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

项目摘要 硫磺芥末(SM)和氯已重新成为对军事和平民人口的潜在威胁。 吸入硫磺芥末(SM)和氯可导致急性肺损伤，可导致呼吸系统 衰竭、多器官功能障碍和死亡。肺毒性对全身损伤的作用机制 都不清楚。我们对吸入卤素(氯和溴)的研究表明，严重的心脏和 神经元损伤。我们以前已经发现了在肺床中形成的循环卤素加合物。 并对远端器官损伤和疾病致病性起作用。在我们的吸入性损伤SM模型中，我们 证明了循环因子，如从肺组织和细胞释放的核酸可以 对肺损伤有显著贡献，清除核酸可以减轻损伤和抢救 从死亡中解脱。因此，循环因子在肺和全身损伤中起关键作用。越来越多的证据 这表明，几种这样的因子以货物的形式存在于胞外小泡(EV)的一种胞外小体中。研究 已经表明EV/exosome可能是致病的。我们的初步数据显示，货物的成分 氯染毒大鼠的支气管肺泡灌洗液(BALF)与胞外液不同 取自对照动物的BALF。此外，我们还证明了从猪肺泡灌洗液中分离到的外切体 动物暴露于CEES(2-氯乙基硫化物，又名：半芥末)，一种SM的类似物，当加入细胞时 在培养中呈剂量依赖性地增加炎症细胞因子和促凝血基因，这是重要的成分 在SM和CEES诱导的损伤的发病机制中起重要作用。在我们的活体研究中，SM BALF外切体 含量与BALF蛋白相关，BALF蛋白是泄漏的肺泡屏障的标志。这些研究引导我们提出了假设 接触有毒化学物质会导致致病外切体的释放，从而激活 炎症和凝血途径以及阻止它们的生物发生或吸收可以减轻损伤和 防止急性发病和死亡。因此，我们将(A)鉴定来源于 暴露于Cl2和CEES的大鼠的BALF和血浆，(B)确定EV/Exosome从这些EV/Exosome 接触有毒化学物质的动物会造成伤害，以及(C)评估来自有毒化学品的EV/Exosome的致病性 接触化学物质的动物是测试效力或生物发生或吸收的抑制剂。这些研究将描绘出 EV/Exosome影响肺损伤/疾病严重程度和消退的机制 确定外切体是否可以作为潜在的治疗靶点，以减轻由 吸入有毒化学物质。

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.