Alveolar responses to viral lung infection

项目总结/摘要 意义三分之一的甲型流感病毒（IAV）严重肺部感染患者会发生继发性 吸入性金黄色葡萄球菌感染。IAV和SA的共感染导致约30%的死亡率， 疗法目前还不清楚IAV如何促进继发性SA感染，特别是在肺泡中。这个问题 重要的是，因为肺泡是致命性SA诱导的急性肺损伤（ALI）的解剖部位，但肺泡 防御机制，包括肺泡壁液（AWL）分泌，应防止SA稳定， 共感染启动。这项建议的长期目标是确定肺泡对IAV的反应， 促进肺泡中的继发性SA感染，导致SA诱导的肺泡损伤、ALI和死亡率。的 假设IAV肺部感染抑制AWL分泌，AWL分泌是肺泡清除吸入的 粒子抑制导致SA和分泌的SA毒素α溶血素（Hla）的肺泡滞留。的 保持增强了肺泡与SA和Hla的接触，促进SA相对于肺泡壁的稳定， HLA诱导的肺泡液体屏障丧失，导致肺泡水肿和致命的ALI。除了直接支持 假设，初步数据表明IAV肺部感染引起：（A）去磷酸化，因此灭活 肺泡囊性纤维化跨膜传导调节因子（CFTR）Cl-通道， AWL分泌;和（B）CFTR-去磷酸化蛋白，蛋白磷酸酶2A（PP 2A）的甲基化 催化亚基（PP 2Ac），其可以促进PP 2Ac-CFTR相互作用。具体目标如下。要求1 将定义CFTR在吸入SA的肺泡滞留中的作用。目的2将定义PP 2Ac甲基化的作用 在IAV诱导的AWL分泌抑制中。由于我们的初步数据表明CFTR和PP 2Ac- 甲基化酶亮氨酸羧基甲基转移酶1（LCMT 1）可能代表新的治疗靶点， 恢复IAV感染的肺中AWL分泌，目标3将测试CFTR-和LCMT 1-的治疗潜力。 有针对性的方法，以防止合并感染引起的肺泡损伤，ALI和死亡率。这些目标 将使用我们建立的方法，其中包括细胞培养，ALI小鼠模型，和实时 活的、完整的小鼠和人肺的共聚焦显微镜。在IAV感染小鼠中的测定将包括 测量：（1）SA的肺泡保留;（2）AWL分泌;（3）肺泡CFTR磷酸化状态; (4)（5）SA-和Hla-肺泡上皮损伤和肺泡屏障丧失;和 (6)SA诱导的肺水肿和死亡率。我们将使用：用途：（i）用肺泡巨噬细胞抑制剂处理的野生型小鼠， PP 2Ac-CFTR和PP 2Ac-LCMT 1相互作用，包括药物抑制剂、编码突变蛋白的质粒DNA， 和siRNA;和（ii）缺乏肺泡上皮CFTR和LCMT 1表达的转基因小鼠。这项建议是 有望对IAV破坏关键肺泡功能的分子机制获得新的见解 导致致命性ALI，并建立AWL分泌的恢复-即“AWL拯救”-作为一种新的治疗方法， 方法：对IAV-SA混合感染引起的ALI进行治疗。因此，本提案涉及NHLBI的使命。

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.