Airway Epithelial Barrier Dysfunction In Response to Respiratory Syncytial Virus

 描述（由申请人提供）：最近的研究表明，呼吸道病毒，包括呼吸道合胞病毒（RSV），破坏上皮屏障结构和功能，但 气道炎症的机制和后果仍然知之甚少。我们研究的长期目标是更好地了解病毒感染如何影响气道上皮细胞屏障结构和功能，以及如何修改相关机制，恢复屏障完整性和抑制炎症反应。我们最近的出版物揭示，在细胞培养中，RSV感染引起紧密连接结构和功能的破坏。我们的目标是在这个特殊的应用程序是调查的分子机制和信号通路参与RSV诱导的屏障破坏。我们的方法的优势在于，我们利用四种高度互补的模型，包括一个良好表征和可操作的人支气管上皮细胞系，原代人类和小鼠气道上皮细胞，以及尖端的小鼠模型;每个模型都用于研究所提出的机制的不同关键方面。中心假设是RSV感染以蛋白激酶D（PKD）依赖性方式通过顶端连接复合物（AJC）的内吞作用促进气道上皮屏障功能障碍。在强有力的初步数据的指导下，将通过追求两个具体目标来检验该假设：1）检验PKD依赖性内吞作用对于RSV诱导的气道上皮屏障功能障碍是必不可少的假设; 2）检验RSV以PKD依赖性方式引起持续的“气道渗漏”的假设。在目标1中，我们将阐明RSV引发的内吞途径、细胞骨架重塑和PKD激活。在目标2中，我们将使用野生型的良好表征的小鼠模型和PKD的尖端条件性缺失来研究连接功能障碍、肺部炎症和病毒复制的关联。我们的建议是创新的，因为它是第一个全面分析RSV感染和PKD途径在肺中的作用。此外，它将通过使用具有强大转化潜力的吸入甘露醇来提供外/内气道渗透性的创新测定。这项拟议的研究意义重大，因为它将提供有关临床相关病毒的基本知识，而这些病毒的发病机制尚不清楚。整个K 08应用程序旨在建立过渡到独立所需的技术，智力和领导技能的基础。职业发展计划将帮助我加强我在病毒学和屏障免疫学方面的知识，发展研究细胞信号传导和内吞作用的专业知识，获得利用转基因小鼠的基本技能，并将我的利基定义为呼吸生物学领域的独立医生科学家。我的导师和咨询委员会将监督是否遵守详细的生产力和发展进度时间轴。我对研究事业的承诺，强有力的指导，儿科部门在培养下一代医生科学家方面的独特奉献精神，以及罗切斯特大学呼吸道病毒研究的杰出合作环境，将为我的职业生涯提供必要的工具。

 DESCRIPTION (provided by applicant): Recent studies indicate that respiratory viruses, including Respiratory Syncytial Virus (RSV), disrupt epithelial barrier structure and function, but the mechanisms involved and consequences for airway inflammation remains poorly understood. The long-term goal of our studies is to better understand how airway epithelial cell barrier structure and function are affected by viral infection and how modifying the involved mechanisms restore barrier integrity and dampen inflammatory responses. Our recent publication revealed that, in cell culture, RSV infection caused disruption of tight junction structure and function. Our objective in this particular application is to investigate the molecula mechanism and signaling pathways involved in RSV-induced barrier disruption. The strength of our approach is that we utilize four highly complementary models including a well-characterized and manipulable human bronchial epithelial cell line, primary human and mouse airway epithelial cells, and cutting-edge mouse models; each serves to examine different key aspects of proposed mechanisms. The central hypothesis is that RSV infection prompts airway epithelial barrier dysfunction via endocytosis of apical junctional complexes (AJC) in a Protein Kinase D (PKD)-dependent manner. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Test the hypothesis that PKD- dependent endocytosis is essential for RSV-induced airway epithelial barrier dysfunction; 2) Test the hypothesis that RSV causes sustained "leaky airway" in a PKD-dependent manner. In Aim 1, we will elucidate endocytic pathways, cytoskeleton remodeling, and PKD activation triggered by RSV. In Aim 2, we will use a well-characterized mouse model of wild type, and cutting-edge conditional deletion of PKD to investigate association of junction dysfunction, lung inflammation, and viral replication. Our proposal is innovative because it is the first comprehensive analysis of role of RSV infection and PKD pathway in the lung. Furthermore, it will provide an innovative assay of outside/in airway permeability by using inhaled mannitol, which has strong translational potential. The proposed research is significant, because it will provide essential knowledge about a clinically relevant virus with poorly understood pathogenesis. The overall K08 application is designed to build a foundation of technical, intellectual and leadership skills required to transition into independence. The career development plan will help me strengthen my knowledge in virology, and barrier immunology, develop expertise in studying cell signaling, and endocytosis, acquire essential skills in utilizing genetically modified mice, and define my niche as an independent physician scientist in the field of respiratory biology. My mentors and advisory committee will monitor adherence to the detailed productivity and developmental progress timeline. My commitments to a research career, strong mentorship, unique dedication of Pediatrics Department in training the next generation of physician-scientists, and outstanding collaborative environment for respiratory virus research at the University of Rochester will provide the necessary tools with which I can build my career.