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.

GSK3 is a constitutively active serine/threonine kinase that is a critical modulator of innate and adaptive immunity through the regulation of several transcription factors important in the production of cytokines and inflammation, including NF-kB, CREB, AP-1 and STATs [16] . We have previously shown that the GSK3 inhibitor lithium is prophylactic and therapeutic in EAE [17] . Recovery from EAE in lithium treated mice was associated with reduced demyelination, reduced microglia activation, and reduced CD4 + T cell infiltration in the spinal cord. We also found that treatment of mice in vivo with the GSK3 inhibitor lithium, inhibited myelin oligodendrocyte glycoprotein peptide (MOG 35–55 )-specific T cell proliferation and significantly reduced MOG 35–55 -specific production of IFN-γ, IL-6, and IL-17 from splenocytes [17] . GSK3 has been shown to facilitate IFN-γ mediated activation of macrophages [18] . Furthermore inhibition of GSK3 in macrophages suppresses activation of STAT3 and STAT5, and constrains the synergistic activation by IFN-γ and lipopolysaccharides (LPS) of STAT3 [19] , [20] . However the mechanism of the therapeutic action of lithium in neuroinflammation in vivo is still unresolved. In this study we tested the hypothesis that lithium is beneficial in EAE through GSK3 regulation of IFN-γ signaling. Our results show that lithium suppresses Th1 but not Th17 neuroinflammation, and through inhibition of GSK3 tunes IFN-γ-STAT1 signaling for optimal therapeutic efficacy in EAE.

We have recently found that relapsing-remitting MS segregates into a Th1 or a Th17 disease and that each form of disease is differentially responsive to type I IFN therapy [15] . Thus the elucidation of signaling pathways regulating the production and expansion of specific Th effector cells in EAE and MS is a necessary goal to identify new specific targets for therapeutic intervention. A lot is known about the transcription factors and cytokines that are determinant for the differentiation of Th1 and Th17 effector cells, but the mechanisms regulating their production, expansion and pathogenic function in disease are still largely undefined.

The IFN-γ-STAT1 signaling axis has an important pleiotropic role, both pathogenic and protective, in autoimmune diseases including MS and its mouse model, EAE [11] . Both Th1 and Th17 cells are independently capable of inducing autommunity in mouse models and they not only play a role in regulating one another, but that they have a more complex, both overlapping and differential, role in tissue inflammation [4] , [12] , [13] . There is also increasing evidence of the plasticity/instability of the Th17 cell phenotype; Th17 cells may acquire Tbet expression, gaining the ability to secrete IFN-γ in addition to IL-17 [14] . These dual cytokine expressing Th17 cells may ultimately lose the ability to secrete IL-17 and convert into Th1-like cells. Thus the finding that Th17 cells can turn into Th1 cells highlights the importance of controlling the effector function of Th1 cells once disease is established.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease in which both adaptive and innate immunity play a role. CD4 + T cells, believed to be early effector cells in the disease, migrate to the central nervous system (CNS), leading to demyelination, axonal loss, and neurological disability. The cells of the innate immune system are also involved both in the initiation and progression of MS by influencing the effector function of T cells [1] , [2] . Both Th1 and Th17 cells are involved in the pathogenesis of MS, and are the primary effector cells in experimental autoimmune encephalomyelitis (EAE), the most common animal model of MS [3] – [6] . These lineages have distinct effector functions and are characterized by the expression of specific transcription factors and cytokines. The differentiation of naïve CD4 + T cells to interferon-γ (IFN-γ)-producing T helper (Th1) cells is dependent on IFN-γ and interleukin (IL)-12, activation of STAT1 and STAT4, respectively, and the transcription factor Tbet [7] . TGF-β and IL-6, and STAT3 drive IL-17-producing T helper (Th17) cell differentiation in a process that is dependent on the transcription factor ROR-γt [8] , [9] . Although IL-23 is not needed for differentiation, it has an essential role in pathogenicity of Th17 cells perhaps by promoting expansion and stability [10] .

Spinal cords were isolated from EAE mice perfused with PBS on day 20 post-immunization and snap frozen using dry ice in ethanol. RNA was extracted using Trizol reagent (Invitrogen) and cleaned up using RNeasy Mini Kit (Qiagen). cDNA synthesis was performed using SuperScript VILO cDNA synthesis kit (Invitrogen) per manufacturer’s instruction. Gene expression was assayed using Taqman Gene Expression Assays (Applied Biosystems) in combination with Taqman Fast Advanced Master Mix (Applied Biosystems). Taqman assay IDs include: Hprt (Mm00446968_m1), Il10 (Mm00439614_m1), Il27 (Mm00461164_m1), Nos2 (Mm00440502_m1) and Ifnb1 (Mm00439552_s1). Expression data are normalized to Hprt and expressed as 2 −ΔC T ; [2 −C T gene of interest −C T hprt ].

For adoptive transfer of EAE, donor mice were immunized with a s.c. injection of 150 µg MOG 35–55 emulsified in complete Freund’s adjuvant. Mononuclear cells from spleens and dLNs of MOG 35–55 -immunized mice were restimulated with 10 µg/ml MOG 35–55 under either Th1 or Th17 polarizing conditions (described above). Lithium-treated recipient mice were fed lithium chow 6–10 days prior to transfer. Cells (4–6×10 6 ) were injected i.v. into 350 rad irradiated untreated or lithium-treated recipient mice. Cells secreting IL-17F were isolated from IL-17F-Thy1.1 mice, cultured under Th17 polarization conditions (as above), labeled with biotin anti-rat CD90/MUCD90.1 (OX-7; Biolegend) and magnetically sorted using Dynabeads biotin binder (Invitrogen Dynal). Enriched cells (3–6×10 5 ) were injected i.v. as above. Mononuclear cells were isolated, following perfusion with PBS, from the spinal cords of untreated and lithium-treated Th1 animals. Spinal cords were incubated with 2 mg/ml collagenase D (Roche) and 5 U/ml DNase (Sigma-Aldrich) for 1 h at 37°C. Mononuclear cells from the spinal cord were purified by two-step Percoll gradient centrifugation as done previously [15] , [17] , and described in detail in [21] .

Mononuclear cells from spleens and draining lymph nodes (dLNs) of MOG 35–55 -immunized WT mice were restimulated with 10 µg/ml MOG 35–55 (CPC Scientific) for 24 h. For the generation of encephalitogenic Th1 cells, cells were cultured with 10 µg/ml MOG 35–55 , 20 ng/ml IL-12 (Biolegend) and 1 µg/ml anti-IL-4 neutralizing antibody for 3 days. On day 2, 2.5 ng/ml IL-2 (Biolegend) was added to the culture. Where indicated, Th1 cells were restimulated with anti-CD3 and anti-CD28 (1 µg/ml, each) for 8 h and supernatants were assayed for IFN-γ production by ELISA (Biolegend). For the generation of encephalitogenic Th17 cells, cells were cultured with 20 ng/ml IL-23 (Biolegend), 10 µg/ml anti-IFN-γ and 1 µg/ml anti-IL-4 neutralizing antibodies for 3 days. Where indicated, polarized Th17 cells were cultured without or with LiCl for additional 24 h and supernatants were assayed for IL-17A and GM-CSF production by ELISA (eBioscience).

For active EAE, male mice were immunized with a s.c. injection of 50 µg MOG 35–55 emulsified in incomplete Freund’s adjuvant (Difco) containing 125 µg M. tuberculosis (H37Ra; Difco). Immunized mice were monitored for classical disease using a standard scale of 0 to 6∶0, no clinical signs; 1, loss of tail tone; 2, flaccid tail; 3, incomplete paralysis of one or two hind legs; 4, complete hind limb paralysis; 5, moribund (animals were humanly euthanized); 6, death. Atypical EAE in Ifngr1 −/− mice was scored on a 0–6 scale: 0, no disease; 1, slight head tilt; 2; severe head tilt, 3; slight axial rotation/staggered walking, 4; severe axial rotation/spinning; 5, moribund; 6, death. Scores reported for Ifngr1 −/− mice are classical and atypical combined.

Cells were stained with anti-CD4 (RM4-5; Biolegend), fixed, permeabilized with Phosflow Perm Buffer III (BD Pharmingen) and stained with an antibody against phospho-STAT1-Y701 (p-STAT1) (58D6, Cell Signaling). For intracellular cytokine staining, cells were incubated with Brefeldin A, 1X as recommended by manufacturer (Biolegend), 50 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich) for 4 h. Cells were stained with anti-CD4 (RM4-5; eBioscience), fixed, permeabilized (Biolegend) and then stained with antibodies against IFN-γ (XMG1.2; eBioscience) or IL-17A (TC11-18H10.1; Biolegend). Data was collected on an LSRII (BD) and analyzed using FlowJo (TreeStar).

Macrophages were lavaged from the peritoneum on day 4 after injection with Brewer’s thioglycollate. Mononuclear cells were isolated from spleen using the standard protocol of first mashing the spleen through a cell strainer, then lysing red blood cells by using ACK (Ammonium-Chloride-Potassium) Lysing Buffer, and then washing well the cells with PBS, and re-suspending them in culture medium. Mononuclear cells were stimulated with 5 U/ml IFN-γ or 100 U/ml IFN-β (Biolegend) and/or 1.25 µg/ml anti-CD3 (145-2C11). Where indicated, cultures were supplemented with GSK3 inhibitors LiCl (5 mM-20 mM; Sigma) or TDZD-8 (5 µM; Calbiochem).

C57BL/6 mice were purchased from Frederick Cancer Research. B6.129S7-Ifngr1 tm/Agt /J (Ifngr1 −/− ) mice were purchased from the Jackson Laboratory and backcrossed onto C57BL/6 background for 10–12 generations. C57BL/6 Stat1 −/− , Ifnar1 −/− , and IL-17F-Thy1.1 reporter mice [14] were kind gifts from R. Lorenz, J.D. Mountz, and C. Weaver, respectively (UAB). For lithium treatment, lithium was administered in pelleted food containing 0.2% lithium carbonate (Harlan-Teklad) as previously described [17] . This lithium administration is used to achieve serum levels equivalent to those attained therapeutically in human patients.

All experimental animal work in this study was conducted in strict accordance with the National Institutes of Health and University of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC) guidelines. The protocol was approved by the IACUC of the University of Alabama at Birmingham (approval number 111208672). All Surgery was performed under isofluorane anesthesia, and all efforts were made to minimize suffering.

Results and Discussion