The Mechanism Of Tim-3 Regulating NK Cell Subsets And CXCR1 Expression In Primary Biliary Cholangitis

Objective:T-cell immunoglobulin domain and mucin domain-containing molecule(Tim)-3 is a newly discovered costimulatory molecule in recent years,which is specifically expressed on T helper 1(Th1)cells.Soluble Tim-3(sTim-3),the soluble protein form of Tim-3,is related to the development of diseases.Tim-3 is also expressed on various innate immune cells,such as monocytes,natural killer(NK)cells and dendritic cells.The overexpression of Tim-3 on these cells has a linkage to the aggravation of diseases.The frequency of NK cells increased significantly in primary biliary cholangitis(PBC),and NK cells are considered as the initiation of multiple autoimmune diseases.Under the pathological conditions,accumulation of NK cells in the liver may be associated with the chemotaxis of CXCR1 on its surface.Tim-3 is involved in the development of a variety of autoimmune diseases,but the mechanism in PBC disease is not clear.Therefore,we established a PBC murine model induced by 2OA-BSA To investigate the expression of Tim-3 and its natural ligand Galectin-9(Gal-9)and observe the changes of serum sTim-3 level.The distribution of NK cells and its surface molecules in PBC were analyzed.Given different treatment,the modulation of Tim-3 is investigated by detacting the surface molecules expression and secretory function of NK cells.Here the upstream regulation mechanism of NK cell chemotaxis is elucidated,which could provide theoretical basis for clinical diagnosis and treatment to find new targets.MethodsPart Ⅰ:Expression of Tim-3 and Gal-9 in primary biliary cholangitis murine model①Establishment of PBC murine model:2-octynoic acid-bovine serum albumin combined with poly I:C was injected intraperitoneally into C57BL/6 female mice to induce PBC murine model.②The serum of mice was collected and the biochemical profiles of liver were detected by automatic biochemical analyzer.③The level of serum sTim-3 and AMA-M2 was detected by ELISA.④The total mRNA of spleen,liver and NK cells in liver of mice was harvested,and the mRNA levels of Tim-3 and Gal-9 were analyzed by qRT-PCR.⑤Liver inflammation and bile duct lesions were observed by histopathology.⑥The expression of Tim-3 and Gal-9 in liver and spleen was investigated by immunohistochemistry.Part Ⅱ:The study of NK cell frequency and subsets in primary biliary cholangitis①Peripheral blood,spleen and liver mononuclear cells were isolated,and then labeled with APC-CD3,PerCP-NK1.1,PE-Tim-3 and FITC-CXCR3 fluorescent antibodies.The distribution of T lymphocytes,NK cells and NKT cells in different tissues aboved was analyzed by flow cytometry.②The proportion of Tim-3+NK cells and CXCR3+NK cells in NK cells fromdifferent sources were analyzed.Part Ⅲ:Study on the modulation of NK cell subsets and function by Tim-3①NK cells from liver and spleen were isolated and purified by NK cell negative Isolation Kit.105 NK cells were seeded in 12 well plates and cultured with a complete culture medium prepared by RPMI 1640,which contained 10%fetal bovine serum,1%penicillin and streptomycin,200U/ml recombined mouse IL-2.②The total mRNA of liver and liver NK cells of mice was harvested,and the mRNA levels of CXCR1 was analyzed by qRT-PCR.③Mononuclear cells isolated from PBC mice liver were cultured and divided into four groups.The control group was treated without intervention,and the other groups were treated with 50μg/ml recombined mouse Tim-3 Fc,recombined mouse Gal-9 protein and lipopolysaccharide respectively.The phrequencies of Tim-3+NK cells and CXCR3+NK cells were analyzed by flow cytometry.④NK cells isolated from C57BL/6 mice spleen were cultured and divided into three groups.The control group was treated without intervention,and the other groups were treated with 50 μg/ml lipopolysaccharide and 50μg/ml lipopolysaccharide combined with recombinant mouse Tim-3 Fc respectively.The total mRNA of NK cells was harvested and the mRNA level of CXCR1 was analyzed by qRT-PCR.The level of IFN-γ in culture supernatant was detected by ELISA.ResultsPart Ⅰ:Abnormal expression of Tim-3/Gal-9 in primary biliary cholangitis①The murine model immunized by 2OA-BSA mimic human primary biliary cholangitis disease,which is characterized by inflammation in portal area and around bile duct,appearance of specific autoantibody AMA-M2 and elevation of serum alkaline phosphatase.②In PBC,the expression of was higher in liver,while no significant difference of Gal-9.However,Gal-9 was highly expressed in the inflammatory area of liver portal area.③Compared with control group,the expression of Tim-3 and Gal-9 increased in spleen,mainly expressing in the white pulp,just a little in the red pulp.④Serum sTim-3 in PBC mice was lower than that in control group.sTim-3 may be involved in the immune regulation of PBC.Part Ⅱ:Changes of NK cell frequency and subsets in primary biliary cholangitis①In PBC models,the phrequencies of T lymphocytes in liver and peripheral blood was significantly increased,but decreased in spleen.②The phrequencies of NK cells from PBC mice liver were significantly increased,but there were no significant difference in peripheral blood and spleen.③Compared with control group,the proportion of NKT cells in peripheral blood and liver of PBC mice was significantly increased.④In PBC,Tim-3+ NK cells in liver and peripheral blood increased,but decreased in spleen.⑤In PBC,CXCR3+ NK cell were the main subsets in peripheral blood.Part Ⅲ:Study on the modulation of NK cell subsets and function by Tim-3①Treatment with recombined mouse Gal-9 or lipopolysaccharide stimulated the expression of Tim-3 on NK cells.②Blockade Tim-3 pathway with recombined mouse Tim-3 Fc or lipopolysaccharide stimulation promoted the expression of CXCR3 on NK cells,while stimulation with recombined mouse Gal-9 inhibited the expression of CXCR3.③In PBC,the level of CXCR1 mRNA in liver and liver NK cells were significantly increased.④Lipopolysaccharide stimulation up-regulate CXCR1 expression on NK cells and induced the secretion of IFN-γ.Combined with Blockade Tim-3 pathway,the expression of CXCR1 and the secretion of IFN-γ were inhibited.Conclusions①Dysfunction of Tim-3/Gal-9 is involved in the pathogenesis of PBC.②The phrequency of T lymphocytes,NK cells and NKT cells infiltrating the liver in PBC diseases was significantly increased,and the population of T lymphocytes and NKT cells from peripheral blood was also significantly increased.③The number of Tim-3+NK cell subsets from peripheral blood and liver increased during PBC,and CXCR3+NK cell(resemble the human CD56bight NK cell population)were the main subsets in peripheral blood.④The expression of CXCRI was up-regulated in liver immune microenvironment and involved in the development of PBC disease.⑤The chemotactic factor receptor refering to the directional migration of NK cells to inflammatory areas are mainly CXCR1 rather than CXCR3.⑥Tim-3 pathway can up-regulate the expression of CXCRI oin NK cells and IFN-γsecretion,while inhibiting the immune regulation of CXCR3.⑦The expression of CXCR3 on NK cells may be related to immune tolerance.