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M2 macrophages promote wound-induced hair neogenesis
Accepted Manuscript Title: M2 macrophages promote wound-induced hair neogenesis Authors: Akira Kasuya, Taisuke Ito, Yoshiki Tokura PII: DOI: Reference:
S0923-1811(18)30216-0 https://doi.org/10.1016/j.jdermsci.2018.05.004 DESC 3381
To appear in:
Journal of Dermatological Science
Received date: Revised date: Accepted date:
20-12-2017 18-4-2018 8-5-2018
Please cite this article as: Kasuya Akira, Ito Taisuke, Tokura Yoshiki.M2 macrophages promote wound-induced hair neogenesis.Journal of Dermatological Science https://doi.org/10.1016/j.jdermsci.2018.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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M2 macrophages promote wound-induced hair neogenesis Short title: Wound-induced hair neogenesis
Department of Dermatology, Hamamatsu University School of Medicine, Japan
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Akira Kasuya1 MD/PhD, Taisuke Ito1 MD/PhD and Yoshiki Tokura1 MD/PhD.
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Correspondence: Akira Kasuya, Department of Dermatology, Hamamatsu University
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School of Medicine, 1-20-1 Handayama, Higashi-Ku, Hamamatsu 431-3192, Japan.
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E-mail: [email protected]
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M2 macrophages promote wound-induced hair neogenesis Akira Kasuya, Taisuke Ito, and Yoshiki Tokura Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan
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High lights
We established a mouse model of wound-induced hair neogenesis. M2 macrophages infiltrated in the tissue 2 weeks after wounding in mice. M2 macrophages produced various growth factors. Administration of one of the upmodulated growth factors (Igf1 or Fgf2) promoted wound-induced hair neogenesis.
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Background: De novo hair regeneration occurs in scars of normal adult mice. This interesting phenomenon is termed as wound-induced hair neogenesis (WIHN). We hypothesized that M2 macrophages are crucially involved in WIHN. Objective: To clarify the contribution of M2 macrophages to WIHN. Method: We established a mouse model of WIHN. A full thickness skin excision was implemented on the back of C57BL/6 (B6) mice. Newly developing hair follicles were detected by a whole-mount assay. WIHN took place 2 weeks after wounding.
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Results: At first, flow cytometry revealed increased infiltration of CD11b+/CD206+ M2 macrophages at the 2nd and 3rd week after wounding. Immunohistochemistry also showed the existence of CD206+ M2 macrophages in the vicinity of regenerated hair follicles. Secondly, the productions of growth factors were confirmed by culturing M2 macrophages isolated from the skin in a comparison with CD11b+ spleen cells. Array for 84 genes revealed increased expressions of various growth factors including Igf1 and Fgf2. Thirdly, we verified the effect of the growth factors on WIHN. WIHN was increased by 2 folds in mice treated with Fgf2 (p=0.05) or by 1.5 folds with Igf1 (p=0.05). Finally, we used B6.Tg(ITGAM-DTR) mice in which macrophages are ablated by diphtheria toxin. We depleted macrophages at one to 2 weeks after wounding
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when M2 macrophages were dominant. WIHN was attenuated to one third (P=0.05) by the ablation of macrophages. Conclusion: Our study suggests that M2 macrophages could promote WIHN through producing a panel of growth factors.
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Key words: wound healing, hair neogenesis, M2 macrophage, growth factor
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INTRODUCTION In humans, full-thickness skin excision usually leaves hairless scars, but the definitive evidence for de novo hair regeneration has been shown in skin scars of genetically normal adult mice [1]. This phenomenon was termed wound induced hair neogenesis (WIHN). The fact that hair follicles can be regenerated in vivo in scarred skin without the use of ES cells or iPS cells has high impact on tissue engineering. Dermal γδ T cells are suggested to promote WIHN through the production of Fgf9, a crucial growth factor [2]. However, it remains unknown whether other cell types are
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involved in WIHN. Here, we hypothesized that M2 macrophage could be involved in WIHN, because M2 macrophages produces various growth factors in the process of wound healing. Macrophages in the wound bed display different functional phenotypes, which can roughly be divided into two groups: M1 (classically activated) and M2 (alternatively activated) macrophages [3, 4]. While M1 macrophages contribute to the acute inflammation immediately after wounding, M2 macrophages, called “wound healing” macrophages, infiltrate at the late stage of wound healing and produce various growth factors [5-7]. Growth factors such as fibroblast growth factors (Fgf), insulin-like growth factor (Igf), epidermal growth factor (Egf)-related ligands, hepatocyte growth
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factor (Hgf), and platelet-derived growth factor (Pdgf) have been shown to be crucial for the regulation of the hair cycle and hair growth [8, 9]. In this study, we verify whether M2 macrophages could promote WIHN by means of growth factors.
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METHOD Animals C57BL/6N wild-type mice (WT) were obtained from SLC Inc. (Hamamatsu, Japan). The transgenic mice of B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J (Tg) were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were healthy, fertile, and did not display any evidence of infection or disease. Female mice (5- to 6-week-old) were
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used for all the experiments. All mice were housed in specific pathogen-free barrier facility and screened regularly for pathogens. Mice were individually housed in plastic cages to prevent to prevent tampering with the resultant ulcer by other mice. All studies and procedures were approved by the Committee on Animal Experimentation of Hamamatsu University School of Medicine. A transgenic construct containing sequence encoding DTR/GFP under the control of the human ITGAM (integrin alpha M) promoter was introduced into fertilized FVB/N donor eggs. The mice were backcrossed onto C57BL/6 for 6 generations. So the control strain suggested by Jackson laboratory is C57BL/6.
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Wounding and injection of growth factors C57BL/6N and B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J mice were anaesthetized with isoflurane, and a full thickness excision of skin with a diameter of 1.5 cm was made on the mid back of mice, as previously reported [1]. Either saline, Igf1 (100 g/body, Biolegend, San Diego, CA), Fgf2 (10 g/body, Kaken, Tokyo, Japan) were intraperitoneally administered at days 9, 11, and 13 in the experiments of growth factor injection.
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Whole-mount hair follicle neogenesis assay To detect newly developing hair follicles in the wound, whole-mount hair follicle neogenesis assay was performed [1]. We incubated the sampled skin in 1M NaCl at 37 °C overnight. Then, we gently peeled the epidermis off under a dissecting microscope (SZ61, Olympus Tokyo, Japan). The epidermis was fixed in 3.5% paraformaldehyde for 1 h, rinsed with PBS, blocked with buffer (Blocking One®, Nakalai Tesque, Kyoto, Japan). Immunostaining was performed for K17 using polyclonal rabbit antibody (Abcam, Cambridge, UK). Histofine Simple Stain MAX-PO
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(Nichirei Co., Tokyo, Japan) was used for second antibody. The dermis was used to detect alkaline phosphatase activity in the hair follicle. We fixed the dermis in acetone at 4 °C overnight, rinsed it with PBS, and then incubated with NBT/BCIP (Roche, Basel, Switzerland) for 30 min at 37 °C. We stopped the reaction with 20 mM EDTA in phosphate-buffered saline (PBS, pH 7.4).
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Histological examination and immunohistochemistry After mice were sacrificed, the area of scar was harvested. The sample was cut into halves, fixed in 3.5% paraformaldehyde, and embedded in paraffin. Sections were stained with hematoxylin & eosin (HE). Immunohistochemistry was performed using
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polyclonal rabbit antibodies specific for CD206 (Thermo Fisher Scientific Inc., Waltham, MA), Fgf2 (Abcam, Cambridge, UK) and monoclonal antibody for Igf1 (Acris, Rockville, MD). Alexa Fluor Secondary Detection Reagents (Life Technologies Co., Carlsbad, CA) was used for secondary antibody.
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Flow Cytometric Analysis Skin-infiltrating inflammatory cells were isolated and subjected to flow cytometric analysis. The scar of back skin was harvested. Subcutaneous tissue was removed by scalpel and was minced with the surface of frosted glass in PBS. The tissue was washed with PBS. The fluid was filtered through a 70-μm nylon mesh to obtain a single-cell
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suspension. The following primary antibodies were used: phycoerythrin (PE)-labeled anti-mouse CD206 monoclonal antibodies (Biolegend, San Diego, CA), fluorescein isothiocyanate (FITC)-labeled anti-mouse CD11b monoclonal antibodies (Biolegend). All antibodies were used at 1:200 dilution according to manufacturer instructions. Incubation was performed for 15 min at room temperature, followed by two washes in PBS supplemented with 5% fetal calf serum (FCS) and 0.02% sodium azide. FACSCanto II
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(Japan BD, Tokyo, Japan) was used to obtain fluorescent profiles. Data analysis was performed using Flowjo software (Treestar, Inc. Ashland, OR).
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Real-time PCR Total RNAs were extracted from skin samples using QIAGEN RNase spin columns ® (QIAGEN Ltd., Crawley, UK) and digested with DNase I (QIAGEN Ltd.) to remove chromosomal DNA in accordance with the manufacturer’s protocols. Total RNA was reverse transcribed to cDNA using a Reverse Transcription System with random hexamers (Promega, Madison, WI). Real-time PCR was performed using the TaqMan
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system (Applied Biosystems, Foster City, CA) on an ABI Prism 7000 Sequence Detector (Applied Biosystems) according to the manufacturer’s instructions. The 96 well RT² Profiler PCR Arrays® (Mouse Wound Healing; QIAGEN Ltd.) are used in combination with the RT² SYBR Green qPCR Mastermixes® (QIAGEN Ltd.). To analyze the PCR-array data, online analysis was performed on the manufacturer’s website (http://www.qiagen.com). Relative expression of real-time PCR products was determined using the ΔΔCt technique. Briefly, each set of samples was normalized using the difference in threshold cycle (Ct) between the target gene and housekeeping gene: ΔCt. (Ct target gene -Ct GAPDH). Relative mRNA levels were calculated by the expression 2-ΔΔCt, where ΔΔCt = ΔCt sample-ΔCt calibrator.
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Sorting of monocytes and differentiation towards M2 macrophages The spleens from C57BL/6N mice were minced in PBS. The fluid was filtered through a 70-μm nylon mesh to obtain a single-cell suspension. For the isolation of CD11b+ cells, cells were magnetically labeled with CD11b MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, the autoMACS Pro Separator ® (Miltenyi Biotec, Bergisch Gladbach, Germany) was used according to the manufacturer’s protocol. Isolated monocytes were placed in culture medium (M2-Macrophage Generation Medium DXF ®; PromoCell, Heidelberg, Germany) supplemented with M-CSF for 7 days at 37°C and 5% CO2 according to the manufacturer’s protocol according to the
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manufacturer’s protocol [10-12]. Then, 20 ng/ml of IL-4 (Biolegend) was added to trigger M2 polarization. Two days later, M2 macrophages were harvested and assessed by flow cytometry and real time PCR. Bone Marrow Translation (BMT) C57BL/6-Tg(CAG-EGFP)C57BL/6 transgenic 12-week-old female mice were purchased from SLC Inc. (Hamamatsu, Japan). The mice ubiquitously express enhanced
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green fluorescent protein (GFP). The bone marrow (BM) cells were isolated from the femur bones. Eight-week-old female C57BL/6N mice were used as recipients. Recipients was intravenously injected with 5×106 BM cells from GFP transgenic mice, immediately after the irradiation of 7.5 Gy X-rays. Experiments were performed in the BMT mice at least 6 weeks after the BMT [13].
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Ablation of macrophage in the mild stage of wound healing Diphtheria toxin (DT; Sigma-Aldrich, St. Louis, MI) was intraperitoneally administered at days 7, 9, 11 and 13 at 25 ng/g body to B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J
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mice or C57BL/6N[14]. These timepoints for administration were chosen to ablate M2 macrophages that appears in the late phase of wound healing.
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Statistical analysis The two-tailed unpaired Student’s t-test was used to determine the level of significance of differences between the sample means. Tukey's test was used for multiple comparisons. A p-value <0.05 was considered statistically significant.
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RESULT Wound-healing induced hair neogenesis Scar formation was completed 2 weeks after wounding (Figure 1a). Three weeks after wounding, WIHN took place as assessed by alkaline phosphatase activity and K17 positivity (Figure 1b, 1c).
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Infiltration of M2 macrophages after wounding Flow cytometry analysis of cells infiltrating in the wounded tissue showed increased percentage of CD11b+/CD206+ M2 macrophages at the 1st and 2nd week after wounding (Figure 2a). Immunohistochemistry for CD206 (red) in C57 mice translated with BM from GFP mice showed that GFP+/CD206+ cells (merged color, yellow) infiltrated into the wounded tissue (Figure 2b, arrowheads), suggesting that these infiltrated M2 macrophages are derived from BM. Some of GFP+/CD206+ M2
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macrophages infiltrated in the vicinity of neogenerated follicle (Figure 2b, white arrowheads). Productions of Fgf2 and Igf1 were confirmed in CD206+ M2 macrophages in the scar tissues (Figure 3e). Cultured M2 macrophages produce growth factors CD11b+ cells were isolated from mouse spleen cells by auto MACS and were
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differentiated to CD206+ M2 macrophages by cultivation (Figure 3a). Microscopic image exhibited the fried egg appearance of M2 macrophages (Figure 3b). By immunohistochemistry, CD206+ M2 macrophages were positive for Fgf2 and Igf1,
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representative macrophage-secreted growth factors (Figure 3c). Gene expression array for wound healing-associated molecules in cultured M2 macrophages showed increased expression of a number of growth factors such as Igf1, Fgf2, Fgf7, Fgf10, Pdgf and Egf, compared to undifferentiated CD11b+ spleen cells.
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Administration of growth factor promotes WIHN Fgf2 or Igf1 was administered intraperitoneously at day9, day13 and day21. The injection of Fgf2 or Igf1 during the late phase of wound healing promoted WIHN (Figure 4).
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Ablation of macrophages inhibits WIHN B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J mice were used to examine the effect of
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depletion of M2 macrophage (Figure 5a). The number of M2 macrophages increases in the late phase of the wound healing. We therefore initiated injection of DT (25
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ng/mouse i.p.) at day 9 and subsequent 3 times during wounding in Tg mice (Figure 5b). Flow cytometry analysis and immunohistochemistry of infiltrating cells confirmed that DT injections ablated M2 macrophages in the ulcer (Figure 5c). While DT
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injections changed neither the healing state (Figure 5d) nor the extent of scar area (Figure 5e), they decreased the WIHN (Figure 5f, 5g).
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DISCUSSION Wound healing is processed with an inflammatory phase. In this phase, M1 macrophages work for the killing of bacteria and the wound debridement, including clearing of neutrophils and damaged tissues [3, 15]. The inflammatory phase ceases within 3 days after wounding. In the late inflammatory phase, a shift of M1 to M2
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macrophages takes place, followed by a late phase of wound healing. In fact, direct conversion of M1 to M2 macrophages was also observed [16]. M2 macrophages produce various growth factor and promote regeneration and remodeling of the tissues [3, 17]. In this study, we first confirmed the existence of M2 macrophage at 2 weeks after wounding, in which WIHN was reported to occur [1]. With this circumstantial evidence, it is tempting to speculate that M2 macrophages are associated with neogeneration of hair follicles.
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Using Tg mice, we ablated M2 macrophages by DT during the proliferation phase of wound healing. The ablation of M2 macrophages attenuated WIHN, suggesting contribution of M2 macrophage to WIHN. However, the limit of this strategy is that DT depletes both M1 and M2 macrophages. To ablate mainly M2 macrophage, we thus administered DT in the proliferation phase of wound healing, at days 7, 9, 11 and 13, when M2 macrophages are dominant compared to M1 macrophage. Usually the infiltration of M1 macrophages peaks at day 1 and rapidly decreases as wound healing progresses. As a mechanism underlying the promotion of WIHN by M2 macrophages, we expected
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the involvement of macrophage-derived growth factors. In confirmation of the previous studies, M2 macrophages produced various growth factors, including Fgf2 and Igf1. The administration of Fgf2 or Igf1 promoted WIHN. Fgf2 and Igf1 participate in Wnt signaling pathway [18, 19]. Since Wnt signaling plays an important role in WIHN [2], it may intervene between Fgf2 and Igf1, and WIHN. We have shown the involvement of M2 macrophages on WIHN, however, our study fails to show how M2 macrophages specifically promote hair neogenesis in the context of wound healing. To suggest that M2 macrophage promote WIHN through Igf1 or Fgf2, knockdown of Igf1 and Fgf2 should be performed. The present study suggests that M2 macrophages play a crucial role in hair growth as
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well as wound healing. In rodents where hair follicles are not completely abrogated even in scar tissue, the wound-associated inflammatory alteration can induce hair neogenesis by virtue of M2 macrophages.
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ACKNOWLEDGEMENT We thank Yuki Yamada for the handling of animals.
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Reference
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[1] M. Ito, Z. Yang, T. Andl, C. Cui, N. Kim, S.E. Millar, G. Cotsarelis, Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding, Nature. 447(7142) (2007) 316-320. [2] D. Gay, O. Kwon, Z. Zhang, M. Spata, M.V. Plikus, P.D. Holler, M. Ito, Z. Yang, E. Treffeisen, C.D. Kim, A. Nace, X. Zhang, S. Baratono, F. Wang, D.M. Ornitz, S.E. Millar, G. Cotsarelis, Fgf9 from dermal gammadelta T cells induces hair follicle
M
A
N
U
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neogenesis after wounding, Nat Med. 19(7) (2013) 916-923. [3] B. Mahdavian Delavary, W.M. van der Veer, M. van Egmond, F.B. Niessen, R.H. Beelen, Macrophages in skin injury and repair, Immunobiology. 216(7) (2011) 753-762. [4] A. Mantovani, S. Sozzani, M. Locati, P. Allavena, A. Sica, Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes, Trends Immunol. 23(11) (2002) 549-555. [5] D.A. Rappolee, D. Mark, M.J. Banda, Z. Werb, Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping, Science. 241(4866) (1988) 708-712. [6] N. Jetten, S. Verbruggen, M.J. Gijbels, M.J. Post, M.P. De Winther, M.M. Donners,
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Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo, Angiogenesis. 17(1) (2014) 109-118. [7] T.J. Koh, L.A. DiPietro, Inflammation and wound healing: the role of the macrophage, Expert Rev Mol Med. 13 (2011) e23. [8] D. Peus, M.R. Pittelkow, Growth factors in hair organ development and the hair growth cycle, Dermatol Clin. 14(4) (1996) 559-572. [9] R. Hoffmann, W. Eicheler, A. Huth, E. Wenzel, R. Happle, Cytokines and growth factors influence hair growth in vitro. Possible implications for the pathogenesis and treatment of alopecia areata, Arch Dermatol Res. 288(3) (1996) 153-156. [10] S. Tauber, B.A. Lauber, K. Paulsen, L.E. Layer, M. Lehmann, S. Hauschild, N.R.
A
Shepherd, J. Polzer, J. Segerer, C.S. Thiel, O. Ullrich, Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity, PLoS One. 12(4) (2017) e0175599. [11] K.D. Phipps, S. Gebremeskel, J. Gillis, P. Hong, B. Johnston, M. Bezuhly, Alternatively activated M2 macrophages improve autologous Fat Graft survival in a mouse model through induction of angiogenesis, Plast Reconstr Surg. 135(1) (2015) 140-149.
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[12] K.B. Challagundla, P.M. Wise, P. Neviani, H. Chava, M. Murtadha, T. Xu, R. Kennedy, C. Ivan, X. Zhang, I. Vannini, F. Fanini, D. Amadori, G.A. Calin, M. Hadjidaniel, H. Shimada, A. Jong, R.C. Seeger, S. Asgharzadeh, A. Goldkorn, M. Fabbri, Exosome-mediated transfer of microRNAs within the tumor microenvironment and neuroblastoma resistance to chemotherapy, J Natl Cancer Inst. 107(7) (2015). [13] K. Tamai, T. Yamazaki, T. Chino, M. Ishii, S. Otsuru, Y. Kikuchi, S. Iinuma, K. Saga, K. Nimura, T. Shimbo, N. Umegaki, I. Katayama, J. Miyazaki, J. Takeda, J.A. McGrath, J. Uitto, Y. Kaneda, PDGFRalpha-positive cells in bone marrow are mobilized by high mobility group box 1 (HMGB1) to regenerate injured epithelia, Proc
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Natl Acad Sci U S A. 108(16) (2011) 6609-6614. [14] J.S. Duffield, S.J. Forbes, C.M. Constandinou, S. Clay, M. Partolina, S. Vuthoori, S. Wu, R. Lang, J.P. Iredale, Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair, Journal of Clinical Investigation. 115(1) (2005) 56-65. [15] F. Porcheray, S. Viaud, A.C. Rimaniol, C. Leone, B. Samah, N. Dereuddre-Bosquet, D. Dormont, G. Gras, Macrophage activation switching: an asset for the resolution of inflammation, Clin Exp Immunol. 142(3) (2005) 481-489. [16] S. Gordon, Alternative activation of macrophages, Nat Rev Immunol. 3(1) (2003) 23-35.
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[17] N. Osaka, T. Takahashi, S. Murakami, A. Matsuzawa, T. Noguchi, T. Fujiwara, H. Aburatani, K. Moriyama, K. Takeda, H. Ichijo, ASK1-dependent recruitment and activation of macrophages induce hair growth in skin wounds, J Cell Biol. 176(7) (2007) 903-909. [18] V.M. Ding, L. Ling, S. Natarajan, M.G. Yap, S.M. Cool, A.B. Choo, FGF-2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3-K/GSK3beta signaling, J Cell Physiol. 225(2) (2010) 417-428. [19] C. Desbois-Mouthon, A. Cadoret, M.J. Blivet-Van Eggelpoel, F. Bertrand, G. Cherqui, C. Perret, J. Capeau, Insulin and IGF-1 stimulate the beta-catenin pathway through two signalling cascades involving GSK-3beta inhibition and Ras activation,
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Figure 1. Wound-induced hair neogenesis model. Chronological change of macroscopic appearance of the wound (a). HE image of neogenerated follicles in the scar tissue (b), (scale bar 100 µm). Newly regenerated hair follicles in the scar are active for alkaline phosphatase (AP) and stained for K17 (c), (dashed circle, scale bar 1 mm).
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Figure 2. Infiltration of M2 macrophages in the scar tissue. Flow cytometry analysis of infiltrating cells (a). Increased percentage of CD11b+/CD206+ M2 macrophages is observed in the 2nd week (lower panel) after wounding as compared to the 1st week (upper panel). Immunohistochemistry for CD206 (red) in the mouse translated with BM from GFP mouse (b). GFP positive cells (green) are BM-derived cells. GFP+CD206+ M2 macrophages (arrowheads; merged color, yellow) are also observed. Some GFP+CD206+ M2 macrophages (white arrowheads) are located in the vicinity of neogenerated hair follicle (surrounded by dashed line), suggesting a possible interaction of M2 macrophages and hair follicles. Scale bar, 100μm.
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Figure 3. Expression of growth factors in cultured M2 macrophages differentiated from mouse spleen cells. Flow cytometry analysis for CD11b+CD206+ M2 macrophages from CD11b+ spleen cells (a). Microscopic image of fried egg-shaped M2 macrophages (b). Scale bar, 10 μm. Immunohistochemistry for growth factors in CD206+ M2 macrophages (c). Scale bar, 100 μm. Gene expression array for wound healing in the cultured M2 macrophages (d). The scatter plot represents the mRNA expression. Relative expression of real-time PCR products was determined using the ΔΔCt technique with GAPDH and -actin as internal control. As control, samples from undifferentiated CD11b+ spleen cells were used. Productions of Fgf2 and Igf1 were
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confirmed in CD206+ M2 macrophages in the scar tissues (e, scale bar 50μm, arrow Fgf2 positive M2 macrophages, arrow head Igf1 positive M2 macrophages).
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Figure 4. Effects of Fgf2 and Igf1 on hair follicle neogenesis. Data represent the mean ± SD. Asterisks indicate significant differences compared with control (*P<0.05).
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Figure 5. Effect of macrophage ablation in the late phase of wound healing on WIHN. Tg: B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J mice. WT: Wild type mice. DT: Diphtheria toxin. Procedure for ablation of macrophages by DT (25 ng/mouse i.p.) (a). Protocol for ablation (b). Flow cytometry analysis and immunohistochemistry of infiltrating cells confirmed that DT injections ablated M2 macrophages in the ulcer (c, scale bar 10 μm). DT injections did not change the healing curve (d) and the largeness of scar area (e). DT injections decreased the WIHN (f, g). AP: alkaline phosphatase staining. K17: keratin 17 immunostaining.
S0923-1811(18)30216-0 https://doi.org/10.1016/j.jdermsci.2018.05.004 DESC 3381
To appear in:
Journal of Dermatological Science
Received date: Revised date: Accepted date:
20-12-2017 18-4-2018 8-5-2018
Please cite this article as: Kasuya Akira, Ito Taisuke, Tokura Yoshiki.M2 macrophages promote wound-induced hair neogenesis.Journal of Dermatological Science https://doi.org/10.1016/j.jdermsci.2018.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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M2 macrophages promote wound-induced hair neogenesis Short title: Wound-induced hair neogenesis
Department of Dermatology, Hamamatsu University School of Medicine, Japan
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Akira Kasuya1 MD/PhD, Taisuke Ito1 MD/PhD and Yoshiki Tokura1 MD/PhD.
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Correspondence: Akira Kasuya, Department of Dermatology, Hamamatsu University
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School of Medicine, 1-20-1 Handayama, Higashi-Ku, Hamamatsu 431-3192, Japan.
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E-mail: [email protected]
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M2 macrophages promote wound-induced hair neogenesis Akira Kasuya, Taisuke Ito, and Yoshiki Tokura Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan
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High lights
We established a mouse model of wound-induced hair neogenesis. M2 macrophages infiltrated in the tissue 2 weeks after wounding in mice. M2 macrophages produced various growth factors. Administration of one of the upmodulated growth factors (Igf1 or Fgf2) promoted wound-induced hair neogenesis.
Ablation
of
macrophages
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genetically
engineered
mice
decreased
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wound-induced hair neogenesis.
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Background: De novo hair regeneration occurs in scars of normal adult mice. This interesting phenomenon is termed as wound-induced hair neogenesis (WIHN). We hypothesized that M2 macrophages are crucially involved in WIHN. Objective: To clarify the contribution of M2 macrophages to WIHN. Method: We established a mouse model of WIHN. A full thickness skin excision was implemented on the back of C57BL/6 (B6) mice. Newly developing hair follicles were detected by a whole-mount assay. WIHN took place 2 weeks after wounding.
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Results: At first, flow cytometry revealed increased infiltration of CD11b+/CD206+ M2 macrophages at the 2nd and 3rd week after wounding. Immunohistochemistry also showed the existence of CD206+ M2 macrophages in the vicinity of regenerated hair follicles. Secondly, the productions of growth factors were confirmed by culturing M2 macrophages isolated from the skin in a comparison with CD11b+ spleen cells. Array for 84 genes revealed increased expressions of various growth factors including Igf1 and Fgf2. Thirdly, we verified the effect of the growth factors on WIHN. WIHN was increased by 2 folds in mice treated with Fgf2 (p=0.05) or by 1.5 folds with Igf1 (p=0.05). Finally, we used B6.Tg(ITGAM-DTR) mice in which macrophages are ablated by diphtheria toxin. We depleted macrophages at one to 2 weeks after wounding
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when M2 macrophages were dominant. WIHN was attenuated to one third (P=0.05) by the ablation of macrophages. Conclusion: Our study suggests that M2 macrophages could promote WIHN through producing a panel of growth factors.
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Key words: wound healing, hair neogenesis, M2 macrophage, growth factor
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INTRODUCTION In humans, full-thickness skin excision usually leaves hairless scars, but the definitive evidence for de novo hair regeneration has been shown in skin scars of genetically normal adult mice [1]. This phenomenon was termed wound induced hair neogenesis (WIHN). The fact that hair follicles can be regenerated in vivo in scarred skin without the use of ES cells or iPS cells has high impact on tissue engineering. Dermal γδ T cells are suggested to promote WIHN through the production of Fgf9, a crucial growth factor [2]. However, it remains unknown whether other cell types are
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involved in WIHN. Here, we hypothesized that M2 macrophage could be involved in WIHN, because M2 macrophages produces various growth factors in the process of wound healing. Macrophages in the wound bed display different functional phenotypes, which can roughly be divided into two groups: M1 (classically activated) and M2 (alternatively activated) macrophages [3, 4]. While M1 macrophages contribute to the acute inflammation immediately after wounding, M2 macrophages, called “wound healing” macrophages, infiltrate at the late stage of wound healing and produce various growth factors [5-7]. Growth factors such as fibroblast growth factors (Fgf), insulin-like growth factor (Igf), epidermal growth factor (Egf)-related ligands, hepatocyte growth
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factor (Hgf), and platelet-derived growth factor (Pdgf) have been shown to be crucial for the regulation of the hair cycle and hair growth [8, 9]. In this study, we verify whether M2 macrophages could promote WIHN by means of growth factors.
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METHOD Animals C57BL/6N wild-type mice (WT) were obtained from SLC Inc. (Hamamatsu, Japan). The transgenic mice of B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J (Tg) were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were healthy, fertile, and did not display any evidence of infection or disease. Female mice (5- to 6-week-old) were
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used for all the experiments. All mice were housed in specific pathogen-free barrier facility and screened regularly for pathogens. Mice were individually housed in plastic cages to prevent to prevent tampering with the resultant ulcer by other mice. All studies and procedures were approved by the Committee on Animal Experimentation of Hamamatsu University School of Medicine. A transgenic construct containing sequence encoding DTR/GFP under the control of the human ITGAM (integrin alpha M) promoter was introduced into fertilized FVB/N donor eggs. The mice were backcrossed onto C57BL/6 for 6 generations. So the control strain suggested by Jackson laboratory is C57BL/6.
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Wounding and injection of growth factors C57BL/6N and B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J mice were anaesthetized with isoflurane, and a full thickness excision of skin with a diameter of 1.5 cm was made on the mid back of mice, as previously reported [1]. Either saline, Igf1 (100 g/body, Biolegend, San Diego, CA), Fgf2 (10 g/body, Kaken, Tokyo, Japan) were intraperitoneally administered at days 9, 11, and 13 in the experiments of growth factor injection.
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Whole-mount hair follicle neogenesis assay To detect newly developing hair follicles in the wound, whole-mount hair follicle neogenesis assay was performed [1]. We incubated the sampled skin in 1M NaCl at 37 °C overnight. Then, we gently peeled the epidermis off under a dissecting microscope (SZ61, Olympus Tokyo, Japan). The epidermis was fixed in 3.5% paraformaldehyde for 1 h, rinsed with PBS, blocked with buffer (Blocking One®, Nakalai Tesque, Kyoto, Japan). Immunostaining was performed for K17 using polyclonal rabbit antibody (Abcam, Cambridge, UK). Histofine Simple Stain MAX-PO
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(Nichirei Co., Tokyo, Japan) was used for second antibody. The dermis was used to detect alkaline phosphatase activity in the hair follicle. We fixed the dermis in acetone at 4 °C overnight, rinsed it with PBS, and then incubated with NBT/BCIP (Roche, Basel, Switzerland) for 30 min at 37 °C. We stopped the reaction with 20 mM EDTA in phosphate-buffered saline (PBS, pH 7.4).
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Histological examination and immunohistochemistry After mice were sacrificed, the area of scar was harvested. The sample was cut into halves, fixed in 3.5% paraformaldehyde, and embedded in paraffin. Sections were stained with hematoxylin & eosin (HE). Immunohistochemistry was performed using
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polyclonal rabbit antibodies specific for CD206 (Thermo Fisher Scientific Inc., Waltham, MA), Fgf2 (Abcam, Cambridge, UK) and monoclonal antibody for Igf1 (Acris, Rockville, MD). Alexa Fluor Secondary Detection Reagents (Life Technologies Co., Carlsbad, CA) was used for secondary antibody.
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Flow Cytometric Analysis Skin-infiltrating inflammatory cells were isolated and subjected to flow cytometric analysis. The scar of back skin was harvested. Subcutaneous tissue was removed by scalpel and was minced with the surface of frosted glass in PBS. The tissue was washed with PBS. The fluid was filtered through a 70-μm nylon mesh to obtain a single-cell
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suspension. The following primary antibodies were used: phycoerythrin (PE)-labeled anti-mouse CD206 monoclonal antibodies (Biolegend, San Diego, CA), fluorescein isothiocyanate (FITC)-labeled anti-mouse CD11b monoclonal antibodies (Biolegend). All antibodies were used at 1:200 dilution according to manufacturer instructions. Incubation was performed for 15 min at room temperature, followed by two washes in PBS supplemented with 5% fetal calf serum (FCS) and 0.02% sodium azide. FACSCanto II
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(Japan BD, Tokyo, Japan) was used to obtain fluorescent profiles. Data analysis was performed using Flowjo software (Treestar, Inc. Ashland, OR).
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Real-time PCR Total RNAs were extracted from skin samples using QIAGEN RNase spin columns ® (QIAGEN Ltd., Crawley, UK) and digested with DNase I (QIAGEN Ltd.) to remove chromosomal DNA in accordance with the manufacturer’s protocols. Total RNA was reverse transcribed to cDNA using a Reverse Transcription System with random hexamers (Promega, Madison, WI). Real-time PCR was performed using the TaqMan
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system (Applied Biosystems, Foster City, CA) on an ABI Prism 7000 Sequence Detector (Applied Biosystems) according to the manufacturer’s instructions. The 96 well RT² Profiler PCR Arrays® (Mouse Wound Healing; QIAGEN Ltd.) are used in combination with the RT² SYBR Green qPCR Mastermixes® (QIAGEN Ltd.). To analyze the PCR-array data, online analysis was performed on the manufacturer’s website (http://www.qiagen.com). Relative expression of real-time PCR products was determined using the ΔΔCt technique. Briefly, each set of samples was normalized using the difference in threshold cycle (Ct) between the target gene and housekeeping gene: ΔCt. (Ct target gene -Ct GAPDH). Relative mRNA levels were calculated by the expression 2-ΔΔCt, where ΔΔCt = ΔCt sample-ΔCt calibrator.
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Sorting of monocytes and differentiation towards M2 macrophages The spleens from C57BL/6N mice were minced in PBS. The fluid was filtered through a 70-μm nylon mesh to obtain a single-cell suspension. For the isolation of CD11b+ cells, cells were magnetically labeled with CD11b MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, the autoMACS Pro Separator ® (Miltenyi Biotec, Bergisch Gladbach, Germany) was used according to the manufacturer’s protocol. Isolated monocytes were placed in culture medium (M2-Macrophage Generation Medium DXF ®; PromoCell, Heidelberg, Germany) supplemented with M-CSF for 7 days at 37°C and 5% CO2 according to the manufacturer’s protocol according to the
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manufacturer’s protocol [10-12]. Then, 20 ng/ml of IL-4 (Biolegend) was added to trigger M2 polarization. Two days later, M2 macrophages were harvested and assessed by flow cytometry and real time PCR. Bone Marrow Translation (BMT) C57BL/6-Tg(CAG-EGFP)C57BL/6 transgenic 12-week-old female mice were purchased from SLC Inc. (Hamamatsu, Japan). The mice ubiquitously express enhanced
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green fluorescent protein (GFP). The bone marrow (BM) cells were isolated from the femur bones. Eight-week-old female C57BL/6N mice were used as recipients. Recipients was intravenously injected with 5×106 BM cells from GFP transgenic mice, immediately after the irradiation of 7.5 Gy X-rays. Experiments were performed in the BMT mice at least 6 weeks after the BMT [13].
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Ablation of macrophage in the mild stage of wound healing Diphtheria toxin (DT; Sigma-Aldrich, St. Louis, MI) was intraperitoneally administered at days 7, 9, 11 and 13 at 25 ng/g body to B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J
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mice or C57BL/6N[14]. These timepoints for administration were chosen to ablate M2 macrophages that appears in the late phase of wound healing.
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Statistical analysis The two-tailed unpaired Student’s t-test was used to determine the level of significance of differences between the sample means. Tukey's test was used for multiple comparisons. A p-value <0.05 was considered statistically significant.
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RESULT Wound-healing induced hair neogenesis Scar formation was completed 2 weeks after wounding (Figure 1a). Three weeks after wounding, WIHN took place as assessed by alkaline phosphatase activity and K17 positivity (Figure 1b, 1c).
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Infiltration of M2 macrophages after wounding Flow cytometry analysis of cells infiltrating in the wounded tissue showed increased percentage of CD11b+/CD206+ M2 macrophages at the 1st and 2nd week after wounding (Figure 2a). Immunohistochemistry for CD206 (red) in C57 mice translated with BM from GFP mice showed that GFP+/CD206+ cells (merged color, yellow) infiltrated into the wounded tissue (Figure 2b, arrowheads), suggesting that these infiltrated M2 macrophages are derived from BM. Some of GFP+/CD206+ M2
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macrophages infiltrated in the vicinity of neogenerated follicle (Figure 2b, white arrowheads). Productions of Fgf2 and Igf1 were confirmed in CD206+ M2 macrophages in the scar tissues (Figure 3e). Cultured M2 macrophages produce growth factors CD11b+ cells were isolated from mouse spleen cells by auto MACS and were
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differentiated to CD206+ M2 macrophages by cultivation (Figure 3a). Microscopic image exhibited the fried egg appearance of M2 macrophages (Figure 3b). By immunohistochemistry, CD206+ M2 macrophages were positive for Fgf2 and Igf1,
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representative macrophage-secreted growth factors (Figure 3c). Gene expression array for wound healing-associated molecules in cultured M2 macrophages showed increased expression of a number of growth factors such as Igf1, Fgf2, Fgf7, Fgf10, Pdgf and Egf, compared to undifferentiated CD11b+ spleen cells.
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Administration of growth factor promotes WIHN Fgf2 or Igf1 was administered intraperitoneously at day9, day13 and day21. The injection of Fgf2 or Igf1 during the late phase of wound healing promoted WIHN (Figure 4).
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Ablation of macrophages inhibits WIHN B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J mice were used to examine the effect of
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depletion of M2 macrophage (Figure 5a). The number of M2 macrophages increases in the late phase of the wound healing. We therefore initiated injection of DT (25
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ng/mouse i.p.) at day 9 and subsequent 3 times during wounding in Tg mice (Figure 5b). Flow cytometry analysis and immunohistochemistry of infiltrating cells confirmed that DT injections ablated M2 macrophages in the ulcer (Figure 5c). While DT
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injections changed neither the healing state (Figure 5d) nor the extent of scar area (Figure 5e), they decreased the WIHN (Figure 5f, 5g).
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DISCUSSION Wound healing is processed with an inflammatory phase. In this phase, M1 macrophages work for the killing of bacteria and the wound debridement, including clearing of neutrophils and damaged tissues [3, 15]. The inflammatory phase ceases within 3 days after wounding. In the late inflammatory phase, a shift of M1 to M2
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macrophages takes place, followed by a late phase of wound healing. In fact, direct conversion of M1 to M2 macrophages was also observed [16]. M2 macrophages produce various growth factor and promote regeneration and remodeling of the tissues [3, 17]. In this study, we first confirmed the existence of M2 macrophage at 2 weeks after wounding, in which WIHN was reported to occur [1]. With this circumstantial evidence, it is tempting to speculate that M2 macrophages are associated with neogeneration of hair follicles.
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Using Tg mice, we ablated M2 macrophages by DT during the proliferation phase of wound healing. The ablation of M2 macrophages attenuated WIHN, suggesting contribution of M2 macrophage to WIHN. However, the limit of this strategy is that DT depletes both M1 and M2 macrophages. To ablate mainly M2 macrophage, we thus administered DT in the proliferation phase of wound healing, at days 7, 9, 11 and 13, when M2 macrophages are dominant compared to M1 macrophage. Usually the infiltration of M1 macrophages peaks at day 1 and rapidly decreases as wound healing progresses. As a mechanism underlying the promotion of WIHN by M2 macrophages, we expected
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the involvement of macrophage-derived growth factors. In confirmation of the previous studies, M2 macrophages produced various growth factors, including Fgf2 and Igf1. The administration of Fgf2 or Igf1 promoted WIHN. Fgf2 and Igf1 participate in Wnt signaling pathway [18, 19]. Since Wnt signaling plays an important role in WIHN [2], it may intervene between Fgf2 and Igf1, and WIHN. We have shown the involvement of M2 macrophages on WIHN, however, our study fails to show how M2 macrophages specifically promote hair neogenesis in the context of wound healing. To suggest that M2 macrophage promote WIHN through Igf1 or Fgf2, knockdown of Igf1 and Fgf2 should be performed. The present study suggests that M2 macrophages play a crucial role in hair growth as
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well as wound healing. In rodents where hair follicles are not completely abrogated even in scar tissue, the wound-associated inflammatory alteration can induce hair neogenesis by virtue of M2 macrophages.
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ACKNOWLEDGEMENT We thank Yuki Yamada for the handling of animals.
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Reference
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[1] M. Ito, Z. Yang, T. Andl, C. Cui, N. Kim, S.E. Millar, G. Cotsarelis, Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding, Nature. 447(7142) (2007) 316-320. [2] D. Gay, O. Kwon, Z. Zhang, M. Spata, M.V. Plikus, P.D. Holler, M. Ito, Z. Yang, E. Treffeisen, C.D. Kim, A. Nace, X. Zhang, S. Baratono, F. Wang, D.M. Ornitz, S.E. Millar, G. Cotsarelis, Fgf9 from dermal gammadelta T cells induces hair follicle
M
A
N
U
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neogenesis after wounding, Nat Med. 19(7) (2013) 916-923. [3] B. Mahdavian Delavary, W.M. van der Veer, M. van Egmond, F.B. Niessen, R.H. Beelen, Macrophages in skin injury and repair, Immunobiology. 216(7) (2011) 753-762. [4] A. Mantovani, S. Sozzani, M. Locati, P. Allavena, A. Sica, Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes, Trends Immunol. 23(11) (2002) 549-555. [5] D.A. Rappolee, D. Mark, M.J. Banda, Z. Werb, Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping, Science. 241(4866) (1988) 708-712. [6] N. Jetten, S. Verbruggen, M.J. Gijbels, M.J. Post, M.P. De Winther, M.M. Donners,
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Anti-inflammatory M2, but not pro-inflammatory M1 macrophages promote angiogenesis in vivo, Angiogenesis. 17(1) (2014) 109-118. [7] T.J. Koh, L.A. DiPietro, Inflammation and wound healing: the role of the macrophage, Expert Rev Mol Med. 13 (2011) e23. [8] D. Peus, M.R. Pittelkow, Growth factors in hair organ development and the hair growth cycle, Dermatol Clin. 14(4) (1996) 559-572. [9] R. Hoffmann, W. Eicheler, A. Huth, E. Wenzel, R. Happle, Cytokines and growth factors influence hair growth in vitro. Possible implications for the pathogenesis and treatment of alopecia areata, Arch Dermatol Res. 288(3) (1996) 153-156. [10] S. Tauber, B.A. Lauber, K. Paulsen, L.E. Layer, M. Lehmann, S. Hauschild, N.R.
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Shepherd, J. Polzer, J. Segerer, C.S. Thiel, O. Ullrich, Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity, PLoS One. 12(4) (2017) e0175599. [11] K.D. Phipps, S. Gebremeskel, J. Gillis, P. Hong, B. Johnston, M. Bezuhly, Alternatively activated M2 macrophages improve autologous Fat Graft survival in a mouse model through induction of angiogenesis, Plast Reconstr Surg. 135(1) (2015) 140-149.
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[12] K.B. Challagundla, P.M. Wise, P. Neviani, H. Chava, M. Murtadha, T. Xu, R. Kennedy, C. Ivan, X. Zhang, I. Vannini, F. Fanini, D. Amadori, G.A. Calin, M. Hadjidaniel, H. Shimada, A. Jong, R.C. Seeger, S. Asgharzadeh, A. Goldkorn, M. Fabbri, Exosome-mediated transfer of microRNAs within the tumor microenvironment and neuroblastoma resistance to chemotherapy, J Natl Cancer Inst. 107(7) (2015). [13] K. Tamai, T. Yamazaki, T. Chino, M. Ishii, S. Otsuru, Y. Kikuchi, S. Iinuma, K. Saga, K. Nimura, T. Shimbo, N. Umegaki, I. Katayama, J. Miyazaki, J. Takeda, J.A. McGrath, J. Uitto, Y. Kaneda, PDGFRalpha-positive cells in bone marrow are mobilized by high mobility group box 1 (HMGB1) to regenerate injured epithelia, Proc
M
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Natl Acad Sci U S A. 108(16) (2011) 6609-6614. [14] J.S. Duffield, S.J. Forbes, C.M. Constandinou, S. Clay, M. Partolina, S. Vuthoori, S. Wu, R. Lang, J.P. Iredale, Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair, Journal of Clinical Investigation. 115(1) (2005) 56-65. [15] F. Porcheray, S. Viaud, A.C. Rimaniol, C. Leone, B. Samah, N. Dereuddre-Bosquet, D. Dormont, G. Gras, Macrophage activation switching: an asset for the resolution of inflammation, Clin Exp Immunol. 142(3) (2005) 481-489. [16] S. Gordon, Alternative activation of macrophages, Nat Rev Immunol. 3(1) (2003) 23-35.
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[17] N. Osaka, T. Takahashi, S. Murakami, A. Matsuzawa, T. Noguchi, T. Fujiwara, H. Aburatani, K. Moriyama, K. Takeda, H. Ichijo, ASK1-dependent recruitment and activation of macrophages induce hair growth in skin wounds, J Cell Biol. 176(7) (2007) 903-909. [18] V.M. Ding, L. Ling, S. Natarajan, M.G. Yap, S.M. Cool, A.B. Choo, FGF-2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3-K/GSK3beta signaling, J Cell Physiol. 225(2) (2010) 417-428. [19] C. Desbois-Mouthon, A. Cadoret, M.J. Blivet-Van Eggelpoel, F. Bertrand, G. Cherqui, C. Perret, J. Capeau, Insulin and IGF-1 stimulate the beta-catenin pathway through two signalling cascades involving GSK-3beta inhibition and Ras activation,
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Oncogene. 20(2) (2001) 252-259.
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Figure 1. Wound-induced hair neogenesis model. Chronological change of macroscopic appearance of the wound (a). HE image of neogenerated follicles in the scar tissue (b), (scale bar 100 µm). Newly regenerated hair follicles in the scar are active for alkaline phosphatase (AP) and stained for K17 (c), (dashed circle, scale bar 1 mm).
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Figure 2. Infiltration of M2 macrophages in the scar tissue. Flow cytometry analysis of infiltrating cells (a). Increased percentage of CD11b+/CD206+ M2 macrophages is observed in the 2nd week (lower panel) after wounding as compared to the 1st week (upper panel). Immunohistochemistry for CD206 (red) in the mouse translated with BM from GFP mouse (b). GFP positive cells (green) are BM-derived cells. GFP+CD206+ M2 macrophages (arrowheads; merged color, yellow) are also observed. Some GFP+CD206+ M2 macrophages (white arrowheads) are located in the vicinity of neogenerated hair follicle (surrounded by dashed line), suggesting a possible interaction of M2 macrophages and hair follicles. Scale bar, 100μm.
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Figure 3. Expression of growth factors in cultured M2 macrophages differentiated from mouse spleen cells. Flow cytometry analysis for CD11b+CD206+ M2 macrophages from CD11b+ spleen cells (a). Microscopic image of fried egg-shaped M2 macrophages (b). Scale bar, 10 μm. Immunohistochemistry for growth factors in CD206+ M2 macrophages (c). Scale bar, 100 μm. Gene expression array for wound healing in the cultured M2 macrophages (d). The scatter plot represents the mRNA expression. Relative expression of real-time PCR products was determined using the ΔΔCt technique with GAPDH and -actin as internal control. As control, samples from undifferentiated CD11b+ spleen cells were used. Productions of Fgf2 and Igf1 were
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confirmed in CD206+ M2 macrophages in the scar tissues (e, scale bar 50μm, arrow Fgf2 positive M2 macrophages, arrow head Igf1 positive M2 macrophages).
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Figure 4. Effects of Fgf2 and Igf1 on hair follicle neogenesis. Data represent the mean ± SD. Asterisks indicate significant differences compared with control (*P<0.05).
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Figure 5. Effect of macrophage ablation in the late phase of wound healing on WIHN. Tg: B6.FVB-Tg(ITGAM-DTR/EGFP)34Lan/J mice. WT: Wild type mice. DT: Diphtheria toxin. Procedure for ablation of macrophages by DT (25 ng/mouse i.p.) (a). Protocol for ablation (b). Flow cytometry analysis and immunohistochemistry of infiltrating cells confirmed that DT injections ablated M2 macrophages in the ulcer (c, scale bar 10 μm). DT injections did not change the healing curve (d) and the largeness of scar area (e). DT injections decreased the WIHN (f, g). AP: alkaline phosphatase staining. K17: keratin 17 immunostaining.