-ketoglutarate produced from glutaminolysis supports M2 ......Supplementary Figure 1 α-ketoglutarate produced from glutaminolysis supports M2 phenotype but inhibits M1 activation.

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


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


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.).



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).



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


(g, h; mean ± s.d.).



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.).


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


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


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


-ketoglutarate produced from glutaminolysis supports M2 ......Supplementary Figure 1 α-ketoglutarate produced from glutaminolysis supports M2 phenotype but inhibits M1 activation.

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