Role Of CX3CL1/CCL26-CX3CR1 Pathway In The Immunological Mechanism Of Primary Biliary Cholangitis

Objectives:To investigate the role of CX3CL1/CCL26-CX3CR1 pathway in the immunological mechanism of primary biliary cholangitis(PBC).Methods:Peripheral blood and liver tissue samples from patients with PBC and healthy controls(HCs)were collected.The plasma level of CX3CL1,CCL26 and their relevant cytokines were determined by ELISA.Flow cytometry was conducted to measure the percentage of CX3CR1+ and CCR3+ cells in different subsets of peripheral blood mononuclear cells(PBMCs).In vitro chemotaxis assay was performed to evaluate the chemotactic activity of CX3CL1 and CCL26 towards various subsets of PBMCs.The expression of CX3CL1 and CCL26 in liver tissues were revealed by immuno-histochemistry.Using ELISA and flow cytometry,the change of CX3CL1/CCL26-CX3CR1 pathway expression in human intrahepatic biliary epithelial cell(HIBEC)was studied upon stimulation of various cytokines and LPS.Results:Forty patients with PBC and 18 HCs were included.The plasma level of CX3CL1 was significantly higher in patients with PBC(0.690±0.271 ng/ml)than HCs(0.540±0.101 ng/ml,P = 0.044).An increased tendency was observed for plasma level of CCL26 in patients with PBC compared with HCs(8.94±4.09 pg/ml vs 6.75± 1.86 pg/ml,P = 0.104),which positively correlated with peripheral eosinophils,basophils and C reactive protein level.In comparison with HCs,the expression of CX3CR1 were significantly higher in NKT-like cells(63.5±25.4%vs 78.6± 18.0%,P＝0.026)and CD4+ T cells(5.5±5.4%vs 13.7±9.0%,P = 0.003)in patients with PBC.Such difference existed in both CD28+ and CD28-subsets of CD4+ T cells.The expression of CCR3 decreased significantly in patients with PBC than HCs(1.0±0.6%vs 3.2±2.0%,P＝0.001),which attribute to its CD28+subset.CX3CL1 exhibited potent chemotactic activity towards CD8+ T cells,NK cells,and NKT-like cells in a dose-response manner,whereas CCL26 showed no chemotactic effect towards these cells.The intrahepatic biliary epithelial cell in liver biopsy samples of PBC showed high expression of both CX3CL1 and CCL26.In contrast,HCs’ only showed mild expression of CX3CL1 and no CCL26 expression.Upon stimulation of IFN-γ,HIBEC significantly increase its expression of CX3CL1 in intro,while IL-4 and IL-13 significantly increased the expression of CCL26.The expression of CX3CR1 was also up-regulated upon IFN-y stimulation.Conclusion:The intrahepatic biliary epithelial cell in PBC may increase its expression of CX3CL1 upon stimulation of IFN-γ,and its expression of CCL26 upon IL-4 and IL-13.Meanwhile,their receptor CX3CR1 was up-regulated in peripheral NKT-like cells and CD4+ T cells.CX3CL1 and CCL26 may cooperate via CX3CR1 to mediate the injury of small bile duct in PBC.