EPITHELIAL BARRIER DYSFUNCTION AND MUCOSAL INFLAMMATION IN ASTHMA

描述（由申请人提供）：肺部不断受到吸入颗粒和抗原的轰击，并进化出许多防御机制，以防止由于其他无害物质而导致的炎症发展。这些防御机制包括粘膜纤毛清除、粘膜免疫系统和上皮顶端连接复合物（AJC）。近年来，我们对连接复合体结构和功能的理解有了重大进展。然而，迄今为止，大多数研究都是在模型上皮和非肺组织中进行的，关于粘膜表面上皮连接复合物的功能仍存在许多问题。屏障结构、上皮内树突状细胞（DC）的抗原采样和 粘膜危险性需要进一步研究。有趣的是，气道上皮连接功能障碍越来越多地与哮喘相关，但涉及的潜在机制和气道炎症的后果仍然知之甚少。此外，在临床应用中没有良好的气道外/内渗透性测定。在这里，我们表明，Th 2细胞因子，合成的双链RNA（dsRNA），和呼吸道合胞病毒（RSV）诱导气道AJC惊人的解体。我们发现了一种新的信号通路，将dsRNA和RSV与蛋白激酶D（PKD）和细胞骨架重塑联系起来，导致气道上皮连接功能障碍。关于PKD在肺中的表达和功能知之甚少，但新出现的证据表明，这种多功能信号分子可能在一般粘膜免疫中起重要作用。接下来，我们使用了无内毒素的卵清蛋白（Ova）的粘膜致敏小鼠模型，当与dsRNA polyI：C一起吸入时，在单独用Ova攻击后两周后导致过敏型炎症。使用相互骨髓嵌合小鼠，在放射抗性（基质）细胞中需要toll样受体3（TLR 3）表达，以使dsRNA充当促变应性吸入佐剂。在这里，我们将建立在 这些数据，并提出了一个转化的研究计划，将提供新的见解气道上皮屏障功能和上皮：DC串扰的相关性呼吸道病毒感染和过敏性气道炎症。我们提出了四个具体目标，将：（1）确定上皮PKD和dsRNA传感器在粘膜致敏期间的确切作用，（2）测试闭合蛋白过表达抑制粘膜变应原致敏的假设，（3）测试RSV感染以PKD依赖性方式引起气道上皮屏障持续功能障碍的假设，和（4）开发漏肺测试，一种临床上有用的和非侵入性的外/内气道渗透性测定。

DESCRIPTION (provided by applicant): The lung is constantly bombarded by inhaled particles and antigens, and has evolved numerous defense mechanisms that guard against the development of inflammation due to otherwise innocuous substances. These defense mechanisms include mucociliary clearance, the mucosal immune system, and epithelial apical junctional complexes (AJC). In recent years there have been significant advances in our understanding of junctional complex structure and function. However, most research to-date has been conducted with model epithelia and in non-pulmonary tissues, and many questions remain about the function of epithelial junctional complexes at mucosal surfaces. The relationship between barrier structure, antigen sampling by intraepithelial dendritic cells (DC), and sensing of mucosal danger needs further study. Interestingly, airway epithelial junction dysfunction is increasingly associated with asthma, but the underlying mechanisms involved and consequences for airway inflammation remain poorly understood. Furthermore, there are no good assays of outside/in airway permeability in clinical use. Here we show that Th2 cytokines, synthetic double stranded RNA (dsRNA), and respiratory syncytial virus (RSV) induce striking disassembly of airway AJC. We identified a novel signaling pathway linking dsRNA and RSV with protein kinase D (PKD) and cytoskeletal remodeling resulting in airway epithelial junction dysfunction. Very little is known about the expression and function of PKD in the lung, but emerging evidence suggests that this versatile signaling molecule may play an important role in mucosal immunity in general. We next used a mouse model of mucosal sensitization with endotoxin-free ovalbumin (Ova), which when inhaled together with the dsRNA polyI:C, results in allergic-type inflammation two weeks later following challenge with Ova alone. Using reciprocal bone marrow chimeric mice, toll-like receptor 3(TLR3) expression was required in radioresistant (stromal) cells in order for dsRNA to act as a pro-allergic inhaled adjuvant. Here we will build on these data and propose a translational research program that will provide new insights into airway epithelial barrier function and epithelial:DC cross-talk of relevance to respiratory viral infections and allergic airway inflammation. We propose four specific aims that will: (1) Define the precise roles of epithelial PKD and dsRNA sensors during mucosal sensitization, (2) Test the hypothesis occludin overexpression inhibits mucosal allergen sensitization, (3) Test the hypothesis that RSV infection causes sustained dysfunction of the airway epithelial barrier in a PKD-dependent manner, and (4) Develop the Leaky Lung Test, a clinically useful and non-invasive assay of outside/in airway permeability.