DOI: 10.1126/science.1250684, (2014);345 Science

et al.Shih-Chin Chengfor trained immunity

mediated aerobic glycolysis as metabolic basis−αmTOR- and HIF-1

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RESEARCH

26 SEPTEMBER 2014 • VOL 345 ISSUE 6204 1579SCIENCE sciencemag.org

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 expo-

sure to certain infections or vaccinations.

The underlying mechanism of trained im-

munity is represented by epigenetic pro-

gramming through histone modifications,

leading to stronger gene transcription

upon restimulation. However, the specific

mTOR- and HIF-1α–mediated aerobic glycolysis as metabolic basis for trained immunity

IMMUNOGENETICS

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*

RESEARCH ARTICLE SUMMARY

Aerobic glycolysis as metabolic basis for trained immunity. In naïve macrophages dur-

ing 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 dec tin-1–Akt–mTOR–HIF-1α pathway.

Glucose Glucose

Pyruvate Pyruvate

Lactate

LactateOxidative

phosphorylation

Aerobic glycolysis

(Warburg efect)Oxidativephosphorylation

Naïve monocyte(resting monocyte)

Akt mTor

HIF-1α

trained monocyte

Susceptible Protected

Dead

C. albicans

S. aureus sepsis S. aureus sepsis

Dectin-1

β-glucan

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 com-

ponent of Candida albicans, that was

previously shown to induce nonspecific

protection against both infections and ma-

lignancies. Genome-wide transcriptome

and histone modification profiles were

performed and pathway analysis was ap-

plied to identify the cellular processes

induced during monocyte training. Biolog-

ical validations were performed in human

primary monocytes and in two experimen-

tal models in vivo.

RESULTS: In addition to immune signaling

pathways, glycolysis genes were strongly up-

regulated 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 meta-

bolic 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 im-

munity. Mice receiving

metformin, an adeno-

sine monophosphate–

activated protein kinase

(AMPK) activator that

subsequently inhibits

mTOR, lost the trained

immunity–induced protection against le-

thal C. albicans infection. The role of the

mTOR–HIF-1α pathway for β-glucan–

induced innate immune memory was fur-

ther validated in myeloid-specific HIF-1α

knockout (mHIF-1α KO) mice that, unlike

wild-type mice, were not protected against

Staphylococcus aureus sepsis.

DISCUSSION: The shift of central glucose

metabolism from oxidative phosphoryla-

tion 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 in-

nate 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

Read the full article at http://dx.doi.org/10.1126/science.1250684

ON OUR WEB SITE

Published by AAAS

RESEARCH ARTICLE◥

IMMUNOGENETICS

mTOR- and HIF-1a–mediatedaerobic glycolysis as metabolicbasis for trained immunityShih-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, confersnonspecific protection from secondary infections. Using histone modification profiles ofhuman monocytes trained with the Candida albicans cell wall constituent b-glucan, togetherwith a genome-wide transcriptome, we identified the induced expression of genes involvedin glucose metabolism.Trained monocytes display high glucose consumption, high lactateproduction, and a high ratio of nicotinamide adenine dinucleotide (NAD+) to its reducedform (NADH), reflecting a shift in metabolism with an increase in glycolysis dependent onthe 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 monocyteinduction of trained immunity, whereas the adenosine monophosphate–activated proteinkinase activator metformin inhibited the innate immune response to fungal infection. Micewith a myeloid cell–specific defect in HIF-1a were unable to mount trained immunity againstbacterial sepsis. Our results indicate that induction of aerobic glycolysis through anAkt–mTOR–HIF-1a pathway represents the metabolic basis of trained immunity.

In classical descriptions of host defense mech-anisms, innate immune responses that arerapid, are nonspecific, and lack memory aredistinguished from specific T and B cell–dependent immune responses, which are

highly specific and have the capacity to buildimmunological memory. The hypothesis thatthe innate immune system is incapable of mount-ing adaptive responses (1) is contradicted bystudies showing that organisms lacking a specificimmune system, such as plants or insects, are

able to respond adaptively to infection (2, 3) andthat innate immune cells, such as macrophages,have adaptive characteristics (4). In line with theproposal that there are nonspecific adaptiveresponses in the innate immune system, T andB cell–independent protective effects of mono-cytes and natural killer (NK) cells have beendemonstrated in models of bacterial and viralinfections, respectively (5, 6). Furthermore, epi-genetic reprogramming at the level of histoneH3 methylation has been proposed as the mo-lecular mechanism responsible for long-termmemory of innate immunity (5, 7), and thisprocess has been termed trained immunity.Initiation of innate immune memory through

trained immunity is likely to be responsible for thenonspecific protective effects of certain vaccines(8). Furthermore, the increased inflammatory re-sponsiveness ofmonocytes andmacrophages due totrained immunity appears to play a central role ininflammatory diseases (9). From this perspective,the capacity of innate immunity to mount adaptiveresponses both redefines the function of innate im-munity and identifies a potential therapeutic targetinhumandiseases. It is thus essential to understandthe cellular and molecular mechanisms that medi-ate trained immunity, in hopes of harnessing theirtherapeutic potential. Although epigenetic modifi-cations are known to underlie information storageduring innate immune memory in both plants (10)

and mammals (7), less is known regarding the mo-lecular pathways and downstream mechanismsthat lead to trained immunity.

Transcriptome and epigeneticsof monocytes

Candida albicans and its main cell wall constit-uent, b-glucan, induce trained innate immunememory both in vitro and in vivo (7). We per-formed an unbiased assessment of whole-genomemRNA expression, histone methylation, and acet-ylation patterns after training human primarymonocytes with b-glucan, the major Candidacell wall structure that mediates trained immu-nity, which induces nonspecific protection againstboth infections and malignancies (11). An invitro experimental model of b-glucan–inducedtrained immunity was established in monocytes(Fig. 1A). b-Glucan training of cells induced apotentiated cytokine production upon restim-ulation with lipopolysaccharide (LPS) 7 dayslater (Fig. 1B). An enhanced response was alsoobserved after stimulation with the TLR2 ligandPam3Cys or with nonrelated Gram-negative andGram-positive bacteria (fig. S1). Assessment ofhistone 3 Lys4 trimethylation (H3K4me3) andhistone 3 Lys27 acetylation (H3K27Ac) identi-fied promoters that were specifically induced byb-glucan training (Fig. 1C). Pathway analysis ofthe promoters potentiated by b-glucan identi-fied innate immune and signaling pathways up-regulated in trained cells that are responsiblefor the induction of trained immunity (7, 12).In addition to immune signaling pathways,

epigenetic profiling of trained monocytes on thebasis of both methylation and acetylation pat-terns identified a signature associated with cen-tral metabolism (fig. S2) and an increase in thepromoters of genes encoding enzymes involvedin glycolysis and itsmaster regulatormTOR (mam-malian target of rapamycin) (Fig. 1, D and E).Furthermore, after priming of monocytes withb-glucan, genes involved in glycolysis, such ashexokinase and pyruvate kinase, were epigenet-ically up-regulated 1 week later (Fig. 1F and fig.S3). The gene expressing mTOR and the glyco-lytic genes that are targets of the transcriptionfactorHIF1awere also enhancedby b-glucan (fig.S4). In line with this, HIF-1a activation was in-creased in b-glucan–trained monocytes (fig. S5).In addition, glycolysis geneswere also up-regulatedin vivo in mice challenged with b-glucan, as re-vealed by total RNA sequencing analysis in spleno-cytes of these mice (Fig. 1G and fig. S6).

Glycolysis and monocytes

Monocytes from peritoneal exudates rely on gly-colysis as a main energy source (13). The role ofglucose as an energy substrate for monocytes isdemonstrated by the blockade of monocyte stim-ulation and trained immunity by incubation of cellswith 2-deoxy-D-glucose, a glucose analog that can-not be metabolized by the cells and inhibits gly-colysis (fig. S7). This is in linewith observations thatactivatedmacrophages, dendritic cells, andTH1 andTH17 lymphocytes undergo a switch from oxidativephosphorylation to aerobic glycolysis (14).

RESEARCH

SCIENCE sciencemag.org 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204 1250684-1

1Department of Internal Medicine, Radboud University MedicalCenter, 6525 GA Nijmegen, Netherlands. 2Department ofMicrobiology and Immunology, Geisel School of Medicine atDartmouth, Hanover, NH 03755, USA. 3Department ofMolecular Biology, Faculties of Science and Medicine, NijmegenCentre for Molecular Life Sciences, Radboud University, 6500HB Nijmegen, Netherlands. 4Department of Genetics, Universityof Groningen, University Medical Center Groningen, Groningen,Netherlands. 54th Department of Internal Medicine, Universityof Athens Medical School, 12462 Athens, Greece. 6Departmentof Biochemistry, Faculties of Science and Medicine, NijmegenCentre for Molecular Life Sciences, Radboud University, 6500HB 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 IntegrativeBiology and Gastrointestinal Unit, Massachusetts GeneralHospital, Harvard School of Medicine, Boston, MA 02114,USA. 10Broad Institute of MIT and Harvard, Cambridge, MA02142, USA.*Corresponding author. E-mail: mihai.netea@radboudumc.nl

Consistent with these findings, monocytestrained with b-glucan showed a reduced baselineoxygen consumption on day 7 relative to naïvecells; this finding is compatiblewith the hypothesisthat these cells underwent a shift from oxidativemetabolism toward glycolysis. Moreover, trainedcells showed a decreasedmaximal rate of oxygenconsumption after complete uncoupling with car-bonyl cyanidep-trifluoromethoxyphenylhydrazone(FCCP), a chemical substrate that permeabilizesmitochondrialmembranes anduncouples electrontransport systems from the oxidative phospho-rylation systems (Fig. 2, A and B), whereas the rateof proton leak–dependent oxygen consumption

wasnot altered (table S1). The latter result indicatesa reduction of the capacity of the mitochondrialelectron transport chain (ETC) as observed after aperiod of hypoxia (15). Hypoxia decreases the activ-ity of the ETC complexes I and IV through HIF-1a(16). This hypothesis was reinforced by observa-tions of increased glucose consumption (Fig. 2C),lactate production (Fig. 2D), and ratio of nicotin-amide adenine dinucleotide (NAD+) to its reducedform (NADH) (Fig. 2E) in trained monocytes.Differences in glucose consumption did not

offset the high glucose concentrations in theRPMI medium, which suggests that glucoseavailability is not the limiting factor for the ob-

served training phenotype (fig. S8). In addition,the training effect induced by b-glucan was likelynot due to the presence of pyruvate in the culturemedium, an intermediatemetabolite in glycolysis,because training occurred even when mediumdevoid of pyruvate was used during the trainingprocess (fig. S9).Earlier studies have shown that a high cellu-

lar NAD+/NADH ratio acts through sirtuin-1 todecrease the mitochondrial content (17). Thismechanism may explain the observed b-glucan–induced reduction in ETC capacity. In contrast,LPS stimulation leads to a strong but transientincrease in the glycolytic process in monocytes

1250684-2 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204 sciencemag.org SCIENCE

Fig. 1.Trained immunity in monocytes. (A) Sche-matic of in vitro trained immunity experimentalsetup. (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) Genome-wide H3K4me3 (red) and H3K27Ac (blue) epige-netic modifications 7 days after b-glucan treatment.Ratios of b-glucan/RPMI for both H3K4me3 andH3K27Ac modification were calculated for eachpromoter. The promoters that display significantlyhigher or lower ratio (P ≤ 0.05, t test) relative tomedian values are called b-glucan–induced pro-moters and b-glucan–repressed promoters, re-spectively. Box plots show distributions of thesequence read density (reads per kilobase) forall promoters, b-glucan–induced promoters, andb-glucan–repressed promoters in each data set.In each box plot, the band inside each box (mid-point) represents the median value, and upper andlower borders of the box represent the Q3 (thirdquartile) and Q1 (first quartile) values, respectively.The upper line represents the maximum value with-in the upper bound [Q3 + 1.5 × (Q3 – Q1)]; thelower line represents the minimum value withinthe lower bound [Q1 – 1.5 × (Q3 – Q1)]. Dots rep-resent observed points outside the upper andlower bound. (D) Epigenetic modifications in thepromoter regions of the genes involved in glycol-ysis and mTOR pathways. The box plots wereanalyzed as in Fig. 1C. (E) Schematic representa-tion of the up-regulated enzymes (red) in the gly-colysis pathway. (F) Representative screen shotsof H3K4me3 (red) and H3K27Ac (blue) modifica-tions in the promoter region of pyruvate kinase(PKM) and hexokinase. (G) Differential gene ex-pression analysis between the b-glucan–treatedgroup and the control group. Genes in the gly-colysis pathway that are up-regulated by theb-glucan training are highlighted in the box atright. The colors in the heat map represent thenormalized RNA levels of identified differentialexpressed genes (false discovery rate = 0.01,relative change ≥ 1.5) in three mice per group.

RESEARCH | RESEARCH ARTICLE

(fig. S10); this finding supports the suggestionthat, although the acute response of monocytesto LPS is characterized by glycolysis (18), at latertime points this response switches to oxidativephosphorylation—a process that subsequently in-duces immune tolerance by activation of sirtuin-1and sirtuin-6 histone deacetylases (19). In contrastto LPS-induced tolerance, b-glucan training inhib-ited the expression of Sirtuin1 (fig. S11). Moreover,the addition of resveratrol, a sirtuin-1 activator,during the first 24 hours of b-glucan training par-tially inhibited the enhanced interleukin-6 (IL-6)production (fig. S11). This suggests that sirtuin de-acetylases play a role in the modulated monocytefunctional phenotype and highlights the complexinteraction between the intermediate metabo-

lites and subsequent immune responses throughchromatin-modifying enzymes (20).mTOR acts as a sensor of the metabolic envi-

ronment (21) and functions as amaster regulatorof glucose metabolism in activated lymphocytes(22). Epigenetic signals at promoters of genesin the mTOR pathway were significantly higherin b-glucan–trained monocytes (paired t test,P < 0.001) than in cells exposed to culture me-dium (Fig. 3A). Target genes of mTOR, such asEIF4EBP1, displayed a similar pattern (Fig. 3B).In line with this finding, mTOR phosphorylationwas up-regulated in trained monocytes as as-sessed by Western blot (Fig. 3C). Monocytes iso-lated from patients with a complete deficiency indectin-1 (23) failed to activate mTOR upon stim-

ulation with b-glucan (Fig. 3D) and failed to en-hance tumor necrosis factor (TNF) productionupon LPS restimulation (fig. S12), supporting thehypothesis that mTOR phosphorylation is de-pendent on the dectin-1 C-type lectin receptor.

Glycolysis in trained immunity

As the data presented above demonstrate activa-tion of mTOR and glycolysis in trained monocytes,we next investigated the causality between thesetwo processes by blocking glycolysis during b-glucantraining. Inhibition of mTOR with rapamycinduring the first day of stimulation resulted ina dose-dependent inhibition of the trainingeffect induced by b-glucan (Fig. 3E). Indirect in-hibition of mTOR with AICAR, an adenosine

SCIENCE sciencemag.org 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204 1250684-3

Fig. 2. Physiology afterb-glucan treatment. (A)Representative oxygen con-sumption rate of untreated(RPMI, black) and b-glucan–trained (red) monocytes asdetermined by high-resolutionrespirometry (Oxygraph;OROBOROS Instruments,Innsbruck). (B) Baseline(basal oxygen consumptionbefore oligomycin treatment,upper panel) and maximumoxygen consumption rate(maximum oxygen consump-tion upon FCCP treatment,lower panel) of untrained(open bar) and b-glucan–trained (solid bar) monocytesdetermined by respirometryand normalized to the leakoxygen consumption. (C andD) Kinetic changes of glucoseconsumption (C) and lactateproduction (D) from days 1, 3,and 7 of untreated andb-glucan–trained monocytes.(E) Kinetics of NAD+/NADHratio determined at days 1, 3,and 7. In (B) to (E), dataare means T SEM (n = 5 to 8,*P < 0.05, Wilcoxon signed-rank test).

RESEARCH | RESEARCH ARTICLE

monophosphate–activated protein kinase (AMPK)activator, had similar effects (Fig. 3F). On the basisof observations that mTOR induction of glycolysisis mediated through activation of HIF-1a andstimulation of glycolytic enzymes (24) and thatrapamycin inhibits HIF-1a expression (25), weassessed the effect of a HIF-1a inhibitor on mono-cyte training. We found that the HIF-1a inhibitorascorbate also blocked trained immunity in adose-dependent manner (Fig. 3F).We further investigated the link betweenmeta-

bolic effects and epigenetic changes by assess-ing the effects of the epigenetic inhibitors MTA(methylthioadenosine, amethyltransferase inhib-itor) and ITF (ITF2357, a histone deacetylaseinhibitor) during the training setup on the lac-tate measurements. As expected, the epigeneticinhibitors had no effect on lactate production inthe acute phase (24 hours after b-glucan stim-ulation; fig. S13). However, lactate productionwas significantly reduced in the trained mono-cytes on day 7 when MTA was added to mono-cytes with b-glucan during the first 24 hours inthe incubation period (fig. S13), which suggeststhat histonemethylation also partiallymodifies theinduction of glycolysis in the trained monocytes.

Monocyte mTOR activation

Activation of mTOR by insulin or colony-stimulating factors such as GM-CSF (granulocyte-macrophage colony-stimulating factor) ismediatedby intermediary activation of the Akt-PI3K (phos-phatidylinositol 3-kinase) pathway (26). A similarsignal route is induced in monocytes by b-glucan,as stimulation with b-glucan induced a strongphosphorylation of Akt (Fig. 4A). This effect wasagain dectin-1–dependent, being absent inmono-cytes isolated from dectin-1–deficient patients(Fig. 4B). Inhibition of Akt phosphorylation alsoresulted in down-regulation of mTOR activation(Fig. 4C), demonstrating the relationship betweenAkt and mTOR activation. Finally, the Akt inhib-itor wortmannin inhibited monocyte training byb-glucan in a dose-dependent manner (Fig. 4D).Epigenetic reprogramming of monocytes by

trained immunity has been reported as a mech-anismof nonspecific protection in differentmodels.Mice were protected from lethal disseminatedcandidiasis after an initial nonlethal Candidaalbicans infection (7). Similarly, b-glucan alsoinduced protection against infection with a lethalStaphylococcus aureus inoculum (27), while BacillusCalmette-Guérin (BCG) vaccination protectedmicefrom systemic candidiasis (5). We first assessedwhether metformin—which acts through AMPKactivation and subsequently mTOR inhibition(28) and is commonly used for the treatment oftype 2 diabetes (29)—abrogates the protectiveeffects in these experimental models. In vitro,metformin suppressed trained immunity in-duced by b-glucan (Fig. 4E), and administrationof metformin to mice during and after primaryinfectionwith a low-inoculumC. albicans inhibitedthe protective effects induced by it against sec-ondary disseminated candidiasis (Fig. 4F), dem-onstrating that mTOR-mediated effects mounta protective trained immunity in vivo.

1250684-4 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204 sciencemag.org SCIENCE

Fig. 3. mTOR signaling in b-glucan–treatedmonocytes. (A) Schematic representation of up-regulatedenzymes (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 atbottom), the main target of mTOR, in both RPMI- and b-glucan–treated monocytes. (C) Western blotfrom cell lysate harvested at day 7 after RPMI or b-glucan treatment. Antibodies specific for endogenousphospho-mTOR (p-mTOR), total mTOR, phospho-AMPK, AMPK, and actin were used to blot the totaland phospho proteins, respectively. Representative blots of five independent experiments are shown.Thep-mTOR/mTOR ratio is shown as a bar chart (n = 5, P = 0.0625,Wilcoxon signed-rank test). (D to F) Theendogenous p-mTOR status of dectin-1–deficient patients was determined by Western blot (D) from celllysate harvest at day 7 after RPMI of b-glucan treatment and probed with antibodies to p-mTOR andtotal mTOR, respectively. The p-mTOR/mTOR ratio is shown as a bar chart. Relative cytokine productionwas determined from cells incubated with rapamycin (mTOR inhibitor) (E) and with AICAR (AMPKinhibitor) and ascorbate (HIF-1a inhibitor) (F) in a dose-dependent manner. In (E) and (F), data aremeans T SEM (n = 6, *P < 0.05, Wilcoxon signed-rank test).

RESEARCH | RESEARCH ARTICLE

We assessed whether the effects of mTORwere mediated at the level of innate immunitybut not at the level of adaptive T and B cellimmunity elicited during vaccination. An exper-imental model of b-glucan–induced protectionagainst S. aureus sepsis can be observed inmyeloid cell–specific HIF-1a conditional knock-

out mice (mHIF-1a KO) (30). These mice are un-able tomount glycolysis specifically in cells of themyeloid lineage. We assessed the metabolic ac-tivity of wild-type andmHIF-1aKOmacrophageswhen stimulated with b-glucan. mHIF-1a KOmacrophages showed increased chemical reduc-tion of the metabolic indicator resazurin (Fig.

4G), consistent with the hypothesis that HIF-1a induces the switch to aerobic glycolysis inresponse to b-glucan. In this model, the cells donot undergo the switch in the absence of HIF-1a and are “metabolically” dysregulated.Whereasb-glucan increased the survival of wild-type miceinfected with S. aureus from 40% to 90%, the

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Fig. 4. Akt–mTOR–HIF-1apathway downstream ofb-glucan stimulation. (A)Monocytes were treated witheither RPMI or b-glucan inthe presence or absence ofwortmannin, a PI3K inhibitor.GM-CSF stimulation wasincluded as a positivecontrol. Cell lysates wereharvested at 5, 15, 30, 60,and 120 min. Akt phospho-rylation and actin level wereblotted with specific anti-bodies to p-Akt and actin.Representative blots fromthree independentexperiments are shown.(B) Akt phosphorylation andp-Akt/Akt ratio induced byb-glucan from dectin-1 weredetermined by Western blotby specific antibodies top-Akt, total Akt, and actin.(C) Effects of PI3K inhibitorson Akt and mTOR phospho-rylation in b-glucan–treatedmonocytes were determinedby Western blot by probingwith specific antibodies top-AKT and p-mTOR. (D)Monocytes were treatedwith b-glucan in the presenceof wortmannin in a dose-dependent manner. Cytokinelevels after 7 days weredetermined by enzyme-linked immunosorbent assay.(E) Relative cytokine produc-tion was determined fromcells incubated with metfor-min (AMPK inhibitor) in adose-dependent manner.(F) Survival of wild-typeC57BL/6J mice infected withlive C. albicans after trainingwith PBS or b-glucan. Metforminor PBS was given from 1 daybefore the first nonlethal doseof live C. albicans challenge until3 days after challenge on a dailybasis. (G) Wild-type (WT) andHIF-KO alveolar macrophagesat a concentration of 8 × 104

were incubated with PBS orcurdlan (100 mg/ml) for 1 hour.Resazurin was added and absorbancewas recorded every 30min for 24 hours. Inset (*) shows absorbance values at the 20-hour time point. Data are representative ofthreebiological replicates. (H)Survival curveofwild-typeormHIF-1aKOmiceprimedwith b-glucanandchallengedwith a lethal doseofS. 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).

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induction of trained immunity was completelyabrogated in mHIF-1a KO mice (Fig. 4H). Tofurther dissect which pathways are modulatedin the mHIF-1a KOmice, we performed RNA se-quencing and compared the differential RNAexpression profiles of wild-type andmHIF-1a KOmice. Several interesting genes were specificallyup-regulated inwild-type but not inmHIF-1aKOmice (fig. S14 and table S2), including those en-coding beclin-1 (an autophagy-related protein),STK11 (an AMPK-related serine-threonine kinase),JHDM1D (jumonji C domain containing histonedemethylase), and the FOXO4 transcription fac-tor involved in Akt-PI3K stimulation. Thus, theseresults demonstrate that stimulation of HIF-1a–mediated glycolysis in myeloid cells is crucial formounting trained immunity in vivo.

Discussion

The role of histone methylation as a mediatorof short-term innate immunological memoryin macrophages has been described (7) and hasbeen referred to as a latent enhancer for the epi-genetic elements that mediate this phenomenon(31). In this study, whole-genome epigenetic pro-filing of histonemodifications andRNA sequenc-ing analysis have identified both immunologic andmetabolic pathways stimulated during trainedimmunity. A cyclic adenosine monophosphate–dependent pathway mediating trained immu-nity in monocytes has also been described inan accompanyingmanuscript (12). In the presentstudy, we identified the metabolic pathways in-duced in trained monocytes, demonstrating ametabolic switch toward aerobic glycolysis, whichis in turn crucial for the maintenance of trainedimmunity (Fig. 5).A metabolic switch toward aerobic glycoly-

sis was earlier reported to be a feature of cellactivation and proliferation [such an effect wasfirst described in neoplastic cells and termed theWarburg effect (32)] while also playing a role ineffector T helper lymphocytes (33) and activatedmacrophages (34). The elevated glycolysis metab-olism observed in trained monocytes might benecessary to equip and prepare cells to respondto the intruding pathogens in a robust and rapid

manner through proinflammatory cytokine pro-duction and possibly also through enhanced phago-cytosis capacity (35).Although we observed trained immunity in

monocytes, this response should not be restrictedto cells in the monocyte lineage. Recently, adapt-ive features of NK cells have been demonstratedto be involved in resistance to reinfection withviruses (6, 36). The specific NKmemory cells, likeT cells, rapidly proliferate, degranulate, and pro-duce cytokineuponactivation.However, it remainsto be determined whether metabolic rewiringalso plays a role in NK or in other innate im-mune cells, such as dendritic cells. In addition,it is of interest to determine whether trainingis contact-dependent or could also be inducedby soluble mediators. This is an important ques-tion in the field of autoinflammatory and auto-immune diseases, because these diseases areworsened by the unregulated cytokine produc-tion. Our results suggest that proinflammatorycytokines such as IL-1b could also induce trainedimmunity in monocytes in vitro (fig. S15). Thishypothesis is further supported by nonspecificprotective effects induced by IL-1b, even wheninjected several days before an experimental in-fection is induced (37).One important aspect to note is that the mo-

lecular mechanisms investigated in the presentstudy focused on trained immunity in the first7 days after the initial stimulus. This is thecrucial period during which trained immunityoffers protection in newborn children againstperinatal sepsis (38) and thus is relevant from abiological and clinical point of view. Longer-lasting in vivo effects of trained immunity havebeen demonstrated in humans (5), and it isimportant to assess whether these later effectsare mediated through similar mechanisms. How-ever, any such later effects are also likely to beexerted at the level of bone marrow myeloid cellprogenitors, as recently demonstrated in the caseof Toll-like receptor (TLR)–induced tolerance (39).Our study introduces an interesting prelimi-

nary step in understanding the glycolytic processin trained immunity. Hypoxia and glycolysis en-hance the proliferative response of macrophages

to CSF-1 (40) and sustain the survival of activateddendritic cells (41). Soluble b-glucan fromGrifolafrondosa induces macrophage proliferation (42),although we were not able to observe these ef-fectswith trainedmonocytes byCandida b-glucan(7). However, epigenetic profiling has identifieda cell cycle activation signal in b-glucan–trainedcells (12), and it is tempting to speculate thattrained monocytes are not only capable of in-creased cytokine production but also primed torespond to proliferative signals, although thisremains to be demonstrated. Finally, the identi-fication of glycolysis as a fundamental process intrained immunity further highlights a key regu-latory role for metabolism in innate host defenseand also defines a novel therapeutic target inboth infectious and inflammatory diseases (9).

Materials and methods

Isolation of primary human monocytes

Blood was collected from human healthy volun-teers and two dectin-1–deficient patients afterwritten informed consent (Ethical CommitteeNijmegen-Arnhem, approval no. NL32357.091.10).Peripheral blood mononuclear cells (PBMCs)were isolated by differential centrifugation usingFicoll-Paque (GE Healthcare, Diegem, Belgium)from buffy coats obtained from Sanquin Blood-bank, Nijmegen, Netherlands. Monocytes werepurified by MACS depletion of CD3-, CD19-, andCD56-positive cells from the PBMCs; CD3Micro-Beads (130-050-101), CD19 MicroBeads (130-050-301), and CD56 were purchased from MiltenyiBiotec (Leiden, Netherlands) and used accordingto the manufacturer’s protocol. Efficacy of de-pletionwas controlled by flow cytometry (FC500;Beckman-Coulter, Woerden, Netherlands) andwas higher than 98%.

Genome-wide sequence analysis

For chromatin immunoprecipitation (ChIP) anal-ysis or RNA sequencing, 10 × 106 CD3–CD19–CD56–

pure monocytes were plated on 100-mm dishes.Monocytes were preincubated with cell culturemedium (RPMI) or b-glucan (5 mg/ml) for 24 hoursin a total volumeof 10ml. After 24hours, cellswere

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Fig. 5. Model of metabolic activation of trained monocytes, characterizedby a shift toward increased aerobic glycolysis and decreased oxidative

phosphorylation. Upon b-glucan/dectin-1 recognition, the AKT–mTOR–HIF-1a pathway is ac-tivated 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.Thepotential role of rapamycin and metformin in inhibition of trained immunity is also depicted. The metabolic differencesbetween a trained monocyte and naïve monocytes are summarized at the right.

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washed to remove the stimulus and were resus-pended in RPMI supplementedwith 10% humanpool serum. Monocytes were collected before and6 days after the incubation for ChIP or RNA se-quencing. For RNA sequencing, monocytes werecollected in TRIzol reagent (Invitrogen, Bleiswijk,Netherlands). The purifiedmaterials were then pro-cessed to generate genomic DNA for WGBS, RNA(Trizol extraction according to manufacturer in-structions; Agilent BioAnalyser RIN >8), and chro-matin by fixing the cells in 1% formaldehyde.

Reagents

Candida albicans b-1,3-(D)-glucan (b-glucan) waskindly provided by D. Williams (East TennesseeState University). Reagents used were as follows:LPS (E. coli 0B5/B5, Sigma, Diegem, Belgium),rapamycin (Sigma, R0395), metformin (R&D, AF1730, Abingdon, UK), AICAR (Sigma, A9978), as-corbate (Sigma, A4034), wortmannin (InvivoGen,tlrl-wtm, Toulouse, France). C. albicans ATCC MYA-3573 (UC 820) cells were heat-inactivated for30 min at 95°C.

Stimulation experiments

For training, monocytes were preincubatedwith b-glucan (10 mg/ml) for 24 hours. After 7 days,cells were restimulated with various microbialligands: 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°Cuntil cytokine measurement. All the cytokine mea-surements presentedwere from at least six donors.To address the HIF-1a–AMPK–mTOR pathway

in trained immunity, we added the specific inhib-itors together with b-glucan for the first 24 hoursin different doses as follows: rapamycin from 1 to100 nM, metformin from 0.3 to 30 mM, AICARfrom 5 to 500 nM, and ascorbate at 5 and 50 mM.

ChIP-seq data analysis

H3K4me3 and H3K27ac ChIP, sequencing, andprocessing of the data were performed as de-scribed (7). The detailed data have been depos-ited in the GEO database with accession numberGSE57206. Sequenced reads of 42-bp length weremapped to human genome (NCBI hg19) usingbwa-alignment package mapper (43). ChIP-seqdata sets were normalized as described (44), andthe sequenced reads were directionally extendedto 300 bp, corresponding to the original lengthof sequenced DNA fragments. For each base pairin the genome, the number of overlapping se-quence reads was determined, averaged over a10-bp window, and visualized in UCSC browser(http://genome.ucsc.edu). These normalized trackswere used to generate the genome browser screenshots. Putative H3K4me3- and H3K27ac-enrichedregions in the genome were identified by usingMACS (45) with P < 10−8. All the transcriptionstart sites (T1 kb) of genes with significant H3K4me3signal were regarded as active promoters. H3K4me3and H3K27ac signals at all active promoters wereestimated, and log2 ratios of ChIP-seq signalbetween treatment and control samples werecalculated. Promoters that showed an absolute

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 countedfrom the normalized ChIP-seq data sets wereused to generate the box plots.

Metabolite measurements

Culture mediumwas collected at days 1, 3, and 7.The glucose and lactate concentrations within themediumwere determined by Glucose ColorimetricAssay Kit (K686-100; Biovision, Milpitas, CA) andLactate Colorimetric Assay Kit (K627-100, Biovi-sion), respectively.NAD+andNADHconcentrationwere determined by NAD/NADH QuantificationColorimetric Kit (Biovision, K337-100) from thecell lysate according to manufacturer’s protocol.All the metabolite measurement data presentedwere from at least six donors.

Oxygen consumption measurement

Culture medium was collected from 1 millioncells treated with either RPMI or b-glucan. Afterstimulation, the cells were trypsinized, washed,and resuspended in 60 ml of the collected culturemedium. The cell suspensionswere then used forcellular O2 consumption analysis. Oxygen consump-tion was measured at 37°C using polarographicoxygen sensors in a two-chamber Oxygraph(OROBOROS Instruments, Innsbruck, Austria).First, basal respiration (baseline oxygen con-sumption) was measured. Next, leak respira-tion was determined by addition of the specificcomplex V inhibitor oligomycin A (OLI). Then,maximal electron transport chain complex (ETC)capacity (maximum oxygen consumption) wasquantified by applying increasing concentrationsof themitochondrial uncoupler FCCP (1 to 14 mMfinal maximal concentration). Finally, minimalrespiration was assessed by adding a maximal(0.5 mM) concentration of the specific complex Iinhibitor rotenone (ROT; 0.5 mM) and the com-plex III inhibitor antimycin A (AA; 0.5 mM).After establishment of the baseline oxygen con-

sumption rate, cells were treatedwith the ATP syn-thase inhibitor oligomycin to determine the rate ofproton leak–dependent oxygen consumption, afterwhich the baseline rate value was normalized tothe valueof the leak rate.Next, the cellswere treatedwith FCCP to determine themaximumoxygen con-sumption rate. For normalization, the maximumFCCPvaluewas ratioed to the leakvalue. Theoxygenconsumption measurement was repeated in mono-cytes isolated from five healthy individuals.

Western blot

For Western blotting of AMPK, mTOR, Akt(total and phosphorylated), and actin, trainingwas performed as described in stimulation ex-periments. Adherent monocytes were trained in24-well plates. After training and the resting period,cells were lysed in 150 ml of lysis buffer. Equalamounts of protein were subjected to SDS-PAGEelectrophoresis 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-conjugatedanti-mouse antibody at a dilution of 1:5000 in5% (w/v) BSA/TBST was used for 1 hour at roomtemperature. Quantitative assessment of band in-tensity was performed by Image Lab statisticalsoftware (Bio-Rad, CA, USA). The following anti-bodies were used: actin antibody (Sigma, A5441),mTOR antibody (Cell Signaling, #2972, Leiden,Netherlands), phospho-mTOR antibody (Ser2448)(Cell Signaling, #2971), AMPKa antibody (Cell Sig-naling, #2532), phospho-AMPKa (Thr172) (Cell Sig-naling, #2531), Akt antibody (Cell Signaling, #9272),phosphor-Akt (Ser473) (Cell Signaling, #9271). Atleast four different individual experiments wererepeated for each Western blot experiment.

Analysis of RNA sequencing data

Sequencing reads were mapped to the mousegenome (mm10 assembly) using STAR (version2.3.0). The aligner was provided with a file con-taining junctions from Ensembl GRCm38.74. Intotal, there were 507.5 million reads from 12 sam-ples. Htseq-count of the Python package HTSeq(version 0.5.4p3) was used to quantify the readcounts per gene based on annotation versionGRCm38.74, using the default union-countingmode (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 frombioconductor. The statistically significant threshold[false discovery rate (FDR) = 0.05] was applied.For visualization, relative changes larger than 1.5and FDR of 0.01 were used to plot the expressionlevel of protein-coding genes.

Animal experimental models

The metformin experiment was done at the Uni-versity of Athens with the approval of the EthicsCommittee on Animal Experiments of the Univer-sity of Athens (approval no. 2550). C57BL/6J femalemice (8 to 12 weeks) were used (Jackson Labora-tories, Bar Harbor, ME, USA). Mice were injectedwith liveC. albicansblastoconidia (2× 104CFU permouse) or pyrogen-free phosphate-buffered sa-line (PBS) alone. Seven days later, mice wereinfected intravenously with a lethal dose of liveC. albicans (2 × 106 CFUpermouse). Survival wasmonitored daily. To assess the involvement of theAMPK-mTORpathway in the training,metformin(250 mg/kg) or PBS was given via intravenousinjection from 1 day before the first nonlethaldose of live C. albicans challenge until 3 days afterchallenge on a daily basis.Wild-type (Cre +/+, HIF flox/flox) andHIF-KO

mice 8 to 10 weeks old were trained with 200 mlintraperitoneally (i.p.) of either 1 mg of b-glucanparticles or sterile PBS on days –7 and –4 prior totail vein inoculation with 200 ml of 5 × 106 S.aureus strain RN4220 on day 0. Mice were mon-itored three times daily for survival for 14 days.Data presented are the combined survival data(Kaplan-Meier) from two independent experiments.Therewere fivemice per group in the first survivalexperiment and seven mice per group in thesecond survival experiment. A log-rank test wasused to assess the statistical significance between

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the groups. For the RNA sequence analysis, bothwild-type and mHIF1a-KO mice were injectedwith PBS or b-glucan i.p. and the total RNA wasextracted from splenocytes at day 4. The total geneexpression profiles were accessed byRNA sequenc-ing. This study was carried out in accordance withthe recommendations of the National ResearchCouncil (46). The protocol was approved by theDartmouth IACUC (approval no. cram.ra.2).

Metabolic activity assay

Alveolar macrophages were isolated from 6- to10-week-old HIF-KO and wild-type mice by flush-ing lungs 10 times with 1 ml of PBS containing0.5 mM EDTA. Alveolar macrophages were addedand allowed to adhere for 1 hour to a 96-wellplate at a concentration of 8 × 104 in 200 ml ofCO2-independent media (Leibovitz’s L-15, LifeTechnologies) supplemented with 10% FCS,5 mM HEPES buffer, 1.1 mM L-glutamine, pen-icillin (0.5 U/ml), and streptomycin (50 mg/ml).To the media, 10% Resazurin dye (Sigma) wasadded and the plate was incubated at 37°C for24 hours, with readings recorded every 30 minat 600 nm. A 690-nm reference wavelength wassubtracted from the 600-nm wavelengths andthe data were normalized to wells without cells.Curdlan (100 mg/ml, Sigma) was used as a stim-ulator of metabolic activity.

Statistical analysis

The differences between groups were analyzedusing the Wilcoxon signed-rank test (unless other-wise stated). Statistical significance of the survivalexperiment was calculated using the productlimit method of Kaplan andMeier. The level of sig-nificance was defined as a P value of <0.05. Cyto-kine production as well as the band intensity ratiofor Western blot were plotted as a bar chart withmean T SEM. Replicate numbers of the experi-ments performed are reported in the figure legends.

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ACKNOWLEDGMENTS

S.-C.C., J.Q., and M.G.N. were supported by a Vici grant ofthe Netherlands Organization of Scientific Research and ERCConsolidator grant 310372 (both to M.G.N). C.W. is supportedby funding from the European Research Council underthe European Union’s Seventh Framework Programme(FP/2007-2013)/ERC grant agreement 2012-322698). Y.L. issupported by Veni grant 863.13.011 of the NetherlandsOrganization for Scientific Research. R.A.C. and K.M.S.were supported by National Institute of General MedicalSciences grant 5P30GM103415-03 (William Green, PI) and1P30GM106394-01 (Bruce Stanton, PI), and National Institute ofAllergy 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 byDK43351, DK097485, Helmsley Trust, and JDRF. The detaileddata have been deposited in the GEO database with accessionnumber GSE57206.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/345/6204/1250684/suppl/DC1Figs. S1 to S15Tables S1 and S2

10 January 2014; accepted 28 August 201410.1126/science.1250684

1250684-8 26 SEPTEMBER 2014 • VOL 345 ISSUE 6204 sciencemag.org SCIENCE

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