Figure 6.

Inhibition of NO restored CD4 T cell proliferation but did not alter the ability of MIS416 to inhibit EAE or modulate spleen myeloid subsets. (a) Splenocytes (1 × 10⁶/ml) were isolated from mice 15 days post MIS416 treatment initiation, labelled with CSFE, and stimulated in vitro with Con A (3 μg/ml) +/− MIS416 (20 μg/ml) in the presence of absence of aminoguanidine (AG; 200 mM) for 48 hours. The % of CD4⁺ cells that divided was quantified by flow cytometry. Shown are the means and SEM of individual mice (n = 8–10/group) from 2 independent experiments. ***p < 0.001 and **p < 0.01 by two-way ANOVA with Sidak’s multiple comparison test. (b,c) Mice were treated with MIS416 weekly (i.v.; 100 μg/mouse) starting on day 0, immunized to induce EAE, and scored daily for EAE (b) with the cumulative disease shown as area under the curve (AUC; c). Aminoguanidine (AG; 100 mM) was administered in the drinking water starting on day 12 (dotted line). Shown are the means and SEM of individual mice (n = 9–10/group) from 2 independent experiments. ***p < 0.001 and *p < 0.05 by two-way ANOVA with Sidak’s multiple comparisons test (b) and ***p < 0.001 and *p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. (d–f) Splenocytes and serum were isolated from EAE mice treated with MIS416 (weekly; starting day 0) and/or AG (100 mM; starting day 12) 22 days post immunization. Serum NO (d) was assessed directly, and the total number of red pulp macrophages (RPMφ; e) and expression of PDL-1 (geoMFI; f) was determined by flow cytometry. Shown are the means and SEM from individual mice (n = 4–5/group) from one experiment. (g–i) Splenocytes and serum were isolated from healthy mice treated with MIS416 and/or AG (100 mM; starting day 0) 15 days post MIS416 treatment initiation. Serum NO (g) was measured directly, and the total number of red pulp macrophages (RPMφ; h) and expression of PDL-1 (geoMFI; i) were determined by flow cytometry. Shown are the means and SEM from individual mice from two independent experiments (h; n = 7–8/group), and one experiment (i; n = 5/group). (d–i) ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test.