THE MOLECULAR REGULATION OF NEUTROPHIL CALCIUM SIGNALING IN ACUTE LUNG INJURY

 描述(申请人提供)：急性肺损伤(ALI)是一种毁灭性的疾病，其特征是肺泡上皮屏障破坏，导致肺水肿和低氧血症。目前还没有有效的疾病修正疗法，支持性护理仍然是医疗管理的支柱。最终，ALI会导致多达30%的患者出现多器官功能障碍和/或死亡。中性粒细胞对宿主抵抗细菌具有重要作用，但中性粒细胞中毒性介质如活性氧自由基、颗粒酶和中性粒细胞胞外陷阱也通过引起内皮、实质和肺泡损伤而参与肺损伤的发病机制。中性粒细胞是操纵炎症反应的主要靶点，因此了解引导中性粒细胞反应的分子机制至关重要。克莱门斯博士的长期研究目标是了解细胞内信号通路如何在急性肺损伤和其他炎症性疾病期间调节中性粒细胞的激活。钙是一种进化上保守的信号信使，是多种信号通路的中心组成部分。在免疫细胞中，胞浆内钙的增加是通过存储操作钙内流(SOCE)控制的，当内质网(ER)存储耗尽时，钙释放激活的钙(CRAC)通道允许细胞外钙内流。STIM蛋白是一种内质网钙“传感器”，它可以感知钙离子的耗竭，并直接门控CRAC通道的一个孔亚基ORAI。对STIM、ORAI或中性粒细胞钙信号的分子调控知之甚少。该方案的目的是确定调节中性粒细胞SOCE的分子途径，并确定这些途径如何在ALI过程中调节中性粒细胞的激活。核心假设是：1)STIM钙感受器和ORAI钙通道协同调节中性粒细胞钙信号；2)钙依赖的中性粒细胞激活在ALI的诱导过程中起关键作用。AIMS 1和AIMS 2中提出的研究将确定中性粒细胞SOCE所需的分子机制，并使用一组独特的中性粒细胞特定缺失的STIM或ORAI基因来确定中性粒细胞钙信号在急性肺损伤中的作用。目标 3将利用一种新的方法，用人源化的小鼠在人类中性粒细胞中测试这些假设。该项目与NIH和NIAID的任务相关，因为这些研究将 阐明以前未被探索的中性粒细胞钙信号通路，这可能导致治疗急性肺损伤和其他炎症过程的新的治疗方法。

 DESCRIPTION (provided by applicant): Acute lung injury (ALI) is a devastating disease characterized by alveolar-epithelial barrier disruption and resultant pulmonary edema and hypoxemia. There are currently no effective disease-modifying therapies and supportive care remains the mainstay of medical management. Ultimately, ALI leads to multi-organ dysfunction and/or death in up to 30% of patients. Neutrophils are clearly important for host defense against bacteria, however toxic neutrophil mediators such as reactive oxygen radicals, granule enzymes and neutrophil extracellular traps also contribute to the pathogenesis of lung injury by causing endothelial, parenchymal and alveolar injury. Neutrophils are prime targets for manipulating the inflammatory response and therefore it is critical to understand the molecular mechanisms that guide neutrophil responses. Dr. Clemens' long-term research goal is to understand how intracellular signaling pathways regulate neutrophil activation during acute lung injury and other inflammatory diseases. Calcium is an evolutionarily conserved signaling messenger that is a central component of multiple signaling pathways. In immune cells, increases in cytoplasmic calcium are controlled via store operated calcium entry (SOCE), where calcium release-activated calcium (CRAC) channels allow influx of extracellular calcium when endoplasmic reticulum (ER) stores are depleted. STIM proteins are ER calcium "sensors" which sense calcium depletion and directly gate ORAI, a pore subunit of the CRAC channel. Little is known about STIM, ORAI or the molecular regulation of calcium signaling in neutrophils. The objectives of this proposal are to define the molecular pathways that regulate SOCE in neutrophils and to determine how these pathways modulate neutrophil activation during ALI. The central hypotheses are: 1) STIM calcium sensors and ORAI calcium channels cooperate to regulate neutrophil calcium signaling, and 2) calcium-dependent neutrophil activation is critical for induction of ALI. The studies proposed in Aims 1 and 2 will identify the molecular machinery required for SOCE in neutrophils and determine the role of neutrophil calcium signaling in acute lung injury using a unique set of mice with neutrophil specific deletion in Stim or Orai genes. Aim 3 will utilize a novel approach with humanized mice to test these hypotheses in human neutrophils. This project is relevant to the missions of the NIH and NIAID since these studies will elucidate previously unexplored pathways of calcium signaling in neutrophils that may lead to new therapeutic approaches to treat acute lung injury and other inflammatory processes.