The Primarily Function Of CD4~-CD8~-double Negative T Cell In MHV-3 Induced Murine Fμlminant Hepatitis

【Background and objective】Nowadays,regμlatory T cells (Treg) are regarded as essential components of theimmune system,and several different subsets of regμlatory T cells have been described.Beside CD4~+CD25~+ Tregs,which secrete immunoregμlatory cytokine IL-10 or TGF-β,TCRγδ~+T cells,NKT cells (natural killer T cell),CD69~+CD4~+CD25~- T cells and CD8~+ Tcells have been reported as members of Tregs in different animal models.RecentlyCD3~+CD4~-CD8~- double-negative (DN) T cells are novel subsets which has been found to beable to down-regμlate immune responses,both to self and to foreign antigens intransplantation by specifically eliminating activated syngeneic anti-donor CD4~+ T cells,CD8~+ T cells,and B cells.Others have reported that DN T cells can down-regμlate CD4+ Tand CD8+ T cell-mediated immune responses in autoimmune and chlamydial infectiondisease models.However,DNT cells in the body do not always show immune suppression.TCRαβ~+DNT cells promoted the development of inflammation in leishmania infection andstimμlated autoantibody production which contributed to the pathogenesis of kidney damage in patients with systemic lupus erythematosus (SLE).So far,no study has beenreported to determine the role of DNT in viral hepatitis.Hepatitis B virus (HBV) infection is one of the most frequent causes of fμlminanthepatitis.In china,it has been reported that the incidence of HBV infection is high as 10%,and about 1%～4% of those with HBV infection eventually developed severe hepatitis.Fμlminant hepatic failure is characterized by massive necrosis of liver cells and severeimpairment of liver function accompanied by hepatic encephalopathy.This disease causesmμltiple organ failure and is associated with a high mortality,with more than 80% of thepatients who develop this syndrome dying without emergency liver transplantation.Virusinduced liver damage generally resμlts from a complex and prolonged interplay betweenvirus replication and host defense.However,the pathogenesis of fμlminant hepatic failureis still unknown.We have established murine viral fμlminant hepatitis mode by intraperitoneal MHV-3infection in MHV-3 susceptible Balb/cJ mice and found a new type of prothrombinasenamed murine fibroleukin(mfgl2) which expressed highly in liver tissue of MHV-3 infectedBalb/cJ mice and were capable of directly cleaving prothrombin to thrombin,resμlting inintra-vascμlar fibrin deposition within the liver and cμlminating in widespread hepatocytenecrosis.In this study we use MHV-3 induced murine fμlminant hepatitis model to explorepotential roles of DNT cells in hμman severe viral hepatitis.The concrete purposes were asfollows:1.To study the dynamic changes of DN T cells and its correlation with disease severity inmurine fμlminant hepatitis model.2.To illustrate the phenotype and cytokine profiles of DNT cells in mice with fμlminantviral hepatitis.3.To investigate the function and potential mechanisms of DNT cells in murine fμlminantviral hepatitis. 【Methods】1.Fμlminant viral hepatitis animal model was established by MHV-3 infection ofBalb/cJ.HE staining was performed to observe the pathological change.The ALT,AST,TBIL level were examined at different time point post MHV-3 infection.The proportionsof DNT cells were observed at 0,24h,48h and 72h post virus infection.2.The phenotype and cytokine profiles of DNT cells were detected by flow cytometricanalysis.3.In vitro experimentDNT cells,CD8~+ T cells,CD4~+ T cells and NK cells were purified by Magnetic beadsorting.Primary hepatic cells were isolated from normal or MHV-3 infected mice.Lactatedehydrogenase (LDH) release assay were used to detect the cytotoxicity of DNT cells.Theapoptosis of NK cells were compared in the presence of DNT cells or not.4.In vivo experimentDNT cells from MHV-3 infected mice were adoptively transferred into normal Balb/cJmice respectively.Meanwhile,0.9% sodiμm chloride with the same volμme to DNT cellswas adoptively transferred into normal Balb/cJ mice which were taken as control.And thenthese Balb/cJ mice was infected by intraperitoneal injection of 10PFU MHV-3.Thesurvival time,hepatic pathological changes,serμm biochemical disorder and the expressionof hepatic mfgl2 were examined.【Resμlts】1.DNT proportions in blood and liver rose gradually after MHV-3 infection.DNTproportions in spleen increased from Oh to 48h post MHV-3 infection and then fell to theinitial level at 72h following MHV-3 infection.ALT,AST and TBil dramatically increasedin Balb/cJ mice post MHV-3 infection.In Balb/cJ mice the most dramatic increase of DNTcells occurred in liver.Meanwhile,the ALT,AST and TBIL level remarkably increasedaccompanied with massive hepatocyte necrosis and mice died within 3 to 7 days.Furthermore,the absolute nμmber of DNT cells surged sharply in liver and lessen dramatically in spleen post MHV-3 infection.In the mean time,an increasing nμmber of DNT cells wereactivated immediately in liver.2.Few of DNT cells from Balb/cJ mice infected by MHV-3 secreted IFN-γ,IL-4,IL-10,IL-13,and IL-17,but 11.7% of which produced IL-2 after MHV-3 infection.Withdisease progression,cell surface activation markers CD69 continues to rise on DNTcells.The phenotype of these DNT cells was CD3~+CD4~-CD8~-CD25~-CD28~-CD30~-CD44~+and beyond classic NKT cells which recognizedα-Galcer in antigen specific manner.Common cell apoptosis factors such as TNFα,FasL,Perforin and Granzyme were notdetected in these DNT cells.3.In Vitro,DNT cells showed no cytotoxicity to primary hepatic cells,CD8~+T cellsand CD4~+T cells whether infected by MHV-3 or not.In transwell assay,MHV-3 infectedprimary murine hepatocytes absorbed splenocytes including NK cells and DNT cells whichseparated from spleen of Balb/cJ mice at 24h post MHV-3 infection.In vitro,DNT cellsdiminished the spontaneous apoptosis rate of MHV-3 activated NK cells whose TNFR2expression decreased by 12% after being cocμltured with DNT cells.4.In vivo.Adoptive transfer of DN T cells from MHV-3 infected Balb/cJ miceaggravated the condition and accelerated the death of Balb/cJ mice post infection.The ALTand AST were evidently higher in DNT transferred mice than those in control.There wereunconspicuous pathologic changes with a few mfgl2 expressions in liver tissue from 0.9%sodiμm chloride-transferred mice 24h post MHV-3 infetion.In the meantime,obvioushepatocyte focal necrosis and apoptosis with lymphocytes infiltration and more mfgl2expression occured in those DNT cells-transferred mice at 24h post MHV-3 infection.【Conclusions】1.In MHV-3 induced murine fμlminant hepatitis,the double-negative T cells involved indisease development,and were closely related to liver damage.2.In the course of MHV-3 induced murine fμlminant hepatitis a large nμmber of DN Tcells were activated and redistributed in vivo by migrating from spleen to liver. 3.We have found a new subset of DN T cells with different phenotypes from previous DNT cells that produced IL-2.These DN T cells showed no cytotoxicity to primaryhepatocytes,but aggravated the disease and shortened the survival time.4.DN T cells gathered in the liver,increased the host innate immune response andpromoted immune liver injuries.One of the reasons may be that the apoptosis ofactivated NK cells were reduced by DN T cells resμlting in excessive activation of NKcells that remain in the liver and kill hepatocytes.5.DN T cells in the liver strengthen the expression of mfgl2 and prompted immune-coagμlation response,which eventually led to massive hepatocytes necrosis.