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Macrophage-Mediated Inflammation in Skin Wound Healing

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Abstract and Figures

Macrophages are key immune cells that respond to infections, and modulate pathophysiological conditions such as wound healing. By possessing phagocytic activities and through the secretion of cytokines and growth factors, macrophages are pivotal orchestrators of inflammation, fibrosis, and wound repair. Macrophages orchestrate the process of wound healing through the transitioning from predominantly pro-inflammatory (M1-like phenotypes), which present early post-injury, to anti-inflammatory (M2-like phenotypes), which appear later to modulate skin repair and wound closure. In this review, different cellular and molecular aspects of macrophage-mediated skin wound healing are discussed, alongside important aspects such as macrophage subtypes, metabolism, plasticity, and epigenetics. We also highlight previous studies demonstrating interactions between macrophages and these factors for optimal wound healing. Understanding and harnessing the activity and capability of macrophages may help to advance new approaches for improving healing of the skin.
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Citation: Hassanshahi, A.; Moradzad,
M.; Ghalamkari, S.; Fadaei, M.;
Cowin, A.J.; Hassanshahi, M.
Macrophage-Mediated Inflammation
in Skin Wound Healing. Cells 2022,
11, 2953. https://doi.org/10.3390/
cells11192953
Academic Editor: Alexander V.
Ljubimov
Received: 16 August 2022
Accepted: 16 September 2022
Published: 21 September 2022
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cells
Review
Macrophage-Mediated Inflammation in Skin Wound Healing
Alireza Hassanshahi 1, Mohammad Moradzad 2, Saman Ghalamkari 3, Moosa Fadaei 3, Allison J. Cowin 1, *
and Mohammadhossein Hassanshahi 4, *
1Regenerative Medicine, Future Industries Institute, University of South Australia, Adelaide, SA 5095, Australia
2Department of Clinical Biochemistry, Faculty of Medicine, Kurdistan University of Medical Sciences,
Sanandaj 66179-13446, Iran
3Department of Biology, Islamic Azad University, Arsanjan 61349-37333, Iran
4Vascular Research Centre, Adelaide Medical School, Faculty of Health and Medical Sciences,
University of Adelaide, North Terrace, Adelaide, SA 5005, Australia
*Correspondence: allison.cowin@unisa.edu.au (A.J.C.); mohammadhossein.hassanshahi@adelaide.edu.au (M.H.)
Abstract:
Macrophages are key immune cells that respond to infections, and modulate pathophysiolog-
ical conditions such as wound healing. By possessing phagocytic activities and through the secretion
of cytokines and growth factors, macrophages are pivotal orchestrators of inflammation, fibrosis,
and wound repair. Macrophages orchestrate the process of wound healing through the transitioning
from predominantly pro-inflammatory (M1-like phenotypes), which present early post-injury, to anti-
inflammatory (M2-like phenotypes), which appear later to modulate skin repair and wound closure.
In this review, different cellular and molecular aspects of macrophage-mediated skin wound healing
are discussed, alongside important aspects such as macrophage subtypes, metabolism, plasticity, and
epigenetics. We also highlight previous studies demonstrating interactions between macrophages and
these factors for optimal wound healing. Understanding and harnessing the activity and capability of
macrophages may help to advance new approaches for improving healing of the skin.
Keywords: macrophages; inflammation; wound healing
1. Introduction
Wound healing is a complex but finely-tuned process, which initiates immediately
following injury and can continue for many months or years following wound closure. It
is a multi-step process that requires coordination of four distinct but overlapping physio-
logical stages which include hemostasis, inflammation, proliferation, and remodeling [
1
].
Wounds are divided into two categories of acute and chronic wounds. Acute wounds heal
at a predictable and expected rate of healing, while wounds that fail to heal within 6 weeks
and exhibit inefficient cellular and molecular functions are termed chronic wounds, which
may lead to limb amputation if left without proper treatment [
2
4
]. It has been suggested
that about 188 proteins are expressed more than twofold in chronic wounds, which may
cause chronic inflammation, impaired angiogenesis, and dampened cell survival [5].
The inflammatory response is known as the first of several overlapping stages that
constitute wound healing [
6
]. Inflammation has been reported to delay wound healing and
cause increased scarring [
6
,
7
]. Macrophages, which constitute an important immunomodu-
latory cell type, play a key role in regulating inflammation and wound healing. They play
important roles in protecting the host through multiple mechanisms such as phagocytosis,
inflammation initiation and resolution, and growth factor secretion for cell proliferation
and tissue recovery in wounds [
8
]. Macrophages release growth factors such as epidermal
growth factor, keratinocyte growth factor, and tumor growth factor-
α
(TGF-
α
) to stimulate
fibroblast and keratinocyte proliferation and production of collagen and extracellular matrix
(ECM) proteins, leading to wound granulation and re-epithelialization [
8
]. In addition,
macrophages secrete autocrine pro-resolving lipid mediators (SPMs) such as omega-6 (e.g.,
Cells 2022,11, 2953. https://doi.org/10.3390/cells11192953 https://www.mdpi.com/journal/cells
Cells 2022,11, 2953 2 of 14
lipoxins) and omega-3 (e.g., resolving, protections, and maresins) [
9
]. SPMs regulate in-
flammatory responses and inflammation resolution. Interestingly, macrophages secrete
IL-10, which prevents extra invasion of macrophages [
10
]. In addition, macrophages bal-
ance proangiogenic and antiangiogenic signals in wounds to manage angiogenic response
during tissue granulation and scar resolution [
11
]. Generally, circulating monocytes mi-
grate into the wound and differentiate into macrophages; these macrophages remove dead
neutrophils through a mechanism named efferocytosis [
12
15
]. Along with monocyte-
derived macrophages, resident macrophages concurrently stimulate inflammatory reactions
by releasing hydrogen peroxide that attracts blood neutrophils and monocytes. Overall,
migrating monocytes/macrophages and tissue-resident macrophages are believed to be the
main regulatory cells that play critical roles in managing inflammation [
16
,
17
]. Therefore,
a better understanding of how macrophages function in wounds can expand our current
knowledge about macrophages’ contributions in the process of wound healing.
2. Macrophages and Inflammation in Wounds
Skin macrophages arise from the two different developmental pathways: yolk sac-
derived primitive hematopoiesis, which then develop to tissue-resident macrophages, and
macrophages that originate from definitive hematopoiesis, which arises from aorta-gonad-
mesonephros/fetal liver embryonically and bone marrow postnatally [1823]. Macrophages
possess distinct functional phenotypes as a reaction to microenvironmental stimuli and signals,
referred to as macrophage polarization [
24
]. Macrophages polarity promotes or inhibits the
inflammatory stage of wound repair [
25
,
26
].
In vitro
wound healing studies have shown
that macrophages are classically divided into two groups based on their phenotype and role:
(i) the “classically-activated” macrophages, pro-inflammatory, or “M” (CD86
+
) macrophages
that release cytokines including IL-12, IL-1
β
, IL-6, TNF
α
, and induced nitric oxide syn-
thase (iNOS), and are involved in pathogen elimination, inflammatory cytokines release,
and creating Th1-type reaction [
27
,
28
]; and (ii) the alternatively-activated” macrophage,
anti-inflammatory, or “M2” (CD206
+
) macrophages that promote angiogenesis, ECM repair,
anti-inflammatory cytokines release, and inflammation resolution [
29
] (Figure 1). When
macrophages phagocytose neutrophils, their phenotypes change from M1 to M2, a process
regulated via mediators secreted from neutrophils [
30
]. In the inflammatory stage of wound
healing, macrophages are attracted into the wound, where they present a polarity of M1 and
M2 phenotypes that are regulated through cytokines, oxidants, lipids, and growth factors
secreted by the macrophages [
11
,
31
,
32
]. Previous studies have suggested that macrophage
plasticity plays a major role in wound healing [
33
]. As discussed later, macrophage plasticity is
regulated epigenetically via histone modifications, DNA modifications, and microRNA; also,
macrophage polarization is influenced through interaction with other cells such as adipocytes,
infiltrating immune cells (polymorphonuclear neutrophils and T cells), and keratinocytes [
33
].
Previous studies have shown several medicators produced by macrophages which pos-
sess autocrine activity that can affect inflammatory response of M1 phenotype macrophages.
Production of IL-1
β
and NLR family pyrin domain containing 3 (NLRP3) in macrophages
are the main stimuli of inflammation and the M1 phenotype, leading to wound healing
dysregulation [
34
]. Inflammation caused by macrophages requires two signals: a priming
signal to transcript immature IL-1
β
[
35
] and a danger signal to produce mature IL-1
β
to
release [
34
]. Glycoprotein Nmb (GPNMB) expressed by macrophages develop polarity of
the macrophage from the M1 phenotype into the M2 [
36
]. It has been also reported that
deficiency in Notch signaling induces high expression of TNF
α
, IL6, IL12, and iNOS, and
increases inflammation through the effect on the Toll-like receptor and nuclear factor kappa
B (NF-
k
B) pathways, as seen in diabetic conditions [
37
]. Another function of macrophage
is the production of matrix metalloproteinases (MMPs), enzymes that degrade matrix
and non-matrix proteins. MMPs are considered important modulators for switching the
phenotype and function of macrophages [
38
]. For example, macrophages secrete a high
concentration of MMP-9 (gelatinase-B) after invading the wound site [
39
]. This MMP can
cleave macrophage integrin beta-2 (CD18) to switch the macrophages phenotype [40].
Cells 2022,11, 2953 3 of 14
Cells2022,11,xFORPEERREVIEW3of15
Figure1.M1andM2polarizationofmacrophages.M1macrophagesproduceproinflammatory
cytokines,mediateresistancetopathogens,andpossessstrongmicrobicidalproperties.M2macro
phages,ontheotherhand,areantiinflammatorymacrophagesthatmediateinflammationresolu
tionandcontributetowoundhealingbypromotingangiogenesis.
Previousstudieshaveshownseveralmedicatorsproducedbymacrophageswhich
possessautocrineactivitythatcanaffectinflammatoryresponseofM1phenotypemacro
phages.ProductionofIL1βandNLRfamilypyrindomaincontaining3(NLRP3)inmac
rophagesarethemainstimuliofinflammationandtheM1phenotype,leadingtowound
healingdysregulation[34].Inflammationcausedbymacrophagesrequirestwosignals:a
primingsignaltotranscriptimmatureIL1β[35]andadangersignaltoproducemature
IL1βtorelease[34].GlycoproteinNmb(GPNMB)expressedbymacrophagesdevelop
polarityofthemacrophagefromtheM1phenotypeintotheM2[36].Ithasbeenalsore
portedthatdeficiencyinNotchsignalinginduceshighexpressionofTNFα,IL6,IL12,and
iNOS,andincreasesinflammationthroughtheeffectontheTolllikereceptorandnuclear
factorkappaB(NF
k
B)pathways,asseenindiabeticconditions[37].Anotherfunctionof
macrophageistheproductionofmatrixmetalloproteinases(MMPs),enzymesthatde
gradematrixandnonmatrixproteins.MMPsareconsideredimportantmodulatorsfor
switchingthephenotypeandfunctionofmacrophages[38].Forexample,macrophages
secreteahighconcentrationofMMP9(gelatinaseB)afterinvadingthewoundsite[39].
ThisMMPcancleavemacrophageintegrinbeta2(CD18)toswitchthemacrophagesphe
notype[40].
However,invivostudiesshowthatmacrophagesrepresentdifferentfeaturesthan
justclassicM1/M2phenotypesseeninvitro[41,42].Whilemacrophagesareclassifiedus
ingthebroadF4/80andCD11bmarkers,empiricalevidencesuggeststheprevalenceof
multiplemacrophagesubtypesexpressingcombinationsofmacrophagespecificmarkers
withvariedontology.AseminalstudybyTamoutounouretal.revealedthecomplexhet
erogeneityofskinmacrophages[23].Thisstudyidentifiedapopulationofcellsinhealthy
skinofdermalCD11b
+
nonDCmacrophages,includingCCR2
andCCR2
+
cells.CCR2
macrophageswerefurtherclassifiedintoLy6C
Lo
MHCII
andLy6C
Lo
MHCII
+
subsets,
whichwerebothCD64
Hi
MerTK
+
andshowedsimilarcharacteristics,includingtranscrip
tionalprofile,havingfoamycytoplasm,andcellcyclekineticssimilartoothertissuemac
rophages[23].CCR2
+
macrophagesincludedLy6C
Hi
MHCII
,Ly6C
HitoLo
MHCII
+
,and
Ly6C
Lo
MHCII
+
subpopulations,ofwhichthelattertwoexhibitedintermediatemorphol
ogybetweenmacrophagesanddendriticcells.Thedifferentiationofthesesubtypeswas
suggestedtooccurthroughCSF1Rsignaling.Transcriptomicandfunctionalanalyses
Figure 1.
M1 and M2 polarization of macrophages. M1 macrophages produce pro-inflammatory cy-
tokines, mediate resistance to pathogens, and possess strong microbicidal properties. M2 macrophages,
on the other hand, are anti-inflammatory macrophages that mediate inflammation resolution and
contribute to wound healing by promoting angiogenesis.
However,
in vivo
studies show that macrophages represent different features than
just classic M1/M2 phenotypes seen
in vitro
[
41
,
42
]. While macrophages are classified
using the broad F4/80 and CD11b markers, empirical evidence suggests the prevalence
of multiple macrophage subtypes expressing combinations of macrophage specific mark-
ers with varied ontology. A seminal study by Tamoutounour et al. revealed the com-
plex heterogeneity of skin macrophages [
23
]. This study identified a population of cells
in healthy skin of dermal CD11b
+
non-DC macrophages, including CCR2
and CCR2
+
cells. CCR2
macrophages were further classified into Ly6C
Lo
MHCII
and Ly6C
Lo
MHCII
+
subsets, which were both CD64
Hi
MerTK
+
and showed similar characteristics, including
transcriptional profile, having foamy cytoplasm, and cell cycle kinetics similar to other tissue
macrophages [
23
]. CCR2
+
macrophages included Ly6C
Hi
MHCII
, Ly6C
Hi-to-Lo
MHCII
+
,
and Ly6C
Lo
MHCII
+
subpopulations, of which the latter two exhibited intermediate mor-
phology between macrophages and dendritic cells. The differentiation of these subtypes
was suggested to occur through CSF1R signaling. Transcriptomic and functional analy-
ses demonstrated specialization of dermal macrophages. For instance, those with high
phagocytic activity expressed the genes C4b, CD209f, Tlr5, Pdgfc, Itga9, and the scavenger
receptors Stabilin-1 and CD36. Further analyses revealed dendritic cells developed from
Flt3-dependent and CCR2-independent pathways. Ly6C
Hi
blood monocytes generated der-
mal CCR2
+
Ly6C
Hi
MHCII
, CCR2
+
Ly6C
Hi-to-Lo
MHCII
+
, and CCR2
+
Ly6C
Lo
MHCII
+
cells,
whereas CCR2
Ly6C
Lo
MHCII
and CCR2
Ly6C
Lo
MHCII
+
subpopulations consistent of
both embryonic and adult hematopoietic cells. Dermal wound macrophages actively and
constantly alter their phenotype from pro-inflammatory to reconstructive [
43
,
44
]. For exam-
ple, it has been recently shown that the level of CX3CR1 expression by macrophages play
a critical role in wound healing [
45
]. This study categorizesmacrophages into two CX3CR1
Hi
vs. CX3CR1
Med/Lo
subtypes, and suggest that a reduction of CX3CR1
Hi
macrophages in
type 2 diabetes leads to delayed wound healing [
45
]. Moreover, in
in vivo
wound heal-
ing, there is another category for macrophages, including tissue-resident macrophages vs.
monocyte-derived macrophages [
14
]. Several studies classify M2 macrophages based on
their function in the wound healing process, and sub-classify them into three macrophage
subsets: M2a, M2b, and M2c. M2a is activated upon stimulation with IL-4 or IL-13, which
subsequently results in macrophages secreting high concentrations of arginase-1, PDGF,
insulin-like growth factor-1 (IGF-1), and other cytokines [
46
]. M2a also contribute to angio-
genesis, proliferation, migration, and differentiation of fibroblasts [
47
]. M2b macrophages
modulate anti-inflammatory and pro-inflammatory functions through the secretion of pro-
inflammatory cytokines (e.g., TNF
α
, IL-6, and IL-1) and anti-inflammatory cytokines (e.g.,
Cells 2022,11, 2953 4 of 14
IL-10 and IL-12) [48,49]. Therefore, this subset of macrophages can be a status between M1
and M2a polarity [
50
]. M2c macrophages have strong anti-inflammatory activity following
stimulation with IL-10, TGF-
β
, or glucocorticoid [
51
53
]. Additionally, they contribute to
angiogenesis by stimulating high endothelial cell migration and tube establishment [
54
,
55
].
They produce MMP-9 to absorb vessel and blood-derived stem cells in injured sites [
56
],
phagocytize wound debris, and deposit ECM components [47].
An important point regarding the role of macrophages in wound healing is to know the
contribution of tissue-resident macrophages and non-bone marrow-derived macrophages
in modulating inflammation and wound healing. Currently there are insufficient studies to
investigate the extent of the contribution of both bone marrow and non-bone marrow derived
macrophages in wound healing. Previous studies have reported that chemotherapy and/or
irradiation can cause significant bone marrow damage, leading to delay in hematopoiesis
recovery and, thus, migration of monocytes into the circulation [
57
60
]. Therefore, it is
important to interrogate whether non-bone marrow derived macrophages can compensate the
delay in migration of circulating monocytes into the injury sites to regulate wound healing.
3. Macrophage Metabolism and Plasticity in Diabetic Wounds
The viability of immune cells is associated with their metabolism [
61
]. Therefore, it might
be hypothesized that macrophage metabolism is altered in diabetic wounds. Macrophages
utilize a different source of energy to produce adenosine triphosphate (ATP), and glucose has
a pivotal role in orchestrating the ATP production in macrophages [
62
]. Glucose provides
precursors for histone acetylation and methylation, which are known to be two major epige-
netic processes altering macrophage plasticity and function [
63
,
64
]. Nicotinamide adenine
dinucleotide phosphate (known as NADPH), which is important in producing reactive oxygen
species (ROS), is generated within glycolysis [
65
]. Dysregulation of glucose metabolism is
common in diabetes, resulting in changes in the number and type of macrophage-induced
cytokines such as IL-1
β
, which leads to a dampening of glycolysis in macrophages in diabetic
wounds [
66
]. Although the mechanism of compromised glycolytic capacity of macrophages is
not fully understood, it seems that monocyte-driven macrophage function originating from
bone marrow is highly affected by alterations in glucose metabolism in comparison with tissue
resident macrophages [
66
]. In addition, the alternative activation of macrophages depends
on others energy sources such as lipid synthesis and converting arginine to proline in the
proliferation and remodeling stages of wound healing [62,67].
In diabetes, macrophages are known to have a pro-inflammatory phenotype, which is
suggested to contribute to the pathogenesis of different diabetic complications [
66
]. This
could influence macrophage metabolism in diabetes as M1 and M2 macrophage phenotypes
rely on glycolysis, oxidative phosphorylation and tricarboxylic acid-dependent mitochon-
dria in order to produce ATP [
68
,
69
]. It has also been hypothesized that ROS-induced
mitochondrial damage in macrophages might prevent switching of M1 to M2 pheno-
type [
70
]. Furthermore, Zhang et al. demonstrated that in diabetes, improper functions of
macrophages depend on glucose metabolism where under high glucose-availability, over
activation of NLRP3 inflammasome is followed by increased expression of IL-1
β
, which
subsequently leads to increased induction of M1 macrophages and elevated production
of pro-inflammatory cytokines, which are detrimental for diabetic wound healing [
71
].
Membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) promotes glycolysis in
macrophages via hypoxia-inducible factor-1 (HIF-1) reported by Sakamoto et al. [
72
]. Here
it was suggested that HIF-1 regulates oxygen availability in diabetic wounds and medi-
ates macrophages metabolism [
73
,
74
]. Additionally, in diabetic wounds increasing IL-1
β
because of overexpression of TRL4 through mixed-lineage leukemia 1 (MLL1)-a histone
methyltransferase at Histone H3K4 in promotor of TLR4 could change the metabolism of
macrophage [
75
]. Although previous studies showed that glycolysis was used to produce
ATP for M1 macrophages, glycolysis in general is a core physiological process that provides
ATP for both M1/M2 macrophages [
62
]; thus, any changes in glycolysis and contributing
factors are likely to change macrophage performance, and consequently affect healing of
Cells 2022,11, 2953 5 of 14
wounds in diabetes. Taken together, as glycolysis drives the metabolism of macrophages in
diabetic and normal conditions, diabetes may cause significant alteration of macrophages’
metabolism, plausibly through alteration of glycolysis.
Compared to normal wounds, which transition from M1 (pro-inflammatory) macrophages
to M2 (pro-healing) macrophages in a fine-tuned manner, diabetic wounds display dysregu-
lated and persistent M1 macrophage polarization, resulting in prolonged inflammation and
delayed wound healing [
76
]. Although the mechanisms by which macrophage function
is altered in diabetes remain unclear, it is simplistic to consider hyperglycemia as the sole
cause of disrupting macrophage plasticity in diabetes patients. In a study by Davis et al., it
is suggested that the cyclooxygenase 2/prostaglandin E2 (COX-2/PGE2) pathway which
regulates macrophage-mediated inflammation is highly activated in human and murine
wound macrophages [
77
]. Using single-cell RNA sequencing of human wound tissue, this
study showed (COX-2/PGE2)-mediated NF
κ
B-activated inflammation of M1 macrophages.
Another study showed that monocytes are exposed to oxidative stress, which consequently
activate NF-
κ
B via Toll-like receptor 2 in diabetic wounds [
78
]. Other studies suggested
the important role of vitamin D in the downregulation of NF
κ
B downstream signaling
pathways, and showed that NF-
κ
B-activated IL-1
β
, IL-6, and TNF-
α
pro-inflammatory
cytokines were downregulated in diabetic mice [
79
]. Apart from NF
κ
B, chemokine ligand2
(CCL2), a pro inflammatory chemokine, has been shown to be an important molecule
that regulates macrophage function in diabetic wounds [
80
]. Studies have shown that
the level of CCL2 in diabetic wounds negatively correlates with prevalence of M2-like
macrophages. [
81
,
82
]. Similarly, others have reported that CCL2 is an important factor in
maintaining the presence of M1-like macrophages in wounds [83,84].
4. Factors Affecting Macrophage Activity
During wound healing, the macrophage phenotype is regulated by epigenetic mod-
ifications (e.g., histone modification and DNA modification), miRNA activities, ATP-
dependent remodeling, and cellular interactions, as addressed below and shown in Figure 2.
Cells2022,11,xFORPEERREVIEW6of15
Figure2.Differentdiseasesandpathologicalconditionssuchasdiabetesandobesity,epigenetic
elements,anddifferentcellularactivitiescaninduceinflammationbyaffectingmacrophagesfunc
tionswhichpromoteM1macrophagesactivities.
4.1.EpigeneticModifications
Epigeneticregulatorsareinvolvedintheprocessesofskinwoundhealingandare
capableofdynamicallyregulatingproliferationandmigrationofdifferentcelltypes,in
cludingkeratinocytesandendothelialcells[85].Asdiscussedhere,studieshavealsoil
lustratedthatepigeneticfactorsregulatemacrophagesbiologythroughaseriesofcom
plexmodulatorymechanismsthatupregulateordownregulategeneactivationtotransi
entlyaltercellularphenotypeandfunction.
4.1.1.HistoneModification
Twoadditionalhistonemodificationeventsthatplayasignificantroleinthepolar
izingandswitchingofmacrophagesaremethylationanddemethylation.Theseoccurvia
histonemethyltransferasesandhistonedemethylases.Histonemethylationcanactivate
orsuppresstranscriptionfactorsbasedonthepositionofthelysineandthenumberofthe
methylgroupsaddedtothelysineresidue[86,87].MLL1isonemethyltransferasere
quiredformacrophagepolarity,whichincreasesthegeneexpressionofproinflammatory
macrophagesduringtheinflammatorystageofwoundhealing.Furtherstudieshave
shownthatMLL1knockoutdelayswoundhealingandreducesproinflammatorycyto
kinesinamurinemodelofobesityandtype2diabetes[88].Twoothermainmechanisms
thatplayrolesinmacrophagepolarityduringwoundhealingincludedhistoneacetylation
anddeacetylation.Intheacetylationprocess,histoneacetyltransferasestransmitstheac
etylgroupsfromacetylCoAtothelysineresidueonthehistonetail.Thisprocessinflu
encestherelationshipbetweentheDNAandhistone,andleadstogeneexpression[89].
Ahistoneacetyltransferasecalledmalesabsentonthefirst(knownasMOF)increasesin
type2diabetescondition,andpromotesinflammatorygenes[90].Sirtuin1(knownas
SIRT1),aclassofdeacetylaseenzymes,controlsmacrophageinflammatoryreactionsby
deacetylationoftheIFNregulatoryfactor8(IRF8)[91].Furthermore,sirtuin3affects
Figure 2.
Different diseases and pathological conditions such as diabetes and obesity, epigenetic
elements, and different cellular activities can induce inflammation by affecting macrophages functions
which promote M1 macrophages activities.
Cells 2022,11, 2953 6 of 14
4.1. Epigenetic Modifications
Epigenetic regulators are involved in the processes of skin wound healing and are
capable of dynamically regulating proliferation and migration of different cell types, includ-
ing keratinocytes and endothelial cells [
85
]. As discussed here, studies have also illustrated
that epigenetic factors regulate macrophages biology through a series of complex modu-
latory mechanisms that upregulate or downregulate gene activation to transiently alter
cellular phenotype and function.
4.1.1. Histone Modification
Two additional histone modification events that play a significant role in the polariz-
ing and switching of macrophages are methylation and demethylation. These occur via
histone methyltransferases and histone demethylases. Histone methylation can activate
or suppress transcription factors based on the position of the lysine and the number of
the methyl groups added to the lysine residue [
86
,
87
]. MLL1 is one methyltransferase
required for macrophage polarity, which increases the gene expression of proinflammatory
macrophages during the inflammatory stage of wound healing. Further studies have
shown that MLL1 knockout delays wound healing and reduces proinflammatory cytokines
in a murine model of obesity and type 2 diabetes [
88
]. Two other main mechanisms that
play roles in macrophage polarity during wound healing included histone acetylation and
deacetylation. In the acetylation process, histone acetyltransferases transmits the acetyl
groups from acetyl CoA to the lysine residue on the histone tail. This process influences the
relationship between the DNA and histone, and leads to gene expression [
89
]. A histone
acetyltransferase called males absent on the first (known as MOF) increases in type 2 dia-
betes condition, and promotes inflammatory genes [
90
]. Sirtuin 1 (known as SIRT1), a class
of deacetylase enzymes, controls macrophage inflammatory reactions by deacetylation of
the IFN-regulatory factor 8 (IRF-8) [
91
]. Furthermore, sirtuin 3 affects macrophage polarity
during inflammation of the wound [
92
]. Therefore, histone modifications play a major role
in switching macrophages phenotype during wound healing.
4.1.2. DNA Methylation
DNA methylation is associated with macrophage plasticity [
93
]. DNA methylation
of the peroxisome proliferator-activated receptor (PPAR)
γ
1 promoter leads to raised num-
bers of M1 macrophages and decreased M2 macrophages in diabetes [
94
]. Yu et al. sug-
gested that PPARγ1 decreases chronic inflammation through increasing the number of M2
macrophages [
95
]. Therefore, manipulation of PPAR
γ
1 may have a significant effect in dia-
betic wound healing by transitioning M1 macrophages to M2 macrophages. Further studies
in the domain of epigenetic mechanisms have revealed that methylation of specific sites
in histones affect macrophage polarization, allowing macrophage alterations depending
on environment and tissue site [
96
]. The activation of Jumonji domain-containing protein
3 (JMJD3) as histone demethylase could act in favor of activation of both M1 and M2 pheno-
types [
97
]. Likewise, methylation of CpG islands is involved in macrophage polarization in
which the activation of both M1 and M2 phenotypes are developed [98].
DNA methylation mainly leads to the suppression of transcription factors that bind to
DNA. This process occurs via DNA methyltransferases (DNMTs) that transmit a methyl
group to the cytosine ring of DNA. DNMT1 controls macrophage phenotype towards
the M1 [
99
,
100
]. When DNMT1 is inhibited by 5-aza-2
0
-deoxycytidine, macrophages are
induced to take on a more M2 phenotypesand reduce inflammation [
100
]. Interestingly, the
level of DNMT1 increases in T2D disease in mice, potentially contributing to the prevalence
of M1 macrophages’ prolonged inflammatory state, and DNMT1 inhibition was found to
promote wound healing in the mice [
99
]. It has also been shown that DNMT3b increases in
macrophages of diet-induced obese mice. The DNMT3b also induces macrophage polarity
towards the M1 phenotype, and DNMT3b inhibition also induces macrophage polarity
towards an M2 phenotype [
101
]. Therefore, DNA methylation influences macrophage
polarity and contributes to wound repair.
Cells 2022,11, 2953 7 of 14
4.2. miRNA Regulation
Apart from methylation as major epigenetic affecting macrophage plasticity, recent studies
have illustrated that miRNAs have the potential to impact on macrophage performance in dia-
betic wounds [
102
]. In this regard, miRNA-497 has a protective role against pro-inflammatory
cytokines such as IL-1
β
, IL-6, and TNF-
α
inducing prolonged inflammation in diabetic wounds
where M1 phenotype dominates [
103
]. Moreover, miRNA155 promotes wound repair by en-
hancing M2 phenotype, and miRNA21 has been shown to have a multifunctional role in
wound healing, affecting the inflammatory and remodeling phases [104]. This may be partic-
ularly important as miRNA21 is affected by hyperglycemia, a metabolic condition affecting
healing of diabetic wounds [
105
]. In the early phase of injury miRNA21 can induce polar-
ization of M1 macrophages under hyperglycemic conditions [
106
] and drives the transition
to M2 phenotype in later stages of healing [
107
]. Overall, this suggests that hyperglycemia
has an important role in macrophages plasticity which is mediated through alterations in
epigenetic or molecular signature (e.g., miRNAs and inflammatory signals) of macrophages.
miRNAs alter macrophage polarity by affecting gene expression [
108
]. MiR-146a ex-
pression is reduced in M1 macrophages, while it increases in M2 macrophages and MiR-146a
can inhibit pro-inflammatory cytokines and exert protective effects on macrophages [
109
].
The expression of MiR-155 induces an M1 macrophage phenotype and inflammatory re-
sponse [
110
]. In diabetic wound macrophages, MiR-21 overexpression is linked with the
upregulation of pro-inflammatory genes including IL-1
α
, TNF-
α
, iNOS, IL-6 and IL-8, and
induces the polarity of macrophages towards M1 phenotype [105].
4.3. ATP-Dependent Remodelling
Recent studies have shown that nanoliposome-encapsulated-ATP can improve wound
healing [
111
113
]. This treatment affects macrophage polarization, progenitor cell recruit-
ment, leukocyte chemotaxis, increased platelet, increased monocyte activity, monocyte
differentiation to macrophages, increased macrophage proliferation, changes in RNA ex-
pression patterns, enhance collagen production by fibroblasts, and balancing between cell
proliferation and regression. All cellular processes involved in wound healing require
consuming cellular energy [
114
]. Thus, impairment in intracellular ATP can disrupt wound
healing and lead to inflammation.
4.4. Cellular Interaction
The wound microenvironment can also regulate macrophage phenotype. Phenotype
alterations of keratinocytes, adipocytes, T cells, and neutrophils have all been reported in
diabetic wounds [
115
119
], which may affect their interactions with wound macrophages.
4.4.1. Adipocytes
Dermal adipocytes can produce palmitic acid and oleic acid, as well as monocyte
chemoattractant protein-1 and TNF. These adipocyte-produced biomolecules can change
the macrophage inflammatory phenotype [
90
]. Palmitate can increase JMJD3 expression in
macrophages that leads to the induction of inflammatory genes [
90
]. A study by Shook et al.
showed that dermal adipocytes undergo lipolysis after injury, and contribute to skin
wound healing through the recruitment of macrophages to the wound. This study fur-
ther showed that adipocyte lipolysis impairment significantly compromised the number of
macrophages in a wound, resulting in delayed revascularization and re-epithelialization of
the wound bed [
120
]. Adipocyte-derived fatty acids and biomolecules can directly affect
macrophages’ functions, as macrophages express multiple fatty acid receptors and trans-
porters [
121
,
122
]. Intriguingly, previous
in vitro
studies illustrated that heat-inactivated,
adipocyte-conditioned media can enhance monocyte/macrophage migration, suggesting
that adipocyte-derived lipids may stimulate macrophage migration [
123
]. In addition,
obesity-induced changes in macrophages and adipocytes lead to chronic inflammation and
insulin resistance [
124
]. Obesity-related insulin resistance has been reported to correlate
with elevated levels of pro-inflammatory cytokines such as TNF-
α
, IL-1
β
and IL-6 [
125
].
Cells 2022,11, 2953 8 of 14
These cytokines are secreted by adipocytes due to increased release of pro-inflammatory
factors during the development of obesity. These factors include free fatty acid, triglycerides,
resistin, leptin, retinol binding protein 4, IL-6, TNF-
α
, and IL-1
β
[
125
]. These studies suggest
that adipocytes can play a significant role in macrophage-mediated skin wound healing.
4.4.2. Keratinocytes
Keratinocytes secrete different cytokines/chemokines and play an important role in
cutaneous immunity. In chronic wound inflammation, keratinocytes release cytokines and
interferons by regulating NF-
K
B, which affects immune cell inflammatory profiles [
126
]. In
addition, increased proliferation of keratinocytes in chronic wound margins is observed
compared to normal wounds [
127
]. A study by Villarreal-Ponce et al. showed that Ccl2
release by keratinocytes prompts macrophage trafficking and production of epidermal
growth factor by macrophages in the wound [
128
]. Interestingly, macrophage-released epi-
dermal growth factor stimulates keratinocytes proliferation. In a similar study, Zhou et al.
showed exosome-mediated crosstalk between keratinocytes and macrophages in cuta-
neous wound healing. This study found that exosomes released by keratinocytes affected
macrophage plasticity with pro-inflammatory macrophages exhibiting an M1 phenotype
of pro-inflammatory resolution macrophages [
129
].
In vivo
inhibition of keratinocyte-
released exosomes resulted in a significant increase in the prevalence of pro-inflammatory
macrophages in skin wounds [
129
]. This suggests that the fate and function of macrophages
in the wound bed can be modified by other cell types, particularly keratinocytes.
4.4.3. Immune Cells
Immune cells that infiltrate the wound are also shown to regulate macrophage phe-
notype in the wound healing process. Neutrophils are the first cells present in the wound
that release neutrophil extracellular traps (NETs). In diabetic wounds, NETs are present
at much higher levels than in normal healthy wounds. NETs induce inflammation and
IL-1
β
secretion by macrophages [
130
]. In addition, growth factors and protease activity (i.e.,
matrix metalloproteases 2, 8, and 9) are elevated due to increased levels of neutrophils, serine
elastase, and inflammatory macrophages, leading to prolonged inflammation [
2
,
35
,
83
,
131
].
Also, lymphocytes are known to play an important role in macrophage polarity [
132
]. It has
been shown that T cells, especially gamma, delta, and Th17 cells, increase in numbers in
diabetic wounds [
133
]. Th17 cells produce IL-17 that can regulate macrophage polarity. IL-17
elimination ameliorates wound healing in a diabetic mouse via decreased M1 macrophages
and increased M2 macrophages [
134
]. These studies suggest that infiltrated immune cells
in wounds can influence macrophage polarity.
5. Recent Studies and Conclusions
A better understanding of how macrophages function in wounds can provide better
therapeutic approaches for skin wound healing. In a study by Theocharidis et al. it has
been shown that murine macrophages or their secretome delivered in alginate dressings
enhance impaired wound healing in diabetic mice [
135
]. In clinical practices, a study
by Mao et al. discusses recent advances in biomaterials that balance the phenotypes of
macrophages in wound healing [
136
]. Moreover, it has been reported that wounds treated
with macrophages illustrated better cell recruitment and enhanced transition of healing
process from inflammation to tissue repair [
137
]. This has led to the development of a novel
hypothesis, which suggests that controlling macrophages modulation and recruitment
in wounds may provide a better therapeutic outcomes compared to the approach that
inhibits macrophages activities [
137
]. Recent studies have examined whether changes in
macrophages polarization can improve skin wound healing. For instance, recent studies
show that exosomes-laden self-healing injectable hydrogel increased diabetic wound heal-
ing by modulating macrophage polarization to improve skin angiogenesis [
138
]. Similarly,
others have investigated whether mechanical stimulation plays a vital role in regulating
macrophage polarization in the wound healing context [139].
Cells 2022,11, 2953 9 of 14
Although important in different stages of skin wound healing, macrophages possess
diverse biological features that help both the development and resolution of inflammation
in wound repair. Macrophage subtypes have different physiological features with different
molecular signatures. These molecular differences can induce or prevent various biologi-
cal activities including inflammation, angiogenesis, and skin re-epithelization. In addition,
macrophage metabolism and plasticity are affected in different conditions, such as obesity,
aging, and diabetes, in which the wound microenvironment is distinctly altered. Moreover,
the activity of other cell types including keratinocytes, adipocytes, and other immune cells can
affect macrophage functions in wound healing. Collectively, macrophages are key players in
skin wound healing, and further studies are required to elaborate how targeting macrophages
can effectively improve skin wound healing in different pathophysiological conditions.
Author Contributions:
Conceptualization, M.H. and A.J.C.; writing—original draft preparation,
A.H., M.M., S.G., M.F. and M.H.; writing—review and editing, M.H. and A.J.C. All authors have read
and agreed to the published version of the manuscript.
Funding:
M.H. is supported by The Channel 7 Children’s Research Foundation 22-20658466. A.J.C.
is supported by NHMRC Senior Research Fellowship GNT#1102617.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is available upon request to the corresponding authors.
Acknowledgments:
A.H. is supported by the University President’s Scholarship and School of
Pharmacy and Medical Sciences Scholarship, University of South Australia. A.J.C. is supported by the
NHMRC Senior Research Fellowship (GNT #1102617). M.H. is supported by Channel 7 Children’s
Research Foundation.
Conflicts of Interest: The authors declare that there are no conflicts of interest.
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