ALBUMIN TRANSPORT TRIGGERS MICROGLIAL ACTIVATION BEFORE BBB BREAKDOWN IN EARLY STAGES OF CEREBRAL MALARIA

Cerebral malaria (CM) is a severe P. falciparum complication. Blood-brain barrier (BBB) breakdown, observed in later stages, is associated with endothelial damage and serum leakage into the brain parenchyma. The role of brain endothelial cells (BEC) in early stages of CM and the interaction of transported serum proteins with brain parenchyma cells remain poorly understood. Recent findings show that even in low concentrations in brain, serum proteins can contribute to cognitive deterioration in neurological disorders. This study aimed to verify the role of BEC in early CM and the interaction of transported serum proteins with microglial cells. CM was induced in C57BL/6 mice by injecting 10⁶ P. berghei ANKA-infected erythrocytes. BBB stability was evaluated 3–5 days post-infection (p.i.). (CEUA 032/23) In vitro assays were performed using mouse microglial cells lineage (BV-2). As expected, Evans blue dye assay and endogenous IgG deposition showed BBB breakdown in mice on days 4–5 p.i. However, FITC-labeled albumin accumulated in brain tissue as early as day 3 p.i. which correlated with increased microglial activation revealed by increased IBA-1 marker. Nystatin (8 mg/kg), a caveolae-mediated transport inhibitor, reduced in vitro FITC-albumin uptake, attenuated microglial activation, improving BBB stability and behavioral outcomes (n=4). In, BV-2 cells, increasing albumin concentrations reduced FITC-albumin endocytosis and induced morphological changes in a dose- and time-dependent manner (n=3). High albumin (HA, 20 mg/mL) decreased FITC-albumin binding and uptake while enhancing IBA-1 and MHC II expression. Interestingly, while lipopolysaccharide-induced neuroinflammation polarized BV-2 cells toward a pro-inflammatory phenotype marked by iNOS expression, HA also induced M2 markers such as mannose receptor expression. Moreover, HA stimulated cytokine release, including IFN-γ, TNF, IL-4 and IL-10 production. Inhibition of cytokine secretion by Brefeldin A, did not alter HA effects on endocytosis, suggesting cytokines do not directly mediate this response. Together, our results suggest that BEC protein transport through a caveolae-dependent manner precedes BBB breakdown in CM and that serum albumin can interact with microglial cells promoting activation, highlighting new insights into CM pathogenesis.