Lack of Galanin Receptor 3 Alleviates Psoriasis by Altering Vascularization, Immune Cell Infiltration, and Cytokine Expression  Felix Locker, Silvia Vidali, Barbara S. Holub, Julia Stockinger, Susanne M. Brunner, Sabine Ebner, Andreas Koller, Andrea Trost, Herbert A. Reitsamer, David Schwarzenbacher, Roland Lang, Barbara Kofler  Journal of Investigative Dermatology  Volume 138, Issue 1, Pages 199-207 (January 2018) DOI: 10.1016/j.jid.2017.08.015 Copyright © 2017 The Authors Terms and Conditions

Figure 1 GAL3-R, α-SMA, and CD31 staining of human skin. Representative images of immunofluorescent staining of (a) CD31 (white), (b) α-SMA (red), (c) GAL3-R (green), and (d) merge of all channels, in normal male skin. Scale bar = 50 μm. GAL3-R, galanin receptor 3; SMA, smooth muscle actin. Journal of Investigative Dermatology 2018 138, 199-207DOI: (10.1016/j.jid.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Immunohistochemical staining for GAL3-R and nestin in human skin. (a, d) Representative images of immunofluorescent staining of GAL3-R (red) and (b, e) nestin (green). (c, f) Merge of channels. (a–c) Normal male skin, (d–f) psoriatic male skin. Arrows in a and c indicate GAL3-R–positive blood vessels in normal skin negative for nestin. Arrows in d indicate GAL3-R–positive blood vessels in psoriatic skin, arrows in e indicate nestin-positive blood vessels, and arrows in f indicate overlapping staining of GAL3-R and nestin in capillaries of the papillary dermis of psoriatic skin (psoriasis vulgaris). Scale bar = 50 μm. GAL3-R, galanin receptor 3. Journal of Investigative Dermatology 2018 138, 199-207DOI: (10.1016/j.jid.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Severity of IMQ induced inflammation in GAL3-KO mice. Semiquantitative scoring of (a) erythema, (b) scaling, (c) thickening of the skin, and (d) cumulative scoring over time after IMQ treatment of WT and GAL3-KO mice. (e) The thickness of epidermis was measured in hematoxylin and eosin-stained skin samples of vehicle and IMQ-treated mice on the indicated time points. (f) Splenomegaly, indicated as total spleen weight, was analyzed in vehicle (ctrl) and IMQ-treated mice on days 4 and 7. (g) Representative images from days 4, 5, and 6. Values represent mean ± standard error of the mean. (a–d) n = 18–23, (e–f) n = 13–22 per group. ∗P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.001 versus WT mice, °°°P < 0.001 vs. vehicle-treated (ctrl) mice. d, days; GAL3-KO, galanin receptor 3 knockout mice; IMQ, imiquimod; WT, wild type. Journal of Investigative Dermatology 2018 138, 199-207DOI: (10.1016/j.jid.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Determination of vascular density and vascularization-related transcripts. (a) CD31-positive vessels in skin sections of vehicle-treated (ctrl) and IMQ-treated WT and GAL3-KO mice on days 4 and 7. GAL3-KO and WT mice show no differences in mRNA levels of (b) VEGF, (c) ANGPT I, and (d) ANGPT II in skin sections of IMQ-treated WT and GAL3-KO mice. GAL3-KO mRNA was calculated relative to the levels in vehicle-treated WT animals. Values represent mean ± standard error of the mean. (a) n = 11–15, (b) n = 12–15 per group. ∗∗P < 0.01 versus WT mice, °°°P < 0.001 versus vehicle-treated (ctrl) mice. ANGPT I, angiopoietin I; ANGPT II, angiopoietin II; ctrl, control; d, day; GAL3-KO, galanin receptor 3-knockout mice; IMQ, imiquimod; VEGF, vascular endothelial growth factor; WT, wild type. Journal of Investigative Dermatology 2018 138, 199-207DOI: (10.1016/j.jid.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 5 MPO activity and the amount of macrophages, neutrophils, and mast cells in murine psoriasiform skin. (a) MPO concentration (U/ml) in 8-mm skin punch biopsy samples from vehicle-treated (ctrl) and IMQ-treated mice on days 4 and 7. Number of (b) F4/80+, (c) NIMP-R14+, and (d) toluidine blue-stained cells in paraffin-embedded skin sections of vehicle-treated and IMQ-treated WT and GAL3-KO mice on day 4 and day 7, respectively. Values represent mean ± standard error of the mean. (a) n = 13–18, (b–d) n = 11–14 per group. ∗∗P < 0.01 versus WT mice, °°°P < 0.001 versus vehicle-treated (ctrl) mice. ctrl, control; d, day; GAL3-KO, galanin receptor 3 knockout mice; IMQ, imiquimod; MPO, myeloperoxidase; n.q., no quantification possible due to sparse presence of neutrophils in healthy skin; WT, wild type mice. Journal of Investigative Dermatology 2018 138, 199-207DOI: (10.1016/j.jid.2017.08.015) Copyright © 2017 The Authors Terms and Conditions

Figure 6 mRNA expression analysis. Relative mRNA levels of IL-17A, IL-22, IL-23, and TNF-α in skin sections of IMQ-treated GAL3-KO mice on days (a) 4 and (b) 7; mRNA levels of GAL3-KO mouse skin are delineated relative to the expression levels in IMQ-treated WT animals. Values represent mean ± standard error of the mean, n = 12–15 per group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus WT mice. GAL3-KO, galanin receptor 3 knockout mice; TNF, tumor necrosis factor; WT, wild type mice. Journal of Investigative Dermatology 2018 138, 199-207DOI: (10.1016/j.jid.2017.08.015) Copyright © 2017 The Authors Terms and Conditions