Wednesday, January 15, 2025
1/15/2025
Exposures to chlorine (Cl2) or bromine gas (Br2) may occur during industrial accidents and in the military arena. Acute lung injury (ALI), followed by chronic lung injury (CLI) characterized by airway reactivity, fibrosis and remodeling are significant concerns following halogen gas exposure; however, the mechanisms underlying this transition to CLI after exposure to Cl2 or Br2 remain to be determined. An important tenet in CLI mechanisms is protease dependent degradation and remodeling of the lung with neutrophil elastase (NE) considered a key mediator. However, α1-anti-trypsin (α-1AT), an endogenous inhibitor of NE is typically present at higher levels than NE raising the question how can NE mediate CLI in vivo. We recently discovered a novel mechanism where NE is also bound to the surface of small exosome-sized extracellular vesicles (EVs) released by PMNs during their activation and degranulation. Importantly, when associated with EV’s, NE is resistant to inhibition by α-1AT explaining how NE can induce pathogenic effects in the face of abundant α-1AT. We showed that NE+EVs are proteolytically active in vitro and in vivo; administration of NE+EV’s to naïve mouse airways led to reactive airway disease and a progressive destruction of the airways in an NE activity dependent manner. Thus, NE+EVs are novel mediators of CLI; a role for these proteolytic EVs in halogen gas toxicity has not been investigated. We hypothesize that PMN-derived NE+EVs mediate chronic lung injury after halogen gas exposure. Preliminary data supporting this hypothesis include demonstrating that NE+EVs are elevated in the airways post Cl2 gas exposure, and importantly cause progressive lung injury when administered to naïve mice. We propose two aims: Specific Aim 1: Determine the role of PMN-derived EVs in mediating CLI after halogen gas exposure. Proposed studies will determine the temporal formation of EVs and changes in NE activity following exposure of male and female mice, or rats, to either Cl2 or Br2 gas and relate these to the development of CLI assessed by reactive airways, fibrosis and airway remodeling (morphometry). In addition, we will isolate EVs from lungs after Cl2 or Br2 gas exposure, and then transfer them to naïve mice to evaluate their potential to cause CLI. Finally, PMN depletion experiments will be conducted to confirm that formation of pathogenic EVs after halogen gas exposure are derived from PMNs. Specific Aim 2: Determine the role of NE activity associated with EVs in mediating CLI after halogen gas exposure. We will determine the role of NE by comparing Cl2 or Br2 toxicity in WT or littermate NE-/- mice and the lung injury causing potential of EVs from these mice will be compared. Finally, to validate and identify EV associated NE activity as a novel target for countermeasure development, we will develop and test a strategy that specifically inhibits NE activity on EVs and then determine the ability of the latter to cause CLI after transfer to naïve mice. Collectively, the proposed studies will delineate a new and common mechanism for Cl2 and Br2 toxicity mediated by PMN-derived EVs and identify EV-associated NE activity as novel target for future countermeasure development.
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