Epicutaneous Allergic Sensitization by Cooperation between Allergen Protease Activity and Mechanical Skin Barrier Damage in Mice  Sakiko Shimura, Toshiro Takai, Hideo Iida, Natsuko Maruyama, Hirono Ochi, Seiji Kamijo, Izumi Nishioka, Mutsuko Hara, Akira Matsuda, Hirohisa Saito, Susumu Nakae, Hideoki Ogawa, Ko Okumura, Shigaku Ikeda  Journal of Investigative Dermatology  Volume 136, Issue 7, Pages 1408-1417 (July 2016) DOI: 10.1016/j.jid.2016.02.810 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Epicutaneous administration of papain and tape stripping cooperatively promoted dermatitis. Papain was painted (days 0, 3, 7, and 11 [day 10 in c and d] onto ears with (TS+) or without (TS-) tape stripping. (a) Ear swelling. Data represent the mean ± standard deviation of four mice per group. ∗P < 0.05 versus the vehicle and #P < 0.05 vs papain (TS-) by Mann-Whitney U test. (b) Histology after the first (12-, 24-, and 72-hour) and second (84 - and 96-hour [12 and 24 hours after day 3, respectively]) administrations. Scale bar = 50 μm except for 72 hours, scale bar = 100 μm). (c, d) Gene expression 24 hours after the (c) first and (d) last administration. ∗P < 0.05 by analysis of variance and #P < 0.05 versus the vehicle alone by the Mann-Whitney U test. AREG, amphiregulin; h, hour; HB-EGF, heparin-binding epidermal growth factor-like growth factor; mRNA, messenger RNA; TNF-α, tumor necrosis factor-α; TS–, without tape stripping; TS+, with tape stripping. Journal of Investigative Dermatology 2016 136, 1408-1417DOI: (10.1016/j.jid.2016.02.810) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Epicutaneous administration of papain induced IgE/IgG1 responses and differentiation of Th2 and Th17 cells. Papain was applied to the surface of the right ear with (TS+) or without (TS-) tape stripping (fays 0, 3, 7, and 11). (a–c) Serum antibody response. ∗P < 0.05 versus vehicle and #P < 0.05 versus papain (TS-) by the Mann-Whitney U test. Data represent the mean ± standard deviation of four or eight mice per group. (d) Cytokine release in draining lymph node cells recovered on day 12 and restimulated in vitro with E64-treated papain. ∗P < 0.05 versus without restimulation by the Mann-Whitney U test. Data represent the mean ± standard deviation of four mice per group. OD, optical density; Th, T helper cell; TS–, without tape stripping; TS+, with tape stripping. Journal of Investigative Dermatology 2016 136, 1408-1417DOI: (10.1016/j.jid.2016.02.810) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Dermatitis and IgE/IgG1 responses depended on the protease activity of epicutaneously administered papain. Papain or that treated with E64 (E64-Papain), which was prepared as described in Supplementary Methods online, was applied to the surface of the right ear with tape stripping (TS+) (days 0, 3, 7, and 11). (a) Ear swelling. (b–d) Serum antibody response. ∗P < 0.05 versus vehicle and #P < 0.05 versus E64-papain by the Mann-Whitney U test. Data represent the mean ± standard deviation of four mice per group. (e) Histology (day 12). Scale bar = 50 ±m. OD, optical density; TS–, without tape stripping; TS+, with tape stripping. Journal of Investigative Dermatology 2016 136, 1408-1417DOI: (10.1016/j.jid.2016.02.810) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Papain showed adjuvant activity in antibody responses against co-administered OVA. OVA was administered alone or with papain to the surface of the right ear with tape stripping (days 0, 3, 7, and 11). (a) Ear swelling. (b) Histology. Scale bar = 50 μm. Representative data are shown. (c–g) Serum antibody response. ∗P < 0.05 versus vehicle and #P < 0.05 versus OVA by the Mann-Whitney U test. Data represent the mean ± standard deviation of four mice per group. OD, optical density; OVA, chicken ovalbumin; TS+, with tape stripping. Journal of Investigative Dermatology 2016 136, 1408-1417DOI: (10.1016/j.jid.2016.02.810) Copyright © 2016 The Authors Terms and Conditions

Figure 5 IL-33 was dispensable for responses in the model of epicutaneous sensitization to papain. Papain was applied to the surface of the right ear with tape stripping in wild-type C57BL/6N and IL-33–/– mice (days 0, 3, 7, and 11). (a) Ear swelling. (b) Histology. Scale bar = 50 μm. (c–e) Serum antibody response. ∗P < 0.05 versus vehicle (wild type) by the Mann-Whitney U test. Data represent the mean ± standard deviation of four or five mice per group, except for one group (vehicle, IL-33–/–: n = 2). ns, not significantly different; OD, optical density; TS+, with tape stripping. Journal of Investigative Dermatology 2016 136, 1408-1417DOI: (10.1016/j.jid.2016.02.810) Copyright © 2016 The Authors Terms and Conditions

Figure 6 IL-33 contributed to atopic march and maintenance of IgE levels induced by papain inhalation after epicutaneous sensitization. (a) Serum antibody levels on day 12 after epicutaneous papain sensitization. Papain (1 or 10 mg/ml) was applied to the surface of both ears with tape stripping in wild-type C57BL/6N and IL-33–/– mice (days 0, 3, 7, and 10). (b) Serum antibody levels and (c) allergic airway inflammation after the intranasal challenge with papain (day 21). A low dose of papain (2.5 μg) was intranasally administered (twice, days 14 and 17) to mice presensitized with papain. ∗P < 0.05 by the Mann-Whitney U test. Data represent the mean ± standard deviation of 5–7 mice per group. i.n., intranasal administration; OD, optical density. Journal of Investigative Dermatology 2016 136, 1408-1417DOI: (10.1016/j.jid.2016.02.810) Copyright © 2016 The Authors Terms and Conditions