Aryl hydrocarbon receptor negatively regulates dendritic cell immunogenicity via a kynurenine-dependent mechanism - PubMed (original) (raw)

Aryl hydrocarbon receptor negatively regulates dendritic cell immunogenicity via a kynurenine-dependent mechanism

Nam Trung Nguyen et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Although an immunoregulatory role of aryl hydrocarbon receptor (Ahr) has been demonstrated in T cells and macrophages, little is known about its function in dendritic cells (DC). Here, we show that lipopolysaccharide (LPS) and CpG stimulate Ahr expression in bone marrow-derived dendritic cells (BMDC). Furthermore, we found that Ahr is required to induce indoleamine 2,3-dioxygenase (IDO) expression, an immunosuppressive enzyme that catabolizes tryptophan into kynurenine (Kyn) and other metabolites in DC. In the presence of LPS or CpG, Ahr-deficient (Ahr(-/-)) mature BMDC induced immune responses characterized by reduced Kyn and IL-10 production compared with results observed with tolerogenic mature WT BMDC. In a coculture system with LPS- or CpG-stimulated BMDC and naive T cells, Ahr(-/-) BMDC inhibited naive T-cell differentiation into regulatory T (Treg) cells, which likely facilitated Th17 cell development and promoted naive T-cell proliferation. Addition of synthetic L-Kyn to the coculture system skewed the differentiation of naive T cells to Treg cells rather than Th17 cells. Taken together, our results demonstrate a previously unknown negatively regulatory role for Ahr in DC-mediated immunogenesis in the presence of LPS or CpG, which, in turn, alters the Kyn-dependent generation of Treg cells and Th17 cells from naive T cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

LPS and CpG stimulated Ahr expression in BMDC. Bone marrow cells were cultured in the presence of GM-CSF for 9 d. MACS-sorted CD11c+ BMDC were then harvested and stimulated with LPS or CpG for 24 h. (A) Ahr mRNA expression was examined using qPCRs. (B) Cells were lysed and analyzed by Western blotting for Ahr and G3PDH. Representative data were obtained from one of two experiments.

Fig. 2.

Fig. 2.

IL-10 production was inhibited in Ahr−/− BMDC stimulated with LPS or CpG. WT and Ahr−/− BMDC were stimulated with LPS or CpG and culture supernatants were harvested after 24 h. Levels of IL-6, TNF-α, IL-12p40, TGF-β1, and IL-10 were measured using ELISA. Data show mean ± SD from three independent experiments. **P < 0.01; ***P < 0.005.

Fig. 3.

Fig. 3.

IDO was not expressed in Ahr−/− BMDC stimulated with LPS or CpG. (A) WT and Ahr−/− BMDC were stimulated with LPS or CpG either alone or in combination with TCDD for 24 h. IDO mRNA expression was examined using qPCR. (B) To measure Kyn levels, WT and Ahr−/− BMDC were cultured in complete RPMI medium 1640 plus L-Trp (100 μM). Cells were then stimulated with LPS or CpG, and culture supernatants were harvested after 18 h. Kyn levels were measured in culture supernatants using a colorimetric assay. Data show mean ± SD from three independent experiments. *P < 0.05; n.s., not significant.

Fig. 4.

Fig. 4.

Absence of Ahr in BMDC inhibited Treg development and facilitated Th17 cell generation from naive T cells. WT and Ahr−/− BMDC were stimulated with LPS or CpG for 24 h. MACS-sorted naive T cells from WT mice were then cocultured with or without stimulated BMDC for 4 d. Synthetic L-Kyn (50 μM) was added to some samples as indicated. (A) After 4 d coculture, Foxp3 expression in T cells was examined using FITC-conjugated anti-mouse Foxp3 antibodies and stained cells were analyzed using FACS. (B) After 4 d coculture, T cells were stimulated with PMA/ionomycin for 5 h; GolgiStop was added for the final 2 h. T cells were then intracellularly stained for IL-17 and IFN-γ and analyzed using FACS. Data are shown as dot blots. (C) Levels of IL-17 in coculture supernatants of LPS- or CpG-stimulated WT and Ahr−/− BMDC with naive T cells after 4 d. Supernatants were harvested from BMDC–T-cell coculture after 4 d. IL-17 was measured in coculture supernatants using ELISA. These results are representative of three independent experiments.

Fig. 5.

Fig. 5.

Absence of Ahr in BMDC promoted naive T-cell proliferation. MACS-sorted naive T cells from WT mice were stained with 3 μM CFSE at 37 °C for 10 min. WT and Ahr−/− BMDC were stimulated with LPS or CpG for 24 h. CFSE-labeled naive T cells were cocultured with or without stimulated BMDC. L-Kyn (50 μM) was added to some samples as indicated. After 4 d, cell proliferation was examined based on CFSE dilution using FACS. Equivalent percentage of proliferation in each generation of cells is shown. These results are representative of three independent experiments.

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