Purinergic regulation of Innate Immunity to promote Venous Homeostasis

摘要 深静脉血栓形成(DVT)和继发性肺栓塞(VTE)影响到90万人 每年导致60,000人死亡。越来越多的证据支持这一概念 炎症和凝血之间的串扰，放大了静脉血栓栓塞术中的血栓炎症反应。 被激活的、受损的和垂死的细胞会释放核苷酸，形成嘌呤能光环的“危险”信号，从而破坏 血管动态平衡。位于白细胞和内皮细胞表面的血管 胞外核糖核酸酶，CD39迅速磷酸水解三磷酸腺苷，以消除这些“危险”信号和 促进动态平衡。我们发现CD39是一种有效的血栓炎症反应的抑制因子 在深静脉血栓治疗中。我们的新的初步数据显示，CD39抑制循环中的白细胞-血小板相互作用，并且 限制程序性先天免疫反应的激活，包括炎症小体组装和 中性粒细胞胞外陷阱(Net)的形成，是血栓不断增长的关键驱动因素。推动这一点的假设 研究表明，CD39在先天免疫和血栓形成的交叉点上提供了一个关键的血管检查点 以阻止无情的静脉血栓-炎症循环。使用独特的细胞谱系(髓系、中性粒细胞、 我们已经建立的内皮)特异性CD39基因缺失的小鼠，以及DVT的补充模型，我们将： 1)阐明CD39在静脉血栓形成和成熟过程中的细胞募集作用。这些 实验将确定谱系特异性CD39对血细胞募集的影响，以及空间动力学 在静脉血栓形成过程中的相互作用；2)确定髓系CD39的机制(S 预防静脉血栓炎症。这些实验将确定 巨噬细胞和中性粒细胞CD39对静脉血栓形成的影响。体外和体内互补研究 转基因小鼠将阐明CD39抑制的分子信号传递过程 血栓形成过程中炎性小体的激活；3)CD39缺乏对微泡的影响 静脉血栓形成过程中的表型、功能和动力学。这些研究将揭示功能 改变全局和谱系特异性CD39对微泡介导的血栓炎症的影响 发信号。这些研究应该会对作为检查点的电子核苷酸酶如何发挥作用产生新的见解 在血栓和炎症的交界处，可能会被用来改善静脉治疗 血栓形成。

ABSTRACT Deep venous thrombosis (DVT) and secondary pulmonary embolism (together, VTE) affect 900,000 Americans, and result in >60,000 deaths each year. A growing body of evidence supports the concept of crosstalk between inflammation and coagulation which amplifies the thrombo-inflammatory response in VTE. Activated, injured, and dying cells release nucleotides that form purinergic halo of “danger” signals that disrupt vascular homeostasis. Located on the surface of leukocytes and the endothelium, the vascular ectonucleotidase, CD39 rapidly phosphohydrolyzes ATP, and ADP to extinguish these “danger” signals and promote homeostasis. We have found that CD39 is a potent suppressor of the thrombo-inflammatory response in DVT. Our new preliminary data show that CD39 inhibits circulating leukocyte-platelet interactions, and restricts activation of programmed innate immune responses, including inflammasome assembly and neutrophil extracellular trap (NET) formation, key drivers of the growing thrombus. The hypothesis driving this work is that CD39 provides a critical vascular checkpoint at the intersection of innate immunity and thrombosis to arrest the relentless venous thrombo-inflammatory cycle. Using unique cell lineage (myeloid, neutrophil, endothelial)-specific CD39 gene-deleted mice we have generated, and complementary models of DVT, we will: 1) Elucidate the role for CD39 on cellular recruitment during venous thrombogenesis and maturation. These experiments will define the effect of lineage-specific CD39 on blood cell recruitment, and the spatial dynamics of their interactions during venous thrombogenesis; 2) Determine the mechanism(s) by which myeloid CD39 protects against venous thrombo-inflammation. These experiments will determine the contributions of macrophage and neutrophil CD39 to venous thrombosis. Complementary in vitro and in vivo studies with genetically-modified mice will elucidate the molecular signaling processes by which CD39 inhibits inflammasome activation during thrombogenesis; 3) Determine the effect of CD39-deficiency on microvesicle phenotype, function, and kinetics, during venous thrombosis. These studies will reveal the functional consequences of altering global- and lineage-specific CD39 on microvesicle-mediated thrombo-inflammatory signaling. These studies should yield new insights into how an ectonucleotidase that functions as a checkpoint at the intersection of thrombosis and inflammation may be exploited to improve treatments in venous thrombosis.