ClC-3 ion transport modulates NADPH oxidase-dependent PMN priming by endotoxin

描述（由申请人提供）：革兰氏阴性脓毒症的死亡率源于脓毒症综合征的发展与不受控制的全身炎症。多形核白细胞（pmn）是先天免疫反应的关键细胞成分，但在完全激活时也负责宿主组织损伤。存在多种调控机制来控制PMN的激活水平，包括启动。启动刺激调节PMN的表型，从而对后续刺激的反应被大大放大，包括NADPH氧化酶活性的增强。内毒素是革兰氏（-）细菌的一种促炎成分，在体外和体内诱导PMN启动，在脓毒症患者的循环中发现了启动的PMN。阴离子转运体ClC-3是正常NADPH氧化酶活性所必需的，缺乏ClC-3的小鼠似乎存在先天免疫缺陷。此外，炎症细胞因子引发的氧化还原信号在ClC-3缺陷小鼠的平滑肌细胞中明显受损。该建议的总体假设是阴离子转运体ClC-3在内毒素启动过程中调节NADPH氧化酶依赖的反应。这一假设得到了强有力的初步数据的支持，这些数据表明，缺乏ClC-3的PMNs在内毒素刺激后明显损害了启动反应。该项目的长期研究目标是更好地了解Gram（-）败血症期间的PMN启动，以增强调节宿主防御的治疗选择。该假设的方法将包括首先评估ClC-3和NADPH氧化酶对内毒素启动PMN的个体贡献，然后重点研究它们在启动过程中的相互作用机制，具体目的如下：1)探索ClC-3和NADPH氧化酶衍生的ROS在启动表型产生中的作用2)表征ClC-3和NADPH氧化酶在LOS启动过程中的相互作用。为了探索这些目标，将利用PMN亚细胞组分的生化分析与中性粒细胞功能分析相结合，包括测量NADPH氧化酶活性、脱颗粒和细胞表面受体表达。共聚焦和电子显微镜研究将定义启动刺激产生氧化剂的定位和动力学，并探索可能参与这一过程的相关离子。人类和小鼠PMNs以及分化的PLB细胞的全细胞膜片钳分析将用于直接观察相关电导，并允许使用分子工具直接关注ClC-3的作用。用ClC-3和NADPH氧化酶抑制剂治疗的人pmn将被用于慢性肉芽肿疾病患者的pmn和小鼠ClC-3缺陷pmn。

DESCRIPTION (provided by applicant): The mortality from Gram-negative sepsis arises from development of the sepsis syndrome with uncontrolled systemic inflammation. Polymorphonuclear leukocytes (PMNs) are critical cellular elements of the innate immune response, but are also responsible for host tissue damage when fully activated. Diverse regulatory mechanisms exist to control the level of PMN activation, including priming. A priming stimulus modulates the phenotype of the PMN so that the response to subsequent stimuli is greatly amplified, including enhancement of NADPH oxidase activity. Endotoxin, a pro-inflammatory component of Gram (-) bacteria induces PMN priming in vitro and in vivo with primed PMNs identified in the circulation of patients with sepsis. The anion transporter ClC-3 is required for normal NADPH oxidase activity and mice deficient in ClC-3 appear to have a defect in innate immunity. In addition, redox signaling elicited by inflammatory cytokines is markedly impaired in smooth muscle cells from ClC-3 deficient mice. The overall hypothesis of this proposal is that the anion transporter ClC-3 modulates NADPH oxidase-dependent responses during endotoxin priming. This hypothesis is supported by strong preliminary data demonstrating that PMNs lacking ClC-3 have markedly impaired priming responses after stimulation with endotoxin. The long-term research goal of this project is to better understand PMN priming during Gram (-) sepsis in order to enhance therapeutic options to modulate host defense. The approach to the hypothesis will include first an assessment of the individual contributions of ClC-3 and the NADPH oxidase to PMN priming by endotoxin, followed by a focused investigation of the mechanism of their interaction during the priming process with the following specific aims: 1) To explore the roles of ClC-3 and NADPH oxidase-derived ROS in the generation of the primed phenotype 2) To characterize the interaction between ClC-3 and the NADPH oxidase during LOS priming. To explore these aims biochemical analyses of PMN subcellular fractions in combination with neutrophil functional assays including measurement of NADPH oxidase activity, degranulation, and cell surface receptor expression will be utilized. Confocal and electron microscopy studies will define the localization and kinetics of oxidant generation by priming stimuli, and explore the relevant ions that might participate in this process. Whole cell patch clamp analysis of human and murine PMNs along with differentiated PLB cells will be employed to directly observe the relevant conductances, and will allow the use of molecular tools to focus directly on the role of ClC-3. Human PMNs treated with inhibitors of ClC-3 and the NADPH oxidase will be used in addition to PMNs from patients with chronic granulomatous disease, and murine ClC-3 deficient PMNs. PUBLIC HEALTH RELEVANCE: Sepsis continues to be a major challenge in medicine and a profound health care burden with unacceptably high mortality. The requirement for normal neutrophil function as a component of the innate immune response to bacterial pathogens has been unequivocally demonstrated. The anion transporter ClC-3 appears to have a role in several aspects of basic neutrophil function including modulation of the NADPH oxidase. The studies of neutrophil priming by bacterial products that are proposed will advance our fundamental knowledge of the function of neutrophils in the maintenance of normal immune system function and in the pathogenesis of Gram-negative sepsis.