Alveolar responses to viral lung infection

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PROJECT SUMMARY / ABSTRACT Significance. One-third of patients with severe lung infection by influenza A virus (IAV) develop secondary infection by inhaled Staphylococcus aureus (SA). Coinfection by IAV and SA causes about 30% mortality despite therapy. It remains unclear how IAV promotes secondary SA infection, particularly in lung alveoli. This issue is important because alveoli are the anatomical site of fatal SA-induced Acute Lung Injury (ALI), but alveolar defense mechanisms, including alveolar wall liquid (AWL) secretion, should prevent SA stabilization and coinfection initiation. The long-term objective of this proposal is to determine alveolar responses to IAV that promote secondary SA infection in alveoli, resulting in SA-induced alveolar damage, ALI, and mortality. The hypothesis is IAV lung infection inhibits AWL secretion, a homeostatic mechanism by which alveoli clear inhaled particles. The inhibition causes alveolar retention of SA and the secreted SA toxin, alpha hemolysin (Hla). The retention enhances alveolar contact with SA and Hla, promoting SA stabilization against the alveolar wall and Hla-induced alveolar fluid barrier loss, leading to alveolar edema and fatal ALI. In addition to directly supporting the hypothesis, preliminary data indicate IAV lung infection caused: (A) dephosphorylation, hence inactivation of the alveolar cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, the critical protein for AWL secretion; and (B) methylation of the CFTR-dephosphorylating protein, protein phosphatase 2A (PP2A) catalytic subunit (PP2Ac), which may promote PP2Ac-CFTR interactions. Specific Aims are as follows. Aim 1 will define the role of CFTR in alveolar retention of inhaled SA. Aim 2 will define the role of PP2Ac methylation in IAV-induced inhibition of AWL secretion. Since our preliminary data suggest CFTR and the PP2Ac- methylating enzyme, leucine carboxyl methyltransferase 1 (LCMT1) may represent new therapeutic targets to restore AWL secretion in IAV-infected lungs, Aim 3 will test the therapeutic potential of CFTR- and LCMT1- targeted approaches to protect against coinfection-induced alveolar damage, ALI, and mortality. These Aims will be achieved using our established methods, which include cell culture, mouse models of ALI, and real-time confocal microscopy of live, intact mouse and human lungs. Determinations in IAV-infected mice will include measures of: (1) alveolar retention of SA; (2) AWL secretion; (3) alveolar CFTR phosphorylation status; (4) alveolar PP2Ac methylation status; (5) SA- and Hla- alveolar epithelial damage and alveolar barrier loss; and (6) SA-induced pulmonary edema and mortality. We will use: (i) wild type mice treated with inhibitors of alveolar PP2Ac-CFTR and PP2Ac-LCMT1 interactions, including drug inhibitors, plasmid DNA encoding mutant proteins, and siRNA; and (ii) transgenic mice lacking alveolar epithelial CFTR and LCMT1 expression. This proposal is expected to achieve new insights into the molecular mechanisms by which IAV disrupts critical alveolar function leading to fatal ALI, and to establish restoration of AWL secretion – that is, “AWL rescue” – as a new therapeutic approach for ALI caused by IAV-SA coinfection. Therefore, this proposal addresses the NHLBI mission.