Dynamic Imaging in Viral Encephalitis Defines Unique Roles for Chemoattractants.

背景-病毒性脑炎，最常见的原因是单纯疱疹病毒(HSV)，是一种破坏性的脑部炎症疾病，其特征是血脑屏障(BBB)的完整性被破坏，免疫细胞，特别是早期的中性粒细胞，迁移到大脑中，在血管周围形成血管周围的“袖带”。由于没有建立免疫疗法，因此许多患者死亡，大多数幸存者脑损伤。导致广泛免疫抑制的治疗方法有可能导致病毒复制失控。因此，迫切需要靶向治疗来缓解有害的免疫细胞迁移和血脑屏障的破坏。免疫细胞的迁移是由称为趋化因子的吸引蛋白决定的，但越来越多的非典型吸引蛋白被识别出来。然而，这在脑炎及其在脑中性粒细胞行为中的作用，如中性粒细胞-细胞外陷阱(NETs)的产生，以及对血脑屏障通透性的影响尚不清楚。合作-尽管英国和日本的PI都在免疫学中心(MGH/哈佛)完成了博士后培训，但他们注意到，需要的研究技能具有显著的附加值，以解决他们重叠的学科问题。英国PI建立了病毒性脑炎的多光子活体显微镜(MP-IVM)模型，作为实时显示血脑屏障破坏和免疫细胞迁移的有力工具。日本PI在非中枢神经系统炎症方面具有丰富的MP-IVM经验，确定了非典型吸引蛋白的新角色，并建立了直接可视化吸引蛋白和中性粒细胞行为。具体目标-我们将确定典型/非典型吸引蛋白对中性粒细胞迁移到大脑的相对贡献以及推动HSV脑炎BBB破坏的迁移后行为。假设-HSV感染后，特定典型/非典型吸引蛋白的协调使中性粒细胞迁移到大脑，并通过净产生驱动BBB分解。方法和途径-使用MP-IVM来确定与BBB分解有关的中性粒细胞迁移行为以及典型/非典型吸引蛋白及其受体的表达。-询问这些蛋白质的去除对中性粒细胞行为和BBB破坏的影响。-使用MP-IVM来可视化Net迁移后的产生和相关的过程，并使用单细胞方法来探索更广泛的上调。重要：病毒脑炎迫切需要目标免疫疗法来减轻中性粒细胞迁移和改善BBB破坏，而不允许不受控制的病毒复制。KEY目的和目标-在HSV脑炎小鼠模型中建立与BBB破坏相关的特定的中性粒细胞迁移行为。-确定驱动这种行为的典型/非典型吸引蛋白-受体相互作用的相对贡献。-询问Net的不同产生。在BBBOBJECTIVES的完整性分解中的相关流程-在英国和日本的PI之间分享现有的经验和技能，包括脑部MP-IVM的开颅窗手术，以及吸引蛋白和中性粒细胞行为的实时可视化；为未来的合作研究建立强有力的伙伴关系。-用早期职业研究人员在英国和日本的实验室环境中接触这些技术，开始建设能力，为更大规模的赠款奠定基础，建立免疫细胞迁移生物学领域的领先实验室。-使用MP-IVM(日本)和BBB建模(英国)的互补平台来展示这两种方法的相加作用，这具有广泛的潜力，作为探索免疫细胞迁移以应对感染和自身免疫的基础。

ABSTRACTBackground-Viral encephalitis, most commonly due to Herpes simplex virus (HSV), is a devastating disease of brain inflammation characterised by break down in the integrity of the blood-brain barrier (BBB) and the migration of immune cells, particularly early neutrophils, into the brain where they form perivascular 'cuffs' around blood vessels. No immune therapy is established therefore many patients die and most survivors have brain injury. Therapy which causes broad immune suppression risks uncontrolled viral replication. Therefore, targeted therapy which mitigates deleterious immune cell migration and BBB breakdown is desperately required. The migration of immune cells is determined by attractant proteins, termed chemokines, but increasingly atypical attractant proteins are recognised. However, this has not be investigated in encephalitis and their role in neutrophil behaviour in the brain, such as the production of neutrophil-extracellular traps (NETs), and the impact on BBB permeability are not known.Collaboration-Whilst both UK and Japanese PIs were completing their Post-Doctoral training in the Center for Immunology (MGH/Harvard) they noted significant added value in the investigatory skills required to address their overlapping disciplines. The UK PI established a multiphoton intravital microscopy (MP-IVM) model of viral encephalitis as a powerful tool to visualize BBB breakdown and immune cell migration in real-time.The Japanese PI has extensive experience of MP-IVM in non-CNS inflammation identifying novel roles for atypical attractant proteins and has established direct visualisation attractant proteins and also neutrophil behaviour.Specific aims-We will determine the relative contribution of typical/atypical attractants proteins on neutrophil migration into the brain and the post-migration behaviour driving BBB breakdown in HSV encephalitis.Hypotheses-Following HSV infection, co-ordination of specific typical/atypical attractant proteins cause neutrophils to migrate into the brain and drive BBB breakdown through NET production.Methodology and approach-- Use MP-IVM to determine neutrophil migratory behaviour in relation to BBB breakdown and the expression of typical/atypical attractants proteins and their receptors. - Interrogate the impact of the abrogation of these proteins on neutrophil behaviour and BBB breakdown.- Use MP-IVM to visualise the post-migration production of NETs and associated processes and use single cell approaches to explore wider up-regulation.IMPORTANCETargeted immune therapy which mitigates neutrophil migration and ameliorates BBB breakdown without allowing uncontrolled viral replication is desperately needed for viral encephalitis.KEY AIMS AND OBJECTIVES AIMS- Establish the specific neutrophil migration behaviour which correlates with BBB breakdown in a murine model of HSV encephalitis.- Determine the relative contribution of typical/atypical attractant protein-receptor interactions which drive this behaviour.- Interrogate the differential production of NETs and associated processes in the break down in integrity of the BBBOBJECTIVES- Share existing experience and skills between UK and Japanese PIs, including cranial windowing surgery for MP-IVM of the brain and also real-time attractant protein and neutrophil behaviour visualisation; building a strong partnership for future collaborative research.- Exchange Early Career Researchers for exposure of these techniques in UK and Japanese laboratory settings to begin to build capacity which lays the ground work for larger grants to establish leading laboratories in the field of immune cell-migration biology.- Use the complementary platforms of MP-IVM (Japan) and BBB modelling (UK) to demonstrate the additive roles of the two approaches which has broad potential as a basis for exploring immune cell migration in response to infection and autoimmunity.