The AHR is a ligand-dependent transcription factor that mediates a range of critical cellular events in response to halogenated aromatic hydrocarbons and non-halogenated polycyclic aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR expression is ubiquitous in vertebrate cells, suggesting important and widespread roles, but the physiological role of AHR is not yet understood. Mice with a targeted mutation of the Ahr gene provided unequivocal evidence that the AHR is crucial to TCDD-induced toxicity and suggested a function for AHR in liver development.

Gene array analysis of CD4+ effector T-cell subsets showed that the T_H17 CD4+ T-cell subset, in addition to the lineage-defining transcription factor Rorc (encoding ROR-&ggr;t, an isoform of ROR-&ggr; expressed in the thymus), expresses Ahr. Quantitative polymerase chain reaction (PCR) analysis of CD4+ effector T-cell subsets from wild-type and AHR-deficient C57BL/6 (B6) mice established that the lineage-defining transcription factors T-bet, Gata3 and Rorc are expressed in a comparable manner as are the marker cytokines interferon (IFN)-&ggr;, IL-4, IL-17A and IL-17F (Fig. 1a). There is also similar Foxp3 expression in CD4+ T cells from B6 and AHR-deficient mice (Supplementary Fig. 1b). AHR was only induced under T_H17-cell-inducing conditions (IL-6 and transforming growth factor (TGF)-&bgr;) with levels of expression similar to liver (Fig. 1a, d). Under our conditions we found no AHR expression in cultures with TGF-&bgr; alone (induced T-regulatory cells; iT_reg) or IL-6 alone and no expression in natural T_reg cells (Supplementary Fig. 1a). Notably, AHR expression is also found in human T_H17 cells (Fig. 1b).

Although CD4+ T cells from AHR-deficient mice could differentiate into T_H17 cells, they lacked the expression of IL-22 (Fig. 1c). IL-22, originally defined as a hepatocyte-stimulating factor, is co-expressed with IL-17 by T_H17 T cells and its expression is thought to be enhanced by dendritic-cell-derived IL-23 (refs 8, 9). The biological functions of IL-22 are not fully understood: on the one hand IL-22 seems to be pro-inflammatory, inducing dermal inflammation and pro-inflammatory gene expression in the skin, on the other hand IL-22 delivery ameliorates T-cell-mediated liver injury in T-cell-mediated hepatitis.

AHR resides in the cytoplasm in complex with Hsp90 until binding of ligand triggers conformational changes resulting in an exchange of Hsp90 for the nuclear translocation component ARNT (reviewed in ref. 1). Arnt was found expressed in all CD4+ T-cell subsets (data not shown). Ligation of AHR by 6-formylindolo[3,2-b]carbazole (FICZ), a tryptophan-derived photoproduct that is thought to be an endogenous ligand with high affinity for the AHR receptor, upregulates genes encoding xenobiotic metabolizing cytochrome P450 enzymes such as Cyp1a1 (ref. 13).

To test whether exposure of T cells to FICZ influences differentiation of naive CD4+ T cells to effector cells, we added FICZ during the in vitro differentiation of CD4+ effector T-cell subsets. The addition of FICZ did not induce Ahr expression or its downstream target Cyp1a1 in T_H0, T_H1, T_H2 and iT_reg T-cell subsets (Fig. 2a) and did not alter their expression of Ifng, Il4, Il21 nor their lineage-defining transcription factors (Supplementary Fig. 1c). However, the presence of FICZ during T_H17-cell-inducing conditions led to strong upregulation of Il17a, Il17f and particularly of Il22 mRNA expression (Fig. 2a and Supplementary Fig. 2a).

Comparison of T_H17 differentiation in CD4+ T cells from wild-type B6 and AHR-deficient mice by intracellular staining showed that exposure of B6 CD4+ T cells to FICZ under T_H17-cell-inducing conditions strongly enhanced IL-17A and IL-17F production and increased the proportion and staining intensity of cells producing IL-22 (Fig. 2b, top panels and Supplementary Fig. 2b). In contrast, IL-17A and IL-17F production was attenuated in T_H17 cells from AHR-deficient mice and no IL-22 was detectable whether FICZ was present or not (Fig. 2b, bottom panels). A similar response was seen with another AHR ligand, &bgr;-naphthoflavone, which has lower affinity for AHR and consequently requires about a 10-fold higher dose compared with FICZ (Supplementary Fig. 2c). Similar to mouse, human T_H17 cells reacted to AHR ligation with increased expression of IL17A, IL17F and IL22 as well as induction of CYP1A1 (Fig. 2c).

To test whether Ahr expression on its own is essential and sufficient to drive IL-22 expression, we performed retroviral transduction of sorted naive AHR-deficient CD4+ T cells with an AHR–green fluorescent protein (GFP) construct or a GFP vector control construct. Transduction under neutral, T_H1, T_H2 or iT_reg conditions did not reconstitute IL-22 expression even in the presence of FICZ (Supplementary Fig. 3). However, under T_H17-cell-inducing conditions, reconstitution of AHR expression by retroviral transduction induced expression of IL-22 (Fig. 3a, top-right panel) and increased the proportion of IL-17-producing cells (Fig. 3b, top-right panel). Exposure to FICZ resulted in a substantial increase in IL-22 (Fig. 3a, bottom-right panel) as well as the enhanced expression of IL-17A (Fig. 3b, bottom right panel). PCR with reverse transcription (RT–PCR) analysis of cultured Ahr-transduced T_H17 cells confirmed the increase in Il17 expression, the enhancement of Il22 as well as the induction of the Ahr target Cyp1a1 on exposure to FICZ (Fig. 3c). Thus, Ahr expression is only functional in CD4+ T cells that have differentiated to the T_H17 lineage.

Next we immunized B6 mice with myelin oligodendrocyte peptide 35–55 (MOG_35–55) in complete Freund’s adjuvant (CFA), removed draining lymph nodes 7 days later and isolated CCR6+CD4+ T cells (which are enriched in T_H17 cells) to test whether Ahr is also expressed in T_H17 cells generated in vivo. CCR6+CD4+ T cells expressed Ahr, Il17a, Il17f, Il22 and Rorc, whereas the CCR6-CD4+ T-cell fraction lacked expression of Ahr and T_H17 markers, confirming that physiological differentiation in vivo recapitulates in vitro differentiation (Fig. 4a).

T_H17 T cells have a prominent role in the pathology of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE), which is induced by immunization with MOG peptide 35–55 and CFA. Analysis of spinal cord at the height of the EAE response on day 18 showed increased numbers of CD4+ T cells producing IL-17 and IL-22 in B6 mice treated with MOG_35–55/CFA and FICZ, whereas AHR-deficient mice showed reduced numbers of IL-17-producing T cells and minimal numbers of IL-22 producers (Fig. 4b). There was no difference in the numbers of Foxp3-expressing regulatory T cells in spinal cord in the three groups of mice.

Whereas B6 mice developed EAE with a mean day of onset of 13.9, AHR-deficient mice developed EAE with delayed kinetics (mean day of onset 15.6) in line with the attenuated T_H17 differentiation seen in vitro. Despite the delayed onset of EAE, most AHR-deficient mice succumbed to disease eventually (Fig. 4c, e). Stimulation of AHR by inclusion of FICZ in the antigen emulsion accelerated the onset (day 11.7) and increased the severity of EAE in B6 mice, but, as expected, did not influence the onset or severity of EAE in AHR-deficient mice (day 15.8) (Fig. 4c, e). To assess the influence of AHR deficiency in haematopoietic or non-haematopoietic cells, we also induced EAE in chimaeras constructed either by injection of AHR-deficient bone marrow into irradiated wild-type mice (AHR-B6) or by injection of B6 wild-type bone marrow into irradiated AHR-deficient mice (B6-AHR). AHR-B6 chimaeras showed attenuated EAE like AHR-deficient mice, whereas B6-AHR chimaeras developed EAE with kinetics and severity similar to wild-type mice (Fig. 4d).

Studies on the effect of AHR stimulation on the immune system have so far focused exclusively on TCDD as a ligand because of its toxicological relevance. Although adverse effects of TCDD on immune responses are well documented, no direct measurements of AHR expression on highly purified polarized subsets of CD4+ T cells have been reported. TCDD, which cannot be metabolized and therefore causes prolonged stimulation of AHR in many cells of the body, is known to induce profound suppression of immune responses in wild-type, but not AHR-deficient, mice, but despite decades of research the underlying mechanisms for this profound toxicity remain unclear. More recently, it has been suggested that TCDD promotes the generation of regulatory T cells while causing the premature decline of activated CD4+ T cells. In our hands, stimulation of AHR by FICZ did not influence the number of regulatory T cells during MOG_35–55-induced EAE responses (see Fig. 4b). It is conceivable that the high toxicity of TCDD for effector cells causes a proportional shift in regulatory T cells—which are, in general, more resistant to many depletion regimes—due to the death of other cells, rather than an actual expansion in numbers. Although TCDD may be the classical AHR ligand used in the toxicology field to analyse the effects of AHR activation, it is clear that it is not a natural ligand and it is widely recognized that pollutants such as TCDD are unlikely to have provided the evolutionary pressure for the function of this highly conserved system. There is ongoing debate in the toxicology field about what is the most relevant physiological ligand for AHR. Nevertheless, compelling indirect evidence shows that ultraviolet photoproducts of tryptophan, such as the high-affinity ligand FICZ studied in our paper, could be synthesized in vivo as exposure of human skin to ultraviolet light induces CYP1A1 (ref. 21). Future studies focusing on additional physiological ligands of AHR that can be metabolized by the CYP1 enzymes and therefore cause only transient AHR activation may give further insights into the consequences of AHR stimulation in T_H17 cells.

Our data show that AHR, in addition to promoting the expression of IL-22, enhances T_H17 cell development and the expression levels of IL-17A and IL-17F, and consequently increases autoimmune pathology. Blockade of IL-17A with an auto-vaccine as well as neutralizing antibody can completely prevent the development of EAE, emphasizing the central role of IL-17A in the pathogenesis of this autoimmune disease. Although it has been suggested that IL-22 may contribute to autoimmune pathology because, like IL-17, it disrupts blood–brain barrier tight junctions, IL-22-deficient mice do not seem to have altered susceptibility to EAE induction, suggesting that this cytokine may be dispensable for the development of pathology in EAE. Thus, the enhancement of IL-17 production by AHR ligation may be more crucial than the induction of IL-22 in determining disease severity.

It is currently thought that IL-22 expression depends on induction by IL-23 (refs 8, 9). Re-stimulation of lymph node cells from mice immunized with MOG_35–55/CFA or MOG_35–55/CFA and FICZ in the presence or absence of IL-23 showed that IL-23 increased the proportion of IL-17-producing cells both from wild-type and AHR-deficient mice (Supplementary Fig. 4, left), but only had a small effect on the production of IL-22 (Supplementary Fig. 4b, right), suggesting that IL-23 has some effect independent of AHR ligation, although clearly stimulation of AHR seems to be the dominant pathway for IL-22 production.

How AHR interacts with the T_H17 pathway is currently unknown, but there is a substantial amount of literature (reviewed in ref. 26) describing interactions of AHR with other key regulatory proteins including nuclear factor-&kgr;B, which has a role in the induction of EAE. The core nucleotide sequence to which the nuclear AHR complex binds, also termed the ‘xenobiotic responsive element’, occurs frequently in the mammalian genome and is also represented in IL-17A, IL-17F, IL-22 and ROR-&ggr;t. Although basal expression of AHR and IL-22 is detectable in T_H17 T cells in the apparent absence of a ligand, there are numerous endogenous agents that can activate AHR, such as prostaglandins, bilirubin at high concentration, modified low-density lipoprotein and various modifications of tryptophan, whose ultraviolet-light-irradiated photoconversion into the high-affinity ligand FICZ is only one example (reviewed in ref. 20).

Autoimmune diseases are multifactorial, depending on intrinsic components such as genetics, hormones or age, and environmental factors, including infections, diet, drugs and chemicals. The increasing prevalence of certain autoimmune diseases in highly industrialized countries is probably connected to such environmental factors.

Our data linking a transcription factor responsive to environmental pollutants to the T_H17 programme open intriguing possibilities regarding the potential of such factors to initiate or augment autoimmune conditions, and warrant closer examination of a possible role of AHR in human autoimmune diseases.