Mechanisms of Alveolar Mitochondrial Damage and Resolution in Pneumonia

摘要 急性肺损伤（ALI）每年困扰美国约20万人， 这些病人会死ALI的特征是严重的肺泡炎症，主要由 肺炎、败血症和吸入，导致肺泡I型上皮细胞损失和肺泡 屏障功能ALI的新治疗方法已经失败，部分原因是损伤的异质性， 不确定合适的药物靶点。ALI延迟消退与肺泡上皮细胞 细胞（AEC）死亡;然而，在肺炎/ALI中存在超过 细胞死亡的程度。我们已经将ALI缓解与线粒体损伤的遗传机制联系起来， 质量控制（MQC）。MQC通过协调诱导细胞内线粒体损伤， 线粒体生物发生和线粒体自噬，由诱导型转录因子，如核转录因子， 呼吸因子1（NRF-1）及其伴侣PGC-1 β。NRF-1调节线粒体的关键基因 DNA转录和复制。线粒体DNA（mtDNA）损伤破坏MQC，并逃逸 细胞线粒体DNA的损伤是炎症性的，与ALI中受损的线粒体自噬和MQC一致。 线粒体功能也随着年龄的增长而下降，并且年龄与肺功能下降相关， 更严重的急性肺损伤因此，mtDNA释放可能是一种有用的生物标志物。我们建议肺炎/ALI 增加AEC中的mtDNA损伤，并损害降低线粒体氧化剂的MQC活性 在某些实施方案中，本发明的组合物通过刺激炎性小体的产生和炎性小体的组装，促进AEC细胞存活并改善ALI消退。 将在第二个模型（高氧）中测试这些机制的激活，并在第二个模型中测试这些机制的减弱效应。 肺老化伴长期和多种环境氧化剂暴露和获得性缺陷 线粒体DNA中。为了验证这一假设，我们提出了三个具体目标： 目的1：鉴定肺AEC中线粒体DNA氧化（8-OHdG）， 线粒体储备，海马技术呼吸，荧光技术细胞氧化剂产生 探针、MQC蛋白诱导分析以及共聚焦AEC MQC定位和细胞死亡 显微镜下观察S.金黄色葡萄球菌肺炎/ALI 目的2：研究线粒体DNA和线粒体储备的丢失及其在炎症小体中的作用 激活、线粒体自噬和坏死性凋亡。金黄色葡萄球菌肺炎/老年小鼠的ALI。我们将使用 线粒体过氧化氢酶（mCAT）小鼠保护线粒体DNA和测量肺部炎性小体的变化 组装和细胞死亡。 目的3：检测线粒体DNA氧化的频率和MQC在AECs中的分布， 与年龄匹配的健康对照组相比，死于ALI/ARDS的人类患者。我们将使用 免疫荧光分布和确定损伤与年龄、氧剂量和 持续的炎症反应。 这些目标将支持MQC招募和mtDNA预防的原理证明 氧化作为肺细胞防御的关键参与ALI的消退，决定肺衰老的影响 对MQC和ALI分辨率的影响，并为新的生物标志物和药理学奠定分子基础 MQC的干预措施，以促进ALI的解决。

Abstract Acute Lung Injury (ALI) afflicts ~200,000 people in the United States every year and >30% of these patients die. ALI is characterized by severe alveolar inflammation caused primarily by pneumonia, sepsis, and aspiration, causing loss of alveolar type I epithelial cells and failure of alveolar barrier function. New treatments for ALI have failed, partly because of the injury heterogeneity and uncertainty about appropriate drug targets. Delayed resolution of ALI is related to alveolar epithelial cell (AEC) death; however there is excessive mitochondrial damage in pneumonia/ALI that exceeds the degree of cell death. We have linked ALI mitigation to genetic mechanisms of mitochondrial quality control (MQC). MQC eliminates mitochondrial damage from cells by coordinated induction of mitochondrial biogenesis and mitophagy, regulated by inducible transcription factors, such as nuclear respiratory factor 1 (NRF-1) and it partner, PGC-1. NRF-1 regulates key genes for mitochondrial DNA transcription and replication. Mitochondrial DNA (mtDNA) damage disrupts MQC, and escape of mtDNA from cells is inflammatory and consistent with impaired mitophagy and MQC in ALI. Mitochondrial function also declines with age, and age correlates with declining lung function and more severe ALI. Thus, mtDNA release may be a useful biomarker. We propose that pneumonia/ALI increases mtDNA damage in AECs and impairs MQC activity that lower mitochondrial oxidant production and inflammasome assembly, promotes AEC cell survival and improves ALI resolution. These mechanisms will be tested for activation in a second model (hyperoxia) and for waning effect in aging lung with prolonged long and multiple environmental oxidant exposures and acquired defects in mtDNA. To test the hypothesis, we propose three Specific Aims: Aim 1: To identify mitochondrial DNA oxidation (8-OHdG) in lung AECs with changes in mitochondria reserve, respiration by Seahorse technology, cell oxidant production by fluorescent probes, MQC protein induction analysis, and AEC MQC localization and cell death by confocal microscopy during S. aureus pneumonia/ALI in young adult mice Aim 2: To examine loss of mtDNA and mitochondrial reserve and its role in inflammasome activation, mitophagy and necroptosis in S. aureus pneumonia/ALI in older mice. We will use mitochondrial catalase (mCAT) mice to protect mtDNA and measure changes in lung inflammasome assembly and cell death. Aim 3: To examine the frequency of mtDNA oxidation and MQC distribution in AECs in human patients dying of ALI/ARDS compared with healthy age-matched controls. We will use immunofluorescence distributions and determine relationships of damage to age, oxygen dose, and ongoing inflammatory response. These Aims will support proof-of-principle for MQC recruitment and prevention of mtDNA oxidation as a critical lung cell defense involved in ALI resolution, determine the impact of lung aging on MQC and ALI resolution, and lay a molecular basis for new biomarker and pharmacological interventions for MQC to promote resolution of ALI.