Tolerance Disorder Of Anergic B Cells In The Pathogenesis Of Systemic Lupus Erythematosus

AIMS Systemic lupus erythematosus(SLE)is a chronic autoimmune disease with multiple systems involved.The pathogenesis of SLE is the result of the interaction of heredity,immunity and environment.The key in the pathogenesis of SLE is the activation of autoreactive B cells,which produces a large number of autoimmune antibodies against nucleus,cytoplasm and other cellular components.Anergic B cells are self-reactive B lymphocytes in the peripheral which achieve immune tolerance by clone anergy.Anergic B cells have the potential to become pathogenic autoreactive B cells.BND cells with CD19+CD27-Ig D+Ig M-/low as surface marker are recognized as anergic B cell markers at present.It has been reported that the BND cells play a role in the pathogenesis of autoimmune diseases such as rheumatoid arthritis and type 1 diabetes mellitus.However,there are few studies on BND cells in patients with SLE.In this study,we aimed to explore the role of BND cells in the pathogenesis of SLE.METHODS 1.The frequency of BND cells in peripheral blood of SLE patients and healthy controls was detected by flow cytometry,and the correlation between the frequency of BND cells and clinical indexes was analyzed.2.Flow cytometry was used to detect the CD markers on the surface of BND cells in peripheral blood of SLE patients.The level of calcium influx and the increase of intracellular tyrosine phosphorylation after BCR stimulation were tested by flow cytometry.These were in order to evaluate the immune tolerance stat of BND cells in peripheral blood of SLE patients.3.BND cells and Ig M+ naive B cells from SLE patients and healthy controls were sorted by flow cytometry and sequenced to understand the transcriptional characteristics of BND cells.4.The cytokines that regulate BND cells were identified by plasma cross-culture tests,plasma cytokine difference detection between SLE patients and HC and Th1/Th2/Th17 cytokines stimulation tests.5.The correlation analysis between plasma IL-4 expression level and clinical indexes in SLE patients and the whole genome association analysis(GWAS)was used to determine whether IL-4 was related to the pathogenesis of SLE.6.To establish the model of anergic B cells in vitro in order to study the function of anergic B cells.7.The model of anergy B cells in vitro was used to explore whether IL-4 could regulate anergic B cells.8.Immunoconfocal microscopy,indirect labeling flow cytometry,fluorescence quantitative PCR and Western blot were used to explore the mechanism of IL-4 in regulating anergic B cells.RESULTS 1.The frequency of BND cells in peripheral blood of SLE patients was decreased than that of healthy controls.And the frequency of BND cells in SLE patients was negatively correlated with disease activity.Glucocorticoid therapy could up-regulate the frequency of BND cells in SLE patients.2.The surface CD80,CD86,CD95 and CD71 of BND cells in SLE patients were not significantly different from those of healthy controls.The calcium influx level and tyrosine phosphorylation of BND cells in SLE patients were higher than those in healthy controls after BCR stimulation.The results showed that the immune tolerance of BND cells in SLE patients was breached and in a relative activation state.3.Transcriptome sequencing analysis showed that the expression of genes related to cell activation,proliferation and differentiation in BND cells of SLE patients was different from those of healthy controls.Function and pathway analysis showed that BND cells in SLE patients were different from healthy controls in cell activation,immune function regulation,cytokine-cytokine receptor interaction and so on.4.IL-4,IL-6,IL-10 and IFN-γ were highly expressed in plasma of SLE patients,and only IL-4 could decrease the frequency of BND cells in vitro.5.The level of plasma IL-4 in SLE patients was positively correlated with SLEDAI score and negatively correlated with complement C3 and C4.GWAS analysis indicated that IL-4 was associated with SLE.6.Anti-CD79 b monoclonal antibody could induce naive B cells to become anergic B cells in vitro.7.IL-4 could rescue the surface Ig M of anergic B cells and reverse the immune tolerance stat of anergic B cells.8.IL-4 rescued the surface Ig M of anergic B cells by promoting the recycling of internalized Ig M to the cell surface and promoting the new synthesis of Ig M,and these effects were mediated by p-STAT6.CONCLUSIONS Our study confirmed that the frequency of BND cells in peripheral blood of SLE patients decreased than that of healthy controls,and the frequency of BND cells was related to the disease activity of SLE,which could be a potential index to evaluate the disease activity.The response ability of BCR signaling pathway in BND cells of SLE patients was higher than that of healthy controls,which indicated that the immune tolerance of BND cells in SLE was disordered and relatively activated.The transcriptional study of BND cells in healthy people and SLE patients was carried out for the first time,and the anergy related gene was proposed,which laid a foundation for further exploring the mechanism of immune tolerance of anergic B cells.In addition,we found that IL-4,which is highly expressed in SLE plasma,plays an important role in the decrease of the frequency of BND cells.IL-4 could rescue surface Ig M of anergic B cells by promoting the recycling of internalized Ig M to the cell surface and promoting the new synthesis of Ig M,and these effects were mediated by p-STAT6.Our study found a new strategy for the treatment of SLE,that is,the disease progression of SLE can be controlled by inducing peripheral B cell anergy or targeting blocking IL-4 signaling pathway.