Selective M2 Macrophage Depletion Leads to Prolonged Inflammation in Surgical Wounds
Kerstin Klinkert a Derek Whelan a Anthony J.P. Clover a, b Anne-Laure Leblond a Arun H.S. Kumar a Noel M. Caplice a
a Centre for Research in Vascular Biology and b Department of Plastic Surgery, University College Cork, Cork, Ireland
Keywords
Macrophages · Surgical wound · Wound healing Abstract
Background: A prolonged inflammatory phase is seen in aberrant wound healing and in chronic wounds. Macrophages are central to wound healing. Distinct macrophage subtypes have differing roles both in initial inflammation and in later tissue repair. Broadly, these cells can be divided into M1 and M2 macrophages. M2 macrophage proliferation and differentia- tion is regulated by colony-stimulating factor 1 (CSF-1) signalling and can be blocked by GW2580, a competitive cFMS kinase inhibitor, thereby allowing for analysis of the effect of M2 blockade on progression of surgical wounds. Materials and Methods: Macrophage Fas-in- duced apoptosis (MaFIA) transgenic mice with a macrophage-specific promoter used to ex- press green fluorescent protein (GFP) were used to allow for cell tracking. The animals were treated by oral gavage with GW2580. Surgical wounds were created and harvested after 2 weeks for analysis. Results: GW2580-treated mice had significantly more GFP+ cells in the sur- gical scar than vehicle-treated animals (GW2580, 68.0 ± 3.1%; vehicle, 42.8 ± 1.7%; p < 0.001), and GW2580 treatment depleted CD206+ M2 macrophages in the scar (GW2580, 1.4%; vehicle, 19.3%; p < 0.001). Treated animals showed significantly higher numbers of neutrophils (vehicle, 18.0%; GW2580, 51.3%; p < 0.01) and M1 macrophages (vehicle, 3.8%; GW2580, 12.8%; p < 0.01) in the scar compared to vehicle-treated animals. The total collagen content in the area of the scar was decreased in animals treated with GW2580 as compared to those treated with vehicle alone (GW2580, 67.1%; vehicle, 79.9%; p < 0.005). Conclusions: Depletion of M2 macrophages in surgical wounds via CSF-1 signalling blockade leads to persistent inflammation, with an in- crease in neutrophils and M1 macrophages and attenuated collagen deposition.
© 2017 S. Karger AG, Basel
Anthony J.P. Clover
Centre for Research in Vascular Biology, University College Cork Biosciences Institute, College Road
Cork (Ireland)
E-Mail j.clover @ ucc.ie
Introduction
Skin wound healing involves a complex sequence of cellular and biological events. Cells are recruited into the wound in a sequence of haemostasis, inflammation, remodelling, and repair, and ultimately scar formation. When wound healing becomes aberrant, it can lead to substantial morbidity and cost [1]. Macrophages are essential for normal wound healing and have multiple functions in both the inflammatory phase and in promoting the proliferative/
repair phase by attracting endothelial cells and fibroblasts and promoting angiogenesis and extracellular matrix deposition [2]. Prolongation of the inflammatory phase by infection, wound debris, ischemia, or repetitive trauma may inhibit progression to the proliferative/
repair and remodelling phases, resulting in a chronic wound [3]. Both chronic wounds and impaired acute wounds demonstrate excess inflammation in the form of leucocytosis and reduced matrix deposition [4].
Macrophages have roles in the phagocytosing of wound debris and as secreting factors essential for the switch between initial inflammation and tissue repair and remodelling [5]. Distinct macrophage subsets have differing roles in inflammation and subsequent remod- elling and repair [6], and these subsets change over time [7, 8]. These cell subsets can be broadly divided into M1 and M2 macrophages, which can be differentiated by surface receptors amongst other functional attributes [9]. M1 macrophages secrete high levels of the proinflammatory cytokines TNFα, IL-1, and IL-6 and have an enhanced microbicidal/phago- cytic function suggestive of a role in the early inflammatory response. Conversely, M2 macro- phages may be essential for early tissue repair, dampening proinflammatory cytokine release and secreting extracellular matrix [10].
The temporal sequence of M1 and M2 phenotypic effects has begun to be elucidated in vivo; total macrophage abrogation by selective targeting of CD11b-positive cells with diph- theria toxin causes delayed wound healing [5]. Selective depletion of macrophages at different stages of wound healing leads to differential effects; early depletion, about 3 days after injury, is associated with severely altered wound healing. Depletion of macrophages on day 5 leads to poorly formed granulation tissue and diminished wound contraction, abro- gating the transition into the phase of tissue maturation [8]. Whether this effect is due to a specific subset of macrophages has not yet been proven, but it has been posited that this effect may be due to alteration in the macrophage phenotype and that the late-stage effects may be due to a decrease in M2 macrophages. The effect of selective depletion of M2 macro- phages on the phenotype of surgical wounds has not yet been described. In order to block M2 macrophage transition within an evolving wound repair model, we used GW2580, a cFMS kinase inhibitor.
Macrophage differentiation from monocyte precursors is primarily driven by colony- stimulating factor 1 (CSF-1) signalling [11]. CSF-1 binding to its receptor results in autophos- phorylation of receptor cFMS kinase and activation of downstream pathways involved in the regulation of proliferation, differentiation, and survival of the M2 phenotype of monocytes and macrophages [12]. Macrophages primed by granulocyte-macrophage colony-stimulating factor (GM-CSF) display an M1 phenotype [12–15]. M2 macrophage transition can be dimin- ished by treatment with a cFMS kinase inhibitor, such as GW2580 [16, 17], acting through competitive inhibition of ATP binding to the CSF-1 receptor [18].
Macrophage Fas-induced apoptosis (MaFIA) mice are transgenic mice with a macro- phage-specific colony-stimulating factor 1 receptor (Csf1r) promoter used to express human FK506-binding protein 1A and green fluorescent protein (GFP) allowing for macrophage tracking in vivo. The transgene is expressed constitutively in Csf1r-expressing cells such as macrophages and some dendritic cells, but not significantly in T or B cells [19], allowing the monitoring of myeloid cell populations in mice. In this model, we investigated the impact and
function of the M2 subtype of monocytes and macrophages in wound healing, in the presence or absence of GW2580, a cFMS kinase inhibitor which has been previously shown to specifi- cally deplete M2 macrophages [11, 20, 21].
In this paper, we show that abrogation of M2 macrophages in surgical scars by treatment with a cFMS kinase inhibitor results in a prolonged inflammatory phase of wound healing.
Methods
Ethical approval (#2009/16) was obtained from Animal Experimental Ethics Committee, University College Cork, and a licence obtained from the Department of Health and Children (B100/4236).
Experiments were performed on transgenic MaFIA (C57BL/6J-Tg(Csf1r-GFP, NGFR/FKBP12)2Bck/J) [19] and wild-type C57BL/6J mice. A total of 49 animals were used in this study.
Creation of Surgical Wounds
Male MaFIA mice were treated once daily with 160 mg/kg GW2580 (Biorbyt, UK) diluted in a vehicle solution (0.5% hydroxy propyl methylcellulose and 0.1% Tween 80). The drug was administered by oral gavage in a volume of 200 μl (using a graduated syringe) for 1 week prior to surgery and for 2 weeks after surgery [11].
GW2580 is a potent small-molecule ATP-competitive cFMS kinase inhibitor that has been shown to selectively inhibit monocyte/macrophage growth by inhibition of the cFMS pathway [11]. GW2580 is specific to cFMS kinase, preventing its autophosphorylation, and does not inhibit JAK kinase, a major component of the GM-CSF signalling pathway, a promoter of M1 differentiation, and therefore depletes M2-type macro- phages) [11].
The effect of GW2580 treatment on tissue-resident macrophages depletion does not depend on the genetic background of the mice and will deplete macrophage/monocyte populations in both MaFIA and wild- type C57BL/6J mice [20].
Surgical wounds were created under general anaesthesia on the left side of the thoracic cage. Briefly, anaesthesia was induced by intraperitoneal injection of ketamine (90 mg/kg), xylazine (10 mg/kg), and urethane (1.25 mg/kg). Wounds were left lateral thoracotomy incisions and were closed with single-layer 6-0 nylon (Ethicon). To prevent aggravating the mice and subsequent disturbance of sutures from scratching, the wounds were not dressed and the animals were housed individually and left to recover. Animals were provided food and drink ad libitum and were housed under standard laboratory conditions with a 12-h light- dark cycle. Wounds were harvested after 2 weeks and collected for analysis.
Histological Assessment
To assess the wound healing, postoperative scar tissue and normal skin were collected. Collected tissues were fixed in 4% PFA for 1 h at 4°C and then incubated in 20% sucrose overnight before being embedded in OCT or paraffin.
For immunohistochemistry, 10-μm-thick sections were obtained using a microcryotome and mounted on Menzel Superfrost slides. These were fixed in 4% PFA for 10 min at 4°C and stained with 4′,6-diamidino- 2-phenylindole (DAPI) (1:2,000 in water) for 10 min in the dark for GFP quantification. For determination of M1, M2, and neutrophil populations, sections were stained with Ly6G (Serotec, 0.2 mg/mL), CD206 (Santa Cruz, 100 μg/mL), and Gr-1 (Serotec, 1 mg/mL). Goat anti-rat secondary antibody (Alexa Fluor 546, Invit- rogen) 2 mg/mL was used. Images of sections were acquired at 60× magnification with a confocal microscope (Nikon eC1 plus, TE2000E) and stored for later analysis.
For cell subtype analysis of GFP-positive cells, neutrophils were considered as GFP+/Ly6G+ and M2 macrophages were considered as GFP+/CD206+. As the Gr-1 antibody used (clone RB6-8C5) reacts with both Ly6G and Ly6C surface proteins present on both M1 macrophages and neutrophils, the M1 population was calculated by counting the percentage of GFP+/Gr-1+ cells, minus the percentage of neutrophils (Ly6G). Several attempts were made to better characterise this M1 population using a specific Ly6C antibody (online suppl. Fig. 1, see www.karger.com/doi/10.1159/000451078); however, no positive staining was detected in the tissue samples.
The numbers of GFP-fluorescent cells, macrophages, and neutrophils were calculated, as were the numbers of cell nuclei stained with DAPI, and standardised to the total area of cells in the section. Five sections per wound of each animal were analysed by manual counting (approximately 10,000 nuclei per animal).
Fig. 1. a GFP+ myeloid cell quan- tification in normal skin and scar 2 weeks after wound injury in an- imals with or without GW2580 treatment. b Representative skin and scar sections from vehicle- and GW2580-treated animals. c Cell number quantification by 4′,6-diamidino-2-phenylindole (DAPI) staining on skin and scar sections. Treatment with GW2580 decreases the number of myeloid cells in uninjured skin; however, these cells are seen in greater number in the scars of treated an- imals (a, b). There is an increase in total cell number in the scars of treated animals (c) (n = 13). * p < 0.05, ** p < 0.01. HPF, high-power
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Paraffin sections were used to assess total cell numbers and collagen staining. These were stained with Masson’s trichrome using a standard protocol [22], and 20× images were taken to assess the cell density in the scar tissue. Nuclei of 5 areas per image were counted on 5 sections per animal, and the cell number was expressed over the analysed area. Collagen staining was semiquantified by colorimetric analysis of blue staining in sections stained with Mason’s trichrome using the NIS Elements software (Nikon).
Statistics
Data are presented as mean ± SEM. Nonparametric tests were used to determine differences between groups (n ≤ 30) as follows: The Mann-Whitney test was used for 2-group comparison and the Kruskal-Wallis test was used for ≥3 groups, with subsequent pairwise comparisons using Dunn’s test (GraphPad Prism version 4; GraphPad Software, Inc., San Diego, CA, USA). A p value <0.05 was considered significant. All results are presented as means with error bars representing the SEM.
Results
Treatment with the cFMS kinase inhibitor GW2580 led to a reduction in the numbers of GFP-positive cells in the normal dermis, but not in the surgical scar. In the normal dermis of animals treated with delivery vehicle only, the numbers of myeloid cells, characterised as GFP-positive cells, was 12.9 ± 1.7%, whereas this decreased to 4.3 ± 1.2% (p < 0.05, n = 12) in animals treated with GW2580. However, GW2580-treated mice had significantly more GFP+ cells in the surgical scar than vehicle-treated animals (GW2580, 68.0 ± 3.2%; vehicle, 42.8 ± 1.7%; p < 0.001, n = 10) (Fig. 1a, b). Histological characterisation of the scar confirmed a higher number of cells per high-power field in GW2580-treated animals 2 weeks after surgery compared to vehicle-treated controls (Fig. 1c) (skin, 9.9 ± 0.5; GW2580, 91.2 ± 2.5; vehicle, 50.6 ± 1.2; p < 0.001, n = 13).
To determine the nature of the increased cell numbers in treated animals, the numbers of neutrophils, M1, and M2 macrophages were assessed both in normal skin (adjacent to the wound) and in animals treated with GW2580. Staining of tissue sections with antibodies against Ly6G for neutrophils, Gr-1 for M1 monocytes/macrophages, and CD206 for M2 macro- phages revealed that GW2580 treatment significantly depleted CD206+ M2 macrophages in the scar (GW2580, 1.4 ± 0.3%; vehicle, 19.3 ± 1.7%; p < 0.001, n = 10). Furthermore, treated animals showed significantly higher numbers of neutrophils (vehicle, 18.0 ± 3.2%; GW2580, 51.3 ± 2.5%; p < 0.01, n = 10) and M1 macrophages (vehicle, 3.8 ± 0.25%; GW2580, 12.8 ± 0.8%; p < 0.01, n = 10) in the scar compared to vehicle-treated animals (Fig. 2).
Collagen staining in the surgical scar was assessed using Masson’s trichrome (Fig. 3). Total collagen staining in the area of the scar was significantly decreased in animals treated with GW2580 as compared to those treated with vehicle alone (GW2580, 67.1 ± 2.5%; vehicle, 79.9 ± 2.4%; p < 0.005, n = 14).
Discussion
In this study, the role of M1 and M2 macrophage population subsets in the healing of surgical wounds was investigated. We found that depletion of M2 macrophages with the cFMS kinase inhibitor GW2580 results in maintenance of the proinflammatory phase of wound healing characterised by persistence of neutrophils and M1 macrophages. In contrast, in vehicle-treated mice, M2 macrophages were the dominant cell type in the scar 2 weeks after injury. In this study, we used a dosage of 160 mg/kg of GW2580, which had been previously shown to give an optimal blood plasma concentration of 1 μM [21] and which we have previ- ously demonstrated to effectively deplete M2-type macrophages in vivo [20]. Others have used lower dosages, such as 80 mg/kg; however, as the drug is rapidly metabolised, plasma concentrations fall below 1 μM after 12 h with this approach [11]. Treatment of GW2580 was found to have no significant effect on neutrophils or M1 macrophages in normal skin. However, we did find a depletion in M2-type macrophages, presumably resident tissue macrophages.
Skin
CD206 Gr-1 Ly6G
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Scar
CD206 Gr-1 Ly6G
Fig. 2. a Normal skin sec- tions from animals treated with GW2580 or vehicle. Sections were probed with either CD206 (M2 marker), Gr-1 (M1 marker), or Ly6G (neutrophil marker) an- tibodies (red, Alexa Fluor 546), nuclei in blue (4′,6-diamidi- no-2-phenylindole, DAPI), while green fluorescence shows GFP+ myeloid cells. b Scar sections
from animals treated with GW2580 or vehicle (sections as
b
in a). Images were taken within Skin *** Scar
the dermis at 60× magnification. c Quantification of the percent- age of cell types in skin sections based on co-staining in a and b. The increase in total cell number in GW2580-treated animals is due to an increase in neutrophils and M1 macrophages. * p < 0.05, *** p < 0.001. Neutrophils, GFP+/
Ly6G+; M1 macrophages, GFP+/
Gr-1+ cells minus percentage of neutrophils; M2 macrophages,
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In normal wound healing, neutrophils do not persist in the tissue in large numbers; however, we found increased neutrophil persistence in the wounds of M2-depleted animals [2]. Whether the inflammation is prolonged due to impaired transition of M1 into M2 macro- phages, ongoing recruitment of neutrophils, or lack of signalling to initiate neutrophil apo- ptosis needs to be further investigated. By assessing wound healing in surgical scars, it is
Vehicle GW2580
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Vehicle GW2580
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Fig. 3. Masson’s trichrome stain- ing of skin sections. Collagen staining, in blue, is decreased in the scars of animals treated with GW2580. a Representative imag- es of scars stained with Masson’s trichrome. b Magnified sections contained in the boxes in a. c Col- orimetric analysis of blue stain in- dicative of collagen content. * p <
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0.05, ** p < 0.01, *** p < 0.001.
possible to gain insight into the contribution of M2 macrophages to resolution of inflam- mation and to collagen deposition.
Similar to Th1/Th2 responses, macrophages can be polarised into either M1 or M2 states. M1 polarisation or classically activated macrophages can be induced by various stimuli such as IFNγ, LPS, or GM-CSF and are associated with greater levels of inflammatory cytokines such as TNFα, IL-1β, and increased microbicidal and phagocytic activity. M2 (or alternatively activated) macrophages can be polarised by IL-4 and IL-13 and are characterised by increased expression of IL-10, reduced TNFα, and altered metabolism [23]. Other inducers of M2 pheno- types are IL-21 and IL-33 [24, 25]. M1 and M2 polarisation states represent in vitro extremes of what is believed to be a dynamic continuum of functional states in vivo [26].
Functionally, it is believed that biphasic accumulation of different monocyte subsets occurs with inflammatory monocytes providing cells with the capacity to phagocytose dead cells and pathogens. As the wound evolves, a reparative phase occurs, with the accumulation of M2 macrophages presumed to be from the differentiation of Ly6C non-inflammatory mono- cytes. These cells induce proliferation of fibroblasts and myofibroblasts with subsequent synthesis of collagen, increasing wound strength and promoting wound closure. Recent work by Hilgendorf et al. [27] suggests that Ly6C proinflammatory monocytes may be responsible for the majority of M2 phenotype macrophages through M1 to M2 transition.
What is becoming increasingly clear is that changes in these population subsets evolve during wound repair and that any aberration can affect the quality and progress of wound healing. Depletion of M1 phenotype macrophages or depletion of their mediators has been shown to reduce scar formation [5]. Conversely, reduced macrophage wound infiltration due to impaired recruitment or conditional depletion also impairs granulation tissue formation and wound closure, indicating that the M1 macrophages can alter both early- and late-stage inflam- matory responses [28], adding further weight to their role in transitioning to the M2 phenotype.
Helper T cell responses play a role in the polarisation of macrophages; Th2-related cytokine IL-4 induces signal transducer and activator of transcription 6 (STAT6), IRF4, and peroxisome proliferator-activated receptor gamma (PPARγ). These factors induce fatty acid oxidation, inhibiting proinflammatory cytokine production. CD18 or β2 integrin-dependent phagocytosis of neutrophils by M1-type macrophages is also a driver of M2 phenotype and is biologically important [29]. Deficiency of CD18 leads to spontaneous development of hard to heal skin ulcers and delayed wound closure through reduced TGF-β1-dependent myofibro- blast differentiation [29].
M2 macrophages are a predominant source of TGF-β, which promotes many aspects of wound repair including angiogenesis, interactions with wound fibroblasts, and the production of collagen. We found significantly less collagen in the scar after M2 monocyte/macrophage depletion, consistent with the previous findings in macrophage-depleted DTR mice (which express human diphtheria toxin under a CD11b promoter, allowing selective ablation of macrophage population), which also exhibited reduced collagen deposition [5]. Moreover, using diphtheria toxin, Lucas et al. [8] and others were able to deplete macrophages at various time points during the wound healing process. They found that ablation of macrophages in the inflammatory period delayed healing and resulted in a reduction in re-epithelialisation, granulation tissue, and collagen deposition within the wound [5, 8].
Depletion of macrophages at different time points can have varying and opposing effects, indicating a dynamic interaction between macrophage subsets and the functionality of these populations [30, 31]. Depletion of macrophages at early time points (M1 phenotype) in various models leads to poor wound healing, with reduced vascularisation, phagocytosis, and granulation tissue [30, 31].
Collagen synthesis during wound healing is a critical component, but can have both positive and negative effects. Previously we have shown that M2 depletion is associated with
reduced collagen deposition, which may reduce strength and is also associated with left ventricular dilation and wall thinning in myocardial infarct models [31]. Application of allo- geneic macrophages has been shown to improve collagen content and increase wound ten- sile strength [32].
However, if there is an excessive or prolonged inflammatory response, this may result in increased TNFα and further monocyte recruitment, leading to exaggerated M2 activation, excessive TGF-β release, and consequently increased collagen deposition and fibrosis. In this instance, reduction of M2 macrophages may inhibit excessive collagen formation, as suggested by previous ablation of late-stage macrophages in a liver fibrosis model [30].
A prolonged inflammatory phase does not necessarily mean that the overall outcome of wound healing is impaired. In mice deficient for IL-10, which is mainly produced by M2 macrophages, wound closure was accelerated, with increased thickness and organisation of collagen bundles in the scar [33]. However, prolonged inflammation is implicated in abnormal scar formation [34, 35] and in the aetiology of keloid scars [36]. Thus, abrogating the M1 phenotype in wounds may decrease the development of scarring [37]. While the relationship between the ratios of M1 and M2 macrophages in human wounds and the presence of keloid scarring remains ambiguous, keloid scar tissue has been noted to have a higher number of M2 macrophages as well as increased numbers of T and B cells [38]. Thus, the relative fibrotic and reparative roles of the M2 macrophage are complex and require further elaboration.
In acute wounds, including surgical incisions, trauma, and burns, the promotion of rapid healing and the prevention of scarring are desirable clinical objectives. Both chronic wounds and impaired acute wounds demonstrate excess inflammation and reduced matrix depo- sition [4]. Macrophages are important to wound repair, contributing to the initial inflam- mation, tissue remodelling, and repair [6]. The phenotype of wound-infiltrating macrophages is dynamic, suggesting that different subpopulations of macrophages have complementary roles in the diverse phases of skin repair [7, 8]. The transition of macrophage phenotype from a predominantly M1 phenotype to a predominantly M2 phenotype is observed in wound healing in skeletal muscle [39] and cardiac tissue [40, 41] and is also implicated in cutaneous wound healing [8].
The balance of macrophage phenotypes in the wound is an area of increasing clinical interest, especially in diabetes where chronic inflammation is a characteristic feature of wound healing [42]. Unrestrained M1 activation due to haemochromatosis (chronic iron overload) also results in chronic inflammation and impairs wound healing both in animal models and in humans [10]. Cutaneous wounds in diabetic mice exhibit an increase in M1 type cells (Ly6C+/CD206–), with a decrease in M2-type cells (Ly6C+/CD206+) [43]. This balance of macrophages is associated with elevated levels of proinflammatory cytokines (TNFα, IL-6) and decreased expression of insulin-like growth factor 1, TGF-β1, and vascular endothelial growth factor [7, 44].
Our current study demonstrates the effect of myeloid phenotype within the wound and supports the view that transient inflammation is integral to successful healing. Inflammation supports key functions such as debris removal and needs to be resolved in a timely manner to allow for progression of the healing process [45]. Abrogating M2 macrophages in surgical wounds is associated with persistence of inflammation, reinforcing the role of M2 macro- phages in the transition between inflammation and repair. Tilting the balance from inflam- mation to repair by altering the relative proportions of macrophages in wounds could be of benefit in conditions such as diabetes, where wounds characteristically demonstrate increased and prolonged inflammation. Conversely, decreasing the matrix deposition from M2 macro- phages could be of benefit in conditions characterised by excess scarring. These data add to our understanding of the importance of macrophage subtypes in wound healing and the dynamic transition that is required for normal healing. It demonstrates that the M2 macro-
phage response is important and required for progression of wound healing. Clinically, if it were possible to accentuate the M2 response with the use of a targeted small molecule/drug or biologics, a therapeutic response might be achieved particularly in M1-driven pathologies such as chronic wounds. Surgical scars represent a major concern for both patients and surgeons alike, and better understanding the biology of wound healing will allow us to better design therapies to minimise disfiguring scars due to surgery. Knowledge gained from this simple model may also be relevant to more complex wounds and pathologies, such as burns and chronic wounds.
Acknowledgements
This work was supported by PRTLI-5 and the Irish Blood Transfusion Service. N.M. Caplice is supported by Science Foundation Ireland (R11482 and RFP06-NMC).
Disclosure Statement
The authors declare no conflict of interest.
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