Immune Cell Regulation by Macrophage Derived Small Extracellular Vesicles in Pain

Abstract

BackgroundDevelopment of chronic pain is related to aberrant neuroimmune signaling. Small extracellular vesicles (sEVs) are 30‐150nm size particles of endosomal origin. They play an important role in neuroimmune crosstalk facilitating transfer of biomolecular cargo between cells. Our studies show that sEVs from RAW 264.7 macrophage cells can be therapeutic or prophylactic in mouse model of inflammatory pain. We investigated the mechanistic basis of how sEVs from lipopolysaccharide (LPS) stimulated (Exo+) and unstimulated (Exo‐) cells impact primary glial cells, dendritic cells (DCs) and adaptive immune cells such as CD4+T cells. We hypothesize that macrophage derived sEVs can potentiate T cell activation directly, or indirectly by the upregulation of major histocompatibility complex‐II (MHC‐II) and co‐stimulatory molecules on DCs, attenuating pain hypersensitivity. We will also test if sEVs confer protection to glial cells in the context of inflammation.MethodsPrimary mouse DCs, microglia and astrocytes were incubated with 1µg Exo‐/+sEVs for 24 hours. Splenocyte derived CD4+ T cells were treated with 0.1ug Exo‐/+ sEVs for 72 hours. Flow cytometry and qPCR was used for analysis.ResultsCharacterization of RAW sEVs showed the presence of sEV markers CD81 and Alix. sEV treatment of DCs resulted in an increase in CD80+/CD86+ MHCIIhiCD11c+/hi DCs. Treatment of CD4+T cells with sEVs increased Tgf‐βexpression. Pretreatment with Exo+ sEV increased Tfgf‐β expression in glial cells upon LPS stimulation. An increase in PPAR (Peroxisome proliferator activated receptor) expression was observed upon sEV treatment of glial cells.ConclusionUpregulation of MHC‐II and costimulatory molecules on DCs as well as PPAR upregulation in glial cells upon sEVs treatment indicates that sEVs from RAW 264.7 cells can modulate immune response. These results indicate sEVs modulate innate cells such as microglia and DCs and adaptive immune cells such as CD4+ T cells may contribute to therapeutic effect in attenuating chronic pain.