Alveolar responses to viral lung infection

肺泡对病毒性肺部感染的反应

基本信息

批准号：
10503083
负责人：
Jaime Lynn Hook
金额：
$ 55.38万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-20 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10503083
关键词：
Accounting Acute Lung Injury Address Adult Air Alveolar Alveolar wall Alveolus Anatomy Apical Bacteria Bioenergetics COVID-19 pandemic Catalytic Domain Cell Culture Techniques Cells Cessation of life Child Complex Confocal Microscopy Convection Critical Illness Cultured Cells Cystic Fibrosis Transmembrane Conductance Regulator Data Defense Mechanisms Edema Enzymes Epithelial Excess Mortality Film Future Goals Hemolysin Hospitalization Hour Human Image In Situ In Vitro Influenza Influenza A virus Inhalation Ion Channel Leucine Lipids Liquid substance Lung Lung infections Measures Mediating Membrane Membrane Proteins Methods Methylation Methyltransferase Mission Modeling Molecular Mus National Heart, Lung, and Blood Institute Particle Size Pathogenesis Pathologic Patients Persons Pharmaceutical Preparations Phosphorylation Physiology Population Positioning Attribute Protein Dephosphorylation Protein Methylation Protein phosphatase Proteins Pulmonary Edema Pulmonary alveolar structure Research Resistance Respiratory Failure Role Secondary to Small Interfering RNA Staphylococcus aureus Staphylococcus aureus infection Structure Surface Testing Time Toxin Transgenic Mice United States Ursidae Family Viral Viral Interference Virus Wild Type Mouse alveolar epithelium aqueous base co-infection influenza infection inhibitor insight knock-down lung injury mortality mouse model mutant new therapeutic target novel therapeutic intervention pandemic disease particle plasmid DNA prevent protective effect protein expression real-time images response restoration secondary infection success therapeutic evaluation

项目摘要

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.

项目概要/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出现继发性感染。吸入金黄色葡萄球菌 (SA) 感染 IAV 和 SA 混合感染导致约 30% 的死亡率。目前尚不清楚 IAV 如何促进继发性 SA 感染，特别是在肺泡中。很重要，因为肺泡是 SA 引起的致命性急性肺损伤 (ALI) 的解剖部位，但肺泡防御机制，包括肺泡壁液体 (AWL) 分泌，应防止 SA 稳定和该提案的长期目标是确定肺泡对 IAV 的反应促进肺泡继发性 SA 感染，导致 SA 诱导的肺泡损伤、ALI 和死亡。假设 IAV 肺部感染抑制 AWL 分泌，这是肺泡清除吸入的一种稳态机制这种抑制会导致 SA 和分泌的 SA 毒素、α 溶血素 (Hla) 滞留在肺泡中。保留增强了肺泡与 SA 和 Hla 的接触，促进 SA 对肺泡壁的稳定， Hla 诱导肺泡液屏障丧失，导致肺泡水肿和致命的 ALI。根据假设，初步数据表明 IAV 肺部感染引起： (A) 去磷酸化，从而失活肺泡囊性纤维化跨膜电导调节因子 (CFTR) Cl-通道，是肺泡囊性纤维化的关键蛋白 AWL 分泌；和 (B) CFTR 去磷酸化蛋白、蛋白磷酸酶 2A (PP2A) 的甲基化催化亚基 (PP2Ac)，可促进 PP2Ac-CFTR 相互作用。具体目标如下。将定义 CFTR 在吸入 SA 的肺泡滞留中的作用目标 2 将定义 PP2Ac 甲基化的作用。由于我们的初步数据表明 CFTR 和 PP2Ac- 存在 IAV 诱导的 AWL 分泌抑制。甲基化酶亮氨酸羧基甲基转移酶 1 (LCMT1) 可能代表新的治疗靶点恢复 IAV 感染肺部的 AWL 分泌，目标 3 将测试 CFTR- 和 LCMT1- 的治疗潜力采取有针对性的方法来预防合并感染引起的肺泡损伤、ALI 和死亡率。将使用我们既定的方法来实现，包括细胞培养、ALI 小鼠模型和实时对感染 IAV 的活体、完整小鼠和人类肺部进行共聚焦显微镜检查。测量：(1) SA 的肺泡滞留；(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：(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。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的 ALI，并建立 AWL 分泌的恢复——即“AWL 救援”——作为一种新的治疗方法因此，该提案涉及 NHLBI 的使命。