The Role Of Abnormal B Cell Activation And Differentiation In The Pathogenesis Of Bullous Pemphigoid

Background:Bullous pemphigoid(BP)is a common autoimmune skin disease characterized by subepidermal blister formation,inflammatory cell infiltration,and autoantibodies and complement deposition in the basement membrane zone.The etiology of BP is complex.The genetic factors and imbalance of immune system are involved in the occurrence and development of BP.After the exposure of autoantigens in the basal membrane zone,the autoreactive B cells and T cells are activated and participated in humoral and cellular immunity to mediateautoantibodies production and skin injury.B cells are the critical cells which could produce pathogenic autoantibodies.A number of clinical studies have shown that the elimination of B cells by anti-CD20 monoclonal antibody has a good therapeutic effect on patients with severe BP,confirming the important roles of B cells in the pathogenesis of BP.However,the abnormality of B cells and its specific roles in the pathogenesis of BP remain to be further studied.B cells are the critical effector cells of humoral immunity,and participate in adaptive immune response by producing autoantibodies,presenting antigens,secreting cytokines and providing help for T cells.The activation and differentiation of B cells and autoantibody production are improtant steps in pathogenesis of autoimmune diseases.Moreover,the B cell activation and differentiation are regulated by many factors,including B cell surface receptor activation,antigen stimulation,and induction of cytokines and transcription factors,etc.For example,the chemokine receptor CXCR4 on the surface of B cells is involved in the migration and differentiation of B cells.In addition,a large number of studies have reported the crosstalk between neutrophils and B cells in inflammatory sites or immune organs.In addition,neutrophils can form neutrophil extracellular traps(NETs)which could promote B cell proliferation,activation and differentiation.However,the key B cell subsets in circulation and skin lesions of BP patients and the upstream mechanisms of abnormal B cell activation and differentiation in BP patients remain unclear.Based on these findings,this study focused on the abnormal activation and differentiation of B cells which induced by abnormally overexpression of CXCL12/CXCR4 axis and NETs in BP patients and its underline molecular mechanism.Objectives:1.To investigate the critical pathogenic B cell subsets in peripheral blood and skin lesions of patients with BP.2.To identify the role of upregulated CXCL12/CXCR4 axis in regulating abnormal B cell differentiation and the underlying mechanisms.3.To identify the role of neutrophil extracellular traps in mediating B cell activation and differentiation and its specific mechanisim in the pathogenesis of BP.Methods:1.Analysis on the B cell subsets in peripheral blood and skin lesions of BP patients.We collected the peripheral blood from BP patients and sex-and age-matched healthy controls,and then the frequencies of B cell subsets were analyzed by flow cytometry.Correlation analysis was performed between the frequencies of B cell subsets and autoantibody titers in patients with BP.Each B cell subpopulations from BP patients and healthy controls were sorted by flow cytometry,and then these B cells subsets were cultured in vitro.The supernatants from B cell subsets were collected and the antibody titers in the supernatants were detected by ELISA to dientify the critical B cell subsets which produce pathogenic autoantibodies.The blister fluid samples of BP patients were collected and the proportions of different immune cells in blister fluid weredetected by flow cytometry.Immunohistochemistry was used to analyze the B cells or plasma cells expression in the skin lesions of BP patients.Tissue immunofluorescence staining were employed to dertimine the critical B cell subsets infiltrated in the skin lesions of BP patients.2.Analysis on the differentially expressed genes of B cells from BP patients and healthy controls.The peripheral blood from 3 BP patients and 3 healthy controls were collected.After sorted by magnetic beads,CD19~+B cells were employed by RNA sequencing analysis and the differentially expressed genes were analyzed.GO functional clustering and KEGG pathway analysis were used to compare the differentially expressed genes.Then the highly expressed gene CXCR4 were verified by quantitative real-time PCR and flow cytometry.Tissue immunofluorescence co-localization were used to dertimine the expression and function of CXCR4~+B cells in skin lesions of BP patients.3.Analysis on the role of CXCL12/CXCR4 axis in chemotaxis of B cells in skin lesions of BP patients.The serum and blister fluid samples were collected from BP patients and healthy controls.The levels of CXCL12 in serum and blister fluid were detected by ELISA.Tissue immunofluorescence staining were used to observe the CXCL12-producing cells in the skin lesions of BP patients.Transwell experiment was used to study the effect of CXCL12 and blister fluid from BP patients on CXCR4~+B cells chemotaxis.In addition,CXCL12 neutralizing antibody was used to block CXCL12/CXCR4 axis and then chemotaxis of CXCR4~+B cells was detected in vitro.4.Analysis on the effect of CXCL12/CXCR4 axis in mediating B cell differentiation and the underlying mechanisms.CD19~+B cells from peripheral blood of BP patients and healthy controls were sorted by magnetic beads.Then,CD19~+B cells were stimulated with recombinant CXCL12 protein for 7 days in vitro,and the frequencies of B cell subsets were analyzed by flow cytometry and the titers of autoantibody in the supernatant were detected by ELISA,respectively.In the functional inhibition experiment,CD19~+B cells were pretreated with CXCR4 inhibitor and then stimulated with CXCL12.Then,B cell subsets and autoantibody titers were detected by flow cytometry and ELISA.In addition,RNA-sequencing analysis was used to analyze the differentially expressed transcription factors related to B cell differentiation in B cells,and real-time quantitative PCR was performed to identify the differentially expressed transcription factors.After CD19~+B cells were pretreated with CXCL12 or CXCR4 inhibitor,real-time quantitative PCR,western blot,cell immunofluorescence were used to determine the key transcription factor related to CXCL12/CXCR4 axis in meditaing B cell activation and differentiation.Further,CD19~+B cells were pretreated with c-Myc inhibitor,and then B cell subsets were detected by flow cytometry and autoantibody titers were detected by ELISA.5.Analysis the expression NETs in circulation and skin lesions of BP patients.Tissue immunofluorescence staining was used to detect the expression of NETs in skin lesions of BP patients.The levels of MPO-DNA complex and cf DNA in serum and blister fluid of BP patients were detected by ELISA.Correlation analysis was performed between the levels of MPO-DNA complex in serum and blister fluid and disease severity of BP patients.6.Analysis on the NETs formation induced by immune complex and the underlying mechanisms.We collected the peripheral blood from BP patients and healthy controls.Then the neutrophils were isolated using magnetic beads positive selection system.The spontaneous formation of NETs in the resting condition was observed by cell immunofluorescence analysis.Neutrophils were stimulated with serum and blister fluid from BP patients and healthy controls,and the amount of NETs was detected by immunofluorescence and ELISA.Then,BP180-NC16A protein and anti-BP180-NC16A Ig G were purified to form BP180-NC16A ICs.After neutrophils were stimulated with BP180-NC16A ICs,NETs formation was detected by immunofluorescence and ELISA.Neutrophils were pretreated with Fc R inhibitor,NADPH inhibitor or PAD4 inhibitor,respectively,and then stimulated with BP180-NC16A ICs to detect the effects of key pathways or receptors in mediating NETs formaiton.7.Analysis on the role of NETs in promoting B cell differentiation and antoantibodies generation and the underlying mechanisms.NETs formation was induced by neutrophils of BP patients stimulated with BP180-NC16A ICs,and then NETs components were extracted.CD19~+B cells from BP patients and healthy controls were isolated by magnetic beads.In our in vitro condition,CD19~+B cells were stimulated with NETs for 7 days,and B cell subsets and autoantibody titers were detected by flow cytometry and ELISA.The activation of signal pathways after B cells treated with NETs were observed by flow cytometry analysis.Further,CD19~+B cells were pretreated with MAPK P38 signaling pathway inhibitors to detect the effect of blocking the critical signaling pathway on B cell differentiation and autoantibody production.Results:1.The alteration of B cell subsets in BP patients and antibody secreting B cells are expanded in the peripheral blood and skin lesions of BP patients.The flow cytometry results showed that that DN(CD19~+CD27~-Ig D~-),switched memory(CD19~+CD27~+Ig D~-,SWM)B cells and plasma cells(CD19~+CD138~+)were significantly increased,whereas na?ve(CD19~+CD27~-Ig D~+)B cells were decreased in BP patients compared to those in healthy controls,and no differences were detected in the unswitched memory(CD19~+CD27~+Ig D~+,USM)B cells.The levels of autoantibodies in the culture supernatant of the above five B cell subsets were detected,and the ELISA data showed that plasma cells and DN B cells produced more anti-BP180 NC16A antibodies than other B cell subsets,suggesting that plasma cells and DN B cells are the main ASCs that expand in the peripheral blood of BP patients.Additionally,the frequencies of DN B cells and CD19~+CD138~+plasma cells showed strong positive correlations with the titers of anti-BP180-NC16A antibodies,the indicator of BP severity.Immunohistochemical analyses revealed that CD19~+B cells in 84%samples.Immunofluorescence revealed that most infiltrated B cells in BP skin lesions were CD19~+CD138~+plasma cells.2.CXCR4 are overexpressed in B cells of BP patients.By using RNA sequencing analysis,we detected 579 differentially expressed genes in BP B cells compared to the healthy controls,including 171 up-regulated genes and 408down-regulated genes.The most upregulated genes were associated with B cell responses,including B cell activation and antigen presentation,immunoglobulin biosynthesis,pro-inflammatory cytokines,and B cell chemokine receptors.GO analysis identified enriched differentially expressed genes involved in immune response,cellular response to cytokine stimulus,and cell chemotaxis.Furthermore,the KEGG enrichment analysis identified cytokine-cytokine receptor interactions as the most enriched signaling pathway.Consistent with the RNA-seq analysis,q RT-PCR and flow cytometry results showed that CXCR4 was dramatically increased in the peripheral CD19~+B cells of BP patients when compared with that in healthy controls.Immunofluorescence co-staining analysis identified that CD19~+CXCR4~+B cells infiltrated skin lesions of BP patients,and these cells exhibited the characteristics of antibody secreting cells in the skin lesions of BP patients.3.CXCL12 mediates the accumulation of CXCR4~+B cells in BP lesional sites.ELISA analysis showed that CXCL12 level was significantly higher in the serum and blister fluid of BP patients than in those of healthy controls.Immunofluorescence staining revealed that CXCL12 was overexpressed in the dermis of BP patients,and CXCL12-producing cells infiltrating BP skin lesions were mainly CD3~+T cells.The transwell experiment showed that CXCL12 induced the migration of peripheral CXCR4~+B cells in a dose-dependent manner and the blister fluid from BP patients also induced the chemotaxis of CXCR4~+B cells.Additionally,CXCL12 neutralizing antibody pretreated with CXCR4~+B cells inhibited the CXCL12/CXCR4 axis mediated B cell chemotaxis.4.CXCL12/CXCR4 axis promotes B cell differentiation and antibody production via activating transcription factor c-Myc.The flow cytometry results showed that the combination of CXCL12 with anti-Ig M induced a significant increase in the proportion of ASCs in BP patients and healthy controls,including DN B cells and CD19~+CD138~+plasma cells,compared with the induction with anti-Ig M alone.The CXCR4 inhibitor reduced the CXCL12-induced B cell differentiation to DN B cells and plasma cells in vitro.Meanwhile,the anti-BP180 NC16A titers in the supernatant of BP B cells were increased by the stimulation of CXCL12 with anti-Ig M,but significantly reduced by CXCR4 inhibitor.RNA-seq analysis and q RT-PCR data confirmed the overexpression of c-Myc m RNA in B cells of BP patients.In our in vitro conditions,treatment of CXCL12 induced a high expression of c-Myc in B cells,which was nearly completely blocked by CXCR4 inhibitor.Furthermore,B cells pretreated with c-Myc inhibitor attenuated the CXCL12/CXCR4 axis mediated B cell differentiation and autoantibodies production.5.NETs levels are elevated in serum and blister fluids of BP patients and correlate with disease activity.Immunofluorescence results showed that the amount of web-like structures associated with histones and neutrophil MPO increased in lesional skin tissues of BP patients.The levels of cf DNA and MPO-DNA complexes in circulation and blister fluid were higher in BP patients compared to healthy controls.Additionally,the MPO-DNA complexes levels in serum and blister fluid were positively correlated with titers of anti-BP180-NC16A antibodies.Following treatment with corticosteroids,serum levels of MPO-DNA complexes and cf DNA in BP patients were decreased compared with their respective pre-emission levels.6.BP180-NC16A immune complex-mediated induction of NETosis in BP neutrophils is dependent on FcγR,NADPH,and PAD4.Neutrophils isolated from BP patients produced more NETs than those obtained from healthy controls under resting conditions or PMA sitimulation.As shown by immunofluorescence,serum and blister fluid from BP patients induced higher NETosis in neutrophils compared with that induced by control serum.In addition,we observed that BP180-NC16A immune complexes s induced NETs formation in a dose-dependent manner.Our immunofluorescence results showed that NADPH inhibition via DPI,PAD4 inhibition via Cl-amdine,and FcγR blockade decreased NETosis in BP neutrophils stimulated with BP180-NC16A immune complexes.7.NETs promote B cell differentiation and antibody production via activation of MAPK p38 cascades.We found that the level of anti-BP180 antibodies and total Ig G levels in supernatants of B cells obtained from BP patients were increased in the NETs-stimulated group,but not in DNase pretreated group.Flow cytometric analysis showed that NETs promoted B cell differentiate into CD19~+CD138~+plasma cells in vitro.In addition,we found that stimulation with NETs strongly induced activation of MAPK p38 phosphorylation in B cells isolated from BP patients.Furthermore,we used the chemical inhibitor of MAPK P38 to block this downstream signaling pathway in B cells,and found that MAPK P38 inhibitor decreased B cell differentiation and autoantibody production under NETs stimulation.Conclusion:In the present study,we show for the first time that CXCL12/CXCR4 is up-regulated in the peripheral blood and skin lesions of BP patients,and CXCL12 could mediate the accumulation of CXCR4~+B cells in BP lesional sites.Moreover,CXCL12 promote B cell differentiation into antibody secreting cells and produce pathogenic autoantibodies via activating the transcription factor c-Myc.Furthermore,we demonstrate that BP180-NC16A immune complexes induce neutrophils to produce more NETs,and elevated NETs levels triggers the activation of MAPK P38 signaling in B cells,thereby promoting differentiation of B cells into antibody-secreting cells;this cascade enhances antibody production and amplifies autoimmune responses in BP development.Our study also highlights the role and related mechanism of abnormal B cell activation and differentiation in the pathegenesis of BP,providing new strategies of targeting CXCL12/CXCR4 or NETs for the clinical treatment of BP in the future.