Genetic Ablation of PARP-1 Protects Against Oxazolone-Induced Contact Hypersensitivity by Modulating Oxidative Stress Attila Brunyánszki, Csaba Hegedűs,

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Genetic Ablation of PARP-1 Protects Against Oxazolone-Induced Contact Hypersensitivity by Modulating Oxidative Stress Attila Brunyánszki, Csaba Hegedűs, Magdolna Szántó, Katalin Erdélyi, Katalin Kovács, Valérie Schreiber, Szabolcs Gergely, Borbála Kiss, Éva Szabó, László Virág, Péter Bai Journal of Investigative Dermatology Volume 130, Issue 11, Pages 2629-2637 (November 2010) DOI: 10.1038/jid.2010.190 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 PARP-1−/-, but not PARP-2−/-, mice are protected against the oxazolone-induced CHS reaction. (a) Ear thickness was measured before and 24hours after challenge by a caliper, and ear swelling was expressed as the difference of the two values. (b) At 24hours after OXA challenge, ears were removed and relative MPO activity was determined. *P<0.05; ***P<0.001, significant difference between cohorts. CHS, contact hypersensitivity; MPO, myeloperoxidase; OXA, oxazolone; PARP, poly(ADP-ribose) polymerase. Journal of Investigative Dermatology 2010 130, 2629-2637DOI: (10.1038/jid.2010.190) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 PARP-1−/-, but not PARP-2−/-, mice are protected against the PMA-induced irritative dermatitis. PMA (10μl, 0.05% w/v) was smeared onto both sides of the ears of female mice (six animals per group). After 24hours, (a) ear swelling, (b) MPO, and (d) MMP activities were determined. (c) Formalin-fixed, paraffin-embedded tissue sections were stained with HE. Scale bar=200μm. HE, hematoxylin and eosin; MMP, matrix metalloproteinase; MPO, myeloperoxidase; PARP, poly(ADP-ribose) polymerase; PMA, 12-O-tetradecanoyl-phorbol 13-acetate. Journal of Investigative Dermatology 2010 130, 2629-2637DOI: (10.1038/jid.2010.190) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Genetic ablation of poly(ADP-ribose) polymerase-1 (PARP-1) suppresses inflammatory cell immigration. Formalin-fixed, paraffin-embedded tissue sections were stained with (a) hematoxylin and eosin (HE) and, (b) for neutrophil elastase, with 4,6-diamidino-2-phenylindole (DAPI) counterstain. The arrows point at the neutrophil elastase-positive cells (linear contrast adjustment was applied on image b). Scale bars=200μm for a and 20μm for b. Journal of Investigative Dermatology 2010 130, 2629-2637DOI: (10.1038/jid.2010.190) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Expression of proinflammatory genes is suppressed in the PARP-1−/- mice. Expression of (a) a set of proinflammatory cytokines, (b) adhesion molecules, (c) MMP-9 and TIMP-2 were determined with qRT-PCR. (d) Transcription factor activation was determined using the TransFactor kit. *P<0.05; ***P<0.001, significant difference between cohorts. ATF-2, activating transcription factor-2; MCP-1, monocyte chemotactic protein-1; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; PARP, poly(ADP-ribose) polymerase; qRT-PCR, quantitative real-time reverse transcriptase-PCR; TIMP-2, tissue inhibitor of metalloproteinase-2; TNF-α; tumor necrosis factor-α. Journal of Investigative Dermatology 2010 130, 2629-2637DOI: (10.1038/jid.2010.190) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Reduced nitrosative stress in poly(ADP-ribose) polymerase (PARP)-1−/- mice. (a) Inducible and endothelial nitric oxide synthase (iNOS and eNOS) expression was determined by quantitative real-time reverse transcriptase PCR (qRT-PCR). (b) Nitrotyrosine immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections. NS, nonsensitized. *P<0,05; ***P<0.001, significant difference between cohorts (linear contrast adjustment was applied on image b). Scale bar=20μm. Journal of Investigative Dermatology 2010 130, 2629-2637DOI: (10.1038/jid.2010.190) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Reduced oxidative stress, DNA damage, and poly(ADP-ribose) polymerase (PARP) activity in PARP-1−/- mice. (a) 4-Hydroxy-2-nonenal (HNE), (b) protein carbonyl, and (c) 8-OHdG were determined using commercial kits. (d) TUNEL assay and (e) PAR immunohistochemistry were performed on formalin-fixed, paraffin-embedded tissue sections. NS, nonsensitized. The arrows point at PAR-positive nuclei. *P<0,05; ***P<0.001, significant difference between cohorts (linear contrast adjustment was applied on images d and e). Scale bar=20μm. Journal of Investigative Dermatology 2010 130, 2629-2637DOI: (10.1038/jid.2010.190) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions