Mitochondria-Targeted OGG1 remodels Alveolar Epithelial Transitional States and Immune-Epithelial Communication after Chlorine Lung Injury

Abstract

Chlorine (Cl2) inhalation causes acute epithelial and endothelial injury, pulmonary edema, inflammation, and impaired gas exchange, yet no approved therapy directly targets the mechanisms that sustain lung injury after exposure. We recently showed that oxidative mitochondrial DNA (mtDNA) damage drives chlorine-induced lung injury and that post-exposure delivery of a fusion protein containing mitochondria-targeted 8-oxoguanine DNA glycosylase-1 (mOGG1) mitigates lung injury and improves survival. However, the cell populations and transcriptional states through which mtDNA injury shapes lung damage and repair remain unclear. Here, we used single-nucleus RNA sequencing to define early cellular responses to distinct epithelial and immune cell populations. C57BL/6 mice (4/group; 2 males and 2 females per group) were exposed to air or Cl2 (500 ppm, 30 min), returned them to room air and mOGG1 (1 mg/kg, intranasal) was administered 1 h after exposure; the mice were sacrifice and their lungs were collected at 24 h post exposure. Integrated analysis resolved 39 transcriptionally distinct epithelial, immune, endothelial, stromal, and vascular populations. Although the major cellular compartments remained broadly preserved, Cl2 induced marked remodeling of epithelial composition and transcriptional states. Subclustering of the AT1/AT2 transitional compartment identified four Differential Transcriptomic Adaptation Phases (DTAPs): an acute stress-response state (DTAP_D), a proliferative state (DTAP_P), a Krt8 damage-associated transitional state (DTAP_T), and an AT1-like resolution state (DTAP_A). Pseudotemporal analyses suggested that chlorine exposure increased heterogeneity in alveolar and airway epithelial states, with injury-associated transitional programs expanding along the inferred trajectories. Ligand-receptor analysis further predicted that Cl2 increased the complexity of IL-1 communication among neutrophils, macrophages, and epithelial populations. mOGG1 did not globally suppress this network but instead redistributed signaling interactions, including the participation of selected DTAP populations. Together, these findings identify heterogeneous epithelial transitional states associated with Cl2 injury and support a model in which mOGG1 promotes recovery by reshaping epithelial injury-repair programs and immune-epithelial communication. Keywords: Chlorine inhalation; mitochondrial DNA damage; mitoOGG1; single-nucleus RNA sequencing; alveolar epithelial repair; epithelial transitional states; epithelial-immune communication.