Fig 1.
Reduced levels of zonula occludens 1 (ZO-1), claudin-1, and claudin-4 proteins in the epidermis of patients with atopic dermatitis (AD).
Skin biopsies were taken from healthy subjects (Nos. 1–3) and patients with AD (Nos. 4, 5, 6). Nonlesional sites and lesional sites were biopsied in all patients with AD. (a) Double-immunostaining for claudin-1 [fluorescein isothiocyanate (FITC), green], claudin-4 (Cy3, red), occludin (FITC, green), and ZO-1 (Cy3, red). Scale bars = 50 μm. (b) Western blot analysis of claudin-1, claudin-4, ZO-1, occludin, β-actin and GAPDH syntheses.
Fig 2.
IL-17 impairs the tight junction (TJ) barrier in monolayer cultures of keratinocytes and in a skin-equivalent model.
(a) Monolayer cultures of human keratinocytes were incubated with TNF-α, IL-4, IL-17, and IL-22, and the transepithelial electric resistance was then measured. Results are shown as the mean ± standard deviation. Results of a representative experiment are shown. *P < 0.05, **P < 0.01. (b) Human skin-equivalent model specimens were incubated with IL-17 and subjected to a TJ permeability assay by using sulfo-NHS-LC-Biotin (Texas Red; red) and concomitantly stained with occludin (fluorescein isothiocyanate; green). Sulfo-NHS-LC-biotin is identified beneath the sites where occludin is localized. (c) Human skin-equivalent model specimens were incubated with TNF-α, IL-4, and IL-22 and then subjected to the TJ permeability assay. Scale bars = 50 μm.
Fig 3.
IL-17 decreases zona occludens 1 (ZO-1), claudin-1, and claudin-4 synthesis in a skin-equivalent model.
(a) Double-immunostaining of claudin-1 (fluorescein isothiocyanate; green) and claudin-4 (Cy3, red) was performed in skin-equivalent model specimens incubated with IL-17. Scale bars = 50 μm. (b) Western blot analysis of the synthesis of claudin-1, claudin-4, ZO-1, and occludin in the skin-equivalent model incubated with IL-17.
Fig 4.
IL-17 alters filaggrin localization in skin-equivalent model.
(a) Hematoxylin and eosin staining of the skin-equivalent model incubated with or without varying concentrations of IL-17. (b) Double-immunostaining of loricrin (fluorescein isothiocyanate, green) and filaggrin (RRX, red). Skin-model specimens were incubated with or without varying concentrations of IL-17. Scale bars = 50 μm.
Fig 5.
IL-17 alters profilaggrin processing in skin-equivalent model.
(a) qRT–PCR analysis of filaggrin mRNA expression. (b) Western blot analysis of filaggrin synthesis.(c) Amino acid content of the SC. Amino acids extracted from SC were measured using the OPA method. *P < 0.05.