Development of a Human Primary Intestine Chip and Identification of Species-Specific Microbiome Metabolites Enhancing Enterohemorrhagic E. Coli Pathogenesis

The human intestine and its microbiome play an extremely important role in regulating human pathologies such as inflammatory disease and bacterial infections. Due to the complexity of human studies there has been a great effort in developing in vitro models mimicking the complex human intestinal physiology, such as Gut Chip. The Gut Chip is a microfluidic device that emulates normal tissue-tissue interface and mimics the complex physiological, physical and biochemical environment of a living intestine. This model has been previously established utilizing cell lines, such as Caco-2 or HT-29 cells, that were isolated from tumors. These cells harbor several genetic mutations and have altered expression of some receptors for bacterial toxins (i.e. Shiga Toxin), compared to normal colonic epithelium. Therefore, these Gut Chips do not fully recapitulate normal intestinal functions and would not be applicable to the investigation of important human diseases such as bacterial infections. Thus, in this study, we developed a primary human Small Intestine Chip (Intestine Chip) containing primary endothelial cells and primary epithelial cells isolated from biopsies. Epithelial cells, harvested from intestinal tissue, are expanded as intestinal organoids, dissociated, and cultured on a extracellular-matrix-coated porous membrane, in one channel of the Intestine Chip. The channel laying on the opposite side of the porous membrane is lined with primary human endothelial cells and the Chip is kept under fluidic flow and cyclic deformation. The intestinal epithelium cultured in the Intestine Chip gives rise to villi-like projections formed by polarized epithelial cells that differentiate in several lineages similar to what occurring in intestinal organoids. However, in the Intestine Chip, the apical side of epithelial cells is exposed to an accessible lumen subject to fluidic flow, compared to the closed lumen of intestinal organoids. Transcriptomics analysis indicates that the Intestine Chip more closely mimics human duodenum in vivo compared to intestinal duodenal organoids. Thanks to the fluidic flow, the lumen of the Intestine Chip can be collected and used to measure mucus secretion, nutrients digestion, and intestinal barrier function over an extended period of time. Furthermore, presence of fluidic flow and the separation between vascular and intestinal luminal compartment allow the investigation of the effect of microbiome metabolites and intestinal luminal pathogens. Intestinal pathogens differentially affect different host species and, in some cases, commensal bacteria in a specie can be extremely harmful to a different host. Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic E. coli (EHEC) infection, while mice are relatively resistant. Studying the contribution of the microbiome to this differential susceptibility is difficult due to complex hostpathogen- microbiome interactions. Here, we optimized the Intestine Chip model to include pathogenic bacteria, microbiome metabolites and colonic epithelial cells (Colon Chip), more relevant in the context of EHEC infection. We show that epithelial injury is greater when the Colon Chip is exposed to human microbiome metabolites compared to murine. Using a multiomics approach we identified four specific human microbiome metabolites that are sufficient to mediate this effect. Thus, the greater injury seen in human could be partially explained by the presence of specific microbiome metabolites. These metabolites preferentially induce the expression of flagellin, increase bacteria motility, and worsen epithelial injury. The study described in this thesis provides a new model to investigate colonic infectious disease and the role played by microbiome metabolites. Additionally, it offers new insights in EHEC pathogenesis mediated by human microbiome metabolites, that could partially explain increased susceptibility to EHEC in certain human population, such as children. These findings also provide the basis to therapies aimed at modulating the intestinal content in order to provide protection from life-threatening pathogens