Regulation of CNS leukocyte extravasation

虽然白细胞进入中枢神经系统（CNS）是脑梗死发病机制的基础， 许多神经炎性疾病，调节该过程的最后和最关键步骤的机制， 跨内皮迁移（TEM）仍然不清楚。但最近两个不同领域的系列发现可能 揭示了TEM如何发生以及在何处发生的新颖而重要的线索。紧密连接（TJ）异位表达 已经描述了在患有神经炎性疾病复发的患者的循环白细胞上的蛋白质。 多发性硬化症（MS）。炎症状态下白细胞TJ蛋白表达升高， 以及干细胞和恶性细胞，可能赋予这些细胞更高的能力， - 可能是通过“拉链”机制与内皮TJ蛋白的瞬时相互作用。二，纳米- 大小的细胞外囊泡（EV），例如，外泌体和微泡，携带连接蛋白， 已经报道了内皮细胞。EV在炎症（包括MS）期间在血液中升高，穿梭 蛋白质和RNA，并与各种免疫细胞相互作用，以改变它们的粘附和迁移 特性.这些共同的发现可能表明EV将TJ蛋白转移到白细胞，并作为白细胞的一部分。 缺少功能性连接白细胞、TEM和神经炎症的TJ蛋白的链接。具体而言，由 通过将TJ货物从内皮细胞转移到白细胞，EV可以使粘附 白细胞瞬时接合相应的血管TJ蛋白，从而促进TEM穿过 血脑屏障（BBB）的高度限制性内皮细胞。利用最先进的技术，我们将专注于 对展示紧密连接蛋白5（CLN-5）（BBB的主要TJ蛋白）的白细胞进行分析，并检验以下假设： 白细胞通过新的相互作用利用CLN-5和内皮源性EV外渗穿过 神经炎症期间的BBB。Aim 1将使用高分辨率3D荧光成像和FACS检测 CLN-5+-白细胞，并在血液中的不同时间鉴定其免疫表型和活化状态， 患有实验性自身免疫性脑脊髓炎（EAE）的小鼠的不同CNS区域，MS的模型Aim 2， 将使用新的内皮条件性eGFP-CLN-5小鼠或CLN-5敲除小鼠来确定白细胞 内源性表达CLN-5或从内皮细胞外源性获得CLN-5。目标3，将使用3D荧光 成像/FACS以分析CLN-5货物（蛋白质和/或mRNA）从内皮衍生的EV转移至内皮细胞的方法。 白细胞，和一种新的CLN-5肽模拟物Pep 5，以确定这种作用是否是CLN-5依赖性的。目标4，威尔 使用公认的体外BBB模型和Pep 5来检查白细胞CLN-5和EV在 促进TEM。这些研究将是第一个系统和有条理的方法来解决 白细胞TJ蛋白和EV之间的关系及其在神经炎症中的协同作用。类似 这些过程可能在其他细胞外渗的情况下普遍运作，这些研究应该揭示 常见的致病机制，并强调新的，针对EV的治疗方法。 !

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. !