mTOR- and HIF-1α−mediated aerobic glycolysis as metabolic basis
for trained immunity
Shih-Chin Cheng et al.
Science 345, (2014);
DOI: 10.1126/science.1250684
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RES EA RC H
induced during monocyte training. Biological validations were performed in human
primary monocytes and in two experimental models in vivo.
RESEARCH ARTICLE SUMMARY
IMMUNOGENETICS
mTOR- and HIF-1α–mediated aerobic
glycolysis as metabolic basis for
trained immunity
Shih-Chin Cheng, Jessica Quintin, Robert A. Cramer, Kelly M. Shepardson, Sadia Saeed,
Vinod Kumar, Evangelos J. Giamarellos-Bourboulis, Joost H. A. Martens,
Nagesha Appukudige Rao, Ali Aghajanirefah, Ganesh R. Manjeri, Yang Li,
Daniela C. Ifrim, Rob J. W. Arts, Brian M. J. W. van der Meer, Peter M. T. Deen,
Colin Logie, Luke A. O’Neill, Peter Willems, Frank L. van de Veerdonk,
Jos W. M. van der Meer, Aylwin Ng, Leo A. B. Joosten, Cisca Wijmenga,
Hendrik G. Stunnenberg, Ramnik J. Xavier, Mihai G. Netea*
INTRODUCTION: Trained immunity refers
to the memory characteristics of the innate
immune system. Memory traits of innate
immunity have been reported in plants and
invertebrates, as well as in mice lacking
functional T and B cells that are protected
against secondary infections after exposure to certain infections or vaccinations.
The underlying mechanism of trained immunity is represented by epigenetic programming through histone modifications,
leading to stronger gene transcription
upon restimulation. However, the specific
cellular processes that mediate trained
immunity in monocytes or macrophages
are poorly understood.
METHODS: We studied a model of trained
immunity, induced by the β-glucan component of Candida albicans, that was
previously shown to induce nonspecific
protection against both infections and malignancies. Genome-wide transcriptome
and histone modification profiles were
performed and pathway analysis was applied to identify the cellular processes
Glucose
Glucose
Akt
Pyruvate
C. albicans
Lactate
Pyruvate
HIF-1α
DISCUSSION: The shift of central glucose
Aerobic glycolysis
(Warburg efect)
Oxidative
phosphorylation
Lactate
trained monocyte
S. aureus sepsis
S. aureus sepsis
Susceptible
pathways, glycolysis genes were strongly upregulated in terms of histone modification
profiling, and this was validated by RNA
sequencing of cells from β-glucan–treated
mice. The biochemical characterizations of
the β-glucan–trained monocytes revealed
elevated aerobic glycolysis with reduced
basal respiration rate, increased glucose
consumption and lactate production, and
higher intracellular ratio of nicotinamide
adenine dinucleotide (NAD+) to its reduced
form (NADH). The dectin-1–Akt–mTOR–
HIF-1α pathway (mTOR, mammalian target
of rapamycin; HIF-1α, hypoxia-inducible
factor–1α) was responsible for the metabolic shift induced by β-glucan. Trained
immunity was completely abrogated in
monocytes from dectin-1–deficient patients.
Blocking of the mTOR–HIF-1α pathway by
chemical inhibitors inhibited trained immunity. Mice receiving
metformin, an adenoON OUR WEB SITE
sine monophosphate–
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activated protein kinase
at http://dx.doi
(AMPK) activator that
.org/10.1126/
science.1250684
subsequently inhibits
mTOR, lost the trained
immunity–induced protection against lethal C. albicans infection. The role of the
mTOR–HIF-1α pathway for β-glucan–
induced innate immune memory was further validated in myeloid-specific HIF-1α
knockout (mHIF-1α KO) mice that, unlike
wild-type mice, were not protected against
Staphylococcus aureus sepsis.
β-glucan
Dectin-1
Oxidative
phosphorylation
Naïve monocyte
(resting monocyte)
mTor
RESULTS: In addition to immune signaling
Protected
Dead
Aerobic glycolysis as metabolic basis for trained immunity. In naïve macrophages during aerobic conditions, glucose metabolism is mainly geared toward oxidative phosphorylation
providing adenosine triphosphate (ATP) as the energy source. In contrast, long-term functional
reprogramming during trained immunity requires a metabolic shift toward aerobic glycolysis
and is induced through a dectin-1–Akt–mTOR–HIF-1α pathway.
SCIENCE sciencemag.org
metabolism from oxidative phosphorylation to aerobic glycolysis (the “Warburg
effect”) meets the spiked need for energy
and biological building blocks for rapid
proliferation during carcinogenesis or
clonal expansion in activated lymphocytes.
We found that an elevated glycolysis is the
metabolic basis for trained immunity as
well, providing the energy and metabolic
substrates for the increased activation of
trained immune cells. The identification
of glycolysis as a fundamental process in
trained immunity further highlights a
key regulatory role for metabolism in innate host defense and defines a potential
therapeutic target in both infectious and
inflammatory diseases. ■
The list of author affiliations is available in the full article online.
*Corresponding author. E-mail: mihal.netea@radboudumc.nl
Cite this article as S.-C. Cheng et al., Science 345, 1250684
(2014). DOI: 10.1126/science.1250684
26 SEP TEMBER 2014 • VOL 345 ISSUE 6204
Published by AAAS
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R ES E A RC H
RESEARCH ARTICLE
◥
and mammals (7), less is known regarding the molecular pathways and downstream mechanisms
that lead to trained immunity.
IMMUNOGENETICS
Transcriptome and epigenetics
of monocytes
mTOR- and HIF-1a–mediated
aerobic glycolysis as metabolic
basis for trained immunity
Candida albicans and its main cell wall constituent, b-glucan, induce trained innate immune
memory both in vitro and in vivo (7). We performed an unbiased assessment of whole-genome
mRNA expression, histone methylation, and acetylation patterns after training human primary
monocytes with b-glucan, the major Candida
cell wall structure that mediates trained immunity, which induces nonspecific protection against
both infections and malignancies (11). An in
vitro experimental model of b-glucan–induced
trained immunity was established in monocytes
(Fig. 1A). b-Glucan training of cells induced a
potentiated cytokine production upon restimulation with lipopolysaccharide (LPS) 7 days
later (Fig. 1B). An enhanced response was also
observed after stimulation with the TLR2 ligand
Pam3Cys or with nonrelated Gram-negative and
Gram-positive bacteria (fig. S1). Assessment of
histone 3 Lys4 trimethylation (H3K4me3) and
histone 3 Lys27 acetylation (H3K27Ac) identified promoters that were specifically induced by
b-glucan training (Fig. 1C). Pathway analysis of
the promoters potentiated by b-glucan identified innate immune and signaling pathways upregulated in trained cells that are responsible
for the induction of trained immunity (7, 12).
In addition to immune signaling pathways,
epigenetic profiling of trained monocytes on the
basis of both methylation and acetylation patterns identified a signature associated with central metabolism (fig. S2) and an increase in the
promoters of genes encoding enzymes involved
in glycolysis and its master regulator mTOR (mammalian target of rapamycin) (Fig. 1, D and E).
Furthermore, after priming of monocytes with
b-glucan, genes involved in glycolysis, such as
hexokinase and pyruvate kinase, were epigenetically up-regulated 1 week later (Fig. 1F and fig.
S3). The gene expressing mTOR and the glycolytic genes that are targets of the transcription
factor HIF1a were also enhanced by b-glucan (fig.
S4). In line with this, HIF-1a activation was increased in b-glucan–trained monocytes (fig. S5).
In addition, glycolysis genes were also up-regulated
in vivo in mice challenged with b-glucan, as revealed by total RNA sequencing analysis in splenocytes of these mice (Fig. 1G and fig. S6).
Shih-Chin Cheng,1 Jessica Quintin,1 Robert A. Cramer,2 Kelly M. Shepardson,2
Sadia Saeed,3 Vinod Kumar,4 Evangelos J. Giamarellos-Bourboulis,5 Joost H. A. Martens,3
Nagesha Appukudige Rao,3 Ali Aghajanirefah,3 Ganesh R. Manjeri,6 Yang Li,4
Daniela C. Ifrim,1 Rob J. W. Arts,1 Brian M. J. W. van der Meer,4 Peter M. T. Deen,7
Colin Logie,3 Luke A. O’Neill,8 Peter Willems,6 Frank L. van de Veerdonk,1
Jos W. M. van der Meer,1 Aylwin Ng,9,10 Leo A. B. Joosten,1 Cisca Wijmenga,4
Hendrik G. Stunnenberg,4 Ramnik J. Xavier,9,10 Mihai G. Netea1*
Epigenetic reprogramming of myeloid cells, also known as trained immunity, confers
nonspecific protection from secondary infections. Using histone modification profiles of
human monocytes trained with the Candida albicans cell wall constituent b-glucan, together
with a genome-wide transcriptome, we identified the induced expression of genes involved
in glucose metabolism. Trained monocytes display high glucose consumption, high lactate
production, and a high ratio of nicotinamide adenine dinucleotide (NAD+) to its reduced
form (NADH), reflecting a shift in metabolism with an increase in glycolysis dependent on
the activation of mammalian target of rapamycin (mTOR) through a dectin-1–Akt–HIF-1a
(hypoxia-inducible factor–1a) pathway. Inhibition of Akt, mTOR, or HIF-1a blocked monocyte
induction of trained immunity, whereas the adenosine monophosphate–activated protein
kinase activator metformin inhibited the innate immune response to fungal infection. Mice
with a myeloid cell–specific defect in HIF-1a were unable to mount trained immunity against
bacterial sepsis. Our results indicate that induction of aerobic glycolysis through an
Akt–mTOR–HIF-1a pathway represents the metabolic basis of trained immunity.
n classical descriptions of host defense mechanisms, innate immune responses that are
rapid, are nonspecific, and lack memory are
distinguished from specific T and B cell–
dependent immune responses, which are
highly specific and have the capacity to build
immunological memory. The hypothesis that
the innate immune system is incapable of mounting adaptive responses (1) is contradicted by
studies showing that organisms lacking a specific
immune system, such as plants or insects, are
I
1
Department of Internal Medicine, Radboud University Medical
Center, 6525 GA Nijmegen, Netherlands. 2Department of
Microbiology and Immunology, Geisel School of Medicine at
Dartmouth, Hanover, NH 03755, USA. 3Department of
Molecular Biology, Faculties of Science and Medicine, Nijmegen
Centre for Molecular Life Sciences, Radboud University, 6500
HB Nijmegen, Netherlands. 4Department of Genetics, University
of Groningen, University Medical Center Groningen, Groningen,
Netherlands. 54th Department of Internal Medicine, University
of Athens Medical School, 12462 Athens, Greece. 6Department
of Biochemistry, Faculties of Science and Medicine, Nijmegen
Centre for Molecular Life Sciences, Radboud University, 6500
HB Nijmegen, Netherlands. 7Department of Physiology,
Radboud University Medical Center, 6525 GA Nijmegen,
Netherlands. 8School of Biochemistry and Immunology,
Trinity Biomedical Sciences Institute, Trinity College Dublin,
Dublin 2, Ireland. 9Center for Computational and Integrative
Biology and Gastrointestinal Unit, Massachusetts General
Hospital, Harvard School of Medicine, Boston, MA 02114,
USA. 10Broad Institute of MIT and Harvard, Cambridge, MA
02142, USA.
*Corresponding author. E-mail: mihai.netea@radboudumc.nl
SCIENCE sciencemag.org
able to respond adaptively to infection (2, 3) and
that innate immune cells, such as macrophages,
have adaptive characteristics (4). In line with the
proposal that there are nonspecific adaptive
responses in the innate immune system, T and
B cell–independent protective effects of monocytes and natural killer (NK) cells have been
demonstrated in models of bacterial and viral
infections, respectively (5, 6). Furthermore, epigenetic reprogramming at the level of histone
H3 methylation has been proposed as the molecular mechanism responsible for long-term
memory of innate immunity (5, 7), and this
process has been termed trained immunity.
Initiation of innate immune memory through
trained immunity is likely to be responsible for the
nonspecific protective effects of certain vaccines
(8). Furthermore, the increased inflammatory responsiveness of monocytes and macrophages due to
trained immunity appears to play a central role in
inflammatory diseases (9). From this perspective,
the capacity of innate immunity to mount adaptive
responses both redefines the function of innate immunity and identifies a potential therapeutic target
in human diseases. It is thus essential to understand
the cellular and molecular mechanisms that mediate trained immunity, in hopes of harnessing their
therapeutic potential. Although epigenetic modifications are known to underlie information storage
during innate immune memory in both plants (10)
Glycolysis and monocytes
Monocytes from peritoneal exudates rely on glycolysis as a main energy source (13). The role of
glucose as an energy substrate for monocytes is
demonstrated by the blockade of monocyte stimulation and trained immunity by incubation of cells
with 2-deoxy-D-glucose, a glucose analog that cannot be metabolized by the cells and inhibits glycolysis (fig. S7). This is in line with observations that
activated macrophages, dendritic cells, and TH1 and
TH17 lymphocytes undergo a switch from oxidative
phosphorylation to aerobic glycolysis (14).
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Fig. 1. Trained immunity in monocytes. (A) Schematic of in vitro trained immunity experimental
setup. (B) TNF-a levels after 7 days in b-glucan–
treated cells. Data are means T SEM (n = 8, *P <
0.05, Wilcoxon signed-rank test). (C) Genomewide H3K4me3 (red) and H3K27Ac (blue) epigenetic modifications 7 days after b-glucan treatment.
Ratios of b-glucan/RPMI for both H3K4me3 and
H3K27Ac modification were calculated for each
promoter. The promoters that display significantly
higher or lower ratio (P ≤ 0.05, t test) relative to
median values are called b-glucan–induced promoters and b-glucan–repressed promoters, respectively. Box plots show distributions of the
sequence read density (reads per kilobase) for
all promoters, b-glucan–induced promoters, and
b-glucan–repressed promoters in each data set.
In each box plot, the band inside each box (midpoint) represents the median value, and upper and
lower borders of the box represent the Q3 (third
quartile) and Q1 (first quartile) values, respectively.
The upper line represents the maximum value within the upper bound [Q3 + 1.5 × (Q3 – Q1)]; the
lower line represents the minimum value within
the lower bound [Q1 – 1.5 × (Q3 – Q1)]. Dots represent observed points outside the upper and
lower bound. (D) Epigenetic modifications in the
promoter regions of the genes involved in glycolysis and mTOR pathways. The box plots were
analyzed as in Fig. 1C. (E) Schematic representation of the up-regulated enzymes (red) in the glycolysis pathway. (F) Representative screen shots
of H3K4me3 (red) and H3K27Ac (blue) modifications in the promoter region of pyruvate kinase
(PKM) and hexokinase. (G) Differential gene expression analysis between the b-glucan–treated
group and the control group. Genes in the glycolysis pathway that are up-regulated by the
b-glucan training are highlighted in the box at
right. The colors in the heat map represent the
normalized RNA levels of identified differential
expressed genes (false discovery rate = 0.01,
relative change ≥ 1.5) in three mice per group.
Consistent with these findings, monocytes
trained with b-glucan showed a reduced baseline
oxygen consumption on day 7 relative to naïve
cells; this finding is compatible with the hypothesis
that these cells underwent a shift from oxidative
metabolism toward glycolysis. Moreover, trained
cells showed a decreased maximal rate of oxygen
consumption after complete uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone
(FCCP), a chemical substrate that permeabilizes
mitochondrial membranes and uncouples electron
transport systems from the oxidative phosphorylation systems (Fig. 2, A and B), whereas the rate
of proton leak–dependent oxygen consumption
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was not altered (table S1). The latter result indicates
a reduction of the capacity of the mitochondrial
electron transport chain (ETC) as observed after a
period of hypoxia (15). Hypoxia decreases the activity of the ETC complexes I and IV through HIF-1a
(16). This hypothesis was reinforced by observations of increased glucose consumption (Fig. 2C),
lactate production (Fig. 2D), and ratio of nicotinamide adenine dinucleotide (NAD+) to its reduced
form (NADH) (Fig. 2E) in trained monocytes.
Differences in glucose consumption did not
offset the high glucose concentrations in the
RPMI medium, which suggests that glucose
availability is not the limiting factor for the ob-
26 SEPTEMBER 2014 • VOL 345 ISSUE 6204
served training phenotype (fig. S8). In addition,
the training effect induced by b-glucan was likely
not due to the presence of pyruvate in the culture
medium, an intermediate metabolite in glycolysis,
because training occurred even when medium
devoid of pyruvate was used during the training
process (fig. S9).
Earlier studies have shown that a high cellular NAD+/NADH ratio acts through sirtuin-1 to
decrease the mitochondrial content (17). This
mechanism may explain the observed b-glucan–
induced reduction in ETC capacity. In contrast,
LPS stimulation leads to a strong but transient
increase in the glycolytic process in monocytes
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RE S E ARCH | R E S E A R C H A R T I C L E
Fig. 2. Physiology after
b-glucan treatment. (A)
Representative oxygen consumption rate of untreated
(RPMI, black) and b-glucan–
trained (red) monocytes as
determined by high-resolution
respirometry (Oxygraph;
OROBOROS Instruments,
Innsbruck). (B) Baseline
(basal oxygen consumption
before oligomycin treatment,
upper panel) and maximum
oxygen consumption rate
(maximum oxygen consumption upon FCCP treatment,
lower panel) of untrained
(open bar) and b-glucan–
trained (solid bar) monocytes
determined by respirometry
and normalized to the leak
oxygen consumption. (C and
D) Kinetic changes of glucose
consumption (C) and lactate
production (D) from days 1, 3,
and 7 of untreated and
b-glucan–trained monocytes.
(E) Kinetics of NAD+/NADH
ratio determined at days 1, 3,
and 7. In (B) to (E), data
are means T SEM (n = 5 to 8,
*P < 0.05, Wilcoxon signedrank test).
(fig. S10); this finding supports the suggestion
that, although the acute response of monocytes
to LPS is characterized by glycolysis (18), at later
time points this response switches to oxidative
phosphorylation—a process that subsequently induces immune tolerance by activation of sirtuin-1
and sirtuin-6 histone deacetylases (19). In contrast
to LPS-induced tolerance, b-glucan training inhibited the expression of Sirtuin1 (fig. S11). Moreover,
the addition of resveratrol, a sirtuin-1 activator,
during the first 24 hours of b-glucan training partially inhibited the enhanced interleukin-6 (IL-6)
production (fig. S11). This suggests that sirtuin deacetylases play a role in the modulated monocyte
functional phenotype and highlights the complex
interaction between the intermediate metaboSCIENCE sciencemag.org
lites and subsequent immune responses through
chromatin-modifying enzymes (20).
mTOR acts as a sensor of the metabolic environment (21) and functions as a master regulator
of glucose metabolism in activated lymphocytes
(22). Epigenetic signals at promoters of genes
in the mTOR pathway were significantly higher
in b-glucan–trained monocytes (paired t test,
P < 0.001) than in cells exposed to culture medium (Fig. 3A). Target genes of mTOR, such as
EIF4EBP1, displayed a similar pattern (Fig. 3B).
In line with this finding, mTOR phosphorylation
was up-regulated in trained monocytes as assessed by Western blot (Fig. 3C). Monocytes isolated from patients with a complete deficiency in
dectin-1 (23) failed to activate mTOR upon stim-
ulation with b-glucan (Fig. 3D) and failed to enhance tumor necrosis factor (TNF) production
upon LPS restimulation (fig. S12), supporting the
hypothesis that mTOR phosphorylation is dependent on the dectin-1 C-type lectin receptor.
Glycolysis in trained immunity
As the data presented above demonstrate activation of mTOR and glycolysis in trained monocytes,
we next investigated the causality between these
two processes by blocking glycolysis during b-glucan
training. Inhibition of mTOR with rapamycin
during the first day of stimulation resulted in
a dose-dependent inhibition of the training
effect induced by b-glucan (Fig. 3E). Indirect inhibition of mTOR with AICAR, an adenosine
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monophosphate–activated protein kinase (AMPK)
activator, had similar effects (Fig. 3F). On the basis
of observations that mTOR induction of glycolysis
is mediated through activation of HIF-1a and
stimulation of glycolytic enzymes (24) and that
rapamycin inhibits HIF-1a expression (25), we
assessed the effect of a HIF-1a inhibitor on monocyte training. We found that the HIF-1a inhibitor
ascorbate also blocked trained immunity in a
dose-dependent manner (Fig. 3F).
We further investigated the link between metabolic effects and epigenetic changes by assessing the effects of the epigenetic inhibitors MTA
(methylthioadenosine, a methyltransferase inhibitor) and ITF (ITF2357, a histone deacetylase
inhibitor) during the training setup on the lactate measurements. As expected, the epigenetic
inhibitors had no effect on lactate production in
the acute phase (24 hours after b-glucan stimulation; fig. S13). However, lactate production
was significantly reduced in the trained monocytes on day 7 when MTA was added to monocytes with b-glucan during the first 24 hours in
the incubation period (fig. S13), which suggests
that histone methylation also partially modifies the
induction of glycolysis in the trained monocytes.
Monocyte mTOR activation
Fig. 3. mTOR signaling in b-glucan–treated monocytes. (A) Schematic representation of up-regulated
enzymes (red) in mTOR signaling pathway in b-glucan–trained monocytes. (B) Screen shot of H3K4me3
(red) and H3K27Ac (blue) modification in the promoter region of EIF4EBP1 (coding region denoted at
bottom), the main target of mTOR, in both RPMI- and b-glucan–treated monocytes. (C) Western blot
from cell lysate harvested at day 7 after RPMI or b-glucan treatment. Antibodies specific for endogenous
phospho-mTOR (p-mTOR), total mTOR, phospho-AMPK, AMPK, and actin were used to blot the total
and phospho proteins, respectively. Representative blots of five independent experiments are shown. The
p-mTOR/mTOR ratio is shown as a bar chart (n = 5, P = 0.0625, Wilcoxon signed-rank test). (D to F) The
endogenous p-mTOR status of dectin-1–deficient patients was determined by Western blot (D) from cell
lysate harvest at day 7 after RPMI of b-glucan treatment and probed with antibodies to p-mTOR and
total mTOR, respectively. The p-mTOR/mTOR ratio is shown as a bar chart. Relative cytokine production
was determined from cells incubated with rapamycin (mTOR inhibitor) (E) and with AICAR (AMPK
inhibitor) and ascorbate (HIF-1a inhibitor) (F) in a dose-dependent manner. In (E) and (F), data are
means T SEM (n = 6, *P < 0.05, Wilcoxon signed-rank test).
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Activation of mTOR by insulin or colonystimulating factors such as GM-CSF (granulocytemacrophage colony-stimulating factor) is mediated
by intermediary activation of the Akt-PI3K (phosphatidylinositol 3-kinase) pathway (26). A similar
signal route is induced in monocytes by b-glucan,
as stimulation with b-glucan induced a strong
phosphorylation of Akt (Fig. 4A). This effect was
again dectin-1–dependent, being absent in monocytes isolated from dectin-1–deficient patients
(Fig. 4B). Inhibition of Akt phosphorylation also
resulted in down-regulation of mTOR activation
(Fig. 4C), demonstrating the relationship between
Akt and mTOR activation. Finally, the Akt inhibitor wortmannin inhibited monocyte training by
b-glucan in a dose-dependent manner (Fig. 4D).
Epigenetic reprogramming of monocytes by
trained immunity has been reported as a mechanism of nonspecific protection in different models.
Mice were protected from lethal disseminated
candidiasis after an initial nonlethal Candida
albicans infection (7). Similarly, b-glucan also
induced protection against infection with a lethal
Staphylococcus aureus inoculum (27), while Bacillus
Calmette-Guérin (BCG) vaccination protected mice
from systemic candidiasis (5). We first assessed
whether metformin—which acts through AMPK
activation and subsequently mTOR inhibition
(28) and is commonly used for the treatment of
type 2 diabetes (29)—abrogates the protective
effects in these experimental models. In vitro,
metformin suppressed trained immunity induced by b-glucan (Fig. 4E), and administration
of metformin to mice during and after primary
infection with a low-inoculum C. albicans inhibited
the protective effects induced by it against secondary disseminated candidiasis (Fig. 4F), demonstrating that mTOR-mediated effects mount
a protective trained immunity in vivo.
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RE S E ARCH | R E S E A R C H A R T I C L E
Fig. 4. Akt–mTOR–HIF-1a
pathway downstream of
b-glucan stimulation. (A)
Monocytes were treated with
either RPMI or b-glucan in
the presence or absence of
wortmannin, a PI3K inhibitor.
GM-CSF stimulation was
included as a positive
control. Cell lysates were
harvested at 5, 15, 30, 60,
and 120 min. Akt phosphorylation and actin level were
blotted with specific antibodies to p-Akt and actin.
Representative blots from
three independent
experiments are shown.
(B) Akt phosphorylation and
p-Akt/Akt ratio induced by
b-glucan from dectin-1 were
determined by Western blot
by specific antibodies to
p-Akt, total Akt, and actin.
(C) Effects of PI3K inhibitors
on Akt and mTOR phosphorylation in b-glucan–treated
monocytes were determined
by Western blot by probing
with specific antibodies to
p-AKT and p-mTOR. (D)
Monocytes were treated
with b-glucan in the presence
of wortmannin in a dosedependent manner. Cytokine
levels after 7 days were
determined by enzymelinked immunosorbent assay.
(E) Relative cytokine production was determined from
cells incubated with metformin (AMPK inhibitor) in a
dose-dependent manner.
(F) Survival of wild-type
C57BL/6J mice infected with
live C. albicans after training
with PBS or b-glucan. Metformin
or PBS was given from 1 day
before the first nonlethal dose
of live C. albicans challenge until
3 days after challenge on a daily
basis. (G) Wild-type (WT) and
HIF-KO alveolar macrophages
at a concentration of 8 × 104
were incubated with PBS or
curdlan (100 mg/ml) for 1 hour.
Resazurin was added and absorbance was recorded every 30 min for 24 hours. Inset (*) shows absorbance values at the 20-hour time point. Data are representative of
three biological replicates. (H) Survival curve of wild-type or mHIF-1a KO mice primed with b-glucan and challenged with a lethal dose of S. aureus infection. In (D) and (E),
data are means T SEM (n = 6, *P < 0.05, Wilcoxon signed-rank test). In (F) and (H), a log-rank test was used to assess significance of the survival curves (*P < 0.05).
We assessed whether the effects of mTOR
were mediated at the level of innate immunity
but not at the level of adaptive T and B cell
immunity elicited during vaccination. An experimental model of b-glucan–induced protection
against S. aureus sepsis can be observed in
myeloid cell–specific HIF-1a conditional knockSCIENCE sciencemag.org
out mice (mHIF-1a KO) (30). These mice are unable to mount glycolysis specifically in cells of the
myeloid lineage. We assessed the metabolic activity of wild-type and mHIF-1a KO macrophages
when stimulated with b-glucan. mHIF-1a KO
macrophages showed increased chemical reduction of the metabolic indicator resazurin (Fig.
4G), consistent with the hypothesis that HIF1a induces the switch to aerobic glycolysis in
response to b-glucan. In this model, the cells do
not undergo the switch in the absence of HIF1a and are “metabolically” dysregulated. Whereas
b-glucan increased the survival of wild-type mice
infected with S. aureus from 40% to 90%, the
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Fig. 5. Model of metabolic activation of trained monocytes, characterized
by a shift toward increased aerobic glycolysis and decreased oxidative
phosphorylation. Upon b-glucan/dectin-1 recognition, the AKT–mTOR–HIF-1a pathway is activated and shifts the glucose metabolism from oxidative phosphorylation to aerobic glycolysis.
The activated glycolysis state prepares b-glucan–trained monocytes to respond to stimulation in a robust manner. The
potential role of rapamycin and metformin in inhibition of trained immunity is also depicted. The metabolic differences
between a trained monocyte and naïve monocytes are summarized at the right.
induction of trained immunity was completely
abrogated in mHIF-1a KO mice (Fig. 4H). To
further dissect which pathways are modulated
in the mHIF-1a KO mice, we performed RNA sequencing and compared the differential RNA
expression profiles of wild-type and mHIF-1a KO
mice. Several interesting genes were specifically
up-regulated in wild-type but not in mHIF-1a KO
mice (fig. S14 and table S2), including those encoding beclin-1 (an autophagy-related protein),
STK11 (an AMPK-related serine-threonine kinase),
JHDM1D (jumonji C domain containing histone
demethylase), and the FOXO4 transcription factor involved in Akt-PI3K stimulation. Thus, these
results demonstrate that stimulation of HIF-1a–
mediated glycolysis in myeloid cells is crucial for
mounting trained immunity in vivo.
Discussion
The role of histone methylation as a mediator
of short-term innate immunological memory
in macrophages has been described (7) and has
been referred to as a latent enhancer for the epigenetic elements that mediate this phenomenon
(31). In this study, whole-genome epigenetic profiling of histone modifications and RNA sequencing analysis have identified both immunologic and
metabolic pathways stimulated during trained
immunity. A cyclic adenosine monophosphate–
dependent pathway mediating trained immunity in monocytes has also been described in
an accompanying manuscript (12). In the present
study, we identified the metabolic pathways induced in trained monocytes, demonstrating a
metabolic switch toward aerobic glycolysis, which
is in turn crucial for the maintenance of trained
immunity (Fig. 5).
A metabolic switch toward aerobic glycolysis was earlier reported to be a feature of cell
activation and proliferation [such an effect was
first described in neoplastic cells and termed the
Warburg effect (32)] while also playing a role in
effector T helper lymphocytes (33) and activated
macrophages (34). The elevated glycolysis metabolism observed in trained monocytes might be
necessary to equip and prepare cells to respond
to the intruding pathogens in a robust and rapid
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manner through proinflammatory cytokine production and possibly also through enhanced phagocytosis capacity (35).
Although we observed trained immunity in
monocytes, this response should not be restricted
to cells in the monocyte lineage. Recently, adaptive features of NK cells have been demonstrated
to be involved in resistance to reinfection with
viruses (6, 36). The specific NK memory cells, like
T cells, rapidly proliferate, degranulate, and produce cytokine upon activation. However, it remains
to be determined whether metabolic rewiring
also plays a role in NK or in other innate immune cells, such as dendritic cells. In addition,
it is of interest to determine whether training
is contact-dependent or could also be induced
by soluble mediators. This is an important question in the field of autoinflammatory and autoimmune diseases, because these diseases are
worsened by the unregulated cytokine production. Our results suggest that proinflammatory
cytokines such as IL-1b could also induce trained
immunity in monocytes in vitro (fig. S15). This
hypothesis is further supported by nonspecific
protective effects induced by IL-1b, even when
injected several days before an experimental infection is induced (37).
One important aspect to note is that the molecular mechanisms investigated in the present
study focused on trained immunity in the first
7 days after the initial stimulus. This is the
crucial period during which trained immunity
offers protection in newborn children against
perinatal sepsis (38) and thus is relevant from a
biological and clinical point of view. Longerlasting in vivo effects of trained immunity have
been demonstrated in humans (5), and it is
important to assess whether these later effects
are mediated through similar mechanisms. However, any such later effects are also likely to be
exerted at the level of bone marrow myeloid cell
progenitors, as recently demonstrated in the case
of Toll-like receptor (TLR)–induced tolerance (39).
Our study introduces an interesting preliminary step in understanding the glycolytic process
in trained immunity. Hypoxia and glycolysis enhance the proliferative response of macrophages
26 SEPTEMBER 2014 • VOL 345 ISSUE 6204
to CSF-1 (40) and sustain the survival of activated
dendritic cells (41). Soluble b-glucan from Grifola
frondosa induces macrophage proliferation (42),
although we were not able to observe these effects with trained monocytes by Candida b-glucan
(7). However, epigenetic profiling has identified
a cell cycle activation signal in b-glucan–trained
cells (12), and it is tempting to speculate that
trained monocytes are not only capable of increased cytokine production but also primed to
respond to proliferative signals, although this
remains to be demonstrated. Finally, the identification of glycolysis as a fundamental process in
trained immunity further highlights a key regulatory role for metabolism in innate host defense
and also defines a novel therapeutic target in
both infectious and inflammatory diseases (9).
Materials and methods
Isolation of primary human monocytes
Blood was collected from human healthy volunteers and two dectin-1–deficient patients after
written informed consent (Ethical Committee
Nijmegen-Arnhem, approval no. NL32357.091.10).
Peripheral blood mononuclear cells (PBMCs)
were isolated by differential centrifugation using
Ficoll-Paque (GE Healthcare, Diegem, Belgium)
from buffy coats obtained from Sanquin Bloodbank, Nijmegen, Netherlands. Monocytes were
purified by MACS depletion of CD3-, CD19-, and
CD56-positive cells from the PBMCs; CD3 MicroBeads (130-050-101), CD19 MicroBeads (130-050301), and CD56 were purchased from Miltenyi
Biotec (Leiden, Netherlands) and used according
to the manufacturer’s protocol. Efficacy of depletion was controlled by flow cytometry (FC500;
Beckman-Coulter, Woerden, Netherlands) and
was higher than 98%.
Genome-wide sequence analysis
For chromatin immunoprecipitation (ChIP) analysis or RNA sequencing, 10 × 106 CD3–CD19–CD56–
pure monocytes were plated on 100-mm dishes.
Monocytes were preincubated with cell culture
medium (RPMI) or b-glucan (5 mg/ml) for 24 hours
in a total volume of 10 ml. After 24 hours, cells were
sciencemag.org SCIENCE
RE S E ARCH | R E S E A R C H A R T I C L E
washed to remove the stimulus and were resuspended in RPMI supplemented with 10% human
pool serum. Monocytes were collected before and
6 days after the incubation for ChIP or RNA sequencing. For RNA sequencing, monocytes were
collected in TRIzol reagent (Invitrogen, Bleiswijk,
Netherlands). The purified materials were then processed to generate genomic DNA for WGBS, RNA
(Trizol extraction according to manufacturer instructions; Agilent BioAnalyser RIN >8), and chromatin by fixing the cells in 1% formaldehyde.
Reagents
Candida albicans b-1,3-(D)-glucan (b-glucan) was
kindly provided by D. Williams (East Tennessee
State University). Reagents used were as follows:
LPS (E. coli 0B5/B5, Sigma, Diegem, Belgium),
rapamycin (Sigma, R0395), metformin (R&D, AF
1730, Abingdon, UK), AICAR (Sigma, A9978), ascorbate (Sigma, A4034), wortmannin (InvivoGen,
tlrl-wtm, Toulouse, France). C. albicans ATCC MYA3573 (UC 820) cells were heat-inactivated for
30 min at 95°C.
Stimulation experiments
For training, monocytes were preincubated
with b-glucan (10 mg/ml) for 24 hours. After 7 days,
cells were restimulated with various microbial
ligands: LPS (10 ng/ml), Pam3Cys (10 g/ml),
and heat-killed S. aureus or heat-killed E. coli
(both at 106 microorganisms/ml). After 24 hours,
supernatants were collected and stored at –20°C
until cytokine measurement. All the cytokine measurements presented were from at least six donors.
To address the HIF-1a–AMPK–mTOR pathway
in trained immunity, we added the specific inhibitors together with b-glucan for the first 24 hours
in different doses as follows: rapamycin from 1 to
100 nM, metformin from 0.3 to 30 mM, AICAR
from 5 to 500 nM, and ascorbate at 5 and 50 mM.
ChIP-seq data analysis
H3K4me3 and H3K27ac ChIP, sequencing, and
processing of the data were performed as described (7). The detailed data have been deposited in the GEO database with accession number
GSE57206. Sequenced reads of 42-bp length were
mapped to human genome (NCBI hg19) using
bwa-alignment package mapper (43). ChIP-seq
data sets were normalized as described (44), and
the sequenced reads were directionally extended
to 300 bp, corresponding to the original length
of sequenced DNA fragments. For each base pair
in the genome, the number of overlapping sequence reads was determined, averaged over a
10-bp window, and visualized in UCSC browser
(http://genome.ucsc.edu). These normalized tracks
were used to generate the genome browser screen
shots. Putative H3K4me3- and H3K27ac-enriched
regions in the genome were identified by using
MACS (45) with P < 10−8. All the transcription
start sites (T1 kb) of genes with significant H3K4me3
signal were regarded as active promoters. H3K4me3
and H3K27ac signals at all active promoters were
estimated, and log2 ratios of ChIP-seq signal
between treatment and control samples were
calculated. Promoters that showed an absolute
SCIENCE sciencemag.org
deviation of 2 times the median (median T 2 ×
MAD) of the ratio of ChIP-seq signal (treatment/
control) were regarded as regulated promoters
(induced or repressed). Sequence reads counted
from the normalized ChIP-seq data sets were
used to generate the box plots.
Metabolite measurements
Culture medium was collected at days 1, 3, and 7.
The glucose and lactate concentrations within the
medium were determined by Glucose Colorimetric
Assay Kit (K686-100; Biovision, Milpitas, CA) and
Lactate Colorimetric Assay Kit (K627-100, Biovision), respectively. NAD+ and NADH concentration
were determined by NAD/NADH Quantification
Colorimetric Kit (Biovision, K337-100) from the
cell lysate according to manufacturer’s protocol.
All the metabolite measurement data presented
were from at least six donors.
Oxygen consumption measurement
Culture medium was collected from 1 million
cells treated with either RPMI or b-glucan. After
stimulation, the cells were trypsinized, washed,
and resuspended in 60 ml of the collected culture
medium. The cell suspensions were then used for
cellular O2 consumption analysis. Oxygen consumption was measured at 37°C using polarographic
oxygen sensors in a two-chamber Oxygraph
(OROBOROS Instruments, Innsbruck, Austria).
First, basal respiration (baseline oxygen consumption) was measured. Next, leak respiration was determined by addition of the specific
complex V inhibitor oligomycin A (OLI). Then,
maximal electron transport chain complex (ETC)
capacity (maximum oxygen consumption) was
quantified by applying increasing concentrations
of the mitochondrial uncoupler FCCP (1 to 14 mM
final maximal concentration). Finally, minimal
respiration was assessed by adding a maximal
(0.5 mM) concentration of the specific complex I
inhibitor rotenone (ROT; 0.5 mM) and the complex III inhibitor antimycin A (AA; 0.5 mM).
After establishment of the baseline oxygen consumption rate, cells were treated with the ATP synthase inhibitor oligomycin to determine the rate of
proton leak–dependent oxygen consumption, after
which the baseline rate value was normalized to
the value of the leak rate. Next, the cells were treated
with FCCP to determine the maximum oxygen consumption rate. For normalization, the maximum
FCCP value was ratioed to the leak value. The oxygen
consumption measurement was repeated in monocytes isolated from five healthy individuals.
Western blot
For Western blotting of AMPK, mTOR, Akt
(total and phosphorylated), and actin, training
was performed as described in stimulation experiments. Adherent monocytes were trained in
24-well plates. After training and the resting period,
cells were lysed in 150 ml of lysis buffer. Equal
amounts of protein were subjected to SDS-PAGE
electrophoresis using 7.5% polyacrylamide gels.
Primary antibodies [1:500 and 1:50 000 (actin)]
in 5% (w/v) BSA/TBST (5% bovine serum albumin/
TBST) were incubated overnight at 4°C. HRP-
conjugated anti-rabbit antibody or HRP-conjugated
anti-mouse antibody at a dilution of 1:5000 in
5% (w/v) BSA/TBST was used for 1 hour at room
temperature. Quantitative assessment of band intensity was performed by Image Lab statistical
software (Bio-Rad, CA, USA). The following antibodies were used: actin antibody (Sigma, A5441),
mTOR antibody (Cell Signaling, #2972, Leiden,
Netherlands), phospho-mTOR antibody (Ser2448)
(Cell Signaling, #2971), AMPKa antibody (Cell Signaling, #2532), phospho-AMPKa (Thr172) (Cell Signaling, #2531), Akt antibody (Cell Signaling, #9272),
phosphor-Akt (Ser473) (Cell Signaling, #9271). At
least four different individual experiments were
repeated for each Western blot experiment.
Analysis of RNA sequencing data
Sequencing reads were mapped to the mouse
genome (mm10 assembly) using STAR (version
2.3.0). The aligner was provided with a file containing junctions from Ensembl GRCm38.74. In
total, there were 507.5 million reads from 12 samples. Htseq-count of the Python package HTSeq
(version 0.5.4p3) was used to quantify the read
counts per gene based on annotation version
GRCm38.74, using the default union-counting
mode (The HTSeq package, www-huber.embl.
de/users/anders/HTSeq/doc/overview.html).
Differentially expressed genes were identified
by statistics analysis using the edgeR package from
bioconductor. The statistically significant threshold
[false discovery rate (FDR) = 0.05] was applied.
For visualization, relative changes larger than 1.5
and FDR of 0.01 were used to plot the expression
level of protein-coding genes.
Animal experimental models
The metformin experiment was done at the University of Athens with the approval of the Ethics
Committee on Animal Experiments of the University of Athens (approval no. 2550). C57BL/6J female
mice (8 to 12 weeks) were used (Jackson Laboratories, Bar Harbor, ME, USA). Mice were injected
with live C. albicans blastoconidia (2 × 104 CFU per
mouse) or pyrogen-free phosphate-buffered saline (PBS) alone. Seven days later, mice were
infected intravenously with a lethal dose of live
C. albicans (2 × 106 CFU per mouse). Survival was
monitored daily. To assess the involvement of the
AMPK-mTOR pathway in the training, metformin
(250 mg/kg) or PBS was given via intravenous
injection from 1 day before the first nonlethal
dose of live C. albicans challenge until 3 days after
challenge on a daily basis.
Wild-type (Cre +/+, HIF flox/flox) and HIF-KO
mice 8 to 10 weeks old were trained with 200 ml
intraperitoneally (i.p.) of either 1 mg of b-glucan
particles or sterile PBS on days –7 and –4 prior to
tail vein inoculation with 200 ml of 5 × 106 S.
aureus strain RN4220 on day 0. Mice were monitored three times daily for survival for 14 days.
Data presented are the combined survival data
(Kaplan-Meier) from two independent experiments.
There were five mice per group in the first survival
experiment and seven mice per group in the
second survival experiment. A log-rank test was
used to assess the statistical significance between
26 SEPTEMBER 2014 • VOL 345 ISSUE 6204
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the groups. For the RNA sequence analysis, both
wild-type and mHIF1a-KO mice were injected
with PBS or b-glucan i.p. and the total RNA was
extracted from splenocytes at day 4. The total gene
expression profiles were accessed by RNA sequencing. This study was carried out in accordance with
the recommendations of the National Research
Council (46). The protocol was approved by the
Dartmouth IACUC (approval no. cram.ra.2).
Metabolic activity assay
Alveolar macrophages were isolated from 6- to
10-week-old HIF-KO and wild-type mice by flushing lungs 10 times with 1 ml of PBS containing
0.5 mM EDTA. Alveolar macrophages were added
and allowed to adhere for 1 hour to a 96-well
plate at a concentration of 8 × 104 in 200 ml of
CO2-independent media (Leibovitz’s L-15, Life
Technologies) supplemented with 10% FCS,
5 mM HEPES buffer, 1.1 mM L-glutamine, penicillin (0.5 U/ml), and streptomycin (50 mg/ml).
To the media, 10% Resazurin dye (Sigma) was
added and the plate was incubated at 37°C for
24 hours, with readings recorded every 30 min
at 600 nm. A 690-nm reference wavelength was
subtracted from the 600-nm wavelengths and
the data were normalized to wells without cells.
Curdlan (100 mg/ml, Sigma) was used as a stimulator of metabolic activity.
Statistical analysis
The differences between groups were analyzed
using the Wilcoxon signed-rank test (unless otherwise stated). Statistical significance of the survival
experiment was calculated using the product
limit method of Kaplan and Meier. The level of significance was defined as a P value of <0.05. Cytokine production as well as the band intensity ratio
for Western blot were plotted as a bar chart with
mean T SEM. Replicate numbers of the experiments performed are reported in the figure legends.
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AC KNOWLED GME NTS
S.-C.C., J.Q., and M.G.N. were supported by a Vici grant of
the Netherlands Organization of Scientific Research and ERC
Consolidator grant 310372 (both to M.G.N). C.W. is supported
by funding from the European Research Council under
the European Union’s Seventh Framework Programme
(FP/2007-2013)/ERC grant agreement 2012-322698). Y.L. is
supported by Veni grant 863.13.011 of the Netherlands
Organization for Scientific Research. R.A.C. and K.M.S.
were supported by National Institute of General Medical
Sciences grant 5P30GM103415-03 (William Green, PI) and
1P30GM106394-01 (Bruce Stanton, PI), and National Institute of
Allergy and Infectious Diseases grant R01AI81838 (R.A.C., PI).
R.A.C./K.M.S. thank B. Berwin for the S. aureus. R.J.X funded by
DK43351, DK097485, Helmsley Trust, and JDRF. The detailed
data have been deposited in the GEO database with accession
number GSE57206.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/345/6204/1250684/suppl/DC1
Figs. S1 to S15
Tables S1 and S2
10 January 2014; accepted 28 August 2014
10.1126/science.1250684
sciencemag.org SCIENCE