PROJECT SUMMARY: The use of beneficial bacteria to promote intestinal health and to limit inflammation is widely practiced, although experimental evidence corroborating the efficacy of many bacteria promoted with such claims remains limited. We directly address this gap by identifying a newly characterized beneficial bacterium that potently dampens injury-induced intestinal inflammation. In a screen of potential beneficial bacteria, we identified Lactococcus lactis subsp. cremoris as a bacterium that elicited potent anti-inflammatory activity in the mouse intestine. In order to substantiate the use of this strain as a therapeutic to treat IBD, which is required ‘Early Clinical Trial with Live Biotherapeutic Products’, we will take a rigorous approach to generate critical pre- clinical data using relevant mouse models of colitis. Our preliminary data show that feeding of L. lactis elicits lower Disease Activity Index (DAI) scores during chronic DSS treatment, and a significantly faster rate of recovery following the withdrawal of DSS. Mechanistically, we show that L. lactis activates the cytoprotective NRF2 signaling pathway in intestinal tissue, and show here that L. lactis-mediated protection, or Nrf2 gene activation does not occur in Tlr2 & Myd88-null mice. Importantly, culture supernatant of L. lactis was sufficient to accelerate healing in an in vitro epithelial cell wound healing model in cultured epithelial cells, indicating that a factor secreted by L. lactis elicits the beneficial effects. Because phylogenetically related L. lactis ATCC strains do not elicit the same beneficial effects, we hypothesize that a specific factor, bacterial exopolysaccharide, within L. lactis is responsible for eliciting its powerful anti-inflammatory and pro-restitutive effects via MyD88-dependent signaling. In addition, we show novel preliminary data that L. lactis activates the expression of a specific set of microRNAs in intestinal tissue that are associated with the regulation of NRF2 pathway signaling. Therefore, our central hypothesis is that the specific element(s) released by L. lactis induces cytoprotective and anti-inflammatory effects in the intestine, and can modulate the pathobiology of IBD. We will test our hypothesis in the following specific aims: (1) to characterize the pro-restitutive effects of L. lactis subsp. cremoris in colitis, (2) to identify the factor produced and released by L. lactis subsp. cremoris that induces its beneficial effects and activates NRF2 signaling, and (3) to determine the role of key L. lactis subsp. cremoris-induced microRNAs that regulate NRF2 signaling in modulating the cytoprotective effects of L. lactis subsp. cremoris. Together, we identify an effector microorganism that beneficially influences host phenotype, with clear favorable effects on dampening colitis-induced inflammation. We will discover the underlying mechanisms of action, which has become foci of microbiome research, and which is essential for the ...