BM-MSCS Alleviate The Development Of ESS By Enhancing The Suppressive Function Of MDSCs

Objective:In this study,we tried to explore the effect of BM-MSCs on the progression of experimental Sj?gren syndrome(ESS),and then,the regulation of BM-MSCs on the immunosuppressive function of MDSCs and its molecular mechanism were further explored.Methods:(1)ESS mouse model was established and BM-MSCs were adoptively transferred to ESS mice on the 18th and 25th day after the first immunization.The saliva flow rates were measured.Autoantibodies against SG Ags,ANA,and anti-M3R Abs were analyzed in the serum of mice by ELISA.Proportions of Th1 cells and Th17 cells were measured in spleens(SP)and cervical lymph node(CLN)by flow cytometry(FCM).(2)After BM-MSCs were adoptively transferred to ESS mice,proportions of MDSCs PMN-MDSCs and M-MDSCs were measured in SP and CLN of mice by FCM.CD11b~+Gr-1~+MDSCs in the spleens of ESS mice with different treatments were sorted by immunomagnetic beads.The suppressive capacity of MDSCs on the proliferation of CFSE-labeled CD4~+T cells was detected by FCM.The activity of Arg-1 was measured by colorimetric assay and the expression of NO was measured by Griess assay.(3)MDSCs isolated from the spleens of ESS mice were cocultured with BM-MSCs.The suppressive capacity of MDSCs on the proliferation of CFSE-labeled CD4~+T cells was detected by FCM.The activity of Arg-1 was measured by colorimetric assay and the expression of NO was measured by Griess assay.Surface molecules including CD40,CD80,CD86 and MHC-II on MDSCs were analyzed by FCM.(4)To further explore the molecular mechanisms for the regulation of BM-MSCs on MDSCs,real time fluorescene quantitative PCR(qRT-PCR)and FCM were used to analyze the predominant molecules regulating the function of MDSCs(TGF-β1).Anti-TGF-β1 antibody was used to block the TGF-β1 pathway in the co-culture of BM-MSCs and MDSCs,then the suppressive capacity of MDSCs on the proliferation of CFSE-labeled CD4~+T cells was detected by FCM,the activity of Arg-1 was measured by colorimetric assay and the expression of NO was measured by Griess assay.Results:(1)Compared to the control group,the saliva flow rate was increased in the BM-MSCs treated group(p<0.01).Levels of autoantibodies against total SG Ags,ANA,and anti-M3R Abs were significantly decreased after BM-MSCs treatment(p<0.01).The proportion of Th1cells in SP of mice treated with BM-MSCs was 4.15±0.65%,the proportion of Th17 cells was4.83±0.30%,and the proportion of Th1 cells in SP of control mice was 9.03±0.83%,the proportion of Th17 cells was 8.25±0.35%.The proportion of Th1 cells in CLN of mice treated with BM-MSCs was 0.99±0.05%,the proportion of Th17 cells was 1.01±0.18%,and the proportion of Th1 cells in CLN of control mice was 3.50±0.32%,the proportion of Th17cells was 2.01±0.29%.Both Th1(p<0.01)and Th17(p<0.05)cell responses were also inhibited after the treatment of BM-MSCs.(2)After BM-MSCs were adoptively transferred to ESS mice,the proportion of MDSCs in SP of mice was 2.77±0.28%,the proportion of MDSCs in CLN of mice was 1.23±0.11%.The proportion of MDSCs in SP of control mice was 1.31±0.27%,and the proportion of MDSCs in CLN of ctrl mice was 0.45±0.04%.The proportion of MDSCs in SP and CLN of BM-MSCs treated group was significantly increased(p<0.01).In addition,proportions of PMN-MDSCs and M-MDSCs were also significantly increased(p<0.01).MDSCs from mice treated with BM-MSCs had a stronger ability to inhibit the proliferation of CD4~+T cells,and the levels of Arg1 and NO were higher than the control group(p<0.01).Additionally,BM-MSCs had the ability to upregulate the suppressive function of two subpopulations of MDSCs in vivo.(3)MDSCs treated with BM-MSCs showed a stronger ability to inhibit the proliferation of CD4~+T cells in vitro,and the levels of Arg1 and NO were higher than the untreated group(p<0.01).And,BM-MSCs had the ability to upregulate the suppressive function of two subpopulations of MDSCs in vitro.Besides,the expression of CD40,CD80,CD86 and MHCII on MDSCs were decreased after BM-MSCs treatment.(4)BM-MSCs expressed high level of TGF-β1 cocultured with MDSCs.After blocking TGF-β1 pathway in the co-culture of BM-MSCs and MDSCs,the regulation of BM-MSCs on MDSCs was diminished.The suppressive capacity of MDSCs on the proliferation of CD4~+T cells was weak,and the levels of Arg1 and NO were decreased(p<0.05).Conclusion:(1)BM-MSCs can effectively alleviate the development of ESS by enhancing the suppressive function of MDSCs and their subpopulations in vitro and in vivo.(2)BM-MSCs enhance the suppressive function of MDSCs mainly by secreting TGF-β1.