Innate Immune Mechanisms of Primary Graft Dysfunction after Lung Transplantation

 描述(申请人提供)：每年有超过3,000人接受肺移植，否则很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)是一种肺缺血-再灌注损伤，发生在移植后即刻，与早期发病率和死亡率及随后的慢性同种异体排斥反应有关。需要更好地了解肺移植后PGD的发病机制，以确定新的途径，从而为新的治疗方法提供信息。在缺血再灌注损伤中，中性粒细胞被显著地招募到肺内，最近观察到中性粒细胞将其染色质释放到细胞外空间，其染色质被颗粒蛋白装饰--被称为中性粒细胞外陷阱(NETs)的结构。我们假设Net在肺缺血-再灌流过程中形成，并直接导致肺屏障破坏导致PGD。我们将使用最先进的小鼠模型、用肺活体显微镜实时跟踪免疫事件以及使用来自患有和不患有PGD的肺移植受者的生物样本来验证这一假说。在目标1中，我们将确定在PGD小鼠单肺原位移植模型中Net的空间和时间形成。我们将通过聚焦于激活的血小板和从缺血肺释放的与损伤相关的分子模式来定义净触发因素，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(PAD4-/-)和有过度净积累的小鼠(DNase1-/-)来确定Net在PGD模型中的致病性。我们将针对可能与网络介导的肺毒性有关的网络组件(细胞外组蛋白、中性粒细胞蛋白酶)。在目标3中，我们将使用一项对人类肺移植受者的前瞻性队列研究来测试患有和不患有PGD的受试者移植后获得的生物样本。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DNase1活性对Net的体内调节以及对PGD严重程度的影响。这些实验和翻译研究将为Net在PGD中的作用提供确凿的证据，并将为未来对一种没有有效治疗方法的疾病-PGD-的临床试验奠定基础。

 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.