Dermal Fibroblasts Promote Alternative Macrophage Activation Improving Impaired Wound Healing  Rubén A. Ferrer, Anja Saalbach, Mike Grünwedel, Nadine Lohmann, Inka Forstreuter, Susann Saupe, Elke Wandel, Jan C. Simon, Sandra Franz  Journal of Investigative Dermatology  Volume 137, Issue 4, Pages 941-950 (April 2017) DOI: 10.1016/j.jid.2016.11.035 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Dermal fibroblast shift differentiation from inflammatory macrophages toward alternative macrophages. (a) Monocyte differentiation toward inflammatory macrophages alone (Ma(GM)) or dFb coculture (Ma(GM)/dFb) or MSC coculture (Ma(GM)/MSC), compared with alternative differentiation with M-CSF (Ma(M)). Polarization characterization: TNF/IL-12/IL-10 release + CD163 expression after LPS stimulation. (b) As in (a) and dFb from passage 1 (Ma(GM)/dFb(p1)), 3 (Ma(GM)/dFb(p3)), or 6 (Ma(GM)/dFb(p6)) for cocultures. (c) As in (a), and dFb were separated into ABCB5+ and ABCB5− populations (Ma(GM)/ABCB5+; Ma(GM)/ABCB5−; Ma(GM)/dFb) for cocultures. Data are presented as mean ± SD and derived from: (a) n = 7 different experiments, at least n = 3 monocyte donors and n = 3 MSC or dFb donor; (b) n = 4 different experiments, at least n = 4 monocyte donors and n = 3 dFb donors; (c) n = 5 different experiments, at least n = 3 monocyte donors and n = 5 dFb donors. Analysis of variance, pairwise multiple comparison with Ma(GM): *P < 0.05; **P < 0.01; ***P < 0.001. ABCB5, ATP-binding cassette member B5; dFb, dermal fibroblasts; LPS, lipopolysaccharide; M-CSF, colony-stimulating factor macrophage; MSC, mesenchymal stromal cell; SD, standard deviation; TNF, tumor necrosis factor. Journal of Investigative Dermatology 2017 137, 941-950DOI: (10.1016/j.jid.2016.11.035) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Macrophage-immunomodulation by dFb is mediated by soluble dFb-derived factors on inflammatory activation. Experimental setting as in Figure 1. (a) Monocytes were either cocultured directly atop dFb (Ma(GM)/dFb) or cocultured in presence of dFb but without physical contact by using a transwell insert (transwell Ma(GM)//dFb). (b) Inflammatory-macrophage differentiation performed with control medium (Ma(GM)) or conditioned medium (CM) derived from dFb stimulated for 24 hours with TNF/IL-1b (Ma(GM)/CM-dFb(IL-1b/TNF)) or from none stimulated dFb (Ma(GM)/CM-dFb(non-stim.)) or in stimulation medium containing TNF/IL-1b (Ma(GM)/SN(IL-1b/TNF)). Data are presented as mean ± SD and derived from (a) at least n = 9 different experiments, at least n = 4 monocyte donors and n = 5 dFb donors; (b) at least n = 12 different experiments, at least n = 6 monocyte donors and n = 6 dFb donors. The unpaired t-test compared with Ma(GM): *P < 0.05; **P < 0.01; ***P < 0.001. dFb, dermal fibroblasts; SD, standard deviation; SN, supernatant; TNF, tumor necrosis factor. Journal of Investigative Dermatology 2017 137, 941-950DOI: (10.1016/j.jid.2016.11.035) Copyright © 2016 The Authors Terms and Conditions

Figure 3 dFb activation induces expression/release of TSG6 and Cox2/PGE2. (a–c) Analysis of gene expression (a) and levels of released mediators (b/c) of dFb cultured without stimulation (non stim.) or with IL-1b/TNF or with GM-CSF or in coculture (Ma(GM)/dFb): not-transfected dFb (non), siRNA-transfected dFb (scramble; siTSG6; siCox2). (d) Cocultures as in Figure 1: nontransfected (Ma(GM)/dFb), siRNA-transfected (Ma(GM)/dFb-siCox2; Ma(GM)/dFb-siTSG6; Ma(GM)/dFb-siCox2/siTSG6; Ma(GM)/dFb-scramble) dFb. Data are presented as mean ± SD, derived from: (a) at least n = 3 dFb donors; (b) at least n = 3 different experiments, n = 3 monocyte donors and n = 3 dFb donors; (c) at least n = 7 different experiments, n = 3 monocyte donors, and n = 4 dFb donors. The unpaired t-test compared with non stim. (a), Ma(GM) (b,d), or scramble (c): *P < 0.05; **P < 0.01; ***P < 0.001; unpaired t-test to Ma(GM)/dFb: ###P < 0.001; n.dec., not detectable; dFb, dermal fibroblasts; GM-CSF, colony-stimulating factor granulocyte macrophage; IDO-1, indoleamine 2,3-dioxygenase 1; PGE2, prostaglandin E2; SD, standard deviation; siRNA, small interference RNA; TNF, tumor necrosis factor; TSG6, tumor necrosis factor-inducible gene 6 protein. Journal of Investigative Dermatology 2017 137, 941-950DOI: (10.1016/j.jid.2016.11.035) Copyright © 2016 The Authors Terms and Conditions

Figure 4 dFb reduce inflammation and promote M2 polarization in vivo. Sterile peritonitis in mice was induced by intraperitoneal (i.p.) injection of thioglycollate (TG) while control mice received PBS. Fifteen minutes after TG administration mice were treated (i.p. injection) with dFb (TG/dFb) or sham (TG). Injected dFb were either untransfected or treated with siRNA as in Figure 3d (TG/dFb-siCox2, TG/dFb-siTSG6, TG/dFb-siCox2/siTSG6, TG/dFb-scramble). Analysis followed 24 hours after peritonitis induction. (a) IL-1b and TNF levels in peritoneal fluid. (b) Immune cell infiltration in peritoneal cavity (PC) (Supplementary Figure S5). (c) Isolated F4/80+ cells (Supplementary Figure S6) were cultured for 4 hours and activated with LPS for extra 20 hours: IL-1b, TNF, and IL-10 secretion and Retnla gene expression. Data are presented as mean ± SD. Each symbol represents one mouse. unpaired t-test: *P < 0.05; **P < 0.01; ***P < 0.001. dFb, dermal fibroblasts; LPS, lipopolysaccharide; PBS, phosphate buffered saline; PMN, polymorphonuclear leucocytes; SD, standard deviation; siRNA, small interference RNA; TNF, tumor necrosis factor; TSG6, tumor necrosis factor-inducible gene 6 protein. Journal of Investigative Dermatology 2017 137, 941-950DOI: (10.1016/j.jid.2016.11.035) Copyright © 2016 The Authors Terms and Conditions

Figure 5 dFb injection improves impaired wound healing. Six-millimeter dorsal wounds received intradermal dFb injection or PBS (non) in wound margins 1 day post wounding (1 dpw). Injected dFb were either untransfected (dFb) or treated with siRNA (dFb-siCox2, dFb-siTSG6, dFb-scramble). Analysis of wounds 10 dpw. (a) Appearance of wounds. (b) Wound closure = epithelial-tip distance in pan-cytokeratin immunofluorescence staining (IF). (c) Wound HE staining (scale bar 100 μm). Arrow heads mark wound margins. (d) Pan-cytokeratin/aSMA-IF (scale bar 100 μm). Granulation tissue = aSMA positive area. (e) CD31-IF (scale bar 100 μm). Angiogenesis = CD31 distribution/wound area. (f) Wound gene expression and protein levels of IL-1b/TNF. (g) Wound IL-10 and Retnla expression and numbers of Ym-1+/F4/80+cells in IF (scale bar 20 μm). Arrows mark Ym-1+/F4/80+ cells. Each symbol represents one wound. Unpaired t-test: *P < 0.05; **P < 0.01; ***P < 0.001; e, epidermis; f, fat tissue; g, granulation tissue; s, scab; dFb, dermal fibroblasts; PBS, phosphate buffered saline; siRNA, small interference RNA; TNF, tumor necrosis factor; TSG6, tumor necrosis factor-inducible gene 6 protein. Journal of Investigative Dermatology 2017 137, 941-950DOI: (10.1016/j.jid.2016.11.035) Copyright © 2016 The Authors Terms and Conditions