Saturday, September 7, 2024
9/7/2024
PROJECT SUMMARY Chronic inflammation is associated with most non-communicable diseases, which affect over 40% of individuals in the U.S.. While drugs are available that target suppression of inflammatory effector pathways, none target resolution of chronic inflammation, in part due to a lack of understanding of how to re-establish immune homeostasis. Bacteria in the colon play a key role in regulating the balance between acute and chronic inflammation, and understanding how to re-establish immune homeostasis is paramount in preventing chronic inflammatory disease development. Enterotoxigenic Bacteroides fragilis (ETBF) is representative of one species that is responsible for diarrhea, colitis and colon cancer. Yet, the commensal non-toxigenic strain of B. fragilis (NTBF) appears to prevent inflammation. Our research group and others have documented a major communication tool of bacteria are extracellular small RNAs, which can dampen or active the immune system. Little is known, however, about the contribution of sRNA species in governing microbe-host communication. Recently, bacterial small RNA (sRNA) species were identified within outer membrane vesicles (OMVs) that are shed by bacteria. Small RNAs represent a critical trigger governing whether host cells respond with pro- or anti-inflammatory signals. Intriguingly, OMVs from the commensal B. fragilis and E. coli prevent chemically induced murine colitis, while those from pathogenic E. coli induce systemic inflammation. Preliminary evidence from our lab indicate that OMVs from ETBF and NTBF differentially activate the TLR pathway, and their OMVs carry distinct sRNA cargo. Results from our preliminary research demonstrate that OMV-derived small RNAs are differentially abundant in OMVs from ETBF as compared to NTBF. We also show that OMVs from ETBF specifically activate TLR7 as compared to OMVs from NTBF. In this study, we hypothesize that the differential inflammatory response to OMVs from toxic and non-toxigenic Bacteroides fragilis is governed, in part, by sRNA engagement with TLRs, and that this mechanism partially explains the prevention or development of chronic inflammation. The purpose of this proposal is to determine if OMVs or OMV-derived sRNA from the pathogenic ETBF either prevent or induce chronic inflammation as compared to the commensal strain NTBF. Our specific aims will 1) Elucidate the differential activation of the innate immune response between commensal and enterotoxigenic OMVs from B. fragilis using an established tri-culture system, 2) Identify the mechanism of immune- stimulating intracellular uptake and sub-cellular localization from commensal vs. enterotoxigenic B. fragilis OMVs into host cells., and 3) Determine the commensal vs. enterotoxigenic B. fragilis small RNA host targets in silico using bioinformatics tools and validate in vitro using tri-culture and nanolipovesicle transfection. Confirmation of this hypothesis would resolve an outstanding gap in the field to explain how OMVs from commensal vs pathogenic strains induce or prevent inflammation.
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