TGF-β-induced CD4+FoxP3+regulatory T cell-derived extracellular vesicles modulate Notch1 signaling through miR-449a and prevent collagen-induced arthritis in a murine model

CD4^(+)FOXP3^(+)Treg cells are central to the maintenance of self-tolerance and can be defective in autoimmunity.In autoimmune rheumatic diseases,dysfunctional self-tolerance,is to a large extent,caused by insufficient Treg-cell activity.Although nTregs have therapeutic effects in vivo,their relative scarcity and slow rate of in vitro expansion hinder the application of nTreg therapy.It was previously reported that EVs contribute significantly to the suppressive function of FOXP3^(+)Treg cells.Considering that the stability and plasticity of nTregs remain major challenges in vivo,we established EVs derived from in vitro TGF-β-induced Treg cells(iTreg-EVs)and assessed their functions in a murine model of autoimmune arthritis.The results demonstrated that iTreg-EVs preferentially homed to the pathological joint and efficiently prevented the imbalance in Th17/Treg cells in arthritic mice.Furthermore,we found that miR-449a-5p mediated Notch1 expression modulation and that miR-449a-5p knockdown abolished the effects of iTreg-EVs on effector T cells and regulatory T cells in vitro and in vivo.Taken together,our results show that iTreg-EVs control the inflammatory responses of recipient T cells through miR-449a-5p-dependent modulation of Notch1 and ameliorate the development and severity of arthritis,which may provide a potential cell-free strategy based on manipulating iTreg-EVs to prevent autoimmune arthritis.