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

Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal... BRIEF DEFINITIVE REPORT Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal diff erentiation of Th17 T cells 1 1 2 2 Marc Veldhoen , Keiji Hirota , Jillian Christensen , Anne O ’ Garra , and Brigitta Stockinger 1 2 Division of Molecular Immunology and Division of Immunoregulation, Medical Research Council National Institute for Medical Research, London NW7 1AA, England, UK Th17 cell differentiation is dependent on interleukin (IL)-6 and transforming growth factor (TGF)-  , 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 devel- opment from wild-type mice is suboptimal in the presence of the AhR antagonist CH- 223191, similar to the situation in AhR-defi cient 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 activa- tion. However, the most commonly used medium, RPMI, supports very low levels of Th17 polarization, whereas Iscove ’ s modifi ed 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. Diff erentiation of the new CD4 eff ector subset it induces expression of IL-22 ( 3 ). Despite the CORRESPONDENCE Brigitta Stockinger: Th17 requires the presence of IL-6 and TGF-  fact that AhR binds many toxins, its evolution- [email protected] and is further enhanced by IL-1  and -21 ( 1, 2 ). ary conservation ( 8 ) suggests that this is not its In addition, Th17 polarization is promoted by primary function. Although invertebrate AhR stimulation of the aryl hydrocarbon receptor shares high protein sequence homology with (AhR) ( 3 ), a ligand-dependent transcription the vertebrate receptor, it does not recognize factor that responds to a wide range of ligands. xenobiotic ligands such as TCDD or  -naph- Ligands include environmental toxins, such as thofl avone, which indicates that the AhR has halogenated aromatic hydrocarbons represented physiological functions that are not restricted to by tetrachlorodibenzo- p -dioxin (TCDD) and recognition of environmental pollutants ( 5 ). polycyclic aromatic hydrocarbons (e.g., 3-meth- Involvement of an endogenous AhR ligand ylcholanthrene), as well as potential endogenous in Th17 development in vivo is suggested by the ligands, including dietary components, heme fact that AhR-defi cient mice have an attenuated metabolites, indigoids, and tryptophan metabo- Th17 program, with fewer Th17 cells acti- lites ( 4 ). The tryptophan metabolite 6-formylin- vated upon immunization with MOG peptide/ dolo[3,2-b] carbazole (FICZ) was shown to CFA, resulting in milder pathology in the EAE have very high affi nity for AhR, comparable to model and the absence of IL-22 production (3 ). that of TCDD ( 5 – 7 ). Exposure of CD4 T cells to FICZ under Th17-polarizing conditions en- © 2009 Veldhoen et al. This article is distributed under the terms of an Attribu- tion–Noncommercial–Share Alike–No Mirror Sites license for the fi rst six months hances Th17 development and functions such after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncom- as autoimmune pathology in EAE, which is mercial–Share Alike 3.0 Unported license, as described at http://creativecommons substantially exacerbated by AhR ligation, and .org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 206 No. 1 43-49 43 www.jem.org/cgi/doi/10.1084/jem.20081438 The Journal of Experimental Medicine The attenuated Th17 development from naive CD4 T cell tions show increased Stat5 phosphorylation. Given that IL-2 precursors of AhR-defi cient mice is also noticeable in vitro. dampens Th17 diff erentiation via the induction of Stat5 ( 10 ), There are several studies in the toxicology fi eld suggest- it is not surprising that Th17 diff erentiation is diffi cult to ob- ing that high affi nity ligands for AhR are generated in culture tain in this medium. Our results emphasize the important medium and play a role in the baseline activation of AhR in role AhR agonists play in the modulation of the Th17 pro- hepatocyte cell lines, resulting in activation of genes encod- gram and illustrate that diff erentiation of this eff ector T cell ing xenobiotic metabolizing cytochrome P450 enzymes such subset is inhibited by factors that are conducive to generation as CYP1A (for review see reference [ 9 ]). These findings of the other T cell subsets, a fact that is refl ected in the for- prompted us to test whether such endogenous AhR activity mulation of some culture media that support Th1, Th2, and might infl uence the diff erentiation of Th17 eff ector cells in regulatory T (T reg) cell diff erentiation, but lead to ineffi - vitro. Indeed, our studies show that addition of an AhR an- cient Th17 development. tagonist during Th17 polarization decreases Th17 polariza- tion, whereas it does not infl uence diff erentiation of other RESULTS AND DISCUSSION eff ector T cell subsets. Interestingly, we also found that there AhR antagonist CH-223191 impairs Th17 differentiation are substantial diff erences in regard to generation of endoge- Because tryptophan metabolites generated by light exposure, nous AhR ligands in diff erent culture media, with RPMI such as FICZ, are potent enhancers of Th17 diff erentiation and having far less activity than IMDM. This could explain the there are reports suggesting that light exposure of culture me- puzzling variability in the literature in regard to Th17 polar- dium generates AhR ligands that contribute to background ization, which is generally in the range of 5 – 20%, whereas AhR activation ( 11 ), we set out to assess Th17 development our values usually range from 40 – 60%. RPMI is by far the from naive precursors in the absence or presence of an AhR an- most commonly used culture medium, but as we show here, tagonist. Several AhR antagonists found in food components, IMDM supports better Th17 polarization from both mouse such as fl avones and resveratrol have been previously described and human CD4 T cells. We also observed that in RPMI, ( 12 ), but all of them seem to exert AhR agonist activity at high but not IMDM, CD4 T cells cultured under Th17 condi- concentrations ( 13 ). In contrast, screening of a chemical library Figure 1. Increased Th17 polarization caused by endogenous AhR ligands. (A) Intracellular staining for IL-17 versus IL-22 of CD4 T cells from con- trol B6 (top) or AhR-defi cient (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-  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 expres- sion after activation of naive CD4 T cells under Th17 conditions. Expression in CD4 T cells activated under neutral conditions is shown for comparison. The fi gure shows mRNA levels normalized to Hprt expression and is representative of three independent experiments. 44 AHR AGONISTS IN CULTURE MEDIUM SHAPE TH17 RESPONSE | Veldhoen et al. BRIEF DEFINITIVE REPORT identifi ed CH-223191 as an antagonist that lacked agonist activ- ity even at high doses ( 14 ). Th17 diff erentiation of naive CD4 T cells from B6 mice was markedly inhibited in the presence of the AhR antagonist ( Fig. 1 A, left). CD4 T cells from AhR-defi - cient mice showed attenuated Th17 development, as previously described ( 3 ), and this was not infl uenced by the presence of AhR antagonist ( Fig. 1 A, right). IL-22 expression is low, but detectable, in Th17 cells from wild-type B6 mice, but abrogated in the presence of AhR antagonist and absent in Th17 cells from AhR-defi cient mice. In contrast to Th17 cells, the generation of Th1 or induced T reg (iT reg) eff ector cells was not inhibited in the presence of AhR antagonist ( Fig. 1 B ), which is in line with our fi nding that they do not express AhR ( 3 ). It has been suggested that AhR ligation by TCDD, a high affi nity, nonmetabolized AhR ligand, induces regulatory T cells ( 15, 16 ). However, it re- mains to be investigated whether the AhR ligand directly af- fected regulatory T cells, as so far only changes in percentage of T reg cells were measured under conditions in which other T cell subsets, such as Th17, underwent apoptosis; this would result in a proportional shift toward T reg cells, especially if these cells are not aff ected by AhR ligands. The presence of endogenous AhR agonists in culture medium was additionally supported by kinetic analysis of the expression of AhR and CYP1A1, which is an AhR-dependent enzyme whose ex- pression is induced upon AhR activation. Under Th17 condi- tions, AhR expression was induced  12 h after the onset of culture and, concordantly, induction of CYP1A1 expression was observed, followed by expression of IL-17 and -22 ( Fig. 1 C ). It should be emphasized that because we culture highly FACS- purifi ed naive CD4 T cells without the presence of APCs, there is no source of IL-23 in these cultures, so the induction of IL-22 is independent of this cytokine. Thus, our results indicate that there are endogenous AhR agonists in culture medium, resulting in AhR activation, which plays an important role in shaping Th17 polarization, whereas it does not play a role for Th1 or iT reg cells. The molecular mechanisms involved in AhR modulation of the Th17 program are currently not well defi ned. Our recent data indicated that AhR expression on its own does not drive expression of IL-17 or -22 ( 3 ). Furthermore, forced expres- sion of either ROR  t or AhR does not impact on the ex- pression of the other transcription factor ( Fig. 2 A ). Even Figure 2. Independent ROR  t and AhR regulation in Th17 cells . cotransduction of both transcription factors is insuffi cient to (A) FACS-sorted naive CD4 T cells retrovirally transduced with RV-GFP aff ect the characteristic Th17 modulation with IL-22 induc- control, AhR-GFP, or ROR  t-GFP vector as indicated on abscissa were tion ( Fig. 2 B ), suggesting that other pathways are involved. cultured in the presence (shaded bars) or absence (open bars) of FICZ. As a Thus, it was recently suggested that AhR interacts with Stat1 comparison, FACS-sorted naive CD4 T cells cultured under Th17 condition in the presence (shaded bars) or absence (open bars) of FICZ are shown. and Stat5 and that it may regulate Th17 development by The fi gure shows mRNA expression for the indicated genes normalized for modifying the activation of these two negative regulators of HPRT mRNA expression. The fi gure is representative of three independent Th17 generation ( 17 ). experiments. (B) FACS-sorted naive CD4 T cells were retrovirally trans- duced with either control vector containing truncated human CD4 Reduced endogenous AhR ligands in RPMI medium (RV-hCD4  cyto) together with RV-GFP control vector (top) or with We noticed in the course of experiments on Th17 polarization Ahr-hCD4  cyto together with ROR  t-GFP (bottom) and cultured in the that diff erent individuals consistently obtained diff erent levels of presence of FICZ. Expression of hCD4 versus GFP is shown on the left. Th17 polarization, despite our using the same pool of FACS- Intracellular IL-22 expression in gated populations is shown on the right. sorted naive CD4 T cells as a starting population. Eventually, it Results represent three independent experiments. JEM VOL. 206, January 19, 2009 45 became clear that some researchers used RPMI as culture me- tenuated Th17 polarization irrespective of the source of cul- dium, like most immunological laboratories, whereas IMDM, a ture medium ( Fig. 3 A ). The AhR antagonist CH-223191 richer culture medium developed by Norman Iscove at the Basel strongly reduced Th17 development from B6 CD4 T cells in Institute for Immunology ( 18 ), was typically used by former Ba- sel Institute researchers and was the medium used in the original studies showing that TGF-  and IL-6 drive Th17 diff erentiation ( 19 ). It is noteworthy that IMDM contains three- to fi vefold higher amounts of aromatic amino acids, such as tryptophan, tyrosine, and phenylalanine (Table S1, available at http://www .jem.org/cgi/content/full/jem.20081438/DC1), than RPMI medium, which could give rise to diff erent levels of endogenous AhR ligands in the two types of media. Th17 diff erentiation in RPMI medium was consistently lower and similar to that of AhR-defi cient cells that show at- Figure 3. Suboptimal Th17 differentiation in RPMI medium. (A) Scatter plots showing the percentage of IL-17 polarization from indi- vidual experiments with CD4 T cells from B6 (fi lled triangles) or AhR- defi cient 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 ex- periments are shown. (right) Percentage of IFN-  producers (fi lled dia- Figure 4. Substitution of RPMI with tryptophan improves Th17 dif- monds) or Foxp3 expression (open squares) in B6 CD4 T cells cultured ferentiation. (A) Scatter plot showing the percentage of IL-17 producers in under Th1 or iT reg cell conditions, respectively in IMDM or RPMI. P value CD4 T cells from wild-type B6 (fi lled triangles) or AhR-defi cient mice (open for iT reg cell generation in IMDM versus RPMI is P < 0.01. (B) Total CD4 circles) cultured under Th17-polarizing conditions in IMDM (left) or RPMI T cells purifi ed from PBMCs of human blood and stimulated under Th17 (right) with or without addition of 11 mg/liter of L- tryptophan. P values are conditions in IMDM (left) or RPMI (right) medium in the absence (top) or shown in the graph. Data from four independent experiments are shown. presence (bottom) of AhR antagonist. IL-17 versus IFN-  intracellular (B) IL-17/IL-22 expression in CD4 T cells cultured under Th17 conditions in staining is shown in the dot plots. The scatter plot shows the percentage either IMDM (left) or RPMI (right) in the absence or presence of AhR agonist of IL-17 producers from three different individuals (representing three FICZ after 4 d of culture or (C) in the presence of neutralizing antibodies to independent experiments) obtained in IMDM versus RPMI medium. IFN-  and IL-4. Dot plots are representative of three independent experiments. 46 AHR AGONISTS IN CULTURE MEDIUM SHAPE TH17 RESPONSE | Veldhoen et al. BRIEF DEFINITIVE REPORT IMDM, but even in RPMI medium, antagonizing AhR re- sulted in a further reduction of Th17 diff erentiation. In contrast to its eff ect on development of Th17 cells, Th1 po- larization was not aff ected by the choice of culture medium or the presence of AhR antagonist. The development of iT reg cells was even increased in RPMI medium. Human Th17 cells also express AhR ( 3 ), and, similar to mouse T cells, the expansion of human Th17 cells from total CD4 T cells in pe- ripheral blood was markedly better in IMDM compared with RPMI medium and strongly reduced in the presence of the AhR antagonist ( Fig. 3 B ). Supplementation of tryptophan partially restores Th17 polarization To test whether the increased amount of tryptophan in IMDM is involved in the enhanced Th17 polarization in IMDM ver- sus RPMI medium, we supplemented RPMI medium with l -tryptophan to adjust levels to those in IMDM. Tryptophan supplementation of RPMI signifi cantly improved Th17 po- larization, but levels were still much lower than those in IMDM. On the other hand, Th17 polarization of AhR-defi - cient cells was not infl uenced by tryptophan ( Fig. 4 A ). There could be several AhR ligands generated in IMDM, which is considerably richer in aromatic amino acids than RPMI. One of them may be derived from tryptophan, an essential amino acid that functions as the precursor to many substances, in- cluding melatonin, serotonin, or kynurenic acid. The light and culture medium – related activation of AhR has been ob- served as early as 1976 ( 20 ). More recently, it was shown that the tryptophan metabolite FICZ is produced in culture me- dium not only under UV irradiation but also under normal laboratory light conditions, and is stable during storage in the refrigerator for 3 d ( 11 ). In this respect, it is notable that the other aromatic amino acids that could be metabolic precur- sors for AhR ligands, l- tyrosine and l- phenylalanine, are also represented in substantially lower amounts in RPMI medium (Table S1). Furthermore, it has been suggested that oxidiza- tion of histidine could also generate an AhR ligand, and this amino acid is likewise underrepresented in RPMI compared with IMDM ( 21 ). However, we found that supplementation with these other components did not improve Th17 diff er- entiation beyond that seen with tryptophan supplementation (unpublished data). Thus, it appears that the relative paucity of endogenous AhR ligands only partially explains the defec- tive Th17 development in RPMI medium. We previously reported that addition of FICZ, a trypto- phan-derived AhR agonist with high affi nity, substantially Figure 5. IL-2 blockade together with AhR activation restored increases Th17 polarization and induces IL-22 production Th17 differentiation. (A) Intracellular IL-17/IL-22 in Th17 cells develop- ( 3 ). To test whether FICZ can overcome the defect in Th17 ing in IMDM (left) or RPMI medium (right) without (top) or with addition polarization in RPMI medium, we diff erentiated naive CD4 of neutralizing antibodies to IL-2 (bottom). Dot plots are representative of T cells to Th17 in the presence of FICZ. This improved 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 (represen- (C) IL-17/IL-22 expression in Th17 cells cultured in IMDM (left) or RPMI tative of three independent experiments) of gated CD4 T cells showing (right) in the presence of neutralizing antibodies to IL-2 and FICZ. Dot plots phospho-Stat5 expression at 48 h after onset of culture are shown. are representative of three independent experiments. JEM VOL. 206, January 19, 2009 47 Th17 polarization and, importantly, resulted in some IL-22 onist that is likely to play a role in vivo because exposure of expression. However, similar to tryptophan, supplementation human skin to UV-B induces CYP1A1 expression ( 23 ), but of FICZ did not fully restore the defect ( Fig. 4 B ). the number and nature of endogenous AhR agonists are still a matter of debate ( 5 ). Here, we describe that endogenous IL-2 depletion restores Th17 differentiation, but not IL-22 AhR activity present in culture medium has a strong infl u- production, in RPMI medium ence on Th17 polarization. Thus, Th17 diff erentiation repre- Because the diff erentiation of Th17 cells is strongly inhibited sents an alternative biological system in which the eff ects of by cytokines that support development of the other CD4 T potential AhR agonists or antagonists can be directly tested. cell subsets, we tested whether addition of anti – IFN-  and Whereas the assessment of CYP1A1 reporter activity in he- – IL-4, which are frequently used in Th17 polarization to in- patocyte or other cell lines, as currently used in the toxicol- hibit Th1 and Th2 polarization ( 22 ), would have a positive ogy fi eld, is a reliable and convenient readout for AhR infl uence on generation of this eff ector T cell subset. How- activity, the connection of AhR activation to the Th17 pro- ever, blockade of IFN-  and IL-4 had little eff ect on Th17 gram opens a wide range of possibilities in regard to testing diff erentiation under our culture conditions ( Fig. 4 C ). Given the infl uence of AhR agonists and antagonists on biological that our cultures contain highly purifi ed T cells without APCs, processes dependent on these cells. this is perhaps not surprising, as blocking IFN-  and IL-4 is In the immunology fi eld, obtaining improved Th17 po- eff ective mainly in mixed cultures with insuffi ciently purifi ed larization in vitro will facilitate their further molecular char- T cells that may contain Th1 or Th2 eff ector cells and residual acterization that depends on methods such as gene array APCs that may promote Th1 development via secretion of analysis or qPCR, which are not reliable unless the popula- IL-12. In contrast, neutralization of IL-2 markedly increased tions analyzed are relatively pure. Furthermore, the analysis Th17 polarization in RPMI medium to levels similar to those of human Th17 development, which can only be assessed in obtained in IMDM ( Fig. 5 A ). In contrast, blockade of IL-2 vitro, will benefi t from improved culture conditions. Our had only a marginal eff ect on Th17 diff erentiation in IMDM. data show that not all media formulations that had been used We checked IL-2 production in the two media and could not successfully over decades to support the development of CD4 fi nd a notable diff erence. Furthermore, careful analysis of IL-2 eff ector T cell subsets are conducive to Th17 diff erentiation mRNA induction and expression of CD25 did not establish through a combination of lacking AhR agonists and contain- any diff erences in the two media (unpublished data). ing components that interfere with Th17 diff erentiation. All Despite recovery of Th17 diff erentiation in RPMI by in all, our data emphasize that activation of AhR by a poten- blockade of IL-2, this was not suffi cient to rescue IL-22 ex- tially diverse range of endogenous agonists has to be consid- pression. However, IL-22 production in RPMI medium was ered an essential co-factor in the optimal diff erentiation of obtained by a combination of IL-2 neutralization and AhR Th17 eff ector T cell subset. ligation, emphasizing the important role for AhR activation MATERIALS AND METHODS in induction of IL-22 production ( Fig. 5 C ). Mice. C57BL/6 and B6 BRA AhR KO (AhR-defi cient B6) mice were bred It has been shown that Th17 diff erentiation is inhibited by and maintained under specifi c pathogen – free conditions, and all animal ex- IL-2 signaling via induction of Stat5 ( 10 ). In line with these periments were carried out according to institutional guidelines approved by fi ndings, we observed considerably more Stat5 phosphoryla- the local ethical panel and by a project licence from the UK Home Offi ce. tion during Th17 culture in RPMI medium (80.5%) com- In vitro T cell diff erentiation and intracellular staining. Naive CD4 pared with IMDM (54.3%), whereas blockade of IL-2 reduced T cells were isolated by FACS sorting using a MoFlo sorter of lymph nodes Stat5 phosphorylation and the addition of FICZ caused a re- lo  + cell suspensions for CD44 CD25 CD4 cells. The culture mediums used duction in Stat5 phosphorylation, albeit less dramatic than that were IMDM (Sigma-Aldrich) or RPMI 1640 (Sigma-Aldrich), both supple- mented with 2 × 10 M l- glutamine, 100 U/ml penicillin, 100 μ g/ml seen with anti – IL-2 ( Fig. 5 B ). It is currently unknown how streptomycin, 5 × 10 M  -mercaptoethanol, and 5% FCS. In some cases, AhR interacts with the Th17 program. We have shown that RPMI medium was supplemented with 11 mg/liter l -tryptophan (Invitro- there is no direct interaction between AhR and ROR  t gen) to adjust it to the concentrations found in IMDM. ( Fig. 2), but it is conceivable that AhR interacts with other Th17 cells were diff erentiated on plates coated with 2 μ g/ml anti-CD3 transcription factors that positively or negatively infl uence + 5 μ g/ml anti-CD28 with a cytokine cocktail of 50 ng/ml IL-6, 1 ng/ml Th17 diff erentiation. The latter is in agreement with data by TGF-  , and 10 ng/ml IL-1. Neutralizing antibodies to IFN-  , IL-4, or IL-2 were added at 10 μ g/ml in some experiments. Th1 cells were stimulated in Kimura et al. ( 17 ) showing direct interaction of AhR with the presence of 3 ng/ml IL-12, and iT reg cells were generated by adding Stat1 and Stat5 protein. Nevertheless, the interactions between 10 ng/ml TGF-  . The AhR antagonist CH-223191 (Calbiochem) was added IL-2, AhR, and Stat5 are yet to be defi ned on a molecular level. at 3 μ M at the start of culture. FICZ was added at 300 nM at the start of Collectively, Th17 development is clearly controlled not some cultures. For measurements of intracellular cytokines, T cells were re- only by multiple cytokines, but also by modulation via acti- stimulated with 500 ng/ml phorbol dibutyrate and 500 ng/ml ionomycin in vation of the AhR, whose involvement in shaping Th17 the presence of brefeldin A for 4 h on day 5 after initiation of cultures. Mea- surement of Stat5 phosphorylation was done with antibodies to pStat5 (BD) differentiation in vivo is highlighted by the phenotype of according to the manufacturer ’ s instructions. AhR-defi cient mice that show decreased IL-17 development and absence of IL-22 production ( 3 ). The tryptophan metab- qPCR analysis. RNA was extracted using 1-bromo-3-chloro-propane olite FICZ has been suggested to be an endogenous AhR ag- (Sigma-Aldrich) and reverse transcribed with oligo d(T) (Applied Biosystems) 48 AHR AGONISTS IN CULTURE MEDIUM SHAPE TH17 RESPONSE | Veldhoen et al. BRIEF DEFINITIVE REPORT according to the manufacturer ’ s protocol. The cDNA served as template for 10 . Laurence , A. , C.M. Tato , T.S. Davidson , Y. Kanno , Z. Chen , Z. Yao , the amplifi cation of genes of interest and the housekeeping gene (Hprt) by R.B. Blank , F. Meylan , R. Siegel , L. Hennighausen , E.M. Shevach , and J. O ’ Shea . 2007 . Interleukin-2 signaling via STAT5 constrains T helper real-time PCR, using TaqMan Gene Expression Assays and Applied Biosys- 17 cell generation. Immunity . 26 : 371 – 381 . tems 7900HT Fast Real-Time PCR System. The primers obtained from 11 . Oberg , M. , L. Bergander , H. Hakansson , U. Rannug , and A. Rannug . Applied Biosystems are as follows: Hprt, Mm00446968_m1; AhR, 2005 . Identifi cation of the tryptophan photoproduct 6-formylin- Mm00478930_ml; IL-17A, Mm00439619_m1; IL-22, Mm00444241_m1; dolo[3,2-b]carbazole, in cell culture medium, as a factor that con- CYP1A1, Mm00487217_m1. trols the background aryl hydrocarbon receptor activity. Toxicol. Sci. 85 : 935 – 943 . Human Th17 polarization. CD4 T cells from PBMCs were purifi ed by 12 . Amakura , Y. , T. Tsutsumi , K. Sasaki , T. Yoshida , and T. Maitani . 2003 . magnetic cell sorting and cultured on plates coated with 1 μ g/ml anti-CD3 Screening of the inhibitory eff ect of vegetable constituents on the aryl and 2.5 μ g/ml anti-CD28 in either IMDM or RPMI supplemented with hydrocarbon receptor-mediated activity induced by 2,3,7,8-tetrachlo- 10% serum replacement factor (Invitrogen) in the presence of 0.5 ng/ml rodibenzo-p-dioxin. Biol. Pharm. Bull. 26 : 1754 – 1760 . TGF-  , 30 ng/ml IL-6, 10 ng/ml IL-1  , and 10 ng/ml IL-23. 13 . Zhang , S. , C. Qin , and S.H. Safe . 2003 . Flavonoids as aryl hydrocar- bon receptor agonists/antagonists: eff ects of structure and cell context. Statistics. P values were determined using two-tailed Student ’ s t test. Environ. Health Perspect. 111 : 1877 – 1882 . 14 . Kim , S.H. , E.C. Henry , D.K. Kim , Y.H. Kim , K.J. Shin , M.S. Han , Online supplemental material. Table S1 is a comparison of the amino T.G. Lee , J.K. Kang , T.A. Gasiewicz , S.H. Ryu , and P.G. Suh . 2006 . acid content in IMDM and RPMI. Online supplemental material is avail- Novel compound 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl- able at http://www.jem.org/cgi/content/full/jem.20081438/DC1. 4-o-tolylazo-phenyl)-amide (CH-223191) prevents 2,3,7,8-TCDD- induced toxicity by antagonizing the aryl hydrocarbon receptor. Mol. We are grateful to George Kassiotis for comments on the manuscript. Pharmacol. 69 : 1871 – 1878 . This work was funded by the Medical Research Council UK. 15 . Quintana , F.J. , A.S. Basso , A.H. Iglesias , T. Korn , M.F. Farez , E. The authors have no confl icting fi nancial interests. Bettelli , M. Caccamo , M. Oukka , and H.L. Weiner . 2008 . Control of T(reg) and T(H)17 cell diff erentiation by the aryl hydrocarbon receptor. Submitted: 3 July 2008 Accepted: 3 December 2008 Nature . 453 : 65 – 71 . 16 . Funatake , C.J. , N.B. Marshall , L.B. 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Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal differentiation of Th17 T cells

Veldhoen, Marc; Hirota, Keiji; Christensen, Jillian; O'Garra, Anne; Stockinger, Brigitta
The Journal of Experimental Medicine , Volume 206 (1) – Jan 19, 2009
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Pubmed Central
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© 2009 Veldhoen et al.
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0022-1007
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1540-9538
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10.1084/jem.20081438
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Abstract

BRIEF DEFINITIVE REPORT Natural agonists for aryl hydrocarbon receptor in culture medium are essential for optimal diff erentiation of Th17 T cells 1 1 2 2 Marc Veldhoen , Keiji Hirota , Jillian Christensen , Anne O ’ Garra , and Brigitta Stockinger 1 2 Division of Molecular Immunology and Division of Immunoregulation, Medical Research Council National Institute for Medical Research, London NW7 1AA, England, UK Th17 cell differentiation is dependent on interleukin (IL)-6 and transforming growth factor (TGF)-  , 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 devel- opment from wild-type mice is suboptimal in the presence of the AhR antagonist CH- 223191, similar to the situation in AhR-defi cient 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 activa- tion. However, the most commonly used medium, RPMI, supports very low levels of Th17 polarization, whereas Iscove ’ s modifi ed 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. Diff erentiation of the new CD4 eff ector subset it induces expression of IL-22 ( 3 ). Despite the CORRESPONDENCE Brigitta Stockinger: Th17 requires the presence of IL-6 and TGF-  fact that AhR binds many toxins, its evolution- [email protected] and is further enhanced by IL-1  and -21 ( 1, 2 ). ary conservation ( 8 ) suggests that this is not its In addition, Th17 polarization is promoted by primary function. Although invertebrate AhR stimulation of the aryl hydrocarbon receptor shares high protein sequence homology with (AhR) ( 3 ), a ligand-dependent transcription the vertebrate receptor, it does not recognize factor that responds to a wide range of ligands. xenobiotic ligands such as TCDD or  -naph- Ligands include environmental toxins, such as thofl avone, which indicates that the AhR has halogenated aromatic hydrocarbons represented physiological functions that are not restricted to by tetrachlorodibenzo- p -dioxin (TCDD) and recognition of environmental pollutants ( 5 ). polycyclic aromatic hydrocarbons (e.g., 3-meth- Involvement of an endogenous AhR ligand ylcholanthrene), as well as potential endogenous in Th17 development in vivo is suggested by the ligands, including dietary components, heme fact that AhR-defi cient mice have an attenuated metabolites, indigoids, and tryptophan metabo- Th17 program, with fewer Th17 cells acti- lites ( 4 ). The tryptophan metabolite 6-formylin- vated upon immunization with MOG peptide/ dolo[3,2-b] carbazole (FICZ) was shown to CFA, resulting in milder pathology in the EAE have very high affi nity for AhR, comparable to model and the absence of IL-22 production (3 ). that of TCDD ( 5 – 7 ). Exposure of CD4 T cells to FICZ under Th17-polarizing conditions en- © 2009 Veldhoen et al. This article is distributed under the terms of an Attribu- tion–Noncommercial–Share Alike–No Mirror Sites license for the fi rst six months hances Th17 development and functions such after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncom- as autoimmune pathology in EAE, which is mercial–Share Alike 3.0 Unported license, as described at http://creativecommons substantially exacerbated by AhR ligation, and .org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Exp. Med. Vol. 206 No. 1 43-49 43 www.jem.org/cgi/doi/10.1084/jem.20081438 The Journal of Experimental Medicine The attenuated Th17 development from naive CD4 T cell tions show increased Stat5 phosphorylation. Given that IL-2 precursors of AhR-defi cient mice is also noticeable in vitro. dampens Th17 diff erentiation via the induction of Stat5 ( 10 ), There are several studies in the toxicology fi eld suggest- it is not surprising that Th17 diff erentiation is diffi cult to ob- ing that high affi nity ligands for AhR are generated in culture tain in this medium. Our results emphasize the important medium and play a role in the baseline activation of AhR in role AhR agonists play in the modulation of the Th17 pro- hepatocyte cell lines, resulting in activation of genes encod- gram and illustrate that diff erentiation of this eff ector T cell ing xenobiotic metabolizing cytochrome P450 enzymes such subset is inhibited by factors that are conducive to generation as CYP1A (for review see reference [ 9 ]). These findings of the other T cell subsets, a fact that is refl ected in the for- prompted us to test whether such endogenous AhR activity mulation of some culture media that support Th1, Th2, and might infl uence the diff erentiation of Th17 eff ector cells in regulatory T (T reg) cell diff erentiation, but lead to ineffi - vitro. Indeed, our studies show that addition of an AhR an- cient Th17 development. tagonist during Th17 polarization decreases Th17 polariza- tion, whereas it does not infl uence diff erentiation of other RESULTS AND DISCUSSION eff ector T cell subsets. Interestingly, we also found that there AhR antagonist CH-223191 impairs Th17 differentiation are substantial diff erences in regard to generation of endoge- Because tryptophan metabolites generated by light exposure, nous AhR ligands in diff erent culture media, with RPMI such as FICZ, are potent enhancers of Th17 diff erentiation and having far less activity than IMDM. This could explain the there are reports suggesting that light exposure of culture me- puzzling variability in the literature in regard to Th17 polar- dium generates AhR ligands that contribute to background ization, which is generally in the range of 5 – 20%, whereas AhR activation ( 11 ), we set out to assess Th17 development our values usually range from 40 – 60%. RPMI is by far the from naive precursors in the absence or presence of an AhR an- most commonly used culture medium, but as we show here, tagonist. Several AhR antagonists found in food components, IMDM supports better Th17 polarization from both mouse such as fl avones and resveratrol have been previously described and human CD4 T cells. We also observed that in RPMI, ( 12 ), but all of them seem to exert AhR agonist activity at high but not IMDM, CD4 T cells cultured under Th17 condi- concentrations ( 13 ). In contrast, screening of a chemical library Figure 1. Increased Th17 polarization caused by endogenous AhR ligands. (A) Intracellular staining for IL-17 versus IL-22 of CD4 T cells from con- trol B6 (top) or AhR-defi cient (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-  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 expres- sion after activation of naive CD4 T cells under Th17 conditions. Expression in CD4 T cells activated under neutral conditions is shown for comparison. The fi gure shows mRNA levels normalized to Hprt expression and is representative of three independent experiments. 44 AHR AGONISTS IN CULTURE MEDIUM SHAPE TH17 RESPONSE | Veldhoen et al. BRIEF DEFINITIVE REPORT identifi ed CH-223191 as an antagonist that lacked agonist activ- ity even at high doses ( 14 ). Th17 diff erentiation of naive CD4 T cells from B6 mice was markedly inhibited in the presence of the AhR antagonist ( Fig. 1 A, left). CD4 T cells from AhR-defi - cient mice showed attenuated Th17 development, as previously described ( 3 ), and this was not infl uenced by the presence of AhR antagonist ( Fig. 1 A, right). IL-22 expression is low, but detectable, in Th17 cells from wild-type B6 mice, but abrogated in the presence of AhR antagonist and absent in Th17 cells from AhR-defi cient mice. In contrast to Th17 cells, the generation of Th1 or induced T reg (iT reg) eff ector cells was not inhibited in the presence of AhR antagonist ( Fig. 1 B ), which is in line with our fi nding that they do not express AhR ( 3 ). It has been suggested that AhR ligation by TCDD, a high affi nity, nonmetabolized AhR ligand, induces regulatory T cells ( 15, 16 ). However, it re- mains to be investigated whether the AhR ligand directly af- fected regulatory T cells, as so far only changes in percentage of T reg cells were measured under conditions in which other T cell subsets, such as Th17, underwent apoptosis; this would result in a proportional shift toward T reg cells, especially if these cells are not aff ected by AhR ligands. The presence of endogenous AhR agonists in culture medium was additionally supported by kinetic analysis of the expression of AhR and CYP1A1, which is an AhR-dependent enzyme whose ex- pression is induced upon AhR activation. Under Th17 condi- tions, AhR expression was induced  12 h after the onset of culture and, concordantly, induction of CYP1A1 expression was observed, followed by expression of IL-17 and -22 ( Fig. 1 C ). It should be emphasized that because we culture highly FACS- purifi ed naive CD4 T cells without the presence of APCs, there is no source of IL-23 in these cultures, so the induction of IL-22 is independent of this cytokine. Thus, our results indicate that there are endogenous AhR agonists in culture medium, resulting in AhR activation, which plays an important role in shaping Th17 polarization, whereas it does not play a role for Th1 or iT reg cells. The molecular mechanisms involved in AhR modulation of the Th17 program are currently not well defi ned. Our recent data indicated that AhR expression on its own does not drive expression of IL-17 or -22 ( 3 ). Furthermore, forced expres- sion of either ROR  t or AhR does not impact on the ex- pression of the other transcription factor ( Fig. 2 A ). Even Figure 2. Independent ROR  t and AhR regulation in Th17 cells . cotransduction of both transcription factors is insuffi cient to (A) FACS-sorted naive CD4 T cells retrovirally transduced with RV-GFP aff ect the characteristic Th17 modulation with IL-22 induc- control, AhR-GFP, or ROR  t-GFP vector as indicated on abscissa were tion ( Fig. 2 B ), suggesting that other pathways are involved. cultured in the presence (shaded bars) or absence (open bars) of FICZ. As a Thus, it was recently suggested that AhR interacts with Stat1 comparison, FACS-sorted naive CD4 T cells cultured under Th17 condition in the presence (shaded bars) or absence (open bars) of FICZ are shown. and Stat5 and that it may regulate Th17 development by The fi gure shows mRNA expression for the indicated genes normalized for modifying the activation of these two negative regulators of HPRT mRNA expression. The fi gure is representative of three independent Th17 generation ( 17 ). experiments. (B) FACS-sorted naive CD4 T cells were retrovirally trans- duced with either control vector containing truncated human CD4 Reduced endogenous AhR ligands in RPMI medium (RV-hCD4  cyto) together with RV-GFP control vector (top) or with We noticed in the course of experiments on Th17 polarization Ahr-hCD4  cyto together with ROR  t-GFP (bottom) and cultured in the that diff erent individuals consistently obtained diff erent levels of presence of FICZ. Expression of hCD4 versus GFP is shown on the left. Th17 polarization, despite our using the same pool of FACS- Intracellular IL-22 expression in gated populations is shown on the right. sorted naive CD4 T cells as a starting population. Eventually, it Results represent three independent experiments. JEM VOL. 206, January 19, 2009 45 became clear that some researchers used RPMI as culture me- tenuated Th17 polarization irrespective of the source of cul- dium, like most immunological laboratories, whereas IMDM, a ture medium ( Fig. 3 A ). The AhR antagonist CH-223191 richer culture medium developed by Norman Iscove at the Basel strongly reduced Th17 development from B6 CD4 T cells in Institute for Immunology ( 18 ), was typically used by former Ba- sel Institute researchers and was the medium used in the original studies showing that TGF-  and IL-6 drive Th17 diff erentiation ( 19 ). It is noteworthy that IMDM contains three- to fi vefold higher amounts of aromatic amino acids, such as tryptophan, tyrosine, and phenylalanine (Table S1, available at http://www .jem.org/cgi/content/full/jem.20081438/DC1), than RPMI medium, which could give rise to diff erent levels of endogenous AhR ligands in the two types of media. Th17 diff erentiation in RPMI medium was consistently lower and similar to that of AhR-defi cient cells that show at- Figure 3. Suboptimal Th17 differentiation in RPMI medium. (A) Scatter plots showing the percentage of IL-17 polarization from indi- vidual experiments with CD4 T cells from B6 (fi lled triangles) or AhR- defi cient 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 ex- periments are shown. (right) Percentage of IFN-  producers (fi lled dia- Figure 4. Substitution of RPMI with tryptophan improves Th17 dif- monds) or Foxp3 expression (open squares) in B6 CD4 T cells cultured ferentiation. (A) Scatter plot showing the percentage of IL-17 producers in under Th1 or iT reg cell conditions, respectively in IMDM or RPMI. P value CD4 T cells from wild-type B6 (fi lled triangles) or AhR-defi cient mice (open for iT reg cell generation in IMDM versus RPMI is P < 0.01. (B) Total CD4 circles) cultured under Th17-polarizing conditions in IMDM (left) or RPMI T cells purifi ed from PBMCs of human blood and stimulated under Th17 (right) with or without addition of 11 mg/liter of L- tryptophan. P values are conditions in IMDM (left) or RPMI (right) medium in the absence (top) or shown in the graph. Data from four independent experiments are shown. presence (bottom) of AhR antagonist. IL-17 versus IFN-  intracellular (B) IL-17/IL-22 expression in CD4 T cells cultured under Th17 conditions in staining is shown in the dot plots. The scatter plot shows the percentage either IMDM (left) or RPMI (right) in the absence or presence of AhR agonist of IL-17 producers from three different individuals (representing three FICZ after 4 d of culture or (C) in the presence of neutralizing antibodies to independent experiments) obtained in IMDM versus RPMI medium. IFN-  and IL-4. Dot plots are representative of three independent experiments. 46 AHR AGONISTS IN CULTURE MEDIUM SHAPE TH17 RESPONSE | Veldhoen et al. BRIEF DEFINITIVE REPORT IMDM, but even in RPMI medium, antagonizing AhR re- sulted in a further reduction of Th17 diff erentiation. In contrast to its eff ect on development of Th17 cells, Th1 po- larization was not aff ected by the choice of culture medium or the presence of AhR antagonist. The development of iT reg cells was even increased in RPMI medium. Human Th17 cells also express AhR ( 3 ), and, similar to mouse T cells, the expansion of human Th17 cells from total CD4 T cells in pe- ripheral blood was markedly better in IMDM compared with RPMI medium and strongly reduced in the presence of the AhR antagonist ( Fig. 3 B ). Supplementation of tryptophan partially restores Th17 polarization To test whether the increased amount of tryptophan in IMDM is involved in the enhanced Th17 polarization in IMDM ver- sus RPMI medium, we supplemented RPMI medium with l -tryptophan to adjust levels to those in IMDM. Tryptophan supplementation of RPMI signifi cantly improved Th17 po- larization, but levels were still much lower than those in IMDM. On the other hand, Th17 polarization of AhR-defi - cient cells was not infl uenced by tryptophan ( Fig. 4 A ). There could be several AhR ligands generated in IMDM, which is considerably richer in aromatic amino acids than RPMI. One of them may be derived from tryptophan, an essential amino acid that functions as the precursor to many substances, in- cluding melatonin, serotonin, or kynurenic acid. The light and culture medium – related activation of AhR has been ob- served as early as 1976 ( 20 ). More recently, it was shown that the tryptophan metabolite FICZ is produced in culture me- dium not only under UV irradiation but also under normal laboratory light conditions, and is stable during storage in the refrigerator for 3 d ( 11 ). In this respect, it is notable that the other aromatic amino acids that could be metabolic precur- sors for AhR ligands, l- tyrosine and l- phenylalanine, are also represented in substantially lower amounts in RPMI medium (Table S1). Furthermore, it has been suggested that oxidiza- tion of histidine could also generate an AhR ligand, and this amino acid is likewise underrepresented in RPMI compared with IMDM ( 21 ). However, we found that supplementation with these other components did not improve Th17 diff er- entiation beyond that seen with tryptophan supplementation (unpublished data). Thus, it appears that the relative paucity of endogenous AhR ligands only partially explains the defec- tive Th17 development in RPMI medium. We previously reported that addition of FICZ, a trypto- phan-derived AhR agonist with high affi nity, substantially Figure 5. IL-2 blockade together with AhR activation restored increases Th17 polarization and induces IL-22 production Th17 differentiation. (A) Intracellular IL-17/IL-22 in Th17 cells develop- ( 3 ). To test whether FICZ can overcome the defect in Th17 ing in IMDM (left) or RPMI medium (right) without (top) or with addition polarization in RPMI medium, we diff erentiated naive CD4 of neutralizing antibodies to IL-2 (bottom). Dot plots are representative of T cells to Th17 in the presence of FICZ. This improved 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 (represen- (C) IL-17/IL-22 expression in Th17 cells cultured in IMDM (left) or RPMI tative of three independent experiments) of gated CD4 T cells showing (right) in the presence of neutralizing antibodies to IL-2 and FICZ. Dot plots phospho-Stat5 expression at 48 h after onset of culture are shown. are representative of three independent experiments. JEM VOL. 206, January 19, 2009 47 Th17 polarization and, importantly, resulted in some IL-22 onist that is likely to play a role in vivo because exposure of expression. However, similar to tryptophan, supplementation human skin to UV-B induces CYP1A1 expression ( 23 ), but of FICZ did not fully restore the defect ( Fig. 4 B ). the number and nature of endogenous AhR agonists are still a matter of debate ( 5 ). Here, we describe that endogenous IL-2 depletion restores Th17 differentiation, but not IL-22 AhR activity present in culture medium has a strong infl u- production, in RPMI medium ence on Th17 polarization. Thus, Th17 diff erentiation repre- Because the diff erentiation of Th17 cells is strongly inhibited sents an alternative biological system in which the eff ects of by cytokines that support development of the other CD4 T potential AhR agonists or antagonists can be directly tested. cell subsets, we tested whether addition of anti – IFN-  and Whereas the assessment of CYP1A1 reporter activity in he- – IL-4, which are frequently used in Th17 polarization to in- patocyte or other cell lines, as currently used in the toxicol- hibit Th1 and Th2 polarization ( 22 ), would have a positive ogy fi eld, is a reliable and convenient readout for AhR infl uence on generation of this eff ector T cell subset. How- activity, the connection of AhR activation to the Th17 pro- ever, blockade of IFN-  and IL-4 had little eff ect on Th17 gram opens a wide range of possibilities in regard to testing diff erentiation under our culture conditions ( Fig. 4 C ). Given the infl uence of AhR agonists and antagonists on biological that our cultures contain highly purifi ed T cells without APCs, processes dependent on these cells. this is perhaps not surprising, as blocking IFN-  and IL-4 is In the immunology fi eld, obtaining improved Th17 po- eff ective mainly in mixed cultures with insuffi ciently purifi ed larization in vitro will facilitate their further molecular char- T cells that may contain Th1 or Th2 eff ector cells and residual acterization that depends on methods such as gene array APCs that may promote Th1 development via secretion of analysis or qPCR, which are not reliable unless the popula- IL-12. In contrast, neutralization of IL-2 markedly increased tions analyzed are relatively pure. Furthermore, the analysis Th17 polarization in RPMI medium to levels similar to those of human Th17 development, which can only be assessed in obtained in IMDM ( Fig. 5 A ). In contrast, blockade of IL-2 vitro, will benefi t from improved culture conditions. Our had only a marginal eff ect on Th17 diff erentiation in IMDM. data show that not all media formulations that had been used We checked IL-2 production in the two media and could not successfully over decades to support the development of CD4 fi nd a notable diff erence. Furthermore, careful analysis of IL-2 eff ector T cell subsets are conducive to Th17 diff erentiation mRNA induction and expression of CD25 did not establish through a combination of lacking AhR agonists and contain- any diff erences in the two media (unpublished data). ing components that interfere with Th17 diff erentiation. All Despite recovery of Th17 diff erentiation in RPMI by in all, our data emphasize that activation of AhR by a poten- blockade of IL-2, this was not suffi cient to rescue IL-22 ex- tially diverse range of endogenous agonists has to be consid- pression. However, IL-22 production in RPMI medium was ered an essential co-factor in the optimal diff erentiation of obtained by a combination of IL-2 neutralization and AhR Th17 eff ector T cell subset. ligation, emphasizing the important role for AhR activation MATERIALS AND METHODS in induction of IL-22 production ( Fig. 5 C ). Mice. C57BL/6 and B6 BRA AhR KO (AhR-defi cient B6) mice were bred It has been shown that Th17 diff erentiation is inhibited by and maintained under specifi c pathogen – free conditions, and all animal ex- IL-2 signaling via induction of Stat5 ( 10 ). In line with these periments were carried out according to institutional guidelines approved by fi ndings, we observed considerably more Stat5 phosphoryla- the local ethical panel and by a project licence from the UK Home Offi ce. tion during Th17 culture in RPMI medium (80.5%) com- In vitro T cell diff erentiation and intracellular staining. Naive CD4 pared with IMDM (54.3%), whereas blockade of IL-2 reduced T cells were isolated by FACS sorting using a MoFlo sorter of lymph nodes Stat5 phosphorylation and the addition of FICZ caused a re- lo  + cell suspensions for CD44 CD25 CD4 cells. The culture mediums used duction in Stat5 phosphorylation, albeit less dramatic than that were IMDM (Sigma-Aldrich) or RPMI 1640 (Sigma-Aldrich), both supple- mented with 2 × 10 M l- glutamine, 100 U/ml penicillin, 100 μ g/ml seen with anti – IL-2 ( Fig. 5 B ). It is currently unknown how streptomycin, 5 × 10 M  -mercaptoethanol, and 5% FCS. In some cases, AhR interacts with the Th17 program. We have shown that RPMI medium was supplemented with 11 mg/liter l -tryptophan (Invitro- there is no direct interaction between AhR and ROR  t gen) to adjust it to the concentrations found in IMDM. ( Fig. 2), but it is conceivable that AhR interacts with other Th17 cells were diff erentiated on plates coated with 2 μ g/ml anti-CD3 transcription factors that positively or negatively infl uence + 5 μ g/ml anti-CD28 with a cytokine cocktail of 50 ng/ml IL-6, 1 ng/ml Th17 diff erentiation. The latter is in agreement with data by TGF-  , and 10 ng/ml IL-1. Neutralizing antibodies to IFN-  , IL-4, or IL-2 were added at 10 μ g/ml in some experiments. Th1 cells were stimulated in Kimura et al. ( 17 ) showing direct interaction of AhR with the presence of 3 ng/ml IL-12, and iT reg cells were generated by adding Stat1 and Stat5 protein. Nevertheless, the interactions between 10 ng/ml TGF-  . The AhR antagonist CH-223191 (Calbiochem) was added IL-2, AhR, and Stat5 are yet to be defi ned on a molecular level. at 3 μ M at the start of culture. FICZ was added at 300 nM at the start of Collectively, Th17 development is clearly controlled not some cultures. For measurements of intracellular cytokines, T cells were re- only by multiple cytokines, but also by modulation via acti- stimulated with 500 ng/ml phorbol dibutyrate and 500 ng/ml ionomycin in vation of the AhR, whose involvement in shaping Th17 the presence of brefeldin A for 4 h on day 5 after initiation of cultures. Mea- surement of Stat5 phosphorylation was done with antibodies to pStat5 (BD) differentiation in vivo is highlighted by the phenotype of according to the manufacturer ’ s instructions. AhR-defi cient mice that show decreased IL-17 development and absence of IL-22 production ( 3 ). The tryptophan metab- qPCR analysis. RNA was extracted using 1-bromo-3-chloro-propane olite FICZ has been suggested to be an endogenous AhR ag- (Sigma-Aldrich) and reverse transcribed with oligo d(T) (Applied Biosystems) 48 AHR AGONISTS IN CULTURE MEDIUM SHAPE TH17 RESPONSE | Veldhoen et al. BRIEF DEFINITIVE REPORT according to the manufacturer ’ s protocol. 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Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Jan 19, 2009

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