CCR6 regulates the migration of inflammatory and regulatory T cells

Abstract

Th17 and regulatory T (Treg) cells play opposite roles in autoimmune diseases. However, the mechanisms underlying their proper migration to inflammatory tissues are unclear. In this study, we report that these two T cell subsets both express CCR6. CCR6 expression in Th17 cells is regulated by TGF-beta and requires two nuclear receptors, RORalpha and RORgamma. Th17 cells also express the CCR6 ligand CCL20, which is induced synergistically by TGF-beta and IL-6, which requires STAT3, RORgamma and IL-21. Th17 cells, by producing CCL20, promote migration of Th17 and Treg cells in vitro in a CCR6-dependent manner. Lack of CCR6 in Th17 cells reduces the severity of experimental autoimmune encephalomyelitis and Th17 and Treg recruitment into inflammatory tissues. Similarly, CCR6 on Treg cells is also important for their recruitment into inflammatory tissues. Our data indicate an important role of CCR6 in Treg and Th17 cell migration.

FIGURE 1

CCR6 is highly expressed by Th17 cells. A, In vitro-differentiated Th1, Th2, and Th17 cells were restimulated with anti-CD3 for 4 h for real-time RT-PCR analysis. The data are expressed as the mean ± SD of triplicate samples. Data shown were repeated twice with consistent results. B, FACS-sorted naive T cells were cultured with the indicated cytokines for 1, 2, and 5 days. After restimulation with anti-CD3, CCR6 and IL-17 expression was analyzed by real-time PCR. The data are expressed as the mean ± SD of triplicate samples. Data shown were repeated twice with consistent results. C, Naive CD4⁺ T cells from RORγ KO, RORα/γ double mutant, IL-21 KO, or WT mice were differentiated under Th17 conditions for 4 days. CCR6 expression was analyzed by real-time PCR. Real-time PCR results in A–C were normalized to Actb gene expression and relative gene expression levels are indicated. The data are expressed as the mean ± SD of triplicate samples. A–C, The expression level in Th0 of WT or KO cells was set at 1. Data shown were repeated twice with consistent results. D, CCR6 expression was analyzed with allophycocyanin-labeled anti-CCR6 or allophycocyanin-labeled rat IgG in Th1, Th2, or Th17 cells. E, CCR6 expression on Th17 cells in the spleen, LN, and CNS of mice with EAE diseases. C57BL/6 mice were immunized with MOG peptide to induce EAE. The cells from spleen, LN, and CNS of mice that developed EAE were restimulated with MOG peptide for 1 day. CCR6 expression was analyzed with allophycocyanin-labeled anti-IFN-γ and PE-labeled IL-17A in IL-17⁺ or IFN-γ⁺ CD4⁺ T cells. The data are expressed as the mean ± SD of three mice. The results shown are a representative of at least two independent experiments.

FIGURE 2

CCR6 expression on Treg cells. A, CCR6 or Foxp3 expression was analyzed in cDNA of naive CD4⁺ T cells, nTreg, Th0, and iTreg cells by real-time PCR. Results were normalized to Actb gene expression and the relative gene expression levels are indicated. The expression level in Th0 was set at 1. The data are expressed as the mean ± SD of triplicate samples. Data shown were repeated twice with consistent results. B, iTreg cells were analyzed for the expression of CCR6 in gated CD4⁺CD25⁺ cells. Data shown were repeated twice with consistent results. C, The expression of CCR6 in nTreg cells was analyzed in gated CD4⁺CD25⁺ cells in the spleen and popliteal LN. The results shown are a representative of two independent experiments.

FIGURE 3

CCL20 is specifically expressed by Th17 cells. A, Th1, Th2, Th17, nTreg, and iTreg cells were analyzed for CCL20 expression by real-time PCR. Real-time PCR results were normalized to Actb gene expression and relative gene expression levels are indicated. The expression level in iTreg was set at 1. The data are expressed as the mean ± SD of triplicate samples. Data shown were repeated twice with consistent results. B, In vitro-differentiated Th17 cells were restimulated with anti-CD3 for up to 72 h. The cell culture supernatants were analyzed for CCL20 production by ELISA at the indicated time points. The data are expressed as the mean ± SD of triplicate wells. Data shown were repeated twice with consistent results. *, p < 0.01. C, Naive T cells were cultured with the indicated cytokines for 5 days. After restimulation with anti-CD3, CCL20 expression was analyzed by real-time PCR. The expression level in naive T cell was set at 1. The data are expressed as the mean ± SD of triplicate samples. Data shown were repeated twice with consistent results. D–F, Naive CD4⁺ T cells from STAT3 KO (D), RORγ KO (E), RORα/γ KO (E), or IL-21 KO mice (F) and their appropriate control mice were cultured for Th17 differentiation for 4 days. CCL20 expression was analyzed by real-time PCR after restimulation of T cells with anti-CD3. E, CCL20 expression was assessed in CD4⁺ T cells overexpressing RORγt with real-time PCR. Real-time PCR results in C–F were normalized to Actb gene expression and relative gene expression levels are indicated. The expression levels in Th0 of WT cells were set at 1. The data are expressed as the mean ± SD of triplicate samples. The results shown (D–F) are a representative of two independent experiments.

FIGURE 4

CCL20 does not regulate the differentiation or cytokine production of Th17 cells. A, FACS-sorted naive CD4⁺ T cells were stimulated with anti-CD3 (3 μg/ml) and anti-CD28 (2 μg/ml) under Th1 conditions (20 ng/ml IL-12 and 5 μg/ml anti-IL-4) or Th17 conditions (10 ng/ml TGF-β, 50 ng/ml IL-6, 30 ng/ml IL-23, 5 μg/ml anti-IFN-γ, and 5 μg/ml anti-IL-4) in the presence or absence of CCL20 (10 ng/ml) for 4 days. Intracellular cytokine staining was performed with allophycocyanin-labeled anti-IFN-γ and PE-labeled IL-17 after permeabilization. The results are representative of three independent experiments. B, In vitro- differentiated Th17 cells were restimulated with anti-CD3 (3 μg/ml) with or without CCL20 (10 ng/ml) for 3 days. The production of IL-17 and IL-17F in the culture supernatants was measured by ELISA. The data has no statistical difference between T cells in the presence of CCL20 and absence of CCL20 (IL-17, p = 0.322; IL-17F, p = 0.391). Similar results were obtained from three independent experiments. C, Naive CD4⁺ T cells from WT or CCR6^−/− mice were differentiated to Th17 cells with anti-CD3 (3 μg/ml) and anti-CD28 (2 μg/ml) in the presence of TGF-β (10 ng/ml), IL-6 (50 ng/ml), IL-23 (30 ng/ml), anti-IFN-γ (5 μg/ml), and anti-IL-4 (5 μg/ml). Four days after culture, the cells were analyzed for Th17 differentiation in flow cytometry by staining with allophycocyanin-labeled IFN-γ and PE-labeled IL-17. The data show no statistical difference between T cells from WT mice and CCR6^−/− mice (IFN-γ⁺ cells, p = 0.050; IL-17⁺ cells, p = 0.144). Data shown are based on three mice in each group. The results are representative of two independent experiments.

FIGURE 5

The migration of Th17 and Treg cells is mediated by CCR6 and CCL20 interaction. A, Transwell chemotaxis assay on Th1, Th2, and Th17 cells in the presence of CCL20. The migrated cells were analyzed at 7 h of culture. Data shown were repeated twice with consistent results. B, The migration of naive CD4⁺ T cells and nTreg cells was evaluated in response to CCL20. Data shown were repeated twice with consistent results. C, WT or CCR6-deficient Th1, Th17, and Treg cells were examined in chemotaxis assays in response to CCL20. Data shown were repeated twice with consistent results. D, The culture supernatants of anti-CD3-stimulated Th17 cells were used for chemotaxis assay. The migration of Th1, Th17, or Treg cells from C57BL/6 or CCR6 KO mice was measured. The result shown is representative of two independent experiments. All data are expressed as the mean ± SD of triplicate wells. *, p < 0.01.

FIGURE 6

CCR6 deficiency in lymphocytes leads to inhibited EAE diseases. A, CCR6^−/− or C57BL/6 mice (five mice in each group) were immunized with MOG peptide to induce EAE. Disease scores in each group were evaluated. The data are shown as the mean ± SD. The data represent one of two experiments with similar results. B–K, Rag1^−/− chimeric mice reconstituted with BM cells from C57BL/6 or CCR6^−/− mice were immunized with MOG peptide to induce EAE. B, Disease scores in five mice in each group were evaluated. The data are expressed as the mean ± SD of five mice. The data represent one of two experiments with similar results. C and D, The CNS-infiltrating cells were analyzed by staining with PerCP-labeled anti-CD4 and FITC-labeled anti-CD11b. The analysis was done on five mice in each group. E, The population of CD4⁺CD25⁺Foxp3⁺ cells was analyzed in the cells from the CNS. The analysis was done on five mice in each group. F–H, IL-17⁺ or IFN-γ⁺ cells in mononuclear cells in the CNS were analyzed in a CD4⁺ gate. The analysis was done on five mice in each group. Representative data from flow cytometry are shown in F. The data of cell percentage are expressed as the mean ± SD of five mice. The absolute cell numbers of IL-17⁺ cell and IFN-γ⁺ cells were shown in G and H. I–K, IL-17⁺ or IFN-γ⁺ cells in spleen cells were analyzed in a CD4⁺ gate. The data from flow cytometry was shown in I. The analysis was done on five mice in each group. The data of cell percentages are expressed as the mean ± SD of five mice. The absolute cell numbers of IL-17⁺ cell and IFN-γ⁺ cells were shown in J and K. The analysis was done on five mice in each group. B–J, Circles represent individual mice. The horizontal lines represent the mean. The results shown are representative of two independent experiments. *, p < 0.01 and **, p < 0.05.

FIGURE 7

CCR6^−/− Th17 cells fail in induction of EAE. A, IL-23-cultured MOG-reactive CD4⁺ T cells from C57BL/6 or CCR6 KO mice were adoptively transferred into five C57BL/6 mice in each group. The mice were immunized with MOG to induce EAE. A, Disease scores in five mice in each group were evaluated. The data are shown as the mean ± SD of five mice. B and C, CD4⁺ (B) or CD11b⁺ cells (C) in the mononuclear cells from CNS were analyzed in five mice in each group by staining with PerCP-labeled anti-CD4 and FITC-labeled anti-CD11b. D, CD4⁺CD25⁺Foxp3⁺ cells were analyzed in the cells from the CNS of five mice in each group. E–G, IL-17⁺ or IFN-γ⁺ cells in the CNS of five mice in each group were analyzed in mononuclear cells in a CD4⁺ gate. The data from flow cytometry are shown in E. The data of cell percentages are expressed as the mean ± SD of five mice. The absolute cell numbers of IL-17⁺ cell and IFN-γ⁺ cells in the CNS of five mice in each group were shown in F and G. H–J, IL-17⁺ or IFN-γ⁺ cells in spleen cells of five mice in each group were analyzed in a CD4⁺ gate. The data from flow cytometry are shown in H. The data of cell percentages are expressed as the mean ± SD of five mice. The absolute cell numbers of IL-17⁺ cells and IFN-γ⁺ cells in spleen cells of five mice in each group are shown in I and J. B–J, Circles represent individual mice. The horizontal lines represent the mean. All results are representative of two independent experiments. *, p < 0.01 and **, p < 0.05.

FIGURE 8

CCR6 regulates the distribution of Treg cells in EAE. A and B, WT naive CD4⁺ T cells were mixed with either WT or CCR6^−/− Treg cells and transferred into five RAG1 ^−/− mice in each group. The mice were immunized with MOG peptide to induce EAE. A, The absolute cell number of CNS-infiltrating Treg cells was analyzed in CD4⁺Foxp3⁺ gate. The data of absolute cell number are expressed as the mean ± SD of five mice. B, The absolute cell number of Treg cells in the spleen was analyzed in the CD4⁺Foxp3⁺ gate. The data of absolute cell number are expressed as the mean ± SD of five mice. C and D, The distribution of Treg cells in the CD45.1⁺ congenic mice developing EAE. CD45.2⁺ WT Treg cells (1 × 10⁶/mouse) or CCR6^−/− CD45.2⁺ Treg cells (1 × 10⁶/mouse) were transferred into CD45.1⁺ congenic mice. The mice were immunized with MOG peptide to induce EAE. C, The absolute cell number of CNS-infiltrating CD45.2⁺ Treg cells derived from WT or CCR6^−/− mice were analyzed by staining with anti-CD45.2 and anti-Foxp3 in CD4⁺ gate. The data of absolute cell number are expressed as the mean ± SD of two mice. D, The absolute cell number of CD45.2⁺ Treg cells from WT or CCR6^−/− mice in the spleen was analyzed by staining with anti-CD45.2 and anti-Foxp3 in the CD4⁺ gate. The data of absolute cell number are expressed as the mean ± SD of two mice. E, Foxp3-GFP mice (three mice in each group) were immunized with MOG/CFA to induce EAE. After the mice reached EAE disease score 3, the cells in the CNS were harvested and analyzed for CCR6 and IL-17 expression. All data shown are representative of two independent experiments. *, p < 0.01 and **, p < 0.05.