ACUTE AIRWAY EFFECTS OF TLR7 AND TLR8 STIMULATION IN HEALTH AND DISEASE

描述(申请人提供)：病毒单链RNA通过Toll样受体(TLR)-7和TLR-8被细胞感知。我们最近发现，刺激TLR-7和TLR-8可以松弛气道平滑肌，防止因各种刺激而引起的支气管收缩。这种影响是深远的，可以导致几乎完全的平滑肌松弛。我们已经在豚鼠和小鼠身上以及在人类呼吸道组织中证明了这一效应。在豚鼠和人的呼吸道中，TLR7依赖的支气管扩张是由一氧化氮的产生介导的，而TLR8依赖的支气管扩张则不是。我们的初步数据表明，TLR8介导的支气管扩张是由前列腺素的产生和大电导钙激活钾通道的开放所介导的。我们还发现，抗原致敏和急性病毒感染都会严重损害TLR依赖的支气管扩张。在这一应用中，我们提出了三个特定的目的：具体目的1)利用TLR7(-/-)小鼠以及新生的TLR8(-/-)和TLR7(-/-)TLR8(-/-)小鼠，更深入地研究和建立这些受体对呼吸道神经元和平滑肌影响的信号通路，B)检测这些受体是否通过一氧化氮、前列腺素和体外人呼吸道大电导、钙激活钾通道(BKCa)来传递信号，以及c)在原代培养的人气道副交感神经细胞和气道平滑肌细胞中，探讨一氧化氮和前列腺素以及细胞内钙离子的变化在这些受体快速信号传递中的作用。特异性目标2：检测抗原致敏后TLR7和/或TLR8的减少是否介导了TLR介导的支气管扩张，测试了AIM#1中确定的气道平滑肌信号通路。由于这些TLR存在于副交感神经以及聚集到气道神经的嗜酸性粒细胞上，我们还将测试TLR7和TLR8是否改变致敏的呼吸道的神经控制。特异性目的3：检测TLR7和/或TLR8信号通路的减少是否通过TLR7和/或TLR8信号通路的减少介导病毒感染后TLR介导的支气管扩张，检测目标1中确定的所有第二信使信号通路。我们还将研究副交感神经功能改变的作用。我们提出的实验结果将对建立TLR7和TLR8激动剂作为哮喘和其他呼吸道疾病治疗药物的潜力具有重要意义。此外，由于这些受体对病毒RNA有反应，了解刺激TLR7受体在呼吸道的作用，以及在哮喘模型中这些作用的丧失，将有助于我们理解病毒诱导哮喘发作的病理生理学。 公共卫生相关性：5%-10%的美国人患有哮喘。哮喘患者所经历的喘息和呼吸短促是由于呼吸道收缩所致。我们最近发现，药物通过正常识别病毒的受体起作用，会导致呼吸道松弛。在这个项目中，我们将研究这些药物是如何放松呼吸道的，以及这种反应在哮喘中可能会受到怎样的破坏，为我们将这类药物开发成哮喘治疗奠定基础。

DESCRIPTION (provided by applicant): Viral single standed RNA is sensed by cells via toll-like receptor (TLR)-7 and TLR-8. We have recently shown that stimulating TLR-7 and TLR-8 relaxes airway smooth muscle and prevents broncho-constriction in response to a variety of stimuli. The effect is profound, and can lead to virtually complete relaxation of smooth muscle. We have demonstrated this effect in guinea pigs and mice, and in human airway tissues. TLR7 dependent broncho-dilation in guinea pigs and in human airways is mediated by production of nitric oxide, while TLR8 dependent broncho-dilation is not. Our preliminary data suggest that TLR8 mediated broncho-dilation is mediated by production of prostaglandins and opening of the large conductance, calcium activated potassium channel. We have also shown that both antigen sensitization and acute viral infection substantially impair TLR- dependent broncho-dilation. In this application, we propose three specific aims: Specific Aim #1. To A) use TLR7(-/-) mice, as well as newly generated TLR8(-/-) and TLR7(-/-)TLR8(-/-) mice, to more thoroughly investigate and establish the signaling pathways responsible for the effects of these receptors on airway neurons and smooth muscle, B) test whether these receptors signal through nitric oxide, prostaglandins, and the large conductance, calcium activated potassium channel (BKCa) in human airways in vitro, and C) explore the role of nitric oxide and prostaglandins and changes in intracellular calcium in rapid signaling by these receptors in primary cultures of human airway parasympathetic neurons and airway smooth muscle cells. Specific Aim 2: To test whether decreased TLR mediated broncho-dilation after antigen sensitization is mediated by decreased TLR7 and/or TLR8, testing signaling pathways in airway smooth muscle identified in Aim #1. Since these TLRs are on parasympathetic nerves, as well as on eosinophils recruited to the airway nerves, we will also test whether TLR7 and TLR8 change neural control in sensitized airways. SPECIFIC AIM #3: To test whether decreased TLR mediated broncho-dilation after viral infection is mediated by decreased TLR7 and/or TLR8 pathways, testing all second messenger signaling pathways in airway smooth muscle identified in aim one. We will also investigate the role of changes in parasympathetic function. The results of the experiments we propose will be important in establishing the potential of TLR7 AND TLR8 agonists as treatments for asthma and other airway diseases. In addition, because these receptors respond to viral RNA, understanding the effects of stimulating TLR7 receptors in the airways, as well as the loss of these effects in models of asthma, will help us understand the pathophysiology of virus induced asthma attacks. PUBLIC HEALTH RELEVANCE: Asthma affects 5 - 10% of Americans. The wheezing and shortness of breath that people with asthma experience is due to constrictions of the airways. We have recently found that drugs working through a receptor that normally recognizes viruses causes the airways to relax. In this project, we will study how these drugs relax the airways, and how this response may be damaged in asthma, to lay the groundwork to allow us to develop this class of drugs into asthma treatments.