Modulation of NF-kB Signaling by Immunoprobiotics

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DESCRIPTION (provided by applicant): The overall hypothesis of this proposal is that glycerol/glycerophospholipid metabolism is a focal point for determining the relative capacities of probiotics to suppress NF-?B and MAP kinase signaling in mammalian cells. Ultimately, suppression of key signaling pathways results in down-regulation of pro-inflammatory cytokine production, and selective depletion of activated immune cells. Targeted and random mutagenesis strategies will be used to generate insertion mutants of probiotics that gain or lose the ability to suppress NF-?B activation or MAP kinase signaling. Bioreactors with defined combinations of intestinal bacteria will be used in order to simulate gut bacterial ecosystems. Myeloid and epithelial cells will be combined in co-culture models in order to provide simplified models of the gut mucosa. Primary candidate immunoregulatory factors produced by lactobacilli include glycerophospholipid derivatives that may down-regulate pro-inflammatory responses. Finally, selected probiotic mutants will be introduced into a defined microbiota-containing mouse model in order to study effects of a defined microbiome on NF-?B activation and MAP kinase immune signaling pathways in vivo. These studies will occur within the context of well-characterized fluctuations of the intestinal microbiota so that microbial and host contributions to immunoregulation can be investigated in parallel using one mouse model. 1. Generate insertional mutants of immunoprobiotic Lactobacillus reuteri and identify key bacterial genes that regulate factors affecting NF-?B and MAP kinase signaling pathways. 2. Investigate regulation of NF-?B and MAP kinase signaling in a simulated gut mucosa by probiotics/mutants in the milieu of a defined microbiota. 3. Introduce wild type and isogenic probiotic mutants into IL-10-deficient mice with a defined microbiome in order to study probiotic effects on NF-?B and MAP kinase signaling pathways in vivo. PUBLIC HEALTH REVELANCE: The investigator seeks to understand how beneficial bacteria may regulate inflammation in the intestine. This project includes a simulation of the intestinal microbial community so that investigations take place in the context of conditions similar to the complex intestinal environment. Finally, a mouse model of intestinal inflammation will be used to explore which genes of beneficial bacteria are important for regulating intestinal immune responses in mammals.