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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: [email protected]
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