Vitamin D Analog Calcipotriol Suppresses the Th17 Cytokine–Induced Proinflammatory S100 “Alarmins” Psoriasin (S100A7) and Koebnerisin (S100A15) in Psoriasis  Zuzana Hegyi, Stephanie Zwicker, Daniela Bureik, Mark Peric, Sarah Koglin, Aleksandra Batycka-Baran, Jörg C. Prinz, Thomas Ruzicka, Jürgen Schauber, Ronald Wolf  Journal of Investigative Dermatology  Volume 132, Issue 5, Pages 1416-1424 (May 2012) DOI: 10.1038/jid.2011.486 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Psoriasin (S100A7) and koebnerisin (S100A15) are differentially induced and secreted in psoriatic skin and by psoriatic T cell–derived Th17 cytokines in epidermal keratinocytes. (a) Immunofluorescent co-staining of frozen sections of normal and lesional psoriatic back skin stained for psoriasin (S100A7, green) and koebnerisin (S100A15, red). Nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI; blue). Bar=100μm. (b) Human keratinocyte-derived HaCaT cells were treated with supernatants from T cells isolated from psoriatic plaques and (c) primary human keratinocytes were treated with IL-17A (10ngml−1), IL-22 (10ngml−1), and tumor necrosis factor-α (TNF-α; 100ngml−1), alone or in combination. (b, c) Expression of psoriasin (S100A7) and koebnerisin (S100A15) alternate mRNA isoforms was analyzed after 24hours by quantitative real-time reverse transcription-PCR. Data are mean±SEM of three independent experiments performed in triplicate; *P<0.05 determined by Student's t-test. (d) Immunoblots of supernatants from cultured primary human keratinocytes using specific antibodies against psoriasin (S100A7) or koebnerisin (S100A15). Numbers show S100 ratios normalized for β-actin from corresponding lysates. Representative pattern from experiments performed in triplicate. NC, negative control. Journal of Investigative Dermatology 2012 132, 1416-1424DOI: (10.1038/jid.2011.486) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Psoriasin (S100A7) and koebnerisin (S100A15) are differently regulated by Th17-derived cytokines in epidermal keratinocytes. (a–c) Differential regulation of psoriasin (S100A7) and koebnerisin (S100A15) alternate mRNA isoforms (S100A15L, S100A15S) in primary human keratinocytes after treatment with (a) IL-17A, (b) tumor necrosis factor (TNF)-α, or (c) IL-22 at indicated concentrations over time and analyzed by quantitative real-time reverse transcription-PCR. Data are mean±SEM of three independent experiments performed in triplicate; *P<0.05 determined by Student's t-test. NC, negative control. Journal of Investigative Dermatology 2012 132, 1416-1424DOI: (10.1038/jid.2011.486) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 T cell–derived IL-17A binds to normal and psoriatic epidermis and is a principal inducer of psoriasin (S100A7) and koebnerisin (S100A15) in skin keratinocytes. (a) Immunofluorescent staining of frozen sections of healthy and lesional psoriatic skin stained for IL-17A (yellow) detecting IL-17A bound to epidermal keratinocytes. Arrow marks IL-17A-producing immunocytes in the dermis of psoriatic skin. Bar=100μm. (b, c) Human keratinocyte-derived HaCaT cells were treated with supernatants from T cells isolated from psoriatic plaques in the presence of (b) IL-17AR neutralizing antibody (Ab) or (c) tumor necrosis factor (TNF)-α neutralizing Ab (golimumab, Simponi) compared with non-immune IgG. (d) Primary human keratinocytes were treated with IL-17A (10ngml−1) in the presence of IL-17AR blocking Ab compared with non-immune IgG. Cells were harvested after 24hours and S100 transcript levels were analyzed by quantitative real-time reverse transcription-PCR. Data are mean±SEM of three independent experiments from individual patients performed in triplicate; *P<0.05 determined by Student's t-test. Journal of Investigative Dermatology 2012 132, 1416-1424DOI: (10.1038/jid.2011.486) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The “alarmins” psoriasin (S100A7) and koebnerisin (S100A15) synergize to regulate proinflammatory cytokines and antimicrobial peptides in epidermal keratinocytes. (a–f) Primary human keratinocytes were treated with psoriasin (S100A7, 100ngml−1) and koebnerisin (S100A15, 100ngml−1) alone or in combination and expression of indicated proinflammatory mediators and antimicrobial peptides was analyzed by quantitative real-time reverse transcription-PCR after 1hour. Data are mean±SEM of three independent experiments performed in triplicate; *P<0.05 determined by Student's t-test. TNF, tumor necrosis factor. Journal of Investigative Dermatology 2012 132, 1416-1424DOI: (10.1038/jid.2011.486) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Calcipotriol treatment suppresses the increased expression of psoriasin (S100A7) and koebnerisin (S100A15) in psoriatic skin. (a) Immunofluorescent staining of frozen sections of psoriatic skin before and after treatment with calcipotriol for psoriasin (S100A7, green) and koebnerisin (S100A15, red; left and middle panel, antibody 3090; right panel, antibody 3923). Nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI; blue). Bar=100μm. (b) Production and (c) expression of psoriasin (S100A7) and koebnerisin (S100A15) in psoriatic plaques before and after treatment with calcipotriol. Punch biopsies from psoriatic plaques were taken from patients (n=5) before and after treatment with vitamin D analog calcipotriol (0.005%; applied twice daily for 5–7 days). Expression of psoriasin (S100A7) and koebnerisin (S100A15) alternate mRNA isoforms was analyzed by quantitative real-time reverse transcription-PCR and production of both proteins was quantified in the skin sections as stated in the Materials and Methods section. Shown are relative changes of S100 expression and production after calcipotriol treatment in psoriatic skin; *P<0.05 determined by Student's t-test. (d) Lysates from normal and psoriatic skin were subjected to immunoblotting by incubation with monoclonal anti-hS100A7 (psoriasin) or affinity-purified polyclonal anti-hS100A15 (koebnerisin) antibody; shown are representative data from four independent patients. Journal of Investigative Dermatology 2012 132, 1416-1424DOI: (10.1038/jid.2011.486) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 1,25-Dihydroxyvitamin D3 (1,25D3) inhibits the Th17-induced expression of psoriasin (S100A7) and koebnerisin (S100A15) and suppresses the S100-mediated proinflammatory loop in psoriasis. (a–d) Primary human keratinocytes were treated with IL-17A (10ngml−1) alone or in combination with IL-22 (10ngml−1), tumor necrosis factor-α (TNF-α; 100ngml−1) in the presence of vitamin 1,25 D3 (10−9M) or vehicle control. Expression of psoriasin (S100A7) and koebnerisin (S100A15) alternate mRNA isoforms was analyzed by (a, c) quantitative real-time reverse transcription-PCR and (b, d) immunoblot analysis. Data are mean±SEM of three independent experiments performed in triplicate; *P<0.05 determined by Student's t-test. (e) Pro-psoriatic T cell–derived Th17 cytokines, mainly IL-17A, induce the expression of psoriasin (S100A7) and koebnerisin (S100A15) in epidermal keratinocytes. Their increased release from keratinocytes allows them to synergize as extracellular chemoattractants for leukocytes and “alarmins” on resident skin cells and thus amplify the inflammatory response in psoriasis. Vitamin D analogs suppress the increased production of psoriasin (S100A7) and koebnerisin (S100A15) and thus interfere with the S100-mediated proinflammatory feedback loop in psoriasis (modified from Wolf et al., 2010a). Journal of Investigative Dermatology 2012 132, 1416-1424DOI: (10.1038/jid.2011.486) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions