EPITHELIAL BARRIER DYSFUNCTION AND MUCOSAL INFLAMMATION IN ASTHMA

描述(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，以防止因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体(AJC)。近年来，我们对连接复合体的结构和功能的理解有了很大的进步。然而，到目前为止，大多数研究都是在模型上皮细胞和非肺组织中进行的，关于粘膜表面上皮连接复合体的功能仍然存在许多问题。屏障结构、上皮内树突状细胞(DC)抗原采集与感觉功能的关系 粘膜危险有待进一步研究。有趣的是，呼吸道上皮连接功能障碍与哮喘的相关性越来越大，但其涉及的潜在机制和对呼吸道炎症的后果仍知之甚少。此外，临床上还没有良好的外/内呼吸道通透性测定方法。在这里，我们展示了Th2细胞因子、合成双链RNA(DsRNA)和呼吸道合胞病毒(RSV)诱导呼吸道AJC显著分解。我们发现了一条新的信号通路，将dsRNA和RSV与蛋白激酶D(PKD)和细胞骨架重塑联系在一起，导致呼吸道上皮连接功能障碍。对PKD在肺中的表达和功能知之甚少，但越来越多的证据表明，这种多功能的信号分子可能在粘膜免疫中发挥重要作用。接下来，我们使用了无内毒素卵白蛋白(OVA)的小鼠粘膜致敏模型，当与dsRNA PolyI：C一起吸入时，两周后单独用OVA攻击后会导致过敏性炎症。在相互的骨髓嵌合小鼠中，Toll样受体3(TLR3)需要在抗辐射(基质)细胞中表达，才能使dsRNA作为亲过敏的吸入佐剂。在这里，我们将在 这些数据，并提出了一项转化研究计划，将提供对呼吸道上皮屏障功能和上皮：DC与呼吸道病毒感染和过敏性呼吸道炎症的相关性的新见解。我们提出了四个具体目标：(1)明确上皮PKD和dsRNA传感器在粘膜致敏过程中的确切作用；(2)检验封闭蛋白过度表达抑制粘膜变应原致敏的假说；(3)以PKD依赖的方式检验RSV感染导致呼吸道上皮屏障持续功能障碍的假说；(4)发展漏肺试验，这是一种临床上有用的非侵入性测定呼吸道内外通透性的方法。

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.