Extracellular vesicle-mediated transfer of miR-196b-5p from Cryptococcus neoformans-stimulated microglia activates astrocytes via targeting FAS

Background Cryptococcal meningitis is characterized by prominent neuroinflammation, with microglia and astrocytes being the primary glial cells involved in this process. However, the molecular mechanisms underlying the microglia-astrocyte interactions mediated by extracellular vesicles (EVs) during cryptococcal infection have not yet been fully elucidated. Methods We treated human HMC3 microglia with heat-killed Cryptococcus neoformans (strain WM148) and purified EVs via ultracentrifugation. EV morphology and markers were assessed by TEM, NTA, and Western blot. Dye-labeled EV uptake by HA1800 astrocytes was assessed by confocal microscopy. Functional effects on astrocytes were evaluated using CCK-8, Annexin V/PI staining, ELISA, RT-qPCR, and Western blotting. miRNA sequencing identified differentially expressed miRNAs, and mechanistic studies were performed using miRNA inhibition, luciferase reporter assays, and gene silencing. Results Our findings show that astrocytes internalize morphologically intact EVs derived from HMC3/WM148. EV uptake reduces astrocyte viability, induces apoptosis, and increases secretion of IL-1β, IL-6 and TNF-α. GW4869-mediated inhibition of EV biogenesis largely reverses these harmful effects. miRNA profiling identifies high enrichment of miR-196b-5p in EVs. Functional assays verify miR-196b-5p as a pro-inflammatory mediator; its inhibition rescues astrocyte apoptosis and cytokine release. Mechanistically, miR-196b-5p directly targets FAS in HEK293T. Consistently, FAS knockdown abrogates the protective effect of miR-196b-5p inhibition in HA1800 astrocytes. Conclusions Cryptococcus neoformans stimulate reprograms microglial-derived EVs to promote astrocyte inflammation and injury through the miR-196b-5p-FAS axis. These findings identify the EV-associated miR-196b-5p/FAS axis as a candidate mechanism of glial inflammatory communication that warrants validation in primary-cell, live-infection, and in-vivo models.