Topical Application with a New NF-κB Inhibitor Improves Atopic Dermatitis in NC/NgaTnd Mice  Akane Tanaka, Susumu Muto, Kyungsook Jung, Akiko Itai, Hiroshi Matsuda  Journal of Investigative Dermatology  Volume 127, Issue 4, Pages 855-863 (April 2007) DOI: 10.1038/sj.jid.5700603 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Clinical symptoms of AD in NC/NgaTnd mice topically treated with IMD-0354. (a and b) Clinical skin (a) severity scores and (b) scratching frequency in NC/NgaTnd mice treated with 1% IMD-0354 ointment for 2 weeks were significantly reduced as well as those in FK506-treated mice. Each point represents means±SE of 10 mice in each group. *P<0.05, when compared with mice applied with ointment base alone. (c and d) Representatives of (c) clinical and (d) histological (H and E staining, bar=200μm) features of NC/NgaTnd mice treated with ointment base alone (left) or with 1% IMD-0354 ointment (right). Journal of Investigative Dermatology 2007 127, 855-863DOI: (10.1038/sj.jid.5700603) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effect of IMD-0354 on infiltration of inflammatory cells and NF-κB activation at affected skin sites. (a and b) Typical (a) histological features and (b) numbers of CD4+ cells, mast cells, and eosinophils in dorsal skins removed from mice treated with ointment base alone, with 0.1% FK506 ointment, or with 1% IMD-0354 ointment for 2 weeks. The total number of the cells in five high-power fields (bar=50μm) from eight individual skin specimens was counted under a microscope. Data represent means±SE of eight mice. *P<0.05, when compared with mice applied with ointment base alone. (c) Suppressive effect of IMD-0354 on NF-κB activation in skin lesions. Phosphorylation of IκBα in epidermal cells and infiltrating cells was obvious in the skin of placebo-treated mice, but not in the skin of IMD-0354-treated mice (bar=50μm) (left row). Positive staining of NF-κB p50 was remarkable in the nucleus of epidermal cells in the skin of placebo-treated mice (the upper of right row). On the other hand, translocation of NF-κB to the nucleus was suppressed in the skin of IMD-0354-treated mice on 2 weeks after the topical treatment (bar=20μm) (the lower of right row). Journal of Investigative Dermatology 2007 127, 855-863DOI: (10.1038/sj.jid.5700603) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of IMD-0354 on cytokine expression in lymph nodes and affected skins. (a and b) Cytokine expressions in (a) axillary lymph nodes and (b) skins of NC/NgaTnd mice treated with 0.1% FK506 ointment or 1% IMD-0354 ointment. (c) Effect of IMD-0354 on PWM-induced in vitro cytokine production in lymphocytes isolated from axillary lymph nodes conventional NC/Nga mice with severe dermatitis. Lymphocytes were incubated with or without PWM (3μg/ml) in the presence or absence of IMD-0354 (1μM) for 24hours. After reverse transcription, the real-time PCR analysis was performed with a SYBR Green assay system. The amount of gene expression that was normalized to β-actin was given by 2−ΔΔCT. Each value was presented as the expression value that was calculated ointment base alone as 1. Data represent means±SE of three different experiments with five mice. *P<0.05, when compared with mice applied with ointment base alone. Journal of Investigative Dermatology 2007 127, 855-863DOI: (10.1038/sj.jid.5700603) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Inhibitory effect of IMD-0354 on proliferation of various cells. (a) Inhibitory effect of IMD-0354 on NF-κB activation in PAM212 keratinocytes was confirmed by a reporter assay. Luciferase activity was normalized to the activity of vector transfectants. *P<0.05, when compared with the activity of cells cultured without IMD-0354. (b) Western blot analysis revealed that IMD-0354 abolished spontaneous (PAM 212 keratinocytes) and IgE-mediated (BMCMC) phosphorylaion of IκBα at 1μM. (c) Effect of IMD-0354 on proliferation of cell lines originated from mouse keratinocytes (PAM212), mast cells (P815), B cells (BCL1), and T cells (BW5147). Data represent means±SE of three different experiments with triplication. *P<0.05, when compared with cells applied with DMSO alone. Journal of Investigative Dermatology 2007 127, 855-863DOI: (10.1038/sj.jid.5700603) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effect of IMD-0354 on various cell functions correlating to allergic skin conditions. (a) Inhibitory effect of IMD-0354 on neurite outgrowth of PC12 cells after incubated with NGF (50ng/ml) for 48hours. *P<0.05, when compared with cells applied with DMSO alone. (b) Suppression of IgE production by splenic B cells stimulated by IL-4 (200U/ml) and CD40 ligand (100ng/ml). After 9 days, culture supernatants were collected and IgE levels were measured by an ELISA. *P<0.05, when compared with cells applied with DMSO alone. (c) Decrease in β-hexosaminidase release from mast cells at 60minutes after IgE-mediated activation. We used pyrrolidine dithiocarbamate, another NF-κB inhibitor, as a positive control. *P<0.05, when compared with β-hexosaminidase release (%) from IgE-sensitized BMCMC that were stimulated with DNP-BSA without any inhibitor. (d) Negative regulation of the de novo TNF-α production at 1 and 3hours after IgE-mediated activation of BMCMC. Journal of Investigative Dermatology 2007 127, 855-863DOI: (10.1038/sj.jid.5700603) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions