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Abstract

Cerebral malaria (CM) is a severe neurovascular complication of Plasmodium falciparum infection, characterized by vasogenic edema and brain swelling caused by the disruption of the blood-brain barrier (BBB). While postmortem samples have shown that P. falciparuminfected red blood cells (iRBC), as well as different immune cell populations, accumulate in the microvasculature of CM patients, the exact pathogenic mechanisms of BBB disruption in CM remain unknown. I utilized an in vitro 3D-BBB model in combination with single-cell RNA sequencing (scRNAseq) to dissect the pathogenic contribution of P. falciparum-iRBC and P. falciparum-activated immune cells. P. falciparum-iRBC-egress products induced a transcriptional downregulation of endothelial junction genes, resulting in barrier disruption. Furthermore, I observed an increased expression of genes related to interferon type I response, JAK–STAT pathway, antigen presentation, and ferroptosis in all BBB cell types. Through the perfusion of different iRBC stages, parasite egress was identified as the primary iRBC-related pathogenic driver, inducing localized transcriptional changes associated with areas with high levels of iRBC-egress. P. falciparum stimulation of immune cells significantly enhanced their microvascular binding affinity in the 3D-BBB model, with the strongest increase in activated CD8+ and gd T cells. Adhesion of P. falciparum-activated immune cells triggered BBB disruption and activation of inflammatory, cytoskeletal, and apoptotic transcriptional modules. BBB inflammation could mainly be explained by an increased secretion of TNF-a and IFN-g. BBB disruption was shown to be dependent on the binding of P. falciparum-activated immune cells and correlated significantly with granzyme B and IFN-g released by NK cells and gd T cells. Integration of the two acquired scRNAseq datasets showed that both P. falciparum and innate immune cells activate JAK–STAT signaling at the BBB. Moreover, the analysis disentangled parasite- from host immune response-driven effects and identified a potential detrimental feedback loop between microvascular P. falciparum and immune cell accumulation. This multimodal analysis provides new mechanistic insights into the contribution of different pathogenic drivers in CM and highlights potential host-directed therapeutic strategies.