IL-4-Primed Microglial Extracellular Vesicles Attenuate Rotenone-Induced Cell Death in SH-SY5Y Cells: A Contributory Role for miR-191-5p

Microglia contribute to central nervous system homeostasis and neuroprotection partly through the release of small extracellular vesicles (sEVs) carrying regulatory cargoes such as microRNAs. Interleukin-4 (IL-4) alters microglial state and secretory output; however, whether sEVs released from IL-4-treated microglia protect neurons against toxic injury, and which cargoes mediate these effects, remains unclear. Here, we investigated the protective effects of sEVs derived from the IL-4-treated HMC3 human microglial cell line in a rotenone-induced injury model in the SH-SY5Y cell line and examined the contribution of microRNA-191-5p to neuroprotection. Small RNA sequencing revealed a distinct miRNA profile in IL-4-sEVs, with microRNA-191-5p emerging as the most statistically significant upregulated candidate. Its enrichment was confirmed by RT-qPCR. PKH67-labeled sEV-associated fluorescence was detected in SH-SY5Y cells, indicating uptake of microglia-derived sEVs by recipient cells. Functionally, pretreatment with IL-4-sEVs significantly reduced rotenone-induced cell death and preserved cell morphology compared with untreated and control sEV-treated cells. To assess the contribution of microRNA-191-5p, IL-4-sEVs were loaded with a microRNA-191-5p antagomir, which reduced microRNA-191-5p levels and partially attenuated the protective effect of IL-4-sEVs. Together, these findings suggest that sEVs derived from the IL-4-treated HMC3 microglial cell line mitigate rotenone-induced injury in the SH-SY5Y cell line in vitro and that microRNA-191-5p contributes, at least in part, to this effect.