Chronic Alcohol Consumption Inhibits CD8~+T Cell Immune Response Against Hepatitis B Virus

Objective In this study,HBV-carrier mice were created by hydrodynamic injection of p AAV/HBV1.2 plasmid via tail vein,and Lieber-De Carli alcohol or a control liquid diet was used to establish a mouse chronic alcoholic liver model.We explored the effects of alcohol on hepatitis B virus(HBV)and CD8~+T cell anti-HBV immune response,and further study the immunomodulatory effects of NK cells on CD8~+T cell and then provide some research evidence for pathogenesis and immune-targeted therapy in HBV.Methods Male C57BL/6J mice kept under specific pathogen-free condition.The mouse models were divided into 6μg HBV tolerance model and 20μg HBV acute infection mouse model,the difference of two models were the amount of plasmid injection.The mice were adapted for 3 days,they were randomly divided into control groups(HBV mice),the mice were injected with HBV1.2 plasmid(6μg or 20μg)into the tail vein and fed with control liquid diet;experimental group(1)(HBV+5%Et OH mice),mice were injected with HBV1.2 plasmid and fed alcohol liquid diet containing 5%(v/v)alcohol;and experimental group(2)(HBV+5%Et OH+anti-As GM1 mice),mice were injected with HBV plasmid 1.2 and fed 5%Alcohol,while intraperitoneal injection of 50μg anti-As GM1 antibody targeted to eliminate NK cells in mice to explore the role of NK cells in the immune response of mice against HBV.Modeling for 10 days,the weight of the mice and the consumption of liquid feed were recorded daily.Immunoradioanalysis(IRMA)was used to quantitatively detect mouse HBV surface antigen(HBs Ag),Q-PCR was used to detect the copy number of HBV DNA and RNA,and HBV core antigen(HBc Ag)in liver tissue was detected by immunohistochemistry,thereby understand the changes in the state of HBV in mice.Liver pathology methods were used to assess the severity of liver steatosis in mice after alcohol consumption.The phenotype and function of mouse liver mononuclear cells(MNCs)were studied by flow cytometry.In addition,the metabolism in mouse liver tissues was detected based on ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry(UHPLC-QTOFMS).Results Long-term intake of alcohol promoted HBV to stay longer in mice,which was manifested as the number of copies of HBV genome in the liver was significantly higher than that in the control group.In the HBV tolerance model induced by high-pressure injection of 6μg HBV1.2 plasmid,long-term alcohol intake can induce a significant decrease in the number of CD8~+T cells in the liver of HBV mice,and a significant increase in the expression of the negative regulatory factor LAG-3 on the surface of CD8~+T cells.It is manifested in a state of immune function exhaustion.Further mechanisms have found that NK cells play an important role in maintaining the state of CD8~+T cell exhaustion,and depletion NK cells can significantly restore part of the CD8~+T function.In the model of acute HBV infection in mice induced by high-pressure injection of 20μg HBV1.2 plasmid,long-term alcohol intake induces the ability of CD8~+T cells in the liver of HBV mice to secrete interferon gamma(IFN-gamma)weakened,and the number the of effector CD8~+T cells(CD44~+CD62L~-)has dropped significantly.In order to further verify the effect of alcohol on the function of CD8~+T cells,we transferred the same number of CD8~+T cells from the two groups of mice to immune-deficient HBV-carrier mice to observe the HBV clearance ability of the donor immune cells.The results showed that CD8~+T cells derived from mice fed alcohol for a long time had a significantly weakened ability to clear HBV in recipient mice,which was manifested by more HBV-related genome copies and higher serum HBs Ag content.In order to further study the immune response of NK cells to HBV in mice,we eliminated NK cells in HBV acutely infected mice with alcohol consumption,and found that the elimination of HBV-related genes was accelerated.Meanwhile,alcohol increased the proportion of liver-resident NK cells in HBV acutely infected mice and decreased the level of conventional NK cells.Meanwhile,the ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry method was used to detect multiple sets of different metabolites in the liver tissues of the two groups of mice,result shows ester and ribose reserves were significantly reduced,and cysteine,which helps synthesize glutathione(GSH),was significantly reduced,which may aggravate oxidative stress and reactive oxygen species(ROS)levels in most cells.Conclusion The long-term intake of alcohol induces the exhaustion of CD8~+T cell function and inhibits the anti-HBV immune response of CD8~+T cell,thereby increasing the viral load of HBV in mouse liver.At the same time,the elimination of NK cells by antibodies can promote the ability of mice to eliminate HBV from the liver.The mechanism may be related to the negative immune regulation of the liver--resident NK on CD8~+T cells induced by alcohol.In addition,the down-regulation of energy metabolism and the up-regulation of oxidative stress may also contribute to the failure of CD8~+T cells.