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Casein Kinase 2 Is a Critical Determinant of the Balance of Th17 and Treg Cell Differentiation

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Casein Kinase 2 Is a Critical Determinant of the Balance of Th17 and Treg Cell Differentiation OPEN Experimental & Molecular Medicine (2017) 49, e375; doi:10.1038/emm.2017.132 Official journal of the Korean Society for Biochemistry and Molecular Biology www.nature.com/emm ORIGINAL ARTICLE Casein kinase 2 is a critical determinant of the balance of Th17 and Treg cell differentiation Sung Woong Jang, Soo Seok Hwang, Hyeong Su Kim, Keoung Oh Lee, Min Kyung Kim, Wonyong Lee, Kiwan Kim and Gap Ryol Lee Th17 cells promote inflammatory reactions, whereas regulatory T (Treg) cells inhibit them. Thus, the Th17/Treg cell balance is critically important in inflammatory diseases. However, the molecular mechanisms underlying this balance are unclear. Here, we demonstrate that casein kinase 2 (CK2) is a critical determinant of the Th17/Treg cell balance. Both the inhibition of CK2 with a specific pharmacological inhibitor, CX-4945, and its small hairpin RNA (shRNA)-mediated knockdown suppressed Th17 cell differentiation but reciprocally induced Treg cell differentiation in vitro. Moreover, CX-4945 ameliorated the symptoms of experimental autoimmune encephalomyelitis and reduced Th17 cell infiltration into the central nervous system. Mechanistically, CX-4945 inhibited the IL-6/STAT3 and Akt/mTOR signaling pathways. Thus, CK2 has a crucial role in regulating the Th17/Treg balance. Experimental & Molecular Medicine (2017) 49, e375; doi:10.1038/emm.2017.132; published online 8 September 2017 INTRODUCTION T cells and ultimately activates mTOR complexes (mTORC1 Autoimmune diseases are regulated by the balance between T and mTORC2).15 mTORC1, which is indirectly activated helper 17 (Th17) cells, which promote inflammatory reactions by Akt phosphorylated on Thr308 upon TCR signaling, by secreting interleukin (IL)-17A, IL-17F, IL-21 and IL-22,1–4 translocates RORγt to the nucleus in a S6K1/2-dependent and regulatory T (Treg) cells, which suppress effector T cells manner and induces Th17 differentiation.16 Then, mTORC2 and regulate self-tolerance.5,6 Despite their conflicting roles, phosphorylates Akt on Ser473, which inactivates the FoxO Th17 and Treg cells share a precursor cell (naive CD4 T cells) proteins that positively regulate Foxp3 expression.17 Thus, and an inducing signal (transforming growth factor β (TGFβ)). mTOR-deficient T cells differentiate into Foxp3+ cells upon Depending on other environmental conditions, such as the TCR stimulation, even in the absence of exogenous cytokines.18 presence of specific antigens, T cell receptor (TCR) binding, Casein kinase 2 (CK2) is a highly conserved serine–threonine and the amount of IL-2, IL-6, IL-21 and IL-23, naive kinase that contains two catalytic α-subunits (α, α′) and two CD4 T cells differentiate into either Th17 or Treg cells.7–10 regulatory β-subunits that form a holoenzyme tetramer.19 CK2 Therefore, the differentiation of Th17 and Treg cells is regulates many targets, such as the NF-κB, Wnt,20 JAK/STAT controlled reciprocally, and the balance between them has a (including STAT3)21,22 and PI3K/Akt/mTOR23 signaling critical role in autoimmune responses. pathways, in different types of cells. A recent study showed IL-6, together with TGF-β, triggers the differentiation of that CK2 is important for the Th2-restraining function of Th17 cells by inducing retinoid-related orphan receptor Treg cells.24 However, the role of CK2 during differentiation (ROR)-γt, the key transcription factor that determines Th17 of other CD4 T cell subsets is unclear. As CK2 regulates the lineage.11,12 STAT3, which is phosphorylated and activated by JAK/STAT pathway and the PI3K/Akt/mROR pathway, we IL-6, is an important component of IL-6-mediated Th17 cell hypothesized that CK2 may regulate CD4 T cell differentiation differentiation. STAT3-deficient T cells fail to produce IL-17 and, in particular, the Th17/Treg balance. and instead induce Foxp3, even in the presence of IL-6 Here, we examined the role of CK2 in determining the and TGF-β.13,14 Phosphoinositide 3-kinase (PI3K), Akt and Th17/Treg balance. We found that the inhibition of CK2 by a mammalian target of rapamycin (mTOR) complexes are also specific pharmacological inhibitor, CX-4945, or by shRNA- important regulators of Th17 and Treg cell differentiation. The mediated knockdown suppressed Th17 cell differentiation and PI3K–Akt axis works downstream of the TCR and CD28 in induced iTreg cell differentiation in vitro.CK2antagonists Department of Life Science, Sogang University, Seoul, Korea Correspondence: Professor GR Lee, Department of Life Science, Sogang University, 35 Baekbeom-ro, Mapo-ku, Seoul 04107, Korea. E-mail: [email protected] Received 17 November 2016; revised 7 March 2017; accepted 22 March 2017 CK2 determines Th17/Treg balance SW Jang et al 2 exerted this effect by inhibiting the IL-6/STAT3 and mTOR Intracellular staining of cytokines and transcription factors signaling pathways. Moreover, treatment with CX-4945 For cytokine staining, the cells were re-stimulated with 1 μM ameliorated symptoms of experimental autoimmune encepha- ionomycin and 10 nM PMA (both from Sigma-Aldrich, St Louis, lomyelitis (EAE), an animal model of a Th17-mediated disease MO, USA) in the presence of Brefeldin A (BioLegend) for 4 h and then stained with an Intracellular Fixation & Permeabilization Buffer and reduced Th17 cell infiltration into the central nervous Set (eBioscience). Intracellular Foxp3 staining was performed using a system (CNS), thereby demonstrating that inhibiting CK2 may Foxp3 Fix/Perm Buffer Set (BioLegend). To detect the STAT3 be a potential potent therapeutic strategy for Th17-mediated phosphorylation, the cells were re-stimulated with IL-6 (100 ng ml − 1; diseases. eBioscience), fixed and permeabilized with IC Fixation buffer (eBioscience) before staining. Flow cytometric analyses were per- MATERIALS AND METHODS formed using a FACSCalibur flow cytometer (BD Biosciences; Mice Franklin Lakes, NJ, USA). Female C57BL/6 mice (6–8 weeks old) were purchased from Taconic Biosciences (Daehan Bio Link, EumSeong, ChungBuk, Korea) and RNA isolation and quantitative RT-PCR maintained under specific pathogen-free conditions during the in vivo The total RNA was isolated from cells using TRI Reagent (Molecular experiments. All animal experiments were approved by the Sogang Research Center; Cincinnati, OH, USA) according to the University Institutional Animal Care and Use Committee. manufacturer’s protocol. Reverse transcription was performed using TOPscript Reverse Transcriptase (Enzynomics; Daejeon, Korea). In vitro culture of CD4 T cells Quantitative real-time PCR was then performed using HiFast Probe Naive CD4+ T cells were enriched from the spleen of C57BL/6 mice Lo-ROX, HiFast SYBR Lo-ROX master mix (PCR Biosystems; using the anti-NK1.1 (#108712, BioLegend; San Diego, CA, USA), London, UK) and a Roche LightCycler 96 (Roche, Basel, Switzerland). anti-CD25 (#102014, BioLegend), anti-I-A/I-E (#107610, BioLegend), anti-CD8α (#100716, BioLegend), BioMag goat anti-rat IgG (Qiagen; Cell viability assay Venio, Netherlands) and BioMag goat anti-mouse IgG (Qiagen) Cell viability was measured using an EZ-Cytox Cell viability assay kit ’ antibodies. Then, the cells were sorted using the biotinylated anti- (DaeilLab Service; Seoul, Korea) according to the manufacturer s CD62L antibody (#104404, BioLegend) and anti-biotin microbeads protocol. Cultured cells were collected and seeded into a 96-well (Miltenyi Biotec; Bergisch-Gladbach, Germany). The cells were then microplate containing assay reagent. After a 3 h incubation at 37 °C, activated by incubation with the plate-bound anti-CD3ε (10 μgml− 1; the absorbance was measured at 450 nm using a microplate reader 2C11) and anti-CD28 (10 μgml− 1; 37.51) antibodies in RPMI-1640 (Bio-Rad; Hercules, CA, USA). medium supplemented with 5% fetal bovine serum, 2-mercaptoethanol, MEM amino acids, non-essential MEM amino acids and penicillin– Mouse EAE model – streptomycin (all from Gibco Life Technologies, Carlsbad, CA, USA). Female mice (8 10-weeks old) were immunized by a subcutaneous μ Differentiation of Th1 and Th2 cells was induced as previously injection with 200 g of myelin oligodendrocyte glycoprotein (MOG) 25 − 1 35–55 (Peptron; Daejeon, Korea) emulsified in complete Freund’s described. Mouse recombinant IL-6 (50 ng ml ; eBioscience; − − adjuvant containing 5 mg ml 1 heat-killed Mycobacterium tuberculosis Santa Clara, CA, USA), human recombinant TGF-β1(2ngml 1; − (Chondrex; Redmond, WA, USA) (day 0). Pertussis toxin (200 ng; List eBioscience), mouse recombinant IL-1β (2 ng ml 1; eBioscience), Biological Laboratories; Campbell, CA, USA) was then injected mouse recombinant TNFα (1 ng ml − 1; eBioscience), anti-IFNγ Ab intraperitoneally into the mice on days 0 and 2. Clinical signs were (XMG1.2; 10 μgml− 1) and anti-IL4 Ab (11B11; 5 μgml− 1) were assessed daily and scored as follows: 0, no symptoms; 1, limp tail; added to the culture medium to induce Th17 cell differentiation. 2, weakness of hind legs; 3, complete paralysis of hind legs; 4, complete Mouse recombinant IL-2 (1 ng ml − 1), human recombinant TGF-β1 hind leg and partial front leg paralysis. The mice were killed on the (5 ng ml − 1), XMG1.2 Ab (5 μgml− 1) and 11B11 Ab (5 μgml− 1) were indicated days, and the brain and spinal cord were isolated and added to the medium to induce Treg cell differentiation. CX-4945 homogenized. Mononuclear cells were isolated by gradient centrifuga- (Selleckchem; Houston, TX, USA) was added to the culture medium tion with a 30/70% Percoll gradient (GE Healthcare, Little Chalfont, throughout the study at the indicated concentrations. UK).
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