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 Freunds 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/706465v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@e49aa4org.highwire.dtl.DTLVardef@648c9forg.highwire.dtl.DTLVardef@14c3cc6org.highwire.dtl.DTLVardef@2a17f0_HPS_FORMAT_FIGEXP M_FIG C_FIG