Human primary organoid-derived epithelial monolayers as a novel strategy for the study of adherent invasive escherichia coli pathogenicity and the effects of postbiotics on intestinal epithelial function

Resumen

Inflammatory bowel disorders – such as Inflammatory Bowel Disease (IBD) – are rising worldwide. A well-known feature of IBD is dysbiosis of the gut microbiota, characterized by a significant reduction of beneficial strains and a sharp increase in facultative anaerobes, as is the case of Adherent Invasive E. coli (AIEC). Even though the implication of the microbiota in persistent inflammation has been studied for years, a direct causal relationship between dysbiosis and IBD has not been established. To date, several strategies, such as the use of probiotics, have been proposed to counteract this condition. Nevertheless, probiotics are thought to impair the return of the indigenous microbiome, and to aggravate inflammation in immune compromised patients. Recently, postbiotics – bacterial-free metabolites secreted by probiotic strains – have been proposed as a better and safer strategy to counterbalance the effects of intestinal inflammation. The intestinal epithelium is the first layer the luminal bacteria interact with. It is composed by a thin monolayer of cells that form a protective barrier against potential detrimental antigens along the whole gut. Thus, the study of epithelial responses to bacteria or their derived metabolites is of great importance to understand intestinal health and disease. Recent advances in primary epithelial cell culture using intestinal organoid-derived cells as starting material offers a more faithful representation of the human gut compared to immortalized cell lines. In the first study of this thesis, we describe for the first time the use of a primary epithelial bidimensional (2D) model derived from freshly isolated human intestinal crypts as a successful tool to study AIEC infection and its effects on the host epithelium at early and extended infection periods. We proved that this ex vivo culture adopts an appropriate cell polarization and orientation, thus becoming a promising resource to study the interactions of the luminal content with host epithelial cells. Importantly, we show a strain and timespecific response of primary human intestinal epithelial cells when infected with AIEC and non-AIEC strains thus supporting the use of our system to study the functional consequences of AIEC infection on the intestinal epithelium. This 2D primary cell culture system derived from intestinal organoids was also employed in the second part of this thesis to explore the putative beneficial properties of postbiotics in the epithelial response to inflammatory stimuli. Transcriptomic analysis of 2D cultures pretreated with postbiotics points towards an effect of the metabolite-cocktail in reverting the inflammatory status of the intestinal epithelium. Moreover, postbiotics induced the differential expression of several genes on intact primary epithelial cells, demonstrating their potential in contributing to maintenance of homeostasis. Overall, the human primary organoid-derived monolayer culture provides a promising tool for elucidating the potentially detrimental or beneficial mechanisms underlying the crosstalk of bacteria and its metabolites with the intestinal epithelium. Moreover, the capacity of this culture to mimic the pro-inflammatory environment in vitro, may expand its use in modeling bacteria-host interactions in the context of intestinal inflammatory disorders, such as IBD.