Supplementary Figure 1

α-ketoglutarate produced from glutaminolysis supports M2 phenotype but inhibits M1 activation.

(a) An illustration of α-ketoglutarate metabolic pathway controlled by genes listed in (Fig. 1d). (b, c) Quantitative PCR

(qPCR) analysis of relative mRNA expression of M2 marker genes in BMDMs stimulated with IL-4 (b) and M1 marker

genes in BMDMs stimulated with LPS (c) in glutamine-replete media supplemented with indicated treatments for 6h.

Compound 968 (10µM); Dimethyl-α-ketoglutarate (DM-αKG; 1mM). (d, e) qPCR analysis of the relative mRNA

expression of M2 markers genes (d) and arginase activity (e) in BMDMs stimulated with or without IL-4 in glutamine-

depleted media plus escalating doses of DM-αKG for 6h. (f, g) qPCR analysis of the relative mRNA expression of M1

markers genes (f) and ELISA of IL-1β and TNFα cytokine production (g) in BMDMs stimulated with or without LPS in

glutamine-depleted media plus escalating doses of DM-αKG for 6h. (h) The effects of glutaminase 1 inhibitor (BPTES;

10mM) and supplementation of DM-aKG (1mM) on IL-1β secretion in LPS stimulated macrophages in the presence of

Nigericin (left panel) or MSU (monosodium urate crystal) (right panel) were analyzed by ELISA. *P < 0.05 is determine

by unpaired, two-tailed Student's t-test. Data shown are representative from three independent experiments (b, c, d, e, f;

mean ± s.d.), from two independent experiments (g; mean ± s.d.) and cumulative results of two independent experiments

(h; mean ± s.d.).

Nature Immunology doi:10.1038/ni.3796

Supplementary Figure 2

αKG does not modulate IL-4-induced STAT6 activation.

(a) Immunoblot analysis of phosphor-STAT6 and STAT6 in BMDMs stimulated with IL-4 in glutamine-replete (+Gln.)

or glutamine-depleted (-Gln) media for 0-2h. -actin served as loading control. (b) Immunoblot analysis of phosphor-

STAT6 and STAT6 in BMDMs stimulated with IL-4 in the presence of absence of BPTES in glutamine-replete media for

0-2h. (c) Immunoblot analysis of phosphor-STAT6 and STAT6 in BMDMs activated with IL-4 in in glutamine-depleted

media supplemented with control vehicle (Ctrl) or DM- KG (1mM). (d, e) qPCR analysis of relative mRNA expression

of M2 marker genes (d) and arginase activity (e) in BMDMs stimulated with IL-4 in the indicated cultures for 6h. (f)

Immunoblot analysis of Jmjd3 and -actin 6 in BMDMs derived from Cas9-LysM-Cre mice transduced with lentivirus

harboring control sgRNAs or Jmjd3-targeting sgRNAs. Data shown are representative from three independent

experiments (a, b, c, d, e) and from two independent experiments (f).

Nature Immunology: doi:10.1038/ni.3796

Supplementary Figure 3

αKG/succinate ratio generated by macrophage activation regulates macrophage immune responses.

(a) The expression of intracellular succinate (Suc.) and -ketoglutarate ( KG) in BMDMs with IL-4 or LPS for 18h,

followed by metabolite extraction mentioned in methods and then measured with mass spectrometry. (b) Arginase activity

in BMDMs stimulated with IL-4 in glutamine-depleted media supplemented with diethyl-succinate (DE-Suc.; 5mM) plus

indicated concentration of DM- KG for 6h. (c) ELISA of IL-1 in BMDMs stimulated with LPS in glutamine-depleted

media supplemented with diethyl-succinate (DE-Suc.; 5mM) plus indicated concentration of DM- KG for 6h. (d)

Arginase activity in BMDMs stimulated with IL-4 in glutamine-depleted media supplemented with DM- KG plus

indicated concentration of DE-Suc. for 6h. (e) ELISA of IL-1 in BMDMs stimulated with LPS in glutamine-depleted

media supplemented with DM- KG plus indicated concentration of DE-Suc. for 6h. *P < 0.05 is determine by unpaired,

two-tailed Student's t-test. Data shown are representative from three independent experiments (a, b, d; mean ± s.d.) and

from two independent experiments (c, e; mean ± s.d.).

Nature Immunology: doi:10.1038/ni.3796

Supplementary Figure 4

Glutaminolysis suppresses NF-κB nuclear translocation upon LPS treatment.

(a) Immunoblot analysis of nuclear NF- B p65 in BMDMs stimulated with LPS plus control vehicle (Ctrl) or BPTES in

glutamine-replete media for 0-60 mins, followed by nuclear fraction isolation. Lamin A/C served as loading control for

nuclear fraction lysate and -actin served as loading control for whole cell lysate (WCL). (b) Immunoblot analysis of

IKK in BMDMs left untransduced (Ctrl) or transduced with lentivirus harboring green fluorescence protein (GFP)-fused

wild type (Wt) or P191A mutant form (P191A) of IKK . -actin served as loading control. Data shown are representative

from three independent experiments (a) and from two independent experiments (b).

Nature Immunology: doi:10.1038/ni.3796

Supplementary Figure 5

Glutaminolysis during LPS priming is essential for induction of endotoxin tolerance via impairment of the NF-κB

pathway.

(a) A schematic diagram of in vitro endotoxin tolerance experimental design. (b) qPCR analysis of relative mRNA

expression in BMDMs stimulated with or without LPS (10ng/ml) in glutamine-replete media supplemented with control

vehicle (Ctrl) or BPTES for 18h and then re-challenged with LPS (10ng/ml) for another 6h. (c) A schematic diagram of in

vitro endotoxin tolerance experimental design. BMDMs were stimulated with or without LPS (10ng/ml) in glutamine-

depleted (Gln.-depleted) media supplemented with or without DM- KG for 18h and then re-challenged with LPS

(10ng/ml). (d, e) IL-1 (d) and TNF (e) production in BMDMs stimulated with LPS as described in (c), followed by

ELISA analysis. (f) A heat map showing expression of genes encoding tissue repairing and antimicrobial molecules in

BMDMs stimulated with LPS for 18h in glutamine-replete media (ET w/ Gln.), glutamine-depleted media (ET w/o Gln.)

or glutamine-depleted media supplemented with 1mM DM- KG (ET w/o Gln.+ DM- KG), assessed by RNA-

sequencing. (g) A heat map showing metabolome changes associated with induction of endotoxin tolerance, based on

metabolite expression between non-tolerant macrophage (LPS w/ Gln.; LPS stimulation for 6h in glutamine-replete

media) and tolerant macrophages (ET w/ Gln.; endotoxin tolerant macrophages stimulated with LPS for 6h in glutamine-

replete media). The metabolites of this list in BMDMs stimulated with LPS in glutamine-depleted media (ET w/o Gln.) or

glutamine-depleted media supplemented with DM- KG (ET w/o Gln.+ DM- KG) for 18h and then re-activated with LPS

for another 6h were also included in this heat map. (h) The expression of lactate and pyruvate in BMDMs of indicated

groups were determined by mass spectrometry. *P < 0.05 is determine by unpaired, two-tailed Student's t-test. Data

shown are representative from three independent experiments (b; mean ± s.d.), cumulative results from two independent

experiments (d, e, f; mean ± s.d.) and combined from three independent experiments and normalize to LPS w/ Gln. group

Nature Immunology: doi:10.1038/ni.3796

(g, h; mean ± s.d.).

Nature Immunology: doi:10.1038/ni.3796

Supplementary Figure 6

Glutamine metabolism during LPS priming prevents reactivation of proinflammatory genes in a Jmjd3-

independent manner.

(a, b, c) Immunoblots of proximal signaling pathways of toll-like receptor 4 in BMDMs treated as described in figure 6a

(a) or treated in glutamine-replete media supplemented with control vehicle (Ctrl) or BPTES (b), or treated in glutamine-

depleted media supplemented with or without DM- KG (c), for 18h and then re-stimulated with second exposure of LPS

for 0-60 mins. -actin served as loading control. (d) Immunoblots of NF- B p65 in BMDMs treated with LPS in

glutamine-replete media supplemented with or without BPTES for 18h, followed by re-stimulated with second exposure

of LPS for 0-60 mins. (e) Immunoblots of NF- B p65 in BMDMs treated with first exposure of LPS in glutamine-replete

(+Gln.) or glutamine-depleted (-Gln.) media plus control vehicle or DM- KG for 18h. After washing, BMDMs were then

re-stimulated with second exposure of LPS for indicated period. Those BMDMs did not receive first LPS treatment

represent control groups for intact responsiveness to LPS-induced nuclear translocation of NF- B. (f) qPCR analysis of

relative mRNA expression of M1 marker genes in Cas9-expressing BMDMs transduced with lentivirus harboring control

sgRNAs or Jmjd3-targeting sgRNAs. These BMDMs were treated with or without LPS in the indicated conditions for 18h

and then re-challenged with LPS re-exposure in glutamine-replete media for another 6h. (g) A schematic diagram of in

vivo endotoxin tolerance assay. *P < 0.05 is determine by unpaired, two-tailed Student's t-test. Data shown are

representative from three independent experiments (a, b, c, d, e, f; mean ± s.d.).

Nature Immunology: doi:10.1038/ni.3796

SUPPLEMENTARY TABLES

Supplementary Table 1. Information for quantitative PCR primers.

CPT1a-F TGCTTTACAGGCGCAAACTG CPT1a-R GCAGATGTGTCAGGACCGAGT

CCL22-F AGGTCCCTATGGTGCCAATGT

CCL22-R CGGCAGGATTTTGAGGTCCA Hr-F CCCCTGTGAACGGCATTGT Hr-R CCCCTCCAAAAGGGAGCAG Ii4il-F CAGAAGGTGGTAGTGGTTGGT

Il4il-R TCATCCCGGAAAGTGAAGATA Arg1-F CTCCAAGCCAAAGTCCTTAGAG Arg1-R AGGAGCTGTCATTAGGGACATC Ym1-F AGAAGGGAGTTTCAAACCTGGT Ym1-R GTCTTGCTCATGTGTGTAAGTGA Retnla-F CTGGGTTCTCCACCTCTTCA Retnla-R TGCTGGGATGACTGCTACTG Irf4-F TCCGACAGTGGTTGATCGAC Irf4-R CCTCACGATTGTAGTCCTGCTT Mrc1-F CTCTGTTCAGCTATTGGACGC Mrc1-R CGGAATTTCTGGGATTCAGCTTC CD36-F GAGGCATTCTCATGCCAGT CD36-R ACGTCATCTGGGTTTTGCAC β-actin-F TCCATCATGAAGTGTGACGT β-actin-R TACTCCTGCTTGCTGATCCAC Tnfa-F ACGGCATGGATCTCAAAGAC Tnfa-R AGATAGCAAATCGGCTGACG Mmp9-F CAAGTGGGACCATCATAACATCA Mmp9-R GATCATGTCTCGCGGCAAGT IL1b-F TACGGACCCCAAAAGATGA IL1b-R TGCTGCTGCGAGATTTGAAG IL6-F TAGTCCTTCCTACCCCAATTTCC IL6-R TTGGTCCTTAGCCACTCCTTC

Nature Immunology: doi:10.1038/ni.3796

Supplementary Table 2. Information of primers for ChIP assay.

Retnla-Chip-F TGCAATTCTTTGATGCTGTGTCT Retnla-Chip-R TTGTGTCCCTTGGCTACATGAA ArgI-Chip-F TGAACAGGCTGTATTAGCCAACA ArgI-Chip-R AGCACCCTCAACCCAAAGTG Mrc1-Chip-F TTTCACTTGAAGGTAAACCATCTG Mrc1-Chip-R CCAGAGTTCAACAAGAATTAAGA Chi3l3-Chip-F CATTTGCCCTGCCTTTGG Chi3l3-Chip-F TCTTTCATGGATATTGATTTCCTAAGAG

Nature Immunology: doi:10.1038/ni.3796

Supplementary Table 3. Information of primers for cloning.

JMJD3 gRNA1-F CACCAGAGTTCAGAGGTCGCGGAGT JMJD3 gRNA1-R TAAAACTCCGCGACCTCTGAACTCT JMJD3 gRNA2-F CACCGAACTATGGGGCTAAGCGAGT JMJD3 gRNA2-R TAAAACTCGCTTAGCCCCATAGTTC JMJD3 gRNA3-F CACCGCTGCAAGCGGCCAATCCGGT JMJD3 gRNA3-R TAAAACCGGATTGGCCGCTTGCAGC JMJD3 gRNA4-F CACCAAGCTTCCTCCATAGCGAAGT JMJD3 gRNA4-R TAAAACTTCGCTATGGAGGAAGCTT

IKKβ P191A mutation-F CTCCAGAAGCTCTGCCGCCAGGTATTGCA IKKβ P191A mutation-R TGCAATACCTGGCGGCAGAGCTTCTGGAG

Nature Immunology: doi:10.1038/ni.3796