Understanding cerebral inflammation in viral encephalitis - how does neuron-glial signalling drive blood-brain barrier permeability?

脑炎症，又称“脑炎”，是一种毁灭性的疾病，通常由感冒疮病毒（单纯疱疹病毒-HSV）引起。炎症的发生是由于血流中的天然屏障的破坏和免疫细胞的迁移。尽管有抗病毒药物，炎症仍在继续。因此，大多数患者死亡或有严重的脑损伤。有时患者给予类固醇，但其广泛的免疫抑制作用的风险增加病毒，所以需要有针对性的治疗。我在血脑屏障（BBB）破坏最严重和预后最差的患者中发现了几种这样的蛋白质，并开发了一种小鼠模型，通过观察大脑中免疫细胞的运动来研究这一点。目前尚不清楚BBB破坏在多大程度上导致免疫细胞迁移，反之亦然，也不知道受感染的神经如何与其他脑细胞交流，了解这种相互作用的网络对于开发靶向免疫疗法至关重要，这种疗法可以减少脑部炎症并改善患者的预后，而不会增加病毒的数量。建立与HSV脑炎中BBB破坏最密切相关的炎症蛋白，并且这些蛋白是/不是类固醇反应性的2。探究阻断这些炎症蛋白如何改变BBB分解、免疫细胞迁移和病毒控制3。确定这些免疫蛋白是如何在受感染的神经、其他脑细胞和BBB细胞之间进行交流的。1.从一个接受类固醇或安慰剂治疗的HSV脑炎患者的独特队列中，我收集了血液和脊髓液。a.我将通过寻找特异性炎症蛋白和评估所有蛋白b来分析这些。我将比较它们与BBB渗透性的程度，从：i.从血液中渗漏到脊髓液中的白蛋白量i.脑扫描中脑肿胀的体积（磁共振成像）2.通过确定关键炎症蛋白的作用，在我建立的小鼠模型中，使用缺乏这些炎症蛋白受体的小鼠和/或使用中和抗体阻断炎症蛋白，并确定：a.使用已建立的评分，发病率是否降低？B.通过测量静脉内染料向脑中的渗漏，是否减少了BB B的分解？c.使用空斑试验，病毒控制是否受到影响？d.使用活体显微镜观察麻醉小鼠脑中真实的免疫细胞运动是否改变了免疫细胞迁移？3.在这个模型中，我将分析随着时间的推移，大脑和BBB中每种细胞类型的炎症蛋白和相关信号的产生，以确定关键炎症蛋白的上游和下游通信网络。在对单个细胞和不同细胞培养物的HSV感染的反应中，我将询问阻断这些通信是否会改变BBB的破坏和免疫细胞的迁移。优点这很重要，这样我就可以开发出有针对性的免疫治疗方法来控制炎症、BBB破坏和白细胞迁移，而不会有病毒复制失控的风险。通过对HSV的建立，它为广泛的其他脑感染开辟了治疗机会。在建立了从临床样本和小鼠模型和脑感染的BBB模型产生假设的能力之后，我将非常适合确定对HSV和其他脑感染的免疫反应之间的共性和差异。因此，我将能够推动靶向治疗，并将处于有利地位，成为该领域的领导者。

CONTEXTBrain inflammation, termed 'encephalitis', is a devastating disease commonly caused by the cold-sore virus (herpes simplex virus-HSV). Inflammation occurs due to breakdown of the natural barrier with the bloodstream and the migration of immune-cells in. Despite antivirus medication the inflammation continues. Consequently, most patients die or have significant brain injury.Sometimes patients are given steroids but their broad immune-suppression action risks increasing the virus, so targeted treatments are needed.Immune-cells are attracted by inflammation-proteins; I identified several such proteins in patients with the greatest blood-brain barrier (BBB) breakdown and worst outcome, and developed a mouse-model to study this by visualising immune-cell movement in the brain.To what extent BBB breakdown leads to immune cell migration or vice versa is not clear and it is not known how infected nerves communicate with other brain cells, and BBB cells.Understanding this network of interactions is critical to develop targeted immune therapy which reduces brain inflammation and improves patient outcome, without increasing the amount of virus.AIMS1. Establish the inflammation proteins which most closely associated with BBB breakdown in HSV encephalitis and which are/are not steroid responsive2. Interrogate how blocking these inflammation proteins alters BBB breakdown, immune cell migration and viral control3. Determine how these immune proteins communicate between infected nerves, the other brain cells and the BBB cellsOBJECTIVES1.From a unique cohort of HSV encephalitis patients who have received either steroids or placebo, I have collected blood and spinal fluid.a.I will analyse these by both looking for specific inflammation proteins and also by assessing all proteinsb.I will compare them to the degree of BBB permeability from:i.Amount of albumin protein which has leaked from the blood into the spinal fluidii.The volume of brain swelling on the brain scan (magnetic resonance imaging)2.Take forward key inflammation proteins by determining their action, in a mouse model I established, using mice lacking the receptor for these inflammation proteins and/or block the inflammation protein using a neutralising antibody and determine:a.Is morbidity decreased, using an established score?b.Is BBB breakdown reduced, measuring the leakage of an intravenous dye into the brain?c.Is viral control affected, using a plaque assay?d.Is immune cell migration altered, using intravital microscopy to visualise immune cell movements in real time in the brain of an anaesthetised mouse?3. In this model, I will analyse the generation of inflammation proteins and associated signals from each cell type in the brain and BBB over time to determine networks of communication up-stream and down-stream to key inflammation proteins.a.I will then confirm these findings by examining the interaction between nerve cells, other brain cells, and BBB cells in a BBB modeli.In response to HSV infection of single cells and cultures of different cells which may interactii.I will interrogate whether blocking these communications alters BBB breakdown and immune cell migrationPOTENTIAL APPLICATIONS AND BENEFITSThis is important so that I can develop targeted immune treatments to control inflammation, BBB breakdown, and leucocyte migration, without risking uncontrolled viral replication. By establishing this for HSV it opens up treatment opportunities for a wide range of other brain infections.Having built the capacity to generate hypotheses from clinical samples and with both a mouse model and a BBB model of brain infection, I will be excellently placed to determine the commonalities and differences between the immune response to HSV and other brain infections. Consequently, I will be able to drive forward targeted therapies and will be in a strong position to become a leader in the field.