The Mechanism Of IL-10、IL-35 Secreted By CD4~+CD25~+ Regulatory T Cells In The Pathogenesis Of Allergic Asthma

Background and ObjectivesAsthma is a multifactorial disease of airway inflammation, with the rapid development of immunology and molecular biology, the study of the pathogenesis of allergic asthma has also made great process. All the studies showed that there were a variety of immune cells and other factors which were involved in the immune molecules of asthma disease. The traditional opinions showed that the imbalance of T1/T2 and Ig E-mediated sensitization allergenicity were the main reasons of the asthma, but many other studies show that: T cells, mast cells, eosinophils, basophils, dendritic cells, interleukins, transforming growth factor and nitric oxide were involved in the release of inflammatory mediators, which lead to the occurrence and development of asthma disease.Asthma is a global issue. although a lot of money had been invested as well as many research staffs are working on it, and risk factors concerning excitation and exacerbations of asthma have been identified, statistics showed that the incidence and mortality rates of asthma were still rising, especially among children. So, the study of asthma remains a major challenge for global medical research staff.In recent years, with the rapid development of immunology, a large number of medical researchers study this area in depth from the perspective of the immunological mechanisms, and it’sfound that IL-10, IL-35, which are secreted by Treg cells, were highly correlated to the mechanisms of asthma, but the exact influence of them are unclear now.CD4 + CD25 + Tregs are the latest discovered T cells which have the function of adjustment and immune inhibition; they are indispensable roles in the immune system, mainly by inhibiting the secretion of cytokines related to the immune suppression. Only by combining Treg(CD4 + CD25 + T) and Teff(CD4 + CD25-T) T cells effectively under the circumstance of anti-CD3 antibody and anti-CD28, could the density of Teff cells be controlled. Studies have shown that Treg cells especially IL-10, IL-35 and TGF-β play an immunosuppressive cytokine function. IL-10 is an important component of immune suppression, and is closely related to the effects of the cytokine, but it is not the only inhibitory cytokines.IL-35 is an important factor discovered in recent years, which is a member of the IL-12 family, and is composed by EBI3 / IL-27β and IL12α / p35 heterodimer. It can act directly to effector T cells, and induce naive T cells to produce a new type of regulatory T cells-T35 cells(i TR35). Studies have shown that if Treg and Teff are cultivated together, they can increase the expression of EBI3 and IL12α m RNA’s. This suggests that it is possible to enhance the secretion of IL-35 by cultivating Treg and Teff together and then enhance the immune function. TGF-β is also very important as a cell-mediated immunosuppressive factor for Treg. These immunosuppressive factors may also play a role as an important mediator in the negative adjustment of immune in allergic asthma. MethodsMethods1. Randomly divide the SPF grade female BALB / c mice into model group and the control group, sensitize them with saline and OVA respectively.2. Separate the spleen of the mouse and fully polished, and separate mononuclear cells from lymphocytes. Use MACS(Magnetic Activated Cell Sorting) technology to divide mononuclear cells into Treg and Teff.3. Determinate the purity of Treg and Teff by flow cytometry.4. Add Treg and Teff cells with anti-CD28 in 96-well cell culture plate coated with anti-CD3 e, then cultivate Treg and Teff separatly and mixed. Repeat three times foreach one group.5. Cultured in a carbon dioxide incubator for 48 hours, the supernatant, containing IL-10 and IL-35, under the different ways were determined with ELISA kits, Collect cell for the extraction of RNA, reverse transcripte c DNA,and quantify the gene expression of IL10, EBI3 and P35 with real time PCR.6. The statistical dates were analyzed by SPSS 17.0 to make the various charts with Graph Pad Prism 5.Results1. Murine asthma model and control model have been established successfully.2. Extract mononuclear cells and get Treg(CD4+ CD25+ T) and Teff(CD4+ CD25-T) cells successfully with MACS technology. Sort the Treg and Teff primary cells successfully.3. Compared with the control group, the relative quantity of IL-10, EBI3 and P35 genes decreased, but not too obviously(0.5<2-ΔΔCt<2). IL-10, EBI3 and P35 of Treg + Teff group and Treg + Teff + αCD3/CD28 groupdecreased significantly, while Treg + Teff + αCD3 / CD28 group was significantly decreased IL-10((2-ΔΔCt<0.5)4. Compared with the control group, the expression levels of the IL-10 and IL-35 protein have declined relatively, but there was no significant difference(p>0.05).Conclusions The results of this study show that Treg cells secrete IL-10 and IL-35 and It may has a close relationship with allergic asthma, the decrease of IL-10 IL-35 expression levels may reduce the pathological process in allergic asthma. The simultaneous presence of Treg and Teff may cause the secretion of IL-10 and IL-35. Treg, IL-10 and IL-35 could be a potential reason to the treatment of allergic asthma.