Abstract
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.
Highlights
Extracellular vesicles (EVs), known as exosomes, are bilayered lipid membrane particles secreted from cells that play an important role in intercellular communication under physiological and pathological conditions
In agreement, using cultured astroglia cells, we have shown that ethanol treatment increases the release of EVs and enriches their content in inflammatory-related proteins and miRNAs, effects that are associated with Toll-like receptor 4 (TLR4) immune response activation [10]
We wondered whether physiologically relevant concentrations of ethanol can activate the BV2 microglial cell line
Summary
Extracellular vesicles (EVs), known as exosomes, are bilayered lipid membrane particles secreted from cells that play an important role in intercellular communication under physiological and pathological conditions. These nanovesicles (30–150 nm in diameter) carry specific proteins, RNAs and lipids [1]. Studies have shown that microglia increase EV secretion after a stimulus [3,4], and the content of microglia-derived EVs differs depending on the nature of the stimuli, triggering either pro- or anti-inflammatory responses [5,6,7]. Ethanol was reported to increase the content of pro-inflammatory cytokines and chemokines in microglia-derived EVs in a dose-dependent manner [9]
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