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Title: Modelling barrier function of the villous and follicle-associated epithelium in 3D
Author: Thompson, Louise Miranda
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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The intestinal epithelium acts as a barrier that excludes external molecules from the tissues beneath. Defects in this barrier function are increasingly associated with pathological states, including inflammatory bowel disease, irritable bowel syndrome, coeliac disease, obesity and food allergies. Understanding of epithelial biology and barrier function has been limited by a lack of physiologically representative in vitro models. Identification of intestinal epithelial stem cells and recent methodological advances have allowed intestinal stem cells to be cultured so that they proliferate, differentiate and self-assemble to mimic in vivo tissue architecture, forming a continuous epithelium around a hollow central lumen, known as an enteroid. The initial aim of this study was to evaluate the extent to which mouse small intestinal enteroids simulate their parent tissue. The second intention was to investigate whether this system can be used to model barrier function of both villous epithelium and specialised areas of epithelium overlying gut-associated lymphoid tissues, where antigen sampling occurs due to the presence of rare phagocytic M cells. Enteroids were shown to contain all major epithelial cell lineages and to retain region-specific differences in the distribution of cell types that are observed in vivo. An assay based on leakage of a fluorescent was developed to allow investigation of epithelial barrier function in 3D enteroids. Peptide fragments of dietary gliadin were used to probe the ability of enteroids to model barrier function, since they are involved in the pathogenesis of coeliac disease and have been linked to increased epithelial permeability. A peptide spanning α-gliadin amino acids 56-68 induced rapid, dose-dependent luminal fluid secretion via activation of an apical chloride channel, the cystic fibrosis transmembrane regulator. In contrast, a second peptide had no effect on fluid secretion. Regarding modelling the follicle-associated epithelium, it was confirmed that M cell differentiation is stimulated by treatment of enteroids with the cytokine RANKL. Alternative pathway NF-κB signalling was required, as M cell-associated gene expression was not upregulated in RelB- or NF-κB2-deficient enteroids. As previously suggested, co-culture with B cells also appeared to promote M cell differentiation, although their presence was insufficient to induce mature M cells. Finally, plasmid constructs were generated to express a fluorescently labelled bacterial surface adhesion molecule for microinjection into the enteroid lumen. As a tool, this will allow modelling of bacterial adhesion to, and translocation by, enterocytes and M cells. This work supports a growing body of evidence showing that enteroids accurately reproduce in vitro many characteristics of the small intestinal epithelium. It is demonstrated that enteroids can be used to model barrier function, fluid secretion and the role of epithelium-intrinsic factors in differentiation of a rare cell lineage. Further assay developments will likely result in more widespread use of enteroids for various applications, including basic research, therapeutic screening and drug development.
Supervisor: Campbell, Barry ; Duckworth, carrie Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral