Figure 1.
Risperidone dose-dependently reduces the severity of EAE.
a. Mice were treated with risperidone (1 or 3 mg/kg/day; n = 10 and 5, respectively) or vehicle (n = 9) in their drinking water from the time of immunization and scored daily (0; normal – 5; moribund). Shown are the means and SEM of individual mice. Two mice from the vehicle group were euthanized on day 19 (indicated by †) and were excluded from analysis. p<0.05 by two-way ANOVA (vehicle vs risperidone 3 mg). b. Mice treated with 3 mg/kg/day risperidone (n = 20) have significantly reduced peak disease score compared to vehicle-treated mice (n = 18). Shown are the medians and values of individual mice from 4 experiments. **p = 0.01 by Mann-Whitney test. c. Risperidone (3 mg/kg/d) significantly reduces cumulative disease as assessed by the area under the curve (AUC) of individual animals 42 days post-immunization. Shown are the means and SEM of individual mice (n = 9 vehicle-treated and 5 risperidone-treated) from 2 experiments. *p<0.05 by unpaired Student's t test. d. Risperidone (3 mg/kg/day) reduces lesion area in the spinal cords of mice. Spinal cord lesions were assessed on H & E stained tissue (see Figure S5 for images) and expressed as lesion area per 100 µM area of tissue examined. Shown are the medians and values of individual mice (n = 9 vehicle and 15 risperidone-treated) from three experiments. *p<0.05 vehicle versus drug by Mann-Whitney test.
Table 1.
Effect of risperidone treatment on splenocyte subpopulations.
Figure 2.
Risperidone treatment sustains antigen-specific IFN-γ responses and depresses IL-17a and IL-4 responses in EAE-immunized mice.
Splenocytes were isolated from risperidone- and vehicle-treated, unimmunized and immunized mice 15 days post-immunization and stimulated in vitro with MOG peptide for 72 hours (a-f) or ConA for 48 hours (g-l). IL-17a (a and g), IFN- γ (b and h), IL-4 (c and i), IL-6 (d and j), IL-2 (e and k), IL-13 (f and l) production from individual mice was assessed by CBA. Shown are the means and SEM of individual mice from three experiments (n = 10–15 per group). * p<0.05 by one-way ANOVA with Newman-Keul's multiple comparison test.
Figure 3.
Risperidone reduces IL-12p40 and nitric oxide production and enhances IL-10 production by LPS-stimulated BMMΦ (a and b) and modifies their ability to bias CD4 T cells (c and d).
a. Risperidone inhibited IL-12p40 production by LPS-stimulated BMMΦ in a concentration-dependent manner and at high concentrations affected viability as assessed after 24 hours by MTT assay. Values are expressed as % control (i.e. compared to LPS + vehicle), and shown are the means and SEM of values from 5 experiments. *p<0.05, **p<0.01, and ***p<0.001 by one-way ANOVA with Dunnett's post-test (compared to LPS + vehicle). b. Risperidone (20 µg/ml) significantly reduced IL-12p40 and NO but enhanced IL-10 production by LPS-stimulated BMMΦ. Shown are the means and SEM of replicate wells from 1 of at least 3 experiments (IL-12p40 and NO) or combined from 2 experiments (IL-10). **p<0.01 and ***p<0.001 by unpaired Student's t test. c & d. LPS-stimulated BMMΦ activated purified MOG-specific 2D2 T cells and 4 hour pretreatment (d) or continuous exposure (c) of BMMΦ to risperidone (50 µM) for 4 hour before addition of T cells modified T cell biasing as assessed by IFN-γ, IL-17a, and CD124 expression. Shown are the means and SEM of triplicate wells from 4 (c) or 2 (d) experiments. *p<0.05, **p<0.01, and ***p<0.001 by one-way ANOVA with Newman-Keul's post test (IFN-γ) or by paired Student's t test (IL-12, IL-10, IL-17a, IL-2, and CD124).
Figure 4.
Another atypical antipsychotic agent, clozapine, also reduces the severity of EAE.
a & b. Clozapine (a) but not sulpiride (b) inhibited IL-12p40 production by LPS-stimulated BMMΦ in a concentration-dependent manner and at higher concentrations affected viability as assessed after 24 hours by MTT assay. Values are expressed as % control (i.e. compared to LPS + vehicle), and shown are the means and SEM from 1 of 3 (a) or 2 (b) experiments. *p<0.05, **p<0.01, and ***p<0.001 by one-way ANOVA with Dunnett's post-test (compared to LPS + vehicle). c. Mice were treated with clozapine (5–60 mg/kg/day) or vehicle in their drinking water from the time of immunization and scored daily (0; normal – 5; moribund). Shown are the means and SEM of individual mice (n = 5 per group) from 1 of 4 dose-response experiments. d. Clozapine (30 mg/kg/day; n = 10) reduces EAE disease to a similar extent as risperidone (3 mg/kg/day; n = 20) but sulpiride (100 mg/kg/day; n = 5) is ineffective compared to vehicle treatment (n = 27). Shown are the means and SEM of individual mice from 4 experiments. e. Blocking dopamine signaling with a specific D1 antagonist (LE300) or D2 antagonist (L741,626) did not affect IL-12 production by RAW264.7 macrophages but D2 antagonism did reduce viability as assessed by MTT assay. Values are expressed as % control (i.e. compared to LPS + vehicle), and shown are the means and SEM of values from 1 of 2 experiments. *p<0.05, **p<0.01, and ***p<0.001 by one-way ANOVA with Dunnett's post-test (compared to LPS + vehicle). f & g. Dopamine inhibited IL-12p40 production by RAW264.7 macrophages without affecting viability (f) and risperidone enhanced the effects of dopamine on IL-12 but not IL-10 production (g). Shown are the means and SEM combined from 7 experiments. *p<0.05, **p<0.01, and ***p<0.001 by one-way ANOVA with Dunnett's post-test (compared to LPS + vehicle).
Figure 5.
Treatment with risperidone in vivo does not alter LPS-stimulated splenic cytokine production.
Splenocytes were isolated from risperidone- and vehicle-treated, unimmunized and immunized mice 15 days post-immunization and stimulated in vitro with LPS for 24 hours. TNF-α (a), IL-6 (b), IL-10 (c), IL-17a (d), IFN- γ (e), IL-4 (f) production from individual mice was assessed by CBA. Shown are the means and SEM of individual mice from three experiments (n = 10-15 per group). *** p<.05 by one-way ANOVA with Newman-Keul's multiple comparison test.
Figure 6.
Risperidone treatment does not change the percentage or activation of splenic myeloid populations during EAE.
a. Gating strategy for the major splenic myeloid populations. b. Red pulp macrophages (RP MΦ) and DC but not white pulp macrophages express MHC II and CD40. Shown are representative plots from a vehicle-treated, immunized mouse. c. Immunization induces an increase in neutrophils and red pulp macrophages and risperidone alone enhances splenic DC. Splenocytes were isolated from risperidone- and vehicle-treated, unimmunized and immunized mice 15 days post-immunization and gated as shown in a. Shown are the means and SEM of individual mice from three experiments (n = 10–15 per group). **p<0.01 and ***p<.001 by one-way ANOVA with Newman-Keul's multiple comparison test. d. No difference in the number of total splenocytes is observed. Splenocytes were isolated from risperidone- and vehicle-treated, unimmunized and immunized mice 15 days post-immunization and live cells counted by the trypan blue exclusion assay. Shown are the means and SEM of individual mice from three experiments (n = 10–15 per group). e–f. Immunization increases the expression of I-A on RP MΦ (e) and DC (f) and CD40 on DC (h) and risperidone does not alter these levels. Splenocytes were isolated from risperidone- and vehicle-treated, unimmunized and immunized mice 15 days post-immunization and gated as shown in a. The expression (ΔMFI compared to isotype controls) of I-A and CD40 are expressed as % of vehicle-treated, unimmunized group. Shown are the means and SEM of individual mice from three experiments (n = 10–15 per group). *p<0.05 and **p<0.01 by two-way ANOVA to distinguish overall drug and immunization effects.
Figure 7.
Risperidone treatment significantly reduces microglial activation in the CNS of immunized mice during EAE.
a. Iba-1 expression (deep pink) in the cerebellum was assessed by immunohistochemistry and counterstained with hematoxylin (light purple). Shown are representative sections from unimmunized and immunized, vehicle and risperidone-treated mice as well as the “Iba-1 score” and “disease score” at time of euthanasia. b & c. Iba-1 expression in the cerebellum (b) and all brain and spinal cord regions (c) assessed (cerebellum, hippocampus, brain stem, olfactory bulb, and spinal cord). Shown are the means and SEM of individual mice from one of two experiments (n = 3 per unimmunized group; 4–5 per immunized group). **p<0.01 and ***p<0.001 by one-way ANOVA with Newman-Keul's multiple comparison test. d & e. Risperidone reduces the level of F4/80 (d) and the number of F4/80+ foci (e) in the cerebellum of immunized mice compared to vehicle treatment. F4/80 expression in the cerebellum was assessed by immunohistochemistry and shown are the means and SEM of individual mice (3 section per mouse) from 3 per unimmunized group and 4–5 per immunized group. * p<0.05, **p<0.01, and ***p<0.001 by one-way ANOVA with Newman-Keul's multiple comparison test. f & g. Risperidone reduces the expression of I-A (f) and CD40 (g) on microglia and macrophages in the CNS. CD45+ cells were isolated from the spinal cords of risperidone- and vehicle-treated, unimmunized and immunized mice 15 days post-immunization and the expression of I-A (f) and CD40 (g; ΔMFI compared to isotype controls) expressed as % of vehicle-treated, immunized group. Shown are the means and SEM of individual mice from three experiments (n = 10–15 per group). **p<0.01 and ***p<0.001 by one-way ANOVA with Newman-Keul's multiple comparison test (microglia) or unpaired Student's t test (macrophages) compared to vehicle-treated, immunized group.