Membrane potential and Calcium Signaling in Neutrophil Development and Inflammation

项目总结 中性粒细胞对于宿主抵抗细菌是必不可少的，然而有毒的中性粒细胞介质，如 活性氧自由基、颗粒酶和中性粒细胞胞外陷阱参与了糖尿病的发病机制。 急慢性炎症性疾病。而中性粒细胞吞噬和杀死病原体的能力 已经知道100多年了，我们对引导中性粒细胞激活的分子机制的了解 显著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。我们的长期目标是定义离子通道和 相关的信号通路调节中性粒细胞的激活，并利用这一知识来修改 疾病。中性粒细胞需要通过存储操作钙内流(SOCE)启动的钙信号 激活。细胞膜电位直接影响带正电荷的钙离子的内流。而当 细胞膜电位在兴奋性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。这一建议是基于我们实验室的四个基本观察结果：1)ORAI1和 在金黄色葡萄球菌感染过程中，ORAI2钙通道对中性粒细胞SOCE和宿主防御起关键作用。2) 小鼠中性粒细胞的钙反应是异质性的，钙反应的大小 部分地受细胞膜电位的不同调节。3)钙离子的表达- 激活的钾通道KCa3.1(Kcnn4)驱动细胞超极化和增强的SOCE 中性粒细胞。4)膜电位-SOCE关系在中性粒细胞发育和 紧急粒系造血术。此外，我们还观察到金黄色葡萄球菌的一种成孔毒素能操纵 中性粒细胞膜电位和SOCE。这些观察结果表明，膜电势 是钙依赖的中性粒细胞功能的关键调节剂，提示该途径是一个战略靶点。 指人类致病细菌，其分泌形成孔洞的细胞毒素以克服宿主的中性粒细胞防御。这个 这项建议的目的是表征膜电位调节SOCE的机制 在中性粒细胞发育和炎症过程中。我们将检验中心假说，即膜 在成熟的中性粒细胞中，电势是中性粒细胞钙信号的关键修饰物，动态平衡和 紧急粒系造血术。在目标1和目标2中，我们将研究KCa3.1在中性粒细胞SOCE和 成熟和发育中的中性粒细胞的钙依赖激活。在目标3中，我们将把这些研究扩展到 探讨细菌致孔毒素对细胞膜电位的外源调控 破坏钙依赖的中性粒细胞功能。从这些研究中得出的见解预计将 为调节中性粒细胞依赖的感染和炎症提供新的临床机会 疾病，并有可能使我们更广泛地了解免疫细胞功能中的膜电位。

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.