Regulation of CNS leukocyte extravasation

而白细胞进入中枢神经系统(CNS)是糖尿病发病的基础。 许多神经炎症状态，调节这一过程的最后也是最关键的一步的机制， 跨内皮细胞迁移(TEM)，目前仍不清楚。但最近来自不同领域的两个系列发现可能 揭示新的和重要的线索，如何以及在哪里发生瞬变。紧密连接的异位表达(TJ) 神经炎性复发患者循环白细胞上的蛋白质已被描述 疾病多发性硬化(MS)。炎症条件下白细胞TJ蛋白表达升高， 以及干细胞和恶性细胞，可能赋予这些细胞跨越组织屏障的能力 -可能通过拉链机制实现与内皮TJ蛋白的瞬时相互作用。第二，纳米- 大小的胞外小泡(EV)，例如外体和微泡，携带连接蛋白并通过 血管内皮细胞已有报道。肠道病毒在炎症(包括多发性硬化症)、穿梭期间血液中升高 蛋白质和RNA在细胞之间，并与各种免疫细胞相互作用，改变其黏附和迁移 属性。这些集体发现可能表明EV将TJ蛋白转移到白细胞并作为 缺少连接白细胞上的TJ蛋白、透射电子显微镜和神经炎症的功能环节。具体地说，通过 以旁分泌方式将TJ货物从内皮细胞转移到白细胞，EVS可能使黏附 白细胞瞬间与相应的血管TJ蛋白结合，从而促进横跨 血脑屏障(BBB)的高度限制性内皮细胞。使用最先进的技术，我们将专注于 在显示Claudin 5(CLN-5)的白细胞上，CLN-5是BBB的主要TJ蛋白-并检验以下假设： 白细胞利用CLN-5和内皮来源的EV通过新的相互作用跨血管外渗 神经炎症时的血脑屏障。AIM 1将使用高分辨率3D荧光成像和FACS来检测 CLN-5+-白细胞，鉴定其免疫表型和活化状态 实验性自身免疫性脑脊髓炎(EAE)小鼠不同中枢神经系统区域的变化 将使用新的内皮条件，EGFP-CLN-5小鼠或CLN-5基因敲除小鼠来确定白细胞 CLN-5内源性表达或从内皮细胞外源性获得。目标3，将使用3D荧光 成像/流式细胞仪分析CLN-5货物(蛋白质和/或信使核糖核酸)从内皮来源的EVS转移到 以及一种新型的CLN-5肽模拟物Pep5，以确定这一作用是否依赖于CLN-5。目标4，意志 使用公认的体外血脑屏障模型和Pep5来检验白细胞CLN-5和EVS在血管内皮细胞中的功能作用(S) 推广瞬变电磁法。这些研究将是第一个系统和系统的方法来解决 白细胞TJ蛋白与EVS的关系及其在神经炎症中的协同作用相似的 这些研究应该揭示，这些过程可能在其他细胞外渗的情况下普遍起作用 常见的致病机制，并强调针对EVS的新的治疗方法。 好了！

While the entry of leukocytes into the central nervous system (CNS) is fundamental to the pathogenesis of many neuroinflammatory conditions, mechanisms regulating the last and most critical step of this process, transendothelial migration (TEM), remain obscure. But two recent series of findings from separate fields could reveal novel and vital clues to how and where TEM takes place. One, ectopic expression of tight junction (TJ) proteins has been described on circulating leukocytes in patients with relapses of the neuroinflammatory disease multiple sclerosis (MS). Elevated expression of TJ proteins by leukocytes in inflammatory conditions, as well as by stem and malignant cells, might endow these cells with heightened ability to cross tissue barriers – possibly by enabling transient interactions with endothelial TJ proteins via a ‘zipper’ mechanism. Two, nano- sized extracellular vesicles (EVs), e.g., exosomes and microvesicles, bearing junctional proteins and shed by endothelial cells have been reported. EVs are elevated in blood during inflammation (including MS), shuttle protein and RNA between cells, and interact with various immune cells to alter their adhesion and migration properties. These collective findings could suggest EVs transfer TJ proteins to leukocytes and serve as the missing links functionally connecting TJ proteins on leukocytes, TEM and neuroinflammation. Specifically, by transferring TJ cargo from endothelial cells to leukocytes in a juxtacrine manner, EVs might enable adherent leukocytes to transiently engage corresponding vascular TJ proteins and, thereby, foster TEM across the highly restrictive endothelium of the blood-brain barrier (BBB). Using state-of-the-art technologies, we will focus on leukocytes displaying claudin 5 (CLN-5) – a major TJ protein of the BBB – and test the following hypothesis: Leukocytes exploit CLN-5 and endothelial-derived EVs via novel interactions to extravasate across the BBB during neuroinflammation. Aim 1 will use high-resolution 3D fluorescence imaging and FACS to detect CLN-5+-leukocytes and identify their immunophenotypes and activation states at various times in the blood and different CNS regions of mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. Aim 2, will use novel endothelial conditional, eGFP-CLN-5 mice or CLN-5 knockdown mice to determine if leukocytes express CLN-5 endogenously or acquire it exogenously from endothelial cells. Aim 3, will use 3D fluorescence imaging/FACS to analyze transfer of CLN-5 cargo (protein and/or mRNA) from endothelial-derived EVs to leukocytes, and a novel CLN-5 peptidomimetic, Pep5, to establish if this action is CLN-5-dependent. Aim 4, will use a recognized in vitro BBB model and Pep5 to examine the functional role(s) of leukocyte CLN-5 and EVs in promoting TEM. These studies will be the first systematical and methodical approach to address the relationship between leukocyte TJ proteins and EVs, and their concerted role in neuroinflammation. As similar processes might generically operate in other instances of cell extravasation, these studies should reveal common pathogenic mechanisms and highlight new, therapeutic approaches that target EVs. !