Immunological Characteristics Of B10 Cells In Schistosoma Japonicum Infected C57BL/6 Mice

Schistosomiasis is one of the zoonoses caused by parasitic flatworms of the genus schistosoma. The regulatory B cells(Bregs)producing cytokines IL-10, TGF-β and so on exert an immunosuppressive role in autoimmune disease, chronic inflammation, cancer and parasitic infections. Several studies have indicated that Schistosoma mansoni infection can induce B cells secreting IL-10(B10 cells)and play an essential role in autoimmune diseases. Previous study demonstrates that soluble egg antigen (SEA) can induce B10 cells both in vivo and in vitro whereas little date are available on the dynamics of B10 cells at different stage of Schistosoma japonicum infection. Moreover, it’s still uncertain that what surface markers the B10 cells express in mediating host immune response in schistosoma infection. In addition, the underlying mechanism of SEA activating Bregs remains undiscovered.Our study could be divided into three parts.Part I. Observation of the changes in B10 cells levels at different stage of Schistosoma japonicum infectionAims:To explore dynamics of regulatory B cells during Schistosoma japonicum infection.Methods:At 0,3,6 and 13 week post infection, mice were sacrificed and the SEA specific IgG, IgGl and IgG2a antibodies in mouse serum samples were detected by standard ELISA. CD19+B cells were sorted by beads and stimulated by SEA or LPS in vitro, the cytokines IL-10, IL-4 and IFN-y levers in cultured supernatants were detected by ELISA. CD19+IL-10+B cells ratios in splenocytes were analyzed by flow cytometry. Estimation of worm in thoracic aorta and egg burdens and histopathological examination in livers were observed at 13 week post infection.Results:The levels of SEA specific IgG, IgGl and IgG2a antibodies in mouse serum significantly increased. The levels of IL-10 in cell culture supernatants and serum samples showed significant increase at 6 and week post infection yet IF-y and IL-4 showed no significant change. Meanwhile, the proportion of CD19+IL-10+B cells followed the similar trends as IL-10 in serum. Adult worms could be collected and there were eggs and granulomas deposition in livers at 13 week post infection.Conclusions:At 6 and 13 week in Schistosoma japonicum infection, Schistosoma japonicum could induce B10 cells secreting IL-10 in mice spleen.Part Ⅱ. Immunomodulation role and phenotype of B10 cells in Schistosoma japonicum infectionAims:To verify the phenotype and regulatory function of B10 cells and probe into which subunit of these cells are involved in mediating host immune response in Schistosoma japonicum infection.Methods:Mice were sacrificed 13 weeks post infection and the spleen mononuclear cells were isolated and then stained with conjugated antibodies. The expression of surface markers CD19+CD1d+CD5+ and CD19+Tim-1+ were analysed by FACS. B10 cells were sorted according to the two pheno types and then cultured in the presence of SEA or LPS. IL-10 in cell culture supernatants were detected by ELISA. B cells secreting IL-10 were sorted by FACS and then co-cultured with CD4+T cells isolated from mouse splenic cells in the presence of medium or anti-CD3 plus anti-CD28 for 72h, then cytokines IL-4, IFN-y and IL-17 levers in culture supernatants were detected by ELISA.Results:CD19+Tim-1+B cells can secrete IL-10 and raised IL-4 levels secreted by activated CD4+T cells and decreased the IL-17 production.Conclusions:In Schistosoma japonicum infection, CD19+Tim-1+B10 cells have a regulatory effect on Th2 and Th17 cells.PartⅢ. Preliminary investigation into the underlying mechanism of SEA activating B10 cellsAims:To study the underlying mechanism of SEA activating B10 cells.Methods:CD19+B cells were isolated from normal C57BL/10 mice and C57BL/10 TLR4-/- mice respectively and then stimulated by SEA or LPS in vitro.After 3 days, the mRNA expression of TLR2, TLR4 and IL-10 of B cells were evaluated by Real-Time Quantitative PCR(qPCR). Meanwhile, the percentages of IL-10+B cells were analysed by FACS and cytokines IL-10 were detected by ELISA.Results:In contrast to control groups, the mRNA expression of IL-10 and TLR4 were significantly increased in SEA and LPS induced B10 cells. Compared to normal group, proportion of IL-10+B cells and levels of IL-10 were significantly lower in the TLR4-/- group.Conclusions:SEA may activate B10 cells in a TLR4-dependent pathway.Our findings will significantly contribute to understanding of the immunologic mechanism in Schistosoma japonicum infection and may provide us a potential new method for developing schistosomiasis vaccine.