B Cells Induced By Schistosoma Japonicum Infection Display Diverse Phenotypes And Modulate CD4~+ T-cell Responses

Background and Objectives:Chronic worm infections are often associated with the reduction in the incidence of inflammatory diseases and allergic diseases.It has been reported that worm infection can alleviate allergic inflammation by inducing Regulatory B cells(Breg),and IL-10 plays a key role in this process.In recent years,several subsets of Breg cells with the different immunosuppressive functions have been identified in the experimental animal models and patients with autoimmune and infectious diseases,such as:Transitional 2 marginal zone B precursor(T2-MZP)and marginal zone B cells(MZB).In addition,many studies have shown that Breg cells can exert immunoregulatory effects by secreting a variety of inhibitory cytokines.However,studies on the differentiation and mechanism of B-cell subsets with potential immunomodulatory function in Schistosoma(S.)japonicum infection are insufficient.Thus,we studied the differentiation of B-cell subsets and the expression of immunosuppressive molecules of B cells during the experimental S.japonicum infection and explored the immune regulation mechanism of B cells induced by S.japonicum infection.The polyclonal activation of B cells induced by schistosome antigens is one of the important immune responses in the hosts of schistosome infection.Schistosome infection can also induce the proliferation and the immune regulation ability of B cells.However,there are few reports on the mechanism of restricting excessive activation of B cells during schistosome infection.In order to further understand the changes in the activation and proliferation of B cells during schistosomiasis,we screened the differentially expressed genes by comparing the gene expression profiles of splenic B cells from the normal and schistosome-infected mice,and carried out bioinformatic analysis to determine the significance of differential gene expression.Materials and Methods:BALB/c mice were percutaneously infected with cercariae for investigating the profile of B-cell subsets during S.japonicum infection.B cells isolated from spleen or peritoneal cavity were analyzed for the regulatory phenotype after stimulation with soluble egg antigens(SEA)in vitro.CD4~+T cells were then cocultured with B cells pretreated with or without anti-PD-L1 antibody for investigating the role of B cells from infected mice on regulating CD4~+T cells.Furthermore,the in vivo administration of anti-PD-L1 antibody was conducted to investigate the role of PD-L1 in regulating host immunity during infection.BALB/c mice were percutaneously infected with cercariae of S.japonicum and after eight weeks of infection the splenic B cells of the uninfected and infected mice were isolated for the comparative transcriptome analysis.Reverse transcription?quantitative PCR were used to verify the selected differential expression genes.Results:The proportions of B cell subsets and peritoneal B-1a cells failed to change at 3weeks post-infection.There were no changes in the percentages of T2-MZP cells and follicular B cells at eight or twelve weeks of infection.The proportion of MZB cells at both acute and chronic stages of infection was significantly lower than that of uninfected mice.The proportions of splenic and peritoneal B-1a cells were declining at eight and twelve weeks of infection.The expression of PD-L1 in splenic B cells was increased at eight and twelve weeks after infection.The expression of PD-L1 in peritoneal B cells was downregulated in mice at twelve weeks post-infection.The acute and chronic infection enhanced the expression of PD-L1 on FOB cells,B-1a cells,T1 B cells and T2-MZP cells.The percentage of splenic B cells expressing IL-10 was increased at twelve weeks post-infection,while TGF-?in splenic CD19~+B cells was elevated at eight weeks after infection but declined thereafter.In addition,schistosome infection induced elevated percentages of IFN-?-expressing splenic B cells at both acute and chronic stages.There were no differences in IL-10,TGF-?or IFN-?expression of peritoneal CD19~+B cells between infected and uninfected mice.Moreover,higher secretion of IL-6,TNF and IFN-?were also detected in culture supernatant of splenic B cells isolated from mice at eight weeks post infection compared to splenic B cells from uninfected mice.The splenic B cells stimulated by SEA for 24 h in vitro expressed high level of CD5.CD5 expression was not increased in peritoneal B cells stimulated by SEA.Soluble worm antigen(SWA)failed to affect the expression of CD5 and PD-L1 in splenic B cells in vitro.Additionally,SEA stimulation in vitro enhanced the expression of surface PD-L1and CD23 of B cells.SEA stimulation for 24 h did not significantly enhance IL-10production of splenic B cells,but drove intracellular IL-10 expression in peritoneal B cells.SEA treatment in vitro did not affect the intracellular IFN-?expression of splenic and peritoneal B cells..Intraperitoneal injection of SEA into mice increased the expression of PD-L1 in splenic and Per C B cells,and enhanced the expression of TGF-?in splenic B cells.In contrast,SWA injection did not affect the expression of PD-L1 and TGF-?in B cells.The frequencies of T-bet~+,Gata3~+and Fox P3~+in CD4~+T cells,as well as percentages of CD4~+T cells producing IFN-?,IL-4 and IL-10,significantly decreased after co-culture with B cells from infected mice.Additionally,B cells from infected mice were prone to generate fewer CD4~+T effector memory cells than B cells from uninfected mice.In contrast,B cells from infected mice enhanced the expression of Bcl6 in CD4~+T cells.Anti-PD-L1 antibody pre-treatment did not recover T-bet,Gata3,Fox P3,IFN-?and IL-10 expression of CD4~+T cells,whereas IL-4 production and Bcl6 expression in CD4~+T cells were significantly increased.Also,treatment with anti-PD-L1 antibody did not affect suppression in CD4~+T effector memory cells mediated by B cells from infected mice,and interaction of B cells from uninfected mice with CD4~+T cells.PD-L1 blocking significantly increased the frequency of splenic Gata3~+CD4~+T cells and promoted the proliferation of CD4~+T cells.CD4~+T cells from infected mice treated with anti-PD-L1 antibody exhibited higher level of Bcl6,and secreted more IL-4.Following anti-PD-L1 treatment,the expressions of T-bet,Fox P3,IFN-?,IL-10 and TGF-?in CD4~+T cells from infected mice were similar to those found in infected mice treated with isotype antibody.The hepatic pathology was not affected by anti-PD-L1antibody treatment started after the egg deposition in liver.A distinctive signature profile was defined for the splenic B cells from infected mice by their increased expression of genes(Aicda,Ada,Atad5,and Exo1)related to somatic hypermutation and class switch recombination.Schistosome infection also enhanced the expression of genes(Cdk1,Ccna2,Ccnb1,and Cdkn1a)in splenic B cells related to the cell cycle.Besides,splenic B cells from infected mice showed high expression levels of proapoptotic gene Pawr and low expression level of the antiapoptotic gene Bcl2.Among the differentially expressed BCR signaling molecules,levels of Ighv12-3 and most of Igkv4 transcripts were up-regulated in B cells from infected mice.Overall,we found that splenic B cells from uninfected and infected mice had distinct expression profiles for genes encoding modifiers of the cell-cycle status and B cell receptor(BCR)signaling molecules.Conclusion:Our findings revealed that the percentages of B-1a cells and MZB cells decreased at eight and twelve weeks after S.japonicum infection.The expression of TGF-?in splenic B cells increased at eight weeks and returned to normal level at twelve weeks after infection.The expression of PD-L1 on splenic B cells increased at eight and twelve weeks after infection.In vitro,SEA enhanced the expression of PD-L1 and CD5on splenic B cells,but had no effect on peritoneal B cells.In vivo,the splenic and peritoneal B cells stimulated by SEA had the enhanced expression of IL-10 and TGF-?.In addition,the splenic B cells from the infected mice inhibited the function of Th1 and Th2 cells in vitro,but enhanced the expression of Tfh cells transcription factor Bcl6;blocking the PD-L1 of the splenic B cells from the infected mice before co-culture further enhanced Bcl6 expression in CD4~+T cells.Anti-PD-L1 antibody treatment enhanced the Th2 response in the infected mice and the expression of Bcl6 in CD4~+T cells.RNA-Seq analysis showed that the transcription of pro-apoptotic genes(Pawr and Tnfrsf21)increased in B cells from the infected mice,but the transcription of anti-apoptotic gene Bcl2 decreased.Therefore,although schistosome infection promoted the proliferation of B cells,it regulated the transcription of genes related to apoptosis,which may be why chronic schistosome infection fails to cause B-cell lymphoma.The increased transcription of Ighv12-3 and Igkv4 genes in B cells from the infected mice may increase the possibility of the combination of Ighv12-3 and Igkv4,thereby affecting the development of B-1 cells.In summary,this study reveals that Schistosoma japonicum infection regulates the differentiation of B-cell subsets and the expression of immunosuppressive molecules in B cells that have the capability to affect the CD4~+T-cell response.Our research contributes to better understand the immune response of B cells during schistosomiasis,and provides the basis for fully elucidating the activation,proliferation,and apoptosis of B cells during schistosome infection.