Unravelingthe Impact of TLR4 and Sex on Chronic AlcoholConsumption-Induced Lipidome Dysregulation in Extracellular Vesicles

The lipids that form extracellular vesicles (EVs) play critical structural and regulatory roles, and cutting-edge bioinformatics strategies have shown the ability to decipher lipid metabolism and related molecular mechanisms. We previously demonstrated that alcohol abuse induces an inflammatory immune response through Toll-like receptor 4 (TLR4), leading to structural and cognitive dysfunction. This study evaluated how TLR4 and sex as variables (male/female) impact the lipidome of plasma-resident EVs after chronic alcohol exposure. Using a mouse model of chronic ethanol exposure in wild-type and TLR4-deficient mice, enrichment networks generated by LINEX² highlighted significant ethanol-induced changes in the EV lipid substrate–product of enzyme reactions associated with glycerophospholipid metabolism. We also demonstrated ethanol-induced differences in Lipid Ontology enrichment analysis in EVs, focusing on terms related to lipid bilayer properties. A lipid abundance analysis revealed higher amounts of significant lipid subclasses in all experimental comparisons associated with inflammatory responses and EV biogenesis/secretion. These findings suggest that interrogating EV lipid abundance with a sensitive lipidomic-based strategy can provide deep insight into the molecular mechanisms underlying biological processes associated with sex, alcohol consumption, and TLR4 immune responses and open new avenues for biomarker identification and therapeutic development.