| ObjectTo explore the distribution, relative amount of interleukin-17+regulatory T (IL-17+Foxp3+Treg) cells in the liver of biliary atresia patients and animal models, and research the regulatory mechanisms of these cells and the function.Material and Methods35 cases of BA, obtained the liver tissues during the Kasai procedure, and 30 cases of other non-BA patients as controls. Some were extracted the supernatant through livers homogenate and tested the concentration of cytokines such as IL-6, IL-17, IL-1?, IL-12p40, TGF-?, IL-10 and IL-23 by ELISA; some tissues were used to exam the expression status of Foxp3 and ROR-yt by RT-qPCR and Western Blot; some tissues were immunofluorescences stained to show the distribution of IL-17+Foxp3+Treg cells; some tissues were density gradient centrifuged to extract CD4+T cells and flow cytometry (FACS) was used to study the relation amount (ratio) of L-17-Foxp3+Treg cells, IL-17+Foxp3+Treg cells and IL-17+Th17 cells, then sorted these three kinds and tested the methylation level of CpG islands among the promotor region of Foxp3 gene by Bisulfite Genomic Sequence (BSP); meanwhile, some IL-17+Foxp3+Treg cells were co-cultured with antigen presentation cells (APC) for proliferation assay to explore the function of IL-17+Foxp3+Treg cells.BA animal models were made by Rotavirus and separated the newborn BALB/c mice randomly into three groups:?RRV group:RRV was injected intraperitoneally (i.p.) within 24 hours after birth;?Control group:Culture medium i.p. within 24 hours after birth; ?Methylation intervention group (Aza group):RRV i.p. within 24 hours after birth, and methylation inhibitor Aza i.p. everyday after RRV infection. Recorded the physiological states and pathological appearance everyday and harvested the liver tissues at Day 4,7, and 10 after birth, then performed procedures as followed: supernatant of livers homogenate was retrieved and examined the concentration of cytokines such as IL-6, IL-17, IL-1?, IL-12p40, TGF-? and IL-23 by ELISA, the expression status of Foxp3 and ROR-yt were tested by RT-qPCR and Western Blot; the distribution of IL-17+Foxp3+Treg cells in the liver tissue was explored by immunofluorescences staining; CD4+T cells were sorted from liver tissues by density gradient centrifuging and flow cytometry was used to study the relation amount (ratio) of L-17-Foxp3+Treg cells, IL-17+Foxp3+Treg cells and IL-17+Th17 cells, then sorted these three kinds and tested the methylation level of CpG islands among the promotor region of Foxp3 gene by BSP.To explore the effect of inflammatory micro environment on the differentiation of IL-17+Foxp3+Treg cells, we sorted CD4+naive T cells in the mice liver, and cultured in the 3 kinds of medium:Treg culture system, Treg adds Aza culture system, Th17 culture system for 7 days, then tested the concentration of IL-10, IL-17 in the supernatant by ELISA, counted the cell ratio of IL-17-Foxp3+Treg cells? IL-17+Foxp3+Treg cells and IL-17+Th17 cells by FACS, then examined the methylation status of Foxp3 and histone acetylation level of ROR-yt gene in the three culture system.ResultsCompared with control group, IL-17+Foxp3Treg cells were elevated significantly in the portal area of livers from BA patient and animal models, so as the cell ratio. The changes of IL-17+Foxp3Treg cell ratio in BA mice liver were associated with the time after RRV infection, and at 7 days after RRV infection (dpi), the IL-17+Foxp3+Treg cell ratio reached top. At the same time, the jaundice incidence (85.3±12.36%) and acholic stool occurrence (80.6±15.22%) was reached top as well, the mice started to die, and the survival rate at 14dpi was 20.5±3.24%. The concentrations of IL-12p40 (associated with Th1 cells), IL-23, IL-17 (associated with Th17 cells), IL-10, TGF-?1 (associated with Treg cells) and IL-6 were elevated significantly compared with control group, and the inflammatory cytokines, such as IL-12p40, IL-23, IL-17, IL-6, had higher content in the BA liver compared with anti-inflammatory cytokines such as IL-10 and TGF-p.IL-17+Foxp3+Treg cells have remarkable pro-inflammation effect which were derived from BA mice through the experiments of co-culture with effector T cells (Teff), and the methylation level of CpG islands in the promotor region of Foxp3 gene of BA group was significantly higher than the controls (Human:73.2±16.17% vs.2.9± 0.85%; mouse:70.3±10.51% vs.13.5±4.67%, p<0.01).In the intervene experiments, the jaundice rate (60.3±12.34%) and acholic stool rate (57.5±11.25%) were lower in the Aza group, and the day when the BA mice started to die (8.3±0.58) was delayed and the survival rate at 14dpi (59.3±9.52%) was remarkable elevated. The IL-17+Foxp3+Treg cells infiltrated in the portal area of BA liver were less and cell ratio decreased, accompanied by the decreased concentration of inflammatory cytokines. The methylation level of Foxp3 fell and the expression level was ascended.Immunofluorescences staining showed IL-17+Foxp3+Treg cells and IL-17+Th17 cells infiltrated in the portal area were increased sharply, the call ratios acquired by FACS were elevated significantly and the gene transcription, protein production level were went up, too.In vitro experiments, Foxp3 expression was enhanced in Treg cell culture system after 7 days of culture, the ratio of IL-17-Foxp3+Treg cells was the highest (76.4±14.2%) and which of IL-17+Foxp3+Treg cells was 3.7±0.49%. Methylation degree of Foxp3 gene is 9.4±3.7%. Under the intervention of Aza, the concentration of IL-10, TGF-?1 in the medium were elevated, the ratio of IL-17- Foxp3+Treg cells increased to 85.6± 18.49%, Foxp3 methylation level dropped to 6.2Q2.45%. In the Th17 cell culture system, ROR-yt gene expression was enhanced and the IL-17+Th17 cell ratio was the highest (58.2±10.2%), the ratio of IL-17+Foxp3+Treg cells was 11.6±3.79%, and IL-17+ Foxp3+Treg cell ratio was the lowest (1.45±0.28%).Conclusion1. Around the portal area of BA patients or mice livers, the concentrations of inflammatory cytokines are elevated, which create a pro-inflammatory micro environment, IL-17+Foxp3+Treg cell and IL-17+Th17 cell ratios elevate, and at the obstruction phase of bile duct (7dpi), the ratio of IL-17+Foxp3+Treg cell reach to top, with the decrease of IL-17Treg cells, which shows that IL-17+Foxp3+Treg cells take part in the immunological inflammatory process of BA disease.2. In the micro environment of BA liver, IL-17+Treg cells have capacity to activate effector T cells, which shows pro-inflammatory function, and promote the disease development of immunological inflammation in BA liver.3. The proliferation and differentiation of IL-17+Foxp3+Treg cells in BA are regulated by the methylation status of Foxp3 promotor CpG islands. When the methylation of Foxp3 is inhibited, the gene expression is enhanced with the down regulation of ROR-yt gene, the differentiation of IL-17+Foxp3+Treg cell is inhibited and of IL-17Treg cell is enhanced.4. IL-17+Foxp3+Treg, along with IL-17-Treg and IL-17+Th17 cells, take part in the immunological inflammatory process of biliary atresia. By inhibition of proliferation and differentiation on IL-17+Foxp3+Treg cells, the BA mice can have alleviated liver inflammation, and lower jaundice accidence, prolonged survival rate, and this kind of cell maybe the immunological therapeutic target to cure the BA disease. |
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