Macrophage-derived extracellular vesicles promote T cell-dependent inflammatory pain resolution

Inflammatory pain resolution is increasingly recognized as an active, immune-regulated process, yet the adaptive immune mechanisms that govern this process remain poorly defined. We previously demonstrated that intrathecal administration of macrophage-derived small extracellular vesicles (sEVs) from unstimulated (sEV) or LPS-stimulated (sEV⁺) RAW 264.7 cells accelerate resolution of complete Freund's adjuvant (CFA)-induced inflammatory pain in male mice. However, the immunological mechanisms underlying this effect remain undefined. Given growing evidence that T cells regulate inflammatory pain resolution, we investigated whether macrophage-derived sEVs engage adaptive immune pathways to promote recovery. In vitro, both sEV and sEV⁺ enhanced T cell activation, with sEV⁺ exhibiting greater immunostimulatory capacity. Direct effects on T cells were modest; instead, sEV⁺ induced robust antigen-presenting cell (APC)-dependent T cell activation characterized by increased costimulatory molecule expression and enhanced Th1 polarization. Loss-of-function and rescue studies in Rag2 ^-/- mice demonstrate that T cells are required for late-phase sEV⁺-mediated inflammatory pain resolution. In vivo, sEV⁺ elicited immunostimulatory responses in intrathecal-draining cervical and CFA-draining sacral/internal iliac lymph nodes. Together, these findings identify adaptive immune engagement as a critical mediator of sEV⁺-induced pain resolution and position macrophage-derived sEVs as a cell-free immunotherapeutic modality that harnesses endogenous T cell-dependent mechanisms of active inflammatory pain resolution.