Figure 3

Neuroprotective concentrations of dimethyl fumarate (DMF) suppress cytokine production in activated splenocytes from two different mouse strains without altering viability. 10 μM DMF does not significantly affect cell viability of splenocytes from C57BL/6 mice (A) or SJL mice (A’) while 100 μM is toxic (A) and (A’). Cells were treated for 24 h with the indicated concentrations of DMF or vehicle. Cell viability was measured by flow cytometry quantitating 7-AAD and Annexin V-positive cells. B) and B’) DMF concentration-dependently reduced TNFα production from anti-CD3-stimulated splenocytes from C57BL/6 (B) and SJL mice (B’). Primary splenocytes from seven mice were treated with the indicated concentrations of DMF for 48 h and co-stimulated with 1 μg/ml anti-CD3 for the same time before TNFα was measured in the supernatants by ELISA. C) and C’) DMF decreases anti-CD3-induced production of IL-17 and IL-2 in splenocytes from C57BL/6 mice (C) and of the production of IF-γ, IL-2, IL-4, IL-5, IL-6 and IL-17 in splenocytes from SJL mice (C’). Primary splenocytes from seven mice were treated with 10 μM of DMF for 24 h and costimulated with 0.5 μg/ml anti-CD3 for the same time before interferon-gamma, IL-2, IL-4, IL-5, IL-6 and IL-17 were measured using ELISPOT technology. Graphs of all experiments represent the means ± standard error of the mean (SEM) of three independent experiments performed in triplicate.*P < 0.05, paired Student’s t-test in (C) and (C’) and two-way analysis of variance (ANOVA) with Bonferroni post hoc test in (A), (A), (B), and (B’).