adhesion molecule-1 MHC II Major histocompatibility complex-II MLN Mesenteric lymph node mRNA Messenger ribonucleic acid NF-?B Nuclear factor kappa light chain enhancer of activated B cells RANK Receptor activator of nuclear factor kappa-? RANKL Receptor activator of nuclear factor kappa-? ligand NLRP3 NLR family pyrin domain containing-3 NLR Nod-like receptor NSCLC Non-small cell lung cancer OPG Osteoprotegerin PAMP Pathogen-associated molecular pattern PD-1 Programmed cell death-1 PD-L1 Programmed cell death ligand-1 PERK Protein kinase RNA like ER kinase PIP Phosphatidylinositol 4,5-bisphosphate PLC Protein-phospholipase PPR Pathogen pattern recognition receptor PSR Picrosirius red qPCR Quantitative real-time polymerase chain reaction RAG Recombination activating gene Rho Ras homolog gene RIG-I Retinoic acid-inducible gene-1 RLR Retinoic acid-inducible gene 1 like receptor ROS Reactive oxygen species SCCHN Squamous cell carcinoma of the head and neck SELE E-selectin SMA? Smooth muscle actin-? T (reg) T regulatory TCR T-cell receptor TDAG8 T cell death-associated gene 8 Th-1,2,17 T helper-1,2,17 TLR Toll-like receptor TNF Tumor Necrosis Factor UC Ulcerative colitis VCAM-1 Vascular cell adhesion molecule-1 VEGF Vascular endothelial growth factor VEGFR2 Vascular endothelial growth factor receptor-2 WT Wild type X-BP-1 X box-binding protein-1 Chapter I: Introduction A. Inflammatory response The body is protected by innate and adaptive immunity systems. An innate immunity system is comprised of barrier surface protection (skin and mucous membranes), complement system, inflammatory response, and innate immune cells such as neutrophils and macrophages. An adaptive immunity system is comprised of immune cells response towards a specific invader mainly through antibody production and T cell response. These two systems work in harmony to create a homeostasis and maintain bodily protection [1]. Inflammation is considered part of the body’s normal healing process. Microbial infection, parasites, foreign bodies, direct tissue injury, tissue stress, or disturbances in homeostasis lead to a physiological inflammatory response that, when the body's homeostasis is maintained, resolves naturally. Nonetheless, this homeostasis may be disrupted, leading to chronic inflammation if the insult burden overweighs the homeostatic balance. If this inflammatory response further persists, it may lead to tissue damage, autoimmune disease, allergies, fibrosis, and cancer [1]. During the 1st Century A.D., nearly 2,000 years ago, Celsus described the four cardinal signs of inflammation: rubor (redness), calor (heat/warmth), tumor (swelling), and dolor (pain), caused by exudate formation as a result of inflammation [2]. A fifth pillar was added by Rudolph Virchow "The father of Pathology," in the 19th century, describing the disruption in function as a result of inflammation [3]. These cardinal signs are local alterations resulting from acute inflammation that is greatly attributed to alterations in local vasculature [4]. Currently, the consensus of the inflammatory immune response describes it as an innate and adaptive immune response. In this context, a mediator or an insult such as infection or injury is required, followed by a sensor which is the innate immune response from cells such as neutrophils, macrophages, dendritic cells, natural killer cells, and/ or mast cells. This is considered the first response to infection or injury. After cells detect the insult, immune cells such as the professional antigen presenting cells (APCs), macrophages and dendritic cells, use their pathogen pattern recognition receptors (PPRs) to recognize specific conserved pathogenic structures such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). In turn, APCs travel to nearby lymph nodes to activate CD4+ T cells via two signals, they present major histocompatibility complex II (MHC II) to bind T-cell receptor (TCR) on CD4+ T cells, they also present costimulatory molecules Cluster of Differentiation (CD) 80 or CD86 to bind CD28 on CD4+ T cells and in turn those activated CD4+ T cells secrete stimulatory cytokines. Depending on the secreted molecules/inflammatory mediators (cytokines) they produce, they are able to activate naïve CD4+ T cells differentiation into different T helper (Th) cells subsets, for example, (Interferon gamma (IFN?) and Interleukin (IL)-12) activate Th1, (Tumor Growth Factor beta (TGF?) and interleukin (I.L.) IL-6) activate Th17, (TGF? and IL-2) activate T regulatory (T- reg) and (IL-4 and IL-2) activate Th2 cell subset. Th cell subsets can be identified in the inflamed tissue via their cytokine signature, for example Th1 cells secrete (IL-2, IFN?, Tumor Necrosis Factor (TNF)-? or ?, and granulocyte-macrophage colony stimulating factor (GM-CSF)), Th17 secrete (IL-17A, IL-17F, IL-21, and IL-22), Treg secrete (IL-10), and Th2 secrete (IL-4, IL-5 and IL-13). Moreover, the cytokines and chemokines produced by sensor cells can be detected by effector cells such as the endothelium [1, 4, 5]. These cytokines can also act locally on the tissue blood vessels causing activation of the endothelial lining that leads to vasodilation to increase the blood flow to the affected area, which causes redness and heat. They also cause vascular permeability or leakiness leading to plasma seeping from blood vessels into the external local 2 tissue causing edema or swelling. Altogether, these effects cause the site of inflammation to be painful [4, 6]. The role of endothelial activation is very crucial in this process as it first leads the endothelium to further produce proinflammatory mediators and become more adhesive to immune cells causing their extravasation towards the inflamed site. Endothelial-leukocytes adhesion is the determining process for immune extravasation and a determinant of the intensity of the acute inflammatory response, as initially neutrophils and monocytes are the first responders to the site of injury or insult, they extravasate through the endothelium, also later on this process is a major determinant for the severity of the adaptive immune response. Several molecules are expressed on endothelial cells are responsible for this process, selectins, (e.g. E-selectin and P-selectin), are lectin-like adhesion molecules that mediate leukocyte rolling, followed by mucosal addressin cell adhesion molecule 1 (MAdCAM-1), intercellular adhesion molecule (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), are immunoglobulin-like adhesion molecules that are responsible for firm adhesion and ensuing transendothelial migration of leukocytes [4, 7, 8]. B. Inflammatory bowel disease (IBD) and immune-checkpoint inhibitors mediated colitis (IMC) Inflammatory bowel disease (IBD) is comprised of two main subtypes, Ulcerative Colitis (UC) and Crohn's Disease (CD). This disease is characterized by phases of activity and relapse of inflammation. CD’s inflammation can occur in any part of the intestinal tract, forms foci of inflammation, is not restricted to mucosal inflammation, and can extend to be transmural, whereas UC's Inflammation is a continuous mucosal inflammation that is restricted to the colon, most severe in the rectum area, with rare occurrence of terminal ileum inflammation. IBD symptoms 3 include diarrhea, blood in stool, constipation, body weight loss, bloating, cramping, flatulence, and bleeding ulcers [9-11]. The etiology of IBD is yet to be uncovered; nonetheless, it is widely accepted that IBD inflammation results from a defect in mucosal barrier response or immune response to luminal flora [12-15]. The interplay of several factors contributes to these defects, such as diet, environmental factors, genetic susceptibility, host microbiome, or infections [16]. Microbial dysbiosis is a key player in promoting IBD, where a decrease in the beneficial microbiome is overweighed by an increase in a pathogenic one, activating an inflammatory response in the gut of both UC and CD patients [17]. Microbial dysbiosis is one of the hallmarks observed in IBD patients, however, It is debatable whether microbial dysbiosis is a cause or a consequence of IBD [18]. Pathogenesis of IBD is attributed to imbalances in one or more of the components of the mucosal immune system, such as intestinal epithelial cells (IEC), innate lymphoid cells (ILC), components of innate (monocytes/macrophages, neutrophils, natural killer cells, and dendritic cells) and adaptive (T-cells and IgA producing B-cells) immune system, and their associated mediators (cytokines and chemokines) [19, 20]. When bacterial antigens come in contact with mucosal components, an activation of the innate immune response may be attained through the activation of immune sensor receptors, also known as PRRs, such as toll-like receptors (TLRs), Nod-like receptors (NLRs), leucine-rich repeat (LRR) receptors, C-type lectin receptors (CLRs), and retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) [21]. Antigen-presenting cells such as macrophages and dendritic cells then orchestrate the differentiation of naïve CD4+ T-cells into T-reg and effector Th cells, such as Th1, Th2, and Th17, consequently controlling gut homeostasis and depending on the cytokine profiles presented possibly promoting IBD [22-24]. 4 Genome-Wide Association Studies (GWAS) studies have identified over 200 genetic risk foci for IBD, such as NOD2, TLR2, GPR65, IL23R, JAK2, CDH1, or HLA genes [25-29]. Mutations in CDH1 or C1orf106 genes cause the disruption of IEC tight junctions via downregulating E- cadherin [29, 30], allowing luminal bacterial content to seep into the lamina propria and its immune components [31, 32]. In turn, this activates an innate immune response in IBD patients where PRRs such as TLR4, NOD1, and NOD2 expression levels are increased in resident macrophages and dendritic cells. Which activates downstream pathways nuclear factor kappa light chain enhancer of activated B cells (NF-?B) and NOD-like receptor (NLR) family of proteins, respectively [33- 35]. In turn, further monocytic and neutrophilic recruitment occurs to the lamina propria, which produces anti-pathogenic reactive oxygen species (ROS) [36]. Consequently, proinflammatory effector cytokines such as IL-1?, IL-6, IL-18, tumor necrosis factor (TNF), IL-12, IL-23, and IFN- ? are secreted in both UC and CD [33, 37, 38]. IL-12 activates CD4+ Th1 cells [39], while IL-1? activate IL-17A-secreting ILCs which recruit CD4+ Th17 cells [40, 41]. CD4+ effector T-cells upregulate the chemokines CCL3 and CCL5 [42, 43]. Thus recruiting ?4?7 integrin expressing circulating T cells which infiltrate intestinal tissues via binding vascular MAdCAM-1 (mucosal addressin-cell adhesion molecule 1) expressed on colon endothelial cells [44]. Although previous reports have linked CD to Th1 and UC to Th2 cell cytokines profiles, current knowledge identified a link to Th1 and Th17 cells for both disease subtypes [45-47]. In addition, both activated CD4+ T-helper cells and cytotoxic CD8+ T-cells are detected in the peripheral blood of IBD human patients and associated with disease activity [48-51]. The vast contributions of T-cell activation in colitis pathogenesis are linked to Immune- checkpoint inhibitors mediated Colitis (IMC) [52], which presents with clinical and pathologic resemblance to IBD [53, 54]. IMC occurs as a side effect of immune-checkpoint inhibitors (ICIs) 5 cancer therapies. Monoclonal anti-programmed cell death-1 (PD-1), anti-programmed cell death ligand-1 (PD-L1), and anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4), antibodies are termed immune checkpoint inhibitors. Currently, they are considered the treatment of choice for advanced-stage cancer patients, such as metastatic melanoma, non-small cell lung cancer (NSCLC), and colon cancer [55-57]. While PD-1, PDL-1, and CTLA-4 are functional immune brakes expressed on T-cells to resolve physiological inflammation, their acquired expression on tumor cells allows them to evade immune surveillance. Thus, the treatment with monoclonal antibodies that target these checkpoints render tumor cells detectable to immune cells for elimination, and may cause an overall immune response in the body. In turn, this immune response can be non-specific and lead to several toxicities in different organs. These toxicities are commonly termed immune-related adverse events (irAEs). Common irAEs, such as diarrhea, Colitis, hepatitis, rash, endocrinopathy, and pneumonitis, and rare but fatal ones, such as hematologic, neurologic, and cardiovascular toxicities, have been reported [58-60]. IrAE severity is clinically graded using National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE), which categorizes toxicities into five grades, mild (grade 1), moderate (grade 2), severe (grade 3), life-threatening (grade 4), and death (grade 5) [61]. 90% of patients develop one or more irAEs consequent to their ICI treatments [62, 63]. According to CTCAE, IrAEs range from mild (50-90%) to severe (10-50%). IMC, in most cases, requires treatment cessation and could lead to 3 out of 10 fatal irAEs [64, 65]. Similar to IBD patients, an increased incidence of activated CD4+ and CD8+ T-cells was reported in the peripheral blood of anti-CTLA-4 antibodies treated patients [66, 67]. Moreover, in concordance with the immune infiltration of IBD patients, IMC patients treated with combination therapy (both anti-PD-1 and anti-CTLA-4 antibodies) show increased neutrophilic, CD4+, and 6 CD8+ cells in the colonic mucosa [50, 68-70]. An imbalance between Treg/ Th17 subsets is another hallmark of IBD and given that ICI treatment will dampen the anti-inflammatory role of Treg, evidently, Treg-altered profiles were observed in IMC patients receiving combination therapy [70, 71]. The close resemblance in clinical and immune profiles between IMC and IBD reinforces the use of similar diagnostic and treatment modalities [53]. C. Acidic microenvironment and the immune response in IBD and tumorigenesis An acidic microenvironment plays a role in immune homeostasis. Studies have shown that chronically inflamed tissue is associated with acidosis which consequently leads to immune and cytokine production modulation [72]. Extracellular acidification has been linked to neutrophil chemotaxis. Neutrophils function as the first responders to inflammatory stimuli. The main inflammatory stimulus is the accumulation of bacterial by-products of short-chain fatty acids, creating an acidic environment at the site of inflammation [73, 74]. Neutrophils and macrophages can attempt to eliminate these harmful bacteria through respiratory bursts, which can further acidify the microenvironment [75]. Interestingly, varying reports find that the colon lumen of patients with IBD is more acidic than the normal colon, although a few results are conflicting [36- 40]. The expected source of acidity in the inflammatory microenvironment of IBD patients is bicarbonate loss in the colon, as IBD patients commonly suffer from diarrhea [76, 77]. Increased immune infiltrates quickly deplete available O2 and switch from aerobic to anaerobic glycolysis. This metabolic switch is driven by Hypoxia-inducible factor 1-alpha (HIF-1?) since it regulates tissue homeostasis in response to hypoxia which includes increased vascularization in those areas in an attempt to increase O2 [78]. Hypoxia is another major factor contributing to the acidic environment at sites of inflammation. This hypoxic environment favors a highly glycolytic phenotype producing large amounts of lactic acid into the inflammatory loci to further acidification 7 [79]. Small vessel damage, as well as metabolic activity resulting from respiratory bursts infiltrating leukocytes at the site of inflammation, promote hypoxic conditions and the production of reactive oxygen species (ROS). ROS are highly reactive small short-lived oxygen-producing molecules that contribute to cell damage or even cell death [80]. In normal cells, ROS production functions as a sensor for cell damage and initiates a signal transduction cascade before ROS elimination [81]. ROS in low to moderate levels activates physiological functions such as cell angiogenesis, proliferation, migration, and invasion [82]. In contrast, ROS in high levels cause oxidative damage to cellular DNA and may be cytotoxic; it also plays a role in the initiation and progression of cancer [82, 83]. In tumorigenesis, increased ROS production is needed to maintain the rapid proliferative nature of these cells and their high metabolic turnover [84]. Oxidative stress occurs as a result of a disturbance in the homeostasis of ROS production and elimination [85]. When ROS production rate supersedes its elimination, cells develop a resistance mechanism by which they shift their metabolic pathway to producing lactic acid rather than employing aerobic mitochondrial respiration, rendering the environment acidic [81]. Both inflammatory and tumor environments are infamous for being acidic, which is an essential driver of tumor progression. Chronic inflammation in IBD promotes colitis-associated colorectal cancer (CAC) initiation through the stimulation of oxidative stress-related mutations [86, 87]. Tumor formation occurs in a step-wise fashion starting from initiation, promotion, and progression. A single-cell mutation may or may not lead to neoplastic shifts, depending on the type of that genetic alteration. Nevertheless, the microenvironment is a major determinant of the progression rate and clinical phenotype of neoplasia [88]. The extracellular pH of the tumor is renounced for being acidic, mainly due to the production of lactic acid through aerobic glycolysis switch, known as “Warburg Effect” [89, 90]. Recently, the attribution of cancer stem cells (CSS) to drug resistance and disease 8 re-emission has caught researchers’ attention. The acidic microenvironment is one of the factors that contribute to the positive selection of cancer stem cell growth, leading to changes in the phenotype and genetics of the tumor [91]. Lactic acid production confers an immune suppressive role through inhibition of cytotoxic T cells proliferation and function, via disrupting their metabolism [92]. Lactate suppresses dendritic cell activation during antigen-specific autologous T-cell stimulation [93]. It also enhances the motility of tumor cells and inhibits monocyte migration and cytokine release [94]. Elevated lactic acid production has been linked to many malignancies [95-97]. Tumor-derived lactate exacerbates angiogenesis, inflammation and immune deficiency of tumor cells. Lactate enhances the expression of vascular endothelial growth factor (VEGF), which promote tumor angiogenesis [98]. Lactate increases interleukin (IL)-17A production by T-cells and macrophages, giving rise to the promotion of chronic inflammation in tumor microenvironments [99]. Moreover, initial environmental acidosis in the tumor site is linked to avascular in situ carcinoma (localized) [100]. It is expected that this increase in acidity results in tumor vascularization and invasion due to the “Warburg effect” glycolytic shift [101]. This shift occurs as a coping mechanism to overcome extreme acidic conditions, hypoxia, and nutrient deprivation [102]. Even after cells have been provided sources of oxygen and nutrients, it continues producing metabolic acids. G-protein coupled receptors and Acid-sensing ion channels (ASIC) have a pivotal role in sensing pH changes and cellular adaptation to these changes [103]. D. Carcinogenesis-promoting-inflammation: Colitis Associated Colorectal Cancer (CAC): (Inflammation-carcinoma axis) Chronic inflammation resulting from bacterial, or viral infections, environmental irritants or autoimmune disease has been connected to promoting cancer [104]. Inflammation resulting from 9 infections, such as Helicobacter pylori (H.pylori), Hepatitis B virus (HBV) and Hepatitis C virus (HCV), Schistosoma Bacteroides, have been linked to gastric, liver, and bladder cancer, respectively [105, 106]. The inflammatory response towards pathogens is considered part of the normal host defense mechanism to eradicate pathogens. However tumorigenic pathogens may alter this response and develop low grade chronic inflammatory response that results in cancer [107]. Environmental irritants and obesity are other factors that contribute to low-grade chronic inflammation [108, 109]. Tumor-associated inflammation concords with tumor development [110]. Leukocyte infiltration has been linked to tumor-associated inflammation. The first observation of leukocytes in the tumor microenvironment occurred in the 19th century by Rudolf Virchow [111, 112]. This immune-inflammatory response can enhance genomic instability, promote tumor progression, invasion, angiogenesis, and immunosuppression in the tumor microenvironment (TME) [113, 114]. Anticancer agents may initiate a pro-inflammatory response through inducing trauma, tissue injury and necrosis, that cause tumor resistance. Immune-based therapies induce a specific type of inflammation which enhances tumor antigen presentation and hence, it’s eradication by host immune response [104]. These different types of inflammation complicate the tumor microenvironment cellular content. TME contains different cell types including, innate immune cells (macrophages, neutrophils, mast cells, myeloid-derived suppressor cells, dendritic cells, and natural killer cells) and adaptive immune cells (T and B lymphocytes) in addition to the transformed epithelial cells (cancer cells) and their surrounding stroma (which consists of fibroblasts, endothelial cells, pericytes, and mesenchymal cells) [115]. These various types of cells communicate with one another through cytokine and chemokine production or through direct contact. They act in autocrine and paracrine fashion to direct tumor growth patterns [116]. 10 Cytokines are the major means of communication amongst cells in the inflammatory milieu, they can also act as drivers or inhibitors of tumor-associated inflammation. Neoplastic cells produce proinflammatory mediators including proteases, eicosanoids, cytokines, and chemokines. Several cytokines such as macrophage migratory inhibitory factor (MIF), IL-1?, TNF-?, IL-6, IL-17, IL- 12, IL-23, IL-10, and TGF-? have been clinically and experimentally linked to either the promotion or inhibition of tumor development [113, 117]. IBD may be the result of immune over-reaction from the host against commensal microbiota. Normally a localized acute inflammatory immune reaction is warranted as a host defense mechanism to remove all harmful bacteria. The first line of defense is the innate immune cells including neutrophils, macrophages, and dendritic cells. The engulfment of harmful pathogens is sufficient to protect the host against harmful bacteria, yet antigen-presenting cells such as macrophages and dendritic cells may be needed to adjust the interplay between innate and adaptive immunity that has the ability to eradicate the infection and inflammation [107]. The failure to resolve this localized immune-inflammatory reaction results in low-grade chronic inflammation, which leads to IBD. Chronic inflammation associated with IBD is a key player in modulating the intestinal microenvironment, leading to colorectal cancer initiation through oxidative stress caused mutations. These mutations enable transformed cells to resist apoptosis and acquire oncogenic characteristics [118-120]. Meanwhile, breaching the intestinal epithelial barrier causes microorganism invasion which creates a more acidic environment furthering inflammation which create an environment suitable for cancer progression [121, 122]. Intestinal inflammation drives both initiation and promotion of transformed cells leading them to be malignant. Tumor formation in the case of CAC is referred to as; “inflammation-dysplasia-carcinoma” pathway. This pathway is a description of the progression of low-grade dysplasia that results from intestinal inflammation 11 then advance into high-grade dysplasia then carcinoma [123]. The biology behind IBD-derived colorectal cancer is supported by the 2.4-fold higher risk of IBD patients for developing colorectal cancer than the general population [124]. Like other cancer types driven by chronic inflammation, CAC progression involves many diverse key players starting from genetic mutations and ending with cytokines delivering pro-inflammatory queues. ROS initiates CAC through P53 mutations, reduced DNA repair machinery efficiency, increased TNF-?, increased IL-6, increased IL-17A, increased IL-1?, K-RAS mutations, and Adenomatous polyposis coli (APC) mutations [125-127]. E. IBD therapeutic landscape Genetic susceptibility, gut dysbiosis, and Environmental factors, are factors that overlap and lead to inappropriate immune response in IBD patients [128]. Current treatments such as corticosteroids, immune modulators, and anti-TNF-? biologics are used to induce clinical improvement through modulating the immune response in IBD patients [129, 130]. Corticosteroids are successful in inducing quick remission, nonetheless, they are not suitable for long term remission induction. For IBD patients that are refractory to corticosteroids, anti-TNF-? treatments are the current biologics most widely used. Although they have shown great success in inducing disease remission, inducing mucosal healing, and reducing hospitalization rates [130], non- responsive IBD patients have been reported [131]. Moreover, in responders, there is no clear agreement on these treatments’ efficacy in reducing the risk for CAC development or long-term use safety [132, 133]. Adverse events of anti-TNFs, such as infections and cancer development have been reported [134]. Moreover, immune modulators, such as methotrexate and thiopurines, are used mainly to maintain remission as a second line treatment, as they are slow acting [135]. In CD patients, thiopurines are effective in maintaining remission, while they are used to maintain remission in UC patients who were resistant to corticosteroids, or irresponsive to 5-ASAs [135]. 12 Nonetheless, they are not suitable for long-term use as they can carry a risk of cancer development, as well [136]. Additionally, 5-Aminosalicylic acid (5-ASA), and antibiotics, are used in less severe cases of IBD. For mild-to-moderate UC only, but not severe UC and CD, 5-ASAs are effective and well- tolerated [137, 138]. Moreover, ciprofloxacin, metronidazole, or the combination of both, are used to modulate the gut microbiome, that is believed to cause an aberrant immune response to gut bacteria in genetically susceptible patients. They are more beneficial for disease activity involving the colon and modest activity in active luminal CD. They also play a role in maintenance of remission and prevention of post operative recurrence of CD. Their efficacy has not yet been proven in UC. Antibiotics are not recommended for long term use as they may cause bacterial antibiotic resistance [139]. While the previously mentioned line of therapy targets the inappropriate immune response, adhesion molecules on the other hand, modulate immune trafficking aspect, via blocking homing of leukocytes to the inflammation site. Categories of this class are, integrins (?4?1, ?4?7 or ?E?7) mainly expressed on lymphocytes, addressins (ICAM-1 and MAdCAM-1) mainly expressed on endothelial cells, and the chemokine receptor 9-GPCR. Especially, blocking the ?4?7/MAdCAM- 1 interaction is effective in IBD. Despite these advances in IBD therapeutics, non-responsive cases have been reported to corticosteroids, TNF-? antibodies, and anti-adhesion treatments [140]. For the aforementioned efficacy and safety concerns, it is of crucial value to explore more drug targets. F. pH-sensing G- protein-coupled receptors The occurrence of physiological events in a living organism begins with chemical or biochemical signals initiating a response. The signal starts at the cell surface and is sensed through a receptor and translated within milliseconds to hours to its specific biological function. Those 13 biological functions are processes that a living organism undergoes to change its environment; examples are cellular growth, cellular differentiation, wound healing, muscle contraction, secretory functions, sensory functions, and neural communications [141]. The process of receiving and translating a biological signal is termed signal transduction. Membrane receptors or cell surface receptors are transmembrane proteins that contain an extracellular domain that senses stimuli and is activated by ligand binding and an intracellular domain that can activate intracellular proteins to initiate a biological response. G-protein Coupled Receptors (GPCRs), Ion channel receptors, and Enzyme-linked receptors are three main types of cell surface receptors that are activated amid ligands binding; examples of ligands are hormones, lipids, neurotransmitters, and enzymes [141]. GPCRs are the largest and most diverse known family of receptors to transduce extracellular stimuli into intracellular queues. They are 7-transmembrane receptors with an extracellular N-terminus and an intracellular C-terminus, where the intracellular domains interact with heterotrimeric G-proteins to transduce signals [142]. The variety of these receptors lies in the diverse stimuli they respond to and signals they transduce, such as neurotransmitters, light, odors, proteins, biogenic amines, lipids, amino acids, hormones, and chemokines, amongst many others. There are about 18 different human G? proteins that may be coupled with GPCRs [143, 144]. These G? protein subunits form complexes with G? and G? subunits, of which at least 5 and 11 types have been discovered, respectively [143]. In recent years, more than 800 GPCRs have been identified in the human genome [145]. pH-sensing GPCRs are a novel class of these receptors, capable of sensing acidic extracellular environments through the protonation of several histidine residues on their extracellular binding domain [146-153]. Upon activation, these receptors signal through G-proteins to modulate cellular function in response to their environment. Family members of pH-sensing GPCRs include GPR4, TDAG8 (GPR65), and OGR1 (GPR68), which 14 evolve from the same ancestor, GPR132 [146, 147, 150, 154-158]. Although, previous studies showed the pH-sensing properties of GPR132 [148, 159], structural modeling and mutagenesis studies, show that GPR132 does not contain the conserved key proton-sensing residues found in GPR4, GPR65, and GPR68 [158]. GPR4, GPR65, and GPR68 are capable of activation within the physiological pH range (7.32- 7.42). However, the peak activation of these receptors occurs at pH range of (6.4-7.0) [146]. Their activation facilitates the downstream signaling through the Gq/11, Gs, and G12/13 pathways. GPR65, GPR4, and GPR68 have been described to couple to the Gs and G12/13 while GPR4 and GPR68 can also couple to Gq/11 (Figure 1.1.). The great body of evidence suggest that GPR4 receptor is expressed predominately in the vasculature, while GPR65, GPR68 and GPR132 are expressed predominantly in leukocytes [160- 166]. More recently, a wider range of cellular expression of GPR4 is observed in histiocytes (macrophages) [161] fibroblasts [167], chondrocytes [168], neurons [169], and kidney epithelial cells [152]. Similarly, GPR68 expression was observed in airway epithelial and myofibroblast cells [170], fibroblasts [170, 171], osteoblasts [172], and vascular endothelial cells [173]. This family of receptors adjust cellular functions in response to acidosis (increased H+ concentration) in many diseases associated with improper pH homeostasis, such as IBD, cancer, and ischemic disease of the hindlimb, brain, heart, and kidney [6, 7, 27, 147, 152, 160, 163, 174-186]. Each of these receptors is implicated in regulating the inflammatory response upon pH sensing through immune cell or non-immune cell functions. GPR68 and GPR65 are predominantly expressed in immune cells [187]. GPR68 acid sensing role has been described in immune (leukocytes autoimmune response), cardiovascular (cardiomyocyte viability and endothelium mechano-sensing), renal (acid/base homeostasis), respiratory (inflammatory airway remodeling), 15 gastrointestinal (intestinal homeostasis), skeletal (bone acid sensing), and endocrine (insulin secretion) systems, and in cancer (progression and cell proliferation) [188-194]. (Fig. 1.1.) The roles of GPR65 has been studied in the immune (leukocyte functional response), respiratory (asthma), nervous (nociception and panic disorders), and skeletal (bone density) systems [188]. (Fig.1.1.). In an acidic microenvironment, expression of GPR65 by immune cells regulates the inflammatory response by a myriad of differential mechanisms, such as its role in Th1/Th17 differentiation of CD4+ T-cells [195], decreasing leukocyte infiltration to the site of inflammation in IBD and colitis associated colorectal cancer mouse models [166]. GPR65 expression showed a protective role in many systems, such as the brain, heart, and tumors drug response [182, 196-198]. The time-dependent expression of GPR65 during cerebral ischemia reduces the volume of the infarction of ischemic stroke and reduces neurological deficits [199]. In Immunoglobulin A nephropathy (IgAN) patients, a type of glomerulonephritis GPR65 expression on macrophages was negatively correlated to disease severity [200]. Under acidic environment, microglia cells show impaired phagocytic function associated with a decrease in cyclic AMP (cAMP) and protein kinas A (PKA) associated with a decrease in GPR65 expression [201]. Expression of GPR65 in cardiovascular disease confers a protective action against myocardial infarction through transcriptional downregulation of the chemokine CCl20 expression in macrophages which in turn downregulates the proinflammatory IL17A production [202]. It has been reported that rs8005161 polymorphism on GPR65 may alter the response of human monocytes towards acidic pH activation, leading to increased severity of IBD in human patients [203]. GPR65 polymorphism where a substitution of isoleucine to leucine at codon 231, was also identified as a risk factor in colitis, Ile231Leu-GPR65 knock-in mice were challenged with bacterial and T-cell mediated colitis showed increased susceptibility to colitis [204]. Decreased 16 expression of GPR65 led to impaired bacterial phagocytosis and increased proinflammatory signaling via the NLRP3 inflammasome in macrophages, via G12/13 pathway [205]. The lack of GPR65 expression in macrophages resulted in the increase of mRNA expression of proinflammatory cytokines such as IFN-?, TNF, IL-6, and iNOS in murine colonic tissues, leading to a heightened course of IBD [206]. However, there are also some studies suggesting a pro- inflammatory role for GPR65 in an asthma mouse model through increasing eosinophil viability [207]. Furthermore, GPR65 has been found to increase the severity in the experimental autoimmune encephalomyelitis (EAE) mouse model via increasing Th17 pathogenicity in addition to increasing GM-CSF production in CD4 T cells [208, 209]. Differential roles of GPR65 expression on CD4+ T-cells have been described, where in one study, the adoptive transfer of naive CD4+ T-cells isolated from GPR65-/- did not show increased disease severity in the colitis clinical score in the DSS model, while in a different study, the conditional knockout of GPR65-/- in CD4+ T-cells conferred their polarization towards the proinflammatory Th1/Th17 phenotype [195, 206]. Altogether, GPR65 is predominantly expressed in both the myeloid and lymphoid immune systems and showing differential roles that appear to be context dependent, thus further investigations into its role are warranted. GPR4’s proinflammatory role has been established in several organ systems such as the nervous (CO2 chemosensing and neuronal cell death), cardiovascular (coronary artery disease and endothelium physiology), respiratory (COPD and COVID-19), renal (acid-base balance), skeletal system (cartilage damage and osteophyte formation), skin (endothelium integrity), gastrointestinal tract (endothelium-leukocyte interaction) systems and cancer (angiogenesis and proliferation) [6, 7, 161, 177, 188, 210-215]. (Fig. 1.1.). Upon acidic activation, GPR4 mediates its function through to Gs/cAMP, G12/13/Rho, and Gq/11/PLC pathways [216, 217]. In vitro acidic activation of GPR4 17 leads to endothelium activation, regulating their adhesiveness and leukocyte extravasation through G protein pathways including the G(s)/cAMP/Epac pathway [8]. This activation further augments the endothelium proinflammatory gene expression of IL8, CCL20, TNF, COX-2, and IL1A, adhesion molecules VCAM-1, and E-selectin [162], and ER stress response genes, PERK, ATF6, and IRE1 [218]. Moreover, GPR4 also functions through G?12/13/Rho GTPase pathways leading to vascular leakiness due to induced actin stress fibers formation [6]. Under acidic conditions GPR4 was found to be implicated in increasing the expression of RANK/RANKL/OPG in osteoblastic cells [219]. Additionally, GPR4 overexpression promotes the development of osteoarthritis (OA), and the pharmacological inhibition of GPR4 protects the mice against OA via regulating CXCR7/CXCL12 pathway. GPR4 expression was upregulated in IL-33-activated human mast cells, and pharmacological inhibition of GPR4 countered the proinflammatory action of IL-33 by downregulating IL?17, IFN??, and TNF?? [220]. GPR4 was also associated with increased nociception of dorsal root ganglion [221], and cAMP mediated upregulation of the RANK/RANKL/OPG system and neurotrophins by nucleus pulposus cells, thus promoting Intervertebral disc (IVD) degeneration [222]. Transcriptome-wide association study (TWAS) identified GPR4 as a biomarker in Alzheimer’s patients [223]. Both pharmacological inhibition or genetic knockout of GPR4 protects against neuronal cell death through the modulation of PIP2 degradation-mediated calcium signaling, thereby protecting against neurodegenerative disorders such as Parkinson's disease [224]. Moreover, mice Lacking GPR4 showed enhanced glucose tolerance and insulin sensitivity [225], and lower blood pressure [226]. Most recently, a proinflammatory role for GPR4 has been proposed in COVID-19 [214, 215], where GPR4 gene expression was found to be up-regulated in the lung and colon COVID-19 patient samples by 2.3- fold (p = 3.04E-06) and 3.9-fold (p = 0.0074), respectively [213, 214]. Additionally, Kochbeck et 18 al. described a correlation between GPR4 RNA expression obtained from patient test nasal swab at test time and COVID-19 severity at a later stage [215]. In addition, it was observed that GPR4 expression is upregulated in the colons of UC and CD patients [161, 163]. Recently, GPR4 has been positively correlated to increased fibrosis in CD patients [167]. We and others have shown the proinflammatory role of GPR4 in acute and chronic IBD mouse models [7, 161, 227]. GPR4 confers a proinflammatory role in IBD through modulating chemokines, cytokines, and adhesion molecules, such as, VCAM-1 and E-selectin expression on endothelial cells, regulating leukocyte infiltration into intestinal tissues of IBD mouse models [161]. Pharmacological inhibition of GPR4 mitigates IBD in mouse models and downregulates the cytokines such as TNF-?, and IL-10, as well as adhesion molecules, such as VCAM-1, SELE, MAdCAM-1 [7]. Additionally, pharmacologically antagonizing GPR4 confers anti-nociceptive, anti-fibrotic, anti-angiogenic, as well as anti-inflammatory roles [167, 228, 229]. Moreover, GPR4 is essential to maintain acid-base homeostasis in kidney in which GPR4 KO mice show lower plasma bicarbonate concentration at steady state and increased hypercalciuria associated metabolic acidosis in response to acid challenge (oral administration of NH4Cl), compared to WT mice [152]. GPR4 role in physiological and pathological angiogenesis has been established [167, 185, 230, 231]. Moreover, protumorgenic and proangiogenic roles for GPR4 were demonstrated in orthotopic breast and colorectal cancer mouse models [232]. Furthermore, GPR4 was found to be overexpressed in tumor tissue of CRC patients compared to normal tissue, and its overexpression was correlated with poor CRC prognosis in those patients [183]. Additionally, GPR4 was highly expressed in malignant liver tumors and its expression was positively correlated to angiogenesis 19 in hepatocellular carcinoma human specimens [233]. Its proangiogenic role has also been proved in the squamous cell carcinoma of the head and neck (SCCHN) cell lines [185]. G. Rational for evaluating the role of pH sensing GPR65 and GPR4 in inflammation and CAC The aim of the work done in this dissertation is to evaluate the functional roles of the proton (H+)/pH sensing GPR65 and GPR4 in the development of the inflammation driven CAC. In addition, an evaluation of the functional role of GPR4 in IMC, is presented in this dissertation. GPR65 and GPR4 were previously shown to have abundant expression levels in the intestine and that their activation functionally modulates intestinal inflammation. Although, GPR65 and GPR4 are both acid sensing GPCRs, they are distinctive in their cellular expression and show differential functions at organ level. Hence, their exact roles are warranted for further investigations. We previously investigated the functional role for GPR65 in IBD, in reducing intestinal inflammation, a role that has been shown by others as well. GPR65 expression in intestinal immune cells was found to modulate the inflammatory response and show protection against inflammation in the DSS-IBD mouse model [166, 180, 181, 234]. However, its role in spontaneous inflammation-mediated cancer development has not yet been investigated. We hypothesized that GPR65 will confer an antiinflmmatory role during chronic IBD and protects against CAC development. In this dissertation work we provide an insight on the functional role for GPR65 in the development of CAC development under the setting of the mutagen (Azoxymethane) (AOM) and the intestinal irritant (dextran sodium sulfate) (DSS) to mimic human CAC development. Our group also demonstrated GPR4’s role in proinflammatory endothelium activation and leukocyte-endothelium interaction, which serves as the main mechanistic pathway in many disease models including IBD. Where acidic activation of GPR4 on endothelial cells, upregulates 20 chemokines and the adhesion molecules, SELE, ICAM-1 and VCAM-1, thereby allowing leukocytes extravasation into the inflamed tissue [161]. (Fig.1.2.) Given GPR4’s proinflammatory role in IBD, it can be hypothesized that its prolonged activation in light of chronic inflammation represents a high risk for CAC development. Additionally, GPR4 has been described as a proangiogenic in many systems. None of which, directly shows its role in spontaneous cancer development. Herein in the work presented in this dissertation, we study the functional role of GPR4 in inflammation driven cancer development using the spontaneous cancer model AOM/DSS. Finally, based on the role of GPR4 in mediating immune cell extravasation in the intestine that directly contributes to IBD inflammation. We hypothesized that inhibiting GPR4 expression will reduce other forms of colitis similar to its role in IBD, based on our previous observations. We previously observed that both genetic knockout and pharmacological inhibition of GPR4 reduced IBD inflammation in DSS-mouse models [7, 161]. In this dissertation work, we explore the potential role for GPR4 genetic knockout in protecting against intestinal inflammation induced by the use of immune checkpoint inhibitors, thus we will use an IMC mouse model to test this hypothesis. 21 22 Figure 1.1. Roles of proton sensing GPCRs in different systems Adapted and modified from Cell Health and Cytoskeleton 2015:7. 23 24 Figure 1.2. A graphical illustration of GPR4- mediated leukocytes extravsation Adapted from Biochimica et Biophysica Acta 1863 (2017) 569–584. 25 Chapter II: Materials and Methods A. Summary The functional roles of GPR65 and GPR4 in the development of inflammation driven colorectal cancer was assessed by chemically inducing CAC. GPR65 Knockout (KO), and GPR4 KO mice were compared to Wild Type (WT). We used the well-established AOM/DSS model to induce mutagenesis and intestinal irritation to assess the tumor burden in conjunction with clinical disease activity, and macroscopic inflammatory parameters [235]. We also used histopathological evaluation of dysplastic, adenocarcinoma lesions and immunohistochemical analysis for distinct inflammatory or angiogenic tumor features. Furthermore, the functional role of GPR4 in immune mediated colitis was assessed using an IMC mouse model by administering the immune checkpoint inhibitors (ICIs), anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) and anti-PD-1 (Programmed death-1) antibodies, or control isotype in conjunction with DSS to induce colitis as an irAE [236, 237]. GPR4 KO mice were compared to WT. Clinical disease activity, inflammatory indicators, and histopathological evaluation of cellular inflammatory and fibrotic markers were evaluated to comprehensively assess the role of GPR4 in this model. The methods employed in this dissertation work will provide a new insight on the contributions of the proton sensing GPCR family members, GPR65 and GPR4 to colorectal cancer development, driven by chronic intestinal inflammation. It will also reveal a new role for GPR4 in immune checkpoint inhibitors-mediated colitis using an experimental IMC mouse model. B. Materials and Methods used for the investigation of CAC development and IMC B.1. AOM and DSS-induced colitis associated colorectal cancer mouse model Experiments were performed using equal number of 9 to 12 weeks old of male and female WT, GPR65 KO, and GPR4 KO mice. Mice were backcrossed 9 and 10 generations into the C57BL/6 background. Colitis-associated colorectal cancer was induced in mice using a single i.p. injection (10mg/kg) of azoxymethane (AOM, product# A5486, Sigma-Aldrich, Saint Louis, MO) followed by 4% (w/v) DSS (Lot# Q5229 and S0948, MP Biomedical, Solon, OH) in drinking water. The treatment schedule was modified from Thaker, et al. [235] in which mice were given 4% DSS in water for 3 cycles. Each cycle constituted 5 days of 4% DSS followed by 16 days of water. Following the third cycle, mice were given water for the remaining period to the endpoint on the 13-14th week. Mouse body weight was measured each day for the first 14 days of each cycle. All animal experiments were performed according to the randomized block experimental design. B.2. Immune checkpoint inhbitors-mediated colitis (IMC) mouse model 9 to 12 weeks old of equal number of male and female WT and GPR4 KO mice were used in the experiments. GPR4 deficient mice were generated as previously described and were backcrossed into the C57BL/6 background for 11 generations [231]. Animals were maintained under specific pathogen-free conditions and were free from Helicobacter, Citrobacter rodentium, and norovirus. Colitis was induced using three i.p. injections of 100 mg/ml anti-CTLA4 and 250 mg/ml anti-PD-1 antibodies or the corresponding isotypes on days (-3, 0, and 3). 2% (w/v) colitis grade dextran sulfate sodium (DSS) with molecular weight 36,000-50,000 KD (Lot# Q1408, MP Biomedical, Solon, OH) was added within the drinking water of mice on day 0 till day 10 when mice were euthanized for tissue collection [236, 237]. The 2% DSS solution or water was provided to mice ad libitum, as previously described [166]. Mouse body weight and clinical phenotype 27 scores were measured each day [166]. The mouse experiments were approved by the Institutional Animal Care and Use Committee of East Carolina University in accordance with the Guide for the Care and Use of Laboratory Animals (The National Academies Press). B.3. Clinical phenotype scoring Assessment of colitis severity was determined using the clinical parameters of body weight loss and fecal score [161]. Disease activity index represented by body weight loss percentage, fecal score, spleen weight, colon shortening, and lymph node expansion, was measured to assess inflammation in WT and GPR65 mice treated with AOM/DSS. Feces was collected from mice and assessed for presence of blood and consistency. Fecal scoring system consisted of the following: 0= normal, dry, firm pellet; 1= formed soft pellet with negative hemoccult test, 2= formed soft pellet with positive hemoccult test; 3= formed soft pellet with visual blood; 4= liquid diarrhea with visual blood; 5= no colonic fecal content and bloody mucus upon necropsy. Presence of micro blood content was measured using the Hemoccult Single Slides screening test (Beckman Coulter, Brea, CA). B.4. Tissue collection, evaluation, and processing Upon the study endpoint, mice were euthanized followed by necropsy. Colon length was measured from the ileocecal junction to anus. Colon was then removed from cecum and the colon lumen was cleared of fecal content by washing with phosphate buffer saline (PBS) and then opened along the anti-mesenteric border. The colon tissue was then fixed with 10% buffered formalin and cut evenly into distal, mid, and proximal sections for histologic evaluation. The mesenteric lymph node and tumor polyps were collected for histological analysis and the volume was assessed with a caliper using the formula (length × width2) ?/6. Lymph nodes and colon tissues were then fixed with 10% formalin for histological analysis. Mouse spleens were also collected and weighed. 28 B.5. Histopathological analysis Five µm sections of distal, middle, and proximal colon tissue segments were obtained from WT, GPR65 KO and GPR4 KO mice treated with DSS or AOM/DSS and stained with hematoxylin and eosin (H&E) for histological analysis. Sample identification was concealed during histopathological analysis for unbiased evaluation. Board certified medical pathologists (Swati Satturwar, Heng Hong, Deepak Donthi, and Ying Sun, MD) evaluated dysplasia and adenocarcinoma features in the colons of WT, GPR65 KO and GPR4 KO AOM/DSS mice, and (Ahmed Younes, MD) evaluated colitis histopathological features in WT, and GPR4 KO mice treated with DSS, or ICI/DSS colon segments including inflammation, crypt damage, edema, architectural distortion, and leukocyte infiltration in a blinded manner as previously described [7, 161, 238], a score macrophage clusters, and their percent involvement was added to the previous parameters. Each parameter was scored and multiplied by a factor corresponding to total disease involvement. Additional scoring criteria of colonic fibrosis were evaluated as previously described. Briefly, WT, GPR4 KO, GPR65 KO mice treated with DSS, ICI/DSS or AOM/DSS colon segments were stained with picrosirius red for fibrosis analysis and graded in a blinded manner for pathological fibrosis [239]. The sample identification was concealed during scoring. B.6. Immunohistochemistry Colon tissues and mesenteric lymph nodes were embedded in paraffin and serial five µm sections were evaluated for immunohistochemical analysis as previously described [161, 166]. Briefly, antigen retrieval was performed of colon and mesenteric lymph node sections followed by endogenous peroxidase blocking. Tissue segments were blocked with normal serum and stained with stained with anti-Green Florescence Protein (GFP) (ab6673, Abcam, Cambridge, MA), anti- Caspase-3 (#9664S, Cell Signaling Technology, Danvers, MA), anti-CD31 (#77699S, Cell 29 Signaling Technology, Danvers, MA), anti-Ki67 (#ab15580, Abcam, Waltham, MA), anti- VEGFR2 (#9698S, Cell Signaling Technology, Danvers, MA), anti-CD45 (ab25386, Abcam, Cambridge, MA), anti-F4/80 (#70076, Cell Signaling Technology, Danvers, MA), and anti-?SMA (#ab5694, Abcam, Cambridge, MA) primary antibodies. The IHC detection system VECTASTAIN® Elite ABC-HRP Kit, Peroxidase (Rabbit IgG) (Vector laboratories, California, CA), and VECTASTAIN® Elite ABC-HRP Kit, Peroxidase (Goat IgG) (Vector laboratories, California, CA) were used. Following addition of secondary antibody, DAB (3,3'- diaminobenzidine) ImmPACT® DAB Substrate Kit, Peroxidase (HRP) (#SK-4105, Vector laboratories, California, CA) incubation was performed for HRP detection. Pictures were taken using the Zeiss Axio Imager M1 or M2 microscope. B.7. Leukocyte and myofibroblast quantification Distal colon tissue segments were randomly selected from WT and GPR65 KO mice treated with AOM/DSS (n=21). Slides from WT and GPR65 KO AOM/DSS-treated mouse colons were stained for the pan leukocyte marker CD45 and the myofibroblast marker ?-SMA. Picture were taken (n=8-12) sequentially starting from the anus of the distal segment of the colon at 200× magnification. Positively stained immune cells and myofibroblasts were manually quantified using ImageJ 1.53k software per high power field of view (FOV) and then averaged per mouse. The sample identification was concealed during cell counting and scoring. B.8. Tumor necrotic area quantification Hematoxylin and Eosin (H&E) stain was performed on formalin fixed, paraffin embedded tumor sections to assess necrosis in WT and GPR4 KO AOM/DSS mouse colons. Percent of necrotic area per field of view (FOV) was measured using ImageJ 1.53k software, in a blinded manner. Briefly, 200X magnification images were taken for every tumor to capture the total tumor 30 area using (Axio Imager M2, Zeiss Inc.). Percent of necrosis per FOV was calculated using the following equation: % necrotic area = (sum of necrotic areas / total area of FOV) * 100. B.9. Microvessel density quantification CD31+ immunohistochemistry stain was used as a marker for endothelial cells forming blood vessels in the WT and GPR4 KO AOM/DSS colon tumor sections. Image J 1.53k software was used for manual quantification of individual blood vessels/ FOV. 5-8 images were captured using 200X magnification/ polyp and blood vessel numbers were averaged. Images were analyzed in a blinded manner. Data is represented as the averaged FOV/ polyp for the two groups WT and GPR4 KO AOM/DSS. B.10. Tumor proliferation quantification Ki67+ immunohistochemistry stain was used as a proliferation marker in the WT and GPR4 KO AOM/DSS colon tumor sections. Fiji 1.53t software was used for analysis, as previously described [240] with some modifications. Briefly, 4-8 Images were captured using 400X magnification/ polyp. Images were analyzed in a blinded manner. Percent positive cells were quantified, by dividing number of positive cells by the total number of cells/ FOV multiplied by 100 and averaged to produce one percentage per polyp. B.11. Statistical analysis All statistical analysis was performed using the GraphPad Prism 9 software. The unpaired t- test and Mann-Whitney U test were used to compare differences between two groups (WT and GPR65 KO), (WT and GPR4 KO), (WT-ICI DSS and WT-DSS), (WT-ICI DSS and GPR4 KO- ICI DSS), (WT-DSS and GPR4 KO-DSS), and (GPR4 KO-ICI DSS and GPR4-DSS) mice. Chi- square test was used to compare the differences between (WT and GPR65 KO), and (WT and GPR4 KO) tumor lesions. P < 0.05 is considered statically significant. 31 Chapter III: The Role of GPR65 in Colitis-Associated Colorectal cancer (CAC) Portions of this chapter are modified and reprinted from Biochimica et Biophysica Acta - Molecular Basis of Disease, Volume 1868, Issue 1, 166288 (2022). A. Summary GPR65 (also known as T-cell death-associated gene 8, TDAG8) is a proton-sensing GPCR predominantly expressed in immune cells. GWAS have identified GPR65 gene polymorphisms as an emerging risk factor for the development of IBD. Patients with IBD have an elevated risk of developing colorectal cancer when compared to the general population. We and others have previously shown an anti-inflammatory role of GPR65 in IBD. To determine the functional role of GPR65 in inflammation-driven carcinogenesis, we developed a CAC mouse model. CAC was induced in GPR65 KO and WT mice using the well-established AOM/DSS murine model. Clinical parameters observations show an increase of inflammation in GPR65 KO mice colons compared to WT, as reflected by body weight loss, splenomegaly, mesenteric lymph node (MLN) expansion, leukocyte infiltration, intestinal fibrosis and colon shortening. Moreover, tumor burden as reflected by tumor number and volume, was higher in GPR65 KO mice colons compared to WT mice colons. Altogether, our data demonstrate that GPR65 suppresses intestinal inflammation and colitis-associated tumor development in the murine CAC model, suggesting potentiation of GPR65 with agonists may have a therapeutic anti-inflammatory effect in IBD and reduce the risk of developing colitis-associated colorectal cancer. B. Introduction Extracellular acidification in inflammatory milieu activates pH-sensing GPCRs, such as GPR65. GPR65 has been genetically linked to several inflammatory disease such as multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, and IBD (both CD and UC) [241-245]. IBD is a broad term covering both CD and UC [246]. Murine bone marrow derived macrophages lacking GPR65 as well as Hela cells encoding the genetic variant GPR65 I231L linked to IBD, show impaired lysosomal function [247]. In line with these observations, Mercier et al. showed that decreased GPR65 expression on human macrophages impaired their phagocytic function and increased their proinflammatory response via activating the inflammasome pathway NLRP3 [27]. Lysosomal disruption of specialized macrophages responsible for autophagy, explained the impaired bacterial clearance observed in the mice lacking GPR65 IBD-bacterial model [247]. In addition, the lack of GPR65 expression on CD4+ T-cells did not confer a proinflammatory role in an IBD mouse model. In this study, the adoptive transfer of naive CD4+ T-cells isolated from GPR65-/- did not affect the severity of colitis clinical score in murine DSS induced IBD [206]. IBD is characterized by recurrent, aberrant inflammation within the intestinal tissue. These two disease forms are distinct yet have overlapping clinical and histopathological features. The exact etiology is unknown; however, a complex interaction between immunologic, environmental, and genetic constituents is believed to contribute to the disease onset and progression. Chronic inflammation associated with IBD is a key player in modulating the intestinal microenvironment leading to colorectal cancer development [118-120]. CAC can develop from repeated damage to the intestinal epithelium and exposure to the inflammatory microenvironment. As such, IBD patients have a higher risk of developing colorectal cancer than the general population [124]. 33 As aberrant GPR65 function is associated with IBD in patients [27, 203, 248, 249], we sought to utilize the AOM/DSS-induced CAC mouse model to evaluate the role of GPR65 in CAC development. Throughout the study, GPR65 KO mice showed increased inflammation compared to WT, as reflected by increased body weight loss data. This observation was also evident at endpoint where GPR65 KO mice gained less body weight than WT mice. In addition, GPR65 KO mice showed enlarged spleens and mesenteric lymph nodes compared to WT, reflecting an increase in inflammation caused by the lack of GPR65 expression in immune cells in those mice. Moreover, a significant increase in inflammation was reflected by the increased number of immune cells in GPR65 KO in comparison to WT mice colons. In line with these observations of increased inflammation in GPR65 KO colons versus WT, intestinal fibrosis, increased alpha-smooth muscle cells number and consequently, colon shortening were intensified in GPR65 KO mice colons in comparison to WT. The heightened inflammation and fibrosis in GPR65 KO colons were also reflected on tumor development, as GPR65 KO mice developed more tumors with larger volumes compared to WT mice. Our results demonstrate that GPR65 is protective against colonic inflammation, and IBD associated complications such as fibrosis, and CAC development. 34 C. Results C.1. GPR65 KO mice show aggravated intestinal inflammation in the CAC mouse model Inflammation is a driving force for tumorigenesis in the AOM/DSS colitis-associated colorectal cancer model [250, 251]. We assessed the role of GPR65 in the development of CAC using the AOM/DSS mouse model. WT and GPR65 KO mice were injected with AOM followed by DSS administration for the development of inflammation-associated colorectal cancer in the colon. Lymph node expansion, spleen enlargement, colon shortening, body weight loss and fecal blood score can be used as parameters to assess inflammation in colitis and colitis associated colorectal cancer [252-255]. Body weight loss percent was calculated in reference to initial body weight for each mouse and normalized to untreated mice body weight gain. Generally, GPR65 KO mice treated with AOM/DSS showed increased body weight loss percentage in comparison to WT mice, signifying an increase in disease progression. On the second and third day of the first cycle of DSS administration, a statistically significant body weight percent loss in WT in comparison to GPR65 KO AOM/DSS mice was observed, whereas there was no observable difference between them for the remainder of cycle one. Starting on the 28th day, GPR65 KO AOM/DSS show a steady trend in body weight loss (average range of ~17.6%) compared to WT AOM/DSS (average range of ~14.6%) (Fig.3.1.A). Interestingly, fecal blood and diarrhea score did not show significant differences between GPR65 AOM/DSS and WT AOM/DSS mice (Fig.3.1.B.). Moreover, at the terminal point of the experiment, macroscopic disease indicators were evaluated such as spleen size, mesenteric lymph node expansion, and colon shortening. Spleen size corresponding to the extent of inflammation has been assessed, where GPR65 KO AOM/DSS mice spleens were significantly larger in comparison to WT AOM/DSS mice (Fig.3.1.C). Furthermore, spleen sizes were normalized to corresponding mice weights, and the trend stood with GPR65 KO AOM/DSS 35 spleen 2.5-fold larger than WT AOM/DSS mice (Fig.3.1.D). MLN volume was significantly increased in GPR65 KO AOM/DSS treated mice, mean ~ 53 mm3 compared to WT AOM/DSS with mean ~24 mm3, showing 2.2 -fold expansion, indicating the inflammation of GPR65 KO AOM/DSS was more severe (Fig.3.1.E). Finally, colon length was measured to assess the degree of shortening, which corresponds to heightened inflammation. GPR65 KO AOM/DSS mice had significant colon shortening (mean ~7.6 cm2) in comparison to WT AOM/DSS mice (mean ~8.1 cm2) (Fig.3.2.A-B). Colon shortening, mainly due to tissue fibrosis secondary to intestinal inflammation, was consistent with collagen staining by picrosirius red (PSR). PSR positive areas (red color) demonstrated that the levels of fibrosis were higher in the colon of GPR65 KO AOM/DSS mice in comparison to WT AOM/DSS mice throughout the distal, mid and proximal colon (Fig.3.2.C-D). These observations are in concordance with myofibroblasts expansion observed via ?-SMA+ cells (Fig.3.2.E). ?-SMA+ cells in the distal area were observed to have increased by ~2 folds in GPR65 KO AOM/DSS mice than WT (Fig.3.2.F). To assess leukocyte infiltration throughout the colon, tissue sections were stained for CD45, a pan-leukocyte marker. Significant increase of leukocyte infiltration in the distal, middle, and proximal colon of GPR65 KO-AOM/DSS mice was observed when compared to WT-AOM/DSS mice (Fig.3.3.A-B). Altogether, these results suggest that intestinal inflammation is aggravated in the GPR65 KO- AOM/DSS mice. 36 37 C D Spleen Size Spleen/Body Weight Ratio 0.6 0.020* * 0.015 0.4 0.010 0.2 0.005 0.0 0.000 T O T O W K W K 65 65 PR P R G G E Mesenteric Lymph Node (MLN) 200 ** 150 100 50 0 T O W K 65 PRG 38 Lymph Node Volume (mm3) Weight (gms) Spleen/Body Weight (gm/gm) Figure 3.1. Macroscopic parameters of colitis in WT and GPR65 KO AOM/DSS mice. GPR65 KO AOM/DSS mice have elevated inflammation parameters compared to WT AOM/DSS mice. Macroscopic colitis indicators include (A) body weight loss, (B) fecal blood and diarrhea score, (C) spleen size, (D) spleen/body weight ratio, and (E) mesenteric lymph node (MLN) expansion. Data are presented as the mean ± SEM (N=21 WT and N=21 GPR65 KO mice, with 11 males and 10 females for each) and statistical significance was determined using the Mann- Whitney U test between WT and GPR65 KO groups (*P < 0.05, **P < 0.01). 39 B Colon shorteningA * WT 10 9 8 GPR65 KO 7 6 5 WT AOM/DSS GPR65 KO AOM/DSS C D Colon Fibrosis WT KO 20 *** *** *** 15 10 5 0 T O T O T O W K W K W K Proximal Mid Distal E F WT KO (?-SMA+) myofibroblasts in distal colon 200 ** 150 100 50 0 WT AOM/DSS GPR65 KO AOM/DSS 40 Colon length (cm) (?-SMA+) myofibroblasts/ (FOV) Score of Severity F igure 3.2. Colon shortening, fibrosis, and myofibroblast expansion in AOM/DSS mice. The colons of GPR65 KO AOM/DSS mice show reduced length, increased fibrosis, and heightened myofibroblast expansion compared to WT AOM/DSS mice. (A, B) Colon length, (C) representative images of pathological fibrosis by Picrosirius Red staining (blue arrow heads indicate fibrotic areas), (D) fibrosis score of severity (N=21 WT and N=21 GPR65 KO mice, with 11 males and 10 females for each), (E) immunohistochemistry of ?-SMA+ myofibroblasts in mouse colon tissues, and (F) graphical presentation of ?-SMA+ myofibroblasts. Scale bar is 100µm for microscopic images. Scale bar is 1cm for macroscopic representation. Data are presented as the mean ± SEM and statistical significance was determined using the Mann-Whitney U test between WT AOM/DSS and GPR65 KO-AOM/DSS mice (*P < 0.05, ***P < 0.001). 41 A B C CD45+ Cells in AOM/DSS Colon 400 ** * ** 300 200 100 0 T O T O T O W K W K W K Proximal Mid Distal 42 Cell Count (FOV) Figure 3.3. Leukocyte infiltration in colons of AOM/DSS mice. Leukocyte infiltration in colon tissues is increased in GPR65 KO AOM/DSS mice when compared to WT AOM/DSS mice. CD45+ immunohistochemistry of leukocytes (blue arrow heads) in (A) WT and (B) GPR65 KO AOM/DSS mouse colon sections, and (C) cell count of leukocyte infiltration in distal, middle, and proximal colon segments (WT N = 21 and GPR65 KO N = 21, with 11 males and 10 females for each). Scale bar is 100 ?m. Data are presented as the mean ± SEM and statistical significance was determined using the unpaired t-test between WT and GPR65 KO AOM/DSS mice (*P < 0.05, **P < 0.01). 43 C.2. Genetic knockout of GPR65 increases intestinal tumorigenesis in the AOM/DSS-CAC model Chronic intestinal inflammation is associated with a higher risk of developing colorectal cancer in IBD patients [256-261]. Using the previously discussed AOM/DSS CAC mouse model, we assessed tumor burden in GPR65 KO AOM/DSS compared to WT AOM/DSS. Tumor burden was represented by both polyp/tumor incidence and tumor volume in the mouse colon. A significant increase of ~2-fold in polyp/tumor numbers were observed in GPR65 KO AOM/DSS when compared to WT-AOM/DSS mouse colon tissues. The majority of tumor polyps were observed in the distal colon with decreasing tumor numbers in middle and proximal colon segments (Fig.3.4.A, C-D). Similarly, the total volume of tumor polyps in GPR65 KO AOM/DSS mice were elevated by ~2-fold when compared to WT AOM/DSS mice (average total volume of ~ 66 mm3/colon vs. 31 mm3/colon, respectively) (Fig.3.4.B). Histological analyses of the colon sections harboring tumor polyps revealed colon dysplasia and adenocarcinoma were induced in both WT AOM/DSS and GPR65 KO AOM/DSS mice (Fig.3.4.E-F). While the total number of lesions observed was higher in GPR65 KO AOM/DSS mouse colons, the relative distribution of colon dysplasia and adenocarcinoma was similar between WT and GPR65 KO AOM/DSS mice (Fig.3.4.I). 44 A B Tumor Volume Tumor Number 20 300* * 15 200 10 100 5 0 0 T O T O W K5 W K 6 65 PR PR G G C D E F G H 45 Tumor Number/Colon Tumor Volume/Colon (mm3) I AIS/ Dys in tumors 150 AIS ns Dys 100 50 0 WT GPR65 KO 46 % AIS/Dys in tumors 81 76 F igure 3.4. Effects of GPR65 on tumorigenesis in the CAC mouse model. Tumor burden is increased in the colons of GPR65 KO AOM/DSS mice (N = 21, with 11 males and 10 females) compared to WT-AOM/DSS mice (N = 21, with 11 males and 10 females). Red arrows indicate polyps and tumors. (A) Polyp/tumor number, (B) polyp/tumor volume, (C–D) representative pictures of distal colons bearing polyps/tumors in (C) WT and (D) GPR65 KO, (E– H) histopathological representation of dysplasia and adenocarcinoma (blue arrow heads) in (E, G) WT and (F, H) GPR65 KO AOM/DSS mouse colons, and (I) Distribution of dysplasia (Dys) and adenocarcinoma in situ (AIS) in GPR65 KO and WT AOM/DSS mouse colons. Data are presented as the mean ± SEM and statistical significance was determined using the unpaired t-test and chi square between WT and GPR65 KO AOM/DSS mice. (*P < 0.05). 47 D. Discussion In this study we investigated the functional role of GPR65 in the AOM/DSS-induced colitis associated colorectal cancer mouse model. Our results indicate that GPR65 provides a protective role against CAC development. Our observations of GPR65 function are concordant with previous studies that demonstrate an anti-inflammatory role of GPR65 in a variety of inflammatory diseases [197, 249, 262, 263] and provide new insights into the function of GPR65 in CAC development. GPR65 is expressed predominately in leukocytes and can regulate the inflammatory response of immune cells [264-267]. Downstream effectors of the GPR65-coupled G?s/cAMP demonstrate anti-inflammatory effects in a diverse set of processes [268]. G?s/cAMP/PKA/CREB pathway has been shown to reduce granulocyte, macrophage, and monocyte inflammatory programs [269]. Additionally, cAMP can reduce dendritic cell function in lymph nodes, T cell activation, and can increase T regulatory cell activity. These data are consistent with reports that GPR65 activation can inhibit inflammatory profiles in macrophages, microglia, neutrophils, and T cells [264-267, 270]. Additionally, the anti-inflammatory role of GPR65 was demonstrated in immune-mediated murine disease models such as arthritis, lipopolysaccharide (LPS)-induced acute lung injury, myocardial infraction, ischemic stroke, and intestinal inflammation [197, 249, 262, 263, 271]. However, there are also some studies suggesting GPR65 expression in eosinophils promotes inflammation through increasing eosinophil viability in an asthma mouse model [207]. Furthermore, recent studies found GPR65 is a regulator for Th17 pathogenicity and increases the severity in the EAE mouse model as well as increases GM-CSF production in CD4 T cells [208, 209]. Pertaining to Th17 cell pathogenicity, reports have provided evidence for both protective and pathogenic roles in the context of intestinal inflammation [195, 272]. Lin et al. reported a proinflammatory role of GPR65 in IBD, where ectopic expression of GPR65 on CD4+T-cells 48 obtained from peripheral blood of IBD patients and healthy controls promoted their polarization towards Th1/Th17 phenotypes [195]. Moreover, Lin et al shows that adoptive transfer of the conditional GPR65 CD4 knockout protect against trinitrobenzene sulfonic acid (TNBS)-induced colitis in RAG1-/- mice [195]. In addition to these in vitro and in vivo animal studies, recent GWAS have identified single nucleotide polymorphisms (SNPs) of GPR65 associated with several human inflammatory diseases such as multiple sclerosis, asthma, heparin-induced thrombocytopenia, spondyloarthritis, and IBD [208, 248, 273-277]. As previously mentioned, IBD-associated GPR65 genetic variant (I231L) was investigated within a bacteria-induced colitis mouse model and found that this GPR65 gene variant confers reduced GPR65 activity as well as impaired lysosomal function [249]. Our study focuses on the functional role of GPR65 in the regulation of inflammation-associated carcinogenesis in a chemically induced chronic colitis mouse model which further provides evidence that GPR65 functions to inhibit inflammation in colitis. We have previously demonstrated that GPR65 is expressed in infiltrated leukocytes within the colon of inflamed intestinal tissues using GFP as a surrogate marker in GPR65 KO mice [166]. There is a discernible increase in GFP positive leukocytes in the DSS treated mouse colon tissues compared to the untreated tissues indicating GPR65 expression is increased in inflamed tissues compared to non-inflamed intestinal tissues [166]. Consistently, GPR65 gene expression is increased in human colitis and Crohn’s intestinal lesions compared to non-inflamed intestinal tissues [166]. It is likely the increased expression of GPR65 in IBD intestinal samples is partly due to the increase of infiltrated leukocytes, which have high endogenous GPR65 expression. We previously reported that pathological fibrosis was increased in GPR65 KO DSS mice compared to WT DSS mice in the chronic colitis mouse model [166]. Fibrosis is a serious consequence of recurrent intestinal inflammation and can lead to complications such as intestinal 49 strictures and obstruction [278-280]. Collagen can be produced by several cellular constituents within the intestinal tissues. Some such cells include fibroblasts, sub-epithelial myofibroblasts, smooth muscle cells, and pericytes. Additionally, fibroblasts, smooth muscle cells, fibrocytes, endothelial cells, pericytes can undergo epithelial/endothelial- mesenchymal transition into myofibroblasts for wound healing functions [278-280]. Myofibroblasts are described as a major contributor of pathological extracellular matrix deposition within the inflamed intestine [278-280]. As such, GPR65 KO mice showed an increase in the number of SMA+ myofibroblasts in the mucosa than WT mice of AOM/DSS CAC model. which is supported by the observed increased fibrotic deposition in the chronic DSS-treated GPR65 KO colons [166]. It remains to be determined how GPR65 regulates fibrosis in the chronic colitis model and CAC. Interestingly, however, a recent study demonstrates that GPR65 regulates macrophage CCL20 expression, ??T cell infiltration, and fibrosis in a myocardial infarction mouse model [197]. IBD patients have a higher risk of developing colorectal cancer than the general population. The cumulative incidence of colorectal cancer is 2.5–8.0% and 7.5–18.0% in IBD patients with 20 and 30 years of disease duration, respectively [281]. It has been well demonstrated that the inflamed intestinal microenvironment is a driving force for colon epithelial transformation [256- 261, 282]. In the inflamed milieu, inflammatory cells generate reactive oxygen species (ROS), cytokines, chemokines, and pro-angiogenic factors that induce gene mutations, promote cell proliferation and survival, and stimulate angiogenesis. Chronic intestinal inflammation drives tumor initiation and progression of CAC, a process referred to as the “inflammation-dysplasia- carcinoma” pathway. To further build upon our previous results showing GPR65 dampens intestinal inflammation, we utilized the AOM/DSS mouse model to assess the role of GPR65 in CAC development [283, 284]. We observed the DSS insult induced elevated disease activity scores 50 in the GPR65 KO mice when compared to WT mice in the AOM/DSS-induced CAC mouse model. Several macroscopic and microscopic disease indicators were aggravated in the GPR65 KO mice when compared to the WT in the AOM/DSS-induced CAC model. Consistent with heightened intestinal inflammation scores, colonic polyp and tumor formation was elevated in the AOM/DSS- treated GPR65 KO mice than when compared to WT. Chronic, unresolved intestinal inflammation driven by leukocyte infiltration in the colon tissues can drive dysplasia from preneoplastic lesions [285]. We observed increased leukocyte infiltration in peri-tumoral areas of GPR65 KO AOM/DSS mice when compared to WT. Our results demonstrate that the loss of GPR65 expression in AOM/DSS mice elevates leukocyte infiltration in the colon and suggests activation of GPR65 can suppress intestinal inflammation and colitis associated colorectal cancer development. The development of CAC in IBD patients depends on the duration and severity of intestinal inflammation [286]. This makes surveillance colonoscopy as a preventative measure, crucial for IBD patients to detect dysplastic lesions. Our study provides support for an anti-inflammatory role of GPR65 in colitis and suggests GPR65 could be a potential target for therapeutic intervention for CAC prevention. Currently, IBD treatment options are limited and predominately consist of steroids, anti-TNF? monoclonal antibodies, and anti-integrin monoclonal antibodies [287]. The efficacy of these anti-inflammatory treatments against CAC development in IBD patients are inconclusive [288]. However, earlier epidemiological studies suggest that non-steroidal anti- inflammatory treatments could have a protective role for CAC development [289, 290]. Similarly, TNF? inhibitors were found to reduce tumor burden of CAC and associated inflammation in experimental mouse models [291]. This warrants the development of more effective approaches for IBD treatments. The GPR65 agonist BTB09089 has been developed and recently investigated 51 for anti-inflammatory properties. BTB09089 was shown to activate GPR65 in vitro but provided weak activity in vivo according to one study [267]. An additional study has shown in vivo efficacy of BTB09089 using an ischemic stroke murine disease model [271]. Further studies must be done to develop highly efficacious GPR65 agonists for potential use in IBD treatment and CAC prevention. 52 Chapter IV: The Role of GPR4 in colitis-associated colorectal cancer (CAC) A. Summary GPR4 is a proton-sensing G protein-coupled receptor expressed in endothelial cells and other cell types. GPR4 functions as a gate keeper for transendothelial leukocyte extravasation into local inflamed tissue. GPR4 expression is upregulated in the intestinal tissues of IBD and colorectal cancer patients and is associated with a worse prognosis. Herein, we studied the role of GPR4 in CAC development by inducing CAC in GPR4 KO and WT mice using AOM/DSS. Disease activity was reduced in GPR4 KO AOM/DSS compared to WT AOM/DSS mice, signifying dampening of inflammation in GPR4 KO mice colons compared to WT, as reflected by body weight loss, fecal blood and diarrhea score, and MLN expansion data. In addition, reduced tumor burden was observed in the GPR4 KO AOM/DSS mice compared to WT AOM/DSS. Angiogenesis and tumor proliferation were significantly reduced within GPR4 KO AOM/DSS compared to WT AOM/DSS tumors. Consequently, necrosis was increased in GPR4 KO tumors compared to WT tumors. Our data demonstrate that the lack of GPR4 KO alleviates intestinal inflammation and reduces angiogenesis, and tumor development in the CAC mouse model. B. Introduction GPR4 is a proton sensing GPCR, activated by protonation of several histidine residues on its extracellular and transmembrane domains. GPR4 is mainly expressed on endothelial cells amongst other cell types. We and others observed that GPR4 expression is upregulated in the colons of UC and CD patients [161]. A proinflammatory role for GPR4 was also established in IBD mouse models [7, 227]. GPR4 confers this role in IBD through endothelium activation by modulating chemokines, cytokines, and adhesion molecules, on endothelial cells, thus regulating leukocyte trafficking and infiltration into intestinal tissues of IBD mouse models [7, 161]. Our group has demonstrated that GPR4 activation by acidic microenvironment increases the expression of endothelial proinflammatory molecules, such as IL-8, IL-1A, COX-2, VCAM-1 and E-selectin [162]. Moreover, intestinal fibrosis is a serious complication in IBD [279, 292, 293], and GPR4 expression is positively correlated to fibrogenic genes expressed in highly fibrotic lesions obtained from terminal ileums of CD patients [167]. In addition to its proinflammatory, profibrotic and endothelial cell activation roles, GPR4 has been implicated in both physiological and pathological angiogenesis [167, 185, 230, 231]. Wyder et al, showed a role for GPR4 in promoting angiogenesis and tumor growth [232]. Its expression has been associated with increased angiogenesis in hepatocellular, head and neck, breast and colorectal cancers [232, 233, 294]. Increased GPR4 expression was observed in colorectal cancer patients tumors compared to adjacent normal tissue and was associated with decreased overall survival [183]. Therefore, GPR4 may contribute to tumorigenesis in the colon through reinforcing the inflammatory and angiogenesis processes in IBD. Herein, using WT and GPR4 KO mouse CAC models, we investigated the role of GPR4 in inflammation driven colorectal cancer. 54 C. Results C.1. Genetic knockout of GPR4 reduces intestinal inflammation in the AOM/DSS- CAC mouse model To study the role of GPR4 on the development of CAC, we used the well-established AOM/DSS murine model [235, 253]. Disease severity indicators were measured during the experiment by monitoring body weight loss, and fecal blood and diarrhea score. WT AOM/DSS mice showed more significant body weight loss when compared to GPR4 KO AOM/DSS mice starting at the end of the first cycle, and throughout the second and third cycles. Both WT and GPR4 KO AOM/DSS mice reached an average body weight loss of ~17% by day 9 during the first cycle, followed by a partial recovery in that body weight loss. This body weight loss rescue was more significant in the GPR4 KO mice than WT (Fig.4.1.A). The severity of fecal blood and diarrhea score was also significantly higher in WT AOM/DSS in comparison to GPR4 KO AOM/ DSS mice starting mid cycle in the first, second and third cycles (Fig.4.1.B). Upon terminal point of the experiment, macroscopic disease indicators were evaluated such as final body weight, mesenteric lymph node expansion and colon length shortening. GPR4 KO AOM/DSS mice showed a significant increase (~2.5 fold) in body weight recovery compared to WT (Fig.4.1.C). Mesenteric lymph node volume of WT mice were ~2 fold larger than that of GPR4 KO mice, indicating a more severe inflammation in WT mice (Fig.4.1.D). No significant differences were observed in colon length between WT and GPR4 KO AOM/DSS-treated mice (Fig.4.1.E). 55 Day0 10mg/kg AOM i.p. 4% 4% 4% H O H O H O DSS 2 DSS 2 DSS 2 100 days (14 weeks) A Body weight change 10 GPR4 KO AOM/DSS WT AOM/DSS 0 * -10 * ** * * * ** * -20 Cycle 1 Cycle 2 Cycle 3 3 6 9 12 15 24 27 30 33 36 45 48 51 54 57 Days of measurement B Fecal blood and diarrhea 4 GPR4 KO AOM/DSS WT AOM/DSS 3 2 1 Cycle 1 Cycle 2 Cycle 3 0 0 3 6 9 12 15 24 27 30 33 36 45 48 51 54 57 Days of measurement 56 Body weight % Fecal score **** * ***** ***** * ** ** ** ** ** ** **** * **** ** ****** ** **** ** ** **** * * ** * ** * *** * *** ** * ******** *** **** ** **** **** ** **** ** C Final body weight change D Mesenteric lymph node size 60 ?? 60 *** 40 GPR4 KO AOM/DSS WT AOM/DSS 20 40 0 20 -20 -40 0 WT GPR4 KO WT GPR4 KO E Colon length 15 P=0.85 10 5 0 WT GPR4 KO 57 Colon length (cm) Body weight % Lymph node volume (mm3) Figure 4.1. Clinical indicators of intestinal inflammation in the WT and GPR4 KO CAC mice. WT AOM/DSS (n=16) and GPR4 KO AOM/DSS (n=16), with 8 males and 8 females for each, were used for this analysis. WTAOM/DSS show increased body weight loss and fecal blood and diarrhea score throughout treatment cycles when compared to GPR4 KO-AOM/DSS mice. (A) Percentage of body weight change, and (B) fecal blood and diarrhea score. At the endpoint, disease parameters such as percentage of final body weight change, colon length and lymph node expansion were measured. WT AOM/DSS show less body weight gain and increased lymph node expansion when compared to GPR4 KO AOM/DSS mice. (C) final body weight change percent, (D) colon shortening, and (E) lymph node size Data are presented as the mean ± SEM. Statistical significance was determined using the unpaired t-test between WT AOM/DSS and GPR4 KO AOM/DSS mice. (*P<0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). 58 C.2. GPR4 deficiency reduces tumor burden in the CAC mouse model A significant increase in colon tumor number was observed in WT mice when compared to GPR4 KO mice suggesting GPR4 promotes CAC development. WT mice has an average tumor number of 7.4 whereas GPR4 KO mice had an average of 5.1, demonstrating a 45.7% increase in tumor burden in the WT over GPR4 KO AOM/DSS mice (Fig.4.2.A-B). Moreover, the volume of the detected observed polyps was 115 mm3 in WT mice and 55 mm3 in GPR4 KO AOM/DSS mice, respectively, showing a ~2 fold increase in the WT mice (Fig.4.2.C). Histological analyses of the colon tissue sections revealed colon dysplasia and adenocarcinoma in situ (AIS) were induced in both WT and GPR4 KO AOM/DSS mice. Interestingly, the distribution of dysplasia and adenocarcinoma in WT and GPR4 KO AOM/DSS mice showed a 9% decrease in adenocarcinomas in GPR4 KO mice compared to WT (Fig.4.2.E). These data suggest absence of GPR4 delays progression from low grade dysplasia to adenocarcinoma in the inflamed colon tissues. Moreover, minimal residual inflammation was observed in the colons of both experimental groups, indicating the resolution of active inflammation at this experimental time point. 59 A WT GPR4 KO B Tumor volume AOM/DSS C Tumor number AOM/DSS * 20 500 * 400 300 200 15 100 10 50 5 0 0 WT GPR4 KO WT GPR4 KO D E AIS/ Dys in tumors WT GPR4 KO 150 AIS ? Dys 100 50 0 WT GPR4 KO 60 mm3 # visible polyps % AIS/Dys in tumors 94.0 85.0 Figure 4.2. Effects of GPR4 on tumorigenesis in the CAC mouse model. Tumor burden was evaluated using clinical observation of tumor number and volume at endpoint for WT AOM/DSS (n=16) mouse colons compared to GPR4 KO AOM/DSS mice, with 8 males and 8 females for each used. Tumor burden was found to be increased in WT AOM/DSS mouse colons compared to GPR4 KO AOM/DSS (n=16) mice. (A) Representative pictures of distal colons bearing tumors (blue arrow heads), (B) tumor volume, (C) tumor number, (D) histopathological representation of adenocarcinoma (yellow arrowheads) in AOM/DSS mouse colons, and (E) distribution of adenocarcinoma in situ and low-grade dysplasia in the WT AOM/DSS and GPR4 KO AOM/DSS mouse tumors. Data are presented as the mean ± SEM and statistical significance was determined using the unpaired t-test and chi-squared test between WT AOM/DSS and GPR4 KO AOM/DSS mice. (*P<0.05). 100 µM scale bar used for histology representation. 1 cm scale bar used for macroscopic representation. 61 C.3. GPR4 is highly expressed in the tumor blood vessels of AOM/DSS mice We next characterized the expression profile of GPR4 in the AOM/DSS mouse model. Due to the lack of a reliable antibody for GPR4, that can be validated using GPR4 KO mouse tissues. We performed immunohistochemistry for green fluorescence protein (GFP) which functions as a surrogate marker for GPR4 expression. GPR4 KO mice were generated by replacing the GPR4 coding region with an internal ribosome entry site (IRES)-GFP cassette under the control of the endogenous GPR4 gene promoter as previously described [231]. We were able to detect GFP signal in GPR4 KO AOM/DSS, but not WT AOM/DSS colon tissues. In line with our previous observations [161, 179], we observed higher levels of expression of GFP in vascular endothelial cells (Fig.4.3.A) and lower levels in macrophages. Furthermore, using a double fluorescent stain with CD31 and GFP, GFP expression was predominately detected in the endothelial cells (ECs) of blood vessels in the tissues of GPR4 KO AOM/DSS colons (Fig.4.3.B). 62 A W T GPR4 KO B WT CD31 GFP CD31 GFP DAPI GPR4 KO CD31 GFP CD31 GFP DAPI 63 Figure 4.3. GFP signal and double labeling with CD31 in the colon tumor tissues of the CAC mouse model. GFP knock-in signal under the control of GPR4 promoter serves as a surrogate marker for endogenous GPR4 expression in GPR4 KO mice. GFP signal could be detected in GPR4 KO mice (A) GFP signal in GPR4 KO AOM/DSS versus negative signal in WT AOM/DSS tumors, and (B) double labeling of GFP and CD31. Blood vessels show a double positive signal of GFP and CD31 in GPR4 KO AOM/DSS versus only CD31 positive signal in the WT AOM/DSS tumors. Scale bar is 100µm. 64 C.4. GPR4 knockout decreases angiogenic blood vessel formation in the tumors of AOM/DSS mice During tumorigenesis, angiogenesis is a fundamental process in the tumor development and progression [295]. We analyzed the blood vessel density in the tumors of WT AOM/DSS and GPR4 KO AOM/DSS using the endothelial marker CD31. The deficiency of GPR4 in the GPR4 KO AOM/DSS mice caused a significant reduction in blood vessel density in the tumors of these mice by ~2.6 fold when compared to WT AOM/DSS (Fig.4.4.A-B). Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is a cell membrane tyrosine kinase receptor that regulates and potentiates angiogenesis in both physiological and pathological settings [296]. VEGFR2 protein levels were decreased in the lung tissues of GPR4 deficient mice [232]. We sought to compare the protein expression levels of VEGFR2 in the blood vessels of tumor tissue sections obtained from WT AOM/DSS and GPR4 KO AOM/DSS mice colons using IHC (Fig.4.4.C). Our semiquantitative analysis show a 30% decrease in VEGFR2 signal intensity of blood vessels in GPR4 KO AOM/DSS tumors compared to WT AOM/DSS (Fig.4.4.D). 65 A B WT GPR4 KO Microvessel density in tumors 500 ???? 400 300 200 100 0 WT GPR4 KO C WT GPR4 KO D VEGFR2+ endothelial cells in tumors 250 ??? 200 150 100 50 0 WT GPR4 KO 66 # vessels/ FOV Mean color density/cell Figure 4.4. Micro-vessel density and VEGFR2 protein expression assessment in the blood vessels of CAC mice. Using CD31 Immunohistochemistry (IHC) stain, blood vessels numbers in WT AOM/DSS (n=20 tumors/ 16 mice) and GPR4 KO AOM/DSS (n=19 tumors/ 16 mice) of CAC mice tumors. WT AOM/DSS showed increased number of blood vessels compared to GPR4 KO AOM/DSS tumors. (A) Representative pictures of WT and GPR4 KO AOM/DSS tumors CD31+ blood vessels (red arrows), and (B) quantification of number of vessels per averaged field of view/ tumor. ImageJ was used for quantification. VEGFR2+ endothelial cells protein expression levels using IHC were assessed in WT AOM/DSS (n=69 endothelial cells ROI) and GPR4 KO AOM/DSS (n=90 endothelial cells ROI) of CAC mice tumors. WT AOM/DSS blood vessels showed increased VEGFR2+ protein expression compared to GPR4 KO AOM/DSS. (C) Representative images of VEGFR2+ IHC stained tumor tissues for WT AOM/DSS and GPR4 KO AOM/DSS mice, (D) quantification of color density/ cell for VEGFR2+ endothelial cells per field of view for WT AOM/DSS and GPR4 AOM/DSS mice tumors. Data for VEGFR2 was quantified using Fiji using region of interest (ROI) method. Data are presented as the mean ± SEM and statistical significance was determined using the unpaired student t-test (****P<0.0001). Scale bar is 100µm. 67 C.5. GPR4 KO increases necrosis and cell death in the tumors of AOM/DSS mice Tumor necrosis is considered as a common feature of solid tumors which occurs as a consequence of nutrient and oxygen deprivation [297]. Areas in the tumors where tissue was identified to be morphologically necrotic with H&E stain or there were apparent gaps formed from transformed crypts loss were identified as necrotic and were assessed (Fig.4.5.A). The percentage area of tumor necrosis between WT and GPR4 KO AOM/DSS tumors and observed a 2.4 fold increase in tumor necrosis area in GPR4 KO AOM/DSS compared to GPR4 WT AOM/DSS tumors (Fig.4.5.B). Next, we used Ki67 as a proliferation marker to assess tumor proliferation in the WT and GPR4 KO AOM/DSS mice. We quantified Ki67 positive tumor cells using immunohistochemistry between WT and GPR4 KO AOM/DSS mice. WT AOM/DSS tumors show a more proliferative phenotype when compared to GPR4 KO AOM/DSS (Fig.4.5.C). Quantification of Ki67 positive cells revealed a 2 fold decrease in proliferating tumor cells when compared GPR4 KO AOM/DSS mice (Fig.4.5.D). Using immunohistochemistry, we assessed the protein expression of cleaved caspase 3 in WT and GPR4 KO AOM/DSS tumors. The expression of cleaved caspase 3 was predominantly detected in the necrotic tumor area in addition to positive epithelial cells scattered within the tumor (Fig.4.5.E). 68 A B WT GPR4 KO Tumor necrosis in tumors 40 ??? 30 20 10 0 WT GPR4 KO C WT GPR4 KO D E WT GPR4 KO Proliferative cells in tumors ???? 100 80 60 40 20 0 WT GPR4 KO 69 % Ki67 + cellls % necrosis F igure 4.5. Tumor necrosis and cell death in the tumor tissues of the CAC mouse model. H&E stain was used to assess the morphology of necrotic areas in WT AOM/DSS (n=20 polyps) and GPR4 KO AOM/DSS (n=19 polyps) of CAC mice tumors. WT AOM/DSS showed decreased percent of tumor necrosis when compared with GPR4 KO AOM/DSS. (A) Representative images of H&E-stained tumor tissues showing areas of necrosis (blue arrow heads) for WT AOM/DSS and GPR4 KO AOM/DSS mice, and (B) quantification of percent of tumor necrosis per averaged field of view/ polyp for WT AOM/DSS and GPR4 AOM/DSS mice tumors. Cell death was assessed using cleaved caspase-3 IHC in the tumor tissues of WT AOM/DSS and GPR4 KO AOM/DSS, (C) Representative images of cleaved caspase-3+ cells (red arrows) in WT AOM/DSS and GPR4 AOM/DSS mice tumor tissue. Tumor proliferation was assessed using Ki67 IHC in the tumor tissues of WT AOM/DSS and GPR4 KO AOM/DSS, (D) Representative images of Ki67+ cells (blue arrows) in IHC stained tumor tissues for WT AOM/DSS and GPR4 KO AOM/DSS mice, and (E) quantification of percent of tumor proliferation per averaged field of view/polyp for WT AOM/DSS and GPR4 AOM/DSS mice tumors. ImageJ was used for quantification. Proliferation assessment in the tumor tissues of the (CAC) colitis associated colorectal cancer (CAC) mouse model. Ki67+ IHC was used to assess proliferation in WT AOM/DSS (n=20 polyps) and GPR4 KO AOM/DSS (n=19 polyps) of CAC mice tumors. WT AOM/DSS showed increased percent of proliferative cells when compared with GPR4 KO AOM/DSS. Data was quantified using Image J & Fiji as described in materials and methods. Data are presented as the mean ± SEM and statistical significance was determined using the unpaired student t-test (****P<0.0001). Scale bar is 100µm. 70 D. Discussion In this study we demonstrate the proinflammatory, and pro-tumorigenic roles of GPR4 in colitis associated colorectal cancer (CAC), using the AOM/DSS CAC murine model. Our observations are in line with previous reports establishing GPR4’s proinflammatory role in several systems such as the brain, heart, kidney, Lung, bone, skin, and the gastrointestinal tract [6, 7, 161, 177, 210-212]. In addition, GPR4 has a protumorigenic role in hepatocellular, head and neck, breast and colorectal cancers [232, 233, 294]. GPR4 is expressed mainly in endothelial cells, and other cell types such as some macrophages, chondrocytes, neurons, epithelial kidney cells, smooth muscle cells, and fibroblasts [6, 152, 161, 167, 168, 227, 298, 299]. GPR4 is activated through the protonation of several histidine residues, which leads to uncoupling Gs and activating cAMP downstream pathway [217]. GPR4 regulate transendothelial leukocyte extravasation by increasing the adhesiveness of endothelial cells through the G(s)/cAMP/Epac pathway [8]. Moreover, acidotic activation of GPR4 cause vascular leakiness through G?12/13/Rho GTPase induced actin stress fibers formation [6]. In addition to increased endothelial adhesiveness, and leakiness, GPR4 activation confers a proinflammatory role through endothelial activation of inflammatory genes such as IL8, CCL20, TNF, COX-2, and IL1A [162]. Moreover, its activation by acidosis induces ER stress response in endothelial cells by activating PERK, ATF6, and IRE1 [218]. Recently, GPR4 has been positively correlated to fibrogenesis genes, ACTA2, COL1A1 and COL3A1 in fibrotic terminal ileum lesions obtained from CD patients [167]. Finally, a significant body of evidence have implicated GPR4 in angiogenesis [167, 185, 230, 231]. All of the previously mentioned proinflammatory and profibrogenic roles for GPR4 are directly implicated in disease severity of UC and CD [279, 292, 293, 300], representing a high-risk factor for developing CAC [301-304]. GPR4 mRNA 71 overexpression is significantly increased in inflamed intestinal tissue of both IBD patients and colitis mice when compared to normal tissue [161, 163]. Similarly, GPR4 expression is upregulated in colorectal cancer patients tumor samples compared to adjacent normal tissue, and correlated to poor disease outcomes [183]. Thus abrogating GPR4 expression or activation reduces IBD disease activity and hence reduces the risk for developing CAC, consistently, we and others have previously observed a reduced disease activity in the acute and chronic DSS murine colitis model, where inflammation was significantly alleviated in the genetic Knock-Out GPR4 DSS mice [161, 163]. Similarly, pharmacological inhibition of GPR4 confers a reduction in mucosal inflammation via downregulating the expression of disease mediators, proangiogenic, profibrogenic and adhesion molecules such as TNF-?, VEGF, procollagen, MAdCAM-1, VCAM- 1, and SELE [7, 167]. In line with these observations, we observe reduced disease activity, represented in body weight loss, fecal blood and diarrhea scores, in addition to decreased mesenteric lymph node expansion in the GPR4 KO AOM/DSS mice compared to WT. This reduced inflammatory response in the GPR4 KO mice led to decreased tumor development in those mice, as reflected by the tumor burden observed in the GPR4 KO AOM/DSS colons when compared to WT. Hence, revoking the previously mentioned proinflammatory as well as proangiogenic roles for GPR4 as anticipated, indeed forestalled malignant transformation in the GPR4 AOM/DSS mice. This is especially reflected by the reduced number of adenocarcinoma in situ lesions versus dysplastic lesions in the GPR4 KO AOM/DSS colons versus WT. In line, Wyder et al showed halted orthotopic breast and colon tumor growth in GPR4 KO mice [184]. In addition Yu et al, observed a significant reduction in the proliferation of GPR4 knock down colorectal cancer cell lines via regulating the YAP1/ hippo pathway [183]. Consistently, we observe decreased tumor proliferation in the GPR4 KO mice tumors associated with increased cell death 72 whereas in WT mice increased tumor proliferation and reduced cell death was evident. This further demonstrates the protumorigenic role of GPR4 in CAC. Using GFP as a surrogate marker for GPR4, Sanderlin et al, observed the endogenous expression of GPR4 on endothelial cells of arteries, veins, and microvessels of the cecum and colon in the GPR4 KO untreated mice [161]. Herein, using GFP as a surrogate marker double stained with the endothelial marker CD31, we observed GPR4 is mainly expressed on endothelial cells in the AOM/ DSS mouse colon tumors, signifying that expression of GPR4 on endothelial cells is the major contributor to this disease phenotype of CAC in the AOM/DSS mouse model. In the Wyder et al study, GPR4 proangiogenic role was directly linked to vascular VEGFR2 expression, so when VEGFR2 receptor activity was inhibited using tyrosine kinase inhibitors, tumor growth in WT mice was reduced to that same level of GPR4 KO mice [184]. In the growth factor implant angiogenesis model, GPR4 KO mice pathological angiogenic formation was resistant to vascular endothelial growth factor treatment compared to WT mice [184]. Interestingly, acidic activation of squamous cell carcinoma of the head and neck (SCCHN)-GPR4 overexpression system increased the expression and secretion of the proangiogenic, IL6, IL8 and VEGFA [185]. Tube formation assay showed longer blood vessels in human microvessel endothelial cells (HMEC-1) treated with conditioned media obtained from the SCCHN-GPR4 overexpression system, this effect was inhibited upon IL6, IL8 and VEGFA inhibition, further indicating the proangiogenic role of GPR4 [185]. Dong et al, previously showed increased expression of IL6, IL8, and VEGFA in human umbilical vein endothelial cells (HUVEC)-GPR4 overexpression system upon acidic activation [162]. This evidence indicates a possible activation of paracrine and autocrine proangiogenic pathways in endothelial cells in which GPR4 maybe implicated [305, 306]. Herein, we observed decreased blood vessel density in the tumors of GPR4 73 KO AOM/DSS mice compared to WT, reflecting reduced angiogenesis in these mice. Consequently, larger necrotic areas in GPR4 KO AOM/DSS tumors were observed. The reduction in angiogenesis could be attributed to the downregulation of VEGFR2 protein expression on endothelial cells in the tumors of GPR4 KO AOM/DSS mice. Consistent with Wyder et al.’s findings, that VEGFR2 protein expression was reduced in GPR4 KO tissue and in GPR4 knockdown on HUVEC cells [184]. Other pathways that may contribute to this reduction in angiogenesis are CXCR7, Notch, and/ or CXCL1 (the functional homologue of human IL8) [307- 311]. In one study, CXCR7 conferred a proangiogenic role in HUVEC cells via VEGFR2 upregulation [312]. Both pharmacologic and genetic abolishment of GPR4 enhanced chondrocyte differentiation and ameliorated osteoarthritis via downregulating the CXCR7/CXCL12 pathway [212]. Moreover, Ren et al., identified Notch1 pathway activation to be responsible for angiogenesis in HMEC-1 cells overexpressing GPR4 [311]. Also, acidic activation of GPR4 promoted angiogenesis in coronary artery patients’ endothelial progenitor cells (EPCs) via activating the signal transducer and activator of transcription 3 (STAT3)/VEGFA pathway [211]. Given the previously discussed potential targets, further investigation is warranted to identify downstream signaling contributing to GPR4 induced angiogenesis in the CAC mouse model. Effective disease management of IBD can confine the symptoms and reduce the risk of developing colitis associated colorectal cancer. Despite the vast advancement in IBD therapies starting from aminosalicylates, corticosteroids, immune modulators and reaching to biologics targeting TNF?, interleukins, and adhesion molecules, colorectal cancer incidence is increasing in younger individuals [313]. While impeding inflammation has a major role for halting tumor development, more targets are needed to protect against carcinogenic development due to chronic inflammations. Targeting angiogenesis is an additive attractive therapeutic approach, nonetheless 74 using antiangiogenic therapy causes the risk of tumor progression due to increased hypoxia [314]. Thus, normalizing the angiogenic process in tumors is believed to be an effective strategy to further reduce tumor development, and hence more drug targets should be explored in this area. Collectively, GPR4 knock out halts cancer development via alleviating chronic inflammation, in addition to interluding colitis associated colorectal cancer (CAC) progression via reducing angiogenesis. The therapeutic benefit for GPR4 allosteric modulation has been described in reducing pain, inflammation, and angiogenesis [228, 229]. We and others have shown the therapeutic value for GPR4 antagonism in IBD pre-clinical mouse models [7, 167]. Therefore, we propose GPR4 as an attractive target to be inhibited for the treatment of IBD and during early- stage CAC, which may serve as a prophylactic against CAC development in IBD patients through alleviating chronic intestinal inflammation and reducing pathological angiogenesis in those patients. 75 Chapter V: The Role of GPR4 in Immune-checkpoint Inhibitors Mediated Colitis (IMC) A. Summary Immune-checkpoint inhibitors (ICI) have revolutionized cancer therapeutics. However, systemic immunostimulation by ICIs may lead to immune related adverse events (irAEs) in some patients that range from mild to fatal. Colitis is one such irAE that could present as mild, nonetheless causing treatment cessation if not resolved. More severe forms of colitis, on the other hand, may result in fatal intestinal perforation. The pathology of IMC and IBD overlap, so do treatment modalities. We and others have previously identified GPR4 as a candidate drug target for IBD treatment. In both human and mouse IBD, GPR4 is overexpressed in inflamed tissues, signifying its involvement in intestinal inflammation. Using a GPR4 antagonist, we previously observed a reduced disease activity of IBD in the DSS experimental mouse model. Hence, we proposed that GPR4 knock out would have similar effects in IMC. We employed an IMC pre- clinical mouse model, where GPR4 KO and WT mice treated with ICI or the isotype control in addition to DSS, were used to assess the role of GPR4 in intestinal inflammation mediated by ICI. We observed a significant reduction in clinical disease activity in the GPR4 KO mice reflected in reduced body weight loss, and fecal blood and diarrhea scores. At endpoint necropsy, we observed a decrease in mesenteric lymph node volume and spleen sizes of the GPR4 KO-ICI DSS mice compared WT-ICI DSS. Additionally, GPR4 KO-ICI DSS colons had reduced fibrotic and histopathological scores compared to WT-ICI DSS. Interestingly, ICI treated mice show increased histiocytic (macrophages) clusters in distal, mid and proximal segments. These clusters were less observed in the WT-DSS, GPR4 KO-ICI DSS or GPR4 KO-DSS mice. B. Introduction Immune checkpoint inhibitors have advanced cancer therapies in hematologic and solid cancers such as melanoma, non-small cell lung cancer, and many others [315]. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), programmed cell death protein-1 (PD-1), and programmed cell death ligand-1 (PD-L1), are checkpoints that cancer hijack to induce immune- tolerance and thereby evade T-cells attacks in favor of cancer proliferation. T-cell proliferation and activation requires two main signals, the first one is antigen presentation through MHC receptors presented by APCs to bind a TCR expressed on T-cells, and the second one is a co- stimulatory signal, where CD80 or CD86, presented on APCs bind the costimulatory CD28, presented on T-cells. Both CTLA-4 and PD1 are inhibitory receptors and the homologues of CD28, all typically expressed by T-cells, can block the second co-stimulatory signal. CTLA-4 competes with CD28 ligands, CD80 and CD86 and produce co-inhibitory response, upon binding. PD-1, on the other hand bind to its ligand PDL-1, typically expressed by APCs and blocks T-cell activation. ICIs are monoclonal antibodies used to inhibit these checkpoint pathways used by cancer cells by either blocking ligands or receptors, which would lead to anti-cancer host immunity activation, and proliferation allowing T cells to demolish cancer cells [315]. This T-cell activation is systemic, thus allowing for nonspecific immune responses in many organ systems which mediates ICI-related adverse events, also called irAEs [316]. ICI-mediated adverse events are commonly endocrine, dermatological, and gastrointestinal (G.I.) in addition to further less common effects in other organs [315]. G.I. side effects represent 30% of all irAEs reported in patients using CTLA-4 antibodies and 20% of PD-1/PDL-1 antibodies [317]. Diarrhea and colitis are among the side effects commonly reported which depending on their grade may 77 result in treatment interruption or a fatal consequence due to intestinal perforation [318-321]. Clinically, IMC and IBD appear similar in endoscopy, showing features of UC [320-322]. Additionally, non-pathologic lymph node enlargement in a patient with IMC was associated with diarrhea, and increase intestinal wall thickness, consistent with features of IBD [11, 323-325]. Microscopically, IMC histopathological characteristics may overlap with IBD in which neutrophilic inflammation, increased intraepithelial leukocytes, and crypts architectural distortion were evident [326]. On a cellular level, CD8+ and CD4+ T-cell intestinal infiltration was described in IMC as well as UC and CD patients [327]. Macrophage tissue infiltration was described in IMC and IBD colon biopsies [328, 329]. In addition, several reports reported that microbial dysbiosis is associated to pathogenesis of both IBD and IMC [330, 331]. Thus, similar treatment modalities are used for both IMC and IBD. Corticosteroids, 5-aminosalicylic acid, immunosuppressants, anti- adhesion molecules and antibodies targeting TNF-? are the current treatments for IBD [11, 332]. Where corticosteroids are the first line of treatments for IMC [323, 333-336], followed by TNF-? monoclonal antibodies if resistant or recurrent to preserve long term remission [323]. In addition anti-mobility therapy is used for immune checkpoint-mediated diarrhea [323]. Anti-adhesion therapy is subcategorized to integrins (?4?1, ?4?7 or ?E?7), addressins (ICAM-1 and MAdCAM- 1), and the GPCR, the chemokine receptor 9, is used in IBD to blocks homing of lymphocytes. Especially, blocking the ?4?7/MAdCAM-1 interaction is effective in IBD, and already indicated for IMC patients’ refractory to corticosteroids [332, 337, 338]. Since the mucosal addressin-cell adhesion molecule 1 (MAdCAM-1) ligand, expressed in the endothelial venules in the small intestine, in the Peyer's patches and the colon interacts with ?4?7 integrin, that is expressed on the lymphocytes colonizing the gut and gut-associated lymphoid tissues [339, 340]. Despite these 78 advances in IBD therapeutics, non-responsive IMC cases have been reported to corticosteroids, TNF-? antibodies, and anti-adhesion treatments [140]. GPR4 increased expression has been detected in both UC and CD patients as well as in colitis mice [161, 163]. Acidic activation of GPR4 on endothelial cells increased adhesion molecules, VCAM-1, Intracellular adhesion molecules-1 (ICAM-1), and E-selectin (CD62 or SELE) expression [8]. We previously proposed GPR4 antagonism as an attractive treatment for IBD, that reduced disease activity, histopathology of IBD, and decreased TNF-? and MAdCAM-1 expression in the colons of colitis mice [7]. Moreover, both pharmacological inhibition and GPR4 genetic knockout show reduced adhesion molecules (VCAM-1 and E-SELE) protein expression in colitis mice colons [7, 161], and in the hindlimb ischemia reperfusion mouse model [6]. Collectively, knocking out GPR4 alleviated intestinal inflammation in experimental mouse models [161, 163]. Thus, we proposed GPR4 as an attractive target, and employed an IMC mouse model to study the role of GPR4 in colitis as a side effect of immune checkpoint inhibitors, using GPR4 KO and WT mice, injected with ICI in conjunction to the administration of DSS [236, 237]. 79 C. Results C.1. GPR4 genetic knockout decreases disease severity index in the IMC mouse model To study the role of GPR4 in remediating intestinal inflammation side effects of the immune- checkpoint inhibitors, anti-PD1 and anti-CTLA4, we employed an IMC mouse model using WT and GPR4 KO mice [236, 237], as described in the materials and methods section. WT and GPR4 KO mice received a total of three i.p. injections of a combination of anti-PD1 and antiCTLA4 (ICI) or the corresponding isotype control, mice also received 2%DSS in the drinking water three days after the first injection of either ICI or isotype control. Disease severity indicators were measured during the experiment by monitoring body weight loss, and fecal blood and diarrhea score. WT- ICI DSS mice showed more body weight loss (mean ~ -3.8%) when compared to GPR4 KO-ICI DSS (mean ~ -2%), WT-DSS (mean ~ -3%), and GPR4KO-DSS (mean ~ -2.2%), mice starting day 8, and the trend persisted till the end of the experiment. On the last day of the experiment, a statistically significant body weight loss in WT-ICI DSS (mean ~ -7.1%) compared to GPR4 KO- ICI DSS (mean ~ -2.7%), similarly in WT-DSS (mean ~-5.3%), and GPR4KO-DSS (mean ~ - 2.4%) (Fig.5.1.A-C). Interestingly, fecal blood and diarrhea score in the ICI treated group was significantly increased after the first ICI injection and prior to DSS administration in both WT-ICI DSS (mean ~ 1) compared to GPRKO-ICI DSS (mean ~ 0.2). In contrast, the isotype injection for WT-DSS (mean ~ zero) compared GPR4 KO-DSS (mean ~zero) did not increase the score of severity for fecal blood and diarrhea. Throughout the experiment, we observed a statistically significant difference in the score of severity for fecal blood and diarrhea between WT-ICI DSS and GPR4 KO-ICI DSS group and a similar between WT-DSS and GPR4 KO-DSS groups. On the last day of the experiment, the score of severity for fecal blood and diarrhea in the WT-ICI DSS (mean ~3.4) compared to GPR4 KO-ICI DSS (mean ~3), and WT-DSS (mean ~2.8), 80 compared to GPR4KO-DSS (mean ~2.3) (Fig.5.1.B-D). At terminal necropsy, we measured mesenteric lymph node (MLN) expansion, colon length and spleen weights. We observed a ~2 fold increase in MLN expansion of WT-ICI DSS in comparison with to GPR4 KO-ICI DSS, and consistently, a ~1.33 fold increase in WT-DSS MLNs, compared to GPR4KO-DSS (Fig.5.1.E). Moreover, we measured colon length for all four treatment groups and did not observe a statistically significant difference between groups, although there was a trend for WT-ICI DSS and GPR4 KO-ICI DSS mice having shorter colons than WT-DSS and GPR4 KO-DSS, respectively (Fig.5.1.F). In addition, spleen weights of WT-ICI DSS were ~1.5 fold heavier than GPR4 KO-ICI DSS spleens. Interestingly, but not surprisingly WT-ICI DSS spleens showed a significant increase (~1.4 fold) in weight compared to WT-DSS. Moreover, WT-DSS spleens showed a trend of increase compared to GPR4 KO-DSS (Fig.5.1.G). 81 A B Body Weight Change 4 Fecal blood and diarrhea score 4 WT ICI + 2% DSS 2 GPR4 KO ICI + 2% DSS ICI Injection 3 * -2 2 * -4 * -6 1 -8 -3 -2 -1 1 2 3 4 5 6 7 8 9 10 11 -10 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 Days since 2% DSS administration Days since 2% DSS administration C Body Weight Change D 4 Fecal blood and diarrhea score 4 2 3 * WT Isotype + 2% DSS GPR4 KO Isotype + 2% DSS -2 2 Isotype Injection -4 1 -6 -3 -2 -1 1 2 3 4 5 6 7 8 9 10 11 -8 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 Days since 2% DSS administration Days since 2% DSS administration 82 % change % change Score of severity Score of severity ** ** ** ** ** ** **** ** ** E Mesenteric lymph node volume ?? 30 ? ns ns 20 10 0 S S S S SS S S I- D -D -D -D C T IC O I W I K T O 4 W K PR4 R GP G F Colon length ns 9 ns ns ns 8 7 6 5 SS SS S SS -D -D S I -D - D C T I O I W T I C K W O 4 K P R 4 R GP G G Spleen weight ? 0.3 ? ns ns 0.2 0.1 0.0 S S S S S S D D S S I- - -D - D C T I I W IC O K T O 4 W K PR 4 G PR G 83 mm3gm cm Figure 5.1. Clinical indicators of intestinal inflammation in IMC mouse model. WT-ICI DSS (n=12), WT-Isotype control IgG DSS (WT-DSS) (n=12), GPR4-ICI DSS (n=12), and GPR4 KO-Isotype control IgG DSS (GPR4 KO-DSS) (n=12), with 6 males and 6 females for each, were used for this analysis. WT-ICI DSS show increased body weight loss and fecal blood and diarrhea scores compared to GPR4 KO-ICI DSS mice. WT-DSS show increased body weight loss and fecal blood and diarrhea scores compared to GPR4 KO-DSS mice. (A-C) Body weight change percent, and (B-D) fecal blood and diarrhea scores. At the endpoint, disease parameters such as Mesenteric Lymph Node volume (MLN), colon length and spleen weight were measured. WT-ICI DSS show increased MLN and spleen size when compared to WT-DSS and GPR4 KO- ICI DSS mice. (E) MLN, (F) colon shortening, and (G) spleen size. Data are presented as the mean ± SEM. Statistical significance was determined using the unpaired t-test between each pair of the following: (WT-ICI DSS and WT-DSS), (WT-ICI DSS and GPR4 KO-ICI DSS), (WT-DSS and GPR4 KO-DSS), and (GPR4 KO-ICI DSS and GPR4-DSS) mice. (*P<0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). 84 C.2. Lack of GPR4 expression reduces Immune checkpoint inhibitors mediated colitis histopathological inflammation We further assessed the role of GPR4 in immune checkpoint inhibitors induced intestinal inflammation using histopathological analysis. Given the close resemblance of histopathologic features of IBD and IMC [326], we used a pre-established scoring criteria, as previously described [7, 161, 238], with one modification of adding macrophage aggregates (clusters) score and percent involvement as a unique histopathologic feature observed in our IMC mouse model. We quantified the degree of histological features of colitis between WT and GPR4 KO mice treated with immune- checkpoint inhibitors (ICI) or isotype control in conjunction with DSS treatment. Distal, middle, and proximal segments of the colon were examined for histopathological features of colitis, such as inflammation, macrophages aggregates (clusters), area of infiltration, edema, crypt damage, and architectural distortion. Our observations indicate a trend of ~1.4, 1.5, and 1.6- mean fold increase in histopathological score in ICI DSS treated WT mice compared to the DSS group in distal, mid and proximal colon segments, respectively. Whereas distal, mid and proximal colon segments of GPR4 KO-ICI DSS colons showed ~ 6, 6, and 5- mean fold reduction in the inflammation as reflected by the histopathology score compared to WT-ICI DSS. In addition, a ~ 3, 5, and 5-fold decrease in GPR4 KO-DSS score was observed in comparison to WT-DSS (Fig.5.2.A-F). . Upon histopathological analysis, immune clusters that form foci like aggregates were observed in the ICI treated colons with higher prevalence compared to DSS treated ones. Those aggregate showed histiocytic (macrophage) like morphology. To confirm that the aggregate’s immune subtype, we used immunohistohemistry for anti-body-based identification. F4/80+ macrophages were indeed the main immune cell component of those aggregates. This feature was independently analyzed by a trained pathologist based on number of clusters as well as percent involvement in 85 each segment. Interestingly, the WT-ICI DSS group had a ~2 fold increase in the score of severity compared to WT-DSS, GPR4 KO-ICI-DSS and GPR4 KO-DSS groups (Fig.5.3.A-F). . Fibrosis is common pathological feature of both forms of IBD, crohn’s dieseae and ulcerative colitis. Fibrosis occurs as result of scar tissue formation due recurrent inflammation which can lead to life-threatening complications in IBD patients. We stained colon segments with picrosirius red (PSR) to assess the accumulation of collagen fibers in mice colons to reflect the degree of fibrosis. WT-ICI DSS treated group colons showed a ~2.2, 1.7, and 1.2 fold increase in fibrosis score compared to WT-DSS group for the distal, mid and proximal segments. GPR4 KO-ICI DSS colons had a reduced fibrosis score of ~3.3, 2.8, and 1.5 fold when compared to WT-ICI DSS colons for all three segments, respectively. Moreover, a trend of decreased fibrosis in the distal area GPR4 KO-DSS colons compared to WT-DSS colons was observed. Whereas a ~2.8 fold decrease in fibrosis score for both mid and proximal segments was observed in the GPR4 KO-DSS mice colons (Fig.5.4.A-F). . 86 A WT- WT- ICI DSS GPR4 KO- GPR4 KO- ICI DSS B Histopathology in distal colon 40 ??? ns ??? ns 30 20 10 0 SS SS SS D D D S S I- - I- -D IC T W IC O T KO 4 W 4 K PR R GP 87 G Score of severity C WT- WT- ICI DSS GPR4 KO- GPR4 KO- ICI DSS D Histopathology in mid colon ???? ns ??? 40 ns 35 30 25 20 15 15 10 5 0 S S S S S S S -D -D S I T I- D -D C O I W C I T KO 4 W K PR 88 4 G PR G Score of severity E WT- WT- ICI DSS GPR4 KO- GPR4 KO- ICI DSS F Histopathology in proximal colon 40 ???? 35 30 ns ???? 25 ns 20 15 15 10 5 0 SS SS SS S -D -D D S I T I- - D IC W I C O T KO 4 W K R 4 PG PR G 89 Score of severity F igure 5.2. Histopathological analysis of IMC mice colons. Histological features of colitis were examined to further assess the degree of disease activity in the mice by an independent trained pathologist using the following criteria for scoring colitis severity; Inflammation, macrophages aggregates (clusters), area of infiltration, edema, crypt damage, and architectural distortion. Overall, GPR4 KO-DSS and GPR4 KO-ICI DSS mice had reduced histopathological scores distal, mid, and proximal colon segments when compared to WT-DSS and WT-ICI DSS mice. (A-C-E) Representative images of Hematoxylin and Eosin (H&E) stained sections of distal, mid, and proximal colons for WT-ICI DSS (n=12), WT-DSS (n=12), GPR4 KO- ICI DSS (n=12), and GPR4 KO-DSS (n=12), with 6 males and 6 females for each, using a 20X microscope objective, and (B, D, F) Pathologist assessment of colon histopathology. Data are presented as mean ± SEM and analyzed for statistical significance using the Mann-Whitney U test between each pair of the following: (WT-ICI DSS and WT-DSS), (WT-ICI DSS and GPR4 KO- ICI DSS), (WT-DSS and GPR4 KO-DSS), and (GPR4 KO-ICI DSS and GPR4-DSS) mice. (*P<0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Scale bar is 100 µM. 90 A WT- WT- ICI DSS GPR4 KO- GPR4 KO- ICI DSS B Macrophage clusters in distal colon 4 ??? ? ns ns3 2 1 0 SS SS I IC SS DI -D - D C T O - -I W K O T 4 KR 4 W P R G PG 91 Score of Severity C WT- WT- ICI DSS GPR4 KO- GPR4 KO- ICI DSS D Macrophage clusters in mid colon p= 0.08 3 p= 0.08 ns ns 2 1 0 SS SS I C SS D -DI - I O -DT IC- W 4 K O T K4 W P R G P R G 92 Score of severity E WT- WT- ICI DSS GPR4 KO- GPR4 KO- ICI DSS F Macrophage clusters in proximal colon ? 3 p= 0.06 ns ns 2 1 0 I SS SS S D D -I C S I - O -DC T -I W 4 K O T K4 W P R R G PG 93 Score of severity Figure 5.3. Pathological histiocytic (macrophage) clusters analysis in IMC mice colons. The score of severity for macrophage aggregates (clusters) replacing crypts structures, as indicated by blue stars was assessed by an independent trained pathologist as an indication of disease severity. Overall, WT-DSS, GPR4 KO-DSS and GPR4 KO-ICI DSS mice had reduced fibrosis scores in distal, mid, and proximal colon segments when compared to WT-ICI DSS mice. (A-C- E) Representative images of PSR stained sections of distal, mid, and proximal colons for WT-ICI DSS (n=12), WT-DSS (n=12), GPR4 KO-ICI DSS (n=12), and GPR4 KO-DSS (n=12), with 6 males and 6 females for each, using a 200X microscope objective, and (B, D, F) Score of severity assessment of colon fibrosis. Data are presented as mean ± SEM and analyzed for statistical significance using the Mann-Whitney U test between each pair of the following: (WT-ICI DSS and WT-DSS), (WT-ICI DSS and GPR4 KO-ICI DSS), (WT-DSS and GPR4 KO-DSS), and (GPR4 KO-ICI DSS and GPR4-DSS) mice. (*P<0.05, and ***P < 0.001). Scale bar is 100 µM. 94 A WT- WT- ICI DSS GPR4 KO- GPR4 KO- ICI DSS B Fibrosis score in distal colon 15 ?? ? ns ns 10 5 0 S S S S S S D D S S I- - -D -D C T I I W IC OT K W O 4 K4 P R PR G G 95 Score of Severity C WT- WT-DSS ICI GPR4 KO- GPR4 KO- ICI DSS D Fibrosis score in mid colon ??? 8 ? ns ns 6 4 2 0 SS SS SS S -D -D S I D DT I- - I C W C O T I KO 4 W 4 K PR PR G 96 G Score of Severity E WT- WT-DSS ICI GPR4 KO- GPR4 KO- ICI DSS F Fibrosis score in proximal colon 8 ns ns ? ns 6 4 2 0 SS SS SS SS I-D -D D D C T I - - I W O T I C K O 4 W 4 K PR PR G G 97 Score of Severity Figure 5.4. Pathological fibrosis analysis of IMC mice colons. Degree of fibrosis was examined to further assess the degree of disease activity in the mice using Picro Sirius Red (PSR) stain. Overall, GPR4 KO-DSS and GPR4 KO-ICI DSS mice had reduced fibrosis scores in distal, mid, and proximal colon segments when compared to WT-DSS and WT- ICI DSS mice. (A-C-E) Representative images of PSR (blue arrow heads) stained sections of distal, mid, and proximal colons for WT-ICI DSS (n=12), WT-DSS (n=12), GPR4 KO-ICI DSS (n=12), and GPR4 KO-DSS (n=12), with 6 males and 6 females for each, using a 200X microscope objective, and (B, D, F) Score of severity assessment of colon fibrosis. Data are presented as mean ± SEM and analyzed for statistical significance using the Mann-Whitney U test between each pair of the following: (WT-ICI DSS and WT-DSS), (WT-ICI DSS and GPR4 KO-ICI DSS), (WT-DSS and GPR4 KO-DSS), and (GPR4 KO-ICI DSS and GPR4-DSS) mice. (*P<0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Scale bar is 100 µM. 98 D. Discussion In this study we demonstrate that GPR4 knockout alleviates immune checkpoint inhibitors mediated colitis in an experimental mouse model, which further proves the proinflammatory involvement of GPR4 activation in IMC, consistent with its previously described pro- inflammatory role in the gut and many systems such as the brain, heart, kidney. Lung, bone, and skin [6, 7, 161, 177, 210-212]. IBD and IMC’s pathophysiology is overlapping, clinically, [320- 322], microscopically [326], microbially [330, 331], and also in cellular components [327-329]. Mechanistically, the most prominent cell types identified to contribute to pathogenesis of both IMC and IBD are Th17 and Th1 CD4+ T-helper cells, for patients who received anti-CTLA4 and anti-PD-1, respectively [52, 341-343]. Combination therapy of anti-CTLA-4 and anti-PD-1 stimulated both myeloid cell populations and lymphoid cytotoxic effector T cells (CD8+ T-cells) in IMC patients compared to normal controls [70]. One distinctive feature is that CD8+/ CD4+ ratio in IMC was significantly higher than in UC and CD [327], which indicates that despite the many similarities between IMC and IBD, some features are ICI related. Other distinctive features discovered, such as, histiocytic response to crypts rapture was reported in patients receiving PD-1 inhibitors, as an uncommon distinctive feature for anti-PD-1-induced IMC [344]. For all the previously mentioned similarities and differences, developing IMC specific pre-clinical models are crucial to further delineate features of IMC versus IBD, which will aid in uncovering new drug targets. Recently, Wang et al. developed an IMC mouse model, where anti-PD-1/anti-CTLA4 antibodies are injected in conjunction to supplying DSS in the drinking water to C57BL/6 mice [237]. They show that depleting gram positive gut microbiome aid in the ICI mediated intestinal inflammation [237]. In addition, they demonstrate that Oral administration of the gram positive, Lactobacillus reuteri inhibited ICI mediated colitis in those mice by decreasing the mucosal 99 numbers of Group 3 innate lymphoid cells (ILC3s) [237]. These findings are consistent with another study where they show that Bifidobacterium administration mitigates anti–CTLA-4 mediated colitis in mice [236]. Thus, we employed an IMC mouse model, where WT and GPR4 KO mice received combination anti-CTLA-4/ anti-PD-1 antibodies or the control isotype prior and during mice receiving DSS in the drinking water to study the role of GPR4 in ICI mediated colitis. GPR4 is expressed mainly in endothelial cells, and other cell types amongst are macrophages and fibroblasts [6, 152, 161, 167, 168, 227, 298, 299]. Acidic activation of GPR4 on endothelial cells upregulates several proinflammatory pathways such as IL8, CCL20, TNF, COX-2, and IL1A [159], as well as ER stress pathways PERK, ATF6, and IRE1 [276]. GPR4 is activated via the protonation of several histidine residues, leading to uncoupling Gs and activating cAMP [217], inducing the downstream Epac mediated transendothelial leukocyte extravasation by upregulating VCAM-1, ICAM-1, and SELE in vitro [8]. In vivo, abrogating GPR4 reduces TNF-?, VEGF, procollagen, MAdCAM-1, VCAM-1, and SELE expression [7, 167]. Both genetic and pharmacological inhibition of GPR4 confers a reduced intestinal inflammation phenotype reflected by reducing disease activity, histopathology score and leukocyte infiltration in DSS colitis mouse models [7, 161, 163]. Additionally, pharmacological inhibition of GPR4 exhibits anti- inflammatory, anti-nociception and anti-angiogenic properties [228, 229]. In line with these observations, we observe reduced disease activity, represented in body weight loss, fecal blood and diarrhea scores, in addition to end point parameters such as decreased mesenteric lymph node (MLN) expansion and spleen size in the GPR4 KO-DSS and GPR4-ICI DSS compared to WT- DSS and WT-ICI DSS mice. Similarly, a reduction in the histopathological score of severity in GPR4 KO-DSS and GPR4-ICI DSS compared to WT-DSS and WT-ICI DSS mice, was observed. One interesting feature we observed, was the significant increase in immune aggregates (clusters), 100 in the WT-ICI DSS mice, which are primarily consistent of histiocytes (macrophages) in all three segments of the colon. Macrophages are described as the gatekeepers in intestinal homeostasis [345]. Contrary to the consensus that CTLA4 and PD-1 confer their inhibitory role primarily on T-cells, studies have shown evidence of their expression in human monocytes [346-348]. Additionally, human M1 polarized monocyte derived macrophages (MDMs) and rheumatoid arthritis MDMs treated with CTLA4-Ig in vitro shifted from M1 (alternative proinflammatory subtype) to M2 (classical anti-inflammatory subtype) macrophages [349]. Mice deficient of CTLA4 showed lethal multiorgan failure caused by macrophage and T-cell proliferation inducing myocarditis and pancreatitis [350]. CTLA-4 was found to mediate transendocytosis of the CD28 ligands CD80/86 (expressed on dendritic cells, macrophages, and other cell types), where Anti? CTLA?4 IgG may inhibit this transendocytosis [351, 352], thus allowing for macrophages and dendritic cells stimulation. In addition, CD25+CD45RBlowCD4+ T-regs identified as essential for both intestinal inflammation and autoimmunity, were demonstrated to exert their protective role through CTLA-4 signaling. Thus, blocking CTLA-4 signaling using monoclonal antibodies resulted in intestinal inflammation in mice [353]. One such mechanism could be the depletion of high-CTLA-4 T-regs via antibody?dependent cellular cytotoxicity (ADCC) resulting from the engagement of anti?CTLA?4 IgG1 antibody to Fc?Rs expressed on immune effector cells (NK cells, monocytes/macrophages) via its Fc region [352]. From previous observations, it can be concluded that CTLA4 may confer an inhibitory role in monocytes/macrophages. In line, with the afore mentioned CTLA4 role on macrophage polarization, observations from in vitro polarization experiments for peripheral CD14+ monocytes obtained from women’s decidual tissues (womb tissues), indicate that PD-1 blockade promoted dominance of the M1 phenotype over M2. Moreover, PD-1 polarized macrophages showed enhanced phagocytic activity, which was 101 decreased with PD-1 blockade [354]. Moreover, bone marrow derived macrophages (BMDMs) from PD-1-KO mice showed an increased shift towards M1 phenotype when stimulated with LPS + IFN-? or IL-4, as shown by increased the levels of inducible nitric oxide synthase (iNOS) mRNA were compared with control BMDMs. Also, spinal cord injured PD-1 KO mice showed M1-type macrophages/microglia accumulation associated with poor locomotor recovery compared with wild-type mice. PD-1 suppression of M1 macrophage/microglial polarization was mediated by reducing the phosphorylation of signal transducer and activator of transcription 1 (STAT1) while mediation of M2 macrophage/microglial polarization was attained by increasing STAT6 phosphorylation. As for the phagocytic ability, upon LPS + IFN-? or IL-4 stimulation, Macrophages and microglial cells showed opposite phagocytic phenotypes where M2 macrophages showed better phagocytic ability than M1 macrophages, and the opposite for microglial cells. Consistent to its role in M1 macrophage polarization, deficiency in PD-1 decreased the phagocytic ability of macrophages. In contrast, its deficiency increased microglial phagocytosis ability [355]. Interestingly contrary to previous observation, PD-1 was shown to regulate macrophage polarization form the alternative less inflammatory subtype M2 to the classical proinflammatory subtype M1, in the Sprague–Dawley rats ischemia–reperfusion in vivo model and in the in vitro rat alveolar macrophages culture [356]. Furthermore, PD-1 was found to be highly expressed on peritoneal macrophages in a sepsis mouse model and in the monocytes of sepsis patients. PD-1 expression on macrophages was observed to be upregulated especially during sepsis, disrupting their bactericidal capacity. In septic PD-1?/? macrophages, this bactericidal capacity was restored showing a more protective action against sepsis inflammatory effects. Also, macrophage depletion in septic PD-1?/? mice was detrimental to their survival, concluding that macrophages are the primary cell type in this bacterial model [357]. Collectively, these 102 observations imply that that PD-1 role on macrophage polarization and phagocytic ability, maybe context dependent. Because colitis is associated with microbial dysbiosis, both macrophages’ proinflammatory polarization as well as bactericidal function are of great value in this disease. Thus, further macrophage subtyping is required for the IMC disease model to delineate the exact mechanism for exacerbated colitis phenotype we observed using CTLA4 and PD-1 blockade. We observed a significant increase in macrophages clusters, associated with a more severe colitis in the WT-ICI DSS colons, as confirmed by F4/80+ IHC. This feature was scarce in the WT-DSS, GPR4 KO- DSS, and GPR4 KO-ICI DSS. Fibrosis is a prominent feature of colitis that is associated with severity and chronicity [292, 293]. Extracellular matrix (ECM) producing cells such as, fibroblasts, myofibroblasts, stellate cells, bone marrow-derived cells, fibrocytes and pericytes are among the cells contributing to intestinal fibrosis [358, 359]. GPR4 has recently been identified as a profibrogenic in CD, positively correlated to fibrogenesis genes, ACTA2, COL1A1 and COL3A1 [167]. In addition, both genetic and pharmacologic inhibition of GPR4 decreased collagen disposition in the chronic DSS mouse model [167]. Since the mouse model used in this study is thirteen days long, mimicking an acute colitis model, we did not expect to observe a high score of severity for fibrosis as a disease indicator. Nonetheless, we observed a significant difference in fibrosis score in the WT-ICI DSS, being the most severe compared to WT-DSS, GPR4 KO-DSS and GPR4 KO-ICI DSS. Collectively, we propose GPR4 as an attractive drug target to inflammation of the gut as a response to immune checkpoint inhibitor treatments. As previously, discussed several antagonists, such as antagonist 13 and antagonist 39c have been developed and proved effective in be delivered 103 via oral route and allosterically modulate GPR4, reducing inflammation in preclinical models [228, 229]. We and others have shown a significant therapeutic efficacy for the use of GPR4 antagonists in colitis mouse models in reducing disease activity, inflammatory mediators, and fibrosis [7, 167]. Thus, we propose the use of GPR4 pharmacological modulation in treating IMC. 104 Chapter VI: General Discussion The work in this dissertation aimed to assess the functional roles of GPR65 and GPR4 in inflammation driven colorectal cancer development and to further evaluate role of GPR4 in immune checkpoint inhibitors mediated colitis. Mice lacking GPR65 showed aggravation of inflammation represented in increased colitis disease activity, indicated by increased body weight loss; similarly, endpoint parameters showed increased mesenteric lymph node expansion, spleen size, and colon shortening in the AOM/DSS Colitis Associated Colorectal Cancer model. In line with the previously stated disease indicators, colon fibrosis, reflected by increased collagen deposition, which justifies the increased colon shortening observed in the GPR65 KO AOM/DSS mice compared to WT. Justifiably, we observed an increase in the number of myofibroblasts in the GPR65 KO mice, as one of the contributors to intestinal fibrosis. We further observed a significant increase in immune infiltration in the colons of GPR65 KO compared to WT AOM/DSS mice. Collectively, the increase in inflammatory indicators contributed to the increased tumor burden observed in GPR65 KO AOM/DSS mice. Both tumors number and volume were significantly higher in GPR65 KO compared to WT AOM/DSS mice. This study was based on the premise that chronic IBD may lead to CAC development via an “inflammation-dysplasia- carcinoma” axis. We built our hypothesis that GPR65 expression may protect against CAC development in mouse model on our previous observations of the anti-inflammatory role for GPR65 in the chronic DSS-colitis mouse model [166, 234]. In that model, GPR65 KO mice show increased inflammatory markers such as body weight loss, increased MLN expansion, increased histopathology score, increased fibrosis, and increased isolated lymphoid follicles [166, 234]. Using green fluorescence protein (GFP) as a surrogate marker for GPR65, we showed that it’s mainly expressed in the interstitial leukocytes within colon mucosa, transverse folds, and intestinal isolated lymphoid follicles. Based on morphology these colon leukocytes appear to be predominately macrophages, neutrophils, and lymphocytes. Within the MLN, high GFP expression was detected in histocytes and lymphocytes within the sinus regions, B cell follicles/germinal centers, and paracortical/interfollicular T cell zone. We also observed a discernable increase of GFP positive leukocytes within the inflamed colon mucosa and transverse folds when compared to non-inflamed colon tissues [166, 234]. GPR65 immune cell localization as confirmed by our previously mentioned observations conclude that the increase in the number of isolated lymphoid follicles, infiltrated macrophages and T-cells observed in the chronic DSS- GPR65 KO mice colons is due to the lack of GPR65 expression on those cells, based on previous reports showing that GPR65 activation can inhibit inflammatory profiles in macrophages, microglia, neutrophils, and T cells [264-267, 270]. Several studies, show the anti-inflammatory roles for GPR65 in the myeloid system, phenotypically reducing the activity of in different systems such as IBD mouse models, Tcymbarevich et al. observed an increase in neutrophils and monocytes/macrophages and their associated proinflammatory mediators, iNOS, IFN-?, and IL-6 in the acute and chronic DSS-colitis GPR65 KO mice compared to WT [206]. Moreover, Mercier et al. showed that GPR65 knockdown impaired phagocytic ability in macrophages and mediated the upregulation of the inflammasome pathway NLRP3 in human THP-1-derived macrophages [27]. Additionally, impaired GPR65 was shown to disrupt the endo-lysosomal phagocytic ability of macrophages [180], and in both GPR65 106 I231L and GPR65 KO Bone Marrow Dendritic Cells (DMCDs) [204], while showing impaired bacterial clearance in GPR65 KO mice [180], and in GPR65 I231L mice [204]. On the other hand, the assessment of the role for GPR65 in the lymphoid system as an anti- inflammatory mediator in IBD, was based on observations from global GPR65 KO systems [204]. Tcymbarevich et al. had previously reported that the adoptive transfer of naive CD4+ T-cells isolated from GPR65 KO mice did not affect the colitis clinical score and showed a more significant role for macrophages in the increased inflammation observed in the GPR65 KO-DSS colitis mice [206]. Most recently, Lin et al. reported a proinflammatory role of GPR65 in CD4+T- cells polarizing them towards Th1/Th17 phenotypes and that adoptive transfer of the conditional GPR65 CD4 knockout showed protection against TNBS colitis in RAG1-/- mice [195]. Other studies described GPR65 as a regulator for Th17 pathogenicity and in an experimental autoimmune encephalomyelitis (EAE) mouse model, adoptive transfer of CD4+ T cells caused more severe disease phenotype [208, 209]. Recently, Wirasinha et al show a more severe EAE in GPR65 KO mice, mainly dependent on (invariant natural killer T) iNKT cells not CD4+ T cells [182]. Regarding Th17 cell pathogenicity, reports have provided evidence for both protective and pathogenic roles in the context of intestinal inflammation and colitis associated colorectal cancer development [195, 272, 360, 361]. The previously mentioned observations underscore the need for further investigations to delineate the exact role of GPR65 in different lymphoid subsets. Yet, we can propose that the major functional role for GPR65 in IBD inflammation may be mediated at the innate immune response level as per previous findings [180, 203, 206]. Using the CAC AOM/DSS model, we present a new functional role for GPR65 in which it can halt inflammation-driven cancer development in a spontaneous tumor model. The acidic environment is one common environment in the inflammatory and neoplastic phases of this model, 107 where GPR65 plays a major role as an acid sensor. With GPR65’s effect on reducing immune infiltration, fibrosis, myofibroblasts number in the colons of WT mice, which halts cell transformation, it was associated with a decrease in tumor burden. After cellular transformation, initially acidosis in the tumor site is linked to in situ carcinoma (localized) tumor formation [100]. At this point of evaluation, our observation shows a great value for GPR65 agonism. Interestingly, a new study recently reported, GPR65 protein expression to be significantly upregulated in primary human lung fibroblasts cells isolated from patients with a diagnosis of idiopathic pulmonary fibrosis (IPF), compared to non-fibrotic areas. They also report that upon GPR65 siRNA knockdown in the primary human lung fibroblasts, myofibroblasts differentiation was significantly reduced in response to TGF-? treatment. They also show similar results using the GPR65 allosteric agonist BTB, where a dose dependent decrease of myofibroblasts differentiation was shown in both non-fibrotic and IPF primary human lung fibroblasts. They do show that this effect was GPR65 independent and was mediated through RhoA dependent mechanism [362]. Collectively, we propose that GPR65 agonists maybe beneficial in IBD remission and as prophylaxis against CAC development at the initiation stage. Yet, a question might arise about the possible roles for GPR65 proton sensing in the complex tumor microenvironment. An increase in acidity results in tumor progression from the in- situ type to a more invasive type due to the “Warburg effect” glycolytic shift [101]. Thus, acid sensing receptors, such as GPR65 are expected to have a complicated role in this heterogenous microenvironment. Tumor-associated macrophages (TAMs) play a crucial role in the tumor development, progression, immune suppression and are associated with poor prognosis. Making them great targets for immunotherapeutic therapies. Most recently, a published abstract has proposed GPR65 as an immune checkpoint expressed on tumor associated macrophages [363], 108 conferring an immune suppressive action on TAM, via upregulating the inducible cAMP early repressor (ICER), a mechanism previously demonstrated by Bohn et al. [364]. In the abstract, cancer patients with GPR65 (I231L) were described to have better survival compared to other genotypes. They tested this survival advantage in patients with highly glycolytic tumors that are usually resistant to immunotherapy and according to the study, the association was maintained [364]. Admittedly, further studies are needed to evaluate the different roles of GPR65 in different cancer types, subtypes, and stages. We further evaluated the role of GPR4 in the development of colitis associated colorectal cancer using the AOD/DSS model. Mice lacking GPR4 showed a significant decrease in disease activity parameters such as body weight loss, and fecal blood and diarrhea score compared to WT AOM/DSS. At endpoint, a significant recovery in body weight was observed in GPR4 KO mice compared to WT OAM/DSS. Additionally, a significant decrease of lymph node expansion was observed in the GPR4 KO mice to compared to WT AOM/DSS. These parameters indicate an overall decrease in inflammation in the GPR4 KO AOM/DSS mice compared to WT, which reflected on CAC development, as we observed that GPR4 KO mice showed decreased numbers and volumes of tumors compared to WT. We observed delayed adenocarcinoma development in the GPR4 KO AOM/DSS compared to WT mice, as reflected by higher percent of dysplastic lesion compared to adenocarcinoma is those mice, based on the premise of “inflammation-dysplasia- adenocarcinoma” axis. We next observed decreased blood vessel density in the tumors of GPR4 KO AOM/DSS mice, showing less angiogenesis in those tumors when compared to WT AOM/DSS tumors. We then observed a reduction in the protein expression of the pro-angiogenic receptor, vascular endothelial growth factor receptor-2 (VEGFR2) in the tumor vasculature of GPR4 KO AOM/DSS mice compared to WT mice. This decrease in angiogenesis in the GPR4 KO 109 AOM/DSS tumors was associated with increased tumor cell death, as represented by increased necrotic areas and increased cleaved-caspase-3 positive cells. Additionally, a significant reduction in the proliferation of the transformed epithelial cells in the GPR4 KO AOM/DSS tumors was observed compared to WT tumors. This work represents new findings on the role of GPR4 in inflammation-driven tumorigenesis and is an extension of our group’s previous findings of the proinflammatory role of acidity on GPR4 activation in endothelial cells (ECs), upregulating cytokines CSF2, IL1A, IL1?, IL6, IL8, IL23A, chemokines CCL2, CCL5, CCL7, CCL20, CXCL1, CXCL2, CXCL3, CXCL6, CX3CL1, CXCR7, Adhesion molecules MADCAM1, VCAM-1, E-selectin, ICAM-1, and other inflammatory pathways such as TNF, prostaglandins, NF-??, NOD2, Endoplasmic Reticulum (ER)-stress pathways. This activation is mediated through G protein pathways including the Gs/cAMP/Epac pathway [8, 218, 365]. We have also demonstrated that acidotic activation of GPR4 stimulates paracellular gap formation in human pulmonary artery endothelial cells (HPAECs), human colon microvascular endothelial cells (HMVEC-Colon), and human lung microvascular endothelial cells (HMVEC-Lung). Using gain and loss of function assays in human umbilical vein endothelial cells (HUVECs), we demonstrate that this effect is mediated through the G?12/13/Rho GTPase pathway [6]. The consensus in the literature has been related to the role of GPR4 in endothelial activation via upregulating proinflammatory cytokines, chemokines, and adhesions molecules. Moreover, its functional role in IBD has been directly linked to its role in immune extravasation to the site of inflammation [161]. Additionally, GPR4 has been identified to promote both physiological and pathological angiogenesis [184, 185, 312]. Wyder et al show that mice lacking GPR4 have decreased VEGFR2 protein expression and consistently knocking down GPR4 in HUVEC cells was associated with 110 lower expression of VEGFR2 [184]. They also show that GPR4 KO mice were resistant angiogenesis induced by vascular endothelial growth factor treatment compared to WT mice [184]. We show a similar observation in our spontaneous AOM/DSS CAC mouse model, where VEGFR2 protein expression is reduced in the GPR4 KO tumors. Yet the exact mechanism of how GPR4 regulates VEGFR2 is still unknown. One possible mechanism could be through cAMP-mediated transcriptional downregulation of Ras-related protein (R-RAS), increasing VEGFR2 mediated angiogenesis in tumor vasculature [366, 367]. Sawada et al. demonstrated an increase in both phosphorylated and total VEGFR2 protein expression in the blood vessels of the subcutaneous tumor models, Lewis lung cancer (LLC) and B16F10 melanoma injected in the R-Ras KO mice compared to WT mice [366]. Another possible mechanism, could be through GPR4 activation of the yes-associated protein (YAP) which binds the signal transducer and activator of transcription factor 3 (STAT3) and together promote the transcription of VEGF in endothelial cells, thus inducing an autocrine activation of endothelial angiogenesis [183, 368]. The activation of endothelial angiogenesis is also supported by increased tube formation in the human microvessel endothelial cells (HMEC-1) treated with conditioned media obtained from the SCCHN-GPR4 overexpression system. This phenotype was proven to be due to secreted IL6, IL8 and VEGFA, as a result of GPR4 acidic activation in SCCHN tumor cells, an effect abolished by IL6, IL8 and VEGFA inhibition, indicating the involvement of GPR4 in angiogenesis [185]. Consistently, our group previously observed IL6, IL8, and VEGFA upregulation as a result of GPR4 activation in HUVEC cells [162]. Moreover GPR4’s angiogenic role, could be through the unfolded protein response (UPR)/ER-stress pathways activation. A UPR is an adaptive process that occurs as a response to an increase in unfolded or misfolded proteins in the ER during protein synthesis to preserve cell 111 viability and function. A mild response maintains cell homeostasis, while a prolonged one will result in pathologic ER stress leading to cell death and inflammation. Most recently, ER stress genes have been shown to also confer proangiogenic roles. Dong et al. showed acidosis to upregulate all three arms of the ER stress/unfolded protein response (UPR) pathways in HUVECs, HPAECs, and HMVEC-Lung primary cell lines, they show increased protein expression of phosphorylated eIF2? (eukaryotic initiation factor 2?), phosphorylated IRE1? (inositol-requiring enzyme 1?), and cleaved-activating transcription factor (ATF6) upon acidic pH treatment (pH6.4) compared to pH7.4. In the GPR4 overexpression system (HUVEC/GPR4), upon acidic pH treatment, they show further increase in cleaved-ATF6, ATF3, ATF4, pIRE1? and spliced XBP-1 (X box-binding protein 1), compared to HUVEC/vector. They also show that GPR4 knock down and pharmacological inhibition inhibited this acidosis ER-stress response through the downregulation of peIF2?, ATF3, ATF4, cleaved-ATF6, pIRE1?, spliced XBP-1 mRNA and C/EBP homologous protein (CHOP) in HUVECs [218]. UPR signaling has been implicated in embryonic angiogenesis where IRE1? knockout mice showed embryonic lethality after 12.5 days of gestation as described by Iwawakia et al. By using the luciferase gene instead of GFP in their ERAI transgenic mouse, they were able to detect IRE1? activity predominantly in the placenta of mice showing developmental defects, they also showed that lack of IRE1? caused reduction in mRNA and protein levels of vascular endothelial growth factor-A in the placental leading to placental labyrinth dysfunction in these mice [369]. Zeng et al, demonstrated using qPCR and western blot that VEGF treatment of HUVECs transiently activated XBP1 mRNA splicing and IRE1? phosphorylation at serine724 with a peak at 30 to 60 minutes, and 15 minutes, respectively. VEGF activation of its receptor VEGFR2 resulted in its internalization and physical association of VEGFR2 with unspliced XBP1 and the 112 inositol requiring enzyme 1 ? in the endoplasmic reticulum, leading to inositol requiring enzyme 1 ? phosphorylation and XBP1 mRNA splicing leading to endothelial cell proliferation. Furthermore, in EC-specific XBP1 knockout mice, retinal vasculogenesis was halted in the first 2 postnatal weeks and ischemia-associated angiogenesis was reduced [370]. Thus, it may be proposed that GPR4 diverse proinflammatory roles through modulating barrier function and ER stress response in ECs contributes to their migration, proliferation as well as angiogenesis. Also, the previously discussed mechanisms indicate that GPR4 may promote angiogenesis through the VEGF/VEGFR2 pathway regulation, whether directly regulating VEGFR2 receptor function and expression or by autocrine activation of the receptor by regulating VEGF expression. Additionally, the role of GPR4 was predominantly described in EC, However, a lower level of expression has been demonstrated in macrophages and fibroblasts [7, 371]. Recently, Weder et al. demonstrated that GPR4 is expressed in fibroblasts obtained from GPR4 knock-in (Gpr4Cre-/+ x R26pCAGeGFP-/+) reporter mice treated with Dextran Sodium Sulfate (DSS) to induce colitis. They used GFP immunohistochemistry (IHC) stain as a surrogate marker for GPR4, they also stained similar areas with ACTA2 as a marker for activated fibroblasts (myofibroblasts). Additionally, they demonstrate increased GPR4 mRNA expression in the primary murine fibroblasts obtained from WT mice at basal levels and upon acidic activation [167]. They also, show a positive correlation between GPR4 and the fibrogenesis genes, ACTA2, COL1A1 and COL3A1, in fibrotic lesions obtained from CD patients’ ileums [167]. Previously, Riemann et al. detected GPR4 and GPR68 expression in Normal rat kidney fibroblasts, nonetheless, they ruled out a pro-inflammatory role for them based on the lack of cAMP production or free calcium accumulation upon acidic stimulation in these cells [371]. Yet, Weder et al argued that baseline levels of GPR4 may be ample in promoting inflammation and fibrosis. Moreover, 113 they propose that the increased number of GPR4 positive fibroblasts may not only be due to increased migration, proliferation, and differentiation of fibroblasts, but also due to endothelial- to-mesenchymal transition (EndoMT), a process previously linked to fibrosis and tumor angiogenesis [167, 372]. Collectively, the role of GPR4 in fibroblasts remains to be investigated. Finally, we used an immune checkpoint-mediated colitis (IMC) mouse model to study the functional role of GPR4 in colitis as a side effect towards the anti-cancer immune checkpoint inhibitors (ICI) therapy. GPR4 KO and WT mice were injected with a combination of anti-PD-1 and anti-CTLA4 antibodies (ICI) or control isotype prior to DSS administration to induce the IMC mouse model. We observed increased overall disease activity as presented by increased body weight loss percentage, fecal blood and diarrhea scores, LN size, spleen size, histopathology scores, macrophage clusters, and fibrosis score in the WT-ICI DSS mice compared to WT-DSS, GPR4 KO-ICI DSS, and GPR4 KO-DSS. Which indicates that GPR4 genetic knockout is alleviates IMC consistent with previous observations for IBD. We believe that this role is mediated primarily through GPR4 mediated leukocyte-endothelial cells extravasation regulation. Additionally, we anticipate further contributions of the proinflammatory role of GPR4 in endothelial cells, as mediated by cytokines, chemokines, barrier function, and ER stress, in IMC disease severity. The most prominent feature of ICI treatment was macrophage clusters sometimes forming granuloma like features, these clusters were mainly observed in WT-ICI DSS colons. Our group and others previously showed that GPR4 was expressed in macrophages, with observed increased expression in inflamed intestinal tissue compared to normal tissue [161, 163]. Also, GPR4 deficient DSS mice showed reduced Interferon gamma IFN? mRNA expression [163]. IFN? possibly promotes the observed increased macrophages in IBD and their proinflammatory M1 polarization [161, 373]. Whether GPR4 expression on macrophages is implicated in the 114 observed phenotype of increased macrophage clusters in WT-ICI DSS colons but not GPR4 KO- ICI DSS colons, is unknown. However, it is a possible hypothesis for the mechanism of IMC inhibition in the GPR4 KO ICI-DSS mice. Interestingly but not surprisingly, the observed macrophage clusters overlapped with fibrotic lesions found in the colons of WT-ICI DSS mice. This observation is consistent with previous reports describing an association between macrophage infiltration and extracellular matrix production indirectly linking macrophage to fibrosis lesions, possibly through cytokine mediated myofibroblasts activation [374-376]. A recent report shows a direct link between macrophages and fibrosis through collagen deposition. Using unbiased transcriptomics, Simões et al. show an upregulation of collagens in both zebrafish and mouse macrophages following heart injury. They observed enhanced scar formation via production of collagen upon adoptive transfer of macrophages, from either collagen-tagged zebrafish or adult mouse GFPtpz-collagen donors [377]. Nonetheless, whether our observed macrophage clusters are directly related to the increased collagen deposition in the WT-ICI colons is yet to be explored. In conclusion, this dissertation work evaluated the differential roles for the proton sensing GPR65 and GPR4 in CAC, and the functional role for GPR4 in IMC. The functional roles of the proton sensor GPR65, mainly expressed on immune cells indicates that its expression confers protection against CAC development. We conclude that this effect is predominantly mediated by reducing colitis inflammation, likely through decreasing immune cell proinflammatory programs activation. On the other hand, we show a differential role for the proton sensing GPR4, mainly expressed on endothelial cells, in that it confers a proinflammatory action in intestinal inflammation via inducing leukocyte-endothelial adhesion and upregulating immune extravasation to inflamed colons, which promotes colitis and hence promotes CAC development. We also show 115 a proangiogenic role for GPR4 in CAC tumors that sustain their development possibly through a VEGFR2 mediated angiogenic mechanism. Finally, we show a proinflammatory role for GPR4 in IMC, mainly associated with macrophage infiltration regulation. Expectedly, through the previously discussed role for GPR4 in mediating immune extravasation to the inflamed sites. Thus, we propose that the use of a GPR65 agonist, such as the previously discussed BTB09089, may cause IBD remission which will protect against CAC development, possibly through the modulation of immune function and reduction of fibrosis. Additionally, GPR4 allosteric modulation through the use of antagonist 13 [229] or antagonist 39c [228] is proposed to induce intestinal inflammation remission in both IBD as we and others previously demonstrated [7, 167], also an expected outcome in IMC. 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Nature Communications, 2020. 11(1): p. 600. 142 Appendix A: Animal use protocol Appendix B: Elsevier license to publish Appendix C: BioRender license to publish Appendix D: BioRender license to publish