Roles Of Regulatory B Cells In The Negative Regulation Of Immunity In Chronic Hepatitis B Virus Infection

BackgroundHepatitis B virus(HBV)infection is one of the most prevalent chronic virus infections in the world and it’s a great hazard to human’s health.The current therapy of chronic HBV infection is antiviral treatment.But antiviral treatment can’t eradicate the virus.The mechanisms of HBV persistence are not fully elucidated but the impairment of cellular immune responses is important.There is increasing evidence that B cells may involve in the immune control of chronic HBV infection.Recently published data indicate that regulatory B cells(Breg)may play a crucial role in the negative regulation of immunity.Previous study demonstrated that Breg cells may be involved in the pathologic process of chronic HBV infection via the Toll-like receptors(TLRs)pathway.In the present study,we aim to explore the change characteristics of the percentage of Breg cells and the expression of TLRs on Breg cells in the peripheral blood of different immune status of chronic HBV infection and reveal their clinical significances.And we will investigate the influences of pathogen associated molecular pattern(PAMP)on Breg cells expansion and its functions as well as the expressions of TLRs on Breg cells.We will also clarify the impact of Breg cells on T cells in chronic HBV infection.These studies will reveal the important roles and mechanisms of Breg cells in the negative regulation of immunity in chronic HBV infection and provide the basis for new targets for immunotherapy of chronic HBV infection.Materials and MethodsOne hundred and sixty-six patients with chronic HBV infection in different immune phases from the outpatient service of infectious department of Nanjing drum tower hospital during November 2013 and July 2016 were enrolled in this study.Among them 43 are in immune tolerant phase(IT),71 are in immune active phase(IA)and 52 are in inactive HBV carrier state(IC).And 50 healthy donors were also included as control in this study.The frequencies of B cells and Breg cells(CD19+CD24hiCD38hi B cells)in peripheral blood were determined by flow cytometry.The expressions of TLR2,LTR4,TLR6,TLR9,IL-10 and IL-35 on Breg cells were also determined by flow cytometry.Serum IL-10 levels were detected using Luminex and serum B cell activating factor(BAFF)levels were detected using ELISA in part of the chronic hepatitis B(CHB)patients.To investigate the influences of HBV antigens to the Breg cells expansion and its functions.Peripheral blood mononuclear cells(PBMC)were isolated from CHB patients and healthy controls and then cultured with the stimulation of HBsAg or HBcAg.The changes of the frequencies of Breg cells in PBMC were determined with flow cytometry and the levels of IL-10 in supernatant were detected using ELISA.To investigate the influences of Breg cells to T cells,Breg cells were isolated from healthy donors and co-cultured with homologous T cells.The changes of the subsets of T cells and the expressions of CD69,PD-1 on T cells were determined with flow cytometry and the levels of cytokines secreted by T cells were detected using ELISA.Results(1)The 166 CHB patients comprise 104 males and 62 females and the median age of these patients is 29.The levels of ALT and AST in IA phase were significantly higher than that in IT,IC and HC groups.(2)Compared with healthy controls,the frequencies of CD19+total B cells in peripheral blood of CHB patients were elevated and especially in IA phase.Besides,the frequencies of Breg cells were also elevated in CHB patients.Furthermore,the frequencies of Breg cells in IA phase were higher than that in IT and HC groups.Meanwhile,IC group showed a higher Breg cells than healthy controls.(3)Compared with healthy controls,the expressions of TLR9 in B cells and Breg cells both reduced in CHB patients,on contrast,the expressions of TLR2 on Breg cells increased in CHB patients.However,there were no differences of the expressions of TLR4,TLR6,IL-10 and IL-35 on B cells or Breg cells between CHB patients and healthy controls.(4)Compared with healthy controls,the secretion of IL-10 in serum in CHB patients,especially in IA phase,were significantly increased.And the levels of IL-10 in IA phase showed significantly positively correlations with both ALT and AST levels.In contraste,the levels of BAFF in CHB patients,especially in IT phase,were significantly decreased.And the levels of BAFF in IA phase showed significantly negatively correlations with both ALT and AST levels.(5)The frequencies of Breg cells in CHB patients were positively correlated with ALT and AST levels.However,there were no significantly correlations between Breg cells and HBV DNA,HBsAg,HBeAg levels.(6)Breg cells and IL-10 introduction could be induced by HBcAg in CHB patients,while it couldn’t be induced in healthy donors.Breg cells couldn’t be induced by HBsAg no matter in CHB patiens or in healthy donors.(7)The co-culture model of Breg cells and T cells showed that Breg cells can suppress the expansion and promote the apoptosis of CD4+T cells.And Breg cells can inhibit the secretion of IL-2,IFN-y,IL-10,IL-8 and granzyme B from T cells.Conclusions(1)Compared with healthy controls,the frequencies of Breg cells in peripheral blood and the production of IL-10 in serum were increased in CHB patients.Moreover,the different immune status of CHB patients showed different frequencies of Breg cells and levels of IL-10.Breg cells may be seen as the indicator of immune active phase.The expressions of TLR9 and TLR2 on Breg cells were different between CHB patients and healthy controls,but whether HBV infection can affect the expansion and function of Breg cells through TLRs pathway deserves further investigation.(2)HBcAg stimulation could induce the proliferation of Breg cells and the production of IL-10 in CHB patients.(3)Breg cells could suppress the proliferation of T cells and the production of cytokines from T cells.