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.