Deletion of the Major Bullous Pemphigoid Epitope Region of Collagen XVII Induces Blistering, Autoimmunization, and Itching in Mice Tiina Hurskainen,

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Deletion of the Major Bullous Pemphigoid Epitope Region of Collagen XVII Induces Blistering, Autoimmunization, and Itching in Mice Tiina Hurskainen, Nina Kokkonen, Raija Sormunen, Joanna Jackow, Stefanie Löffek, Raija Soininen, Claus-Werner Franzke, Leena Bruckner-Tuderman, Kaisa Tasanen Journal of Investigative Dermatology Volume 135, Issue 5, Pages 1303-1310 (May 2015) DOI: 10.1038/jid.2014.443 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Generation and characterization of ΔNC14A mice. (a) Cre recombination removes the loxP-flanked PGK-neoR cassette, and the reading frame is restored in the targeted Col17a1 locus lacking the 216-bp exon 18. Exons are marked as rectangles and primers with arrows. Bottom: genomic DNA PCR products. (b) Immunoblotting of skin extracts of adult mice. The domain–specific antibody NC14A revealed the absence of the NC14A domain. EC-7 binds to the extracellular domain and shows lower molecular weight of ColXVII in the ΔNC14A mouse sample. A shed ectodomain, approximately 110–120 kDa, was detected in both WT and ΔNC14A skin. GAPDH was used as a loading control. (c) Relative ColXVII protein levels quantified from immunoblots showed variation between individuals. Bars, mean values. ColXVII, collagen XVII; NC, noncollagenous; WT, wild type. Journal of Investigative Dermatology 2015 135, 1303-1310DOI: (10.1038/jid.2014.443) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Appearance of the ΔNC14A mice. (a) The fur of ΔNC14A mice appears to turn gray, and it is thinner than the fur of the WT mice at the same age (13 weeks). (b–d) Because of scratching, ΔNC14A mice develop large erosions and crusts in their skin. Symptomatic skin is also seen on the ears (c) and paws (d) of ΔNC14A mice (b=13, c=20, d=9 weeks). (e) The number of mice in time point (age in weeks) when scratching was initiated. The age when mice were observed to start scratching was typically 10–16 weeks (n=39). In total, 82 ΔNC14A mice out of 102 were detected to scratch at varying age, a few (n=8) not until >52 weeks old. NC, noncollagenous; WT, wild type. Journal of Investigative Dermatology 2015 135, 1303-1310DOI: (10.1038/jid.2014.443) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Histopathological and ultrastructural analysis of skin. (a–f) Hematoxylin–eosin-stained skin sections. (a) Newborn WT skin. (b) Subepidermal microblisters (white arrow) in the skin of the newborn ΔNC14A mouse. (c–f) Thickening of the epidermis, subepidermal blistering (white arrows), hyperkeratosis, and eosinophils (black arrows), as well as hyperproliferation of fibroblasts (asterisk), were detected in the perilesional skin of scratching ΔNC14A mice. Scale bars=50 μm. (g, h) Transmission electron microscopy of adult WT (g) and ΔNC14A (h) mouse skin. (h) Malformed hemidesmosomes (white arrows) and basement membrane zone (arrowheads) in the skin of adult ΔNC14A mouse. (i, j) Immunoelectron microscopy with ectodomain-specific antibody (EC-7) showed ColXVII localization (black arrows) in hemidesmosomes and at the dermo-epidermal junction in WT (i) and ΔNC14A (j) skin. Scale bars=500 nm. ColXVII, collagen XVII; ECM, extracellular matrix; NC, noncollagenous; WT, wild type. Journal of Investigative Dermatology 2015 135, 1303-1310DOI: (10.1038/jid.2014.443) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Increased immunoglobulin levels in ΔNC14A mice. (a, b) Skin extracts of WT and ΔNC14A mice were analyzed by immunoblotting with anti-IgG, anti-IgG1, and anti-IgE-specific antibodies. GAPDH was used as a loading control. (c) Quantified immunoglobulin levels in skin compared with one WT sample and reported as fold-change values. (d) Total IgE concentration in serum samples analyzed by ELISA. NC, noncollagenous; WT, wild type. Journal of Investigative Dermatology 2015 135, 1303-1310DOI: (10.1038/jid.2014.443) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Scratching ΔNC14A mice have circulating autoantibodies. (a–c) Frozen skin sections were double-immunostained with anti-IgG, anti-IgG1, and ColXVII-specific antibody (EC-7). IgG-specific staining was not seen in WT (age 9 weeks) (a), whereas linear IgG and IgG1 deposition was detected in the DEJ of ΔNC14A skin (b, c) (b=8 weeks, c=35 weeks). (d) Direct IF (DIF) with anti-IgA showed similar DEJ deposition in the skin of ΔNC14A mice (8 weeks) as IgG. (e, f) Indirect IF (IIF) with scratching ΔNC14A mouse serum (e=13 weeks, f=8 weeks) and anti-IgG showed linear staining in the DEJ and colocalized with ColXVII in WT untreated (e) and salt-split (f) skin, both signals on epidermal side of the blister (asterisk). Arrowheads, basement membrane zone. Scale bars=20 μm. ColXVII, collagen XVII; DEJ, dermo–epidermal junction; IF, immunofluorescence; NC, noncollagenous; SSS, salt-split skin; WT, wild type. Journal of Investigative Dermatology 2015 135, 1303-1310DOI: (10.1038/jid.2014.443) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Antigen detection with scratching ΔNC14A mouse sera. (a) WT and scratching ΔNC14A mouse sera (age 62 weeks) were used as primary antibodies in immunoblotting to detect the autoantigen in WT mouse keratinocytes. Anti-mouse IgG was used as a secondary antibody. A 180-kDa protein was detected with scratching mouse serum and ColXVII-specific antibody (EC-7) but not with WT serum. The target protein was not detected in collagenase-treated cell extract. (b) ColXVII-specific antibody (EC-7) was used in the immunoprecipitation of WT keratinocyte extract. The immunoprecipitated 180-kDa protein was detected with scratching ΔNC14A mouse serum (age 62 weeks) and anti-IgG but not with WT serum. ColXVII, collagen XVII; NC, noncollagenous; WT, wild type. Journal of Investigative Dermatology 2015 135, 1303-1310DOI: (10.1038/jid.2014.443) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions