Gut-associated lymphoid tissues such as Peyer’s patches (PP) are inductive sites for immune response in the intestine. Unlike other peripheral lymphoid tissues, gut-associated lymphoid tissues lack afferent lymphatics and can directly sample mucosal antigens by specialized epithelial cells in the follicular associated epithelia (FAE), known as M cells. M cells derive from Lgr5+ intestinal stem cells in intestinal crypts, where the daughter cells of Lgr5+ cells differentiate into M cells after stimulation from the cytokine receptor activator of nuclear factor-κB ligand (RANKL). RANKL is produced by stromal cells within the sub-epithelial dome (SED) residing below the FAE. The transcytosis of antigens across the FAE by M cells is an important initial step in the induction of efficient mucosal immune responses against certain pathogenic bacteria as well as the commensal bacterial flora. However some pathogens, for example orally-acquired prions, may also exploit M cells to infect the host. M cells have been implicated in the uptake of orally acquired prions from the gut lumen. After oral exposure, the accumulation of prions in PP is important for their efficient spread to the nervous system. Previous studies have also shown that pathogen-induced inflammation increases M cell density and this effect can be mimicked by exogenous administration of RANKL. This has led to the hypothesis tested in this thesis that inflammation-related enhancement of M cell differentiation aids the delivery of prions into the lamina propria of villi. The administration of RANKL resulted in increased M cell density in the gut epithelium of mice. Consequently, RANKL treatment enhanced the accumulation of orally-administered prions in PP, decreased disease incubation time and increased prion disease susceptibility. These data indicate the importance of M cells in prion disease pathogenesis and highlight the potential of M cells as vaccine targets against prion disease. The fate and terminal differentiation of distinct intestinal epithelial cell lineages from their uncommitted precursors is dependent on their intrinsic expression of one or more specific transcription factors during their development. Alongside inducing M cell differentiation, RANKL stimulation can also induce the nuclear translocation of the NF-κB transcription factor subunit c-Rel. A comparison of the genes encoding the individual NF-κB subunits c-Rel, Rel-A and Rel-B revealed that they were expressed at the mRNA level in the FAE and by M cells. A c-Rel-deficiency in mice did not influence the expression of RANKL or RANK in PP. The subsequent induction of M cell maturation in the FAE was also unaffected in, indicating that c-Rel is dispensable for the RANKL-mediated differentiation and functional maturation of M cells. The factors implicated in Lgr5+ intestinal stem cell proliferation and their differentiation into M cells are poorly understood. Some reports have indicated that crypt-associated macrophages may provide extrinsic factors that assist Lgr5+ intestinal stem cell proliferation. In this thesis, the ablation of macrophages in the gut resulted in dysregulation of crypt microarchitecture, depleting Paneth cells and the Lgr5+ stem cells. This adversely affected the subsequent differentiation of intestinal epithelial cell lineages and impeded the functional development of M cells. These data reveal a previously unknown role for macrophages in the maintenance of intestinal crypts and intestinal stem cell proliferation and differentiation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:738707 |
| Date | January 2017 |
| Creators | Sehgal, Anuj |
| Contributors | Mabbott, Neil ; Donaldson, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/28738 |
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