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

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.