IL-33 and ST2 in Atopic Dermatitis: Expression Profiles and Modulation by Triggering Factors Terhi Savinko, Sampsa Matikainen, Ulpu Saarialho-Kere, Maili.

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IL-33 and ST2 in Atopic Dermatitis: Expression Profiles and Modulation by Triggering Factors Terhi Savinko, Sampsa Matikainen, Ulpu Saarialho-Kere, Maili Lehto, Guoying Wang, Sari Lehtimäki, Piia Karisola, Timo Reunala, Henrik Wolff, Antti Lauerma, Harri Alenius Journal of Investigative Dermatology Volume 132, Issue 5, Pages 1392-1400 (May 2012) DOI: 10.1038/jid.2011.446 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 ST2 and IL-1RAcP are increased in lesional skin of patients with atopic dermatitis (AD). (a) mRNA levels of ST2, IL-33, and IL-1RAcP and (b) proinflammatory T-helper type (Th)2 and Th1 cytokines were analyzed by real-time reverse transcription-PCR in lesional and nonlesional skin of AD patients and in the skin of healthy volunteers. Columns and error bars represent means±SEMs (n=13–15). Immunohistological staining of IL-33 in (c) nonlesional and (d) lesional skin of AD patients (n=8). (e) Keratinocytes and (f) endothelial cells were IL-33+. Immunohistological staining of ST2 in (g) nonlesional and (h) lesional skin of AD patients (n=4). ST2+ cells were found from the (i) epidermis and (j) dermal inflammatory cell infiltrates. *P<0.05; **P<0.01; and ***P<0.001. Bar=200μm in c, d, g, and h; 50μm in e, f, i, and j. RU, relative units; TNF, tumor necrosis factor. Journal of Investigative Dermatology 2012 132, 1392-1400DOI: (10.1038/jid.2011.446) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 ST2 and IL-33 mRNA expressions in mouse atopic dermatitis (AD) skin and activated mast cells. (a) ST2 and IL-33 expressions in mouse ovalbumin (OVA)-sensitized skin after the first and third sensitization week were analyzed by real-time reverse transcription-PCR, n=8 mice per group, *P<0.05, **P<0.01, and ***P<0.001. (b) ST2 and IL-33 were increased in 38-week-old ft/ft mice compared with 4- or 22-week-old ft/ft mice. (c) ST2 and IL-33 expressions are shown in IgE+allergen-stimulated bone marrow–derived murine mast cells. The columns and the error bars represent means±SEMs of three replications. PBS, phosphate-buffered saline; RU, relative units. Journal of Investigative Dermatology 2012 132, 1392-1400DOI: (10.1038/jid.2011.446) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 ST2 and IL-33 mRNAs in human atopic dermatitis skin after house dust mite (HDM) and staphylococcal enterotoxin B (SEB) exposure. mRNA expression of ST2 and IL-33 after (a) HDM atopy patch test and (b) SEB patch test was analyzed by real-time reverse transcription-PCR. Control mRNA expressions are adjusted to 1, and other expressions are shown as the fold change compared with the control. The columns and the error bars represent means±SEMs, n=10 patients per group; *P<0.05. Journal of Investigative Dermatology 2012 132, 1392-1400DOI: (10.1038/jid.2011.446) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Staphylococcal enterotoxin B (SEB) increases the expression of ST2, IL-33, tumor necrosis factor (TNF)-α, IFN-γ, IL-4, and IL-13 in murine skin. (a) ST2 mRNA and (b) IL-33 mRNA are dose dependently increased after topical SEB exposure in murine skin. Topical SEB exposure increases the expression of (c) proinflammatory cytokine TNF-α, (d) T-helper type (Th)1 cytokine IFN-γ, and Th2 cytokines (e) IL-4 and (f) IL-13. The columns and the error bars represent means±SEMs. *P<0.05; **P<0.01; and ***P<0.001; n=8 mice per group. PBS, phosphate-buffered saline; RU, relative units. Journal of Investigative Dermatology 2012 132, 1392-1400DOI: (10.1038/jid.2011.446) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Tacrolimus downregulates the expression of ST2 and IL-33 in murine atopic dermatitis skin. (a) ST2 and (b) IL-33 expressions in ovalbumin (OVA)/staphylococcal enterotoxin B (SEB)-sensitized skin and control skin after topical corticosteroid (betamethasone-17-valerate) and tacrolimus treatment were analyzed. Phosphate-buffered saline with acetone was used as a vehicle. The columns and the error bars represent means±SEMs. *P<0.05; **P<0.01; and ***P<0.001; n=8 mice per group. RU, relative units. Journal of Investigative Dermatology 2012 132, 1392-1400DOI: (10.1038/jid.2011.446) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 IL-33 protein is produced by stimulated dermal fibroblasts, HaCaT keratinocytes, and primary macrophages. (a) Primary human dermal fibroblasts, HaCaT keratinocytes, and macrophages express IL-33 mRNA after stimulation with both tumor necrosis factor (TNF)-α and IFN-γ, and (b) poly(I:C) transfection further increases IL-33 mRNA expression in fibroblasts. (c) Stimulated dermal fibroblast extracts were western blotted, and SYPRO Ruby staining of the immunoblot confirmed equal loading and transfer of proteins from cell lysate samples. Culture medium was used as a control. RU, relative units. Journal of Investigative Dermatology 2012 132, 1392-1400DOI: (10.1038/jid.2011.446) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions