Abstract
BackgroundCurrent 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.MethodsCortical 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.ResultsWe 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.ConclusionsThese 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.
Highlights
Current evidence indicates that extracellular vesicles (EVs) participate in intercellular signaling, and in the regulation and amplification of neuroinflammation
The ethanol-triggered secretion of EVs by astrocytes is dependent on the Toll-like receptor 4 (TLR4) response To evaluate whether ethanol is able to increase the secretion of EVs from astroglial cells, the cultured cortical astrocytes from the WT or TLR4-KO mice were treated with or without ethanol (40 mM) for 24 h
Flow cytometry and the SYTO green fluorescent nucleic acid stain, which labels the RNA contained in EVs, confirmed that ethanol increased the number of the SYTOpositive nanoparticles in astrocyte-derived EVs (Fig. 1d)
Summary
Current evidence indicates that extracellular vesicles (EVs) participate in intercellular signaling, and in the regulation and amplification of neuroinflammation. 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. Astrocytes, one of the most abundant cells in the central nervous system, have emerged as important regulators of brain function as they participate under physiological and pathological conditions. Recent evidence supports the role of exosomes in different physiological processes, such as intercellular communication, proliferation, and immune response, and under neuropathological conditions, such as neurodegenerative diseases like Alzheimer’s, Parkinson’s, or prion diseases [7,8,9]
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