Mucosal M Cell Endocytosis: Specificity and Mechanisms

描述（由申请人提供）：M细胞是一种特化上皮细胞，可诱导其在粘膜淋巴泡上分化，如上呼吸道的鼻相关淋巴组织（NALT）和肠道的Peyer's Patches （PP）。这些细胞通过选择性地捕获微粒并将其通过上皮运送到潜在的抗原提呈细胞和淋巴细胞，从而提供重要的免疫监视功能。利用淋巴毒素/肿瘤坏死因子家族配体诱导M细胞分化的一些密切相关的细胞培养模型，并与M细胞在体内的功能进行比较，我们发现了一种涉及紧密连接蛋白claudin4的颗粒胞吞的新机制。有趣的是，这一机制显示出对微生物和合成微珠的优先吸收有明显的层次结构，其中金黄色葡萄球菌>大肠杆菌>>酿酒葡萄球菌=葡聚糖=苯乙烯微珠。在目前的建议中，我们将完善细胞培养模型，以确定胞吞机制的关键组成部分，具体目标有三个：(1)我们将在转染细胞中使用基因敲低和显性阴性结构来确定特定候选蛋白成分在胞吞机制中的重要性。(2)我们将开发颗粒摄取和胞吞作用的定量和定性分析，以确定颗粒摄取的明确相对选择性。该试验将用于检测有限的微生物和合成微球，这些微球呈现与病原体相关的分子模式，包括从不同微生物中提取的肽聚糖、脂磷壁酸和内毒素。(3)我们将使用噬菌体展示文库来鉴定通过内吞机制选择性摄取的肽。这些展示肽将被用作初步尝试的试剂，以确认细胞用于选择性颗粒内吞作用的已知受体的作用，并识别以前方法尚未识别的新受体。这些研究将为进一步研究M细胞特异性在粘膜免疫监测中的分子基础提供重要的基础，也为开发M细胞选择性摄取疫苗的粘膜免疫治疗策略提供有用的信息和材料。流感、霍乱、炭疽等许多重大传染病都是通过粘膜表面进入人体的，因此了解免疫系统如何能够在粘膜表面捕获和识别这些生物并引发免疫反应是很重要的。粘膜M细胞被认为是主要的特化上皮细胞，用于捕获和运输感染因子穿过粘膜屏障到达免疫系统细胞，拟议的研究有望为这些细胞的功能提供见解。这些见解不仅有助于了解粘膜免疫的生物学，而且为开发有效的粘膜疫苗提供了潜在的策略。

DESCRIPTION (provided by applicant): M cells are specialized epithelial cells that are induced to differentiate over mucosal lymphoid follicles such as Nasal Associated Lymphoid Tissue (NALT) in the upper airways and Peyer's Patches (PP) in the intestine. These cells provide an important immune surveillance function by selectively trapping microparticles and transporting them across the epithelium to underlying antigen presenting cells and lymphocytes. Using a number of closely related cell culture models of M cell differentiation induced by lymphotoxin/TNF family ligands and comparisons to M cell function in vivo, we have found a novel mechanism of particle transcytosis involving the tight junction protein claudin 4. Interestingly, this mechanism shows a distinct hierarchy in the preferential uptake of microbes and synthetic microbeads with preference for S. aureus > E. coli >> S. cerevisiae = Dextran = styrene beads. In the present proposal, we will refine the cell culture model to identify critical components of the transcytosis mechanism within three specific aims: (1) We will establish the importance of specific candidate protein components of the endocytosis mechanism using genetic knockdown and dominant negative constructs in transfected cells. (2) We will develop quantitative and qualitative assays for particle uptake and transcytosis to establish clear relative selectivity in the uptake of particles. This assay will be applied to testing a limited selection of microbes and synthetic beads presenting pathogen associated molecular patterns including peptidoglycan, lipoteichoic acid, and endotoxin extracted from different microbes. (3) We will use phage display libraries to identify peptides showing selective uptake by the endocytosis mechanism. These display peptides will be used as reagents for initial attempts to confirm the role of known receptors used by the cells for selective particle endocytosis, and identify new receptors not yet identified by previous approaches. The studies will provide an important foundation for continuing studies on the molecular basis of M cell specificity in mucosal immune surveillance, and also provide useful information and materials for developing therapeutic strategies for mucosal immunization by targeting selective uptake of vaccines by M cells. A great number of major infectious diseases such as influenza, cholera, and anthrax enter the body through mucosal surfaces, so it is important to understand how the immune system is able to capture and identify such organisms at mucosal surfaces and trigger immune responses. Mucosal M cells are considered the major specialized epithelial cell for trapping and transporting infectious agents across the mucosal barrier to the cells of the immune system, and the proposed studies are expected to provide insights to the function of these cells. Such insights will not only help understand the biology of mucosal immunity, but also provide potential strategies for developing effective mucosal vaccines.