Fig. 6

IL-33/ST2 signaling in macrophages protects against BBB leakage in vitro in response to oxygen-glucose deprivation. (A) Primary mouse brain microvascular endothelial cells (BMECs) in cell culture inserts were exposed to 6 h of OGD. Reperfused BMECs were then cocultured with PBS-treated or IL-33-treated (25 µg/mL) WT or ST2 KO macrophages for 6 h. The diffusion of 40 kDa FITC-dextran from the luminal to abluminal chamber was measured over time. (B-C) Primary mouse BMECs in cell culture plates were exposed to 6 h of OGD. Reperfused BMECs were then treated for 4 h with conditioned media collected from PBS-treated or IL-33-treated WT or ST2 KO macrophages. (B) Representative western blot images and quantification of ZO-1. β-Actin was used as the loading control. (C) Representative images of BMECs immunolabeled for VE-cadherin (green) or ZO-1(magenta), and counterstained with DAPI (blue) for nuclear labelling. Images are representative of 3 independent experiments. (D) PCR arrays were used to measure immune regulatory changes in IL-33 or PBS-treated macrophages. The black line indicates fold-changes (2 ^ ^(-ΔCt)) relative to one. The purple lines indicate two-fold or negative two-fold changes in gene expression. Two arrays/group in two independent experiments. (E) RT-PCR was used to quantify the expression of IL10 and IL13ra2 in PBS or IL-33 treated macrophages. (F) BMECs in cell culture inserts were exposed to 6 h of OGD followed by reperfusion and treatment with PBS or IL-33 for 6 h. The diffusion of FITC-dextran from the luminal to abluminal chamber was measured over time. (G-H) BMECs in cell culture plates were exposed to 6 h of OGD followed by reperfusion and treatment with PBS or IL-33 for 4 h. (G) Representative western blot images and quantification of ZO-1. (H) Representative images of BMECs immunolabeled for VE-cadherin (green) or ZO-1(magenta), and counterstained with DAPI (blue) for nuclear labelling. Images are representative of 3 independent experiments. *P < 0.05. **P < 0.01. Two-tailed Student’s t test (E), one-way (B, G) or two-way (A, F) ANOVA followed by post hoc Bonferroni test