Rapid Toll-like receptor 7-mediated nitric oxide production in the airway

Toll 样受体 7 介导的气道中快速一氧化氮的产生

• 批准号: 9304273
• 负责人: Matthew G. Drake
• 金额: $ 15.88万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9304273
• 关键词: Agonist; Antiviral Agents; Award; Bathing; Calcium; Clinical Research; Contracts; Data; Development Plans; Disease; Dose; Environment; Epithelial; Fluorescent Probes; Functional disorder; Funding; Genetic Transcription; Goals; Health Sciences; Human; Imiquimod; Immune response; Immunofluorescence Immunologic; In Vitro; Inflammatory; Interferons; Isoenzymes; Knock-out; Laser Scanning Confocal Microscopy; Lead; Lung; Maps; Mediating; Mentors; Mus; Muscle relaxation phase; NCI Scholars Program; Nerve; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthetase Inhibitor; Oregon; Organ; Pathway interactions; Pharmacology; Physiological; Physiology; Preparation; Primary Cell Cultures; Production; RNA; Relaxation; Research; Research Institute; Research Personnel; Resolution; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Smooth Muscle; Source; Stains; TLR7 gene; Testing; Time; Tissue Stains; Tissues; Transcriptional Regulation; Translational Research; Universities; Viral Genome; Virus; Virus Receptors; Work; airway epithelium; airway hyperresponsiveness; career; career development; cytokine; design; experimental study; new therapeutic target; novel; omega-N-Methylarginine; public health relevance; respiratory smooth muscle; respiratory virus; response; skill acquisition; therapeutic target

DESCRIPTION (provided by applicant): This proposal will map expression of Toll-like receptor 7 (TLR7) in the airway and identify the mechanism of novel rapid TLR7-mediated bronchorelaxation. TLR7 is vital to human pulmonary immune responses in that TLR7 detects single-stranded RNA, common to many respiratory virus genomes, and triggers transcription of antiviral interferons, and inflammatory cytokines via NF-κB. My initial data suggest TLR7 also signals through a transcription-independent pathway, to induce nitric oxide production and cause bronchorelaxation within seconds of activation. This novel mechanism has not been described for any other TLR. Furthermore, preliminary data indicate TLR7 is expressed on airway nerves, but not smooth muscle, despite TLR7's profound relaxant effect on contracted airway tissues. The central hypothesis is that TLR7 is located on airway nerves and relaxes airway smooth muscle through production of nitric oxide by a non-classical TLR signaling pathway. The current work will define TLR7 expression in human and murine airways using immunofluorescence staining of tissue whole mount and primary cell culture preparations. TLR7 expression will be localized to specific airway nerve subtypes, and colocalized with nitric oxide synthase isozymes using high-resolution laser-scanning confocal microscopy. TLR7's rapid bronchorelaxant mechanism will be characterized using pharmacologic antagonists of potential TLR7 signal transduction pathways in wild-type and knockout murine airway tissues, and in deceased donor human airway tissues in organ bath experiments in vitro. In vitro findings will be confirmed in primary cell culture experiments using nitric oxide- and intracellula calcium-sensing fluorescent probes. This proposal's findings have significant potential to expand our understanding of TLR7's signal transduction and function in the airways, and may provide novel therapeutic targets for human airway diseases. These experiments are one component of the principle investigator's K08 career development plan that demonstrates a logical scientific progression towards research independence with the ultimate goal of obtaining R01 level funding by the end of the award period. A detailed plan for hands on scientific skill development and obtaining proficiency as a mentor are also included in this proposal. Lending additional evidence to the feasibility of this proposal is the supportive research environment at Oregon Health and Science University. The principle investigator's research progress and career development will be overseen by a select group of senior investigators, and supported by the Oregon Clinical and Translational Research Institute Scholar's Program for early career investigators.

描述(申请人提供)：这项建议将绘制Toll样受体7(TLR7)在呼吸道中的表达图，并确定TLR7介导的新型快速支气管松弛的机制。TLR7对人类的肺部免疫反应至关重要，因为TLR7可以检测许多呼吸道病毒基因组中常见的单链RNAs，并通过NF-κB触发抗病毒干扰素和炎性细胞因子的转录。我的初步数据表明，TLR7还通过一条转录不依赖的途径发出信号，在激活后几秒钟内诱导一氧化氮的产生和引起支气管松弛。这一新的机制还没有被描述用于任何其他TLR。此外，初步数据表明，尽管TLR7的S对收缩的呼吸道组织有深刻的松弛作用，但TLR7在呼吸道神经上表达，而不是在平滑肌上表达。中心假说是，TLR7位于呼吸道神经上，通过非经典的TLR信号通路产生一氧化氮，起到松弛气道平滑肌的作用。目前的工作将使用组织整体和原代细胞培养准备的免疫荧光染色来确定TLR7在人和小鼠呼吸道中的表达。TLR7的表达将定位于特定的呼吸道神经亚型，并使用高分辨率激光扫描共聚焦显微镜与一氧化氮合酶同工酶共同定位。在体外器官浴实验中，将利用TLR7信号转导通路的药理拮抗剂，在野生型和基因敲除的小鼠呼吸道组织以及死亡的供体人呼吸道组织中研究TLR7的S快速松弛支气管炎的机制。在体外，使用一氧化氮和细胞内钙感应荧光探针的原代细胞培养实验将证实这一发现。本研究结果对进一步了解TLR7的S信号转导途径及其在呼吸道中的作用具有重要意义，并可能为人类呼吸道疾病的治疗提供新的靶点。这些实验是首席研究员K08职业发展计划的一个组成部分，该计划展示了朝着研究独立的合乎逻辑的科学进展，最终目标是在获奖期结束前获得R01水平的资金。这份提案中还包括了一份详细的计划，内容涉及到科学技能的发展和作为导师获得熟练程度。俄勒冈健康与科学大学的支持性研究环境为这一提议的可行性提供了额外的证据。首席调查员的研究进展和职业发展将由一组精选的高级调查员监督，并得到俄勒冈州临床和翻译研究所早期职业调查员学者计划的支持。