Fig. 2

Ibrutinib disrupts brain endothelial integrity and inhibits ABC transporter function. Brain endothelial cell viability is not affected by ibrutinib treatment at varied doses (a). Dose-dependently, ibrutinib decreases brain endothelial cell–cell impedance, significantly 2 h after treatment seen with 5 and 10 µM with subsequent cell index plateau (*p < 0.05, **p < 0.005) (b). Bicellular junction protein ZO-1 and tricellular junction protein MarvelD2 was significantly decreased 2 h after 10 µM ibrutinib treatment (c). Junctional mRNA expression also decreased from 2 to 24 h after 10 µM treatment, without a washout period seen in tjp1 (ZO-1), MarvelD2 (tricellulin), Ocln (occludin), Cldn5 (claudin-5), Lsr (lipolysis stimulated lipoprotein receptor/angulin-1), and cldn3 (claudin-3) (d). High baseline BTK expression seen in both cytoplasm and nucleus of brain endothelial cells. Confirmatory immunostaining of ZO-1 expression demonstrated decreased tight junctional linear staining at 4 h, with rearrangement closer to baseline regarding adhesion expression by 24 h. (e). Silencing of BTK with siBTK results in decreased cell–cell impedance transiently and impaired tight junction gene expression (***p < 0.0005, ****p < 0.0001) (f, g). Ibrutinib dose-dependently inhibited Abcb1 function to increase rhodamine accumulation with higher FITC-H fluorescence measurement causing a shift of amplitude to the right, comparative to valspodar (ABCB1 inhibitor) treated cells (h). Monolayer endothelial cells treated with ibrutinib on transwells resulted in approximately 26% higher sodium fluorescein permeability compared with control treatment 24 h later (****p < 0.0001) (i)