MCPIP1 RNase Is Aberrantly Distributed in Psoriatic Epidermis and Rapidly Induced by IL-17A  Ester Ruiz-Romeu, Marta Ferran, Ana Giménez-Arnau, Beata Bugara, Barbara Lipert, Jolanta Jura, Edwin F. Florencia, Errol P. Prens, Antonio Celada, Ramon M. Pujol, Luis F. Santamaria-Babí  Journal of Investigative Dermatology  Volume 136, Issue 8, Pages 1599-1607 (August 2016) DOI: 10.1016/j.jid.2016.04.030 Copyright © 2016 The Authors Terms and Conditions

Figure 1 IL-17A induces the up-regulation of ZC3H12A expression in keratinocytes. (a) Keratinocytes were cultured and stimulated at confluence with a range of doses of IL-17A, IL-17C, IL-17F, TNF-α, IL-23, or IFN-γ. ZC3H12A mRNA expression was evaluated at 15 hours of treatment (n = 3). (b) mRNA from IL-17A–stimulated (1, 10, and 100 ng/ml) keratinocytes was extracted at various early and late time points for ZC3H12A expression analysis (n = 3). Data are presented as mean ± standard deviation. Significant differences were analyzed with two-way analysis of variance t test versus untreated ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. h, hours; TNF, tumor necrosis factor. Journal of Investigative Dermatology 2016 136, 1599-1607DOI: (10.1016/j.jid.2016.04.030) Copyright © 2016 The Authors Terms and Conditions

Figure 2 ZC3H12A induction by supernatants enriched with psoriasis-associated inflammatory mediators depends mainly on the effect of IL-17A. (a) ZC3H12A mRNA expression was analyzed in keratinocytes stimulated with supernatants derived from SE-activated co-cultures with CLA+ or CLA– T cells and autologous epidermal lesional cells (nine psoriatic patients and five control subjects; both are represented by dots). Horizontal bar shows median induction. Differences were analyzed with the Mann-Whitney U test (∗P < 0.05). (b) The supernatants from five psoriasis patients with neutralized IL-17A, TNF-α, and IFN-γ were used for subsequent activation of keratinocytes and ZC3H12A mRNA analysis. Bar chart represents the percentage of ZC3H12A expression in relation to that found in untreated keratinocytes expression (mean ± standard error of the mean). A Student t test was used to check differences versus isotype conditions (∗∗P < 0.01). (c) Correlation between IL-17A, TNF-α, or IFN-γ contents of supernatants derived from SE-activated CLA+/EPI co-cultures and respective ZC3H12A mRNA up-regulation in keratinocytes. (d) Correlation between ZC3H12A mRNA up-regulation in keratinocytes induced by supernatants from SE-activated CLA+/EPI co-cultures with anti-streptolysin O blood levels from respective patients. RNA extraction was done at 15 hours after stimulation. ASO, anti-streptolysin O; CLA, cutaneous lymphocyte-associated antigen; EPI, epidermal cells; IU, international units; SE, Streptococcus pyogenes extract; sup, supernatant; TNF, tumor necrosis factor. Journal of Investigative Dermatology 2016 136, 1599-1607DOI: (10.1016/j.jid.2016.04.030) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Psoriatic skin shows aberrant MCPIP1 expression. MCPIP1 localization by immunohistochemistry visualized by fluorescence microscopy. Skin samples from healthy controls and from psoriatic and atopic dermatitis patients were fixed with 4% formaldehyde for paraffin embedding. Tissue sections were stained with hematoxylin and eosin (left column) or were deparaffinized for immunostaining without primary antibody (center column) and with specific anti-MCPIP1 primary antibody (right column). Scale bars = 100 μm. H&E, hematoxylin and eosin; MCPIP1, monocyte chemotactic protein-induced protein 1. Journal of Investigative Dermatology 2016 136, 1599-1607DOI: (10.1016/j.jid.2016.04.030) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Abnormal MCPIP1 expression in psoriatic skin is distributed in highly and mid-differentiating suprabasal epidermal population subsets and is inducible by IL-17A. (a) Intracellular MCPIP1 detection by flow cytometry in epidermal cell suspension from a representative healthy skin sample. (b) Gating strategy for the evaluation of MCPIP1 expression in basal (CD29) and suprabasal differentiated (KRT10) cells (1 = epidermal cells, 2 = KRT10/CD29 quadrant, 3 = MCPIP1 gating within each quadrant). (c) Distribution of MCPIP1+ cells in epidermal cell subsets in psoriatic (n = 4) and control (n = 3) samples. (d) Epidermal cell suspensions were cultured for 4 hours with or without IL-17A (0, 10, or 50 ng/ml), and MCPIP1 expression was analyzed by flow cytometry. Paired t test comparison analysis was used between stimuli conditions (∗∗P < 0.01). (e) MCPIP1 induction in KRT10+ subsets in one representative psoriatic sample after 4 hours of IL-17A activation. Data are presented as mean ± standard error of the mean. FSLin, forward scatter linear; KRT10, keratin 10; MCPIP1, monocyte chemotactic protein-induced protein 1; SS Log, side scatter logarithmic. Journal of Investigative Dermatology 2016 136, 1599-1607DOI: (10.1016/j.jid.2016.04.030) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Inhibition of STAT3 by AZD1480 reduces MCPIP1 induction. Primary keratinocytes were incubated with 1 μmol/L of pharmacological inhibitor AZD1480 or DMSO (0.02%) before stimulation with (a) IL-17A or (b) IL-6 for 15 hours, and phosphorylated STAT3 and MCPIP1 protein were detected and quantified by densitometry. Statistical analysis was done on the basis of results from three independent experiments. A Student t test was used for data analysis. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (c) ZC3H12A expression from IL-6–stimulated (1, 10, and 100 ng/ml) keratinocytes (n = 3) was analyzed at various early and late time points. Significant differences were analyzed with two-way analysis of variance t test versus untreated. ∗∗P < 0.01, ∗∗∗P < 0.001. Data are presented as mean ± standard deviation. h, hours; MCPIP1, monocyte chemotactic protein-induced protein 1; p-STAT, phosphorylated signal transducer and activator of transcription; STAT, signal transducer and activator of transcription. Journal of Investigative Dermatology 2016 136, 1599-1607DOI: (10.1016/j.jid.2016.04.030) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Induction of Zc3h12a in imiquimod-induced psoriasiform skin in mice is abrogated by topical corticosteroid and is dependent on IL-17R signaling. (a) Balb/c mice were left untreated (n = 3) or treated with Aldara (imiquimod) cream on dorsal skin for 6 days (n = 4). A group was additionally treated with corticosteroid-containing Clovate cream (n = 4). (b) Wild-type and Il17ra–/– C57BL/6 mice (minimum of two animals per group) were treated with Aldara cream for 5 days. Relative units of mRNA expression of Zc3h12a in skin samples were assessed by quantitative real-time PCR. Data are presented as mean ± standard deviation. Journal of Investigative Dermatology 2016 136, 1599-1607DOI: (10.1016/j.jid.2016.04.030) Copyright © 2016 The Authors Terms and Conditions