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

描述（由申请人提供）：该提案将绘制气道中Toll样受体7（TLR 7）的表达图，并鉴定新型快速TLR 7介导的支气管舒张的机制。 TLR 7对人类肺部免疫应答至关重要，因为TLR 7检测许多呼吸道病毒基因组共有的单链RNA，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，TLR 7也通过转录非依赖性途径发出信号，诱导一氧化氮产生，并在激活后几秒钟内引起支气管舒张。 这种新机制尚未在任何其他TLR中描述。 此外，初步数据表明，TLR 7在气道神经上表达，而不是平滑肌，尽管TLR 7对收缩的气道组织具有显著的松弛作用。 中心假设是TLR 7位于气道神经上，并通过非经典TLR信号传导途径产生一氧化氮来松弛气道平滑肌。 目前的工作将确定TLR 7表达在人类和小鼠气道使用免疫荧光染色的组织整体安装和原代细胞培养制备。 TLR 7的表达将被定位到特定的气道神经亚型，并与一氧化氮合酶同工酶共定位，使用高分辨率激光扫描共聚焦显微镜。 TLR 7的快速支气管舒张机制将在体外器官浴实验中使用野生型和敲除鼠气道组织中以及死亡供体人气道组织中潜在TLR 7信号转导途径的药理学拮抗剂来表征。 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敏感荧光探针进行确认。 这一发现对扩大我们对TLR 7在气道中的信号转导和功能的理解具有重要意义，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K 08职业发展计划的一个组成部分，该计划表明了朝着研究独立性的逻辑科学进展，最终目标是在奖励期结束时获得R 01级资金。 本提案还包括一个详细的科学技能开发计划和作为导师获得熟练程度的计划。 俄勒冈州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，并由俄勒冈州临床和转化研究所学者计划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-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.