| 【BACKGROUND&OBJECTIVE】Viral hepatitis refers to the acute and chronic liver diseases induced by hepatotropicviruses. Viral hepatitis is characteristics with continuous liver injury. It is generally thoughthepatitis viruses themselves are not cytopathogenic and do not kill hepatocytes. Previousstudies have extensively reported the centrol role of activity of T cell-mediated immuneresponses, include CD4~+T cells, CD8+T cells, natural killer T (NKT) cells, and regulatoryT cells (Treg cells), in induction of hepatocellular injury during viral infection. Recentreports have shown that non-specific immune system do play roles in induction ofhepatocellular injury during viral infection. In mouse model of fulminant hepatic failure(FHF) induced by murine hepatitis virus strain3(MHV-3), activated macrophage producedproinflammatory mediators such as TNF and IL-1as well as the unique procoagulantactivity fgl2prothrombinase, whic subsequent exacerbate the liver micro-environment andled to hepatocyte apoptosis and necrosis. Zou Y et al demonstrated that NK cells viaFas/FasL and NKG2D/NKG2D ligand pathway induced hepatocyte injury in patients withFHF and acute-on-chronic liver failure (ACLF).A small population of CD4-CD8-(double negative, DN) T cells has been shown to beinvolved in several diseases, including autoimmune diseases, immunodeficiency diseases, infectious disease, and tumor. In both mice and humans, these cells compose about1-5%ofall peripheral T lymphocytes, and express a specific set of cell surface molecules and acharacteristic cytokine profile. In mice infected with Francisella tularensis live vaccinestrain, lung DN T cells were found to expand into two subsets that produced two essentialcytokines, IFN-γ and IL-17A. TCRγδ~+DN T cells from Leishmania-infected individualspresent a regulatory profile, producing immunoregulatory cytokine IL-10.Interestingly, DN T cells have also been reported in the livers of healthy humans andmice and those with diseases. Norris et al. demonstrated that DN T cells ranged from2.7to29%of total hepatic T lymphocytes in healthy humans. Seki et al. reported that the liverwas probably an important organ for activation and expansion of TCRγδ~+DN T cells inhumans and mice, especially under tumor-bearing conditions. In the portal areas of livercirrhosis patients, the majority of the γδ+T cells were TCRγδ~+DN T cells. These findingssuggest a potential role for liver DN T cells in the pathological process of liver disease.However, very little information about the potential role of DN T cells in viral hepatitis hasbeen reported.In this study we use MHV-3induced murine hepatitis model to explore potential rolesof liver DN T cells in viral hepatitis. Therefore the purposes of this study are as the follows:1. To examine the proportions of TCRγδ~+DN T cells and TCRαβ+DN T cells in liver DNT cells in MHV-3-induced hepatitis mice model.2. To illustrate the phenotype and cytokine profiles of liver TCRγδ~+DN T cells in MHV-3induced fulminant hepatitis mice model.3. To investigate the effect of of liver TCRγδ~+DN T cells on hepatocytes and its potentialmechanisms in MHV-3-induced hepatitis mice model.【METHODS】1. Viral hepatitis animal model was established by MHV-3infection of C3H/HeJ. Theproportions of TCRγδ~+DN T cells and TCRαβ+DN T cells in liver DN T cells inMHV-3-induced hepatitis mice model was examined. The proportions and number ofliver TCRγδ~+DN T cells were observed on day0,3,7,10,15,20, and35post virus infection.2. The phenotype and cytokine profiles of liver TCRγδ~+DN T cells were detected by flowcytometric analysis.3. In vitro experimentLiver TCRγδ~+DN T cells were purified by Magnetic bead sorting. Primary hepatic cellswere isolated from normal or MHV-3infected mice. Lactate dehydrogenase (LDH)release assay were used to detect the cytotoxicity of liver TCRγδ~+DN T cells oninfected hepatocytes or uninfected hepatocytes. A Transwell experiment was performedto determine whether the cytotoxicity required cell-cell contact. The cytotoxic effects ofliver TCRγδ~+DN T cells on infected hepatocytes were examined in the presence ofthree neutralizing Abs (anti-TNF-α, anti-IFN-γ, anti-IL-17A mAbs) or not.4. In vivo experimentLiver TCRγδ~+DN T cells from MHV-3infected C3H/HeJ mice (3×106cell/mouse)were adoptively transferred to C3H/HeJ mice5days post MHV-3infection. PBSinjected into mice5days post MHV-3infection was used as control group. Thesurvival rates, hepatic pathological changes, serum ALT and AST levels of two groupswere examined.【RESULTS】1. Predominant population of DN T cells in the liver of healthy or MHV-3-infected miceexpressed TCRγδ+. The proportion and number of TCRγδ~+DN T cells in the liverincreased markedly and peaked on day10post infection, compared with normalcontrols (19.7±3.22%vs.6.38±1.1%,6.51±1.29×105vs.0.76±0.11×105), and remainedhigh thereafter.2. The surface phenotype of liver TCRγδ~+DN T cells was as TCRγδ+CD3~+CD4~-CD8~-CD25-CD28-CD30-CD44+. TCRγδ~+DN T cells did not recognize α-Galcer,demonstrating that these cells differed from NKT cells. The proportion of liver TCRγδ+DN T cells expressing CD69increased significantly post MHV-3infection, peaked onday10(83.45±5.32%), and then decreased gradually. These cells revealed a classicalinflammatory cytokine profile, with high production of TNF-α, IFN-γ, IL-17A and IL-2, but no IL-4, IL-10, IL-21or IL-22was observed.3. In Vitro, infected liver TCRγδ~+DN T cells had cytotoxic effect on infected hepatocytes.In transwell assay, the cytotoxic effect was still observed at a comparable level to thatobserved without the Transwell. TCRγδ~+DN T cell cytotoxicity decreased markedlywhen TNF-α was blocked, but neither neutralizing IFN-γ nor IL-17A alone was able toinhibit TCRγδ~+DN T cell-mediated cytotoxicity.4. In vivo, adoptive transfer of liver TCRγδ~+DN T cells led to a dramatic decrease insurvival of infected mice, from41.7%down to8.33%, and resulted in furtherdeterioration in liver histopathology. Hydropic degeneration, inflammatory cellinfiltration, and bridge necrosis in livers were more aggravated, accompanied byrobustly elevated blood levels of aspartate aminotransferase (AST) and alanineaminotransferase (ALT).【CONCLUSION】1. Liver of healthy C3H/HeJ mice contains abundant TCRγδ~+DN T cells, which markedlyincreased after MHV-3infection.2. Liver TCRγδ~+DN T cells bearing a specific phenotype and highly activated afterMHV-3infection. These cells were distinct from NKT cells and produced theinflammatory cytokines TNF-α and IL-17A, and the Th1cytokines IFN-γ and IL-2, butnot Th2cytokines.3. Highly activated liver TCRγδ~+DN T cells were cytotoxic to MHV-3-infectedhepatocytes in vitro and this effect did not require cell-cell contact. Moreover, thecytotoxic effect of liver TCRγδ~+DN T cells against hepatocytes involves TNF-αpathway, but not IL-17A or IFN-γ.4. Adoptive transfer of TCRγδ~+DN T cells led to dramatically decreased survival inMHV-3-infected mice, accompanied by deteriorated histopathology and elevated ALTand AST levels. |
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