Extracellular vesicles as mediators of injury in inhaled exposures to toxic chemicals.

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Project Summary Sulfur mustard (SM) and chlorine have reemerged as a potential threat to both military and civilian populations. Inhaled exposures to sulfur mustard (SM) and chlorine cause acute lung injury, which can lead to respiratory failure, multiorgan dysfunction and death. Mechanisms by which pulmonary toxicity contributes to systemic injury are not clear. Our studies with inhaled halogens (chlorine and bromine) have demonstrated serious cardiac and neuronal injury. We have previously identified circulating adducts of halogens that form in the pulmonary bed and contribute towards distant organ damage and disease pathogenicity. In our inhaled SM models of injury, we demonstrated that circulating factors such as nucleic acids released from pulmonary tissues and cells can contribute significantly to lung damage and that scavenging the nucleic acids can alleviate injury and rescue from mortality. Therefore, circulating factors are critical to pulmonary and systemic injuries. Growing evidence suggests that several such factors are carried as cargo in exosomes a type of extracellular vesicles (EV). Studies have shown that EV/exosomes could be pathogenic. Our preliminary data shows that the composition of cargoes from the bronchoalveolar lavage fluid (BALF) of chlorine exposed rats are distinct from the exosomal cargo obtained from the BALF of control animals. Further, we demonstrate that exosomes isolated from the BALF of animals exposed to CEES (2-chloroethyl ethylsulfide, aka: half mustard), an analog of SM, when added to cells in culture dose-dependently increased inflammatory cytokines and procoagulation genes, important components in the pathogenesis of SM-induced and CEES-induced injuries. In our in vivo studies the SM BALF exosome content correlated with the BALF protein, a marker of leaky alveolar barrier. These studies led us to hypothesize that toxic chemical exposures result in the release of pathogenic exosomes that causes activation of the inflammatory and coagulation pathways and that blocking their biogenesis or uptake can mitigate injury and protect from acute morbidity and mortality. Accordingly, we will (a) characterize EV/exosomes derived from the BALF and plasma of rats exposed to Cl2 and CEES, (b) identify mechanisms by which EV/exosomes from these toxic chemical-exposed animals cause injury, and (c) evaluate pathogenicity of EV/exosomes derived from toxic chemical-exposed animals are test efficacy or biogenesis or uptake inhibitors. These studies will delineate mechanisms by which EV/exosomes influence pulmonary injury/disease severity and resolution and also determine whether exosomes can serve as potential therapeutic targets in mitigating organ injury caused by inhaled toxic chemicals.