The Role Of CXCR3 And Toll-like Receptor 3 Signaling Pathways In The Induction Of Primary Biliary Cirrhosis

Background:Primary biliary cirrhosis(PBC) is an organ-specific autoimmune disease that predominantly affects women and is characterized by chronic,progressive destruction of small intrahepatic bile duct with portal inflammation and ultimately fibrosis,leading to liver failure in the absence of treatment.The serologic hallmark of PBC is the presence of autoantibodies to mitochondria.PBC is an autoimmune liver disease with profound changes in different compartments of the immune system,including those involved in innate,and adaptive immunity.Current theories on the pathogenesis of PBC favor the hypothesis that T lymphocytes play a pivotal role in the autoimmune response in PBC. Once this adaptive response develops,it appears that much of the subsequent pathology is exacerbated by innate responses.The chemokine receptor CXCR3 is found predominantly on T cells,particularly prominent on cells with a T helper cell type 1 bias.CXCR3 mediates chemotaxis in response to its ligands,CXCL9,CXCL10 and CXCL11.CXCR3 and its ligands play a critical role in the recruitment of T cells to inflammatory sites and regulate T cell activation in several diverse models of diseases.Toll-like receptor 3,one of the intracellular members of TLR family,recognizes dsRNA and poly I:C,triggers an innate response independent of MyD88.TLR3 initiates its response depending only on TRIF,activating NF-κB and IRF-3,leading to the induction of type 1 IFN and cytokine/chemokine production.Up to now,there were no report about CXCR3 and TLR3 signaling pathways in the induction of primary biliary cirrhosis.Objective:Generate an autommune cholangitis animal model.Investigate whether CXCR3 and TLR3 signaling pathway were involved in the pathogenesis of PBC.Study the immunopathology of primary biliary cirrhosis,including innate and adaptive immunity.Methods:Female C57BL/6 wide-type and CXCR3^-/- mice were injected with 5mg/kg of poly I:C intraperitoneally twice a week for 28 consecutive weeks.Liver specimens were collected to evaluate the degree of cell infiltration. Autoantibodies,including AMA,were assayed by IIF and ELISA.Differences in pathology and immunohistochemistry were compared between CXCR3^-/- mice and wide-type mice.Lymphocytes subsets in liver and spleen were analyzed using flow cytometry.The liver expressions of TLR3,TRIF and p- NFκB p65 in mice model and control mice were evaluated by immunoblotting.Results:Ⅰ.CXCR3 might contribute to the development of primary biliary cirrhosis in murine model.ⅰ.Wide-type mice PBC model was compared with control model:a.Autoantibodies,including AMA,were detected in the sera from all poly I:C injected mice.b.Flow cytometry analysis demonstrates a significantly higher proportion of CXCR3+ cells of CD3+,CD4+ and CD8+ cells in the liver than the spleen.Compared to control model,the proportion of cells positive for CXCR3+ was increased in the intrahepatic infiltrates of PBC model,chiefly on CD8+ T cells.It is suggested that high expression of CXCR3 is an important event in migration of T cell to sites of inflammation.c.Considerable numbers of infiltrating cells were detected at the portal areas 8 weeks after the start of poly I:C injection,which progressed up to 24 weeks.In addition to lymphocytic infiltration,small bile ducts showed irregular luminal shape and epithelial injury.In 24 weeks period,the interlobular bile ducts were lost and bileplug were evident.Up to 28 weeks period,fibrosis formation was developed with lymphocytic infiltration.d.With comparison to control model,the proportion of cells positive for CD3+ cells in mononuclear cells was increased in the intrahepatic infiltrates of PBC model.There was no significant difference of T cells proportion between PBC WT mice and CXCR3^-/- mice model.e.Immunohistochemical examination results indicated the types of cells that infiltrated the liver.In the animal model,CXCR3+ mononuclear cells were found mainly in the enlarged periportal areas,with a similar distribution of CD4+ and CD8+ cells.f.Immunohistochemical examination results suggested that CD4+ and CD8+ mononuclear cells were manily localized in the portal tract and CD8+ cells were localized near the bile ducts.With the progressment of mononuclear cells infiltration, CD4+ and CD8+ cells were detected increased by degrees.g.Compared to control model,CXCL10 serum level were increased in PBC animal model.With the disease progression,CXCL10 serum level was elevated gradually.ⅱ.Wide-type mice PBC model was compared with CXCR3^-/- mice PBC model:a.There was no difference of AMA and ALP level between WT and CXCR3^-/- mice PBC model.b.Pathologic result indicated CXCR3^-/- mice developed a delayed onset of cellular infiltration and inflammation of PBC animal model,suggesting blockade of CXCR3 might prevent clinical progression of PBC.c.There was no significant difference of T cells proportion between PBC WT mice and CXCR3^-/- mice model.d.In comparison with wide-type mice,CXCR3^-/- mice model had a higher serum level of CXCL10.In 24 weeks period,there was significant difference of CXCL10 serum level between wide-type mice and CXCR3^-/- mice model.Ⅱ.The role of TLR 3 signaling pathways in the induction of primary biliary cirrhosis.a.The liver protein levels of TLR3 were elevated in PBC mice.There were no significant difference of TLR3 level between different periods or between WT mice and CXCR3^-/- mice.The levels of TLR3 were decreased in PBC CXCR3^-/- mice of 16 weeks and 24 weeks period.b.There were no significant difference of TRIF level between PBC mice and control mice or between WT mice and CXCR3^-/- mice.c.There were no significant difference of p- NFκB p65 level between PBC mice and control mice.The level of p- NFκB p65 in CXCR3^-/- PBC mice was higher than control,and that in 16 weeks period was increased evidently.Conclusions:1.In conclusion,we show an early development of a PBC-like cholangitis in a genetically susceptible mouse strain because of poly I:C administration.2.CXCR3 might contribute to the development of primary biliary cirrhosis in murine model.CXCR3 and its ligands play an important role in the migration of T cells to inflammatory sites,especially CD8+ cells.3.CXCR3 had no great effect on T cells proportion in liver mononuclear cells.CXCR3 might contribute to T cells activation in the development of PBC murine model. 4.Given the role that CXCR3 play in induction of hepatic damage in PBC animal model,prevention of CXCR3 engagement may be beneficial not only for the treatment of the early lymphocytes infiltrating period but also for treatment during the early phases of fibrosis.5.The protein levels of TLR3 were elevated in PBC mice.There were no significant change of levels of TRIF and p- NFκB p65 in PBC mice.6.NF-κB might not be the key transcription factor in TLR3 signaling pathways in the induction of PBC.In the PBC mice model,IRF3 might play a critical role in TLR3 signaling pathways by inducting production of type 1 IFN.7.CXCR3 have no great effect on TLR3 signaling pathways in the induction of PBC.