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

描述（由申请人提供）：本提案将绘制toll样受体7 （TLR7）在气道中的表达，并确定TLR7介导的新型快速支气管舒张的机制。TLR7对人类肺部免疫应答至关重要，因为TLR7检测许多呼吸道病毒基因组中常见的单链RNA，并通过NF- B触发抗病毒干扰素和炎症细胞因子的转录。我的初步数据表明，TLR7也通过转录独立的途径发出信号，诱导一氧化氮的产生，并在激活的几秒钟内引起支气管松弛。这种新机制尚未在其他TLR中被描述。此外，初步数据表明，尽管TLR7对收缩的气道组织具有深远的松弛作用，但TLR7在气道神经而非平滑肌上表达。核心假设是TLR7位于气道神经上，通过非经典TLR信号通路产生一氧化氮来放松气道平滑肌。目前的工作将通过组织全载和原代细胞培养的免疫荧光染色来确定TLR7在人和小鼠气道中的表达。TLR7的表达将定位于特定的气道神经亚型，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定位。TLR7的快速支气管松弛机制将在体外器官池实验中利用TLR7潜在信号转导途径的药物拮抗剂在野生型和敲除小鼠气道组织以及死亡供体人气道组织中进行表征。体外研究结果将在原代细胞培养实验中使用一氧化氮和细胞内钙感应荧光探针进行证实。本研究的发现有可能扩大我们对TLR7在气道中的信号转导和功能的理解，并可能为人类气道疾病提供新的治疗靶点。这些实验是主要研究者K08职业发展计划的一个组成部分，该计划展示了一个合乎逻辑的科学进展，朝着研究独立的方向发展，最终目标是在奖励期结束时获得R01水平的资助。该建议还包括一份详细的科学技能发展计划和作为导师获得熟练程度的计划。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。主要研究者的研究进展和职业发展将由一组精选的高级研究者监督，并由俄勒冈临床和转化研究所学者项目为早期职业研究者提供支持。

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-?B. 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.