Mechanisms of Alveolar Mitochondrial Damage and Resolution in Pneumonia

摘要 急性肺损伤(ALI)每年在美国困扰着大约20万人，而30%的人 这些病人会死。ALI的特征是严重的肺泡炎，主要由 肺炎、败血症和吸入，导致肺泡I型上皮细胞丧失和肺泡衰竭 屏障功能。ALI的新治疗方法失败了，部分原因是损伤的异质性和 关于合适的药物靶点的不确定性。ALI延迟消退与肺泡上皮细胞的关系 细胞(AEC)死亡；然而，在肺炎/ALI中存在过度的线粒体损伤 细胞死亡的程度。我们已经将ALI的缓解与线粒体的遗传机制联系起来 质量控制(MQC)。MQC通过协同诱导细胞线粒体损伤 线粒体生物发生和有丝分裂，由可诱导的转录因子调节，如核 呼吸因子1(nrf-1)及其伴侣pGC-1。NRF-1调控线粒体关键基因 DNA转录和复制。线粒体DNA(MtDNA)损伤扰乱MQC，逃逸 细胞线粒体DNA是炎性的，与ALI中丝裂原吞噬和MQC受损相一致。 线粒体功能也随着年龄的增长而下降，年龄与肺功能下降和 更严重的ALI。因此，线粒体DNA的释放可能是一个有用的生物标志物。我们建议肺炎/ALI 增加血管内皮细胞线粒体DNA损伤，削弱降低线粒体氧化剂的MQC活性 生产和炎症体组装，促进AEC细胞存活，提高ALI的分辨率。 这些机制将在第二个模型(高氧)中进行激活测试，并在 长期多次接触环境氧化剂和获得性缺陷的老化肺 在线粒体DNA中。为了检验这一假设，我们提出了三个具体目标： 目的1：鉴定肺血管内皮细胞线粒体DNA氧化(8-OHdG) 线粒体储备、海马技术的呼吸作用、细胞氧化剂的荧光生产 探针、MQC蛋白诱导分析和共聚焦定位AEC MQC和细胞死亡 幼年小鼠金黄色葡萄球菌肺炎/ALI期间的显微镜观察 目的2：检测线粒体DNA和线粒体储备的丢失及其在炎症体中的作用 老年小鼠金黄色葡萄球菌肺炎/ALI的活化、有丝分裂和坏死性下垂我们将使用 线粒体过氧化氢酶(MCAT)小鼠保护线粒体DNA和测量肺炎性小体的变化 组装和细胞死亡。 目的3：检测动脉内皮细胞线粒体DNA氧化频率和MQC的分布 将死于ALI/ARDS的人类患者与年龄匹配的健康对照组进行比较。我们将使用 免疫荧光分布和确定损伤与年龄、氧气剂量和 持续的炎症反应。 这些目标将支持MQC招募和预防线粒体DNA的原则证明 氧化作为参与ALI解决的关键肺细胞防御，决定了肺老化的影响 研究MQC和ALI的分辨率，为寻找新的生物标志物和药理作用奠定分子基础 MQC干预促进ALI消退。

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.