Mucosal Immune Barrier in Infection and Immunity

描述（由申请人提供）：计划项目概述：这是重新提交的计划项目资助（PPG）申请，用于研究感染和炎症中的粘膜屏障。95%的感染源通过暴露的粘膜表面进入，如呼吸道、胃肠道、乳腺和泌尿生殖道。粘液感染包括细菌性和病毒性肺炎、SARS、TB、AIDS和其他性传播疾病、许多机会性、新出现和重新出现的感染以及生物战剂/恐怖分子制剂。粘膜表面衬有上皮细胞，通常为单层。上皮细胞层是感染因子、过敏原和其他有害抗原进入的主要屏障。从根本上说，上皮层是先天性粘膜免疫系统的最基本组成部分。在这个计划项目中，我们专注于两个广泛的和相互关联的领域，上皮层如何在粘膜免疫保护中发挥其功能，特别强调肺上皮。项目1（Mostov）、项目2（Engel）和项目3（罗森）研究上皮伤口愈合的机制、硫酸酯（修饰硫酸乙酰肝素蛋白聚糖的硫酸酯酶）在上皮对损伤的反应中的作用以及极化细胞与细菌病原体的相互作用。项目4（Werb）的重点是炎症细胞如何穿过上皮屏障进入器官内腔。项目1使用两种新的上皮伤口愈合的三维细胞培养模型。项目2的重点是机会致病菌，铜绿假单胞菌，作为上皮病原体相互作用的模型。项目3研究硫如何参与上皮伤口愈合。项目4的重点是蛋白酶和硫在调节粘膜组织中炎性细胞的募集和迁移中的作用。核心A在行政上支持计划项目。Core B（Matthay）提供单层培养物、三维模型和肺切片中的原代人类和啮齿动物肺上皮细胞，以及小鼠中的功能研究。Core C有一个由四个显微镜组成的阵列，适用于体外研究以及活体麻醉小鼠实验的三维和四维细胞动力学分析。大多数传染性病原体通过暴露的粘膜表面进入人体。这些表面由上皮细胞排列，最常见的是单层。我们正在研究这种上皮屏障如何保护我们免受感染。 项目1：三维上皮伤口愈合（Mostov，K） 项目1描述（由申请人提供）：超过95%的传染性病原体通过暴露的粘膜表面进入，如呼吸道、胃肠道和泌尿生殖道。这些疾病包括艾滋病毒、性传播疾病、许多机会性感染、结核病、许多新出现和重新出现的感染以及生物战剂/恐怖分子制剂，如炭疽、鼠疫耶尔森氏菌和天花。大多数粘膜表面衬有单层极化上皮细胞，其形成感染因子进入的主要屏障。本质上，上皮层可以被认为是先天粘膜免疫系统的最基本组成部分。有些病原体通过破坏上皮层而穿过上皮层，另一些病原体则利用单层的破坏，这可能是由继发于炎症、创伤的组织损伤引起的，也可能是单层内细胞死亡或分裂引起的。为了维持其作为感染屏障的功能，上皮组织已经开发出有效的伤口愈合机制。伤口愈合是粘膜防御感染的核心。上皮屏障必须尽快恢复，以尽量减少感染因子进入的机会。一些感染因子，如铜绿假单胞菌，不仅利用预先存在的伤口，而且还阻碍伤口愈合过程。我们正在研究通过在细胞外基质的三维培养物中生长上皮细胞来愈合上皮伤口，这使得细胞更接近体内条件。在目标1中，我们使用形成肺泡样囊肿的人原代肺泡II型细胞系统作为研究对急性肺损伤/急性呼吸窘迫综合征的反应的模型。在目标2中，我们使用了一个三维系统的分化良好的人气道细胞系，形成囊肿和小管内衬假复层上皮，作为模型来研究气道损伤的反应。在目标3中，我们正在研究基质金属蛋白酶和硫酸酯酶（从硫酸乙酰肝素蛋白聚糖中去除6-O-硫酸酯基团的酶）在我们的三维培养系统中的作用。这项工作将与项目2、3和4合作，并得到核心B和C的支持。大多数传染性病原体通过内衬内部器官（如肺）的细胞层进入。细胞层的损伤使病原体更容易进入，我们正在研究细胞层如何自我修复。

DESCRIPTION (provided by applicant): Overview of Program Project: This is a resubmission of a renewal of a Program Project Grant (PPG) application to study the mucosal barrier in infection and inflammation. Ninety-five percent of infectious agents enter through exposed mucosal surfaces, such as the respiratory, gastrointestinal, mammary and urogenital tracts. Mucosal infections include bacterial and viral pneumonia, SARS, TB, AIDS and other sexually transmitted diseases, numerous opportunistic, emerging and re-emerging infections, and biological warfare/terrorist agents. Mucosal surfaces are lined by epithelial cells, usually in a monolayer. The epithelial cell layer is the principal barrier to entry of infectious agents, allergens and other noxious antigens. Fundamentally, the epithelial layer is the most basic component of the innate mucosal immune system. In this Program Project we focus on two broad and inter-related areas of how the epithelial layer performs its functions in mucosal immune protection, with a particular emphasis on pulmonary epithelium. Projects 1 (Mostov), 2 (Engel) and 3 (Rosen) study mechanisms of epithelial wound healing, the roles of sulfs (sulfatases which modify heparan sulfate proteoglycans) in epithelial response to injury and the interaction of polarized cells with a bacterial pathogen. Project 4 (Werb) is focused on how inflammatory cells cross the epithelial barrier to enter the lumen of the organ. Project 1 uses two novel three dimensional cell culture models of epithelial wound healing. Project 2 focuses on the opportunistic pathogen, Pseudomonas aeruginosa, as a model for epithelial pathogen interaction. Project 3 examines how sulfs participate in epithelial wound healing. Project 4 focuses on the role of proteases and sulfs in regulating inflammatory cell recruitment and migration in mucosal tissues. Core A supports the Program Project administratively. Core B (Matthay) provides primary human and rodent lung epithelial cells in monolayer cultures, three-dimensional models and lung slices, as well as functional studies in mice. Core C has an array of four microscopes suitable for three- and four-dimensional analysis of cell dynamics for in vitro studies as well as experiments in living, anesthetized mice. Most infectious agents enter the body through exposed mucosal surfaces. These surfaces are lined by epithelial cells, most commonly in a single layer. We are studying how this epithelial barrier protects us against infection. PROJECT 1: Epithelial wound healing in 3 dimensions (Mostov, K) Project 1 Description (provided by applicant): Greater than 95% of infectious agents enter through exposed mucosal surfaces, such as the respiratory, gastrointestinal and genitourinary tracts. These include HIV, sexually transmitted diseases, numerous opportunistic infections, TB, many emerging and re-emerging infections, and biological warfare/terrorist agents, such as anthrax, Yersinia pestis and smallpox. Most mucosal surfaces are lined by a monolayer of polarized epithelial cells, which forms the principal barrier to entry by infectious agents. In essence, the epithelial layer can be considered the most basic component of the innate mucosal immune system. Some pathogens cross the epithelial layer by disrupting it. Other pathogens exploit disruptions in the monolayer, which can be caused by tissue injury secondary to inflammation, trauma, or may result from cell death or division within the monolayer. To maintain their function as a barrier to infection, epithelial tissues have developed efficient wound healing mechanisms. Wound healing is central to mucosal defense against infection. The epithelial barrier must be restored as quickly as possible, to minimize the opportunity for entry of infectious agents. Some infectious agents, such as Pseudomonas aeruginosa, not only exploit pre-existing wounds, but also impede the wound healing process. We are studying epithelial wound healing by growing epithelial cells in three-dimensional cultures of extracellular matrix, which causes the cells to more closely resemble in vivo conditions. In Aim 1 we are using a system of human primary lung alveolar type II cells, which form alveolar-like cysts, as a model to study response to acute lung injury/acute respiratory distress syndrome. In Aim 2, we are using a three-dimensional system of a well-differentiated human airway cell line, which forms cysts and tubules lined by pseudostratified epithelium, as a model to study airway response to injury. In Aim 3 we are studying the roles of matrix metalloproteinases and sulfatases (enzymes that remove 6-O-sulfate groups from heparan sulfate proteoglycans) in our three dimensional culture systems. This work will be in collaboration with Projects 2, 3 and 4, and supported by Cores B and C. Most infectious agents enter through the layer of cells that lines internal organs, such as the lung. Injuries to this cell layer make it much easier for pathogens to enter and we are studying how the cell layer heals itself.