The Regulatory Role Of CD24 For Hepatic NKT In Acute Liver Injury

Objects and contents:This work was performed to study the regulation of CD24 in hepatic NKT cells in acute liver injury and inflammation,including cell number,activity and expression of cytokines.This study had three sections.Firstly,we detected wether WT and CD24knock-out(CD24^-/-)mice had different hepatic NKT cell number in normal physiological state.Secondly,we established the acute liver injury mouse model induced by Concanavalin A?Con A?.We compared the degree of liver inflammation,the cell number and activity of hepatic NKT in acute liver injury induced by Con A in WT and CD24^-/-mice.Thridly,intraperitoneal injection of ?-GalCer??-Galactosylceramide?to activate NKT cells,we detected the degree of liver inflammation and difference between WT and CD24^-/-mice and the cell number and activity of hepatic NKT cells.Methods and results:This study can be divided into three sections:Section One: Comparison the number of NKT cell between WT and CD24^-/-mice in normal physiological state,including two experiments:1.Identification of CD24^-/-miceWe detected the expression of CD24 on cells in blood sample or splenocytes by Flow Cytometry.Results showed that CD24 cann't be detected in CD24^-/-mice.We bred CD24^-/-mice normally.2.Regulation of CD24 in hepatic NKT cells in normal physiological state emmWe detected hepatic NKT cell number in normal physiological state with Flow Cytometry in WT and CD24^-/-mice.Results showed that NKT cell was significantly decreased in CD24^-/-mice which indicated that CD24 may play an improtant role on liver NKT cell.Section Two: In acute liver injury induced by Con A,we detected the degree of liver injury and inflammation,difference of cell number and activity of NKT in WT and CD24^-/-mice.1.The regulatory role of CD24 in Con A induced acute liver injuryWT and CD24^-/-mice were injected respectively with Con A?10mg/kg per mouse?through tail vein.After 8-10 hours,we detected the level of ALT in mouse serum and the degree of liver injury and inflammation with H&E staining.We established acute liver injury induced by Con A in mice.The degree of liver injury and inflammation in CD24^-/-mice was significantly severe than in WT.It indicated that CD24 might serve as a protective role in acute liver injury.2.Regulation of CD24 in NKT cell in acute liver injury induced by Con A?1?CD24 regulated the number of hepatic NKT cell in acute liver injuryWe detected the change of hepatic NKT cell number in acute liver injury by Flow Cytometry,and compared the number in total NKT cells and its subsets,including NKT1,NKT2 and NKT17.Results showed that the number of hepatic NKT cell was increased in mouse treated with Con A.NKT1 accounted for the largest proportion of NKT cells and changed most significantly in liver.The number of NKT1 was increased,however,NKT2 and NKT17 were decreased in CD24^-/-mice compared with WT.The number of NKT cells was measured immediatedly by immunofluorescence staining with mouse monoclonal antibody CD1d: ?-GalCer Dimer.Results showed that liver NKT cells were increased treated with Con A especially in CD24^-/-mice.?2?CD24 regulated the activity of hepatic NKT cell in acute liver injuryThe expression of cytokines and surface markers of NKT cells and liver tissue were detected by intracellular staining and Real-time PCR,including CD69,TNF-?,IFN-?,IL-4 and PLZF?zbtb16?.Results showed that the expression of cytokines and surface markers were significantly increased in CD24^-/-mice liver treated with Con A.Section Three: we detected the activation of NKT cells after injecting ?-GalCer and compared the degree of liver inflammation,cell number and activity of hepatic NKT in WT and CD24^-/-mice.1.The regulatory role of CD24 in ?-GalCer induced liver inflammationMice was treated with ?-GalCer i.p.?2?g per mouse,12h?,we detected thedifference of degree of liver inflammation in WT and CD24^-/-mice with H&E staining.The level of serum ALT was also detected.Results showed that inflammatory cell infiltration and serum ALT were decreased significantly in CD24^-/-mice which indicated liver inflammation relieved in CD24^-/-mice.2.The regulatory role of CD24 in NKT cell in ?-Gal Cer induced liver inflammation?1?CD24 regulated the number of hepatic NKT cell in liver inflammationTreated with ?-GalCer,we detected the number of hepatic NKT cell and its subsets,like NKT1,NKT2 and NKT17.Results showed that the number of hepatic NKT cell was significantly increased in mouse treated with ?-GalCer.However,no matter in normal physiological state or after activation,both liver total NKT cell and the subsets were decreased in CD24^-/-mice.The number of NKT cell was detected by immunofluorescence staining.Results showed that hepatic NKT cell was decreased in CD24^-/-mice which in consistent with results detected by Flow Cytometry.?2?CD24 regulated the activity of hepatic NKT cell in liver inflammationExpression of cytokines and surface markers in hepatic NKT cells and liver tissue were detected by intracellular staining and Real-time PCR,including CD69,TNF-?,IFN-?,IL-4 and PLZF?zbtb16?.Results showed that expression of CD69,TNF-?,IFN-?,IL-4 and PLZF was decreased in CD24^-/-mice.Conclusion:1.CD24^-/-mice are bred normally.In normal physiological state,hepatic NKT cell is decreased in CD24^-/-mice.2.In acute liver injury induced by Con A,CD24^-/-mice exhibit more severe liver injury and inflammation,hepatic NKT cell number,especially NKT1,and expression of inflammatory cytokines in liver tissue are increased significantly.3.In acute liver injury induced by ?-GalCer,liver injury and inflammation is alleviated,the number of hepatic NKT cell,especially NKT1,and expression ofinflammatory cytokines in liver tissue are decreased in CD24^-/-mice.4.Different mechanisms may exist in CD24 regulates NKT cells in acute liver injury and in its own development.