Articles

a-ketoglutarate orchestrates macrophage activation

through metabolic and epigenetic reprogramming
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

Pu-Ste Liu1,2, Haiping Wang1,2, Xiaoyun Li1,2, Tung Chao1,2, Tony Teav3, Stefan Christen4,5, Giusy Di Conza1,2,
Wan-Chen Cheng1,2, Chih-Hung Chou6,7, Magdalena Vavakova1,2, Charlotte Muret1,2, Koen Debackere8,9,
Massimiliano Mazzone8,9, Hsien-Da Huang6,7, Sarah-Maria Fendt4,5, Julijana Ivanisevic3 & Ping-Chih Ho1,2

Glutamine metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses;
however, the underlying mechanisms regulated by glutamine metabolism to orchestrate macrophage activation remain unclear.
Here we show that the production of a-ketoglutarate (aKG) via glutaminolysis is important for alternative (M2) activation of
macrophages, including engagement of fatty acid oxidation (FAO) and Jmjd3-dependent epigenetic reprogramming of M2
genes. This M2-promoting mechanism is further modulated by a high aKG/succinate ratio, whereas a low ratio strengthens the
proinflammatory phenotype in classically activated (M1) macrophages. As such, aKG contributes to endotoxin tolerance after
M1 activation. This study reveals new mechanistic regulations by which glutamine metabolism tailors the immune responses of
macrophages through metabolic and epigenetic reprogramming.

M1 macrophages elicit rapid proinflammatory responses to infec-
tion and tissue damage by sensing microbial components such as
lipopolysaccharide (LPS) and damage-associated molecular patterns
released from injured tissues. In contrast, M2 macrophages induced
by interleukin 4 (IL-4) and IL-13 show anti-inflammatory and repara-
tive activities to resolve inflammation and promote tissue repair 1,2.
Macrophages are thought to engage the M1 phenotype initially in
infection, to orchestrate host immunity, and to then obtain the M2
phenotype to restrain proinflammatory responses and repair dam-
aged tissues. However, deregulated activation and a phenotypic switch
in macrophages can lead to ineffective immune responses against
infections and severe tissue damage3. For example, failure to restrain
excessive production of proinflammatory cytokines in macrophages
in response to systemic bacterial infection results in septic shock
accompanied by tissue and organ damage. In contrast, after initial
infection, the prolonged restriction of M1 activation can paralyze the
immune system against subsequent microbial infections3–5. Therefore,
a sophisticated regulatory network in macrophages is critical to guid-
ing activation in the context of infection, cytokine milieu and other
microenvironment cues.
The different regulatory mechanisms that orchestrate macrophage
activation, including signaling cascades and epigenetic programming,
are increasingly understood. Among these regulatory mechanisms,
differences in the bioenergetic demands of M1 and M2 macrophages
are emerging as regulatory circuits that adjust macrophage behavior
in response to nutrient states in its habitation and the infected tissues.
M1 macrophages rely on aerobic glycolysis for ATP generation and
increased glucose and glutamine consumption, but they suppress oxi-
dative metabolism6–9. M1 macrophages have a broken tricarboxylic
acid (TCA) cycle, allowing accumulation of citrate and succinate.
Accumulated citrate can be used for production of fatty acids and
itaconate, an antimicrobial molecule6–8. Succinate acts as a proinflam-
matory metabolite that stabilizes transcription factor HIF-1α through
inhibition of prolyl hydroxylase (PHD) activity and promotion of
reactive oxygen species (ROS) production8,10. In contrast, M2 macro-
phages maintain an intact TCA cycle and favor oxidative metabolism,
especially FAO, as a mode of ATP production11,12. Similarly to block-
ing electron transport chain (ETC) activity, inhibition of lysosomal
lipolysis or fatty acid oxidation impairs IL-4-induced anti-inflamma-
tory responses and the reparative ability of M2 macrophages, which
suggests that oxidative phosphorylation (OXPHOS) and FAO con-
trol the regulatory circuits of M2 activation11,12. M2 macrophages
consume more glucose and glutamine than naive macrophages. This
glycolytic metabolism strengthens M2 activation by generating fatty
acids to fuel FAO and serving as an acetyl-CoA donor for epigenetic
reprogramming13,14. However, it remains unclear how glutamine
metabolism supports M2 activation15. These findings reveal the
importance of metabolic remodeling during macrophage activation,
but whether or not these metabolic activities intervene in canonical
signaling cascades and epigenetic reprogramming for macrophage

1Department of Fundamental Oncology, Faculty of Biology and Medicine, University of Lausanne, Epalinges, Switzerland. 2Ludwig Lausanne Branch, Epalinges,
Switzerland. 3Metabolomics Unit, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland. 4Laboratory of Cellular Metabolism and Metabolic
Regulation, Center for Cancer Biology, VIB, Leuven, Belgium. 5Laboratory of Cellular Metabolism and Metabolic Regulation, Department of Oncology, KU Leuven and
Leuven Cancer Institute (LKI), Leuven, Belgium. 6Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsin-Chu, Taiwan. 7Department
of Biological Science and Technology, National Chiao Tung University, Hsin-Chu, Taiwan. 8Laboratory of Tumor Inflammation and Angiogenesis, Vesalius Research
Center, VIB, Leuven, Belgium. 9Laboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, Leuven, Belgium. Correspondence should
be addressed to P.-C.H. (ping-chih.ho@unil.ch).

Received 2 May; accepted 21 June; published online 17 July 2017; doi:10.1038/ni.3796

nature immunology VOLUME 18 NUMBER 9 SEPTEMBER 2017 985

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activation remains unclear. Most importantly, the therapeutic poten- stimulation than BMDMs activated in glutamine-replete conditions
tial of fine-tuning deregulated macrophage activation via metabolic (Fig. 1c). Because glutamine is catabolized mainly into αKG, which
targeting is still largely unexplored. enters the TCA cycle to replenish TCA cycle intermediates via glutami-
We report here that αKG generated from glutaminolysis promotes nolysis16, we assessed the transcriptomes of naive and LPS- and IL-4-
M2 activation via Jmjd3-dependent metabolic and epigenetic repro- stimulated BMDMs to determine the expression of genes involved in
gramming. In contrast, αKG impairs proinflammatory responses glutaminolysis and αKG metabolism. Treatment with LPS, but not IL-4,
in M1 macrophages by suppressing the nuclear factor-κB (NF-κB) inhibited the expression of genes promoting αKG production (Fig. 1d
pathway through PHD-dependent proline hydroxylation of protein and Supplementary Fig. 1a). To test whether glutamine promotes
kinase IKKβ. Although glutaminolysis restricts M1 polarization, we M2 but suppresses M1 activation through production of αKG, we
found that αKG produced from glutaminolysis during LPS stimula- treated BMDMs with BPTES, an inhibitor of glutaminase 1 (GLS1),
tion has a crucial role in promoting LPS-induced endotoxin tolerance. to suppress glutaminolysis in the absence or presence of dimethyl-
These results highlight the mechanisms controlled by glutaminolysis αKG (DM-αKG), a cell-permeable analog of αKG, during IL-4 or LPS
to fine-tune macrophage activities and suggest that modulation of stimulation17. Treatment with BPTES suppressed expression of M2-
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

glutamine metabolism would be an attractive strategy for harnessing specific genes and arginase 1 activity in IL-4-treated BMDMs, but the
macrophage-mediated immune responses. addition of DM-αKG restored the M2 phenotype (Fig. 1e,f). In con-
trast, BPTES treatment boosted proinflammatory gene expression and
RESULTS cytokine production in LPS-stimulated BMDMs, and the presence of
aKG modulates macrophage polarization DM-αKG impaired this induction (Fig. 1g,h). The effects of targeting
To investigate how glutamine metabolism regulates macrophage acti- glutaminolysis in both M1 and M2 macrophages were confirmed with
vation, we first determined the effect of glutamine deprivation in compound 968, another GLS1 inhibitor (Supplementary Fig. 1b,c).
mouse bone-marrow-derived macrophages (BMDMs) treated with Furthermore, DM-αKG augmented M2 marker gene expression and
IL-4 or LPS. As reported15, glutamine deprivation impaired expres- arginase 1 activity (Supplementary Fig. 1d,e) but impaired the M1
sion of M2-specific marker genes, including Arg1, Ym1 (Chil3), Retnla proinflammatory phenotype (Supplementary Fig. 1f–h) in a dose-
and Mrc1, and suppressed arginase 1 activity (Fig. 1a,b). In contrast, dependent manner. Together, these results indicate that αKG gener-
BMDMs deprived of glutamine showed higher expression of M1-spe- ated by glutaminolysis is crucial for supporting an anti-inflammatory
cific marker genes, including Il1b, Tnf, Il6 and Il12 (Il12b), upon LPS phenotype in macrophages.

a Argl Yml Retnla Mrcl b c Il1b Tnf Il6 Il12

w/o Gln w/o Gln 1.2 * 1.2 * 1.5 1.5 w/o Gln
Relative fold change in

* * 5 *
Relative fold change in

2.0 3.0
Relative fold change in

3.0 2.0 *
mRNA expression

* w/ Gln w/ Gln * w/ Gln

mRNA expression

arginase activity

4
* 1.5 0.8 0.8 1.0 1.0
2.0 2.0 3
1.0 1.0 2 0.4 0.4 0.5 0.5
1.0 1.0
0.5 1
0 0 0 0 0 0 0 0 0

d Ctrl LPS IL-4 e Argl Yml Retnla Mrcl f 1.5 *

2.0 * 2.0
Relative fold change in

2.0 2.0
Ccbl1 *
Relative fold change in

* * * *
arginase activity
mRNA expression

Got1 1.5 1.5 *

1.5 * 1.5
* 1
Gpt2
Idh1 1.0 1.0 1.0 1.0
Idh2 0.5
0.5 0.5 0.5 0.5
Gpt1
Idh3g 0 0 0 0 0
Idh3b BPTES – + + BPTES – + + BPTES – + + BPTES – + + BPTES – + +
DM-αKG – – + DM-αKG – – + DM-αKG – – + DM-αKG – – + DM-αKG – – +
Bcat1
Glud1 g Il1�
*
Tnf Il6 Il12
h
* * *
* *
12 2.5
Gls1 40 * * 3 * * *
900 *
Relative fold change in

Got2 60
mRNA expression

2.0
Idh3a 30
IL-1β (pg/ml)

8
TNF (pg/ml)

2 1.5 40 600
Bcat2
20
Ccbl2 1.0
4 1 20 300
Psat1 10 0.5
Z-score 0
0 0 0 0 0
–1.5 +1.75
e BPTES – + + BPTES – + + BPTES – + + BPTES – + + BPTES – + + BPTES – + +
DM-αKG – – + DM-αKG – – + DM-αKG – – + DM-αKG – – + DM-αKG – – + DM-αKG – – +

Figure 1 Glutamine metabolism modulates macrophage activation via αKG production. (a–c) qPCR analysis of mRNA expression of M2 marker
genes (a), arginase activity (b) or M1 marker genes (c) in BMDMs stimulated with IL-4 (a,b) or LPS (c) under various culture conditions for 6 h.
(d) Expression of genes encoding metabolic enzymes contributing to αKG metabolism in untreated (ctrl) or LPS- or IL-4-treated BMDMs, assessed
by next-generation RNA-seq. (e–h) qPCR analysis of relative mRNA M2 marker gene expression (e), arginase activity (f), M1 marker gene expression
(g) or production of proinflammatory cytokines (h) in BMDMs treated with IL-4 (e,f) or LPS (g,h) under various culture conditions for 6 h. *P < 0.05,
unpaired, two-tailed Student’s t-test. Data are representative of 3 independent experiments with 3 samples per group (a–c, e–h; mean ± s.d.) or
cumulative results from 2 independent experiments (d).

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a w/ Gln
w/ Gln+IL-4
b c w/ Gln
w/ Gln+IL-4
d n.s.
w/o Gln+IL-4 80 * w/o Gln+IL-4 60 n.s.
w/o Gln+DM-αKG+IL-4 * w/o Gln+DM-αKG+IL-4
UK n.s.
Rot/AA

SRC (pmol/min/mg protein)

* 180
180 60

(pmol/min/mg protein)
UK-sensitive OCR
160 40
160
OCR (pmol/min/mg protein)

OCR (pmol/min/mg protein)

140 Rot/AA
140 Oligo
Oligo 40
120 120
FCCP SRC 100
100 FCCP
20
80 20 80

60 60
40 40
0 0
20 DM-αKG – – – + 20 DM-αKG – – – +
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

0 Gln + + – – 0 Gln + + – –
6 18 30 42 54 66 78 90 IL-4 – + + + 6 18 30 42 54 66 78 90 IL-4 – + + +
Time (min) Time (min)

e w/ Gln f g * h
w/ Gln+IL-4 60 * 10 * 14 *
w/o Gln+IL-4 * *
w/o Gln+DM-αKG+IL-4 * * 12

Bodipy C12 uptake (MFI × 103)

160 Eto 8 *

Cpt1a mRNA expression

Etomoxir-sensitive OCR

Relative fold change in

(pmol/min/mg protein)

10
140 40
OCR (pmol/min/mg protein)

6
120 8
Oligo Rot/AA
100 6
4
80 FCCP 20
4
60 2
2
40

20 0 0 0
DM-αKG – – – + DM-αKG – – – + DM-αKG – – – +
0 Gln + + – – Gln + + – – Gln + + – –
6 18 30 42 54 66 78 90
IL-4 – + + + IL-4 – + + + IL-4 – + + +
Time (min)

Figure 2 αKG promotes metabolic changes induced by M2 activation. (a,b) OCR (a) and SRC (b) of BMDMs under various culture conditions, with or
without 6 h IL-4 stimulation before treatment with oligomycin (oligo), FCCP, and rotenone plus antimycin A (Rot/AA). (c) OCR of BMDMs treated as in
a followed by addition of FCCP, UK5099 (UK) and Rot/AA. (d) UK-sensitive OCR of BMDMs treated as in a, calculated as the reduction of OCR after
UK5099 treatment. (e) OCR of BMDMs treated as in a then treated with oligomycin, FCCP, etomoxir (eto) and Rot/AA. (f) Etomoxir-sensitive OCR of
BMDMs treated as in a, calculated as the reduction of OCR after etomoxir treatment. (g,h) Cpt1a mRNA expression (g) and uptake of BODIPY-labeled
C12-fatty acids (h) in BMDMs treated as indicated for 6 h. *P < 0.05, unpaired, two-tailed Student’s t-test. Data are representative of 3 independent
experiments with 3 samples per group (mean ± s.d.).

Glutaminolysis supports metabolic reprogramming in but it was restored by DM-αKG supplementation (Fig. 2e,f), sug-
M2 macrophages gesting that αKG serves as a regulator governing the engagement
M2 activation induces metabolic remodeling to promote oxygen of FAO in M2 macrophages. Further, we found that M2 activation
consumption and drive OXPHOS as the major mode of ATP pro- promotes the expression of carnitine palmitoyltransferase 1a (Cpt1a),
duction11,12. However, the metabolic checkpoints that determine a rate-limiting enzyme in FAO, and increases the rate of fatty acid
the engagement of OXPHOS in M2 macrophages are unknown. To uptake in an αKG-dependent manner (Fig. 2g,h). Collectively, these
investigate whether glutamine metabolism provides such metabolic results indicate that glutamine metabolism is crucial for supporting
checkpoints, we determined the oxygen-consumption rates (OCR) of metabolic reprogramming of M2 macrophages through αKG-medi-
IL-4-stimulated BMDMs using a Seahorse extracellular flux analyzer. ated regulations.
As expected, IL-4-treated BMDMs showed higher spare respiratory
capacity (SRC) than naive BMDMs11. Whereas IL-4 treatment did not M2 polarization relies on the aKG–Jmjd3 pathway
increase SRC in glutamine-deprived BMDMs, the addition of DM-αKG We next investigated whether glutaminolysis promotes M2 activa-
allowed glutamine-deprived BMDMs to acquire this metabolic change tion by influencing activation of transcription factor STAT6. Neither
(Fig. 2a,b), suggesting that αKG is a metabolic checkpoint produced glutamine deprivation nor BPTES treatment suppressed IL-4-induced
by glutaminolysis to support metabolic reprogramming of M2 mac- STAT6 phosphorylation in BMDMs; DM-αKG supplementation in
rophages. We next investigated whether αKG selectively boosts spe- also failed to increase STAT6 phosphorylation in glutamine-deprived
cific nutrient-mediated OXPHOS by treating BMDMs with UK5099, BMDMs (Supplementary Fig. 2a–c), ruling out the possibility that
which blocks mitochondrial import of pyruvate18, or etomoxir, which glutaminolysis promotes M2 activation by boosting STAT6 activa-
suppresses FAO7,11. IL-4 stimulation and glutamine deprivation did tion. In addition to STAT6 signaling, epigenetic reprogramming
not affect pyruvate-dependent OCR in macrophages (Fig. 2c,d). orchestrates M2 activation and sustains macrophage polarization
In contrast, IL-4 treatment markedly augmented FAO-depend- flexibility2,19. We next examined trimethylation of histone H3 K27
ent OCR in glutamine-replete conditions. In glutamine-deprived (H3K27me3), a repressive epigenetic mark that prevents expression
BMDMs, FAO-dependent OCR was not induced by IL-4 stimulation, of M2 marker genes19, on the promoters of M2 marker genes Arg1,

nature immunology VOLUME 18 NUMBER 9 SEPTEMBER 2017 987

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a b Arg1 Ym1
*
Retnla Mrc1
*
* *
Arg1 Ym1 Retnla Mrc1 * *
n.s. * *
n.s.
0.4 n.s. n.s.
* 0.8 * 1.5 * 0.5 0.5 0.8

H3K27 me3 enrichment

0.3 4
H3K27 me3 enrichment

* 1.2 0.3 0.4 0.4 * 0.6

* 0.6 3 * *

(% of input)
*
(% of input)

0.2 * 0.9 * * 0.3 0.3 *

0.4 2 * 0.2 * * 0.4
0.6 0.2 0.2
0.1
0.2 1 0.1 0.2
0.3 0.1 0.1

0 0 0 0 0 0 0 0
Gln + + – Gln + + – Gln + + – Gln + + – Gln – – + + – – Gln – – + + – – Gln – – + + – – Gln – – + + – –
IL-4 – + + IL-4 – + + IL-4 – + + IL-4 – + + DM-αKG – – – – + + DM-αKG – – – – + + DM-αKG – – – – + + DM-αKG – – – – + +
GSK-J4 – + – + – + GSK-J4 – + – + – + GSK-J4 – + – + – + GSK-J4 – + – + – +

c Ctrl
Jmjd3-sgRNA
d Ctrl
Jmjd3-sgRNA
e Ctrl
Jmjd3-sgRNA
Argl Yml Rentla Mrcl Arg1 Ym1 Retnla Mrc1
4 9 * *
10 16 * 20
in mRNA expression

*
Relative fold change

Relative fold change

2.5

in arginase activity
20 3 1.5
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

8 12 3 15 2
** ** * * * *
6

H3K27 me3
15

(% of input)
enrichment
6 1.5 2 1
8 2 n.s. 10
4 n.s. 10
3 1
4 1 n.s. 5 1 0.5
2 n.s. n.s. 0.5 5
0 0 0 0 0 0 0 0 0
DM-αKG – + – + DM-αKG – + – + DM-αKG – + – + DM-αKG – + – + DM-αKG – + – + Gln + – – + – – Gln + – – + – – Gln + – – + – – Gln + – – + – –
DM-αKG – – + – – + DM-αKG – – + – – + DM-αKG – – + – – + DM-αKG – – + – – +

f Ctrl
Jmjd3-sgRNA
g Ctrl Jmjd3-sgRNA w/ Gln h Ctrl Jmjd3-sgRNA
Ccl22 Hr Il4il Irf4 Rot/AA w/ Gln+IL-4
100 100 50 * 50
* w/o Gln+IL-4 * *
Relative fold change in

1.5 1.5
**
2.0 1.2 ** Rot/AA

SRC (pmol/min/
*
mRNA expression

OCR (pmol/min/
n.s. 80 40 40
* n.s. 80 w/o Gln+ n.s.

mg protein)
Oligo
mg protein)
n.s. 1.5 n.s. n.s. DM-αKG+IL-4
1.0 1.0 n.s. * 0.8 60 60 Oligo 30 30 n.s. n.s.
n.s. n.s. FCCP
1.0 40 FCCP 20 20
40
0.5 0.5 0.4
0.5 20 20 10 10
0.0 0.0 0.0 0.0 0 0 0 0
Gln + – – + – – Gln + – – + – – Gln + – – + – – Gln + – – + – – 18 36 54 72 18 36 54 72 DM-αKG – – – + DM-αKG – – – +
DM-αKG – – + – – + DM-αKG – – + – – + DM-αKG – – + – – + DM-αKG – – + – – + Time (min) Gln + + – – Gln + + – –
Time (min)
IL4 – + + + IL4 – + + +

Figure 3 αKG promotes IL-4-induced epigenetic changes in a Jmjd3-dependent manner. (a,b) Chromatin-immunoprecipitation (ChIP) analysis of
H3K27me3 in BMDMs with or without IL-4 stimulation (a) or stimulated with IL-4 with or without DM-αKG or GSK-J4 (b) after real-time qPCR of
promoter regions on M2 marker genes. Data are normalized to input. (c–e) Relative mRNA expression (c) or H3K27me3 (e) of promoter regions on M2
marker genes and arginase activity (d) in Jmjd3-deficient (Jmjd3-sgRNA) or control (ctrl) IL-4-stimulated BMDMs with or without DM-αKG (1 µM).
(f) Relative mRNA expression in IL-4 stimulated BMDMs under various culture conditions. (g) OCR (g) or SRC (h) of BMDMs stimulated with IL-4
under various conditions for 6 h then treated with oligomycin (oligo), FCCP and rotenone plus antimycin A (Rot/AA). *P < 0.05, unpaired, two-tailed
Student’s t-test. Data are representative of 3 independent experiments (a,b,e–h; mean ± s.d.) or from 2 independent experiments with 3 samples per
group (c,d; mean ± s.d.).

Ym1, Retnla and Mrc1. IL-4 treatment decreased H3K27me3 on the Jmjd3-deficient BMDMs (Fig. 3c,d). Furthermore, supplementation
promoters of these genes in a glutamine-dependent fashion (Fig. 3a), of glutamine or DM-αKG did not augment demethylation of
which indicates that glutamine metabolism may support M2 activa- H3K27me3 in Jmjd3-deficient BMDMs (Fig. 3e).
tion by facilitating demethylation of H3K27. Given that Jmjd3 is a Next, we compared the expression of IL-4-induced genes, including
key enzyme for demethylation of H3K27 and controls M2 activa- Ccl22, Hr, Il4i1 and Irf4, which have been reported to be suppressed
tion through epigenetic regulations19,20, we asked whether glutamine by glutamine deprivation15. Deprivation of glutamine suppressed
metabolism supports M2 activation by promoting Jmjd3-dependent the expression of these genes, and supplementation with DM-αKG
demethylation of H3K27 on the promoters of M2-specific marker restored it in BMDMs. However, supplementation of glutamine or
genes. DM-αKG promoted loss of H3K27me3 in glutamine-deprived DM-αKG did not promote expression of these genes in Jmjd3-defi-
BMDMs, and treatment with GSK-J4, a Jmjd3 selective inhibitor 21, cient BMDMs (Fig. 3f). Next, we found that Jmjd3-deficient BMDMs
suppressed demethylation of H3K27 in the presence of glutamine did not increase oxygen consumption in response to IL-4 in the pres-
or DM-αKG (Fig. 3b). Moreover, GSK-J4 suppressed expression of ence of glutamine or DM-αKG (Fig. 3g,h), which suggests that the
M2-specific marker genes and arginase 1 activity (Supplementary αKG–Jmjd3 signaling pathway is responsible for metabolic repro-
Fig. 2d,e) in BMDMs stimulated with IL-4 in glutamine-replete gramming in M2 macrophages. Together, these results indicate that
conditions or with DM-αKG supplementation, but not in BMDMs the αKG–Jmjd3 pathway functions as a metabolic checkpoint by
cultured in glutamine-depleted conditions. This suggests that αKG which glutamine metabolism supports macrophage M2 activation.
produced from glutamine metabolism promotes M2 activation via
Jmjd3-dependent epigenetic reprogramming. To further test whether aKG/succinate ratio modulates macrophage activation
DM-αKG augments M2 activation in a Jmjd3-dependent manner, we αKG is a co-stimulator factor for Jmjd3, and succinate is an inhibi-
transduced BMDMs from mice in which Cas9 is selectively expressed tor; therefore, a high αKG/succinate ratio induces Jmjd3-dependent
in macrophages (LysM-Cre Cas9 mice)22 with a lentivirus harboring demethylation of H3K27 to support pluripotency of embryonic stem
singe-guide RNAs (sgRNAs) targeting Jmjd3 or scramble sgRNAs to cells17,23. To test whether αKG/succinate ratios modulate macrophage
generate Jmjd3-deficient BMDMs or control BMDMs, respectively. activation, we measured the αKG/succinate ratios in naive, M1 and
Jmjd3 expression was lower in Jmjd3-deficient BMDMs than in con- M2 BMDMs and found that IL-4 stimulation resulted in a higher
trol BMDMs (Supplementary Fig. 2f), and DM-αKG treatment did αKG/succinate ratio than LPS treatment, (Fig. 4a and Supplementary
not promote M2-specific gene expression or arginase 1 activity in Fig. 3a), suggesting that regulation of αKG dehydrogenase activity

988 VOLUME 18 NUMBER 9 SEPTEMBER 2017 nature immunology

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a 2.5 *
* b *
c Argl Yml Retnla Mrcl
2.0
5 * * 4 * 2.5 *

αKG dehydrogenase
3 *

in mRNA expression
Relative fold change
in mRNA expression
Relative fold change

in mRNA expression
Relative fold change
2.0 *

in mRNA expression
Relative fold change
αKG/succinate

*
arbitrary ratio

1.5 4 2.0
*

activity (fold)
3 n.s.
1.5

Relative
3 2 1.5
1.0 n.s. 2 n.s.
1.0 2 n.s. 1.0
1
0.5 0.5 1 1 0.5
0.0 0.0 0 0 0 0.0
Ctrl IL-4 LPS IL-4 LPS DE-Suc. – + + + DE-Suc. – + + + DE-Suc. – + + + DE-Suc. – + + +
DM-αKG (mM) – – 0.1 1 DM-αKG (mM) – – 0.1 1 DM-αKG (mM) – – 0.1 1 DM-αKG (mM) – – 0.1 1

d II1b e Argl Yml Retnla Mrcl f II1b

* * *
* * * * *
4
* * 3
* * 3 * * 4
* *
1.2

in mRNA expression
Relative fold change
4
in mRNA expression
Relative fold change

in mRNA expression
Relative fold change
in mRNA expression
Relative fold change
in mRNA expression
in mRNA expression

Relative fold change

Relative fold change
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

3 3 3
2 2 0.8
n.s.
2 2 2
n.s.
1 1 0.4
1 1 1

0 0 0 0 0 0.0
DE-Suc. – + + + DM-αKG – + + + DM-αKG – + + + DM-αKG – + + + DM-αKG – + + + DM-αKG – + + +
DM-αKG (mM) – – 0.1 1 DE-Suc. (mM) – – 0.1 1 DE-Suc. (mM) – – 0.1 1 DE-Suc. (mM) – – 0.1 1 DE-Suc. (mM) – – 0.1 1 DE-Suc. (mM) – – 0.1 1

Figure 4 Integration of αKG/succinate ratio in macrophage determines macrophage immune responses. (a) Intracellular αKG/succinate ratio in BMDMs
stimulated with vehicle (ctrl), IL-4 or LPS for 18 h, measured by MS after metabolite extraction (Online Methods). (b) Activity of mitochondrial αKG
dehydrogenase in BMDMs stimulated with IL-4 or LPS for 18 h. (c,d) qPCR analysis of relative mRNA expression of M2 marker genes in glutamine-
deprived BMDMs stimulated with IL-4 (c) and Il1b mRNA in BMDMs stimulated with LPS (d) under various conditions for 6 h. (e,f) qPCR analysis of
relative mRNA expression of M2 marker genes in glutamine-deprived BMDMs stimulated with IL-4 (e) and Il1b mRNA in BMDMs stimulated with LPS
(f) for 6 h under various conditions. *P < 0.05, unpaired, two-tailed Student’s t-test. Data are representative of 3 independent experiments with
3 samples per group (a–f; mean ± s.d.).

may differ between M1 and M2 macrophages to affect αKG-to- NF-κB signaling targets, such as Cd80 and Sele, in LPS-stimulated
succinate conversion and establish different αKG/succinate ratios. BMDMs but not in BMDMs treated with control vehicle (Fig. 5c),
Indeed, LPS-stimulated BMDMs showed higher αKG dehydrogenase which suggests that αKG derived from glutaminolysis may impair
activity than IL-4-treated BMDMs (Fig. 4b), which indicates that M2 proinflammatory responses by restraining the NF-κB signaling path-
macrophages may favor αKG accumulation by suppressing αKG dehy- way. Because αKG can interfere with cellular function by activating
drogenase activity. To further examine whether αKG/succinate ratios PHD enzymes24, we examined whether αKG suppresses M1-specific
affect M2 activation, we treated glutamine-deprived BMDMs with a marker gene expression in a PHD-dependent manner. Treatment with
fixed concentration of diethyl-succinate (DE-Suc), a cell-permeable dimethyloxalyglycine (DMOG), an inhibitor of PHD activity, restored
succinate, in the presence of increasing doses of DM-αKG. Increasing M1 marker gene expression in DM-αKG-treated BMDMs (Fig. 5d).
αKG/succinate ratios in this manner promoted M2-specific marker BPTES treatment in LPS-stimulated BMDMs increased the amount
gene expression (Fig. 4c) and arginase 1 activity (Supplementary of phosphorylated IKK, whereas DM-αKG reduced it (Fig. 5e).
Fig. 3b). In contrast, although DE-Suc acts as a proinflammatory Co-treatment with DMOG restored IKK phosphorylation in DM-
metabolite to stimulate Il1b gene expression and cytokine produc- αKG-treated BMDMs, suggesting that αKG impedes IKK activation
tion in LPS-treated macrophages8, treatment with escalating doses of by augmenting PHD activity. PHD has been reported to inhibit IKKβ
DM-αKG gradually abolished IL-1β expression in BMDMs (Fig. 4d activation through hydroxylation of IKKβ on P191 (refs. 25,26). We
and Supplementary Fig. 3c). In addition, increasing doses of DE-Suc next overexpressed wild-type IKKβ or P191A-IKKβ, a proline-to-
impaired DM-αKG-induced M2 marker gene expression (Fig. 4e) alanine substitution mutant that cannot be hydroxylated by PHD, in
and arginase 1 activity (Supplementary Fig. 3d). However, DE-Suc BMDMs (Supplementary Fig. 4b) and treated them with LPS. DM-
did not restore IL-1β expression in DM-αKG-treated macrophages αKG did not suppress LPS-induced M1 marker gene expression in
(Fig. 4f and Supplementary Fig. 3e). Together, these data indicate BMDMs transduced with P191A-IKKβ, in contrast to non-transduced
that αKG/succinate ratio affects macrophage polarization programs, BMDMs and BMDMs overexpressing wild-type IKKβ (Fig. 5f).
and manipulation of this ratio may allow tailoring of macrophage Similarly, DM-αKG did not suppress LPS-induced nuclear translo-
immune responses. cation of NF-κB in P191A-IKKβ−transduced BMDMs, as assessed by
imaging flow cytometry (Fig. 5g,h). These results suggest a role for
aKG suppresses IKKb activation αKG in restricting M1 activation by intervening with the NF-κB path-
We tested whether αKG inhibits production of proinflammatory way via PHD-mediated post-translational modification of IKKβ.
cytokines by suppressing a conserved upstream signal responsible
for M1 activation. Both glutamine deprivation and BPTES treatment Priming-phase glutaminolysis modulates endotoxin tolerance
in BMDMs promoted nuclear translocation of transcription factor Glutamine uptake in macrophages is greatly enhanced by LPS treat-
NF-κB in response to LPS treatment, an essential event to drive broad ment27, while exposure to LPS induces endotoxin tolerance, in which
proinflammatory functions (Fig. 5a and Supplementary Fig. 4a). macrophages become resistant to subsequent exposure after reso-
Further, DM-αKG suppressed nuclear translocation of NF-κB in lution of the acute response, to prevent excessive proinflammatory
glutamine-deprived BMDMs (Fig. 5b). As assessed by a PCR array, responses3,28,29. To test whether the engagement of glutamine metabo-
supplementation with DM-αKG suppressed expression of many lism during initial LPS activation contributes to the establishment

nature immunology VOLUME 18 NUMBER 9 SEPTEMBER 2017 989

 Articles

a +Gln –Gln c
LPS (min) 0 15 30 60 15 30 60 2
Nuclear NF-κB 1
fraction Lamin-A/C 0

Fold regulation
–1
NF-κB –2
WCL –3
β-actin
–4
–5
b Ctrl DM-αKG
LPS (min) 0 15 30 60 15 30 60
–6
–20
Nuclear NF-κB –24

Csf1
Irf1
Ifnb1

CxcI1

Icam1

Vcam1

Nfkb1

BcI2I1

Stat1

Akt1

Nqo1

Ccnd1
Lta

II1a

Nfkbia

Bcl2a1a

Rela

Cdkn1a

II2ra
II1m
Cd74

Adm
Sele

II12b

Ltb

Cfb

Gadd45b
Fas

Pdgfb

Selp
Relb
Tnfrsf1b

Xiap

II1b

Csf2rp
Stat5b
Plau
Cd80

Myd88

F8
Ifng

Birc3

Cxcl3

Cd83

C3

Ncoa3

Tnfsf10
Cxcl9
Myc

Cd40
Stat3

Trp53

Csf3

Cxcl10

Mmp9

F3
II6

Map2k6
Egr2

Csf2
Birc2

II1r2
Nr4a2

CcI12

Nfkb2
CcI22

Sod2
Ptgs2
Aldh3a2
Traf2
Ccr5

II15
CcI5
Rel

Egfr
Tnf

Mitf
fraction Lamin-A/C

NF-κB
WCL
β-actin

d e f
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

II1b Tnf II1b Tnf II6 II12

** * * Ctrl
3 3.0 * BPTES – + + + – + + + 2.5 1.5 * 1.6 n.s. 1.5
* WT IKKβ

in mRNA expression
Relative fold change
n.s.
in mRNA expression
Relative fold change

* * DM-αKG – – + + – – + + * P191A-IKKβ
2.5
n.s. DMOG – – – + – – – + 2.0
* * 1.2 * n.s. 1.2 1.2
*
2 * 2.0 LPS 30 min 60 min 1.5 0.9 0.9
n.s.
1.5 0.8
p-IKKα/β 1.0 0.6 0.6
1 1.0
0.5 0.3 0.4 0.3
0.5 IKKα/β
0 0.0 β-actin 0.0 0.0 0.0 0.0
DMOG(mM) – – – 0.5 1 DMOG(mM) – – – 0.5 1 DM-αKG – + – + – + DM-αKG – + – + – + DM-αKG – + – + – + DM-αKG – + – + – +
DM-αKG – – + + + DM-αKG – – + + +
BPTES – + + + + BPTES – + + + +
g h
II6 II12 WT IKKβ P191A-IKKβ n.s. DAPI NF-κB Merge
* * * Relative similarity index 1.2
4 * * 3.0 * * Ctrl
in mRNA expression
Relative fold change

36.5% 46.4% Ctrl

n.s. * Ctrl 1.0
DM-αKG WT IKKβ
3
2.0
0.8 DM-αKG
2
1.0
1 0.6
26.3% 48.1% 0.3 Ctrl
0 0.0 DM-αKG P191A-IKKβ
DMOG(mM) – – – 0.5 1 DMOG(mM) – – – 0.5 1 0.0
DM-αKG
Kβ

Kβ

DM-αKG – – + + + DM-αKG – – + + +
IK

IK

BPTES – + + + + BPTES – + + + +
A-
T
W

91
P1

Figure 5 αKG suppresses M1 activation through a PHD-dependent post-translational regulation of IKKβ. (a,b) Immunoblot analysis of NF-κB p65 in
BMDMs stimulated with LPS in medium with or without glutamine (a) or in glutamine-depleted medium supplemented with vehicle (ctrl) or DM-αKG
(1 mM) (b). WCL, whole-cell lysate. (c) qPCR array analysis of NF-κB target gene expression in BMDMs stimulated with LPS in glutamine-depleted
medium with or without DM-αKG (1 mM) for 6 h. Genes showing significant differences (P < 0.05) after DM-αKG treatment are shown in red.
(d) qPCR analysis of M1 marker gene expression in BMDMs stimulated with LPS in glutamine-replete medium with or without DM-αKG and/or BPTES.
DMOG, dimethyloxalyglycine. (e) Immunoblot analysis of phosphorylated (p-) IKKα/β and IKKα/β in BMDMs stimulated with LPS under various culture
conditions. (f,g) qPCR analysis of M1 marker gene expression (f) and imaging flow analysis of NF-κB (g) in untransduced (ctrl) BMDMs or BMDMs
overexpressing wild-type (WT) IKKβ or P191A-IKKβ, stimulated with LPS in glutamine-depleted medium. (g) Left, representative histograms of
similarity profiles of NF-κB nuclear translocation. Right, combined quantitative results of similarity index. (h) Representative images of cells from g.
*P < 0.05, unpaired, two-tailed Student’s t-test. Data are representative of 3 (a,b,d,e; mean ± s.d.)), 4 (g,h; mean ± s.d.) or 2 (f) independent
experiments or are cumulative results from 4 independent experiments (c) with 3 (a,b,d–f) or 4 (c) samples per group.

of endotoxin tolerance, we deprived BMDMs of glutamine during genes2,3,28. RNA sequencing of BMDMs primed with LPS in
first LPS priming and then re-challenged them with LPS to examine glutamine-replete medium, or glutamine-depleted medium with or
production of proinflammatory genes, including Il1b, Tnf, Il6 and without DM-αKG, showed that glutamine deprivation during LPS
Il12 (Supplementary Fig. 5a). Macrophages primed with LPS in priming suppressed expression of reparative and antimicrobial gene
glutamine-deprived conditions show high expression of proinflam- expression, while DM-αKG treatment sustained the expression of
matory genes compared to BMDMs primed in glutamine-replete most of this gene set (Supplementary Fig. 5f). Further comparison
conditions (Fig. 6a), which indicates that LPS-induced endotoxin of global gene expression patterns showed that glutamine depriva-
tolerance requires glutamine. Furthermore, inhibition of glutami- tion altered the expression patterns of more than 1,200 genes in LPS-
nolysis by BPTES during the LPS priming phase sustained BMDM’s primed BMDMs, and supplementation with DM-αKG restored the
ability to produce proinflammatory genes upon re-challenge with LPS expression patterns of 506 genes in this set (Fig. 6d). Furthermore,
(Supplementary Fig. 5b), which suggests that glutaminolysis dur- gene cluster analysis revealed that those genes regulated by αKG and
ing initial LPS treatment is essential to induce endotoxin tolerance. glutamine metabolism controlled biological processes involved in
Glutamine-deprived BMDMs treated with DM-αKG during LPS prim- inflammatory responses, exosome release and mitochondrial activ-
ing (Supplementary Fig. 5c) showed suppression of proinflammatory ity (Fig. 6e). Because mitochondrial activity controls energy pro-
gene expression (Fig. 6b) and cytokine production (Supplementary duction and metabolic profiles, and immunometabolic paralysis—a
Fig. 5d,e) upon re-exposure to LPS. However, DE-Suc treatment did global impairment of major metabolic pathways—has been reported
not restore endotoxin tolerance in glutamine-deprived macrophages to be a metabolic phenotype during endotoxin tolerance in human
(Fig. 6c), implying that αKG promotes endotoxin tolerance independ- monocytes30, we next used metabolomics analysis to determine
ently of its conversion into succinate. whether glutamine metabolism during LPS priming supports immu-
In addition to resolving proinflammatory responses, endotoxin- nometabolic paralysis. As expected, tolerant macrophages showed a
tolerant macrophages express tissue-repairing and antimicrobial systemic reduction of metabolic activities compared to nontolerant

990 VOLUME 18 NUMBER 9 SEPTEMBER 2017 nature immunology

 Articles

a II1b Tnf II6 II12

c II1b
n.s.
Tnf
n.s.
II6
n.s.
II12
DE-Suc.
2.5 3.0 2.0 2.0 2.5
3.0 4.5 w/ Gln 1.6 n.s. Ctrl
in mRNA expression
Relative fold change

in mRNA expression
Relative fold change
2.5 w/o Gln n.s. n.s. n.s. 2.0
2.5 2.0 1.5
1.2 1.5
2.0 3.0 2.0 1.5 n.s.
1.5
* * 1.0
1.5
1.0
1.5 * 0.8 1.0
1.0
1.0 1.5 * 1.0
0.4 0.5 0.5 0.5
0.5 0.5 0.5
0 0 0 0 0 0 0 0
2nd LPS + + + + 2nd LPS + + + + 2nd LPS + + + + 2nd LPS + + + + 2nd LPS + + + + 2nd LPS + + + + 2nd LPS + + + + 2nd LPS + + + +
1st LPS – + – + 1st LPS – + – + 1st LPS – + – + 1st LPS – + – + 1st LPS – + – + 1st LPS – + – + 1st LPS – + – + 1st LPS – + – +

b 2.5
II1b
n.s. * 2.0
Tnf
*
2.0
II6
n.s. * 2.5
II12
*
in mRNA expression
Relative fold change

2.0 * * n.s. 2.0 n.s.

* * 1.5 1.5 * * * w/ Gln + Ctrl
1.5 1.5 * w/ Gln + DM-αKG
1.0 1.0 w/o Gln + Ctrl
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

1.0 1.0
w/o Gln + DM-αKG
0.5 0.5 0.5
0.5
0 0 0 0
2nd LPS + + + + + + + + 2nd LPS + + + + + + + + 2nd LPS + + + + + + + + 2nd LPS + + + + + + + +
1 LPS – + – + – + – +
st 1 LPS – + – + – + – +
st 1 LPS – + – + – + – +
st 1 LPS – + – + – + – +
st

d e Cell adhesion Chemotaxis Wound healing Response to LPS

Integral component of
plasma membrane Mitochondrion
0 0 0
Fold changes

Fold changes
Upregulated genes Downregulated genes 2
2 –0.5

Cluster 2
–0.5
2 –1
1 1 –1 –1
1
–2 –1.5
–1.5
Cluster 1

0 0 0
184 202 55 589 304 124
Extracellular exosome Protein binding Cell surface

w ln
ln

ln

M n

ln

M n
KG

KG

KG
l

l
ET / G

/G

ET / G

G
-α

-α

-α
/o

/o

ln w/o
w

w
Fold changes

M
3 3

w
2

ET

ET

ET
D

D
ET
+

+
Gln supplementation Gln supplementation 2 2

ln

ln
G

G
DM-αKG supplementation DM-αKG supplementation 1

/o

/o

/o
1 1

w
Overlap Overlap

ET

ET

ET
0 0 0
ln

ln

ln

ln

/o ET Gln

M n
KG

KG

KG
l
/G

/G

G
-α

-α

-α
/o

/o

/
/o
w

w
M

M
w

w
ET

ET

ET
D

D
ET

ET
+

+
ln

ln

ln
G

G
/o

/o
w

w
ET

ET

ET

Figure 6 Glutamine metabolism supports induction of endotoxin tolerance in an αKG-dependent manner. (a) qPCR analysis of relative mRNA expression
in LPS-stimulated BMDMs with or without LPS priming in glutamine-replete or glutamine-depleted medium. (b,c) qPCR analysis of relative mRNA
expression of M1 marker genes in BMDMs stimulated with LPS after priming in glutamine-depleted medium with or without DM-αKG (1 mM) (b) or
DE-Suc (5 mM) (c). (d) Numbers of unique and overlapping genes significantly (P < 0.05) upregulated (left) or downregulated (right) in BMDMs
stimulated with LPS for 18 h in glutamine-depleted medium then supplemented with glutamine or DM-αKG. (e) Clustering of differentially expressed
genes in BMDMs primed with LPS in glutamine-replete (ET w/ Gln) glutamine-depleted (ET w/o Gln) medium or in ET w/o Gln medium + DM-αKG
(centered to the mean expression of ET w/o Gln group). Black lines represent expression patterns of individual genes. Red lines indicate mean changes
of indicated gene clusters. Data are representative of 3 independent experiments (a–c; mean ± s.d.; *P < 0.05, unpaired, two-tailed Student’s t-test) or
are cumulative from 2 independent experiments with 3 samples per group (d,e).

macrophages (Supplementary Fig. 5g), while glutamine deprivation LPS (Supplementary Fig. 6a,b). Moreover, the addition of DM-αKG
during initial LPS treatment prevented induction of endotoxin tol- during LPS priming did not affect activation of proximal signaling
erance but did not affect reduction of global metabolic activities in pathways in glutamine-deprived BMDMs (Supplementary Fig. 6c),
BMDMs. In agreement with published findings30, LPS-primed BMDMs which indicates that glutamine metabolism affects establishment of
produced lower amounts of lactate and pyruvate upon re-exposure to endotoxin tolerance by mechanisms independent of LPS proximal
LPS than did naive BMDMs exposed to LPS (Supplementary Fig. 5h); signaling pathways. Next, we examined expression of negative regu-
however, nontolerant, glutamine-deprived BMDMs also generated lators of the NF-κB signaling pathway, which are known to induce
lower amounts of lactate and pyruvate during initial LPS treatment, endotoxin tolerance3. Glutamine-deprived BMDMs expressed lower
which suggests that glutamine metabolism may not impede the induc- amounts of these regulators during LPS priming than did BMDMs
tion of endotoxin tolerance by preventing immunometabolic paralysis. primed with LPS in the glutamine-replete cultures. Notably, this phe-
Together, these data demonstrate that glutamine metabolism during nomenon was prevented by DM-αKG treatment (Fig. 7a). Moreover,
M1 activation acts as a metabolic checkpoint to induce endotoxin glutamine deprivation and BPTES treatment during LPS priming
tolerance and regulate the transcriptomic landscape associated with sustained NF-κB nuclear translocation upon re-exposure to LPS in
endotoxin tolerance in macrophages. BMDMs (Fig. 7b and Supplementary Fig. 6d). In contrast, DM-αKG
treatment diminished the capacity of glutamine-deprived BMDMs
Glutaminolysis impairs the NF-kB pathway in tolerant macrophages to enhance NF-κB nuclear translocation upon re-challenge with LPS
Impairment of LPS-activated proximal signaling pathways is a hall- (Fig. 7c). Of note, supplementation of DM-αKG altered nuclear trans-
mark event in the induction of endotoxin tolerance3,31. Glutamine location of NF-κB only in BMDMs primed with LPS in glutamine-
deprivation and BPTES treatment during LPS priming did not prevent deprived cultures (Supplementary Fig. 6e). These results suggest that
the impairment of proximal signaling pathways upon re-exposure to αKG modulates endotoxin tolerance by affecting NF-κB-mediated

nature immunology VOLUME 18 NUMBER 9 SEPTEMBER 2017 991

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a b c

+
KG ln
-α G
ln
Ctrl DM-αKG

ln

M /o
G
+Gln –Gln

/G

D Tw
/o
1st LPS 1st LPS + + + + + + +

E
– – – – + + + + + +

ET

ET
2nd LPS (min) 0 15 30 60 15 30 60 15 30 60 2nd LPS (min) 0 15 30 60 15 30 60
Trim30a NF-κB NF-κB
Nuclear Nuclear
Irf7
fraction fraction Lamin-A/C
Irf4 Lamin-A/C
Trafd1
NF-κB NF-κB
Zfp36 WCL
Socs3 WCL
β-actin β-actin
Inpp5d
Irf3
Maf1
Homer1
Ltbp1
d 1.5
Arg1
2.0
Mmp9
1.5
Mrc1
* Ctrl
* * *

Relative fold change in

II1rl1 Jmjd3-sgRNA

mRNA expression
Bcl3 1.5
1.0 * 1.0
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.

Dusp1 *
Socs1 1.0
Nlrc5
Tfnaip8l2 0.5 0.5
Kremen1 0.5
Tnaip3
Nr4a2 0 0 0
Nlrx1 Gln + – – + – – Gln + – – + – – Gln + – – + – –
Mt2
Dusp4 DM-αKG – – + – – + DM-αKG – – + – – + DM-αKG – – + – – +
Z-score
–1.8 +1.8
e ET f g ET

ET-BPTES * ET-BPTES + DM-αKG ET-BPTES

Septic shock ** ET-BPTES ET-BPTES + DM-αKG
100 100
100
80 80
80
Survival (%)

Survival (%)

Survival (%)
60 60
60
40 40 P < 0.001 40
20 20 20
0 0 0
0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8
Time (h) Time (h) Time (h)

Figure 7 αKG supports endotoxin tolerance in macrophages by modulating NF-κB signal and Jmjd3-dependent regulation. (a) Expression of genes
encoding regulators contributing to induction of endotoxin tolerance in BMDMs stimulated with LPS for 18 h in glutamine-replete (ET w/ Gln) or
glutamine-depleted (ET w/o Gln) medium or in ET w/o Gln medium plus DM-αKG, assessed by RNA-seq. (b,c) Immunoblot analysis of LPS-primed (1st
LPS) or unprimed BMDMs cultured with or without glutamine (b) or DM-αKG (c) after re-stimulation with LPS in glutamine-replete medium. (d) qPCR
analysis of relative mRNA expression in control (ctrl) or Jmjd3-deficient (Jmjd3-sgRNA) BMDMs after LPS stimulation under various culture conditions
for 18 h. (e) Kaplan–Meier survival curves of mice after injection with vehicle (septic shock), LPS (ET) or LPS plus BPTES (ET-BPTES) followed
by injection of LPS 18 h later. *P < 0.02; **P < 0.005, Mantel–Cox test (n = 9–10 mice per group). (f) Kaplan–Meier survival curves of mice after
injection with ET-BPTES or ET-BPTES + DM-αKG (ET-BPTES-DM-αKG) followed by injection with LPS 18 h later (n = 10 mice per group).
(g) Kaplan–Meier survival curves of macrophage-depleted mice after injection with LPS (ET), ET-BPTES or ET-BPTES-DM-αKG followed by injection
with LPS 18 h later (n = 8–11 mice per group). Data are representative of 3 independent experiments (b–d; mean ± s.d.; *P < 0.05, unpaired,
two-tailed Student’s t-test) or are cumulative from 2 (a,e,f) or 3 (g) independent experiments.

transcriptional events. Because LPS priming can stimulate Jmjd3 To further assess the physiological relevance of endotoxin tol-
expression32, and Jmjd3 promotes expression of IRF4, which is an erance regulated by glutamine metabolism, we pre-injected mice
important regulator for establishment of endotoxin, we tested whether with PBS, a low dose of LPS or a low dose of LPS plus BPTES to
the αKG–Jmjd3 pathway regulates the establishment of endotoxin induce endotoxin tolerance, then we re-challenged the mice with a
tolerance. Supplementation with glutamine and DM-αKG treatment lethal dose of LPS 18 h later. Pre-exposure to LPS resulted in pro-
suppressed induction of proinflammatory genes in Jmjd3-deficient longed survival due to the establishment of endotoxin tolerance,
BMDMs upon re-exposure to LPS (Supplementary Fig. 6f), which as expected, whereas treatment with BPTES abrogated endotoxin
suggests that αKG promotes tolerance on LPS-induced proinflam- tolerance-induced protection (Fig. 7e). However, the addition of
matory gene expression in a Jmjd3-independent manner. In contrast, DM-αKG to the primary injection of LPS with BPTES markedly
supplementation with glutamine and DM-αKG treatment increased improved survival (Fig. 7f). Because BPTES and DM-αKG treat-
the expression of some genes, including Arg1, Mmp9 and Mrc1, which ments might affect multiple cell types in these assays, we examined
regulate reparative activity, in endotoxin-tolerant control BMDMs but induction of endotoxin tolerance in mice injected with clodronate
not in Jmjd3-deficient BMDMs (Fig. 7d), which suggests that their liposome to deplete macrophages and monocytes before first LPS
induction by DM-αKG was Jmjd3-dependent. Together, these data challenge 5 (Online Methods). BPTES treatment with or without
suggest that αKG produced by glutamine metabolism in macrophages DM-αKG during primary injection of LPS resulted in similar sur-
controls induction of endotoxin tolerance via different mechanisms vival rates as those seen with control vehicle treatment (Fig. 7g),
for proinflammatory genes and reparative genes. which suggests that targeting glutamine metabolism in macrophages

992 VOLUME 18 NUMBER 9 SEPTEMBER 2017 nature immunology


may modulate induction of endotoxin tolerance in vivo, as we
observed here. Together, these results demonstrate that glutamine
metabolism during M1 activation acts as a metabolic checkpoint
to induce endotoxin tolerance by affecting NF-κB-mediated
transcriptional regulation.
DISCUSSION
Our study shows that αKG produced by glutaminolysis is an anti-
inflammatory metabolite that augments M2 activation and controls
metabolic reprogramming of M2 macrophages through Jmjd3-
dependent regulations. Similarly to a metabolic checkpoint used
in embryonic stem cells to determine differentiation and pluripo-
tency17,23, glutamine metabolism supported either proinflammatory
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
or anti-inflammatory activation in response to the αKG/succinate
ratio. Furthermore, glutamine metabolism restricted M1 activation by
hampering the NF-κB pathway via an αKG–PHD-dependent mecha-
nism to modulate IKKβ activity. Last, targeting glutamine metabolism
could prevent induction of endotoxin tolerance and sustain a macro-
phage’s proinflammatory potential.
Epigenetic and metabolic reprogramming orchestrate macro-
phage polarization and contribute to macrophage functional plas-
ticity2,6. However, the mechanisms by which macrophages integrate
these complicated cellular activities have not been established. Our
study reveals that αKG prevents M1 activation by suppressing IKKβ
activation, and this mechanism is controlled via PHD-dependent
proline hydroxylation on IKKβ. PHDs belong to the dioxygenase
proteins, in which enzyme activity is positively regulated by αKG
but antagonized by succinate24. We found that succinate did not
antagonize αKG-induced impairment of the IKKβ–NF-κB pathway.
Although the molecular details remain unclear, this may result from
the different affinity of PHD isoforms for succinate binding and
association with IKKβ25. In addition, we showed that αKG acts as a
metabolic regulator that instructs M2 macrophages to augment FAO.
Jmjd3 has been reported to promote formation of brown adipocytes
through epigenetic regulation of genes involved in mitochondrial
biogenesis and FAO33. In support of this result, our data indicate
that the αKG–Jmjd3 pathway modulates M2 macrophage meta-
bolic reprogramming. It is also interesting to note that succinate can
stabilize HIF-1α, but αKG destabilizes it8. Therefore, in M1 macro-
phages, the reduced αKG/succinate ratio might further strengthen
the metabolic program of M1 macrophages by boosting HIF-1α-
supported aerobic glycolysis.
We showed that αKG production during the LPS priming phase
determines the induction of endotoxin tolerance on proinflam-
matory genes; however, this was independent of Jmjd3. It is likely
that other epigenetic regulators in the dioxygenase protein family
are responsible for αKG-induced endotoxin tolerance. For example,
DNA demethylase Tet2, a target of αKG, controls proinflammatory
responses in macrophages34. Future studies are needed to examine
whether Tet2 or other dioxygenase proteins regulate αKG-induced
endotoxin tolerance. The results of such studies could warrant the
development of novel approaches to fine-tune macrophage immune
responses during sepsis and immune paralysis induced by endotoxin
tolerance. Notably, glutaminolysis supports β-glucan-induced trained
immunity, a nonspecific immune response that protects against re-
infection35,36. Because LPS cannot induce trained immunity but
promotes endotoxin tolerance35, it will be of interest to compare the
metabolic demands and metabolic fate of glutamine in β-glucan-
and LPS-treated macrophages. The results of these comparisons
may uncover how these macrophages elicit distinct consequences on
immune responses.
nature immunology VOLUME 18 NUMBER 9 SEPTEMBER 2017
Articles
Tumor-associated macrophages have been suggested to behave sim-
ilarly to endotoxin tolerant macrophages because of their elevated tis-
sue-repairing activity and defective proinflammatory activation37–39.
Because glutaminolysis supports proliferation and the integrity of TCA
cycle in tumor cells40, the antitumor effects of glutaminase inhibitors
are under intensive investigation in animal models and clinical trials41.
It will be important to explore whether glutaminase inhibitor treat-
ment can affect the phenotypes of tumor-associated macrophages and
stimulate antitumor immune responses in the tumor microenviron-
ment. Furthermore, 2-hydroxyglutarate (2-HG), a chemical analog
of αKG, is an oncometabolite produced by mutated isocitrate dehy-
drogenase to promote tumor progression42. Given that 2-HG could
affect Jmjd3 (ref. 43), it will be interesting to investigate whether
production of 2-HG by tumor cells can drive M2-like phenotypes of
tumor-associated macrophages. Notably, PHD activity impairs the
tumoricidal functions of tumor-specific T cells44. Given that αKG is
the activator of PHD enzymes and inhibiting glutaminolysis can sup-
press production of αKG, it will be worthwhile to further investigate
how glutaminase inhibition affects antitumor responses by T cells in
the tumor microenvironment. The results of such studies would pro-
vide important information for development of combination therapies
using glutaminase inhibitors with cancer immunotherapy.
Methods
Methods, including statements of data availability and any associated
accession codes and references, are available in the online version of
the paper.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
We thank F. Cottard and C.-P. Lin for technical help and P. Romero and
C. Hess for discussion. Supported by Swiss National Science Foundation
project grant (31003A_163204), the Swiss Institute for Experimental Cancer
Research (26075483), the Harry J. Lloyd Charitable Foundation, the Swiss
Cancer Foundation (KFS-3949-08-2016) and a Melanoma Research Alliance
Young Investigator Award to P.-C.H. S.-M.F. is supported by a Flanders Research
Foundation (FWO) research grant and by Eugène Yourassowsky Schenking.
J.I. is supported by the University of Lausanne. H.-D.H. is supported by the
Ministry of Science and Technology of Taiwan (MOST105-2627-M-009-007 and
MOST103-2628-B-009-001-MY3). T.C. is supported by a University of Lausanne
FBM PhD fellowship. M.V. is supported by the National scholarship program
of the Slovak Republic.
AUTHOR CONTRIBUTIONS
P.-S.L., H.W., X.L., T.C., T.T., S.C., G.D.C.,W.-C.C., M.V., C.M., K.D. and J.I.
performed experiments. P.-S.L., H.W., S.C., C.-H.C., M.M., H.-D.H., S.-M.F., J.I.
and P.-C.H. analyzed results. P.-S.L., H.W. and P.-C.H. designed the studies. P.-S.L.
and P.-C.H. wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html. Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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VOLUME 18 NUMBER 9 SEPTEMBER 2017 nature immunology
 ONLINE METHODS
Cell culture and differentiation of BMDMs. OT-1 CD8+ T cells were isolated
from splenocytes of OT-1 mice and cultured in RPMI medium with 10% FBS
and β-mercaptoethanol. To activate OT-1 cells, OT-1 splenocytes were treated
with OVA-peptide and IL-2 for 3 d, then cultured in the presence of IL-2 for
another 3 d before adoptive transfer into tumor-bearing mice. Bone marrow
cells were collected and cultured in DMEM supplemented with 10% FBS and
20% L929 cell culture supernatant for macrophage differentiation for 7 d. On
day 7, differentiated BMDMs were re-plated with DMEM (without L929 cell
culture supernatant) overnight and then stimulated with 10 ng/ml LPS for
M1 activation and 20 ng/ml IL-4 (Bioconcept; 214-14) for M2 activation with
indicated duration.
RNA purification, RT-PCR, qPCR, NF-kB signal target gene quantitative
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
PCR array, RNA-seq and bioinformatic analysis. Total RNAs from BMDMs
were isolated with TRIzol reagent (Life Technologies). 1 µg total RNA was con-
verted into cDNA using First Strand cDNA synthesis kit (Life Technologies).
qPCR was performed in triplicate on a LightCycler 480 Instrument II machine
(Roche Life Science) using SYBR Green PCR mixture (KAPA Biosystems)
for quantification of the target gene expression. Relative expression was nor-
malized to β-actin for each sample. The primers for qRT-PCR amplification
are summarized in Supplementary Table 1. For NF-κB signal target gene
PCR array, RNA was extracted by using RNA Miniprep Plus Kit (Direct-
zol, ZymoResearch) and then converted into cDNA with RT 2 First Strand
Kit (Qiagen). RT2 Profiler qPCR array for NF-κB signaling targets was pur-
chased from Qiagen (PAMM-225Z). Real-time PCR was performed according
to the manufacturer’s instructions with a Roche Light Cycler 480 detector. Data
were analyzed by ∆∆Ct method to normalize with GAPDH expression. Fold
regulation in gene expression was calculated using the analysis web software
from Qiagen.
For next-generation RNA-seq, total RNA was extracted with TRIzol and
RNeasy Mini Kit (Qiagen). mRNAs were then isolated from purified DNA-free
RNA for library construction. Libraries were next sequenced on an Illumina
HISEQ 2500 (Illumina). Mappable results were analyzed using the DESeq2
package. A minimum expression of each gene (mean of counts > 20) was
applied as a cutoff before analysis. Significantly differentially expressed genes
was defined as a 1.5-fold change with a false discovery ratio (FDR) ≤ 0.05.
Chromatin immunoprecipitation (ChIP). ChIP was performed using the
ChIP Assay Kit (Millipore) according to the manufacturer’s instructions.
Briefly, 1 × 107 macrophages were fixed with 10% formaldehyde to cross-link
histones to DNA. Cells were lysed with hypotonic buffer (0.3% NP40, 0.1 mM
EDTA, 10 mM HEPES (pH 7.9), 10 mM KCl) to enrich nuclei. Chromatin was
sheared by micrococcal nuclease (Mnase; NEB (Bioconcept); M0247S) for
10 min at 37 °C, and the reactions were stopped by addition of sonication buffer
(1% Triton X-100, 1 mM EDTA, 50 mM HEPES (pH 7.9), 0.4 M NaCl and
proteinase inhibitor cocktail). The soluble chromatin supernatant was immu-
noprecipitated with anti-H3K27me3 (Millipore 07-449). Immunoprecipitated
DNA and input DNA were analyzed by q-RT-PCR, and results are presented
as percentage of input. The primers were used for amplification of promoters
of M2 gene are summarized in Supplementary Table 2.
Plasmids, reagents and antibodies. The retro-gfpIkkb-puro vector was
purchased from Addgene (#58251). The P191A-IKKβ mutant was generated
using Agilent QuikChange II site-directed mutagenesis kit according to the
manufacturer’s instructions. The lentiviral JMJD3 sgRNA expression vector
was constructed using a BbsI site on pKLV-U6gRNA(BbsI)-PGK-puro2ABFP
(Addgene #50946). The primers used for cloning the IKKβ P191A mutation
and Jmjd3 sgRNA vectors are listed in Supplementary Table 3. The follow-
ing chemical reagents were used in this study: LPS (Sigma; L4391), mouse
IL-4 (Peprotech; 214-14), BPTES (Sigma; SML0601), glutaminase inhibitor
968 (Sigma; SML1327), DMOG (Sigma; D3695), GSK4-J4 (Sigma; SML0701),
diethyl-succinate (Sigma; 112402), dimethyl αKG (Sigma; 349631), etomoxir
(Sigma; E1905), oligomycin A (Sigma; 75351), FCCP (Sigma; C2920), roten-
one (Sigma; R8875), and antimycin A (Sigma; A8674). Antibodies used in
this study were purchased from the following sources: NF-κB p65 (Santa
Cruz; sc-8008, 1:1,000), lamin A/C (Santa Cruz; sc-6215, 1:5,000), β-actin
doi:10.1038/ni.3796
 nature immunology
(Sigma; A5316, 1:5,000), IKKβ (Cell Signaling; 2678P, 1:1,000), phospho-
IKKα/β (Cell Signaling; 2694P, 1:1,000), JNK (Cell Signaling ; 9252, 1:2,000),
phospho-JNK (Cell Signaling; 9251, 1:1,000); p38α (Cell Signaling; 9218,
1:2,000), phospho-p38 (Cell Signaling; 9211, 1:1,000), phospho-p44/42 ERK
(Cell Signaling; 4370S, 1:5,000), ERK (Cell Signaling; 4696, 1:5,000), and Jmjd3
(Cell Signaling; 3457, 1:1,000).
Imaging flow analysis of NF-κB translocation. After stimulation, macro-
phages were fixed with 4% paraformaldehyde at room temperature for 10 min
and then incubated with anti-NF-κB p65 (sc-8008, Santa Cruz) in permea-
bilization buffer for 30 min. After washing, cells were then incubated with
anti-mouse antibody conjugated with Alexa Fluor 647 (A-21235, Thermo
Fisher Scientific) and anti-GFP conjugated with Alexa Fluor 488 (338008,
BioLegend) with DAPI. These cells then were resuspended in 1% paraformal-
dehyde, and NF-κB nuclear translocation was analyzed by an ImageStreamX
(Amnis Corporation) flow cytometer. The quantification of nuclear transloca-
tion was done with IDEAS Software.
Animals, in vitro and in vivo endotoxin tolerance assays. C57BL/6,
LysM-Cre (B6.129p2-Lyz2tm1(cre)lfo/J) and Cas9 knock-in (B6J.129(B6N)-
Gt(ROSA)26Sortm1(CAG-cas9-EGFP)Fezh/J) mice were purchased from Jackson
Laboratory and maintained at the animal facility of University of Lausanne.
We generated macrophage-specific Cas9 knock-in mice by crossing
LysM-Cre mice (myeloid-specific overexpression of Cre recombinase) with
Cas9 knock-in mice. All experiments were performed in accordance with
Swiss federal regulations and procedures approved by veterinary authority
of Canton Vaud.
For the in vitro endotoxin tolerance assay, BMDMs were pretreated with
10 ng/ml LPS under various culture conditions. After 18 h, cells were washed
with PBS once then incubated in glutamine-replete medium with 10% FBS.
After 1 h recovery, cells were restimulated with 10 ng/ml LPS to examine their
proinflammatory responses. For in vivo endotoxin tolerance, C57BL/6J mice
were pre-injected with PBS or BPTES (12.5 mg per kg body weight). In the
DM-αKG treatment, mice also received control vehicle or DM-αKG (0.6 g per
kg body weight). 1 h later, mice were injected intraperitoneally with LPS (0.1 µg
per 25 g body weight), to induce endotoxin tolerance, or PBS, as control
treatment. After 18 h, mice were injected intraperitoneally with LPS (100 µg
per 25 g of body weight) plus d-galactosamine (0.5 mg/g of body weight)
to induce acute septic shock. In the experiment with macrophage depletion,
mice were injected intravenously with 200 µl clodronate-containing liposomes
(ClodronateLiposomes). 24 h later, mice were injected intraperitoneally with
100 µl clodronate-containing liposomes. 6 h later, mice were treated with
control vehicle, BPTES or BPTES + DM-αKG. After 1 h, mice were treated to
examine in vivo endotoxin tolerance as described above.
Fatty acid uptake assay and seahorse extracellular flux analysis. BMDMs
were plated in 24-well plates and allowed to attach overnight. After stimulation
with IL-4 for 24 h, cell were washed with DMEM and then incubated with or
without 0.5 µM BODIPY 500/510 C1, C12 (Thermo Fisher; D3823) in DMEM
with 10% dialyzed FBS for 30 min at 37 °C incubator. Fatty acid uptake was
determined by measuring fluorescence intensity within cells with flow cytom-
etry and analyzed by FlowJo. For extracellular flux assay, 1 × 105 BMDMs were
plated in a Seahorse Bioscience culture plate for overnight. Cells were then
activated for 6 h. OCR was measured by an XF96 Seahorse Extracellular Flux
Analyzer following the manufacturer’s instruction. In seahorse assay, cells
were treated with oligomycin (4 µM), FCCP (1.6 µM), rotenone (0.5 µM),
antimycin A (0.5 µM), UK5099 (3.6 µM) or etomoxir (18 µM). Each condition
was performed with 4–6 replicates, and the readings of OCR of each well were
normalized to protein amount.
Untargeted and targeted metabolomic analyses. To assess targeted metabo-
lite levels, treated BMDMs were washed once with 0.9% saline and quenched in
liquid nitrogen. The cells were then extracted with water–methanol and chlo-
roform as described45. The dried samples with the polar metabolite fraction
were derivatized for 90 min at 37 °C with 20 mg/ml methoxyamine in pyridine.
Subsequently, 7.5 µl was transferred into glass vials and derivatized for 60 min
at 60 °C with 15 µl N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide,
 with 1% tert-butyldimethylchlorosilane. 1 µl of sample was injected into a
7890A GC system (Agilent Technology) combined with a 5975C Inert MS
system (Agilent Technology) to determine expression levels of metabolites.
For untargeted metabolic profiling, cells were extracted using a pre-cooled
methanol and H2O (4:1, v/v) solvent mixture to extract a broad range of polar
and nonpolar metabolites. After lysing cells and spinning down protein pellets,
the resulting supernatant was collected and dehydrated in a vacuum con-
centrator (LabConco). The dry metabolome extracts were reconstituted in
acetonitrile and H2O (1:1, v/v), sonicated for 30 s, and centrifuged 15 min at
13,000 r.p.m. and 4 °C to remove insoluble debris. The supernatants were ana-
lyzed by HILIC ESI-Q-TOF-MS in MS only and auto MS/MS (data-depend-
ent analysis (DDA)) acquisition mode on LC-MS 6550 iFunnel Q-TOF mass
spectrometer interfaced with 1290 UPLC system (Agilent Technologies). Raw
LC/MS data were converted to mzXML files using ProteoWizard and then
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
uploaded to the XCMS Plus server platform for data processing including peak
detection, retention time correction, profile alignment and isotope annotation.
Data were processed as a two-group and multi-group experiments and the
parameter settings were as follows: centWave for feature detection (∆m/z =
15 p.p.m., minimum peak width = 5 s and maximum peak width = 30 s); obi-
warp settings for retention time correction (prof Step = 1); and parameters for
chromatogram alignment, including mzwid = 0.015, minfrac = 0.5 and bw = 5.
The relative quantification of metabolite features was based on EIC (Extracted
Ion Chromatogram) areas. An orthogonal partial least-squares discriminant
nature immunology
analysis (OPLS-DA) was employed for modeling the discrimination among
groups and to determine the metabolite features (VIP variables) that drove the
separation. Multivariate analysis was performed using both SIMCA-P+ version
12 (Umetrics AB) and MetaboAnalyst 3.0. In addition, one-way analysis of
variance (ANOVA) was used to filter out the significantly altered metabo-
lite features among groups. Accurate masses were searched against HMDB
and METLIN databases. Targeted MS/MS analyses were further performed
to obtain the high-quality MS/MS data for significantly altered metabolite
features (or ions) of interest. Finally, metabolite identification was carried
out by matching the acquired MS/MS data from macrophage cell extracts
against recorded MS/MS data for standards, assembled in METLIN database
(https://metlin.scripps.edu/index.php).
Statistical analysis. All results are presented as mean ± s.d. and analyzed for
statistical significance by an unpaired Student’s t-test. P < 0.05 was considered
statistically significant.
Data availability. Data have been deposited in the Gene Expression Omnibus
under accession code GSE99296. Other data that support the findings of this
study are available from the corresponding author upon request.
45. Christen, S. et al. Breast cancer-derived lung metastases show increased pyruvate
carboxylase-dependent anaplerosis. Cell Rep. 17, 837–848 (2016).
doi:10.1038/ni.3796