The Immunoregulation Of Myeloid-derived Suppressor Cells In Experimental Autoimmune Myocarditis And Its Mechanistic Study

Part Ⅰ The dynamic expression of Myeloid-derived suppressor cells in experimental autoimmune myocarditis miceObjective To investigate the dynamic expression of myeloid-derived suppressor cells(MDSCs)in experimental autoimmune myocarditis(EAM).Methods The model of experimental autoimmune myocarditis was established by subcutaneous immunization with cardiac α-myosin heavy chain(My HC-α)dissolved in saline and emulsified 1:1 in complete Freund’s adjuvant.MDSCs in peripheral blood,spleen,bone marrow,lymph nodes and heart of mice were detected by flow cytometry.Simultaneously,the heart tissues of mice were stained with hematoxylin and eosin(H&E),to analyse the the correlation between the percentage of MDSCs in spleen and the clinical myocarditis scores.Results As shown in H&E staining,the cardiomyocytes of EAM mice appeared to be swelling and necrotic from day 10 after immunization,with the blood vessels and pericardium infiltrated with inflammatory cells.On day 14,the inflammation of myocarditis was further aggravated and fibrosis was observed in a small part of the myocardium.Inflammation responses started to decrease with increased myocardial fibrosis from day 21.The percentages of MDSCs in the bone marrow and spleen were maximized on days 14 and 21,respectively.Correlation analysis showed that there was a positive correlation between the percentages of MDSCs in the spleen and the clinical scores of myocarditis.Conclusion This study suggests that MDSCs were released from the bone marrow into blood and migrated to the heart and secondary lymphoid tissues,such as spleen and lymph nodes during myocarditis,which confirms the close involvement of MDSCs in the development of EAM.Part Ⅱ The regulation of MDSCs in Th17/Tregs balance of myocarditisObjective Having shown that MDSCs are closely involved in the development of EAM,we observed the development of EAM and the balance of Th17/Treg cell through different ways of MDSCs regulation,in order to explore its immunomodulatory effects in EAM.Methods we controlled MDSC numbers via adoptive transfer(AT)of purified MDSCs by magnetic beads or depletion by intraperitoneal gemcitabine administration in murine EAM model,to observe the percentages of Th17 cells and Tregs in spleen by flow cytometry,the inflammatory infiltration of heart sections by H&E staining and the expression of inflammatory cytokines in spleen tissues by real-time quantitative polymerase chain reaction(RT-q PCR).Results The percentage of MDSCs by flow cytometry confirmed the success of administration.In the early stages of myocarditis(ie,d0 and d7 after immunization),adoptive transfer of MDSCs or depletion of MDSCs decreased the percentage of Th17 cells and reduced inflammatory infiltrations of the myocardium.Adoptive MDSCs increased the percentage of Tregs,while MDSCs depletion had no significant effect on the percentage of Tregs.Correspondingly,the expression levels of TNF-α,IL-10 and TGF-β were significantly decreased compared with EAM mice,except IL-1β.However,adoptive transfer of MDSCs after MDSCs depletion by gemcitabine,could significantly upregulated the percentage of Th17 cells and accelerate clinical scores of myocarditis,with no change of the percentage of Tregs.Compared with TNF-α,TGF-β and IL-1β,only the level of IL-10 was significantly increased after adoptive transfer.Conclusion The role of MDSCs in myocarditis is bi-directional.MDSCs have a protective effect on heart at a high level of inflammation in vivo,while MDSCs are pathogenic when the body is in a low-level inflammatory state,which,to some extent,depends on the balance of Th17/Tregs.Part Ⅲ The effect of MDSCs on T cell proliferation and differentiationObjective Through above-mentioned experiments,it is known that MDSCs modulate the development of EAM and may participate in the regulation of Th17/Tregs balance.To further study the effect of MDSCs on T cell response,we detected the proliferation and differentiation of T cells via co-culture system,and explored its relevant mechanism.Methods Purified MDSCs and splenic mononuclear cells were co-culturing at different ratios(0: 1,1: 8,1: 4,1: 2,1: 1 and 2: 1),and the level of T cell proliferation was assessed by a CFSE dye dilution assay.The administration of blocking arginase 1(Arg-1)and inducible NO synthase(i NOS),is helpful to investigate the mechanism of MDSCs in the regulation of T cell proliferation.At the ratio of 1:4,we detected the percentages of Th17 cells and Tregs,and the levels of IFN-γ,IL-1β,IL-6,IL-10 and TGF-β by enzyme-linked immunosorbent assay(ELISA).To further study the effect of MDSCs on T cell differentiation,we co-culture MDSCs and T cells in the Th17-or Tregs-skewing conditions at the same ratio of 1:4,and explore the differentiation of T cells.Results With the addition of purified MDSCs,the suppression of T cell proliferation reached the maximum at a ratio of 1: 4.However,the another rise of MDSCs gradually restored the T cell proliferation.At the ratio of1: 4,the block of Arg-1 partially alleviated their suppressive function.The percentages of Th17 cells and Tregs in T cells increased at the ratio of 1:4,and the levels of IFN-γ,IL-1β,IL-6 and IL-10 in the supernatant increased.However,MDSCs were found to induce differentiation of Th17 cells in the Th17-skewing condition and inhibited the expression of Foxp3 in T cells in the Tregs-skewing conditions.Conclusion The effect of MDSCs immunosuppression on T cell proliferation is double-faced,which is partly dependent on Arg-1.Compared with the induction of Tregs,MDSCs are more likely to promote Th17 cell differentiation.