Primary Study On The Mechanism Of Immunopathology Mediated By Influenza A Virus Infection In Mice

Influenza is an epidemic, acute respiratory disease that has a high rate of mortality, especially among the elderly and children, and a large number of deaths due to influenza are reported every year all over the world. In 2009, a novel swine-origin influenza virus capable of rapid human transmission was reported. As of 4 April 2010, worldwide more than 213 countries and overseas territories or communities have reported laboratory confirmed cases of pandemic influenza H1N1 2009, including over 17700 deaths. Surveillance network has been build worldwide since March 2009. The recent report on the drug resistance to Oseltamivir phosphate capsules made the prevention and control of pandemic influenza more difficult.Influenza A virus is the most important among the three types of the virus from a clinical point of view. Influenza A viruses belong to the family Orthomyxoviridae. On the basis of the antigenicity of their haemagglutinin (HA) and neuraminidase (NA) molecules, they are classified into 16 HA subtypes (H1–H16) and 9 NA subtypes (N1–N9). The point mutations and reassortment events of the viral genomes contribute to the emergence of new variants or strains with epidemic or pandemic potential. Influenza A viruses have caused several pandemics during the last century, and continue to cause annual epidemics. The pandemic of 1918-1919 killed as many as 50 million people worldwide. Understanding of the mechanisms of increased pathogenicity of fatal influenza A viral infection is thus critical to optimize the antiviral treatment strategy and control the potential pandemic. The uncontrolled and aberrant activation of the innate immune system had been implicated in the mice model of fatal influenza A viral infection. A significantly rapid cell recruitment of macrophages and neutrophils into the lungs were assumed to play a role in the pathogenesis associated with H5N1 highly pathogenic avian influenza virus infection (HPAI). In addition, macrophages and neutrophils were associated with increased secretion and enrollment of some cytokine and chemokines, and the increased levels of cytokines are suggested to mediate influenza A infection signs and may play a role in the severe symptoms of fatal cases of HPAI H5N1 influenza virus infection. However, inhibition of the cytokine response was found not to protect against the lethal influenza A infection, and neutrophil or macrophage depletion initiated after infection in the early stage were also found not to have a significant effect on the disease outcome. These findings suggested complicated biological effect of macrophages and neutrophils in the fatal influenza A viral infection. In addtion, innate immune cells such as macrophages and neutrophils are the targets of influenza A viruses. The direct infection of macrophages and neutrophils may contribute to an inability to mount an adaptive immune response, such as antibody stimulation.In this study, the mechanism of immunopathology mediated by influenza A virus infection was carried out. To discover the role of these complicated factors during the infection, and evaluate the cellular and molecular discrepancy of different virulence virus, the initiation, development and outcome of the pulmonary immunopathology was investigated. Three parts were included in this study. On the first part, immunopatholgy on mice mediated by A/PR/8/34 (H1N1) influenza virus was studied. This part included: (1) The establishment and evaluation of mouse model for immunopathology on mice with influenza A/PR/8/34 (H1N1) virus infection. (2) Primary study on the mechanism of immunopathology on mice mediated by influenza A/PR/8/34 (H1N1) virus. (3) The immune response of mucosal and secondary lymphoid tissue during the pulmonary injury of mice with influenza A/PR/8/34 (H1N1) virus infection. On the second part, preliminary research on the mechanism of immunopothology on mice mediated by A/Beijing/501/2009 (H1N1) virus was studied. This part included: (1) The establishment and evaluation of mouse model for immunopathology on mice with influenza A/Beijing/501/2009 (H1N1) virus infection. (2) Primary study on the mechanism of immunopathology on mice mediated by influenza A/Beijing/501/2009 (H1N1) virus. (3) The immune response of mucosal and secondary lymphocyte tissue during the pulmonary injury of mice with influenza A/Beijing/501/2009 (H1N1) virus infection. In the third part, we compared the discrepancy pulmonary immune injury induced by A/Beijing/501/2009 (H1N1) and A/PR/8/34 (H1N1) virus infection.Results are as follows. First, we successfully established the mouse model for immunopathology on mice with two strains of influenza A virus by monitoring the weight lost, death ratio and histopathology of lung. Viral replication of two strains of influenza A virus in nasal and lung were detected in the early and late stage of infection. Second, complicated immune response was companied with the immunopathology during the infection. Large number of macrophage and neutrophils were accumulated both in the early stage and later stage of infection. Statics analysis shows that there is close correlation between the neutrophils with the viral load, serum and pulmonary antibody response. Close correlation also be found between the antibody responses with the viral load in lung. The infiltration of macrophage and neutrophils promote the cytokine response thereby implicated in the antibody response. Third, beside the robust antibody response, cytokine secreting cells (CSCs) and antibody secreting cells (ASCs) response which regulates the kinetics and magnitude of antibody response were also detected in lung of mice post infection. Fourth, cellular and humoral response in mucosal and second lymphoid organs cooperate with the immune response in lung contributes to the systemic protective response during the infection. Fifth, the different virulence of two influenza A virus strains is demonstrated by lethal dose fifty (LD50), weight lost and death ratio on mice, furthermore, the response initiated by two strains is not similar. Taken together, the response induced by A/Beijng/501/2009 (H1N1) is earlier and stronger than that induced by A/PR/8/34 (H1N1) virus. Sixth, the alignment of the protein sequency among A/Beijing/501/2009(H1N1), A/California/04/2009 and A/PR/8/34(H1N1) virus shows that there are mutations located either in known molecular determinants that confer pathogenicity and antiviral drug resistance and the glycan receptor-binding sites and antigenic loops which should be further characterized of their functional determinants.In conclusion, the process of immunopathology mediated by influenza A virus is multifaceted and highly complicated, involving different arms of the host response, all of which appear to be required for efficient virus clearance. The continuous replication of viral and accumulation of macrophage and neutophils in lung in the early stage of infection are the main reason for immune-mediated pathology. In the late stage of infection, neutrophils cooperate with the antibody response contributed to control the viral burden. Local pulmonary pathologic response is connected with the initiation of system immune response.