Lipidomic landscape of circulating extracellular vesicles isolated from adolescents exposed to ethanol intoxication: a sex difference study.

BACKGROUND: Lipids represent essential components of extracellular vesicles (EVs), playing structural and regulatory functions during EV biogenesis, release, targeting, and cell uptake. Importantly, lipidic dysregulation has been linked to several disorders, including metabolic syndrome, inflammation, and neurological dysfunction. Our recent results demonstrated the involvement of plasma EV microRNAs as possible amplifiers and biomarkers of neuroinflammation and brain damage induced by ethanol intoxication during adolescence. Considering the possible role of plasma EV lipids as regulatory molecules and biomarkers, we evaluated how acute ethanol intoxication differentially affected the lipid composition of plasma EVs in male and female adolescents and explored the participation of the immune response. METHODS: Plasma EVs were extracted from humans and wild-type (WT) and Toll-like receptor 4 deficient (TLR4-KO) mice. Preprocessing and exploratory analyses were conducted after the extraction of EV lipids and data acquisition by mass spectrometry. Comparisons between ethanol-intoxicated and control human female and male individuals and ethanol-treated and untreated WT and TLR4-KO female and male mice were used to analyze the differential abundance of lipids. Annotation of lipids into their corresponding classes and a lipid set enrichment analysis were carried out to evaluate biological functions. RESULTS: We demonstrated, for the first time, that acute ethanol intoxication induced a higher enrichment of distinct plasma EV lipid species in human female adolescents than in males. We observed a higher content of the PA, LPC, unsaturated FA, and FAHFA lipid classes in females, whereas males showed enrichment in PI. These lipid classes participate in the formation, release, and uptake of EVs and the activation of the immune response. Moreover, we observed changes in EV lipid composition between ethanol-treated WT and TLR4-KO mice (e.g., enrichment of glycerophosphoinositols in ethanol-treated WT males), and the sex-based differences in lipid abundance are more notable in WT mice than in TLR4-KO mice. All data and results generated have been made openly available on a web-based platform ( http://bioinfo.cipf.es/sal ). CONCLUSIONS: Our results suggest that binge ethanol drinking in human female adolescents leads to a higher content of plasma EV lipid species associated with EV biogenesis and the propagation of neuroinflammatory responses than in males. In addition, we discovered greater differences in lipid abundance between sexes in WT mice compared to TLR4-KO mice. Our findings also support the potential use of EV-enriched lipids as biomarkers of ethanol-induced neuroinflammation during adolescence.

Lipids represent essential components of extracellular vesicles (EVs), playing structural and regulatory functions during EV biogenesis, release, targeting, and cell uptake. Lipidic dysregulation has been linked to several disorders. We evaluated how acute ethanol intoxication differentially affected the lipid composition of plasma EVs in male and female adolescents and explored the participation of the immune response. Plasma EVs were extracted from humans and wild-type (WT) and Toll-like receptor 4 deficient (TLR4-KO) mice. Preprocessing and exploratory analyses were conducted after the extraction of EV lipids and data acquisition by mass spectrometry. Our analysis of differential abundance demonstrated, for the first time, that acute ethanol intoxication induced a higher enrichment of distinct plasma EV lipid species in human female adolescents than in males. We observed a higher content of the PA, LPC, unsaturated FA, and FAHFA lipid classes in females, whereas males showed enrichment in PI. These lipid classes participate in the formation, release, and uptake of EVs and the activation of the immune response. Moreover, we observed changes in EV lipid composition between ethanol-treated WT and TLR4-KO mice (e.g., enrichment of glycerophosphoinositols in ethanol-treated WT males), and the sex-based differences in lipid abundance are more notable in WT mice than in TLR4-KO mice. All data and results generated have been made openly available on a web-based platform ( http://bioinfo.cipf.es/sal ). Our findings also support the potential use of EV-enriched lipids as biomarkers of ethanol-induced neuroinflammation during adolescence.

Ethanol Induces Extracellular Vesicle Secretion by Altering Lipid Metabolism through the Mitochondria-Associated ER Membranes and Sphingomyelinases.

Recent evidence pinpoints extracellular vesicles (EVs) as key players in intercellular communication. Given the importance of cholesterol and sphingomyelin in EV biology, and the relevance of mitochondria-associated endoplasmic reticulum membranes (MAMs) in cholesterol/sphingomyelin homeostasis, we evaluated if MAMs and sphingomyelinases (SMases) could participate in ethanol-induced EV release. EVs were isolated from the extracellular medium of BV2 microglia treated or not with ethanol (50 and 100 mM). Radioactive metabolic tracers combined with thin layer chromatography were used as quantitative methods to assay phospholipid transfer, SMase activity and cholesterol uptake/esterification. Inhibitors of SMase (desipramine and GW4869) and MAM (cyclosporin A) activities were also utilized. Our data show that ethanol increases the secretion and inflammatory molecule concentration of EVs. Ethanol also upregulates MAM activity and alters lipid metabolism by increasing cholesterol uptake, cholesterol esterification and SMase activity in microglia. Notably, the inhibition of either SMase or MAM activity prevented the ethanol-induced increase in EV secretion. Collectively, these results strongly support a lipid-driven mechanism, specifically via SMases and MAM, to explain the effect of ethanol on EV secretion in glial cells.

Circulating MicroRNAs in Extracellular Vesicles as Potential Biomarkers of Alcohol-Induced Neuroinflammation in Adolescence: Gender Differences.

Current studies evidence the role of miRNAs in extracellular vesicles (EVs) as key regulators of pathological processes, including neuroinflammation and neurodegeneration. As EVs can cross the blood-brain barrier, and EV miRNAs are very stable in peripheral circulation, we evaluated the potential gender differences in inflammatory-regulated miRNAs levels in human and murine plasma EVs derived from alcohol-intoxicated female and male adolescents, and whether these miRNAs could be used as biomarkers of neuroinflammation. We demonstrated that while alcohol intoxication lowers anti-inflammatory miRNA (mir-146a-5p, mir-21-5p, mir-182-5p) levels in plasma EVs from human and mice female adolescents, these EV miRNAs increased in males. In mice brain cortices, ethanol treatment lowers mir-146a-5p and mir-21-5p levels, while triggering a higher expression of inflammatory target genes (Traf6, Stat3, and Camk2a) in adolescent female mice. These results indicate, for the first time, that female and male adolescents differ as regards the ethanol effects associated with the inflammatory-related plasma miRNAs EVs profile, and suggest that female adolescents are more vulnerable than males to the inflammatory effects of binge alcohol drinking. These findings also support the view that circulating miRNAs in EVs could be useful biomarkers for screening ethanol-induced neuroinflammation and brain damage in adolescence.

Exosomes as mediators of neuron-glia communication in neuroinflammation.

In recent years, a type of extracellular vesicles named exosomes has emerged that play an important role in intercellular communication under physiological and pathological conditions. These nanovesicles (30-150 nm) contain proteins, RNAs and lipids, and their internalization by bystander cells could alter their normal functions. This review focuses on recent knowledge about exosomes as messengers of neuron-glia communication and their participation in the physiological and pathological functions in the central nervous system. Special emphasis is placed on the role of exosomes under toxic or pathological stimuli within the brain, in which the glial exosomes containing inflammatory molecules are able to communicate with neurons and contribute to the pathogenesis of neuroinflammation and neurodegenerative disorders. Given the small size and characteristics of exosomes, they can cross the blood-brain barrier and be used as biomarkers and diagnosis for brain disorders and neuropathologies. Finally, although the application potential of exosome is still limited, current studies indicate that exosomes represent a promising strategy to gain pathogenic information to identify therapeutically targets and biomarkers for neurological disorders and neuroinflammation.

TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles.

BACKGROUND: Current evidence indicates that extracellular vesicles (EVs) participate in intercellular signaling, and in the regulation and amplification of neuroinflammation. We have previously shown that ethanol activates glial cells through Toll-like receptor 4 (TLR4) by triggering neuroinflammation. Here, we evaluate if ethanol and the TLR4 response change the release and inflammatory content of astrocyte-derived EVs, and whether these vesicles are capable of communicating with neurons by spreading neuroinflammation. METHODS: Cortical neurons and astrocytes in culture were used. EVs were isolated from the extracellular medium of the primary culture of the WT and TLR4-KO astrocytes treated with or without ethanol (40 mM) for 24 h. Flow cytometry, nanoparticle tracking analysis technology, combined with exosomal molecular markers (tetraspanins) along with electron microscopy, were used to characterize and quantify EVs. The content of EVs in inflammatory proteins, mRNA, and miRNAs was analyzed by Western blot and RT-PCR in both astrocyte-derived EVs and the neurons incubated or not with these EVs. Functional analyses of miRNAs were also performed. RESULTS: We show that ethanol increases the number of secreted nanovesicles and their content by raising the levels of both inflammatory-related proteins (TLR4, NFκB-p65, IL-1R, caspase-1, NLRP3) and by changing miRNAs (mir-146a, mir-182, and mir-200b) in the EVs from the WT-astrocytes compared with those from the untreated WT cells. No changes were observed in either the number of isolated EVs or their content between the untreated and ethanol-treated TLR4-KO astrocytes. We also show that astrocyte-derived EVs could be internalized by naïve cortical neurons to increase the neuronal levels of inflammatory protein (COX-2) and miRNAs (e.g., mir-146a) and to compromise their survival. The functional analysis of miRNAs revealed the regulatory role of the expressed miRNAs in some genes involved in several inflammatory pathways. CONCLUSIONS: These results suggest that astrocyte-derived EVs could act as cellular transmitters of inflammation signaling by spreading and amplifying the neuroinflammatory response induced by ethanol through TLR4 activation.