Inflammatory stimulation remodels microglial extracellular vesicle miRNA cargo and selectively modulates recipient microglial responses

Abstract

Neuroinflammation is a hallmark of neurodegenerative diseases and contributes to progressive neuronal dysfunction. Microglia, the resident immune cells of the Central Nervous System, orchestrate inflammatory responses and can transmit activation signals through extracellular vesicles (EVs). These vesicles carry bioactive molecules, including microRNAs (miRNAs), that may influence recipient cell phenotypes. In this in vitro study, we investigated whether EVs-associated inflamma-miRNAs derived from classically activated microglia contribute to the propagation of inflammatory responses. Microglial cells were stimulated with lipopolysaccharide (LPS) or ATP. EVs were isolated from conditioned medium and characterized by nanoparticle tracking analysis and western blot. Naïve microglia were then exposed to conditioned medium or isolated EVs, and inflammatory markers and miRNA expression were assessed. LPS stimulation induced a pro-inflammatory phenotype, characterized by increased iNOS expression and significant enrichment of miR-155, whereas miR-34a showed an increasing trend in donor cells. A similar increase was observed in their EVs, although this did not reach statistical significance. Inhibition of miR-155 partially attenuated LPS-induced iNOS expression, whereas miR-34a inhibition partially increased TREM2 levels, supporting their involvement in inflammatory and homeostatic pathways. EVs derived from activated microglia induced modest inflammatory changes in recipient cells; however, inhibition of individual miRNAs in donor cells had no effects on EVs-mediated inflammatory propagation. Overall, these findings support a role for miR-155 and miR-34a in the inflammatory response of donor microglia, while suggesting that EVs-mediated inflammatory communication is likely regulated by multiple factors beyond individual miRNAs. The limited impact of miRNA inhibition on EVs-mediated propagation highlights the complexity of EVs-mediated signaling mechanisms. Graphical abstract Created in BioRender: Graphical summary showing that EVs-associated miR-155 and miR-34a released from activated microglia propagate inflammatory responses to recipient cells, increasing iNOS and reducing TREM2. Inhibition of these miRNAs partially attenuates EVs-mediated inflammation.