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

Objectives:To investigate the role of CX3CL1/CCL26-CX3CR1 pathway in the pathogenesis of primary biliary cholangitis(PBC).Methods:Peripheral blood samples were obtained from patients with PBC and healthy controls(HCs).Peripheral blood mononuclear cells(PBMCs)and plasma were separated and stored for further experiments.The expression of CX3CR1 and CCR3 in different subsets of lymphocytes from patients with PBC and HCs was measured by flow cytometry.ELISA was adopted to determine the level of CX3CL1 and CCL26 in plasma.In vitro chemotaxis assays with the Transwell system were conducted to reveal the chemotactic activity of CX3CL1 and CCL26 towards various subsets of PBMCs.The function of membrane-bound CX3CL1,soluble CX3CL1 and CCL26 in stimulating the secretion of IFN-? and IL-4 from various subsets of lymphocytes were evaluated by flow cytometry with intracellular staining.Results:59 patients with PBC and 54 HCs were included.The average age of PBC patients was 51.7±2.8 years old and the ratio of female to male was 53:6.The expression level of CX3CR1 in CD4+ T cells was significantly higher in PBC patients compared to HCs(17.0±13.0%vs 7.8±3.6%,p<0.001)and this discrepancy mainly came from the CD28-subset(65.9±22.3%vs 36.0±22.2%,p=0.005).The percentage of CX3CR1+cells was moderate to high in CD8+ T cells and a significant increase could be seen in PBC patients(44.3±19.8%vs 30.8±20.9%,p=0.012).While CX3CR1 was highly expressed in NK and NKT-like cells,the level was comparable between PBC patients and HCs.In comparison with HCs,a significant increase of CCR3 expression could be found in CD4+T cells and the CD4+CD28+subset in PBC patients(p=0.002,p<0.001,resp).In the aspect of plasma,the levels of both CX3CL1 and CCL26 were significantly higher in PBC patients than HCs(p=0.047,p<0.0001,resp).CX3CL1 exhibited potent chemotactic activity towards CD8+T cells,NK cells and NKT-like cells while no obvious chemotactic effects of CCL26 towards these cells were revealed.Under the stimulation of membrane-bound CX3CL1,CD4+ T cells,CD8+ T cells and NK cells upregulate their secretion of IFN-? but not IL-4.Meanwhile,similar effects were not shown in soluble CX3CL1 and CCL26.Conclusion:CX3CL1 plays an important role in the mechanism of bile duct damage by attracting immune cells into the liver via its receptor CX3CR1.These cells can secrete IFN-y to maintain the Th1 predominant inflammation which induces the expression of CX3CL1 and thus perpetuate the abnormal chemotaxis of immune cells towards the damaged bile duct areas.No evidence supports that CCL26 participates in the pathogenesis of PBC by recruiting lymphocytes.