Significance Of Regulatory And Effector T Cells In The Development Of Autoimmune Inflammation In Experimental Biliary Atresia

Objective:The role of regulatory T cells (Treg) and Th17effector cells (Th17) in the development of immune inflammation of experimental biliary atresia was studied. Adoptive transfer of Treg cell or injection of IL-17neutralizing antibodies, further clarified the role of Treg/Thl7cells in the immunological pathogenesis of biliary atresia mice.Methods:Used RRV to establish mouse model of biliary atresia, newborn Balb/c mice were randomly divided into four groups:1, normal group:intraperitoneal injection of saline in24h after birth;2, rotavirus group:intraperitoneal injection of RRV in24h after birth;3, Treg cells group:adoptive transfer of Treg cells within12h after birth, followed by intraperitoneal infusion of RRV within12h;4, IL-17antibody group:intraperitoneal infusion of RRV in12h after birth, followed by injection of different doses (15μg,30μg,50μg) of IL-17neutralizing antibodies in consecutive two days. The changes of phenotypes and histopathology of all groups at different periods were observed. Real-time quantitative PCR (RT-qPCR) and Western blot were used to detect the expression of mRNA and protein of Foxp3, IL-17and ROR-yt in livers. Immunohistochemistry was used to detect the location and changes of CD4+cells, Foxp3+Treg cells and IL-17+Th17cells in livers of each group. The changes of proportion of Treg cells and Th17cells in livers were detected by flow cytometry. ELISA was used to detect the changes of inflammatory cytokine in liver tissue. And the expression of TLRs for DC cells in livers was observed. DC cells and CD4+T cells were separated and cultured. ELISA was used to detect the expression of IL-6in culture supernatants. The suppressive function of Treg cells on Th17cells was observed with co-culture assays. The change of suppressive function of Treg cells in biliary atresia mice was also observed.Results:None of23normal mice manifested symptoms of cholestasis and all survived to21days except one died in3days.18mice of24in rotavirus group developed biliary atresia (75.0%), showed signs of cholestasis (icterus of the skin not covered with fur and acholic stools) and growth retardation (slow growth or stagnation of weight). Livers showed obvious cholestasis, granular surface. Extrahepatic bile duct showed cable strip and gallbladder showed unclear. The HE staining of livers and bile ducts showed extrahepatic biliary atresia and portal areas infiltrated by a large number of inflammatory cells. The expression of Foxp3, IL-17and ROR-yt mRNA and protein in biliary atresia mice was significantly higher than in normal group, and reached a peak at7days after birth. Immunohistochemistry revealed CD4+cells, Foxp3+Treg cells and IL-17+Th17cells in livers, especially portal areas of BA mice were significantly increased. Flow cytometry found that the proportion of Treg cells was reduced and Thl7cells increased in BA mice. IL-2, IL-4, IL-5, TGF-β1,IL-10, IL-17, IFN-γ, IL-12, IL-23and IL-6in livers of BA mice were increased significantly. The expression of TLR3,7and8of DC cells in B A mice was higher than in the normal control group. IL-6was highly secreted in DC cells of BA mice, while there was no significant difference in the culture supernatant of CD4+T cells between BA mice and normal control group. Co-cultures assays found that Treg cells can suppress the differentiation of Th17cells, and the suppressive function of Treg cells reduced in biliary atresia mice. After adoptive transfer Treg cells, the incidence of biliary atresia decreased to23.1%, hepatic inflammatory manifestations reduced significantly and survival time prolonged. Th17cell-associated mRNAs were reduced significantly compared with biliary atresia mice. In livers, the expression of IL-17+cells decreased by immunohistochemistry. And the proportion of Th17cells reduced in livers compared to BA mice by flow cytometry (P<0.05). With the injection of different doses of IL-17neutralizing antibodies, the incidence of biliary atresia reduced in varying level, and the group of50μg antibody injection was statistically significant (P<0.05). In liver tissue, especially portal area, inflammatory cells reduced significantly. And this can prolong the survival time of mice. Further study found that IL-17protein reduced obviously in50μg IL-17antibody group compared to biliary atresia mice (P<0.05). The expression of IL-17+cells reduced in livers compared to biliary atresia mice by immunohistochemistry (P<0.05).Conclusions:1. After virus infection, the changes of liver micro-environment and TLRs dependent DC cells resulted in the reduction of expression and function of Treg cells, while the increase in Th17cells. The imbalance between Treg and Th17cells plays an important role in the progressive inflammation of bile duct injury in biliary atresia.2. Adoptive transfer of Treg cells can alleviate the phenotype of biliary atresia mice significantly, prolong the survival time of mice, and reduce inflammation. These indicated that Treg cells play an important role in suppressing the inflammatory response after viral infection. And these present a possibility for the prevention and treatment of biliary atresia.3. With the injection of IL-17neutralizing antibodies, the phenotype of biliary atresia mice reduced obviously, survival time prolonged, and inflammation reduced. These suggested that Th17cells can promote the occurrence and development of biliary atresia. It established an experimental basis for the treatment of progressive immune and inflammatory response of biliary atresia after viral infection.