Other type-II activating products stimulate IL-4 production by macrophages.

<p>IFN-γ-primed BMMφ (10⁵/well) from WT BALB/c (a) or C57BL/6 (b) mice were stimulated with LPS (10 ng/ml) in the presence or absence of opsonized SRBC (IC), glatiramer acetate (200 ng/ml), SEA (5 µg/ml) or SEA opsonized with IgG purified from infected mice (SEA:IgG) or control IgG (SEA:CIgG; 5 µg/ml: 20 µg/ml) for 24 hours. Cytokine production was measured in culture supernatants by ELISA. Shown are the means and SEM of triplicate wells from 1 of 2 representative experiments. *p<0.05 compared to LPS alone and **p<0.001 compared to al other groups by one-way ANOVA with Bonferroni’s post test. ***p<0.05 compared to medium by Dunnett’s Multiple Comparison Test.</p

Type II-activated macrophages produce IL-4.

<p>(<b>a</b>) IFN-γ-primed BMMφ (10⁵/well) from WT and IL-4Rα-deficient mice were stimulated with LPS (10 ng/ml) in the presence or absence of opsonized SRBC (IC) for 24 hours. Cytokine production was measured in culture supernatants by ELISA. Shown are the means and SEM of triplicate wells from 1 of 3 experiments. *p<0.05 compared to medium and **p<0.001 compared to medium or IC. (<b>b–d</b>) IFN-γ-primed BMMφ (10⁵/well) from heterozygous G4 mice were stimulated with LPS (10 ng/ml) in the presence or absence of opsonized SRBC (IC) for 24 hours. IL-4 production by F4/80+ macrophages was assessed by flow cytometry (b). ELISA (c), or by fluorescence microscopy (d). IL-4-GFP is found intracellularly after LPS + IC (2 examples shown) but not LPS stimulation in DAPI-counterstained macrophages. Shown are the means and SEM from 3 experiments (b) or representative data from 1 of 3 experiments (c and d). *p<0.05 compared to t = 0 and **p<0.01 compared to medium by Dunnett’s Multiple Comparison Test.</p

GM-CSF plus IL-3-derived BMMφ differentiate in a more IL-4 rich environment and produce higher IL-12p40 and IL-4 levels upon stimulation compared to M-CSF-derived BMMφ.

<p>GM-CSF plus IL-3 or M-CSF-derived BMMφ (10⁵/well) from WT (a–f) and IL-4Rα−/− (a, e, and f) mice were primed overnight with IFN-γ and stimulated with LPS (10 ng/ml) in the presence or absence of opsonized SRBC (IC) for 24 hours. (<b>a</b>) IL-4 in the supernatants of the derivation cultures after harvesting of the mature macrophages was assessed by ELISA. p<0.05 by repeated measures ANOVA. (<b>b–d</b>) IL-4, IL-12, and IL-10 levels were assessed in the culture supernatants of GM-CSF plus IL-3 or M-CSF –derived WT macrophages by ELISA. *p<0.01 compared to all other groups (b) or GM-CSF/IL-3 compared to M-CSF (c and d) by one way ANOVA with Bonferroni’s multiple comparison test. (<b>e and f</b>) M-CSF-derived macrophages from WT and IL-4Rα-deficient mice produce similar levels of IL-12 and IL-10 as assessed by ELISA. (<b>a–f</b>) Shown are the means and SEM of 3 independent experiments.</p

Addition of IL-4 reduces IL-12p40 and IL-10 production by BMMφ whereas neutralization of IL-4 does not alter cytokine production.

<p>(<b>a–c</b>) IFN-γ-primed BMMφ (10⁵/well) from WT (a–c) and IL-4Rα−/− (c) mice were stimulated with LPS (10 ng/ml) in the presence or absence of opsonized SRBC (IC), IL-4 (3 ng/ml), or neutralizing anti-IL-4 mAb (1 µg/ml) for 24 hours. In (c) only cultures with exogenous IL-4 (3 ng/ml) are shown. Cytokine production was measured in culture supernatants by ELISA. Shown are the means and SEM of triplicate wells from 1 of 3 representative experiments. *p<0.001 LPS or LPS + anti-IL-4 versus all other groups, **p<0.001 LPS + IL-4 versus all other groups, and ***p<0.01 LPS versus LPS + IL-4 or LPS + IC by one-way ANOVA with Bonferroni’s post-test. For (c) *p<0.05 compared to medium by Dunnett’s Multiple Comparison Test. (<b>d</b>) IFN-γ-primed BMMφ (10⁵/well) from heterozygous G4 mice were stimulated as above (a–c) and IL-4 production assessed by flow cytometry. % medium  =  (geoMFI of sample/geoMFI of medium)×100. Shown are the means and SEM of triplicate wells from 3 experiments. *p<0.01 compared to medium by Dunnett’s Multiple Comparison Test.</p

F4/80+/CD11b+ but not F4/80-CD11b- cells produce IL-4.

<p>IFN-γ-primed BMMφ (10⁵/well) from heterozygous G4 mice were stimulated with LPS (10 ng/ml) in the presence or absence of opsonized SRBC (IC) for 24 hours. (a) Live cells were selected by FSC vs SSC and this subpopulation divided into F4/80+CD11b+ and F4/80-CD11b- populations. (b) F4/80+CD11b+ cells and not F4/80-CD11b- cells produce IL-4 after stimulation with LPS + IC. Shown are representative data from 1 of 3 experiments.</p

Type II-activated BMMφ from IL-4Rα-deficient mice produce significantly more IL-12p40 and IL-10 than WT BALB/c BMMφ.

<p>IFN-γ-primed BMMφ (10⁵/well) from WT and IL-4Rα-deficient mice were stimulated with LPS (10 ng/ml) in the presence or absence of opsonized SRBC (IC) for 24 hours. Cytokine production was measured in culture supernatants by ELISA. Shown are the means and SEM of triplicate wells from 1 of 3 experiments (a and c) or combined results from 4 experiments (b and d). ***p<0.001 compared to all other groups. **p<0.01 (b & d) LPS versus LPS + IC and (d) BALB/c versus IL-4Rα^−/−. *p<0.05 (c) LPS versus medium or IC or (b) BALB/c versus IL-4Rα^−/−. All p values were calculated by one-way ANOVA with Bonferroni’s post-test.</p

Effect of risperidone treatment on splenocyte subpopulations.

<p>* p&lt;0.05 by one-way ANOVA with Newman-Keul's multiple comparison test; vehicle, immunized compared to risperidone, immunized at that time point.</p><p>** p&lt;0.05 by one-way ANOVA with Newman-Keul's multiple comparison test; immunized compared to unimmunized at that time point.</p

Risperidone treatment significantly reduces microglial activation in the CNS of immunized mice during EAE.

<p><b>a.</b> 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>b &amp; c.</b> Iba-1 expression in the cerebellum (<b>b</b>) and all brain and spinal cord regions (<b>c</b>) 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&lt;0.01 and ***p&lt;0.001 by one-way ANOVA with Newman-Keul's multiple comparison test. <b>d &amp; e.</b> Risperidone reduces the level of F4/80 (<b>d</b>) and the number of F4/80+ foci (<b>e</b>) 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&lt;0.05, **p&lt;0.01, and ***p&lt;0.001 by one-way ANOVA with Newman-Keul's multiple comparison test. <b>f &amp; g.</b> Risperidone reduces the expression of I-A (<b>f</b>) and CD40 (<b>g</b>) 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 (<b>f</b>) and CD40 (<b>g</b>; 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&lt;0.01 and ***p&lt;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.</p

Risperidone dose-dependently reduces the severity of EAE.

<p><b>a.</b> 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&lt;0.05 by two-way ANOVA (vehicle vs risperidone 3 mg). <b>b.</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. <b>c.</b> 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&lt;0.05 by unpaired Student's t test. <b>d.</b> Risperidone (3 mg/kg/day) reduces lesion area in the spinal cords of mice. Spinal cord lesions were assessed on H &amp; E stained tissue (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104430#pone.0104430.s005" target="_blank">Figure S5</a> 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&lt;0.05 vehicle versus drug by Mann-Whitney test.</p

Risperidone treatment does not change the percentage or activation of splenic myeloid populations during EAE.

<p><b>a.</b> Gating strategy for the major splenic myeloid populations. <b>b.</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. <b>c.</b> 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 <b>a</b>. Shown are the means and SEM of individual mice from three experiments (n = 10–15 per group). **p&lt;0.01 and ***p&lt;.001 by one-way ANOVA with Newman-Keul's multiple comparison test. <b>d.</b> 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). <b>e–f.</b> Immunization increases the expression of I-A on RP MΦ (<b>e</b>) and DC (<b>f</b>) and CD40 on DC (<b>h</b>) 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 <b>a</b>. 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&lt;0.05 and **p&lt;0.01 by two-way ANOVA to distinguish overall drug and immunization effects.</p