The aryl hydrocarbon receptor interacts with c-Maf to promote the differentiation of type 1 regulatory T cells induced by IL-27

Abstract

Type 1 regulatory T cells (Tr1 cells ) that produce interleukin 10 (IL-10) are instrumental in the prevention of tissue inflammation, autoimmunity and graft-versus-host disease. The transcription factor c-Maf is essential for the induction of IL-10 by Tr1 cells, but the molecular mechanisms that lead to the development of these cells remain unclear. Here we show that the ligand-activated transcription factor aryl hydrocarbon receptor (AhR), which was induced by IL-27, acted in synergy with c-Maf to promote the development of Tr1 cells. After T cell activation under Tr1-skewing conditions, the AhR bound to c-Maf and promoted transactivation of the Il10 and Il21 promoters, which resulted in the generation of Tr1 cells and the amelioration of experimental autoimmune encephalomyelitis. Manipulating AhR signaling could therefore be beneficial in the resolution of excessive inflammatory responses.

Figure 1. IL-27 upregulates AhR in T_R1 cells

RNA isolated from naïve CD4⁺CD44^loCD62L^hiCD25⁻ cells differentiated into indicated populations in the presence of anti-CD3 and anti-CD28 antibodies was subjected to quantitative real-time PCR (RT-PCR) relative to the expression of mRNA encoding β-actin (2^−ΔCT x100000) to examine expression of Ahr at different time points following activation. a) RT-PCR analysis of Ahr expression at 48 hours in T_H0, T_H1, T_H2, T_H17 and T_R1 cells differentiated with either no cytokines, IL-12, IL-4, TGF-β plus IL-6 or TGF-β plus IL-27 respectively. RT-PCR kinetic analysis of b) Ahr and c) xenobiotic metabolizing cytochrome P450 enzyme Cyp1a1 expression in T_H0 or T_R1 cells differentiated with IL-27 or TGF-β and IL-27. d) RT-PCR kinetic analysis of Ahr and Maf expression in T_R1 cells. Gene expression relative to T_H0 cells is depicted. Representative data from one of three experiments are shown.

Figure 2. AhR regulates IL-10-production in T_R1 cells induced by TGF-β and IL-27

Naïve CD4⁺ T-cells from IL-10 reporter mice (Vert-X mice) were cultured with IL-27 and TGF-β in the absence (Control: Ctrl) or presence of the AhR agonists FICZ (100nM) or TCDD (100nM) a) IL-10.GFP expression was analyzed by flow cytometry after 72 hours of culture. b) IL-10 protein was measured by cytokine bead array analysis at 48 hours. c) Naïve cells from IL-10 reporter mice were cultivated with IL-27 plus TGF-β and transfected with either an irrelevant control siRNA or an siRNA against AhR or c- Maf. Ahr, Maf and IL-10 mRNA expression in T_R1 cells were assessed after 24 hours of culture by quantitative PCR (top) and IL-10.GFP expression was analyzed by flow cytometry after 48 hours (bottom). d) Naïve T cells isolated from wildtype (WT) or c- Maf transgenic (c-Maf-TG) mice were differentiated into T_H0 or T_R1 cells with TGF-β and IL-27 in the absence or presence of FICZ (100nM). After 48 hours of culture, Il10 mRNA expression was assessed by quantitative PCR (left panel) and IL-10 secretion was analyzed by ELISA at 72 hours (right panel). (*p<0.05; **p<0.01)

Figure 3. AhR signaling dictates IL-21 secretion in T_R1 cells

Naïve T cells were differentiated into T_R1 cells without (Ctrl) or with FICZ (100nM) and a) IL-21 cytokine production was assessed by cytokine bead array analysis after 72 hours of culture; b) The transcription factor Maf was quantified by RT-PCR at 48 hours c) and d) Naïve T cells from wild type and Il21r^−/− mice were differentiated into T_R1 cells and IL-21 and IL-10 production were analyzed by cytokine bead array analysis after 48 hours of culture e) mRNA for Maf, Ahr and Tbx21 in the cells described in c) was quantified by RT-PCR relative to the expression of mRNA encoding β-actin. Data are from one of three experiments with similar results. (*p<0.05; **p<0.01)

Figure 4. AhR and c-Maf transactivate the Il10 and Il21 promoters in T_R1 cells

a) AhR and c-Maf binding sites in the Il10 and the Il21 promoters. Schematic representation of the Il10 and the Il21 promoters, AhR binding sites (XRE) are depicted as open boxes and c-Maf binding sites (MARE) are depicted as filled boxes. b) ChIP analysis of the interaction of AhR or isotype control antibody (IgG) to the XRE in the Il10 and c) the Il21 promoter in in vitro differentiated T_R1 or control T_H0 cells. (*p<0.01; **p<0.001 between AhR vs IgG) d) ChIP analysis of the interaction of c-Maf or isotype control antibody (IgG) to the MARE in the Il10 and e) the Il21 promoter in in vitro differentiated T_R1 or control T_H0 cells. (**p<0.001 between c-Maf vs IgG) f) and g) Transactivation of the Il10 or Il21 promoters by c-Maf or AhR. Reporter constructs for the Il10 f) or Il21 g) promoters (Il10-Luc and Il21-Luc, respectively) were co-transfected in EL-4 T cells with vectors coding for AhR and/or c-Maf, and firefly luciferase activity was determined 24 hours later and normalized to the renilla luciferase activity of a co-transfected control. (**p<0.001) h) In vitro differentiated T_H0 or T_R1 cell expression of AhR and c-Maf (immunoblot; IB, left panel). AhR was immunoprecipitated from nuclear extracts with a specific antibody (IP, right panel), c-Maf and AhR complexes immunoblotted (right panel) using an anti-c-Maf antibody.

Figure 5. AhR controls the generation of T_R1 cells in vivo

AhR^d, wild type (WT) or Il27ra^−/− mice were injected i.p. with 20 μg of antibodies to CD3 or an isotype control (IC) once every 3 days, for a total of 3 times. 4 hours after the last injection, mice were sacrificed and the expression of IL-10 in the spleen and the mesenteric lymph nodes (MLN) was analyzed by flow cytometry. a) Representative plots of IL-10 expression in CD4⁺ cells in the spleen and the MLN. b) and c) Frequency of IL-10⁺ CD4⁺ T cells in anti-CD3 or isotype control (IC) treated mice (mean + s.d. of 3–5 mice) in spleen (b) or MLN (c). (*p<0.01)

Figure 6. AhR controls the IL-27-mediated inhibition of EAE

MOG-specific cells from spleen and lymph nodes from wild type (WT) or AhR^d mutant mice were obtained 11 days after immunization and a) the percentage of IL-10-producing CD4⁺ T cells was assessed by flow cytometry after five days of culture with MOG in the presence or absence of IL-27. b) Cells were restimulated in vitro with MOG in the presence of IL-12 either with or without IL-27 and IL-10 and IFN-γ secretion was analyzed 3 days later by cytokine bead array analysis. (*p<0.05) c) and d) WT or AhR^d MOG specific cells prepared as in b) were adoptively transferred into WT mice and recipient animals were monitored for the development of EAE. c) The mean clinical disease score in each group is shown for WT or AhR^d donor cells. d) Linear regression curves of the disease for each group are shown for the experiments depicted in c). The disease course differs significantly between the two treatments (IL-12 versus IL-12 plus IL-27) of WT donor cells but not of AhR^d donor cells. The 95% confidence intervals for each curve are represented with dashed lines. (***p<0.0001).