www.sciencemag.org/content/345/6204/1250684/suppl/DC1

Supplementary Materials for

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

Shih-Chin Cheng, Jessica Quintin, Robert A. Cramer, Kelly M. Shepardson, Sadia Saeed, Vinod Kumar, Evangelos J. Giamarellos-Bourboulis, Joost H. A. Martens, Nagesha Appukudige Rao, Ali Aghajanirefah, Ganesh R. Manjeri, Yang Li, Daniela C. Ifrim, Rob J. W. Arts, Brian M. J.

W. van der Meer, Peter M. T. Deen, Colin Logie, Luke A. O’Neill, Peter Willems, Frank L. van de Veerdonk, Jos W. M. van der Meer, Aylwin Ng, Leo A. B. Joosten, Cisca Wijmenga, Hendrik

G. Stunnenberg, Ramnik J. Xavier, Mihai G. Netea*

*Corresponding author. E-mail: mihai.netea@radboudumc.nl

Published 26 September 2014, Science 345, 1250684 (2014) DOI: 10.1126/science.1250684

This PDF file includes: Figs. S1 to S15

Tables S1 and S2

Supplementary Materials: Figure S1

At day 7 cells were restimulated with RPMI, LPS, pam3Cys, heat killed Staphylococcus aureus or heat killed E. coli for 24 hours. Supernatant was harvested and the proinflammatory cytokines IL-6 and TNF were measured by ELISA.

Figure S2

Pathway analysis of the top 500 upregulated genes according to the epigenetic modification in their promoter region.

Figure S3

Schematic representation of the genes induced (in red) in the glycolysis pathway and gluconeogenesis pathway and the corresponding gene list. (adapted from KEGG pathway analysis)

Figure S4 mTOR

HIF-1α

Representative screenshots of the H3K4me3 (purple) and H3K27ac (blue) modifications in the promoter region of mTOR and HIF-1α.

Figure S5

Monocytes (Day 0) or β-glucan trained monocytes (Day 7) were stimulated with LPS for 2 hours. The HIF-1α level were detected by ELISA from the total cell lysate. The relative HIF-1α expression level was normalized to the RPMI treated control group. The data was pooled from three individual donors.

HIF-1α level

- + - +0

1

2

3

4monocyte (Day 0)β-glucan trainedmonocyte (Day 7)

Stimulation LPS (10 ng/ml)

rela

tive

expr

essi

on le

vel

Figure S6

The upregulated genes upon β-glucan injection in wild-type mice that are involved in glycolysis pathway were colored red in KEGG pathway analysis.

Figure S7

Monocytes were primed with β-glucan in the presence or absence of 2-deoxyglucose (5 mM). Cells were restimulated at day 7 and the IL-6 and TNF productions were determined by ELISA.

Figure S8

Adherent monocytes were primed with β-glucan and washes with PBS after 24 hours. The cells were then in incubated with RPMI for another 6 days. Supernatant was harvested at day 7 and the glucose concentration within the supernatant was determined by the glucose colorimetric assay kit. The data is pooled of 6 different individuals.

Figure S9

Adherent monocytes were primed with β-glucan in the presence or absence of pyruvate and during the entire culturing period. At day 7 the cells were restimulated with LPS for 24 hours. The IL-6 and TNF within the supernatant were determined by ELISA. The data is pooled of 6 different individuals.

Figure S10

The kinetic change of (C) glucose consumption and (D) lactate production were determined by measuring the glucose concentration in the supernatant taken from day 1, day 3 and day 7 of the RPMI and LPS tolerant monocytes.

Figure S11 A

B

A. Adherent monocytes were primed with β-glucan in the presence or absence of resveratrol. At day 7 the cells were restimulated with LPS for 24 hours. The IL-6 and TNF within the supernatant were determined by ELISA. The data is pooled of 6 different individuals. B. Fold changes of Sirtuin-1 mRNA expression in control monocytes (primed with nutrient rich medium) and β-glucan trained-monocytes was analyzed by qRT-PCR in 3 independent individuals.

Figure S12

Adherent monocytes from 2 dectin-1 deficient patients were primed with β-glucan. At day 7 the cells were restimulated with LPS for 24 hours. TNF production was determined by ELISA.

Figure S13 A B

The epigenetic inhibitors effect on lactate production in the β-glucan trained cells. A. Adherent monocytes were primed with β-glucan in the presence of RPMI, MTA or ITF respectively. 24 hours post stimulation supernatant was collected for lactate measurement. B. Cells were washed with PBS once after 24 hours and resting for another 6 days. At day 7 the cells were restimulated with RPMI or LPS for 24 hours. The Δ lacate production ([lactate]LPS

restimulation – [lactate]RPMI group) was shown. The data is pooled of 6 different individuals.

24 hour lactate production

RPMI MTA ITF RPMI MTA ITF0.0

0.2

0.4

0.6

β-glucanRPMI

Trained with

mM

Figure S14 Venn diagram of the genes specifically upregulated by β-glucan in both wild-type and mHIF-1α mice (FDR=0.01, fold change≥1.5). The gene list of Zone A is listed in supplementary Table 2.

β-glucan

vs. PBS

(WT)

β -glucan

vs. PBS

(HIF-1 α KO)

A (35)

C

(624)

B

(95)

Figure 15

Adherent monocytes were primed with different doses of recombinant IL-1β. At day 7 the cells were restimulated with LPS for 24 hours. The IL-6 and TNF within the supernatant were determined by ELISA. The data is pooled of 6 different individuals.

IL-6

RPM

I

0.00

1

0.01 0.

1 1 10 100

0

100

200

300

400

500

600

700

IL-1β (ng/ml)

pg/m

lTNF

RPM

I

0.00

1

0.01 0.

1 1 10 100

0

500

1000

1500

2000

2500

IL-1β (ng/ml)

pg/m

l

Supplementary Table 1

Proton leak-dependent oxygen consumption (pmol/s/million cells)

Cell group Basal Oligomycin sensitive

Oligomycin + FCCP

Rotenoe + Antimycin A

RPMI 3.7 3.49 10.21 3.18

β-glucan 3.7 3.49 10.21 3.18

Oxygen consumption rate of non-trained and β-glucan trained monocytes determined

by high-resolution respirometry with different ETC inhibitors. The proton leak-

dependent oxygen consumption was determined after the addition of ATP-synthase

(CV) inhibitor, Oligomycin. No difference in proton leak-dependent oxygen

consumption was found in non-trained and β-glucan trained monocytes.

Supplementary Table 2 Gene list in Zone A of Fig. S14 (Genes specifically upregulated in wild-type mice upon β-glucan training)

gene.name P-Value Zfand6 3.84E-14 Foxo4 1.56E-11 Ube2b 1.60E-10 Otud5 3.39E-10 Gpcpd1 9.99E-10 Ccndbp1 1.73E-09 Slc25a39 1.77E-09 Fcho2 3.00E-09 Il9r 3.26E-09 Rnf14 1.61E-08 Trafd1 2.58E-07 Hbb-bt 3.12E-07 Nbr1 5.30E-07 Nedd4 5.48E-07 Tor1aip2 6.49E-07 Becn1 8.15E-07 Hbb-bs 1.62E-06 Slc40a1 1.79E-06 Usp7 2.06E-06 Bpgm 2.18E-06 Lpcat1 2.24E-06 Ftl1 2.49E-06 Ppt1 3.19E-06 Trim56 5.46E-06 Stk11 8.36E-06 Wnk1 8.47E-06 Hba-a1 1.79E-05 Hba-a2 6.97E-05 Smc4 7.75E-05 Osbpl8 0.000105656 Uba7 0.000200356 Serpina3g 0.000500946 Sdc3 0.001225769 Jhdm1d 0.001227757 Cst3 0.00126663