Multiomic Investigation of the Human Gut Toward Insight into Childhood Inﬂammatory Bowel Disease Pathogenesis
Inﬂammatory bowel disease (IBD) is an abnormal immune response to the gut microbiome causing chronic debilitating pain in genetically susceptible people. The development of eﬀective therapies for this condition is hampered by a poor understanding of its complex aetiology. Progress in this area will require identiﬁcation of both the immune and epithelial contributions to disease in susceptible individuals as well as detangling the contributions of diﬀerent pathways and genes, all in a cell type speciﬁc context. This thesis summarizes my research into the gene regulatory architecture of IBD in intestinal epithelial cells. First I assess the utility of intestinal epithelial organoids, a model of the human intestine. This system is well established and has been shown to be comparable to in vivo intestinal tissue in many ways. However, my study revealed DNA methylation (DNAm) is generally lost and becomes more variable the longer organoids are cultured. My ﬁndings suggest a major impact of prolonged culturing on global organoid DNAm proﬁles, highlighting the importance of considering time in culture in organoid experiments. Next I explore DNAm, gene expression and genotype to: deﬁne a possible IBD biomarker, explain previously established IBD genotype associations, and identify potential mechanistic candidates for functional follow-up. In both primary epithelial cells and organoids I found widespread diﬀerential DNAm with IBD. Of the CpGs diﬀerentially DNAm in IBD, changes in the major histocompatibility complex class I (MHC I) signaling pathway are particularly interesting. The MHC I pathway represents a promising candidate mechanism of IBD as it is involved in the communication between cells and the immune system. Finally, I follow up on the association of MHC I and IBD in individual intestinal epithelial cells. As in the bulk expression data, I also see increased expression of MHC I in IBD compared to controls. This is consistent across cell types, but interestingly MHC I activity is higher in villus tip cells compared to crypt cells regardless of diagnosis, suggesting activity could be related to exposure to luminal microbiota. I also show MHC I can be activated successfully in organoids with proinﬂammatory cytokine stimulation, meaning organoids could be a useful model to study MHC I pathway function in IBD. In this thesis, I demonstrate that organoids are a valuable model of the human gut, and use them to identify MHC I activation as a potential mechanism of pediatric IBD pathogenesis.
Epigenetics, Inflammatory bowel disease, Organoid, single-cell RNA sequencing
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