Mechanisms of Airway Epithelial Barrier Dysfunction by Respiratory Syncytial Virus and Environmental Stimuli

摘要 呼吸道合胞病毒(RSV)是引起儿童急性下呼吸道感染(ALRI)的主要原因 以及世界各地的高危成年人。我们的数据表明，呼吸道合胞病毒感染通过破坏 上皮尖连接复合体(AJC)，调节呼吸道上皮屏障。我们证明了RSV- AJC的介导性破坏伴随着周围肌动蛋白细胞骨架的分解，以及 下调肌动蛋白--一种关键的肌动蛋白结合蛋白。Cortactin缺乏症此前曾被认为与 破坏血管内皮细胞和肠道上皮屏障的稳定。然而，RSV之间的因果联系- 诱导的渗漏屏障、肌动蛋白细胞骨架重排和皮质蛋白缺乏症尚未建立。在……里面 此外，流行病学报告表明，接触周围颗粒物与 (PM)和ALRI风险增加。纳米颗粒(NP)是极小的PM，具有更大的能力成为 与较小的颗粒相比，它沉积在远端呼吸道，并逃避宿主防御。我们的初步数据 证明支气管上皮细胞预先暴露于NP不仅能增强RSV诱导的AJC 解体和肌动蛋白细胞骨架的破坏，但会增加病毒感染。基于我们新奇的观察， 我们提出的中心假设是：a)呼吸道合胞病毒通过触发 周围肌动蛋白细胞骨架的解聚，并下调皮质肌动蛋白；和b)这种破坏 纳米颗粒对呼吸道上皮屏障的破坏加重了呼吸道合胞病毒诱导的呼吸道上皮损伤。我们将测试我们的 通过以下具体目标的假设：目标1：确定皮质肌动蛋白依赖的肌动蛋白的作用 呼吸道合胞病毒诱导的呼吸道上皮屏障功能障碍中的细丝动力学。使用人的支气管上皮细胞 从儿童供体中分离出来，以及Cortactin缺失的小鼠模型，我们将研究(I)RSV对 肌动蛋白细胞骨架动力学，(Ii)皮质肌动蛋白对肌动蛋白动力学和AJC结构的功能作用，以及(Iii) Rap-1的功能作用。我们还将使用一个详细描述AJC影响的3-D人体肺器官模型 对RSV感染的干扰，这为研究复杂的宿主环境提供了创新的平台 互动。目的2：确定纳米颗粒是否增强呼吸道合胞病毒诱导的呼吸道上皮细胞的破坏 障碍。使用体外和体内模型，我们将(I)表征颗粒大小对屏障完整性的影响， (Ii)研究氧化应激对AJC功能的作用，以及(Iii)确定暴露于NP对AJC的影响 功能障碍。这项拟议的研究对美国国立卫生研究院的任务意义重大，因为我们的目标是探索 呼吸道合胞病毒感染对呼吸道屏障完整性的临床相关后果，以及如何暴露于环境中 污染物会加重呼吸道合胞病毒的感染。我们的方法是创新的，因为它将在 对RSV在AJC分解中的作用以及对NPs在增强RSV-2中的作用的新见解。 诱发AJC中断。已确定的通路将为治疗干预提供新的靶点 有可能对RSV疾病的管理产生积极影响。

ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections (ALRI) in children and high-risk adults worldwide. Our data demonstrate that RSV infection results in a ‘leaky airway’ by disrupting epithelial apical junctional complexes (AJC), which regulate the airway epithelial barrier. We show that RSV- mediated disruption of AJC is accompanied by disassembly of the perijunctional actin cytoskeleton, and downregulation of cortactin - a key actin-binding protein. Cortactin deficiency has been previously implicated in destabilizing the vascular endothelial and intestinal epithelial barrier. However the causal link between RSV- induced leaky barrier, actin cytoskeletal rearrangements and cortactin deficiency have not been established. In addition, epidemiological reports suggest a strong association between exposure to ambient particulate matter (PM) and increased risk of ALRI. Nanoparticles (NPs) are extremely small PM, with the greater ability to become deposited in distal airways and evade host defenses compared to smaller particles. Our preliminary data demonstrate that pre-exposure of bronchial epithelial cells to NP not only enhances RSV-induced AJC disassembly and actin cytoskeleton disruption, but augments viral infection. Based on our novel observations, we formulated the central hypotheses that a) RSV induces disruption of the airway epithelial barrier by triggering depolymerization of the perijunctional actin cytoskeleton, and by downregulating cortactin; and b) that disruption of the epithelial barrier by nanoparticles worsens RSV-induced airway epithelium injury. We will test our hypotheses through the following Specific Aims: Aim 1: To determine the role of cortactin-dependent actin filament dynamics in RSV-induced airway epithelial barrier dysfunction. Using human bronchial epithelial cells isolated from pediatric donors, and a mouse model of cortactin null mice, we will investigate (i) RSV effect on actin cytoskeletal dynamics, (ii) the functional role of cortactin on actin dynamics and AJC structure, and (iii) the functional roles of Rap-1. We will also use a 3-D human lung organoids model detailing the effects of AJC disruption upon RSV infection, which offers an innovative platform to study complex host-environmental interactions. Aim 2: To determine if nanoparticles enhance RSV-induced disruption of the airway epithelial barrier. Using in vitro and in vivo models, we will (i) characterize the effects of particle size on barrier integrity, (ii) study role of oxidative stress on AJC function, and (iii) define the effects of exposure to NP on AJC dysfunction. The proposed research is significant and relevant to the NIH’s mission as we aim to explore the clinically relevant consequences of RSV infection on airway barrier integrity, and how exposure to environmental pollutants worsens RSV infection. Our approach is innovative because it will provide new mechanistic insight in to the roles of RSV in AJC disassembly, as well as novel insight in to the effects of NPs in enhancing RSV- induced AJC disruption. The identified pathways will provide new targets for therapeutic intervention and the potential for positively impacting the management of RSV disease.