Airway Epithelial Barrier Dysfunction In Response to Respiratory Syncytial Virus

 描述(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 有关呼吸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呼吸道上皮细胞屏障的结构和功能，以及如何修改相关机制来恢复屏障的完整性和抑制炎症反应。我们最近发表的文章显示，在细胞培养中，RSV感染导致紧密连接结构和功能的破坏。我们在这一特殊应用中的目标是研究RSV诱导的屏障破坏所涉及的分子机制和信号通路。我们方法的优势在于，我们利用了四个高度互补的模型，包括特征良好且可操作的人支气管上皮细胞系，原代人和小鼠呼吸道上皮细胞，以及尖端小鼠模型；每个模型都用于检查所提出机制的不同关键方面。中心假说是RSV感染通过蛋白激酶D(PKD)依赖的顶端连接复合体(AJC)内吞促进呼吸道上皮屏障功能障碍。在强大的初步数据的指导下，这一假说将通过追求两个特定的目标来检验：1)检验依赖PKD的内吞作用在RSV诱导的呼吸道上皮屏障功能障碍中是必不可少的假说；2)检验RSV以PKD依赖的方式导致持续的“漏气”的假说。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，我们将使用一个特征良好的野生型和前沿条件缺失PKD的小鼠模型来研究连接功能障碍、肺部炎症和病毒复制之间的关系。我们的建议是创新的，因为这是第一次全面分析RSV感染和PKD途径在肺中的作用。此外，它还将通过使用具有很强翻译潜力的吸入甘露醇来提供一种创新的呼吸道外/内通透性测定方法。这项拟议的研究意义重大，因为它将提供关于一种临床相关病毒的基本知识，而这种病毒的发病机制尚不清楚。整个K08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在病毒学和屏障免疫学方面的知识，发展研究细胞信号和内吞作用的专业知识，获得利用转基因小鼠的基本技能，并将我的利基定位为呼吸生物学领域的独立内科科学家。我的导师和咨询委员会将监督对详细的生产率和发展进度时间表的遵守情况。我对研究事业的承诺、强大的导师、儿科在培养下一代内科科学家方面的独特奉献精神，以及罗切斯特大学在呼吸道病毒研究方面出色的合作环境，将为我提供必要的工具来建立我的职业生涯。

 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.