Alveolar responses to viral lung infection

项目摘要/摘要 意义重大。被甲型流感病毒(IAV)严重肺部感染的患者中有三分之一会继发 金黄色葡萄球菌(SA)吸入感染。IAV和SA的混合感染导致约30%的死亡，尽管 心理治疗。目前尚不清楚IAV如何促进继发性金黄色葡萄球菌感染，特别是在肺泡。这个问题是 重要的是肺泡是致死性SA所致急性肺损伤(ALI)的解剖部位，而肺泡 防御机制，包括肺泡壁液体(AWL)分泌，应防止SA稳定和 合并感染启动。这项建议的长期目标是确定肺泡对IAV的反应 促进继发性金黄色葡萄球菌在肺泡的感染，导致金黄色葡萄球菌引起的肺泡损伤、ALI和死亡率。这个 假设是IAV肺部感染抑制AWL的分泌，这是一种使肺泡清洁吸入的动态平衡机制 粒子。这种抑制导致SA和分泌的SA毒素-α溶血素(Hla)的肺泡滞留。这个 固位增强了牙槽与SA和Hla的接触，促进了SA对牙槽壁的稳定和 人类白细胞抗原诱导的肺泡液体屏障丧失，导致肺泡水肿和致死性ALI。除了直接支持 这一假说，初步数据表明，IAV肺部感染导致：(A)去磷酸化，因此失活 肺泡囊性纤维化跨膜电导调节因子(CFTR)氯离子通道是肺泡囊性纤维化的关键蛋白 AWL分泌；以及(B)CFTR去磷酸化蛋白PP2A(PP2A)的甲基化。 催化亚基(PP2Ac)，可能促进PP2Ac-CFTR相互作用。具体目标如下。目标1 将确定CFTR在吸入性SA的肺泡滞留中的作用。目标2将定义PP2Ac甲基化的作用 IAV对AWL分泌的抑制作用。因为我们的初步数据显示CFTR和PP2Ac- 甲基化酶亮氨酸羧甲基转移酶1(LCMT1)可能是治疗的新靶点 恢复IAV感染肺中AWL的分泌，AIM 3将测试CFTR-和LCMT1-的治疗潜力 预防合并感染引起的肺泡损伤、ALI和死亡率的有针对性的方法。这些目标 将使用我们建立的方法来实现，包括细胞培养、ALI的小鼠模型和实时 活的、完整的小鼠和人类肺部的共聚焦显微镜。对感染IAV的小鼠的检测将包括 检测指标：(1)肺泡SA滞留；(2)AWL分泌；(3)肺泡CFTR磷酸化状态； (4)肺泡PP2Ac甲基化状态；(5)SA和HL A上皮损伤及肺泡屏障丧失； (6)SA所致肺水肿和死亡率。我们将使用：(I)用肺泡抑制剂治疗的野生型小鼠 PP2Ac-CFTR和PP2Ac-LCMT1相互作用，包括药物抑制剂，编码突变蛋白的质粒DNA， 和siRNA；以及(Ii)缺乏肺泡上皮CFTR和LCMT1表达的转基因小鼠。这项建议是 有望对IAV扰乱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分泌恢复--即“AWL抢救”--作为一种新的治疗方法 肺炎衣原体与沙门氏菌混合感染致急性肺损伤的探讨因此，这项提案涉及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.