Investigating gene expression and epigenetic regulation in the intestinal epithelium during chronic inflammation.

Willemze, R.A. (Rose) (2013) Investigating gene expression and epigenetic regulation in the intestinal epithelium during chronic inflammation. thesis, Medicine.

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Abstract

Introduction: The intestinal epithelium plays a major role in the gastrointestinal tract as the first line of defence having to balance active protection against bacterial flora and potential toxins, with absorption of nutrients. Inflammatory bowel diseases (IBD) are thought to be, at least in part, caused by an impaired function of this single cell layer. These conditions are characterised by a chronic relapsing pattern of intestinal inflammation. In recent years, epigenetics has emerged as a promising concept with the potential to provide answers to some of the missing links in complex disease pathogenesis including IBD. Hypotheses and Aims: We hypothesise that chronic intestinal inflammation leads to epigenetic alterations in the intestinal epithelium. If these changes become stable, they might alter cellular responses and hence potentially contribute to the development of chronic relapsing inflammation observed in IBD. We therefore aimed to investigate the regulation of gene expression and DNA methylation in the intestinal epithelium during exposure to inflammatory stimuli as well as following a withdrawal period. Materials and Methods: We developed and optimised an in-vitro model to replicate chronic intestinal inflammation by stimulating Caco-2 cells (a colon carcinoma cell line) with inflammatory cytokines (interleukin 1 beta (IL-1), interferon gamma (IFN-) and tumour necrosis factor alpha (TNF-)) over different time periods. Subsequently, gene expression (i.e. mRNA) of four innate immune defence genes was measured with quantitative polymerase chain reaction (q-PCR). The methylation level of selected genes was then investigated by pyrosequencing. Finally, we used DNA methyltransferase (DNMT) inhibitors to investigate the potential role of methylation in regulating gene expression. Results: Comparing Caco-2 cells that had undergone inflammatory pre-stimulation with previously untreated cells, we observed a significant difference in mRNA expression of interleukin 6 (IL-6), interleukin 8 (IL-8), polymeric immunoglobulin receptor (PIGR) and toll-like receptor 3 (TLR 3) in response to treatment with a mix of inflammatory cytokines. Investigating the DNA methylation profile of selected CpG dinucleotides within these genes revealed no differences regardless of whether pre-stimulation was performed or not. However, pre-treatment with the DNMT inhibitor 5-aza-2‘-deoxycytidine (5-AZA-dC) was associated with an altered gene expression of all investigated genes compared to untreated control cells, indicating a potential role of DNA methylation in regulating gene expression in the intestinal epithelium during inflammation. Discussion and Conclusion: The observed changes in intestinal epithelial gene expression in response to inflammatory stimuli following pre-stimulation are exciting and would be in keeping with our initial hypothesis. In spite of the fact that we did not observe any changes in DNA methylation within the selected genes, these results point towards a potential impact of epigenetic mechanisms in regulating gene expression during inflammation. Moreover, demonstrating a significant change in the intestinal epithelial inflammatory response following inhibition of DNMTs further supports this hypothesis. Future work will now focus on identification of the specific epigenetic mechanisms that are involved during chronic intestinal inflammation using a whole genome approach as well as the analysis of patient samples.

Item Type:	Thesis (Thesis)
Supervisor name:	Kallenberg, Prof. dr. C.G.M. and Weersma, R.K.
Supervisor name:	Zilbauer, M. MD PhD MRCPCH and Addenbrooke’s Hospital, Cambridge
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 10:44
Last Modified:	25 Jun 2020 10:44
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/512

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