Phosgene inhalation injury in mice: Modeling human pathophysiology, biomarker discovery, and therapeutic target identification.

Phosgene gas (choking gas (CG), military designation) has been used as a terrorist weapon, in warfare, and has injured many in transportation or industrial accidents. Phosgene inhalation exposure causes severe pulmonary edema and lung injury, with high lethality in exposed victims. Despite these devastating effects, no mechanism-based treatments of phosgene inhalation injury or forensic diagnostic biomarkers have been developed. Additionally, reproducible animal models that recapitulate human acute respiratory distress syndrome (ARDS) after phosgene exposure remain limited. Here, we developed a reproducible mouse model of phosgene inhalation injuries, identified potential forensic diagnostic biomarkers and potential drug targets, and evaluated a potential mechanism-based medical countermeasure. Briefly, 8-9 week old male and female BALB/c mice were exposed to 20 ppm phosgene for 15 minutes using a nose-only exposure manifold. In cohorts of mice, at 8h post-CG exposure, we collected bronchoalveolar lavage fluid (BALF) for total and differential leukocyte counts and pro-inflammatory cytokines; conducted methacholine airway challenge pulmonary functional tests; or collected lung and sensory nerve ganglia tissues for analysis of gene expression, protein expression, and/or histopathology. In a separate cohort of mice, we evaluated the therapeutic efficacy of a potential medical countermeasure, the transient receptor potential vanilloid 4 (TRPV4) ion channel antagonist, GSK2220691. Phosgene exposure significantly increased BALF total protein and albumin, suggesting vascular protein leakage and alveolar-capillary barrier disruption, as well as BALF leukocytes. Gene expression and protein levels of key pro-inflammatory cytokines and markers of vascular injury and coagulation disorder were elevated in phosgene-exposed mice. Additionally, phosgene exposure resulted in altered baseline lung mechanics, airway hyperresponsiveness, and histopathological characteristics consistent with key human ARDS features, such as thickening of alveolar septal walls and intra-alveolar accumulation of neutrophils and proteinaceous debris. Multiplex protein profiling identified 63 differentially expressed biomarkers that distinguished CG-exposed mice from air controls and reflected inflammation, immune activation, endothelial dysfunction, vascular leakage, matrix remodeling, and impaired repair. Many of these proteins are linked to FDA-approved or investigational therapeutics, supporting their utility as diagnostic biomarkers and actionable drug targets. TRPV4 antagonism ameliorated CG-induced pulmonary injury, supporting TRPV4 as a promising medical countermeasure target.