Sex differences in the effects of alcohol consumption on the content of plasma extracellular vesicles: a multi-omics study in humans and mice

Alcohol is a psychoactive substance that can induce dependence and cause serious health and societal consequences. Despite its toxicity and links to mortality, disease, and significant socioeconomic costs, it remains one of the most widely consumed and socially normalized drugs worldwide. Its effects, however, are not uniform: biological sex plays a critical role in shaping vulnerability. Evidence indicates that women exhibit a stronger neuroinflammatory response to both acute and chronic alcohol exposure. In this response, Toll-Like Receptor 4 (TLR4) plays a key role, being activated by alcohol and triggering an immune response. In TLR4-deficient animal models, both sex differences and inflammatory intensity are attenuated. This dissertation investigates the biological mechanisms underlying alcohol consumption, focusing on sex-specific responses and the role of TLR4. Its novelty lies in the study of plasma extracellular vesicles (EVs) and their multi-omic content. EVs and their cargo are central to signaling, inflammation, and pathology, and provide a promising source of non-invasive biomarkers. Two EV components—the lipidome and the miRNome—were analyzed in detail due to their potential to reflect cellular physiological and pathological states. The processing and analysis of these data required the use of specific bioinformatic tools for lipidomic exploration (Chapters 3, 4, and 5) and microRNA transcriptomic analysis approaches (Chapter 6), as well as multi-omic integration approaches (Chapter 6). Acute ethanol exposure in human adolescents produced sex-dependent lipidomic profiles, with women showing higher levels of pro-inflammatory lipid species (Chapter 3). In mice, lipidomic changes depended on both sex and TLR4 expression, highlighting the receptor’s modulatory role (Chapter 3). In adults with Alcohol Use Disorder (AUD), sex-specific alterations involved pathways linked to neuroinflammation, hepatotoxicity, and tumor progression (Chapter 4). Chronic alcohol exposure in mice demonstrated that TLR4 and sex jointly shaped EV lipid composition, affecting processes such as the Lands’ cycle and phospholipid-related enzymatic activity (Chapter 5). A multi-omic integration of lipidomic and miRNA transcriptomic data from AUD patients identified robust diagnostic molecular signatures, including sex-specific profiles that reveal fundamental differences in biological responses to alcohol (Chapter 6). Notably, miRNome analysis revealed opposing functional alterations between males and females in pathways related to inflammation, oxidative stress, and autophagy. Overall, these findings underscore the value of lipidomic research in alcohol studies, suggest a sex-dependent interaction between the Lands’ cycle and TLR4 in alcohol-induced inflammation, and highlight lipid rafts as key hubs of TLR4 signaling. Factors such as sex, age, TLR4 status, and drinking patterns influence EV composition. By integrating multiple bioinformatic approaches, this work provides a more comprehensive view of alcohol’s effects, positioning EVs as powerful tools for biomarker discovery and supporting the development of personalized diagnostic and therapeutic strategies.