| Organ transplant is one of the crucial therapy for kidney failure. With the development of immunosuppressive reagents, survival rate of renal transplantation is over 90%. However, chronic kidney diseases were observed in most of patients who received renal transplant due to loss of function of renal graft. Immune rejection response is the reason for loss of function of renal graft. Therefore, diagnosis and therapy for immune rejection response and inducing immune tolerance are important for regaining function of renal graft. Inducing donor specific immune tolerance is becoming hot spot in organ transplantation and immune research.Various type of lymphocytes including CD8+T cells, γδT cells, NK cells, CD3+CD4- CD8T cells and CD4+CD25+T cells had been demonstrated to be regulator and immumo-suppressor of immune rejection response. Recent study demonstrated their potential to induce immune tolerance. Mechanisms of inducing transplant tolerance including blockade of T cells activation, which leads to apoptosis and incompetent of T cells, activating T-regulatory cells (Tregs), which result in immunesuppression. Numerous studies had proved that Tregsfunction as immune suppressor and regulate auto-reactive T cell to maintain immune tolerance and reduce auto-immune diseases. Revealing function mechanisms of Tregs is important for transplant tolerance and tumor immunology.Interleukin 35 (IL-35) is a member of interleukin 12 (IL-12) protein family and a suppressive cytokine secreted by regulatory T cells (Tregs). IL-35 is the hot issue in immunology. Similar to other members of IL-12 family, IL-35 is a heterodimer and has two subunit, a and p. a subunit is Epstein Barr virus induced gene 3 (EBB). Psubunit is p35 protein.Native T cells develop a novel phenotype of regulatory T cells, iTR35, following IL-35 exposure. The immunosuppressive function of iTR35 is dependent on IL-35, bur not IL-10 and TGF-β. iTR35 induce infectious tolerance and promote T cells mediated tumor development. IL-35 suppress inflammation related diseases and autoimmune diseases by upregulating anti-inflammatory cytokines, inducing generation of regulatory T cells and suppressing CD4+effecter cells and other targeting cells.iTR35 suppress CD4+ and CD8+ cells requires IL-35. Human rhinovirus (HRV) activated dendrite cells induce generation of IL-35 by regulatory T cells, which leads to suppression of mature CD4+ and CD8+ cells. IL-35 increased expression of IL-10 and decreased accumulation of IFN-γ, IL-6 and IL-17 in CD4+CD25+CD39+ cells, in addition, IL-35 inhibits proliferation of CD4+CD25+ cells and protect liver from inflammation injuries. In conclusion, IL-35 is a effective regulator of regulatory T cells. However, the mechanism of suppression mediated by IL-35 in organ transplant, such as renal transplant, remains unclear yet.Interleukin 10 (IL-10) is a Th2 cytokine and suppress generation of IL-2 and IFN-y by Thl cells. Recent studies demonstrate the versatile biological functions of IL-10. IL-10 is a crucial regulator of various type of cells, including thymocytes, T cells, B cells, natural killing cells, macrophages, mast cells, acidophilic cell and neutrophile granulocyte. Recent reports indicate that monocyte-macrophage is target of IL-10. IL-10 inhibits native and specific immune response mediated by monocyte-macrophages, and enhance suppression, immune tolerance and ablation mediated by monocyte-macrophages. In addition, IL-10 increase release of anti-inflammation cytokines, such as antagonistfor IL-1 receptor and cytolytic receptor of TNF-a, which suggest IL-10 down-regulate a lot of crucial cytokines of natural immune system. IL-10 inhibit antigen presenting mediated by monocyte-macrophages and decrease IFN-y induced accumulation of MHC Ilmolecules, stimulator and adhesive molecules. IL-10 directly regulate T cells and doesn’t depend on its’suppression to antigen presenting cells. IL-10 inhibits proliferation of CD4+T cells and generation of Thl (IL-2and IFN-y) and Th2 (IL-4 and IL-5) cytokines. IL-10 prevents apoptosis and promotes proliferation and differentiation of B cells. It also increases secretion of MHC Ilmolecules and promotes transformation of immunoglobulin. IL-10 induce cytotoxic activities of NK cells and increases secretion of cytokines induced by IL-2, such as IFN-y, GM-CSF and TNF-a. IL-10 enhances proliferation of CD56+ NK cells induced by IL-2. In conclusion, IL-10 is a crucial cytokine in maintaining immunotolerance.The aim of this study is investigating the effects and molecular mechanisms of IL-35 induced immune tolerance in allograft renal transplantation. First, we investigated percentages of CD4" T cells, CD8T cells, CD4+CD25+Tregs and CD4+CD25+FOXP3+ in peripheral blood of SD rats following IL-35 treatment. Expression levels of cytokines in peripheral blood, kidney and spleen and pathlogical parameters were analyzed to determine the effects of IL-35 on normal SD rats, which will provide guidance for our further research about effects of IL-35 on renal allograft transplant. Next, we investigated percentages of CD4" T cells, CD8T cells, CD4+CD25+ Tregs and CD4+CD25+FOXP3+ in peripheral blood of renal allograft transplant rat model following IL-35 treatment. Both mRNA and protein expression levels of cytokines, including IL-2, IL-6, IL-10, IL-17, IL-23, IL-35, TGF-β and INF-γ, were examined in peripheral blood, renal graft and spleen. Morphology and pathological changes of renal graft were evaluated to determine effects of IL-35 treatment on renal transplant rat model.Methods:Quantitive real time RT-PCR was performed to investigate mRNA expression of IL-10, TGF-β and FOXP3 in peripheral blood of SD rats. ELISA was used to evaluate protein expression of IL-2, IL-6, IL-10, IL-17, IL-23, TGF-β and INF-y in peripheral blood of SD rat. Western blot was performed to detect protein expression level of FOXP3 and EBB in renal graft and spleen of normal SD rats and renal transplant model. Flow cytometry was applied to detect percentage of CD4-T cells, CD8T cells, CD4+CD25+Tregs and CD4+CD25+FOXP3+Tregs in peripheral blood.Results:Our data from quantitive real time RT-PCR and ELISA indicated that mRNA and protein expression of IL-2, IL-6, IL-10, IL-17, IL-23, TGF-P and INF-y in peripheral blood of nomal SD rat were increased significantly by IL-35 treatment. Results of flow cytometry suggested that IL-35 treatment caused increased percentage of CD4" T cells, CD8"T cells, CD4+CD25+ Tregs and CD4+CD25+FOXP3+ Tregs in peripheral blood of SD rat. Taken together, these data indicated IL-35 regulated both mRNA and protein expressions of cytokines important for immune tolerance and modulating T-regulatory cells which is regulator and wuppressor of immune rejection response. In order to investigate effects of IL-35 on immune tolerance of renal transplant, Wistar→SD rats were used to established renal transplant model. And SD→SD rats were used to established renal transplant false model. Divided into five groups, IL-35+model, PBS+model, IL-35+false model, PBS+false model and normal SD rat. Expression levels of Cr,IL-2, IL-10, IL-17, IL-23, IL-6, IFN-γ and TGF-β were examined at day 5 and day 14 after transplant using ELISA assays. Our results showed at day 5 protein expression of Cr (F=276.869, P<0.001), IL-6 (F=3411.957, P<0.001), IL-17(F=173.206, P<0.001), IL-23(F=109.609, P<0.001), IFN-γ (F=800.145, P<0.001) and TGF-β (F=122.876, P<0.001) were decreased and expression of IL-2 (F=39.471, P<0.001) and IL-10 (F=151.537, P<0.001) were increased in peripheral blood of renal transplant model following IL-35 exposure. At day 14, expression of Cr (F=532.881,P<0.001), IL-6 (F=158.974, P<0.001), IL-17(F=35.642, P<0.001), IL-23(F=87.057, P<0.001), IFN-y (F=190.986, P<0.001) and TGF-β (F=45.276, P<0.001) were decreased and expression of IL-2 (F=133.488, P<0.001) and IL-10 (F=274.343, P<0.001) were increased in peripheral blood of renal transplant model following IL-35 exposure. QPCR results showed mRNA expression of FOXP3 (F=21.289, P<0.001), IL-10 (F=17.737,P<0.001) and TGF-p (F=188.327,P<0.001) in peripheral blood of both renal transplant model and normal SD rats were increased significantly at day 5 following IL-35 treatment. Similar results were obtain at day 14. IL-35 treatment resulted in up-regulation of FOXP3 (F=23.502, P<0.001), IL-10 (F=70.242, P<0.001) and TGF-P (F=11.184, P<0.001) in peripheral blood of both renal transplant model and normal SD rats.Flow cytometry data showed at day 5 the percentage of CD4+ cells of IL-35+model group, PBS+model group, IL-35+false modle group, PBS +false model group and normal SD rat group were 40.5%, 28.19%,43.69%,47.06% and 41.73%; the percentage of CD8+cells of IL-35+model group, PBS+model group, IL-35 group, PBS group and normal SD rat group were 26.18%,18.08%,27.92%,25.59% and 21.77%; the percentage of CD4+CD25+Tregs of IL-35+model group, PBS+model group, IL-35 group, PBS group and normal SD rat group were 5.69%,4.51%,5.06%,5.34% and 5.19%. At day 14, the percentage of CD4+cells of IL-35+model group, PBS+model group, IL-35+false model group, PBS+false model group and normal SD rat group were 48.04%,28.81%,51.74%, 50.9% and 50.49%; the percentage of CD8+ cells of IL-35+model group, PBS+model group, IL-35+false model group, PBS+false model group and normal SD rat group were25.5%,25.08%,23.14%,22.58% and 23.61%; the percentage of CD4+CD25+ Tregs of IL-35+model group, PBS+model group, IL-35+false model group, PBS+false model group and normal SD rat group were 5.44%,4.48%,4.93%, 5.04% and 4.83%. These data suggest that IL-35 increase percentage of CD4+cells, CD8+cells and CD4+CD25+Tregs in peripheral blood of both renal transplant model and normal SD rats.In conclusion, IL-35 induced transplant tolerance through modulating CD4+CD25+FOXP3+Tregs. |
PDF Full Text Request