Lithium controls central nervous system autoimmunity through modulation of IFN-γ signaling.

Inhibitors of glycogen synthase kinase 3 (GSK3) are being explored as therapy for chronic inflammatory diseases. We previously demonstrated that the GSK inhibitor lithium is beneficial in experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. In this study we report that lithium suppresses EAE induced by encephalitogenic interferon-γ (IFN-γ)-producing T helper (Th1) cells but not by interleukin (IL)-17-producing T helper (Th17) cells. The therapeutic activity of lithium required functional IFN-γ-signaling, but not the receptor for type I IFN (IFNAR). Inhibitor/s of GSK3 attenuated IFN-γ dependent activation of the transcription factor STAT1 in naïve T cells as well as in encephalitogenic T cells and Th1 cells. The inhibition of STAT1 activation was associated with reduced IFN-γ production and decreased expansion of encephalitogenic Th1 cells. Furthermore, lithium treatment induced Il27 expression within the spinal cords of mice with EAE. In contrast, such treatment of Ifngr(-/-) mice did not induce Il27 and was associated with lack of therapeutic response. Our study reveals a novel mechanism for the efficacy of GSK3 targeting in EAE, through the IFN-γ-STAT1 axis that is independent IFNAR-STAT1 axis. Overall our findings set the framework for the use of GSK3 inhibitors as therapeutic agents in autoimmune neuroinflammation

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

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

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

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