| [BACKGROUND&OBJECTI VE]Viral hepatitis refers to the acute and chronic liver diseases induced by hepatotropic viruses. Viral hepatitis is characteristics with continuous liver injury. It is generally thought hepatitis viruses themselves are not cytopathogenic and do not kill hepatocytes. Previous studies have extensively reported the centrol role of activity of T cell-mediated immune responses, include CD4+ T cells, CD8+ T cells, natural killer T (NKT) cells, and regulatory T cells (Treg cells), in induction of hepatocellular injury during viral infection. Recent reports have shown that non-specific immune system do play roles in induction of hepatocellular injury during viral infection. In mouse model of fulminant hepatic failure (FHF) induced by murine hepatitis virus strain 3 (MHV-3), activated macrophage produced proinflammatory mediators such as TNF and IL-1 as well as the unique procoagulant activity fg12 prothrombinase, whic subsequent exacerbate the liver micro-environment and led to hepatocyte apoptosis and necrosis. Zou Y et al demonstrated that NK cells via Fas/FasL and NKG2D/NKG2D ligand pathway induced hepatocyte injury in patients with FHF and acute-on-chronic liver failure (ACLF).A small population of CD4- CD8-(double negative, DN) T cells has been shown to be involved in several diseases, including autoimmune diseases, immunodeficiency diseases, infectious disease, and tumor. In both mice and humans, these cells compose about 1-5%of all peripheral T lymphocytes, and express a specific set of cell surface molecules and a characteristic cytokine profile. In mice infected with Francisella tularensis live vaccine strain, lung DN T cells were found to expand into two subsets that produced two essential cytokines, IFN-γand IL-17A. TCRγδ+DN T cells from Leishmania-infected individuals present a regulatory profile, producing immunoregulatory cytokine IL-10.Interestingly, DN T cells have also been reported in the livers of healthy humans and mice and those with diseases. Norris et al. demonstrated that DN T cells ranged from 2.7 to 29% of total hepatic T lymphocytes in healthy humans. Seki et al. reported that the liver was probably an important organ for activation and expansion of TCRγδ+DN T cells in humans and mice, especially under tumor-bearing conditions. In the portal areas of liver cirrhosis patients, the majority of theγδ+T cells were TCRγδ+DN T cells. These findings suggest 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 has been reported.In this study we use MHV-3 induced murine hepatitis model to explore potential roles of 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 DN T cells in MHV-3-induced hepatitis mice model.2. To illustrate the phenotype and cytokine profiles of liver TCRγδ+DN T cells in MHV-3 induced fulminant hepatitis mice model.3. To investigate the effect of of liver TCRγδ+DN T cells on hepatocytes and its potential mechanisms in MHV-3-induced hepatitis mice model.[METHODS]1. Viral hepatitis animal model was established by MHV-3 infection of C3H/HeJ. The proportions of TCRγδ+DN T cells and TCRαβ+DN T cells in liver DN T cells in MHV-3-induced hepatitis mice model was examined. The proportions and number of liver TCRγδ+DN T cells were observed on day 0,3,7,10,15,20, and 35 post virus infection.2. The phenotype and cytokine profiles of liver TCRγδ+DN T cells were detected by flow cytometric analysis.3. In vitro experiment Liver TCRγδ+DN T cells were purified by Magnetic bead sorting. Primary hepatic cells were isolated from normal or MHV-3 infected mice. Lactate dehydrogenase (LDH) release assay were used to detect the cytotoxicity of liver TCRγδ+DN T cells on infected hepatocytes or uninfected hepatocytes. A Transwell experiment was performed to determine whether the cytotoxicity required cell-cell contact. The cytotoxic effects of liver TCRγδ+DN T cells on infected hepatocytes were examined in the presence of three neutralizing Abs (anti-TNF-α, anti-IFN-γ, anti-IL-17A mAbs) or not.4. In vivo experiment Liver TCRγδ+DN T cells from MHV-3 infected C3H/HeJ mice (3×106 cell/mouse) were adoptively transferred to C3H/HeJ mice 5 days post MHV-3 infection. PBS injected into mice 5 days post MHV-3 infection was used as control group. The survival rates, hepatic pathological changes, serum ALT and AST levels of two groups were examined.[RESULTS]1. Predominant population of DN T cells in the liver of healthy or MHV-3-infected mice expressed TCRγδ+. The proportion and number of TCRγδ+DN T cells in the liver increased markedly and peaked on day 10 post infection, compared with normal controls (19.7±3.22% vs.6.38±1.1%,6.51±1.29>×105 vs.0.76±0.11×105), and remained high 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 a-Galcer, demonstrating that these cells differed from NKT cells. The proportion of liver TCRγδ+ DN T cells expressing CD69 increased significantly post MHV-3 infection, peaked on day 10 (83.45±5.32%), and then decreased gradually. These cells revealed a classical inflammatory cytokine profile, with high production of TNF-α,IFN-γ,1L-17A and IL-2, but no IL-4, IL-10, IL-21or IL-22 was 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 that observed without the Transwell. TCRγδ+DN T cell cytotoxicity decreased markedly when TNF-a was blocked, but neither neutralizing IFN-γnor IL-1 A alone was able to inhibit TCRγδ+DN T cell-mediated cytotoxicity.4. In vivo, adoptive transfer of liver TCRγδ+DN T cells led to a dramatic decrease in survival of infected mice, from 41.7% down to 8.33%, and resulted in further deterioration in liver histopathology. Hydropic degeneration, inflammatory cell infiltration, and bridge necrosis in livers were more aggravated, accompanied by robustly elevated blood levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT).[CONCLUSION]1. Liver of healthy C3H/HeJ mice contains abundant TCRγδ+DN T cells, which markedly increased after MHV-3 infection.2. Liver TCRγδ+DN T cells bearing a specific phenotype and highly activated after MHV-3 infection. These cells were distinct from NKT cells and produced the inflammatory cytokines TNF-a and IL-17A, and the Thl cytokines IFN-y and IL-2, but not Th2 cytokines.3. Highly activated liver TCRγδ+DN T cells were cytotoxic to MHV-3-infected hepatocytes in vitro and this effect did not require cell-cell contact. Moreover, the cytotoxic effect of liver TCRγδ+DN T cells against hepatocytes involves TNF-a pathway, but not IL-17A or IFN-y.4. Adoptive transfer of TCRγδ+DN T cells led to dramatically decreased survival in MHV-3-infected mice, accompanied by deteriorated histopathology and elevated ALT and AST levels.
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