Highly purified lipoteichoic acid from gram-positive bacteria induces in vitro blood–brain barrier disruption through glia activation: Role of pro-inflammatory cytokines and nitric oxide

The co-culture of bovine brain capillary endothelial cells and rat primary glial cells was established as an in vitro blood–brain barrier model to investigate the mechanisms by which the Gram-positive bacterial cell wall components lipoteichoic acid and muramyl dipeptide induced injury of blood–brain barrier structure and function. We found that highly purified lipoteichoic acid disrupted blood–brain barrier integrity in a concentration- and time-dependent manner indirectly, through glia activation. Low trans-endothelial electrical resistance and high permeability to fluorescein isothiocyanate-inulin observed in the presence of lipoteichoic acid-activated glial cells were potentiated by muramyl dipeptide and could be reversed only when glial cells were activated by lipoteichoic acid at 10 μg/ml but not with a higher lipoteichoic acid concentration (30 μg/ml). Immunocytochemistry analysis revealed no evident changes in the distribution of the cytoskeleton protein F-actin and tight junction proteins occludin and claudin after lipoteichoic acid treatment. However, the tight junction associated protein AHNAK clearly revealed the morphological alteration of the endothelial cells induced by lipoteichoic acid. Lipoteichoic acid-activated glial cells produced nitric oxide and pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β) that contributed to lipoteichoic acid-induced blood–brain barrier disruption, since the direct treatment of the endothelial monolayer with tumor necrosis factor-α or interleukin-1β increased blood–brain barrier permeability, whereas the pre-treatment of lipoteichoic acid-activated glial cells with antibodies against these two cytokines blocked lipoteichoic acid effects. Additionally, nitric oxide was also involved in blood–brain barrier damage, since the nitric oxide donor itself (diethylenetriamine-nitric oxide adduct) increased blood–brain barrier permeability and inducible nitric oxide synthase inhibitor (1400W) partially reversed lipoteichoic acid-induced trans-endothelial electrical resistance decrease.