Glucocorticoids Shape Macrophage Phenotype for Tissue Repair
- 1Institut NeuroMyoGène, Université Claude Bernard Lyon 1, Univ Lyon, CNRS UMR 5310, INSERM U1217, Lyon, France
- 2Institute of Comparative Molecular Endocrinology, University of Ulm, Ulm, Germany
Inflammation is a complex process which is highly conserved among
species. Inflammation occurs in response to injury, infection, and
cancer, as an allostatic mechanism to return the tissue and to return
the organism back to health and homeostasis. Excessive, or chronic
inflammation is associated with numerous diseases, and thus strategies
to combat run-away inflammation is required. Anti-inflammatory drugs
were therefore developed to switch inflammation off. However, the
inflammatory response may be beneficial for the organism, in particular
in the case of sterile tissue injury. The inflammatory response can be
divided into several parts. The first step is the mounting of the
inflammatory reaction itself, characterized by the presence of
pro-inflammatory cytokines, and the infiltration of immune cells into
the injured area. The second step is the resolution phase, where immune
cells move toward an anti-inflammatory phenotype and decrease the
secretion of pro-inflammatory cytokines. The last stage of inflammation
is the regeneration process, where the tissue is rebuilt. Innate immune
cells are major actors in the inflammatory response, of which,
macrophages play an important role. Macrophages are highly sensitive to a
large number of environmental stimuli, and can adapt their phenotype
and function on demand. This change in phenotype in response to the
environment allow macrophages to be involved in all steps of
inflammation, from the first mounting of the pro-inflammatory response
to the post-damage tissue repair.
Macrophages therefore, appear to be an ideal target of
anti-inflammatory drugs due to their central role in inflammation.
Glucocorticoids (GCs) are highly potent anti-inflammatory drugs,
commonly used around the world. GCs have been used for decades to treat a
variety of inflammatory diseases such as rheumatoid arthritis, contact
allergy, or pulmonary diseases. Since the first GC therapies during the
1950s, various synthetic GCs have been developed to optimize their
action, and new molecules are still under development to modulate
therapeutic effects vs. the adverse effects of these drugs.
Surprisingly, given the importance of macrophages in the inflammatory
response, the direct effects of GCs on macrophages are less
well-documented. The present review aims at summarizing the knowledge on
macrophage functions during the post-injury inflammatory response, with
a focus on sterile inflammation and tissue repair, discussing how GC
signaling pathways operate in macrophages, and finally on the specific
action of GCs on macrophages.
Macrophages and Tissue Repair—Example of Skeletal Muscle Regeneration
Similar Macrophage Subtypes Are Found in Various Tissues During Repair
Macrophages belong to the innate immune system, however
their role is far more than protecting against pathogens. In the late
nineteenth century, Metchnikoff originally described and named these
cells as “macro” (big) “phage” (eaters) due to their phagocytotic
activity. In the following 100 years, scientists discovered that
macrophages are not only phagocytic cells. Different macrophage subtypes
were described, first in in vitro experiments, based on the main
cytokinic activation of lymphocytes, allowing macrophages to be divided
into different categories. “Classically activated” macrophages are
induced by stimulation with the Th1 cytokine IFNγ and “alternatively
activated” macrophages, involved in anti-inflammatory processes were
observed when using the Th2 cytokine IL-4 (1).
These two activation states were also called M1 (or pro-inflammatory
macrophages) and M2 (or anti-inflammatory macrophages), respectively.
However, this simplistic view of two potential statuses was quickly
expanded on. Macrophages can adopt a very large panel of phenotypes
depending on the inflammatory cues they encounter, even in vitro (2–4). In vivo,
the situation is more complex. The terms M1 and M2, although widely
used, are not appropriate to describe specific and dynamic inflammatory
status that occurs in the inflammatory milieu of a living organism (5, 6).
The Ly6C (Lymphocyte antigen 6 complex, a membrane protein expressed by
monocytes, and macrophages) and CX3CR1 (chemokine (C-X3-C motif)
receptor 1, another transmembrane protein involved in the adhesion and
migration of leukocytes) antigens have been widely used to classify
pro-inflammatory and anti-inflammatory macrophages in the context of
post-injury inflammatory response (7). During sterile inflammation, pro-inflammatory Ly6CposCX3CR1neg(CCR2posF4/80low)
cells infiltrate the injured tissue. After a rather undefined set of
signaling events, a phenotypic switch occurs whereby macrophages lose
Ly6C and CCR2 and gain CX3CR1 and F4/80 (forming Ly6CnegCX3CR1posCCR2low/negF4/80high cells) corresponding to their anti-inflammatory status (8).
This sequence of events from the infiltration of pro-inflammatory
macrophages to the phenotypic switch toward anti-inflammatory activity
appears to be universal. These events have been described after injury
in heart (9), central nervous system (10, 11), liver (12), kidney (13, 14), and skeletal muscle (15–18).
Skeletal Muscle Regeneration
The core cell type within skeletal muscle is the myofiber—a multinucleated cell formed by fusion of precursor cells (19). Skeletal muscle has a high regenerative capacity, after injury, muscle regenerates ad integrum,
where the old damaged cells are replaced by proliferation and
differentiation of satellite cells, which are the muscle resident stem
cells (MuSCs). Skeletal muscle regeneration, therefore, is an ideal
paradigm to study the biological events involved in tissue
repair/regeneration, helped by highly reproducible experimental models
in mouse (20).
Satellite cells are localized under the basal lamina surrounding each
myofiber, in a quiescent state. After an injury, damaged myofibers
undergo necrosis which triggers alteration of the satellite cell niche,
in turn leading to their activation (19).
Activated MuSCs proliferate, in order to produce a critical pool of
cells necessary to repair muscle, after which MuSCs differentiate into
myocytes, that eventually fuse to form new myofibers. While myogenesis
takes place, multiple other biological processes occur simultaneously
during muscle regeneration. Angiogenesis is required for efficient
muscle regeneration. Endothelial cells and MuSCs communicate through
secreted factors to mutually promote myogenesis and angiogenesis (21).
Fibro-Adipogenic Precursors (FAPs) control the extracellular matrix
remodeling during muscle regeneration, depending on the number and
differentiation status of the FAPs (22). Thus, muscle regeneration is a complex process where multiple cell types interact and coordinate to reconstruct the tissue (Figure 1).
Figure 1. Phenotype switch of macrophages regulates
skeletal muscle regeneration. After an injury, monocytes are recruited
from the bloodstream, and infiltrate the damaged area. In the tissue,
monocytes acquire a damaged associated pro-inflammatory phenotype. They
secrete inflammatory cytokines such as IL-1β and IL-6 and exert specific
functions: they stimulate the proliferation of the myogenic precursors
(myoblasts) and trigger fibroblast apoptosis to avoid excessive matrix
deposition. Upon phagocytosis of cell debris that triggers the
activation of AMPK, CEBPβ-CREB axis and P38/MKP1 pathways,
pro-inflammatory macrophages switch their phenotype toward an
anti-inflammatory restorative phenotype. Through the secretion of a
variety of factors, among which anti-inflammatory cytokines IL-10 and
TGFβ, anti-inflammatory macrophages are involved in tissue repair and
regeneration through the stimulation of myoblast differentiation and
fusion, of FAP/fibroblasts for matrix remodeling and of angiogenesis.
Each step of muscle regeneration
is linked to the inflammatory response, which is mainly mediated by
macrophages. Macrophages modulate myogenesis through MuSCs (17), as well as angiogenesis (21), and matrix remodeling (22)
that occur concomitantly. Macrophages represent more than 75% of the
leukocytes present in a regenerating muscle; however other immune cells
are present in lower numbers (16)
and are more prominent during the early steps of muscle regeneration.
Neutrophils are transiently present during the very first days after
injury, but their contribution to muscle regeneration has not been
deciphered yet and may depend on the extent of the injury (23). Eosinophils participate in muscle regeneration through the secretion of IL-4 that activates FAP proliferation (24). Tregs secrete the growth factor amphiregulin that stimulates MuSC expansion and differentiation (25).
Therefore, macrophages are major actors in the regulation of skeletal
muscle regeneration through the establishment of various interactions
with several cell types. While the above-mentioned studies clearly show
how macrophagic populations impact on other cell types, the effect of
those cells on macrophage phenotype and function has not been evidenced
The Inflammatory Phase During Muscle Regeneration
Tissue injury triggers the release of chemoattractants
into the bloodstream that recruit circulating leukocytes. Monocyte entry
into the injured muscle is regulated through the CCL2 (MCP1)/CCR2 axis.
In mouse models of CCR2 or CCL2 depletion, muscle regeneration is
severely hindered (26, 27). Indeed, only circulating Ly6CposCCR2pos monocytes are recruited into the injured muscle (6, 15, 18). In the nur77KO mouse model where CCR2negLy6Cneg monocytes are absent from the circulation, muscle regeneration occurs normally, indicating that circulating CCR2negLy6Cneg monocytes are not recruited into the injured muscle (15, 18).
Once in the tissue, macrophages clear debris from apoptotic and
necrotic cells through efferocytosis. They also potentiate the survival
and growth of MuSCs by establishing direct cell-cell contacts (28, 29). Moreover, pro-inflammatory macrophages secrete factors such as IL-6, IL-1β, or VEGF that stimulate MuSC proliferation (15, 17). Finally, pro-inflammatory macrophages control FAP apoptosis, preventing excess matrix deposition by fibroblastic cells (22, 30).
Macrophage Phagocytosis and the Resolution of Inflammation
At the time of resolution of inflammation, pro-inflammatory macrophages shift toward an anti-inflammatory phenotype (Figure 1).
Signaling pathways involved in this switch are beginning to be
documented in the literature. Currently, 3 main intracellular pathways
have been described: AMPK, p38/MKP1, CREB-C/EBPβ (see below section
“Time and space orchestration of the inflammatory response”). While the
activation of these pathways is required, the activating upstream cues
are still unknown. However, one likely candidate is the phagocytotic
pathway that has been shown to be essential for the acquisition of an
anti-inflammatory phenotype. Efferocytosis, that is the ingestion of
apoptotic cells by macrophages, results in a reduction of
pro-inflammatory markers, and an increase in the expression of
anti-inflammatory markers, suggesting that the death signals of
apoptotic cells may contribute to the generation of an anti-inflammatory
Anti-inflammatory macrophages act on several cell types in regenerating
skeletal muscle, inducing both differentiation, and fusion of MuSCs as
well as growth of the newly regenerated myofibers (15–17).
Anti-inflammatory macrophages promote extracellular matrix remodeling
by inducing fibroblast survival and collagen production through the
secretion of TGF-β (30). In vitro
experimentation has shown that anti-inflammatory macrophages stimulate
endothelial cell sprouting and differentiation, inducing vessel
formation concomitantly to myogenesis, through the secretion of specific
effectors, such as the cytokine Oncostatin M (21).
Accordingly, CCR2 KO mice exhibit defect of vascularization in the
regenerating muscle, as macrophages are not efficiently recruited to the
site of injury (34).
Thus, anti-inflammatory macrophages are a key component of the
regeneration phase. They act on multiple cell types within the muscle,
promoting growth of newly formed muscle cells, remodeling of
extracellular matrix and revascularization all simultaneously, allowing
the full, and importantly functional, recovery of the muscle tissue.
Time and Space Orchestration of the Inflammatory Response
The inflammatory response needs to be tightly
orchestrated to be efficient, and the regulation of macrophage activity
is no exception. Resolution of inflammation is a key step in skeletal
muscle regeneration, that must occur timely. Indeed, when the
pro-inflammatory phase is blunted by the inhibition of the expression of
the pro-inflammatory cytokine IFNγ (35) or reduced by the early administration of anti-inflammatory cytokine IL-10 (36),
muscle regeneration is impaired, resulting in the formation of smaller
myofibers. Similarly, blunting the inflammatory phase by administrating
anti-inflammatory drugs or icing the early injured muscle to prevent the
entry of monocytes is detrimental for muscle regeneration [reviewed in (37)].
AMP-activated protein kinase (AMPK), a key metabolic
regulator is also important for the generation of anti-inflammatory
Similarly, the p38/MKP1 pathway (MAP kinase pathway) modulates the
phenotype of macrophages. Inhibition of the phosphatase MKP1 allows for
an early activation of AKT, leading to a too early acquisition of the
anti-inflammatory status in macrophages, resulting in to an impairment
of muscle regeneration (36).
Finally, blocking the CREB-C/EBPβ cascade prevents the acquisition of
the anti-inflammatory phenotype of macrophages, that also impairs muscle
Given the importance of the process of the resolution of inflammation
for tissue homeostasis, it is likely that other pathways are also
involved in the switch of the inflammatory status of macrophages.
Glucocorticoids: a General Overview
Origins of GCs
The hypothalamic-pituitary-adrenal axis is critical for
the regulation of a variety of biological processes: stress, feeding,
circadian rhythm, growth, and reproduction. GC production is regulated,
via multiple hormonal inputs at all levels of the axis [reviewed in (39, 40)].
The hypothalamus secretes corticotropin releasing hormone (CRH), the
first step in the regulation of GC secretion. CRH is controlled through
input of the nervous system, such as exposure to stress, circulating
hormones like progesterone and adrenaline, but also by GCs. CRH acts on
the pituitary gland to induce the secretion of the Adreno Cortico Tropic
Hormone (ACTH) into the bloodstream. ACTH binds to its receptor on
cells of the adrenal cortex to regulate the secretion of a variety of
hormones, especially the GC cortisol (in humans), and corticosterone (in
mouse). The HPA axis, and therefore GC production is also under control
of the inflammatory response. Using computational modeling and
comparison to clinical data, it was demonstrated that after an
inflammatory trigger, ACTH and cortisol rise within minutes to hours,
slightly after cytokine release. However, this is not maintained for
long, and returns to baseline after 10 h (41).
The homeostatic release of GCs after an inflammatory challenge plays an
important protective role, which without (e.g., through a disrupted HPA
axis) results in relatively mild inflammation becoming deadly [reviewed
Investigation into the potential medical use of GCs started in the
1930s, where Philip Hench, Edward Kendall, and Tadeusz Reichstein showed
the incredible therapeutic potential of these molecules as
anti-inflammatory drugs, and later received the Nobel prize for their
work in 1950. From that point, GC therapies spread all around the world
and are still used today to counter inflammation.
The GC Receptor
GCs act through the Glucocorticoid Receptor (GR), a member of the nuclear receptor superfamily, and first cloned in 1985 (43). The gene encoding GR is located on the locus 5q31.3 in the human genome comprised of 9 exons (43).
GR expression gives rise to the expression of 2 major isoforms: GRα
(777 amino acids) and GRβ (742 amino acids), along with other less
well-expressed (and less well-studied) isoforms (43).
GRα is the active isoform that binds GCs and that regulates target gene
expression. GRβ isoform is a regulator of the α isoform, acting as a
dominant negative (44, 45).
A third isoform of the receptor, GRγ has also been characterized. This
isoform only differs from GRα by one arginine in the DNA Binding Domain
(DBD) that alters the capacity for the isoform to regulate gene
expression, giving GRγ its own transcriptomic profile (46). This altered profile may play a role in GC resistant leukemia (47), however its action during inflammation has not yet been extensively studied.
The 3D structure of GR is comprised of several domains:
the N-terminal domain, the DBD, the hinge region, the Ligand Binding
Domain (LBD) and the C-terminal domain (48–50).
GR, like other nuclear receptors is a ligand regulated
transcription factor, which regulates gene expression by binding either
directly, or indirectly to the genome [review in (51)]:
Activation: after ligand binding in the cytoplasm, GR translocates to
the nucleus, and directly binds specific palindromic regions on DNA
called Glucocorticoid Response Elements (GREs). GREs are present in the
regulatory regions, such as the promoters, enhancers, and even within
the exons or introns of target genes (such as Gilz and Dusp1) and binding of GR dimers induces the transcription of these genes (positive GRE) (51).
Transactivation can also occur by a tethering mechanism, whereby GR
associates with other transcription factors that positively drive gene
expression. Transcription can also be induced by monomeric GR that binds
DNA to a half-site motif (52).
Repression: as with activation, nuclear GR can bind DNA and represses
the transcription of genes. GR can directly act as a monomer in
association with other transcription factors such as NFκB (53) or AP-1 (54) to transrepress gene expression by a tethering mechanism (51).
GR monomer sequestrates transcription factors to prevent their binding
to promoters and so to prevent transcription. Moreover, GR cis-repress
genes by directly binding so called negative GREs or by directly binding
the NFκB or AP-1 response elements (55).
More mechanisms are currently emerging driven by genome wide studies
that are reviewed in detail elsewhere in this Research Topic
(Escoter-Torres et al., Submitted).
Thus, GR is a transcription factor that regulates gene expression through several pathways [reviewed in (45, 49, 56, 57)] and in a tissue dependent manner (58).
Non-genomic effects of GCs, that is GC regulated actions that are
independent from the regulation of gene expression, have been described
in several tissues and that were very recently reviewed in Panettieri et
Adverse Effects of GCs
During the 1960s, it became clear that clinical use of
GCs causes severe metabolic side effects. In 1970, David and colleagues
reviewed 20 years of GC utilization (60).
They discussed side effects that were observed in almost all tissues of
the body. In 1970, it was already known that long exposure to GCs was
responsible for several metabolic disturbances, but more recent studies
have expanded on this, dramatically enhancing our knowledge about GC
effects on metabolic organs. Chronic GC use results in the development
of type 2 diabetes (due to increased gluconeogenesis, hepatosteatosis,
decreased insulin sensitivity, and decreased glucose consumption) (61–63), skin (64, 65), and muscle atrophy (66), and bone mass reduction (both due to induction of catabolism and/or reduction of anabolism) (67).
Moreover, free fatty acids are increased in the bloodstream and in
clinical cases of GC excess—for example Cushing's Disease, this results
in increased adipose tissue mass, but usually localized to the face and
truck, resulting in a “Moon-Face” and “Buffalo Hump” (68, 69).
Although literature documenting GC side effects is very abundant, the
molecular mechanisms involved have not been completely elucidated, in
part due to the complexity of the tissue specific effects of GCs.
Anti-inflammatory effects of GCs were historically
associated with the monomeric form of GR, mainly due to the evidence
that GR can bind and inhibit, and thus transrepresses the inflammatory
transcription factor NFκB, downregulating the expression of
pro-inflammatory cytokines (39, 70).
The metabolic actions of GR were ascribed to the dimer, suggesting that
drugs specific to monomeric, over dimeric GR would exhibit all
beneficial anti-inflammatory effects without having negative side
effects. A mouse model, in which GR dimerization is impaired (GRdim),
has allowed several laboratories to show that GR dimerization is also
required for the anti-inflammatory properties of GCs in several
contexts, such as rheumatoid arthritis (71, 72), septic shock (73, 74), or inflammatory bowel disease (75). Interestingly however, the metabolic side effects of GCs are enhanced in the GRdim
mice. The loss of dimerization can drive increased insulin resistance
and obesity, suggesting that the classical view of monomeric GR only
being associated with the anti-inflammatory actions is not entirely
Therefore, both inflammatory and metabolic regulation by GCs may be
driven by both the dimer and the monomer, depending on the cell type,
the tissue, and the pathology considered.
Glucocorticoids, Macrophages, and Tissue Repair
First investigations into the action of GCs on
macrophages during tissue repair started a few decades ago. One of the
side-effects of chronic GC exposure is the loss of bone mass
(osteopenia/osteoporosis). Bone resorption, that is, the digestion of
existing bone, is more efficient when highly specialized macrophages
involved in bone remodeling, osteoclasts, are in direct contact with the
bone. Resident tissue osteoclasts are derived from myeloid progenitor
cells during development, however they are maintained throughout life by
circulating blood monocytes fusing to existing osteoclasts in the bone (77).
Osteoclasts treated with cortisol are more adherent to bone, more
sensitive to RANKL, and release more calcium useable for bone
resorption, enhancing the bone resorption process (78–80).
GCs also increase osteoclastogenesis by driving the production of
RANKL, the necessary factor for osteoclast differentiation, and
downregulating osteoprotegerin, the decoy receptor for RANKL (81, 82).
It was possible to prevent GC-induced osteoporosis by treating mice
with a RANKL neutralizing antibody, further demonstrating that the
effects of GCs on osteoclasts contribute to the bone loss that occurs
during GC treatment (83).
GCs can also have direct effects on osteoclasts. Using either mice
deficient for GR in osteoclasts or 11BHSD2 overexpressing mice (where
the GC inactivating enzyme is over-expressed in osteoclasts), it was
confirmed that GCs act directly on osteoclasts to modulate bone density,
in part by increasing the life span of osteoclasts (84, 85). Interestingly, chronic treatment with GCs decreases osteoclast life-span, suggesting a temporal effect (67, 86).
A mouse model based on the cre/loxP system was designed to specifically deplete GR in the myeloid lineage where the cre recombinase gene is located at the Lysozyme M locus. These so-called LysMcre;GRfl/fl
mice, delete GR in monocytes, macrophages and neutrophils. In a mouse
model of contact hypersensitivity, the anti-inflammatory effects of GCs
were shown to be mediated through GR in macrophages, rather than other
tissues. Treatment of LysMcre;GRfl/fl mice with GCs failed to repress the cytokines IL1-β, MCP1, MIP2, and IP10. In addition, GRdim mice are also insensitive to GCs, indicating that GR dimerization, likely in macrophages, is required in this context (87). In a model of myocardial infarction, LysMcre;GRfl/fl
mice die earlier after infarction than wild-type animals with full
expression of macrophage GR, probably due to the persistence of Ly6Cpos
macrophages into the infarcted area, leading to a dysregulation of the
resolution of inflammation and a defects in wound healing. This results
in alteration of angiogenesis, abnormal production of TGFβ, decreased
production of IL-1α and finally deregulation of myofibroblast
differentiation leading to scar formation (88). Moreover, in a mouse model of inflammatory bowel disease, macrophages from LysMcre;GRfl/fl
animals show a defect in the acquisition of the anti-inflammatory
status. After 10 days, IL-1β, and IL-6 expression is not repressed and
expression of anti-inflammatory genes (CD163, CD206, and IL-10) is not
induced, leading to a defect in tissue repair (89).
Local availability of GCs also plays an important role in inflammation.
The enzyme 11-β-hydroxysteroid dehydrogenase (type-1) (11bHSD1)
catalyzes the conversion of the inactive cortisone to cortisol, enabling
binding to GR and signaling. Myeloid specific knockouts of 11bHSD1,
preventing endogenous GC signaling in macrophages and neutrophils,
result in a more severe arthritis phenotype (90). This is however not limited to macrophages, inhibition of 11bHSD1 increases neutrophil recruitment during peritonitis (91).
Expansion of GC research into zebrafish models is still
in the early stages, and so appears somewhat contradictory. No effect of
the GC beclomethasone has been observed on the migratory capacity of
macrophages toward the wounding area in an amputation model in zebrafish
(92). However in a separate model of wounding, prednisolone reduced macrophage accumulation in both larvae and adults (93).
This may be due to the different ligands used, as different ligands
have previously been shown to have different transcriptional effects (51).
Thus, in most tissue injuries, GC-GR axis appears to be a central
pathway in macrophages to regulate the resolution of inflammation and to
proceed to tissue repair after injury.
Glucocorticoids and Macrophages—Cellular Aspects
GCs Regulate Survival, Migration, and Proliferation of Macrophages
Maintenance of living immune cells in appropriate
numbers is essential to modulate the inflammatory response, and GCs
appear to play several roles in the regulation of macrophage life-span.
GCs exert anti-apoptotic effects on macrophages: macrophages treated
with dexamethasone are more resistant to lipopolysaccharide
(LPS)-induced apoptosis (94).
Similar results were obtained with other apoptotic stimuli
(staurosporine, actinomycin D, or cyclohexine) where GC effects are
mediated through ERK1/2 phosphorylation in an adenosine receptor
A3-dependent-manner (95, 96). Moreover, macrophages treated with dexamethasone are smaller with less cytoplasmic extensions (97),
which could be related to altered migratory capacity. The capacity of
macrophages to move toward the injured area also shapes the inflammatory
response. Macrophages treated with hydrocortisone (cortisol) show a
decreased capacity to migrate in vitro (98, 99). In vivo,
a similar effect was observed in a model of lung injury induced by
bleomycin, where GCs inhibited macrophage infiltration into the lung (100).
Studies using myeloid like cells and whole bone marrow preparations
showed that GCs decrease proliferation of cells (including macrophages) in vitro (101, 102),
but GC impact on proliferation has never been investigated on
macrophage cultures. GR activation also has potent effects on nitric
oxide (NO) production by macrophages. Initial studies in the J774a.1
macrophage cell line demonstrated that GCs suppress the induction of the
NO-generating enzyme, nitric oxide synthase, thus controlling the level
of NO produced by the cells in response to an inflammatory stimulus (103).
Later studies however, showed that GCs are protective in a mouse model
of stroke through increasing NO production in a non-genomic manner. By
activating PI3K, GCs rapidly induce NO dependent vasodilation (104).
The effects of GCs on NO production were further demonstrated to be
dose dependent, with lower doses eliciting an increase in NO, while
higher doses reducing the production of NO (105).
Thus, GCs promote macrophage survival in order to switch
off inflammation and to sustain late phase of healing. In the following
decades, studies have focused on the understanding of the molecular
aspects of GC signaling pathways.
GCs and Phagocytosis
During inflammation, damaged tissue produces cell
debris, and releases cytoplasmic proteins into the environment due to
cell lysis (106). Before tissue repair can start, debris must be cleared up (106).
The clearing process is mainly performed by neutrophils, then
macrophages, through phagocytosis of tissue debris, i.e., efferocytosis (106).
Since phagocytosis is a major function of macrophages and is an
essential trigger of their inflammatory switch (see above section
“Macrophage phagocytosis and the resolution of inflammation”), the
action of anti-inflammatory treatments on this process is of importance.
GCs were detected very early to have an impact on phagocytic activity
of macrophages (107). Later on, studies showed in in vitro
models using a variety of particles (zymosan, heat-kill yeast,
apoptotic neutrophils, latex beads, bacteria) that dexamethasone
increases the phagocytic activity of monocytes/macrophages (95, 102, 108–115). Some of these studies have also shown, using a GR antagonist (RU486), that GC-dependent phagocytosis is also GR dependent (109, 110). The increased macrophage phagocytic activity by dexamethasone is annexin 1-FRP1 dependent (116). Annexin 1 belongs to the superfamily of annexin protein, which bind acidic phospholipids in the presence of Ca2+ (116). Annexin A1 is described to be a pro-resolving molecule during inflammation (117).
Indeed, when the annexin receptor FRP1 is antagonized by the Boc1
compound or in annexin 1-null macrophages, dexamethasone loses its
effect on phagocytosis (118).
On closer examination of the phagocytic process, it
became clear that GCs induce the up-regulation of several membrane
receptors, such as the scavenger receptor CD163, required to detect and
bind haptoglobin, a product from hemoglobin degradation (111, 113, 114, 119).
The mannose receptor CD206, required for the detection of specific
oligosaccharides on the bacterial wall, is also upregulated in
macrophages treated by GCs (120). Moreover, GCs upregulate the membrane receptor Mer tyrosine kinase (MerTK) (121), in a C/EBPβ dependent-manner (122). When mertk is silenced, dexamethasone-induced phagocytosis is reduced (121).
MerTK belongs to the Tyro3, Axl, MerTK (TAM) family of tyrosine kinase
receptor. It binds to phosphatidyl serine exposed on the surface of
apoptotic cells (121, 122). MerTK is also responsible for the phagocytosis of protein S-opsonized apoptotic neutrophils by GC-treated macrophages (123).
The other members of the TAM family do not seem to be necessary for
GC-induced phagocytosis, as Tyro3 deficient, or Axl deficient mice are
able to successfully clear apoptotic cells in response to GCs (124).
Interestingly, in a model of serum-transfer induced arthritis, Axl,
MerTK, and CD163 upregulation in macrophages requires GR function on
synovial fibroblasts, indicating their regulation through cross-talk
between local cells (72).
Finally, GCs regulate the C/EBPβ-dependent expression of nuclear
receptors (liver X receptor [LXR], retinoid X receptor α [RXRα] and
peroxisome proliferator-activated receptor δ [PPARδ]), which are
required for prolonged phagocytosis of macrophages (122). Thus, GCs act on several steps of phagocytosis and their effects are mediated through various signaling pathways.
Glucocorticoids and Gene Expression in Macrophages—Molecular Aspects
Although the first effects of GCs on macrophages were
reported in 1950, the literature about their specific effects on this
cell type is not abundant (see section GCs on macrophages: expression of
anti-inflammatory effectors). In 1950, Dougherty and colleagues showed
in a model of local inflammation in mice that cortisone treatment
reduces the number of macrophages in the inflamed area (125).
In another model of skin inflammation induced by injection of
turpentine, Spain et al. showed that cortisone inhibits the formation of
granulation in the inflamed area (granulations corresponding to
macrophages according to the authors) and a decrease of carbon particle
phagocytosis when administrated early during the inflammatory response (107).
However, the experiments done by Gell and Hinde on intraperitoneal
macrophages exposed to bacteria showed that cortisone does not alter
either the number of macrophages or their phagocytic capacity (126).
GCs on Macrophages: Expression of Anti-inflammatory Effectors
It is well-known that macrophages can exert pleiotropic
functions through the secretion of a variety of factors. Macrophages are
highly versatile, and may secrete pro-inflammatory, anti-inflammatory,
or other factors necessary at each step of the inflammatory response.
GCs decrease the secretion of the pro-inflammatory cytokines TNFα (94, 127), IL-1, IL-6 in macrophages exposed to IFNγ (100, 113). Monocytes treated with GCs increase their secretion of IL-10 and TGFβ (128, 129) and express high levels of the anti-inflammatory membrane markers CD206 (120), CD163 (95, 111, 113, 114, 119, 130) and CD169 (95, 131).
GC anti-inflammatory effects are partly mediated by Mitogen-activated
protein kinase phosphatase-1 (MKP-1) in macrophages, as it was GC-driven
inhibition of IL-6 expression was abrogated in MKP-1 deficient
Furthermore, macrophages exposed to GCs secrete molecules
which have direct functions on the extracellular matrix and therefore
participate to matrix remodeling during the late phase of the
inflammatory response. The production of elastase, collagenase and
plasminogen activator (whose secretion is elevated in pro-inflammatory
macrophages and which are required to degrade extracellular matrix) is
reduced in macrophages treated with GCs (133, 134).
On the contrary, macrophages exhibiting an alternatively activated
status (i.e., IL-4 driven) secrete more fibronectin when treated with
GCs, participating in matrix remodeling at the time of tissue repair (114, 135, 136).
GC Action on Macrophages: Regulation of Gene Expression
GCs act through either the GR dimer or GR monomer,
entirely depending on the gene regulated. For example, in dermatitis, GR
dimerization is required to shut down the expression of the
pro-inflammatory cytokines IL-1β and MCP-1 whereas TNFα downregulation
induced by GCs does not require GR dimerization (87).
GCs also modulate chromatin architecture, mainly closing down access to
genes involved in inflammation, preventing access to other
transcription factors (137, 138).
Importantly, the gene regulatory actions of GCs depend on
the activation state of macrophages. Indeed, more than 10,000 genomic
GR binding sites are induced by dexamethasone in resting macrophages
with more than 5,400 known GR target genes, while in macrophages
pre-treated with GCs, then LPS, there is a rewiring of GR binding, with
13,000 binding sites and more than 6,400 GR target genes identified (139).
Furthermore, GCs regulate a different set of genes in macrophages
activated with LPS or IFNγ indicating that genes are regulated by GCs
are also dependent on the inflammatory stimulus (130).
LPS stimulation also increases the ability of GR to bind DNA indicating
that pro-inflammatory stimulation potentiates GR DNA binding, likely
through the generation of more potential binding loci (138, 139).
Oh et al. also demonstrated that pre-treatment compared to
post-treatment of GCs with LPS results in a differential effect on gene
regulation. The number and location of GR binding sites and p65 binding
sites were different between the GC pre-treated cells and the cells
treated with LPS first, then GCs (138).
Furthermore, another GR partner, the Glucocorticoid
Receptor-Interacting Protein (GRIP) 1, also known as nuclear receptor
co-activator 2 (NCOA2) is required for the acquisition of the
anti-inflammatory phenotype of macrophages (140).
GRIP1 can be phosphorylated by Cyclin-Dependent Kinase 9 (CDK9) in a GR
dependent-manner. Phosphorylated GRIP1, in association with GR, binds
GREs to induce the expression of anti-inflammatory genes. However,
phosphorylated GRIP1 is not observed in GR repressed sites such as of IL1a or IL1b,
indicating that phosphorylated GRIP1 only acts on positive
transcription of anti-inflammatory genes, and it is likely that the
phosphorylation status of GRIP1 can modulate GR transcriptional activity
Our understanding of the role of GR as an anti-inflammatory
transcription factor is still evolving, and with new technologies, the
actions of GR will become clearer with time.
The GC Effector GILZ in Macrophages
GC-mediated anti-inflammatory effects are known to be
partly mediated through the regulation of the expression of specific
proteins that in turn modulate inflammatory signaling. A very
well-studied example is Glucocorticoid-Induced Leucine Zipper (GILZ).
Originally found expressed in lymphoid tissues (thymocyte, spleen, lymph
nodes) treated by dexamethasone (142),
GILZ is a major regulator of GC effects in a variety of cells. GILZ was
also found to be expressed by macrophages in liver and lung treated by
dexamethasone (143). In the THP-1 macrophage cell line, dexamethasone induces Gilz mRNA expression after only 30 min of treatment (143). GILZ acts by binding the p65 subunit of the NFκB complex to shut down its activity (143).
GILZ also inhibits the expression of the Toll like receptor 2 (TLR2),
thus limiting the recognition of bacterial components and the associated
inflammatory signaling (143). GCs however also enhance the expression of TLR2 in a cell-type specific manner (144, 145),
suggesting that GILZ may act as a homeostatic brake on GC enhanced TLR2
signaling. Furthermore, GC-induced GILZ expression is strongly reduced
in annexin A1 deficient macrophages, therefore preventing the
downregulation of the pro-inflammatory cytokines IL-1, IL-6, and TNFα (146, 147). This regulation is not dependent of the annexin receptor FRP (146), thus, the exact mechanism by which annexin regulates Gilz expression remains to be elucidated.
The effects of GCs on macrophages, especially in the
broader context of resolution of inflammation during tissue repair, are
not as well-understood as one would assume. GCs play key roles in the
regulation of macrophage homeostatic functions, as well as the
macrophage function as innate immunity cells. GR however, does not act
alone. In association with several partners including other
transcription factors (C/EBPβ, PPARs, NFκB) or proteins that modulate
its activity (GRIP1), GR controls the functional properties of
macrophages to resolve inflammation and tissue damage. Finally, GCs
regulate the expression of a huge number of genes that are essential to
relay their anti-inflammatory properties such as Gilz and Annexin a1.
Despite 60 years of work on GCs, we are still discovering further
molecular mechanisms that govern their actions. The role of the
inflammatory context (138, 139) and species differences in GC mediated gene regulation (148)
highlight that further investigation is necessary to decipher, for each
situation, how GCs operate to regulate gene expression, and therefore
control macrophage function.