Single cell interrogation of the blood-brain barrier in human cerebral malaria: a translational approach

背景：恶性疟原虫是最致命的人类疟疾寄生虫，每年造成2亿人感染。这些感染中有一小部分发展为严重疾病，每年造成40万人死于疟疾。大多数是由于脑疟疾（CM），其特征是癫痫发作和昏迷。CM主要发生在非洲儿童中。幸存者中有一半患有脑损伤或学习困难。尽管使用抗疟疾药物进行治疗，这些不良结果仍然存在。需要额外的治疗，但确定因果过程存在许多障碍：寄生虫引发的关键过程发生在大脑中，而大脑在生命中是无法进入的；动物模型再现了人类CM的部分特征，但不是全部；体外模型虽然是可操作的，但必然是简化的。马拉维数十年的合作工作已经确定了导致CM患者死亡的主要过程：血脑屏障破裂导致液体泄漏到大脑而导致的脑肿胀。在没有疾病的情况下，大脑血管内皮细胞之间存在水密连接。当疟疾感染的红细胞（iRBC）粘在内皮细胞上时，这些“紧密连接”就会丢失，液体就会渗漏到大脑中。我们假设血脑屏障的破坏是由iRBC释放有毒物质引起的；iRBC的破裂是寄生虫感染新红细胞的过程。另外，iRBC可能会吸引免疫细胞，通过过度活跃的防御反应导致血脑屏障破裂。这两个过程可能都很重要。每一种都可能成为治疗的目标。但是以前的技术无法解决这种复杂性，因此解决这个问题的尝试都是零零碎碎的——一次只看一个或两个标记物，或者将大脑的片段均质化（因此失去了关键的细胞多样性）。单单元方法（在1000个单个单元上提供1000个数据点）的出现彻底改变了解决此类复杂过程的能力。在这里，我们将使用两种高度互补的单细胞方法来解决导致血脑屏障分解的相互作用：单细胞RNA测序（scRNA-seq），它分析每个细胞中打开和关闭的所有基因，以及成像质量细胞术，它通过激光扫描组织切片上的40个标记来提供高分辨率图像，从而提供组织的细胞图谱。我们提出了一个系统的方法。从检查具有良好特征的患有CM的马拉维儿童死后组织中的宿主-寄生虫相互作用开始：生成大脑细胞图（Obj1），以及它们如何相互作用导致血脑屏障分解（Obj2）；从而预测导致血脑屏障分解的事件，以及我们可以通过治疗来阻止这一点。我们将在实验室培养的脑细胞模型中测试这些预测的治疗方法及其阻止irbc驱动的血脑屏障分解的能力（Obj3）。这些方法将导致新的假设，然后我们将回到患者床边进行测试（Obj4）。创新：CM中对脑和血脑屏障破坏进行全面检查的同样局限性也阻碍了对其他疾病（如脑膜炎、脑癌、COVID-19）中的血脑屏障的研究。这将是这些尖端方法在人类血脑屏障研究中的首次应用，并将在撒哈拉以南非洲率先使用scRNA-seq。我们可能是世界上唯一一个有能力广泛表征CM患者血脑屏障破坏的网站（通过MRI测量脑容量，通过超声测量脑血流，测量眼睛平行血管的泄漏），收集死后脑组织，并在这些组织中进行scRNA-seq的关键步骤。应用和益处：我们的主要目的是确定治疗目标，以改善CM的结果。我们的发现也可以推广到血脑屏障分解是一个关键过程的其他情况。

CONTEXT: Plasmodium falciparum is the deadliest human malaria parasite, causing 200-million infections annually. A small proportion of these infections progress to severe disease, resulting in 400,000 malaria deaths each year. Most are due to cerebral malaria (CM), characterized by seizures and coma. CM occurs mainly in African children. Among survivors, half suffer brain damage or learning difficulties. These poor outcomes persist despite treatment with anti-malarial drugs. Additional treatments are needed, but identifying the causal processes has had many barriers: critical processes triggered by the parasite occur in the brain, which is inaccessible during life; animal models reproduce some features of human CM but not all; in vitro models, while manipulatable, are necessarily reductive.Decades of collaborative work in Malawi have identified the major process leading to death in CM: brain swelling resulting from fluid leak into the brain caused by blood-brain barrier breakdown (BBB). In the absence of disease, there are watertight junctions between the endothelial cells lining blood vessels in the brain. When malaria infected red cells (iRBC) stick to the endothelial cells, those "tight junctions" are lost, and fluid leaks into the brain. We hypothesise that BBB breakdown is caused by iRBC releasing toxic contents; rupture of iRBC is the process by which the parasite infects new red blood cells. Alternatively, iRBC may attract immune cells which cause BBB breakdown through an overactive defence response. Both processes may be important. Each may be a target for treatments. But previous techniques are unable to resolve this complexity and thus attempts to resolve this have been piecemeal - looking at one or two markers at a time or homogenizing pieces of brain (thus losing critical cellular diversity).The advent of single cell approaches (providing 1000s of datapoints on 1000s of individual cells) has revolutionized capacity to resolve such complex processes. Here we will use two highly complementary single cell approaches to resolve the interactions that cause BBB breakdown: single cell RNA sequencing (scRNA-seq), which analyses all the genes that are switched on and off in each cell, and imaging mass cytometry which provides high resolution images by laser-scanning 40 markers in parallel on a slice of tissue -providing a cellular map of the tissue.We propose a systematic approach. Starting by examining host-parasite interactions in postmortem tissue from well-characterized Malawian children with CM to: generate a map of the cells in the brain (Obj1), and how they are interacting to cause BBB breakdown (Obj2); leading to predictions of the events that lead to BBB breakdown, and the points at which we might stop this with treatments. We will test these predicted treatments and their capacity to stop iRBC-driven BBB breakdown in a model of brain cells cultured in the lab (Obj3). These approaches will lead to novel hypotheses that we will then return to the patient bedside to test (Obj4).INNOVATION: The same limitations to a holistic examination of the brain and BBB breakdown in CM have stymied research on the BBB in other diseases (e.g. meningitis, brain cancers, COVID-19). This will be the first application of these cutting-edge approaches to study of the BBB in humans and will pioneer the use of scRNA-seq in sub-Saharan Africa. Ours is possibly the only site in the world with the capacity to extensively characterise BBB breakdown in CM patients (brain volume by MRI, measure by ultrasound blood flow in the brain, measure leak in the parallel vessels in the eye), collect brain tissue postmortem, and conduct the key steps for scRNA-seq in those tissues on site.APPLICATIONS & BENEFITS: Our principal aim is to identify treatment targets to improve outcomes in CM. Our findings may also be generalisable to other conditions in which BBB breakdown is a critical process.