Interleukin-10 Downregulates Anti-Microbial Peptide Expression in Atopic Dermatitis Michael D. Howell, Natalija Novak, Thomas Bieber, Saveria Pastore,

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Interleukin-10 Downregulates Anti-Microbial Peptide Expression in Atopic Dermatitis Michael D. Howell, Natalija Novak, Thomas Bieber, Saveria Pastore, Giampiero Girolomoni, Mark Boguniewicz, Joanne Streib, Cathy Wong, Richard L. Gallo, Donald Y.M. Leung Journal of Investigative Dermatology Volume 125, Issue 4, Pages 738-745 (October 2005) DOI: 10.1111/j.0022-202X.2005.23776.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Decreased anti-microbial gene expression in extrinsic atopic dermatitis (EAD) and intrinsic atopic dermatitis (IAD) patients. RNA was isolated from the skin of normal subjects (▪, n=7), EAD (▴, n=9), IAD (▾, n=7), and psoriasis (♦, n=8), patients and the levels of human β-defensin (HBD)-2 (A) and LL-37 (B) evaluated by real-time PCR. Significant differences between patient groups were determined by a non-parametric Mann–Whitney test. Journal of Investigative Dermatology 2005 125, 738-745DOI: (10.1111/j.0022-202X.2005.23776.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of LL-37 is increased in allergic contact dermatitis but not extrinsic atopic dermatitis (EAD) or intrinsic atopic dermatitis (IAD). (A) Paraffin-embedded skin explants from EAD (n=5), IAD (n=4), and allergic contact dermatitis (n=5) patients were cut into 5 μm sections and stained for LL-37. Scale bars represent 25 μm. (B) The intensity of the immunostaining was visually scored on a scale from 0 to 5, with 0 indicating no staining and 5 the most intense staining. Significant differences between patient groups were determined by a non-parametric Mann–Whitney test. Journal of Investigative Dermatology 2005 125, 738-745DOI: (10.1111/j.0022-202X.2005.23776.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Reduced anti-microbial peptide expression is an acquired defect in primary keratinocytes from atopic dermatitis (AD) patients. The levels of human β-defensin (HBD)-2 (A) and LL-37 (B) were evaluated by real-time RT-PCR in differentiated keratinocytes obtained from normal subjects (♦, n=6), extrinsic AD (EAD) patients (▴, n=3), intrinsic AD (IAD) patients (▪, n=3), and psoriatic patients (•, n=6). HBD-2 was induced by 24 h stimulation with tumor necrosis factor-α (TNF-α) plus interferon-γ (IFN-γ), and LL-37 with heat-inactivated group A Staphylococcus extract (GAS). Samples treated with media alone are indicated by non-treated (NT). Journal of Investigative Dermatology 2005 125, 738-745DOI: (10.1111/j.0022-202X.2005.23776.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Increased interleukin (IL)-10 gene expression in extrinsic atopic dermatitis (EAD) and intrinsic atopic dermatitis (IAD) patients. RNA was isolated from the skin of normal subjects (▪, n=7), EAD (▴, n=9), IAD (▾, n=7), and psoriasis (♦, n=8) patients and the levels of IL-10 evaluated by real-time PCR. Significant differences between patient groups were determined by a non-parametric Mann–Whitney test. Journal of Investigative Dermatology 2005 125, 738-745DOI: (10.1111/j.0022-202X.2005.23776.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Inverse relationship between interleukin (IL)-10 and anti-microbial peptide (AMP) expression in intrinsic atopic dermatitis (IAD) patients. RNA was isolated from the skin of IAD (n=7) patients. The correlation between IL-10 and either human β-defensin (HBD)-2 (A) or LL-37 (B) gene expression was evaluated using linear regression. Journal of Investigative Dermatology 2005 125, 738-745DOI: (10.1111/j.0022-202X.2005.23776.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Anti-microbial expression in HaCaT keratinocytes is downregulated by interleukin (IL)-10 and IL-13. RNA was isolated from keratinocytes stimulated with the supernatants from normal peripheral blood mononuclear cell (n=6) cultured with media or anti-CD3 (100 ng per mL) in the presence or absence of IL-10 and/or IL-13 and the levels of human β-defensin (HBD)-2 (A) and LL-37 (B) were evaluated by real-time RT-PCR. Significant differences between exposure groups were determined by a two-tailed t test. Journal of Investigative Dermatology 2005 125, 738-745DOI: (10.1111/j.0022-202X.2005.23776.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Neutralization of interleukin (IL)-10 and IL-13 in atopic dermatitis (AD) skin augments anti-microbial peptide expression. Lesional skin from extrinsic AD patients (n=5) was cultured in the presence of anti-IL-10 (1 μg per mL) or anti-IL-13 (1 μg per mL) for 24 h and the levels of human β-defensin (HBD)-2 (A) and LL-37 (B) evaluated by real-time RT-PCR. Significant differences between exposure groups were determined by a two-tailed t test. Journal of Investigative Dermatology 2005 125, 738-745DOI: (10.1111/j.0022-202X.2005.23776.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions