Requirement of GATA-binding protein 3 for Il13 gene expression in IL-18-stimulated Th1 cells

International Immunology, Vol. 23, No. 12, pp. 761–772 doi:10.1093/intimm/dxr087 Advance Access publication 29 October 2011 ª The Japanese Society for Immunology. 2011. All rights reserved. For permissions, please e-mail: Requirement of GATA-binding protein 3 for Il13 gene expression in IL-18-stimulated Th1 cells Masakiyo Nakahira and Kenji Nakanishi Department of Immunology and Medical Zoology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan Correspondence to: K. Nakanishi; E-mail: Received 28 June 2011, accepted 3 October 2011 Abstract Keywords: allergy, Gata3, IL-13, IL-18, Th1 cells Introduction CD4+ Th cells play critical roles in controlling adaptive immune response to invading pathogens, allergens and self-antigens. Coffman and Mosmann originally classified Th subsets into Th1 cells and Th2 cells based on their cytokine expression patterns. Th1 cells produce IFN-c, which activates macrophage and eliminates intracellular pathogens, whereas Th2 cells produce IL-4, IL-5 and IL-13, which are crucial for IgE production, eosinophil infiltration and clearance of extracellular parasites. This Th1/Th2 paradigm may help elucidating the mechanism for the development of human immunological diseases. However, this paradigm cannot necessarily explain onsets of autoimmune diseases, such as experimental autoimmune encephalomyelitis and collagen-induced arthritis. Recent studies suggest contribution of Th17 (Th cells producing IL-17) and regulatory T cells (Treg cells) to the development of these diseases. Now, we know that there are at least four Th cell subsets and transcription factors control Th cell differentiation and function. T-box expressed in T-cells (T-bet), GATAbinding protein 3 (Gata3), Retinoid-Related Orphan Receptor c t and forkhead box P3 (Foxp3) are known as the master transcription factors for Th1, Th2, Th17 and Treg cells, respectively (1–4). Th1 cells were regarded to produce IFN-c and IL-2. However, our previous studies revealed that, in response to IL-18, the established Th1 cells become to produce Th2 cytokines as well as their proper Th1 cytokines (5–10), suggesting that Th1 cells have plasticity. They produce Th2 cytokines (IL-9, IL-13), granulocyte macrophage colony-stimulating factor and various chemokines that can recruit granulocytes, macrophages and lymphocytes. However, they cannot produce IL-4. We designated these Th2 cytokines-producing Th1 cells as ‘super Th1 cells’. Recent studies have revealed that Th cells have plasticity in their cytokine expression profile and can be reprogrammed by master transcription factors to generate various types of immune responses (11–14). For example, Lee et al. (15) reported that Th17 cells produce IFN-c in a signal transducer and activator of transcription (Stat) 4–T-betdependent manner, and it is well documented that IFN-c+IL-17+ T cells exist in vivo. Treg cells expressing Foxp3 can also produce IFN-c in parallel with up-regulation of T-bet expression when cultured under Th1 condition or in a Th1-biased inflammatory environment such as Toxoplasma gondii infection (16, 17). Although it is supposed relatively difficult to convert the cytokine profile of Th1 and Th2 cells (13), Hegazy et al. (18) demonstrated the presence of a stable Gata3+T-bet+ cell Recent reports have revealed that CD41 Th cell subsets have the ability to alter their gene expression pattern in response to extracellular stimuli. We previously highlighted the plasticity of Th1 cells by demonstrating that Th1 cells gain the capacity to produce IL-3, IL-9, IL-13 and granulocyte macrophage colony-stimulating factor in response to antigen, IL-2 and IL-18, and based on their unique function, we designated these activated Th1 cells as ‘super Th1 cells’. However, the precise molecular mechanism underlying IL-13 production by super Th1 cells has not been elucidated. Here, we show that the GATA-binding protein 3 (Gata3) is essentially required for Il13 gene expression in super Th1 cells. Gata3 is synergistically induced in T-box expressed in T-cells (T-bet)-expressing Th1 cells when co-stimulated with anti-CD3, IL-18 and IL-4 through the activation of nuclear factor of activated T cells, nuclear factor kappa-light-chain-enhancer of activated B cells and signal transducer and activator of transcription 6, respectively. However, Gata3 induction is not satisfactory, and additional TCR or anti-CD3 signaling is prerequisite for triggering IL-13 production by Gata3 plus T-bet-expressing Th1 cells. These findings suggest that Gata3, which is not originally expressed in Th1 cells, alters the cytokine production profile by Th1 cells. 762 The mechanism for Il13 expression in Th1 cells Methods Mice Wild-type BALB/c and C57BL/6 mice were purchased from Charles River Laboratories Japan, Inc (Yokohama, Japan). Stat6-deficient (Stat6 / ) mice (19) and Il4gfp/gfp mice (20) on a BALB/c background and Il13 / mice on a C57BL/6 background (21) were described previously. All mice were housed under specific pathogen-free conditions. All experiments were performed according to the guidelines of the Institutional Animal Care Committee of Hyogo College of Medicine. Reagents PE-conjugated anti-mouse IL-4 (clone: 11B11; 12-7041-71; eBioscience, San Diego, CA, USA), FITC-conjugated antimouse IFN-c (clone: XMG1.2; 11-7311-82; eBioscience), PEconjugated anti-mouse IL-13 (clone: eBio13A; 12-7133-71; eBioscience), PE-conjugated anti-human/mouse Gata3 (clone: TWAJ; 12-9966-71; eBioscience), Alexa Fluor
647 anti-mouse/human T-bet (clone: eBio4B10; 51-5825-80; eBioscience), PE-conjugated anti-mouse IL-4Ra (clone: mIL4R-M1;5552509; BD Pharmingen, San Diego, CA, USA), biotin-conjugated anti-rat IgG1 antibody (clone: RG 11/39.4; 553890; BD Pharmingen), Streptavidin-PE (13025D; BD Pharmingen), anti-mouse IL-4Ra antibody (clone: mIL4R-M1; 552288; BD Pharmingen), rat IgG2a isotype control (clone: R35-95; 553926; BD Pharmingen), FITC-conjugated anti-rat IgG1 antibody (clone: RG 11/39.4; 553892; BD Pharmingen), anti-Gata3 (clone: HG3-31; sc-268; Santa Cruz, Santa Cruz, CA, USA), anti-mouse CD3e (clone: 145-2C11; 100302; BioLegend, San Diego, CA, USA), anti-mouse CD28 (clone: 37.51; 102102; BioLegend), anti-mouse Fas ligand (FasL) antibody (clone: MFL3; 106608; BioLegend), Hamster IgG isotype control (clone: HTK888; 400916; BioLegend), recombinant mouse IL-12 (210-12; PEPROTECH, Rocky Hill, NJ, USA), recombinant mouse IL-13 (210-13; PEPROTECH), recombinant human IL-2 (200-02; PEPROTECH), recombinant mouse IL-4 (404-ML-010; R&D Systems, Minneapolis, MN, USA), FITC-conjugated anti-mouse IL-18Ra (clone: 112614; FAB1216F; R&D Systems), recombinant mouse IL-18 (B0042; MBL, Nagoya, Japan), nuclear factor of activated T cells (NFAT) inhibitor (480401; Calbiochem, Darmstadt, Germany) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-jB) inhibitor [ammonium pyrrolidinedithiocarbamate (APDC); P8765; Sigma, St Louis, MO, USA] were purchase (...truncated)