Study Of IL-27 Signaling Pathway In The Pathogenesis Of Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is a slowly progressive autoimmune disease of liver characterized by destruction of the small intrahepatic bile ducts, portal inflammation, and the presence of antimitochondrial antibodies (AMA), The loss of bile ducts leads to decreased bile secretion and the retention of toxic substances within the liver, resulting in further hepatic damage, fibrosis, cirrhosis, and eventually, liver failure. This disease primarily affects women, and its peak incidence occurs in the fifth decade of life, and it is uncommon in persons under 25 years of age. Histopathologically, The characteristic lesion of PBC is the asymmetric destruction of the intralobular bile ducts within portal triads, portal inflammation and immune-mediated destruction of the intrahepatic bile ducts. Ursodeoxycholic acid(UDCA) is known as the most effective drug for PBC, it can only provide symptomatic relief of cholestasis but not block disease course. At late stage of PBC, the only selection is liver transplantation.The pathogenesis of PBC remains obscure. Genetic, environmental risk factors, pollution, host susceptibility may be involved in the pathogenetic mechanism of PBC that initiate the immunopathological cascade. Some research suggest that the generation of immune responsiveness to self-antigen can result in pathogenic autoimmune damage of the intrahepatic biliary epithelial cells mediated by both humoral and cellular immune responses. The pathogenetic mechanism is believed to be caused by the breakdown of immunologic tolerance, resulting in cholestasis and the development of PBC. But the mechanism of initiation of immune responsiveness and the self-tolerance breakdown is unclear. The portal cellular infiltrate in PBC contained a preponderance of CD4+T cells in comparison with CD8+T cells, which indicate that CD4+T cells play important roles in the immune inflammation process. In situ nucleic acid hybridization of cytokines in primary biliary cirrhosis showed that IFN-γmRNA positive cells were detected primary around damaged bile ducts. The data indicate that Thl cells are the more prominent T cell subset in the lymphoid infiltrates in PBC.Interleukin-27 is a member of the IL-6/IL-12-family that consists of EBV-induced gene 3 (EBI3)2 and p28. It is the only known ligand for the gp130/WSX-1 receptor.Macrophages and dendritic cells are the main producers of IL-27, and the IL-27 signaling cascade includes activation of Jakl, STAT-1, STAT-3, STAT-4, and STAT-5 in T cells, STAT-1 and STAT-3 in monocytes, and STAT-3 in mast cells IL-27 may exert both pro-and anti-inflammatory functions It can augment T cell proliferation and enhance synthesis of IFN-γthrough the induction of T-bet and the activation of STAT-4. IL-27 can activate the transcription of T-bet independently from STAT-1, and such induction of T-bet appears to be mainly responsible for IL-27-mediated IFN-γproduction. IL-27 is critical in initial Thl differentiation via STAT1-mediated T-bet activation. IL-27 suppresses production of a group of proinflammatory cytokines by T cells. IL-27 induces IL-10 production by activated T cells for its anti-inflammatory effects.On the basis of status described above, in order to explore these hypothesis, four sections were included in our experiments:1) to measure the expression levels of IL-27 in PBMC from PBC patients, and observe the relationship with function of liver; 2) to explore the role of IL-27 on CD4+T in PBC patients; 3) to detect the effect of IL-27 on monocytes by Western blot, flow cytometry real-time in PBC patients.Partl The role of IL-27 in patients with primary biliary cirrhosis and its clinical significanceIL-27 is a new cytokine of the IL-6/IL-12 family and play a key role in regulating T cell differentiation and function, it consists of IL-12 p40-related protein (EBI3) and a newly discovered IL-12 p35-related protein (p28), and it is the only known ligand for the IL-27 receptor complex formed by WSX-1 and gp130 subunits. IL-27 is predominantly produced by activated macrophages and dendritic cells, while IL-27 receptor complex is mainly expressed on the surfaces of T cells, NK cells, monocytes, B cells and dendritic cells. IL-27 was an early signal for the induction of Thl responses, and depending on cell type and activation state can activate Jak-STAT signal transduction pathway in a context dependent manner, More recently, some studies found that IL-27 signal pathway can provide a deeper insight into the mechanisms of immune-mediated disease and increase the chances of exploring the causes of human autoimmune diseases such as PBC. This part we detect the expression of IL-27 in PBC patients and the possible involvement of IL-27 signal pathway in PBC. This part was designed to measure the gene transcription and protein expression levels of IL-27 in patients with PBC, chronic hepatitis B(CHB) group and healthy controls(HCs) by real-time PCR, ELISA, Flow cytometry and immunohistochemistry. AST, ALP, ALT, TBIL, GGT were determined and their correlation with IL-27 was also analyzed. Results show that IL-27 was significantly elevated in patients with PBC and IL-27 is present in the liver tissues of patients with PBC. Expression of IL-27 on CD4+T cells was increased in patients with PBC(72.4±6.22%) and CHB(59.4±7.03%) compared with HCs(1.7±0.55%, P<0.01). Expression of IL-27 protein is increased in patients with PBC(126.25±36pg/ml) compared with CHB(51.81±23.3pg/ml, P<0.01) and HC(34.19±9.7pg/ml, P<0.01), and it was positively correlated with GGT(r=0.554, P<0.01) and TBIL(r=0.559, P<0.01), but no correlation with AST, ALT, ALP.In conclusion, the expression of IL-27 was elevated in peripheral blood and intrahepatic portal area of PBC patients, and the expression of IL-27 were positively correlated with liver function parameters, These fact indicated that the key role of IL-27 in the immune inflammatory reaction in patients with PBC.Part 2 IL-27 vigorously induces STAT1 and STAT3 in CD4+T cell from patient with PBCOur previous studies have shown that the gene and protein lever of IL-27 in PBC patients increased significantly than HC group, These fact indicated that the key role of IL-27 in the immune inflammatory reaction in patients with PBC. How IL-27 involved in the development of PBC? Therefore, in this part, we detected the effect of IL-27 on CD4+T cells in patients with PBC.Result showed that IL-27 can induced tyrosine phosphorylation of both STAT1 and STAT3 in freshly isolated CD4+T cells in patients with PBC, CHB and HCs, with phosphorylation of STAT1 and STAT3 more marked in patients with PBC, than HCs. IL-27 strongly induced the expression of T-bet and suppressed the GATA3 in patients with PBC. Besides, culture supernatant was collected and analyzed for IL-2, IFN-γand IL10 production by ELISA, and the results showed that IL-27 can promote cytokine production such as IL-2 and IFN-γ, but suppresses IL-10 production in patients with PBC. Moreover, CD4+T cells proliferation measurements were obtained by using the Cell Counting Assay Kit-8 assay technique. When CD4+T cells were stimulated with plate-coated anti-CD3 and anti-CD28 in the presence or the absence of IL-27 for 3 days, we initially noticed that cell number in the presence of IL-27 appeared to be more than absent of IL-27.The results showed that the important role of IL-27 is to promote the differentiation and proliferation of T cells, The CD4+T cells of PBC patients secreted larger amounts of IL-2 and IFN-y at the present of IL-27, which induced Thl cell responses, and resulted in the excessive immunological reaction and the breakdown of self-tolerance.Part3 IL-27 activates predominantly STAT1 and STAT3 in monocytes of patients with PBCPrevious studies have shown that the expression of IL-27 in peripheral blood and liver of patients with PBC was higher than HC, and the IL-27 can change the differentiation of CD4+T cells. This part we tested the effect of IL-27 on monocytes. Like the resoponses of CD4+T cells, the test results showed that IL-27 induced tyrosine phosphorylation of both STAT1 and STAT3 in freshly isolated human monocytes. The production of pro-inflammatory cytokines after stimulation with IL-27 was measured using the ELISA kit. IL-27-stimulated monocytes of patients with PBC were found to produce significantly higher amounts of IL1 (P<0.01), TNF-α(P<0.01) except for IL8(P>0.05).In conclusion, IL27 can change the expression of inflammatory cytokines IL-1 and TNF-α, this might be involved in in the pathogenesis of PBC, This provides clues for finding a better treatment proposal.