Mechanisms Of Influenza A Virus Disrupt The Intercelluar Junctions Of Alveolar Epithelial Cells

Influenza A Virus(IAV),a member of the Orthomyxoviridae family,is one of the most common pathogens that causes infections in the respiratory tract.Up to 500,000 individuals worldwide die of seasonal influenza annually.Due to insufficient immunity to new IAV subtypes,the influenza pandemic in 2009 resulted in 150-600,000 deaths.New IAV subtypes such as H5N1 and H7N9 have always been a potential risk for influenza pandemics.The highly pathogenic avian influenza virus causes a high mortality rate infected in chickens that seriously endangers the poultry industry.IAV not only causes great economic losses to poultry industry,but also poses a global threat to human health.The onset of influenza A infection in some patients leads to acute respiratory distress syndrome,which is characterized by disruption of alveolar epithelial and endothelial barriers.This barrier impairment leads to increased alveolar permeability.The resulting alveolar protein-like effusion and edema contain cellulose,red blood cells,and inflammatory cells.Alveolar edema and accumulation of contents in the alveoli impair gas exchange and cause dyspnea.These pathologic changes in lung tissue can lead to viral pneumonia and secondary bacterial infections.Multiple organ failure,which has a high death rate,may develop in these patients.Tight junction is a fundamental junctional complex of the adjacent epithelial cells,with multiple functions including the barrier effect regulating intercellular permeability and maintaining cellular polarity,prevent large molecules and water from freely passing through the epithelial space,and also prevent bacteria and viruses entry into host cells.A variety of signaling molecules can regulate tight junction assembly,decomposition,and integrity maintenance.Tight junction proteins can be regulated at a transcriptional level by Glil,a transcription factor in the Shh signaling pathway.Alternatively,they can be regulated by ubiquitin ligase Itch at a post-translational level.Recent studies have shown that the MAP and PI-3 kinase pathways can cross-activate Glil.H1N1 virus activates PI-3 kinase signaling pathway,whereas H5N1 virus does not activate the PI-3 kinase pathway.In the present study,we investigated if H1N1 and H5N1 virus could disrupt epithelial junctional structure by different mechanisms.Mechanisms of H1N1 virus-induced disruption of the intercelluar junctions of alveolar epithelial cellsA549 cells and C57BL/6 mice were used to investigate the mechanism of H1N1(PR8 and CA09)viruses-induced disruption of the intercelluar junctions of alveolar epithelial cells.Here we report that PR8 and CA04 viruses increased the levels of Glil,Snail,and Slug,decreased the levels of tight junction proteins(E-cadherin,Occludin,and ZO-1)expression in a dose-dependent manner.Immunofluorescence staining revealed that H1N1 PR8 virus infection dramatically induced Glil expression in both the cytoplasm and nuclei,and Snail expression in nuclei,decreased Occludin and ZO-1 expression in the cell membrane of A549 cells.We next tested if H1N1 virus regulated Glil expression at a transcriptional level.We analyzed the promoter activity of the Glil gene by conducting a Glil promoter-driven luciferase reporter assay and measured Glil mRNA levels by qRT-PCR.H1N1 virus infection led to increased Glil promoter activity and Glil transcription.We next determined the effect of GANT61,a Glil-specific transcription factor,on the expression of the epithelial junction proteins.GANT61 blocked H1N1 virus-induced Glil,Snail,and Slug expression and restored the levels of E-cadherin,ZO-1 and Occludin.Immunofluorescence staining revealed that GANT61 restored the expression of Occludin and ZO-1 on the cell membrane of H1N1-infected A549 cells.H1N1 infection significantly decreased TEER values in a time-dependent manner.GANT61 did not alter TEER values of uninfected cells but significantly prevented the decrease of TEER values in IAV-infected A549 cells.ERK1/2 and AKT phosphorylation was increased in a dose-dependent manner in A549 cells infected with H1N1 virus.U0126,a MEK inhibitor,and LY294002,a PI-3 kinase inhibitor,partially inhibited H1N1 virus-induced ERK1 phosphorylation and AKT phosphorylation,inhibited the expression of Snail and Slug,partially restored the levels of E-cadherin,ZO-1 and Occludin expression in H1N1 virus-infected A549 cells.Immunofluorescence intensity analysis revealed that H1N1 virus infection significantly decreased the Occludin and ZO-1 signals located in the intercellular junctions,which was largely reversed by U0126 and LY294002.TEER assay revealed that inhibition of the MAP kinase pathway by U0126 and inhibition of the PI-3 kinase pathway by LY294002 moderately prevented the decrease of electricresistance in H1N1 virus-infected A549 cells.We tested whether the effect of IAV virus on Glil activation and the expression of junction proteins could be observed in vivo in mice infected with the H1N1 virus.Western blot analysis revealed that AKT and ERK phosphorylation was significantly increased in the lung tissues from C57BL/6 mice infected with H1N1 virus for 48 hr than from those uninfected mice.Glil and Snail expression was also significantly increased,whereas E-cadherin,Occludin and ZO-1 expression were significantly decreased.H1N1 virus infection also led to the lung tissue injury,as evidence by edema and infiltration of inflammatory cells such as macrophages and neutrophils.GANT61 treatment blocked H1N1 virus-induced Glil and Snail expression in the lung tissues and restored the expression of E-cadherin and Occludin but did not significantly change ZO-1 expression.H&E staining revealed that there was widespread edema and inflammatory cell infiltration in the lung tissues of mice infected with H1N1 virus.GANT61 treatment dramatically blocked the pathological changes in H1N1 virus-infected lungs.These data suggest for the first time have shown that H1N1 virus activates the MAP and PI-3 kinase pathways can cross-activate transcription factors Glil and Snail,decreased the expression of the epithelial junction proteins,leading to increased alveolar epithelial cells permeability.Our study unveiled a novel role of Glil in the pathogenesis of H1N1 virus infection and suggests that Gllil could be a potential target for the control of IAV-induced lung tissue damage.Mechanisms of H5N1 virus-induced disruption of the intercelluar junctions of alveolar epithelial cellsH5N1 viruses decreased the levels of transcription factors Glil,Snail and Slug,decreased the levels of tight junction proteins E-cadherin,Occludin,Claudin-1 and ZO-1 expression in a dose-dependent manner by western blot.ERK1/2,p38,JNK and TAK1 phosphorylation was increased in a dose-dependent manner in A549 cells infected with H5N1 virus.U0126,a MEK inhibitor,SB202190,a p38 inhibitor and 5Z-7-oxozeaenol,a TAK1 inhibitor partially inhibited H5N1 virus-induced ERK1 phosphorylation,p38 phosphorylation and TAK1 phosphorylation,partially restored the levels of E-cadherin,Claudin,ZO-1 and Occludin expression in H5N1 virus-infected A549 cells.SP600125,a JNK inhibitor is unable to restored he levels of E-cadherin,Claudin-1,ZO-1 and Occludin expression in H5N1 virus-infected A549 cells.Immunofluorescence intensity analysis revealed that H5N1 virus infection significantly decreased the Occludin and ZO-1 signals located in the intercellular junctions,which was largely reversed by U0126,SB202190 and 5Z-7-oxozeaenol.TEER assay revealed that inhibition of the MAP kinase pathway by U0126 and inhibition of the p38 kinase pathway by SB202190 and inhibition of the TAK1 pathway by 5Z-7-oxozeaenol moderately prevented the decrease of electricresistance in H5N1 virus-infected A549 cells.TAK1 silencing alone slightly increased E-cadherin,Claudin-1,Occludin and ZO-1 expression in uninfected A549 cells.TAK1 silencing largely restored the levels of E-cadherin,ZO-1,Claudin-land Occludin expression in H5N1-infected A549 cells.TAK1 silencing did not significantly change the levels of the NP and NS1 proteins of H5N1 virus.H5N1 virus infection induced the expression of E3 ubiquitin ligase Itch,proteasome inhibitor MG 132 restored the levels of tight junction protein E-cadherin,ZO-1,Claudin-1 and Occludin expression.We next detected whether the ubiquitination of Occludin was affected by H5N1 virus.Co-immunoprecipitation experiment showed that the ubiquitination level of Occludin was remarkably increased in H5N1 virus-infected A549 cells.SB202190 and 5Z-7-oxozeaenol decreased the ubiquitination level of Occludin in H5N1 virus-infected A549 cells.Itch silencing significantly decreased the ubiquitination level of Occludin.We tested whether the effect of IAV virus on Glil activation and the expression of junction proteins could be observed in vivo in mice infected with the H5N1 virus.W estern blot analysis revealed that TAK1 and ERK phosphorylation was significantly increased in the lung tissues from C57BL/6 mice infected with H5N1 virus for 48 hr than from those uninfected mice.Glil and Snail expression was also significantly decreased,whereas E-cadherin,Occludin,Claudin-1 and ZO-1 expression were significantly decreased.Itch expression was also significantly increased.H5N1 virus infection also led to the lung tissue injury,as evidence by edema and infiltration of inflammatory cells such as macrophages and neutrophils.Our present study have shown that H5N1 virus activates the MAP and TAK1 pathways decreased the expression of the epithelial junction proteins,leading to increased alveolar epithelial cells permeability.Our findings indicate that the ubiquitin-proteasome pathway may participate H5N1 virus disrupt the intercelluar junctions of alveolar epithelial.Thus,ubiquitin E3 ligase Itch could be a potential target for the control of IAV-induced lung tissue damage.In summary,our study demonstrated that cross-activation of Glil by IAV H1N1-activated PI-3 and MAP kinase pathways led to the disruption of the intercellular junction structure and subsequently increased paracellular permeability of the alveolar epithelial monolayer.In contrast,H5N1 decreased the levels of transcription factors Glil and Snail but activated TAK1,leading to ERK and p38 MAPK activation.MAPK activation induced the expression of Itch and promoted ubiquitination and degradation of the epithelial junction proteins,which led to increased alveolar epithelial cells permeability.