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

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），自身侵袭性T细胞进入中枢神经系统（CNS），引起炎症和脱髓鞘。免疫细胞进入中枢神经系统是由形成血脑屏障的高度特化的内皮细胞控制的。人源化抗α 4-整合素抗体natalizumab抑制免疫细胞在血脑屏障上的运输是治疗复发-缓解型多发性硬化症的一种成功的治疗方案，但伴随着发展为进行性多灶性白质脑病（PML）的风险，PML是一种由少突胶质细胞JC病毒感染引起的中枢神经系统致命疾病。拟议研究项目的总体目标是确定涉及T细胞多步骤募集的新分子途径，这将允许唯一阻断破坏性免疫细胞的CNS募集，同时使保护性免疫细胞迁移到CNS不受影响。虽然已经描述了介导T细胞阻滞、极化和爬行的第一批分子，但涉及T细胞跨越血脑屏障的分子机制仍然几乎未被探索。在这里，我们将重点研究连接粘附分子- b （JAM-B）作为α 4整合素的替代配体的个体贡献，以及最近描述的分子如ALCAM， MCAM和Ninjurin在体外和体内血脑屏障上的T细胞极化，阻滞，爬行和渗透。由于JAM-B是α - 4整合素的相互作用伙伴，因此在EAE中是一个有趣的靶点，第一个目的是通过研究JAM-B-/- C57BL/6和SJL小鼠的疾病发展来阐明JAM-B在EAE发病机制中的作用。浸润的白细胞将从中枢神经系统中分离出来，并使用流式细胞术确定亚型。此外，通过体外血脑屏障模型，分析JAM-B、ALCAM、MCAM和Ninjurin对血流条件下血脑屏障完整性和T细胞外渗的贡献。第二个目的是描述血脑屏障的特殊屏障特性是否有利于T细胞亚群的跨细胞或细胞旁浸润，并确定JAM-B是否参与相应的过程。我们将通过活体显微镜和双光子活体显微镜在JAM-B-/-小鼠中研究不同T细胞与中枢微血管的相互作用。此外，使用VE-cadherin-GFP敲入小鼠，内皮连接可见，可以根据个体粘附分子的依赖性来评估有利的粘附途径。第三个目标是通过使用小鼠TU标记的转录谱分析来识别特定细胞类型中的新粘附分子。最后，第四点是检验MK2是否参与JAM-B和ALCAM的生物合成。

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.