Studies Of CD19~+Tim-1~+IL-10~+ Regulatory B Cells Participating In Schistosoma Japonicum Infection And Hepatopathy

Schistosomiasis japonicum is an important zoonotic parasitic disease,and remains one of the major public health problems in China.One of the basic characteristics of schistosomiasis infection immunity is that the host gradually shows a down-regulation of immunity after the adult spawning,which makes the infection appear chronic.Previous studies have suggested that this negative immunoregulatory is associated with regulatory T cells(Tregs),DC,and M(p.Regulatory B cell(Bregs)is a class of B cells with regulatory functions,which play an immunosuppressive role in autoimmune diseases,chronic inflammation,cancer and parasite infection.MicroRNAs(miRNAs)are endogenetic,small RNAs with a length of about 20-25 nucleotides that do not encode proteins but have many important regulatory roles in the cell.Recent studies have found that microRNAs play a key role in the activation,differentiation,and function of B cells,but it is still unclear whether miRNAs play an important role in the regulatory functions of B cells.Our previous studies have confirmed that soluble egg antigen(SEA)can induce the production of Bregs and secretion of high level IL-10 in vitro and in vivo,and also induce the production of CD19+Tim-1+ IL-10-secreting B cells in the process of Schistosoma japonicum infection.It is still unclear whether miRNAs are involved in the development and differentiation of Bregs induced by Schistosoma japonicum infection.The main pathological change of Schistosomiasis japonicum is liver granuloma,which leads to liver fibrosis in the chronic stage.Studies have shown that B cells play an important role in the formation of liver granuloma.However,it is still unclear whether regulatory B cells are involved in the pathological changes of the liver.Therefore,we conducted a preliminary study on the production and role of regulatory B cells in mice after Schistosoma japonicum infection.Our study was divided into three parts.Part ?.The role of miRNA-21 in the differentiation of regulatory B cells induced by Schistosoma japonicum.Aims:To explore the role of miRNA-21 in the differentiation of regulatory B cells after Schistosoma japonicum infection in mice and the production of regulatory B cells after SEA stimulation in vitro.Methods:The C57BL/6 mouse model of Schistosoma japonicum infection was established.The CD19+ B cells of spleen were collected from 13W-infected and normal mice respectively and detected miRNA-21 and IL-10 mRNA expression levels by Real-Time Quantitative PCR(qPCR).In vitro,,the CD19+ B cells of normal mice were stimulated with SEA and LPS for 72 h respectively.The mRNA levels of miRNA-21 and IL-10 in CD19+ B cells were detected by qPCR.CD19+ B cells of normal mice were stimulated with SEA and LPS for 72 h following transfected with miRNA-21-Inhibitor in vitor.The proportion of IL-10+ B cells was analysed by FACS and the IL-10 in culture supernatant was detected by ELISA.Results:After Schistosoma japonicum infection,the mRNA expression of miRNA-21 and IL-10 in spleen B cells of mice was significantly higher than those in normal mice.The mRNA levels of miRNA-21 and IL-10 in CD19+ B cells stimulated by SEA and LPS in vitro were significantly higher than those in normal mice.The proportion of IL-10+ B cells and the secretion of IL-10 were significantly decreased after stimulated by SEA and LPS for 72 h respectively following miRNA-21-Inhibitor transfected CD19+ B cells of normal mice in vitro.Conclusions:MiRNA-21 is involved in the production of regulatory B cells and the secretion of IL-10 after Schistosoma japonicum infection.Part ?.Dynamic changes of regulatory B cells during liver granuloma induced by Schistosoma japonicum infectionAims:To observe the dynamic changes of CD19+Tim-1+IL-10+ B cells in different stages of Schistosoma japonicum in vivo.Methods:The C57BL/6 mouse model of Schistosoma japonicum infection was established.Spleen PBMCs and liver lymphocytes of mice were respectively isolated at OW,3W,6W,and 13W after infection,and the proportion of CD19+Tim-1+IL-10+ B cells was analysed by FACS.At the same time,the changes of CD19+ Tim-1+ cells in liver were analyzed by immunohistochemistry at different stages of infection.Results:The proportion of CD19+Tim-1+IL-10+ B cells in spleen significantly increased in the 6W and 13W after Schistosoma japonicum infection.The proportion of CD19+Tim-1+IL-10+B cells in liver was significantly increased at 3W and 6W,which was significantly decreased at 13 W after infection.The liver immunohistochemical analysis revealed that there was serious egg granuloma in the liver at 6W after infection,accompanied by a large number of CD19+ Tim-1+cells recruited around the granuloma.The granuloma size was reduced at 13W after infection,accompanied by significantly decreased CD19+ Tim-1+ cells recruited around granuloma.Conclusions:In the early and acute stages,CD19+Tim-1+IL-10+ B cells were involved in the occurrence and development of liver granuloma induced by Schistosoma japonicum infection.Part ?.Preliminary study on the effect of regulatory B cells induced by SEA on macrophagesAims:To preliminary investigate the effect of regulatory B cells on macrophages in vitro.Methods:CD19+ B cells of WT mouse were isolated by microbeads and co-cultured with mouse macrophages RAW264.7 following stimulated by LPS and SEA in vitro.The levels of NO and IL-10 in the supernatants were measured.The mRNA levels of iNOS and Arg-1 in RAW264.7 cells were detected by qPCR after co-cultured.Results:Compared with the negative control group,there were no significant changes in the expression of NO and mRNA levels of iNOS after RAW264.7 cells co-cultured with SEA-stimulated B cells.The expression of IL-10 and mRNA levels of Arg-1 were increased significantly.Conclusion:B cells stimulated by SEA can induce macrophage to differentiate into M2 macrophages in vitro.