Abstract
Situated between the intestinal lumen and underlying tissue, the intestinal epithelium acts as a physical barrier, protecting against inflammatory antigens, microbial toxins, and pathogenic attack. Cells that make up the intestinal barrier sample luminal antigens and constantly communicate with the gut microbiota and host immune cells to regulate inflammatory status. Upon detection of pathogenic stimuli, a large and rapid inflammatory response is launched in the gut. Neutrophils are recruited from the vasculature by pro-inflammatory chemoattractants and are first to reach the site of infection. Whilst a strong inflammatory response is crucial to protect the host, perturbations of the balance between pro- and anti-inflammatory pathways in the gut may lead to chronic activation of the immune response and development of inflammatory diseases, such as inflammatory bowel disease (IBD). IBDs, encompassing Crohn’s disease and ulcerative colitis, are debilitating, life-long conditions, which significantly reduce patient quality of life and are associated with high health care costs. Current treatments focus on amelioration of inflammatory symptoms, without treating the underlying cause of inflammation. Additionally, these treatments only benefit a subset of patients, with a significant proportion ultimately submitting for intestinal surgery. In ulcerative colitis, a massive infiltration of neutrophils into the intestinal lumen correlates with intestinal inflammatory symptoms and disease activity. A disrupted gut barrier is another hallmark of IBD. The mechanisms underpinning aberrant intestinal inflammation have not yet been fully characterised.The intestinal epithelium is protected by the efflux pump P-glycoprotein (P-gp), which effluxes toxins and anti-inflammatory endocannabinoids (eCB), including anandamide (AEA) and oleoylethanolamide (OEA). Intestinal inflammation in IBD is associated with reduced expression or function of P-gp. This thesis describes the development and validation of a liquid chromatography-mass spectrometry method to detect eCBs at the epithelial surface. OEA was observed to diminish in line with P-gp in inflamed colonic tissue. To further understand why a consortium of eCBs is secreted, their individual roles at the intestinal epithelium were probed in vitro. AEA was shown to have multiple, distinct roles in maintaining intestinal homeostasis, including promotion of epithelial wound healing and inhibition of neutrophil infiltration. Interestingly, OEA was also found to suppress neutrophil migration, hinting at a complex synergy between eCBs. Preliminary data revealed OEA facilitates epithelial cell polarisation, suggesting a key role in epithelial barrier regeneration. Through western blotting and fluorescent microscopy studies, a tentative link between OEA and regulation of the key junctional proteins occludin and E-cadherin is described. Furthermore, a novel role for OEA in promoting expression of P-gp was observed. These findings highlight important contributions of eCBs in restoring intestinal homeostasis following injury or inflammation.
| Date of Award | 24 May 2023 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Randy Mrsny (Supervisor), A T Lubben (Supervisor) & Shaun Reeksting (Supervisor) |