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

ClC-3 离子转运通过内毒素调节 NADPH 氧化酶依赖性 PMN 启动

• 批准号: 7795734
• 负责人: JESSICA G MORELAND
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7795734
• 关键词: Anions; Bacteria; Biochemical; Biological Assay; Blood Circulation; Catheters; Cell Surface Receptors; Cells; Charge; Chloride Ion; Chlorides; Chronic Granulomatous Disease; Collaborations; Cytosol; Data; Defect; Development; Disease; Electron Microscopy; Elements; Endotoxemia; Endotoxins; Exposure to; Failure; Generations; Goals; Gram-Negative Bacteria; Healthcare; Host Defense; Human; Immune; Immune response; Immune system; In Vitro; Incidence; Individual; Infection; Inflammation; Inflammatory; Invaded; Investigation; Ion Transport; Ions; Kinetics; Knowledge; Laboratories; Location; Maintenance; Measurement; Mediating; Medicine; Membrane; Microscopy; Molecular; Mus; NADPH Oxidase; Natural Immunity; Nature; Neutrophil Activation; Oxidants; Oxidases; Oxidation-Reduction; Particulate; Pathogenesis; Patients; Peptide Hydrolases; Phagocytes; Phenotype; Positioning Attribute; Process; Production; Proteins; Reaction Time; Reactive Oxygen Species; Research; Role; Sepsis; Sepsis Syndrome; Signal Transduction; Smooth Muscle Myocytes; Stimulus; Subcellular Fractions; TNF gene; Testing; Therapeutic; Tissues; antiporter; body system; care burden; cytokine; expectation; improved; in vivo; inhibitor/antagonist; innovation; interest; killings; microbicide; mortality; neutrophil; novel; patch clamp; pathogen; public health relevance; receptor expression; research study; response; tool

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.

描述（由申请人提供）：革兰氏阴性败血症的死亡率是由于全身炎症不受控制的败血症综合征的发展引起的。多形核白细胞 (PMN) 是先天免疫反应的关键细胞元件，但在完全激活时也会造成宿主组织损伤。存在多种调节机制来控制 PMN 激活水平，包括启动。启动刺激可调节 PMN 的表型，从而大大增强对后续刺激的反应，包括 NADPH 氧化酶活性的增强。内毒素是革兰氏 (-) 细菌的促炎成分，可在体外和体内诱导 PMN 启动，并在败血症患者的循环中鉴定出启动的 PMN。正常的 NADPH 氧化酶活性需要阴离子转运蛋白 ClC-3，而缺乏 ClC-3 的小鼠似乎存在先天免疫缺陷。此外，ClC-3 缺陷小鼠的平滑肌细胞中炎症细胞因子引发的氧化还原信号显着受损。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 在内毒素刺激后的启动反应明显受损。该项目的长期研究目标是更好地了解革兰氏（-）脓毒症期间中性粒细胞的启动，以增强调节宿主防御的治疗选择。该假设的方法将首先评估 ClC-3 和 NADPH 氧化酶对内毒素引发 PMN 的各自贡献，然后重点研究它们在引发过程中相互作用的机制，具体目标如下： 1) 探索 ClC-3 和 NADPH 氧化酶衍生的 ROS 在引发表型生成中的作用 2) 表征 ClC-3 和 NADPH 氧化酶之间的相互作用 LOS 启动期间的 NADPH 氧化酶。为了探索这些目标，将利用中性粒细胞亚细胞部分的生化分析与中性粒细胞功能测定相结合，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通过启动刺激来定义氧化剂产生的定位和动力学，并探索可能参与这一过程的相关离子。对人类和小鼠 PMN 以及分化的 PLB 细胞进行全细胞膜片钳分析，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。除了来自慢性肉芽肿病患者的 PMN 和 ClC-3 缺陷的鼠 PMN 之外，还将使用用 ClC-3 和 NADPH 氧化酶抑制剂治疗的人 PMN。 公共卫生相关性：脓毒症仍然是医学领域的一项重大挑战，也是沉重的医疗保健负担，死亡率高得令人无法接受。正常的中性粒细胞功能作为对细菌病原体的先天免疫反应的一个组成部分的要求已被明确证明。阴离子转运蛋白 ClC-3 似乎在中性粒细胞基本功能的多个方面发挥作用，包括 NADPH 氧化酶的调节。所提出的细菌产物引发中性粒细胞的研究将增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败血症发病机制中的功能的基本了解。