The Effect And Underlying Mechanisms Of BAFF For The Secretion Of IL-35 From Regulatory B/T Cells In Systemic Lupus Erythematosus

Part 1.The effect of exogenous BAFF on the IL-35 production of Bregs or Tregs derived from MRL-Faslpr/lpr mice[Purpose] The purpose of this study was to investigate whether BAFF has an effect on IL-35 production by splenic regulatory B cells or T cells and their underlying mechanisms in lupus.[Methods] Splenic MZ & FO B cells,CD4+CD25-T cells and CD4+CD25+ Tregs were sorted by magnetic isolation.The production of p35 and EBI3 were evaluated by flow cytometry,Western-blotting and real time RT-PCR,and the secretion level of IL-35 in culture supernatant was confirmed by ELISA.Furthermore,the phenotype of BAFF-induced IL-35-producing regulatory B cells,the expression of BAFF receptors on Tregs and the frequency of IL-35-producing Tregs in different treatment groups were also analyzed by flow cytometry.In addition,CD5+CD1dhiFc?RIIbhi B cells were sorted and co-cultured with CD4+CD25-T cells or CD4+ T cells at a ratio of 1:1.Then,the secretion of IFN-?,TNF-?and IL-35 by CD4+ T cells were determined by flow cytometry.[Results] We found that,compared with the control group,MRL-Faslpr/lpr mice showed higher IL-35 production in splenic B cells and exogenous BAFF could induce IL-35 production by splenic B cells from MRL-Faslpr/lpr mice and control group under certain concentrations.BAFF-induced IL-35-producing B cells were mainly from the marginal zone B-cell subset and exhibited a CD5+CD1dhiFc?RIIbhi phenotype.These IL-35-producing CD5+CD1dhiFc?RIIbhi regulatory B-cell subsets exhibited regulatory effects on both CD4+CD25-T cells and CD4+CD25+ regulatory T cells partly through inhibiting the secretion of IFN-? and TNF-? by CD4+CD25-T cells and promoting proliferation and IL-35 production of Tregs,orchestrating a positive feedback loop to exert an immunosuppressive effect.Furthermore,we identifeied that BAFF-R and TACI were expressed in Tregs and exogenous BAFF could promote Tregs to produce IL-35 directly.[Conclusions] Collectively,our study identified that BAFF,a key pathogenic factor in systemic lupus erythematosus,also a dichotomous regulator for B-cell immune responses,could induce IL-35 production by a previously unreported CD5+CD1dhiFc?RIIbhi regulatory B-cell subset and Tregs in lupus.Part 2.The underlying mechanisms of BAFF on the production of IL-35 by Bregs in vitro[Purpose] To ascertain which receptor and related signal pathway mainly contributes to the BAFF-induced IL-35 production by splenic B cells derived from MRL-Faslpr/lpr mice.[Methods] Splenic MZ B cells derived from MRL-Faslpr/lpr mice were sorted by magnetic isolation and pretreated with anti-BAFFR,anti-TACI antibodies or NF-?B pathway inhibitors before BAFF stimulation.Then,the production of p35 and EBI3 were evaluated by flow cytometry and Western-blotting.Furthermore,the secretion level of IL-35 in culture supernatant was confirmed by ELISA.[Results] Blocking TACI resulted in significant reduction of p35 and EBI3 expression,although their expressions were only slightly decreased when BAFFR blockage was used.Similar results were obtained by flow cytometric analysis.The secretion level of IL-35 reduced significantly in the culture supernatant of both anti-BAFFR and anti-TACI antibody-treated groups.the expression of p35 and EBI3 and the extracellular level of IL-35 were obviously reduced when both total NF-?B and classical NF-?B1 signaling pathways were blocked.[Conclusions] Collectively,these results indicated that BAFF-TACI interaction induces MZ Bcells to produce IL-35 mainly through the classical NF-?B1 pathway.Part 3.Identifing the effect of BAFF on the IL-35 production of splenic Bregs or Tregs derived from MRL-Faslpr/lpr mice in vivo[Purpose] To investigated whether BAFF enables B cells to produce IL-35 and related mechanisms in lupus in vivo.[Methods] we intravenously injected PBS or anti-BAFF m Ab into MRL-Faslpr/lpr mice(n=5).Before and 10 days after injection,the sera were collected.ELISA was used to examine the secretion levels of BAFF and IL-35.the expression of IL-35 in different cell subsets from spleen,including CD19+B cells and CD4+T cells were evaluated by flow cytometric analysis,which was further confirmed by the confocal microscopy analysis.[Results] Compared with MRL/Mp J mice used as controls,MRL-Faslpr/lpr mice had distinctly elevated serum BAFF and IL-35 levels and the serum IL-35 level correlated positively with serum BAFF level.We also observed that the proportion of IL-35-producing CD19+B cells,CD4+CD25+Foxp3+Tregs and IL-35-producing CD4+T cells were elevated significantly in MRL-Faslpr/lpr mice compared with control group.BAFF blocking resulted in a significantly declining level of BAFF,whereas no difference in serum IL-35 level was observed.Meanwhile,blocking BAFF resulted in significantly reduced frequencies of IL-35-producing CD19+B cells,CD5+CD1dhiFc?RIIbhi B cells,Tregs and IL-35-producing CD4+T cells.Furthermore,the IL-35-producing B cells expectedly exhibited a CD21+CD23-phenotype similar to MZ B cells.[Conclusions] These data definitely suggested that BAFF could induce MZ B cells to produce IL-35 in MRL-Faslpr/lpr mice and this IL-35-producing B-cell subset exhibited a a previously unreported CD5+CD1dhiFc?RIIbhi phenotype.Furthermore,BAFF inhibition resulted in significantly reduced frequencies of IL-35-producing Bregs and Tregs,which could partly explain the unsatisfactory clinical efficacy of BAFF-targeted therapy and providing an advanced understanding of the unknown regulatory effect of BAFF in autoimmune diseases.Part 4.Identifing the effect of BAFF on the IL-35 production of Bregs in the cutaneous lesions of both lupus-prone mice and SLE patients in vivo[Purpose] To further evaluate whether BAFF stimulation is linked to the generation of IL-35-producing B cells in the cutaneous lesions of both lupus-prone mice and SLE patients.[Methods] The animal experiments in vivo were divided into systematic and local intralesional anti-BAFF treatment.Meanwhile,25 specimens of cutaneous lesions from SLE patients and seven normal skin specimens from healthy control subjects were collected.we investigated the expression levels of BAFF,EBI3,p35,CD19,CD5,CD1 d,and Fc?RIIb in the normal skin and skin lesions by monitoring the immunohistochemistry results under microscope?Furthermore,the integral optical density analysis was obtained by mage Pro Plus,version 6.0.[Results] IHC staining showed significantly elevated expressions of BAFF,EBI3,p35,CD19,CD5,CD1 d,and Fc?RIIb in the cutaneous lesions of MRLFaslpr/lpr mice compared with those in the normal skin of both MRL-Faslpr/lpr mice and control group,which is consistent with previous observation in spleen.The integral optical density analysis showed that EBI3 and p35 were strongly correlated.Meanwhile,BAFF and p35,as well as BAFF and EBI3 also showed powerful correlations.Although systemic anti-BAFF treatment seems to have no obvious effect on the production of IL-35 and IL-35-producing CD5+CD1dhiFc?RIIbhi B cells in the cutaneous lesions of MRL-Faslpr/lpr mice,IHC analysis showed that intralesional injection of anti-BAFF m Ab into the cutaneous lesions of MRL-Faslpr/lpr mice resulted in significantly reduced expression of BAFF,EBI3,p35,CD19,CD5,CD1 d,and Fc?RIIb compared with those in PBS control.Furthermore,we found that the expressions of BAFF,EBI3,p35,CD5,CD1 d,and Fc?RIIb were significantly increasedin the cutaneous lesions of SLE patients than those of healthy control subjects.Prominent CD19+B-cell infiltrates were also detected in the cutaneous lesions of SLE patients.There are also strong correlations between EBI3 and p35,BAFF and p35 and BAFF and EBI3,which further strengthened the evidence that BAFF is able to induce IL-35-producing CD5+CD1dhiFcg RIIbhiB cells in the cutaneous lesions of both lupus-prone MRL-Faslpr/lpr mice and SLE patients.[Conclusions] Consistent with previous observation in spleen,BAFF is positively related to IL-35-producing CD5+CD1dhiFc?RIIbhi B cells in the cutaneous lesions of both lupus-prone MRL-Faslpr/lpr mice and SLE patients,indicating that BAFF could induce CD5+CD1dhiFc?RIIbhi B cells to produce IL-35 both in system and skin topical immunity.