Effects And Characteristics Of B10 Cells Affecting Local Immunity During Experimental Periodontitis

Objective: Periodontitis is a periodontopathogen induced disease that leads to periodontal ligament destruction,alveolar bone resorption,and even teeth loss.Periodontitis often accompanies by gingivitis,bleeding of probing,clinical attachment loss,and other problems.Therefore,periodontitis has become the main reason which results in loss of chewing function.In addition,periodontitis can also affect the appearance,pronunciation,and even psychological health of its host.Accordingly,periodontologists and dentists pay more and more attention to this disease.Studies have confirmed that B cells mediated adaptive immune response results in periodontal tissue injury during periodontitis.As one of the functional subsets of B cells,B10 cells are characterized by a high expression of IL-10.Although the function of B10 cells is not completely clarified,it has been demonstrated that this subpopulation plays an important role in the pathological process of infectious diseases,autoimmune diseases,and even tumors.B10 cells regulate periodontitis probably based on two manners: the first is the expression of IL-10 which affects alveolar bone homeostasis;the second is the regulation of other immune cells which affect immune response.However,the changes of B10 cells in periodontitis and its regulatory effect and mechanism on local immunity are still needed to clear.In the present study,we set experimental periodontitis models.How periodontopathogen affects B10 cells was established and how B10 cells regulate Th17/Treg during periodontitis were analyzed by ELISA,Flow Cytometry,q PCR,and Western Blot.Also,we performed cell culture to furtherly clarify the potential mechanisms of B10 cells on regulating Th17/Treg.Methods: Our research contained three parts.1.Periodonto-pathogens affect IL-10 + B cellsWe set an experimental periodontitis model for the present research.The changing characteristics of IL-10 + B cells in different tissues were analyzed.In addition,healthy splenic B cells were cultured for one week independently or with Pg-LPS.The expression of IL-10 was evaluated by flow cytometry,ELISA,and q PCR.2.The effect of IL-10 + B cells regulating Th17 / Treg during periodontitis B10 cells activated by periodontal pathogens were used to establish the adoptive metastasis model by tail vein injection at a dose of 106.Periodontitis models were established in both healthy animals and adoptive transfer animals,and healthy animals(without periodontitis)were used as controls.The proportion levels of Th17 / Treg and expression of related cytokines RANKL,TGF-β,IL-6,IL-10,and IL-17 A in local lesions and peripheral blood were evaluated by the immunoassay techniques,to clarify the regulation of B10 cells on localhost immunity across the development of periodontitis.3.The mechanism of B10 cells regulating Th17 / TregB10 cells and Th0 cells were isolated from healthy animals and co-cultured independently or with by Pg-LPS.The proportions of CD4 + IL-17 + cells,CD4 + IL-10+ cells and CD4 + TGF-β + cells in each group were detected by flow cytometry.The concentrations of IL-10,TGF-β,and IL-17 A in each culture medium were analyzed by ELISA.The m RNA expression levels of IL-10,TGF-β,and IL-17 A were detected by q PCR.The expression of IL-10 R α and its relationship with the above indexes were evaluated by Western blot.Results:1.B10 cells are detected in gingiva and peripheral blood of both healthy individuals and periodontitis ones.In periodontitis,the proportion of B10 cells in local lesions was significantly increased,but not in peripheral blood.B10 cells presented a rapid increase in the early stage of periodontitis,but a smoothed change in the later stage.When cocultured with periodontopathogens,the proportion of B10 cells was significantly increased,and the IL-10 m RNA level and the concentration of IL-10 in the medium were also increased.2.The adoptive transfer of B10 cells downregulated the proportion of Th17 and remedied Tregs in the local lesions of recipients.In gingiva,B10 cells significantly downregulated the expression of RANKL and IL-17 A and up-regulated the expression of IL-10.Compared with the normal periodontitis model,the levels of RANKL and IL-17 A in the gingival crevicular fluid decreased and IL-10 increased in the adoptive transfer group.3.B10 cells induces the differentiation of CD4 + IL-10 + cells and CD4 + TGF-β +cells,prohibits the differentiation of CD4 + IL-17 + cells,increase the concentration of IL-10 and TGF-β in culture medium and their m RNA level,decrease the IL-17 A and its m RNA level in culture medium and cells.In the presence of Pg-LPS,B10 cells decreased the proportion of CD4 + TGF-β + cells,but not the concentration of TGF-β in the culture medium.Pg-LPS also promoted the differentiation of Th17 and the expression of IL-17,accompanied by the increase of IL-10Rα expression and phosphorylation.Conclusion:1.Periodontopathogens promote the differentiation and function of B10 cells.2.B10 cells regulate the local Th17/Treg during periodontitis.