Innate Immune Mechanisms of Primary Graft Dysfunction after Lung Transplantation

 描述（由申请人提供）：肺移植每年在3,000多人中进行，否则他们可能死于终末期肺部疾病。肺移植后的临床结果正在改善，但这些结果落后于其他实体器官移植。原发性移植物功能障碍（PGD）是一种发生在移植后即刻的肺缺血-再灌注损伤，并与大量早期发病率和死亡率以及随后的慢性同种异体移植物排斥反应相关。需要更好地了解肺移植后PGD的发病机制，以确定新的治疗方法。在缺血-再灌注损伤期间，中性粒细胞显著地被募集到肺中，并且最近已经观察到中性粒细胞将它们的染色质释放到细胞外空间中，所述染色质被称为中性粒细胞胞外陷阱（NET）的颗粒蛋白质修饰。我们假设NET在肺缺血-再灌注中形成，并且直接负责导致PGD的肺屏障破坏。我们将使用最先进的小鼠模型，肺活体显微镜实时跟踪免疫事件，以及使用来自肺移植受者的生物样本（有或无PGD）来验证这一假设。在目的1中，我们将确定NET在小鼠原位单肺移植模型中的空间和时间形成。我们将通过关注活化的血小板和从缺血性肺释放的损伤相关分子模式来定义NET触发，然后测试与这些途径相关的治疗策略。在目标2中，我们将通过测试不能产生NET的小鼠（PAD 4-/-）以及具有过量NET积累的小鼠（DNase 1-/-）来确定NET在PGD模型中的致病性。我们将靶向可能导致NET介导的肺毒性的NET组分（细胞外组蛋白，中性粒细胞蛋白酶）。在目标3中，我们将使用人肺移植受者的前瞻性队列研究来测试在具有和不具有PGD的受试者中移植后获得的生物样品。我们将确定血浆和支气管肺泡灌洗液中NET和NET触发物的存在，并测试它们与PGD的相关性。我们还将确定DNase 1活性对NET的体内调节以及对PGD严重程度的影响。这些实验和转化研究将提供关于NET在PGD中的作用的明确证据，并将为未来的PGD临床试验奠定基础。

 DESCRIPTION (provided by applicant): Lung transplantation is performed in over 3,000 persons annually who would otherwise likely die of end-stage lung diseases. Clinical outcomes are improving after lung transplantation, but these outcomes lag behind other solid organ transplants. Primary graft dysfunction (PGD) is a form of lung ischemia-reperfusion injury that occurs in the immediate post-transplant period and is associated with substantial early morbidity and mortality and subsequent chronic allograft rejection. A better fundamental understanding of the pathogenesis of PGD after lung transplantation is needed to identify novel pathways to inform new therapeutic approaches. Neutrophils are prominently recruited to the lung during ischemia-reperfusion injury, and recently neutrophils have been observed to release into the extracellular space their chromatin decorated with granular proteins- structures termed neutrophil extracellular traps (NETs). We hypothesize that NETs are formed in lung ischemia-reperfusion and are directly responsible for lung barrier disruption leading to PGD. We will test this hypothesis using state-of-the art mouse modeling, real-time tracking of immune events with lung intravital microscopy, and the use of biological samples from lung transplant recipients with and without PGD. In Aim 1, we will determine the spatial and temporal formation of NETs in a mouse orthotopic, single-lung transplantation model of PGD. We will define the NET trigger by focusing on activated platelets and also damage-associated molecular patterns that are released from the ischemic lung and then test therapeutic strategies related to these pathways. In Aim 2, we will determine the pathogenicity of NETs in the PGD model by testing mice that are incapable of producing NETs (PAD4-/-) and also mice that have excessive NET accumulation (DNase1-/-). We will target components of NETs (extracellular histones, neutrophil proteases) that might be responsible for NET-mediated lung toxicity. In Aim 3, we will use a prospective cohort study of human lung transplant recipients to test biological samples obtained post-transplantation in subjects with and without PGD. We will determine the presence of NETs and NET-triggers in plasma and bronchoalveolar lavage fluid and test for their association with PGD. We will also determine the in vivo regulation of NETs by DNase1 activity and the influence on PGD severity. These experimental and translational studies will provide definitive evidence on the role of NETs in PGD and will set the stage for future clinical trials for a condition-PGD-that has no effective therapies.