Role of anion transporters in the regulation of PMN chemotaxis

阴离子转运蛋白在中性粒细胞趋化性调节中的作用

• 批准号: 7893266
• 负责人: Alexandra Paige Davis Volk
• 金额: $ 12.74万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7893266
• 关键词: 1-Phosphatidylinositol 3-Kinase; Address; Affect; Anions; Biological; Biological Assay; Catheters; Cause of Death; Cell Volumes; Cells; Charge; Chemotactic Factors; Chemotaxis; Chloride Ion; Chlorides; Collaborations; Complement; Complement 5a; Complex; Confocal Microscopy; Data; Education; Effector Cell; Electron Microscopy; Electrons; Electrophysiology (science); Employee Strikes; Endotoxins; Environment; Equilibrium; Event; Family; Flow Cytometry; Fluorescence Microscopy; Fostering; Functional disorder; Generations; Goals; Homologous Gene; Host Defense; Host Defense Mechanism; Human; Hydrogen Peroxide; Image Analysis; Immune response; Immunofluorescence Immunologic; Indium; Infection; Inflammation; Inflammatory Response; Intensive Care Units; Investigation; Ion Channel; Ions; Knowledge; Laboratories; Leukocyte Cell Biology; Leukocytes; Leukotrienes; Link; Mediating; Medical; Mentors; Microscopic; Mission; Modeling; Molecular; Molecular Immunology; Movement; Mus; NADPH Oxidase; Natural Immunity; Null Lymphocytes; Oxidants; Pathway interactions; Patients; Phosphoric Monoester Hydrolases; Physicians; Positioning Attribute; Process; Production; Proteins; Regulation; Research; Resources; Role; Science; Scientist; Secretory Vesicles; Sepsis; Shapes; Signal Pathway; Signal Transduction; Signaling Protein; Site; Stimulus; Swelling; Technical Expertise; Techniques; Therapeutic; Tissues; Transfection; Translating; United States; United States National Institutes of Health; Work; antiporter; body system; burden of illness; career; career development; cell motility; chemokine; cytokine; disability; experience; extracellular; genetic manipulation; human MAPK14 protein; improved; in vivo; innovation; interest; intravital microscopy; killings; member; methionyl-leucyl-phenylalanine; microbial; microbicide; migration; neutrophil; novel; pathogen; programs; research study; response; skills; tensin; therapeutic target; two-photon

DESCRIPTION (provided by applicant): The goal of this proposal is to foster the career development of Dr. A. Paige Volk while undertaking the investigation of a fundamental component of innate host defense, neutrophil (PMN) chemotaxis, i.e. directed motility. Dr. Volk's long term career goal is become an independent physician-scientist with the education, research skills and experience necessary to make a significant contribution to bio-medical science. Her more short-term goals are: 1) to advance Dr. Volk's expertise in cellular and molecular immunology through both coursework and molecular biological experimentation; and 2) to define mechanisms by which anion transporters and oxidants regulate PMN chemotaxis. Dr. Volk's career development would take place in the nurturing environment of the NIH-supported Inflammation Program, with Dr. Jessica Moreland as her mentor, and Dr. Fred Lamb as co-mentor. This environment provides Dr. Volk full access to the resources and scientific expertise required to make a significant contribution to the study of innate host defense. Dr. Volk began to investigate the role of the anion transporter, ClC-3, in PMN function after mice lacking this protein demonstrated impaired innate immunity and deficits in PMN function. The hypothesis of this proposal is that cell volume regulation via the swelling-induced chloride current, IClswell, is mediated by oxidant signaling and required for chemotaxis to select stimuli. This hypothesis will be addressed with the following aims: 1) To characterize mechanisms of cell volume regulation in PMN chemotaxis; and 2) To characterize the oxidant-sensitive signaling interactions that distinguish chemotaxis to select stimuli and determine the role of ClC-3 in this process. To explore these aims a combination of molecular analyses and functional assays of human and murine PMN will be used. Localized volume changes and chloride movement will be studied using advanced 2D/3D microscopic image analysis, confocal fluorescence microscopy and electrophysiology techniques. Regulation of chemotaxis signaling pathways by ClC-3- modulated oxidants will be assessed by confocal microscopy and flow cytometry using a novel competitive chemotaxis assay. The role of ClC-3 and intracellular oxidants in chemotaxis will be studied in vivo using advanced two-photon intravital microscopy techniques. This proposal is highly innovative in that we will elucidate a previously unappreciated role for intracellular oxidants in chemotaxis signaling. The requirement for normal PMN function in the innate immune response during sepsis has been demonstrated unequivocally and the anion transporters ClC-3 and IClswell are required for normal PMN chemotaxis. Uncontrolled PMN activation and migration into healthy tissues during severe sepsis leads to multiple organ system dysfunction. The current project will significantly advance the understanding of neutrophil migration mechanisms that can ultimately be translated into therapeutic strategies to reduce the burden of illness and disability due to sepsis

描述(由申请人提供)：本提案的目标是促进A.Paige Volk博士的职业发展，同时从事天然宿主防御的一个基本组成部分-中性粒细胞(PMN)趋化性的研究，即定向运动。沃尔克博士的长期职业目标是成为一名独立的内科科学家，拥有必要的教育、研究技能和经验，为生物医学做出重大贡献。她更短期的目标是：1)通过课程和分子生物学实验提高Volk博士在细胞和分子免疫学方面的专业知识；2)确定阴离子转运体和氧化剂调节PMN趋化性的机制。沃尔克博士的职业发展将在NIH支持的炎症项目的培育环境中进行，杰西卡·摩兰博士是她的导师，弗雷德·兰姆博士是共同导师。这种环境为Volk博士提供了充分的资源和科学专业知识，为研究宿主固有防御做出了重大贡献。Volk博士开始研究阴离子转运蛋白ClC-3在PMN功能中的作用，此前缺乏该蛋白的小鼠表现出先天免疫功能受损和PMN功能缺陷。这一设想的假设是，通过肿胀诱导的氯电流IClswell调节细胞体积是由氧化剂信号介导的，是趋化性选择刺激所必需的。这一假说的目的如下：1)研究中性粒细胞趋化作用中细胞体积调节的机制；2)研究氧化敏感的信号相互作用，以区分趋化作用以选择刺激，并确定ClC-3在这一过程中的作用。为了探索这些目标，将使用人和小鼠PMN的分子分析和功能分析相结合的方法。将使用先进的2D/3D显微图像分析、共聚焦荧光显微镜和电生理学技术来研究局部体积变化和氯化物移动。共聚焦显微镜和流式细胞术将使用一种新的竞争性趋化分析来评估ClC-3调节的氧化剂对趋化信号通路的调控。CLC-3和细胞内氧化剂在趋化作用中的作用将使用先进的双光子活体显微镜技术在体内进行研究。这一建议具有很高的创新性，因为我们将阐明细胞内氧化剂在趋化信号中以前未被认识到的作用。 败血症时天然免疫反应中需要正常的PMN功能，而阴离子转运蛋白ClC-3和IClswell是正常PMN趋化所必需的。严重脓毒症时，PMN不受控制地激活和迁移到健康组织中，导致多器官系统功能障碍。目前的项目将极大地促进对中性粒细胞迁移机制的理解，这些机制最终可以转化为治疗策略，以减轻败血症引起的疾病和残疾的负担。