PERSPECTIVE ARTICLE

Macrophages: A review of their role in wound healing and

their therapeutic use
Robert J. Snyder, DPM, MSc, CWS1; John Lantis, MD, FACS2; Robert S. Kirsner, MD, PhD3;
Vivek Shah, PhD4; Michael Molyneaux, MD, MBA5; Marissa J. Carter, PhD, MA6
1. Department of Clinical Research, Barry University School of Podiatric Medicine, Miami Shores, Florida,
2. Department of Surgery, Mount Sinai St. Luke’s Roosevelt Hospital Center, New York, New York,
3. Department of Dermatology and Cutaneous Surgery, University of Miami School of Medicine, Miami, Florida,
4. Research and Development, Macrocure Ltd, Tenafly, New Jersey,
5. Macrocure Ltd, Petach Tivka, Israel,
6. Strategic Solutions Inc, Cody, Wyoming

Reprint requests: ABSTRACT

Robert Snyder, DPM, MSc, CWS,
Barry University School of Podiatric
Medicine, Paul & Margaret Brand
Research Center, 11300 NE Second
Avenue, Miami Shores, FL 33161.
Tel: 305-899-3098;
Fax: 305-899-3253;
E-mail: drwound@aol.com
Manuscript received: August 26, 2015
Revised: April 20, 2016.
Accepted in final form: April 24, 2016
DOI:10.1111/wrr.12444
Macrophages are mononuclear phagocytes established during embryogenesis and
derived from the yolk sac or the fetal liver but also recruited from the blood and
bone marrow under proliferative inflammatory conditions (such as tissue repair).
Most importantly, they take on distinct phenotypes and functions crucial to healing
upon localization in the wound. The objective of this review is to summarize recent
findings in regard to the cellular mechanisms of macrophages and chronic wounds.
Advances in the potential use of macrophage therapy have arisen based, in part, on
the fact that early recruitment of macrophages is critical to wound healing. Higher
quality evidence is needed to support the use of macrophage therapy for chronic
wound types, as is a better understanding of the signaling related to macrophage
polarization, activation of macrophages, and their effect of mechanisms of repair.
An evaluation of the currently available research on mechanism of action may lead
to a better understanding of the signaling processes of the many macrophage
phenotypes, as well as their roles and outcomes in wound healing, which could then
guide the development and eventual widespread use of macrophage therapies.

INTRODUCTION
Chronic wounds represent a worldwide problem affecting
6.5 million people in the United States (US) alone.1 Com-
mon chronic wounds include venous leg ulcers (VLUs)
with a prevalence of 2% in developed countries,2 pressure
ulcers (PUs) with an incidence of 0.4–38% in acute care
settings and 0–24% in long-term and home care set-
tings,3–5 and diabetic foot ulcers (DFUs), which affect up
to one in four patients with diabetes mellitus (DM).
Chronic wounds have an alarming impact on society. For
example, in 2007, 8.1% of the US Medicare beneficiary
population with DM had a DFU and 1.8% had a resulting
lower extremity amputation (LEA).6 The overall lifetime
incidence for a DFU among those with DM is approxi-
mately 6%, and an estimated 0.5% will undergo an LEA.7
In addition to their pain and reduced quality of life,
patients with chronic wounds have increased mortality. For
example, three out of five of patients with a diabetic LEA
will die within 5 years of their first amputation.8,9
However, the chronic wound burden on global society
has, thus, far been under-appreciated. For example, in
2008, the US Congress authorized $48 billion over a 5-
year period to combat the priority global diseases of HIV/
AIDS, malaria, and tuberculosis10—roughly $9.6 billion in
annual spending—but no funding for chronic wounds. In
2009, Sen et al. estimated that more than $25 billion is
spent on direct costs in the US each year on treating
chronic wounds, more than 150% of the annual US
budget allocated for the top 3 global diseases.1 Others sug-
gest that the cost is even higher, as DFUs alone in the US
have been estimated to cost at least $38.6 billion a year.11
Moreover, chronic wound costs are on the rise as health
and home care costs increase, the population ages, and
DM becomes more prevalent (in part because of increased
obesity rates), especially in developing countries.1,12
As a consequence of poor healing rates in chronic
wounds treated with standard of care, adjunctive therapy is
often needed. Wound care practice is increasingly looking
toward cell-based treatments as an adjunct therapy in addi-
tion to standard of care for chronic wounds, as the break-
down in the orderly sequence of normal wound healing
(hemostasis, inflammation, proliferation, and remodeling)
becomes better understood).13,14 Some key cells involved
in wound healing are platelets, neutrophils, macrophages,
and fibroblasts. In part, the release of cytokines and
growth factors are thought to promote wound healing.
Early work on macrophages in the 1970s confirmed that
they are critical to wound healing,15,16 and although the
definition of their role remains incomplete,17–21
macrophage-based therapies appear beneficial for some

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groups, such as the elderly and patients with hard-to-heal matrix (ECM), facilitated by epidermal growth factor
wounds.22–28 (EGF), keratinocyte growth factors (KGFs) and TGF-
The objective of this review is to summarize recent a.13,14 Keratinocytes migrate from the wound edges
findings on the cellular mechanisms of chronic wounds in between the dermis and fibrin facilitated by production of
the context of macrophages, as well as advances made to collagenase and other proteases in the epidermis.29 Fibro-
date on their potential therapeutic uses. blasts migrate, proliferate, and produce ECM in the wound
bed, resulting in early granulation and tissue formation.29
CELLULAR MECHANISMS INVOLVED IN Epithelialization is characterized by the exit of inflamma-
tory cells, a decrease in growth factor release, an increase
WOUND HEALING
in the ratio of collagen deposition to fibroblasts, and the
Over the past few decades, while advances have been cross-linking and organization of collagen molecules.
made in the understanding of the biochemistry of the Remodeling is the final phase that takes place over a much
chronic wound, detailing all the cellular events that can longer period of time in which the newly formed tissue is
actually delay wound healing remains a work in progress. reorganized for higher tensile strength.13,14 Thus, the nor-
Normally, the signaling, timing, and regulation of cellular mal wound healing process constitutes a delicate balance
events must take place in an orchestrated manner.13,14,19 of cells secreting and regulating the many cytokines, che-
The process starts with platelet activation and initiation of mokines, proteins (Table 1), and growth factors (Table 2)
the coagulation cascade, stimulating the release of various which in turn regulate cellular activity.
growth factors, such as platelet derived growth factor A chronic wound is defined as an ulcer open for 2
(PDGF), transforming growth factors (TGFs; especially weeks or more in a patient with at least 1 underlying co-
TGF-b), fibroblast growth factors (FGFs), and vascular morbidity such as diabetes.59 Chronic wounds are charac-
endothelial growth factors (VEGFs). Within 24 hours, terized by dysfunctional cellular events and aberrant cyto-
inflammatory cells (neutrophils, macrophages, and T-lym- kine and growth factor activity.13,15,29,60–65 For example,
phocytes) accomplish phagocytosis and the removal of fibroblast senescence, leading to replicative senescence, is
bacteria, other foreign materials, cellular debris, and dam- common; both secretion of and response to growth factors
aged tissue. In addition to phagocytosis, these inflamma- in fibroblasts may be impaired. Levels of neutrophils, ser-
tory cells continue to secrete cytokines and growth factors. ine elastase, and inflammatory macrophage activity also
The next phase of healing, proliferation, involves angio- increase, leading also to the differential expression of
genesis (new blood vessel formation), the migration of growth factors and sustained protease activity at elevated
fibroblasts, which deposit new extracellular collagen levels (i.e., matrix metalloproteases 2, 8, and 9), thus, pro-
longing inflammation.13,14,29,60,63,66 Neutrophils also
release free oxygen radicals that induce considerable oxi-
DFU Diabetic foot ulcer dative stress, which, if not mediated, further contributes to
DM Diabetes mellitus chronic inflammation.20
DT Diphtheria toxin While most wounds are colonized by bacteria to varying
ECM Extracellular matrix degrees, the hypoxia associated with chronic wounds
EGF Epidermal growth factor
impairs leukocyte bactericidal action leading to an
FGF Fibroblast growth factor
(GAPDH) Glyceraldehyde-3-phosphate dehydrogenase
increased and persistent proliferation of bacteria, including
hAMS Human activated macrophage in
anaerobes, at the wound site.39 More polymorphonuclear
suspension leukocytes respond to the bacterial colonization, resulting
IL Interleukin in inflammation from the prolonged cytokine and protein
INF- c Interferon gamma activity and release of free oxygen radicals.
KGF Keratinocyte Inappropriate cytokine spatial distribution in the wound
LEA Lower extremity amputation may be one cause of inhibited epithelialization, with cyto-
LHALS Living human activated leukocyte kines trapped in perivascular fibrin deposits,67 but much
suspension more evidence is needed to strengthen this concept. In the
LysMCre/iDTR Lysozyme M promoter/hetero-/homozygous first comparative proteome analysis of wound exudates
for inducible diphtheria toxin receptor gene from normal wounds and VLUs, 23 proteins out of 149
MOA Mechanism of action total were found exclusively in normal wounds vs. 26 in
PDGF Platelet derived growth factor the chronic wounds, and the distribution of these wound-
PU Pressure ulcer
type specific proteins seemed to drive prognosis.68 A more
RCT Randomized controlled trial
recent proteome analysis identified 188 proteins in chronic
rhGM-CSF Recombinant human granulocyte macro-
phage colony-stimulating factor
wounds that were differentially expressed by more than
SBG Soluble b-1,3/1,6-glucan twofold and were involved in prolonged inflammation,
TGF Transforming growth factor reduced angiogenesis, and increased cell mortality.69 Addi-
Th T helper tionally, margins of chronic ulcers display high prolifera-
TLR Toll-like receptor tion of keratinocytes (demonstrable with the proliferation
TNF-a Tumor necrosis factor alpha marker Ki67), which is not expressed in epithelialization
US United States of normal wounds.70 Inhibited keratinocyte migration in
VEGF Vascular endothelial growth factor patients with DM might be due to reduced insulin-
VLU Venous leg ulcer regulated LM-3A32, the unprocessed, precursor of the
alpha3 of laminin 5 essential to migrating epithelium.70

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Table 1. The functions and roles of cytokines, chemokines, and proteases in wound healing

Inflammatory and Tissue Expressed

antimicrobial Proliferation remodeling in/induced by
Description function function function macrophages References

IL-8  Induces phagocytosis  Is involved in Yes 14,30

angiogenesis
 Stimulates keratino-
cyte migration and
proliferation
IL-23  Promotes up-regula-  Increases Yes 18
tion of MMP-9 angiogenesis
IL-1b  Proliferates cells Yes 18,19,31,32
 Differentiates cells
 Is involved in
apoptosis
IL-1RA  Inhibits IL-1a and Yes 20
IL-1b
IL-13  Mediates type-2 Yes 17–21,30,31,33,34,
cytokine response
 Inhibits inflamma-
tory cytokine
production
 Induces cell-
mediated immunity
 Stimulates baso-
phils and mast
cells to produce
Th2 cytokines
IL-17A  Recruits inflamma- Yes 35,36
tory cells
 Enhances nitric oxide
production
MIP-1a/CCL3,  Recruit leukocytes to Yes 17,30,37,38
MIP-1b/CCL4 the site of
inflammation
 Chemoattract natural
killer cells
 Coactivate macro-
phages in concert
with IFN-c
 Promote the antibody
response
 Induce superoxide
production by
neutrophils
MPP-1, 27, 29  Promote extracellular Yes 19,39
matrix breakdown
RANTES/ CCL5  Recruits leukocytes  Recruits Yes 37
into inflammatory sites macrophages

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Table 1. Continued.

Inflammatory and Tissue Expressed

antimicrobial Proliferation remodeling in/induced by
Description function function function macrophages References

 Induces proliferation
and activation of nat-
ural killer cells
 Has microbicidal
activity
GRO-a  Induces exocytosis  Stimulates prolifera- Yes 40–42
(CXCL1) and the respiratory tion of keratinocytes
burst in neutrophils and endothelial cells
 Is critical for neutro-
phil mobilization and
degranulation
IFN-c  Increases antigen Yes 17–19,33,37,43
presentation and
lysosome activity in
macrophages
 Activates inducible
nitric oxide synthase
 Promotes natural
killer cell activity
 Activates
macrophages
ANGPT1  Contributes to blood Yes 44–46
vessel maturation
and stability
 Promotes
angiogenesis
MCP-1/ CCL2  Recruits monocytes,  Accumulates Yes 47–49
memory T cells, lymphocytes
mast cells, and den-  Is involved in re-
dritic cells epithelialization
 Contributes to endo-
thelial- cell locomo-
tion during
angiogenesis
 Involved in matrix
(collage fiber)
deposition
TIMP-1,  TIMP-1 promotes  Inhibit MMP and Yes 14
TIMP-2, proliferation tissue degradation
TIMP-3,  Are involved in
extracellular matrix
remodeling
 TIMP-1 and TIMP-2
bind to active MMP-
7 to inhibit MMP-7
Endostatin No 50

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Table 1. Continued.
Inflammatory and
antimicrobial Proliferation
Description function function
 Inhibits
angiogenesis
Thrombo-spondin 1  Inhibits
angiogenesis
IP-10 (CXCL10)  Chemoattracts mono-  Promotes
cytes /macrophages, angiogenesis
T cells, natural killer
cells, and dendritic
cells
ANGPT1, angiopoietin-1; CCL, chemokine (C-C motif) ligand; GRO, growth-related oncogene; IFN-c, Interferon gamma; IL, inter-
leukin; IP, interferon-inducible protein; MCP, monocyte chemotatic protein; MIP, macrophage-inflammatory protein; MMP,
matrix metalloproteinase; RANTES, Regulated on Activation, Normal T Cell Expressed and Secreted; TIMP, tissue inhibitor of
metalloproteinase.
THE ROLE OF MACROPHAGES IN WOUND
HEALING
Macrophages are mononuclear phagocytes established
during embryogenesis and derived from the yolk sac or
the fetal liver but also recruited from the blood and
bone marrow under inflammatory conditions (such as
the tissue repair that is part of wound healing).18,71
Most importantly, they take on distinct phenotypes and
functions crucial to healing.18 Macrophages are
involved in host defense, the initiation and resolution
of inflammation, growth factor production, phagocyto-
sis, cellular proliferation, and tissue restoration in
wounds.
During inflammation, macrophages are recruited to the
wound site, where they display impressive plasticity, as
they express a polarization of classic and alternative acti-
vation phenotypes, which are mediated by cytokines, oxi-
dants, lipids, and growth factors released by the same
macrophages.17,18,21 These cells can modulate their
responses to the changing wound environment, and they
participate in multiple, overlapping healing phases. They
are especially effective in neutrophil removal from the
wound.20 As macrophages ingest neutrophils, they take on
anti-inflammatory phenotypes, which are actively modu-
lated by mediators released by the neutrophils.17 During
remodeling, macrophages either die or migrate from the
wound to draining lymph nodes, contributing to matrix
remodeling and resolved fibrosis. They might provide bal-
anced proangiogenic and antiangiogenic signals in wounds,
thus regulating the early angiogenic response during tissue
granulation and scar resolution.17
Recent scientific opinion, drawing mainly from murine
models, generally agrees that macrophages possess
dynamic plasticity that influence the entire wound healing
process, displaying different phenotypes with different
functions and capacities that are associated with both clas-
sical and alternative activation and change during the heal-
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Review of macrophages in wound healing
Tissue Expressed
remodeling in/induced by
function macrophages References
Yes 20
 Accumulates Yes 20
lymphocytes
ing process.17–20,31,72–75 Impairment of the macrophage
function can lead to delayed healing.18
Macrophage classification
The classically activated macrophage polarizes to its cyto-
toxic, microbicidal phenotype after stimulation with
interferon-c (INF-c) and lipopolysaccharide (LPS). The
alternatively activated macrophage is present with parasitic
infections, allergic reactions, and reduced interleukin
(IL)24 and IL-3. The traditionally broad classifications of
M1 for classically activated macrophages and M2 for alter-
natively activated macrophages were chosen because they
respectively promote T helper (Th) 1 and Th2 responses,
secretion of IFN-c and IL-4, for example, which then
down-regulate M1 and M2 activity in turn33,43 (Figure 1).
The antimicrobial and antitumoral M1 phenotype is acti-
vated upon wound formation by inflammatory signals from
IFN-c and TFN-a, or when pathogen-associated molecular
patterns or endogenous danger signals, such as heat shock
proteins, are recognized.18,19,21,31,76 These macrophages
release IL-12 and promote strong pro-inflammatory Th1
immune responses early on in the healing process, with
host defense being their main role.34
Conversely, the M2 phenotype, which can be formed in
response to IL-4 and IL-13,77 down-regulates inflammation
and initiates tissue repair by releasing anti-inflammatory
cytokines, such as IL-10, which polarize the macrophages
to the alternative activation anti-inflammatory state that
promotes tissue repair.18,19,21,31,33,34 As apoptosis occurs
among neutrophils in the wound, they release cytokines
that promote macrophage recruitment.17 These macro-
phages ingest the apoptotic neutrophils and synthesize
mediators critical to remodeling and angiogenesis, includ-
ing TGF-b, VEGF, and EGF.16 Bioactive lipids (15d-
PGJ2), lipoxins, and resolvin help mediate M2 activity.18
Thus, M2 macrophages are present later in the healing pro-
cess with granulation tissue formation functions.34
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Table 2. The functions and roles of growth factors expressed in or induced by macrophages during wound healing

Inflammatory and Tissue

antimicrobial Proliferation remodeling
Description function function function References

PDFG-AA  Effect activity of matrix  Promote angiogenesis  Are involved in 14,19,20

PDFG-BB metalloproteinases  Accelerate extracellular remodeling
matrix and collagen
formation
 Activate and proliferate
fibroblasts
 Recruit bone marrow
derived endothelial pro-
genitor cells
TFG-b  Recruits macrophages  Promotes angiogenesis  Contributes to re- 13,14,17,18
and induces their  Is chemotactic for epithelialization and
secretion of cytokines fibroblast remodeling
 Promotes proliferation
 Is involved in matrix
(collagen fiber) deposi-
tion
 Is involved in granula-
tion tissue formation
GM-CSF  Stimulates leukocyte  Influences fibroplasia  Is involved in re- 19
production epithelialization
 Enhances microbicidal
activity, oxidative
metabolism, and neu-
trophil and macrophage
phagocytic activity
 Improves cytotoxicity
of neutrophils and
macrophages
 Stimulates in granulo-
cytes the release of
arachidonic acid metab-
olites and increased
generation of ROS
BDNF  Is a factor of 18
angiogenesis
 Promotes neuron
survival
 Encourages the growth
and differentiation of
new neurons
 Is an angiogenic factor
 Is a factor of neuronal
neovascularization
 Is a neutrophic factor
 Induces endothelial
progenitor recruitment

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Table 2. Continued.

Inflammatory and Tissue

antimicrobial Proliferation remodeling
Description function function function References

and postnasal
vasculogenesis
 Is a factor of paracrine
survival
VEGF-D  Promotes angiogenesis 14,17–20,31
 Promotes
lymphangiogenesis
 Maintains differenti-
ated lymphatic
endothelium
 Promotes endothelial
cellular growth
HGF  Proliferates epithelial 51–53
and endothelial cells
 Is mitogenic for endo-
thelial and epithelial
cells, melanocytes,
and keratinocytes
PIGF-1  Promotes angiogenesis 54,55
 Promotes
vasculogenesis
 Is involved in monocyte
active migration and pro-
duction of inflammatory
cytokines and VEGF
EGF  Stimulates epithelial  Is involved in re- 14,18
cell growth, prolifera- epithelialization
tion, and differentiation
 Is mitogenic for kerati-
nocytes and fibroblasts
 Is involved in keratino-
cyte migration
 Forms granulation
tissue
FGF-2  Is involved in fibroblast  Is involved in wound 14,30
chemotaxis contraction
 Proliferates fibroblasts  Is involved in re-
and keratinocytes epithelialization
 Is involved in keratino-
cyte migration
 Promotes angiogenesis
 Is involved in matrix
(collagen fibers)
deposition
SDF-1a (CXCL12)  Promotes angiogenesis  Is involved in re- 56
epithelialiazation

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Table 2. Continued.

Inflammatory and Tissue

antimicrobial Proliferation remodeling
Description function function function References

NGF  Regenerates injured  Promotes re- 57

neurons epithelialization
 Induces fibroblast
migration
 Is involved in matrix
(collage fibers)
deposition
 Promotes angiogenesis
SCF  Promotes hematopoi- 58
etic progenitors’ prolif-
eration, migration,
survival, and
differentiation

bNGF, beta nerve growth factor; BDNF, brain-derived neurotrophic factor; EGF, epidermal growth factor; FGF, fibroblast growth
factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HGF, hepatocyte growth factor; PDGF, platelet-derived
growth factor; PIGF, placental growth factor; ROS, reactive oxygen species; SDF, stromal cell-derived factor; SCF, stem cell fac-
tor; TGF-b, tumor growth factor-beta; VEGF, vascular endothelial growth factor.

The polarization of the M2-like phenotype is key to pre-
venting the delayed healing that results from chronic
inflammation. Diabetic wounds in both humans and mice
were found to have macrophages with an inflammatory
phenotype that secreted IL-1b, which was discovered for
the first time to be part of a pro-inflammatory positive
Figure 1. Simplified characterization of M1 and M2 macro-
phage phenotypes. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
feedback loop that blocked the activation of the M2 phe-
notype.32 By introducing a neutralizing antibody to inhibit
IL-1b in vivo, IL-1b expression was down-regulated and
wound healing growth factors were up-regulated, switching
the macrophages to the M2 phenotype with concurrent
resumption of the healing process.32 It has also been found
that IL-6 programs macrophages for IL-4-dependent M2
polarization by inducing IL-4 receptor expression.78 In
mice with conditional inactivation of the Il6ra-gene in
myeloid cells, there was a tendency to develop obesity-
induced inflammation and glucose intolerance as well as
an exaggerated response to LPS-induced endotoxemia.
This was an unexpected beneficial result as IL-6 is gener-
ally regarding as pro-inflammatory. The data suggest that
further exploration of IL-6 signaling in macrophages in
regard to wound healing be undertaken.
Consensus regarding macrophages markers has not yet
been reached, and knowledge of their functions and impli-
cation on wound healing remains imperfect.20 Generally,
M1 markers include tumor necrosis factor alpha (TNF-a),
IL-12, and IL-6 and are expressed during early inflamma-
tion, while M2 markers, including CD206, arginase 1,
Dectin 1, IL-10, IL-4RA, and TGF-b1 are more highly
expressed during late inflammation and granulation.75,79
Ironically, macrophage cytokines can still promote M2
polarization, which challenges the M1/M2 activation para-
digm.80,81 In both human and murine macrophages, IL-6
enhanced M2 polarization with IL-4 and IL-13, based on
the expression of arginase-1, Ym1 (chitinase 3-like 3), and
CD206.80 The macrophage-identifying role of markers was
also different in a murine wound model that lacked IL-4
and yet still highly expressed Ym1, a marker of IL-4-
induced M2 macrophages.81 Thus, the expression of Ym1

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in situ might not necessarily indicate an M2-like wound
healing macrophage, indicating that macrophage polariza-
tion and activation is much more dynamic and complex
than previously thought.
Macrophage phenotype markers: a conundrum
between in vitro and in vivo models
It has been challenging to pinpoint macrophage phenotype
markers, because in vivo phenotypes are expressed differ-
ently than in vitro phenotypes, as the former exhibit classi-
cally and alternatively activated markers.20,31 This is
compounded by the scarcity of in vitro studies. Combina-
torial gene expression profiles can clearly identify murine
macrophage markers, but these are different in humans
and remain a debatable issue over whether they can even
be identified due to complexity of many stimuli pres-
ent.82,83 For example, the resistin-like molecule alpha is
highly induced by IL-4 and IL-13 in mice only. Chitinase-
3-like-12 is not expressed in mice, while Chitinase-3-like-
1 is not expressed in humans. Neither nitric oxide synthase
2 nor Arginase 1 are readily and/or strongly expressed in
humans.82
There are also conflicting opinions and evidence on the
further classification of macrophages17 beyond the classi-
cally and alternatively activated phenotypes, as these 2
polarized states are increasingly thought to oversimplify
the cellular processes.18,80,81 It is now accepted that multi-
ple discrete and hybrid macrophages can be localized
simultaneously in the wound.20,21,31 Wound macrophages
have hybrid M1/M2 activation phenotypes that enable their
plasticity and ability to switch between functions during
the healing process.34,75,79 In intestinal wounds of mice,
both undifferentiated macrophages and M2 macrophages
contributed to tissue repair, supporting the new view that
M2 phenotypes may not be the sole macrophage popula-
tion responsible for tissue repair.84 New subset macro-
phage classifications have been utilized based on the
versatile role of macrophages in the healing process and
include: 1 classically activated macrophage (M1) observed
after stimulation with LPS or IFN-c, and 3 M2 subsets
(M2a, M2b, and M2c), also known as wound-healing mac-
rophages, which produce high levels of IL-10 and low lev-
els of IL-12. M2a occurs after stimulation with IL-4 or IL-
13 while M2b is dependent on the immune complex, IL-
1B, and LPS. The third regulatory macrophage population,
M2c, is dependent on IL-10, TGF-b, or glucocorticoid
stimulation.85–87 It has recently been found that A2A recep-
tor stimulation by adenosine in the presence of toll-like
receptor (TLR) agonists transforms the M1 phenotype into
an angiogenic M2-like phenotype.88,89 Thus, a fourth, dis-
tinct M2 subset has also been proposed, M2d, as a subtype
of M2-like macrophages. Finally, another
“noninflammatory” macrophage subset was identified from
wound granulation tissue—a Ly6c(lo)MHCII(hi) that
increased in proportion as well as absolute numbers and
was absent in Ob/Ob and MYD88–/– models of delayed
healing.35 IL-17 was the principal cytokine distinguishing
this population from pro-inflammatory macrophages with
inhibition or use of IL-17A-/- mice accelerating normal
and delayed healing.
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Review of macrophages in wound healing
Macrophage phenotypes: a potential paradigm shift
Another wound-healing macrophage classification of 4
phenotypes has been suggested using the macrophage phe-
notypes proposed for tissue repair.19,34 These are pro-
inflammatory–like macrophages present during early
inflammation; anti-inflammatory M2-like macrophages
during late inflammation that synthesize mediators to
reduce inflammation (IL-10) and promote tissue repair
(TGF-b1); profibrotic M2-like macrophages present during
tissue formation that produce growth factors and ECM;
and fibrolytic M2-like macrophages in the ischemic scar
milieu that secrete proteases and promote tissue remodel-
ing.19 These new macrophage subpopulation classifications
are still not comprehensive and inclusive of all types of
macrophages and continue to be modified and expanded
upon as more evidence is produced that further clarifies
their wound healing role. A small group of macrophage
biologists met at the International Congress of Immunol-
ogy in Milan in August 2013 and proposed a set of stand-
ards for the field encompassing three principles: the source
of macrophages, definition of the activators, and a consen-
sus collection of markers to describe macrophage activa-
tion, with the goal of unifying experimental standards for
diverse experimental scenarios.90 The classifications are
complicated and based on a more comprehensive set of
markers than we listed in Figure 1. However, Martinez
and Gordon91 suggest that perhaps a classification system
for macrophages is not viable, as hybrid cells respond to
interacting pathways in the tissue that form and change
their phenotypes. Whether more complicated schemes
based on markers will be accepted in the research commu-
nity as a means of further differentiating macrophage types
will perhaps depend on their utility.
Early stage macrophages are critical for wound
healing
Although the macrophage wound-healing role remains to
be completely elucidated,17–21 the recent use of genetically
modified mice has allowed for a selective and specific
depletion of wound macrophages, advancing the functional
understanding of these cells and emphasizing their critical
role in the beginning and middle of the healing pro-
cess.20,72,73,88,92,93 Nevertheless, as in all animal studies,
translation of findings to humans must be regarded as
tentative.
When transgenic mice expressing the human diphtheria
toxin (DT) receptor under the control of the CD11b pro-
moter were treated with DT, macrophage recruitment to
wounds was delayed, while neutrophils and monocytes still
infiltrated the wound bed, resulting in stalled healing with
increased levels of TNF-a and reduced TGF-b1 and
VEGF.73 Another murine wound model used myeloid cell-
specific lysozyme M promoter/hetero-/homozygous for
inducible DT receptor gene (LysMCre/iDTR) mice with
macrophage-depletion and controls (LysMCre mice) study-
ing different stages of the healing process.72 Both groups
of mice received DT. In the inflammatory phase, depletion
of macrophages reduced granulation and epithelialization,
whereas in the proliferation phase of wound healing there
was severe hemorrhage in the wound tissue and no pro-
gression to the maturation phase. Five days postinjury, the
621
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Table 3. Summarized results of preclinical and clinical studies in animals and humans using macrophage-based therapies or
therapies designed to manipulates macrophages
Benefit (References)
Therapy Animals
Activated monocytes/ TNF-a/IFN-c: yes101
macrophages (diabetic IL-4/IL-10: no102
wounds)
Embryonic stem cell-derived No114
M2-like macrophages80
Human activated macrophage Diabetic wounds: yes103
in suspension (hAMS)
GM-CSF Numerous—not cited
Glycans Aminated beta-1,3-D-glucan: yes102
Rapid recruitment and activa- Excisional wounds: yes109
tion of macrophages genes Burns: yes110
encoding for cytokines (a-gal
liposome/a-gal nanoparticle
treatments)
wound area was reduced by 25% in the LysMCre/iDTR
mice vs. a 50% reduction in the controls. Complete epithe-
lialization was observed in both groups after 10 days, but
LysMCre/iDTR mice displayed a thinner neoepithelium
that was partially detached from the dermis. After 14 days,
there was almost no indication of scar formation in the
LysMCre/iDTR mice. Macrophages recruited early on dur-
ing inflammation impacted all healing phases in mice, con-
trolling both vascularization and wound contraction and
alternatively activated macrophages were present, as indi-
cated by the expression of Fizz1 and Yml, during early
healing stages and to a lesser extent mid-stage. However,
it appears that macrophages present at the late stage of
repair do not impact tissue maturation or scar formation.72
In another in vivo murine wound model,92 the regulatory
role of macrophages in the production of collagen and
control of fibrosis was confirmed during the early healing
phases, as evidenced by the significantly reduced collagen
1a2 transcription from D4 as a result of macrophage
depletion.
Finally, gene profiling analysis with regard to macro-
phages conducted by Nassiri et al.94 in patients with
chronic diabetic foot ulcers showed promise as a diagnos-
tic aid. A score was defined as the ratio of the sum of the
raw values of M1 gene expression relative to the house-
keeping gene glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) to the sum of the raw values of M2 gene
expression relative to GAPDH (M1/M2), such that its
value was higher for M1 macrophages and lower for M2
macrophages. Initial scores were significantly higher for
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Snyder et al.
Humans Comments
—
—
Deep sternal wounds: yes23 Multiple applications
Refractory ulcers: yes28 needed in many
PUs: yes25 instances
Burns: yes105–107 Weekly injections
Chronic leg ulcers: yes107
Leprosy ulcers: yes107
Pressure ulcers: yes104
Diabetic ulcers: yes104
b-1,3/1,6-glucan: yes99 Multiweek injections
over several weeks
—
wounds that healed with the score decreasing for healing
ulcers and increasing for nonhealing ulcers. At 4 weeks
following standard of care the scores were 90 times higher
for nonhealing wounds compared to healing wounds.
Thus, this evidence supports that early recruitment of
macrophages to murine wounds is necessary for the normal
healing process; the limited work conducted in human dia-
betic wounds suggests that this might also be true and that
M1 gene expression is also critical if the wound is to heal.
Monocytes as potential therapeutic targets
Although most research has been devoted to macrophage
differentiation and manipulation, the recruitment of mono-
cyte subpopulations into a site of tissue injury is a subject
that should not be overlooked. For example, evidence from
a recent murine study suggests that monocytes are
recruited early concomitantly with neutrophils to the
wound bed.94 In terms of therapeutic potential, Majmudar
et al.95 also developed a method of silencing CCR2, the
chemokine receptor that governs inflammatory Ly-6C1
monocyte subset traffic, to reduce infarct inflammation in
apoE2/2 mice postmyocardial infarction. Results showed
that monocyte numbers were reduced with concurrent
improved resolution of inflammation in the infarcted mice
hearts. Other researchers have demonstrated that CCR2
signaling is essential to recruiting Ly6C1 inflammatory
circulating blood monocytes into skin wounds, which give
rise to a highly proangiogenic, VEGF-expressing macro-
phage during the early stage of the repair response.96
Wound Rep Reg (2016) 24 613–629 V
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Snyder et al.
Furthermore, CCR2 gene deletion in myeloid cells attenu-
ated angiogenesis in skin wounds. Another key point is
that systemic chronic inflammation, often present in under-
lying conditions associated with some chronic wounds,
induces intrinsic defects in monocytes via chromatin modi-
fications that may induce monocytes to become a pro-
inflammatory phenotype, whereas in the local wound envi-
ronment inhibition of differentiation leads to a pro-healing
phenotype.97 While monocytes certainly are a potential
therapeutic target, better animal wound models incorporat-
ing methods to manipulate the differentiation of monocytes
or CCR2 signaling in such a manner as to improve angio-
genesis are needed before clinical applications can be
contemplated.
THE USE OF MACROPHAGES IN CLINICAL
PRACTICE
Macrophage-based therapy
Wound therapy research increasingly looks toward molecu-
lar, cell-based therapy to induce cell recruitment and tran-
sition the healing process from inflammation to tissue
repair.98 Macrophage wound therapy, via in situ activation,
recruitment, or application of exogenous macrophages to
wounds, has been explored for its use in chronic wounds
through stimulation of proliferation and angiogenesis,
influence on protease imbalances, and increased phagocy-
tosis.20,99,100 A current trend in macrophage therapy
research shifts the focus from inhibiting macrophages to
the wound site to emphasizing controlled recruitment and
modulation.98 Recent studies focusing on macrophage ther-
apy include 1 rat study,101 1 murine model,102 1 guinea
pig model.103 and a nonparallel controlled cohort trial.25
Evidence is thus limited to support the use of macrophage
therapy in the clinical setting. Another wound therapy that
targets macrophages to make them more effective is mac-
rophage stimulation, which has stronger current evidence
supporting its use in clinical practice, including four recent
RCTs,99,104–106 1 systematic review,107 one phase 1
trial,108 and multiple animal studies and in vitro models
(Table 3).100,109–113
Macrophage therapy in preclinical studies
Murine studies101,102 have addressed the effect of macro-
phage therapy on impaired healing in diabetic wounds. Gu
et al. tested the application of activated autologous mono-
cytes/macrophages—including with TNF-a plus IFN-c
stimulation—on 60 rats in a case control study involving a
rat model of diabetes.101 The macrophage therapy (com-
posed of 4 3 106 cells) was applied via injection into four
sites of the wound bed. When TNF-a plus IFN-c-
stimulated autologous monocytes/macrophages were
administered early on to the diabetic wounds, healing was
significantly improved with the stimulation of angiogenesis
and reepithelialization, as well as the correction of the
impaired VEGF expression after 7 days.101 This study sup-
ported the early, critical role of macrophages in the wound
healing process. Both VEGF and IFG-1 levels significantly
increased after administering the TNF-a plus IFN-c stimu-
lated macrophages.
Wound Rep Reg (2016) 24 613–629 V
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Review of macrophages in wound healing
In a mouse model,102 IL-4- or IL-10-stimulated M2
macrophages or nonpolarized macrophages or saline were
injected into the wounds of C57BL/6 or diabetic mice.
After 15 days, delayed reepithelialization and persistence
of neutrophils were observed in the M2 macrophage-
treated diabetic wounds, implying that the topical applica-
tion of M2 macrophages may not be beneficial.102 A simi-
lar finding on the use of ex vivo generated M2-like
macrophages was reported by Dreymueller et al.114 It was
thought that perhaps exogenous M2 macrophages induced
the M1 macrophage in situ leading to premature anti-
inflammatory activity that then impaired the healing
process.102
The efficacy of a human activated macrophage in sus-
pension (hAMS) on resolving acute and chronic sulfur
mustard wounds was explored in a hairless guinea pig
model.103 The hAMS procedure is a wound therapy
involving the use of an allogeneic suspension of live
human macrophages prepared from healthy blood donors
in a cost-effective, closed, sterile system, which is acti-
vated by hypo-osmotic shock and then applied to the
wound by injection or direct deposition. When 1 injection
of hAMS was administered within 6 hours of wound for-
mation, there was less damage observed in the epidermis
at 1 day, but there was no further effect noted after 2–4
weeks. Repeated hAMS injections at 15 minutes, 2 days,
and 7 days, significantly decreased the wound area and
improved the wound barrier function (based on trans-
epidermal water loss) for up to 10 days (p < 0.001). In
humans, the authors previously observed that only 1
hAMS injection was needed,28 likely due to the long life-
span of human macrophages of 2–3 months, but up to
three injections were needed for deep sternal wound infec-
tions.23 The macrophages did not survive from 3 to 7 days
in the hairless guinea pig model, thus, warranting the mul-
tiple injections.103 The authors were uncertain whether
macrophage therapy should be applied early in the healing
process or during scar formation and remodeling.
Recent clinical research of macrophage therapy
A nonparallel, two-arm, open controlled trial compared the
effect of hAMs injections with standard of care for 216
stage III and IV PUs in 100 elderly patients at a
university-affiliated tertiary hospital.25 There were 66 sub-
jects (66%) allocated to the hAMS group and 38 subjects
(38%) allocated to receive standard of care only. The
hAMS were injected at the wound bed (0.1 mL/injection)
by a sterile disposable 2 mL syringe with a 25G needle,
1 cm between injection points, with repeated injections
given typically in 4-week intervals, according to the condi-
tion of each wound. If the wound was too deep or if injec-
tion was not possible, the hAMS was poured directly on
the wound. Wound closure rates were significantly better
in the hAMS group for all subjects, in subjects with DM,
in subjects with leg ulcers, and in wounds with a baseline
area 15 cm2 (p < 0.001). There were no adverse or seri-
ous adverse events related to hAMS, thereby supporting
the safety of this treatment.25 The hAMS were both safe
and effective on PUs, although its effect on larger wounds
remains to be explored.
623
Review of macrophages in wound healing
Figure 2. Example of wounds treated with sham control (A) and living human activated leukocyte suspension (B) in an athy-
mic rat at day 14 posttreatment (trichrome staining). Collagen density is identified with blue staining. Collagen organization is
identified by yellow arrows. Image is at 203 magnification. Data are unpublished. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
Macrophage stimulation
Granulocyte macrophage colony-stimulating factor (GM-
CSF) stimulates the production of granulocytes and
monocytes by stem cells in which the monocytes become
macrophages upon localization in tissue.104–107 The use
of recombinant human GM-CSF (rhGM-CSF) in the
treatment of chronic wounds has been investigated in
eight randomized controlled trials (RCTs) and 23 clinical
studies and case reports.107 A systematic review con-
ducted by Hu et al.107 concluded that topically applied
rhGM-CSF had a positive therapeutic effect on deep
partial-thickness burn wounds, chronic leg ulcers, and
leprosy ulcers, and a possible beneficial effect on PUs
and cancer-related ulcers. The three most recent RCTs
support the use of rhGM-CSF on PUs, varicose ulcers,
DFUs, and burns.104–106 A single center, three-arm,
randomized Chinese study of 60 patients with PUs, vari-
cose ulcers, and DFUs found that the application of algi-
nate dressing with rhGM-CSF resulted in a significantly
faster healing rate and lower pain score (p < 0.01) com-
pared to patients treated with rhGM-CSF alone or a con-
ventional dressing.104 However, analysis was limited to a
small sample size. The application of an rhGM-CSF
hydrogel to deep second-degree burns was found to sig-
nificantly improve healing (p < 0.01), without any side
effects in a multicenter, randomized, double-blind, and
placebo-controlled clinical trial of 90 patients.105
Another RCT had similar beneficial and safe results
when rhGM-CSF hydrogel was compared to a placebo in
93 deep partial thickness wounds.106
After early work in mice with potent immunomodulator
b-glucans115 and obtaining good outcomes with its use on
surgical wounds in diabetic mice,100 a randomized, double
blind, two-center, placebo-controlled study investigated the
safety and efficacy of soluble b-1,3/1,6-glucan (SBG) in
DFUs in 60 patients, 54 of whom completed the study.99
The subjects were given SBG or methylcellulose, which
were applied topically to their wounds three times weekly
up to 12 weeks, in addition to standard of care. At 12
weeks, more SBC-treated DFUs were healed compared to
the placebo (59 vs. 37%), with a significantly higher heal-
ing rate at week 8 (44 vs. 17%, p 5 0.03). The serious
adverse event incidence was also significantly lower
624
Snyder et al.
among the SBG group. The authors concluded that the use
of SBC on DFUs was safe and effective.99
There is also the potential to use other macrophage-
stimulating therapies, such as a-gal liposome and a-gal
nanoparticle treatments, which were found to enhance the
healing of acute burn wounds in mice109,110 by activating
the genes of wound macrophages that encode pro-healing
cytokines.104,109 Anecdotally, intralesional injections may
avoid the aberrant wound bed environment and therefore
facilitate the healing of chronic wounds.
The potential benefits and flaws of macrophage
therapy in current research and their implications for
the future
The intralesional injection application method is a poten-
tial benefit of macrophage-related therapy because of
potential cost and time savings. As demonstrated in the
hAMS studies,25,103 injections were only required every 4
weeks, which translates to less cost and time. Cost-
effectiveness research is still needed for macrophage ther-
apy25 to better understand the feasibility and practicality of
this therapeutic option in the clinical setting.
Cracking the environmental signaling mechanisms that
direct the activation of different macrophages at the wound
site and understanding better the divergent outcomes of
each phenotype will be essential to enhancing potential
macrophage therapies.98,101 For example, is a phenotype
transformed and activated from environmental in situ
response, or as a result of predisposed monocyte popula-
tions that are recruited to the wound?20
Recent studies have shown that the timing of macro-
phage polarization is necessary for good healing, but it
remains unclear if this finding is applicable to the timing
of application of macrophage therapy.103
The potential beneficial use of biomaterials and their
effect on wound macrophages in drug delivery and tissue
engineering merits further investigation.116 For example,
there is little evidence that explores that effect of polariza-
tion after synthetic or naturally derived biomaterials are
implanted in the wound.98
Another complication that inhibits the development of
future macrophage therapies is that most in vitro studies
do not include cells representative of different wound
Wound Rep Reg (2016) 24 613–629 V
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Snyder et al.
locations.20 Similarly, human macrophages, which differ
from those of mice and rats, are studied much less fre-
quently.17 Thus, it should be reiterated that this findings in
macrophage murine research might have limited implica-
tions in clinical practice. However, a recent, unpublished
mechanism of action (MOA) study evaluated the wound
healing effects of living human activated leukocyte suspen-
sion (LHALS) in an athymic rat splinted full-thickness
dermal wound model. The study evaluated the effects of
LHALS treatment on wound closure, reepithelialization,
tissue regeneration, modeling, and maturation. In Figure 2
(unpublished data), examples of a sham control wound site
(Panel A) and a LHALS-treated wound site (Panel B) are
shown with trichrome staining. LHALS-treated wound
sites contained thick and dense layer of collagen as com-
pared to sham control wound sites. Additionally, LHALS-
treated wound sites contained organized collagen bundles
indicated by yellow arrows in Figure 1 compared to sham
wound sites that contained loosely organized thin collagen
fibers. In addition to collagen density, the study also
reported positive results for angiogenesis, blood vessel
maturation, and granulation tissue thickness. The results
from the MOA study demonstrated that injected macro-
phages “mimic” normal wound healing physiology.
LHALS is currently under evaluation in phase 3, multina-
tional, randomized, controlled, double-blind clinical trials
for the treatment of DFUs and VLUs. The findings to date
on LHALS support activated macrophage treatment as a
promising wound care therapy.
The transition of macrophage therapy research to the
widespread, clinical use of macrophages in wound care
will require both a more complete understanding of the
role of macrophages in wound healing and strong evidence
on the safety and efficacy of their application on wounds.
Advanced technology, including laser capture micro-
dissection followed by genomic and proteomic analysis of
human macrophages, or alternative humanized mice mod-
els enabling the investigation of human wound macro-
phages should provide better understanding of the
molecular processes of human macrophages in the
wound,17 which in turn would enhance macrophage ther-
apy. The limited, recent evidence on the use of macro-
phages in wound therapy shows them to be safe and
effective. Larger and more robust RCTs evaluating the use
of macrophage therapy on VLUs and DFUs are currently
ongoing.25
DISCUSSION
The role of the macrophage in wound healing remains
enigmatic, and yet, it is understood that macrophages are
involved in all healing stages. The persistence of classi-
cally activated macrophages and the delayed activation/
recruitment of the alternative activating macrophages can
lead to chronic wounds.20,72,73,88,92,93 The key to unlocking
the mystery is in the regulatory signaling between these
cells and the cytokines, chemokines, proteases, and growth
factors that they secrete during the healing process.13,14
Simultaneous, multiple discrete and hybrid wound macro-
phages can, in fact, switch functions at the same time dur-
ing the healing process.20,21,31,34,75,79 Thus, the traditional
M1/M2 activation paradigm is too simple and does not
explain the plasticity nor comprehensively include many
Wound Rep Reg (2016) 24 613–629 V
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Review of macrophages in wound healing
phenotypes expressed by wound macrophages.18,80,81,91
However, a comprehensive and complete classification is
lacking and may not be possible or applicable due to the
difficulty in identifying distinct molecular markers for
each phenotype and the dynamic signaling pathways
wound macrophages respond to that seem to drive each
phenotypical function.105 The lack of in vitro studies and
the differences between murine and human macrophages
and the expression of in vivo and in vitro macrophage phe-
notypes complicate the identification of these
markers.20,31,82,83
Research in macrophage therapy has shifted from mac-
rophage inhibition and depletion to the promotion of con-
trolled recruitment and modulation.98 Recent evidence
supports the idea that early recruitment of macrophages is
critical to wound healing, but it remains unclear if this
finding translates to the need for early application of mac-
rophage therapy on patients with chronic
wounds.20,72,73,88,92,93,101,103
Unfortunately, the mechanism of potential action of acti-
vated macrophage has fallen short when being translated
into modifying patient wound outcomes. Recently, two
large (pivotal) phase III trials—one in diabetic foot ulcer
and one in venous leg ulcer; using locally injected macro-
phage therapy; have failed to meet their primary endpoints
versus standard of care in those wound types.
Further elucidation around mechanism of action, dosing
strategy and route of dose is still necessary to allow for
the outlined properties to be uniformly deliverable. It
would appear that a better understanding of the signaling
processes of the many macrophage phenotypes, as well as
their roles and outcomes in wound healing, could then
more specifically guide the development and eventual clin-
ical use of macrophage therapies.
Acknowledgments
The authors would like to thank Kristen Eckert (Strategic
Solutions) for her help in literature research and assistance
with drafting the manuscript.
Sources of Funding: The writing of this manuscript was
partly funded by Macrocure.
Conflict(s) of Interest: Snyder: consultant to Macrocure;
Lantis: consultant to Macrocure; Kirsner: consultant to
Macrocure; Shah: employee of Macrocure with stock in
Macrocure; Molyneaux: employee of Macrocure with stock
in Macrocure; Carter: consultant to Macrocure.
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