Introduction

It is not very common that the signalling pathway of a single molecule attracts enough interest to merit international conferences of its own. The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor present in many cells, belongs to the rare elite of such molecules. At the “7th Düsseldorf symposium on immunotoxicology” the fascinating aspects of AhR were discussed by more than 100 attendees. The meeting was organized by the Leibniz Research Institute for Environmental Medicine (IUF) and took place at the University of Düsseldorf, Germany. Alvaro Puga from the University of Cincinnati started with an overview of 30 years of AhR research, which helped put into context what the talks of the next two and a half day would cover: the AhR is striking in its breadth of activities, ranging from dioxin toxicology to biology of the skin, neuronal system or immune system to such curious findings as rejection of unknown food. Moreover, the conference looked at ways how to use AhR and AhR ligands in therapeutic and preventive settings.

AhR and dioxin toxicity

AhR was first discovered as the mediator of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decades ago and has been of ongoing scientific interest, as evident by an unbroken stream of publications in Archives of Toxicology and many other journals (Reggiani 1978; Schrenk et al. 1991; Vogel et al. 1997). AhR is responsible for inducing the xenobiotic metabolizing enzymes needed for the detoxification of polycyclic aromatic hydrocarbons (Nagayama et al. 1985; Schmidt and Bradfield 1996). Over the past years, stringent regulations of industrial processes have led to emission reduction. However, other emission pathways exist, including wood burning, hospital waste incineration, accidental exposure and others. Dioxins are environmentally and biologically stable and, as a result, exposure remains wide spread (Kulkarni et al. 2008). The majority of low-level human exposure is via the food chain (Yaktine et al. 2006), and curiously, exposure of rats to TCDD also diminishes their interest in unknown food in an AhR-dependent manner (Raimo Pohjanvirta (University of Helsinki, Finland; Lensu et al. 2011). Several presentations were about novel findings in dioxin toxicity. It has been known for long that chloracne in humans exposed to TCDD is caused by disturbance of keratinocyte differentiation (Reggiani 1978; Baccarelli et al. 2005). Tom Sutter (Memphis, USA) now reported that filaggrin and other genes of the epidermal differentiation complex are direct targets of TCDD, and that in utero exposure accelerates the formation of the epidermal barrier (Sutter et al. 2011). Alvaro Puga suggested that AhR might be a key mediator of TCDD-mediated cardiovascular malformations due to its interference with homeobox transcription factor Nkx2.5 gene expression and other cardiac markers (Wang et al. 2010). Christopher Bradfield (University of Wisconsin, Madison, USA) stressed that the level of induction of AhR, that is, ligand concentration and availability, governs the outcome of AhR signalling and showed data differentiating hepatocellular toxicity and cancer from developmental changes upon TCDD exposure.

Nonetheless, the conference made clear that the research focus has shifted from the role of AhR in TCDD-mediated toxicity to its intriguing physiological roles and to its use as a promising drug target.

AhR signalling pathways, and interaction with retrotransposons

The major signalling pathway of AhR upon ligand binding is translocation to the nucleus, dimerization with ARNT (AhR nuclear translocator) and binding to the so-called dioxin-responsive element 5′-GCGTG-3′. However, as described by Christoph Vogel (University of Davis, California), ligand-activated AhR can tie into the NFκB pathway and bind/activate RelA or RelB (Vogel and Matsumura 2009; Wu et al. 2011a). This has consequences for toll-like receptor signalling by dendritic cells in inflammatory conditions (Wu et al. 2011a) or, as discussed by Herbert Strobl (University of Vienna, Austria), impairs the differentiation of dendritic cells, both situations where NFκB signalling is critical (Platzer et al. 2009). Ravan Arsenescu (University of Lexington, USA) analysed interactions of AhR with the angiotensin/adiponectin system and found that treatment of angiotensin-infused apolipoprotein E−/− mice (a model for atherosclerosis) with the AhR ligand polychlorinated biphenyl-77 led to a higher incidence of atherosclerotic lesions. He concluded that the AhR pathway triggers inflammatory processes and thereby is associated with overexpression of angiotensin (Arsenescu et al. 2011). In humans, a role for AhR in adipogenesis is possible as well, and Karin Fehsel (University of Düsseldorf, Germany) showed data indicating that a common drug used for treatment of schizophrenia patients, clozapine, might cause hyperplasia of pre-adipocytes in an AhR-dependent fashion. Weight gain is a side-effect of therapy with clozapine. Crosstalk of AhR with the epidermal growth factor receptor, the glucocorticoid receptor and the oestrogen receptor exists as well, either influencing the induction level of CYP4501A1, or endocrine functions (Takemoto et al. 2004; Bolt and Degen 2002). This was discussed by Hasan Mukhtar (University of Wisconsin, Madison, USA), Zdanek Dvorak (University of Olomouc, Czech Republic) and others. Newly discovered are interactions with the β-catenin pathway, as discussed by Jan Vondracek (University of Brno, Czech Republic) and Albert Bräuning (University of Tübingen, Germany). They studied liver cells and their progenitors. β-catenin binds physically to AhR and β-catenin acts cooperatively with AhR ligands, enhancing the transactivation potential of AhR in the induction of CYP4501A1 (Braeuning et al. 2011). The activation of AhR downregulates expression of members of the β-catenin/WNT pathway during liver development. This in turn leads to alterations in the proliferative and differentiation status of liver progenitor cells (Prochazkova et al. 2011). A very intriguing new aspect of AhR in genetics was presented by Pedro Fernandez-Salguero (University of Extremadura, Badajoz, Spain). More than half of the mammalian genome is composed of repetitive sequences interspersed with many retrotransposons, whose sheer abundance suggests an important biological function. The B1 SINE (short interspersed nuclear element) retrotransposon is highly abundant (over 14,000 instances in the mouse genome). Approximately 10% of these retrotransposons have a dioxin-responsive element, separated by 35 bp short spacer from a Slug transcription factor binding DNA element 5-CAGTG-3′ and were thus named B1-X35S. Both activated AhR and Slug bind in combination to these elements. The work of Fernandez-Salguero demonstrated elegantly that these elements form chromatin barriers and enforce a switch between polymerase II and III at their sites. B1-X35S elements thus ensure that gene transcription is insulated and restricted to a given gene (Roman et al. 2011a, b) and does not overflow to adjacent genes.

AhR in brain development, brain tumours, and neuronal tissues

AhR is expressed in the central nervous system of a variety of species including the mouse. Modulation of the AhR by stimulation or knockdown causes defects during neurodevelopment in lower organisms like C. elegans, in zebrafish and mammals like mice and rats. Ellen Fritsche (IUF, Düsseldorf & RWTH Aachen, Germany) showed work from her co-worker Kathrin Gassmann that developmentally derived murine neural progenitor cells (NPCs), which grow as neurospheres and develop in culture, maintain their functional AhR. Mouse AhR activation or inhibition impairs NPC proliferation and migration, respectively. In strong contrast, human NPC development is not affected by AhR modulation. This lack in response is explained by absence of AhR- and AhR-dependent signalling (Gassmann et al. 2010). These data indicate that during the early foetal period human NPCs are protected against detrimental effects of PAH by downregulation of AhR (Gassmann et al. 2010). Aline Chevallier (INSERM, Paris, France, group of Xavier Coumoul) showed gene expression profiling data from AhR-deficient mice versus wild-type mice. More than 2000 genes were dysregulated when AhR is not expressed. Curiously, AhR-deficient mice display also behavioural impairment such as not being able to balance as well on a rod as wild-type mice. Similar phenomena had been described in TCDD exposed rats (Thiel et al. 1994). The brain is also an important site for the circadian clock. Clock proteins belong to the same protein family as AhR, the PAS-bHLH proteins. As discussed by Shelley Tischkau (University of Southern Illinois, Springfield, USA), AhR/ARNT signalling leading is co-regulated with the clock protein BMAL1, a homolog of ARNT. AhR-deficient mice loose their day–night-triggered rhythmicity. In this context, it is intriguing that important physiological ligands of AhR are light-induced metabolites of tryptophan. Tischkau thus suggested a role for AhR in physiological modulation of circadian rhythms (Mukai et al. 2008). Finally, a very exciting story regarding AhR in the brain was presented by Michael Platten (University of Heidelberg, Germany). He showed that brain tumours express tryptophan-2,3-dioxygenase, and thereby produce significant levels of kynurenins from tryptophan. Kynurenins are high-affinity AhR ligands and Platten could demonstrate that the kynurenine-activated AhR in the vicinity of brain tumours drives tumour progression (Opitz et al. 2011). The associated poster by Christiane Opitz showed data on TDO expression levels in patient tumours and correlated them with patient survival. This study was awarded the poster prize of the meeting. Therapeutic interventions inhibiting AhR activity are the next logical step of this work.

AhR in the immune system: stem cells

Research regarding the involvement of AhR in the immune system has exponentially increased since 2009, when several papers showed a role in the differentiation of T cells (Stockinger et al. 2011). Also in this meeting, new findings regarding AhR and immunity were reported. Many immune cells express AhR. Tom Gasiewicz (University of Rochester, USA) and Michael Laiosa (University of Milwaukee, USA) reported that AhR regulated critical genes that allow hematopoietic stem cells (HSC) to respond to the environment. In AhR-deficient mice the capacity of HSC to migrate and cycle is impaired, leading to retention of these cells in the bone marrow and loss of function. Thus, AhR is a negative regulator of HSC proliferation in mice (Casado et al. 2011; Singh et al. 2011). Complementing work was presented by Michael Laiosa who used the in vitro differentiation system of lineage negative foetal liver cells (i.e. the hematopoietic progenitors) grown on OP9 stromal cells transduced with Notch. This system also reveals a high sensitivity of HSC against AhR activation by TCDD, and a physiological role for AhR in lineage decision of HSC towards T cells and B cells.

AhR in the immune system: barrier organs

Important roles for AhR in immune cell development and homoeostasis, with direct relevance to human health, were presented by Andreas Diefenbach (University of Freiburg, Germany) and Marc Veldhoen (Babram Institute, Cambridge, United Kingdom). Diefenbach demonstrated elegantly that AhR signals are indispensable for the expansion and maintenance of RORγt+ innate lymphoid cells (ILC) in the gut. These cells are required for the development of cryptopatches and ultimately the intestinal T cells. Diefenbach showed that dietary AhR ligands (indoles and glucosinolates) present, for example, in cruciferous plants are needed by mice, and that c-kit is an AhR-target gene, necessary for RORγt+ ILC homoeostasis (Kiss et al. 2011). C-kit is a cell surface–bound tyrosine kinase known as a growth factor for various stem cells, including melanocyte and mast cell precursors. Diefenbach showed further that mice either lacking AhR or put on a long-term diet without diet-indoles are highly susceptible to infection with the intestinal pathogen Citrobacter rodentium, a model for the human EHEC infection. The underlying reason is the failure to produce enough IL-22 by the diminished gut T cells. Interestingly, c-kit is necessary for the expansion of innate γδ T cells in the skin as well, as reported by Charlotte Esser (IUF, Düsseldorf, Germany). AhR-deficient mice do not expand innate γδ T cells in the skin, which are lost within a few weeks after birth. γδ T cells from AhR-deficient mice have a very low/absent c-kit expression (Kadow et al. 2011). γδ T cells in the skin are critical for immunosurveillance of this barrier organ and contribute to wound healing (Girardi et al. 2006). Marc Veldhoen found that not only in the skin, but also in the gut of AhR-deficient mice innate γδ T cells are strongly reduced. They are a major source of IL17. Diet intervention studies performed by Veldhoen proved that food-derived AhR ligands are needed to maintain a stable and functional gut immune system by γδ T cells. In an approach similar to the one used by Diefenbach, Veldhoen used diet intervention in the model of dextran-sulfate-induced colitis to show that removal of AhR ligands from the diet increases the susceptibility towards this inflammatory and destructive disease (Li et al. 2011). Thus, the old recommendation “eat your veggies” has a strong molecular basis. In this context, the findings of Heike Weighardt and Irmgard Förster were relevant as well. They reported the generation of an AhR-Repressor (AhRR) reporter mouse. Using this mouse, they could extend older data on tissue-specific AhRR expression (Bernshausen et al. 2006) and demonstrated that AhRR is mainly expressed in dendritic cells of the gut and skin. AhRR expression is inducible by stimulation of AhR, either by normal ligands or via stimulation of toll-like receptors, which sense bacterial surface molecules. Christoforos Vlachos (Radboud University, Nijmegen, The Netherlands) worked on AhR ligands produced by the commensal skin yeast Malassezia spec., which can turn pathogenic. He and his colleagues characterized various ligands (indoles, indirubin, tryptanthrin, malassezin) and found them to be potent suppressors of dendritic cells, capable of reducing the production of inflammatory cytokines IL-6 and IL12 by these cells in the presence of toll-like receptors. This fits well with the observations that DC need AhR and NFκB for induction of the immunosuppressive enzyme IDO, which was reported by several groups (Nguyen et al. 2010; Vogel et al. 2008). Together, all these studies strongly indicate that the AhR/AhRR system is very important for immune homoeostasis of barrier organs and shaped by the environment, both by plant-derived and microorganism-derived ligands.

AhR in the immune system: allergy

Mast cells (MC) belong to the immune system and line epithelia, for example, underneath the epidermis or the gut epithelium, where they respond rapidly to bacterial products, venoms, chemical injury or physical stress. They release histamine, IL-17, and other potent mediators, which are also major players in allergic inflammatory reactions. In the conference, two papers presented work on the role of AhR in MC for the first time. Giorgia Gri (University of Udine, Italy) showed that human and murine MC express AhR protein, and treatment of MC with AhR ligands and/or toll-like receptors induces IL6 and IL17 secretion. MC degranulate faster and more strongly if triggered for histamin release by treatment with IgE and its appropriate antigen. Similarly, passive systemic anaphylaxis is exacerbated by AhR stimulation. Yufeng Zhou (Johns Hopkins University, Baltimore, USA) extended these observations, showing that also production of reactive oxygen species and intracellular calcium is enhanced by AhR ligands, which signals to ERK, eventually triggering the degranulation of MC. Moreover, he reported that AhR-deficient mice have impaired MC proliferation. His data demonstrated that STAT1, 3, and 5 levels are constitutively low in AhRd/d mice (a mouse strain with a low affinity AhR), and that AhR-deficient mice lack MC in their skin and various other tissues, including the gut. MC from AhR-deficient or AhRd/d mice was refractory to IgE-mediated activation. Together, the data from Gri and Zhou suggest that chemical challenges are registered by MC and contribute to allergy or pulmonary mucosa-related inflammatory diseases, especially those that are epidemiologically related to AhR ligands. Smoking immediately comes to the mind, as cigarette smoke contains potent AhR ligands. In addition, disease might become exacerbated by AhR-mediated mitochondrial dysfunction and apoptosis of lung fibroblasts (Carolyne Baglole, McGill University, Montreal, Canada; Rico de et al. 2011). It will be very interesting to observe these facets of AhR-related adverse effects further.

AhR in the immune system: T cells

The sensitivity of T cells towards TCDD exposure has been one of the first immune-related phenomena described by immunotoxicologists (Vos 1977; Esser et al. 2009; Kerkvliet et al. 1990; Neubert et al. 1990). The direct AhR-dependent induction or increase of regulatory T cells is controversial, and some suggest activated AhR targets DC, which in turn shift the differentiation of naïve T cells towards regulatory T cells (rather than helper T cells or cytotoxic T cells). Veronica Schulz (University of Utrecht, The Netherlands) from the group of Raymond Pieters had triggered food allergy against peanuts in mice treated with TCDD. She demonstrated in her talk that ablation of regulatory T cells in this model by anti-CD25 antibodies during the sensitization reversed the TCDD-mediated suppression of peanut allergy (Schulz et al. 2011). However, further studies are needed to elucidate the underlying mechanism, for example, a role for DC or—in the light of the new findings described above—MC. Paige Lawrence (University of Rochester, USA) discussed these issues. Lawrence studies the model of influenza infection in AhR-deficient mice with a focus on cytotoxic T cells and how functional subsets of DC shape their function. She reported a complex picture how distinct subsets DC need AhR activation for trafficking to lymph nodes and proper stimulation of naïve CD8+ T cells. AhR-mediated changes in gene expression of DC underlie the alterations during infection (Jin et al. 2010). The issue of AhR and the induction of regulatory T cells is currently unresolved and of ongoing interest (Funatake et al. 2008; Marshall and Kerkvliet 2010; Apetoh et al. 2010; Mezrich et al. 2010). Roopali Gandhi (Harvard Medical School, Boston, USA) talked about her research trying to dissect direct and indirect effects of AhR on regulatory T cells. She used a reporter mouse, where green fluorescent protein is under the control of the FoxP3 promoter (and thus indicates regulatory T cells, which are FoxP3+), and showed that AhR signalling participates in the differentiation of FoxP3+ regulatory T cells in vivo. Molecular mechanisms require coordinated action of AhR with other transcription factors, Aiolos and Smad1. However, the group also found that AhR ligands act on DC to induce tolerogenicity (Gandhi et al. 2010; Wu et al. 2011b).

AhR, AhR ligands and translational medicine

The brief sketches above immediately show that the AhR is a highly interesting drug target. Thus, it may prove beneficial to suppress AhR activity. For instance, as described above, AhR activity in brain tumours is associated with a bad prognosis. Also, AhR exacerbated allergic responses. Another example is AhR-impaired nucleotide excision repair in UVB-irradiated keratinocytes. This was reported by Thomas Haarmann-Stemmann (IUF, Düsseldorf), who showed that AhR is part of the UVB damage response. AhR-inhibitors derived from plants, such as epigallocatechin-3-gallate enhance nucleotide excision repair of DNA. His data also demonstrated that in AhR-negative keratinocytes significantly less cyclobutan dimers are generated in the DNA by UVB irradiation. Or, it may desirable to enhance AhR activity, for example, to expand regulatory T cells, for instance in combating autoimmunity. These challenges were discussed at the meeting, with a focus on the role of the ligands. It is clear that (beyond cell type and temporal considerations) the outcome of AhR activation depends on ligand characteristics, such as stability, concentration, and affinity to AhR. It is also evident that TCDD—despite its stability and effectivity—can never become a possible or marketable pharmakon. A major physiological ligand, which has been identified by the group of Agneta Rannug (Karolinska Institute, Stockholm, Schweden), is FICZ (Formylindolo-[2,3]b-carbazol), a photoproduct generated by light/UV light from tryptophan in the skin. The affinity of FICZ to AhR is very high, comparable to TCDD. FICZ is currently used by many research groups as a model AhR ligand. Rannug reported on the ongoing chemical and biological characterization of this molecule, which may be active at femtomolar concentrations, and influences CYP4501A1 induction and thus clearance of other AhR ligands. She stressed that the “vitamin-like” ubiquity of FICZ in the body must be taken into consideration in in vivo and in vitro studies of AhR activity (Luecke et al. 2010). Both Stephen Safe (Texas A&M University, Houston, USA), Siva Kolluri (Oregon State University, Eugene, USA) and Gary Perdew (Penn State University, USA) reported data using agonists, antagonists and selective AhR modulators (SAhRM) in cancer and inflammation. They used various cancer lines and identified new genetic phenomena such as a role for micro RNA (e.g. miR-335) in anti-cancerogenic effects of AhR ligands (Zhang et al. 2012) or of HDAC1 in gene repression. The bottom line is that biological outcome and molecular mechanisms depend very much on the cell type and the ligand. While the studies underscore the complexity of AhR modulation, they also promise the possibility of a cell-type-specific treatment.

Concluding remarks

The meeting highlighted that AhR research has moved from its focus on toxic effects of dioxins and other environmental pollutants to the biological role of this evolutionarily conserved receptor. AhR is a sensor of small molecular molecules present in the environment and orchestrates appropriate metabolic responses, in particular through induction of phase I and phase II enzymes. It was previously not recognized that the body also needs AhR ligands from the environment, either provided in food or generated from sunlight in the skin, for development and homoeostasis of cells, notably for immune cells but possibly for many more. AhR contributes as well to such essential functions as insulation of transcription for proper gene expression. These are exciting new findings. In his introduction, Alvaro Puga challenged the audience with the question “Is there a future for AhR research?” The uncontested answer at the end of the conference was “YES”.