MINI REVIEW
published: 12 June 2019
doi: 10.3389/fimmu.2019.01330
Insulin Signaling and Insulin
Resistance Facilitate Trained
Immunity in Macrophages Through
Metabolic and Epigenetic Changes
Eleftheria Ieronymaki 1,2 , Maria G. Daskalaki 1,2 , Konstantina Lyroni 1,2 and
Christos Tsatsanis 1,2*
1
Laboratory of Clinical Chemistry, University of Crete Medical School, Heraklion, Crete, Greece, 2 FORTH, Institute of
Molecular Biology and Biotechnology, Heraklion, Crete, Greece
Edited by:
Nancie J. MacIver,
Duke University, United States
Reviewed by:
Tara Marlene Strutt,
University of Central Florida,
United States
Liza Makowski,
University of Tennessee Health
Science Center, United States
*Correspondence:
Christos Tsatsanis
tsatsani@uoc.gr
Specialty section:
This article was submitted to
Immunological Memory,
a section of the journal
Frontiers in Immunology
Received: 14 March 2019
Accepted: 28 May 2019
Published: 12 June 2019
Citation:
Ieronymaki E, Daskalaki MG, Lyroni K
and Tsatsanis C (2019) Insulin
Signaling and Insulin Resistance
Facilitate Trained Immunity in
Macrophages Through Metabolic and
Epigenetic Changes.
Front. Immunol. 10:1330.
doi: 10.3389/fimmu.2019.01330
Adaptation of the innate immune system has been recently acknowledged, explaining
sustained changes of innate immune responses. Such adaptation is termed trained
immunity. Trained immunity is initiated by extracellular signals that trigger a cascade
of events affecting cell metabolism and mediating chromatin changes on genes that
control innate immune responses. Factors demonstrated to facilitate trained immunity
are pathogenic signals (fungi, bacteria, viruses) as well non-pathogenic signals such as
insulin, cytokines, adipokines or hormones. These signals initiate intracellular signaling
cascades that include AKT kinases and mTOR as well as histone methylases and
demethylases, resulting in metabolic changes and histone modifications. In the context
of insulin resistance, AKT signaling is affected resulting in sustained activation of
mTORC1 and enhanced glycolysis. In macrophages elevated glycolysis readily impacts
responses to pathogens (bacteria, fungi) or danger signals (TLR-driven signals of tissue
damage), partly explaining insulin resistance-related pathologies. Thus, macrophages
lacking insulin signaling exhibit reduced responses to pathogens and altered metabolism,
suggesting that insulin resistance is a state of trained immunity. Evidence from Insulin
Receptor as well as IGF1Receptor deficient macrophages support the contribution of
insulin signaling in macrophage responses. In addition, clinical evidence highlights altered
macrophage responses to pathogens or metabolic products in patients with systemic
insulin resistance, being in concert with cell culture and animal model studies. Herein,
we review the current knowledge that supports the impact of insulin signaling and other
insulin resistance related signals as modulators of trained immunity.
Keywords: macrophages, metabolism, epigenetic, Akt, mTOR, trained immunity
INTRODUCTION
Obesity induces metabolic inflammation, a chronic low-grade inflammation characterized by
systemic increased levels of pro-inflammatory factors and insulin resistance. Energy storage organs
like adipose tissue, liver and muscle exceed their capacity to preserve homeostasis, leading to
metabolic stress. this condition is capable of altering responses mediated by innate immune
cells. Macrophages are central mediators of inflammatory responses and are causally linked to
Frontiers in Immunology | www.frontiersin.org
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Ieronymaki et al.
Trained Immunity in Insulin Resistance
Insulin Signaling Modulates TLR
Responses in Macrophages Facilitating
Trained Immunity
obesity-related pathologic conditions, including Type 2 diabetes
(T2D) and cardiovascular disease (1).
During recent years, the dogma that only adaptive immunity
is responsible for immune memory has been challenged and
the concept of innate immune training has emerged. In
plants, invertebrates and mammals, cells of innate immunity
have been shown to possess a form of memory, also termed
“Trained Immunity.” Trained immunity is defined as persistent
alteration of innate immune responses, which depends on
prior exposure to a signal. For example, exposure to βglucan, infection or vaccination, results in elevated proinflammatory cytokines after a secondary infection. Exposure
to insulin or Saturated Fatty Acids (SFAs) also results
in altered TLR responses (2, 3) Trained immunity also
describes altered responses to pathogens occuring after severe
inflammation, such as endotoxin tolerance or Compensatory
Anti-inflammatory Response Syndrome (CARS) (4). Innate
immune cells shown to be part of “trained immunity” are
monocytes, NK cells and dendritic cells (5, 6). This review will
focus on macrophage trained immunity, which includes bone
marrow-derived monocytes/macrophages and tissue resident
macrophages that are self-renewing embryo-derived (7–10).
Innate immune training is not only associated with infectious
stimuli but also to non-infections conditions, such as obesity and
insulin resistance (3, 11, 12).
Insulin is a hormone produced from pancreatic beta
cells to regulate glucose homeostasis, fat metabolism and
cell growth. Insulin levels change in the context of diabetes
and it is now established that insulin signals in immune
cells modulate their function (13–15). Insulin mediates its
signal through insulin receptor (IR), but also through its
highly homologous Insulin-like growth factor 1 receptor
(IGF1R). Binding of insulin on IR or IGF1R results in
the phosphorylation of insulin receptor substrate 1/2
(IRS1/2) at its tyrosine residues and in the subsequent
activation of two main pathways, the Phosphoinositide3kinase(PI3K)/AKT pathway and the mitogen activated
protein kinase (MAPK) pathway (16, 17). AKT is a family
of serine/threonine kinases encoded by three highly homologous
genes (Akt1, Akt2, and Akt3) (18). It is phosphorylated by
phosphoinositide-dependent protein kinase-1(PDK1) and
mammalian target of rapamycin complex 2(mTORC2) to
regulate glucose transporters (Glut), glycogen synthase kinase
3(GSK3), forkhead box O1 transcription factor (FoxO1), and
mammalian Target of Rapamycin Complex 1 (mTORC1), and
involved in glucose uptake, glycogen synthesis, and protein
synthesis. Activation of mTORC1 from AKT is mediated
through Tuberous Sclerosis-2(TSC2) phosphorylation and
inactivation. TSC2 is a tumor suppressor that forms a complex
with TSC1 and inactivation of the TSC1/TSC2 complex
activates mTORC1 with its specific protein Raptor (19–21)
(Figure 1).
In this review we present current knowledge on
insulin signaling in innate immune cells focusing on
macrophages and how changes in insulin signaling affect
their responsiveness and provide a form of innate
immune training.
Frontiers in Immunology | www.frontiersin.org
During obesity, insulin signaling pathway is deregulated and
a state of low grade systemic inflammation is established.
Macrophages react to both TLR and insulin signals shaping
their responses. Hyperlipidemia and increased levels of SFAs
can trigger inflammation through direct binding to TLR2 and
TLR4 receptors (22). SFAs promote inflammation through
metabolic reprogramming of macrophages resulting in activation
of mTOR and lipid metabolism in vivo altering TLR4-dependent
signals (2), thus promoting training of macrophages. Priming of
macrophages can also occur by circulating bacterial endotoxin
originating from altered gut microbiome due to increased
intestinal permeability occurring in obesity (23). In addition,
increased levels of free fatty acids results in incomplete
metabolism and accumulation of fatty acid intermediates, like
diacylglycerol (DAG) and ceramides that are able to activate
several protein kinases in macrophages such as Protein Kinase
C (PKC), Janus Kinase (JNK), and IκB Kinaseβ (IKKβ) resulting
in subsequent inactivation of IRS-1 and thus inhibition of
insulin signaling both in culture and in vivo (24). Increased
insulin, amino acids and pro-inflammatory cytokines due to
chronic overnutrition in the context of obesity activate mTORC1
that mediates feedback inhibition to PI3K/AKT pathway (21).
AKT signals also regulate CCAAT-Enhancer-Binding Protein
b(CEBPb), a transcription factor that controls anti-inflammatory
gene expression and expression of the TLR signaling regulator
IRAK3 (25, 26) (Figure 1). Thus, signals initiated by metabolic
factors modulate molecules that control TLR responses.
Type 2 diabetes and obesity result in hyperglycemia, which
readily impacts macrophage responses, providing training
in macrophages. Advanced glycation end products (AGEs),
glycated proteins or lipids formed non-enzymatically due to
increased glucose concentration are recognized by receptor
of AGEs (RAGE). Stimulation of RAGE results in increased
ROS production and subsequent NF-kB activation (27). In
addition to oxidative stress, endoplasmic reticulum (ER) stress
is also induced in obesity. Stimulation of Unfolded Protein
Response(UPR) signaling pathways as a consequence of ER
stress results in activation of protein kinases IKKβ and JNK (28,
29), also impacting TLR signaling. Adipokines promote innate
immune training, such as adiponectin that suppresses TLR4
responses via induction of IRAKM in culture and in vivo (25, 30).
Adiponectin also enhances insulin sensitivity via IRS2 (31, 32).
Finally, cytokines released by activated immune cells further
contribute to inflammation and impairment of insulin signaling
in macrophages, altering innate immune responses (33).
Changes in Cell Metabolism Shape
Macrophage Training in the Context of
Insulin Signaling
Macrophage metabolism is tightly linked to their capacity to
respond to TLR signals and characterizes their polarization
status. M1 activation of macrophages, the state of activated
pro-inflammatory macrophages, is characterized by increased
2
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Ieronymaki et al.
Trained Immunity in Insulin Resistance
FIGURE 1 | Crosstalk of TLR and Insulin signals. TLR triggering initiates a cascade of events that results in activation of NFkB and AKT leading to pro- and antiinflammatory gene expression as well as genes regulating metabolism. Insulin signals also activate AKT and result in regulation of metabolic gene expression and at
the same time crosstalk with TLR signaling.
affecting innate immune responses and therefore contributing
to training.
rates of glycolysis, induction of Pentose Phosphate Pathway
(PPP), arginine conversion to nitric oxide (NO) by iNOS
and fatty acid synthesis and oxidation, essential for proinflammatory signaling and NLRP3 inflammasome activation.
Anti-inflammatory macrophages, broadly termed as M2type, display increased glutamine uptake and catabolism,
arginine metabolism by arginase-1, oxidative phosphorylation,
enhanced fatty acid oxidation and glycolysis. The importance
of glycolysis for development of M1-phenotype is supported
by the fact that deletion of Solute Carrier2A1 (Slc2A1),
which encodes Glucose Transporter1 (GLUT1), results in
reduced LPS responses and M2-like phenotype (34), while
its overexpression enhanced response to LPS and Reactive
Oxygen Species (ROS) production (35). Upon M1 activation,
expression of Fatty Acid Transporter1 (FATP1) is reduced
promoting glycolysis, as demonstrated in Fatp1−/− or FATP1overexpressing macrophages (36). GLUT1 expression controls
efferocytosis and removal of apoptotic debris. At the same time
Solute Carrier16A1 (Slc16A1) facilitates lactate release, the
by-product of aerobic glycolysis known to promote M2-type
polarization (37). Glutamine metabolism is also important
for the propagation of epigenetic modifications evident in
trained macrophages (38). Therefore, changes in macrophage
metabolism are tightly linked to their polarization status
and training.
During obesity, Adipose Tissue Macrophages (ATMs) acquire
a unique metabolic profile not characteristic of M1-type or
M2-type activation displaying both increased glycolysis and
oxidative phosphorylation. Glycolysis was found to regulate the
secretion of pro-inflammatory factors (39). In the context of
obesity, peripheral macrophages also become resistant to insulin
exhibiting increased glycolysis, important for the regulation of
their M2-like polarization status (3). Thus, insulin resistance is
associated with a distinct metabolic phenotype in macrophages
Frontiers in Immunology | www.frontiersin.org
Insulin Resistance Confers Innate Immune
Training and an M2-Like Phenotype
Macrophages express all components of insulin signaling,
indicating a functional insulin signaling cascade and develop
insulin resistance in the context of systemic insulin resistance
(3, 40). Resident macrophages obtain M1 or M2 phenotypes,
depending on the microenvironment and the stimuli present,
such as this of insulin that induces AKT kinase signals (3, 41).
In the context of insulin resistance, peripheral macrophages
acquire an anti-inflammatory M2-like phenotype, characterized
by increased expression of M2 markers and reduced secretion of
pro-inflammatory factors upon stimulation (3, 42). In addition,
insulin resistant macrophages produce reduced levels of NO
upon TLR stimulation in vivo and in culture and exhibit reduced
bactericidal capacity in vivo (3).
ATMs acquire an M1-type pro-inflammatory phenotype
in the inflamed adipose tissue during obesity (43–45). This
M1-like phenotype includes expression of both oxidative and
glycolytic genes (39, 46). M1-like ATMs express the scavenger
receptor CD36, ATP-binding cassette transporter1 (ABCA1) and
Perilipin (PLIN) as membrane markers, regulated by Peroxisome
Proliferator-activated Receptor-γ(PPARγ), and do not express
M1 markers (47).
In contrast to adipose tissue macrophages, M2-like polarized
macrophages were also found in other conditions associated
with systemic insulin resistance (obesity and type 2 diabetes),
such as cancer and cardiovascular disease (48, 49). In cancer,
for example, Tumor-Associated-Macrophages (TAMs) possess
an M2-like phenotype and support tumor growth (50). In
atherosclerosis, foam cells display M2-like properties and are
found in atheromatic plaques (11, 51). In animal models, genetic
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Trained Immunity in Insulin Resistance
deletion of IR in macrophages results in an anti-inflammatory
behavior (52) and protects against diet-induced obesity (53).
IGF-1R protein is suppressed in M1-type activated macrophages
(54, 55) and genetic ablation of IGF-1R results in suppression
of M1 responses and enhanced M2 responses in the context of
skin inflammation (42). In addition, myeloid restricted deletion
of IGF1R favors an M2-like polarization and protects mice from
sepsis and inflammatory bowel disease (3, 56, 57). Moreover,
IRS2, a substrate of both IR and IGF1R, suppresses alternative
activation of macrophages in vivo (58). All the above findings
support that inhibition of insulin signals promote an M2-like
phenotype in peripheral macrophages.
Deletion of the downstream insulin signaling components,
AKT kinases, results in differential polarization status of
macrophages. AKT2 is the predominant isoform that participates
in insulin signaling cascade (18, 59). Genetic ablation of Akt2
renders macrophages insulin resistant (3) and promotes an M2like phenotype in vivo (56). Indirect activation of mTORC1 from
AKT is important in propagating insulin signals. Deletion of
Raptor from macrophages, elevates M2 macrophage population
in LysMCre Rptorfl/fl mice (60), consistent with the effect of
TSC1 deletion, which results in sustained activation of mTORC1
and decrease of M2 population (60, 61). In contrast, lack
of TSC2 in macrophages enhanced the expression of M2
polarization markers (62). Insulin resistant macrophages, namely
macrophages from obese mice or lacking AKT2, exhibit increased
mTORC1 basal activity. Basal mTORC1 is important for their
activation status, since treatment with the mTOR inhibitor
rapamycin abrogated their M2-like phenotype and at the same
time changed their metabolism in culture (3). Hence, deletion of
molecules participating in insulin signaling such as AKT2, or the
mTORC1 regulators TSC1 or TSC2, revealed the role of insulin
signaling in modulating macrophage responses and support the
role of insulin signaling in trained immunity.
sustained alteration in gene expression regulated by DNA and
histone modifications (63). Information on DNA modifications
associated with trained immunity is limited to modifications
of the TNF locus in the context of endotoxin tolerance (64),
while most of the information available is focused on histone
modifications (65). Upon primary stimulation of myeloid cells,
a cascade of transcriptional signals ensures tight regulation
of inflammatory genes, through recruitment of transcription
factors, such as NF-kB, AP-1 and Signal Transducer and
Activator of Transcription (STAT) family members, to enhancers
and gene promoters. These transcription factors in turn recruit
chromatin modifying enzymes to enhance genome accessibility
via histone modifications. The persistence of such histone
modifications may itself affect secondary responses (8). De novo
enhancers acquire histone modifications, such as H3K4me1,
only after a primary stimulus (8).
Epigenetic reprograming is mediated by inflammatory signals
and lead to histone modifications that alter gene expression
patterns (66). Among inflammatory signals that epigenetically
reprogram macrophages are NLR Pyrin Domain Containing
Protein 3 (NLRP3) signals (6, 67). In addition, trained immunity
can be triggered by oxidized low-density lipoprotein (oxLDL)
in monocytes. OxLDL is being phagocytosed by macrophages
resulting in foam cell formation and atherosclerosis. Exposure
of cells to oxLDL leads to trimethylation of lysine 4 at
histone 3 (H3K4) in promoter regions of tnfα, il-6, il-18, the
Matrix Metalloproteinase genes mmp2, mmp9, and the scavenger
receptor cd36, which all contribute to foam cell formation in vivo
(68). Epigenetic reprogramming induced by oxLDL was found to
be regulated through mTOR-dependent oxidative stress (12). In
obesity and diabetes, hyperglycemia can trigger “hyperglycemic
memory,” characterized by sustained NF-kB gene activity due
to epigenetic marks, like increased H3K4 and reduced H3K9
methylation (69). In this context, high glucose levels induce
epigenetic changes and training of macrophages (70).
Epigenetic modifications are regulated by both immune
signaling and metabolic pathways, since metabolites such as
SAM, acetyl-CoA, NAD+ , and ATP are used as substrates or
cofactors for chromatin modifying enzymes (6, 71). As a result,
changes in metabolite concentrations regulate gene expression by
modulating the epigenome and alter chromatin dynamics (72).
Epigenetic Regulation of Trained Immunity
and the Role of Insulin Signaling
and Metabolism
The molecular basis of trained immunity is mediated by
transcriptional and epigenetic reprogramming, based on
FIGURE 2 | Obesity and insulin resistance promote macrophage training. Macrophages chronically exposed to high levels of insulin as well as SFAs, adipokines,
inflammatory cytokines, and low levels of endotoxin, all associated with obesity, obtain changes in Akt signaling, cell metabolism and epigenetic alterations in
inflammatory genes that result in altered responses, described as innate immune training.
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Trained Immunity in Insulin Resistance
and this is phenotype is also observed in macrophages from
healthy individuals treated with oxidized LDL (68, 81, 82). Oral
supplementation with the metabolic byproduct butyrate, also
decreases training of peripheral mononuclear macrophages by
oxidized LDL in obese humans with metabolic syndrome (83).
Cancer development has been associated with obesity and
insulin resistance. There is evidence that innate immune
training initiated by vaccination lowers cancer incidence (84).
Treatment with anti-diabetic drugs such as metformin has
been linked to lower risk for cancer development and improve
outcome in cancer patients (85, 86). The action of insulin
sensitizers on cancer is complex since they do not only
affect low grade systemic inflammation and cell metabolism
that impacts the disease but also directly affect cancer cell
homeostasis and survival (85). Nevertheless, utilizing trained
immunity as a therapeutic approach in cancer, cardiovascular
and infectious diseases has been proposed (87). In this context,
insulin sensitization may contribute in training macrophages for
therapeutic purposes.
For example, α-ketoglutarate produced as an intermediate by the
TCA cycle via glutaminolysis promotes alternative activation of
macrophages via epigenetic changes in M2 related genes through
JMJD3 H3K27 demethylase activity (73).
Fumarate, a key metabolite for the induction of innate
immunity, is mediated through induction of H3K4me3 and
H3K27Ac epigenetic marks on promoters of genes encoding
pro-inflammatory cytokines. Accordingly, fumarate positively
regulated the transcription of genes encoding the H3K4
demethylase KDM5 isoforms in culture and in vivo (74).
As a result, BCG-trained macrophages exhibited enhanced
mRNA levels of key glycolysis genes like hk2 and pfkp, the
promoter of which acquired more H3K4me3 and less H3K9me3
methylation marks. Therefore, cells displayed a shift toward
glycolysis and oxygen consumption and reduced glutamine
metabolism (75). In contrast, in β-glucan trained human
macrophages, a Warburg effect was observed displaying high
glycolytic rate and decreased oxidative phosphorylation (76),
supporting the hypothesis that individual training stimuli
utilize distinct metabolic programs. For example, mevalonate
alone induced trained immunity in macrophages through
changes in H3K27ac of inflammatory gene enhancers (77).
Thus, elevated glycolysis and the accumulation of the TCA
intermediate metabolites fumarate and glutamate, control
H3K4me3, and H3K27ac, forming the metabolic basis of
essential metabolo-epigenetic pathways mediating trained
immunity (78).
CONCLUSIONS
The concept of trained immunity has emerged in recent
years to explain sustained changes observed in innate
immune responses following exposure to pathogenic or
environmental stimuli. Among those, insulin signaling and
insulin resistance has been shown to promote training
of cells that may partly explain altered responses and
pathologies associated with obesity and insulin resistance
(Figure 2). The detailed molecular mechanism through which
insulin signaling contributes to trained immunity is yet to
be explored.
Insulin Resistance and Trained Immunity: A
Clinical Perspective
Dysregulated glucose metabolism and overutilization of glucose
have been shown to promote inflammation in monocytes and
macrophages from patients with atherosclerotic coronary artery
disease (CAD) (79). The cause of inflammation originates from
nutrient over-supply, glucose overutilization and imbalanced
ROS generation (79). It is well-established that monocytes from
T2D patients display enhanced activation of the inflammasome
(80). As a result the hyperglycemic environment in T2D patients
promotes training of monocytes and macrophages. Endogenous
metabolic products can also promote immunological training.
For example, monocytes derived from humans with high
levels of Lipoprotein A(Lpa), a cardiovascular risk factor
that carries oxidized phospholipids, have increased capacity
of producing pro-inflammatory cytokines upon stimulation
AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual
contribution to the work, and approved it for publication. EI,
MGD, KL, and CT drafted the manuscript.
FUNDING
This work was supported by Greek and European Union funds
under E1BM34 program of Education and Lifelong learning
EΣ5A2014-2020 Grant No MIS-5006300.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Ieronymaki, Daskalaki, Lyroni and Tsatsanis. This is an openaccess article distributed under the terms of the Creative Commons Attribution
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