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

描述（由申请方提供）：革兰氏阴性脓毒症的死亡率由脓毒症综合征伴不受控制的全身性炎症的发展引起。多形核白细胞（PMN）是先天性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。存在多种调节机制来控制PMN激活的水平，包括引发。引发刺激调节PMN的表型，使得对后续刺激的反应被极大地放大，包括NADPH氧化酶活性的增强。内毒素是革兰氏（-）菌的促炎成分，在体外和体内诱导PMN启动，并在脓毒症患者的循环中鉴定出启动的PMN。阴离子转运蛋白ClC-3是正常NADPH氧化酶活性所必需的，ClC-3缺陷的小鼠似乎具有先天免疫缺陷。此外，由炎症细胞因子引起的氧化还原信号传导在来自ClC-3缺陷小鼠的平滑肌细胞中显著受损。该提议的总体假设是阴离子转运蛋白ClC-3在内毒素引发期间调节NADPH氧化酶依赖性反应。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PMN在用内毒素刺激后具有明显受损的引发反应。本项目的长期研究目标是更好地了解革兰氏（-）脓毒症过程中的PMN启动，以增强调节宿主防御的治疗选择。该假设的方法将首先包括评估ClC-3和NADPH氧化酶对内毒素引起的PMN引发的个体贡献，然后重点研究引发过程中它们相互作用的机制，具体目标如下：1）探索ClC-3和NADPH氧化酶衍生的ROS在引发表型产生中的作用3和NADPH氧化酶在LOS引发过程中。为了探索这些目标，将利用PMN亚细胞组分的生化分析结合中性粒细胞功能测定，包括测量NADPH氧化酶活性、脱粒和细胞表面受体表达。共聚焦和电子显微镜的研究将定义的本地化和动力学的氧化剂产生的启动刺激，并探讨可能参与这一过程的相关离子。将采用人和鼠PMN沿着分化的PLB细胞的全细胞膜片钳分析来直接观察相关电导，并且将允许使用分子工具来直接关注ClC-3的作用。除了来自慢性肉芽肿病患者的PMN和鼠ClC-3缺陷型PMN之外，还将使用用ClC-3和NADPH氧化酶抑制剂处理的人PMN。 公共卫生相关性：脓毒症仍然是医学上的一个重大挑战，也是一个严重的卫生保健负担，死亡率高得令人无法接受。已经明确证明，正常中性粒细胞功能是对细菌病原体的先天免疫应答的组成部分。阴离子转运蛋白ClC-3似乎在中性粒细胞基本功能的几个方面具有作用，包括NADPH氧化酶的调节。提出的细菌产物对中性粒细胞致敏的研究将推进我们对中性粒细胞在维持正常免疫系统功能和革兰氏阴性脓毒症发病机制中的功能的基础知识。

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.