Research ArticleSIGNAL TRANSDUCTION

GLK-IKKβ signaling induces dimerization and translocation of the AhR-RORγt complex in IL-17A induction and autoimmune disease

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Science Advances  12 Sep 2018:
Vol. 4, no. 9, eaat5401
DOI: 10.1126/sciadv.aat5401
  • Fig. 1 Lck-GLK Tg mice display autoimmune phenotypes and selectively increased serum IL-17A levels.

    (A) Hematoxylin and eosin (H&E)–stained sections of the indicated organs from 16-week-old mice. Scale bars, 100 μm. (B) Levels of serum autoantibodies from 20-week-old mice were determined by ELISAs. The levels are presented relative to the value from one of the wild-type (WT) mice. WT, n = 7; Lck-GLK, n = 8. (C) The serum levels of cytokines in 4-week-old mice were determined by ELISAs. WT, n = 20; Lck-GLK, n = 16. (D) The serum levels of autoantibodies in 20-week-old Lck-GLK and Lck-GLK/IL-17A KO mice were determined by ELISAs. The levels are presented relative to the value from one of the Lck-GLK mice. n = 6 per group. (E) IL-17A expression was attenuated by GLK shRNA. Murine primary splenic T cells were transfected with green fluorescent protein (GFP)–human GLK shRNA and a control GFP vector. The transfected T cells were stimulated with anti-mouse CD3 antibodies for 3 hours and then determined by flow cytometry at day 3 after transfection. Data show the events of IL-17A–producing T cells (GFP-gated). WT, wild-type littermate controls; Lck-GLK, T cell–specific GLK Tg mice; Lck-GLK/IL-17A KO, Lck-GLK;IL-17A–deficient mice; ANA, antinuclear antibody; α–double-stranded DNA (dsDNA), anti-dsDNA antibody; RF, rheumatoid factor; APC, allophycocyanin. Data shown are representative of three independent experiments. *P < 0.05, **P < 0.01 (two-tailed Student’s t test).

  • Fig. 2 GLK enhances IL-17A expression by inducing AhR and RORγt.

    (A) Murine IL-17A mRNA levels in peripheral blood T cells from mice were analyzed by real-time polymerase chain reaction (PCR). The expression levels of IL-17A were normalized to Mrpl32 levels. The fold changes are presented relative to the value of WT mice. Means ± SEM are shown. n = 4 per group. (B) Luciferase reporter activity of the IL-17A promoter. Jurkat T cells were cotransfected with the plasmid encoding GLK or GLK kinase-dead (GLK-K45E) mutant plus the IL-17A promoter (2 kb) construct. Means ± SEM are shown. (C) Schematic diagram of transcription factors on the IL-17A promoter. bp, base pair. (D) The binding of AhR, RORγt, STAT3, IRF4, KLF4, or BATF to the IL-17A promoter in T cells from mice was analyzed by chromatin immunoprecipitation (IP) (ChIP)–PCR using immunocomplexes from individual IP experiments. (E) Luciferase reporter activity of the IL-17A mutant promoters. Jurkat T cells were cotransfected with empty vector or GLK plasmid plus the IL-17A promoter construct containing a mutated binding element for AhR, RORγt (−877), or STAT3. (F) Luciferase reporter activity of AhR, RORγt (−877), and STAT3 response element (XRE-Luc, RORγt-Luc, and SIE-Luc) in Jurkat T cells cotransfected with empty vector or plasmid encoding GLK. XRE, xenobiotic response element; SIE, sis-inducible element. WT, wild-type littermate controls; Lck-GLK, T cell–specific GLK Tg mice. Data shown are representative of three independent experiments. Means ± SEM of three independent experiments are shown (B, E, and F). *P < 0.05, **P < 0.01 (two-tailed Student’s t test).

  • Fig. 3 PKCθ phosphorylates AhR and induces its nuclear translocation.

    (A) The serum levels of cytokines from 4-week-old WT, Lck-GLK Tg, and Lck-GLK Tg/AhR cKO mice were determined by ELISAs. n = 8 per group. Means ± SEM are shown. WT, wild-type littermate controls; Lck-GLK, T cell–specific GLK Tg mice; Lck-GLK;AhRf/f;CD4-Cre, T cell–specific GLK Tg mice bred with AhR cKO mice. ***P < 0.001 (two-tailed Student’s t test). (B) Confocal microscopy analysis of subcellular localization of AhR in murine splenic T cells without stimulation. An anti-AhR antibody (clone RPT9, Abcam) was used. Original magnification, ×630; scale bars, 10 μm. (C) Immunoblotting analyses of AhR, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and histone 3 in cytoplasmic and nuclear fractions of primary splenic T cells from WT and Lck-GLK Tg mice. (D) Immunoblotting analysis of p-AhR (Ser36), AhR, and GLK in primary splenic T cells of WT and Lck-GLK Tg mice. (E) Immunoblotting analysis of AhR phosphorylation and indicated kinases in in vitro kinase assays using Flag-GLK, Flag-PKCθ, Flag-IKKβ, Flag-IKKα, and hemagglutinin (HA)–AhR (as the substrate) isolated from individually transfected HEK293T cells. (F) Coimmunoprecipitation of endogenous AhR with PKCθ from lysates of primary splenic T cells from WT and Lck-GLK mice without stimulation. N.S., normal serum. (G) Proximity ligation assays (PLAs) of interaction between endogenous PKCθ and AhR in peripheral blood T cells from WT or Lck-GLK Tg mice. Each red dot represents a direct interaction. T cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Images were captured with ×400 original magnification by a Leica DM2500 fluorescence microscope. (H) In vitro kinase assays of purified HA-tagged AhR plus either Myc-tagged PKCθ WT or PKCθ kinase-dead (K409W) mutant proteins. (I) Immunoblotting analysis of phosphorylated AhR (Ser36), AhR, GLK, and PKCθ in primary splenic T cells of WT, Lck-GLK Tg, and Lck-GLK Tg mice bred with PKCθ KO mice. (J) Confocal microscopy analysis of subcellular localization of AhR and PKCθ in primary splenic T cells of indicated mice. Original magnification, ×630; scale bars, 10 μm. WT, wild-type littermate controls; Lck-GLK, T cell–specific GLK Tg mice; Lck-GLK;PKCθ−/−, Lck-GLK Tg mice bred with PKCθ KO mice. Data shown are representative of three independent experiments.

  • Fig. 4 GLK induces RORγt binding to the IL-17A promoter through AhR and RORγt interaction.

    (A) Binding of RORγt to the IL-17A promoter in T cells from indicated mice was analyzed by ChIP-PCR. (B) Coimmunoprecipitation of endogenous AhR with RORγt using lysates of primary splenic T cells from WT and Lck-GLK mice without any stimulation. IB, immunoblotting. (C) Confocal microscopy analysis of subcellular localization of AhR and RORγt in primary T cells of WT, Lck-GLK Tg, Lck-GLK;PKCθ−/−, and Lck-GLK;AhRf/f;CD4-Cre mice. Original magnification, ×630; scale bars, 10 μm. (D) Binding of AhR and RORγt to the IL-17A promoter in T cells from mice was analyzed by ChIP-PCR using anti-RORγt immunocomplexes. (E) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt in peripheral blood T cells from WT, Lck-GLK Tg, and Lck-GLK;PKCθ−/− mice. Original magnification, ×630; scale bars, 10 μm. (F) Coimmunoprecipitation experiments of HA-tagged AhR and Flag-tagged RORγt using lysates of HEK293T cells cotransfected with GLK–cyan fluorescent protein (CFP), PKCθ-Myc, IKKβ-CFP, or IKKα-Myc plasmid. (G) GST pulldown assays of purified Flag-tagged RORγt and GST-tagged AhR proteins. Flag-tagged RORγt proteins were eluted with Flag peptides using lysates of HEK293T cells cotransfected with Flag-RORγt plus either CFP-IKKβ or vector. For PLA, each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Data shown are representative of three independent experiments.

  • Fig. 5 IKKβ phosphorylates RORγt Ser489, leading to RORγt binding to AhR.

    (A) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt in peripheral blood T cells from WT, Lck-GLK Tg, and Lck-GLK;IKKβf/f;CD4-Cre mice. (B) Confocal microscopy analysis of subcellular localization of AhR and RORγt in primary splenic T cells of WT, Lck-GLK Tg, and Lck-GLK;IKKβf/f;CD4-Cre mice. Original magnification, ×630; scale bars, 10 μm. (C) The serum levels of cytokines in 8-week-old mice were determined by ELISAs. IKKβf/f, n = 6; Lck-GLK, n = 6; Lck-GLK; IKKβf/f, n = 5. Means ± SEM are shown. *P < 0.05 (two-tailed Student’s t test). (D) Direct interaction between recombinant proteins of RORγt and IKKβ. GST or His pulldown assays of purified His-tagged RORγt and GST-tagged IKKβ proteins. (E) In vitro kinase assays of immunoprecipitated Flag-tagged RORγt and either IKKβ or IKKβ kinase-dead (K44M) mutant proteins from individual HEK293T transfectants. (F) Tandem MS (MS/MS) fragmentation spectra of the tryptic peptides of RORγt contain the phosphorylation of Ser489. m/z, mass/charge ratio. (G) Antibody specificity of anti–phospho-RORγt (Ser489) was demonstrated by immunoblotting using HEK293T cells cotransfected with CFP-tagged IKKβ plus either Flag-tagged RORγt WT or RORγt-S489A mutant. (H) Immunoblotting analyses of p-RORγt (Ser489), RORγt, p-IKKβ (Ser180/181), and IKKβ in primary splenic T cells of WT, Lck-GLK Tg, and Lck-GLK;IKKβf/f; CD4-Cre mice. (I) Coimmunoprecipitation experiments of HA-tagged AhR and either Flag-tagged RORγt WT or RORγt-S489A mutant using lysates of HEK293T cells cotransfected with vector or IKKβ-CFP plasmid. WT, wild-type littermate controls; Lck-GLK, T cell–specific GLK Tg mice; Lck-GLK;IKKβf/f;CD4-Cre, T cell–specific GLK Tg mice bred with IKKβ cKO mice. For PLA, each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Data shown (A, B, D, E, and G to I) are representative of three independent experiments.

  • Fig. 6 TCR signaling induces RORγt phosphorylation and subsequent AhR-RORγt interaction.

    (A) Immunoblotting analysis of p-RORγt (Ser489), RORγt, p-IKKβ (Ser180/181), and IKKβ in primary splenic T cells. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). (B) Coimmunoprecipitation of endogenous AhR with RORγt from lysates of murine primary splenic T cells stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). (C) Immunoblotting analysis of p-RORγt (Ser489), RORγt, and IKKβ in primary splenic T cells of IKKβf/f or CD4-Cre;IKKβf/f mice. T cells were stimulated with anti-CD3 antibodies plus streptavidin (3 μg each per milliliter). (D) Confocal microscopy analysis of PLAs for the interaction between endogenous AhR and RORγt (left) or between AhR and Ser489-phosphorylated RORγt (right) in primary T cells of IKKβf/f or IKKβf/f;CD4-Cre mice. T cells were stimulated as in (C). Each red dot represents a direct interaction. T cell nucleus was stained with DAPI (blue). Original magnification, ×630; scale bars, 10 μm. (E and F) ELISA of various cytokines in supernatants of primary splenic T cells from IKKβf/f or IKKβf/f;CD4-Cre mice (E), as well as RORγtf/f or RORγtf/f;CD4-Cre mice (F). T cells were stimulated with plate-bound anti-CD3 antibodies (2 μg each per milliliter) for 3 days. Means ± SD are shown. n = 3 per group. (G) Immunoblotting of RORγt and GAPDH proteins from primary splenic T cells of RORγtf/f or RORγtf/f;CD4-Cre mice. Data shown (A to G) are representative of three independent experiments. (H) Schematic model of IL-17A transcription induced by the AhR-RORγt complex in GLK-overexpressing or TCR-stimulated T cells. GLK overexpression in T cells of T cell–specific GLK Tg (Lck-GLK Tg) mice induces AhR Ser36 phosphorylation through PKCθ and also induces RORγt Ser489 phosphorylation through IKKβ. Once RORγt is phosphorylated, RORγt interacts directly with AhR. Phosphorylated AhR is responsible for transporting RORγt into cell nucleus. The AhR-RORγt complex binds to both the RORγt-binding element (−877 to −872) and the AhR-binding element (−254 to −249) of the IL-17A promoter, leading to induction of IL-17A transcription. In normal T cells, TCR stimulation also induces GLK kinase activity and downstream signaling, including IKKβ activation, RORγt Ser489 phosphorylation, and the AhR-RORγt interaction. Besides NF-κB, other critical transcription factors [such as nuclear factor of activated T cell 1 (NFAT1) or activator protein 1 (AP-1)] are also required for the transcriptional activation of IL-2, IFN-γ, IL-4, IL-6, and TNF-α in T cells. “Others” denotes other critical transcription factors (table S1). NF-κB is required for TCR-induced production of multiple cytokines; however, the GLK–IKKβ–NF-κB cascade alone is not sufficient for the induction of multiple cytokines. Collectively, GLK overexpression or TCR signaling induces IL-17A transcription through AhR and RORγt in T cells.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/9/eaat5401/DC1

    Fig. S1. Normal T cell and B cell development in Lck-GLK Tg mice.

    Fig. S2. Inflammatory phenotypes and enhanced TH17 differentiation in Lck-GLK Tg mice.

    Fig. S3. Autoimmune responses in Lck-GLK Tg mice are abolished by IL-17A deficiency.

    Fig. S4. GLK transgene does not regulate IL-23 receptor expression, STAT3 phosphorylation, and RORγt-binding element at the −120 region of the IL-17A promoter.

    Fig. S5. PKCθ controls Ser36 phosphorylation–mediated AhR nuclear translocation and AhR-mediated autoimmune responses.

    Fig. S6. PKCθ directly interacts with AhR in the cytoplasm of Lck-GLK T cells.

    Fig. S7. Autoimmune responses in Lck-GLK Tg mice are reduced by PKCθ KO.

    Fig. S8. TCR signaling induces in vivo interaction between AhR and RORγt.

    Fig. S9. Schematic model of AhR/RORγt-mediated IL-17A transcription in T cells of Lck-GLK Tg mice with different gene-KO backgrounds.

    Table S1. Transcription factors of NF-κB–mediated cytokines.

    References (58, 59)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Normal T cell and B cell development in Lck-GLK Tg mice.
    • Fig. S2. Inflammatory phenotypes and enhanced TH17 differentiation in Lck-GLK Tg mice.
    • Fig. S3. Autoimmune responses in Lck-GLK Tg mice are abolished by IL-17A deficiency.
    • Fig. S4. GLK transgene does not regulate IL-23 receptor expression, STAT3 phosphorylation, and RORγt-binding element at the −120 region of the IL-17A promoter.
    • Fig. S5. PKCθ controls Ser36 phosphorylation–mediated AhR nuclear translocation and AhR-mediated autoimmune responses.
    • Fig. S6. PKCθ directly interacts with AhR in the cytoplasm of Lck-GLK T cells.
    • Fig. S7. Autoimmune responses in Lck-GLK Tg mice are reduced by PKCθ KO.
    • Fig. S8. TCR signaling induces in vivo interaction between AhR and RORγt.
    • Fig. S9. Schematic model of AhR/RORγt-mediated IL-17A transcription in T cells of Lck-GLK Tg mice with different gene-KO backgrounds.
    • Table S1. Transcription factors of NF-κB–mediated cytokines.
    • References (58, 59)

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