Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis

Abstract

Topical application of coal tar is one of the oldest therapies for atopic dermatitis (AD), a T helper 2 (Th2) lymphocyte-mediated skin disease associated with loss-of-function mutations in the skin barrier gene, filaggrin (FLG). Despite its longstanding clinical use and efficacy, the molecular mechanism of coal tar therapy is unknown. Using organotypic skin models with primary keratinocytes from AD patients and controls, we found that coal tar activated the aryl hydrocarbon receptor (AHR), resulting in induction of epidermal differentiation. AHR knockdown by siRNA completely abrogated this effect. Coal tar restored filaggrin expression in FLG-haploinsufficient keratinocytes to wild-type levels, and counteracted Th2 cytokine-mediated downregulation of skin barrier proteins. In AD patients, coal tar completely restored expression of major skin barrier proteins, including filaggrin. Using organotypic skin models stimulated with Th2 cytokines IL-4 and IL-13, we found coal tar to diminish spongiosis, apoptosis, and CCL26 expression, all AD hallmarks. Coal tar interfered with Th2 cytokine signaling via dephosphorylation of STAT6, most likely due to AHR-regulated activation of the NRF2 antioxidative stress pathway. The therapeutic effect of AHR activation herein described opens a new avenue to reconsider AHR as a pharmacological target and could lead to the development of mechanism-based drugs for AD.

Figures

Figure 1. Coal tar induces epidermal differentiation via AHR signaling.

(A) AHR staining of coal tar–treated or untreated keratinocytes. Nuclei were counterstained using DAPI. Scale bar: 100 μM. (B) Semiquantitative analysis of nuclear and cytoplasmic fluorescence intensity (FI) measured by ImageJ software. *P < 0.05, **P < 0.01, and ***P < 0.001, relative to untreated keratinocytes. Bars indicate mean ± SEM (n = 3). (C) CYP450 mRNA expression levels after 48 hours of stimulation with TCDD or a coal tar concentration series. Expression levels are relative to untreated (control) keratinocytes. *P < 0.05 and **P < 0.001. Bars indicate mean ± SEM (n = 3). (D) mRNA expression levels of epidermal differentiation genes after 48 hours of stimulation with TCDD or a coal tar concentration series. Expression levels are relative to untreated (control) keratinocytes. Bars indicate mean ± SEM (n = 3). (E) Filaggrin and hornerin staining of 2% coal tar–treated or untreated keratinocytes. Nuclei were counterstained using DAPI. Scale bar: 100 μM. (F) Western blot analysis of filaggrin protein in lysates of coal tar–treated and untreated keratinocytes from 2 keratinocyte donors. (G) Filaggrin staining of 2% coal tar–treated or untreated keratinocytes. Nuclei were counterstained using hematoxylin. Scale bar: 100 μM. (H) AHR and CYP1A1, and (I) FLG and HRNR mRNA expression levels after siRNA-mediated AHR knockdown and subsequent 2% coal tar stimulation. Expression levels are relative to mock-treated, coal tar–stimulated keratinocytes. Bars indicate mean ± SEM (n = 3).

Figure 2. Accelerated epidermal differentiation by coal tar.

(A) H&E staining of human skin equivalents cultured in the presence or absence (control) of coal tar during the entire air-liquid interface culture. Bracket indicates the thickness of the stratum corneum. (B) Immunohistochemical staining of filaggrin, loricrin, and hornerin in skin equivalents at day 4 of the air-liquid interface culture when cultured in the presence or absence (control) of coal tar. Images are representative of 3 keratinocyte donors, and the experiment was replicated twice. Scale bar: 100 μM. (C) Filaggrin and loricrin Western blotting of skin equivalents harvested on day 4 or day 10 of air-liquid interface culture, treated with or without coal tar during the entire culture period.

Figure 3. Restored filaggrin and hornerin protein expression by coal tar.

Skin equivalents generated from keratinocytes from AD patients harboring a heterozygous FLG mutation (FLG–/+; n = 3) and healthy control wild-type (WT; n = 3) keratinocytes were treated with coal tar during the last 3 days of air-liquid interface culture. Skin equivalents were stained for (A) filaggrin, and (B) hornerin protein expression analysis. (C) Immunohistochemical analysis of filaggrin and hornerin protein expression in skin biopsies from AD patients receiving coal tar therapy (n = 3). Scale bar: 100 μM. (D) Immunohistochemical analysis of loricrin and involucrin protein expression in skin biopsies from AD patients receiving coal tar therapy (n = 3). Scale bar: 100 μM.

Figure 4. Coal tar attenuates Th2 cytokine–induced AD hallmarks.

(A) H&E staining of skin equivalents stimulated with Th2 cytokines for 3 days, or treated with coal tar thereafter (Th2 → coal tar), or simultaneously stimulated with Th2 cytokines and coal tar (Th2 + coal tar). Closed arrowheads indicate spongiosis. Images are representative of 3 keratinocyte donors. The experiment was replicated twice. Scale bar: 100 μM. (B) TUNEL assay on matching skin equivalents as depicted in A, showing apoptotic cells indicated by open arrowheads. Scale bar: 100 μM. (C) FAS mRNA expression levels in skin equivalents after Th2 cytokine stimulation and coal tar treatment. Expression levels are relative to untreated (control) keratinocytes. Bars indicate mean ± SEM (n = 3). (D) mRNA expression levels of epidermal differentiation genes after Th2 cytokine stimulation and coal tar treatment. Expression levels are relative to untreated (control) keratinocytes. Bars indicate mean ± SEM (n = 3). *P < 0.05 and **P < 0.01. (E) Immunohistochemical staining of loricrin and involucrin in skin equivalents stimulated with Th2 cytokines for 3 days, or treated with coal tar thereafter (Th2 → coal tar). Images are representative of 3 keratinocyte donors. The experiment was replicated twice. Scale bar: 100 μM. (F) Loricrin, involucrin, and filaggrin Western blotting of skin equivalents are depicted in E. (G) CCL26 mRNA expression levels after Th2 cytokine stimulation and coal tar treatment by qPCR analysis. Expression levels are relative to untreated (control) keratinocytes. Bars indicate mean ± SEM (n = 3). ***P < 0.001.

Figure 5. STAT6 dephosphorylation and NRF2 activation by coal tar.

(A) Western blot analysis of phosphorylated STAT6 (pSTAT6) and total STAT6 levels in lysates of 2 keratinocyte donors. Keratinocytes were stimulated for 12 hours with differentiation medium (–/–), Th2 cytokines (+/–), or 2% coal tar (–/+), and with Th2 cytokines during the first 6 hours, followed by the addition of 2% coal tar and stimulation for another 6 hours (+/+). Thin black line indicates that the lanes were run on the same gel, but were noncontiguous. (B) Immunofluorescence staining of NRF2 in coal tar–treated or untreated keratinocytes. Nuclei were counterstained using DAPI. Images are representative of 3 keratinocyte donors. Original magnification, ×400. (C) qPCR analysis of NRF2 and NQO1 mRNA expression levels in keratinocytes. Expression levels are relative to untreated (control) keratinocytes. *P = 0.002 relative to control condition. Bars indicate mean ± SEM (n = 3). qPCR analysis of mRNA expression levels of AHR (D) and NQO1 (E) after siRNA-mediated AHR knockdown and subsequent coal tar stimulation by qPCR analysis. Expression levels are relative to mock-treated, coal tar–stimulated keratinocytes. *P = 0.006 and **P = 0.032, relative to mock-treated keratinocytes. Bars indicate mean ± SEM (n = 3).

Figure 6. Model of the molecular mechanism of coal tar therapy in AD.

Th2 cytokines IL-4 and IL-13 activate the STAT6 signaling cascade, leading to downregulated expression of epidermal differentiation proteins, induction of the eosinophilic chemoattractant CCL26, and histopathological features such as spongiosis and apoptosis. Under normal conditions, PTPN1 dephosphorylates STAT6, resulting in a regulatory feedback loop. However, oxidative stress caused by Th2 cytokines inactivates PTPN1, leading to sustained STAT6 signaling. Coal tar–mediated activation of the AHR signaling pathway leads to enhanced epidermal differentiation and possible improvement of epidermal barrier function, thereby attenuating allergen exposure and reducing inflammatory cues. Importantly, coal tar activates the AHR/NRF2 signaling pathway, enabling detoxification of reactive oxygen species. This may prevent the oxidative inactivation of PTPN1 and lead to decreased STAT6 signaling, normalization of skin barrier protein expression, and downregulation of CCL26 expression.