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. 2010 Jul;246(1-2):18-28.
doi: 10.1016/j.taap.2010.03.013. Epub 2010 Mar 27.

Functional and phenotypic effects of AhR activation in inflammatory dendritic cells

Jaishree Bankoti  1 , Ben Rase, Tom Simones, David M Shepherd
Affiliations

Functional and phenotypic effects of AhR activation in inflammatory dendritic cells

Jaishree Bankoti et al. Toxicol Appl Pharmacol. 2010 Jul.

Abstract

Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immune suppression. Dendritic cells (DCs) are key antigen presenting cells governing T cell activation and differentiation. However, the consequences of AhR activation in DCs are not fully defined. We hypothesized that AhR activation alters DC differentiation and generates dysfunctional DCs. To test this hypothesis, inflammatory bone marrow-derived DCs (BMDCs) from C57Bl/6 mice were generated in the presence of vehicle or TCDD. TCDD decreased CD11c expression but increased MHC class II, CD86 and CD25 expression on the BMDCs. The effects of TCDD were strictly AhR-dependent but not exclusively DRE-mediated. Similar effects were observed with two natural AhR ligands, 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid (ITE). TCDD increased LPS- and CpG-induced IL-6 and TNF-alpha production by BMDCs but decreased their NO production. TCDD decreased CpG-induced IL-12p70 production by BMDCs but did not affect their secretion of IL-10. TCDD downregulated LPS- and CpG-induced NF-kB p65 levels and induced a trend towards upregulation of RelB levels in the BMDCs. AhR activation by TCDD modulated BMDC uptake of both soluble and particulate antigens. Induction of indoleamine-2,3-dioxygenase (IDO) and TGF-beta3 has been implicated in the generation of regulatory T cells following AhR activation. TCDD increased IDO1, IDO2 and TGF-beta3 mRNA levels in BMDCs as compared to vehicle. Despite the induction of regulatory mediators, TCDD-treated BMDCs failed to suppress antigen-specific T cell activation. Thus, AhR activation can directly alter the differentiation and innate functions of inflammatory DCs without affecting their ability to successfully interact with T cells.

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Figures

Figure 1
Figure 1. TCDD alters the expression of differentiation markers on BMDCs
Bone marrow cells were induced to differentiate into immature DCs with GM-CSF in the presence of vehicle (represented by dotted line in the histograms) or 10 nM TCDD (thick line as indicated in the histograms). Isotype staining is shown in gray lines. Non-adherent cells were harvested on day 7 and stained with CD11c as a DC lineage marker. MHC class II, CD86, CD54, CD11a and CD25 expression was determined on CD11c+ BMDCs by FACS analysis. Data shown represents non-adherent cells obtained from 4 separate flasks per treatment group and is representative of 3 independent experiments. *p<0.05 indicates significant differences between vehicle- and TCDD-treated groups.
Figure 2
Figure 2. Low concentrations of TCDD alter BMDC differentiation
Bone marrow cells were generated in the presence of GM-CSF and vehicle or varying TCDD concentrations (0.001nM-10nM). Non-adherent cells were harvested on day 7 and stained for CD11c, MHC II and CD86. Expression of MHC II and CD86 was determined on the CD11c+-gated population. Data represents mean ± SEM of 6 samples at each concentration. *p<0.05 indicates significant differences between the vehicle- and TCDD-treated groups.
Figure 3
Figure 3. Natural AhR agonists alter BMDC differentiation
Bone marrow-derived dendritic cells were grown in presence of vehicle or the natural AhR agonists, FICZ and ITE. Non-adherent cells were harvested on day 7 and stained for CD11c, MHC II and CD86. Expression of MHC II and CD86 was determined on CD11c+ BMDCs. *indicates significant differences between treated and respective vehicle control groups. Data represents mean ± SEM of 4 samples per treatment group and is representative of 2 independent experiments.
Figure 4
Figure 4. The effects of TCDD on BMDCs are AhR-dependent and partially DRE-dependent
BMDCs from AhR+/+, AhRdbd/-, AhRdbd/dbd or AhRnls/nls mice were treated with vehicle or TCDD. Non-adherent cells were harvested on day 7 and stained for CD11c, MHC II and CD86. Expression of MHCII and CD86 was determined on the CD11c+-gated population and analyzed by flow cytometry. Data represent mean ± SEM of non-adherent cells obtained from 3-4 samples per treatment group. *p<0.05 indicates significant differences between respective vehicle and TCDD-treated groups.
Figure 5
Figure 5. Effects of TCDD on LPS- and CpG–induced MHC Class IIhigh expression
BMDCs were grown in vehicle or TCDD and non-adherent cells were harvested on day 7. BMDCs were purified and treated with LPS or CpG for 24 hours. Cells were harvested after 24 hours and MHC class II expression was assessed. Data represents mean ± SEM of 9 samples per treatment group and is representative of 3 independent experiments. *p<0.05 indicates significant differences between the respective vehicle- and TCDD-treated groups. #p<0.05 indicates significant differences between TLR-stimulated and respective untreated groups.
Figure 6
Figure 6. LPS- and CpG-induced p65 but not Rel B activity is reduced by TCDD
Vehicle- or TCDD-treated BMDCs were stimulated with LPS (1 μg/ml) or CpG (0.5μM) for 0.5 hours. Cells were harvested and nuclear proteins isolated. NF-kB p65 and RelB activity was measured using the TransAM activity ELISA. * indicates significant differences between LPS- or CpG-stimulated groups compared to respective vehicle-treated control groups. # represents significant differences between LPS- or CpG-stimulated groups compared to respective untreated control group. Data represents mean ± SEM of 3 samples per treatment group.
Figure 7
Figure 7. TCDD alters antigen uptake by BMDCs
Vehicle- or TCDD-treated BMDCs were purified and their ability to take up FITC-conjugated whole ovalbumin (FITC–Ova), low-density lipoprotein (LDL-FITC) and latex beads was assessed. Cells were exposed to FITC-conjugated ovalbumin overnight, LDL-FITC for 1.5 hours or FITC-latex beads for 6 hours prior to harvesting. Data represents mean ± SEM of 9-12 samples per treatment group for 2 independent experiments. * p≤ 0.05 indicates significant differences between vehicle- and TCDD-treated samples.
Figure 8
Figure 8. Ability of ova-laden BMDCs to activate ova-specific T cells in vivo
CD4+ T cells from OT II/Thy1.1 mice were adoptively transferred into congenic, CD45.1+ host mice on day -1 relative to immunization. On day 0, vehicle- or TCDD-treated BMDCs that were loaded with whole ovalbumin were injected into adoptively transferred mice. On day 4 post-immunization, popliteal and brachial lymph nodes were harvested from host mice and analyzed by flow cytometry. Analysis was based on the differences in the phenotypic expression of cell surface molecule between donor DCs (CD45.2+/CD11c+) and donor OT II T cells (CD4+/V-alpha2+/Thy1.1+) from host mice (CD45.1+/Thy1.2+). Data are representative of 4-5 animals per treatment group. * p ≤ 0.05 indicates significant differences between vehicle- and TCDD–treated groups. Dashed line represents the donor OT II T cell control levels in the brachial lymph nodes.
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