| Backgroud&Objective:Progressive autoimmune inflammatory injury of bile duct occupies an important place in the pathogenesis of biliary atresia (BA). From our previous group study in BA, we found a massive infiltration of Foxp3+Treg in the microenvironment of the intra-hepatic bile duct. Interestingly, these Foxp3+Tregs were proven to be heterogeneous, containing a subset of pro-inflammatory IL-17+FoxP3+Treg. Recent literature reports suggest that the epigenetic modification of Foxp3plays a vital role in the differentiation and regulation of Foxp3+Treg. Thus we can inferred that the variation of microenvironment cytokines, such as TGF-β and IL-6, could promote epigenetic modification of FoxP3/ROR-yt gene, inducing IL-17+FoxP3+Treg differentiation, which secreted IL-17and mediate bile duct injury. Regarding to the above findings, the aims of this research were to;(1) observe the dynamic cytokine (TGF-β, IL-6, etc.) Expression and the Treg cell Foxp3gene promoter DNA methylation status, as well as Foxp3gene expression, affecting the differentiation of Treg cell subsets in the liver microenvironment of BA mice model;(2)observe the effect of the DNA methylation inhibitor5-aza-2’-deoxycytidine Foxp3gene modification (5-aza-2’-deoxycytidine, Aza) on the Treg cell subsets in different functional states and their correlation in the inflammatory bile duct injury in BA mouse model; specifically, the IL-17+FoxP3+Treg formation and its mechanisms involved in bile duct injury.Methods:The first part:SPF level Balb/C mice were randomly divided into control group and BA group. In the BA group, within12hours after birth, the newborn mice were injected rhesus rotavirus (RRV) intraperitoneal to established model of biliary atresia. The liver from the RRV infected and normal control newborn mice were harvested on4th,7th and10th days of life. The inducing-inflammatory cytokines (INF-y, IL-6, IL-17A, IL-23) and suppressing-inflammatory cytokine (IL-10, TGF-β) changes were measured at each time point in each groups; IL-17+FoxP3+Treg cells, IL-17-Foxp3+Treg cells in the cells distribution and the ratio of the absolute number of CD4+T cells were compared, together with the levels of Foxp3gene promoter CpG island methylation.The second part:SPF level Balb/C mice were randomly divided into3groups;(1) normal control group,(2) BA group, where the new born mice were infected by RRV virus to induce BA, and (3) Aza intervention group, where the new born mice were infected by RRV virus to induce BA and were injected intraperitoneal with Foxp3gene methyltransferase inhibitor (5-aza-2’-deoxycytidine, Aza) for4consecutive days. The liver from each group were harvested on the4th,7th and10th days of life. The inflammatory cytokines changes, the Foxp3gene promoter CpG island methylation levels and the proportions IL-17+FoxP3+Treg cells, IL-17-Foxp3+Treg cells and Th17cells in liver tissue were compared, as well as the survival rate of the mice in each group were compared.Results:The first part:in the BA group, we detected a high increased expression of cytokines on4th day. The maximum peaked expression was registered on the7th day with IL-6(31-fold), IL-10(6-fold), INF-γ (11-fold), and TGF-β (3.2-fold), statistically significant difference (P <0.001, P<0.01, P<0.05, P<0.05), which slightly decreased on the10th days. However, the expression values at each time point was significantly higher than those in the control group (P<0.05). The maximum peaked expression of the IL-17(6-fold) and IL-23(4-fold) were registered on the10th day, but the expression values at each time point were still significantly higher than those in the control group (P<0.05). From the Immunohistochemistry fluorescent results in the BA group, we observed a few IL-17+FoxP3+Treg cell infiltration in periportal area on the4th days, but on the7th days there were a lot IL-17+FoxP3+Treg cells and Th17cell infiltration, and we observed a slight decrease of the IL-17+FoxP3+Treg cell infiltration on the10th days. The percentage IL-17+FoxP3+Treg cells in CD4+cell in the BA group was highest in the7th days, followed by a rapid decline, however it was still significantly higher than those in the control group (P <0.05) at each time point. In the BA group, the absolute value of IL-17+FoxP3+Treg cells and IL-17-Foxp3+Treg cells was significantly higher than the control group (P<0.05). Similarly, at each time point, the Foxp3gene promoter CpG island methylation levels were higher than the normal control group (P<0.05).The second part:In the Aza intervention group, the significant decline in the cytokine expression was detected from7th days; inflammatory cytokines IL-6, INF-γ and IL-17, IL-23were statistically significant (P<0.05); and the Foxp3+Treg related cytokines (IL-10and TGF-β) expression was significantly higher (P<0.05) as from the7th days and reached its peaked by the10th days. In the Aza intervention group, the percentage of IL-17+FoxP3+Treg cells and Th17cells in CD4+cell began to decline significantly (P<0.05) on the7th days, and the absolute value of IL-17+FoxP3+Treg cells and IL-17-Foxp3+Treg cells were significantly lower than BA group (P<0.05) on the7th and10th days. At each time point, the Foxp3gene promoter CpG island methylation levels were significantly lower in the same period for the Aza intervention group compared to the BA group (P<0.05). From the HE staining pathological results, we noted that the damages caused by BA were significantly reduced in the Aza intervention group. Survival rates in the intervention group compared with mice BA group increased.Conclusion:(1) In the intrahepatic cytokine inflammatory microenvironment of RRV BA-induced mouse model, the FoxP3+Treg cells and IL-17+FoxP3+Treg cells, and Foxp3gene DNA methylation status change dynamically,:Increased first and then gradually decreased. Wherein, the INF-γ, IL-6, IL-10and TGF-β cytokine reach their peak earlier than IL-17and IL-23cytokine. But Foxp3gene continued to maintain a high DNA methylation status in the BA group.(2) The IL-6can up-regulate the Foxp3promoter DNA methylation, which may promote the IL-17+FoxP3+Treg cell differentiation and the formation of inflammatory Treg cells in the intra-hepatic microenvironment, causing injury to the bile duct in BA.(3) The DNA methylation inhibitor (Aza) can reduce the level of Foxp3gene methylation expression, which in turn reduce the IL-17+FoxP3+Treg cells and inhibit the generation of the inflammatory cytokines (IL-17A), thereby reducing the bile duct injury in BA mice. |
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