Impact Of Circadian Rhythm Disturbance On T Cell Function And HBV Clearance In Mice

ObjectivesCircadian rhythm disturbance of C57BL/6 mice was achieved by changing the zeitgeber time,so as to explore the impact of circadian rhythm disturbance on T cell functions of these mice.Besides,HBV transfected mouse model was constructed through hydrodynamic injection of pAAV/HBV1.2 plasmid,so as to observe whether circadian rhythm disturbance also influence the clearance of HBV.Materials and methods1.The 5-week old male C57BL/6 mice adjusted the environment for one week.Then,these mice were divided into four groups randomly,including advanced circadian rhythm group,delayed circadian rhythm group,reversed circadian rhythm group,and normal circadian rhythm group.Mice in these groups experienced different circadian rhythm regulation or just keep in the normal circadian rhythm.2.When experienced above circadian rhythm patterns respectively for two weeks,four mice were randomly sacrificed in each group.Then lymphocytes in livers and spleens of these mice were separated,while associated immune cell frequencies were detected through flow cytometry.3.When experienced above circadian rhythm patterns respectively for two weeks,transfection of HBV in all of the rest mice were achieved via hydrodynamic injection of pAAV/HBV1.2 plasmid.Blood samples were collected from ophthalmic vein at different time points,then serum were separated and levels of HBsAg,HBsAb,HBeAg,HBeAb and HBcAb were tested.4.Data from flow cytometry were analyzed through FlowJo 10 software,and statistical analysis were conducted by SPSS 20.0 software,while tables and figures were made via Graphpad prism 7 software.Results1.Compared with C57BL/6 mice kept in normal circadian rhythm,CD4~+T cell frequencies both in liver and spleen,CD8~+T cell frequencies in spleen,as well as CD4~+CD44~+CD62L~-T cell frequencies both in liver and spleen decreased in mice from advanced,delayed,and reversed circadian rhythm groups.However,CD8~+T cell frequencies in liver of these mice increased.2.Compared with mice kept in normal circadian rhythm,CD4~+TNF-α~+T cell and CD8~+TNF-α~+T cell frequencies both in liver and spleen increased in mice from advanced,delayed,and reversed circadian rhythm groups.Meanwhile,CD8~+IFN-γ~+T cell frequencies increased in liver of mice from advanced and delayed circadian rhythm groups.What’s more,CD4~+Eomes~+T cell,CD8~+Eomes~+T cell,CD4~+Tbet~+T cell and CD8~+Tbet~+T cell frequencies increased in liver of mice from advanced,delayed,and reversed circadian rhythm groups.3.Compared with mice kept in normal circadian rhythm,CD4~+CD107a~+T cell and CD8~+CD107a~+T frequencies increased in liver of mice from advanced circadian rhythm group.While CD4~+CD107a~+T cell and CD8~+CD107a~+T cell frequencies decreased in liver of mice from delayed and reversed circadian rhythm groups.Besides,both CD4~+CD107a~+T cell and CD8~+CD107a~+T cell frequencies decreased in spleen of mice from advanced,delayed,and reversed circadian rhythm groups.4.Compared with mice kept in normal circadian rhythm,the clearance rate of HBsAg and HBeAg reduced in mice from advanced,delayed,and reversed circadian rhythm groups,the production of HBsAb was also delayed accordingly in these mice.However,the levels of HBeAb and HBcAb were not significantly affected.Conclusions1.Advanced,delayed,and reversed circadian rhythm decreased CD4~+T cell frequencies both in liver and spleen,CD8~+T cell frequencies in spleen,as well as CD4~+CD44~+CD62L~-T cell frequencies both in liver and spleen of C57BL/6 mice.Although T cells,which expressed proinflammatory cytokines such as TNF-α,INF-γ,Eomes and Tbet,increased in livers of mice.However,CD4~+CD107a~+T cell and CD8~+CD107a~+T cell frequencies both in liver and spleen decreased.This indicated that circadian rhythm disturbance apparently impact both T cell frequencies and functions in mice.2.Advanced,delayed,and reversed circadian rhythm prevented the clearance of HBV in HBV transfected mice.Variation of T cell functions caused by circadian rhythm disturbance may be involved in the delayed clearance of HBV,while the specific mechanisms need more researches.