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

背景：恶性疟原虫是人类最致命的疟疾寄生虫，每年造成2亿人感染。这些感染中的一小部分发展为严重疾病，导致每年40万人死于疟疾。大多数是由脑型疟疾(CM)引起的，以癫痫和昏迷为特征。CM主要发生在非洲儿童中。在幸存者中，有一半患有脑损伤或学习困难。尽管接受了抗疟疾药物的治疗，但这些糟糕的结果仍然存在。还需要其他治疗方法，但识别因果过程有很多障碍：寄生虫触发的关键过程发生在大脑中，在生命中无法接触到；动物模型再现了人类CM的一些特征，但不是全部；体外模型，虽然可以操作，但必然是简化的。马拉维的数十项合作工作已经确定了导致CM死亡的主要过程：血脑屏障破裂(BBB)引起的液体泄漏到大脑导致的脑肿胀。在没有疾病的情况下，大脑血管内皮细胞之间有水密的连接。当感染疟疾的红细胞(IRBC)附着在内皮细胞上时，这些“紧密连接”就会消失，液体就会泄漏到大脑中。我们假设血脑屏障的破裂是由红血球释放有毒物质引起的；红血球破裂是寄生虫感染新的红细胞的过程。或者，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的破坏。这两个过程可能都很重要。每一个都可能是治疗的目标。但以前的技术无法解决这种复杂性，因此解决这一问题的尝试是零碎的--一次查看一个或两个标记，或者使脑片同质化(从而失去关键的细胞多样性)。单细胞方法的出现(在数千个单个细胞上提供数千个数据点)彻底改变了解决这种复杂过程的能力。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破坏的相互作用：单细胞RNA测序(scRNA-seq)，它分析每个细胞中所有开启和关闭的基因；成像质量细胞术，它通过激光扫描组织切片上的40个标记物来提供高分辨率图像-提供组织的细胞图谱。从研究患有CM的马拉维儿童身体组织中宿主和寄生虫的相互作用开始：生成脑细胞的地图(Obj1)，以及它们是如何相互作用导致BBB崩溃的(Obj2)；导致预测导致BBB崩溃的事件，以及我们可以通过治疗阻止这一过程的时间点。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驱动的血脑屏障破坏的能力(Obj3)。这些方法将产生新的假设，然后我们将回到患者床边进行测试(目标4)。创新：对大脑的全面检查和CM中的血脑屏障分解的相同限制阻碍了对其他疾病(例如脑膜炎、脑癌、新冠肺炎)中血脑屏障的研究。这将是这些尖端方法首次应用于人类血脑屏障的研究，并将率先在撒哈拉以南非洲地区使用scRNA-seq。我们可能是世界上唯一有能力广泛描述CM患者血脑屏障破坏特征的网站(通过MRI测量脑容量，测量脑内超声血流，测量眼睛平行血管中的泄漏)，在现场收集脑组织，并在这些组织中进行scRNA-seq的关键步骤。应用与优点：我们的主要目标是确定治疗靶点，以改善CM的结果。我们的发现也可以推广到其他情况，在这些情况下，血脑屏障的分解是一个关键的过程。

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.