L’homéostasie des muqueuses intestinale et cutanée dépend des interactions complexes entre le microbiote, l’épithélium et le système immunitaire de l’hôte. Des mécanismes régulateurs divers coopèrent afin de maintenir l’équilibre physiologique, et un défaut dans ces mécanismes entrainent des situations pathologiques. Le glucagon like peptide 2 (GLP-2) est un neuropeptide caractérisé par des propriétés prolifératives et anti-inflammatoires. Le potentiel thérapeutique des analogues de GLP-2 est actuellement évalué dans des essais cliniques pour des maladies digestives. Les effets du GLP-2 dans l’intestin sont médiés par son récepteur GLP-2 receptor (GLP-2R). Malgré la présence des nombreuses études évaluant l’expression cellulaire et la distribution tissulaire du GLP-2R, celles-ci restent controversées, que ca soit chez l’homme ou les rongeurs. Il est admis que l’expression du GLP-2R était confinée au tube digestif, principalement à l’intestin proximal, malgré des études évoquant une expression extra-intestinale. Une meilleure compréhension de l’expression et de la distribution de GLP-2R est nécessaire pour élucider les fonctions biologiques de GLP-2. Nous avons réalisé une cartographie de l’expression de GLP-2R dans différents organes murins ainsi que dans des lignées immortalisées humaines et murines. Nous avons également évalué l’expression intestinale du GLP-2R dans 2 modèles de colites induites chez la souris et dans des biopsies intestinales des patients atteints de maladies inflammatoires chroniques de l’intestin (MICI). Nous avons démontré que GLP-2R était exprimé en dehors du tube digestif notamment dans la vessie, le système nerveux central, le mésentère, les ganglions mésentériques, la rate et le foie. Nous avons également montré que la plus forte expression de GLP-2R dans le tube digestif était détectée dans le colon proximal et le rectum. Dans les modèles murins de colites et chez les patients atteints de MICI, l’expression du GLP-2R était diminuée, notamment dans les zones inflammatoires. Ceci pose la question le rôle physiopathologique des analogues de GLP-2 dans les maladies inflammatoires digestives. En conclusion, les hypothèses précédentes considérant une expression du GLP-2R majoritaire dans le tube proximal doivent être reconsidérées. Les fonctions physiologiques extra-intestinales du GLP-2 doivent être explorés afin d’anticiper des effets indésirables extra-digestifs des analogues de GLP2. / Intestinal and skin physiologies are quite similar as far as the homeostasis in both depends on complex interactions between the microbiota, the epithelium and the host immune system. Diverse regulatory mechanisms cooperate to maintain the equilibrium, and a breakdown in these pathways may precipitate pathological conditions. Glucagon like peptide 2 (GLP-2) represents one of the hot topics in research in intestinal physiology. Its dual function as an anti-inflammatory agent and growth factor has led to its consideration in therapeutic strategies and GLP-2 analogs are currently in clinical trials for several digestive diseases. The integrative responses to GLP-2 are mediated via the GLP-2 receptor (GLP-2R). Despite extensive research, precise tissue distribution of GLP-2R expression remains controversial both in rodents and humans. It is widely believed that GLP-2R expression is restricted to the gastrointestinal tract, mainly to the proximal bowel, despite the presence of few studies reporting extra-intestinal expression. Thus, to enhance our knowledge concerning the potential functions of GLP-2 analogs, a better understanding for GLP-2R expression is considered necessary. We therefore realized a panel of GLP-2R expression in mice tissues and in several human, murine and rat cells lines. Given the therapeutic beneficial effects of GLP-2 analogs in intestinal disorders, we investigated the intestinal expression of GLP-2R in mice models of chemically-induced colitis and in inflammatory bowel disease (IBD) patients. We demonstrated that GLP-2R is more widely expressed than expected with significant expression in several mice tissues including bladder, central nervous system, mesenteric adipose tissue, mesenteric lymph nodes, spleen, and liver. We also showed that the expression of GLP-2R in the gastrointestinal tract follows an increasing gradient toward the distal gut with highest expression in the colon and rectum. Interestingly, the intestinal expression of GLP-2R is significantly decreased in experimental mice models of colitis and in IBD patients which raised the point of the physiological role of GLP-2 analogs in digestive disease patients. Overall, previous hypotheses limiting GLP-2R expression and function to proximal bowel need to be revisited, and further studies should address the extra-intestinal biological function of GLP-2.

Introduction. Inflammatory bowel disease (IBD) is a condition affecting more than 3 million people in Europe and the USA combined. Patients report pain as one of the most severe and debilitating symptoms leading to a lower quality of life. Current analgesics lack efficacy for the treatment of visceral pain or produce unacceptable side effect profiles. New targets are needed. Aims and methods. The aim of this thesis was to examine the activation of primary visceral afferents in C57BL/6 mice in response to biopsy supernatants from paediatric patients with IBD (Crohn's disease and ulcerative colitis) and functional abdominal pain syndrome (FAPS). By comparing the expression of pro-inflammatory and pro-nociceptive mediators in these biopsy samples with patient pain scores and afferent nerve recording activation, we identified putative mediators likely to be responsible for causing pain. The ability of inflammatory mediators to drive visceral nociception was then examined by their exogenous application in recordings of mouse and human visceral nociceptor activity. Results. Nerve activation increased significantly in response to biopsy supernatants from FAPS, CD, and UC patients, when compared to controls. Supernatant IL-8, TNFα, IL-6 and IL-1β, levels were increased in IBD samples compared with control patients. Analysis of mRNA expression also showed high levels of pro-inflammatory cytokines and raised MMP-1, MMP-3, MMP-9, MMP-12, and MMP-19 in IBD samples. The expression of MMP-12 in biopsy samples from Crohn's patients significantly correlated with afferent firing suggesting a causative role. This was confirmed by exogenously application of MMP-12 stimulated afferent firing and sensitised responses to mechanical stimulation and inflammatory mediators. UC samples showed TIMP-1 as an effective inhibitor of afferent firing. Conclusion. Data from this study demonstrates that the bowel of patients with IBD and FAP releases pro-nociceptive mediators which stimulate visceral afferents. MMP's play an important role in the afferent activation mediated by IBD samples suggesting that exploiting the endogenous inhibitor TIMP-1 could be a key target for future therapeutic strategies.

Background: Not only does IBD lead to nutritional deficiencies, but also nutrients influence its pathophysiology: exclusive enteral nutrition (EEN) is an effective primary treatment in Crohn's disease; and vitamin D (VitD) is involved in its pathogenesis and course. Aims: We hypothesised that nutrients impact on the course of IBD. We therefore studied the effect of EEN i) on long term clinical course in children; ii) on CD58, a costimulatory molecule at the intestinal epithelial cell (IEC) lines, iii) adults with Crohn's disease. We examined the possible effect of serum vitamin D levels on the course of IBD and also the possible role of ethnicity in our paediatric and adult populations that were treated with EEN but also in our general adult population. Results Chapter II: 56 paediatric patients with Crohn's disesase were followed up for 5 years. 57% of patients achieved remission after 6 weeks of EEN. Achievement of clinical remission within 6 weeks of EEN was significantly associated with a longer time to relapse and to treatment escalation. VitD deficiency was common; and those patients who were deficient were significantly more likely to require corticosteroids and also needed thiopurines sooner. Chapter III: CD58 was expressed in the IEC isolated from IBD patients and healthy controls. EN down-regulated the expression of CD58 on IEC lines. Chapter IV: 22 adult patients with Crohn's disease with a mean age of 30.8 years were given EEN and followed up for a mean time of 1.9 years. 22.7% of patients went into clinical remission and 77.3% experienced a clinical response. By the end of follow up 63.6% (14/22) of patients had clinically relapsed and 36.4% required surgery during their follow up. There was no difference between South Asian and Caucasian patients in the disease outcomes after administration of EEN. Chapter V: Bangladeshis were more often vitamin D deficient than white Caucasian patients; however vitamin D status was not associated with the course of IBD. Bangladeshis developed perianal disease and required thiopurines earlier in their disease course. Bangladeshi patients with UC had more extensive disease. Conclusions: EEN, when successful, improves the long term outcome of Crohn's disease in children, possibly in part, by down-regulating CD58 on the IEC. VitD deficiency may influence the clinical course of IBD; however our results were contradictory between children and adults and significantly limited by the assessment of the vitamin D level at a single time point.

INTRODUCTION: Reactive oxygen species are responsible for the mediation of physiologic and pathologic cellular responses. The tissue damage occurring in all inflammatory disorders, including that observed in patients with inflammatory bowel disease (IBD), is mediated by reactive oxygen species (ROS) generated and released by activated immunocompetent cells. When present in sufficient concentration, these oxidative ROS are toxic to both real or perceived infectious or allergic threats, as well as native tissues in the context of autoimmune disease. The diagnosis and interval assessment of patients with IBD currently rely on expensive and invasive procedures that create cost and logistic drawbacks for both patients and the larger health care system. Thus, there is a pressing need for the development of reliable, cost-effective, and noninvasive methods to better diagnose and manage patients with IBD. OBJECTIVES: The goal of this present study was to assess the relationship between disease activity and ambient oxidative state in the stool of patients with and without IBD. METHODS: Patients admitted to Boston Children’s Hospital (Boston, MA) were recruited and consented for participation in the study. Stool samples were collected, and the redox potential (mV) was assessed using three different redox status measuring systems. The samples were collected between November 2018 and March 2020. RESULTS: Data demonstrated that reliable measurements could be made of redox status in stool samples collected from patients with and without IBD. Data collected from patients with IBD displayed an inverse correlation between relative redox status and disease activity. CONCLUSION: The measurement of relative redox status in the stool of patients with and without IBD is a reliable tool for indicating clinical disease status. Furthermore, the initiation of an improved method for the collection and processing of stool samples from consented patients appears to increase study accrual and data collection. Data from this study can be used as the basis for future studies that assess the clinical impact of pharmacologic, lifestyle, and dietary approaches to managing fecal redox in patients with IBD.

Ulcerative colitis (UC) and Crohn’s disease (CD) are the two common forms of inflammatory bowel disease (IBD). They share similar clinical and demographic features as well as harbor key differences in tissue damage and prognosis. Previous studies indicated that they contributed to the increased rick to Colorectal cancer (CRC). However, whether UC and CD share inflammatory signatures still remains controversial. In addition, no inflammatory signatures have been reported on CRC. To answer these questions, a comprehensive study has been conducted on collected microarray datasets. Our analysis suggests that although CD and UC share common inflammatory pathways, they also present difference. Especially, CD patients are likely to have type I response, while UC patients are inclined to undergo type II response. Pathway enrichment analysis on CRC uncovered two potential CRC-specific inflammatory pathways.

There is an unmet need for novel biomarker discovery in Inflammatory Bowel Diseases (IBD) to aid clinical management in several clinical settings including diagnosis and prognosis. With an ever-advancing repertoire of biological therapies on the horizon, it is important to personalise treatments at an early stage. The aim of this thesis is to explore the clinical utility of novel blood-based biomarkers in diagnosis, disease classification and prognosis in 2 cohorts: newly diagnosed IBD and acute severe colitis. Investigating the circulating methylome, 290 probes exhibited Holm significant IBD-associated methylation differences, including VMP1/MIR21 (p=7.5×10-14) and RPS6KA2 (1.1×10-19) and were consistent within the European cohort. 11 Differentially methylated positions (DMPs) predicted treatment escalation after Holm adjustment (top probe p=0.003). A panel of 6 probes identified 2 patient subgroups that have significantly different disease courses (Hazard Ratio (HR) 10.5, 95%CI: 4.3-25.6; logrank p=1.5×10-24). The 6 probe marker outperformed conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7-5.8), logrank p=0.0004 and Alb < 36g/L, HR 2.9(1.5-5.6), p=0.0001). Within the same cohort, a novel proximity extension assay (PEA) was then utilised to identify novel diagnostic and prognostic protein markers. 61 proteins were significantly associated with IBD including MMP12 (Holm-adjusted p=4.1×10-26). A total of 9 proteins predicted disease course in this cohort. Using a panel of 7 randomly selected top prognostic probes, 2 patient groups were identified that had significantly different disease courses: logrank p=2.2×10-10, HR 5.6(2.0-15.6), outperforming conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7- 5.8), logrank p=0.0003 and Alb < 36g/L, HR 2.7(1.4-5.2), p=0.0004). In a subcohort, serum calprotectin (SC) and conventional blood markers were investigated for their utility in diagnosis and prognosis in IBD. SC performed at par with CRP at differentiating IBD from controls with an area under the curve (AUC) of 0.87 (CI 0.81-0.92). For prognostication, both albumin and SC remained significant predictors of treatment escalation in IBD (logrank test p=5.1×10-5). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. Using small RNA sequencing in acute severe colitis (ASUC) and healthy controls (HC), 10 serum-based miRNA markers were significantly associated with acute severe colitis, including miR-30a-5p. Validating the findings using qPCR, miR-30a-5p was downregulated in ASUC (p=0.003). Furthermore, miR30a-5p remained a significant predictor of eventual colectomy in acute colitis (logrank test p=0.0014). These data highlight the translational potential for methylation, miRNA and proteomic biomarkers in diagnosing and prognosticating in IBD.

Epigenetic alterations including DNA methylation and microRNAs may provide important insights into gene-environment interaction in complex immune diseases such as inflammatory bowel disease (IBD). An integrative genome-wide approach was used to analyse whole blood genetic, DNA methylation and gene expression data in 240 newly diagnosed IBD patients and 190 controls. Using the Illumina 450k array, differences in whole blood DNA methylation were observed in IBD cases versus controls including 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs). The top DMP (RPS6KA2, discovery Holm adjusted p=1.22×10-16, replication p=1×10-9) and DMRs (VMP1, ITGB2, TXK) were replicated in an independent cohort using pyrosequencing. Paired genetic and epigenetic data allowed the identification of methylation quantitative trait loci (meQTL); two of the five DMRs (VMP1, ITGB2) demonstrated significant association with genetic polymorphisms. Methylation in the VMP1/microRNA-21 region was significantly associated with two single nucleotide polymorphisms (cg18942579 -rs10853015 [meQTL FDR adjusted p=9.4 × 10-5], cg16936953 - rs8078424 [meQTL FDR adjusted p=8.8 × 10-5]), both of which are in linkage disequilibrium with a known IBD susceptibility variant (rs1292053). Separated leukocyte methylation data highlight the cell type of origin of epigenetic signals seen in whole blood. IBD-associated hypermethylation within the TXK gene transcription start-site negatively correlated with gene expression in whole blood and CD8+ T-cells, but not other cell types, highlighting that cell-specificity and gene location-specificity of DNA methylation change is critical when associating methylation and gene expression. These data offer significant translational potential as diagnostic biomarkers. Least absolute shrinkage and selection operator (lasso) modelling identified 30 methylation probes can be used to accurately discriminate IBD cases from controls (Area under receiver operating characteristic curve = 0.898, sensitivity = 90.6%, specificity = 84.7%). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. MiRNAs have been increasingly implicated in many of the important IBD pathogenic pathways including autophagy, intestinal epithelial barrier integrity and the Th17 pathway. In common with all epigenetic mechanisms, miRNA expression is dynamic and cell-specific. Small RNA sequencing (RNA-seq) was performed on RNA extracted from CD14+, CD4+ and CD8+ cells isolated from 8 newly diagnosed cases of ileal or ileocolonic CD and 8 age and sex matched controls. There was a median of 2.4 million reads per sample (range 132,800-12.8 million reads per sample). One microRNA was differentially expressed in CD compared with controls (hsa-miR-503-5p log fold change = 0.7, FDR adjusted p = 9.1 × 10-5) in CD4+ lymphocytes, however this finding did not remain significant when alternative normalisation methods were used. The small number of cases used in microRNA analyses raises the possibility of both type I and II error, and limits the ability to draw firm conclusion from this series of experiments. Site-specific differences in DNA methylation in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression. This is the most detailed characterisation of the epigenome carried out in IBD to date. The findings strongly validate this approach in complex disease, are replicable, and provide clear translational opportunities.

Inflammatory Bowel Disease (IBD) is a debilitating, life- long disease that affects about 1.4 millions Americans. Little is known about the pathogenesis of IBD and an effective cure still remains to be discovered. While there are numerous T cell targeting therapies for IBD, more research is still needed. Bispecific T Cell Engagers, BiTES, is a modified protein capable of engaging two antigens simultaneously; it is capable of activating T cells by circumventing the MHC protein molecule. This provides an alternative to the current molecular therapies for IBD. In addition to monoclonal therapy research, there has been a plethora of research on immunomodulatory molecules, such as β- glucan. The benefit of β-glucan has been shown with supplements and food sources alike in animal models. In this study, we used BiTES, CMPD-1, with an anti-CD3/ Dectin-1 epitopes capable of engaging T Cells and β-glucan in beads and fungi cell wall. CMPD-1 is capable of engaging Splenic and Lamina Propia T Cells from a C57BL/6 mice. Likewise, CMPD-1 engaged T cells to hyphae of C. albicans and A. fumigatus, which have a higher concentration of β-glucan than in the candida form. The data show a delayed in hyphae growth in yeast with CMPD-1 and a decrease in yeast growth for the first four hours when compared to non- BiTES molecules. Additionally, qualitative analysis of CMPD-1 shows a decrease A. fumigatus growth after a 72-hour incubation period. Splenic T cells from mice lacking Dectin-1 and Wild-type (WT) mouse strains where incubated with BiTES compound and yeast for 23 hours followed by a PrestoBlue killing assay to assess yeast cell viability. The PrestoBlue assay showed that CMPD-1 killed more A. fumigatus in both T cell subsets; although, the difference lacked statistical significance. The applications of this molecule as a therapeutic agent for IBD are promising, although, still in its infancy. An alternative use for this molecule is to train the immune system with the BiTES molecule in conjunction with β-glucan supplements to build immunity against opportunistic pathogens such as A. fumigatus and C. albicans that often cause havoc in IBD patients as a result of the changes in microbiota, and compromised integrity of the GI tract. / 2017-06-16T00:00:00Z

Introduction: Inflammatory bowel disease is a common, complex relapsing disorder characterised by immune dysregulation, altered intestinal permeability and microbial insult. Limited knowledge is available regarding the metabolic changes observed during progression of the disease, and limited biomarkers of disease available that have been validated and shown to be of sound clinical value. Aim of Study: A two stage metabolomics approach was adopted to determine if metabolic signature profiles, could distinguish inflammatory bowel disease Crohn’s disease (CD) patients from ulcerative colitis (UC) patients and from healthy controls. Methods: A serum metabolomics approach was undertaken to define metabolic changes associated with UC and CD. Serum samples from a discovery study of 30 UC, 30 CD and 29 ethnically, age and gender matched controls were analysed by ultra-performance liquid chromatography mass spectrometry. A subsequent validation study was preformed using 28UC, 31CD, and 29 gender matched controls were also analysed using UPLC-MS.ResultsClasses of metabolites, identified as biologically interesting and at significantly different levels (p<0.05) in comparisons of control and CD and UC cohorts included: steroids and steroid derivatives, phosphocholine, Vitamin D metabolites, fatty acids and conjugates, glycerolipids, isoprenoids, amino acids, and phosphosphingolipids. There were fewer discriminatory metabolites differentiating the CD and UC cohorts. Conclusion: Serum Metabolomic profiling may represent a novel technology which could be used to distinguish individuals with CD from those with UC and healthy controls.

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