Interactions between commensal obligate anaerobes and human intestinal cells : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Manawatu, New Zealand

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Massey University
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The human intestinal epithelium is formed by a single layer of epithelial cells which regulates intestinal barrier permeability. Increased permeability can result in the entry of potentially harmful compounds into the body, and is implicated in autoimmune, inflammatory and atopic diseases. The intestinal tract is inhabited by an estimated 1014 microbes and it is increasingly evident that they affect intestinal barrier function. However, over 90% of commensal intestinal bacteria are obligate anaerobes, making it difficult to co-culture them with oxygen-requiring mammalian cells in vitro. To investigate the interactions between obligate anaerobes and epithelial cells that regulate the intestinal barrier, an apical anaerobic model of the human intestinal epithelium, which utilises a dual-environment co-culture chamber, was developed and validated. The chamber allowed for polarised monolayers of the intestinal cell line Caco-2 to be grown such that the apical (luminal) side was exposed to an anaerobic environment, while maintaining an aerobic basal side. The cell viability and barrier function of Caco-2 monolayers was unaffected by culture in the apical anaerobic model for at least 12 hours. Global gene expression analysis predicted upregulation of cell survival and proliferation in Caco-2 cells cultured in the apical anaerobic model, compared to Caco-2 cells grown under conventional conditions, suggesting an adaptation of the Caco-2 cells to a lower supply of oxygen. The apical anaerobic model was used to co-culture the commensal obligate anaerobe Faecalibacterium prausnitzii with Caco-2 cells. The survival of F. prausnitzii was improved in the anaerobic apical environment compared to when cultured in an aerobic atmosphere. Live F. prausnitzii, but not non-viable (UV-killed) F. prausnitzii, were shown to increase permeability across Caco-2 monolayers. Furthermore, global gene expression analysis suggested that live F. prausnitzii cells have more profound effects on Caco-2 cells than non-viable F. prausnitzii, illustrating the importance of maintaining viability of obligate anaerobes in an in vitro co-culture system. The apical anaerobic model can be used to gain insights into the mechanisms of crosstalk between commensal obligate anaerobic bacteria and intestinal cells, and new knowledge generated using this model will assist in the development of strategies to improve intestinal barrier function.
Epithelial cells, Anaerobic bacteria, Intestines, Microbiology