Project 3: Membrane-bound mucins on the airway surface ensure efficient mucus clearance and lung health

健康的呼吸道上皮粘膜屏障是强大的先天免疫系统的中心，它保护 肺部表面受到吸入的感染性和有毒物质的持续攻击。然而， 粘液清除系统的异常是许多呼吸道疾病的特征，包括囊性纤维化， 慢性阻塞性肺病和哮喘。这些疾病的发病机制是多因素的，但似乎 包括一个常见的引发疾病的步骤，减少粘液清除。我们开发了一部小说 描述粘液运输系统的范式，被称为“双凝胶”模型，它突出了 呼吸道表面的两个含粘蛋白的层：1)粘液层，含有分泌的粘蛋白 结合吸入的颗粒；以及2)跨膜系带粘蛋白-一层被称为睫状层- 排列在呼吸道表面的糖萼(或PCL-G)。我们之前已经证明，未能对两者进行监管 层数与粘液纤毛清除量的减少有关。然而，目前尚不清楚纤毛和 PCL-G的组织是为了促进粘液从肺部有效地清除，并防止吸入 病原体。PPG项目3的总体目标是应用新的理论概念和实验 回答关于PCL-G在粘液运输方面的多重作用的关键问题的方法 以及保护呼吸道表面所需的宿主屏障防御活动。首先，我们试图了解如何 粘液层实际上是运输的，即动量是如何从纤毛转移到粘液层的。在AIM 1，我们将测试假设，在远端呼吸道中，粘蛋白太低，不能产生粘弹性凝胶， 水从睫毛周围“抽出”，提供足够的水动力来输送稀释液。 粘液层。这一目标的研究旨在量化纤毛敲打产生的抽水力， 评估它们是否足以推动低粘度粘液穿过呼吸道表面，并了解 重力的影响。在目标2中，我们将测试假设，在较大的呼吸道中，粘蛋白浓度为 足以形成粘弹性凝胶，需要第二种模式的纤毛-粘液动量转移才能有效 粘液运输。在这里，我们假设纤毛顶端的高度糖基化的粘蛋白形成 纤毛动力打击期间与粘液凝胶的弱/瞬时相互作用，并在物理上“推动”粘液凝胶 往前走。这里的研究将评估调节系留粘蛋白对粘液凝胶转运效果的影响。 以及它在疾病中是如何改变的。目标3中的研究旨在了解密集包装的 系留的粘蛋白在呼吸道保护中发挥作用。我们将验证这样一种假设，即呼吸道内有系留的粘蛋白 表面形成物理屏障，防止吸入的病原体，包括病毒，穿透PCL-G 太空。我们预计，这些研究的结果将导致我们对如何 粘液清除在健康中保持，并有助于发现治疗策略，可以 加速/恢复黏膜阻塞性肺病患者的清除。

The healthy airway epithelial mucosal barrier is the center of a powerful innate immune system that protects the pulmonary surfaces from the constant onslaught of inhaled infectious and noxious substances. However, abnormalities in the mucus clearance system characterize a number of airway diseases, including cystic fibrosis, chronic obstructive lung disease, and asthma. The pathogenesis of these diseases is multi-factorial, but appears to include a common disease-initiating step, a reduction of mucus clearance. We have developed a novel paradigm describing the mucus transport system, referred to as the “two-gel” model, which highlights the role of the two mucin-containing layers on the airway surface: 1) the mucus layer, containing secreted mucins which bind inhaled particles; and 2) membrane-spanning tethered mucins – a layer referred to as the periciliary layer- glycocalyx (or PCL-G) which lines the airway surface. We have previously shown that the failure to regulate both layers is associated with a reduction in mucociliary clearance. However, it is currently unknown how the cilia and PCL-G are organized to facilitate the efficient clearance of mucus out of the lungs and protect against inhaled pathogens. The overarching goal of Project 3 of this PPG is to apply novel theoretical concepts and experimental approaches to answer key questions about the multiple roles of the PCL-G with respect to both mucus transport and the host barrier defense activities required to protect the airway surfaces. First, we seek to understand how the mucus layer actually is transported, i.e., how momentum is transferred from cilia to the mucus layer. In Aim 1, we will test the hypothesis that in the distal airways, where mucins are too low to generate a viscoelastic gel, water is “pumped out” of the periciliary space, providing hydrodynamic force sufficient to transport the dilute mucus layer. Studies in this aim are directed at quantifying the water pumping forces generated by cilia beating, assess whether they are sufficient to propel a low-viscosity mucus across airway surfaces, and understand the impact of gravity. In Aim 2, we will test the hypothesis that in the larger airways, where mucin concentrations are sufficient to form a viscoelastic gel, a second mode of cilia-mucus momentum transfer is required for efficient mucus transport. Here, we hypothesize that the highly glycosylated tethered mucins on the tips of cilia form weak/transient interactions with the mucus gel during the cilia power stroke and physically “thrust” the mucus gel forward. Studies here will assess the impact of modulating tethered mucins on the efficacy of mucus gel transport and how it is altered in disease. Studies in Aim 3 are directed at understanding the role that the densely packed tethered mucins play in airway protection. We will test the hypothesis that tethered mucins lining the airway surface form a physical barrier to prevent inhaled pathogens, including viruses, from penetrating the PCL-G space. We anticipate that results from these studies will lead to paradigm shifts in our understanding of how mucus clearance is maintained in health and aid in the discovery of therapeutic strategies which can accelerate/restore clearance in persons with muco-obstructive lung diseases.