Aryl hydrocarbon receptor regulates Stat1 activation and participates in the development of Th17 cells

Abstract

IL-17-producing T helper cells (Th17) have been recently identified as a previously undescribed subset of helper T cells. Here, we demonstrate that aryl hydrocarbon receptor (Ahr) has an important regulatory function in the commitment of Th17 cells. Ahr was robustly induced under Th17-polarizing conditions. Ahr-deficient naïve T cells showed a considerable loss in the ability to differentiate into Th17 cells when induced by TGF-beta plus IL-6. We were able to demonstrate that Ahr interacts with Stat1 and Stat5, which negatively regulate Th17 development. Whereas Stat1 activation returned to its basal level in Ahr wild type naïve T cells 24 h after stimulation with TGF-beta plus IL-6, Stat1 remained activated in Ahr-deficient naïve T cells after stimulation. These results indicate that Ahr participates in Th17 cell differentiation through regulating Stat1 activation, a finding that constitutes additional mechanisms in the modulation of Th17 cell development.

Fig. 1.

Ahr is specifically expressed in Th17 cells. Isolated naïve T cells were cultured with anti-CD3/CD28 beads and the indicated cytokines for 2 days. (A) Gene expression profiles in nonstimulated and stimulated naïve T cells were compared by DNA microarray. (B) The indicated cells were lysed and subjected to Western blot analysis for the expression of Ahr and actin. Data are from one representative of three experiments.

Fig. 2.

Ahr deficiency reduces IL-17 production in naïve T cells. (A) Purified naïve T cells were stimulated with anti-CD3/CD28 beads in the presence of IL-6 or TGF-β, either alone or combined. Supernatants were collected 4 days after stimulation, and IL-17 production was measured by means of ELISA. Data show means ± SE of three independent experiments. (B and C) Dot plots show intracellular staining for IFN-γ and IL-17. (B) Isolated naïve T cells from Ahr WT, He, and KO splenocytes were cultured with anti-CD3/CD28 beads and the indicated cytokines for 4 days. (C) Naïve T cells isolated from Ahr WT and KO mice were stimulated with anti-CD3/CD28 beads and TGF-β plus IL-6 in the presence or absence of TCDD or FICZ for 3 days. (D) Naïve T cells isolated from Ahr WT and KO mice were stimulated with anti-CD3/CD28 beads and the indicated cytokines for 2 days. Total RNA and cDNA were prepared as described in Methods. RORγ and RORα induction was examined by using RT-PCR. (B–D) These results are representative of three independent experiments.

Fig. 3.

Different pattern of IL-17 production between CD4⁺CD62L⁻ and CD4⁺CD62L⁺ cells. CD4⁺CD62L⁻ and CD4⁺CD62L⁺ cells isolated from WT mice were stimulated with anti-CD3/CD28 beads and TGF-β plus IL-6. (A) Three days after stimulation, cells were re-stimulated with PMA and ionomycin for 5 h and with GolgiStop (final 2 h), and then subjected to intracellular cytokine staining. Dot plots show intracellular staining for IFN-γ and IL-17. (B) Two days after stimulation, total RNA and cDNA were prepared as described in Methods. RORγ and Ahr induction was examined by using RT-PCR. These results are representative of three independent experiments.

Fig. 4.

Ahr partially participates in the generation of Treg cells by TGF-β. Naïve T cells isolated from Ahr WT and KO mice were stimulated with anti-CD3/CD28 beads and TGF-β with or without Ahr ligands for 2 days. Foxp3 expression was determined by staining with anti-mouse Foxp3 antibody. These data are representative of three independent experiments.

Fig. 5.

Ahr regulates the activation of Stat1 in Th17 cell development. (A) MACS-sorted naïve T cells were cultured with anti-CD3/CD28 beads and stimulated with IL-6 or TGF-β, either alone or combined, for 2 days. Whole cell lysates were immunoprecipitated with anti-Ahr antibody, after which Stat1, Stat3, Stat5, Stat6, and Ahr were detected with Western blotting. IP, immunoprecipitation; IB, immunoblot. (B) Naïve T cells isolated from Ahr WT and He mice were stimulated with anti-CD3/CD28 beads and TGF-β plus IL-6 in the presence or absence of IFN-γ for 3 days, followed by re-stimulation with PMA and ionomycin for 5 h and with GolgiStop (final 2 h), and then staining for intracellular cytokines. Dot plots show intracellular staining for IFN-γ and IL-17. (C and D) Naïve T cells isolated from Ahr WT and KO splenocytes were stimulated with anti-CD3/CD28 beads and TGF-β plus IL-6 for 30 min or 24 h, fixed and permeabilized in 90% methanol, and finally stained with Alexa Fluor 488-conjugated phospho-Stat1 and PE-conjugated phospho-Stat3. Intracellular levels of phospho-Stat1 (C) and Stat3 (D) were measured by means of flow cytometry. These results are representative of three independent experiments.