IL-32 is expressed by human primary keratinocytes and modulates keratinocyte apoptosis in atopic dermatitis Norbert Meyer, MD, Maya Zimmermann, PhD,

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IL-32 is expressed by human primary keratinocytes and modulates keratinocyte apoptosis in atopic dermatitis Norbert Meyer, MD, Maya Zimmermann, PhD, Simone Bürgler, MSc, Claudio Bassin, Dipl Ing FH, Stefan Woehrl, MD, Katharina Moritz, MD, Claudio Rhyner, PhD, Philippe Indermitte, Dipl Ing FH, Peter Schmid-Grendelmeier, MD, Mübeccel Akdis, MD, PhD, Günter Menz, MD, Cezmi A. Akdis, MD Journal of Allergy and Clinical Immunology Volume 125, Issue 4, Pages 858-865.e10 (April 2010) DOI: 10.1016/j.jaci.2010.01.016 Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 IL-32 mRNA is expressed in human primary epidermal KCs and regulated by TNF-α and IFN-γ. KCs were stimulated with TNF-α, IFN-γ, or the combination of TNF-α and IFN-γ for indicated time points, and relative mRNA expression of total IL-32 was analyzed (A). Its isoforms (B) and the ratio between the different IL-32 isoforms (C) were analyzed after 24 hours. Relative IL-32 mRNA expression in human primary KCs derived from patients with AD (AD), patients with psoriasis (PSO), or healthy volunteers (H) was analyzed after incubation with TNF-α or IFN-γ for 24 hours (D and E). Fig 1, A and B: Mean ± SD, n = 3. Fig 1, C: Percentage of total IL-32 for each sample was set 100%, n = 3. Fig 1, D and E: n = 4 for healthy control subjects and patients with AD and n = 3 for patients with psoriasis. ∗P < .05. us, Unstimulated. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 IL-32 production by human primary KCs. IL-32 protein expression in KCs was analyzed by means of flow cytometry after incubation with TNF-α, IFN-γ, or the combination of TNF-α and IFN-γ for the indicated time points (A). IL-32 concentrations of cell lysates or supernatants from KCs stimulated with the combination of TNF-α and IFN-γ for 24 or 72 hours were measured by means of ELISA (B) and Western blotting (C). KCs were incubated with the indicated stimuli for 72 hours; IL-32 was stained with Alexa Fluor 488 (green), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue; D). Fig 2, A and B: Mean ± SD, n = 3. Fig 2, C: One representative Western blot of n = 2. Fig 2, D: One representative staining, n = 3. CL, Cell lysate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; S, supernatant; us, unstimulated. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 TH1 cells induce IL-32 through IFN-γ in KCs. KCs were cultured alone, with the combination of IFN-γ and TNF-α, or with TH1, TH2, TH17, and Treg cells. TH1 cells were added alone or in the presence of anti–IFN-γ–neutralizing mAbs (5 or 0.5 μg/mL). After 72 hours of coculture, IL-32 expression in KCs was analyzed by means of flow cytometry. Mean ± SD, n = 3. us, Unstimulated. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 KC apoptosis induced by IFN-γ and TNF-α is IL-32 dependent. Relative IL-32 mRNA expression was analyzed in unstimulated KCs cultured for 24 hours after transfection with increasing IL-32 siRNA or scrambled (sc) siRNA concentrations. The changes in IL-32 mRNA levels were calculated by setting scrambled siRNA at 100% for each concentration (A). KCs transfected with IL-32 siRNA or scrambled siRNA were incubated with TNF-α and IFN-γ for 72 hours, and IL-32 expression was analyzed (B) and KC apoptosis was determined by means of 7-amino actinomycin D (7-ADD) and annexin-V staining (C). Artificial skin equivalents transfected with IL-32 siRNA or scrambled siRNA were stimulated with TNF-α and IFN-γ for 5 days. IL-32 was stained with Alexa Fluor 488 (green); nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue; D), and terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) staining (Alexa Fluor 488, green) was performed (E, upper panels). Nuclei were stained with DAPI (blue), and merged pictures are presented in the lower panels of Fig 4, E. Fig 4, A: Mean ± SD, n = 3. Fig 4, B and C: Mean percentage ± SD, n = 7 (P < .001 for Fig 4, B, and P = .017 for Fig 4, C). Fig 4, D and E: One representative staining of 2 independent experiments. TUNEL, Terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 IL-32 is expressed in lesional skin of patients with AD. Hematoxylin-stained skin biopsy specimens from patients with AD, healthy subjects, and patients with psoriasis are presented (left panels). Solid rectangles in hematoxylin-stained skin biopsy specimens indicate the area of skin biopsy specimens from which immunostainings are shown. IL-32 was stained with Alexa Fluor 488 (green) in skin biopsy specimens from patients with AD, healthy subjects, and patients with psoriasis (right panels). IL-32 was detected in 5 lesional biopsy specimens from patients with AD and was not expressed in lesional biopsy specimens from patients with psoriasis (n = 4) or healthy donors (n = 3). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Serum IL-32 levels correlate with AD severity. IL-32 serum levels from patients with AD (AD, n = 36), healthy subjects (H, n = 17), patients with psoriasis (PSO, n = 20), and asthmatic patients (AB, n = 24) were analyzed with ELISA. Patients with AD were subdivided into patients with AD without (AD –AB, n = 23) or with (AD +AB, n = 13) concomitant asthma (A). IL-32 serum levels of patients with AD were correlated with the SCORAD values (B) and IgE levels (C). Fig 6, A: ∗P < .05; Fig 6, B: Spearman r = 0.54, P < .001; Fig 6, C: Spearman r = 0.31, P = .066. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

IL-32 isotype controls, unstimulated control, and gating strategy for Fig 2. IgG2A isotype controls for IL-32 staining for each condition (A), unstimulated control stained with anti–IL-32 mAb (B), and gating strategy of KCs (C) are presented. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

IL-32 isotype controls and gating strategy for Fig 3 IL-32 isotype controls and gating strategy for Fig 3. IgG2A isotype controls for IL-32 staining for each condition (A) and gating strategy of KCs (B) are presented. us, Unstimulated. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

IL-32 isotype controls and gating strategy for Fig 4 IL-32 isotype controls and gating strategy for Fig 4. Gating strategy of KCs for IL-32 staining (A) and IgG2A isotype controls for each condition (B) are presented. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

IL-32 isotype control. IgG2A isotype control stained with Alexa Fluor 488 (green) for Fig 5 (skin biopsy of a patients with AD) is shown. Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

IL-4 and IL-13 have no effect on IL-32 mRNA expression in KCs IL-4 and IL-13 have no effect on IL-32 mRNA expression in KCs. KCs were incubated with IL-4 (4 ng/mL) and IL-13 (10 ng/mL) for 24 or 72 hours, and relative IL-32 mRNA expression was analyzed by means of real-time PCR (A). Relative IL-32 mRNA expression was analyzed after incubation with TNF-α (25 ng/mL) and IFN-γ (12.5 ng/mL) with or without IL-4 (4 ng/mL) or IL-13 (10 ng/mL) after 24 or 72 hours (B). Real-time PCR quantification was based on comparative ΔΔCT method by using glyceraldehyde-3-phosphate dehydrogenase as the housekeeping gene and was normalized to the unstimulated control. Mean ± SD, n = 3. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Induction of IL-32 by TNF-α and IFN-γ is not influenced by immunosuppressive drugs. KCs were preincubated with 1 μmol/L dexamethasone (Dexa), 1 μmol/L 13-cis retinoic acid (13-cis RA), or 1 μmol/L FK506 for 30 minutes, and subsequently, TNF-α (25 ng/mL) and IFN-γ (12.5 ng/mL) were added for 72 hours (A). To exclude the involvement of the mammalian target of rapamycin (mTOR) pathway, 10 nmol/L rapamcyin and the according amount of dimethyl sulfoxide (DMSO; for the control) were added, and KCs were stimulated with TNF-α (25 ng/mL) and IFN-γ (12.5 ng/mL) for 72 hours (B). IL-32 expression was analyzed by means of flow cytometry. Mean ± SD, n = 3. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Structure of artificial skin equivalents Structure of artificial skin equivalents. One representative hematoxylin staining of artificial skin equivalents is shown. Journal of Allergy and Clinical Immunology 2010 125, 858-865.e10DOI: (10.1016/j.jaci.2010.01.016) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions