| [BACKGROUND&OBJECTIVE]Viral hepatitis is acute or chronic liver inflammation due to infection with different viruses including five kinds of hepatotropic viruses, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), as well as other viruses that can also induce hepatitis including herpes simplex, cytomegalovirus, Epstein-Barr virus, etc. The most common cause in China is HBV infection, and the incidence rate of HBV infection is as high as 7.18%. Viral hepatitis especially chronic hepatitis B (CHB) is one of the most frequent causes for fulminant viral hepatitis (FVH), which is a rapid development of liver damage and hepatocellular dysfunction, specifically coagulopathy and some other severe complications like mental status disorder, also called encephalopathy, spontaneous bacterial peritonitis, hepatorenal syndrome, etc. in a patient. There is a lack of specific and effective treatment and interference strategy except liver transplantation, thus the mortality has been unacceptably high, being in excess of 70%. Studies have confirmed that murine hepatitis virus strain 3 (MHV-3) can induce fulminant viral hepatitis (FVH), and the course of illness and prognosis are similar to human FVH, so at present it is one of the proper animal models to investigate the pathogenesis of FVH.So far, the immunological pathogenesis of FVH is very complicated and not well defined. Generally speaking, virus induced hepatocyte damage is caused by a complex and prolonged interaction between virus and host immune defense. Most studies showed that the HBV replication does not directly induce hepatocyte death, and the viral protein directly or indirectly triggers activation of immune system, especially cellular response, which then induces primary liver damage. It is suggested that the hepatitis viral antigen-specific cytotoxicity T cell (CTL) may be an important factor in clearing the virus and predicting the disease outcome. However, the role of other innate immune cells in pathogenesis of FVH remains unclear. There are always many lymphocytes including CTL, regulatory T cells (Treg), etc. infiltrating within the liver when patients or mice are infected with hepatitis virus. As an essential component of the immune system, Tregs consist of several different cell subsets, the most common one is CD4+CD25+FOXP3+Tregs secreting immune suppressing cytokine IL-10 or TGF-β.More and more data show that natural killer T cell (NK T), CD4-CD8-double-negative T (DN T) cells,γδT cells are also capable to down-regulate immune responses in different diseases. These cells exhibit various functions. For example, in systemic lupus erythematosus (SLE) DN T cells could promote to produce autoimmune antibodies, but in leishmania infection TCRαβ+DN T cells could induce inflammation. Recently it has been found that CD4+CD25+FOXP3+Tregs contribute to the outcome of FVH through secreted fgl2.γδT cells mainly consist of CD4-CD8-γδT cell subset and are found to play important roles in some infectious or autoimmune diseases.Yet the roles of DN T cells orγδT cells in pathogenesis of FVH remain unclear.Fibrinogen like protein-2 (fgl2), including transmembrane fgl2 and secreted fgl2, is a protein that exhibits various effects. The membrane bound fgl2 showed prothrombinase activity and was mainly expressed on the surface of activated macrophages and endothelial cells. Our previous studies have showed that fgl2 expressed by macrophages can cause fibrin deposition, microcirculatory disturbance and inflammation in the liver which may play an important role in the immunological mechanism of MHV-3 induced FVH. Besides, Tregs were found to produce secreted fgl2 which is a crucial regulator of immune responses. Our previous study showed that after MHV-3 infection, a disparate subset of DN T cells was activated, and expression of mfgl2 on DN T cells increased. However, the roles of DN T cells andγδT cells in precise mechanism of FVH need to be elucidated. Therefore the concrete purposes of this work are as follows:1. To analyze the expression and procoagulant activity of mfgl2 on splenic DN T cells isolated from MHV-3 infected mice.2. To further investigate the mechanism in DN T cells derived mfgl2 contributing to the outcome of MHV-3 induced FVH.3. To primary study the mechanism in hepatic DN T cells promoting activation of NK cells in MHV-3 induced FVH.4. To primarily investigate the role of hepaticγδT cells in pathogenesis of MHV-3 induced FVH.[METHODS]1. FVH mouse model was established through peritoneal infection with 100 PFU MHV-3 into BALB/cJ mice. The pathological changes of hepatic tissue were observed using H E staining, meanwhile the levels of serum AST and ALT were determined.2. Splenic DN T cells were isolated from MHV-3 infected BALB/cJ mice at Oh and 48h by Magnetic Activated Cell Sorting (MACS), and the expression of mfgl2 on DN T cells were detected by confocal microscopy, the procoagulant activity of DN T cells-derived mfgl2 were examined by procoagulant assay in vitro.3. An adenovirus vector expressing miRNA against mfgl2 gene (Ad-mfgl2-miRNA) were introduced into BALB/cJ mice with FVH via tail intravenous injection, and splenic DN T cells isolated from MHV-3 infected BALB/cJ mice were adoptively transferred into these mice. Then the survival rate of mice and the serum ALT, AST were determined, and the pathological changes of liver tissue were detected by H E staining, the mfgl2 mRNA and protein levels in the liver were detected by realtime-PCR and Western blot analysis.4. Splenic DN T cells isolated from MHV-3 infected BALB/cJ mice were adoptively transferred into BALB/cJ mice with FVH, the expression of CD69 on splenic NK cells were analyzed by flow cytometry. Both hepatic DN T cells and NK cells were isolated from MHV-3 infected BALB/cJ mice at 48h and 24h, respectively. Then coculture them in vitro for 12h, and expression of CD69, IFN-y and TNF-a were determined by flow cytometry, the cytotoxic activity of hepatic NK cells against hepatocytes in vitro were examined by a non-radioactive cytotoxicity assay (LDH releasing assay).5. The proportions and CD69 expression ofγδT cells were detected in blood, spleen and liver of BALB/cJ mice infected with MHV-3 at 0h,24h,48h and 72h. The expression of phenotype molecular including CD25, CD28, CD30, CD44 and intracellular cytokines IL-2, IL-4, IL-10, IL-17, FasL, IFN-γ, TNF-α, perform and granzyme B secreting by hepaticγδT cells were determined by flow cytometric analysis. DimerX (recombinant soluble dimeric mouse CD1d:Ig) was used to differentiate DN T cells from NK T cells.6. In vitro cytotoxic activity of hepaticγδT cells aganist MHV-3-infected hepatocytes was detected by LDH releasing assay at different effect-target ratios. To further investigate the possible molecular mechanism involved in the hepaticγδT cells mediated hepatocytes damage, specific antibodies for IFN-γand/or TNF-αwere used to inhibit cytotoxicity of hepaticγδT cells against hepatocytes.[RESULTS]1. BALB/cJ mice with FVH all died within 3 to 7 days, and the serum levels of ALT and AST were remarkably elevated and peaked at 72h post MHV-3 infection (9418.7±23.6 IU/L,8195.3±224.0 IU/L, respectively), meanwhile the H E staining showed massive hepatocytes necrosis and extensive inflammatory cells infiltration in the liver tissue of Balb/cJ mice with FVH.2. mfgl2 was mainly expressed on the membrane of the DN T cells harvested at 48 h after MHV-3 infection by confocal microscopy. A further prothrombinase assay of the membrane mfgl2 showed that functional PCA activity increased significantly in DN T cells harvested from MHV-3 infected mice compared with naive DN T cells (651.51±301.80 mU/ml at 48h,30.30±52.48 mU/ml at 0h).3.5 out of 15 mice (33.33%) pretreated with Ad-mfgl2-miRNA survived. However, adoptive transfer of DN T cells to these Ad-mfgl2-miRNA pretreated mice led to a 100% death within 2 to 3 days post MHV-3 infection. Furthermore, these mice showed a significant increase in serum ALT and AST levels, an average of 18.4-fold higher level of mfgl2 mRNA and an increase in level of mfgl2 protein in the liver at 24 h post MHV-3 infection. The pathologic change was rare in the liver of the mice pretreated with Ad-mfgl2-miRNA, whereas the focal hepatocyte necrosis accompanied by lymphocyte infiltration re-appeared in the liver when DN T cells were adoptively transferred to these mice.4. After adoptive transfer of DN T cells into Balb/cJ mice with FVH, the expression of CD69 on hepatic NK cells were increased significantly. After cocultured with DN T cells in vitro, the expression of CD69, and TNF-a of NK cells remarkably increased by flow cytometric analysis, meanwhile, the cytotoxic activity of hepatic NK cells against hepatocytes was enhanced.5. After MHV-3 infection the proportion of TCRγδ+T cells in the blood, spleen and liver remarkably increased and peaked at 48 h post MHV-3 infection (1.47±0.18%,3.87±0.29% and 14.40±1.47%, respectively).γδT cells were negative for CD25, CD28, CD30, but majority of hepaticγδT cells expressed CD44 (90.37±2.15%). The expression ofα-Galcer onγδT cells was negative, demonstrating these cells are different from NKT cells. Besides, the proportion of hepaticγδT cells expressing CD69 increased significantly post MHV-3 infection, peaking at 72 h (38.27±0.98%). The production of TNF-a and IFN-y by hepatic y8T cells increased significantly and peaked at 48 h (18.37±1.51% and 19.93±2.69%, respectively).6. The cytotoxicity of hepaticγδT cells isolated from Balb/cJ mice with FVH was significantly enhanced at 48 h post infection when the E:T ratios were 10:1 and 20:1 in vitro. Blockage with either TNF-αor IFN-γneutralizing monoclonal antibody inhibited cytotoxic activity of hepaticγδT cells against hepatocytes to some extent post MHV-3 infection.[CONCLUSION]1. After MHV-3 infection, there is a novel subset of DN T cells in Balb/cJ mice which can express transmembrane mfgl2 and might contribute to the outcome of MHV-3 induced FVH.2. DN T cells might directly or indirectly activate NK cells, as well enhanced the production of TNF-a and IFN-y and cytotoxic activity of NK cells, and eventually led to massive hepatocytes necrosis. These findings help to further investigate the immunologic mechanism of MHV-3-induced FVH, and may also introduce new molecular targets for the subsequent intervention treatments.3. Our present work demonstrated that MHV-3 infection can induce remarkable increased proportions and activation ofγδT cells in the liver, and these hepaticγδT cells may contribute to host susceptibility to MHV-3 induced FVH through the effector cytokines TNF-a and IFN-y pathways. This finding provides new insights into the critical role ofγδT cells in the immunologic pathogenesis of MHV-3-induced murine FVH.
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