The aryl hydrocarbon receptor: a molecular pathway for the environmental control of the immune response

Summary

Environmental factors have significant effects on the development of autoimmune diseases. The ligand‐activated transcription factor aryl hydrocarbon receptor (AHR) is controlled by endogenous and environmental small molecules. Hence, AHR provides a molecular pathway by which endogenous and environmental signals can influence the immune response and the development of autoimmune diseases. AHR also provides a target for therapeutic intervention in immune‐mediated disorders. In this review, we discuss the role of AHR in the regulation of T‐cell differentiation and autoimmunity.

Introduction

Genetic susceptibility factors have been identified for multiple sclerosis and other autoimmune diseases, but additional factors such as environmental pollutants,¹ the diet,² the commensal flora³ and exposure to sunlight⁴ also play a role. Recent studies have shown that the transcription factor aryl hydrocarbon receptor (AHR) is an important regulator of the differentiation of murine and human Foxp3⁺ regulatory T cells,^5–9 type 1 regulatory T cells^5,10,11 and T helper type 17 (Th17) cells.^6,12 AHR is activated by endogenous physiological ligands, some of them generated following exposure to UV light, and also by environmental ligands in pollutants, food and products of the commensal flora.¹³ Hence, AHR provides a pathway by which endogenous and environmental signals control multiple sclerosis ‐related immune processes.¹⁴ Moreover, AHR provides a target for the therapeutic manipulation of immunity. In this review, the available information on the role of AHR on the regulation of T‐cell differentiation is discussed.

The aryl hydrocarbon receptor

AHR signalling pathways

The AHR is a ligand‐activated transcription factor with a promiscuous binding pocket that can interact with a broad array of synthetic and natural ligands.¹⁵ AHR was initially identified as a receptor for dioxins like the 2,3,7,8‐tetracholrodibenzo‐p‐dioxin (TCDD). Indeed, much of our understanding of the biology of AHR results from experiments performed using its high‐affinity ligand TCDD.¹⁶ The inactive form of AHR is located in the cytoplasm as part of a protein complex that includes the 90 000 molecular weight heat‐shock protein (hsp 90) and the c‐SRC protein kinase. AHR ligands and hsp 90 interact with overlapping binding sites in AHR.¹⁷ On ligand binding, AHR dissociates from its complex with hsp 90 and c‐SRC, translocates to the nucleus, and interacts with specific sequences (dioxin response elements) in target genes to control their transcriptional activity.¹⁸ Additional mechanisms mediating the biological effects of AHR involve its E3 ubiquitin‐ligase activity¹⁹ and the modulation of the activity of other transcription factors such as nuclear factor‐κB.²⁰

To control the transcriptional activity of its target genes, AHR establishes protein–protein interactions with coactivators and other transcription factors.²¹ The list of transcription factors that interact with AHR includes proteins with well‐characterized functions in the immune system such as signal transducers and activators of transcription (STATs), the retinoic acid receptor (RA), the oestrogen receptor (ER) and nuclear factor‐κB.²¹ The interactions of AHR with other transcription factors result in the recognition of DNA sequences that differ from classical dioxin response elements motifs.²⁰ Strikingly, several AHR protein interactions are only triggered by specific AHR ligands,^22–24 suggesting that some transcriptional partners of AHR are recruited in a ligand‐specific manner.²⁵

Physiological AHR ligands

The aryl hydrocarbon receptor was initially characterized as the receptor for dioxins, environmental pollutants generated by factories and waste‐burning incinerators.^13,26 However, the immune²⁷ and liver defects²⁸ observed in AHR‐deficient mice suggested that natural AHR ligands play a role in normal physiology.

The diet, particularly vegetables, fruits and teas, is an important source of AHR ligands.^13,26 Flavonoids represent the largest group of naturally occurring dietary AHR ligands, which can have either agonist on antagonist activities on AHR activation.^13,26 Usually, dietary AHR ligands have low affinity for AHR, but are converted into high‐affinity ligands by poorly characterized enzymatic reactions. For example, Bradfield's group reported that the d‐amino acid oxidase enzyme generates AHR ligands from the degradation of tryptophan.^29,30 In addition, several indoles, mostly derivatives of tryptophan, are AHR agonists. Examples are two tryptophan‐derived AHR ligands 6‐formylindolo[3,2‐b]carbazole (FICZ)³¹ and 2‐(1′H‐indole‐3′‐carbonyl)‐thiazole‐4‐carboxylic acid methyl ester³² (ITE). Notably, endogenous ligands like ITE do not induce in vivo many of the toxic effects reported for TCDD.^33,34

Role of AHR signalling in the control of 
the T‐cell response

Aryl hydrocarbon receptor signalling plays an important role in the control of several components of the immune system, including T cells, B cells and the innate immune system. In this review, we will focus on the role of AHR in CD4⁺ T cells.

AHR signalling and Foxp3⁺ regulatory T cells

Regulatory T (Treg) cells keep the autoreactive components of the immune system under control.^35,36 A well‐characterized population of CD4⁺ Treg cells is characterized by the expression of the interleukin‐2 (IL‐2) receptor α‐chain (CD25)³⁶ and the transcription factor Foxp3, which controls the development and function of Treg cells.^37,38 The importance of Treg cells for immunoregulation is highlighted by the immune disorders that result from their removal: Treg‐cell depletion from naive animals with depleting antibodies,³⁹ as a result of thymectomy of 3‐day‐old newborns^40,41 or by acute ablation with a toxin in Treg‐cell‐specific toxin receptor knock‐in mice,⁴² results in the development of autoimmune inflammation. As deficits in the function of CD4⁺ CD25⁺ Foxp3⁺ Treg cells have been reported in autoimmune diseases such as multiple sclerosis,^43–46 the induction of functional Foxp3⁺ Treg cells is viewed as a potential approach for the treatment of human autoimmune disorders.⁴⁷

During the course of our studies on zebrafish adaptive immunity we identified a zebrafish Foxp3 homologue that shared molecular and functional features with its mammalian counterpart.⁷ Strikingly, a phylogenetic footprinting analysis identified conserved dioxin response elements within the zebrafish, mouse and human Foxp3 gene, and functional studies showed that AHR controls Foxp3 expression in zebrafish,⁷ suggesting that AHR might also be involved in the control of FoxP3 expression in other vertebrates. Indeed, Funatake et al.⁴⁸ reported that AHR activation by TCDD induces CD4⁺ CD25⁺ T cells with suppressive activity.

We⁴⁹ and subsequently others,^50–54 found that AHR activation by its high‐affinity ligand TCDD in vivo results in the expansion of the CD4⁺ CD25⁺ Foxp3⁺ Treg‐cell compartment. These CD4⁺ CD25⁺ Foxp3⁺ Treg cells are functional and suppress the development of experimental autoimmune encephalomyelitis (EAE),⁴⁹ experimental autoimmune uveoretinitis,⁵⁴ colitis^50,53 and spontaneous autoimmune diabetes.⁵¹ Several mechanisms have been involved in the expansion of Foxp3⁺ Treg cells by AHR activation, including the direct trans‐activation of Foxp3 expression,⁶ the inhibition of STAT‐1 signalling⁸ and changes in the epigenetic status of the Foxp3 locus.⁵³ However, although TCDD is a valuable tool to investigate the immunological effects of AHR activation, TCDD is not a natural AHR ligand and its toxic properties rule out its use to treat human autoimmune disorders. Moreover, although these studies did not detect toxicity, it is not clear to what extent the expansion of Foxp3⁺ Treg cells resulted from preferential toxic effects of TCDD on effector T‐cell populations.⁵⁵

Further support for a physiological role of AHR signalling in Foxp3⁺ Treg cells was provided by experiments that tested the effects of non‐toxic AHR ligands, such as the endogenous mucosal ligand ITE. The oral or parenteral administration of ITE expands the Foxp3⁺ Treg‐cell compartment and treats EAE.⁸ Conversely, AHR‐deficiency or inhibition results in decreased Foxp3⁺ Treg‐cell differentiation.^6,8,52,56 Taken together these data suggest that AHR signalling triggered by physiological ligands plays a role in the regulation of Foxp3⁺ Treg cells, particularly at mucosal sites where AHR can be activated by endogenous and dietary ligands, and also by bacterial products. Indeed, bacterial AHR ligands might be responsible for the AHR‐dependent beneficial effects of Lactobacillus bulgaricus OLL1181 in colitis.⁵⁷ In addition, the tolerogenic effects of AHR signalling might also participate in some pathological conditions, as it has been recently reported that AHR signalling is activated by tumours to evade protective immunity.⁵⁸

In vivo, the promotion of Foxp3⁺ Treg‐cell differentiation by AHR signalling involves AHR activation not only in T cells, but also in dendritic cells (DCs). The DCs stimulate and polarize T cells,⁵⁹ and so balance regulatory and effector adaptive immunity. We⁸ and others^50,56,60,61 found that AHR activation induces murine tolerogenic DCs that produce decreased pro‐inflammatory cytokines and promote regulatory T‐cell differentiation. Several molecular events seem to be responsible for these effects, as AHR activation in DCs was associated with a reduction in the production of several Th1 and Th17 polarizing cytokines. In addition, this tolerogenic activity and the ability to promote the differentiation of Foxp3⁺ Treg cells involved the production of retinoic acid⁸ and tolerogenic kynurenins.^56,61

We have recently used nanoparticles to activate AHR signalling and induce tolerogenic DCs that promote the differentiation of Foxp3⁺ Treg cells.⁶² Nanoparticles (NPs) have been used for in vivo tumour detection and targeting,⁶³ for the delivery of anti‐angiogenic compounds⁶⁴ and also for the induction of pathogen‐specific immunity in vaccination regimens.^65,66 More recently, NPs have been used to deliver short‐interfering RNAs to silence ccr2 expression and prevent the accumulation of inflammatory monocytes at sites of inflammation.⁶⁷ We used NPs to co‐administer the non‐toxic AHR ligand ITE and the T‐cell epitope from myelin oligodendrocyte protein located between residues 35 and 55 (MOG_35–55), to promote the generation of central nervous system‐specific Treg cells by DCs. The NP‐treated DCs displayed a tolerogenic phenotype and promoted the differentiation of Treg cells in vitro. Moreover, NPs carrying ITE and MOG_35–55 expanded the Foxp3⁺ Treg‐cell compartment and suppressed the development of EAE, an experimental model of multiple sclerosis. The effects of NPs in vivo might also involve AHR activation in macrophages, as it has been previously shown that AHR signalling limits the inflammatory response of these cells.^68,69 Hence, NPs are potential new tools for the simultaneous delivery of T‐cell antigens and the activation of AHR signalling in DCs to induce antigen‐specific Treg cells and treat autoimmune disorders.

In mice, Foxp3 is a specific marker for Treg cells, and forced expression of Foxp3^37,38 or its induction with transforming growth factor‐β₁ (TGF‐β₁)⁷⁰ promotes the differentiation of functional Foxp3⁺ Treg cells. In humans, however, FOXP3 expression is not always linked to regulatory function: activated T cells transiently express FOXP3,^71,72 and forced over‐expression of FOXP3⁷³ or its induction with TGF‐β₁⁷⁴ does not result in the differentiation of suppressive FOXP3⁺ Treg cells. Hence, additional signals besides those controlled by FOXP3 are required for the generation of human functional FOXP3⁺ Treg cells. We found that AHR activation in the presence of TGF‐β₁ induces the differentiation of functional human FOXP3⁺ Treg cells that suppress responder T cells via CD39. The induction of functional FOXP3⁺ Treg cells by the concurrent activation of TGF‐β₁ and AHR signalling is mediated, at least partially, by the transcription factors SMAD1 and AIOLOS. SMAD1 alone or in combination with SMAD3/4 interacts and regulates the + 2079 to + 2198 enhancer in the conserved non‐coding sequence 1 of FOXP3⁷⁵ to activate FOXP3 expression. In addition, AIOLOS interacts with FOXP3 through its C‐terminal domain and mediates the repression of IL‐2 expression in FOXP3⁺ Treg cells induced in vitro by the concomitant activation of TGF‐β₁ and AHR signasling. Hence, AHR is a potential target for the generation of functional Treg cells and the treatment of autoimmune disorders.

As we already mentioned, several AHR protein interactions are only triggered by specific AHR ligands,^22,23,24 suggesting that some effects of AHR might be ligand specific. Ligand‐specific effects are well characterized on other nuclear receptors, and are mainly dictated by the structure of the ligand and the cell‐specific expression of receptor‐interacting proteins.^76–79 For example, ligand‐specific effects for the ER are highly relevant for the therapy of tumours: both 17β‐oestradiol and the chemotherapeutic drug tamoxifen are ER ligands; however, tamoxifen is an ER antagonist in breast tumours and an ER agonist in the endometrium whereas 17β‐oestradiol is an ER agonist in both.^80–84 In the case of AHR, ligand‐specific effects have been reported to control its interactions with protein co‐activators.^22–24 Indeed, ligand‐specific effects of AHR on the polarization of Foxp3⁺ Treg cells and other cell types have also been reported,^6,53,56 but the molecular basis for those ligand‐specific effects is still poorly understood.

AHR signalling and IL‐10⁺ type 1 regulatory T cells

The IL‐10⁺ type 1 regulatory cells (Tr1 cells) were first described as suppressive CD4⁺ T cells induced by repeated cycles of activation in the presence of IL‐10 or IL‐10‐conditioned DCs.⁸⁵ Tr1 cells have been shown to prevent the development of colitis and other experimental autoimmune diseases.⁸⁶ However, although Tr1 cells resemble natural Treg cells in some ways, they do not express Foxp3.⁸⁷

Interleukin‐27 promotes the differentiation of Tr1 cells,⁸⁷ and IL‐21 is an autocrine growth factor for Tr1 cells produced in response to IL‐27.⁸⁸ The transcription factor c‐Maf is essential for the induction of IL‐10 by Tr1 cells,⁸⁹ but additional transcription factors involved in the differentiation of Tr1 cells are unknown. We found that AHR is induced by IL‐27 and synergizes with c‐Maf to promote the differentiation of murine and human Tr1 cells.¹⁰ AHR forms a protein complex with c‐Maf, and this AHR/c‐MAF complex transactivates the Il10 promoter. Moreover, we have previously shown that AHR activation up‐regulates IL‐21 production by T cells.⁶ We found that the AHR/c‐Maf complex also binds and transactivates the Il21 promoter in Tr1 cells. Hence, AHR directly controls both the production of the Tr1 signature cytokine IL‐10, and the production of the autocrine Tr1 growth factor IL‐21. In vivo, AHR is required for the differentiation of suppressive TR1 cells capable of halting inflammation in experimental models of multiple sclerosis¹⁰ and lupus.¹¹ Moreover, we also found that AHR was important for the differentiation of human Tr1 cells.⁵ Hence, AHR signalling can modulate the differentiation of murine and human IL‐10‐producing Tr1 cells.

AHR signalling and IL‐17‐producing T cells

Th17 cells, CD4⁺ T cells characterized by the production of IL‐17, IL‐17F, IL‐21 and IL‐22, play an important role in the control of specific pathogens and the development of autoimmune diseases.^90–92 T‐cell activation in the presence of IL‐6^93–95 or IL‐21^96,97 and TGF‐β₁ promotes the differentiation of Th17 cells by STAT‐3‐dependent mechanisms,^98,99 while IL‐21^96,97,100 and IL‐23¹⁰¹ expand and stabilize the phenotype of Th17 cells. The signals initiated in T cells by cytokine receptors induce and activate specific transcription factors that control the transcriptional programme of Th17 cells. The differentiation of Th17 cells is driven by the transcription factors RORγt¹⁰² and RORα,¹⁰³ indeed mice that are deficient in RORγt¹⁰² and RORα¹⁰³ or mice treated with RORγt inhibitors^104,105 show an impaired generation of Th17 cells. In addition to RORγt and RORα, other transcription factors like STAT‐3 and c‐Maf also participate in the differentiation of Th17 cells.

The transcription factor AHR, for example, controls the expression of IL‐21 and IL‐22 and plays an important role in the differentiation of Th17 cells in vivo and in vitro.^{10,52,106–109} We and others reported that AHR expression is also up‐regulated in Th17 cells,^49,109 probably as a result of the direct transactivation of the Ahr promoter by phosphorylated STAT‐3.¹¹⁰ Indeed, AHR ligands can boost the differentiation of Th17 cells.^49,109 The activation of AHR in vivo by its ligand FICZ³¹ boosts the Th17 response and worsens central nervous system autoimmunity.^49,109 Note, however, that similar to what has been reported for Foxp3⁺ Treg cells, ligand‐specific effects have also been described for the differentiation of Th17 cells. Indeed, Mezrich et al.⁵⁶ and Benson and Shepherd.⁵⁰ have both reported inhibitory effects of specific AHR ligands on the differentiation of Th17 cells.

The Th17 cells play an important role in clearing extracellular pathogens; however, an aggressive Th17 response induces severe inflammation,⁹⁰ hence several mechanisms operate to prevent the dysregulated generation of pro‐inflammatory Th17 cells. Interferon‐γ^111,112 and IL‐2^113,114 have been identified as negative regulators of Th17 differentiation in vivo and in vitro.¹¹³ In Th17 cells, the effects of AHR might be mediated through its inhibitory interactions with STAT‐1⁵² and STAT‐5,¹⁰⁸ which might relieve the inhibitory effects of interferon‐γ and IL‐2 on Th17 cell differentiation. In addition, we recently found that under Th17 polarizing conditions AHR together with STAT‐3 promote the expression of the transcription factor Aiolos, which binds to the il2 promoter and induces chromatin modifications that result in il2 silencing. Aiolos‐deficient naive CD4⁺ T cells produce larger amounts of IL‐2 and show an impaired differentiation into Th17 cells, which can be reversed by blocking IL‐2 function. Hence, Aiolos promotes the differentiation of Th17 cells by actively silencing IL‐2 transcription under Th17‐polarizing conditions. In addition to its effects on IL‐21 and IL‐22 production, AHR controls a module in the transcriptional programme of Th17 cells that limits the autocrine inhibitory effects of IL‐2 and thereby promotes Th17 differentiation.

Concluding remarks

Figure 1 summarizes our current knowledge of the role of AHR in CD4⁺ T cells. The identification of AHR as an important player in the development and function of effector and regulatory T cells has both basic and clinical implications: considering the abundance of AHR ligands in environmental pollutants, food and products of the commensal flora, AHR provides a molecular pathway by which the environment can affect the immune response and the development of immune‐mediated disorders. Moreover, AHR constitutes a potential target for the therapeutic modulation of the immune response.

Figure 1.

Role of aryl hydrocarbnon receptor (AHR) signalling on CD4⁺ T cells. AHR signaling in FoxP3⁺ regulatory T (Treg) cells triggers the demethylation of Foxp3 and transactivates its promoter. AHR signalling also interferes with the activation of signal transducer and activator of transcription 1 (STAT‐1), which mediates the inhibitory effects of interferon‐γ (IFN‐γ) on Foxp3⁺ Treg cells. Finally, AHR activation up‐regulates the expression of CD39 and of Aiolos, which then inhibits interleukin‐2 (IL‐2) production. AHR signalling in IL‐10⁺ type 1 regulatory (Tr1) cells triggers the expression of IL‐10 and the Tr1 autocrine growth factor IL‐21. In addition, AHR activation also up‐regulates granzyme B expression. AHR signalling in T helper type 17 (Th17) cells promotes the expression of IL‐21 and IL‐22, and it also limits the activation of STAT‐1 and STAT‐5, which mediate the inhibitory effects of IFNγ and IL‐2 on Th17 cell differentiation, respectively. Finally, AHR activation inhibits the production of IL‐2 through a mechanism dependent on Aiolos.

Disclosure

The author has no financial disclosures or competing interests.