washed to remove the stimulus and were resuspended in RPMI supplemented with 10% human
pool serum. Monocytes were collected before and
6 days after the incubation for ChIP or RNA sequencing. For RNA sequencing, monocytes were
collected in TRIzol reagent (Invitrogen, Bleiswijk,
Netherlands). The purified materials were then processed to generate genomic DNA for WGBS, RNA
(Trizol extraction according to manufacturer instructions; Agilent BioAnalyser RIN >8), and chromatin by fixing the cells in 1% formaldehyde.
Candida albicans b-1,3-(D)-glucan (b-glucan) was
kindly provided by D. Williams (East Tennessee
State University). Reagents used were as follows:
LPS (E. coli 0B5/B5, Sigma, Diegem, Belgium),
rapamycin (Sigma, R0395), metformin (R&D, AF
1730, Abingdon, UK), AICAR (Sigma, A9978), ascorbate (Sigma, A4034), wortmannin (InvivoGen,
tlrl-wtm, Toulouse, France). C. albicans ATCC MYA-
3573 (UC 820) cells were heat-inactivated for
30 min at 95°C.
For training, monocytes were preincubated
with b-glucan (10 mg/ml) for 24 hours. After 7 days,
cells were restimulated with various microbial
ligands: LPS (10 ng/ml), Pam3Cys (10 g/ml),
and heat-killed S. aureus or heat-killed E. coli
(both at 106 microorganisms/ml). After 24 hours,
supernatants were collected and stored at –20°C
until cytokine measurement. All the cytokine measurements presented were from at least six donors.
To address the HIF-1a–AMPK–m TOR pathway
in trained immunity, we added the specific inhibitors together with b-glucan for the first 24 hours
in different doses as follows: rapamycin from 1 to
100 nM, metformin from 0.3 to 30 mM, AICAR
from 5 to 500 nM, and ascorbate at 5 and 50 mM.
ChIP-seq data analysis
H3K4me3 and H3K27ac ChIP, sequencing, and
processing of the data were performed as de-
scribed (7). The detailed data have been depos-
ited in the GEO database with accession number
GSE57206. Sequenced reads of 42-bp length were
mapped to human genome (NCBI hg19) using
bwa-alignment package mapper (43). ChIP-seq
data sets were normalized as described (44), and
the sequenced reads were directionally extended
to 300 bp, corresponding to the original length
of sequenced DNA fragments. For each base pair
in the genome, the number of overlapping se-
quence reads was determined, averaged over a
10-bp window, and visualized in UCSC browser
http://genome.ucsc.edu). These normalized tracks
were used to generate the genome browser screen
shots. Putative H3K4me3- and H3K27ac-enriched
regions in the genome were identified by using
MACS (45) with P < 10−8. All the transcription
start sites (T1 kb) of genes with significant H3K4me3
signal were regarded as active promoters. H3K4me3
and H3K27ac signals at all active promoters were
estimated, and log2 ratios of ChIP-seq signal
between treatment and control samples were
calculated. Promoters that showed an absolute
deviation of 2 times the median (median T 2 ×
MAD) of the ratio of ChIP-seq signal (treatment/
control) were regarded as regulated promoters
(induced or repressed). Sequence reads counted
from the normalized ChIP-seq data sets were
used to generate the box plots.
Culture medium was collected at days 1, 3, and 7.
The glucose and lactate concentrations within the
medium were determined by Glucose Colorimetric
Assay Kit (K686-100; Biovision, Milpitas, CA) and
Lactate Colorimetric Assay Kit (K627-100, Biovision), respectively. NAD+ and NADH concentration
were determined by NAD/NADH Quantification
Colorimetric Kit (Biovision, K337-100) from the
cell lysate according to manufacturer’s protocol.
All the metabolite measurement data presented
were from at least six donors.
Oxygen consumption measurement
Culture medium was collected from 1 million
cells treated with either RPMI or b-glucan. After
stimulation, the cells were trypsinized, washed,
and resuspended in 60 ml of the collected culture
medium. The cell suspensions were then used for
cellular O2 consumption analysis. Oxygen consumption was measured at 37°C using polarographic
oxygen sensors in a two-chamber Oxygraph
(OROBOROS Instruments, Innsbruck, Austria).
First, basal respiration (baseline oxygen consumption) was measured. Next, leak respiration was determined by addition of the specific
complex V inhibitor oligomycin A (OLI). Then,
maximal electron transport chain complex (ETC)
capacity (maximum oxygen consumption) was
quantified by applying increasing concentrations
of the mitochondrial uncoupler FCCP (1 to 14 mM
final maximal concentration). Finally, minimal
respiration was assessed by adding a maximal
(0.5 mM) concentration of the specific complex I
inhibitor rotenone (ROT; 0.5 mM) and the complex III inhibitor antimycin A (AA; 0.5 mM).
After establishment of the baseline oxygen consumption rate, cells were treated with the ATP synthase inhibitor oligomycin to determine the rate of
proton leak–dependent oxygen consumption, after
which the baseline rate value was normalized to
the value of the leak rate. Next, the cells were treated
with FCCP to determine the maximum oxygen consumption rate. For normalization, the maximum
FCCP value was ratioed to the leak value. The oxygen
consumption measurement was repeated in monocytes isolated from five healthy individuals.
For Western blotting of AMPK, mTOR, Akt
(total and phosphorylated), and actin, training
was performed as described in stimulation ex-
periments. Adherent monocytes were trained in
24-well plates. After training and the resting period,
cells were lysed in 150 ml of lysis buffer. Equal
amounts of protein were subjected to SDS-PAGE
electrophoresis using 7.5% polyacrylamide gels.
Primary antibodies [1:500 and 1:50 000 (actin)]
in 5% (w/v) BSA/TBST (5% bovine serum albumin/
TBST) were incubated overnight at 4°C. HRP-
conjugated anti-rabbit antibody or HRP-conjugated
anti-mouse antibody at a dilution of 1:5000 in
5% (w/v) BSA/TBST was used for 1 hour at room
temperature. Quantitative assessment of band in-
tensity was performed by Image Lab statistical
software (Bio-Rad, CA, USA). The following anti-
bodies were used: actin antibody (Sigma, A5441),
mTOR antibody (Cell Signaling, #2972, Leiden,
Netherlands), phospho-m TOR antibody (Ser2448)
(Cell Signaling, #2971), AMPKa antibody (Cell Sig-
naling, #2532), phospho-AMPKa (Thr172) (Cell Sig-
naling, #2531), Akt antibody (Cell Signaling, #9272),
phosphor-Akt (Ser473) (Cell Signaling, #9271). At
least four different individual experiments were
repeated for each Western blot experiment.
Analysis of RNA sequencing data
Sequencing reads were mapped to the mouse
genome (mm10 assembly) using STAR (version
2.3.0). The aligner was provided with a file containing junctions from Ensembl GRCm38.74. In
total, there were 507.5 million reads from 12 samples. Htseq-count of the Python package HTSeq
(version 0.5.4p3) was used to quantify the read
counts per gene based on annotation version
GRCm38.74, using the default union-counting
mode (The HTSeq package, www-huber.embl.
Differentially expressed genes were identified
by statistics analysis using the edgeR package from
bioconductor. The statistically significant threshold
[false discovery rate (FDR) = 0.05] was applied.
For visualization, relative changes larger than 1.5
and FDR of 0.01 were used to plot the expression
level of protein-coding genes.
Animal experimental models
The metformin experiment was done at the University of Athens with the approval of the Ethics
Committee on Animal Experiments of the University of Athens (approval no. 2550). C57BL/6J female
mice (8 to 12 weeks) were used (Jackson Laboratories, Bar Harbor, ME, USA). Mice were injected
with live C. albicans blastoconidia (2 × 104 CFU per
mouse) or pyrogen-free phosphate-buffered saline (PBS) alone. Seven days later, mice were
infected intravenously with a lethal dose of live
C. albicans (2 × 106 CFU per mouse). Survival was
monitored daily. To assess the involvement of the
AMPK-m TOR pathway in the training, metformin
(250 mg/kg) or PBS was given via intravenous
injection from 1 day before the first nonlethal
dose of live C. albicans challenge until 3 days after
challenge on a daily basis.
Wild-type (Cre +/+, HIF flox/flox) and HIF-KO
mice 8 to 10 weeks old were trained with 200 ml
intraperitoneally (i.p.) of either 1 mg of b-glucan
particles or sterile PBS on days –7 and –4 prior to
tail vein inoculation with 200 ml of 5 × 106 S.
aureus strain RN4220 on day 0. Mice were monitored three times daily for survival for 14 days.
Data presented are the combined survival data
(Kaplan-Meier) from two independent experiments.
There were five mice per group in the first survival
experiment and seven mice per group in the
second survival experiment. A log-rank test was
used to assess the statistical significance between