HMGB1/NETs Mediates The Activation Of Dendritic Cells Through Up-Regulated MTOR Channel In The Pathogenesis Of Systemic Lupus Erythematosus

PART ?Objective To investigate the peripheral blood level of HMGB1 and neutrophil extracellular trap net(NETs),as well as the possible association with clinical features in systemic lupus erythematosus(SLE).To identify the phenotypic characteristics and the expression of p-m TOR channel on circulating m DCs and explore the role of p-m TOR channel on circulating m DCs mediate immune responses in patients of SLE.Methods A total of 35 SLE patients and 20 sex-and age-matched normal individuals were enrolled in the prensent study.Polymorphonuclear neutrophils(PMNs)were isolated with human peripheral blood neutrophil separation medium from peripheral blood of subjects.The formation of NETs and the expression of HMGB1 on its in two groups of subjects was evaluated by fluorescence microscopy.At the same time,we adopt quantitative fluorescence photometry to determine the quantity of NETs.Plasma HMGB1 were determined with ELISA.The phenotype of circulating m DCs and the expression of m TOR on m DCs in peripheral blood of patients with SLE by using multi-colour flow cytometry.The possible associations of NETs,HMGB1 and mDCs' phenotype with disease activies,clinical fatures,serum levels of autoantibodies as well as certain labortory parameters were also explored.Result 1.The level of HMGB1,NETs and the expression of CD40,CD86,m TOR on m DCs significantly increased in peripheral blood of SLE patients compared with those of HCs(P<0.05).2.Plasma HMGB1 in SLE patients was positively correlated with NETs,SLEDAI and Anu A,while plasma HMGB1 was inversely correlated with the level of C3,C4 in SLE patients(P<0.05).3.The peripheral blood level of NETs was positively correlated with NETs,SLEDAI and Anu A(P<0.05).4.the expression of CD40 on m DCs was positively correlated with SLEDAI,ESR,hs-CRP,Anti ds-DNA(P<0.05).the expression of CD86 on m DCs was positively correlated with SLEDAI,Anti ds-DNA and Anu A(P<0.05).5.the expression of m TOR on m DCs was positively correlated with HMGB1 and NETs(P<0.05).Conclusion The level of HMGB1,NETs significantly increased in peripheral blood of SLE patients compared with those of HCs,and participate in the pathogenesis of SLE and promote the development of disease.An enhanced activation phenotype of circulating m DCs,and high expression of p-m TOR on m DCs,might contribute to promote the development of SLE.HMGB1 / NETS may mediate the activation of dendritic cells through up-regulated m TOR channel in the pathogenesis of SLE.PART ?Objective To investigate the ability of neutrophil release HMGB1 and their own DNA in NETsosis in patients with systemic lupus erythematosus.Methods The peripheral blood samples of patients with SLE and healthy volunteers were collected.Polymorphonuclear neutrophils were isolated with human peripheral blood neutrophil separation medium from peripheral blood of subjects.We then stimulated the neutrophils with LPS or PMA in vitro.The formation of NETs and the expression of HMGB1 on its in each groups of neutrophils was evaluated by fluorescence microscopy.HMGB1 in supernatants of cultured neutrophils were determined with ELISA.At the same time,we determined the quantity of ds DNA in supernatants of cultured neutrophils with fluorescent quantitative kit.Result 1.The level of HMGB1 significantly increased in each groups of neutrophils from SLE patients compared with those of HCs(P<0.05).2.The LPS-induced NETs and PMA-induced NETs from SLE shared identical property of those from HCs in morphologic characteristics.and the expression of HMGB1 in NETs from SLE is higher than those from HCs by fluorescence microscope.3.The level of ds DNA significantly increased in each groups of neutrophils from SLE patients compared with those of HCs(P<0.05).Conclusion the ability of neutrophil release HMGB1 and their own DNA in NETsosis in patients with systemic lupus erythematosus is stronger than HCs.NETosis may cause high concentration of HMGB1 in peripheral blood of SLE.PART ?Objective Base on m TOR,to explore the role of HMGB1 or NETs in the activation of dendritic cells.Methods The peripheral blood samples of patients with SLE were collected.Peripheral blood monocytes(PBMC)were isolated from peripheral blood of subjects.We then induced m DCs in PBMC to mature with cytokine.At the same time,Polymorphonuclear neutrophils(PMN)were separated from peripheral blood of patients with SLE and NETs were induced.The m TOR channel and its substrate P70S6 K and 4EBP1 expression on dendritic cells after HMGB1/NETs stimulus were determined with RT-PCR,immumofluorescence method.The expression of HLA-DR and costimulatory molecules CD40,CD86 was determined by flow cytometry.The concentrations of IL-1?,IL-6,IL-10 and TNF-? in supernatant were determined by ELISA.Then we blocked m TOR channels with different concentrations of rapamycin(Rapa),The m TOR channel and its substrate P70S6 K and 4EBP1 expression on dendritic cells after HMGB1 stimulus were determined with the same method.The expression of HLA-DR and costimulatory molecules CD40,CD86 was determined by flow cytometry.The concentrations of IL-1?,IL-6,IL-10 and TNF-? in supernatant were determined by ELISA.Result 1.The expression of m TOR channel and its substrate P70S6 K and4EBP1 on dendritic cells is significantly increased after HMGB1 or NETs stimulus(P<0.05).2.The expression of HLA-DR and costimulatory molecules CD40,CD86 is significantly increased after HMGB1 or NETs stimulus(P<0.05).The concentrations of IL-1?,IL-6,IL-10 and TNF-? in supernatant is higher after HMGB1 or NETs stimulus(P<0.05).3.The expression of HLA-DR and costimulatory molecules CD40,CD86 is significantly decreased through Rapa intervention(P<0.05).The concentrations of IL-1?,IL-6,IL-10 and TNF-? in supernatant is lower through Rapa intervention(P<0.05).Conclusion HMGB1 / NETS can mediate the activation of dendritic cells through up-regulated m TOR channel in the pathogenesis of SLE.