The Effects And Mechanism Of Exosomes Derived From Human Umbilical Cord Mesenchymal Stem Cells On Contact Hypersensitivity

Background and objectives:Mesenchymal stem cells(MSCs)are a kind of pluripotent stem cells as immunomodulatory agents for the treatment of immune-related diseases.However,MSCs have some disadvantages as cell therapy.An increasing number of studies have shown that exosomes derived from mesenchymal stem cells(MSC-Exo)can exert the same immunomodulatory effect,and MSC-Exo have more advantages as a cell-free therapy.The efficacy of MSCs on contact hypersensitivity(CHS)has been reported,but the effect of MSC-Exo on CHS has not been deeply reported.Therefore,this study intends to explore the effect and mechanism of MSC-Exo on CHS to provide new preclinical evidence for the development of MSC-Exo as the cell-free immunomodulatory therapy.Methods and results:The morphology of human unbilical cord MSCs(hucMSCs)showed as plastic-adherent cells with a spindle shape at passage three.MSCs had the multi-lineage differentiation ability and the biomarker expression satisfied the minimal definition criteria of the International Society for Cellular Therapy.Exosomes from the serum-free-culture supernatants of hucMSCs were harvested by ultra-speed centrifugation.Electron microscope,nanoparticle tracking analysis and immunoblot analysis were used to identify the characterization of hucMSC-Exo.The results showed that hucMSC-Exo were saucer-like in morphology with most of them were approximately 102nm in diameter.They were identified as positive for CD9 and CD63.A contact hypersensitivity(CHS)mouse model for ACD was established and treated by intravenous MSC-Exo injection.We found that hucMSC-Exo could significantly alleviate the pathology of CHS,including reducing ear swelling and leukocyte infiltration.Injection of MSC-Exo significantly inhibited CD8~+IFN-?~+Tc1 and CD4~+IFN-?~+Th1 immune responses,induced CD4~+CD25~+Foxp3~+regulatory T cells(Tregs),suppressed the generation of TNF-?and IFN-?and increased IL-10 production.In vitro,MSC-Exo also suppressed the development of Tc1 and Th1 and induced the expansion of Tregs,reducd the generation of TNF-?and IFN-?and increased IL-10 production,which changed in a dose-dependent manner in response to treatment with MSC-Exo.Interestingly,PKH26-labeled MSC-Exo were found to be directly internalized by CD3~+T cells,resulting in reduced STAT1 and pSTAT1 protein levels.Conclusions:The isolated particles from hucMSCs were identified as exosomes.MSC-Exo can alleviate CHS by inhibiting Tc1 and Th1 immune responses and inducing the Treg phenotype,and reducing the generation of TNF-?and IFN-?and increasing IL-10 production in vivo and in vitro.The mechanism by which MSC-Exo influence CD3~+T cells might partially involve targeting STAT1.Therefore,MSC-Exo are ideal candidates for cell-free immunomodulatory therapy for T cell-mediated diseases such as ACD.