Amelioration of experimental autoimmune encephalomyelitis by clozapine is not associated with defective CD4 T cell responses

Abstract Atypical antipsychotic agents, such as clozapine, are used for treating psychosis and depression and have recently been found to modulate neuroinflammation. We have shown previously that treatment of mice with the atypical antipsychotic agents, clozapine or risperidone, attenuates disease severity in experimental autoimmune encephalomyelitis (EAE); however, the mechanism by which they are protective is unknown. In this study, we investigated the effects of clozapine on CD4+ T cell responses and found that clozapine did not significantly affect the expansion of myelin-specific T cells, their differentiation into pathogenic subsets, or their encephalitogenic capacity to induce EAE. Interestingly, although clozapine enhanced differentiation of regulatory T (Treg) cells, in vivo neutralization of Tregs indicated that Tregs were not responsible for the protective effects of clozapine during the induction and effector phase of EAE. Taken together, our studies indicate that clozapine does not mediate its protective effects by directly altering CD4 T cells

Type II Activation of Macrophages and Microglia by Immune Complexes Enhances Th17 Biasing in an IL-6-Independent Manner

<div><p>Macrophages can be activated into several distinct activation states. One of these states, type II activation, has a regulatory phenotype characterized by decreased IL-12 and increased IL-10, and has been shown to bias naïve CD4⁺ T cells to a Th2 response. Microglia, the resident macrophage-like cells in the central nervous system (CNS), are important contributors to neuroinflammation and, thus, we investigated if type II activated microglia could bias CD4⁺ T cell responses in a similar manner as type II activated macrophages. Using immune complex ligation in the presence of LPS to induce type II activation, we found that both type II macrophages and type II microglia biased CD4⁺ T cell responses <i>in vitro</i> to express increased levels of IL-17A and CD124. The enhanced IL-17A production occurred independently of IL-6, and IL-10 and IL-12, which were key regulators of IFN-γ production, but were not involved in the increased IL-17A. Finally, we found that another type II-activating compound, glatiramer acetate, did not bias CD4⁺ T cells to produce enhanced IL-17A. Taken together, this study demonstrates that microglia can be type II activated and, similarly to type II macrophages, can bias CD4⁺ T cell responses; however, depending on the type II stimulus, the effect on CD4⁺ T cell subset differentiation may vary.</p></div

IL-10 and IL-12 regulated IFN-γ production but were not responsible for the enhanced Th17 biasing by type II macrophages.

<p>Macrophages were stimulated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164454#pone.0164454.g002" target="_blank">Fig 2</a> in the presence or absence of αIL-10 (JES5-2A5, 2 μg/ml; <b>a, c, e, g</b>), rIL-10 (5 ng/ml; <b>b, d, f, h</b>), αIL-12 (C15.6, 2 μg/ml; <b>i, k, m, o</b>), rIL-12p70 (5 ng/ml; <b>j, l, n, p</b>), or isotype control (rat IgG₁, 2 μg/ml; <b>a, c, e, g, i, k, m, o</b>). After four hours, purified CD4⁺ 2D2 T cells and MOG (25 μg/ml) were added and cultured for 72 hours. IL-12p40 (<b>a, b, i, j</b>), IL-10 (<b>c, d, k, l</b>), IFN-γ (<b>e, f, m, n</b>), and IL-17A (<b>g, h, o, p</b>) were assessed by ELISA. Shown are the means from 2–3 independent experiments (<b>a, c, b, f, h, k, l, m)</b> and means and SEM of triplicate wells from a representative experiment of at 2–5 independent experiments (<b>d, e, g, i, j, n, o, p)</b>. *p<0.05, **p<0.01, and ***p<0.001 by one-way ANOVA with Newman-Keuls’ post-test.</p

Type II microglia biased CD4⁺ T cell responses to a mixed Th17/Th2 phenotype.

<p>(<b>a</b>) Microglia can be type II-activated. Microglia were isolated from the CNS of adult mice (n = 5/experiment) and expanded in the presence of M-CSF (5 ng/ml) for 4 weeks. Microglia were primed with IFN-γ (20 U/ml) overnight before stimulation with LPS (200 ng/ml) with or without IC (10⁶/well) for 24 hours. Shown are the means and SEM of triplicate wells from a representative experiment of 3 independent experiments. **p<0.01 and ***p<0.001 by one-way ANOVA with Newman-Keuls’ post-test. (<b>b-k</b>) Type II microglia enhanced IL-17A production by CD4⁺ T cells. Microglia were isolated and activated as described. After four hours, purified CD4⁺ 2D2 T cells and MOG_35-55 (25 μg/ml) were added for 72 hours. IFN-γ (<b>b</b>), IL-17A (<b>c</b>), IL-2 (<b>d</b>), and IL-6 (<b>e</b>) levels were measured by ELISA and IL-1α (<b>f</b>) and IL-22 (<b>g</b>) levels by CBA. CD124 (<b>h</b>), CD25 (<b>i</b>), CD44 (<b>j</b>), and CD62L (<b>k</b>) were assessed by flow cytometry. Shown are the means 7 (<b>b-d, h-k</b>) or 3 (<b>e & g</b>) independent experiments or the means and SEM of triplicate wells from a representative experiment of 3 independent experiments (<b>b & f</b>). *p<0.05, **p<0.01, and ***p<0.001 by one-way ANOVA with Newman-Keuls’ post-test (<b>a</b>) or Wilcoxon matched pairs signed rank test (<b>b-k</b>).</p

Type II macrophages expressed less IL-12 and more IL-10 than classical macrophages, and enhanced Th17 and Th2 biasing.

<p>(<b>a & b</b>) Macrophages were primed with IFN-γ (20 U/ml) overnight before stimulation with or without LPS (200 ng/ml) in the presence or absence of IC (10 IC/macrophage) for 24 hours. IL-12p40 and IL-10 levels were measured by ELISA. Shown are the means and SEM from 8 (<b>a</b>) or 10 (<b>b</b>) independent experiments. **p<0.01 and ***p<0.001 compared to LPS by one-way ANOVA with Dunnett’s multiple comparison test. (<b>c-h</b>) Type II macrophages biased the CD4⁺ T cell response to reduce Th1 and enhance Th17 cytokines. Macrophages were stimulated as described, and after four hours, purified CD4⁺ 2D2 T cells and MOG (25 μg/ml) were added and cultured for 72 hours. IFN-γ (<b>c & d</b>), IL-17A (<b>e</b>), and IL-2 (<b>f</b>) levels were measured by ELISA, and IL-6 and IL-1α levels by CBA (<b>g & h)</b>. Shown are the means and SEM from triplicate wells from one representative experiment (<b>c</b>) and the means and SEM from 11–15 (<b>d-f</b>) or 4 (<b>g & h</b>) independent experiments. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by one-way ANOVA with Neuman-Keuls’ post-test (<b>c</b>) or paired Student’s t test (<b>d-h</b>).</p

IL-10 and IL-12 are key regulators of IFN-γ in macrophage: T cell cultures while IL-6 does not contribute significantly to T cell biasing in this system.

<p>IL-10 and IL-12 are key regulators of IFN-γ in macrophage: T cell cultures while IL-6 does not contribute significantly to T cell biasing in this system.</p

Type II macrophages do not enhance proliferation of CD4⁺ T cells in the absence of NO.

<p>Macrophages were stimulated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164454#pone.0164454.g002" target="_blank">Fig 2</a> in the presence or absence of 1 mM aminoguanidine (AG). After four hours, purified CFSE-labeled CD4⁺ 2D2 T cells and MOG (25 μg/ml) were added to the cultures and cultured for 72 hours. Proliferation was analysed by flow cytometry, CD4⁺ T cells were gated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164454#pone.0164454.g002" target="_blank">Fig 2</a> and CFSE positive cells were gated as indicated using CFSE unlabeled cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164454#pone.0164454.g002" target="_blank">Fig 2</a>. (<b>a</b>) Representative plots of CFSE staining from the different treatment conditions are shown (Solid line with MOG, filled line without MOG). (<b>b</b>) Percentage of cells that proliferated was calculated. (<b>c</b>) NO levels were measured by Griess reaction. Shown are the means and SEM combined from 3 independent experiments (<b>b</b>) or a representative experiment of 3 (<b>c</b>). *p<0.05 and ***p<0.001 by one-way ANOVA with Neuwman-Keuls’ post-test.</p