SOCS3 Expressed in M2 Macrophages Attenuates Contact Hypersensitivity by Suppressing MMP-12 Production  Kazuyuki Meguro, Daiki Nakagomi, Kotaro Suzuki, Junichi Hosokawa, Tadashi Fukuta, Masaya Yokota, Yuko Maezawa, Akira Suto, Hiroshi Nakajima  Journal of Investigative Dermatology  Volume 136, Issue 3, Pages 649-657 (March 2016) DOI: 10.1016/j.jid.2015.12.010 Copyright © 2015 The Authors Terms and Conditions

Figure 1 IFN-γ induces SOCS3 expression in macrophages in the inflammatory sites of 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity. C57BL/6 mice were sensitized with DNFB, and, 5 days later, the mice were challenged with DNFB on the ear. Four days after DNFB challenge, anti–IFN-γ monoclonal antibody, anti–IL-6 monoclonal antibody, or control IgG was injected intraperitoneally into the mice. Twenty-four hours later, the expression of SOCS3 in MR+ F4/80+ M2 macrophages harvested from the ear was examined. Shown are representative histograms with MFI (left) and mean ± standard deviation of MFI of anti-SOCS3 staining (white column) and isotype-matched IgG staining (gray column). Data are representative of three independent experiments. n = 4 each. **P < 0.01. ns, not significant. MFI, mean fluorescent intensity. Journal of Investigative Dermatology 2016 136, 649-657DOI: (10.1016/j.jid.2015.12.010) Copyright © 2015 The Authors Terms and Conditions

Figure 2 SOCS3 expressed in monocytes/macrophages attenuates contact hypersensitivity in mice. Monocyte/macrophage-specific SOCS3-deficient mice (LysM-Cre SOCS3fl/fl mice [SOCS3Δ/Δ mice]) and control SOCS3fl/fl mice (SOCS3+/+ mice) were sensitized with 2,4-dinitrofluorobenzene (DNFB), and, 5 days after the sensitization, mice were challenged with DNFB on the ear. (a) Ear swelling was quantified by measurements of ear thickness before (baseline) and indicated days after the challenge. Shown are mean ± standard deviation (SD) of the increase in ear thickness from baseline. n = 5 mice in each group. **P < 0.01, significantly different from the mean value of SOCS3+/+ mice. (b, c) Representative (b) photographs and (c) photomicrographs with hematoxylin and eosin staining of SOCS3Δ/Δ mice and SOCS3+/+ mice 5 days after DNFB challenge. n = 5 each. Bars = 200 μm. (d–h) Five days after challenge with DNFB, the numbers of (d) CD4+ cells, (e) CD8+ cells, (f) Ly-6G+ cells, (g) MR– F4/80+ cells, and (h) MR+ F4/80+ cells in the ear were evaluated. Shown are mean ± SD. n = 5 mice in each group. **P < 0.01, significantly different from the mean value of SOCS3+/+ mice. nd, not detectable. (i) Five days after the challenge with DNFB, the level of SOCS3 mRNA in the ear was examined by quantitative real-time PCR analysis. Data are given as mean ± SD of relative expression of SOCS3. Data are representative of three independent experiments. n = 4 each. *P < 0.05. Journal of Investigative Dermatology 2016 136, 649-657DOI: (10.1016/j.jid.2015.12.010) Copyright © 2015 The Authors Terms and Conditions

Figure 3 IFN-γ pretreatment attenuates M2 macrophage-mediated exacerbation of 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity in a SOCS3-dependent manner. C57BL/6 mice were sensitized with DNFB, and, 5 days later, the mice were challenged with DNFB on the ear. M2 macrophages were prepared from bone marrow cells of SOCS3Δ/Δ mice and SOCS3+/+ mice and were treated with or without IFN-γ for 2 hours. After washing, M2 macrophages (5 × 105 cells in 20 μl of phosphate buffered saline) were injected intradermally into the ear 4 hours after the challenge with DNFB. As a control, phosphate buffered saline was injected to the ear. (a) Ear swelling was quantified 48 hours after the challenge with DNFB. Shown are mean ± standard deviation (SD) of the increase in ear thickness from baseline. n = 5 mice in each group. *P < 0.05. ns, not significant. (b–d) Forty-eight hours after the challenge with DNFB, the numbers of (b) Ly-6G+ cells, (c) MR- F4/80+ cells, and (d) MR+ F4/80+ cells in the ear were evaluated. Shown are mean ± SD. n = 5 mice in each group. *P < 0.05. Journal of Investigative Dermatology 2016 136, 649-657DOI: (10.1016/j.jid.2015.12.010) Copyright © 2015 The Authors Terms and Conditions

Figure 4 MMP-12 expression in M2 macrophages is enhanced in SOCS3Δ/Δ mice. (a, b) SOCS3Δ/Δ mice and SOCS3+/+ mice were sensitized with 2,4-dinitrofluorobenzene (DNFB), and, 5 days later, the mice were challenged with DNFB on the ear. (a) Five days after the challenge, the levels of MMP-12 mRNA in the ear were measured by quantitative real-time PCR analysis. Data are given as mean ± standard deviation (SD) of relative expression. n = 5 each. **P < 0.01. nd, not detectable. (b) Five days after the challenge, MR– F4/80+ cells and MR+ F4/80+ cells were isolated from the ear by flow cytometry, and the expression levels of MMP-12 were evaluated by quantitative real-time PCR analysis. Data are given as mean ± SD of relative expression. n = 4. *P < 0.05. (c, d) M1 macrophages and M2 macrophages were prepared from bone marrow (BM) cells of SOCS3Δ/Δ mice and SOCS3+/+ mice. The expression levels of (c) mannose receptor (MR) and (d) MMP-12 were determined by quantitative real-time PCR analysis. Data are given as mean ± SD of relative expression. n = 4. **P < 0.01. Journal of Investigative Dermatology 2016 136, 649-657DOI: (10.1016/j.jid.2015.12.010) Copyright © 2015 The Authors Terms and Conditions

Figure 5 MMP-12 deficiency reduces the enhanced contact hypersensitivity in SOCS3Δ/Δ mice. SOCS3+/+ mice, SOCS3Δ/Δ mice, SOCS3Δ/Δ MMP-12–/– mice, and MMP-12–/– mice were sensitized with 2,4-dinitrofluorobenzene (DNFB), and, 5 days later, the mice were challenged with DNFB on the ear. (a) Ear swelling was quantified by measurements of ear thickness before (baseline) and 48 hours after the challenge. Shown are mean ± standard deviation (SD) of the increase in ear thickness from baseline. n = 5 mice in each group. *P < 0.05, significantly different from the mean value of SOCS3Δ/Δ mice. (b–d) Seventy-two hours after the challenge, the numbers of (b) Ly-6G+ cells, (c) MR– F4/80+ cells, and (d) MR+ F4/80+ cells in the ear were evaluated. Shown are mean ± SD. Data are representative of three independent experiments. n = 5. *P < 0.05. Journal of Investigative Dermatology 2016 136, 649-657DOI: (10.1016/j.jid.2015.12.010) Copyright © 2015 The Authors Terms and Conditions

Figure 6 IFN-γ/SOCS3 pathway suppresses IL-4-induced MMP-12 expression and STAT6 binding to the MMP-12 promoter. Bone marrow-derived macrophages of SOCS3Δ/Δ mice and SOCS3+/+ mice were stimulated with or without IFN-γ for 2 hours. (a) These cells were then stimulated with or without IL-4 for 24 hours, and the expression levels of MMP-12 were evaluated by quantitative real-time PCR analysis. Data are given as mean ± standard deviation (SD) of relative expression. n = 4. *P < 0.05. nd, not detectable; ns, not significant. (b) These cells were then stimulated with IL-4 for 0.5 hour or 2 hours, and whole-cell extracts were subjected to immunoblotting with indicated antibodies. Representative blots from four independent experiments are shown. (c) These cells were then stimulated with or without IL-4 for 2 hours. Chromatin immunoprecipitation-quantitative real-time PCR assay for MMP-12 promoter was performed with anti-STAT6 antibody or control rabbit IgG. Data are given as mean ± SD of percent input for each chromatin immunoprecipitation fraction. n = 4. *P < 0.05. Journal of Investigative Dermatology 2016 136, 649-657DOI: (10.1016/j.jid.2015.12.010) Copyright © 2015 The Authors Terms and Conditions