Aryl Hydrocarbon Receptor in Keratinocytes Is Essential for Murine Skin Barrier Integrity  Katharina Haas, Heike Weighardt, René Deenen, Karl Köhrer, Björn Clausen, Sonja Zahner, Petra Boukamp, Wilhelm Bloch, Jean Krutmann, Charlotte Esser  Journal of Investigative Dermatology  Volume 136, Issue 11, Pages 2260-2269 (November 2016) DOI: 10.1016/j.jid.2016.06.627 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Increased TEWL in full and KC-specific AhR-KO mice. (a, d, g) Basal TEWL and TEWL after tape-stripping of AhR-KO, AhRΔK5 (b, e, h), and (c, f, i) AhRΔLC. The results are shown for mice at ages (upper row) 10 weeks, (middle row) 9 months, and (lower row) 18 months old. n = 7–10/group, mean ± standard error of the mean. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Student t test. h, hours; KO, knockout; TEWL, transepidermal water loss; WT, wild type. Journal of Investigative Dermatology 2016 136, 2260-2269DOI: (10.1016/j.jid.2016.06.627) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Electron microscopy shows structural changes in AhR-KO epidermis. Electron micrographs of back skin epidermis of (a, c, e) WT littermates and (b, d, f) AhR-KO. (a, b) Unstressed skin (original magnification ×3,000) and (c, d) 6 hours after tape-stripping. Original magnification of c × 3,000; original magnification of d × 4,000. (e, f) Same samples as in c, d. Original magnification ×12,000. White arrows indicate desmosomes; red arrows indicate enlargement of KC contacts, bridged by adherens junctions. Scale bars = 20 nm. BM, basal membrane; C, cytoplasm; dC, dead cell; KO, knockout; N, nucleus; SC, stratum corneum; WT, wild type. Journal of Investigative Dermatology 2016 136, 2260-2269DOI: (10.1016/j.jid.2016.06.627) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Gene expression profile differences of WT and AhR-KO epidermis show an AhR battery of barrier genes. AhR-KO and WT littermates were mechanically stressed as described in the Materials and Methods. Skin biopsy samples were analyzed for differential gene expression on an Affymetrix (Santa Clara, CA) Mouse Gene 2.0 ST microarray. (a) n = 3 for each genotype/point of time. Heat map of unstressed skin and skin 6 or 24 hours after tape-stripping. Green indicates decreased signal intensity/expression; red indicates increased signal intensity/expression compared to the median signal intensity across all samples. (b) Stacked bars show the number of significantly up- or down-regulated genes of a given function in AhR-KO versus WT littermates. Genes assigned a relevant function by gene ontology were selected and further identified for the indicated functional involvement via PubMed. Cut-off threshold > 1. For full list see Supplementary Tables S1–S3. h, hours; KO, knockout; WT, wild type. Journal of Investigative Dermatology 2016 136, 2260-2269DOI: (10.1016/j.jid.2016.06.627) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Microbiome differences on the skin of WT versus full and KC-specific AhR-deficient mice. Direct sequencing of bacterial 16S RNA from back skin epidermis of three WT littermates and three AhR-KO mice reveals relative abundance of the main bacterial phyla. (a, b) back skin habitat (c, d) ear skin habitat. KO, AhR-knockout; K5KO, cell-keratinocyte–specific AhR-deficient (i.e., AhRΔK5); WT, wild type. Journal of Investigative Dermatology 2016 136, 2260-2269DOI: (10.1016/j.jid.2016.06.627) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Dietary intervention by AhR ligands impairs or improves skin barrier in WT mice. TEWL measurements during diet intervention with AhR-ligand free diet (NALD, red line) or with the same diet supplemented with AhR ligand-precursor I3C (ALD, purple line). Standard diet fed WT (SDT-WT, black square) and AhR-KO (SDT-AhR-KO, white circle) served as comparative controls. Numbers indicate time points when TEWL kinetics was measured, and graphs are labeled accordingly. (a) Feeding scheme, where diet intervention was started directly after weaning and later switched. Total duration of feeding was 190 days. (b) Feeding scheme where diet intervention was started in 10-week-old adults, discontinued, and then resumed. Total duration was 237 days. Inserts for time points 1 and 3 show relative units of the area under the curve calculated by GraphPad Prism (GraphPad, La Jolla, CA). n = 8; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, Student t test. AUC, area under the curve; KO, knockout; TEWL, transepidermal water loss; WT, wild type. Journal of Investigative Dermatology 2016 136, 2260-2269DOI: (10.1016/j.jid.2016.06.627) Copyright © 2016 The Authors Terms and Conditions