ALD-DNA Regulate The Immunosuppressive Function Of MDSCs In Sle Mice

Objective:To investigate the changes in the proportion and function of myeloid-derived suppressor cells(MDSCs)in systemic lupus erythematosus(SLE)mice and their effects on the progression of SLE.To explore the mechanism of activated lymphocyte-derived DNA(ALD-DNA)regulated the immunosuppressive function of MDSCs.Methods:(1)SLE mouse model was established,and the proportions of MDSCs was detected in spleen,lymph nodes,peripheral blood,and kidney of SLE mice at different periods by FCM.The activity of Arg-1 was measured by colorimetric assay and the expression of NO was measured by Griess assay.FCM detected the suppressive capacity of MDSCs on the proliferation of CD4~+T cells and the expression level of CD80,CD86,CD40 and MHCⅡ.(2)SLE mouse model was established,MDSCs from the spleens of mice at week 5 and week 14were separated by magnetic beads and adopted into mice through the tail vein,respectively.The serum levels of urea nitrogen and creatinine were detected.The serum level of anti-ds DNA antibodies was detected by ELISA,and pathological changes of glomeruli were detected by HE staining,PAS staining,and IF staining.FCM detected the proportion of GCB cells,Tfh cells and plasma cells in spleen and lymph nodes of mice,as well as plasma cells in bone marrow.(3)In order to explore the effect of ALD-DNA on the immunosuppressive function of MDSCs,the effector molecules,the effects of MDSCs on CD4~+T cells proliferation and surface molecules after treatment with ALD-DNA were detected.(4)q RT-PCR detected the changes of ISGs and IFNAR expression in MDSCs after ALD-DNA treatment.FCM and q RT-PCR were used to detect IFN-βexpression in MDSCs.In order to explore the effect of IFN-βsecreted by MDSCs on the immunosuppressive function of MDSCs,the effector molecules,the effects of MDSCs on CD4~+T cells proliferation and surface molecules were detected after blocking IFNAR1.In order to explore the molecular mechanism by which ALD-DNA regulates the secretion of IFN-βby MDSCs,Western Blot detected the phosphorylation level of TBK1 and IRF3 in MDSCs treated with ALD-DNA,and Co-IP detected ubiquitination and K63-Ub level of STING in MDSCs.(5)SLE mouse model was established,q RT-PCR detected the expression level of IFN-βin different cells of mouse spleen.FCM and q RT-PCR detected the expression level of IFN-βin MDSCs at different stages.In order to investigate the effect of IFN-βsecreted by MDSCs on the progression of SLE in vivo,the expression level of IFN-βin MDSCs was interfered by si RNA,and then si IFN-β-MDSCs and si NC-MDSCs were adopted into mice through the tail vein.The serum levels of urea nitrogen,creatinine and anti-ds DNA antibodies were detected,and pathological changes of glomeruli were detected by HE staining,PAS staining and IF staining.FCM detected the proportion of GCB cells,Tfh cells and plasma cells in spleen and lymph nodes of mice,as well as plasma cells in bone marrow.Results:(1)With the progression of SLE disease,the proportion of MDSCs gradually increased,peaking at 5th week of the disease.In addition,compared with the 7th week,the activity of the effector molecule Arg-1 and the content of NO decreased,the inhibitory effect on the proliferation of CD4~+T cells was weakened,and the expressions of CD80,CD86,CD40 and MHCII on the surface of MDSCs were increased in the 14th week.(2)Compared with control mice,adoptive transfer of MDSCs increased disease severity in SLE mice.The serum levels of urea nitrogen,creatinine and anti-ds DNA antibody were increased.The infiltration of glomerular inflammatory cells and the proliferation of stromal cells and mesangial tissues were increased,the accumulation of Ig G in the glomeruli were increased.Compared with control mice,the proportion and number of GCB cells,Tfh cells,plasma cells in spleen and lymph nodes,as well as plasma cells in bone marrow were increased in mice after adoptive transfer of MDSCs.(3)After treatment with ALD-DNA,the activity of the effector molecule Arg-1 and the content of NO of MDSCs decreased,the inhibitory effect on the proliferation of CD4~+T cells decreased,and the expressions of CD80,CD86,CD40 and MHCII on the surface of MDSCs increased.(4)After ALD-DNA treatment,the expression level of ISGs and IFNAR1 in MDSCs increased,and IFN-βsecreted by MDSCs increased significantly.After blocking IFNAR1,the activity of the effector molecule Arg-1 and the content of NO increased,the inhibitory effect on the proliferation of CD4~+T cells was enhanced,and the expression levels of CD80,CD86,CD40 and MHCII on the surface of MDSCs decreased.In addition,ALD-DNA treatment enhanced the phosphorylation levels of TBK1 and IRF3 in MDSCs,and the K63-Ub level of STING in MDSCs increased.(5)With the progression of SLE,and the level of IFN-βexpression in MDSCs in spleen increased and was significantly higher than that of B cells,CD4~+T cells,CD8~+T cells,NK cells and macrophages.In vivo experiments showed that,compared with si NC-MDSCs group,after adoptive transfer of si IFN-β-MDSCs,the serum levels of urea nitrogen,creatinine and anti-ds DNA antibody were decreased.The infiltration of glomerular inflammatory cells and the proliferation of stromal cells and mesangial tissues were decreased,the accumulation of Ig G in the glomeruli were decreased.Compared with si NC-MDSCs group,The proportion and number of GCB cells,Tfh cells,plasma cells in spleen and lymph nodes,as well as plasma cells in bone marrow were reduced in mice after adoptive transfer of si IFN-β-MDSCs.Conclusions:(1)With the progression of SLE,the proportion of MDSCs in spleen,lymph nodes,peripheral blood and kidneys of mice increased,and the immunosuppressive function of MDSCs decreased in the later stage of SLE.(2)ALD-DNA promoted the activation of TBK1-IRF3 signaling pathway and induced the production of IFN-βby enhancing STING K63-Ub level in MDSCs,thus downregulating the autoimmune suppression function of MDSCs.(3)MDSCs can promote the proportion and number of GCB cells,Tfh cells and plasma cells in mice through the secretion of IFN-βto enhance humoral immune response and promote the disease progression of SLE.