Venneri, Tommaso (2022) Role of the Endocannabinoidome – Gut Microbiome Axis in the Development of Inflammatory Bowel Disease and Colorectal Cancer. [Tesi di dottorato]

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Background and aim: Inflammatory bowel disease (IBD) and colorectal cancer (CRC) are the two most important pathologies of the gastrointestinal tract. They affect millions of people around the world, with a higher incidence and prevalence in developed countries, and their prevention and treatment represent a global health concern. The recently discovered endocannabinoidome is a diverse and complex system of lipid mediators and their receptors and enzymes that has been reported to play a role in various physio-pathological processes such as inflammation, immune responses, and energy metabolism. The gut microbiota, which contains trillions of different microorganisms, plays a key role in gut immune homeostasis, and recent evidence suggests that an imbalance in its composition is involved in the onset and development of IBD and CRC. The endocannabinoidome (eCBome) and the microbiome (miBIome) are closely related and are thought to form the eCBome – miBIome axis. In this thesis, by modulating either the eCBome or the miBIome, we explore the possible role of this axis in the development of colon inflammation and tumorigenesis. Materials and methods: We investigated the effect of the eCBome – miBIome axis on IBD and CRC, using receptor ligands, enzymatic inhibitors, genetic deletion of receptors and depletion of gut microbiota in chemically induced models of IBD and CRC. Firstly, we examined the effect of cannabidivarin, an agonist of TRPA1 (ion channel belonging to the eCBome) administered via oral gavage or intraperitoneal injection, either with or without a selective TRPA1 antagonist, in both DNBS- and DSS-induced models of IBD measuring inflammatory parameters, such as colon weight/colon length ratio, histological damage, myeloperoxidase (MPO) activity, intestinal permeability, inflammatory cytokines levels, TRPA1 expression levels and gut microbiota composition; in addition, we investigated the effect of CBDV treatment in biopsies from UC paediatric patients on IL-1β production. Secondly, we investigated the synergistic effect of cannabidiol, an inhibitor of FAAH (a serine hydrolase responsible for the degradation of some eCBome mediators), and fish oil (FO, which contains n-3 PUFAs) administered by oral gavage in the DNBS- and DSS-induced ulcerative colitis models, measuring colon weight/colon length ratio, anxiety-like behaviour, disease activity index (DAI) score, MPO activity, intestinal permeability, inflammatory cytokines levels, gut microbiota composition and eCBome lipid mediator content. Thirdly, we investigated the impact of genetic deletion of Trpm8 (member of the TRPs channel family belonging to eCBome) on colon carcinogenesis and associated changes in gut microbiota composition. Finally, we investigated the effects of altering the gut microbiota using antibiotics or germfree conditions in the DNBS-induced models of IBD by assessing inflammatory parameters in the colon (colon weight/colon length ratio, DAI score, inflammatory cytokines, MPO activity) and associated changes in eCBome lipid mediator levels. Results: Oral administration of cannabidivarin by gavage counteracted intestinal inflammation by reducing, in a TRPA1 dependent manner, signs of colitis induced by DNBS or DSS administration, such as colon weight/colon length ratio, MPO activity, intestinal permeability, histological damage and inflammatory cytokine production. It also alters the profile of the gut microbiota and reduces IL-1β in biopsies from paediatric patients with colitis. Co-administration of per se inactive doses of FO and CBD produced anti-inflammatory effects in DSS-treated mice by decreasing colon weight/colon length ratio, MPO activity, DAI score, intestinal permeability, histological damage and inflammatory cytokines production increased by DSS. In contrast, no effect was observed on anxiety-like behaviour induced by DSS. An altered composition of the gut microbiota was demonstrated. The anti-inflammatory effect of co-administration of FO and CBD was also confirmed in the DNBS-induced model. The synergistic effect was neither due to an increase in CBD bioavailability by FO nor to a change in eCBome mediator levels. Mice with a genetic deletion of Trpm8 showed a lower susceptibility to colon cancer development. Deletion of Trpm8 alters the composition of the gut microbiota in both healthy and AOM-treated mice; in AOM treated mice, the deletion of Trpm8 increases the abundance of CRC-protecting families, such as Ruminococcaceae, Lachnospiraceae and Lactobacillaceae, while reducing the abundance of CRC-related families, such as Burkholderiaceae. Finally, germfree conditions, but not antibiotic treatment, affected inflammatory parameters in DNBS-induced colitis by reducing colon weight/colon length ratio and the expression of inflammatory cytokines. This effect was accompanied by changes in eCBome mediators involved in inflammation, such as oleoylethanolamide, linoleoylethanolamide and docosahexaenoylethanolamide. Conclusions: In conclusion, by studying the eCBome – miBIome axis in experimental colitis and colon cancer, we highlight the crucial role of this axis in the development of IBD and CRC and propose it as an innovative target for the treatment of these diseases.

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