Role of NLRP3 in colitis and colitis associated colorectal cancer

Inflammatory bowel disease (IBD) is an idiopathic group of chronic disorders characterised by inflammation of the small intestine and colon. In Australia, more than 85,000 people live with IBD and by the year 2022 it is expected that this number will have surpassed 100,000. The two major types of IBD are Crohn's disease (CD) and ulcerative colitis (UC). Both diseases are characterised by a series of relapses and remissions where the inflammation in the gastrointestinal tract becomes so severe that patients need hospitalisation or even surgery. A major complication of chronic inflammation (colitis), either by recurrent CD or UC is the development of colitis-associated colorectal cancer (CAC). Crohn and Rosenberg first reported the ulcerative colitis case associated with colorectal cancer development in 1925, thereafter many studies have been published linking gut inflammation as an important predisposing factor for the development of colorectal cancer. However, the understanding of specific underlying mechanism of how chronic inflammation is connected to the initiation and progression of colon cancer is yet to be established. Although the exact aetiology of IBD has yet to be elucidated, a defective innate immune system has been proposed as a primary mechanism in colitis. As an important arm of the innate immune system inflammasomes particularly the well characterised NLRP3, is involved in gut homeostasis and inflammatory pathologies. However, the role of NLRP3 in these processes is not well understood. Investigations on the role of NLRP3 in colitis is still controversial and inconclusive with some studies showing a protective role while other studies demonstrate a detrimental effect of NLRP3 activation. The reason for the discrepancies observed in the Nlrp3\\(^{-/-}\\) chemically induced colitis mouse model phenotype could be due to differences in length and concentration of chemical treatment or baseline differences in the composition of the intestinal microbiota in experimental mice. Thus, the overall aim of the PhD project was to address the above issues and define the role of NLRP3 in the development of colitis. This doctoral research thesis consists of seven chapters. Chapter 1 is an overall introduction to the rationale of the research, hypothesis and aims of the research project. Chapter 2 is a review article that critique the literature and summarises the role of NLRP3 in intestinal inflammation and CAC. Chapter 2a is a detailed description of the Winnie mouse model and methodology of generation and genetic confirmation of the novel mouse model Winnie x Nlrp3\\(^{-/-}\\). Chapter 3 is a review article on background of current experimental NLRP3 inhibitors that have been investigated in IBD experimental models and the potential mechanism of action of these inhibitors. Chapter 4 is a research article that describes the experiments and data generated that detail the effect of MCC950, a specific small molecule inhibitor of NLRP3 inflammasome on colonic inflammation in spontaneous colitis Winnie mice. Chapter 5 details research experiments and data generated that characterise clinical parameters, histopathology, biochemical and cytokine profile of the novel mouse model Winnie x Nlrp3\\(^{-/-}\\) mouse model. In chapter 6, I further explored the molecular mechanisms that lead to CAC by investigating the hyperactive molecular pathways by means of analysing the protein and gene expression, metabolomics and microbiota changes in the CAC mouse model Winnie x Nlrp3\\(^{-/-}\\). Finally, chapter 7 is a comprehensive discussion of all the results obtained from the research objectives and includes potential directions for future work arising from this research thesis. Current treatment regimen for IBD utilise anti-inflammatory drugs, immune system suppressors and antibiotics or a combination of these. However, these therapeutics lead to several adverse effects, remission or significant non-responsiveness leading to colectomy which is an emergency surgery with a high rate of morbidity. Thus, there is an urgent need to develop potent drugs with novel mechanisms of action. Given the evidence that aberrant NLRP3 activation is involved in the progression of IBD, targeting the activation pathway is a promising strategy for the development of novel effective therapeutics for IBD. Therefore, I reviewed past literature and discussed all the experimental NLRP3 inhibitors that has been investigated in IBD experimental animal models. The most salient finding of the review article was that all experiments were conducted in chemically induced colitis models with inhibitors that were nonspecific to the NLRP3 inflammasome and therefore was unable to specifically define its role towards IBD. Therefore, I investigated the therapeutic effect of a NLRP3 inhibitor, MCC950 in a spontaneous chronic colitis mouse model Winnie. Extensive studies in Winnie have proven it to be an appropriate murine model to study IBD and its pathogenesis. MCC950 is a potent, highly specific small molecule inhibitor of both canonical and noncanonical activation of NLRP3 inflammasome and has been evaluated in a multitude of NLRP3 driven inflammatory diseases. However, the effect of MCC950 on colitis has not yet been reported. To my knowledge this is the first time a specific NLRP3 inhibitor has been applied to colitis and I was able to determine the contribution of anti-inflammatory effects resulting exclusively from inhibition of canonical and non-canonical NLRP3 inflammasome activation in colitis. Mice were orally administered with 40 mg/kg of MCC950 for three weeks at chronic stage of colitis. The treatment significantly ameliorated colitis with improved body weight gain, colon length, ratio of colon weight to body weight, and disease activity index. Histopathological scores of MCC950 treated Winnie mice were significantly reduced suggesting not only attenuation of ongoing colitis but also delay of disease onset. MCC950 significantly suppressed IL-1˜í‚⧠and IL-18 cytokine expression at both mRNA and protein levels in Winnie colons. Additionally, MCC950 also effectively suppressed the release of proinflammatory cytokines (IL1-˜í¬±, IL17, TNF-˜í¬± and IFN-˜í‚â•) and chemokine (MIP1a) in mucosal explants. Moreover, MCC950 treatment resulted in a significant decrease of IL-1˜í‚⧠release and activation of caspase-1 in Winnie explants and in vitro macrophage cells isolated from these mice. Taken together, the results illustrate the efficacy of MCC950 in the treatment of murine ulcerative colitis and provides a potential avenue for a novel therapeutic agent for human inflammatory bowel diseases. With the successful discovery of the therapeutic potential of the specific NLRP3 inhibitor MCC950, I hypothesised that the absence of NLRP3 inflammasome in the spontaneous colitis mouse model Winnie would ameliorate colitis. To conduct this work, I generated a novel mouse model by knocking out the Nlrp3 gene in Winnie, with a defined microbiota, to elucidate the functional role of NLRP3 inflammasome in colitis. Interestingly, detailed phenotypical analysis of Winnie x Nlrp3\\(^{-/-}\\) colon at 12 and 16 weeks showed spontaneous multiple colonic tumours. Winnie x Nlrp3\\(^{-/-}\\) mice had significantly shorter colons, and a higher ratio of colon weight to length and colon weight to body weight compared to control groups indicating the severity of colitis and tumorigenesis. Histopathology of Winnie x Nlrp3\\(^{-/-}\\) colon revealed severe crypt distortion and goblet cell depletion with high-grade dysplasia and invasive carcinoma regions. Analyses of colonic tissue homogenates by biochemical assays showed increased activity of myeloperoxidase, Nitric Oxide and serum C-reactive protein consistent with human CAC. RNA was extracted from colonic tissue segments converted to cDNA and analysed for proinflammatory and cancer biomarker gene expression using PCR microarray. Upregulated biomarkers Bcl2, Sod2, Ppar˜í‚â•, Myc, Birc5 and Cdk2 were confirmed by individual qPCR. Colonic organ cultures were performed, and the supernatants were assayed via Bio-Plex and results identified differential expression of proinflammatory cytokines, chemokines and cancer biomarkers. Detailed immunohistochemistry revealed high-grade dysplasia and adenocarcinoma regions with increased expression of DNA damage biomarkers anti-Oxoguanine 8, anti-gamma H2A.x and oxidative stress biomarkers anti-NQ01, anti3-Nitrotyrosine. Immunofluorescence for Ki-67, VEGF and Survivin biomarkers showed an increased expression indicating cell proliferation, angiogenesis, and anti-apoptotic activity respectively, validating Winnie x Nlrp3\\(^{-/-}\\) as a CAC model. Protein analysis of colonic tumours and Western blot results showed upregulation of Wnt/˜í‚â§-catenin and PI3K/ AKT pathways as the potential molecular mechanism of CAC. Faecal microbiota analysis revealed significant increase in colitogenic members such as Akkermensia muciniphila in the phylogenetic architecture in Winnie x Nlrp3\\(^{-/-}\\) mice while metabolomics profiling revealed upregulation of key metabolites and significant decrease of beneficial short chain fatty acids. These results provided confirmation that NLRP3 is a negative regulator of tumorigenesis during CAC. In summary, the study has generated new data and knowledge that defines the NLRP3 inflammasome as a double-edged sword in colitis and CAC. Specific chemical inhibition of an over active NLRP3 inflammasome in chronic colitis attenuated severity of the disease whereas genetic ablation of NLRP3 gene in the same colitis model lead to CAC. This highlights the critical function of NLRP3 inflammasome as an innate immunity guardian in the maintenance of gut homeostasis. Finally, the results stress the importance of evaluating the pharmacokinetics and long-term effect of novel NLRP3 inhibitors designed for chronic inflammatory diseases in clinically relevant experimental models before progr...