associate editor: qiang ma the aryl hydrocarbon receptor...

1521-0081/67/2/259–279$25.00 http://dx.doi.org/10.1124/pr.114.009001PHARMACOLOGICAL REVIEWS Pharmacol Rev 67:259–279, April 2015Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: QIANG MA

The Aryl Hydrocarbon Receptor in Barrier OrganPhysiology, Immunology, and Toxicology

Charlotte Esser and Agneta Rannug

Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany (C.E.); and Institute ofEnvironmental Medicine, Karolinska Institutet, Stockholm, Sweden (A.R.)

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260II. Aryl Hydrocarbon Receptor Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

A. The Canonical Signaling Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261B. Noncanonical Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261C. Aryl Hydrocarbon Receptor–Dependent Transcription. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263D. The Realm of Aryl Hydrocarbon Receptor Ligands: Agonists and Antagonists. . . . . . . . . . . . . 263

III. Role of the Aryl Hydrocarbon Receptor in the Barrier Immune System. . . . . . . . . . . . . . . . . . . . . . . 264A. Differential and Variable Aryl Hydrocarbon Receptor Expression Levels in Cells and

Tissues, Including the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264B. Aryl Hydrocarbon Receptor in the Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

1. The Dermis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2652. The Epidermis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2653. Aryl Hydrocarbon Receptor–Mediated Skin Functions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2654. Aryl Hydrocarbon Receptor in Two Skin-Specific Innate Immune Cells. . . . . . . . . . . . . . . . 2665. High-Affinity Aryl Hydrocarbon Receptor Ligands in the Skin. . . . . . . . . . . . . . . . . . . . . . . . . 267

C. Aryl Hydrocarbon Receptor in the Gut and Consequences for Gut Health and Microbiome . . 2671. The Gut-Associated Immune System: A Paradigm of Immune Activation and

Suppression.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2672. Aryl Hydrocarbon Receptor in Innate Immune Cells of the Gut.. . . . . . . . . . . . . . . . . . . . . . . 2673. Systemic Effects of Oral Aryl Hydrocarbon Receptor Ligand Exposure. . . . . . . . . . . . . . . . . 2684. Role of Dietary Ligands in Development of the Innate Gut Immunity. . . . . . . . . . . . . . . . . 268

D. Aryl Hydrocarbon Receptor in the Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2691. Lung Inflammation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2692. Respiratory Viral Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2693. Lung Fibrosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269

IV. The Aryl Hydrocarbon Receptor in Toxicology and Physiology of the Human Skin, Lung,and Gut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270

V. The Aryl Hydrocarbon Receptor: Promiscuous Sensing of Chemicals or MetabolicControl of the Endogenous High-Affinity Ligand FICZ? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271

VI. Therapeutic Potential of Aryl Hydrocarbon Receptor Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

This research was supported by the German Research Foundation [Grants ES103/6 and ES103/5 (to C.E.)] and the Swedish ResearchCouncil Formas [Grant 216-2011-1364 (to A.R.)].

Address correspondence to: Dr. Charlotte Esser, Leibniz Research Institute for Environmental Medicine, Auf´m Hennekamp 50, 40225Düsseldorf, Germany. E-mail: [email protected]

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Abstract——The aryl hydrocarbon receptor (AhR) isan evolutionarily old transcription factor belonging tothe Per-ARNT-Sim–basic helix-loop-helix protein family.AhR translocates into the nucleus upon binding ofvarious small molecules into the pocket of its single-ligand binding domain. AhR binding to both xenobioticand endogenous ligands results in highly cell-specifictranscriptome changes and in changes in cellularfunctions. We discuss here the role of AhR for immunecells of the barrier organs: skin, gut, and lung. Both

adaptive and innate immune cells require AhR signalingat critical checkpoints. We also discuss the current twoprevailing views—namely, 1) AhR as a promiscuoussensor for small chemicals and 2) a role for AhR asa balancing factor for cell differentiation and function,which is controlled by levels of endogenous high-affinityligands. AhR signaling is considered a promising drug andpreventive target, particularly for cancer, inflammatory,and autoimmune diseases. Therefore, understanding itsbiology is of great importance.

I. Introduction

The environment offers both vital benefits, such asfood and sunlight, and deadly risks, such as infectiouspathogens and toxins. The role of epithelial barrierorgans such as skin, gut, lung, and other mucosal tissuesthat interact with the environment is critical for survival.Furthermore, these barriers are involved in uptake andabsorption of nutrients as well as protection of bodyintegrity against chemical, biologic, and physical stress,andmore. The aryl hydrocarbon receptor (AhR) is a latentcytoplasmic transcription factor that can be activated bycertain low molecular weight chemicals. The AhR ishighly expressed in many barrier organs and in the liver,and its expression pattern conceivably suggests a roleas a sensor of chemicals. Many AhR-activating factors,which cause transcriptional activation, have been identi-fied (Denison and Nagy, 2003). They range from environ-mental pollutants, such as polyhalogenated aromatichydrocarbons (PHAHs), to endogenous amino acid deriv-atives, dietary chemicals such as indoles or glucosinolates,and finally natural substances found in yeasts andbacteria or even marine sponges. As demonstrated byin vitro studies and gene expression profiles, the tran-scriptional changes by the activated AhR are ligandspecific and are highly cell specific. For a given cell type,they may even depend on the tissue milieu, such as anongoing immune response (Sun et al., 2004; Frerickset al., 2007; Beischlag et al., 2008; Tappenden et al.,2013). There appears to be only one ligand bindingpocket in the AhR [in the Per-ARNT-Sim-B domain],and the binding affinity of the best characterized high-affinity ligand, TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), depends on only a few amino acids in the bindingpocket of the AhR, as demonstrated by modeling andmutation studies (Whelan et al., 2010; DeGroot et al.,2012; Wu et al., 2013). The AhR can also be activatedby numerous stress factors and substances that may notfit into the binding pocket, such as hyperoxia, oxidized

low-density lipoproteins, hydrogen peroxide, ozone, ormetals (references in Wincent et al., 2012). Much of thisis not understood because the AhR has not yet beencrystallized, although it was cloned more than 2 decadesago (Burbach et al., 1992).

Research on the AhR has undergone a major paradigmshift in recent years. It has long focused on induction ofgenes coding for metabolizing enzymes, the so-calledAhR-battery genes (Nebert et al., 1991; Köhle and Bock,2007). In particular, cytochrome P450 was of interestbecause of the toxicity associated with its activity inadverse drug–drug interactions and generation of carci-nogenic metabolites from polycyclic aromatic hydrocar-bons (PAHs). Especially in the field of toxicology andpharmacology, the AhR is viewed as a protein that hasdeveloped during evolution to mediate metabolization ofenvironmental small molecules. This view of the AhR asa promiscuous cytosolic sensor of small molecules that isprimarily involved in biotransformation and detoxifica-tion is rapidly changing. As regarded today, the AhRplays an important role in cell development, differenti-ation, and function. Recent evidence from studies withfull and conditional AhR-deficient animal models impliesimportant endogenous roles for the AhR. These includecontrol in perinatal growth, fertility, hepatic and vasculardevelopment, peripheral and intestinal immunity andhematopoiesis, as well as in stem cell expansion andcancer. The specific ligands that are drivers of activationin a given situation are not fully clear.

It is recognized that xenobiotic small chemicals canaberrantly activate AhR because of their structurallikeness to physiologic ligands. By this process, chem-icals may increase the metabolic turnover of physiologicligands and thereby decrease the half-life of the latter.As a result, uncontrolled or persistent activation of theAhR by exogenous small molecules may disturb the tightlycontrolled and transient AhR-regulated cell functions.This could be the underlying cause of the toxicity of

ABBREVIATIONS: AD, atopic dermatitis; AhR, aryl hydrocarbon receptor; AhRE, aryl hydrocarbon receptor responsive element; ARNT,aryl hydrocarbon receptor nuclear translocator; COPD, chronic obstructive pulmonary disease; DC, dendritic cell; DETC, dendritic epidermalT cell; DSS, dextran sodium sulfate; FICZ, 6-formylindolo[3,2-b]carbazole; GNF351, N-(2-(3H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methyl-3-pyridyl)-7H-purin-6-amine; I3C, indole-3-carbinol; ICZ, indolo[3,2-b]carbazole; IEC, intestinal epithelial cell; IEL, intraepithelial lymphocyte;IL, interleukin; ILC, innate lymphoid cell; ITE, 2-(19H-indole-39-carbonyl)-thiazole-4-carboxylic acid methyl ester; LBD, ligand bindingdomain; LC, Langerhans cell; MG132, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; NK, natural killer; PAH, polycyclic aromatic hydrocarbon;PHAH, polyhalogenated aromatic hydrocarbon; SAhRM, selective aryl hydrocarbon receptor modulator; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin (“dioxin”); TCR, T-cell receptor; Th, helper T cell; Treg, regulatory T cell; VAF347, (4-(3-chloro-phenyl)-pyrimidin-2-yl)-(4-trifluoromethyl-phenyl)-amine.

260 Esser and Rannug

ligands such as dioxins and biphenyls. The link betweenenvironment and immunity is particularly intriguing(Kimura et al., 2008; Quintana et al., 2008; Veldhoenet al., 2008; Esser et al., 2009) because it was knownthat allergies, autoimmune diseases, or immunotoxicitycould be caused by chemicals and drugs, although theunderlying molecular mechanism and signaling path-ways were often unclear. Notably, xenobiotic ligandsof the AhR, especially TCDD, are known to adverselyaffect immune cells, which could simply reflect a disrup-tion of endogenous, AhR-driven homeostasis.In any case, the strong flexibility of AhR activation

by chemicals makes it an attractive pharmacologicaltarget, as evident from experiments on stem cell pro-liferation and inhibition of cancer cells (Safe andMcDougal,2002; Lawrence et al., 2008; Singh et al., 2009; Boitanoet al., 2010).Barrier organs—skin, gut, lung, and eyes, and oral

and genital mucosal tissues—specialize in discerningharm from good and can trigger or orchestrate adaptivephysiologic responses. Chemicals from the environment,including small molecules from symbiotic and patho-genic microbes, are first encountered at the barriers.With respect to immunologic functions, a network oftissue-specific immune cells is on watch constantly andattracts immune cells from the blood stream or lymphwhen needed. An important feature of barrier immunityis the distinction between harmful pathogens andharmless microorganisms and protein antigens (e.g.,from food), against which active tolerance must bemaintained. Moreover, we begin to understand that the(symbiotic) microflora and their metabolic products, inturn, shape immunity and tolerance (Schulz et al., 2011;Mavrommatis et al., 2013). Recently, it was discoveredthat AhR can be involved in disease tolerance and canalso be a sensor of bacterial danger by sensing bacterialpigmented virulence factors (Bessede et al., 2014; Moura-Alves et al., 2014).High levels of AhR expression were detected in hemato-

poietic stem cells, in cells of the innate immune system,and also in T-cell subsets and B cells. Exposure to AhRligands changes the function and fate of these cells.Research using various ligands and/or geneticallyengineered mouse models has demonstrated that AhRis involved in both correct development of naïve cellsand in effector functions during an immune response.Here, we review the functions of ligand-activated AhRsignaling in the specialized context of barrier organs,especially regarding the immunologic barrier againstthe environment.

II. Aryl Hydrocarbon Receptor Signaling

A. The Canonical Signaling Pathway

The AhR is a basic helix-loop-helix, Per-ARNT-Sim–

containing ligand-dependent transcription factor. It hasclose structural homology to proteins found in pathways

that regulate hypoxia and circadian rhythms (McIntoshet al., 2010). In the absence of ligand, AhR resides in thecytoplasm as a component of a chaperone complex thatincludes a dimer of Hsp90, together with the cochaper-ones p23, and AIP, the AhR-interacting protein alsoknown as ARA9, and XAP2 (reviewed by Denison et al.,2011). As depicted in Fig. 1, the translocation of AhRinto the nucleus is initiated upon ligand binding andphosphorylation of two protein kinase C sites adjacentto the nuclear localization sequences (Ikuta et al., 2004;McIntosh et al., 2010). AhR dissociates from its chaperonecomplex and forms a heterodimer with ARNT in thenucleus. The AhR–ARNT dimer then binds to upstreamregulatory regions of its target genes (e.g., the cytochromeP450 family 1 gene, CYP1A1). The target genes containcanonical aryl hydrocarbon receptor responsive elements(AhREs, also known as dioxin responsive element orxenobiotic responsive element) having the core sequence59-TNGCGTG-39. The complex with DNA then recruitscoactivators, which alter the chromatin structure into amore accessible configuration through histone acetyltrans-ferase and histone methyltransferase activities. Otherfactors are recruited, including the kinases IKKa, MSK1,andMSK2, the coactivators SP1,NCOA1,NCOA3,NCOA4,and p300, the BRCA1 tumor suppressor protein, and thegeneral transcription factor IIB. The latter is required fortranscriptional initiation by RNA polymerase II (Beischlaget al., 2008; Sartor et al., 2009; Taylor et al., 2009; McIntoshet al., 2010; Kurita et al., 2014).

B. Noncanonical Signaling

Cross-talk between the AhR and other signaling path-ways can lead to noncanonical mechanisms of actions ofthe AhR and AhR ligands. Several types of cross-talkhave been described as a result of molecular interactionbetween activated AhR and other proteins, by competitionfor transcriptional coactivators/repressors, by coactivator-like interactions, or by direct binding (reviewed byDenison et al., 2011). In the nucleus, AhR was foundto associate with the hypophosphorylated form of pRBresulting in growth arrest at the G1/S phase of the cellcycle (Levine-Fridman et al., 2004). AhR binds to thetranscription factor c-Maf, which is important for regula-tory type 1 T-cell differentiation (Apetoh et al., 2010). TheAhR also binds STAT1, resulting in nuclear factor-kBpromoter activity (Kimura et al., 2009). In addition,interactions of AhR have been described for the estrogenreceptor, b-catenin, Nrf2, RelA, and RelB (Kim et al.,2000; Vogel et al., 2004, 2007; Miao et al., 2005; Braeuninget al., 2011; Procházková et al., 2011).

Ligand binding to AhR also gives rise to alternativeactions (nongenomic) such as a rapid increase in intra-cellular Ca2+ concentration leading to downstreamproinflammatory responses mediated by c-src, COX2, andCCL1 (Nebert et al., 1993; N’Diaye et al., 2006; Fritscheet al., 2007; Matsumura, 2009; Zhou et al., 2013). Many ofthe details and the physiologic importance are still unclear.

Aryl Hydrocarbon Receptor in Immunology and Toxicology 261

AhR activity can be modulated by the activity ofother signaling pathways. A relevant example is theWnt/b-catenin pathway. This pathway is important forthe maintenance of cell pools through self-renewal ofhematopoietic stem cells, intestinal and gastric stemcells, hair and melanocyte stem cells, as well as neuraland liver progenitor cells (Clevers, 2006; Tan et al.,2006; Van Camp et al., 2014). It is also essential fordevelopment of early T precursor cells, both for theevents needed to generate mature naïve T cells andfor the peripheral differentiation of naïve T cells intoinflammatory and regulatory T-cell subsets (Clevers,2006; Ma et al., 2012; Xue and Zhao, 2012).Of great scientific interest is the role that the AhR

seems to play in progenitor cell expansion and differen-tiation in which the Wnt/b-catenin pathway is active(Laiosa et al., 2003; Singh et al., 2009; Boitano et al.,2010; Latchney et al., 2011; Procházková et al., 2011).Furthermore, a role for AhR in tissue regeneration hasbeen described in models of liver, fin, and cardiac tissueregeneration (Mathew et al., 2006; Mitchell et al., 2006;Hofsteen et al., 2013).

There is an increased understanding of different typesof cross-talk between the AhR and Wnt/b-catenin path-ways. The Wnt pathway leads to nuclear localization ofb-catenin, which can then interact with the DNA-bindingTCF/LEF proteins to activate transcription ofWnt/b-catenintarget genes. Signaling through b-catenin has beenfound to work cooperatively with AhR signaling in vitroand in vivo. Both basal and TCDD-stimulated CYP1A1expression are influenced by the presence of b-catenin(Braeuning et al., 2011). Wnt/b-catenin signaling is in-volved in both stem cell and cancer cell maintenance andgrowth. It is tightly controlled by the so-called destructioncomplex, and either too much or too little b-catenin hasnegative consequences (Duncan et al., 2005). Notably, theAhR acts as an adaptor protein in the CUL4B:AhRcullin 4B ubiquitin ligase complex (Ohtake et al., 2007). Byactivating the ubiquitin-proteasome system, the ligand-activated AhR targets several proteins for degradationincluding the AhR itself, b-catenin, and some sex steroidreceptors (Kawajiri et al., 2009; Ohtake et al., 2011).

Consistently, rapidly renewing tissues and cells suchas skin, gut, and blood cells are all targets of TCDD

Fig. 1. Scheme of AhR signaling. Events leading from AhR ligand binding to transcription. AhR resides in cytosol in a complex of chaperones and otherproteins (I). Upon ligand binding (I), AhR changes conformation. As a consequence, the complexing proteins are shed (II). c-src can contribute tocytoplasmic signaling on its own (IIa). Second, the nuclear translocation site in the bHLH region is exposed. Release of AIP permits docking of importin,which mediates nuclear import. AhR dimerizes with ARNT or other partners from other signaling pathways (IIIa and IIIb). Finally, the AhR:PARTNER complex binds to DNA at specific elements (either AhREs, NFkB elements, or others) and recruits transcription cofactors (IV). Targetedgenes are transcribed, leading to cell-specific transcriptome changes. Finally, AhR is exported and degraded (V). AhR signaling flexibility is shaped ondifferent levels, especially by formation of complexes with other proteins (cross-talk). AIP, AhR-interacting protein; ER, estrogen receptor; iNOS,inducible nitric oxide synthase; MMP, matrix metalloproteinase; NFkB, nuclear factor-kB; PGHS, prostaglandin synthase; RB, retinoblastoma protein;TNF-a, tumor necrosis factor a; XME, xenobiotic metabolizing enzymes.


toxicity (as detailed below in section III) and are affectedin AhR knockout and AhR-overexpressing mice. In-creased knowledge on the interaction of AhR withthe Wnt/b-catenin pathway is important to increaseour understanding of the role of the AhR in toxicologyas well as for the development of new therapeuticapplications.

C. Aryl Hydrocarbon Receptor–DependentTranscription

The AhR transcriptional induction profile has beenextensively studied. Binding to the AhRE consensussequences initiates transcription of a great number ofgenes. Originally, Daniel Nebert described a battery ofsix AhR-dependent genes coding for xenobiotic metab-olizing enzymes (Nebert, 1989). Today, the number ofgenes that respond to AhR activation by TCDD or otherexogenous compounds is proposed to be on the order of600 (Sartor et al., 2009; Dere et al., 2011). Among themost highly upregulated genes is the AhR repressor,which shares structural homology with AhR and ARNT(Mimura et al., 1999), and TiPARP (MacPherson et al.,2014). Both proteins repress AhR. In addition, the genescoding for the CYP1A and CYP1A2 enzymes, whichcontain multiple functional AhREs in their sharedenhancer region, is highly inducible. CYP1 enzymescan metabolically degrade small endogenous high-affinityligands and can thereby control constitutive AhR-dependent transcription.Transcriptomics and functional analyses, performed

in the presence or absence of exogenous ligands, haverevealed AhR target gene networks that control a broadspectrum of cellular functions. This concerns inter aliathe immune system and nervous system development,embryonic development, eye development, tube mor-phogenesis, angiogenesis, and patterning of blood ves-sels. In the comprehensive genome-wide analysis of theAhR target gene profile performed by Alvaro Puga andcoworkers, an unexpected large number of gene pro-moter regions exhibited significant AhR binding inunstimulated cells (Sartor et al., 2009). They reportedthat their gene ontology analyses gave unanticipatedresults that suggested a prominent role of the AhR inregulatory interactions with the Wnt/b pathway as alsodiscussed above (Sartor et al., 2009).

D. The Realm of Aryl Hydrocarbon Receptor Ligands:Agonists and Antagonists

The enigma of an endogenous or physiologic ligandhas intrigued the AhR research community for a longtime. A three-dimensional structure of the AhR or atleast its ligand binding domain (LBD) is still lacking.However, the approximate dimensions of the ligandbinding pocket and several critical amino acid residuesin the binding pocket are known. This knowledge hasenabled docking studies of proposed AhR ligands (Goryoet al., 2007; Soshilov and Denison, 2014).

By now, there are only a few types of molecules thathave been demonstrated to fit exceptionally well intothe LBD and to activate the receptor at concentrationsin the picomolar or nanomolar range. Among the high-affinity exogenous substances are several planar andlipophilic PHAHs of the dioxin, dibenzofuran, biphenyl,and azoxybenzene types sharing structural propertieswith the highly toxic TCDD (Nguyen and Bradfield,2008). In addition, some planar, lipophilic nonhalogenatedPAHs bind to the AhR with high affinity. These includebenzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene,benzo[k]fluoranthene, chrysene, dibenzo[a,h]anthracene,indeno[1,2,3,c,d]pyrene, 3-methylcholanthrene, andb-naphthoflavone (Till et al., 1999; Nguyen and Bradfield,2008). The bacterial pigment 1-hydroxyphenazine andthree pharmacological agents, StemRegenin 1 andGNF351 [N-(2-(3H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methyl-3-pyridyl)-7H-purin-6-amine] (two potent AhRantagonists), as well as VAF347 [(4-(3-chloro-phenyl)-pyrimidin-2-yl)-(4-trifluoromethyl-phenyl)-amine], havealso been observed to be high-affinity ligands (Lawrenceet al., 2008; Boitano et al., 2010; Smith et al., 2011; Moura-Alves et al., 2014). Many of these exogenous compoundsprobably bear structural similarities to physiologicligand(s).

The search for bona fide endogenous ligands thatinduce AhR signaling under physiologic conditions hasonly produced a small number of candidates. Theseinclude one metabolite of arachidonic acid, lipoxin 4A(Schaldach et al., 1999), and the four indoles, FICZ(6-formylindolo[3,2-b]carbazole) (Rannug et al., 1987),indirubin (Adachi et al., 2001), ITE [2-(19H-indole-39-carbonyl)-thiazole-4-carboxylic acid methyl ester] (Songet al., 2002), and ICZ (indolo[3,2-b]carbazole) (Bjeldaneset al., 1991). In addition, the indolic AhR proligandssuch as indole-3-carbinol (I3C) and tryptamine may beconverted in the body to the high-affinity ligands ICZ orFICZ (Bjeldanes et al., 1991; Vikström Bergander et al.,2012). L-Kynurenine, kynurenic acid, and cinnabarinicacid are other endogenous molecules that are formed viacatalytic breakdown of tryptophan. They can activateAhR signaling at levels that are physiologically orpathophysiologically relevant (DiNatale et al., 2010;Opitz et al., 2011; DeGroot and Denison, 2014; Loweet al., 2014).

Table 1 lists different types of small molecules andother factors that have been identified as AhR activatorsand ligands. In Fig. 2, we display three-dimensionalmodels of high- and low-affinity ligands and an antagonist.Often identification of a chemical as a ligand is accom-plished by assays using AhR-dependent transcription andelectrophoretic band shifts as proxy readouts. In a note ofcaution, such assays may not always reflect AhR ligandbinding in the LBD. In particular, oxidative stress,inflammatory mediators, and many physical stress factors(exposures that strongly suppress CYP1A1 transcrip-tion if added together with inducers such as TCDD or


b-naphthoflavone) have repeatedly been demonstratedto activate AhR-dependent transcription by themselveswithout direct binding (Gonder et al., 1985; Crawfordet al., 1997; Tanaka et al., 2005; Becker et al., 2006;McMillan and Bradfield, 2007; Afaq et al., 2009; Anwar-Mohamed et al., 2009; Wincent et al., 2012). Electro-phoretic band shifts exhibiting formation of AhR-AhREcomplexes have been detected with nuclear extracts fromcells cultured in the absence of added ligands and in cellssubjected to various stressful conditions (e.g., hyperoxia,metals, low temperature, immune cell activators, theproteolysis inhibitor MG132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal), or detached growth (Sadek and Allen-Hoffmann, 1994; Crawford et al., 1997; Monk et al., 2001;Santiago-Josefat et al., 2001; Tamaki et al., 2005; Elbekaiand El-Kadi, 2007). Some authors have suggested thepossibility that AhR-DNA interactions might result fromdisruption of the molecular interaction between thereceptor and the chaperone HSP90, through transientincreases in AhR or ARNT expression, or throughincreased production of endogenous ligands (Pongratzet al., 1992; Sadek and Allen-Hoffmann, 1994; Monket al., 2001; Santiago-Josefat et al., 2001; Kann et al.,2005; Tamaki et al., 2005; Elbekai and El-Kadi, 2007).There are two other mechanisms that are primarily

discussed with regard to AhR activation by factors thatdo not bind at all or that do not fit well into the AhRLBD (discussed further in section V). The first one claimsthat the AhR is promiscuous and AhR signaling can beactivated by structurally very diverse chemicals even atlow affinity (Denison et al., 2011; Soshilov and Denison,2014). The second one claims that seemingly genuineAhR activators inhibit the transcription and/or activity ofCYP1A1 and thereby inhibit the metabolic degradationof the endogenous ligand FICZ (Wincent et al., 2012).Soshilov and Denison (2014) have challenged this latterindirect mechanism. They employed site-directed muta-genesis of the LBD of AhR and describe one particularmutant, I319K, which was activated only by FICZ ina luciferase reporter system. They argue that indirectactivation via FICZ, by some other AhR activators tested,could not be involved because these other substances didnot activate the I319K mutant. However, FICZ-mediatedactivation of the I319Kmutant was only demonstrated ata relatively high concentration of FICZ (0.1 mM) and not

at the picomolar concentrations present in cell culturemedia under normal cell culture conditions (Oberg et al.,2005; Wincent et al., 2009). Therefore, indirect activationvia FICZ cannot be ruled out on the basis of observationswith this mutant.

III. Role of the Aryl Hydrocarbon Receptor in theBarrier Immune System

A. Differential and Variable Aryl HydrocarbonReceptor Expression Levels in Cells and Tissues,Including the Immune System

AhR expression differs significantly between tissues.It is not or only very weakly expressed in muscletissues, testes, kidney, and brain (both in human andin mouse) (Dolwick et al., 1993; Li et al., 1994; Frerickset al., 2007; Veldhoen et al., 2008). Of course, it is possiblethat some subsets of cells in these tissues express AhR athigh levels, which is not detected when analyzing thewhole tissue. Unfortunately, a careful analysis of distinctcell subsets is not available for many tissues. However,constitutive AhR expression is conspicuously high in liverand in the barrier tissues such as skin, lung, gut, andmucosal epithelia as well as in the placenta. These tissuescontain immune cells, many of which express AhR at highlevels. With respect to the adaptive immune system, AhRis low in naïve T cells, helper T cells Th1 and Th2, andregulatory T cells but is high in Th17 cells and both theinterleukin (IL)-17/IL-22–producing and IL-17/IL-22–nonproducing subsets of peripheral gd T cells (Veldhoenet al., 2008; Martin et al., 2009). AhR is present at lowlevels in naïve B cells from the spleen and is inducedupon polyclonal activation (Marcus et al., 1998). Asreviewed elsewhere, T and B cells are targets of AhR-activating factors. For instance, Th17 cells need AhRactivation for IL-22 secretion, and the immunoglobulinlocus can be suppressed by TCDD in an AhR-dependentfashion (Esser et al., 2009; Sulentic and Kaminski, 2011;Quintana and Sherr, 2013). Natural killer (NK) cellsexpress AhR at moderate levels, and AhR activationstimulates antitumor activity as well as resistance toinfections (Wagage et al., 2014). However, the examplesof naïve B cells or NK cells suggest caution on simplifyingprojections such as, “AhR is only relevant when expressedin higher amounts.” AhR can be induced by immunologic

TABLE 1Different types of AhR activators

The term AhR activator (rather than ligand) is used here to indicate that factors that do not bind to the LBD and trigger the canonical signaling pathway can also activateAhR, for instance, via changing the cellular levels of FICZ or modulating chaperones. For a more exhaustive list and references, see Wincent et al. (2012).

AhR Activators withNo or Very Low Binding Potential

Low-Affinity Ligands (Binding Affinity inthe Micromolar to Millimolar Range)

High-Affinity Ligands (Binding Affinity inthe Picomolar to Nanomolar Range)

Inflammatory mediators, glutathione depletion,hydrogen peroxide, hyperoxia, metals andmetalloids, neurotoxins, organic solvents,oxidized lipids, ozone, quartz, shear stressand miscellaneous materials, etc.

Drugs, biochemical model compounds,combustion products, food mutagens, hairdyes, hemes, humic acids, indoles, flameretardants, food additives, phytochemicals,plastic materials and additives, specificantagonists and blockers, etc.

FICZ, GNF351, ICZ, indirubin, ITE, lipoxin 4A,StemRegenin 1, TCDD and some otherPHAHs, some PAHs, VAF347, and1-hydroxyphenazine


stimuli and then become essential for downstream dif-ferentiation events. There are still many unknowns, whichneed to be solved before starting therapeutic manipulationof the receptor in a cell-specific manner. A summary of thecurrent knowledge regarding AhR expression levels inimmune cells is displayed in Fig. 3.Mouse models with no or modified AhR signaling

have been generated and are used in studies of theimmune system (Esser, 2009), and data emerge alsofrom newly bred conditional mouse lines in which AhRdeficiency is restricted to certain cells (Kiss et al., 2011;Di Meglio et al., 2014). AhR deficiency and AhR dys-regulation by xenobiotics have considerable consequencesfor barrier tissue development and function of cells of theadaptive and innate immune system. For example, micewith a constitutively active AhR in keratinocytes areafflicted with inflammatory skin diseases (Tauchi et al.,2005). Together, the findings argue strongly for the viewthat AhR is an evolutionary shaped, signaling path-way for physiologic immune functions. In the immunesystem, it balances and integrates environmental cuesinto an appropriate immune response.

B. Aryl Hydrocarbon Receptor in the Skin

The skin offers protection from environmental stressessuch as dehydration, UV light, mechanical trauma, andinfections. The skin is a layered organ, with the epidermison top, the much thicker dermis underneath, and finallythe subcutis as the innermost layer. Blood and lymphaticvessels reach into the dermis and serve as ports foremigrating and immigrating immune cells. Residentimmune cells are interspersed in both the dermis andepidermis.1. The Dermis. The dermis has high cell diversity;

the structural cells are the fibroblasts, which secretematrix proteins and hyaluronic acid. Interspersed amongthe fibroblasts are macrophages, mast cells, dendriticcells (DCs), and many T cells, all with differing levels ofAhR expression (Esser et al., 2013). For humans, it has

been estimated that more T cells are in the skin than inthe blood (Clark et al., 2006). Dermal fibroblasts expressthe AhR at high levels. Interestingly, they upregulatematrix metalloproteinase-1 but do not upregulate CYP1A1upon AhR ligand exposure. The former result relates tothe role of AhR in UV-mediated skin aging (Ono et al.,2013; Tigges et al., 2014), whereas the latter has beeninterpreted as a protection against generation of reactiveoxygen species in these mostly postreplicative cells.

2. The Epidermis. The structural cells of the epider-mis are the keratinocytes, which differentiate from theinnermost stem cell layer to the outer stratum corneumlayer, with enucleated corneocytes forming a water-tightfinal barrier. Keratinocytes gain AhR and CYP1A1expression along this “outward” differentiation (Jonesand Reiners, 1997; Swanson, 2004). Interspersed inthe epidermis are approximately 5% Langerhans cells(LCs, a subset of DCs) and, in mice, also approxi-mately 5% dendritic epidermal T cells (DETCs, witha skin-typical, invariant gd T-cell receptor (TCR) forwhich the antigen specificity is not known). Togetherwith the keratinocytes, they form an immune networkwith surveillance capacities (Jameson et al., 2004).Similar to dermal fibroblasts, keratinocytes expressAhR; LCs, DETCs, melanocytes, sebocytes, and mast cellsalso express AhR (Esser et al., 2013), as do tissue-residentCD8+ memory T cells (Zaid et al., 2014). Interestingly,a concomitant high constitutive expression of AhR re-pressor has been reported for LCs, DETCs, and fibroblasts(Haarmann-Stemmann et al., 2007; Jux et al., 2009; Tiggeset al., 2013).

3. Aryl Hydrocarbon Receptor–Mediated Skin Functions.It is commonly assumed that the high AhR levels in allskin cell types serve some physiologic purpose (Ma,2011). The search for such physiologic role(s) of AhR inthe epidermis is ongoing, using AhR overactivation onthe one hand and AhR-deficient mouse models on theother hand. Studies involving AhR-deficient mice in-dicated that AhR is needed for proliferation of melano-cytes and thus pigmentation (Jux et al., 2011). In humans,epidemiologic studies strongly suggested that air pollutionfrom soot and traffic (which contains AhR ligands) iscorrelated to increased extrinsic skin aging (Vierkötteret al., 2010). Studies with topical or systemic application ofAhR ligands (e.g., TCDD or FICZ) demonstrated that AhRsignaling is involved in degranulation and cytokine pro-duction of mast cells (Sibilano et al., 2012) and differenti-ation of sebocytes (Ju et al., 2011). In keratinocytes, AhR isinvolved in the UVB stress response (Fritsche et al., 2007)and antiapoptotic signaling in response to UV (Frauensteinet al., 2013). AhR presence in skin can protect againstUV-induced erythema or change the gene expression profileof keratinocytes. Moreover, epithelial-to-mesenchymaltransition and motility of keratinocytes is enhanced inAhR-deficient keratinocytes. Finally, AhR is involvedin UV-induced immunosuppression (Navid et al., 2013;Rico-Leo et al., 2013; Bruhs et al., 2015).

Fig. 2. Space-filling models of some AhR-interacting substances. TCDD,FICZ, and ICZ are all planar molecules that occupy approximately thesame space in the AhR LBD. They are active as AhR agonists in vitro andin vivo when present at 10213 to 1029 M concentrations. The endogenoustryptophan degradation product kynurenine and the synthetic moleculeCH223191, a commonly used AhR antagonist, bind to the AhR with loweraffinity (approximately 1026 M) (Kim et al., 2006; Opitz et al., 2011).Based on their lack of planarity and different size compared with thehigh-affinity agonists TCDD, FICZ, and ICZ, it is most likely that theirinteractions with the AhR also involve other mechanisms. CH223191,2-methyl-2H-pyrazole-3-carboxylic acid(2-methyl-4-o-tolylazo-phenyl)-amide.


4. Aryl HydrocarbonReceptor in Two Skin-Specific InnateImmune Cells. DCs phagocytize antigens and process andpresent them to T cells. In addition, they secrete cytokinesand factors, which provide a proinflammatory or immuno-suppressive milieu, shaping the differentiation patternof T cells into inflammatory or regulatory subsets. LCsare the resident DCs in the epidermis. In contrast withdermal DCs, they presumably are tolerogenic by default(Kaplan et al., 2008). LCs continuously sample skinantigens and migrate to the nearest lymph node topresent their antigen to T cells. Studies in AhR-deficientmice indicated that AhR is necessary for the maturationof LCs and their antigen-presenting capacity (Jux et al.,2009). Expression of maturation markers could be res-cued by GMCSF, a cytokine also produced by DETCs,which are lacking in AhR-deficient mice, as detailed below.As a consequence, AhR-deficient mice do not mount a

strong contact hypersensitivity response when challengedwith chemical haptens, such as fluorescent isothiocyanateor dinitrofluorobenzene.

DETCs are unconventional immune cells, with aninvariant gd TCR. As with all epithelial invariant gdT cells, they are generated exclusively in a tight timewindow in the fetal thymus. gd T cells secrete inflamma-tory cytokines quickly after antigen contact and arepivotal in fighting bacterial infections and killing tumorcells (Hayday et al., 1985; Girardi et al., 2002; Stridet al., 2009). They are an important source of IL-17 andIL-22, and thereby are also involved in autoimmunity(Roark et al., 2008). DETCs are abundant in the murineepidermis but not in the human epidermis. However,humans have a similar population of gd T cells in theirdermis (Holtmeier and Kabelitz, 2005). Apparently, humandermal gd T cells recognize stress-inducible molecules, such

Fig. 3. AhR expression levels in cells of the hematopoietic lineage. All cells of the immune system are derived from the HSC. HSCs differentiate andbranch out into further lineage precursor cells (the common lymphoid and common myeloid precursor). Differentiation is controlled intrinsically and/orextrinsically such as by cytokines or antigen contact. This graph depicts the main lineages and immune cell subsets. Arrows indicate lineagerelationships. Cells behind brackets are different descendants of the cells before the bracket; note that no attempt at depicting detailed lineagerelationships was made. Especially for the ILCs, lineage relationships have not been finally worked out. AhR expression levels in cells of thehematopoietic lineage are usually determined by quantitative polymerase chain reaction or Western blotting. It is difficult or not feasible to consolidateresults for different cell subsets from independent publications. For instance, in the article by Veldhoen et al. (2008), the different Th subsets werecompared side by side, indicating that Th17 cells had significantly higher levels than the other Th cell subsets, but in this study, expression levels ofDCs were not included. AhR expression in the liver is very high compared with other organs. However, some immune cells, such as gd T cells in theskin, can express AhR even higher, as evident when both tissues are tested in the same experiment. Data displayed in the graph are taken from articlescited in this review and in addition from Sherr and Monti (2013) for B cells and plasma cells. Asterisks (*) denote where it is known that AhR playsa role as a transcription factor in lineage-specific differentiation or maturation. Plus signs (1) denote where AhR is known for a functional role inexpressing an effector function of the cell (e.g., cytokine secretion, IDO production or other functions). For details, see the text. CILP, common innatelymphoid precursor; CLP, common lymphoid precursor; CMP, common myeloid precursor; GMP, granulocyte macrophage precursor; HSC, hema-topoietic stem cell; i, invariant; IDO, indolamine 2,3-dioxygenase; NKT, natural killer like T cell; PC, plasma cell; trTm, tissue-resident memory T cell;v, variant.


as MICA/B, as well as other ligands. In mice, DETCs playan important role in the control of inflammatory skinreactions, in wound healing, and in cancer surveillance(Strid et al., 2009). DETCs immigrate into skin shortlybefore birth and expand by proliferation within a fewweeksthereafter. This expansion does not take place in AhR-deficient mice. Their skin niche remains empty (in contrastwith TCRd2/2 mice, for which gd T cells are never made,and whose skin niche becomes filled with ab T cells).Recent work has indicated that the newly recognizedCD8+ tissue-resident memory T cells, which persistafter a viral infection has been resolved, can replaceDETCs in the skin. By contrast, epidermal tissue-resident memory T cells from AhR-deficient mice do notpersist in the epidermis after infection, highlightinga role for the AhR in the competition for space and/orappropriate contact with keratinocytes and LCs byinnate and adaptive T cells in the skin (Zaid et al.,2014). The loss of DETCs and the functional impair-ment of LCs in AhR-deficient mice may lead to barrierimpairment. As indicated for the human epidermis,activated AhR induces production of pivotal barrierproteins by keratinocytes and accelerates epidermalbarrier formation in mouse fetuses (Sutter et al., 2011).Accordingly, we have observed increased transepider-mal water loss in AhR-deficient mice, congruent withbarrier impairment (C. Esser, unpublished data). In-terestingly, an old and efficient therapy for atopicdermatitis (AD) is treatment with medical coal tar, asubstance rich in AhR ligands. Barrier impairmentis a prominent feature of AD. It was demonstrated inorganotypic skin cultures from AD patients that coal taractivated the AhR, induced epidermal differentiation,restored filaggrin expression, and improved skin barrierproteins in an AhR-dependent manner (van den Bogaardet al., 2013).In conclusion, the skin is a site of high AhR ex-

pression and high AhR ligand availability, and theAhR is involved in many skin functions, including theskin immune network and cell homeostasis. This canbe exploited pharmacologically. Examples are the useof medical coal tar described above as well as the use ofAhR antagonists to protect from UVB-associated skincancer (Tigges et al., 2014).5. High-Affinity Aryl Hydrocarbon Receptor Ligands

in the Skin. It is intriguing to think of skin-located,abundant AhR ligands as evolutionary drivers of thesefunctions. In the skin, AhR ligands are formed in situby several sources. UV light generates the high-affinityligand FICZ from tryptophan. In addition, hydrogenperoxide in skin of vitiligo patients can lead to formationof FICZ (Schallreuter et al., 2012). Common skin-residentyeasts, Malassezia spp., produce the AhR ligandsFICZ, ICZ, pityriacitrin, and malassezin (Wille et al.,2001; Gaitanis et al., 2008; Magiatis et al., 2013). LCsproduce AhR-dependently the immunosuppressive enzymeindoleamine 2,3-dioxygenase (Jux et al., 2009; Nguyen

et al., 2010), which converts tryptophan to kynurenines.Kynurenines as AhR ligands thus enhance their ownproduction, generating an immunosuppressive micromilieuin the skin (Mezrich et al., 2010; Nguyen et al., 2010).Kynurenines can also induce regulatory T cells (Tregs)directly (Mezrich et al., 2010). In addition, AhR activatorscan be present in topically applied cosmetics, creams, ordrugs.

C. Aryl Hydrocarbon Receptor in the Gut andConsequences for Gut Health and Microbiome

1. The Gut-Associated Immune System: A Paradigmof Immune Activation and Suppression. The gut is notonly in contact with dietary compounds but is also a siteof contact with numerous organic and inorganic en-vironmental compounds. The small and large intestinesharbor a highly diverse microflora; in humans, it isestimated that approximately 1014 bacteria live in thegut. Many of them are from the phyla firmicutes (65%)and bacteroidetes (30%) (Qin et al., 2010). Of note,food—its proteins, carbohydrates, and lipids—is a richsource of potential antigens, and immune responsesagainst food constituents can lead to allergies andintestinal inflammation. It is thus vital that the gut-associated immune system protects against infectionwhile it maintains tolerance against harmless antigens.The gut-associated immune cells specialize in this dualtask. The gut content is separated from the inner bodyonly by a single epithelial cell layer of intestinal epi-thelial cells (IECs) and a mucus layer secreted by gobletcells. The gut epithelium allows passage of nutrientsfrom the lumen into the blood stream, and the mucushas important functions in protecting the integrity of theepithelium. Interspersed among the IECs are intra-epithelial lymphocytes (IELs), many of them invariant gdT cells, others abTCRCD8aa+ T cells. Underneath theepithelium, in the lamina propria, more immune cellsreside, especially DC subpopulations and innate lym-phoid cells (ILCs). Breakage of the intestinal barrierresults in potentially deadly inflammation and sepsis.

2. Aryl Hydrocarbon Receptor in Innate ImmuneCells of the Gut. Excitingly, a reciprocal relationshipbetween gut bacteria and their metabolites and the gutimmune system exists, in which the AhR also plays arole. AhR-deficient RORgt+ ILCs (a major source ofintestinal IL-22) have reduced IL-22 expression; there-fore, AhR-deficient mice easily succumb to Citrobacterrodentium infection (Qiu et al., 2013). Qiu et al. (2013)indicated that treatment with FICZ (0.5 mg/kg) sig-nificantly increased the accumulation of RORgt+ ILCsin AhR+/2 or AhR+/+ mice but not in AhR2/2 mice. Inanother study, Zelante et al. (2013) analyzed lactobacil-lus species (nonpathogenic gut bacteria) and presentedevidence that these bacilli can generate AhR ligands inthe gut, such as indole-3-aldehyde, from tryptophan andthereby enhance AhR-dependent IL-22 production. In areporter assay, indole-3-aldehyde induced AhR-dependent


transcription but only at high concentration, suggestingthat it is a low-affinity ligand. However, indole-3-acetaldehyde, one of the products formed together withindole-3-aldehyde, can produce the high-affinity ligandFICZ (Rannug et al., unpublished data), and this could berelevant for the effects observed by Zelante and colleagues.IL-22 acts on epithelial cells and induces their productionof antimicrobial peptides (e.g., RegIIIg) and stimulatestissue regeneration. As a result, commensal bacteriamight outcompete pathogenic bacteria and preventcolonization with the fungus Candida albicans (Zelanteet al., 2013). Similar to the situation in skin keratinocytesand skin immune cells, the AhR is expressed highly inIECs and in cells of the gut-associated immune system.AhR-deficient mice have highly reduced IEL numbers inthe small intestine (Chmill et al., 2010; Li et al., 2011;Nakajima et al., 2013), which is associated with reducedlevels of IL-22 and thus a reduction of the antimicrobialpeptides RegIIIb and RegIIIg (measured in the ileum)and a higher microbial load in both the small intestineand colon (Li et al., 2011). The loss of IEL is cell intrinsic,because AhR-deficient bone marrow cells did not re-constitute the intestine in Rag2/2 mice (Li et al., 2011).Moreover, in the gut of AhR-deficient mice, subsets ofILC3s (Spits et al., 2013), ILC22 and CD32NKp46+

lymphoid tissue inducer cells, are lost over time afterbirth (Kiss et al., 2011; Lee et al., 2012). Again, failure ofILC3 to proliferate in AhR-deficient mice is an intrinsiceffect, because the AhR is needed to transcribe a cell-specific proliferation factor, c-kit (Kiss et al., 2011; Leeet al., 2012). As a consequence, no secondary lymphoidstructures, such as cryptopatches or innate lymphoidfollicles, are formed in the intestine of AhR-deficientmice, and they become susceptible to C. rodentiuminfection (a model for attaching and effacing Escherichiacoli EHEC in humans). ILC3s are characterized by IL-17and IL-22 secretion (Diefenbach, 2013; Spits et al., 2013).AhR-deficient mice are not only more susceptible toC. rodentium infection, they are also more susceptible todextran sodium sulfate (DSS)–induced murine colitis.DSS damages gut epithelial integrity and causes in-flammation and bacterial dissemination. AhR-deficientmice constituted with wild-type IELs did not succumb toDSS colitis, demonstrating the importance of IELs inreducing the damage (Lee et al., 2012).3. Systemic Effects of Oral Aryl Hydrocarbon Re-

ceptor Ligand Exposure. The role of the AhR on cellsof the adaptive immune system, especially on T cells,was also reviewed in Pharmacological Reviews (Quintanaand Sherr, 2013). As reviewed there, mice generateprotective Tregs if treated orally with the AhR agonistITE (originally isolated from swine lung) before subject-ing them to a protocol of immunization with myelinoligodendrocyte glycoprotein MOG35–55 to induce exper-imental allergic encephalitis (Gandhi et al., 2010;Quintana et al., 2010). The authors concluded that ITEmediates the effect by inducing CD103+ DCs in the gut,

which then support Treg generation. In another study,however, it was found that TCDD exposure of miceimpaired the capacity to establish stable oral toleranceagainst a food antigen (Chmill et al., 2010), presumablyby inducing an inflammatory state with higher levels ofIL-6 secretion in gut-derived DCs. It is possible and likelythat AhR ligands as such (affinities, degradability, etc.)as well as the exposure regimens are decisive in theoutcome for the tolerogenicity versus immunogenicitybalance in the gut (Duarte et al., 2013).

4. Role of Dietary Ligands in Development of theInnate Gut Immunity. Dietary AhR ligands are neces-sary for constitutive CYP1A1 levels and induction in thegut (Ito et al., 2007). An intriguing set of experimentsdemonstrated the role of dietary AhR ligands for innateimmune cell homeostasis in the gut. Experimental mousediets are for the most part grain based and contain highconcentrations (up to several grams per kilogram) of AhRactivators, such as polyphenols and glucosinolates.Colonies of mice were fed with a synthetic diet of verylow AhR activator content, and then newborn mice wereanalyzed 3 to 4 weeks after birth. Mice had a similarphenotype as AhR-deficient mice: low-level c-kit expres-sion, low ILC3 numbers, and because of ILC3’s tissueinducer capacity, no formation of proper cryptopatchclusters and isolated lymphoid follicles (Kiss et al.,2011). The frequency of lamina propria lymphocytes didnot change at this early stage. The phenotype could berescued by adding the proligand I3C, which is convertedto the high-affinity ligand ICZ in the acid milieu ofstomach, to the diet in wild-type but not AhR-deficientmice, indicating that both ligand and the AhR arenecessary for development of the innate gut immunecells. However, removal of AhR activators from the dietonce the ILC3s are established (approximately 6 weeksafter birth) had no effects on their further homeostasis.In another experiment, feeding adult mice for 3 weekswith a synthetic diet low in AhR activators reducedcyp1a1 expression in the intestine and led to a signifi-cant decrease in IEL (both gd T cells and ab TCR/CD8aa+ T cells). Again, addition of I3C rescued thephenotype (Li et al., 2011). It is not known what theminimal levels of AhR activators in the diet are, whichmight be needed for a “healthy” gut, because dose-response experiments have not been performed. How-ever, the results assert the need for the presence ofdistinct noncaloric micronutrients in the diet, both forformation of innate lymphoid follicles after birth and forhomeostasis of IELs later. In a note of caution, it is alsonot known at what dose “too much of a good thing”would occur. For example, the induction of CYP1A1, orensuing reactive oxygen species formation, could lead toadverse health effects. Experiments in mice indicatedthat TCDD could cause a loss of oral tolerance againstfood proteins (Chmill et al., 2010). In another setting,feeding of encapsulated AhR ligand ITE improved ex-perimental autoimmunity (Yeste et al., 2012). This


suggests that AhR activation in the gut by environmen-tal pollutants or possibly even food additives can haveboth adverse and beneficial effects on oral tolerance.Both findings—the importance of AhR signaling for

the development of innate immune cells and for oraltolerance—have potential therapeutic applications. Plant-derived compounds may be used in the future totherapeutically address diseases such as necrotizingenterocolitis of preterm infants (Neu and Walker, 2011) orinflammatory diseases of the gut. In addition, enhance-ment of oral tolerance might be useful in therapieswhereby autoantigen is given orally to dampen autoim-munity (Thurau et al., 2004; Yeste et al., 2012).

D. Aryl Hydrocarbon Receptor in the Lung

Lung epithelial tissue, similar to the gut, is a mono-layer of cells. Several subsets, including the basal stemcells, the ciliated cells, the goblet cells (for mucusproduction), and the brush cells, line the airways; theepithelium and its mucus layer become thinner towardthe alveoli. Underneath the epithelium is a basal matrixin which immune cells are found (mast cells, lympho-cytes, DCs, and ILC groups 1, 2, and 3). Lung tissueexpresses the AhR at high levels (Li et al., 1994; Frerickset al., 2007).The lung is exposed to AhR activators present in

airborne particulatematter from charcoal or wood burning,industrial exhausts, car emissions, cigarette smoke, orurban dust. PAHs are often part of air pollution particulatematter and have existed as environmental agents longbefore industrialization. Particulate matter has differenteffects depending on chemical composition as well as size,with nanoparticles (defined as 1–100 nm in diameter) evencapable of entering cells and the nucleus (Hemmerich andvon Mikecz, 2013). AhR expression by lung epitheliumallows detection and metabolic elimination of unwantedxenobiotic pollutants through induction of CYP1 enzymes.Another (mechanical) possibility is the regulation of mucussecretion; the genes for MUC5B and MUC5AC could betargeted by the activated AhR in a lung epithelial cell line(Wong et al., 2010; Chiba et al., 2011). In vivo mucusproduction enforces the physical protection of the lungepithelium. In another study, the AhR was demonstratedto protect fibroblasts from apoptosis caused by cigarettesmoke (Rico de Souza et al., 2011). At the same time, AhRactivity could be an integral part of a balanced immuneresponse in the lung. For example, exposure to airborneparticles (urban dust, diesel exhaust, cigarette smoke) viaintranasal administration was shown to increase IL-17expression in the lung of C57BL/6 mice (van Voorhis et al.,2013), and evidence emerges that IL-17 is important inlung diseases in both humans and mice (Ivanov et al.,2005).Epidemiologic studies have linked exposure to air

pollution to immunologic diseases of the respiratorysystem, such as asthma and chronic obstructive pulmonarydisease (COPD), innate immune cells, and polymorphisms

of inflammatory cytokines (Morgenstern et al., 2008;Vawda et al., 2014; Yu et al., 2014). To link air pollutionto these lung diseases, experimental exposures to AhRligands, AhR-deficient mice, and various infection orlung damage models are used to identify underlyingmechanisms and the role of the AhR.

1. Lung Inflammation. Lung inflammation can becaused by AhR activators, such as those present incigarette smoke. Studies in AhR-deficient mice dem-onstrated that exposure to cigarette smoke for onlya few hours on 3 consecutive days before euthanasiaenhanced neutrophil influx and levels of inflammatorycytokines IL-6, macrophage inflammatory protein 2,and prostaglandin E2 and increased tissue damage(Thatcher et al., 2007). The contribution of innate versusadaptive immune cells to lung pathology is still underinvestigation, and many inflammatory mediators havebeen implicated as well. Macrophages, cells critical forthe development of inflammatory processes, express theAhR and can be activated by TCDD to produce modulatorychemokines or release proinflammatory cytokines (Vogelet al., 2007).

2. Respiratory Viral Disease. The AhR is a modula-tor of antiviral immunity in the lung. In particular, thework by the group of Paige Lawrence demonstrated thecomplex relationship of AhR-mediated events in CD8T cells, DCs, neutrophils, and lung epithelium in influenzaA virus–infected mice (Vorderstrasse et al., 2003; Jinet al., 2014). AhR activation by TCDD decreased survivalfrom infection with a nonlethal dose of influenza A virus(Vorderstrasse et al., 2003), accompanied by doubledpulmonary neutrophil influx and suppression of expan-sion and differentiation of virus-specific effector CD8+

T cells (Lawrence et al., 2006). At the same time, AhRactivation decreased the immunostimulatory function ofDCs in lung-draining lymph nodes and reduced theirfrequency in the lungs (Wheeler et al., 2013; Jin et al.,2014). Neutrophil influx is mediated by the AhR in thelung epithelium because conditional AhR-deficient mice(lacking the AhR only in lung epithelium) had noincreased neutrophil influx upon TCDD exposure. BothTCDD and FICZ could modulate the anti–influenzaA response, although FICZ only affected CD8+ cells(Wheeler et al., 2014). Together, the data in mice highlightagain that the AhR is used in a barrier organ toorchestrate an appropriate immune response. It can,however, become unbalanced by persistent and highactivation. It is thus no surprise that epidemiologicevidence found that exposure to environmentally de-rived AhR activators correlates with increased respira-tory infections (Del Donno et al., 2002).

3. Lung Fibrosis. Similar to the skin and the gut,the murine lung harbors a unique population of invariantgd T cells (Vg6/Vd1), which are one source of IL-22production. Mice with either no gd T cells or with thelow-affinity AhR d-allele mount reduced IL-22 levelsin the lung upon Bacillus subtilis infection, leading to


more fibrosis. Administration of IL-22 improves lunginflammation and collagen deposition (Simonian et al.,2010). Together, the data demonstrate that protectiveIL-22 expression in this inflammation-induced pulmo-nary fibrosis is AhR dependent. Of note, unlike in theskin or the gut, gd T cells do not disappear from the lungin AhR-deficient mice (Simonian et al., 2010), indicatingthat intrinsic proliferation requirements and/or inter-cellular interactions are different in the lung tissue thanin skin and gut.

IV. The Aryl Hydrocarbon Receptor inToxicology and Physiology of the Human Skin,

Lung, and Gut

Species differences in TCDD toxicity and AhR signal-ing are well known. It is important to understand AhRsignaling in human biology, both for toxicology and forany possible therapeutic use of the AhR and its ligands.Research draws on results from epidemiology, accidentalenvironment exposures, tissue culture, and patient studies.In this section, we address some AhR events typical forhuman barrier organs and stem cells that go beyondimmune functions.In contrast with some animals, in which dioxins can

cause death after even tiny doses, dioxin-like compoundsare not acutely toxic/deadly in humans even at highexposures, and skin is often the main organ that isaffected by PHAHs. Chloracne, hyperpigmentation, andthickening of the skin on palms and soles have beenreported after exposure to a number of dioxin-like agents.Chloracne, the hallmark of dioxin toxicity in humans, ischaracterized by epidermal hyperplasia, disappearanceof sebaceous glands, and multiple epithelial cysts, whichare now described as hamartomas (Saurat et al., 2012).The same pathologic responses to TCDD, includinghyperkeratosis, epidermal hyperplasia, sebaceous glandinvolution, and intraepidermal keratinous cysts haveearlier been described in mice homozygous for mutationsin the hairless gene (hr/h) but not in other naked mice(Puhvel et al., 1982). The hairless protein is now knownto be a nuclear receptor corepressor that regulates hairregeneration by interacting with Wnt/b-catenin signaling(Beaudoin et al., 2005). Because the sebaceous gland andhair follicle epithelium have the same cellular source forrenewal, chloracne is now suggested to be caused by aswitch from a sebaceous to epidermal type of differenti-ation mediated through transient activation of thenuclear protooncogene c-Myc, which is a target of theWnt/b-catenin pathway (Panteleyev and Bickers, 2006;Saurat et al., 2012). A recent study with TCDD treat-ment of human skin samples exposed ex vivo andcultured human SZ95 sebocytes supports this suggestion.The authors show TCDD- and AhR-dependent shrinkageof sebaceous glands with a switch of sebaceous intokeratinocyte-like differentiation, eventually by targetingsebaceous progenitor cells (Ju et al., 2011). In addition,

hyperpigmentation of human skin and gingiva has beenobserved after exposure to both PAHs and PHAHs (Urabeand Asahi, 1985; Haresaku et al., 2007; Nakamura et al.,2013). The mechanisms behind AhR-mediated pigmenta-tion were first described by us (Luecke et al., 2010; Juxet al., 2011) and were recently further elucidated byNakamura et al. (2013). They provide evidence forinvolvement of both AhR and Wnt/b-catenin signalingin tobacco smoke extract–induced melanocyte activation(Nakamura et al., 2013). In addition, hyperkeratoticskin conditions have been reported in humans exposedto dioxins (Geusau et al., 2000). Correct palmoplantarkeratinization depends on intact Wnt/b-catenin signaling,and palmoplantar keratoses are seen in humans thatcarry mutations in the gene coding for R-spondin, anagonist of Wnt/b-catenin signaling (de Lau et al., 2012).

Induction of the AhR target gene CYP1A1 has beenrepeatedly documented in human skin and culturedhuman keratinocytes. For instance, increased benzo[a]pyrene hydroxylation capacity was observed in skin fromcoal tar–treated patients with dermatological diseases(Bickers and Kappas, 1978). UV light induces CYP1A1 inhuman skin (Katiyar et al., 2000) and cultured humankeratinocytes (Wei et al., 1999; Fritsche et al., 2007).Furthermore, the proposed endogenous AhR ligand FICZhas been detected in UVB-irradiated immortal humankeratinocytes (Fritsche et al., 2007), in skin samples fromvitiligo patients (Schallreuter et al., 2012), and in skinfrom patients with seborrheic dermatitis carrying com-mensal yeasts belonging to the genus Malassezia, whichconverts tryptophan to several AhR-activating com-pounds (Magiatis et al., 2013).

There are also systemic manifestations, which havebeen attributed to the exposure to PHAH in humansexposed in environmental accidents and at the work-place and for human volunteers (Suskind, 1985; Aoki,2001; Saurat et al., 2012). Chronic bronchitis–like symp-toms were observed in over 60% of Yusho patients that hadbeen exposed to cooking oil contaminated with polychlori-nated biphenyls and very potent AhR ligands of thepolychlorinated dibenzofuran type (Aoki, 2001). Epide-miologic reports from the 1976 Seveso accident in Italyhave associated TCDD exposure with amore than doubledincidence of COPD (Consonni et al., 2008). COPD affectsapproximately 200 million people worldwide and isglobally one of the leading causes of death. The diseasestarts with inflammation, influx of macrophages andneutrophils, and oxidative stress, which exacerbatesinflammation (Roca et al., 2013). Smoking, biomassburning, air pollution, and inhalation of fine dust arecausative agents (Brunekreef and Forsberg, 2005). Atmo-spheric particulate matter—known to be carriers of AhRligands—aggravates COPD. Further understanding therole of the AhR in COPD is an important area for futureresearch (van Voorhis et al., 2013).

Furthermore, nausea, vomiting, gastritis, and colitiswith multiple ulcers have been documented in many


epidemiologic studies of PHAH-exposed groups (Kuratsuneet al., 1972; Suskind, 1985; Urabe and Asahi, 1985). Inaddition, the dioxin-poisoned former Ukrainian presidentYushchenko initially suffered from severe gastritis (Sauratet al., 2012). A study performed with nonhuman pri-mates fed chlorinated biphenyls and triphenyls indicatedhyperplasia and dysplasia of the gastric mucosa withreplacement of the gastric acid–secreting parietal cellsby mucus-secreting cells (Allen and Norback, 1973). In-terestingly, constitutively active AhR causes similargastric lesions in mice, including gastric hamartomatoustumors (Andersson et al., 2005).These and other findings indicate important roles of

the AhR in rapidly renewing human tissues anddemonstrate key roles for interactions with the Wnt/b-catenin pathway, which seem to explain the skin dis-orders, including chloracne, that occur in humans exposedto TCDD.A compilation of the facts described in sections III

and IV is displayed in Fig. 4.

V. The Aryl Hydrocarbon Receptor: PromiscuousSensing of Chemicals or Metabolic Control of the

Endogenous High-Affinity Ligand FICZ?

From the evidence reported above and many otherstudies, it is evident that numerous chemicals caninterfere with the AhR signaling system. From this, itwas concluded that the AhR is a promiscuous sensor ofsmall chemicals; in other words, even low-affinity smallchemicals affect physiologic functions through bindingand activation. We think that the evidence is not con-clusive, first, because the very low affinity of many suchputative ligands is not sufficiently taken into account; andsecond, because the presence of the high-affinity ligandFICZ, omnipresent under cell culture conditions andprobably in all tissues, has to be considered in theinterpretation of the data. An influence on the metabolicturnover of the endogenous ligands could be a majorreason for the dysbalance observed with factors thatinduce or inhibit the activity of CYP1 enzymes (Fig. 5).The receptor is highly selective for molecules having

certain characteristics constricted by size, lipophilicity,and shape. A view that the AhR has few endogenousligands is supported by the fact that the AhR isevolutionary conserved and that the protein is presenteven in the most primitive vertebrate species. Thissuggests that it has a fundamental role in cellularphysiology. In addition, several results from studieswith AhR knockout mice have demonstrated functionsthat need the AhR under developmental stages andfor physiologic homeostasis processes without exoge-nously administered ligands (Gasiewicz et al., 2014).According to what is now known about FICZ, ICZ,

and possibly ITE and other indoles, these molecules seemto fulfill the role of endogenous ligands that maintainimportant functions in biologic systems (Ma, 2011). In

particular, FICZ binds to the AhR with the highestaffinity yet reported (Rannug et al., 1987; Nguyen andBradfield, 2008). FICZ also binds to frog and bird AhRswhere TCDD is relatively inactive. This indicates evolu-tionary conservation of the FICZ response in TCDD-insensitive species, suggesting its physiologic importanceas an AhR ligand (Laub et al., 2010). FICZ and ICZare also efficiently autoregulated by the induced CYP1enzymes (Wei et al., 2000; Bergander et al., 2004; Wincentet al., 2009). In fact, the catalytic efficiency of CYP1A1 forFICZ is close to the limit of diffusion (Wincent et al., 2009).FICZ has been identified in human skin (Wincent et al.,2009; Schallreuter et al., 2012; Magiatis et al., 2013), andFICZ-derived sulfate conjugates have been detected inhuman urine (Wincent et al., 2009). The current datasuggest that FICZ can be formed via different path-ways that lead to formation of indole-3-acetaldehyde,the precursor of FICZ, such as enzymatic deaminationof tryptamine and oxidation of tryptophan by intracellularoxidants (Rannug et al., unpublished data). Occurrence ofICZ may be restricted to the gut. It is entirely conceivablethat there are more ligands with similar chemicalstructures that are capable of fulfilling AhR-regulatedfunctions in a tissue-specific manner.

The important role of efficient control over themetabolicturnover of FICZ is illustrated by recent data suggestingthat FICZ can stimulate various signaling pathwaysand thereby affect critical and specific cell functions (seeTable 2).

It is now well recognized that unintended, ill-timed,or prolonged activation of the AhR by many exogenoussmall molecules may lead to expression, synthesis, andactivity of many proteins. This in turn may disturb tightlycontrolled and transient physiologic functions (Mitchellet al., 2006; Stockinger et al., 2014). This seems to explainthe toxicity of TCDD, which is unmatched by any otherhuman-made substance. This fact has to be consideredwhen trying to identify pharmacological agents withtherapeutic potential by interfering with AhR signalingbecause they may disturb such tightly controlled andtransient signals and lead to unwanted toxicity. Thegrowing knowledge about factors that interfere withAhR signaling and the high specificity and flexibility,however, demonstrates ample possibilities for preven-tive actions.

VI. Therapeutic Potential of Aryl HydrocarbonReceptor Ligands

The therapeutic potential of the AhR has been recog-nized early, especially in the context of cancer (Safe et al.,1999, 2013). The effects of AhR ligands as agonists orantagonists are dependent on several factors includingligand structure, specific gene, and cell context–dependentexpression of important cofactors or coactivators. Thus,selective aryl hydrocarbon receptor modulators (SAhRMs)have been developed with a view to clinical applications


(Safe et al., 2013). For instance, 12 weeks of feeding6-methyl-1,3,8-trichlorodibenzofuran inhibited metastasisin a mouse model of prostate tumorigenesis, in partby inhibiting prostatic vascular endothelial growth factorproduction prior to tumor formation (Fritz et al., 2009).SAhRMs aim to modify AhR activation in such a way thatthe beneficial effects outweigh toxic effects (e.g., thoseknown from dioxins). The complexity of AhR signalingoutcomes is evident from the above sections. There areseveral obstacles. First, an AhR ligand or proligand mightbe rapidly metabolized, and both the original compoundand metabolites may be involved in activities independentof AhR signaling. In particular, this is true for anti-oxidants, such as resveratrol and quercetin. Other com-pounds are prone to formation of DNA adducts or proteinadducts, which may cause adverse effects. An exampleof this is glucosinolates, which form DNA adducts(Schumacher et al., 2014). The overall efficiency of aSAhRM will depend on its chemistry and on multiplefactors including the dose, pharmacokinetics, absorption,persistence, and other properties that must be consideredfor development of a new pharmaceutical. For example,poor absorption limits the effectiveness of the AhR ligand,GNF351 (Fang et al., 2014), which exhibited promising

anticancer properties in head and neck cancer cells inculture (DiNatale et al., 2012). Yeste et al. (2012) in-vestigated the delivery to the gut of the ligand ITE, whenITEwas packed in nanoparticles (Yeste et al., 2012). Indeed,this approach may enhance bioavailability.

Two other sources of AhR ligands with pharmaceu-tical potential are currently considered. First, variousdietary phytochemicals were identified as potentialligands. Phytochemicals are non-nutrient plant com-pounds, which can be classified into phenolic compounds,terpenoids, alkaloids, phytosterols, and carotenoids.Phytochemicals are absorbed, metabolized, and effluxedinto the blood stream by IECs. Biotransformation iscatalyzed by enzymes controlled by the AhR battery, thecytochrome P450s and phase II enzymes. For a numberof polyphenols, especially the large group of flavonoids,immunomodulation and AhR activation were demon-strated (Denison and Nagy, 2003; González et al., 2011).Second, AhR ligands are generated in cells and can be,for example, products of amino acid metabolism. Thetryptophan-derived ligand FICZ is discussed above indetail. Other tryptophan derivatives, the kynurenines,and further downstream products such as the newlyidentified cinnabarinic acid (stimulates IL-22) display

Fig. 4. Summary of involvement of AhR signaling in skin, gut, and lung epithelial barrier functions and barrier immunity as they are currently known.In the skin and gut, epithelium immune cells (DCs and specialized T cells) are integrated. In the lung, immune cells are not interspersed but can berecruited. The little cartoons indicate this. For details and references, see the text. AhR is highly expressed in epithelial and various immune cells ofskin, gut, and lung. Endogenous functions of AhR signaling were unraveled through work with either AhR-deficient (full or conditional) mice or byexperimentally activating AhR signaling in healthy or diseased situations. In general, AhR signaling must be leveled appropriately to avoid eithertoxicity or immunologic impairment.


large potential as well (Mezrich et al., 2010; Lowe et al.,2014). Although global gene expression profiles andpromoter studies have shed some light on the activitiesof SAhRMs, AhR antagonists and agonists, and selec-tive modulators for other receptors (Sun et al., 2004;Nohara et al., 2006; Suzuki and Nohara, 2007; Naultet al., 2013), the factors important for the selectivity of aspecific AhR ligand are not well understood and requirefurther investigation.One example highlights the complexities of using

AhR ligand in therapy. With regard to the immunesystem, the capacity of AhR agonists to shift the balancebetween inflammatory, autoimmune-prone Th17 responsesand immunosuppressive responses driven by Tregs is of

considerable interest. Among the Th cell subsets, only Th17cells express AhR at high levels. Under in vitro Th17differentiating culture conditions, AhR ligands (even lowamounts of FICZ produced by light from tryptophanpresent in cell culture medium) promote the generationof the Th17 phenotype, including secretion of IL-17 andIL-22. It was thus unexpected that in mice in vivo, theseverity of two crippling autoimmune diseases (exper-imental allergic encephalitis or experimental allergicarthritis) as well as inflammatory colitis, decreased uponexposure to TCDD, ITE, or FICZ (Quintana et al., 2008;Monteleone et al., 2011; Nakahama et al., 2011). Autoim-munity is driven by Th17 cells and ameliorated orprevented by antigen-specific regulatory T cells. In the

TABLE 2Biologic functions demonstrated to be influenced by FICZ

Biologic End PointReferences

In Vitro In Vivo

Antitumor activity Shin et al., 2013Cell growth and expression of

growth factor genesDu et al., 2005; Fritsche et al., 2007; Kostyuk et al., 2012;

John et al., 2013Smith et al., 2013

Circadian rhythmicity Mukai and Tischkau, 2007Expression of AhR target genes Wei et al., 2000; Oberg et al., 2005; Sciullo et al., 2008; Nair

et al., 2009; Wincent et al., 2009; Lee et al., 2010; Lueckeet al., 2010; Mohammadi-Bardbori et al., 2012; Rico-Leoet al., 2013; Sumida et al., 2013

Jönsson et al., 2009; Laub et al., 2010; Wincentet al., 2012; Odio et al., 2013

Genome rearrangement Okudaira et al., 2010, 2013Immune response Apetoh et al., 2010; Bankoti et al., 2010 Quintana et al., 2008; Veldhoen et al., 2008;

Martin et al., 2009; Monteleone et al., 2011;Jeong et al., 2012; Pauly et al., 2012; Qiu et al.,2012; Sibilano et al., 2012; Duarte et al., 2013;Wheeler et al., 2013; Zhou et al., 2013

Nuclear receptor cross-talk Ekins et al., 2008; Reschly et al., 2008; Bunaciu and Yen,2013

Fig. 5. Hypothetical schemes of FICZ homeostasis changes by unbalanced AhR and cytochrome P450 activity. (A) The high-affinity AhR ligand FICZ isconstantly produced in the organism. It induces CYP1A activity, which in turn degrades FICZ, thereby creating a homeostatic level of FICZ. (B) Other,high-affinity AhR ligands, in particular the nondegradable TCDD, or very high concentrations of low-affinity AhR ligands can compete for AhR binding,resulting in constantly high CYP1A activity and depletion of FICZ. (C) AhR activation can also occur by factors that block/inhibit CYP1A, leading toexceptionally high FICZ levels because FICZ is no longer degraded. In both scenarios (B) and (C), FICZ homeostastic levels are changed, andaberrant transcription of many genes could occur. The events would be similar for other endogenous high-affinity homeostatic ligands, such as ICZin the gut.


same studies, an amelioration of experimental allergicencephalitis and experimental allergic arthritis wasreported for AhR-deficient mice or for AhRd/d mice(which express a low-affinity AhR allele), adding furtherto the puzzle. Originally, the phenomena were explainedby an induction of Tregs, but reports on direct FoxP3induction by TCDD in vivo and in vitro are conflicting(Quintana et al., 2008; Duarte et al., 2013), and noenhancement of Tregs was evident in mice engineeredto have a constitutively active AhR in T cells (Quintanaet al., 2008; Funatake et al., 2009). DCs provide themilieu for T-cell differentiation in vivo, and AhRactivation is involved in their tolerogenicity (Haubenet al., 2008; Nguyen et al., 2010). Conceivably, thesituation in vivo integrates AhR activation in a morecomplex fashion than deduced from in vitro data, andin vitro data must be viewed with caution (Duarteet al., 2013).Although more data are needed, it is evident that

different AhR ligands were beneficial in the experi-mental model autoimmune diseases. In Table 3, wehave listed diseases for which AhR agonists/antagonisthave demonstrated promising results in cell or animalstudies and which have been suggested as havingpotential for clinical applications. Many substances andprocedures are already protected by patents; however,only few clinical trials exist thus far. Of note in thiscontext, there are drugs that had been approved forhuman use (thus need no toxicity evaluation in clinicaltrials) long before their appreciation as AhR agonists orantagonists. Omeprazole and tranilast are examples thatare currently under investigation for new applications inthe context of their AhR activities (Jin et al., 2012). Inaddition, although novel AhR-interacting drugs are notyet approved, AhR-activating nutraceuticals, such as

I3C, resveratrol, and 3,39-diindolylmethane, are beingmarketed.

VII. Conclusions

Organisms must sense and respond to changes in theenvironment in a meaningful way. The AhR is a proteincapable of sensing the (bio)chemical and physical environ-ment. Together with its few high-affinity physiologicligands, such as FICZ and ICZ, it serves functions in cellproliferation, differentiation and cell functions. The signal-ing system can be influenced by low-affinity chemicals andeven physical stressors such as oxidative stress, whichinterfere with turnover of the “true” endogenous ligands. Inaddition, xenobiotic high-affinity ligands can disrupt thesystem, often to an extent that toxicity ensues, bymimicking or competing with endogenous high-affinityligands. Barrier organs come in contact with manyexogenous chemicals, including human-made pollutants,and chemicals characteristic for bacteria and fungi.Barrier organs are characterized by high cell turnoverand express the AhR at high amounts in both theirstructural and immune cells; indeed, barrier organs haveimportant immune functions. The AhR was demon-strated to have many functions in immune cells andother cells; notably, AhR functions are very cell specificand diverse. The high sensitivity and specificity of AhRsignaling are not fully understood. However, availabledata, in animal models, animal and human cell linesand organotypic cultures, as well as human epidemiol-ogy and human studies, suggest high potential for bothpreventive and therapeutic intervention. More researchis needed to understand the full complexity of AhRbiology to avoid risks and master the opportunities oftherapy and prevention.

TABLE 3AhR agonists suggested as potential therapeutic agents built on evidence from cell culture or animal studies

Name of Agonist or Antagonist Disease Involved Cell/AnimalStudies

HumanTrials Reference

E/Z-2-Benzylindene-5,6-dimethoxy-3,3-dimethylindan-1-one

UVB-induced skin damage (cancer,skin aging)

Cells 10 volunteers Tigges et al., 2014

6-Methyl-1,3,8-trichlorodibenzofuran(a so-called selective AhR modulator)

Breast cancer, prostate cancer,metastasis, lung cancer

Cells No Fritz et al., 2009; Zhanget al., 2012

Tranilast Breast cancer Cells, animals No Prud’homme et al., 2010Leflunomide Melanoma Cells No6,29,49-Trimethoxyflavone and GNF351 Head and neck cancer Cells No DiNatale et al., 2012Sulindac, leflunomide, 4-hydroxytamoxifen,

mexiletine, and omeprazoleCancer, bone preservation Cells No Jin et al., 2012

VAF347 Inflammation, transplant acceptance,IL-22 induction

Cells, animals Hauben et al., 2008;Lawrence et al., 2008;Baba et al., 2012

Aminoflavone Breast cancer, renal cancer Phase II Loaiza-Pérez et al., 2004;Callero et al., 2012

ITE Autoimmunity Animals No Yeste et al., 2012FICZ Irritable bowel disease Human cells,

animalsNo Monteleone et al., 2011

Kynurenine inhibitor Glioblastoma Human cells No Opitz et al., 2011FICZ, kynurenine, and 3,39-diindolylmethane NK cell antitumor activity Cells, animals No Shin et al., 2013StemRegenin 1 Expansion of hematopoietic stem cells Cells No Boitano et al., 2010Coal tar AD Skin cultures van den Bogaard et al.,

2013


Acknowledgments

The authors thank Stephen Safe, Paige B. Lawrence, Heike Weighardt,Bettina Jux, Thomas Haarmann-Stemmann, Jean Krutmann, and UlfRannug for critical reading of the manuscript and helpful comments.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Esser,Rannug.

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