Mitochondrial DNA Injury is a Key Contributor to the Development of Chemical Lung Injury

Accidental exposure to chlorine (Cl2) causes potentially fatal lung injury accompanied, in survivors, by reactive airway disease and pulmonary fibrosis resembling symptoms typical of the acute respiratory distress syndrome (ARDS). Our preliminary data also document that the mitochondrial genome–the integrity of which is critical to maintenance of normal bioenergetics and mitochondrial signaling activities– displays oxidative damage after Cl2 gas exposure, similar to that detected in other models of acute and chronic lung injury. Against this background, here we propose to test the hypothesis that an early molecular target in Cl2 gas inhalation is the mitochondrial (mt) genome, in which oxidative damage functions as a molecular sentinel leading to activation of pro-inflammatory pathways that drive acute lung injury and progressive lung remodeling. To address this hypothesis we will perform the following in vivo and in vitro studies outlined in the following three aims: Aim 1. Define the kinetics of lung oxidative mtDNA damage in mice exposed to Cl2 gas and returned to room air and test whether a novel agent to augmenting mtDNA injury decreases mortality and the severity of acute and chronic lung injury. Exp. 1.1 will determine the time course of Cl2 gas-induced lung mtDNA damage and its relation to lung pathophysiology in global (mitochondrial and nuclear) OGG1 knockout mice (OGG1-/-) and their wild type (WT) controls (C57BL/6), along with OGG1 KO mice reconstituted with mito-targeted OGG1 (OGG1-/-+OGG1mt), while Exp. 1.2 will test if intranasal administration of mitoOGG1 at selected time points after termination of Cl2 exposure decreases lung injury pulmonary fibrosis and improves survival. Aim 2: Test the hypothesis that Cl2 gas-induced lung mtDNA damage triggers mobilization of mtDNA Damage Associated Molecular Patterns (DAMPs) that drive nucleic acid-dependent inflammation and serve as biomarkers and causative agents of pneumotoxicity. In Exp. 2.1 will use WT, OGG1-/-, and OGG1-/-+OGG1mt mice to test the hypothesis that OGG1 activity coordinately regulates Cl2 gas-induced mtDNA DAMP accumulation and activation of nucleic acid receptor-mediated pro-inflammatory pathways. Aim 3: Determine key cellular mechanisms by which Cl2 gas causes mtDNA damage-dependent lung cell death and dysfunction. In Exp. 3.1 we will isolate alveolar macrophages (AM) and lung epithelial type II cells (ATII) from WT, OGG1-/-, and OGG1-/-+OGG1mt mice, and measure oxygen consumption rate, reactive oxygen species, mitochondrial membrane potential, ion transport and key indices of cell injury prior to and following exposure to Cl2 gas. Additionally, Exp. 3.2 will define the mtDNA damage-dependent trafficking of mtDNA DAMPs to cytosolic and extracellular compartments and determine how these two mtDNA DAMP pools interact to dictate pro-inflammatory nucleic acid receptor activation. As part of this experiment, we also will determine if exogenous mtDNA DAMPs recapitulate nucleic acid receptor activation and inflammation evoked by Cl2 and use Dnase1 to degrade extracellular mtDNA DAMPs to define the specific effect of mtDNA DAMPs originating from this compartment.