The Role Of Toll Like Receptors And Chemokine Receptor In The Pathogenesis Of Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is a chronic cholestatic and autoimmune liver disease, which caused by abnormal immune responses to the patients’ own livers. This disease occurs at high frequency among middle-aged women. After onset, the following symptoms show up:biliary obstruction and cholestasis, biliary epithelial cells injury, liver fibrosis, and ultimately, cirrhosis and liver failure, which seriously threaten patients’ lives. As it is very difficult to acquire clinical liver samples of patients in the early onset, the pathogenesis studies and therapeutic developments of primary biliary cirrhosis are mainly based on an animal model-dnTGF-βRⅡ mice. The mice spontaneously develop an autoimmune biliary disease closely resembling human PBC. High level of anti-mitochondrial antibodies (AMAs) and increased serum cytokines have been detected in the mice.Histologically, dnTGF-βRⅡ mice manifest extensive CD4and CD8lymphocytic infiltration in portal tracts, associated with biliary destruction. Moreover, dnTGF-β R Ⅱ mice, developing colitis spontaneously as well, could be established as a model to investigate the interactions between hepatic inflammation and intestinal microenvironment.Primary studies based on dnTGF-βRⅡ mouse model about the pathogenesis of autoimmune liver disease in early onset have been reported, including the pathogenic role of CD8T, the regulatory role of Bregs, and the functions of different cytokines. However, the pathogenic mechanisms of this disease have not been fully understood. Many unknown areas still exist in this disease, such as the role of innate immune responses, and the complexity of chemokines and their receptors, which induce inflammatory infiltration.Therefore, in this study, dnTGF-βRⅡ mice and dnTGF-βRⅡ mice lacking either Toll-like receptors or chemokine receptor were established as mouse models to investigate the role of innate immune receptors-Toll-like receptor2and4, as well as chemokine receptors, CXCR3, in the progress of this autoimmune liver disease. Methods used in this study were supported by cellular immunology, molecular biology and medical pathology. The aim of this study is to elucidate the pathogenesis of primary biliary cirrhosis and to search for therapeutic targets. The results obtained in this study are as follows: 1. Changed environmental factors (intestinal flora depletion) affected the severity of primary biliary cirrhosis-like disease in mouse modelWe added antibiotics including metronidazole, ampicillin, neomycin, and vancomycin into drinking water of dnTGF-βR Ⅱ (TG) mice (at SPF level) since the fourth week after birth to deplete the vast majority of the gut symbionts.12weeks later, when15-week-old, the mice were sacrificed and the liver pathology analysis demonstrated that drinking antibiotic water could aggravate cholangitis in TG mice compared with control group without antibiotic water. TG mice in antibiotic water drinking group had increased hepatic inflammatory infiltrations and worse bile duct injury.2. Toll-like receptor2depletion aggravated cholangitis and colitis in dnTGF-βR Ⅱ miceCompared with wild-type (WT) mice, dnTGF-βR Ⅱ (TG) mice have higher expression of tlr2gene in liver. Thus, we crossed TG mice with Toll-like receptor2(TLR2) knockout mice and got TLR2-/-dnTGF-βR Ⅱ (TGT2) mice. First, liver pathology analysis showed that, compared with TG mice, TGT2mice had more severe portal inflammation and bile duct damage in liver. Cytological analysis revealed that, compared with TG mice, infiltrating T cells in TGT2mice liver, especially CD4T cells, were increased. There were also more infiltrating CD4T and CD8T cells presenting effector memory phenotype (CD44highCD62Llow) in TGT2mice than in TG mice. Secondly, intestinal pathology analysis showed aggravated colitis in TGT2mice. Compared with TG mice, the body weight of TGT2mice decreased significantly, but colon weight increased. In the colon of TGT2mice, ulceration with transmural inflammatory cell infiltrates was observed. Moreover, crypts were destroyed and goblet cells were inconspicuous in the colon of TGT2mice, which due to the dense inflammatory cell infiltrates, and accompanied by crypt abscesses and mucin depletion. Again, analysis of cytokines exhibited that compared with TG mice, serum TNF-α and IFN-γ levels were significantly increased and il6gene expression in liver was distinctly up-regulated in TGT2mice. By detecting IL-6related signaling pathway, we found up-regulated phosphorylation of STAT3in the liver of TGT2mice compared with TG mice. Finally, in order to define the role of TLR2in the pathogenesis of primary biliary cirrhosis-like disease in TG mice in another way, we plan to treat TG mice with TLR2specific ligand Pam3CSK4by administrating a single injection at high-dose or a series of injections weekly at low-does. The results will provide us more evidence to elucidate the pathogenic role of TLR2in TG mice.3. Increasing trend of aggravated liver inflammation and colitis in TLR4-/-dnTGF-βRⅡCompared with wide type (WT) mice, tlr4gene expression was up-regulated in TG mice in liver. We obtained TLR4-/-dnTGF-βR Ⅱ (TGT4) mice by crossing TLR4-/-micewith dnTGF-βR Ⅱ mice. Cellular immunology and medical pathology analysis showed that compared with TG mice, TGT4mice had increased proportion of T lymphocytes in liver mononuclear cells. Among these infiltrating T cells, the percentage of CD8T cells was higher than in TG mice. Furthermore, the frequency and absolute number of effector memory CD8T cells in liver of TGT4mice were increased compared with TG mice. Although there was no significant difference in colitis between TG and TGT4mice, increasing trend of aggravated liver inflammation and colitis in TLR4-/-dnTGF-βR Ⅱ mice was observed in both pathological analysis and statistical analysis.4. The exploration of pathogenic role of CXCR3in dnTGF-βR Ⅱ miceFirst, the concentration of chemokine CXCL9and CXCL10was up-regulated in the liver of TG mice compared with wide type mice. Then, the flow cytometry analysis revealed increased CXCR3(receptor of CXCL9and CXCL10) expressing T cells in the liver of TG mice compared with wide type mice. Next, we plan to adoptively transfer T cells from TG mice or CXCR3-/-TG mice, to elucidate the role of CXCR3in T cell migration to liver in TG mice.In summary, this study demonstrated that the absence of TLR2led to aggravated spontaneous cholangitis and colitis in the mouse model of primary biliary cirrhosis. And we also found that CXCR3may participate in guiding T cell migration to inflammatory sites of liver in this mouse model. Our study demonstrated for the first time that depletion of innate immune receptor TLR2aggravated inflammation in liver and colon in TG mice. And we explored the relationship between intestinal micro-environment and liver inflammation in TG mice firstly. All the evidence we provided in this study could shed light on the pathogenic mechanism of primary biliary cirrhosis and make a contribution to the therapeutic developments.