IFN-γ-signaling is required for attenuation of EAE by lithium.

<p>EAE was induced in (A) WT (B), <i>Stat1</i>^−/−, (C) <i>Ifngr1^−/−</i>, and (D) <i>Ifnar1^−/−</i>as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052658#s2" target="_blank">Material and Methods</a>. Arrows indicate first day of administration of lithium. (Mean ± SEM, <i>n</i> = 10–13 mice/group *<i>p</i><0.05, or NS, not significant, from start of treatment until day 30, as determined by Mann-Whitney test). (E) RNA was isolated from spinal cords of WT and <i>Ifngr1^−/−</i> mice on day 20 post-immunization and evaluated for gene expression by real-time PCR, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052658#s2" target="_blank">Materials and Methods</a>. <i>n</i> = 3 for immunized, <i>n</i> = 1 for unimmunized controls. *<i>p</i><0.05, as determined by one-way ANOVA.</p

Analysis of disease parameters for adoptive transfer of EAE induced by Th1, Th17, and IL-17F-Thy1.1 cells in untreated and lithium-treated animals.

<p>Data are presented as mean ± SEM (<i>n</i> = 8–11 mice).</p>a<p><i>p</i><0.05; Lithium-treated Th1 compared to untreated Th1.</p>b<p><i>p</i><0.05; Lithium-treated Th17 compared to untreated Th17.</p

Lithium inhibits Th1-induced, but not Th17-induced EAE.

<p>(A) Th1 cells (4–6×10⁶), (B) Th17 cells (4–6×10⁶), or (C and D) IL-17F-Thy1.1 cells (3–6×10⁵) were adoptively transferred i.v. into naïve untreated or lithium–treated recipient mice to induce EAE (mean ± SEM, <i>n</i> = 8–11 mice/group, *<i>p</i><0.05 or NS, not significant, as determined by Mann-Whitney). (E) Intracellular cytokine staining for IL-17A in CD4⁺ gated cells from MOG_35–55-immunized mice polarized to Th17 for 3 days in the absence or presence of 10 mM LiCl (representative dot plots and percentages are shown, <i>n</i> = 2). (F) 3 days polarized Th17 or Th1 cells from MOG_35–55-immunized mice were cultured for an additional 24 h without or with addition of 5 mM LiCl, and production of IL-17A and GM-CSF was measured by ELISA in culture supernatants (one representative experiment is shown, <i>n</i> = 2; *<i>p</i><0.05). (G) Infiltration of Th1 cells (CD4⁺IFN-γ⁺) into the spinal cord and cerebellum of untreated or lithium pretreated mice with adoptive transferred EAE. Infiltrating cells were isolated (14–15 d post transfer) and characterized by flow cytometry. Bar graphs depict percentage (top) and absolute number (bottom) of CD4⁺IFN-γ⁺ cells. Results are from 2–3 mice pooled per experiment (<i>n</i> = 2).</p

GSK3 promotes IFN-γ and IFN-β-induced STAT1-Y701 phosphorylation in CD4⁺ T cells.

<p>(A and B) Splenocytes were pre-incubated for 1 h in the absence or presence of the GSK3 inhibitors LiCl or TDZD-8, and stimulated without or with IFN-γ (5 U/ml; 25 minutes) or IFN-β (100 U/ml; 45 minutes) and/or anti-CD3 (1.25 µg/ml; 25 minutes in (A), and 45 minutes in (B) as indicated. Mononuclear cells were stained for pSTAT1-Y701 and analyzed by flow cytometry. Representative histograms are gated on CD4⁺ T cells. Induction of pSTAT1-Y701 is normalized to unstimulated cells. Fold induction of pSTAT1-Y701 MFI is normalized to unstimulated cells from combined data of 2-4 experiments. *<i>p</i><0.05, as determined by one-way ANOVA. (C) Thioglycollate-elicited macrophages (left histogram) were pre-incubated for 1 h in the absence or presence LiCl. Cells were then stimulated for 25 minutes with IFN-γ (5 U/ml) or left unstimulated, stained and analyzed for pSTAT1-Y701 in CD11b⁺ gated cells as in (A). CD11b⁺ cells (right histogram) were isolated from dLNs and spleens of MOG_35–55-immunized mice, restimulated for 24 h with MOG_35–55 (10 µg/ml) in the absence or presence of LiCl and evaluated for pSTAT1-Y701. (D) Naïve splenocytes from <i>Ifngr1^−/−</i> mice were pre-incubated without or with LiCl, and stimulated for 25 minutes with IFN-γ (5 U/ml) and/or αCD3 (1.25 µg/ml), as indicated, and evaluated for pSTAT1-Y701. Histograms are gated on CD4⁺ T cells.</p

Lithium attenuates STAT1-Y701 phosphorylation in encephalitogenic CD4⁺ T cells and reduces IFN-γ production.

<p>(A and B) Cells from dLNs and spleen of MOG_35–55 immunized mice (10–21 d post immunization) were either (A) pre-incubated without or with LiCl, and left unstimulated or stimulated for 25 with IFN-γ (5 U/ml) and/or anti-CD3 (1.25 µg/ml) as indicated; or (B) restimulated for 24 h with MOG_35–55 (10 µg/ml) in the absence or presence of LiCl, from onset, or acutely treated for 1 h, as indicated. A subset of cells was stimulated after 24 h with IFN-γ (5 U/ml) for 25 minutes. Cells were gated on CD4⁺ T cells and pSTAT1-Y701 was analyzed as in Fig. 2 and normalized to unstimulated cells from naïve mice. Results are expressed as percent of control, which represent stimulated untreated samples (100%), <i>n</i> = 3. (C) IFN-γ production by 24 h MOG_35–55 restimulated cells (from B). Representative sample shown (<i>n</i> = 3). (D) CD4⁺ T cells from spleens and dLNs of MOG_35–55 immunized mice were polarized under Th1 conditions in the presence or absence of LiCl, from onset or acutely treated for 1 h on day 3. Where indicated cells were stimulated with IFN-γ on day 3. Cells were gated on in CD4⁺, analyzed for pSTAT1-Y701, and normalized to unstimulated cells from naïve mice. Results are expressed as percent of control, which represent stimulated untreated samples (100%), (<i>n</i> = 3) (E), Th1 cells generated by polarization in the absence or presence of LiCl. Dot plots reflect CD4⁺ gated T cells. Representative experiment shown (<i>n</i> = 2). (F) IFN-γ production from Th1 cells (day 3 of polarization) stimulated with anti-CD3 and anti-CD28 (1 µg/ml each) for 8 h in the absence or presence of LiCl was assessed by ELISA. Representative results shown (<i>n</i> = 2). *<i>p</i><0.05, as determined by t-test or one-way ANOVA, as appropriate.</p

Analysis of disease parameters for active EAE induced in untreated and lithium-treated WT, <i>Stat1^−/−</i>, <i>Ifngr1^−/−</i> and <i>Ifnar1^−/−</i> mice.

<p>Data are presented as mean ± SEM (<i>n</i> = 10–28 mice).</p>a<p><i>p</i><0.05; Lithium treated <i>Stat1^−/−</i> compared to untreated <i>Stat1^−/−</i>.</p>b<p><i>p</i><0.05; Lithium treated <i>Ifnar1^−/−</i> compared to untreated <i>Ifnar1^−/−</i>.</p