Fig. 8

Knockdown of S1PR3 in astrocytes reduced cytokine secretion and altered endothelial adhesion. Serum-free culture supernatant from shControl-1 and shS1PR3-1 astrocytes were compared for expression of 36 cytokines and chemokines using a commercial human cytokine array. a Mean spot pixel density was measured of quantifiable spots with ImageJ and plotted which demonstrated a significant reduction in various cytokines (two spots for each cytokine). b The effect of neutralizing antibodies to cytokines on BBB permeability in vitro. See Fig. 5a for experimental schematic. Addition of neutralizing antibodies to IL-6, IL-8, CCL2, CXCL1, and GM-CSF increased TEER resistance as compared to an isotype-matched control antibody. Only anti-IL-6 and anti-CCL2 at 24 h showed a significant increase in TEER (n = 4). c Neutralizing antibodies to cytokines decreased doxorubicin permeability. Permeability of 100 µg mL⁻¹ doxorubicin from luminal to abluminal side of the inserts in the BBB was measured. Anti-IL-6 and anti-CCL2 decreased doxorubicin permeability significantly (n = 4). d Staining of endothelial cells for ZO-1 (green) and VE-cadherin (red) in BBB cultures treated with neutralizing antibodies. Nuclei were DAPI stained (blue) (scale bar = 50 μm). The effect of combined treatment of astrocytes with TY-52156 and anti-IL-6 antibody or anti-CCL2 antibody on TEER. There was no significant augmentation of resistance when IL-6 antibody (e) or CCL2 (f) antibody were included along with TY-52156 in BBB model (n = 4). For panels (b), (c), (e), and (f), each point indicates median fold change, and the bars represent the overall median with the 95% confidence interval, for each BBB culture. Kruskal–Wallis test and Dunn’s multiple comparison test were performed. *P < 0.05, **P < 0.01, ns non-significant