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. 2009 Jan 16;206(1):43-9.
doi: 10.1084/jem.20081438. Epub 2008 Dec 29.

Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells

Marc Veldhoen  1 , Keiji Hirota, Jillian Christensen, Anne O'Garra, Brigitta Stockinger
Affiliations

Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells

Marc Veldhoen et al. J Exp Med. .

Abstract

Th17 cell differentiation is dependent on interleukin (IL)-6 and transforming growth factor (TGF)-beta, and it is modulated by activation of the aryl hydrocarbon receptor (AhR). In this study, we show that differentiation of Th17 cells, but not Th1 or induced regulatory T (iT reg) cells, is increased by endogenous AhR agonists present in culture medium. Th17 development from wild-type mice is suboptimal in the presence of the AhR antagonist CH-223191, similar to the situation in AhR-deficient mice, which show attenuated IL-17 production and no IL-22 production. The presence of natural AhR agonists in culture medium is also revealed by the induction of CYP1A1, a downstream target of AhR activation. However, the most commonly used medium, RPMI, supports very low levels of Th17 polarization, whereas Iscove's modified Dulbecco's medium, a medium richer in aromatic amino acids, which give rise to AhR agonists, consistently results in higher Th17 expansion in both mouse and human cells. The relative paucity of AhR agonists in RPMI medium, coupled with the presence of factors conducive to IL-2 activation and enhanced Stat5 phosphorylation, conspire against optimal Th17 differentiation. Our data emphasize that AhR activation plays an essential part in the development of Th17 cells and provide a rational explanation for the poor in vitro polarization of Th17 cells that is reported in the majority of publications for both mouse and human cells.

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Figures

Figure 1.
Figure 1.
Increased Th17 polarization caused by endogenous AhR ligands. (A) Intracellular staining for IL-17 versus IL-22 of CD4 T cells from control B6 (top) or AhR-deficient (bottom) cells polarized under Th17 conditions in IMDM for 5 d in the absence (left) or presence (right) of AhR antagonist CH-223191. Representative dot plots of four independent experiments are shown. (B) Intracellular staining for IL-17A versus IFN-γ or IL-17A versus Foxp3 of CD4 T cells from B6 mice cultured under Th1 conditions (top) or iT reg cell conditions (bottom) in the absence (left) or presence (right) of AhR antagonist CH-223191. Dot plots are representative of three independent experiments. (C) qPCR kinetic analysis of AhR, IL-17, IL-22, and CYP1A1 expression after activation of naive CD4 T cells under Th17 conditions. Expression in CD4 T cells activated under neutral conditions is shown for comparison. The figure shows mRNA levels normalized to Hprt expression and is representative of three independent experiments.
Figure 2.
Figure 2.
Independent RORγt and AhR regulation in Th17 cells. (A) FACS-sorted naive CD4 T cells retrovirally transduced with RV-GFP control, AhR-GFP, or RORγt-GFP vector as indicated on abscissa were cultured in the presence (shaded bars) or absence (open bars) of FICZ. As a comparison, FACS-sorted naive CD4 T cells cultured under Th17 condition in the presence (shaded bars) or absence (open bars) of FICZ are shown. The figure shows mRNA expression for the indicated genes normalized for HPRT mRNA expression. The figure is representative of three independent experiments. (B) FACS-sorted naive CD4 T cells were retrovirally transduced with either control vector containing truncated human CD4 (RV-hCD4Δcyto) together with RV-GFP control vector (top) or with Ahr-hCD4Δcyto together with RORγt-GFP (bottom) and cultured in the presence of FICZ. Expression of hCD4 versus GFP is shown on the left. Intracellular IL-22 expression in gated populations is shown on the right. Results represent three independent experiments.
Figure 3.
Figure 3.
Suboptimal Th17 differentiation in RPMI medium. (A) Scatter plots showing the percentage of IL-17 polarization from individual experiments with CD4 T cells from B6 (filled triangles) or AhR-deficient mice (open circles) cultured under Th17-polarizing conditions in IMDM or RPMI in the presence or absence of AhR antagonist (left). P value control versus AhR antagonist for B6 are <0.001; between IMDM and RPMI B6 control values P < 0.001. Data from four independent experiments are shown. (right) Percentage of IFN-γ producers (filled diamonds) or Foxp3 expression (open squares) in B6 CD4 T cells cultured under Th1 or iT reg cell conditions, respectively in IMDM or RPMI. P value for iT reg cell generation in IMDM versus RPMI is P < 0.01. (B) Total CD4 T cells purified from PBMCs of human blood and stimulated under Th17 conditions in IMDM (left) or RPMI (right) medium in the absence (top) or presence (bottom) of AhR antagonist. IL-17 versus IFN-γ intracellular staining is shown in the dot plots. The scatter plot shows the percentage of IL-17 producers from three different individuals (representing three independent experiments) obtained in IMDM versus RPMI medium.
Figure 4.
Figure 4.
Substitution of RPMI with tryptophan improves Th17 differentiation. (A) Scatter plot showing the percentage of IL-17 producers in CD4 T cells from wild-type B6 (filled triangles) or AhR-deficient mice (open circles) cultured under Th17-polarizing conditions in IMDM (left) or RPMI (right) with or without addition of 11 mg/liter of l-tryptophan. P values are shown in the graph. Data from four independent experiments are shown. (B) IL-17/IL-22 expression in CD4 T cells cultured under Th17 conditions in either IMDM (left) or RPMI (right) in the absence or presence of AhR agonist FICZ after 4 d of culture or (C) in the presence of neutralizing antibodies to IFN-γ and IL-4. Dot plots are representative of three independent experiments.
Figure 5.
Figure 5.
IL-2 blockade together with AhR activation restored Th17 differentiation. (A) Intracellular IL-17/IL-22 in Th17 cells developing in IMDM (left) or RPMI medium (right) without (top) or with addition of neutralizing antibodies to IL-2 (bottom). Dot plots are representative of three independent experiments. (B) Expression of phospho-Stat5 in CD4 T cells cultured under Th17 conditions in IMDM (top) or RPMI (bottom) in the absence of exogenous AhR agonist (left), with FICZ (middle), or in the presence of neutralizing antibodies to IL-2 (right). Histograms (representative of three independent experiments) of gated CD4 T cells showing phospho-Stat5 expression at 48 h after onset of culture are shown. (C) IL-17/IL-22 expression in Th17 cells cultured in IMDM (left) or RPMI (right) in the presence of neutralizing antibodies to IL-2 and FICZ. Dot plots are representative of three independent experiments.
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References

    1. Bettelli, E., T. Korn, and V.K. Kuchroo. 2007. Th17: the third member of the effector T cell trilogy. Curr. Opin. Immunol. 19:652–657. - PMC - PubMed
    1. Stockinger, B., and M. Veldhoen. 2007. Differentiation and function of Th17 T cells. Curr. Opin. Immunol. 19:281–286. - PubMed
    1. Veldhoen, M., K. Hirota, A.M. Westendorf, J. Buer, L. Dumoutier, J.C. Renauld, and B. Stockinger. 2008. The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins. Nature. 453:106–109. - PubMed
    1. Denison, M.S., and S.R. Nagy. 2003. Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu. Rev. Pharmacol. Toxicol. 43:309–334. - PubMed
    1. Nguyen, L.P., and C.A. Bradfield. 2008. The search for endogenous activators of the aryl hydrocarbon receptor. Chem. Res. Toxicol. 21:102–116. - PMC - PubMed

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