Pulmonary arteriole occlusion by platelet-neutrophil micro-emboli in Acute Chest Syndrome

项目摘要 急性胸部综合征（ACS）是一种急性肺损伤，是镰状细胞肺癌死亡的主要原因之一 细胞疾病（SCD）。触发ACS的病因机制仍然知之甚少。SCD的10-20% 因急性全身疼痛性血管闭塞发作而住院的患者在接下来的几天内发展为ACS， 提示血管闭塞周围的分子事件导致肺损伤。这种流行病学也 提供了一个治疗窗口，以阻止ACS的发展，前提是靶向治疗被确定。在 在R 01的第一个循环中，我们使用真实的时间体内多光子激发显微镜进行了一个新的发现， SCD小鼠急性冠脉综合征继发于嗜血小板微栓塞毛细血管前肺小动脉 集料.这些研究结果已发表在AJRCCM 2019，JCI-Insight 2017 & 2020，Blood Advances 2017 & 2020，实验血液学2020，血液2020和Haematologica 2015。我们发现血小板P- 选择素有助于这些微栓塞细胞聚集体的形成，P-选择素抑制减少 肺血管闭塞约50%。支持我们的小鼠模型的相关性，最近的临床试验报告约50% 减少给予P-选择素Ab治疗的SCD患者的疼痛发作。在我们的五年期的更新中， R 01，我们确定了额外的P-选择素非依赖性病理炎症信号传导事件， 靶向进一步抑制SCD中的肺血管闭塞和ACS。根据我们新的初步发现，我们 假设肝源性中性粒细胞胞外陷阱循环片段（cNET）到达肺， 促进SCD患者血小板聚集激活的肺小动脉微栓塞。我们亦建议 抑制中性粒细胞中的孔形成蛋白Gasdermin-D（GSDMD）依赖性信号传导， ACS的产生和发展。我们将使用我们新开发的模型来测试这一假设。 静脉内血红蛋白诱导的SCD小鼠ACS、活小鼠肺部体内成像、体外微流控 用患者血液和GSDMD或血小板-P-选择素遗传缺陷的SCD小鼠进行的研究。在目标1中，我们 确定血小板-P-选择素遗传缺陷的SCD小鼠是否仅部分保护免于 肺小动脉微栓塞和ACS。在目标2中，我们将确定cNET是否在肝脏中脱落， 促进SCD患者P-选择素非依赖性肺小动脉微栓塞。在目标3中，我们 确定胱天蛋白酶-4/11依赖性激活的嗜中性粒细胞-GSDMD是否促进cNET脱落， ACS在SCD中的发展。这些研究将引入一种新的范式， 肝对肺促进SCD肺损伤，并确定了一个新的GSDMD介导的，P-选择素非依赖性 SCD中ACS的机制。

Project Summary Acute chest syndrome (ACS) is a type of acute lung injury and one of the leading causes of mortality in Sickle Cell Disease (SCD). The etiological mechanism that triggers ACS remains poorly understood. 10-20% of SCD patients hospitalized with acute systemic painful vaso-occlusive episodes develop ACS within next few days, suggesting that molecular events surrounding vaso-occlusion contribute to lung injury. This epidemiology also offers a therapeutic window to halt the development of ACS, provided that targeted therapies are identified. In the first cycle of R01, we used real time in vivo multi-photon-excitation microscopy to make a novel finding that ACS in SCD mice is secondary to micro-embolism of precapillary pulmonary arterioles by neutrophil-platelet aggregates. These findings have been published in AJRCCM 2019, JCI-Insight 2017 & 2020, Blood Advances 2017 & 2020, Experimental Hematology 2020, Blood 2020 and Haematologica 2015. We found that platelet P- selectin contributed to formation of these micro-embolic cellular aggregates and P-selectin inhibition reduced lung vaso-occlusion by ~50%. Supporting the relevance of our mouse model, recent clinical trial reported ~50% reduction in pain episodes in SCD patients given P-selectin Ab therapy. In the present renewal of our 5-year R01, we identify additional P-selectin-independent pathological inflammatory signaling events that can be targeted to further inhibit lung vaso-occlusion and ACS in SCD. Based on our new preliminary findings, we hypothesize that liver-derived circulating-fragments of neutrophil extracellular traps (cNETs) arrive in the lung to promote neutrophil-platelet aggregate-enabled pulmonary arteriole micro-embolism in SCD. We also propose that inhibiting pore forming protein gasdermin-D (GSDMD)-dependent signaling in neutrophils prevents cNETs generation and development of ACS. We will test this hypothesis using our newly developed model of intravenous hemoglobin induced ACS in SCD mice, in vivo imaging of lung in live mice, in vitro microfluidic studies with patient blood, and SCD mice genetically deficient in GSDMD or platelet-P-selectin. In Aim 1, we will determine whether SCD mice genetically deficient in platelet-P-selectin are only partially protected from pulmonary arteriole micro-embolism and ACS. In Aim 2, we will determine whether cNETs shed in the liver, travel to the lung to promote P-selectin-independent pulmonary arteriole micro-embolism in SCD. In Aim 3, we will determine whether caspase-4/11-dependent activation of neutrophil-GSDMD promotes shedding of cNETs and development of ACS in SCD. These studies will introduce a novel paradigm that translocation of DAMPs from liver to lung promotes lung injury in SCD, and also identify a new GSDMD-mediated, P-selectin-independent mechanism of ACS in SCD.