Introduction
Macrophages were initially described as “big eaters” due to their phagocytic nature.
It is now clear that macrophages have many diverse functions not only in innate immunity
and tissue homeostasis but also in metabolism, development, and regeneration. Macrophage
functions are driven largely by tissue-derived and pathogenic microenvironmental stimuli
that help them adapt to changing conditions within tissues and tailor an appropriate
response. The heterogeneity of macrophages has resulted in their classification into
subtypes based on their phenotype and function (1). One major classification, based
on function, is M1 and M2 macrophages, with destructive and healing properties, respectively
(2, 3). As imbalances between M1 and M2 states have been observed in a number of diseases,
an understanding of the molecular mechanisms, signaling pathways, and transcription
factors controlling their polarization has obvious therapeutic implications. Recent
studies have established strong potential for suppressor of cytokine signaling (SOCS)
proteins to regulate M1 and M2 macrophage polarization (4–7). Here, the focus will
be on the evidence for this, and the consequences of altered SOCS expressions on macrophage
function in health and disease. Overall it is proposed that a high SOCS1 to SOCS3
ratio could be a potential marker for M2 macrophages while high SOCS3 expression is
associated with M1 cells.
SOCS Proteins
Suppressor of cytokine signaling proteins are a family of intracellular cytokine-inducible
proteins, consisting of eight members (CIS and SOCS1–SOCS7) (8, 9). SOCS1 and SOCS3
are most widely characterized regarding their roles in shaping M1 and M2 macrophage
polarization (4–6). They show low expression in resting macrophages, but are rapidly
induced on activation. All SOCS family proteins contain an Src homology 2 (SH2) domain,
a variable length amino-terminal domain and a conserved carboxy-terminal SOCS box
motif that interacts with ubiquitin–ligase machinery (8, 9). SOCS are induced by a
variety of stimuli that cause M1 and M2 activation, including cytokines, toll-like
receptor (TLR) ligands, angiotensin II, immune complexes, and high glucose (9). The
most studied signaling pathway regulated by SOCS is JAK/STAT activation. SOCS negatively
regulate JAK/STAT signaling through association with key phosphorylated tyrosine residues
on JAK proteins and/or cytokine receptors, and by degradation of signaling molecules
mediated via the ubiquitin–proteasome pathway (8, 9). SOCS1 and SOCS3 contain a kinase
inhibitory region (KIR) that directly suppresses JAK tyrosine kinase activity. SOCS
proteins also influence ERK (10), PI3K (11), Notch (12), MAPK (13), and NF-κB (14)
signaling cascades that directs M1 and M2 functions.
SOCS1
SOCS1 regulates M1-macrophage activation by inhibiting the interferon gamma-induced
JAK2/STAT1 pathway and TLR/NF-κB signaling (9, 15) (Figure 1). To suppress the latter
pathway, SOCS1 binds to the p65 subunit of NF-κB and the TLR adaptor molecule Mal/TIRAP
as well as IRAK, facilitating its ubiquitin-mediated proteolysis via ubiquitin ligases
recruited by the SOCS box (8, 14–17). SOCS1 indirectly inhibits TLR4 signaling through
secondary mechanisms targeting IRF3 and IFN-β induced JAK/STAT pathways (18, 19).
Thus, SOCS1 mediates a negative feedback mechanism during TLR4 signaling, via control
of both MyD88-dependent and MyD88-independent signaling. SOCS1-deficient mice succumb
to severe systemic autoimmune and inflammatory disease (14, 16) and their M1-macrophages
display an increased capacity to kill intracellular bacterial pathogens, presumably
due to unrestrained IFN-γ/STAT1 and p65 signaling. In line with this, SOCS1 knockout
or knockdown M1-activated macrophages show enhanced levels of IL-6, IL-12, MHC class
II, and nitric oxide suggesting SOCS1 sustains the properties of M1 macrophages at
a less destructive level to prevent overshooting inflammatory responses (4, 18). This
explains why SOCS1 promoter hypermethylation, which results in loss of SOCS1 expression
leads to enhanced secretion of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines
(20). Micro RNA-155 (miR-155) is a critical regulator of innate immunity and TLR signaling
(21–23); miR-155 targets and degrades SOCS1 in M1-activated macrophages (21), thus
miR-155 induction during activation serves to maximize and extend the inflammatory
process.
SOCS1 also regulates M2 macrophage polarization. Expression of macrophage SOCS1, but
not SOCS3, is strongly upregulated in an M2 polarizing environment in vitro and in
vivo, where it has an important role in acquisition of M2 functional characteristics,
such as a high arginase I/low inducible nitric oxide synthase (iNOS) expression ratio
(4). Strikingly, this contrasts with macrophages infiltrating an in vivo inflamed
M1-activating environment, where macrophages with enhanced SOCS3 but not SOCS1 expression
are prominent (5). This suggests that exclusive upregulation of SOCS1, or indeed,
a high SOCS1/SOCS3 expression ratio, has potential as a useful and additional in vivo
biomarker for M2 (see later). Arginase I expression, as an M2 macrophage marker, can
be mediated via activation of either STAT6 (24) or PI3K (25). SOCS1 is important in
controlling PI3K activity, supporting a mechanism for regulating arginase I expression
in M2 cells; SOCS1 also regulates STAT6 phosphorylation (26). Following activation,
SOCS1 knockdown or SOCS1-deficient macrophages show a reciprocal upregulation of SOCS3
expression. SOCS3 inhibits PI3K activation (27), and so the expression of high SOCS1
and low SOCS3 in M2 macrophages could result in greater PI3K activity and more arginase
I induction in these cells. An elevated expression of SOCS1 is important for the arginase
I-induced suppressive nature of M2 macrophages that attenuate lymphocyte proliferation
(28). Moreover, siRNA-mediated knockdown of SOCS1 results in the induction of iNOS
in IL-4-pretreated cells stimulated with IFN/LPS (4). Thus, SOCS1 regulates the iNOS/arginase
I expression ratio in both M1 and M2 macrophages and helps fine-tune key signaling
pathways to mount an appropriate response to changes within the microenvironment.
Figure 1
Role of SOCS1 and SOCS3 in macrophage activation. STAT1 and NFκB drive M1 polarization
and SOCS1 can inhibit these pathways. SOCS3 can regulate TLR signaling and inhibits
IL-6-induced STAT3 activation and SMAD3 and PI3K activity to action an appropriate
destructive effect. STAT3, STAT6, and PI3K can drive M2 activation and SOCS3 inhibits
STAT3 and PI3K. Pathways that trigger SOCS1 in macrophages include STAT1 and NFκB,
while SOCS3 expression can be induced by STAT3, NFκB, NOTCH1, PI3K, and MAP kinase
activation.
SOCS2
An important role for SOCS2 in driving M2 polarization and limiting M1 polarization
has been shown, with IL-4 activation of macrophages, resulting in enhanced SOCS2 expression
(27). Macrophages from SOCS2-/- mice display increased secretion of IFN-γ, IL-1β,
and TNF-α in response to LPS in parallel to an increased pro-inflammatory cytokine
mRNA expression (29). These BMDMs have higher basal levels of p65–NF-κB compared with
macrophages from wild-type mice (29). In another study, SOCS2-deficient macrophages
were hyper responsive to IFN-γ, produced more NO and dealt with infection more efficiently
(30). SOCS2 has also been described as a feedback inhibitor of TLR-induced activation
in dendritic cells (31).
SOCS3
In contrast with SOCS2, a key role for SOCS3 in M1 polarization is proposed (Figure
1). The majority of macrophages activated within an in vivo pro-inflammatory conditioning
environment show strong upregulation of SOCS3 expression and this cell population
co-express the M1 marker, iNOS (5, 6). Without SOCS3, both human and rodent macrophages
have a reduced ability to develop pro-inflammatory features but instead display immunoregulatory
characteristics (5, 6). Notably, mice with a targeted deletion of SOCS3 in macrophages
and neutrophils demonstrate a reduced IL-12 response and succumb to toxoplasmosis
(32). SOCS3 binds to and inhibits gp130-related cytokine receptors and consequently
this abrogates IL-6-induced STAT1 gene expression and IL-6-induced STAT3 anti-inflammatory
effects (33–35). Therefore, in SOCS3-deficient macrophages, IL-6 signals in a similar
manner to the immunosuppressive cytokine IL-10, through prolonged STAT3 activation
and dampening of LPS signaling (33). As a result, mice deficient in SOCS3 in myeloid
cells are resistant to endotoxic shock (35) with reduced production of pro-inflammatory
cytokines. However, one report in the same mice suggests SOCS3 deficiency promotes
M1 macrophage activation in spite of enhanced STAT3 activation (7). The reasons for
this discrepancy in findings are unclear but could relate to differences in dose and
purity of the LPS used in the different studies, as well as and the genes and time-points
analyzed after macrophage activation (7, 35). Moreover, the conflicting results for
the role of SOCS3 in M1 polarization in isolated macrophages in vitro (5–7) could
result from the different technologies and species used (siRNA-mediated knockdown
in rat and human macrophages, which avoids the risk of compensatory effects of other
SOCS genes (5, 6) versus cells from macrophage-specific SOCS3 knockout mice) (7).
Resolving these issues should establish the importance of SOCS3 in modulating macrophage
function in vivo.
Studies of SOCS3-deficient macrophages confirm that SOCS3 positively regulates TLR4
signaling and M1 activation by inhibition of IL-6R-mediated STAT3 activation, as well
as TGF-β-mediated SMAD3 activation, which is critical for the negative regulation
of TLR-induced TNF-α and IL-6 production (5, 6, 33, 36). Since SOCS3 blocks PI3K that
feeds and inhibits TLR responses, this could be an alternative mechanism by which
SOCS3 augments TLR signaling in M1 macrophages (6). Forced activation of Notch signaling
enhances both M1 polarization and anti-tumor activity via SOCS3 induction (12). In
line with this, macrophage-specific SOCS3 knockout animals are resistant to tumor
transplantation due to reduced secretion of tumor-promoting TNF-α and IL-6, together
with elevated MCP2/CCL8 that is anti-tumorigenic (37).
Regulation of SOCS3 in innate cells influences downstream T cell fates. The presence
of SOCS3 in macrophages is important in fine-tuning downstream T effector cell priming
due to both influences in expression of presenting molecules and altered secretion
of T cell polarizing cytokines (6, 7). Mouse SOCS3-deficient dendritic cells display
an analogous reduced potential to drive T effector cell responses and a tolerogenic
phenotype as a result of enhanced TGFβ production and expansion of Foxp3-positive
regulatory T cells (38). These dendritic cells reduce the severity of experimental
autoimmune disease. Therefore, regulation of intracellular signaling pathways by SOCS3
in innate cells is critical for the decision of adaptive responses such as T cell
fates. The depletion of macrophage SOCS3 in a clinical situation would thus be predicted
to dampen both pro-inflammatory innate and adaptive immune responses.
The above studies suggest that macrophage SOCS3 is associated with M1 macrophages
and pro-inflammatory responses and is a potential therapeutic target in inflammatory
diseases. However, a word of caution should be introduced as this may not be the case
in all inflammatory conditions. In diseases, where STAT3 activation exerts a profound
inflammatory and pathogenic response (39, 40) then the effects of SOCS3 targeting
may not be beneficial. For example, in an IL-1/STAT3 model of chronic arthritis where
SOCS3 was deleted in hematopoietic and endothelial cells, animals exhibited more severe
disease. Thus, the pathology needs first to be assessed before SOCS3 manipulation
as a therapy is considered (37).
Macrophage SOCS Expression and Pathology
The heightened expression of macrophage SOCS1 and SOCS3 proteins have been demonstrated
in many pathologies in vivo where this has been proposed, through the molecular mechanisms
described above, to enhance or inhibit pathogenesis.
SOCS and glomerulonephritis
Macrophages are an important feature in glomerulonephritis pathology. Macrophages
infiltrating inflamed glomeruli in experimental models are rapidly polarized to express
either SOCS1 or SOCS3, but rarely both, with most exclusively expressing SOCS3 (5,
6). The proportion of these SOCS3-expressing macrophages correlates strongly with
the severity of immune-mediated injury. Local delivery of IL-4 to inflamed glomeruli
has a major effect on reducing the number of SOCS3-expressing glomerular macrophages,
and this is reflected by a decrease in the severity of nephritis, supporting a role
for SOCS3 in driving M1-mediated injury (5).
SOCS and atherosclerosis
Human atherosclerotic plaques exhibit a high expression of macrophage SOCS1 and SOCS3
in unstable inflammatory shoulder regions as compared to stable fibrous area (41).
SOCS1 and SOCS3 expression is increased in aortic lesion macrophages from apoE(−/−)
mice (42). In human tissue, the percentages of SOCS1-positive, M2 macrophages are
decreased in morphologically stable atherosclerotic plaques, whereas percentages of
SOCS3-positive, iNOS positive, macrophages are increased in unstable, rupture-prone
plaques, suggesting targeting macrophage SOCS3 would be beneficial to dampen inflammation
and plaque vulnerability (43). The differing expression ratio of SOCS1:SOCS3 in atherosclerotic
plaques again suggests that the ratio could be an indicator of the inflammatory status
of human macrophages in vivo. SOCS1 was atheroprotective in mouse models (44) while
the absence of macrophage SOCS3 of apoE(−/−) mice attenuates disease, confirming a
causal link between macrophage SOCS3 and atherosclerosis (45).
SOCS and inflammatory bowel disease
Beneath the gut epithelia, lamina propria macrophages phagocytose bacteria and maintain
an M2 phenotype in the steady state. Approximately 10% of these macrophages express
SOCS3 in healthy individuals, whereas in inflammatory bowel disease (IBD) patients
this increases to 40%, again suggesting SOCS3 expression relates to M1-activated macrophages
(46). Peroxisome proliferator-activated receptor-γ (PPARγ) agonists demonstrate efficacy
in ameliorating intestinal inflammation associated with IBD. PPARγ expression is upregulated
in M2 but not M1 macrophages. In macrophages lacking PPARγ, a significant upregulation
of SOCS3 was noted and this could be important if treating IBD with PPARγ agonists
(47).
SOCS and tumors
In human tumors, SOCS3 expression identifies macrophages with enhanced tumor killing,
whereas SOCS1 expressing macrophages (M2) favor tumor survival (48). Macrophage-specific
deletion of SOCS1 leads to reduced susceptibility to melanoma growth and colon carcinogenesis
through increased anti-tumor responses (49) and a switch to M1 polarization of tumor-associated
macrophage. In contrast, mice with a macrophage-specific deletion of SOCS3, subcutaneously
implanted with melanoma cells, did not show a difference in tumor size, although the
number of metastasis increased in these mice (37). These SOCS3-deficient macrophages
produce less IL-6 and TNF-α upon stimulation with tumor lysates due to aberrant STAT3
activity, again showing a positive link of SOCS and macrophage polarization (37).
SOCS and obesity
SOCS3 restrains macrophage responses to IL-6 and leptin that are systemically upregulated
in obesity (50). SOCS1 inhibits insulin signaling and macrophage cytokine secretion,
resulting in insulin sensitivity in spite of an obese state (17). Moreover, an increase
in SOCS1 expression in mouse macrophages inhibits LPS- and palmitate-induced TLR4
signaling and in so doing prevents systemic inflammation and hepatic insulin resistance
(17).
Conclusion and Perspectives
Given the broad role of SOCS in regulating macrophage functions in health and disease,
the modulation of macrophage-specific SOCS1 and SOCS3 expression provides new opportunities
for therapeutic manipulation of immune and inflammatory responses. However, it is
not only macrophages that are affected by SOCS proteins. Other cell types upregulate
and react to SOCS proteins to shape cellular functions. Targeting SOCS specifically
in macrophages is therefore important as an efficient means of changing the inflammatory
response.
Conflict of Interest Statement
The author declares that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.