A Potent Immunomodulatory Role Of Exosomes Derived From Mesenchymal Stromal Cells In Preventing CGVHD

Background and Objectives:Mesenchymal stromal cells(MSCs)are a promising therapy for preventing chronic Graft-Versus-Host Disease(cGVHD)due to their potent immunomodulatory properties.However,the safety concerns regarding the use of MSCs remain unsolved,and conflicting effects are observed due to the heterogeneity of MSCs.Recently,exosomes were shown to mediate the paracrine effects of MSCs,making it a potential candidate for cell-free therapies.The aim of this study is to investigate the efficacy and safety of MSCs-derived exosomes(MSCs-exo)in an established cGVHD mouse model.Methods:1.After isolation of bone marrow mononuclear cells,bone marrow mesenchymal stem cells were performed to observe the general morphology.Flow cytometry was used to detect the expression of surface markers of mesenchymal stem cells,and mesenchymal stem cells were identified for osteogenic,and adipogenic differentiation.2.The supernatant of mesenchymal stem cells cultured with exosomes-free serum was collected,centrifuged and purified the supernatant by ultracentrifuge,and detected exosome-specific markers CD81,CD63,CD9 antigen by Western-Blot.The expression of the protein was analyzed by Nanosight and transmission electron microscopy to analyze the size and morphology of the exosomes.3.Establish a cGVHD mouse model.BALB/C female mice were accepted the bone marrow cells and spleen cells from major histocompatibility antigen matched,minor histocompatibility antigens mismatched allogeneic B10.D2 male mice,the mouse model that can simulate the occurrence and development of clinical cGVHD.4.cGVHD mice were randomly divided into three groups according to clinical score and body weight:cGVHD control group,Fib-exo treatment group and MSCs-exo treatment group.The clinical manifestations and survival time of cGVHD mice were observed and the pathological changes in target organs of each group were evaluated at the end of observation.The expression ratio of T cell subsets were detected by flow cytometry.Real-time quantitative PCR was used to detect the expression of Th17 and Treg cell-associated transcription factors,and the secretion level of cytokines in peripheral blood supernatant of mice were also detected.5.Sorting out the CD3+ T cells,co-culture the PKH26-labeled exosomes with CD3+ T cells,and detected the cell membrane f’usion ability of exosomes;T cells were cultured in vitro and intervened with Fib-exo and MSCs-exo respectively.The expression ratio of Th17 and Treg cells were detected by flow cytometry.The expression of Th17 and Treg cell-associated transcription factors were detected by real-time quantitative PCR,the secretion level of cytokines in cell culture supernatant were detected simultaneously.6.Statistical analysis:The data of this study were analyzed by SPSS software version 19.0.The data are presented as the mean value ± standard error of the mean(SEM)and were atatistically analyzed by a one-factor analysis of variance(ANOVA).Survival curves were plotted as Kaplan-Meier curves and analyzed with log-rank tests.P values<0.05 were considered statistically significantResults:1.Bone marrow-derived mesenchymal stem cells were successfully cultured.The surface standard makers of mesenchymal stem cells detected by flow cytometry shows that primary cultured MSCs met the literature reports and international identification criteria,and MSCs induced by adipogenesis and osteogenic induction showed fat droplets and calcium nodules2.The western blot results showed that Fib-exo and MSCs-exo were positive for CD9,CD81,and CD63,markers of exosomes,thereby confirming the presence of exosomes.Nanosight and transmission electron microscopy showed that the precipitate obtained through sequential ultracentrifugation were exosomes.3.The cGVHD mouse model was successfully established.The cGVHD mice behaved typical clinical manifestations of cGVHD.The pathological damage of cGVHD occurred in the skin,lung and liver of mice,and the Y chromosome appeared in the cGVHD mice.4.The cGVHD mice treated with MSC-exo improved the clinical manifestations and the survival time.The pathological damage of cGVHD in the skin,lung and liver were alleviated,MSCs-exo treatment suppressed the activation of CD4 T cells and their infiltration into the lung.MSCs-exo alleviated cGVHD by inhibiting pathogenic T cells and inducing regulatory cells.At the same time,the expression of Th17 cell-associated transcription factors and proinflammatory cytokines were significantly decreased in the MSCs-exo treatment group.5.The PKH26-labeled exosomes were co-incubated with CD3+ T cells,showing that CD3+ T cells were stained,indicating that the purified exosomes were uptaken by CD3+ T cells.MSCs-exo supregulated the percentage of CD25 + Foxp3 + CD4+Treg cells,while markedly suppressed the differentiation of Th17 cells.The expression of Th17 cell-associated transcription factors and pro-inflammatory cytokines were significantly decreased in the culture supernatant.Conclusions:Our study suggests that exosomes released from MSCs can effectively ameliorate cGVHD in mice by inhibiting the activation and infiltration of CD4 T cells.Furthermore,MSCs-exo exhibits immunomodulatory potency by inducing regulatory T cells and inhibiting Th17 cells.Thus,MSCs-exo provides a new therapeutic paradigm for cell-free MSCs-based therapies for cGVHD treatment.Our work supports further investigation into understanding the underlying mechanism so that the intended therapeutic effect can be translated and optimized.