Phases of Skin Repair Differential Roles of Macrophages in Diverse

Differential Roles of Macrophages in Diverse Phases of Skin Repair Tina Lucas, Ari Waisman, Rajeev Ranjan, Jürgen Roes, Thomas Krieg, Werner Müller, Axel Roers and Sabine A. This information is current as Eming of September 24, 2021. J Immunol 2010; 184:3964-3977; Prepublished online 22 February 2010; doi: 10.4049/jimmunol.0903356

http://www.jimmunol.org/content/184/7/3964 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2010/02/22/jimmunol.090335 Material 6.DC1 http://www.jimmunol.org/ References This article cites 46 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/184/7/3964.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

by guest on September 24, 2021 • No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Roles of Macrophages in Diverse Phases of Skin Repair

Tina Lucas,* Ari Waisman,† Rajeev Ranjan,* Ju¨rgen Roes,‡ Thomas Krieg,*,x Werner Mu¨ller,{ Axel Roers,‖ and Sabine A. Eming*

Influx of macrophages plays a crucial role in tissue repair. However, the precise function of macrophages during the healing response has remained a subject of debate due to their functional dichotomy as effectors of both tissue injury and repair. We tested the hypothesis that macrophages recruited during the diverse phases of skin repair after mechanical injury exert specific functions to restore tissue integrity. For this purpose, we developed a mouse model that allows conditional depletion of macrophages during the sequential stages of the repair response. Depletion of macrophages restricted to the early stage of the repair response (inflammatory phase) significantly reduced the formation of vascularized granulation tissue, impaired epithelialization, and resulted in minimized scar formation. In contrast, depletion of macrophages restricted to the consecutive mid-stage of the repair response Downloaded from (phase of tissue formation) resulted in severe hemorrhage in the wound tissue. Under these conditions, transition into the subsequent phase of tissue maturation and wound closure did not occur. Finally, macrophage depletion restricted to the late stage of repair (phase of tissue maturation) did not significantly impact the outcome of the repair response. These results demonstrate that macrophages exert distinct functions during the diverse phases of skin repair, which are crucial to control the natural sequence of repair events. The Journal of Immunology, 2010, 184: 3964–3977. http://www.jimmunol.org/ estoration of skin integrity and homeostasis following blasts, which differentiate into myofibroblasts, and the accumu- injury is a vital process. Across different species, this lation of additional macrophages. Deposition of provisional R event requires a complex and dynamic interplay of epi- extracellular wound matrix facilitates cell adhesion, migration, thelial and mesenchymal cells in concert with tissue-resident and and proliferation. Contemporaneously to the formation of granu- recruited hematopoetic cells to accomplish the sequential phases of lation tissue, at the wound edge, complex epidermal-mesenchymal the repair response: inflammation, tissue formation, and maturation interactions stimulate keratinocyte proliferation and migration to (1–4). The early stage of the repair response is dominated by the restore the epidermal barrier (5). Granulation tissue formation inflammatory phase, which is characterized by local activation of continues until the wound space is refilled and the overlaying the innate immune system, resulting in an immediate influx of epidermis is restored. Upon completion of the epidermal barrier, by guest on September 24, 2021 polymorphonuclear leukocytes (neutrophils) followed by sub- the repair response enters the late stage, which is characterized by sequent invasion of blood monocytes, which differentiate into tissue maturation. During the phase of tissue maturation, granu- tissue macrophages. The mid-stage of the repair response consists lation tissue transforms into scar tissue, characterized by attenu- of the phase of tissue formation, which is characterized by the ated cell proliferation, inflammation, neovascularization, and development of granulation tissue that refills the dermal wound replacement of the provisional matrix by deposition of collagen. space. Granulation tissue formation encompasses the invasion of Numerous studies in the past revealed functional consequences endothelial cells resulting in angiogenesis, the influx of fibro- of the innate immune response of the resident cells as well as the recruited inflammatory cells during skin repair (6). They combat x invading microbes, contribute to debris scavenging, but may also *Department of Dermatology and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne; ‖Medical critically support the repair process by releasing a spectrum of Faculty Carl Gustav Carus, Institute for Immunology, Technische Universita¨t Dres- growth factors. However, due to the release of proinflammatory den, Dresden; †First Medical Department, University of Mainz, Mainz, Germany; ‡Department of Immunology and Molecular Pathology, University College London, and cytotoxic mediators, uncontrolled activity of macrophages London; and{Faculty of Life Sciences, University of Manchester, Manchester, United may also be detrimental to tissue repair. Indeed, imbalanced in- Kingdom flammation characterized by increased numbers of macrophages is Received for publication October 13, 2009. Accepted for publication January 25, a hallmark of an attenuated repair response in human diseases 2010. encompassing diabetes mellitus, vascular disease, and aging (7, 8). This work was supported by the Deutsche Forschungsgemeinschaft (SFB829 to S.A.E. Currently, it is not completely understood how macrophages ex- and T.K. and SFB/TR 52 to A.W.). actly impact the physiology of the repair response and how they Address correspondence and reprint requests to Dr. Sabine A. Eming, Professor of Dermatology, University of Cologne, Jospeh-Stelzmann Strasse 9, 50931 Cologne, may contribute to the pathology of the repair response in diseased Germany. E-mail address: [email protected] conditions. Therefore, a more thorough understanding of macro- The online version of this article contains supplemental material. phage-specific functions during the diverse phases of the repair Abbreviations used in this paper: d, dermis; DT, diphtheria toxin; e, epidermis; Fizz1, response might broaden the view of this cell type in skin physi- found in inflammatory zone 1; g, granulation tissue; he, hyperproliferative epidermis/ ology and pathology. epidermal wound edge; HPF, high-power field; iDTR, inducible diphteria toxin receptor; LysM, lysozyme M; pc, panniculus carnosus; rp, red pulp; sc, s.c. fat muscle Neutrophils and macrophages represent the major fraction of layer; sm, s.c. muscle layer; a-SMA, a smooth muscle actin; st, scar tissue; TbRII, inflammatory cells recruited into the wound site. Within a few TGF-b receptor type II; VEGF-A, vascular endothelial growth factor-A; wp, white hours postinjury, neutrophils transmigrate across the endothelial pulp; Ym1/ECF, eosinophil chemotactic factor. cell wall and begin the debridement of devitalized tissue and in- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 fectious agents. Whereas the presence of neutrophils at the wound www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903356 The Journal of Immunology 3965 site is timely restricted to the early stage of the wound healing phycocyanin-conjugated anti-CD115 (eBioscience, San Diego, CA). Cells response, macrophages persist through all stages of the repair were incubated for 30 min at 4˚C and washed three times thereafter in response. Their number increases during the phase of in- PBS (0.5% BSA and 2 mM EDTA). The cells were analyzed on a FACSCalibur using CellQuest Pro Software (BD Biosciences, Heidel- flammation, peaks during the phase of tissue formation, and berg, Germany). gradually declines during the maturation phase (9). Experiments in the 1970s established the concept that under Wound phase-restricted depletion of macrophages sterile conditions, the influx of macrophages is essential for effi- To characterize macrophage function during distinct phases of repair, we cient healing of incisional skin wounds, whereas the influx of inflicted full-thickness excisional skin wounds on the back of LysMCre/ neutrophils might not be crucial (10, 11). This dogma has been iDTR or LysMCre mice (referred to as control mice). To achieve wound phase-restricted depletion of macrophages, LysMCre/iDTR and control challenged by recent reports, thereby arguing against an essential mice were injected with DT according to three regimens. For macrophage role of inflammatory cells in wound repair: early fetal wounds depletion during the inflammatory phase, mice were injected with DT 2 heal with minimal scarring, which is associated with little in- and 1 d prior to wounding as well as 2 and 4 d postwounding (regimen A), flammation (12). Furthermore, wounds in the neonatal PU.1 null the phase of tissue formation mice were injected with DT 3, 4, 6, and 8 mouse, which lacks macrophages and neutrophils (but also B d postinjury (regimen B), and the phase of maturation mice were injected with DT 8, 9, 11, and 13 d postinjury (regimen C). At indicated time cells, mast cells, eosinophils), heal without scar and, surprisingly, points, mice were sacrificed, and the wound tissue was excised for analysis. with a similar time course as wild-type siblings (13). However, the need of macrophages for physiological repair in adults is sup- Wounding and preparation of wound tissues ported in recent wound healing studies in various murine knockout Mice were anesthetized by i.p. injection of Ketanest/Rompun (Ketanest S, models in which impaired wound healing is associated with an Park Davis, Karlsruhe, Germany; Rompun 2%, Bayer, Leverkusen, Downloaded from attenuated number of macrophages at the wound site (14–18). Germany). The back was shaved, and two 6-mm diameter full-thickness Although all of these studies emphasize that leukocytes signifi- wounds were generated using a standard biopsy puncher (Stiefel, Offenbach, Germany). For histological analysis, wounds were excised at cantly affect the quality of the healing response, knowledge of indicated time points. Wounds were bisected in caudocranial direction, these models is limited, because they either do not target pathways and the tissue was either fixed overnight in 4% paraformaldehyde or mediated exclusively by macrophages or they address a neonate embedded in OCT compound (Tissue Tek, Miles, Elkhart, IN). Histo- logical analysis was performed on serial sections from the central portion repair response, which is known to differ from healing in the adult http://www.jimmunol.org/ of the wound. organism (19). Furthermore, the objective of most of the earlier studies was the impact of the global inflammatory response on the Immunohistochemistry outcome of the overall healing response. For immunohistochemical stainings, 5-mm cryosections were fixed either This study tested the hypothesis that macrophages present at the in 4% paraformaldehyde or in acetone and blocked with 10% normal goat wound site during the different stages of skin repair exert specific serum or with 10% FCS in PBS to reduce nonspecific Ab binding. Sections functions. In a mouse model that allows the conditional depletion of were incubated overnight at 4˚C with the primary Abs diluted in Dako- macrophages during distinct phases of the repair response in skin, Cytomation Ab Diluent (DakoCytomation, Carpinteria, CA). Bound primary Ab was detected by incubation with Alexa-conjugated (Invitrogen, we demonstrate in this study differential roles of macrophages in Karlsruhe, Germany) or peroxidase-conjugated (Southern Biotechnology, diverse phases of skin repair and report on macrophage-dependent Birmingham, AL) secondary Abs for 1 h at room temperature, followed by by guest on September 24, 2021 and -independent repair mechanisms that are crucial to restore counterstaining with DAPI, propidium iodide (Invitrogen), or hematoxylin. tissue homeostasis postinjury. As primary Abs, we used rat mAbs against F4/80 (Dianova BMA, Augst, Switzerland), CD31 (Pecam-1, BD Pharmingen, Heidelberg, Germany), Ki-67 (DakoCytomation), and Gr-1 (BD Pharmingen); polyclonal rabbit Materials and Methods Abs against found in inflammatory zone 1 (Fizz1; Peprotech, Paris, Animals France), vascular endothelial growth factor-A (VEGF-A; A-20, Santa Cruz Biotechnology, Santa Cruz, CA), and fibrinogen/fibrin (DakoCytomation); To generate mice in which macrophages can be depleted in a temporally polyclonal goat Abs against Ym-1 (R&D Systems, Wiesbaden, Germany) controlled manner, we bred the inducible diphteria toxin receptor (iDTR) and TGF-b1 (R&D Systems); a rabbit mAb against cleaved caspase-3 mice (C57BL/6 background) (20) with mice expressing the Cre re- (Asp175) (Cell Signaling Technology, Boston, MA) and a mouse mAb combinase (C57BL/6 background) from the myeloid cell-specific lyso- against a smooth muscle actin (a-SMA) coupled to Cy-3 (Sigma-Aldrich). zyme M promoter (LysMCre) [provided by Irmgard Fo¨rster, Du¨sseldorf Specifity of primary Abs was demonstrated by replacing them with irrel- University, Du¨sseldorf, Germany (21)]. Mice were maintained and bred evant isotype-matched Abs. under standard pathogen-free conditions and genotyped by PCR as described earlier (20, 21). Ten- to 12-wk-old mice were used for experiments. Morphometric analysis LysMCre/iDTR mice (hetero- or homozygous for the iDTR gene as in- The macroscopic wound area was quantified by processing of photographs dicated) and control mice (LysMCre) received diphtheria toxin (DT; 25 ng/ taken at various time points and was calculated as the percentage of the g body weight) (Merck, Darmstadt, Germany) injections (i.p. or i.v. as b wound area immediately postsurgery. Immunofluorescence microscopy was indicated) at indicated time points. To generate mice lacking the TGF- conducted at indicated magnifications (Microscope Eclipse 800E; Nikon, receptor type II (TbRII), we bred transgenic mice in which the exon 3 is Du¨sseldorf, Germany). Morphometric analysis was performed on digital flanked by loxP sequences (provided by Ju¨rgen Roes, University College b images using Imaging Software NIS-Elements AR 3.0 (Laboratory Im- London, London, U.K.) with LysMCre mice (LysMCre/T RII) (22). aging, Prague, Czech Republic), as described previously (17). The extent Flow cytometric analysis of epithelialization and granulation tissue formation was determined on H&E paraffin tissue sections using a light microscope at indicated mag- Cells were isolated from the peritoneal cavity by irrigation with ice-cold nifications (Leica DM4000B, Leica Microsystems, Wetzlar, Germany; PBS (0.5% BSA, 2 mM EDTA). For thioglycolate-mediated peritonitis, Diskus 4.50 Software, Diskus, Ko¨nigswinter, Germany). The width of the mice were injected i.p. with thioglycolate (2 ml of 4% in PBS; Sigma- gap between the epithelial tips was measured. The distance between the Aldrich, St. Louis, MO), and peritoneal cells were isolated 4 d thereafter for edges of the panniculus carnosus was determined as a measure of wound FACS analysis. Blood leukocytes were purified from heparinized blood by contraction. Numbers of macrophages, neutrophils, and cells positive for hypotonic lysis of erythrocytes with ACK lysing buffer, followed by PBS activated caspase-3, VEGF-A, or TGF-b1 were determined by counting washes. A total of 106 cells were used for the analysis. For FACS analysis, cells in two representative rectangles of 200 3 160 mm2 in the granulation cells were blocked with mouse seroblock FcR (CD16/CD32; AbD Sero- tissue of wound sections (minimum of three wounds on three mice per time tec, Du¨sseldorf, Germany) and stained with FITC-conjugated anti-F4/80 point for each group). The number of mast cells was determined by (AbD Serotec), PE-conjugated anti–Gr-1 (Miltenyi Biotec, Bergisch counting cells in the entire area of the scar tissue (four wounds on four Gladbach, Germany), allophycocyanin-conjugated anti-CD19 (Miltenyi mice). Ki-67–positive cells were determined by counting positively stained Biotec), PE-conjugated anti-CD11b (Mac-1; Miltenyi Biotec), and allo- cells in two representative rectangles 200 3 160 mm2 within the 3966 MACROPHAGES IN SKIN REPAIR hyperproliferative epidermal wound margins (three wounds on three dif- First, we investigated how macrophage depletion can be con- ferent mice). For quantitative analysis of CD31 and a-SMA expression as trolled by the dose, kinetics, and route of DTapplication (i.p. versus well as for fibrinogen/fibrin excudate, the percentage of the area of gran- i.v.) as well as the DTR gene dose (heterozygous versus homo- ulation tissue, which stained positive for CD31, a-SMA, or fibrinogen/fibrin, was calculated. Organization and maturation of collagen bundles was zygous). As revealed by flow cytometry for macrophages (F4/80 assessed on paraffin sections of day 14 wounds stained with Sirius Red and and CD11b), a single i.p. injection of DT (25 ng/g body weight) in analyzed by polarized light microscopy. All histomorphometric analyses LysMCre/iDTR (heterozygous) mutants resulted in complete de- were performed in a blinded manner by two independent investigators. pletion of peritoneal macrophages 24 h later that persisted for ∼3 Real-time PCR analysis d (Fig. 2A). Interestingly, although the LysM gene is expressed in Peritoneal macrophages from three mice per genotype were isolated, polymorphonuclear leukocytes, DT injection did not result in pooled, and cultured in DMEM supplemented with 10% FCS (106 cells/ depletion of peritoneal neutrophils (Gr-1) as revealed by flow cm2) overnight. Thereafter, the cells were starved in DMEM/1% FCS for cytometry (data not shown). Numbers of peritoneal B cells b 16 h and stimulated with recombinant human TGF- 1 (eBioscience) for (CD19) as identified by flow cytometry were similar in LysMCre 4 h (concentrations as indicated). RNA was isolated using the RNeasy plus mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s and LysMCre/iDTR (heterozygous) mice, demonstrating the instructions and reverse transcribed using the High Capacity cDNA RT specificity of macrophage depletion following DT injection. De- Kit (Applied Biosystems, Foster City, CA). Primers were generated for pletion of tissue-resident macrophages in skin, liver, and spleen TGF-b1(forward:59-TGG AGC AAC ATG TGG AAC TC-39, reverse: was not achieved by a single i.p. injection of DT in LysMCre/ 9 9 9 5 -GTC AGC AGC CGG TTA CCA-3 ) and S18 (forward: 5 -GAT CCC iDTR (heterozygous) mice, as revealed by immunohistochemical AGA CTG GTT CCT GA-39, reverse: 59-GTC TAG ACC GTT GGC CAG AA-39) (Metabion, Martinsried, Germany). Amplification reactions, each staining for F4/80 (data not shown). Also, repetitive i.p. DT in- in triplicate, were set up using PowerSYBR Green PCR Master Mix jections did not result in efficient depletion of resident macro- Downloaded from (Applied Biosystems). Real-time PCR was validated with the 7300 Real phages in these tissues. Therefore, we investigated whether Time PCR system (Applied Biosystems). The comparative method of 2dd increasing the DTR gene dose and i.v. DT application affects the relative quantification (2 Ct) was used to calculate expression level of the target gene normalized to S18. Data are expressed as relative increase efficacy of tissue-resident macrophage depletion. In fact, repetitive of TGF-b1–stimulated expression over controls. i.v. injections of DT (25 ng/g body weight) at 2 consecutive d in Statistical analysis LysMCre/iDTR (homozygous) mice resulted in efficient depletion

of both peritoneal and tissue-resident macrophages in skin, spleen, http://www.jimmunol.org/ Statistical analyses were performed using GraphPad Prism5 (GraphPad, and liver 1 d after DT injection (Fig. 2A,2B). Furthermore, the San Diego, CA). Significance of difference was analyzed using unpaired Student t test. All data are presented as mean 6 SD. p # 0.05 was con- same regimen of DT application resulted 24 h later in efficient sidered significant. depletion of monocytes (CD115, CD11b) and partial depletion of neutrophils (Gr-1, CD115) in the circulation (Fig. 2C). To in- Results vestigate whether myeloid cells can be efficiently depleted under Cell type-specific and timely restricted depletion of inflammatory conditions, LysMCre/iDTR (homozygous) and macrophages in LysMCre/iDTR mice LysMCre mice received two i.v. injections of DT at consecutive To analyze the functional impact of macrophages during diverse days, which 24 h later was followed by a single i.p injection with phases of skin repair, we generated mice in which macrophages can thioglycolate. Thereafter, mice received for 3 consecutive d DT by guest on September 24, 2021 be inducibly ablated using a model of Cre-inducible DT receptor- injections i.p., and at day 4 post thioglycolate application, peri- mediated cell ablation (Fig. 1). This system is based on a Cre-in- toneal lavage cells were analyzed. As revealed by flow cytometry ducible human DT receptor transgenic mouse line (iDTR) in which for macrophages (F4/80, CD11b) and neutrophils (Gr-1, CD115), Cre-mediated excision of a STOP cassette renders naturally DT- only macrophages were effectively ablated, not neutrophils (Fig. resistant mouse cells, DT sensitive. To generate mice in which 2D). Whether increased neutrophil number in LysMCre mice is macrophages can be inducibly ablated, the iDTR mouse line was simply the result of reduced phagocytosis by macrophages or re- crossed to LysMCre mice, reported to express Cre recombinase in sults from the lack of different macrophage-mediated control cells of myeloid origin (21). mechanisms is currently unknown. For the subsequent wound

FIGURE 1. Schematic representation of DT- mediated macrophage depletion in distinct stages of the repair response. To achieve wound phase-restricted depletion of macrophages, LysMCre/iDTR and LysMCre mice were injected with DT (i.v. or i.p.) according to three regimens: DT injection regimen A (A), macrophage depletion during the inﬂammatory phase, DT injections 2 and 1 d prior to wounding as well as at day 2 and 4 postwounding; DT injection regimen B (B), macrophage depletion during the tissue formation phase, DT injections at day 3, 4, 6, and 8 postinjury; DT injection regimen C (C), macrophage depletion during the maturation phase, DT injections at days 8, 9, 11, and 13 postinjury. At indicated time points, mice were sacriﬁced, and the wound tissue was excised for analysis. The Journal of Immunology 3967

FIGURE 2. Selective depletion of macrophages in LysMCre/iDTR mice. A, Analysis of macrophage depletion in peritoneal lavage: single i.p. injection of DT (25 ng/g body weight) in LysMCre/iDTR (heterozygous) or LysMCre/iDTR (homozygous) mice resulted in efficient depletion of peritoneal macrophages (F4/80, CD11b) 24 h after DT injection that lasted for 3 d; specificity of macrophage depletion was identified by a normal B cell number (CD19). DT injection in LysMCre (control) mice Downloaded from had no effect on macrophage number. B, Tissue- resident macrophages (stained by F4/80) in skin, spleen, and liver were efficiently depleted in LysMCre/iDTR (homozygous) mice 24 h following i.v. DT injections (25 ng/g body weight) on 2 consecutive d; tissue-resident macrophages were not http://www.jimmunol.org/ affected by DT injection in LysMCre mice. C, De- pletion of monocytes (CD11b, CD115) and neutrophils (Gr-1, CD115) in the circulation 24 h after two i.v. DT injections on consecutive days. D, Ef- fective depletion of macrophages (F4/80, CD11b) but not neutrophils (Gr-1, CD115) in LysMCre/ iDTR (homozygous) mice after thioglycolate- induced peritonitis. d, dermis; e, epidermis; rp, red pulp; sc, s.c. fat layer; wp, white pulp. by guest on September 24, 2021

healing studies, we used LysMCre/iDTR (homozygous) and LysM- wounds on three to six mice per time point for each group) was Cre (control) mice. excised and analyzed. To simplify the orientation in the wound histology, we included a schematic presentation outlining the Macrophage function during the early stage of repair histological hallmarks of the wound tissue (Fig. 3C). Macrophages recruited during the inflammatory phase of repair At day 5 postinjury, a significantly shorter distance between the induce granulation tissue formation, which results in scar tips of the epithelial tongues was measured for the control wounds formation. As revealed by the macroscopic analysis of wound compared with macrophage-depleted wounds, as demonstrated by closure, depletion of macrophages during the inflammatory phase H&E-stained paraffin sections, representing the longitudinal di- (DT injection regimen A [Fig. 1A]) resulted in significant delay of ameter of the wound (Fig. 3E). Furthermore, as revealed by ex- the early repair response compared with control mice. Whereas at pression of the cell proliferation marker Ki67, the epidermal day 5 postinjury the wound area was reduced to 50% of the margins in control wounds represented a hyperproliferative epi- original wound size in control mice, in macrophage-depleted thelial tongue, whereas the epidermal wound edge in macrophage- wounds of LysMCre/iDTR mice, the wound size was reduced by depleted wounds was short and showed few proliferating kerati- only 25%. However, at later time points, when DT injections were nocytes (Fig. 3D, Supplemental Fig. 1). To analyze dermal repair, discontinued, wounds in LysMCre/iDTR mice demonstrated rapid we determined the amount of granulation tissue formation in wound closure comparable to control wounds (Fig. 3A,3B). These wound tissue of macrophage-depleted and control mice. Differ- macroscopic findings were confirmed by histological assessment. ences in the quantity of granulation tissue were analyzed in H&E- For this purpose, LysMCre/iDTR and control mice were sacrificed stained sections and were shown to be significantly reduced at all on days 5, 10, and 14 postinjury, and the wound tissue (5–12 time points in macrophage-depleted wounds compared with controls 3968 MACROPHAGES IN SKIN REPAIR Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. Macrophage depletion during the early stage of repair attenuates epithelialization, granulation tissue formation, and wound contraction. A, Macroscopic appearance of wounds in LysMCre/iDTR and LysMCre (control) mice after DT injection following regimen A; whereas wounds of control mice had already lost their scab, macrophage-depleted wounds in LysMCre/iDTR mice still carry a firmly adherent scab 7 d postwounding. B, At the time points indicated, the wound area was determined using image analysis and expressed as percentage of the wound area immediately postinjury (n =12 wounds on six mice for each time point and genotype). C, Scheme of a wound section several days following injury: keratinocytes at the wound margin proliferate and migrate underneath the scab and above the granulation tissue and form the hyperproliferative epithelium. D, H&E staining of wounds in LysMCre and LysMCre/iDTR mice at indicated time points postinjury. Original magnification day 5 and 10, 3100; day 14, 350. Whereas in LysMCre mice, the day 5 wound is filled with a vascularized granulation tissue, in LysMCre/iDTR mice, only scarce granulation tissue has formed (black hatched line outlines granulation tissue; white dotted line outlines hyperproliferative epithelial tongue). In day 10 wounds of LysMCre and LysMCre/iDTR mice, the granulation tissue is covered by a complete epithelium; however, the epithelium is detached. Day 14 wounds of LysMCre and LysMCre/iDTR are closed, and scar tissue is minimal in LysMCre/iDTR mice (hatched line outlines scar tissue). Morphometric analysis of wound tissue at different time points postinjury: distance between the epithelial tips (E); distance between the edges of the panniculus carnosus (F); amount of granulation tissue (days 5 and 10 postinjury) or scar tissue (day 14 postinjury) (G). Each dot represents one wound (day 5, two wounds on one mouse; days 10 and 14 one wound per mouse); horizontal bar represents the mean. H, Sirius red staining and examination with polarized light revealed increased scar formation in control wounds when compared with LysMCre/iDTR wounds. Original magnification 3100. Arrows point to the tips of epithelial tongue, white arrowheads indicate wound edges, and black arrowheads indicate edges of panniculus carnosus. d, dermis; e, epidermis; g, granulation tissue; he, hypertrophic epidermal wound edge; pc, panniculus carnosus; sm, s.c. muscle layer; st, scar tissue. The Journal of Immunology 3969

(Fig. 3G). In fact, whereas at day 5 postinjury control wounds showed CD31 within the area of granulation tissue was used as readout for a highly vascularized, cellular, and proliferative granulation tissue, in neovascular processes at the wound site. Wounds in control mice macrophage-depleted wounds, granulation tissue was scarcely vas- revealed a strong vascular response 5 d postinjury, which decreased cularized and showed few proliferating cells (Fig. 3D, Supplemental ∼25% until day 10 postinjury (Fig. 5). In wounds of LysMCre/ Fig. 1). Wound contraction was significantly reduced in LysMCre/ iDTR mice, vascular density was significantly reduced compared iDTR mice when compared with controls (Fig. 3F). with control wounds 5 d postinjury and slightly increased by day At day 10 postinjury, wounds in LysMCre/iDTR and control 10 postinjury. Thereby, in LysMCre/iDTR and control mice, mice were still covered by eschar, and histological analysis wound angiogenesis correlated positively with the presence of revealed that in both, beneath the eschar, a complete neoepithelium macrophages. had formed (Fig. 3D). However, when compared with control Furthermore, to investigate whether the influx of macrophages mice, in LysMCre/iDTR mice, the epithelium appeared fragile, during the phase of inflammation impacts wound contraction, we was thinner, and partially detached from the dermis, indicating measured the distance between the edges of the panniculus car- immature anchorage and basement membrane formation. Fur- nosus at the wound margins in LysMCre/iDTR and control mice thermore, although in these wounds the quantity of granulation receiving DT injections following regimen A. At days 5 and 10 tissue increased when compared with day 5 postinjury, it was postinjury, the distance was significantly shorter in wounds from significantly reduced compared with control wounds (Fig. 3G). control mice compared with wounds of LysMCre/iDTR mice (Fig. Wound contraction remained reduced in LysMCre/iDTR mice 3F), indicating reduced wound contraction in macrophage- when compared with controls (Fig. 3F). depleted wounds. At both time points, a-SMA, the signature

At day 14 postinjury, both wounds in LysMCre/iDTR and control mediator for myoﬁbroblast differentiation, was abundantly ex- Downloaded from mice had lost their eschar and were similar in macroscopic ap- pressed throughout the entire layer of the granulation tissue that pearance (Fig. 3A). In contrast, major differences between wounds had developed beneath the neoepidermis and that extended into in LysMCre/iDTR and control mice became apparent regarding deep dermal layers in control wounds (Fig. 5). In contrast, wounds the extent of scar tissue formation. As revealed by H&E (Fig. 3D) of LysMCre/iDTR mice showed weak a-SMA staining in the and Sirius red staining analyzed in polarized light (Fig. 3H), ﬁne scarce granulation tissue at 5 d following injury, which slightly

collagen bundles characteristic for scar tissue were almost absent increased in intensity within the small amount of granulation http://www.jimmunol.org/ in wounds of LysMCre/iDTR mice. Morphometric quantification tissue at day 10. Immunofluorescent double labeling for CD31 and of scar tissue revealed a significant reduction in LysMCre/iDTR a-SMA indicated in wounds of control and LysMCre/iDTR mice mice when compared with controls (Fig. 3G). Morphological a nonendothelial cell origin of the a-SMA staining and thereby the analysis of the epidermis overlaying the scar tissue revealed presence of myofibroblasts. These data are suggestive for attenu- a slightly hyperproliferative closed epithelium that was similar in ated myofibroblast differentiation and subsequent reduced wound mutant and control wounds (Fig. 3D). contraction in the absence of macrophages during the early stage of tissue repair. Macrophage depletion in wounds of LysMCre/iDTR mice To identify factors that might mediate the accelerated vascular receiving DT injections following regimen A response and the myofibroblast differentiation in control mice, we by guest on September 24, 2021 To analyze whether the changes in the tissue repair response be- stained wound tissue for TGF-b1, a signature mediator of my- tween LysMCre/iDTR and control mice corresponded with mac- ofibroblast differentiation (23), and VEGF-A, one of the most rophage depletion, wound tissue was stained for the macrophage potent angiogenic mediators (24). In control wounds 5 and 10 d marker F4/80. In control wounds 5, 10, and 14 d postinjury, F4/80- postinjury, numerous mononuclear leukocytes were detected that positive cells were present throughout the entire layer of the stained positive for TGF-b1 or VEGF-A within the granulation granulation tissue (Fig. 4). Quantitative evaluation revealed that tissue (Fig. 5C,5D). Double staining for F4/80 and TGF-b1or the number of macrophages peaked at day 5 postinjury and sub- VEGF-A indicated that macrophages present a major fraction of sequently declined to approximately one-third until 14 d post- TGF-b1– or VEGF-A–expressing cells. Consistently, in macro- injury (Fig. 4). In contrast, in LysMCre/iDTR mice receiving DT phage-depleted wounds of LysMCre/iDTR mice 5 d postinjury, injections following regimen A, F4/80-positive cells were absent staining for total TGF-b1 and VEGF-A, but in particular double at day 5 postinjury and present at days 10 and 14 postinjury (Fig. staining for F4/80 and TGF-b1 or VEGF-A, was significantly re- 4). Thus, in LysMCre/iDTR mice, the absence of macrophages duced compared with control wounds. Furthermore, although at day during the early stage of repair corresponded to the time course of 10 postinjury staining for both growth factors increased when DT injections. Furthermore, our data revealed that after DT in- compared with day 5 postinjury, total and double staining for F4/80 jections were discontinued, newly generated macrophages were and TGF-b1 or VEGF-A remained low when compared with control recruited into the wound site during the consecutive phases of wounds. These results suggest that macrophage-derived TGF-b1 tissue formation and maturation. Finally and most important, our and VEGF-A contribute to myofibroblast differentiation and the results demonstrate that macrophages recruited during the in- accelerated angiogenic response in control mice. flammatory phase impact repair mechanisms not only in the early stage of the repair response, but also in the consecutive mid- and Macrophage recruitment during the inflammatory phase of late stages of repair. As revealed by staining for the neutrophil repair promotes alternative activation marker Gr-1 (Fig. 4C) and chloracetatesterase (data not shown), 5 To phenotypically characterize the macrophage infiltrate in wound d postinjury neutrophils were not effectively depleted at the tissue of LysMCre/iDTR and control mice receiving DT injec- wound site by DT injections in this model. tions following regimen A, we analyzed F4/80+ macrophages for the expression of Fizz1/Relm-a and eosinophil chemotactic factor Wound vascularization and contraction is controlled by (Ym1/ECF). Expression of both factors was recently described as macrophage influx during the inflammatory phase of repair a reliable marker of alternatively activated macrophages (25–27). To assess whether the influx of macrophages during the early stage Whereas in wound tissue of control mice at day 5 and 10 d post- of the repair response impacts wound angiogenesis, morphometric injury a large fraction of F4/80+ macrophages stained positive for quantification of the expression of the endothelial cell marker Fizz1, Ym1 was present only until day 5 postinjury (Fig. 6, 3970 MACROPHAGES IN SKIN REPAIR Downloaded from http://www.jimmunol.org/

FIGURE 4. DT-mediated macrophage depletion in LysMCre/iDTR mice following DT injection regimen A. Original magniﬁcation 3200. A, Numerous

macrophages (stained by F4/80, red) are present in granulation tissue of control wounds 5 and 10 d postinjury; in LysMCre mice, macrophages are absent at by guest on September 24, 2021 day 5 postinjury and present 10 d postinjury. B, Morphometric quantiﬁcation of macrophages present in granulation tissue at indicated time points postinjury. C, Left panel: numerous neutrophils (stained by Gr-1, brown) are present in both control and LysMCre/iDTR wounds 5 d postinjury; of note, whereas in controls neutrophils are scattered throughout the granulation tissue, in LysMCre/iDTR wounds, neutrophils are clustered at the wound surface. Right panel: morphometric quantiﬁcation of neutrophils present in granulation tissue of day 5 wounds postinjury 6 SD. n = 3 wounds on three mice for each time point and group. g, granulation tissue.

Supplemental Fig. 2). In contrast, in wounds of LysMCre/iDTR As revealed on H&E-stained paraffin sections 7 d postinjury, all mice 10 d postinjury, the number of cells that stained positive for wounds in control mice and 6 out of 10 wounds in LysMCre/iDTR F4/80 and Fizz1 or Ym1 was significantly reduced (Fig. 6, Sup- mice showed complete wound closure (Fig. 7D). Interestingly, plemental Fig. 2). whereas until day 10 postinjury granulation tissue in all wounds of control animals matured and showed regular transition into a scar Analysis of macrophage depletion during the mid-stage of tissue (Fig. 7C), all wounds in LysMCre/iDTR mice revealed a re- repair gression of granulation tissue maturation and appeared immature Macrophages recruited during the inflammatory phase of tissue (Fig. 7C,7F). Immature appearance of day 10 old macrophage-de- formation control vascular stability and transition of granulation pleted granulation tissue in LysMCre/iDTR mice was reflected by tissue into scar tissue. To characterize the functional impact of severe hemorrhage, fibrin, and serum exudates, which were present macrophages present in granulation tissue, we first inflicted skin in all wounds analyzed (eight wounds on four mice). Hemorrhage wounds on the back of control and LysMCre/iDTR mice, which was assessed by the presence of extravascular erythrocytes (Fig. 7C) was followed by DT injections 3, 4, 6, and 9 d postwounding (DT as well as immunohistochemical staining for fibrinogen/fibrin (Fig. injection regimen B; Fig. 1B). 7G). Morphometric analysis of fibrinogen/fibrin staining revealed The macroscopic analysis of the early wound healing response a significant increase in macrophage-depleted versus control mice. in LysMCre/iDTR mice was similar compared with controls. Furthermore, attenuated functional capacity of granulation tissue at However, DT-mediated macrophage depletion in LysMCre/iDTR days 7 and 10 postinjury in wounds of LysMCre/iDTR mice became mice during the mid-stage of repair significantly delayed the evident by attenuated wound contraction, which did not reach sta- subsequent wound closure rate when compared with controls tistical significance (Fig. 7E). In addition, hypertrophic epidermal (Fig. 7). These macroscopic findings were confirmed by histo- wound edges at day 5 postinjury regressed into atrophic epidermal logical assessment. For this purpose, LysMCre/iDTR and control wound edges, thereby decreasing their wound closure capacity at mice were sacrificed on days 7 and 10 postinjury, and the wound day 10 postinjury (Fig. 7D). tissue (4–10 wounds on three to five mice per time point for each As revealed by staining for F4/80, the morphological and group) was excised. functional alterations in wounds of LysMCre/iDTR mice at day 7 The Journal of Immunology 3971

FIGURE 5. Macrophage depletion during the early stage of repair attenuates angiogenesis and myoﬁbroblast differentiation. LysMCre/iDTR and control mice received DT injections following regimen A. A, CD31 (green) and a-SMA (red) double im- munostaining of day 5 wound tissue in control and LysMCre/iDTR mice; DAPI counterstaining of nuclei (blue). Original magniﬁcation 3100. Dotted line indicates basement membrane;

hatched line outlines granulation tissue. Downloaded from B, Morphometric quantiﬁcation of the area within the granulation tissue that stained positive for CD31 and a-SMA at indicated time points postinjury. Morphometric quantiﬁcation of the area within the granulation tissue that

stained positive for TGF-b1(C)and http://www.jimmunol.org/ VEGF-A (D) at indicated time points postinjury; double-labeled macrophages for TGF-b1 or VEGF-A and F4/80 were counted in high-power ﬁelds (HPFs). Data are expressed as means 6 SD. n = 3 wounds on three mice for each time point and group. Arrow points to the tip of epithelial tongue. d, dermis; e, epidermis; g, granulation tissue; he, hyperproliferative epidermis. by guest on September 24, 2021

and 10 postinjury were characterized by a significant reduction of labeling for CD31 and desmin (marker of pericytes) revealed that macrophages within the granulation tissue compared with controls 10 d postinjury in both granulation tissue of control and LysMCre/ (Fig. 8A). However, whereas in wound tissue of control mice,at iDTR wounds, desmin-positive cells that associated with vascular both time points postinjury neutrophils were minimal or completely structures could easily be identified (data not shown). In contrast, absent, their number was increased in macrophage-depleted defects in endothelial cells themselves were identified by cos- wounds (Fig. 8C,8D). Of interest, whereas hemorrhage was present taining for CD31 and cleaved caspase-3, an apoptosis marker (Fig. in all wounds of LysMCre/iDTR mice, the number of neutrophils 9). Whereas in granulation tissue of control wounds endothelial was only increased in those wounds with incomplete epithelial- cell apoptosis was a rare event, in macrophage-depleted granula- ization. Overall, these data demonstrate that DT-mediated macro- tion tissue, numerous apoptotic endothelial cells could be identi- phage depletion in LysMCre/iDTR mice during the phase of tissue fied. These findings suggest that hemorrhage observed in maturation significantly disturbed the transition of the mid-stage macrophage-depleted granulation tissue in LysMCre/iDTR mice into the late stage of the repair response. Neutrophils appeared was caused by endothelial cell damage rather than altered vessel resistant to DT-mediated cell depletion. maturation. To identify mechanisms that might mediate endothelial cell Endothelial cell damage and apoptosis in macrophage- damage, we stained wound tissue for VEGF-A and TGF-b1; both are depleted granulation tissue signature mediators of vascular homeostasis and endothelial cell To unravel the reason for the severe hemorrhage in macrophage- survival (30, 31). As demonstrated earlier in Fig. 5, in control depleted granulation tissue, we analyzed vessel maturation and wounds 10 d postinjury, double staining for F4/80 and VEGF-A or endothelial cell apoptosis in LysMCre/iDTR and control mice TGF-b1 showed that macrophages present a significant fraction of receiving DT injections following regimen B. Maturation of blood TGF-b1– or VEGF-A–expressing cells in granulation tissue. Con- vessels in healing wounds is reflected by the presence of sur- sistently, depletion of macrophages in LysMCre/iDTR mice during rounding perivascular cells (28, 29). Double immunofluorescent the phase of tissue formation resulted in a significant reduction of 3972 MACROPHAGES IN SKIN REPAIR

Analysis of macrophage depletion during the late stage of repair Macrophages present at the late stage of repair do not impact tissue maturation. To characterize the functional impact of macrophages present during the phase of tissue maturation and after restoration of the epidermal barrier, we first inflicted skin wounds on the back of control and LysMCre/iDTR mice, which was followed by DT injections 8, 9, 11, and 13 d postwounding (DT injection regimen C; Fig. 1C). Depletion of macrophages during the phase of tissue maturation did not result in macroscopic alterations of the wound tissue compared with control wounds. Fourteen days postinjury, both macrophage-depleted and control wounds had lost their eschar and revealed similar scar tissue (Fig. 10A). These macroscopic findings were confirmed by histological assessment. For this purpose, LysMCre/iDTR and control mice were sacrificed on days 10 and 14 postinjury, and the wound tissue (four to seven wounds on four to seven mice per time point for each group) was excised. As revealed on H&E-stained paraffin sections 14 d postinjury, all Downloaded from wounds in control and LysMCre/iDTR mice showed complete wound closure by a slightly hyperproliferative neoepidermis covering scar tissue (Fig. 10C). Sirius Red staining analyzed in polarized light revealed fine collagen bundles typical for scar tissue of both macrophage-depleted and control wounds (data not shown). Morphometric analysis revealed that the amount of scar http://www.jimmunol.org/ FIGURE 6. Fizz1- and Ym1-expressing macrophages in wound tissue of tissue was similar in control and LysMCre/iDTR wounds (Fig. LysMCre/iDTR and control mice. LysMCre/iDTR and control mice re- 10C). Scar tissue stained for F4/80 revealed the presence of ceived DT injections following regimen A. A, In granulation tissue of macrophages in control mice from day 10 until day 14 postinjury control mice at days 5 and 10 postinjury, double labeling for F4/80 (red) and Fizz1 (green) revealed expression of Fizz1 by macrophages; in con- and their significant reduction in DT-injected LysMCre/iDTR trast, in LysMCre/iDTR mice, F4/80- and Fizz1-expressing cells are not mice (Fig. 10D). Of interest, at all time points analyzed, neu- detectable at day 5 postinjury. In day 10 wounds, F4/80-positive cells are trophils were absent in both control and macrophage-depleted negative for Fizz1 staining. Original magnification 3400. B, Morpho- wounds (Supplemental Fig. 3). Furthermore, as revealed by metric quantification of macrophages present in granulation tissue days 5 Giemsa staining at day 14 postinjury, the number of mast cells by guest on September 24, 2021 and 10 postinjury in control and LysMCre/iDTR mice; double-positive present in scar tissue was similar in control and LysMCre/iDTR cells for F4/80 and Fizz1 or Ym1 were counted in HPFs. Arrows point out mice (Supplemental Fig. 3). double-positive cells. Data are expressed as means 6 SD. n = 3 wounds on three mice for each time point and group. g, granulation tissue. Discussion In this study, we show that macrophages play a crucial role during staining for both growth factors (Fig. 9C,9D). Our results suggest skin repair in the adult organism and that their timely restricted that sudden withdrawal of both growth factors due to macrophage depletion during distinct phases of the wound healing response has depletion contributes to endothelial cell damage. profound impact on phase-specific repair mechanisms. Our results To substantiate the hypothesis that a reduction in myeloid cell- show that repair mechanisms controlled by macrophages recruited derived TGF-b1 contributes to the hemorrhagic phenotype in during the early stage of the repair response encompass induction LysMCre/iDTR mice, we wounded mice in which TbRII has been of granulation tissue and myofibroblast differentiation, which depleted on myeloid cells (LysMCre/TbRII). Interestingly, ultimately control the degree of scar formation. During the mid- wounds in LysMCre/TbRII mice at day 7 and 10 postinjury re- stage of the repair response, macrophage function is crucial for vealed a significant increase in hemorrhages revealed by numer- stabilization of vascular structures and transition of granulation ous extravascular erythrocytes (on H&E histologies, data not tissue into scar tissue. In contrast, macrophages present at the late shown) and fibrin exudate when compared with control littermates stage of the repair response do not impact tissue maturation and (Fig. 9E), which was similar to the wound phenotype in macro- scar formation. Therefore, our studies provide evidence that phage-depleted LysMCre/iDTR mice. Earlier studies have shown macrophages exert different roles at diverse stages of the repair that TGF-b1 synthesis in macrophages is induced through binding response and that they orchestrate the natural sequence of repair of TGF-b1toTbRII (32). Consistently, using real-time PCR phases in skin, which are essential to restore solid tissue ho- analysis, we could show that TGF-b1 gene expression is reduced meostasis and integrity postinjury. Overall, our study suggests in peritoneal macrophages of LysMCre/TbRII mice following a crucial and varied role for macrophages in wound healing and stimulation with human TGF-b1 in vitro (Fig. 9E). Furthermore, adds to the previous knowledge. double staining for F4/80 and TGF-b1 of wound tissue in To assess macrophage function at distinct stages of skin repair, LysMCre/TbRII mice revealed reduced TGF-b1 synthesis in we developed a mouse system that allows the cell-type specific and macrophages (data not shown). Therefore, these results suggest timely restricted depletion of macrophages in skin wounds. The a role for macrophage-derived TGF-b1 on vascular cell function role of macrophages during skin repair has remained a subject of during wound healing in skin. Ongoing studies in our group fur- debate due to their functional dichotomy as effectors of both tissue ther investigate the role of TbRII-mediated TGF-b1 synthesis in injury and repair (33). Furthermore, in earlier studies of skin in- macrophages on wound angiogenesis. jury, macrophages have been depleted by administration of The Journal of Immunology 3973 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. Macrophage depletion during the mid-stage of repair abrogates the transition into the phase of tissue maturation. LysMCre/iDTR and control mice received DT injections following regimen B. A, Macroscopic appearance of LysMCre/iDTR wounds in which macrophages were depleted during the tissue formation phase of repair and control mice at indicated time points postinjury; whereas wounds of control mice had already lost their scab, macrophage- depleted wounds still carry a firmly adherent scab 10 d postwounding. B, At the time points indicated, the wound area was determined using image analysis and expressed as percentage of the wound area immediately postinjury (n = 10 wounds on five mice for each time point and genotype. C, H&E stainings of wounds of control and LysMCre/iDTR mice 10 d postinjury. Original magnification 3100. Control wounds reveal a cellular and vascular late granulation tissue covered by a closed hyperproliferative neoepithelium. In contrast, in LysMCre/iDTR wounds, severe hemorrhage, fibrin, and serum exudate are present; the epithelium is not closed, and the epidermal wound edge is thin and detached. Morphometric analysis of wound tissue at different time points postinjury: distance between the epithelial tips (D); distance between the edges of the panniculus carnosus (E); amount of granulation tissue (F). G, Left panel: immunohistochemical stainings of fibrinogen/fibrin and vessles (CD31) in wounds of control and LysMCre/iDTR mice 10 d postinjury. Original magnification 3200. Control wounds reveal a vascular late granulation tissue lacking fibrinogen/fibrin. In contrast, in LysMCre/iDTR wounds, severe fibrinogen/fibrin exudate is present. Right panel: morphometric analysis of fibrinogen/fibrin exsudate in wound tissue at indicated time points postinjury. Each dot represents one wound on one mouse; horizontal bar represents the mean. Data are expressed as mean 6 SD. e, epidermis; g, granulation tissue; sm, s.c. muscle layer. antimacrophage serum and/or hydrocortisone, methods that have activity, reduced phagocytosis by macrophages, and/or short-lived pleiotropic effects and lead to unspecific and partial cell depletion turnover of neutrophils, so that DT might fail to efficiently in- (10, 11). To circumvent these difficulties, we used a transgenic terfere with the high number of neutrophils that infiltrate the mouse line, LysMCre/iDTR, in which minute amounts of DT can wound site (34, 35). These findings are consistent with those of efficiently, specifically, and in a timely restricted manner deplete previous studies showing that DT treatment did not significantly tissue-resident and inflammatory macrophages recruited to the site affect neutrophil numbers (36). of skin injury. It was surprising that neutrophils could not be ef- Our study provides evidence that macrophages exert different ficiently depleted in the presented model by DT, because LysM functions during the distinct phases of skin repair. Specifically, in promoter activity has been reported in neutrophils (21). Inefficient control mice, macrophages recruited during the early stage of the neutrophil depletion might be explained by low LysM promoter repair response induce a vascularized and fibroblast-rich 3974 MACROPHAGES IN SKIN REPAIR Downloaded from

FIGURE 8. DT-mediated macrophage depletion in LysMCre/iDTR mice during the mid-stage of repair. LysMCre/iDTR and control mice received DT injections following regimen B. Original magnification 3200. A, Numerous macrophages (stained by F4/80, red) are present in granulation tissue of control wounds 10 d postinjury, whereas macrophages are almost absent in granulation tissue of LysMCre/iDTR mice. B, Morphometric quantification of mac- http://www.jimmunol.org/ rophages present in granulation tissue at indicated days postinjury. C, Whereas neutrophils are absent in control wounds, numerous neutrophils (stained by Gr-1, brown) are present in wounds of LysMCre/iDTR mice 10 d postinjury. D, Morphometric quantification of neutrophils present in granulation tissue at indicated days postinjury. Dotted line indicates basement membrane. Data are expressed as means 6 SD. n = 3 wounds on three mice for each time point and group. e, epidermis; g, granulation tissue. granulation tissue that promotes dermal as well as epidermal repair. Several activation states and related functions of macrophages have Consistently, wound closure at day 5 postinjury was significantly been described in mice and humans (37–39). The best character- delayed in mice in which macrophages were depleted specifically ized activation states in mice encompass classical activated mac- during the early stage of repair. However, macrophage influx sub- rophages (also called M1), which exert proinflammatory activities, by guest on September 24, 2021 sequent to their depletion rescued the delayed wound closure rate eradicate invading microorganisms, and promote type I immune during the late stage of repair. Yet, the overall amount of granulation responses, and alternatively activated macrophages (also called tissue that developed under these conditions, as well as vasculari- M2), which are hyporesponsive to proinflammatory stimuli and zation, cellularity, contractile force, and, most important, the extent are involved in debris scavenging, angiogenesis, connective tissue of scar formation, remained reduced when compared with control remodeling, and resolution of inflammation (paradigm of M1/M2 wounds. These findings demonstrate that macrophages that infiltrate polarization) (40). The latter is considered to exert repair and the wound site immediately postinjury induce a robust, highly regenerative activities (27, 41). However, conclusive evidence as vascularized granulation tissue associated with myofibroblast dif- to whether the concept of classical/alternative macrophage acti- ferentiation and wound contraction. Although these events ensure vation is operative at the cutaneous wound site and which of the rapid wound closure, they result in significant scar formation. microenvironmental cues might direct macrophage activation is However, a healing response that lacks specifically the initial burst of still missing. Recent studies in mice report on a critical role of macrophage influx results in minimal scarring. These results are alternative macrophage activation for regeneration of skeletal consistent with a recent study published during the time when our muscle and myocardium postinjury (42, 43). manuscript was under revision (36). Based on these findings, it is Our studies reveal the presence of alternatively activated mac- intriguing to speculate that providing a pathogen-free environment rophages (as defined by expression of Fizz1 and Ym1) in control skin and preventing macrophage influx selectively during the early stage wounds particularly during the early stage of the repair response and of repair improves the quality of the wound healing response with to a lesser extent also during the mid-stage of repair. In contrast, less scar formation and without compromising the rate of wound expressionofbothmarkerswasabsentinmacrophagesinfiltratingthe closure. However, one should take into account that scar formation wound site, which was deprived of macrophages during the early and wound contraction in mice differs significantly from scar for- stageofrepair.Thesefindingsindicatethatenvironmentalfactorsthat mation in the human system. Therefore, to validate our ob- induce alternative macrophage activation are primarily present servations, future studies in other model systems that are more during the early stage of repair. Furthermore, a healing response, adequate to study scar formation are needed. which lacks macrophage recruitment during the early stage of repair We have also investigated whether the different repair pheno- in mice, is inefficient in alternative activation. In addition, our data types in control and LysMCre/iDTR mice in which macrophages reveal that alternative macrophage activation correlates positively were exclusively depleted during the early stage of repair might withtheextentofahighlyvascularizedandcellulargranulationtissue originate in different activation states of macrophages present at the as well as ultimately with the degree of scar formation. wound site. The current conceptual model of tissue macrophage In support of a functional relevance of Fizz1- and Ym1-positive activation is based on the hypothesis that macrophages are plastic (and thereby alternatively activated) macrophages for robust an- cells and adapt their response to microenvironmental signals. giogenesis, myofibroblast differentiation, and scar formation is the The Journal of Immunology 3975

FIGURE 9. Macrophage depletion during the phase of tissue formation results in endothelial cell apoptosis. LysMCre/iDTR and control mice received DT injections following regimen B. A, Double labeling for cleaved caspase-3 (red) and CD31 (green) revealed numerous apoptotic endothelial cells in granulation tissue of macrophage- depleted wounds 10 d postinjury; arrowheads indicate double-positive cells. Original magnification 3400. B, Morphometric quantification of the area within the granulation tissue that stained positive for activated caspase-3 and CD31 showed that the number of apoptotic endothelial cells is significantly increased in Downloaded from macrophage-depleted wounds when compared with controls 7 and 10 d postinjury. C and D, Morphometric quantification of the area within the granulation tissue that stained positive for TGF-b1 or VEGF-A at indicated time points postinjury; http://www.jimmunol.org/ double-labeled F4/80+ cells for VEGF-A or TGF-b1 were counted in HPFs. n = 3 wounds on three mice for each time point and group. E, Left panel: immunohistochemical stainings of fibrinogen/fibrin and vessels (CD31) in wounds of control and LysMCre/TbRII mice 7 d postinjury. Control wounds reveal a highly vascular granulation tissue by guest on September 24, 2021 lacking fibrinogen/fibrin. In contrast, in LysMCre/TbRII wounds, severe fibrinogen/fibrin exudate is present. Original magnification 3200. Mid- dle panel: morphometric analysis of fibrinogen/fibrin exsudate in wound tissue at indicated time point postinjury. Right panel: real-time PCR analysis of TGF-b1 in peritoneal macrophages of control or LysMCre/ TbRII mice poststimulation with human TGF-b1. Data are expressed as relative increase of TGF-b1 stimulated expression over controls.

positive correlation of the staining of both markers with VEGF-A ized and cellular granulation tissue, resulted in the abrogation or or TGF-b1. Both growth factors are considered important medi- even retrogression of the physiological repair cycle. This process ators of alternatively activated macrophage function (40). Thus, was evident by severe hemorrhage and fibrin exudate, suspicious our data emphasize that in physiological repair, macrophages re- for destabilization of vascular structures. Indeed, apoptosis of cruited during the early stage of the repair response are alterna- endothelial cells was significantly increased in macrophage- tively activated, resulting in the expression of VEGF and TGF-b1, depleted granulation tissue. Furthermore, epidermal wound which contributes to wound angiogenesis and myofibroblast dif- edges appeared atrophic and detached from the underlying ferentiation. In contrast, macrophages recruited at a later stage of granulation tissue, indicating severe disturbances in epidermal- repair, into a wound site previously deprived of the macrophage dermal interactions. In addition, the number of neutrophils in- influx during the phase of inflammation, do not undergo alterna- creased in macrophage-depleted wounds that had not yet com- tive activation and consistently reveal attenuated expression of pleted epithelialization. During physiological skin repair in our VEGF and TGF-b1, which ultimately attenuates the degree of model, the number of neutrophils declines within the initial 5 granulation tissue and scar formation. d postinjury (35). Thus, high numbers of neutrophils present in Depletion of macrophages during the mid-stage of the repair macrophage-depleted granulation tissue at day 7 and 10 post- response, consecutive to the development of a highly vascular- injury are abnormal. 3976 MACROPHAGES IN SKIN REPAIR Downloaded from http://www.jimmunol.org/

FIGURE 10. DT-mediated macrophage depletion during the late stage of the repair response. LysMCre/iDTR and control mice received DT injections following regimen C. A, At the time points indicated, the macroscopic wound area was determined using image analysis and expressed as percentage of the wound area immediately postinjury (n = 4 wounds on four mice for each time point and genotype). B, H&E stainings of wounds of control and LysMCre/ iDTR mice 14 d postinjury. Wounds of control and LysMCre/iDTR mice were closed and showed a similar epithelium and scar tissue. C, Morphometric analysis of the amount of scar tissue 14 d postinjury. Each dot represents one wound on one mouse; horizontal bar represents the mean. D, Macrophages (stained by F4/80, red) are present in scar tissue of control wounds 14 d postinjury, whereas macrophages are almost absent in granulation tissue of

LysMCre/iDTR mice. E, Morphometric quantiﬁcation of F4/80-positive cells in the granulation tissue in control and LysMCre/iDTR mice at indicated time by guest on September 24, 2021 points post injury. Data are expressed as mean 6 SD. e, epidermis; st, scar tissue.

Several mechanisms underlying the morphological and func- cruited during the early stage of repair significantly control the tional changes associated with macrophage depletion during the degree of scar formation. Furthermore, absence of morphological mid-stage of the repair response might be discussed. As we have alterations in macrophage-depleted day 14 wounds of LysMCre/ shown, macrophages present in granulation tissue of control mice iDTR mice demonstrates that DT-mediated macrophage apoptosis provide a source for VEGF and TGF-b1. Sudden withdrawal of itself does not inevitably cause cellular changes in the wounded these growth factors by macrophage depletion from the metabol- tissue. Therefore, in our model, the altered healing response ob- ically highly active granulation tissue might result in severe al- served in macrophage-depleted wounds during the early or mid- terations in tissue homeostasis. Withdrawal of VEGF and TGF- stage of repair is a consequence of macrophage deficiency and not b1, both of which are potent survival factors for endothelial cells, a sequela of their apoptosis. might explain endothelial cell apoptosis and vessel destabilization In conclusion, our study adds to previous knowledge on the observed in macrophage-depleted wounds (24, 44). Furthermore, function of macrophages in skin repair, but also uncovers several withdrawal of TGF-b1, a potent immunosuppressive mediator novel aspects. First, our findings substantiate previous work, (45), might be responsible for the increased influx of neutrophils demonstrating a crucial role of macrophages in healing skin into the macrophage-depleted granulation tissue. In turn, neu- wounds in the adult organism to achieve tissue homeostasis. trophils are rich in highly active tissue degrading proteases and Secondly, we identified repair mechanisms that are dependent or reactive oxygen species, which could contribute to the severe independent from macrophage function. Third, we revealed that endothelial cell damage observed (46). Although at this stage we macrophages exert distinct functions during the diverse phases of cannot exclude neutrophil-mediated vascular cell damage, we skin repair that are complementary to restore skin integrity. Finally, consider it an unlikely event, because hemorrhage and endothelial our findings suggest that different macrophage functions in skin cell apoptosis were also present in macrophage-depleted granu- repair can originate in different macrophage activation states. lation tissue, which did not present increased neutrophils. Overall, Overall, our findings delineate cellular and molecular mechanisms our data clearly illustrate that macrophages present in granulation that control the kinetics of skin repair and link macrophage function tissue are crucial for the progression of the mid-stage of the repair as the critical force to the dynamics of wound repair. Future studies response into the late stage characterized by tissue maturation. will analyze whether selective modulation of macrophage acti- Finally, depletion of macrophages during the late stage of repair vation and function during specific stages of repair might be an did not cause significant morphological changes, indicating a minor effective therapeutic strategy to normalize tissue regeneration in role of macrophages present during the phase of tissue maturation pathological healing conditions. However, one has to take into and scar formation. Indeed, as outlined above, macrophages re- account that skin repair in mice differs significantly from the human The Journal of Immunology 3977 system. Therefore, we suggest that additional experimental studies 22. Cazac, B. B., and J. Roes. 2000. TGF-beta receptor controls B cell re- sponsiveness and induction of IgA in vivo. Immunity 13: 443–451. in mice and/or other model organisms should be paralleled by 23. Tomasek, J. J., G. Gabbiani, B. Hinz, C. Chaponnier, and R. A. Brown. 2002. analysis of macrophage activation and function during wound Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat. repair in the human system. Rev. Mol. Cell Biol. 3: 349–363. 24. Olsson, A. K., A. Dimberg, J. Kreuger, and L. Claesson-Welsh. 2006. VEGF receptor signalling - in control of vascular function. Nat. Rev. Mol. Cell Biol. 7: Acknowledgments 359–371. 25. Raes, G., P. De Baetselier, W. Noe¨l, A. Beschin, F. Brombacher, and We are grateful to Luca Borradori for critically reading the manuscript and G. Hassanzadeh Gh. 2002. Differential expression of FIZZ1 and Ym1 in alter- Sabine Werner for support in the fibrinogen/fibrin stain. We also thank Joe natively versus classically activated macrophages. J. Leukoc. Biol. 71: 597–602. Leibovich for helpful discussion and advise on macrophage biology and 26. Nair, M. G., D. W. Cochrane, and J. E. Allen. 2003. Macrophages in chronic type Michael Piekarek and Margot Junker for excellent technical assistance. 2 inflammation have a novel phenotype characterized by the abundant expression of Ym1 and Fizz1 that can be partly replicated in vitro. Immunol. Lett. 85: 173– 180. Disclosures 27. Loke, P., I. Gallagher, M. G. Nair, X. Zang, F. Brombacher, M. Mohrs, J. P. Allison, and J. E. Allen. 2007. Alternative activation is an innate response to The authors have no financial conflicts of interest. injury that requires CD4+ T cells to be sustained during chronic infection. J. Immunol. 179: 3926–3936. 28. Benjamin, L. E., I. Hemo, and E. Keshet. 1998. A plasticity window for blood References vessel remodelling is defined by pericyte coverage of the preformed endothelial 1. Gurtner, G. C., S. Werner, Y. Barrandon, and M. T. Longaker. 2008. Wound network and is regulated by PDGF-B and VEGF. Development 125: 1591–1598. repair and regeneration. Nature 453: 314–321. 29. Roth, D., M. Piekarek, M. Paulsson, H. Christ, W. Bloch, T. Krieg, 2. Babcock, D. T., A. R. Brock, G. S. Fish, Y. Wang, L. Perrin, M. A. Krasnow, and J. M. Davidson, and S. A. Eming. 2006. Plasmin modulates vascular endothelial growth factor-A-mediated angiogenesis during wound repair. Am. J. Pathol. 168: M. J. Galko. 2008. Circulating blood cells function as a surveillance system for Downloaded from 670–684. damaged tissue in Drosophila larvae. Proc. Natl. Acad. Sci. USA 105: 10017–10022. 30. Alon, T., I. Hemo, A. Itin, J. Pe’er, J. Stone, and E. Keshet. 1995. Vascular 3. Niethammer, P., C. Grabher, A. T. Look, and T. J. Mitchison. 2009. A tissue- endothelial growth factor acts as a survival factor for newly formed retinal scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. vessels and has implications for retinopathy of prematurity. Nat. Med. 1: 1024– Nature 459: 996–999. 1028. 4. Chen, Y., R. M. Costa, N. R. Love, X. Soto, M. Roth, R. Paredes, and E. Amaya. 31. Carvalho, R. L., F. Itoh, M. J. Goumans, F. Lebrin, M. Kato, S. Takahashi, 2009. C/EBPalpha initiates primitive myelopoiesis in pluripotent embryonic M. Ema, S. Itoh, M. van Rooijen, P. Bertolino, et al. 2007. Compensatory sig- cells. Blood 114: 40–48. nalling induced in the yolk sac vasculature by deletion of TGFbeta receptors in 5. Werner, S., T. Krieg, and H. Smola. 2007. Keratinocyte-fibroblast interactions in mice. J. Cell Sci. 120: 4269–4277. http://www.jimmunol.org/ wound healing. J. Invest. Dermatol. 127: 998–1008. 32. McCartney-Francis, N., D. Mizel, H. Wong, L. Wahl, and S. Wahl. 1990. TGF- 6. Eming, S. A., M. Hammerschmidt, T. Krieg, and A. Roers. 2009. Interrelation of beta regulates production of growth factors and TGF-beta by human peripheral immunity and tissue repair or regeneration. Semin. Cell Dev. Biol. 20: 517–527. blood monocytes. Growth Factors 4: 27–35. 7. Loots, M. A., E. N. Lamme, J. Zeegelaar, J. R. Mekkes, J. D. Bos, and 33. Duffield, J. S. 2003. The inflammatory macrophage: a story of Jekyll and Hyde. E. Middelkoop. 1998. Differences in cellular infiltrate and extracellular matrix of Clin. Sci. (Lond.) 104: 27–38. chronic diabetic and venous ulcers versus acute wounds. J. Invest. Dermatol. 34. Duffield, J. S., S. J. Forbes, C. M. Constandinou, S. Clay, M. Partolina, 111: 850–857. S. Vuthoori, S. Wu, R. Lang, and J. P. Iredale. 2005. Selective depletion of 8. Eming, S. A., T. Krieg, and J. M. Davidson. 2007. Inflammation in wound repair: macrophages reveals distinct, opposing roles during liver injury and repair. J. molecular and cellular mechanisms. J. Invest. Dermatol. 127: 514–525. Clin. Invest. 115: 56–65. 9. Martin, P., and S. J. Leibovich. 2005. Inflammatory cells during wound repair: 35. Kim, M. H., W. Liu, D. L. Borjesson, F. R. Curry, L. S. Miller, A. L. Cheung, the good, the bad and the ugly. Trends Cell Biol. 15: 599–607. F. T. Liu, R. R. Isseroff, and S. I. Simon. 2008. Dynamics of neutrophil in-

10. Simpson, D. M., and R. Ross. 1972. The neutrophilic leukocyte in wound repair filtration during cutaneous wound healing and infection using fluorescence im- by guest on September 24, 2021 a study with antineutrophil serum. J. Clin. Invest. 51: 2009–2023. aging. J. Invest. Dermatol. 128: 1812–1820. 11. Leibovich, S. J., and R. Ross. 1975. The role of the macrophage in wound repair. A 36. Mirza, R., L. A. DiPietro, and T. J. Koh. 2009. Selective and specific macrophage study with hydrocortisone and antimacrophage serum. Am. J. Pathol. 78: 71–100. ablation is detrimental to wound healing in mice. Am. J. Pathol. 175: 2454–2462. 12. Redd, M. J., L. Cooper, W. Wood, B. Stramer, and P. Martin. 2004. Wound 37. Mosser, D. M., and J. P. Edwards. 2008. Exploring the full spectrum of mac- healing and inflammation: embryos reveal the way to perfect repair. Philos. rophage activation. Nat. Rev. Immunol. 8: 958–969. Trans. R. Soc. Lond. B Biol. Sci. 359: 777–784. 38. Martinez, F. O., L. Helming, and S. Gordon. 2009. Alternative activation of 13. Martin, P., D. D’Souza, J. Martin, R. Grose, L. Cooper, R. Maki, and macrophages: an immunologic functional perspective. Annu. Rev. Immunol. 27: S. R. McKercher. 2003. Wound healing in the PU.1 null mouse—tissue repair is 451–483. not dependent on inflammatory cells. Curr. Biol. 13: 1122–1128. 39. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. 14. Nagaoka, T., Y. Kaburagi, Y. Hamaguchi, M. Hasegawa, K. Takehara, Nat. Rev. Immunol. 5: 953–964. D. A. Steeber, T. F. Tedder, and S. Sato. 2000. Delayed wound healing in the 40. Gordon, S. 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3: absence of intercellular adhesion molecule-1 or L-selectin expression. Am. J. 23–35. Pathol. 157: 237–247. 41. Pull, S. L., J. M. Doherty, J. C. Mills, J. I. Gordon, and T. S. Stappenbeck. 2005. 15. Peters, T., A. Sindrilaru, B. Hinz, R. Hinrichs, A. Menke, E. A. Al-Azzeh, Activated macrophages are an adaptive element of the colonic epithelial pro- K. Holzwarth, T. Oreshkova, H. Wang, D. Kess, et al. 2005. Wound-healing genitor niche necessary for regenerative responses to injury. Proc. Natl. Acad. -/- defect of CD18( ) mice due to a decrease in TGF-beta1 and myofibroblast Sci. USA 102: 99–104. differentiation. EMBO J. 24: 3400–3410. 42. Arnold, L., A. Henry, F. Poron, Y. Baba-Amer, N. van Rooijen, A. Plonquet, 16. Werner, S., and R. Grose. 2003. Regulation of wound healing by growth factors R. K. Gherardi, and B. Chazaud. 2007. Inflammatory monocytes recruited after and cytokines. Physiol. Rev. 83: 835–870. skeletal muscle injury switch into antiinflammatory macrophages to support 17. Eming, S. A., S. Werner, P. Bugnon, C. Wickenhauser, L. Siewe, O. Utermo¨hlen, myogenesis. J. Exp. Med. 204: 1057–1069. J. M. Davidson, T. Krieg, and A. Roers. 2007. Accelerated wound closure in 43. Nahrendorf, M., F. K. Swirski, E. Aikawa, L. Stangenberg, T. Wurdinger, mice deficient for interleukin-10. Am. J. Pathol. 170: 188–202. J. L. Figueiredo, P. Libby, R. Weissleder, and M. J. Pittet. 2007. The healing 18. Ishida, Y., J. L. Gao, and P. M. Murphy. 2008. Chemokine receptor CX3CR1 myocardium sequentially mobilizes two monocyte subsets with divergent and mediates skin wound healing by promoting macrophage and fibroblast accu- complementary functions. J. Exp. Med. 204: 3037–3047. mulation and function. J. Immunol. 180: 569–579. 44. ten Dijke, P., and H. M. Arthur. 2007. Extracellular control of TGFbeta sig- 19. Han, M., X. Yang, J. Lee, C. H. Allan, and K. Muneoka. 2008. Development and nalling in vascular development and disease. Nat. Rev. Mol. Cell Biol. 8: 857– regeneration of the neonatal digit tip in mice. Dev. Biol. 315: 125–135. 869. 20. Buch, T., F. L. Heppner, C. Tertilt, T. J. Heinen, M. Kremer, F. T. Wunderlich, 45. Li, M. O., Y. Y. Wan, S. Sanjabi, A. K. Robertson, and R. A. Flavell. 2006. S. Jung, and A. Waisman. 2005. A Cre-inducible diphtheria toxin receptor me- Transforming growth factor-beta regulation of immune responses. Annu. Rev. diates cell lineage ablation after toxin administration. Nat. Methods 2: 419–426. Immunol. 24: 99–146. 21. Clausen, B. E., C. Burkhardt, W. Reith, R. Renkawitz, and I. Fo¨rster. 1999. 46. Ku¨min, A., M. Scha¨fer, N. Epp, P. Bugnon, C. Born-Berclaz, A. Oxenius, Conditional gene targeting in macrophages and granulocytes using LysMcre A. Klippel, W. Bloch, and S. Werner. 2007. Peroxiredoxin 6 is required for blood mice. Transgenic Res. 8: 265–277. vessel integrity in wounded skin. J. Cell Biol. 179: 747–760.