Molecular Mechanism Of GITRL Regulation On Myeloid-Derived Suppressor Cells And Its Role In Mouse Experimental Sj?gren's Syndrome

As a systemic autoimmune disease,primary Sj?gren's syndrome(p SS)is characterised by progressive inflammation and tissue damage of salivary glands and lacrimal glands.Nowadays,several factors are responsible for the development of the disease,such as genetic factors,environmental factors,hormonal factors.It has been reported that SS is associated with the break of the immune balance of Th1/Th2 cells,and Th17 cells are also considered to participate in the development of SS.T cells,B cells and dendritic cells(DCs)are found in the local lesion tissues in patients with pSS.In addition,pSS can not only lead to the lesions of other organs,but also increase the incidence of B-cell non Hodgkin's lymphoma.Myeloid-derived suppressor cells(MDSCs)are a phenotypically heterogeneous cell population with suppressive capacity which derived from bone marrow that includes granulocytic MDSCs(G-MDSCs)and monocytic MDSCs(M-MDSCs).In healthy individuals,MDSCs are generated in the bone marrow and quickly differentiate into mature DCs,macrophages or granulocytes.In pathological conditions,the differentiation of MDSCs into mature myeloid cells is partially blocked,which results in the expansion of this population.Recently,accumulating studies have shown that MDSCs are associated with the development of autoimmune diseases,but the role of MDSCs in autoimmune diseases is still controversial.It is reported that the role of MDSCs in different autoimmune diseases may not be the same,even in the same autoimmune diseases,the effects of MDSC regulation may present the contrary results.It is probably associated with the pathogenic mechanism,internal environment,different subsets of MDSCs and the internal environment in different phases of the disease.Next,the question is what the role of MDSCs and their subsets in pSS is? We thereafter performed the experiments in part I,and the mouse experimental Sj?gren's syndrome(ESS)was used to analyze the proportion and the function of MDSCs in the development of the disease,and then explore the molecular mechanism for the regulation of MDSCs.Part I.The changes of the percentage and the function of MDSCs in the development of mouse experimental Sj?gren's syndromeFirstly,we established the mouse experimental Sj?gren's syndrome model,confirmed the success of the model by measuring the saliva flow rate,auto-antibodies and histopathology of the salivary gland.Compared to the control group,in the ESS mice,the saliva flow rate was reduced,autoantibodies against SG,M3 R and antinuclear antibodies were significantly increased.Moreover,the size of salivary glands and cervical lymph nodes was enhanced.And,the histopathological examination of salivary glands from ESS mice of 20 weeks showed that plenty of lymphocytes were infiltrated in tissues and gland ducts were also destroyed.Thereafter,we confirmed the success of the model establishment.Next,we investigated the proportion and number of MDSCs and their subsets in the development of ESS by flow cytometry.The data showed that proportions of MDSCs in the spleen,lymphoid nodes and blood started to increase at the time of the secretion of saliva decreased,and then reached a peak on day 18 after the first immunization,and finally stayed at a relative high level until the late stage(10 weeks)of the disease.Moreover,MDSCs began to infiltrate in the local salivary gland tissue in the late stage.Meanwhile,the number of M-MDSCs and GMDSCs in spleens was also significantly enhanced with the development of the disease.Next,we further analyzed the function of MDSCs and their subsets.Using microbeads and flow cytometry to isolate the MDSCs and their subsets on day 18 and 10 weeks after the first immunization,we detected the main effector molecules(Arg-1,NO)and surface markers(CD40,CD80,CD86,MHCII)related to the function of MDSCs,and the suppression of MDSCs on CD4+ T cells was also analyzed.The data showed that MDSCs and their subsets from the early stage of ESS had powerful immunue suppressive capacity.However,with the development of the disease,the suppressive capacity of MDSCs from the late stage was significantly decreased.In addition,the expression of suppressive molecules in MDSCs and their subsets from the late stage was decreased and the maturation of the cells was enhanced.Thereafter,the data showed that the suppressive capacity of MDSCs and their subsets was gradually reduced with the development of ESS.By adoptive transfer of MDSCs derived from different stages into ESS mice,we found that MDSCs isolated from the early stage of the disease could significantly enhance the saliva flow rate,decrease the level of auto-antibodies and the percentages of Th1 and Th17 cells,and thus delaying the development of ESS.However,MDSCs isolated from the late stage of the disease did not show any obvious therapeutic effect,which even aggravated the disease.The level of autoantibodies against SG was increased and the saliva flow rate was strongly decreased.Meanwhile,the percentages of Th1 and Th17 cells in draining lymph nodes also enhanced.Next,the question is why the immunosuppression of MDSCs changed with the development of ESS? Thus,we performed the experiments in Part II.Part II.The regulation of GITRL on the function of MDSCsThe ligand of glucocorticoid-induced tumor necrosis factor(GITRL)is a newly discovered member of tumor necrosis factor(TNF)superfamily.According to the past report,GITRL is critically involved in the regulation of immune response via modulating the functions of both effector T cells and regulatory T(Treg)cells.Recent years,accumulating evidences have demonstrated that GITRL is involved the development of different autoimmune diseases.In this study,we found that GITRL expression of cells in the spleen,lymphoid nodes and salivary gland tissues were significantly increased.In addition,we found that GITR was expressed on MDSCs isolated from ESS mice.Our data showed that the immunosuppressive capacity of MDSCs was down-regulated after MDSCs treated with GITRL.Moreover,we transferred GITRL-treated MDSCs into mice,and found that the saliva flow rate did not recover to the normal level,and the related autoantibodies also stayed at a high level.In addition,the proportions of Th1 and Th17 cells were not decreased.All these data demontrates that SG GITRL-treated MDSCs have lost their therapeutic effect as the control group do.To further observe the regulation of GITRL on MDSCs in ESS mice,exogenous GITRL was injected into ESS mice,and the suppressive capacity of MDSCs was down-regulated after GITRL treatment.Furthermore,we used small interference RNA to down-regulate the GITR expression,and then the modified MDSCs were transferred into ESS mice,and the data showed that the modified MDSCs could efficiently enhance the saliva flow rate,decrease the levels of auto-antibodies and the proportions of Th1 and Th17 cells.All these data demonstrate that GITRL could down-regulate the immunosuppressive capacity of MDSCs via binding to GITR on MDSCs,and thus promoting the development of ESS.Taken together,according to the above investigation,we can come to the conclusion that,the increased GITRL can interact with the GITR on MDSCs,and then down-regulate the immunosuppressive capacity of MDSCs in ESS mice,thus finally aggravating the development of the disease.This study reveals the microenvironment regulation on MDSCs in the development of Sj?gren's syndrome from GITRL/GITR aspect,which expands our understanding of the role of MDSCs in autoimmune diseases,and provides new ideas for the clinical treatment of autoimmune diseases.