Influenza A Virus Induced Apoptosis Of Peripheral Blood Mononuclear Cell

Background and ObjectiveInfluenza is an acute respiratory disease caused by influenza virus, it onsets suddenly and spreads fast, results in serious harmness in people especially in children, the elderly and immunodeficiciency patients. Antigen drift, caused by point mutation of influenza virus, result in the seasonal epidemic per year. However, Antigen shift caused by gene reasortment may produce a new subtype strain that results in pandemic because people have no immunity to it.Influenza virus has been shown to induce apoptosis in tissue culture cells and in peripheral blood monocytes. A depletion of lymphocytes due to apoptosis has also been described in mice infected with a highly virulent influenza A virus (IAV) isolated from humans. The immunopathological mechanisms and the role played by the virus infection of leukocytes with respect to disease pathology in general and leukocyte death in particular have not been elucidated. Studies have identified roles for both viral proteins and cell-signaling molecules in the induction of cell death during influenza virus infection. Fas and Fas ligand, tumor necrosis factor, double-stranded RNA-dependent protein kinase R (PKR), nitric oxide, transforming growth factor(TGF), and mitogen-activated protein kinase signaling have been associated with influenzainduced cell death. Despite the identification of many proteins involved in influenza virus-induced apoptosis, studies have not identified common cellular factors or a unifying theory to explain the involvement of all these factors.Apoptosis is essential in many physiological processes, including tissue atrophy, development of the immune system, and tumor biology. It also plays an important role in the pathogenesis of many infectious diseases, including those caused by viruses. Although there are many cellular proteins involved in the induction of apoptosis, a central player is the tumor suppressor protein p53. In general, p53 is an essential component of an emergency stress response that prevents the growth and survival of damaged or abnormal cells. Various genotoxic stresses, including viral infection, increase p53 transcriptional activity, which induces the expression of genes involved in cell cycle arrest and apoptosis. p53 activity is regulated primarily through posttranslational mechanisms, including stabilization of the protein by phosphorylation, increased nuclear localization, and changes in conformation leading to enhanced DNA binding.We inoculated peripheral blood from different health people, and isolated lymphocyte and mononuclear macrophage from peripheral blood. Inoculate H1N1 influenza virus, across PI staining and AnnexinV-PI double-staining to compare the difference of apoptosis between lymphocyte and mononuclear macrophage. Detect the expression of P53, IFN with RT-PCR. According to the result, we inhibit p53 with PFT-αand detect the apoptosis of lymphocyte and mononuclear macrophage.Material and methodsSubtype of influenza A virus has been sequenced previously by our laboratory. To collect the virus as the same PFU=1.2×107 /ml using Hemagglutination assay and Plaque-forming assay. Collect the peripheral blood of volunteer. Use different treatment to make virus absorb to cells: 1. Virus adsorb mononuclear cell direct; 2. Virus adsorb lymphocyte and mononuclear macrophage last for one hour and then lymphocyte and mononuclear are isolated;3. Lymphocyte and mononuclear macrophage are isolated and then adsorbed with virus;4. The apoptosis of lymphocyte and mononuclear macrophage are observed after adsorbed different time. PI staining and AnnexinV-PI double-staining are used to detect apoptosis in order to confirm the difference in all of the treatments. Meanwhile, RNA from cells infected after2h,4h,8h,12h was abstracted to detect the different express level of P53, IFN in lymphocyte and mononuclear macrophage. According to the PCR result, P53 transcription was inhibited with PFT-αand apoptosis of lymphocyte and mononuclear macrophage was then detected by PI staining.Result1. Virus titer decrease from 512 to 256 after culture in MDCK cells2. Plaque-forming assay: PFU=1.2×107/ml3. PI staining Result: the percentage of lymphocyte apoptosis was found increased when the cultivate time in vitro extended except for effected by virus. Before 48h, there are more apoptosis in mononuclear macrophage which adsorbed with virus. While after 48h, the results are opposite to before. The AnnexinV-PI double-staining is confirmed with PI staining.4. Isolate lymphocyte and mononuclear macrophage after adsorb with virus and isolate lymphocyte and mononuclear macrophage before adsorb with virus, the apoptosis of the former lower than the latter.5. Fluorochrome stain: the apoptosis of lymphocyte and mononuclear macrophage has no difference after exposure to virus for 1hour, 2hour, 4four, 8hour.6. PCR result: lymphocyte's expression of P53 was increase after 2 and 4 hours. There is little expression of P53 in 8 hour. The expression of p53 increased after mononuclear macrophage exposure to influenza virus. There is no difference in IFN.7. PI staining after add PFT-α: apoptosis of lymphocyte and mononuclear macrophage disappear and apoptosis is inhibited as well.Conclusion1. Influenza virus adsorb cells include mononuclear cell,lymphocyte and mononuclear macrophage,the difference of apoptosis show that there have interaction between lymphocyte and mononuclear macrophage.2. Influenza virus adsorb cells at different time, the result show that there are no relationship between apoptosis and adsorb time.3. Influenza virus can induce apoptosis of lymphocyte, but it has different effects on mononuclear macrophage at different time, the apoptosis is induced before 48h, and the apoptosis is inhibited after 48h.4. Detect the RNA of IFN and p53.the result show that maybe p53 affect the apoptosis of lymphocyte and mononuclear macrophage.5. The result that we add p53 inhibitor shows that p53 may be impact the apoptosis of lymphocyte and mononuclear macrophage that treat with influenza virus.