A Study Of The Immunogenetic Pathogenesis Of Primary Biliary Cholangitis

BackgroundMAIT cells（Mucosal-Associated Invariant T cell,MAIT）,an indispensable member of human innate immunity,defend against bacterial and viruses and participate in inflammatory biological processes and alergic reactions,etc.MAIT cells also contribute to the military line of immune defense.Of note,MAIT cells get involved in autoimmunity^[1-7],malignancy homeostasis^[8,9],and emerging notorious SARS-COV-2^[10-13],liver diseases^[14-17],and other sicknesses.MAIT cells exist in abundance in humans,represent up to ten percent of total T cells in blood^[18,19],and forty-five percent of liver T cells^[20].Emerging studies suggest MAIT cells play an indispensable role in liver homeostasis and liver diseases.Alcoholic liver disease（ALD）patients liver MAIT cells decrease significantly and are accompanied by severe bacterial infection^[21].In alcoholic liver cirrhosis patients,MAIT cells both remarkably decreased in the peripheral and liver,studies in vivo and in vitro both demonstrate that MAIT cells could promote fibrosis^[22]The number of circulating MAIT cells in patients with NAFLD（Non-Alcoholic Fatty Liver Disease）decreases markedly,while the liver MAIT cells increase,and liver MAIT cells are positively correlated with the NAFLD activity score^[23].Autoimmune liver diseases include PBC（Primary Biliary Cholangitis）,PSC（Primary Sclerosis Cholangitis）,AIH（Autoimmune Hepatitis）,and any overlap syndrome between the three diseases.MAIT cells get involved in chronic hepatitis B virus^[24-26],hepatitis C virus^[27-30]infection autoimmune hepatitis^[5],hepatocellular carcinoma^[9],PBC^[31],and liver fibrosis^[22],and act as an important role.ObjectiveTo explore the potential role of CD3⁺ CD8⁺ CD161 high TCR Vα7.2⁺ MAIT cells in the pathogenesis of Primary Biliary Cholangitis.MethodsWe enrolled 55 PBC patients,69 HCs（healthy controls）,and eight hepatic hemangioma patients.Circulating MAIT cells and their chemokine receptor profilles and cytokine production were quantified using flow cytometry.Liver-resident MAIT cells were examined by immunofluorescence staining.CXCL12-mediated chemotaxis of MAIT cells was measured using a transwell migration assay.Plasma IL-18 was measurec using ELISA,and cytokine production in IL-18-stimulated MAIT cells was detected using flow cytometry.Results1.Peripheral MAIT cells in PBC patients decreased significantly than HCs（3.0±3.2%vs.9.4±8.0%,p<0.01）,and were negatively correlated with alkaline phosphatase（r=-0.3209,p<0.05）.2.MAIT cells in PBC liver were more abundant than controls.3.PBC MAIT cells expressed higher levels of CXCR4 than HCs（84.8±18.0%vs.58.7±11.4%,p<0.01）4.CXCL12,the ligand of CXCR4,demonstrated higher in PBC liver.5.CXCL12 promoted MAIT cell chemotaxis in PBC than HCs（70.4±6.8%vs.52.±3.5%,p<0.01）,and blocking CXCR4 could attenuate chemotaxis.6.PBC MAIT cells produced more IFN-y（88.3±4.2%vs.64.2±10.1%,p<0.01）,TNF-α（93.0±1.1%vs.80.1±5.3%,p<0.01）,Granzyme B（89.3±3.3%vs.72.1±7.0%,p<0.01）and perforin（46.8±6.6%vs.34.8±7.7%,p<0.05）.7.PBC MAIT cells expressed higher levels of IL18-Rα（83.8±10.2%vs.58.3±8.7%,p<0.01）.Plasma IL-18 levels in PBC patients were higher than those in the HCs（286.8±75.7pg/mL vs.132.9±78.1pg/mL,p<0.01）.8.IL-18 could promote the production of IFN-γin PBC MAIT cells（74.9±6.6%vs 54.7±6.7%,p<0.01）,and blocking IL-18 R attenuated the production of IFN-γ（68.6±8.3%vs.43.5±4.2%,p<0.01）.ConclusionMAIT cells from PBC patients accumulated in the liver via CXCL12-CXCR4-mediated chemotaxis,produced proinflammatory cytokines,and contributed to portal inflammation,which was potentially mediated by the elevated IL-18.Targeting MAIT cells might be a therapeutic approach for PBC.BackgroundPrimary biliary cholangitis is an autoimmune disease,characterized by chronic intrahepatic cholestasis,female predominance,imbalance of self-antigen immune tolerance.Serological detection suggests antimitochondrial antibodies can be found positive in more than 90%of patients with PBC.Patients with PBC usually exhibit a significant rise in alkaline phosphatase（ALP）,with or without gamma-glutamyl transferase（GGT）abnormally elevated.PBC is highlighted by chronic lymphocytic non-suppurative granulomatous infiltration and destruction of small hepatic bile ducts.Multiple lymphocyte populations including CD38⁺B cells,CD3⁺T cells,CD4⁺T cells,CD8⁺T cells,NK cells,NKT cells,and macrophages are involved in the pathological injury of PBC.In the preliminary study,we found many susceptibility genes through whole-exome sequencing in two PBC families of the Chinese Han population.After eliminating synonymous mutations and mutations in non-coding regions,we focused on the missense,deletion,and frameshift mutations,and predicted the harmfulness of these genes through bioinformatics analysis and found out candidate co-segregation genes.Finally,we decided to produce out PTK2B knock-in C57BL/6J mice using CRISPR/Cas9 technology to explore whether PTK2B could lead to the spontaneous development of PBC in mice,and explore its potential mechanism contributing to PBC.ObjectiveAfter succeeded in producing out PTK2B Knock-in C57BL/6J mice,this study aimed to explore whether PTK2B Knock-in mice spontaneously developed into abnormality resembling PBC in serology and or histology in a specific pathogen-free environment We observed whether different genotypes（WT/WT,WT/KI,KI/KI）mice exhibited PBC performance,and it depended that further explore the potential mechanism of the PTK2B gene on the pathogenesis of PBC.MethodsThe susceptibility gene of the co-segregation gene PTK2B was previously screened in PBC families by whole exon sequencing technology,and PTK2B Knock-in mice were successfully produced through CRISPR/Cas9 technology.Immunohistochemistry（marked small intrahepatic bile duct CK7,CK19）and H&E staining techniques were used at different ages of different groups to evaluate histology changes among different organs and tissues,including liver,kidneys,heart,spleen,lung,intestine,pancreas,bladders,and further explore subsets of the liver infiltrating cells and molecules expression.Hepatic fibrosis in mice was evaluated by Sirius scarlet staining.The expression levels of anti-nuclear antibodies,anti-mitochondrial antibodies,and changes of liver function indexes were detected,and the expression levels of related cytokines were examined using ELISA.The immunophenotype of peripheral mononuclear cells,spleen cells,and liver cells was evaluated through flow cytometry.A comprehensive conclusion should be made based on the changes of histopathology,serum autoantibodies,cytokines,and liver function indexes to decide whether the mice resembled PBC performance,and potentially explored its pathogenesis.Results1.Serum ANAs level of female C57BL/6J KI/KI mice of 8 weeks was significantly higher than that of WT/KI group（261±35 vs.159±35 U/mL,p<0.05）,but no significant difference found between WT/KI group and WT/WT group（159±35 vs.129±43 U/mL,p>0.05）.2.Serum AMAs level of female KI/KI mice of 12 weeks was significantly higher than that of WT/KI group（3,924±769 vs.1,972±632 U/mL,p<0.05）.The level of AMAs of mice in WT/KI group was not significantly different from that in WT/WT group（1,972±632 vs.1,939±379 U/mL,p>0.05）.As for serum AMAs level in males,no significant differences were found between KI/KI and WT/KI group（3,087±1,532 vs.1,549±588 U/mL,p>0.05）.Comparable serum AMAs levels between male WT/KI group and WT/WT group（1,549±588 vs.1,703±474 U/mL,p>0.05）.3.Female KI/KI mice had obvious intrahepatic bile duct hyperplasia and liver-specific lymphocyte inffiltration,while male KI/KI mice had no significant changes.4.Serum ALP level of female KI/KI mice was significantly higher than that of WT/KI group（236±55 vs.142±43 U/L,p<0.05）,and also higher than that of WT/WT group（236±55 vs.121±37 U/L,p<0.05）.The serum ALP level of WT/KI group and WT/WT group was comparable（142±43 vs.121±37,p>0.05）.5.Immunological abnormalities in liver,spleen,lymph nodes and peripheral blood of female KI/KI mice.The CD19+B cells（44.7±1.7%vs.32.6±2.2%,p<0.05）,CD3+T cells（50.8±1.7%vs.38.9±3.2%,p<0.05）,CD8+ cells（14.3±2.8%vs.7.5±1.3%,p<0.05）and NK cells（MFI:586.1±64.6 vs.469.1±18.6,p<0.05）infiltrated in the liver of female KI/KI mice were significantly higher than those in WT/WT group.Compared with female WT/WT mice,the number of CD3⁺ T cells,CD3⁺ CD4⁺cells,CD3⁺ CD8⁺ cells and CD19⁺ B cells in spleen of female KI/KI mice increased significantly（32.2±6.5%vs.19.6±5.8%,p<0.05）,（23.7±4.9%vs.16.5±3.6%,p<0.05）and（10.6±1.5%vs.6.2±1.6%,p<0.05）,（70.1±9.7%vs.57.8±6.2%,p<0.05）,respectively.Compared with female WT/WT mice,about lymph nodes of female KI/KI mice,CD3⁺ T cells,CD3⁺ CD4⁺cells and CD3⁺ CD8⁺ cells decreased significantly（28.4±3.1%vs.73.1±0.7%,p<0.05）,（12.8±1.2%vs.42.0±3.2%,p<0.05）and（12.3±3.1%vs.28.8±0.7%,p<0.05）,respectively,while CD19+ B cells increased significantly（69.8±3.3%vs.26.4±2.3%,p<0.05）.Compared with female WT/WT mice,CD3+T cells in peripheral blood mononuclear cells of female KI/KI mice decreased significantly（57.6±3.5%vs.78.7±3.3%,p<0.05）,while CD19⁺ B cells increased significantly（40.6±3.6%vs.20.1±3.7%,p<0.05）.CD3⁺ CD4⁺ cells decreased significantly（29.3±1.9%vs.45.1±2.4%,p<0.05）,and CD3⁺ CD8⁺ cells decreased significantly（25.6±2.2%vs.31.3±0.9%,p<0.05）.6.The level of serum MCP-1 in female KI/KI mice was significantly higher than that in WT/WT mice（2,209±412pg/mL vs.1,187±89pg/mL,p<0.05）.7.PTK2B mediated the infiltration of liver lymphocytes in female KI/KI mice,and CD38+B cells performed an indispensable role in the pathogenesis of PBC.The infiltrated cells in the liver of female KI/KI mice include CD20⁺B cells,CD3⁺T cells,CD4⁺cells,CD8⁺cells,F4/80⁺ macrophages,and Ly6G+granulocytes.A huge amount of CD38⁺ CD20⁺ B cells infiltrated around the portal vein area in PBC mice,contributing to the imbalance of liver homeostasis.8.CD38⁺ CD20⁺ B cells enriched in the portal of bile duct of female KI/KI mice.9.In female KI/KI mice,CCR2⁺cells in peripheral blood decreased significantly（33.8±12.8%vs.4.8±6.5%,p<0.05）,while CCR2⁺cells in the liver increased significantly（31.7±6.2%vs.20.4±2.2%,p<0.05）.CCR2⁺ CD20⁺cells of female KI/KI mice were supposed to be primary infiltrated lymphocytes.ConclusionThe co-segregation gene PTK2B knocked-in C57BL/6J mice spontaneously exhibited liver-specific PBC-like performance,serology abnormalities,and histological changes with female predominance.The serum autoantibodies ANA and AMA were positive,and ALP was abnormally elevated in KI/KI mice group.It was in good agreement with clinical PBC performance.Besides,a significant increase in liver CD38⁺ CD20⁺and CCR2⁺ CD20⁺B cell infiltration involved in the pathogenesis of PBC in PTK2B knock-in mice.