Membrane potential and Calcium Signaling in Neutrophil Development and Inflammation

项目摘要 嗜中性粒细胞对于宿主防御细菌是必不可少的，然而，毒性嗜中性粒细胞介质，如 活性氧自由基，颗粒酶和中性粒细胞胞外陷阱有助于发病机制， 急性和慢性炎症性疾病。而中性粒细胞吞噬和杀死病原体的能力 已经知道了100多年，我们的知识的分子机制指导中性粒细胞活化 明显落后于其他免疫细胞，阻碍了战略治疗的发展。 针对中性粒细胞的干预措施。我们的长期目标是确定离子通道和 相关的信号通路调节中性粒细胞活化，并利用这一知识来改变 疾病中性粒细胞需要通过钙库操作的钙进入（SOCE）启动的钙信号， activation.细胞膜电位直接影响带正电荷的钙离子的流入。而 细胞膜电位在兴奋细胞中的功能作用已被广泛研究，但知之甚少 关于在发育过程中膜电位如何改变中性粒细胞的细胞过程 和炎症。该建议基于我们实验室的四个基本观察结果：1）ORAI 1和 在S.金黄色。（二） 小鼠中性粒细胞的钙反应是异质性的， 部分地通过细胞膜电位的差异调节来调节。3)钙的表达- 激活的钾通道KCa3.1（Kcnn 4）驱动细胞超极化和增强的SOCE在一个子集的 中性粒细胞4)膜电位-SOCE关系在中性粒细胞发育过程中受到调节， 紧急粒细胞生成此外，我们还观察到，一个S。金黄色葡萄球菌成孔毒素操纵 中性粒细胞膜电位和SOCE。这些观察结果共同表明， 是钙依赖性中性粒细胞功能的关键调节剂，并表明该途径是一个战略目标， 人类致病细菌分泌孔形成细胞毒素，以克服宿主中性粒细胞防御。的 本研究的目的是描述膜电位调节SOCE的机制 在中性粒细胞发育和炎症期间。我们将检验核心假设， 电位是成熟嗜中性粒细胞中嗜中性粒细胞钙信号传导的关键调节剂， 紧急粒细胞生成在目标1和2中，我们将研究KCa 3.1在中性粒细胞SOCE中的作用， 成熟和发育中的中性粒细胞中的钙依赖性活化。在目标3中，我们将扩展这些研究， 研究细菌致孔毒素如何外源性操纵细胞膜电位 破坏钙依赖性中性粒细胞功能。从这些研究中得出的见解预计将 为调节嗜中性粒细胞依赖性感染和炎性 疾病，并可能为免疫细胞功能中的膜电位提供更广泛的理解。

PROJECT SUMMARY Neutrophils are essential for host defense against bacteria, however toxic neutrophil mediators such as reactive oxygen radicals, granule enzymes and neutrophil extracellular traps contribute the pathogenesis of acute and chronic inflammatory diseases. While the ability of neutrophils to phagocytose and kill pathogens has been known for over 100 years, our knowledge of the molecular mechanisms guiding neutrophil activation lags significantly behind that of other immune cells, precluding the development of strategic therapeutic interventions targeting neutrophils. Our long-term goal is to define the mechanisms by which ion channels and associated signaling pathways regulate neutrophil activation, and to leverage this knowledge to modify disease. Calcium signals initiated via store-operated calcium entry (SOCE) are required for neutrophil activation. The cell membrane potential directly influences influx of positively charged calcium ions. While the functional role of the cell membrane potential has been extensively studied in excitable cells, little is known about how the membrane potential modifies cellular processes in neutrophils in the context of development and inflammation. This proposal is based on four fundamental observations from our laboratory: 1) ORAI1 and ORAI2 calcium channels are critical for neutrophil SOCE and host defense during infection with S. aureus. 2) Calcium responses in mouse neutrophils are heterogenous, with the magnitude of the calcium response modulated in part by differential regulation of the cell membrane potential. 3) Expression of the calcium- activated potassium channel KCa3.1 (Kcnn4) drives cell hyperpolarization and enhanced SOCE in a subset of neutrophils. 4) The membrane potential-SOCE relationship is modulated during neutrophil development and emergency granulopoiesis. Moreover, we have observed that a S. aureus pore-forming toxin manipulates the neutrophil membrane potential and SOCE. Together these observations illustrate that the membrane potential is a key modifier of calcium-dependent neutrophil function, and suggest that this pathway is a strategic target of human pathogenic bacteria that secrete pore-forming cytotoxins to overcome host neutrophil defenses. The objective of this proposal is to characterize the mechanisms by which membrane potential regulates SOCE during neutrophil development and inflammation. We will test the central hypothesis that the membrane potential is a critical modifier of neutrophil calcium signaling in mature neutrophils and homeostatic and emergency granulopoiesis. In Aims 1 and 2 we will investigate the role of KCa3.1 in neutrophil SOCE and calcium-dependent activation in mature and developing neutrophils. In Aim 3 we will expand these studies to investigate how exogenous manipulation of the cell membrane potential by bacterial pore-forming toxins disrupts calcium-dependent neutrophil function. The insight derived from these studies is anticipated to engender new clinical opportunities for modulation of neutrophil-dependent infectious and inflammatory disease, and potentially inform a broader understanding of membrane potential in immune cell function.