Impact of culture medium on maturation of bone marrow-derived murine dendritic cells via the aryl hydrocarbon receptor

Molecular Immunology 51 (2012) 42–50

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Impact of culture medium on maturation of bone marrow-derived murine dendritic cells via the aryl hydrocarbon receptor Anne Ilchmann a,1, Maren Krause a,1, Monika Heilmann a,1, Sven Burgdorf b, Stefan Vieths c, Masako Toda a,∗ a

Junior Research Group “Experimental Allergology”, Paul-Ehrlich-Institut, Langen, Germany Life and Medical Sciences (LIMES) Institute, University of Bonn, Bonn, Germany c Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany b

a r t i c l e

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Article history: Received 12 December 2011 Received in revised form 27 January 2012 Accepted 3 February 2012 Available online 28 February 2012 Keywords: Aryl hydrocarbon receptor Dendritic cells Toll-like receptors Scavenger receptors

a b s t r a c t The aryl hydrocarbon receptor (AhR) plays a role in modulating dendritic cell (DC) immunity. Iscove’s modified Dulbecco’s medium (IMDM) contains higher amounts of AhR ligands than RPMI1640 medium. Here, we examined the influence of AhR ligand-containing medium on the maturation and T-cell stimulatory capacity of bone marrow-derived murine dendritic cells (BMDCs). BMDCs generated in IMDM (BMDCs/IMDM) expressed higher levels of co-stimulatory and MHC class II molecules, and lower levels of pattern-recognition receptors, especially toll-like receptor (TLR) 2, TLR4, and scavenger receptor class A (SR-A), compared to BMDCs generated in RPMI1640 medium (BMDCs/RPMI). Cytokine responses against ligands of TLRs and antigen uptake mediated by SR-A were remarkably reduced in BMDCs/IMDM, whereas the T-cell stimulatory capacity of the cells was enhanced, compared to BMDCs/RPMI. The enhanced maturation of BMDCs/IMDM was attenuated in the presence of an AhR antagonist, indicating involvement of AhR in the maturation. Interestingly, BMDCs/IMDM induced Th2 and Th17 differentiation at low and high concentrations of antigen respectively, when co-cultured with CD4+ T-cells from antigen-specific T-cell receptor transgenic mice. In contrast, BMDCs/RPMI induced Th1 differentiation predominantly in the co-culture. Taken together, optimal selection of medium seems necessary when studying BMDCs, depending on the target receptors on the cell surface of DCs and type of helper T-cells for the co-culture. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Dendritic cells (DCs) are central players in immune responses, and essential to bridge innate and acquired immunity (Steinman and Banchereau, 2007; Joffre et al., 2009). DCs capture microbial and tumor antigens, or allergens, and subsequently undergo maturation and activation induced by these components (innate immunity). Activation of specific T-cell effector mechanisms (acquired immunity) are then triggered by the mature antigen presenting DCs. To perform their function, DCs are equipped with a full

Abbreviations: AhR, aryl hydrocarbon receptor; FICZ, 6-formylindolo [3,2b] carbazole; GM-CSF, granulocyte/macrophage colony-stimulating factor; IDO, indoleamine 2,3-dioxygenase; IMDM, Iscove’s modified Dulbecco’s medium; LPS, lipopolysaccharide; BMDCs, bone marrow derived murine dendritic cells; MR, mannose receptor; SR-A, scavenger receptor class A; TCDD, tetrachlorodibenzo-pdioxin; TLR, toll-like receptor. ∗ Corresponding author at: Research Group 1, “Experimental Allergology“, PaulEhrlich-Institut, Paul Ehrlich Street 59, Langen 63225, Germany. Tel.: +49 0 6103 77 5407; fax: +49 0 6103 77 1258. E-mail address: [email protected] (M. Toda). 1 The authors equally contributed to this work. 0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2012.02.005

array of specialized receptors including co-stimulatory molecules (e.g. CD40, CD80, and CD86) and pattern-recognition receptors (e.g. toll-like receptors, C-type lectin receptors, and scavenger receptors) (Kawai and Akira, 2010; Unger and van Kooyk, 2011; Areschoug and Gordon, 2009). Understanding detailed mechanisms of DC maturation and activation would provide key insights into immune defense and immune disorders. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor from the Per-Arnt-Sim superfamily of proteins, and ubiquitously expressed in vertebrate cells (Stevens et al., 2009; Veldhoen and Duarte, 2010). Ligands for AhR include environmental toxins (e.g. tetrachlorodibenzo-p-dioxin; TCDD), polycyclic aromatic hydrocarbons (e.g. 3-methylcholanthrene), dietary components, heme metabolites, and tryptophan metabolites. Recent findings have indicated that AhR also play modulatory roles in the immune system. For instance, AhR promotes differentiation of Th17 cells and regulatory T-cells in both humans and mice (Funatake et al., 2005; Quintana et al., 2008; Veldhoen et al., 2008; Hauben et al., 2008; Kimura et al., 2008; Ramirez et al., 2010; Nguyen et al., 2010). AhR engagement also influences differentiation and maturation of DCs and Langerhans cells (Jux et al., 2009; Platzer

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et al., 2009; Bankoti et al., 2010; Simones and Shepherd, 2011). Notably, AHR ligands are contained in culture medium (Oberg et al., 2005). Compared to RPMI1640 medium, IMDM contains three- to five-fold higher amount of aromatic amino acids (e.g. tryptophan, phenyalanine, and tyrosine) and generates endogenous AhR ligands (Veldhoen et al., 2009). Indeed, Th17 differentiation, which is promoted by AhR stimulation, was higher in IMDM (Veldhoen et al., 2009). In this study, we aimed to examine whether endogenous AhR ligands in culture medium influence the maturation of bone marrow-derived murine dendritic cells (BMDCs). BMDCs are frequently used to study DC maturation, activation, and function. To generate BMDCs, bone marrow cells are cultured in medium containing granulocyte macrophage colony-stimulating factor (GM-CSF) (Markowicz and Engleman, 1990; Inaba et al., 1992). We found that IMDM enhanced maturation and T-cell stimulatory capacity of BMDCs in AhR dependent manner. BMDCs generated in IMDM expressed higher levels of co-stimulatory and MHC class II molecules and lower levels of pattern-recognition receptors such as toll-like receptors (e.g. TLR2 and TLR4) and scavenger receptors (e.g. scavenger receptor class A and class B), compared to the cells generated in RPMI1640 medium. The enhanced maturation was attenuated if BMDCs were cultured in the presence of an AhR antagonist. This is the first to elucidate a molecular mechanism for an impact of culture medium on maturation and T-cell stimulatory capacity of DCs. 2. Material and methods 2.1. Mice C57BL/6J (B6), and OT-II mice were purchased from Charles River Laboratories International (Kisslegg, Germany) and Jackson Laboratories (Bar Harbor, ME, USA), respectively. Mice were housed under pathogen free conditions and animal experiments were performed in compliance with German legislations. 2.2. Generation of BMDCs Bone marrow cells were seeded at 1 × 106 cells/ml in IMDM, or RPMI1640 (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 0.1 mM 2-mercaptoethanol and 100 ng/ml rGM-CSF (R&D System, Wiesbaden-Nordenstadt, Germany) for eight days to generate BMDCs. In some experiments, BMDCs were generated in the presence of the AhR antagonist CH-223191 (Merck Chemical, Nottingham, UK) at 3 ␮M, or the AhR agonist 6-formylindolo [3,2-b] carbazole (FICZ; Enzo Life Sciences, Lörrach, Germany) at 300 nM.

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with sera of matched animal species for the secondary antibodies (Invitrogen). In addition, the cells were stained with PE-, or FITC-conjugated anti-mouse CD11b and allophycocyanin (APC)conjugated anti-mouse CD11c mAbs to gate the DC population. Fluorescence intensity of CD11b+ CD11c+ cells was measured by a flow cytometry, LSR II (BD Bioscience). Data were analyzed using FlowJo V. 7 (Treestar Inc., Ashland, Ore, USA). 2.4. Assessment of cytokine production by BMDCs BMDCs (1.0–2.0 × 106 cells/ml) were treated with 1.0–1000 ng/ml Pam3CSK4 (Invivogen, Toulouse, France), or 0.01–1000 ng/ml LPS (Sigma Aldrich) in RPMI medium, or IMDM for 20 h. The levels of IL-6, IL-10, and TNF-alpha in the culture supernatant were determined by ELISA (ebioscience). 2.5. Assessment of OVA uptake by BMDCs BMDCs (1.0 × 106 cells/ml) were incubated with FITCconjugated OVA for 15 min. Following incubation, BMDCs were stained with both PE-conjugated anti-mouse CD11b and APCconjugated anti-mouse CD11c mAbs. FITC intensity in CD11b+ CD11c+ cells was analyzed by flow cytometry. 2.6. Assessment of T-cell stimulatory capacity of BMDCs BMDCs were generated in IMDM, or RPMI medium in the presence, or absence of CH-223191, or FICZ as described above. Splenic CD4+ T-cells were isolated from OT-II mice using an isolation kit from Miltenyi Biotec (Bergisch Gladbach, Germany). To evaluate Tcell activation, CD4+ T-cells (8.0 × 105 cells/ml) were co-cultured with BMDCs (1.6 × 105 cells/ml), and stimulated with LPS free OVA (Seikagaku Cooperation, Tokyo, Japan). For the co-culture, IMDM, or RPMI medium, which used for BMMC generation, was selected. CH-223191 and FICZ were not added into the co-culture. The supernatant for measuring IL-2 was harvested after 24 h of co-culture, while that for measuring IFN-gamma, IL-4, and IL-17 was harvested after 72 h of co-culture. The levels of cytokines in the supernatant were determined by ELISA (ebioscience). To evaluate T-cell proliferation, CD4+ T-cells were stained with 10 ␮M carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) and co-cultured with BMDCs in the presence of OVA for 96 h. Cell proliferation was evaluated by measuring the intensity of CFSE in the CD4+ T-cells using flow cytometry. 2.7. Statistical analysis Significant differences between mean values were assessed by ANOVA followed by the Tukey’s HSD multiple comparison test. A P value of <0.05 was considered significant.

2.3. Detection of receptors expressed on BMDCs 3. Results To detect co-stimulatory molecules and pattern recognition receptors expressed on the cell surface of BMDCs, fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD40, CD80, CD86, MHC class I, or class II molecule mAb, phycoerythrin (PE)conjugated rat anti-mouse scavenger receptor class B (SR-B), DEC205, mannose receptor (MR), TLR2, TLR5 (eBioscience, Frankfurt, Germany), or TLR4 (BioLegend, Uithoorn, The Netherland) were used. To detect SR-A expression, the cells were stained with rat anti-mouse scavenger receptor class A (SR-A) Abs (R&D System) followed by Alexa Fluor 488 conjugated goat anti-rat IgG (H+L) Abs (Invitrogen). In all staining, IgG receptors on the cell surface was blocked using mAb against CD16/CD32 (eBioscience), or normal murine IgG antibodies (Santa Cruz biotechnology) in combination

3.1. IMDM promotes maturation marker expression in BMDCs Since IMDM contains higher levels of endogenous AhR ligands than RPMI medium (Veldhoen et al., 2009), we first examined whether the medium influences development and maturation of BMDCs in an AhR dependent manner. Born marrow cells were cultured in IMDM, or RPMI medium containing GM-CSF for 8 days. The frequency of CD11b+ and CD11c+ cells and the total number of the cultured cells on day 8 were almost same between IMDM and RPMI medium (Table 1). However, the expression levels of co-stimulatory and MHC class II molecules on the cell surface of BMDCs generated in IMDM (BMDCs/IMDM) and cells generated in RPMI medium

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Table 1 Influence of medium in the frequency and yield of CD11b+ CD11c+ cells in cultured bone marrow cells. Medium

Treatment

Frequency of CD11b+ CD11c+ cells (%)a

IMDM RPMI IMDM RPMI

None None CH-223191 FICZ

72.78 75.83 71.60 81.95

± ± ± ±

6.1 5.2 3.4 3.3

Cell yield (×106 )b 8.1 9.9 6.3 10.8

± ± ± ±

2.1 4.3 1.3 4.4

The data is the mean ± SEM of four independent experiments. a Bone marrow cells were cultured in IMDM, or RPMI medium containing GM-CSF with, or without CH-223191, or FICZ for 8 days. The frequency of CD11b+ CD11c+ cells was measured by flow cytometry. b Generation from 2 × 107 bone marrow cells.

(BMDCs/RPMI) were different. BMDCs/IMDM expressed higher levels of CD40, CD86, CD274 (B7-H1), and MHC class II molecules than BMDCs/RPMI (Fig. 1 and S1). See Supp Fig. S1 as supplementary file. Supplementary material related to this article found, in the online version, at doi:10.1016/j.molimm.2012.02.005. To examine whether AhR is involved in enhancing the expression of co-stimulatory molecules on the cell surface of BMDCs/IMDM, bone marrow cells were cultured in (i) IMDM in the

presence of the AhR antagonist CH-223191, or (ii) RPMI medium in the presence of the high affinity AhR ligand, 6-formylindolo [3,2b] carbazole (FICZ) (Oberg et al., 2005). Furthermore, BMDCs/RPMI were stimulated with LPS, a TLR4 ligand and a known inducer of DC maturation, as a positive control. CH-223191 reduced the expression of co-stimulatory and MHC class II molecules on the cell surface of BMDCs/IMDM (Fig. 1). In contrast, as well as LPS, FICZ promoted the expression of CD40, CD86, CD274 and MHC class II molecules in BMDCs/RPMI. The

Fig. 1. Influence of medium on expression of co-stimulatory and MHC molecules on the cell surface of BMDCs. BMDCs were generated in IMDM, or RPMI medium containing 100 ng/ml of rGM-CSF in the presence, or absence of 3 ␮M CH-223191, or 300 nM FICZ for 8 days. For LPS stimulation, BMDCs generated in RPMI medium were treated with 10 ␮g/ml of the TLR4 ligand for 18 h. Expression of CD40, CD80, CD86, MHC class I, and class II molecules on the cell surface of BMDCs was measured by flow cytometry. Grey areas represent unstained cells, while dotted lines represent the cells treated cells with an isotype control antibody. The data are representative for three independent experiments.

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Fig. 2. Influence of medium on expression of TLRs on the cell surface of BMDCs. BMDCs were generated in IMDM, or RPMI medium containing 100 ng/ml of rGM-CSF in the presence, or absence of 3 ␮M CH-223191, or 300 nM FICZ for 8 days. For LPS stimulation, BMDCs generated in RPMI medium were treated with 10 ␮g/ml of the TLR4 ligand for 18 h. Expression of TLR2, TLR4, and TLR5 on the cell surface of BMDCs was measured by flow cytometry. Grey areas represent unstained cells, while dotted lines represent the cells treated cells with an isotype control antibody. The data are representative for three independent experiments.

expression of CD80, CD275 (B7-H2) and MHC class I molecule was almost the same among the BMDCs under the different culture conditions. The results suggest that endogenous AhR ligands in IMDM induce maturation of BMDCs. 3.2. IMDM reduces TLR and SR expression in BMDCs Since cell maturation could influence expression of patternrecognition receptors in DCs, we next examined whether the medium influences expression of TLRs (e.g. TLR2, TLR4, and TLR5), scavenger receptors (e.g. SR-A and SR-B), and C-type lectin receptors (e.g. DEC205 and MR) on the cell surface of BMDCs. Interestingly, BMDCs/IMDM expressed lower levels of TLR2, TLR4, SR-A (CD204), SR-B (CD36), DEC205 (CD205), and MR (CD206) than BMDCs/RPMI (Figs. 2 and 3). In the presence of CH-223191, however, BMDCs/IMDM expression of these patternrecognition receptors remained at the levels observed on the cell

surface of BMDCs/RPMI. In contrast, BMDCs/RPMI showed reduced expression of pattern-recognition receptors if the cells were generated in the presence of FICZ. Among the pattern-recognition receptors, SR-A and MR are the receptors mediating uptake of ovalbumin (OVA) by DCs (Burgdorf et al., 2007; Ilchmann et al., 2010). OVA is a model antigen frequently used in immunological assays. We therefore compared the uptake levels of OVA by the BMDCs generated in the different media. Associated with the expression levels of SR-A and MR, the uptake of OVA by BMDCs/IMDM was lower than that by BMDCs/RPMI (Fig. 3). To examine whether reduced cell surface expression of TLRs influences responses of BMDCs to TLR ligands, we assessed cytokine production of BMDCs/IMDM and BMDCs/RPMI upon stimulation with Pam3CSK4, a TLR2 ligand, or LPS, a TLR4 ligand. Concentrations of IL-6, IL-10, and TNF-alpha in the culture supernatants of the stimulated BMDCs were measured by ELISA.

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Fig. 3. Influence of medium on expression of scavenger receptors and C-type rectin receptors on the cell surface of BMDCs. BMDCs were generated in IMDM, or RPMI medium containing 100 ng/ml of rGM-CSF in the presence, or absence of 3 ␮M CH-223191, or 300 nM FICZ for 8 days. For LPS stimulation, BMDCs generated in RPMI medium were treated with 10 ␮g/ml of the TLR4 ligand for 18 h. Expression of SR-A, SR-B, DEC205, and the MR on the cell surface of BMDCs, and uptake of FITC-conjugated OVA by BMDCs were measured by flow cytometry. Grey areas represent unstained cells, while dotted lines represent the cells treated cells with an isotype control antibody. The data are representative for three independent experiments.

The threshold of the TLR ligand concentration needed to induce detectable levels of the cytokines was around 10–100 times higher in BMDCs/IMDM, compared to BMDCs/RPMI (Fig. 4). Collectively, these results suggest that IMDM adversely influences antigen uptake and cytokine expression by BMDCs associated with the pattern-recognition receptors, due to reduced receptor expression.

3.3. IMDM enhances the T-cell stimulatory capacity of BMDCs Next, we examined whether or not the medium influence the T-cell stimulatory capacity of BMDCs. BMDCs were co-cultured with splenic CD4+ T-cells isolated from OT-II mice, a transgenic strain expressing OVA-specific T-cell receptor, in the presence of OVA. OT-II cells showed higher IL-2 production and proliferation in co-culture with BMDCs/IMDM, compared to BMDCs/RPMI (Fig. 5A and B). Higher IL-4 and IL-17 production, but lower IFN-gamma production by CD4+ T-cells was observed in the co-culture with BMDCs/IMDM (Fig. 6A–C). IL-10 was not detectable in the culture supernatant of either co-culture system (data not shown).

To examine whether the differential levels of cytokine production by OT-II cells in the different medium was due to AhR engagement in BMDCs, we used the cells generated in IMDM in the presence CH-223191, or RPMI in the presence of FICZ. The BMDCs were co-cultured with OT-II cells in the medium used for the BMDC generation, but without the AhR antagonist, or the AhR agonist. Interestingly, BMDCs/IMDM generated in the presence of CH-223191 showed lower IL-2 and IL-4 and higher IFNgamma production, but similar IL-17 production, compared to the cells generated in the absence of the AhR antagonist. In contrast, BMDCs/RPMI generated in the presence of FICZ showed higher IL-2, IL-4 and IL-17 production and lower IFN-gamma production, compared to the cells generated in the absence of the AhR agonist. The results suggest that (i) BMDCs/IMDM have a higher T-cell stimulatory capacity than BMDCs/RPMI, and (ii) medium type influences T-cell differentiation via AhR engagement in BMDCs. 4. Discussion Here, we show that culture medium influences maturation and the T-cell stimulatory capacity of BMDCs in an AhR-dependent

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Fig. 4. Influence of medium on cytokine production by BMDCs in response to TLR ligands. BMDCs were generated in IMDM, or RPMI medium containing 100 ng/ml of rGM-CSF for 8 days. The cells were stimulated with 1–1000 ng/ml of Pam3CSK4 (left panels), or 0.01–1000 ng/ml of LPS (right panels) for 20 h. Concentrations of IL-6, TNF-alpha, and IL-10 in the culture supernatant were measured by ELISA. Data indicate mean ± SD of triplicate, and are representative of three independent experiments.*P < .001. **P < .005. n.d., not detectable; IL-6 < 1.25 pg/ml, TNF-alpha < 2.5 pg/ml, IL-10 < 2.5 pg/ml.

manner. Immature DCs reportedly specialize in antigen capture and processing, whereas mature DCs have an increased T-cell stimulatory capacity (Joffre et al., 2009; Kawai and Akira, 2010). Growth in IMDM, which contains higher amounts of endogenous

AhR ligands, generated more matured BMDCs expressing higher levels of co-stimulatory and MHC class II molecules and lower levels of pattern-recognition receptors, especially TLR2, TLR4, and SR-A, compared to culture in RPMI 1640 medium. Furthermore, BMDCs

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Fig. 5. Influence of medium on T-cell stimulatory capacity of BMDCs. BMDCs were generated in IMDM, or RPMI medium in the presence, or absence of 3 ␮M CH-223191, or 300 nM FICZ. (A) CD4+ T-cells were isolated from OT-II mice, stained with CFSE, and co-cultured with the BMDCs in IMDM, or RPMI media in the presence of increasing concentrations of OVA for 96 h. (B) CD4+ T-cells were co-cultured with BMDCs in IMDM, or RPMI media in the presence of increasing concentrations of OVA for 24 h. For the co-culture, IMDM, or RPMI medium, which used for BMMC generation, was selected. Concentrations of IL-2 in the cell supernatant after co-culture were measured by ELISA. The data indicate mean ± SD of triplicate, and are representative of two independent experiments. *P < .001. **P < .0001. n.d., not detectable; IL-2 < 1.25 pg/ml.

generated in IMDM promoted Th2 and Th17 differentiation at low and high concentrations of antigen respectively, when co-cultured with CD4+ T-cells. Our results suggest that selecting optimal culture medium is advisable when generating BMDCs. Especially if the TLRs and SR-A are crucial targets in DC studies, RPMI1640 is useful. IMDM could be optimal for DCs to develop Th2 and Th17 cells. BMDCs generated in IMDM took up lower levels of antigen, but induced higher antigen-specific CD4+ T-cell activation than BMDCs generated in RPMI medium. The enhanced T-cell stimulatory capacity of BMDCs in IMDM is likely associated with higher expression of co-stimulatory and MHC class II molecules on their cell surface. The medium also influenced the CD4+ T-cells’ cytokine production in response to antigen. It has shown that endogenous AhR ligands in IMDM promote Th17 differentiation of both human and murine T-cells (Veldhoen et al., 2009). In agreement, we observed higher IL-17 production by CD4+ T-cells when these were co-cultured with BMDCs in IMDM, rather than BMDCs in RPMI medium. IL-17 production by co-cultured CD4+ T-cells was also promoted in RPMI medium, if T-cell stimulatory capacity of BMDCs was enhanced by stimulation with the AhR agonist FICZ. It seems that Th17 differentiation is associated with T-cell stimulatory capacity of BDMCs in the co-culture. However, Th17 differentiation in IMDM was not attenuated when CD4+ T-cells were co-cultured with BMDCs presenting

lower T-cell capacity, which generated in IMDM in the presence of the AhR antagonist CH-223191. Therefore, direct stimulation of CD4+ T-cells by endogenous AhR ligands could also contribute to Th17 differentiation in IMDM. Interestingly, at low concentrations of antigen, CD4+ T-cells co-cultured with BMDCs in IMDM, but not with BMDCs in RPMI medium, produced detectable levels of IL-4, the Th2 cytokine. T-cell differentiation is influenced by various factors such as cytokine milieu, antigen concentration, and co-stimulatory molecules (Eisenbarth et al., 2003; Zhu et al., 2010; Zygmunt and Veldhoen, 2011). It has been shown that stimulation of CD4+ T-cells at low antigen concentrations tends to induce Th2 cell differentiation (Eisenbarth et al., 2003). The lower uptake of antigen and higher expression of co-stimulatory molecules by BMDCs in IMDM could result in the Th2-oriented cytokine production of CD4+ T-cells in the co-culture system. Notably, the enhanced IL-4 production and reduced IFN-gamma production by CD4+ T-cells in IMDM was attenuated when these were co-cultured with BMDCs generated in the media containing the AhR antagonist. We therefore conclude that Th2 differentiation in IMDM could be due to BMDCs stimulated by endogenous AhR ligands in the medium. Several studies including ours have shown that FICZ, the high affinity AhR ligand, enhances expression of maturation makers on

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Fig. 6. Influence of medium on T-cell differentiation capacity of BMDCs. BMDCs were generated in IMDM, or RPMI medium in the presence, or absence of 300 nM CH223191, or 3 ␮M FICZ. CD4+ T-cells were co-cultured with BMDCs in the presence of increasing concentrations of OVA for 76 h. For the co-culture, IMDM, or RPMI medium, which used for BMMC generation, was selected. CH-223191 and FICZ were not added into the co-culture. (A) IFN-gamma, (B) IL-4, and (C) IL-17 concentrations in the cell supernatant were measured by ELISA. The data indicate mean ± SD of triplicate, and are representative of two independent experiments. *P < .001. **P < .0001. n.d., not detectable; IFN-gamma < 2.5 pg/ml, IL-4 < 2.5 pg/ml, IL-17 < 1.25 pg/ml.

DCs (Bankoti et al., 2010; Simones and Shepherd, 2011). However, molecular mechanisms for AhR-induced DC maturation are still unknown. We detected the enhanced expression of co-stimulatory and MHC class II molecules on the cell surface of BMDCs not at 18 h, but at 40 h after stimulation with FICZ, when the cells were stimulated by the ligand on day 6 of the RPMI culture (data not shown). In contrast, LPS enhanced expression of the maturation markers at 18 h after stimulation of BMDCs. Interestingly, AhR stimulation reduced pattern-recognition receptors expression on the cell surface of BMDCs, whereas LPS stimulation retained expression of all the receptors except TLR4 (see Figs. 2 and 3). These observations suggest that (i) molecular mechanisms for AhR-and TLR4-mediated DC maturation are different, and (ii) the second signal(s), which could be induced by AhR stimulation, might trigger the DC

maturation. At first, we speculated that cytokine(s) produced by AhR-stimulated BMDCs induced the cell maturation. However, treatment of BMDCs with FICZ did not induce detectable levels of GM-CSF, IL-6 (see Fig. S2), TNF-alpha, IL-4 and IL-12 (data not shown), which are capable of influencing DC maturation (Winzler et al., 1997; Son et al., 2002; Park et al., 2004; Trevejo et al., 2001). See Supp Fig. S2 as supplementary file. Supplementary material related to this article found, in the online version, at doi:10.1016/j.molimm.2012.02.005. While the enhancement effect of AhR stimulation on DC maturation has been observed, its adverse effect has also been reported. The effects appear to depend on type of AhR ligands. A small molecule VAF347 decreases CD86 and HLA-DR in human monocyte derived DCs (Lawrence et al., 2008), whereas an environmental

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toxin benzo(a)pyrene (BaP) induced enhanced expression of CD86 and MHC class II molecule on murine BMDCs (Hwang et al., 2007). Dietary constituents, indole-3-carbinol and indirubin-3 -oxime, decreased the expression of CD40, but increased the expression of CD86 and MHC class II molecules (Benson and Shepherd, 2011). Furthermore, it has shown that AhR stimulation of human monocyte derived DCs, or BMDCs with TCDD, another environmental toxin, results in enhanced expression of CD86 and MHC class II molecules, but induces differentiation into tolerogenic DCs via expression of indoleamine 2,3-dioxygenase (IDO) and subsequent generation of regulatory T cells (Vogel et al., 2008; Mezrich et al., 2010; Nguyen et al., 2010; Bankoti et al., 2010; Simones and Shepherd, 2011). IDO catabolizes tryptophan into kynurenine and other metabolites in DCs, and is reportedly involved in generation of tolerogenic DCs, although its detailed mechanisms are still not known (Puccetti and Grohmann, 2007). In our experimental setting, stimulating AhR of BMDCs with FICZ, or culturing the cells in IMDM did not alter AhR and IDO expression (Fig. S3), and did enhance its T-cell stimulatory capacity. The discrepancy among the studies might be due to involvement of receptor(s) other than AhR for TCDD and other ligands, differential binding affinity of these ligands to AhR, or variations in the levels of AHR expression of DCs in different experimental settings. Further study is necessary to elucidate detailed mechanisms of AhR-induced DC maturation and differentiation. See Supp Fig. S3 as supplementary file. Supplementary material related to this article found, in the online version, at doi:10.1016/j.molimm.2012.02.005. Conflict of interest The authors have no financial conflict of interest. Acknowledgements The study was supported by internal funding of the PaulEhrlich-Institut. We thank Prof. Christian Kurts (FriedrichWilhelms University Bonn) and Drs. Matthias Hamdorf and Sabine Schneider (Paul-Ehrlich-Institut) for helpful discussion and Christoph Bock (Johann Wolfgang Goethe University Frankfurt) for technical support. References Areschoug, T., Gordon, S., 2009. Scavenger receptors: role in innate immunity and microbial pathogenesis. Cell Microbiol. 11, 1160–1169. Bankoti, J., Rase, B., Simones, T., Shepherd, D.M., 2010. Functional and phenotypic effects of AhR activation in inflammatory dendritic cells. Toxicol. Appl. Pharmacol. 246, 18–28. Benson, J.M., Shepherd, D.M., 2011. Dietary ligands of the aryl hydrocarbon receptor induce anti-inflammatory and immunoregulatory effects on murine dendritic cells. Toxicol. Sci. 124, 327–338. Burgdorf, S., Kautz, A., Böhnert, V., Knolle, P.A., Kurts, C., 2007. Distinct pathways of antigen uptake and intracellular routing in CD4 and CD8 T cell activation. Science 316, 612–616. Eisenbarth, S.C., Piggott, D.A., Bottomly, K., 2003. The master regulators of allergic inflammation: dendritic cells in Th2 sensitization. Curr. Opin. Immunol. 15, 620–626. Funatake, C.J., Marshall, N.B., Steppan, L.B., Mourich, D.V., Kerkvliet, N.I., 2005. Cutting edge: activation of the aryl hydrocarbon receptor by 2,3,7,8tetrachlorodibenzo-p-dioxin generates a population of CD4+ CD25+ cells with characteristics of regulatory T cells. J. Immunol. 175, 4184–4188. Hauben, E., Gregori, S., Draghici, E., Migliavacca, B., Olivieri, S., Woisetschlager, M., Roncarolo, M.G., 2008. Activation of the aryl hydrocarbon receptor promotes allograft-specific tolerance through direct and dendritic cell-mediated effects on regulatory T cells. Blood 112, 1214–1222. Hwang, J.A., Lee, J.A., Cheong, S.W., Youn, H.J., Park, J.H., 2007. Benzo(a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells. Downregulation of RelB and eIF3 p170 by benzo(a)pyrene. Toxicol. Lett. 169, 82–90. Ilchmann, A., Burgdorf, S., Scheurer, S., Waibler, Z., Nagai, R., Wellner, A., Yamamoto, Y., Yamamoto, H., Henle, T., Kurts, C., Kalinke, U., Vieths, S., Toda, M., 2010. Glycation of a food allergen by the Maillard reaction enhances its T-cell

immunogenicity: role of macrophage scavenger receptor class A type I and II. J. Allergy Clin. Immunol. 125, 175–183. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., Steinman, R.M., 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colonystimulating factor. J. Exp. Med. 176, 1693–1702. Joffre, O., Nolte, M.A., Spörri, R., Reis e Sousa, C., 2009. Inflammatory signals in dendritic cell activation and the induction of adaptive immunity. Immunol. Rev. 227, 234–247. Jux, B., Kadow, S., Esser, C., 2009. Langerhans cell maturation and contact hypersensitivity are impaired in aryl hydrocarbon receptor-null mice. J. Immunol. 182, 67009–67017. Kawai, T., Akira, S., 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nature Immunol. 11, 373–384. Kimura, A., Naka, T., Nohara, K., Fujii-Kuriyama, Y., Kishimoto, T., 2008. Aryl hydrocarbon receptor regulates Stat1 activation and participates in the development of Th17 cells. Proc. Natl. Acad. Sci. U.S.A. 105, 9721–9726. Lawrence, B.P., Denison, M.S., Novak, H., Vorderstrasse, B.A., Harrer, N., Neruda, W., Reichel, C., Woisetschläger, M., 2008. Activation of the aryl hydrocarbon receptor is essential for mediating the anti-inflammatory effects of a novel lowmolecular-weight compound. Blood 112, 1158–1165. Markowicz, S., Engleman, E.G., 1990. Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human peripheral blood dendritic cells in vitro. J. Clin. Invest. 85, 955–961. Mezrich, J.D., Fechner, J.H., Zhang, X., Johnson, B.P., Burlingham, W.J., Bradfield, C.A., 2010. An interaction between kynurenine and the aryl hydrocarbon receptor can generate regulatory T cells. J. Immunol. 185, 3190–3198. Nguyen, N.T., Kimura, A., Nakahama, T., Chinen, I., Masuda, K., Nohara, K., Fujii-Kuriyama, Y., Kishimoto, T., 2010. Aryl hydrocarbon receptor negatively regulates dendritic cell immunogenicity via a kynurenine-dependent mechanism. Proc. Natl. Acad. Sci. U.S.A. 107, 19961–19966. Oberg, M., Bergander, L., Hakansson, H., Rannug, U., Rannug, A., 2005. Identification of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole, in cell culture medium, as a factor that controls the background aryl hydrocarbon receptor activity. Toxicol. Sci. 85, 935–943. Park, S.J., Nakagawa, T., Kitamura, H., Atsumi, T., Kamon, H., Sawa, S., Kamimura, D., Ueda, N., Iwakura, Y., Ishihara, K., Murakami, M., Hirano, T., 2004. IL-6 regulates in vivo dendritic cell differentiation through STAT3 activation. J. Immunol. 173, 3844–3854. Platzer, B., Richter, S., Kneidinger, D., Waltenberger, D., Woisetschläger, M., Strobl, H., 2009. Aryl hydrocarbon receptor activation inhibits in vitro differentiation of human monocytes and Langerhans dendritic cells. J. Immunol. 183, 66–74. Puccetti, P., Grohmann, U., 2007. IDO and regulatory T cells: a role for reverse signalling and non-canonical NF-kappaB activation. Nat. Rev. Immunol. 7, 817–923. Quintana, F.J., Basso, A.S., Iglesias, A.H., Korn, T., Farez, M.F., Bettelli, E., Caccamo, M., Oukka, M., Weiner, H.L., 2008. Control of T(reg) and T(H)17 cell differentiation by the aryl hydrocarbon receptor. Nature 453, 65–71. Ramirez, J.M., Brembilla, N.C., Sorg, O., Chicheportiche, R., Matthes, T., Dayer, J.M., Saurat, J.H., Roosnek, E., Chizzolini, C., 2010. Activation of the aryl hydrocarbon receptor reveals distinct requirements for IL-22 and IL-17 production by human T helper cells. Eur. J. Immunol. 40, 2450–2459. Simones, T., Shepherd, D.M., 2011. Consequences of AhR activation in steady-state dendritic cells. Toxicol. Sci. 119, 293–307. Son, Y.I., Egawa, S., Tatsumi, T., Redlinger Jr., R.E., Kalinski, P., Kanto, T., 2002. A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells. J. Immunol. Methods 262, 145–157. Steinman, R.M., Banchereau, J., 2007. Taking dendritic cells into medicine. Nature 449, 419–426. Stevens, E.A., Mezrich, J.D., Bradfield, C.A., 2009. The aryl hydrocarbon receptor: a perspective on potential roles in the immune system. Immunology 127, 299–311. Trevejo, J.M., Marino, M.W., Philpott, N., Josien, R., Richards, E.C., Elkon, K.B., FalckPedersen, E., 2001. TNF-alpha -dependent maturation of local dendritic cells is critical for activating the adaptive immune response to virus infection. Proc. Natl. Acad. Sci. U.S.A. 98, 12162–12167. Unger, W.W., van Kooyk, Y., 2011. ‘Dressed for success’ C-type lectin receptors for the delivery of glyco-vaccines to dendritic cells. Curr. Opin. Immunol. 23, 131–137. Veldhoen, M., Hirota, K., Westendorf, A.M., Buer, J., Dumoutier, L., Renauld, J.C., Stockinger, B., 2008. The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins. Nature 453, 106–109. Veldhoen, M., Hirota, K., Christensen, J., O’Garra, A., Stockinger, B., 2009. Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells. J. Exp. Med. 206, 43–49. Veldhoen, M., Duarte, J.H., 2010. The aryl hydrocarbon receptor: fine-tuning the immune-response. Curr. Opin. Immunol. 22, 747–752. Vogel, C.F., Goth, S.R., Dong, B., Pessah, I.N., Matsumura, F., 2008. Aryl hydrocarbon receptor signaling mediates expression of indoleamine 2,3-dioxygenase. Biochem. Biophys. Res. Commun. 375, 331–335. Winzler, C., Rovere, P., Rescigno, M., Granucci, F., Penna, G., Adorini, L., Zimmermann, V.S., Davoust, J., Ricciardi-Castagnoli, P., 1997. Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J. Exp. Med. 185, 317–328. Zhu, J., Yamane, H., Paul, W.E., 2010. Differentiation of effector CD4 T cell populations. Annu. Rev. Immunol. 28, 445–489. Zygmunt, B., Veldhoen, M., 2011. T helper cell differentiation more than just cytokines. Adv. Immunol. 109, 159–196.