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

项目摘要 急性胸部综合征是一种急性肺损伤，是镰刀致死的主要原因之一。 细胞病(SCD)。引发急性冠脉综合征的病因机制仍不清楚。10-20%的SCD 因急性全身性疼痛血管闭塞发作而住院的患者在接下来的几天内会发展成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 提供一个治疗窗口来阻止急性冠脉综合征的发展，前提是确定了有针对性的治疗方法。在……里面 在R01的第一个周期，我们使用了实时在体多光子激发显微镜，得到了一个新的发现 SCD小鼠急性冠脉综合征继发于中性粒细胞-血小板对肺毛细血管前小动脉的微栓塞症 集合体。这些发现已经发表在AJRCCM 2019，JCI-Insight 2017&2020，血液进步 2017年和2020年，实验血液学2020年，血液2020年，血液学2015年。我们发现血小板P- 选择素促进了这些微栓子细胞聚集体的形成，P-选择素的抑制作用减弱 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了~50% 接受P-选择素抗体治疗的SCD患者疼痛发作的减少。在本次续订我们的五年期中 R01，我们发现了其他P-选择素非依赖性的病理炎症信号事件，这些事件可以 旨在进一步抑制SCD的肺血管闭塞和急性冠脉综合征。根据我们新的初步调查结果，我们 假设肝脏来源的中性粒细胞胞外陷阱(CNETS)循环片段到达肺 促进SCD患者中性粒细胞-血小板聚集性肺小动脉微栓子形成。我们还提议 抑制中性粒细胞中Gasdermin-D(GSDMD)依赖的孔形成蛋白信号可预防CNETS 先进制造系统的产生和发展。我们将使用我们新开发的模型来检验这一假设 静脉注射血红蛋白诱发SCD小鼠急性冠脉综合征，活体小鼠肺成像，体外微流控 对患者血液和GSDMD或血小板-P-选择素基因缺陷的SCD小鼠进行的研究。在目标1中，我们将 确定血小板-P-选择素基因缺陷的SCD小鼠是否只有部分保护作用 肺小动脉微栓子与急性冠脉综合征。在目标2中，我们将确定CNETS是否在肝脏、旅行 促进P-选择素非依赖性肺小动脉微栓塞术治疗SCD。在《目标3》中，我们将 确定caspase-4/11依赖的中性粒细胞-GSDMD的激活是否促进CNETS和 冠脉综合征在SCD中的发展这些研究将引入一种新的范式，将来自 肝到肺促进SCD肺损伤，并发现一种新的GSDMD介导的P-选择素非依赖性 急性冠脉综合征在SCD中的作用机制。

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.