AD is a highly prevalent inflammatory skin disease caused by skin barrier dysfunction and chronic immune activation (27
). No mechanism-based therapies are available for AD patients and treatment is mainly focused on symptom relief, predominantly targeting dry skin, itch, and inflammation with emollients and corticosteroids. Since the seminal work of the McLean Lab has underscored the importance of skin barrier abnormalities as an etiological factor in AD, we focused on the effects of coal tar on the epidermal biology of this disease (1
). Our results reveal a key role of the AHR signaling pathway in breaching the vicious circle of chronic inflammation by improving skin barrier function and interfering with Th2 cytokine signaling and downstream pathological processes.
The immunopathogenesis of AD has been studied directly on patients or in animal models based on allergic immune responses (29
). We have recently developed in vitro organotypic models for psoriasis and AD (18
), and have shown that the application of genetic techniques in these models could be used to study epidermal aspects of diseases (32
). Others showed that FLG
knockdown in human skin equivalents could mimic AD skin by impaired epidermal differentiation and barrier dysfunction (33
). We have chosen keratinocyte-only human skin equivalents to evaluate the effect of coal tar on the epidermis, thereby excluding the possible effects of coal tar and subsequent AHR activation on other skin cell types such as fibroblasts and immune cells, as these cells could in turn affect epidermal processes. Genetic approaches to modify keratinocyte gene expression were introduced in this system by using an siRNA knockdown approach to prove the role of AHR, or by using patient-derived keratinocytes harboring one FLG
-null allele. These findings illustrate the power of skin equivalents to study human skin diseases and, to a certain extent, to replace mouse models.
In the early seventies, attempts were made to unravel the mechanism of action of coal tar therapy. Suppression of DNA synthesis (35
) and induction of a granular layer using the mouse tail test (37
) have been reported. The mouse tail test has previously demonstrated the effects of coal tar on epidermal differentiation. Since the discovery of FLG
mutations in AD, it is now widely accepted that epidermal barrier dysfunction is key in the pathophysiology of this disease. These new insights are strongly guiding current efforts in AD research and therapy development. The accelerated filaggrin expression and barrier function in fetal mouse skin due to dioxin-mediated AHR activation (17
) suggested to us that there is a potential role for AHR in AD treatment. Here, we show that coal tar activates AHR signaling and found that AHR regulates and induces epidermal differentiation and stimulates filaggrin expression in keratinocytes harboring a monoallelic FLG
loss-of-function mutation. These findings may have a major impact on the reappraisal of coal tar therapy, which is gradually being abandoned by dermatologists due to its cosmetic side effects, safety concerns, and a hitherto unknown mechanism of action.
The AHR signaling pathway is currently of great interest. Though it was previously only considered to be related to immunotoxicity, recent findings have underscored this pathway’s physiological role. AHR appears to be predominantly involved in the development and function of the immune system (14
). Genetic approaches in mice, however, have also revealed that AHR signaling plays a role in epidermal pathophysiology. Deficiency or constitutive expression of AHR interaction partners ARNT (44
) and NRF2 (46
), respectively, has detrimental effects on epidermal differentiation and barrier function, whereas AHR transgenic mice develop inflammatory skin lesions with hyperkeratinization (47
). All these studies show that disturbance of normal AHR signaling, either by genetic approaches or TCDD exposure, leads to epidermal abnormalities. Our study is the first to demonstrate the beneficial therapeutic effects of AHR activation on epidermal differentiation and barrier function in a skin disease characterized by low levels of terminal differentiation proteins and the resultant poor barrier function. The therapeutic use of AHR ligands appears controversial in light of the complete ban on all AHR ligands instituted by the pharmaceutical industry because of the widely known dioxin-related toxic effects of AHR activation. However, recent interest in the AHR signaling pathway has led to a variety of reports describing diverse downstream effects by different exogenous and endogenous AHR agonists. Nowadays, therefore, it is recognized that the specific ligand-receptor interaction determines the downstream effects of AHR activation, and that it is not merely a matter of drug potency. Hence, CYP1A1 induction and/or AHR activation are not synonymous with dioxin-like toxicity.
In addition to the improved epidermal differentiation and filaggrin induction by coal tar–mediated AHR activation, we made the exciting observation that coal tar normalizes histopathological and molecular hallmarks of AD, and found STAT6 to be the key player in this process in keratinocytes. Although we were unable to directly measure the putative phosphatase (PTPN1) activity responsible for inactivating STAT6 activity (24
) in keratinocytes, we do provide evidence that both upstream (NRF2) and downstream (STAT6) pathways are affected by coal tar. Therefore, it is very likely that coal tar inhibits oxidative inactivation of PTPN1, and thereby attenuates STAT6 activation. The Th2 immune response, as observed in atopic diseases like AD, is also driven by STAT6 via IL-4–induced maturation and development of Th2 cells (48
). Based on our data, we believe that AHR has great potential as a pharmacological target in atopic or allergic diseases, especially because it was recently shown that the AHR agonist FICZ (a tryptophan metabolite) suppresses eosinophilia, Th2 cytokine production, and STAT6 activation in allergic asthma in mice (50
). Moreover, PTPN1 deficiency results in exacerbated lung inflammation in mice due to early recruitment of leukocytes and elevated levels of lung CCL26 and Th2 cytokine levels (51
). Since coal tar metabolites are found in urine of patients receiving coal tar therapy (52
), suggesting the systemic entrance of coal tar components following topical application, the therapeutic success of coal tar therapy may, in addition to its epidermal effects, also rely on suppression of the immune response by dampening STAT6 activation in immune cells. The reported role of AHR in Treg development is likely to contribute to the therapeutic efficacy of coal tar in two major skin diseases: psoriasis and AD. The scope of our current investigations, however, was limited to epidermal aspects of AD and the effect of coal tar.
In addition to the recently uncovered role of NRF2 in epidermal barrier function, its main function lies in the defense against oxidative stress, and it is therefore proposed as a potential pharmacological target for chemoprevention (53
). NRF2 has been linked to the AHR signaling pathway mostly in mice liver tissue after dioxin exposure (54
). Here we show that coal tar, but not TCDD, induces NRF2 activation and subsequent expression of NQO1
in human keratinocytes. Upon knockdown of AHR, significantly less NQO1
was expressed, indicating an interaction between AHR and NRF2 in human keratinocytes. The discrepancy between the previously reported TCDD-induced NQO1
levels and the lack of such induction in keratinocytes is in itself not unprecedented, and may be explained by species and cell type differences and ligand-specific effects (55
). Therefore, the induction of a protective mechanism (NQO1 induction) in keratinocytes after coal tar exposure might explain the lack of toxicity and carcinogenicity associated with the medicinal use of coal tar (56
), while AHR activation by TCDD leads to severe symptoms such as chloracne and immunotoxicity, as well as an increased risk of cancer.
Although coal tar therapy is the oldest known dermatological treatment, questions still remain regarding its safety and possible carcinogenicity (57
). A comprehensive study by Roelofzen et al. in 2010 (56
) did not find a relation between an increased risk of skin or non-skin malignancies and coal tar therapy in a large cohort of 13,200 psoriasis and eczema patients. AHR ligands are currently excluded from drug discovery and development pipelines due to the exposure-related toxicity and carcinogenicity of some of its known ligands, such as dioxin and benzo[a]pyrene. The emerging evidence of a more physiological role of AHR, and the therapeutic effect of AHR activation herein described, suggest that it might be the right time to reconsider whether it is justifiable to exclude AHR as a pharmacological target.