Identification of cellular and molecular mechanisms involved in leukocyte trafficking across the blood-brain-barrier in multiple sclerosis

多发性硬化症中白细胞跨血脑屏障运输的细胞和分子机制的鉴定

• 批准号: 246155722
• 负责人: Dr. Silvia Tietz
• 金额: --
• 依托单位: Theodor-Kocher Institut
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/246155722?language=en
• 关键词: Identification; cellular; molecular; mechanisms; involved

In multiple sclerosis and in its animal model, experimental autoimmune encephalomyelitis (EAE), autoaggressive T cells get access to the central nervous system (CNS), causing inflammation and demyelination. Immune cell recruitment into the CNS is controlled by the highly specialized endothelial cells forming the blood-brain barrier (BBB). Inhibition of immune cell trafficking across the BBB with the humanized anti-alpha4-integrin antibody natalizumab is a successful therapeutic regimen for the treatment of relapsing-remitting multiple sclerosis, but comes with a risk of developing progressive multifocal leukoencephalopathy (PML), an often fatal disease of the CNS caused by JC virus infection of oligodendrocytes. The general objective of the proposed research project is to identify novel molecular pathways involved in the multi-step recruitment of T cells that will allow the sole blockade of CNS recruitment of destructive immune cells, while leaving the migration of protective immune cells into the CNS untouched. Whereas the first molecules mediating T cell arrest, polarization and crawling have been described the molecular mechanisms involved in T cell diapedesis across the BBB remain almost unexplored. Here we will focus on investigating the individual contribution of the junctional adhesion molecule-B (JAM-B) as an alternative ligand for alpha4-integrins, as wall as the more recently described molecules such as ALCAM, MCAM and Ninjurin in T cell polarization, arrest, crawling and diapedesis across the BBB in vitro and in vivo. Due to the fact that JAM-B is an interaction partner of alpha4-integrin and therefore is an interesting target in EAE the first aim is dedicated to clarify the role of JAM-B in EAE pathogenesis by studying disease development in JAM-B-/- C57BL/6 and SJL mice. Infiltrated leukocytes will be isolated from the CNS and subtypes will be determined using flow cytometry. Furthermore, using an in vitro model for the BBB the contributions of JAM-B, ALCAM, MCAM and Ninjurin to the BBB integrity and T cell extravasation under flow conditions shall be analysed. The second aim is designed to delineate, if the special barrier characteristics of the BBB favor transcellular or paracellular diapedesis of T cell subsets and to determine, if JAM-B is involved in the respective processes. The interaction of different T cells with the CNS microvasculature will be investigated by means of intravital microscopy and 2-Photon intravital microscopy in JAM-B-/- mice. Moreover, using VE-cadherin-GFP knockin mice, in which endothelial junctions are visualized, the favoured diapedesis route can be evaluated in dependence to individual adhesion molecules. The third aim is to identify new adhesion molecules in specific cell types by transcriptional profiling using mouse TU tagging. Finally, the fourth point is to examine whether MK2 is involved in the biosynthesis of JAM-B and ALCAM.

在多发性硬化症及其动物模型中，实验性自身免疫性脑脊髓炎（EAE），自身攻击性T细胞进入中枢神经系统（CNS），引起炎症和脱髓鞘。免疫细胞向CNS的募集由形成血脑屏障（BBB）的高度特化的内皮细胞控制。用人源化抗α 4-整联蛋白抗体那他珠单抗抑制免疫细胞穿过BBB的运输是治疗复发-缓解型多发性硬化症的成功治疗方案，但伴随着发展进行性多灶性白质脑病（PML）的风险，PML是由JC病毒感染少突胶质细胞引起的CNS的通常致命的疾病。 拟议研究项目的总体目标是确定参与T细胞多步募集的新分子途径，这将允许唯一阻断CNS募集破坏性免疫细胞，同时使保护性免疫细胞迁移到CNS中。尽管已经描述了介导T细胞停滞、极化和爬行的第一个分子，但涉及T细胞跨越BBB的渗出的分子机制仍然几乎未被探索。在这里，我们将重点调查的连接粘附分子-B（JAM-B）作为α 4-整合素的替代配体的个人贡献，以及最近描述的分子，如ALCAM，MCAM和Ninjurin在T细胞极化，逮捕，爬行和跨越血脑屏障在体外和体内渗出。由于JAM-B是α 4-整联蛋白的相互作用伴侣，因此是EAE中令人感兴趣的靶点，因此第一个目的致力于通过研究JAM-B-/-C57 BL/6和SJL小鼠中的疾病发展来阐明JAM-B在EAE发病机制中的作用。将从CNS中分离浸润的白细胞，并使用流式细胞术测定亚型。此外，应使用BBB的体外模型分析JAM-B、ALCAM、MCAM和Ninjurin对血流条件下BBB完整性和T细胞外渗的贡献。第二个目的是划定，如果特殊的屏障特性的血脑屏障有利于跨细胞或细胞旁的T细胞亚群的渗出，并确定，如果JAM-B参与各自的过程。在JAM-B-/-小鼠中，将通过活体显微镜和双光子活体显微镜研究不同T细胞与CNS微血管系统的相互作用。此外，使用VE-钙粘蛋白-GFP敲入小鼠，其中内皮连接可视化，有利的渗出途径可以根据单个粘附分子进行评估。第三个目的是通过使用小鼠TU标签的转录谱来识别特定细胞类型中的新粘附分子。最后，第四点是检查MK2是否参与JAM-B和ALCAM的生物合成。