The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells

A co-culture model of human primary endothelial cells and organoid-derived epithelial cells shows that endothelial cells are abortively infected by influenza A virus but can drive the pro-inflammatory response. Morbidity and mortality from influenza are associated with high levels of systemic inflammation. Endothelial cells play a key role in systemic inflammatory responses during severe influenza A virus (IAV) infections, despite being rarely infected in humans. How endothelial cells contribute to systemic inflammatory responses is unclear. Here, we developed a transwell system in which airway organoid–derived differentiated human lung epithelial cells were co-cultured with primary human lung microvascular endothelial cells (LMECs). We compared the susceptibility of LMECs to pandemic H1N1 virus and recent seasonal H1N1 and H3N2 viruses and assessed the associated pro-inflammatory responses. Despite the detection of IAV nucleoprotein in LMEC mono-cultures, there was no evidence for productive infection. In epithelial–endothelial co-cultures, abundant IAV infection of epithelial cells resulted in the breakdown of the epithelial barrier, but infection of LMECs was rarely detected. We observed a significantly higher secretion of pro-inflammatory cytokines in LMECs when co-cultured with IAV-infected epithelial cells than LMEC mono-cultures exposed to IAV. Taken together, our data show that LMECs are abortively infected by IAV but can fuel the inflammatory response.

[1]  L. Akslen,et al.  Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus , 2022, Frontiers in Cellular and Infection Microbiology.

[2]  G. Boivin,et al.  Viral Interference between Respiratory Viruses , 2022, Emerging infectious diseases.

[3]  T. Bestebroer,et al.  Reduced Replication of Highly Pathogenic Avian Influenza Virus in Duck Endothelial Cells Compared to Chicken Endothelial Cells Is Associated with Stronger Antiviral Responses , 2022, Viruses.

[4]  S. Ebrahimpour,et al.  A brief review of influenza virus infection , 2021, Journal of medical virology.

[5]  Hans Clevers,et al.  SARS-CoV-2 productively infects human gut enterocytes , 2020, Science.

[6]  J. Baillie,et al.  Host susceptibility to severe influenza A virus infection , 2019, Critical Care.

[7]  A. Andiappan,et al.  RNA Sequencing of H3N2 Influenza Virus-Infected Human Nasal Epithelial Cells from Multiple Subjects Reveals Molecular Pathways Associated with Tissue Injury and Complications , 2019, Cells.

[8]  T. Kuiken,et al.  Role of Endothelial Cells in the Pathogenesis of Influenza in Humans , 2019, The Journal of infectious diseases.

[9]  L. Poon,et al.  Risk assessment of the tropism & pathogenesis of the highly pathogenic avian influenza A/H7N9 virus using ex vivo & in vitro cultures of human respiratory tract. , 2019, The Journal of infectious diseases.

[10]  A. Gounder,et al.  Influenza Pathogenesis: The Effect of Host Factors on Severity of Disease , 2019, The Journal of Immunology.

[11]  A. Oudenaarden,et al.  Long‐term expanding human airway organoids for disease modeling , 2019, The EMBO journal.

[12]  K. To,et al.  Differentiated human airway organoids to assess infectivity of emerging influenza virus , 2018, Proceedings of the National Academy of Sciences.

[13]  I. Amit,et al.  Dissection of Influenza Infection In Vivo by Single-Cell RNA Sequencing , 2018, Cell Systems.

[14]  J. Ritchey,et al.  A Three-Dimensional Human Tissue-Engineered Lung Model to Study Influenza A Infection. , 2018, Tissue engineering. Part A.

[15]  H. Feldmann,et al.  1918 H1N1 Influenza Virus Replicates and Induces Proinflammatory Cytokine Responses in Extrarespiratory Tissues of Ferrets , 2018, The Journal of infectious diseases.

[16]  A. Osterhaus,et al.  Proinflammatory Cytokine Responses in Extra-Respiratory Tissues During Severe Influenza , 2017, The Journal of infectious diseases.

[17]  F. Weber,et al.  pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs , 2017, Journal of Virology.

[18]  J. Barnet,et al.  Serum IFN-γ-induced protein 10 (IP-10) as a biomarker for severity of acute respiratory infection in healthy adults , 2017, Journal of Clinical Virology.

[19]  Shun-Hua Chen,et al.  IL-6 ameliorates acute lung injury in influenza virus infection , 2017, Scientific Reports.

[20]  R. Albrecht,et al.  Endothelial cell tropism is a determinant of H5N1 pathogenesis in mammalian species , 2017, PLoS pathogens.

[21]  Matthew Loxham,et al.  Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double‐stranded RNA , 2017, Immunity, inflammation and disease.

[22]  Christine E. Becker,et al.  Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions , 2016, European Respiratory Journal.

[23]  Zhan-Qiu Yang,et al.  The cytokine storm of severe influenza and development of immunomodulatory therapy , 2015, Cellular and Molecular Immunology.

[24]  L. Reperant,et al.  Influenza virus and endothelial cells: a species specific relationship , 2014, Front. Microbiol..

[25]  Stephen S. H. Huang,et al.  Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion. , 2014, Virology.

[26]  R. Gao,et al.  Cytokine and Chemokine Profiles in Lung Tissues from Fatal Cases of 2009 Pandemic Influenza A (H1N1) , 2013, The American Journal of Pathology.

[27]  B. Cao,et al.  Monoclonal antibody against CXCL-10/IP-10 ameliorates influenza A (H1N1) virus induced acute lung injury , 2013, Cell Research.

[28]  T. Moraes,et al.  Influenza Infects Lung Microvascular Endothelium Leading to Microvascular Leak: Role of Apoptosis and Claudin-5 , 2012, PloS one.

[29]  Albert D. M. E. Osterhaus,et al.  Comparison of Temporal and Spatial Dynamics of Seasonal H3N2, Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Virus Infections in Ferrets , 2012, PloS one.

[30]  O. Ramilo,et al.  Plasticity and Virus Specificity of the Airway Epithelial Cell Immune Response during Respiratory Virus Infection , 2012, Journal of Virology.

[31]  T. Tumpey,et al.  Human pulmonary microvascular endothelial cells support productive 1 replication of highly pathogenic avian influenza viruses : possible 2 involvement in the pathogenesis of human H 5 N 1 virus infection , 2011 .

[32]  Robert Peach,et al.  Endothelial Cells Are Central Orchestrators of Cytokine Amplification during Influenza Virus Infection , 2011, Cell.

[33]  H. Hoogsteden,et al.  Highly Pathogenic Avian Influenza Virus H5N1 Infects Alveolar Macrophages without Virus Production or Excessive TNF-Alpha Induction , 2011, PLoS pathogens.

[34]  T. Kuiken,et al.  Comparative Pathology of Select Agent Influenza A Virus Infections , 2010, Veterinary pathology.

[35]  K. McCullough,et al.  Hemagglutinin-Dependent Tropism of H5N1 Avian Influenza Virus for Human Endothelial Cells , 2009, Journal of Virology.

[36]  M. Hosoya,et al.  Human influenza virus infection and apoptosis induction in human vascular endothelial cells , 2008, Journal of medical virology.

[37]  Yan Li,et al.  Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus , 2007, Nature.

[38]  Yi Guan,et al.  Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia , 2006, Nature Medicine.

[39]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[40]  F. Hayden,et al.  Symptom pathogenesis during acute influenza: Interleukin‐6 and Other cytokine responses , 2001, Journal of medical virology.

[41]  G. Kärber,et al.  Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche , 1931, Naunyn-Schmiedebergs Archiv für experimentelle Pathologie und Pharmakologie.

[42]  J. Crapo,et al.  Cell number and cell characteristics of the normal human lung. , 1982, The American review of respiratory disease.