Mechanisms underlying the functional regulation of the gut microbiota by intestinal epithelial cells to maintain intestinal homeostasis - Summary The mutual interactions between the host and the gut microbiota confer health benefits to the host and provide a nutrient-rich environment for the microbial community. However, our knowledge of the direct functional impact of host factors on the gut microbial community remains incomplete. The monolayer of intestinal epithelial cells (IECs) provides the front-line response to the luminal contents, including the gut microbiota, for maintaining intestinal homeostasis. Our previous works and preliminary data have demonstrated a previously unrecognized function of IECs on the gut microbiota: IEC-derived components promote Lactobacillus rhamnosus GG (LGG) growth, the protective effects of LGG on IECs, and production of p40, an anti-inflammatory factor, thereby ameliorating colitis in mice. To elucidate the mechanisms involved in this action, we have used novel transgenic mouse models to demonstrate that IEC-EVs interact with a wide range of microbiota, in addition to Lactobacilli, in the mouse gastrointestinal (GI) tract, and blocking extracellular vesicles (EV) release by IECs increases inflammatory disease severity consistent with the importance of IEC-EVs for enforcing gut homeostasis. We further find that IEC-EVs could be up-taken by LGG and promote LGG growth and protective effects on IECs. Thus, IEC-EVs may functionally mediate the communication between IECs and the gut microbiota. Importantly, we have identified a molecular chaperone, heat shock protein (HSP) 90, particularly HSP90β as a high abundant cargo in IEC-EVs. Blocking HSP90 activity inhibits the increase in LGG growth and p40 production by IEC- derived components and IEC-EVs, indicating that IEC-derived HSP90β has potential clients with functionality in LGG. HSP90 is highly conserved from bacteria to mammals and displays functional overlap. Thus, HSP90β in IEC-EVs is a potential cargo to regulate growth and function of bacteria in the GI tract. We will test the hypothesis that IEC-EVs deliver functional protein cargos, including HSP90, to IEC-EV- targeted bacterial strains in the GI tract to promote their growth and functions in preventing intestinal inflammation. Studies in Aim 1 are proposed to determine whether HSP90 serves as a functional cargo in IEC- secreted EVs to promote LGG growth, promote the beneficial effects of LGG on IECs, and reinforce the efficacy of LGG for ameliorating colitis. Clients of HSP90 and HSP90-regulated pathways in LGG for LGG growth and function will also be investigated. In Aim 2, we will use novel mouse models to identify the microbial profile that are targeted by IEC-EVs and their contents to determine how extensive this mode of host-microbial crosstalk is in mammals during health and its contribution to preventing colitis in mice. Studies in this proposal will advance the field of microbial host mutualistic interactions by defining a novel crosstalk between the microbiota and host via IEC-EVs. Such knowledge will lay a foundation for developing strategies to enhance the effects of health- promoting commensal bacteria such as probiotics for preventing and treating inflammatory bowel disease.
