The Possible Pathogenesis Of Immunopathologic Induced By Hemagglutinin Protein Of H5N1

Rationale:Human Avian Influenza is an acute respiratory disease caused by influenza A virus, with a reported case fatality rate exceeding 60%. Recent studies revealed that the high virus load and excessive inflammatory reaction play a key role in the pathogenic mechanism of the acute respiratory failure caused by Avian Influenza A virus (AIV) infection. AIV-H5N1 targets the alveolar type II cells and macrophage by its outer membrane proein, hemagglutinin (HA) protein, which interacts with sialic residues on host cell surface molecules, leading to the subsequent release of chemokines/cytokines that can promote inflammatory cells migration into the lung tissue, in turn result in severe pulmonary injury characterized by a diffuse alveolar damage. However, few therapeutic options for the management of the immune injuries are under development. We have previously identified a key signal pathway targeted by SARS-CoV and demonstrated that selective inhibition of the key signal molecular, JAK3, protects against experimental pulmonary injury. Since both of AIV and SARS-CoV (coronavirus) have many similarities in the pathophysiological process and clinical manifestations, we speculated that the both might share a common signal pathway that contributes to initiation of host innate immune reaction in response to the viruses infection. The current study is therefore designed to investigate the possible signaling underlying the pathogenesis of immunopathological damage once the viruses invasion.Methods:1.Preparation of the recombinant hemagglutinin protein of AIV H5N1Insect-baculovirus expression system was employed for expression of the recombinant hemagglutinin protein (HA) of AIV H5N1. The recombinant HA was purifid by Nickel affinity Magnet Beads and identified by western-blotting. 2. Investigation of the possible mechanism of immune injury induced by HA protein in the treated A549 cellsThe cultured A549 cells were treated with different concentration of HA protein for detect the expression IRF-1/ IP-10 gene by RT-PCR .After treated for 12 hour,cytokines /chemokines released into the supernatants of the treated cells were measured by liquit Chip system. Western-blotting was performed for testing if the signal pathways of JAK-STAT and NF-κB were activated by HA stimulation.While a JAK3 inhibitor was used to see if it could attenuate the expression of IRF-1/ IP-10 gene and the induction of cytokines /chemokines by HA.3. Evaluation of the role of targeting JAK3 in protection from immune injury in JAK3 knockout mice with the challenge of HA proteinThe JAK3 knockout mice(B6129S4-Jak3tm1Ljb) and JAK3 positive control mice (B6129SF2/J) were randomly divided into PBS Control group and HA group, followed by transtracheal installation of HA protein or PBS. The lung and spleen tissues were isolated from the treated mice 72hrs after transtracheal addition of HA for pathological examination.Results:1. Preparation of the recombinant hemagglutinin protein of AIV H5N1By sequencing, we confirmed the HA gene of A/chicken/Guangdong /191/04(H5N1)(GenBank;AY737289) that was inserted into pFastbacHT-H5HA plasmid vector. As we expect, the PCR product of Bacmid-HA is 4.1Kb. A recombinant HA (64Kda) was purified in the sf9 cells transfected with Bacmid-H5HA and identified by western-blot.2. Investigation of the possible mechanism of immune injury induced by HA protein in the treated A549 cellsAfter HA protein stimulation, the A549 cells became larger, stopped growing with appearance of intracellular vacuoles. The increased expression of IRF-1 and IP-10 gene was detected by RT-PCR. The levels of IL-6, IL-8, MCP-1, MIP-1alpha, MIP-1beta and RANTES were increasing in the supernatants of the HA-treated A549 cells by Liquid Chip assay. Consistently, phosphorylated STAT1, JAK2, JAK3 and NF-κB were observed in the cells once upon the stimulation of HA. In the presence of the JAK3 inhibitor, the expression of IRF-1/IP-10 gene and the induction of chemokines/chemokines by HA was attenuated. Also the JAK3 inhibitor can inhibit the phosphorylation of JAK3 and NF-κB.3. Evaluation of the role of targeting for JAK3 in protection from immune injury in JAK3 knockout mice with the challenge of HA proteinPathologic examination revealed a diffuse alveolar damage in the lung of the mice with wild type JAK3 after challenge with HA, in contract, in the lung of mice with JAK3-/-, the injury is reduced significantly . In addition, the spleen tissue of the mice with wild type JAK3 appeared focal lesions with apoptosis and necrosis of lymphocytes wherest the spleen tissue of JAK-/- mice appeared normal.Conclusion:1. HA protein of H5N1AIV can promote cytokines/chemokines storm through activating JAK/STAT and NF-κBpathway.2. JAK3 is a key target for initiation of the immune inflammation once HA protein of H5N1 stimulation.3. Selective inhibition of JAK3 is able to alleviat H5N1 AIV HA protein–induced immune injury, which will be a new potential drug target for the treatment of virus-related immune damage in the future.