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OPEN
Screening of compounds to identify
novel epigenetic regulatory factors
that affect innate immune memory
in macrophages
Salisa Benjaskulluecha1,2, Atsadang Boonmee2,3, Thitiporn Pattarakankul3,4,
Benjawan Wongprom2,3, Jeerameth Klomsing3 & Tanapat Palaga1,2,3*
Trained immunity and tolerance are part of the innate immune memory that allow innate immune
cells to differentially respond to a second encounter with stimuli by enhancing or suppressing
responses. In trained immunity, treatment of macrophages with β-glucan (BG) facilitates the
production of proinflammatory cytokines upon lipopolysaccharide (LPS) stimulation. For the
tolerance response, LPS stimulation leads to suppressed inflammatory responses during subsequent
LPS exposure. Epigenetic reprogramming plays crucial roles in both phenomena, which are tightly
associated with metabolic flux. In this study, we performed a screening of an epigenetics compound
library that affects trained immunity or LPS tolerance in macrophages using TNFα as a readout.
Among the 181 compounds tested, one compound showed suppressive effects, while 2 compounds
showed promoting effects on BG-trained TNFα production. In contrast, various inhibitors targeting
Aurora kinase, histone methyltransferase, histone demethylase, histone deacetylase and DNA
methyltransferase showed inhibitory activity against LPS tolerance. Several proteins previously
unknown to be involved in innate immune memory, such as MGMT, Aurora kinase, LSD1 and PRMT5,
were revealed. Protein network analysis revealed that the trained immunity targets are linked via
Trp53, while LPS tolerance targets form three clusters of histone-modifying enzymes, cell division and
base-excision repair. In trained immunity, the histone lysine methyltransferase SETD7 was identified,
and its expression was increased during BG treatment. Level of the histone lysine demethylase, LSD1,
increased during LPS priming and siRNA-mediated reduction resulted in increased expression of Il1b
in LPS tolerance. Taken together, this screening approach confirmed the importance of epigenetic
modifications in innate immune memory and provided potential novel targets for intervention.
The innate immune response, as the first line of defense, is generally nonspecific and has been characterized as
an immune response with no memory. Recent evidence, however, strongly indicates that innate immune cells,
such as macrophages, neutrophils, natural killer cells or innate lymphoid cells, can exhibit characteristics of
immune memory by altering the response after previous infection or vaccination1,2. Innate immune memory is
nonspecific, maintained for a relatively short duration, and does not involve specific antigen receptors generated by gene rearrangement, as in adaptive immune memory3,4. Innate memory is classified into two different
types, i.e., “trained immunity”, which is the heightened immune response that can induce nonspecific protection, and “tolerance”, which is the repressed immune response that manifests in cancer and immune paralysis in
sepsis1,2,5,6. In macrophages, trained immunity can be induced by priming with fungal cell wall ß-glucan (BG),
vaccination with Bacillus Calmette–Guérin (BCG), and infection with Candida albicans4,6,7. In contrast, the tolerance response can be induced by primary stimulation with potent inflammatory stimulators, most of which are
pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), including
ligands for Toll-like receptors (TLRs), such as lipopolysaccharide (LPS) and Pam3CysSerLys4 (Pam3CSK4), and
inflammatory cytokines, such as TNFα5,8. Tolerance results in selective repression of a set of tolerizeable genes
1
Medical Microbiology, Interdisciplinary Program, Graduate School, Chulalongkorn University, Bangkok 10330,
Thailand. 2Center of Excellence in Immunology and Immune-Mediated Diseases, Chulalongkorn University,
Bangkok 10330, Thailand. 3Department of Microbiology, Faculty of Science, Chulalongkorn University,
Bangkok 10330, Thailand. 4Center of Excellence in Materials and Bio-Interfaces, Chulalongkorn University,
Bangkok 10330, Thailand. *email: tanapat.p@chula.ac.th
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after restimulation with the same or different stimulants5,9. Trained immunity may provide protection against
unrelated pathogens after vaccination, while unregulated trained immunity may result in maladaptive immune
responses that aggravate chronic inflammatory conditions or autoimmune diseases. Moreover, tolerance may be
pivotal for controlling a heightened immune response during sepsis, but it can have detrimental consequences
during secondary infection after sepsis or in cancer patients1,10.
Mechanistic studies on how innate immune memory is acquired reveal that epigenetic and metabolic reprogramming play essential roles. Regulation of the expression of selective genes in trained and tolerant macrophages
results from changes in chromatin structure5,6. The main mechanism that regulates this process is at the epigenetic
level through histone modification, DNA methylation, and noncoding RNA expression1,2,5. Histone modification
is one of the major epigenetic mechanisms that controls the induction of trained and tolerant macrophages1,2,5,6.
This mechanism relies on histone tail marking, which has a profound impact on gene expression by changing
promoter accessibility or controlling the activity of distal enhancer elements11,12.
Previous studies demonstrated that enhancement of the immune response in β-glucan-trained macrophages
involves the deposition of active histone marks, such as acetylation of lysine 27 (H3K27ac) and monomethylation and trimethylation of lysine 4 on histone H3 (H3K4me1 and H3K4me3, respectively), in the promoters
of targeted genes, including proinflammatory cytokines and intracellular signaling molecules. These modifications allow gene expression by interfering with the histone/DNA interaction, leading to a loosened chromatin structure6,12–14. In contrast, active histone marks in LPS-tolerant macrophages were only observed in
the promoters of inflammatory genes during LPS priming, and these active histone marks were replaced with
repressive epigenetic marks, such as dimethylation of lysine 9 on histone H3 (H3K9me2) and CpG methylation
after restimulation with LPS5,12,14–16. Enzymes that are shown to be involved in epigenetic modifications during
trained immunity or tolerance include histone methyltransferase (HMT), histone acetyl transferase (HAT),
and DNA methyltransferase (DNMT), which are potential targets for therapeutic interventions15–19. Although
various epigenetic regulations have been shown to be involved in innate immune memory, only limited targets
for pharmacological intervention have been reported. Furthermore, the identification of additional epigenetic
modifiers will be beneficial for better understanding how innate immune memory is regulated.
In this study, we performed a screening of an epigenetics compound library to identify additional novel
epigenetic modifications that control trained immunity and tolerance in macrophages using BG-trained or
LPS-tolerant models. We have confirmed the known epigenetic modifying enzymes that have been previously
shown to regulate trained immunity and tolerance in this study. More importantly, we have identified previously
unknown potential novel compounds that have not been previously documented to be involved in innate immune
memory. Detailed studies into the mode of action of these compounds may alter innate immune memory in
macrophages and provide novel intervention strategies.
Materials and methods
Animals. Eight-week-old female C57BL/6 mice were used in this study (Nomura Siam International, Thailand). All experimental procedures involving laboratory animals were approved by the Institutional Animal
Care and Use Committee (IACUC) of the Faculty of Medicine, Chulalongkorn University (approval protocol No.
025/2562). All experiments were performed according to the guidelines issued by the IACUC.
Generation of bone marrow-derived macrophages (BMM).
BMMs were generated from bone marrow cells extracted from the tibia and femur of C57BL/6 mice. Bone marrow cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) (HyClone, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco,
USA), 10 mM HEPES (HyClone, USA), 1 mM sodium pyruvate (HyClone, USA), 100 U/ml penicillin, and
0.25 mg/ml streptomycin (DMEM complete media) with 20% L929 culture supernatant and 5% horse serum
(HyClone, USA), and fresh media was added at day 4. After 7 days in culture, cells were detached with cold PBS
and stored at − 80 °C until use. BMMs were confirmed by flow cytometry using the macrophage cell surface
markers F4/80 and CD11b20.
Induction of beta-glucan (BG)-trained and LPS-tolerant macrophages. For the induction of BGtrained or LPS-tolerant macrophages, BMMs were cultured in complete DMEM and primed with 50 µg/ml
pachyman BG (Megazyme, USA) for trained macrophages or 100 ng/ml Escherichia coli LPS (L2880, Sigma
Aldrich, USA) for tolerant macrophages. After 24 h of priming, the medium was replaced with fresh DMEM
complete medium and the cells were rested for 48 h. The resting step was followed by LPS (10 ng/ml) stimulation for the indicated times. Culture supernatant, RNA or cell lysates were harvested at the indicated times for
analysis. The amount of TNFα in the culture supernatant was measured by a mouse TNFα ELISA kit (Biolegend,
USA) according to the manufacturer’s protocol.
Epigenetics compound library screening. The Epigenetics Compound Library with a unique collection
of 181 epigenetics compounds (Cat L1900, Selleckchem, USA) was used as the inhibitor source. The library was
purchased and obtained in January 2018 and the list of compounds in the library was shown in Supplementary
Table 1. For the screening assay, BG-trained or LPS-tolerant macrophages were pretreated with the inhibitors
at two concentrations of inhibitory concentration 50 (IC50) from the manufacturer’s data for 1 h and during
the priming or stimulation phase. Control cells received vehicle control DMSO and were subjected to the same
priming. Cells were cultured in the presence of inhibitors during priming or stimulation for 24 h. The culture
supernatant was subjected to ELISA to measure TNFα. The relative amount of TNFα was calculated as the fold
change of the inhibitor-treated cells compared with the vehicle control-treated cells. The inhibitors that showed
enhancing or suppressing effects with fold changes of 1.5-fold or higher and 0.75-fold or lower, respectively, were
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chosen for further confirmation. Interaction of the potential targets identified in this study was performed with
STRING version 11.0 (https://string-db.org/)21.
Western blotting. BMMs were treated to become BG-trained or LPS-tolerant macrophages as described
above. Cell lysates were collected at the indicated times using RIPA buffer (50 mM Tris HCl pH 7.4, 150 mM
(for other proteins) or 500 mM (for histone, mTOR and LSD1 extraction) NaCl, 5 mM EDTA, 1% nonidet P-40,
0.5% sodium deoxycholate supplemented with protease and phosphate inhibitors (Cell Signaling Technology,
USA)). The protein concentrations were measured by a bicinchoninic acid assay using the Pierce BCA Protein
Assay Kit (Thermo-Fisher Scientific, USA). Proteins were resolved by 6% (mTOR), 8% (LSD1), 10% (Aurora
kinase or SETD7) or 15% (histone) SDS-PAGE and subjected to Western blot as described elsewhere. The antibodies were diluted in PBS with 3% (w/v) skim milk at the following concentrations: rabbit anti-H3K4me3 antibody, 1:1000; rabbit anti-H3K27me3 antibody, 1:1000; rabbit anti-total H3 antibody, 1:4000; rabbit anti-phospho
mTOR, 1,1000; rabbit anti-mTOR, 1,1000; rabbit anti-phospho Aurora A/Aurora B/Aurora C, 1:1,000, rabbit
anti-LSD1, 1:2000 and goat anti-rabbit IgG HRP, 1:4000 (all antibodies were from Cell Signaling Technology,
USA); mouse anti-actin antibody, 1:10,000 (Merck Millipore, USA), rabbit anti-GAPDH antibody, 1:4000 and
mouse anti-SETD7 antibody, 1:2000 (Bio-Rad, USA); and sheep anti-mouse IgG HRP, 1:4000 (GE Healthcare
Life Sciences, USA). The signal was detected by the ECL chemiluminescent detection method. Relative intensity
was analyzed by ImageJ analysis.
Quantitative reverse transcription realtime-PCR (qRT-PCR). Total RNA of macrophages treated
as indicated was harvested with the TRIzol reagent (Invitrogen, USA) and extracted with direct-zol RNA kits
(Zymo Research, USA). The quality and concentrations of RNA were measured by a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific). One hundred nanograms of RNA per sample was converted to cDNA,
which was used for quantitative PCR using iQ™ SYBR Green SuperMix (Bio-Rad, USA) according to the manufacturer’s instructions. The primers used in this study are shown in Supplementary Table 2. The relative expression of all target genes was normalized to the expression of Actb by the 2−∆∆CT method.
MTT assay. BG-trained or LPS-tolerant macrophages were treated with inhibitors during priming or stimulation as indicated. After 20 h of inhibitor treatment, MTT reagent was added to a final concentration of 0.5 mg/
ml and the cells were further incubated for 4 h. After incubation, 200 µl of DMSO was added to each well to dissolve the MTT formazan pellet. The intensity of the pellet was measured by a microplate reader at a wavelength
of 540 nm.
BrdU cell proliferation assay.
Cell proliferation was detected by BrdU cell proliferation assay (Sigma
Aldrich, USA) according to the manufacturer’s protocol. In brief, BrdU solution was added to unstimulated or
LPS-tolerant macrophages 24 h before detection as indicated. After incubation and fixation, a BrdU detection
antibody and IgG peroxidase-conjugated secondary antibody were added. The level of BrdU incorporation was
measured by a colorimetric method at a wavelength of 450 nm using a microplate reader.
siRNA mediated gene silencing of lsd1. ON-TARGETplus™ SMARTpool siRNA targeted murine
lsd1 or control non-targeting siRNA (NT) were purchased from Dharmacon™ (Horizon Discovery, UK). Lipofectamine 2000 (Promega, Wisconsin, USA) were used as transfection reagent. Lipid-siRNA complex was prepared in warmed Opti-MEM™ I Reducing-Serum Media (Gibco, USA) and incubate for 15 min with gently
rotation before topping up to BMMs in antibiotic free DMEM complete media. The final concentration of siRNA
and Lipofectamine are 50 nM and 0.6%. Following the incubation for 6 h, transfection media were replaced with
fresh BMM media with antibiotic. The reduction of LSD1 mRNA and protein was confirmed at 48 h after siRNA
transfection by qRT-PCR and Western blot as described above.
Chromatin immunoprecipitation (ChIP) assay.
Approximately 7.0 × 106 cells of BMMs were prepared
and activated as indicated. The SimpleChIP® Enzymatic Chromatin IP Kit (Cell Signaling Technology, USA)
was used to perform ChIP according to the manufacturer’s instructions. Samples were subjected to immunoprecipitation using either Rabbit anti-H3K4me3 antibody or a control IgG antibody (Cell Signaling Technology). Fragmented DNAs were purified using spin columns (Cell Signaling Technology) and was used as the
templates for qPCR using indicated primer sets spanning the tnf-α and il6 promoters (Supplementary Table 2).
Fold enrichments were normalized and calculated based on the total amount of 10% input presented in relative
quantification using 2−∆∆ct method.
Statistical analysis. All experiments were performed in triplicate and at least twice independently, except
for the primary screening. Statistical analyses were performed using GraphPad Prism version 9.0. One-way
ANOVA with Dunnett’s or Tukey’s multiple comparison test and two-tailed unpaired t-test (α = 0.05) were used
when comparing the two conditions. This study is reported in accordance with ARRIVE guidelines.
Results
BG-trained or LPS-tolerant macrophages and global changes in histone marks. To generate
BG-trained or LPS-tolerant macrophages, BMMs were primed with BG or LPS for 24 h and allowed to rest in
media for 48 h, as indicated in Fig. 1a. The resting step was followed by LPS stimulation (10 ng/ml) for 24 h. The
amount of TNFα in the culture supernatant was measured by ELISA, and the relative levels were calculated by
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Figure 1. Generating BG-trained and LPS-tolerant macrophages. (a) Protocol to induce BG-trained or
LPS-tolerant macrophages using BMM. (b) TNFα production was detected by ELISA in unstimulated
cells, BG-primed cells, or LPS-primed cells at 24 h and in LPS-stimulated naïve cells (crtl), LPS-stimulated
BG-primed cells (trained) or LPS-stimulated LPS-primed cells (tolerant) after 24 h of stimulation. The relative
fold changes were calculated by normalizing to the amount obtained from LPS-stimulated naïve BMMs (10 ng/
ml). (c) Protocol to induce BG-trained or LPS-tolerant macrophages using BMM and the indicated time for cell
lysate collection. (d–g) Levels of H3K4me3 and H3K27me3 normalized to total histones in BMMs treated as
indicated in (c) as detected by Western blot. *, **, and *** indicate significant differences compared by two-tailed
unpaired t-tests at p < 0.05, p < 0.01 and p < 0.001, respectively.
normalization to the amount of TNFα produced by naïve BMMs without priming that received LPS stimulation
(10 ng/ml). As shown in Fig. 1b, BG priming alone minimally induced TNFα production, whereas priming with
LPS at 100 ng/ml induced significantly higher TNFα than BG priming. Stimulation of BG-primed macrophages
with LPS resulted in 3.26-fold higher TNFα than that in LPS-stimulated macrophages. BG-trained macrophages
also enhanced the RNA expression of the proinflammatory cytokines Tnf, Il6, and Il1b, while no effect was
observed on the level of the anti-inflammatory cytokine Il10 (Supplementary Fig. 1a–d). Furthermore, activation of the mTOR pathway was clearly detected in BG-trained macrophages, consistent with previous studies
indicating the role of the mTOR pathway in the regulation of trained immunity (Supplementary Fig. 2a,b)22.
For LPS-tolerant macrophages, LPS-primed BMMs produced significantly lower TNFα upon LPS stimulation
than naïve BMMs receiving LPS stimulation at 10 ng/ml (0.36-fold, Fig. 1b). This reduction was also observed
at the mRNA level of both proinflammatory and anti-inflammatory cytokine genes (Supplementary Fig. 1a–d).
LPS priming also repressed the expression of other genes that are characterized as tolerizeable genes (T-gene),
such as Cd40 and Serpine1 (Supplementary Fig. 2c,d). This result strongly confirmed that repeated stimulation
with LPS resulted in LPS-tolerant macrophages. In contrast, the expression of non-tolerizeable genes (NT genes),
such as cathelicidin antimicrobial peptide (Camp) and macrophage receptor with collagenous structure (Marco),
was not only repressed but also enhanced in LPS-tolerant macrophages (Supplementary Fig. 2e,f). These results
are consistent with the specific pattern of gene regulation in LPS-tolerant macrophages in a previous study5.
As histone modifications are one of the key mechanisms for regulating innate immune memory, we investigated the global changes in some key histone marks during BG-trained or LPS-tolerant treatment. BMMs were
treated as indicated in Fig. 1c, and the total cell lysates were analyzed for representative active and repressive
histone marks, H3K4me3 and H3K27me3. Priming with LPS or BG did not significantly alter the levels of
H3K4me3 and H3K27me3 compared to the unstimulated condition. During the resting period or LPS stimulation
in LPS-tolerant macrophages, these marks completely disappeared in LPS-tolerant cells. In contrast, these marks
were still detectable but with lower intensity in BG-trained macrophages (Fig. 1d–g and Supplementary Fig. 6).
Taken together, BMM-derived macrophages were successfully conditioned to become BG-trained and LPStolerant macrophages, and drastic changes in global representative histone marks during induction were evident.
Screening of epigenetics compound library. To identify epigenetic modifier(s) that target molecules
with a role in regulating innate immune memory in macrophages, screening assays were performed using an
epigenetics compound library in BG-trained or LPS-tolerant macrophages as described above. The detailed categories of the compounds in the library are listed in Supplementary Fig. 3 and Supplementary Table 1. Among
these compounds, the targets of action included histone modifying enzymes (38%), epigenetic reader domains
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Figure 2. Compounds that showed enhanced or suppressed TNFα production in BG-trained and LPStolerant macrophages. (a) The relative fold changes of TNFα produced from compound-treated BG-trained
macrophages (priming step or stimulation step) were calculated by normalizing to the amount of TNFα
produced from vehicle control treatment of BG-trained macrophages. Average fold changes of two biological
replicates are shown as heatmap format. Only compounds that increased the relative fold changes of TNFα
more than 1.5-fold or lower than 0.75-fold are shown. (b) The relative fold changes of TNFα produced from
compound-treated LPS-tolerant macrophages (priming step or stimulation step) were calculated by normalizing
to the amount of TNFα produced from vehicle control treatment of LPS-tolerant macrophages. The fold changes
are the average of two biological replicates and shown as heatmap format. Only compounds that increased the
relative fold changes of TNFα more than 1.5-fold are shown. (c) Targets of suppressing inhibitors were classified
based on function and time point.
(9%), DNA methyltransferases (4%), the JAK/STAT pathway (12%) and other kinases (13%). The screening
protocol is summarized in Supplementary Fig. 4a,b. The screening aimed to identify compounds that act at the
priming or stimulating step during BG-trained or LPS-tolerant induction.
For treatment at the priming step, cells were pretreated with the compounds or vehicle control DMSO for
1 h before priming with BG (50 μg/ml; trained) or LPS (100 ng/ml; tolerance) for 24 h. After media containing
BG or LPS together with the compounds were removed, fresh media were added, and the cells were allowed
to rest for 48 h. After the resting period, the cells were stimulated with LPS (10 ng/ml) for 24 h. For treatment
during the stimulating step, cells were primed and rested as described above in the absence of compounds. One
hour before LPS stimulation, BG-primed or LPS-primed BMMs were pretreated with the compounds, and LPS
stimulation was carried out as described above in the presence of compounds. The readout for the screening assay
was the amount of TNFα compared to the BG-trained or LPS-tolerant macrophages treated with vehicle control.
The primary screening was performed using two times the IC50 concentration from the manufacturer’s
information for each compound. The secondary screening was performed on those compounds that met the
criteria set for the primary screening. Among the 181 compounds tested, two compounds showed enhancing effects, while only one compound showed an inhibitory effect on the BG-trained responses. PFI-2 HCl,
a histone methyltransferase inhibitor, reduced trained TNFα production when applied during priming. An
O6-methylguanine-DNA methyltransferase (MGMT) inhibitor and a DNA/RNA synthesis inhibitor enhanced
TNFα production when applied during the priming step. However, none of the compounds had an effect when
used during the stimulation phase (Fig. 2a and Supplementary Table 3).
In contrast, 28 compounds showed suppressive effects against LPS tolerance, which resulted in increased
TNFα production after LPS stimulation. A clear inhibitory effect was observed with inhibitors targeting Aurora
kinases, histone methyltransferases (HMT), histone demethylase (HDMT), histone deacetylases (HDAC), sirtuin,
poly (ADP-ribose) polymerase (PARP), DNA methyltransferase and DNA/RNA synthesis. Most inhibitors rescued TNFα production under LPS tolerance conditions when added during the LPS stimulation phase (78.6%).
The HDAC6 inhibitors ricolinostat and nexturastat and the HDAC1 and HDAC3 inhibitors entinostat showed
inhibitory effects when added during the priming step. However, the effect of the enhancer of zeste homolog 2
(EZH2) inhibitor EPZ011989 and the DNA/RNA synthesis inhibitors carboplatin and nedaplatin were detected
when treated at either the priming or the stimulating step (Fig. 2b,c). The compounds that showed enhancing
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Figure 3. Possible interactions among the target proteins for the identified inhibitors. Potential interactions
among target proteins of inhibitors identified in the screening for tolerance (a) and trained immunity (b) by
STRING. The target proteins of inhibitors identified in this study are shown in black text while the potential
functional partner proteins are shown in gray text. The interactions among proteins that are experimentally
determined are linked with pink lines while the interactions predicted from curated database are shown in blue.
Effective targets related to previous studies are represented in blue text.
effects against LPS tolerance were not further validated because the levels of TNFα were already extremely low
in LPS-tolerant macrophages.
Interacting networks of the proteins targeted by the compounds identified in the screening. To investigate the potential interaction (direct interaction or functional interaction) among the protein
targets of the compounds identified by this screening, the STRING database was used to generate a protein–
protein interaction network. As shown in Fig. 3a, the interaction network generated from the targets identified
in LPS-tolerant macrophages revealed the following three distinctive clusters: the Aurora kinase (cell division)related interacting network, the histone modifying enzyme network and the base-excision repair network. In
contrast, for the target proteins of compounds identified in the BG-trained macrophages, the network revealed
a link with Trp53, Foxo3, Suz12, Hist2h3c2 and Msh16, which share common features related to apoptosis,
DNA repair and polycomb repressive complex (PRC) 2 (Fig. 3b). These analyses suggest that proteins involved
in apoptosis, DNA repair, cell division and histone modification may play roles in innate immune memory in
macrophages.
To confirm the effect of suppressive compounds against LPS tolerance, we investigated the level of IL-6 production after treatment with selected inhibitors, as shown in Fig. 4a. The Aurora-B-specific inhibitor barasertib
and the Aurora-B/C inhibitor GSK1070916 significantly increased IL-6 production during LPS stimulation
without reducing cell viability to less than 80% (Fig. 4b,c). Because the majority of the compounds that showed
suppressive effects against LPS tolerance were Aurora kinase inhibitors (25%, Fig. 2b,c) and these inhibitors
suppressed tolerance when using TNFα and IL-6 as readouts, we investigated the expression profiles of Aurora
kinase A, B and C during priming, resting and stimulation in LPS-tolerant macrophages by Western blot. The
protein bands corresponding to phosphorylated Aurora-B and Aurora-C were clearly observed, while phosphorylated Aurora-A was undetectable (Fig. 4d and Supplementary Fig. 7). The level of phosphorylated Aurora-B
was significantly decreased during LPS priming and LPS stimulation, while unstimulated cells and cells in the
resting period maintained Aurora-B at high levels (Fig. 4d,e). Phosphorylated Aurora-C was slightly increased
during LPS priming (Fig. 4d,f). As Aurora kinases play an important role in chromatid segregation during cell
division, we performed a cell proliferation assay using bromodeoxyuridine (BrdU) during DNA synthesis. The
level of BrdU was significantly decreased in a time-dependent manner during LPS tolerance, indicating that cell
proliferation is suppressed during LPS tolerance (Supplementary Fig. 5). These results emphasized the role of
Aurora kinases in the regulation of LPS tolerance, which may not be related to cell cycle progression.
Validating the roles of histone methyltransferase SETD7 and histone lysine demethylase
LSD1 in BG-trained and LPS tolerance. Among the targets identified in the BG-trained response, inhibition of histone lysine methyltransferase SETD7 by PFI-2 HCl showed a suppressive effect. SETD7 has many
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Figure 4. Expression profiles of Aurora kinases in LPS-tolerant macrophages. LPS-tolerant macrophages were
prepared as described above. (a) IL-6 production after treatment with selected inhibitors from Fig. 3a was
measured by ELISA at 24 h after LPS stimulation. (b, c) Cell viability was detected by MTT assay at 24 h after
treatment with inhibitors from (a). (d-f) Phosphorylation of Aurora kinases was analyzed by Western blot. The
relative intensity from Western blot was quantitated by ImageJ analysis and normalized to β-actin. *, **, *** and
**** indicate significant differences compared by one-way ANOVA with Dunnett’s multiple comparison test (a)
and two-tailed unpaired t-test (d) at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively.
target substrates, including histones, and has only recently been implicated in regulating trained immunity23.
We further examined the expression of Setd7 mRNA and SETD7 protein in BG-trained macrophages. As shown
in Fig. 5a, Setd7 mRNA increased significantly at 72 h after BG priming during the rest period, whereas LPS
stimulation significantly decreased its level. In contrast, the protein level of SETD7 clearly increased during BG
priming and was slightly reduced but remained high during resting and LPS stimulation (Fig. 5b,c and Supplementary Fig. 7). The inhibitory effect of SETD7 on BG-trained response was confirmed by cyproheptadine
(CPH), another SETD7 inhibitor. Treatment with CPH decreased BG-trained immune response as indicated by
reduction in both TNFα and IL-6 production in a dose-dependent manner with little impact on cell viability
(Fig. 5d–f). This result agreed with its possible role during BG priming to condition macrophages for trained
responses18.
Next, we focused on LSD1, a lysine demethylase of which inhibitor (OG-L002) attenuated LPS tolerance when
added during LPS stimulation, but not at the priming step (Fig. 2b). The expression profiles of LSD1 during LPS
priming and LPS stimulation revealed increased protein expression during LPS priming while its level declined
after resting period and LPS stimulation (Fig. 6a,b and Supplementary Fig. 8). The effects of LSD1 inhibition by
OG-L002 on TNFα and IL-6 production during LPS tolerance were confirmed to be a dose dependent manner up
to 40 μM (Fig. 6c,d). To understand whether LSD1 inhibition influenced histone modification of H3K4me3 at the
promoters of these two genes, we performed a ChIP-qPCR. As shown in Fig. 6e,f, to our surprise, OG-L002 treatment did not significantly alter the level of H3K4me3 at the promoter of tnf but significantly reduced H3K4me3
association with the Il6 promoter. This result suggests that LSD1 may mediate demethylation of other histone
marks such as H3K9me3 during LPS tolerance that results in attenuated LPS tolerance.
Finally, we performed siRNA-mediated gene silencing of lsd1 to confirm the results obtained by the use
of inhibitor. As shown in Fig. 7a,b and Supplementary Fig. 8, siRNA targeting LSD1 effectively reduced LSD1
protein to roughly 50%. This siRNA treatment was applied to the LPS tolerance regimen described in the Supplementary Fig. 4. siRNA was transfected to BMM 48 h before LPS priming, followed by resting for 48 h and
stimulation by LPS for 6 h. As shown in Fig. 7c, the level of Il1b mRNA significantly increased when LSD1
was silenced in comparison to the control non-targeting siRNA, an indicator that LPS tolerance is suppressed.
Increased mRNA level of inflammatory genes, tnf and Il6 but the difference did not reach statistical significance.
Thus, inhibiting LSD1 by inhibitor or reducing its expression by siRNA treatment consistently rescued LPS tolerance by increasing tolerizable gene expression.
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Figure 5. Expression profiles of SETD7 during BG-trained responses in macrophages. BG-trained macrophages
were prepared as described above. (a) The mRNA expression profile of Setd7 was detected by qRT-PCR. (b, c)
Cell lysates were analyzed by Western blot. The relative intensity of SETD7 from Western blot was quantitated by
ImageJ analysis and normalized to β-actin. The relative intensity was calculated using unstimulated samples as
the baseline. (d, e) Effect of cyproheptadine (CPH) on TNFα and IL-6 production in BG-trained macrophages
after LPS stimulation. (f) Cell viability from the MTT assay in BG-primed macrophages after treatment with
different concentrations of CPH for 24 h. *, **, *** and **** indicate significant differences compared by one-way
ANOVA with Tukey’s multiple test at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively.
Discussion
Trained immunity and tolerance in monocytes and macrophages are part of innate immune memory10. These
innate immune memory phenomena are governed by transcription factors, epigenetic changes and metabolic
rewiring that result in enhanced or suppressed responses in subsequent encounters with stimuli17. Because epigenetic reprogramming plays important roles in regulating innate immune memory, in this study, we aimed to
identify novel epigenetic regulators that play a role in either trained or tolerance responses, which may potentially
be a novel target for the treatment of conditions caused by dysregulated innate immune memory.
BG stimulation through Dectin1, Akt/mTOR and HIF-1α induced metabolic changes coupled with epigenetic
reprogramming. This complicated regulatory network results in higher transcription of trained genes, including Tnf, Il1b and Il6 (Supplementary Fig. 1a–e)22,24. In addition to BG, other microbial stimuli, such as BCG
vaccination, and nonmicrobial stimuli, such as oxidized LDL, can also induce trained immunity in monocytes
and macrophages10. Different trained stimuli utilize common mechanisms with some distinctive features for
inducing trained immune responses. In this study, we used TNFα as a readout for the BG-trained response in
macrophages because TNFα is one of the best characterized representative markers that is under the control of
the trained immune response. Interestingly, only three compounds were identified in our screening assay that
have an effect on the BG-trained immune response. An inhibitor of the histone methyltransferase SETD7, PFI-2
HCl, has a suppressive effect on trained immunity when added during BG priming but not during stimulation.
A lysine methyltransferase, SETD7, has multiple histone and non-histone substrates that have been explored
for targeted treatments of conditions, such as cancer and obesity23. Methylation of H3K4 mediated by SETD7
is associated with increased gene expression. A recent report identified SETD7 as a key enzyme that increases
oxidative phosphorylation in BG-trained macrophages by upregulating key enzymes in the TCA cycle18. This
result supports the validity of an unbiased screening approach in our study.
We also validated the impact of LSD1 inhibition on LPS tolerance by pharmacological and genetic approaches
which yielded consistent outcomes. Both approaches showed that LSD1 play a positive role in regulating LPS
tolerance. LSD1 is the key enzyme that mediates demethylation of mono- and di-methylated lysine, specifically
H3K4 and H3K9 among others25. LSD1 functions downstream of LPS/TLR4 and controls acute inflammatory
response during sepsis in myeloid cells. Deletion of LSD1 resulted in severe cytokine storm and lethality in
sepsis26. In this study, we found that in LPS tolerance, inhibition of LSD1 during LPS stimulation but not LPS
priming rescued LPS tolerance phenotype. However, we could not find changes in H3K4me3 level associated
with the promoter of Tnf upon LSD1 inhibition whereas H3K4me3 level reduced in the Il6 promoter. This result
may indicate that LSD1 may mediate demethylation of other histone marks that have a combined effect on
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Figure 6. Expression profiles of LSD1 during LPS tolerance in macrophages. BMMs were induced to become
LPS tolerance as described above. (a, b) Expression profiles was analyzed by Western blot. The relative intensity
of LSD1 from Western blot was quantitated by ImageJ analysis and normalized to β-actin. (c, d) Effect of
OG-L002 on TNFα and IL-6 production in LPS-tolerant macrophages. (e, f) Effect of OG-L002 on H3K4me3
enrichment in Tnf and Il6 promoter of LPS-tolerant macrophages at 6 h after LPS stimulation. *, **, *** and ****
indicate significant differences compared by one-way ANOVA with Tukey’s multiple test (c,d) and two-tailed
unpaired t-test (e, f) at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively.
LPS tolerance. Other LSD1 substrate(s) that may play a crucial role in regulating LPS tolerance include H3K9,
H3K27, H3K36, and H3K7925.
The other two compounds showed enhancing effects on BG-trained immunity, though lomeguatrib and
carboplatin have not been previously associated with trained immunity. Lomeguatrib is a specific inhibitor of
O6-methylguanine-DNA methyltransferase (MGMT). MGMT is a DNA repair protein that functions during
DNA damage by alkylating agents and plays a role in conferring resistance to cancer cells against some cancer
chemotherapies27. Mgmt knockout mice are susceptible to the lethal effect of alkylating agents28. In addition to
its role in cancer, MGMT has been linked to inflammation, as hypermethylation of its promoter is associated
with chronic inflammatory diseases and chronic infectious diseases29–32. How MGMT functions in epigenetic
regulation and trained immunity requires further investigation.
LPS tolerance is accompanied by gene-specific chromatin modification that results in either suppression of
gene transcription (including inflammatory genes) of tolerized genes and gene activation (including antimicrobial effector genes, Supplementary Figs. 1 and 2) or non-tolerized genes5. Upstream signaling molecules such
as phosphatase SHIP-1 play important roles in reducing the phosphorylation of signal transduction molecules
downstream of TLR33. In tolerized genes, histone deacetylation and certain lysine methylation cooperate to
induce the state of transcriptional silencing34. In our screening assay, various inhibitors showed inhibitory effects
against LPS tolerance, i.e., increasing TNFα production after repeated LPS stimulation. Histone-modifying
enzyme inhibitors are a major group of inhibitors that reverse LPS tolerance. Several suppressive targets of these
compounds have been characterized in previous studies, such as HDAC1 and HDAC335, HDAC636, and G9a and
GLP15,16. Most of these inhibitors showed their effects when added during LPS stimulation after LPS priming. This
result suggests that these molecules may function to rapidly modify epigenetic states that influence responses to
LPS stimulation during the tolerance phase. Interestingly, inhibitors of histone demethylase, OG-L002, JIB-04
and ML-324, only showed an effect when added during LPS stimulation, but not during the LPS priming. This
result indicated that histone demethylase activity may be essential for maintaining methylated histones to suppress chromatin for TNFα expression during LPS stimulation but not during LPS priming. The results from our
screening assay showed that inhibitor function during LPS stimulation may open a window for reversing LPS
tolerance after the first tolerogenic exposure and may be useful for rescuing the immune paralysis observed in
conditions such as sepsis37.
In addition, the EZH2 histone methyltransferase inhibitor EPZ011989 enhanced TNFα production when
added during priming or stimulation. EZH2 is a catalytic subunit of a large protein complex of PRC2 that
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Figure 7. Effect of LSD1 silencing on LPS-tolerant macrophages. BMMs were transfected with 50 nM of
siRNA lsd1 or Non-Target (NT) siRNA as described in “Materials and methods”. (a, b) Level of LSD1 at 48 h
after siRNA transfection was detected by Western blot and qRT-PCR. (c) Expression of pro-inflammatory and
anti-inflammatory cytokines in lsd1 silencing LPS-tolerant macrophages at 6 h after LPS stimulation. *, **, ***
and **** indicate significant differences compared by two-tailed unpaired t-test at p < 0.05, p < 0.01, p < 0.001 and
p < 0.0001, respectively.
regulates the methylation of the repressive histone mark H3K27me3. All EZH2 inhibitors in the current library
(EPZ015666, GSK503, CPI-169, El1 and 3-DZNeP) function as S-adenosyl methionine competitive inhibitor. All of them contain pyridone-benzamide as a core structure and this core stucture is a target for cellular metabolism38. Although GSK503 and CPI-169 slightly rescued the TNFα production in LPS tolerance,
EPZ015666 showed more than 1.5-fold changes of TNFα concentration over that of the vehicle control. Among
these compounds, EPZ015666 is the only EZH2 inhibitor in our library that was designed to prevent oxidation by
metabolism and possibly showed high potency in our assay. Ezh2 also methylates non-histone protein substrates
such as suppressor of cytokine signaling 3 (SOCS3) and the transcription factor GATA439,40. The wide ranges of
the substrates of PRC2/EZH2 imply that PRC2/EZH2 may regulate multiple steps during LPS tolerance.
To our surprise, several DNA/RNA synthesis inhibitors showed inhibitory effects against LPS tolerance and
enhancing effects against the BG-trained immune response. In addition, Aurora kinase inhibitors were among
the compounds that reversed LPS tolerance (Fig. 2b). When protein interaction analysis was performed, apoptosis- and DNA repair-related genes, such as Trp53 and Msh6, were indicated in the trained immunity network
(Fig. 3b). Protein interaction analysis revealed two specific roles of Aurora kinases in the regulation of cell
division and epigenetic regulation (Fig. 3b). Both DNA/RNA synthesis inhibitors and Aurora kinase inhibitors
may not directly link to epigenetic processes but there are evidences supporting that DNA damage may alter
epigenetic states in trained immunity41 while Aurora kinases also classify as enzymes that phosphorylate serine
residue in histone such as H3S10 and H3S2842,43. Therefore, the findings here may uncover novel targets that
modify epigenetics during innate immune memory.
Aurora kinases are well characterized in regulating mitotic processes, and their inhibition results in cytokinesis failure and is one of the targets for cancer therapy44. Because priming with LPS did not induce cell proliferation, Aurora kinases may regulate LPS tolerance through other mechanisms not related to cell cycle regulation,
such as epigenetic regulation (Supplementary Fig. 5). Among the three subtypes of Aurora kinases, Aurora kinase
B has been reported to regulate the deposition of some repressive histone marks, such as phosphorylation of
H3S10, H3S28 and H3K9me342,45. Interestingly, Aurora kinase B was the only subtype that significantly changed
its phosphorylation level during LPS tolerance. Increased phosphorylation of Aurora kinase B during the resting
period of LPS tolerance may regulate the deposition of these repressive histone marks. In addition to its roles during cell division and epigenetic regulation, Aurora kinase A participates in early signaling during T cell activation
by regulating CD3ζ-containing vesicle trafficking46. In one report, Aurora kinase A regulated M1 macrophage
polarization by suppressing NF-κB activation and switched macrophages toward the M2 phenotype47. How these
groups of enzymes regulate LPS tolerance needs further investigation. DNA/RNA synthesis inhibitors included in
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this screening are often used as chemotherapeutics against cancers, such as the platinum derivative carboplatin.
They induce cancer cell death by various mechanisms48. However, the cell cycle and cell death have not been
investigated in terms of training or tolerance in macrophages, but it is possible that innate immune memory is
tightly coupled with epigenetic modification and cell cycle/cell death.
Using STRING database, we uncovered clusters of protein networks of the inhibitor targets identified in
this study. For LPS tolerance response, histone modification and chromatin modifying enzymes formed a large
cluster with targets identified in this study such as LSD1, PRMT3, PRMT5, EZH2, JMJD2 and SIRT1. Most of
these interactions are experimentally determined. Proteins involved in base-excision repair (PARP1/2) and cell
division (Aurka A/B/C) formed small clusters that linked to the histone modification cluster via EZH2, SIRT1 and
HDAC6. For the BG-trained response, two clusters of proteins in chromatin organization and cellular response to
DNA damage were linked together via TRP53 and SETD7. Although TRP53 was not identified in our screening,
methylation of TRP53 by SETD7 (Set7/9) has been reported in cancer settings49. Thus, BG-trained immunity
may involve modification by methyltransferase of non-histone substrates50. Some of the links shown here are
based on the curated database and require further experimental prove for the physical/functional interactions
in innate immune memory.
Tolerance and trained innate immune memory are tightly regulated, and the interaction between the two
events has been reported at multiple levels. BG treatment is able to revert the epigenetic states conditioned by
LPS tolerance, and trained immunity may be a mechanistic link between sepsis and atherosclerosis13,51. Recent
emerging evidence has pointed to the critical roles of innate immune memory in various pathological conditions, including chronic inflammatory diseases and cancer. The use of epigenetic modifying compounds provides
potential interventions for such diseases. The limitation of this study is that the observed effect of each compound
on innate immune memory may be the result of side-effect of the compound and this point needs to be further
validated by genetic approaches. Furthermore, our screening results provide new unappreciated key enzymes/
pathways that may regulate training and tolerance in macrophages.
Received: 15 July 2021; Accepted: 20 January 2022
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Acknowledgements
The authors are grateful to Drs. Nattiya Hirankarn, Asada Leelahavanichkul, Patcharee Rijprajak and Sita Virakul
for sharing reagents and for valuable comments on the manuscript.
Author contributions
S.B. designed and performed all experiments, analyzed all data and prepared all figures and the manuscripts. A.B.,
B.W., and T.P.K. performed ELISA, PCR, and Western blotting and analyzed the data in some experiments. J.K.
helped in analysis and preparation of some of the figures. T.P.G. supervised the overall project, acquired grant
funding, designed all experiments, analyzed data, and prepared the manuscript.
Funding
This work was supported in part by the Ratchadaphisek Somphoch Endowment Fund from Chulalongkorn
University (760001-HR), Thailand Science Research and Innovation (TSRI) Fund (CU_FRB640001_01_23_1)
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and the Program Management Unit for Human Resources & Institutional Development, Research and Innovation—CU B05F630073 and the National Research Council of Thailand to TP. SB is supported by the scholarship
from “The 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship” and also “The 90th
Anniversary Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund)” Chulalongkorn
University. AB is supported by the Second Century Fund ( C2F), Chulalongkorn University.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary Information The online version contains supplementary material available at https://doi.org/
10.1038/s41598-022-05929-x.
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