Dynamic Imaging in Viral Encephalitis Defines Unique Roles for Chemoattractants.

摘要背景：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（尤其是早期中性粒细胞）迁移到大脑中，在血管周围形成血管周围的“袖口”。没有建立免疫疗法，因此许多患者死亡，大多数幸存者有脑损伤。引起广泛免疫抑制的治疗有不受控制的病毒复制风险。因此，迫切需要靶向治疗，以减轻有害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引诱蛋白决定的，但越来越多的非典型引诱蛋白被认识到。然而，这在脑炎中尚未被研究，它们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——当英国和日本的pi都在免疫学中心（MGH/Harvard）完成博士后培训时，他们注意到在解决重叠学科所需的调查技能方面具有显著的附加价值。英国PI建立了病毒性脑炎的多光子活体显微镜（MP-IVM）模型，作为实时可视化血脑屏障分解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中的MP-IVM有丰富的经验，确定了非典型引诱蛋白的新作用，并建立了直接可视化引诱蛋白和中性粒细胞行为。特定目的-我们将确定典型/非典型引诱蛋白在中性粒细胞迁移到大脑中的相对贡献，以及在HSV脑炎中迁移后驱动血脑屏障分解的行为。假设——在HSV感染后，特定的典型/非典型引诱蛋白的协同作用导致中性粒细胞迁移到大脑中，并通过NET的产生驱动血脑屏障的破坏。方法和方法-使用MP-IVM来确定与血脑屏障分解和典型/非典型引诱物蛋白及其受体表达相关的中性粒细胞迁移行为。-探讨这些蛋白的去除对中性粒细胞行为和血脑屏障分解的影响。-使用MP-IVM可视化迁移后的net生产和相关过程，并使用单细胞方法探索更广泛的上调。靶向免疫治疗减轻中性粒细胞迁移和改善血脑屏障破坏而不允许不受控制的病毒复制是病毒性脑炎迫切需要的。主要目的和目的：在小鼠HSV脑炎模型中建立与血脑屏障破坏相关的中性粒细胞特异性迁移行为。-确定驱动这种行为的典型/非典型引诱蛋白-受体相互作用的相对贡献。-在bbb完整性破坏过程中调查NETs的差异产生和相关过程。目标-分享英国和日本pi之间的现有经验和技能，包括脑MP-IVM的颅开窗手术以及实时引诱蛋白和中性粒细胞行为可视化；为未来的合作研究建立强有力的伙伴关系。-交换早期职业研究人员，让他们在英国和日本的实验室环境中接触这些技术，开始建立能力，为获得更大的资助，在免疫细胞迁移生物学领域建立领先的实验室奠定基础。-利用MP-IVM（日本）和BBB建模（英国）的互补平台，展示这两种方法的叠加作用，这两种方法具有广泛的潜力，可作为探索免疫细胞在感染和自身免疫反应中的迁移的基础。

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.