Microglial Epigenetic Memory is Associated with Accelerated Resolution of Inflammatory Pain Induced by Prophylactic Macrophage-Derived Small Extracellular Vesicles.

Introduction: Small extracellular vesicles (sEVs) are cell-released lipid vesicles that facilitate intercellular communication by transferring bioactive cargo to recipient cells. We previously showed that a single intrathecal administration of RAW 264.7 macrophage-derived sEVs, given two weeks prior to complete Freund's adjuvant (CFA)-induced inflammation, resulted in earlier recovery from mechanical and thermal hypersensitivity. How this long-term memory develops, and how sEVs regulate immune responses, are unknown. Recent studies have shown that priming microglia with inflammatory stimuli can enhance or suppress responses to a delayed secondary insult via epigenetic modifications. We hypothesized that prophylactic intrathecal administration of macrophage-derived sEVs confers accelerated resolution of inflammatory pain by reprogramming epigenetic memory in spinal microglia. Methods: Microglia were ablated using the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 prior to sEV administration. Pain behaviors were assessed following CFA-induced inflammation. Chromatin immunoprecipitation sequencing (ChIP-seq) was performed on spinal microglia isolated 14 days after sEV administration. The role of epigenetic modification was evaluated by pharmacological inhibition of the H3K4 mono-methyltransferase SETD7. Results: Prophylactic sEV administration accelerated the resolution of inflammatory pain hypersensitivity. This effect was abolished in mice treated with PLX5622, indicating that microglia are required during sEV exposure. ChIP-seq analysis revealed enrichment of H3K4me1-marked loci in spinal microglia 14 days after sEV administration, consistent with induction of innate immune memory. Inhibition of SETD7 eliminated the protective effect of sEVs, demonstrating a requirement for H3K4 mono-methylation. Discussion: Macrophage-derived sEVs induce a microglia-dependent, epigenetically mediated form of pain prophylaxis. These findings support a model in which sEVs establish a primed, memory-like state in spinal microglia, characterized by enhancer-associated chromatin changes that confer latent regulatory potential and enhance resolution of subsequent inflammatory pain. This work links extracellular vesicles to microglial epigenetic remodeling and suggests a potential strategy for non-addictive, preventive pain therapeutics.