Calcium dependent mechanisms of neutrophil dysfunction that contribute to cystic fibrosis pathobiology

 描述(申请人提供)：囊性纤维化(CF)是一种常染色体隐性遗传病，由囊性纤维化跨膜传导调节因子(CFTR)基因突变引起，其特征是上皮离子转运异常、粘液、慢性细菌感染和过度的呼吸道炎症。引起肺部感染的机会性细菌，包括伯克霍尔德氏菌(B.cenocepacia)，是对CF患者的致命威胁。盲肠弯曲杆菌对抗生素具有抗药性，宿主吞噬细胞无法清除感染，导致严重炎症。中性粒细胞在控制肺部感染和炎症方面起着至关重要的作用，但在CF患者中；中性粒细胞不能根除细菌和促进炎症。CFTR是一种氯离子通道，调节上皮液在肺内的运输。CFTR在吞噬细胞中的功能，以及CFTR缺陷如何影响吞噬细胞的炎症活动尚不清楚。CFTR突变已证实可直接改变呼吸道上皮细胞的钙稳态，但该突变对中性粒细胞钙依赖功能的影响仍有待确定。在中性粒细胞中，我们发现伴随着钙通道TRPM2的钙信号增加，而活性氧(ROS)的产生和内毒素缺乏。因此，我们假设cftr突变导致细胞内钙离子释放增加。钙离子稳态的改变反过来，正向调节钙依赖的炎性中性粒细胞功能，但下调NADPH活性，从而导致中性粒细胞缺陷的细菌杀伤。为了检验这一假设，我们将：1)。确定CFTR突变如何影响中性粒细胞中的钙信号通路2)。确定轴TRPM2/NADPH氧化酶如何影响人和小鼠CF中性粒细胞的抗菌功能。这项研究的成功完成将大大增加我们对中性粒细胞如何参与CF病理生物学的理解，并将有助于通过靶向调节CFTR和NADPH氧化酶的Ca+2信号通路来控制CF患者的感染和炎症，从而帮助定义新的治疗策略。

 DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene and characterized by abnormal epithelial ion transport, viscous mucus, chronic bacterial infection, and exaggerated airway inflammation. Opportunistic bacteria that cause lung infection, including Burkholderia cenocepacia (B. cenocepacia), are lethal threat to CF patients. B. cenocepacia is resistant to antibiotics, and host phagocytes fail to clear the infection causing severe inflammation. Neutrophils play essential roles in controlling lung infection and inflammation, however in CF patients; neutrophils cannot eradicate bacteria and promote inflammation. CFTR is a chloride ion channel known to regulate epithelial fluid transport in the lung. The function of CFTR in phagocytes, and how CFTR deficiency affects the inflammatory activities of phagocytic cells is unknown. CFTR mutation has demonstrated to alter directly calcium (Ca2+) homeostasis in airway epithelia, but the impact of this mutation on neutrophil Ca2+-dependent functions remains to be determined. In CF neutrophils, we have found that Ca2+ signaling along with the Ca2+ channel TRPM2 are increased, whereas the production of reactive oxygen species (ROS), and NETosis are deficient. Hence, we hypothesize that CFTR mutation results in increased cytosolic release of Ca2+. Altered Ca2+ homeostasis in turn, positively regulates Ca2+-dependent inflammatory neutrophil functions, but down regulates NADPH activity contributing to neutrophil defective bacterial killing. To test this hypothesis we will: 1). Determine how CFTR mutation impacts calcium signaling pathways in neutrophils 2). Define how the axis TRPM2/NADPH oxidase affects antimicrobial functions of human and mouse CF neutrophils. The successful completion of this proposal will significantly increase our understanding of how neutrophils contribute to CF pathobiology, and will help define novel therapeutic strategies by targeting Ca+2 signaling pathways that modulate CFTR and NADPH oxidase to control infection and inflammation in CF patients.