Fig. 7

Sequential cytokine addition affects myelin phagocytosis and NOX-mediated ROS production. Data are presented as mean ± SEM; n = 12 (a, b), n = 6 (c). Data were normalized to unstimulated (CTL) microglia (indicated by dashed lines) in the presence of myelin (for phagocytosis) or without myelin (for ROS production). a M2→M1. Microglia were first stimulated with 20 ng/mL IL-4 (M2a) or 20 ng/mL IL-10 (M2c) for 2 h and then with 20 ng/mL IFN-γ + 50 ng/mL TNF-α (I + T; M1) for an additional 22 h. b M1→M2. I + T was added for 2 h, followed by IL-4 (M1→M2a) or IL-10 (M1→M2c) for a further 22 h. For a and b, DiI-labeled myelin (25 μg/mL) was added to cultures 24 h after adding the first cytokine, and both phagocytosis and intracellular ROS levels were assessed 6 h later (as in Figs. 2b,c and 3b,c). c Effect of NOX inhibition on myelin phagocytosis and ROS production in microglia that were stimulated as in a and b. Microglia were incubated with 25 μg/mL myelin for 6 h, with or without the pan-NOX inhibitor, 5 μM VAS2870. Results were analyzed by a one-way ANOVA (for phagocytosis in a) or a two-way ANOVA (for ROS and phagocytosis in c) with Bonferroni’s post hoc test. The comparisons are as follows. Asterisk Differences from unstimulated (CTL) microglia. Dagger sign CTL versus different activation states in the presence of myelin. Number sign Differences within (or among) different activation states. Double dagger sign Effects of myelin (a, b) or VAS2870 (c) within or between activation states. One symbol p < 0.05, two symbols p < 0.01, three symbols p < 0.001