The Pathogenesis Of ALI Caused By Novel Influenza A H1N1 Virus 2009: Immune Regulation In Inflammation And Apoptosis

Pandemic influenza A H1N1 virus swept the globe in 2009, leading to numerou deaths. Because of its’ prone to mutation and genetic ressortmant, it is a continuous threat of new emergence of strains with higher virulence and infectivity. H1N1(2009) can induce severe lung disease and complications in susceptible individuals. Acute lung injury and acute respiratory distress syndrome were the major causes of deaths. Therefore, it is of importance to characterize H1N1-induced host immune response and mechanisms of immune regulation and to explore possible therapeutic targets.A virus strain, A/Nanjing/108/2009(H1N1), was isolated from a clinical throat swab sample. Host responses to H1N1(2009) infection in macropahges and brochial epithelial cells were studied to explore the regulation role of mitogen-activated protein kinase and PI3K/Akt signaling pathways. For exploring the new immune target in the infection with H1N1, PBEF as a novel biomarker in ALI was studied. We aimed to know the role of PBEF in the pathogenesis of H1N1-induced pulmonary microvascular endothelial barrier dysfunction and ALI.Results:1) Compared the sequencing results with selected representative strains of new influenza A H1N1 virus, we confirmed that our isolated strain belongs to the novel H1N1 influenza A viruses.2) H1N1(2009) infection was found to phosphorylate MAPK members, and mediate the regulation of inflammation, apoptosis and anti-viral response. In macrophages infected by H1N1(2009), the induction of IL-6 depended on the ERK activation and partially on JNK activation, and the induction of IL-8 depended on the ERK activation; however, the inhibition of JNK further increased the expression of IL-8 and IL-1β; the induced expression of chemokine CCL-5 and GCSF were dependent on the ERK activation, and CCL-5 expression also depended on JNK activation; and the induction of IL-10 only depended on the ERK activation. The interaction between NFkB signaling pathway and MAPK pathway was observed in the infection of H1N1(2009). ERK activation depended on the NFkB signaling and in turn NFkB signaling transduction was also dependent on the activation of ERK and JNK but not on p38. Pro-apoptotic factor TNF-a was upregulated during the process of the macrophage apoptosis induced by H1N1(2009). Induction of anti-viral factors IFN-β, Mx, and 2’5’-OAS by H1N1(2009) mainly relied on the activation of ERK, and at a lesser extent, on the activation of JNK.3) H1N 1(2009) infection in HBE cells also induced the anti-viral response and the upregulation of inflammatory cytokines and apoptotic factors. The inhibition of Akt in PI3K/Akt pathway upregulated the expression of IL-8 and FasL; on the other hand, the inhibition of JNK reduced the expression of IFN-β, IL-1 β, IL-6, IP-10. In the late stage of infection, dephosphorylation of FoxO1 occurred simultaneously with dephosphorylation of Akt in the process of viral NS1 protein-mediated apoptosis.4) In the human pulmonary micro vascular endothelial cells, H1N1(2009) infection could induce the increased expression of PBEF which is considered as a biomarker in ALI. The inhibition of PBEF with siRN A reduced the expression of some inflammatory cytokines such as IFN-β, TNF-α, IL-β, IL-6, IL-8 and IP-10, and meanwhile decreased the apoptosis through the dwon-regulation of FasL expression. On the other hand, the inhibition of PBEF expression also attenuated the increase of the permeability in human pulmonary microvascular endothelial cell monolayer infected by H1N1(2009) by reducing the level of phosphorylation of VE-cadherin which is the major endothelial adhesion molecule controlling cellular junctions and blood vessel formation. PBEF could also increase the expression of VEGF and VEGFR which could activate the phosphorylation of VE-cadherin during the inflammation. So, PBEF could be a permeability-increasing factor and have a role in the regulation of endothelial barrier dysfunction induced by H1N1 infection.In conclusion, pandemic novel influenza A(H1N1) virus in bronchial epithelial cells, lung microvascular endothelial cells and macrophages, could induce inflammation, apoptosis and antiviral immune responses. MAPK kinase family, PI3K/Akt signaling pathway, and NFkB/IKK pathway play a important regulatory role in the infection. NS1 protein mediated the Fas/FasL apoptosis pathway by dephosphorylation of FoxO1 protein in the late stage of infection and promote apoptosis of infected cells. PBEF is a new PIF factors that may directly or through increased VEGF/VEGFR-mediated expression indirectly increase phosphorylation of VE-cadherin, which is a key adhesion molecule in interendothelial cells and participates in virus-induced lung microvascular endothelial barrier dysfunction; Meanwhile, PBEF was also involved in virus-induced immune responses such as inflammation and apoptosis, suggesting that PBEF may be a potential target in immunotherapy for H1N1(2009)-infected patients.