Environmental stimuli are known to contribute to psoriasis pathogenesis and that of other autoimmune diseases, but the mechanisms are largely unknown. Here we show that the aryl hydrocarbon receptor (AhR), a transcription factor that senses environmental stimuli, modulates pathology in psoriasis. AhR-activating ligands reduced inflammation in the lesional skin of psoriasis patients, whereas AhR antagonists increased inflammation. Similarly, AhR signaling via the endogenous ligand FICZ reduced the inflammatory response in the imiquimod-induced model of skin inflammation and AhR-deficient mice exhibited a substantial exacerbation of the disease, compared to AhR-sufficient controls. Nonhematopoietic cells, in particular keratinocytes, were responsible for this hyperinflammatory response, which involved upregulation of AP-1 family members of transcription factors. Thus, our data suggest a critical role for AhR in the regulation of inflammatory responses and open the possibility for novel therapeutic strategies in chronic inflammatory disorders.

Combining the analysis of psoriasis patient skin biopsies with that of a mouse model of psoriasiform inflammation, we showed that AhR signaling in nonhematopoietic cells plays a central role in preventing excessive skin inflammation.

In order to investigate the potential influence of environmental factors on inflammatory skin disease, we have focused on the ligand-dependent transcription factor aryl hydrocarbon receptor (AhR), which responds to environmental stimuli and plays an important role in the maintenance of intestinal homeostasis. Work from our lab and others has shown that AhR-deficient mice have an inherent weakness of the gut barrier (). The wide expression of AhR in several cell types of the skin suggests a role for AhR signaling also at this barrier organ. The AhR is a member of the bHLH-PAS family of transcription factors best known for mediating the toxic effects of environmental contaminants such as TCDD (dioxin) and a range of other xenobiotic substances. However, its evolutionary conservation from invertebrate species onward points to a physiological role that does not involve xenobiotic stimuli (). Endogenous ligands of AhR are found as indoles and flavonoids either of dietary origin (e.g., indolo[3,2-b]carbazole, ICZ) () or, like the high-affinity ligand 6-formylindolo[3,2-b]carbazole (FICZ), derived from tryptophan metabolism via UV or visible light exposure (), which has been found to be physiologically relevant in human skin ().

The skin is the organ most exposed to environmental insults, and its complex cellular network constitutes an immunological barrier that is crucial for the maintenance of homeostasis (). It is therefore likely that inflammatory disorders of the skin involve environmental factors. One such disorder is plaque-type psoriasis, a disease with complex etiopathogenesis, characterized by epidermal hyperproliferation and prominent immune infiltrates (). Cross-talk between innate, adaptive, and epithelial or stromal cells, such as keratinocytes and fibroblasts, underpins the disease pathology (). A total of 36 disease-associated loci have been identified as contributing to psoriasis (). Environmental risk factors, on the other hand, remain less well defined on a mechanistic basis (). Although no mouse model can fully recapitulate the development and features of psoriasis (), topical application of the imiquimod (IMQ)-containing cream Aldara induces a psoriasiform skin inflammation, which exhibits most of the crucial traits () including acanthosis, parakeratosis, neutrophil recruitment, and involvement of the IL-23-IL-17-IL-22 pathway (), and is thus increasingly used to dissect the mechanisms of psoriasis pathogenesis.

The AhR is thought to be involved in extensive cross-talk with other transcription factors and multiple signaling pathways, which makes the systematic analysis of physiological interactions a challenging task. To gain more insights about how AhR modulates keratinocyte activation, we focused on the early phase of the IMQ treatment and performed microarray analysis of whole skin from Ahror Ahrmice treated for 2 days with IMQ. By using Ingenuity pathway analysis, we identified a number of psoriasis-annotated transcription factors regulated by the IMQ treatment and found an overrepresentation of the activator protein-1 (AP-1) family of transcription factors ( Figure 7 A). AP-1 regulates a range of biological mechanisms, including keratinocyte differentiation and proliferation () and epithelial cell immune activation (). Also, the AP-1 family member JunB, whose activity is transcriptionally regulated (), is increased in psoriasis and localizes to keratinocyte nuclei within the hyperplastic epidermis of psoriasis lesions (). We found the expression of Junb significantly increased in the skin of Ahrmice as compared to Ahrmice in the early phase (day 2) of IMQ-induced inflammation ( Figure 7 B), whereas whole skin isolated from FICZ-treated wild-type mice showed reduced Junb levels ( Figure 7 C), suggesting that AhR can control the expression of this AP-1 family member. Moreover, isolated keratinocytes from Ahrmice significantly upregulated JunB protein ( Figure 7 D) and mRNA ( Figure S6 ) in response to IL-1β and iCM. Finally, inhibition of the AP-1 pathway with the inhibitor Tanshinone IIA () resulted in decreased expression of proinflammatory genes in Ahrkeratinocytes ( Figure 7 E). These findings show that AhR controls the expression of other transcription factors responsible for establishing an inflammatory transcriptional program in keratinocytes, e.g., AP-1, and thus constitutes a crucial regulator in the development of skin inflammatory processes.

Plots show mean ± SEM; n = 3–6 wells or mice per group. Results from one representative experiment of two independent experiments are shown. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

(E) mRNA expression of Csf2 and Csf3 in murine keratinocytes stimulated for 24 hr with recombinant IL-1β (10 ng/ml) with or without Tanshinone (1 μM). Data expressed as fold change over medium control.

(A) Heat map visualization of IMQ-regulated, psoriasis-annotated transcription factors in whole skin of Ahr +/− and Ahr −/− mice on day 2 of IMQ treatment. Green color indicates decreased and red color increased gene expression, expressed as mean fold change in IMQ-treated as compared to corresponding untreated mice. AP-1 family transcription factors are marked in bold. Asterisks indicate genes differentially expressed in Ahr −/− versus Ahr +/− skin at day 2 of IMQ treatment.

Taken together, these results show that nonhematopoietic skin cells, particularly keratinocytes, require AhR to control expression of inflammatory mediators in response to inflammatory stimuli, such as IL-1β. Therefore we conclude that AhR deficiency in both epithelial and stromal cells results in a cell-intrinsic overreaction to inflammatory stimuli, leading to exacerbated skin pathology in vivo.

Furthermore, similar hyperresponsiveness was found in human keratinocytes upon knockdown of AHR expression. Thus, normal primary keratinocytes in which AHR expression was reduced by AHR-SiRNA showed overexpression of proinflammatory mediators compared to keratinocytes transfected with a nontargeting SiRNA (cSiRNA) ( Figures 6 D and S5 B). Similar results were also obtained in the spontaneously transformed keratinocyte cell line HaCaT, in which AHR had been stably silenced (AHR-silenced HaCaT), when compared to cells transfected with an empty vector (EV-HaCaT) ( Figure 6 E;).

Our results showed that physiological AhR activation can ameliorate the inflammatory program in nonhematopoietic skin cells. In order to discriminate between the inflammatory response of epithelial and stromal cells, we assessed the response of Ahr-sufficient or -deficient keratinocytes to the immune activators produced in the early phase of IMQ-induced skin inflammation. We therefore stimulated keratinocytes from Ahrand Ahrmice in vitro with conditioned medium (CM) from in-vitro-reactivated skin cells obtained from either naive (nCM) or 2-day IMQ-treated (iCM) wild-type mice. Ahrkeratinocytes responded to iCM by significantly overexpressing proinflammatory cytokine and chemokine mRNA as compared to heterozygous controls ( Figure 6 A). Ahrfibroblasts also displayed an increased response as compared to their Ahrcounterparts, although to a lesser extent (data not shown). IL-1β is one of the mediators in the early phase of the IMQ-induced skin inflammation model (), and this cytokine was overrepresented in the conditioned medium obtained from wild-type IMQ-treated mice, far exceeding other cytokines tested such as TNF, IL-23, and IL-17A (data not shown). Indeed, recombinant IL-1β could replace iCM, causing comparable upregulation of inflammatory markers on keratinocytes ( Figure 6 B), whereas neutralizing IL-1β abrogated the proinflammatory response to the skin cell conditioned medium ( Figure S5 A). In addition, we also found increased expression of Il1r1 in ex vivo Ahrkeratinocytes ( Figure 6 C).

Results from one representative experiment of two or three independent experiments are shown. Plots show mean ± SEM; n = 3–6 wells per group. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001.

(E) mRNA expression of proinflammatory mediators in human keratinocytes HaCaT cell lines, stable transfected with an empty vector (EV-HaCaT, white bars), or in which AhR had been stable silenced (AhR-silenced HaCaT, black bars), and stimulated for 24 hr with human recombinant IL-1β. Data expressed as fold change over medium control.

(D) mRNA expression of proinflammatory mediators in human primary keratinocytes, transiently transfected for 48 hr with a nontargeting control SiRNA (cSiRNA, white bars) or in which AhR was transiently silenced (AhR-SiRNA, black bars), and stimulated for further 24 hr with human recombinant IL-1β (10 ng/ml).

(A) mRNA expression of proinflammatory mediators in Ahr +/− (white bars) and Ahr −/− (black bars) murine keratinocytes stimulated for 24 hr with conditioned medium nCM and iCM. Data are expressed as fold change over stimulation with iCM over nCM medium.

BM chimeras in which the nonhematopoietic cells were of wild-type origin, whereas the hematopoietic compartment was either Ahr(dAhR) or Ahr deficient (dAhR), did not show increased inflammation above that observed in control mice ( Figures 5 E–5G). Disease severity was not increased in AhrRag1versus AhrRag1(data not shown), emphasizing that the cross-talk between adaptive immune cells and epidermal cells is essential for the exacerbated pathology observed in Ahr-deficient mice. Therefore, AhR deficiency in the nonhematopoietic skin compartment is necessary and sufficient for the development of an exacerbated psoriasiform skin response in the presence of a fully functioning adaptive immune system.

Next, we addressed the contribution of Ahr deficiency in nonhematopoietic skin cells such as keratinocytes and fibroblasts. We generated bone marrow (BM) chimeras in which hematopoietic cells were of wild-type origin, whereas the nonhematopoietic compartment was either Ahr(rAhR) or wild-type (rAhR) by reconstituting AhrRag1and control AhrRag1hosts with BM from Ahr wild-type donors. AhR deficiency in the nonhematopoietic compartment recapitulated the hyperinflammatory phenotype of full AhRmice with exacerbated epidermal pathology ( Figures 5 A and 5B ), increased neutrophil recruitment ( Figure 5 C), overexpression of inflammatory markers, and reduced keratinocyte differentiation ( Figure 5 D). In contrast, expression of both IL-17 and IL-22 was not different in the two experimental groups, making it unlikely that these cytokines are responsible for the hyperinflammatory skin response of Ahrmice. In line with this, treatment of Ahrmice with neutralizing antibody to IL-17A did not improve their exaggerated response ( Figures S4 E–S4G).

Plots show mean + SEM; n = 5–7 mice per group. Results from one representative experiment of two independent experiments per each set of chimeras are shown. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

In order to identify the AhR-expressing cell type responsible for the hyperinflammatory skin response seen in Ahrmice, we generated mice with conditional deletion of AhR in distinct immune cell subsets in the skin. AhrCd11c.Cre or AhrRag1.Cre mice, in which AhR is deleted in DCs and some macrophage subsets or in T and B cells, respectively, were treated with IMQ in order to address the contribution of these cells to the exacerbated skin inflammation seen in complete AhR deficiency. qPCR analysis confirmed the deletion of AhR in DCs or in T cells of these mice ( Figures S4 A and S4C). Lack of AhR in DCs or macrophages did not result in increased inflammation above that observed in control AhrCd11c.Cre mice ( Figures 4 A–4D and S4 B). Lack of AhR in T and B cells resulted in increased skin acanthosis and reduced keratinocyte differentiation, but no difference in skin scaling, expression of the majority of inflammatory mediators, or number of neutrophils when compared to control mice ( Figures 4 E–4H and S4 D). These observations ruled out a role for DCs or macrophages in driving the exacerbated skin inflammation seen in Ahrmice, but left open the possibility that activation of the AhR pathway in T or B lymphocytes is important for skin homeostasis.

Plots show mean + SEM; n = 4–6 mice per group. Results from one representative experiment of two independent experiments per mouse strain are shown. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

AhR Deficiency in Dendritic Cells or in T and B Cells Does Not Recapitulate the Phenotype of Ahr −/− Mice

Figure 4 AhR Deficiency in Dendritic Cells or in T and B Cells Does Not Recapitulate the Phenotype of Ahr −/− Mice

Next, we asked whether deliberate triggering of the AhR pathway would have a beneficial effect on skin pathology as seen in human psoriatic skin. To this end, wild-type mice received intraperitoneal injections of either FICZ or olive oil (vehicle control) daily during the course of IMQ treatment. FICZ administration upregulated expression of Cyp1a1 mRNA in the skin, as compared to mice receiving vehicle only ( Figure 3 A), and resulted in attenuated psoriasiform skin inflammation, with milder parakeratosis and cell infiltration ( Figure 3 B), statistically significant reduction in epidermal and scale thickness ( Figure 3 C), and reduced expression of proinflammatory mediators ( Figures 3 D and S3 ). Thus, activation of the AhR pathway in vivo results in amelioration of psoriasiform skin pathology.

Treatment of AhR-deficient (Ahr) and AhR-heterozygous littermate control (Ahr) mice with IMQ over a 5-day period resulted in scaling and parakeratosis of the Stratum corneum and epidermal acanthosis and widespread inflammatory infiltrates, as seen by visual inspection ( Figure S2 A) and in H&E-stained skin sections ( Figure 2 A). Untreated skin of Ahrand Ahrlittermate controls was histologically indistinguishable, but upon treatment the thickening of both epidermis and Stratum corneum was significantly increased in Ahrmice ( Figure 2 B). Nevertheless, the exacerbated skin pathology elicited by IMQ treatment in the absence of AhR signaling abated after termination of treatment. Quantitative RT-PCR analysis of inflamed skin from Ahrmice revealed statistically significant increased expression of growth factors and chemokines involved in neutrophil attraction (Csf2, Csf3, Cxcl1, Cxcl5) and of antimicrobial peptides typically present in psoriasis lesions (S100a7a, S100a8), as well as reduced expression of the keratinocyte differentiation marker Krt10 ( Figure 2 C). Moreover, mRNA expression of a number of proinflammatory cytokines, including Il17a, Il17c, Il23, Il22, and Il1b, which are crucially involved in psoriatic skin inflammation (), was significantly increased in the skin of Ahr-deficient mice ( Figure 2 D). Upregulation of Il1b mRNA in Ahrmice preceded that of IL-17 ( Figure S2 B) and remained increased at protein level on day 5 ( Figure S2 C). Although there was substantial infiltration of T cells producing IL-17 cell type cytokines, the majority of which were γδ T cells ( Figure S2 D) as previously reported (), absolute numbers of IL-17- and IL-22-producing CD4 and γδ T cells did not differ between Ahrand Ahrmice ( Figure 2 E). In agreement with our earlier finding (), AhR-deficient γδ T cells appear to make more IL-17 on a per cell basis, thus accounting for the increase in IL-17 observed in the skin of Ahrmice ( Figures S2 E and S2F). In line with the increase in neutrophil-recruiting chemokines, there was significantly more neutrophil infiltration into the skin in Ahrmice ( Figure 2 F), whereas the number of both macrophages and dendritic cells (DCs) did not differ between the two groups ( Figure S2 G), and we did not observe substantial infiltration of IL-17-producing CD8 T cells or innate lymphoid cells (data not shown). Thus, absence of AhR signaling led to heightened inflammation and exacerbated skin pathology. This phenotype was not restricted to the psoriasisform inflammation induced by IMQ, but also extended to a model of delayed-type hypersensitivity (DTH) skin reactions in Ahr-deficient mice, which showed enhanced neutrophil infiltration and increased inflammatory chemokine expression (data not shown).

Plots show mean ± SEM, n = 3–5 mice per group or mean and values of individual mice, n = 8 mice per group. Results from one representative experiment of two independent experiments are shown. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

(E) Number of CD4 and γδ T cells expressing IL-17 (left) and IL-22 (right) per cm 2 skin of IMQ-treated Ahr +/− (white bars) and Ahr −/− (black bars) mice obtained by intracellular cytokine staining.

(C and D) mRNA expression of proinflammatory mediators and keratinocyte differentiation marker in whole skin from Ahr +/− (open circles) and Ahr −/− (filled circles) mice at day 5.

(B) Quantification of epidermal (left) and scale thickness (right) of Ahr +/− (open circles) and Ahr −/− (filled circles) at different time points after initiation of IMQ treatment.

In order to address whether the AhR pathway has a role in human skin pathology, we investigated whether AhR activation via the agonist FICZ or inhibition via the antagonist CH-223191 () would cause transcriptional changes in psoriasis-related genes. Full-thickness skin biopsies were obtained from lesional (L) and uninvolved nonlesional (NL) skin of eight psoriatic patients not receiving any systemic treatment and from healthy human subjects (N) serving as control ( Table S1 available online). The biopsies were quartered and one segment was reserved for RNA sequencing without any treatment. The remaining three quarters were cultured with DMSO (vehicle control), FICZ, or CH-223191 for 16 hr. After treatment all samples were subjected to RNA sequencing. Analysis of the untreated L and NL psoriasis skin samples provided us with genes differentially regulated in the two tissue types, or the “psoriasis transcriptome.” The top 25 most significantly regulated genes ( Table S2 ) were in keeping with published data sets () with several members of the S100 protein family significantly upregulated in L versus NL skin. Next, we identified the transcriptional changes induced by exposure to AhR agonist or antagonist. A flowchart describing step by step our filtering criteria is shown in Figure S1 . We identified 884 AhR-modulated genes, defined as genes significantly regulated by either the agonist or the antagonist in at least one out of the three tissue types analyzed (L, NL, H) ( Figure 1 A). As expected, expression of CYP1A and CYP1B1, well-characterized AhR-target genes, were found to be highly upregulated in the presence of agonist and downregulated by the antagonist in all three tissue types ( Figure 1 B). The list of AhR-modulated genes was reduced to 41 genes belonging to the “psoriasis transcriptome,” which were mainly upregulated in untreated L skin ( Figure 1 C, psoriasis-upregulated genes are shown in bold, and Table S3 ). Out of these, 29 (70%) were reduced after FICZ-induced AhR activation. This effect was most prominent in L skin, as confirmed by qRT-PCR showing a downward trend for the top five regulated genes (IFIT, RSAD2, IFIT3, CMPK2, MX2), which reached statistical significance for IFIT1 ( Figure 1 D). Conversely, treatment with AhR antagonist was able to increase expression of these genes in NL skin, with statistically significant fold change increase for all genes validated by qRT-PCR ( Figure 1 E). Ingenuity pathway analysis showed that 26 out of 41 psoriasis-relevant genes modulated by AhR belong to the type I and II IFN pathway, which is known to be upregulated in psoriasis ( Figure S1 ). Thus, AhR appears to play a critical role in modulating the severity of psoriasis. In order to study the influence of AhR in more detail, we employed the mouse model of IMQ-induced psoriasiform inflammation.

(E) qPCR validation of six top modulated genes upregulated in NL skin by antagonist-induced AhR inhibition. Box and whiskers denoting minimum and maximum values are shown. Wilcoxon signed rank test was performed.

(D) qPCR validation for six top modulated genes downregulated in L skin by agonist-induced AhR activation. Box and whiskers denoting minimum and maximum values are shown. Wilcoxon signed rank test (for CMPK2) or paired t test (all other genes) was performed.

(C) Heat map of genes belonging to the “psoriasis transcriptome” (upregulated genes shown in bold) and modulated by FICZ or CH-223191. Genes are sorted by decreasing fold change for agonist effect on L skin.

(B) Heat map of known AhR-target genes modulated by FICZ and CH-223191. Color indicates mean fold change, with green representing decreased and red increased gene expression.

(A) Venn diagram showing genes significantly regulated by either FICZ or CH-223191 in at least one out of the three tissue types analyzed (L, NL, H).

Lesional (L) and nonlesional (NL) skin biopsies from eight psoriasis patients were quartered: one quarter of each was used as baseline and the remaining three quarters were cultured with either vehicle control, the AhR agonist FICZ, or the AhR antagonist CH-223191 for 16 hr. Whole-skin biopsies from five healthy donors (H) were processed in the same way. All samples were subjected to RNA sequencing.

Discussion

Di Meglio et al., 2011 Di Meglio P.

Perera G.K.

Nestle F.O. The multitasking organ: recent insights into skin immune function. Lowes et al., 2013 Lowes M.A.

Russell C.B.

Martin D.A.

Towne J.E.

Krueger J.G. The IL-23/T17 pathogenic axis in psoriasis is amplified by keratinocyte responses. Lowes et al., 2013 Lowes M.A.

Russell C.B.

Martin D.A.

Towne J.E.

Krueger J.G. The IL-23/T17 pathogenic axis in psoriasis is amplified by keratinocyte responses. Pantelyushin et al., 2012 Pantelyushin S.

Haak S.

Ingold B.

Kulig P.

Heppner F.L.

Navarini A.A.

Becher B. Rorγt+ innate lymphocytes and γδ T cells initiate psoriasiform plaque formation in mice. Van Belle et al., 2012 Van Belle A.B.

de Heusch M.

Lemaire M.M.

Hendrickx E.

Warnier G.

Dunussi-Joannopoulos K.

Fouser L.A.

Renauld J.C.

Dumoutier L. IL-22 is required for imiquimod-induced psoriasiform skin inflammation in mice. van der Fits et al., 2009 van der Fits L.

Mourits S.

Voerman J.S.

Kant M.

Boon L.

Laman J.D.

Cornelissen F.

Mus A.M.

Florencia E.

Prens E.P.

Lubberts E. Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. −/− mice. However, our data show that immune cells are not the main cause for the hyperreactivity observed in Ahr-deficient mice. Instead, the response of nonhematopoietic skin cells, primarily keratinocytes but also skin fibroblasts, to inflammatory stimuli was severely dysregulated in the absence of AhR. However, in vivo blockade of this cytokine was not sufficient to dampen the exaggerated skin response of Ahr-deficient mice (data not shown), suggesting that multiple inflammatory pathways are involved. The contribution of environmental factors to the pathogenesis of inflammatory disorders is well known, but the nature and mode of action of such stimuli remains ill defined. We show here that environmental signals transmitted via AhR dampen the inflammatory response in both mouse and human skin. Lack of Ahr causes hyperinflammation, whereas deliberate AhR activation with the endogenous ligand FICZ ameliorates the inflammatory profile in both human psoriasis samples and the mouse model of psoriasiform skin inflammation. Our data emphasize the cross-talk between cells of the immune system and nonhematopoetic cells during inflammation, and it is now widely recognized that such interactions crucially underpin both the homeostasis of the skin environment and its dysregulation in diseases such as psoriasis (). The focus for therapeutic intervention in psoriasis is currently on modulating inflammatory immune parameters such as IL-17, IL-12, IL-23, or TNF (), which are the immune drivers of skin pathology in both human disease and the mouse model. In agreement with the literature (), we found many parameters linked to the IL-17 program highly upregulated in IMQ-induced skin inflammation and even further exacerbated in Ahrmice. However, our data show that immune cells are not the main cause for the hyperreactivity observed in Ahr-deficient mice. Instead, the response of nonhematopoietic skin cells, primarily keratinocytes but also skin fibroblasts, to inflammatory stimuli was severely dysregulated in the absence of AhR. However, in vivo blockade of this cytokine was not sufficient to dampen the exaggerated skin response of Ahr-deficient mice (data not shown), suggesting that multiple inflammatory pathways are involved.

Chodaczek et al., 2012 Chodaczek G.

Papanna V.

Zal M.A.

Zal T. Body-barrier surveillance by epidermal γδ TCRs. −/− mice ( Kadow et al., 2011 Kadow S.

Jux B.

Zahner S.P.

Wingerath B.

Chmill S.

Clausen B.E.

Hengstler J.

Esser C. Aryl hydrocarbon receptor is critical for homeostasis of invariant gammadelta T cells in the murine epidermis. Li et al., 2011 Li Y.

Innocentin S.

Withers D.R.

Roberts N.A.

Gallagher A.R.

Grigorieva E.F.

Wilhelm C.

Veldhoen M. Exogenous stimuli maintain intraepithelial lymphocytes via aryl hydrocarbon receptor activation. Girardi et al., 2006 Girardi M.

Lewis J.M.

Filler R.B.

Hayday A.C.

Tigelaar R.E. Environmentally responsive and reversible regulation of epidermal barrier function by gammadelta T cells. Kadow et al., 2011 Kadow S.

Jux B.

Zahner S.P.

Wingerath B.

Chmill S.

Clausen B.E.

Hengstler J.

Esser C. Aryl hydrocarbon receptor is critical for homeostasis of invariant gammadelta T cells in the murine epidermis. −/− skin although they displayed some abnormalities in the epidermis. All bone-marrow chimeras lacked epidermal γδ T cells because the hosts were Rag1 deficient and epidermal γδ T cells cannot be reconstituted by BM from adult mice, yet only those chimeras with Ahr deficiency in the nonhematopoietic compartment displayed the full phenotype seen in Ahr-deficient mice. It was important to delineate contributions by different cell types because it was suggested, for instance, that the epidermal TCR-γδ population, which interacts with keratinocytes () and is absent in Ahrmice (), may fulfil protective functions in cutaneous inflammation (). However, mice with selective Ahr deficiency resulting from Rag1 Cre-mediated deletion also lack this population, yet did not display the widespread overreaction seen in Ahrskin although they displayed some abnormalities in the epidermis. All bone-marrow chimeras lacked epidermal γδ T cells because the hosts were Rag1 deficient and epidermal γδ T cells cannot be reconstituted by BM from adult mice, yet only those chimeras with Ahr deficiency in the nonhematopoietic compartment displayed the full phenotype seen in Ahr-deficient mice.

Both psoriasis pathogenesis in humans and the IMQ model of psoriasiform skin inflammation in the mouse are T cell dependent and rely on the cross-talk between adaptive immune cells and epidermal cells. Thus, it is not surprising that in the complete absence of effector T cells, such as in Rag1−/− mice, lack of AhR in keratinocytes did not result in increased disease severity.

Wohn et al., 2013 Wohn C.

Ober-Blöbaum J.L.

Haak S.

Pantelyushin S.

Cheong C.

Zahner S.P.

Onderwater S.

Kant M.

Weighardt H.

Holzmann B.

et al. Langerin(neg) conventional dendritic cells produce IL-23 to drive psoriatic plaque formation in mice. −/− mice, indicating that these cells, although important for establishing inflammation in the first place, were not drivers for the overreaction seen in Ahr−/− mice. Of note is the effect of Ahr deletion in different cell types on expression of IL-17 and IL-22. In agreement with previous data ( Martin et al., 2009 Martin B.

Hirota K.

Cua D.J.

Stockinger B.

Veldhoen M. Interleukin-17-producing gammadelta T cells selectively expand in response to pathogen products and environmental signals. Duarte et al., 2013 Duarte J.H.

Di Meglio P.

Hirota K.

Ahlfors H.

Stockinger B. Differential influences of the aryl hydrocarbon receptor on Th17 mediated responses in vitro and in vivo. IL-23-producing myeloid DCs are critical for the IMQ model, whereas plasmacytoid DCs (pDCs) and the type I interferon pathway are dispensable in this mouse model (). We did not detect infiltration of pDCs in the skin (data not shown) and Ahr deficiency restricted to CD11c-expressing antigen-presenting cells did not recapitulate the hyperinflammation of Ahrmice, indicating that these cells, although important for establishing inflammation in the first place, were not drivers for the overreaction seen in Ahrmice. Of note is the effect of Ahr deletion in different cell types on expression of IL-17 and IL-22. In agreement with previous data (), we found that complete Ahr deficiency resulted in higher levels of IL-17 probably due to the predominant infiltration of γδ T cells, which produce more IL-17 in Ahr-deficient mice. This effect, however, was not evident when Ahr deficiency was restricted to T and B cells, suggesting that additional interactions with other Ahr-deficient cells, e.g., APCs, contribute to IL-17 induction. Furthermore, in contrast to our previous demonstration that in-vitro-differentiated Th17 cells or γδ T cells require AhR stimulation for IL-22 production, IL-22 was readily detectable in the skin of Ahr-deficient mice. It is conceivable that the inflammatory milieu in the skin can provide other factors that could override the requirement for AhR. Finally, the systemic application of the AhR ligand FICZ caused a reduction rather than an increase in IL-17 and IL-22. This is in contrast to the effect of FICZ in vitro or in localized application during EAE, but consistent with its suppressive effect when administered systemically ().The complexities of cellular interactions in an inflammatory environment that shape these variables require further dissection. However, in the context of our study, we contend that IL-17 and IL-22, although important for the development of psoriasiform inflammation, are not the reason for the exacerbated response of Ahr-deficient mice.

Bowcock et al., 2001 Bowcock A.M.

Shannon W.

Du F.

Duncan J.

Cao K.

Aftergut K.

Catier J.

Fernandez-Vina M.A.

Menter A. Insights into psoriasis and other inflammatory diseases from large-scale gene expression studies. Nestle et al., 2005 Nestle F.O.

Conrad C.

Tun-Kyi A.

Homey B.

Gombert M.

Boyman O.

Burg G.

Liu Y.J.

Gilliet M. Plasmacytoid predendritic cells initiate psoriasis through interferon-alpha production. The IMQ model of psoriasiform skin inflammation in mice and the pathogenesis process in psoriasis have different kinetics, inflammatory components, and cellular mediators, but nevertheless share critical immunopathological features. Although dispensable for the IMQ model, both type I and II interferon pathways play an important role in human psoriasis (). Type I IFN is critical in the early phases of disease initiation in a clinically relevant skin xenotransplant model where it triggers activation and expansion of autoimmune T cells, leading to fully fledged psoriasis plaque formation ().

Bowcock et al., 2001 Bowcock A.M.

Shannon W.

Du F.

Duncan J.

Cao K.

Aftergut K.

Catier J.

Fernandez-Vina M.A.

Menter A. Insights into psoriasis and other inflammatory diseases from large-scale gene expression studies. Austin et al., 1999 Austin L.M.

Ozawa M.

Kikuchi T.

Walters I.B.

Krueger J.G. The majority of epidermal T cells in Psoriasis vulgaris lesions can produce type 1 cytokines, interferon-gamma, interleukin-2, and tumor necrosis factor-alpha, defining TC1 (cytotoxic T lymphocyte) and TH1 effector populations: a type 1 differentiation bias is also measured in circulating blood T cells in psoriatic patients. Johnson-Huang et al., 2012 Johnson-Huang L.M.

Suárez-Fariñas M.

Pierson K.C.

Fuentes-Duculan J.

Cueto I.

Lentini T.

Sullivan-Whalen M.

Gilleaudeau P.

Krueger J.G.

Haider A.S.

Lowes M.A. A single intradermal injection of IFN-γ induces an inflammatory state in both non-lesional psoriatic and healthy skin. A strong IFN-γ genomic () and cellular () signature is present in psoriasis, and intradermal injection of IFN-γ has been shown to induce several molecular and histological features characteristic of psoriatic lesions in both healthy and psoriatic human skin (). AHR ligation in human skin biopsies strikingly modulated type I and II interferon pathways, particularly normalizing the proinflammatory signature present in L skin. On the other hand, pharmacological blockade of the AHR pathway in ex vivo human skin biopsies and genetic deletion of Ahr in the mouse model resulted in an exacerbation of the inflammatory skin signature, whereas activation of the pathway ameliorated both.

Perhaps not surprisingly, human and mouse data did not show mechanistic similarities in terms of genes or pathways affected by AhR. This could be due to the very different ontogeny of the inflammatory skin response, which is provoked by a single agent and short lasting in the mouse, whereas it is multifactorial and chronic in patients. In contrast, the lack of effect of AhR-mediated activation on other relevant psoriasis gene signatures in human samples, such as the antimicrobial response and tissue remodeling, which are affected in the mouse model, could be the result of the short-term culture of the skin biopsies. However, both murine and human keratinocytes lacking AhR were overreactive to proinflammatory stimuli, suggesting shared aspects of pathology, which may diverge later on in the chronic phase of human psoriasis.

Zenz et al., 2005 Zenz R.

Eferl R.

Kenner L.

Florin L.

Hummerich L.

Mehic D.

Scheuch H.

Angel P.

Tschachler E.

Wagner E.F. Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins. Haider et al., 2006 Haider A.S.

Duculan J.

Whynot J.A.

Krueger J.G. Increased JunB mRNA and protein expression in psoriasis vulgaris lesions. Swindell et al., 2013 Swindell W.R.

Johnston A.

Voorhees J.J.

Elder J.T.

Gudjonsson J.E. Dissecting the psoriasis transcriptome: inflammatory- and cytokine-driven gene expression in lesions from 163 patients. It remains to be clarified what the direct targets of AhR are and how these are linked to the inflammatory networks that are affected. The postulated extensive interaction of AhR with other transcription factors, which may be cell type specific, presents a substantial challenge in identifying direct as well as indirect AhR-mediated effects in inflammatory responses. We found dysregulation of the expression of the AP-1 family member Junb, which was substantially upregulated in the inflamed skin of Ahr-deficient mice and could be readily induced in in vitro keratinocytes stimulated with IL-1β. Moreover, blocking the AP-1 pathway dampened the increase of proinflammatory genes in Ahr-deficient keratinocytes. Whereas deletion of Junb together with Jund in mouse keratinocytes resulted in a skin phenotype resembling psoriasis (), JUNB is upregulated in psoriatic skin () and a recent comprehensive meta-analysis of the psoriasis transcriptome has revealed an enrichment of AP-1-binding sites among psoriasis-increased genes and pinpointed the increased expression of JUNB in psoriasis skin to keratinocytes (), suggesting a role for the AP-1 pathway and JunB as a critical checkpoint of epidermal homeostasis.

Thus, beneficial effects of AhR activation open the possibility of therapeutic intervention in chronic inflammatory skin disease, but further research is needed to understand the mechanism underlying the physiological consequences of AhR signaling in the immune system.