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.

Public Health Relevance

1 This is a revised Merit Review Program to study the principles of regulation of mitochondrial quality control (MQC), which includes mitochondrial biogenesis and mitophagy, and how they result in cell and lung protection through the prevention of mtDNA oxidation and its extracellular release during life-threatening S. aureus infections. The proposal specifically addresses the lack of information on how MQC, a known critical lung cell defense, contributes to cell survival and ALI resolution, and determines the impact of lung aging on MQC and ALI. This work will lay a molecular basis for new MQC biomarker development and biomolecular interventions for MQC activation to improve the resolution of ALI. Upon completing the proposal, we will have novel concepts for host activation of the two main MQC pathways and how they participate in limiting ALI progression and can be translated into treatments to diminish the morbidity, mortality, and health care utilization costs of ICU patients.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Durham VA Medical Center
United States
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