Study On The Underlying Mechanisms Of Regulatory B Cells In Systemic Lupus Erythematosus And Their Relationship

Background:Regulatory B cells(Bregs)is a subset of B cells with negative regulatory function,which can inhibit inflammatory immune response and prevent autoimmunity.Bregs participate in a variety of immunopathological processes.Bregs play an important role in the pathogenesis of many diseases,especially autoimmune diseases.Bregs can secrete a large number of inhibitory cytokines or connect with negative costimulatory factors.However,the distribution of Bregs in the course of SLE and their effect on immune cells during immune regulation remain unclear.Aim:(1)Clarify the expression and distribution characteristics of Bregs subsets in peripheral blood of SLE patients,compare the functional changes of Bregs and analyze the relationship between Bregs subsets and SLE clinical indexes.(2)Clarify the changes of Bregs subsets in peripheral blood of SLE patients before and after treatment.(3)Explore the effects of Bregs and their functional molecules on CD4+ T cells and the possible mechanism of Bregs involved in the occurrence and development of SLE disease.Methods:(1)Newly diagnosed SLE patients(n = 72)were enrolled,aswell as the age and gender matched healthy control(n = 24).The clinical indexes such as SLEDAI-2k,complement,C reactive protein and urinary protein were collected.(2)The distribution of Bregs,Th cells and other B cells subsets in the circulation of SLE patients and healthy controls were analyzed by flow cytometry analysis(FCA).By staining of intracellular cutokines,the functions of Bregs were further analyzed by FCA.(3)The levels of functional cytokines of Bregs and Th subsets,B cell activating factors(BAFF)were determined by enzyme linked immunosorbent assay(ELISA)and cytometric bead array(CBA).(4)The relationship between Bregs subsets and clinical indexes were analyzed.(5)SLE patients underwent immunotherapy were followed up to observe the effect of treatment on the levels of Bregs and the functional cytokines.(6)Transwell co-culture systems were established in vitro,to simulate the microenvironment of Bregs and CD4+T cells,to explore the effect of Bregs on CD4+T cells and its possible mechanism in SLE.Results:(1)The frequencies of circulating IL-10+Bregs and IL-35+Bregs subsets in SLE patients were decreased significantly.Though the frequencies TGF-?1+Bregs did not change.The frequencies of circulating CD19+CD24hiCD38hi transitional Bregs and CD19+CD24hiCD27+memory Bregs subsets in SLE patients were decreased significantly.The poportion of CD19+CD24hiCD38hiBregs were higher than CD19+CD24hiCD27+ Bregs in SLE.The frequencies of circulating IL-10+Bregs were positively correlated with CD19+CD24hiCD27+Bregs.The frequencies of circulating IL-35 +Bregs were positively correlated with CD19+CD24hiCD38hi Bregs and CD19+CD24hiCD27+ Bregs.(2)Plasma levels of IL-35 and TGF-?1 were reduced in SLE patients,whereas the level of IL-10 was elevated.The level of IL-35 was correlated with IL-35+Bregs and CD19+ CD24 hi CD27+ Bregs.(3)Co-cultured in vitro,the levels of IL-10 and IL-35 produced by CD19+CD24hi CD27+Bregs were higher than CD19+CD24hiCD38hi Bregs.(4)The frequencies of circulating Th17 cells were increased in SLE patients.Plasma levels of TNF-?,IL-6,IL-17 A and BAFF were elevated and the levels of IL-2 were reduced in SLE patients.Positive correlations were also found between IL-17 A levels and Th17 cells.(5)The frequencies of circulating CD27+CD38-/lo MB,CD27+CD38hi PB and CD27-CD38 hi TB were increased in SLE patients.The frequencies of circulating CD27-CD38-/lo naive B cells were decreased.The frequencies of circulating IL-35+Bregs were nagetively correlated with CD27+CD38-/loMB and CD27-CD38-/lonaive B cells.The frequencies of circulating TGF-?1+Bregs were nagetively correlated with CD27+CD38hiPB cells.The frequencies of circulating CD19+CD24hiCD38hiBregs were positively correlated with CD27-CD38-/lonaive B cells.The frequencies of circulating CD19+CD24hiCD27+ Bregs were nagetively correlated with CD27-CD38 hi TB cells.(6)The correlational analysis with clinical parameters analysis showed that the frequencies of circulating IL-35+Bregs was correlated with ESR.The frequencies of circulating IL-10+Bregs was correlated with CRP.The frequencies of circulating IL-35+ Bregs and CD19+CD24hiCD38highBregs were correlated with plasma C3 levels.The frequencies of circulating IL-10+Bregs was correlated with plasma C4 levels.The frequencies of circulating IL-10+Bregs and IL-35+Bregs were correlated with SLE disease activity index(SLEDAI).(7)Compared with SLE patients with mild to moderate activity,the frequencies of IL-35+Bregs and CD19+CD24hiCD27+Bregs subsets in SLE patients with severe disease activity were significantly reduced.(8)Compared with patients without renal involvement,the frequencies of IL-10+Bregs,IL-35+Bregs,CD19+CD24hiCD27+Bregs and IL-35 levels in SLE patients with lupus nephritis were significantly reduced.(9)After 12 weeks immunotherapy,the frequencies of IL-10+Bregs,IL-35+Bregs,CD19+CD24hiCD38hiBregs and CD19+CD24hiCD27+Bregs,as well as the levels of IL-10 and IL-35 resumed complying with the alleviation of the disease.(10)Transwell co-culture experiments of CD19+B cells and CD4+T cell found that healthy Bregs inhibited the levels of Th1 and Th17 cells in SLE patients partly depended on the cell-contact.The stimulation of IL-10 m Ab or r IL-35 significantly enhanced the inhibitory effect of Bregs.The inhibitory effect of Bregs in SLE patients on Th cells was impaired.IL-10 m Ab and r IL-35 restored the inhibitory effect s of the impaired Bregs in SLE patients on the level and function of Th1 and Th17 cell.The production of IL-2,IFN-? and IL-6 in Th cells was regulated by IL-10 and IL-35.Conclusions:(1)The levels of IL-10+Bregs,IL-35+Bregs,CD19+CD24hi CD38 hi Bregs and CD19+CD24hiCD27+ Bregs are reduced in SLE.The production of IL-35 and TGF-?1 are weakened.The secretion function of Bregs is mainly based on CD19+CD24hiCD27+Bregs.(2)The decrease of IL-10+Bregs,IL-35+Bregs,CD19+CD24hi CD27+ Bregs and IL-35 levels in peripheral blood may be related to LN in SLE patients.(3)The levels of Bregs and IL-10,IL-35 partly recover in SLE after therapy.(4)Bregs can inhibit the levels of Th1 and Th17 cells in SLE patients,which is not completely dependent on cell-contact.Stimulation of IL-10 m Ab or r IL-35 can significantly enhance the inhibitory effect of Bregs on Th1,Th17 cells levels and functions in SLE patients.(5)The inhibitory effect of Bregs in SLE patients is impaired.IL-10 m Ab and r IL-35 can restore the inhibitory effect s of the impaired Bregs.(6)The production of IL-2,IFN-? and IL-6 in Th cells is regulated by IL-10 and IL-35.