Interrogating the blood brain barrier for translation in cerebral malaria

Perhaps one day malaria will be eliminated, but currently we are nowhere close. Advances from control measures and the development of a vaccine are counterbalanced by resistance in both the parasite (to antimalarial drugs) and the mosquito vector (to insecticides). Malaria still causes >200-million infections and 600,000 deaths annually. The deadliest malaria parasite, Plasmodium falciparum, causes cerebral malaria (CM)—marked by seizures and coma—over 70% of cases in African children. Among survivors, more than half suffer brain damage or learning difficulties. These poor outcomes persist despite antimalarial treatment. Effective adjunctive therapies are needed, but their identification requires understanding the causal processes behind adverse outcomes. Decades of collaborative work in Malawi have identified a key process in CM: brain swelling due to fluid leak into the brain, caused by blood-brain barrier (BBB) breakdown. Normally, tight junctions between endothelial cells prevent such leakage, but when malaria-infected red blood cells (iRBC) adhere to these cells, the junctions are lost, allowing fluid to enter the brain. Aim and Objectives: Our overall aim is to identify potential treatment targets to improve CM outcomes by defining mechanisms of BBB breakdown. We employ a bedside-to-bench-to-bedside framework. In Stage 1, we discovered critical processes in patient postmortem samples. In the renewal phase, we will validate these in vitro (WP1) and return to patients to identify signature proteins in plasma (WP2). Progress: In Stage 1, we analysed postmortem samples from fatal CM cases. Using cutting-edge methods—including single-cell sequencing, spatial transcriptomics, and imaging mass cytometry—we created an exquisitely detailed atlas of brain cells and their states. This enabled computational modelling to make predictions of critical interactions leading to BBB-breakdown. We found a key role for neutrophils via a cell death process called neutrophil extracellular traps (NETs), in which neutrophils release DNA and toxic granules. While NETs trap pathogens, they also damage blood vessels and were closely associated with BBB breakdown. We also observed upregulation of the protein SPP1 in microglia, concentrated around sites of BBB breakdown. SPP1, a pro-inflammatory cytokine, is implicated in other brain diseases and promotes NET formation by recruiting neutrophils. Hypothesis and Next Steps: We hypothesize that SPP1 is essential for BBB-breakdown in CM. Our computational modelling predicts that iRBCs trigger SPP1 upregulation in microglia and immune cells; SPP1 acts on endothelial cells to activate the JAK/STAT pathway, causing BBB breakdown and neutrophil recruitment. Blocking this may offer therapeutic targets. Approach: Because P. falciparum is human-specific, we will use a 3D bioengineered in vitro model developed by project partner, Dr. Bernabeu. This microfluidic model, comprising human brain endothelial cells, pericytes, and astrocytes, mimics key features of brain vessels. Preliminary work shows that iRBC exposure upregulates SPP1 and disrupts the barrier, while JAK/STAT inhibition rescues it. WP1 will assess SPP1’s role and explore other triggers of BBB breakdown using in vitro models and proteomics. WP2 will determine whether proteins released under these conditions are detectable in patient plasma, validating their clinical relevance. Together, these approaches will identify potential treatment targets for BBB breakdown in CM. Our findings and investigative framework are likely to be of major utility for a wide range of diseases in which BBB-breakdown is critical. The renewal also offers key training in 3D-modelling and other methods, alongside courses, coaching and mentorship to support the fellow’s development into a leader in his field.