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A polycomb group protein EED epigenetically regulates responses in lipopolysaccharide tolerized macrophages

Epigenetics & Chromatin volume 17, Article number: 36 (2024) Cite this article

Abstract

Background

To avoid exaggerated inflammation, innate immune cells adapt to become hypo-responsive or “tolerance” in response to successive exposure to stimuli, which is a part of innate immune memory. Polycomb repressive complex 2 (PRC2) mediates the transcriptional repression by catalyzing histone H3 lysine 27 trimethylation (H3K27me3) but little is known about its role in lipopolysaccharide (LPS)-induced tolerance in macrophages.

Result

We examined the unexplored roles of EED, a component of the PRC2, in LPS tolerant macrophages. In Eed KO macrophages, significant reduction in H3K27me3 and increased active histone mark, H3K27ac, was observed. Eed KO macrophages exhibited dampened pro-inflammatory cytokine productions (TNF-α and IL-6) while increasing non-tolerizable genes upon LPS tolerance. Pharmacological inhibition of EED also reduced TNF-α and IL-6 during LPS tolerance. Mechanistically, LPS tolerized Eed KO macrophages failed to increase glycolytic activity. RNA-Seq analyses revealed that the hallmarks of hypoxia, TGF-β, and Wnt/β-catenin signaling were enriched in LPS tolerized Eed KO macrophages. Among the upregulated genes, the promoter of Runx3 was found to be associated with EED. Silencing Runx3 in Eed KO macrophages partially rescued the dampened pro-inflammatory response during LPS tolerance. Enrichment of H3K27me3 was decreased in a subset of genes that are upregulated in Eed KO LPS tolerized macrophages, indicating the direct regulatory roles of PRC2 on such genes. Motif enrichment analysis identified the ETS family transcription factor binding sites in the absence of EED in LPS tolerized macrophages.

Conclusion

Our results provided mechanistic insight into how the PRC2 via EED regulates LPS tolerance in macrophages by epigenetically silencing genes that play a crucial role during LPS tolerance such as those of the TGF-β/Runx3 axis.

Background

It was long believed that the immune memory operated only in adaptive immune cells but this concept has recently changed [1, 2]. Traditionally, innate immune responses are thought to be non-specific and without capability to adapt [3]. Emerging evidence suggested that innate immune cells such as macrophages, monocytes, and natural killer cells, are also able to develop immunological memory to previous encounters such as lipopolysaccharides (LPS) from bacteria [4, 5]. This phenomenon has been linked to metabolic reprogramming and epigenetic changes of the innate immune cells. The outcomes of an innate immune memory manifest as “trained immunity” or “tolerance”, that either enhances or depresses responses to the stimuli after the first encounter [6, 7]. The dampened immunological response to a secondary stimulation is defined as tolerance [2, 8, 9].

It is hypothesized that the induction of innate immune tolerance is a compensatory mechanism to limit an overwhelmed and potentially harmful response to the pathogens or stimuli in the subsequent encounter after the primary infection. The innate immune tolerance is triggered by a systemic inflammation, resulting in immune paralysis that is often seen in sepsis patients that can be lethal upon secondary infection [10]. To date, increasing evidence demonstrates that sepsis is partly controlled by the epigenetic modifications, including DNA methylation and histone modifications [11]. Furthermore, changes in the epigenetic profiles of LPS-induced tolerant macrophages are observed in both acetylation and methylation of the histone tails [12].

Tri-methylation of the histone H3 at lysine 27 (H3K27me3) is a transcriptionally repressive epigenetic mark of enhancers that is associated with gene repression [13, 14]. The Polycomb Repressive Complex 2 (PRC2) catalyzes the tri-methylation of H3K27 through its Enhancer of Zeste Homolog 1 and 2 (EZH1/EZH2) subunit [13]. This modification leads to transcriptional silencing through chromatin compaction [15, 16]. In addition, EZH2 also interacts with Embryonic Ectoderm Development (EED) and SUZ12, the core major subunits of PRC2 to perform the enzymatic activity as methyltransferase. Depletion of these proteins in the PRC2 abrogate its function as histone writer [17,18,19].

PRC2 is shown to play a regulatory role in monocytes/macrophages. Evidence supported that loss of EZH2 decreases pro-inflammatory cytokine productions in macrophage, leading to diminished severity of tissue damages caused by hyperactivated macrophages. The expression of socs3, an anti-inflammatory gene, is accrued in the EZH2 deficient macrophages which leads to inhibition of JAK/STAT signaling pathway [20]. Thus, PRC2 is the potential target for regulatory control of macrophages in response to inflammatory stimuli in chronic inflammation.

The polycomb group protein EED plays a central role in regulating both PRC1 and PRC2 functions. EED binds to H3K27me3 and propagates the tri-methylation of unmodified H3K27 in the vicinity of the initial H3K27me3 [21]. By binding to the H3K27me3, EED recruits PRC2 components to expand the repressive histone marks. On the other hand, EED is also able to recruit PCR1 components to the H3K27me3 loci and enhances PRC1 ubiquitin E3 ligase activity for ubiquitination of H2AK119 [22]. This event leads to chromatin condensation and target gene silencing. Thus, disruption of EED results in loss of H3K27me3 recognition that may lead to abrogation of the trimethylation process mediated by PRC2 and the PRC1 activity. Pharmacological inhibition of EED by EED226 in renal tubular cells suppresses several renal inflammatory cytokines as well as decreasing infiltrated macrophages into injured kidney [23]. These data suggested the disruption of PRC2 by targeting EED interferes with the inflammatory responses in certain settings and may have consequences that differ from EZH1/2 inhibition. Importantly, the targeted deletion of EED in monocytes/macrophages had not been documented and the impact of such is unknown.

Despite the documented detailed involvement of EHZ2/PRC2 in LPS stimulation of macrophages, the involvement of PRC2 in LPS-induced tolerance are not well-understood. Previously, we reported a screening assay to identify epigenetic regulators of innate immune memory, including LPS tolerance and uncovered PRC2 inhibitor as one of the hits [24]. In this study, we investigated how EED, one of the key PRC2 components, regulated LPS-induced tolerized response in macrophages. We uncovered the roles of EED in LPS-induced tolerance and the links with metabolic flux and TGF-β and Runx3 axis.

Methods

Bone marrow-derived macrophages (BMDMs)

BMDMs were harvested from the femurs and tibias of Eedfl/fl; LysM-Cre+/− (Eed KO), Ezh2fl/fl; LysM-Cre+/− (Ezh2 KO), and their littermate control mice by flushing. Ezh2fl/fl and LyM-Cre mice were provided by RIKEN BRC Experimental Animal Division (Ibaraki, Japan) through the National BioResource Project of the MEXT/AMED, Japan. Bone marrow cells were cultured for 7–10 days in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Logan, UT, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, Grand Island, NY, USA), 1% (w/v) sodium pyruvate, 1% (w/v) HEPES, 100 U/ml pen/strep, 20% L929 cell conditioned media and 5% horse serum. On day 7, macrophages were resuspended and plated in the tissue culture plates and allowed to adhere for 16–18 h prior to use. 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) and Chulalongkorn University Laboratory Animal Center (CULAC, approval protocol No. 2073015. All experiments were performed according to the guidelines issued by the IACUC. This study is reported in accordance with the ARRIVE guidelines.

Induction of LPS tolerized macrophages

BMDMs were primed with Salmonella spp. lipopolysaccharide (LPS; Sigma Aldrich, St. Louis, MO, USA) at 100 ng/ml for 24 h. After the 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. Total RNA, culture supernatant or cell lysates were harvested at indicated times after the secondary LPS stimulation for further analyses.

Enzyme-linked immunosorbent assay (ELISA)

The amounts of TNF-α and IL-6 were quantified in the culture supernatants from BMDMs treated as indicated using mouse TNF-α ELISA and IL-6 ELISA kits (BioLegend, CA, USA) following the manufacturer’s instructions. Streptavidin HRP was used to detect bound antibodies, and 3,3’, 5,5”-tetramethylbenzidine (TMB) (Sigma Aldrich) was used as a substrate. The reaction was stopped with 1 M H2SO4. Absorbance at 450 and 620 nm was measured on a microplate reader (Thermo Fisher Scientific).

Reverse transcription and qPCR (RT-qPCR)

Total RNA was isolated using RNeasy Mini Kit (Qiagen) and reverse transcribed using RevertAid reverse transcriptase (Thermo Fisher Scientific) following the manufacturer’s protocol. qPCR was carried out using iQ SYBR® Green Supermix (Bio-Rad Laboratories) using a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories). Relative expression was calculated by normalizing to b-actin or gapdh as housekeeping genes. A list of primers used in this study is shown in Supplementary Table 1. The results were calculated and presented as relative quantifications using the 2−∆∆ct method.

Western blot

BMDMs were treated as indicated and the whole-cell lysates were prepared in RIPA buffer and subjected to SDS-PAGE and Western blot. The antibodies used were mouse anti-EZH2, rabbit anti-EED, rabbit anti-H3K27me3, rabbit anti-H3K27Ac, rabbit anti-GAPDH, rabbit anti-phospho-NF-kB, rabbit anti-phospho-ERK, rabbit anti-phospho-MAPK, rabbit anti-phospho-SAPK-JNK, rabbit anti-NF-kB, rabbit anti-ERK, rabbit anti-MAPK, rabbit anti-SAPK-JNK, mouse anti-actin, HRP-conjugated donkey anti-rabbit IgG and HRP-conjugated sheep anti-mouse IgG (all were purchased from Cell Signaling Technology, Danvers, MA, USA). The signals were detected by chemiluminescence.

Seahorse glycolytic assay

BMDMs were seeded at 1 × 105 cells/well and LPS tolerance were performed as previously described in the Seahorse XF96 Cell Culture Microplate (Agilent Technologies, Santa Clara, CA, USA). The glycolytic activity was measured using the Seahorse XF Glycolysis Stress Test Kit according to manufacturer’s instructions. Briefly, on the assay day, growth media was exchanged with Seahorse Phenol Red-free DMEM, followed by sequential injections with glucose (10 mM), oligomycin (1.0 µM), and 2-deoxy-D-glucose (2-DG; 100 mM). The XF instrument measured the rate of acidification and reports it as the extracellular acidification rate (ECAR). The glycolysis stress test begins by measuring the baseline ECAR in the culture medium without glucose or pyruvate. After the sequential injection of a saturating glucose concentration, cells utilized the glucose, causing a rapid increase in ECAR due to proton production in the surrounding medium. The maximum ECAR was measured after the addition of oligomycin, an ATP synthase inhibitor. Lastly, 2-DG was introduced to inhibit glycolysis, resulting in a reduction of ECAR to baseline levels.

Library preparation and RNA-Sequencing (RNA-Seq)

Total RNA was extracted using RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. All experiments were conducted with three biological replicates. The amounts of total RNA were quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific). RNA quality was assessed using the 2100 Bioanalyzer (Agilent Technology). cDNA libraries were prepared using a TruSeq stranded mRNA library preparation kit (Illumina, San Diego, CA, USA). The quantity and quality of the libraries were determined using a 2100 Bioanalyzer. Sequencing was carried out on a HiSeq (2 × 150 bp paired end reads) at the Omics Sciences and Bioinformatics Center (Chulalongkorn University, Bangkok, Thailand).

RNA-Seq analyses

Sequencing reads were mapped against the Mus musculus reference genome mm10. Reads were mapped and aligned with HISAT2 [25]. Reads were counted by Rsubread [26]. Subsequently, DEGs were compared and analyzed in R version 4.0.3 using the package edgeR [27]. The analyses were conducted from triplicate samples. Genes were considered differentially expressed when the log2 fold change was < -0.75 or > 0.75 (representing down- or upregulation, respectively) and the FDR was < 0.05. Gene Set Enrichment Analysis (GSEA) was performed using predefined gene sets from the Molecular Signatures Database (MSigDB v5.0). GSEA was conducted and applied with the hallmarks (50 gene set, 1000 permutations) [28]. The heat map of the genes enriched at the top or bottom of the gene sets were identified using FDR and ranked by the normalized enrichment score (NES). R script was performed according to https://stephenturner.github.io/deseq-to-fgsea.

Chromatin immunoprecipitation (ChIP) and ChIP-qPCR

Approximately 1 × 106 BMDMs were prepared and treated as described. Cells were cross-linked by 1% formaldehyde. Chromatin immunoprecipitation was performed according to previous protocol with small modification [29]. Samples were subjected to immunoprecipitation using rabbit anti-EED antibody for ChIP-qPCR. DNA was used as a template for qPCR using the indicated primer sets spanning the Runx3, Tnf and Il6 promoter (Supplementary Table 2.) Fold enrichments were normalized and calculated based on the total amount of 5% input and presented as relative quantifications to %input using the 2−∆∆ct method.

CUT&Tag

An approximately 1 × 105 BMDMs together with 10,000 HEK293T cells as spike-in control were counted and resuspended in the culture medium DMEM supplemented with 10% (v/v) fetal bovine serum, 1% (w/v) sodium pyruvate, 1% (w/v) HEPES, 100 U/ml pen/strep, 20% L929 cell conditioned media and 5% horse serum). The CUT&Tag was performed according to the protocol that was previously described [30]. Briefly, the mixed cells were washed and resuspended with PBS prior to a light fixation with 0.1% formaldehyde/PBS. Concanavalin A beads were prepared and washed with the binding buffer and combined with BMDMs-HEK293T mixed cells. The mixture was rotated at room temperature for 20 min. The bead- cells complex was suspended with primary antibody (H3K27me3) in ice-cold antibody for overnight at 4 °C. The mixture was then washed with Dig-wash buffer followed by the addition of secondary antibody for 30 min. The mixture was washed again with Dig-wash buffer and subjected to binding reaction of pAG-Tn5 adaptor complex in Dig-300 buffer for 1 h at room temperature. After washing with Dig-300 buffer, the mixture was resuspended with Tagmentation buffer for tagmentation reaction for 45 min at 37 °C. The tagmented DNA was eluted and purified using SPRI beads (Beckman Coulter). The DNA was amplified by PCR reaction using Q5 Hot Start High-Fidelity 2x Master Mix (NEB). The sequencing was done with paired-end sequencing (75 bp) by Kazusa DNA Research Institute, Japan.

CUT&Tag analyses

Reads were aligned to Mus musculus reference genome mm10 using bowtie2 [31]. The SAM files were converted to the BAM format using Samtools [32]. Picardtools (http://broadinstitute.Github.io/picard) were used to remove PCR duplicates. The BAM files were converted to bigwig files by deepTools for visualization and heatmaps [33]. For camparison across CUT&Tag samples with a spike-in genome, the bigwig files were normalized to spike-in genome coverage (https://yezhengstat.github.io/CUTTag_tutorial/). Genome browser tracks were visualized by Integrative Genomics Viewer (IGV) and pyGenomeTracks [34, 35]. Heatmap clustering was generated using deepTools by k-means clustering. Peak calling was performed using SEACR_1.3 [36]. Peak calling on individual replicates was performed using the spike-in normalized bedgraph with the non stringent parameter. Motif enrichment analysis was conducted using MEME-ChIP [37].

Statistical analyses

Significant differences between two independent samples were determined by an unpaired t-test. One-way ANOVA was used to identify significant differences among samples in one group. p < 0.05 was considered statistical significance.

Results

Targeted deletion of E ed or Ezh2 reduced H3K27me3 while increased H3K27ac in macrophages

To understand the role played by PRC2 components in regulating macrophage responses, we investigated BMDMs from EEDfl/fl; LysM-Cre+/− (Eed KO) and EZH2fl/fl; LysM-Cre+/− (Ezh2 KO) mice, in which Eed or Ezh2 was specifically inactivated in the myeloid lineage cells. We first tested whether loss of EED or EZH2 reduced the global levels of H3K27me3 in BMDMs by Western blot. As expected, Ezh2 or Eed deficient macrophages exhibited globally decreased levels of H3K27me3 as shown in Fig. 1A-B. Unexpectedly, elevated levels of H3K27Ac, which is an active histone mark for the enhancer elements, was observed in BMDMs from both KO macrophages (Fig. 1A-B). The effect of the Ezh2 KO was less dramatic than that of the Eed KO for both decreasing H3K27me3 and increasing H3K27Ac, as previous data has been shown that EZH1-containing PRC2 shows lower histone methyltransferase activity, compared to EZH2-containing PRC2 [38]. In addition to macrophages, the loss of Eed also exhibited a more dramatic reduction of H3K27me3 than Ezh2-deficient ES cells [39]. We further investigated whether the loss of EED also affects other active marks. Although we observed a slight increase in H3K36me3 in Eed KO cells, it was not as pronounced as the increase seen in H3K27Ac (Supplementary Figure S1). However, no alteration in the trimethylation at H3K4 or H3K79 was observed under our condition. Based on our data, we proposed that EED specifically targets H3K27.

Fig. 1
figure 1

PRC2 components and their targeted histone profiles in unstimulated Ezh2 and Eed KO bone marrow-derived macrophages (BMDMs). (A-B) Global protein expression of EZH2, EED and their targeted histone proteins (H3K27me3, H3K27ac) in Ezh2 KO (A) and Eed KO (B) macrophages and their WT controls. β-actin, GAPDH and total H3 proteins were used as internal controls. (C-D) mRNA expression of Ezh1, Ezh2, and Eed profiles of unstimulated Ezh2 KO (C) and Eed KO (D). BMDMs and its littermate WT. **, **** indicate p < 0.01 and p < 0.0001 using unpair t-test. (E) Global protein expression of EZH1, EZH2, EED and their targeted histone proteins upon LPS challenges. BMDMs were treated with LPS (100 ng/ml) or primed with LPS (100 ng/mL) and tolerized with LPS (10 ng/ml) for the indicated durations. Cell lysates were subjected to Western blot. β-actin was used as internal controls. (F) Band intensities of indicated proteins from Figured E were displayed in histograms. The data shown are representative blots of replicates (n ≥ 3). (G) Ezh1, Ezh2, and Eed transcripts upon LPS stimulation at specific time points corresponding to their protein expression. The results shown represent the means ± SEM, n = 3 and p < 0.05

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We next checked whether loss of Eed or Ezh2 affected each other expression or Ezh1 expression. While a significant reduction in Eed and Ezh2 transcripts were observed in Eed KO or Ezh2 KO macrophages, respectively, compared to the WT control, no changes were noticed in the level of Ezh1 expressions (Fig. 1C-D). Therefore, the absence of either Eed or Ezh2 did not impact the expression levels of each other or the Ezh1 transcript. To validate that deletion of either gene using the LysM-Cre system did not interfere with macrophage differentiation in vitro, we examined the expression of F4/80 and CD11b in Eed or Ezh2 BMDM, by flow cytometry. The expression of F4/80 and CD11b in both Eed KO, Ezh2 KO and the littermate WT were comparable, indicating that there was no impact on in vitro differentiation of macrophages in the absence of Eed or Ezh2 (Supplementary Figure S2).

Pharmacological inhibition of EED diminished the immune response in LPS tolererized macrophages

Our previous data has shown that pharmacologically inhibiting PRC2 activity affected how macrophages responded to LPS tolerance [24]. In addition, silencing PRC2 components in fibroblasts require more than 8 days to show significant decrease in H3K27me3 level [40]. Therefore, to mimic Eed KO phenotype, EED inhibitor (EED 226, 10 µM) was used to treat cells for longer period of time by adding at day 4 during macrophage differentiation because in the Eed KO BMDMs, Eed was depleted during this period upon Cre recombinase expression under LyM promoter [41].

We first tested whether EED226-treated macrophages exhibited decreased H3K27me3 as per Eed KO. Interestingly, H3K27me3 was attenuated as observed on day 7 and day 10 after BMDM differentiation, compared to DMSO control while EED was still expressed at comparable level to the control (Supplementary Figure S3). We further assessed their responses to LPS re-stimulation. The schematic stimulation protocol was shown in Supplementary Figure S3. Consistent with the genetic ablation of Eed, a marked reduction in TNF-α and IL-6 production was observed in the EED226-treated macrophages compared to the DMSO control, indicating a phenocopy of the tolerized response in EED226-treated and Eed KO BMDMs.

Loss of Eed, but not Ezh2, restrained inflammatory responses in LPS tolerized macrophages

From the results above, we observed that interfering with PRC2 activity, particularly EZH2 and EED, altered response in LPS tolerance in macrophages. To investigate whether LPS influences their expression, we measured the levels of EZH1, EZH2, EED as well as their targets, H3K27me3 and H3K27Ac, in BMDMs treated with LPS either once or twice (LPS priming that was followed by LPS challenge) as a model of LPS tolerance. As shown in Fig. 1E-F, EZH2 levels initially increased during LPS priming (1–3 h) but dramatically decreased after 24 h compared to its unstimulated control. Subsequently, EZH2 was significantly downregulated during LPS re-stimulation. In contrast, the level of EED remained relatively stable throughout the course of LPS priming; however, EED was significant reduced when exposed to secondary LPS challenge. Conversely, EZH1 levels were constantly expressed throughout stimulation. The levels of their target, H3K27me3, were expressed quite consistently, while the active mark decreased when LPS was introduced and significantly disappeared after the LPS re-challenge.

Apart from protein levels, we also measured their transcripts upon LPS stimulation. Surprisingly, only Ezh2 transcript was initially upregulated during the early phase of LPS priming but progressively declined over the duration of the stimulation while the others were expressed with fluctuation (Fig. 1G).

Based on this observation, we hypothesized that PRC2 (EZH2 or EED) might has a role in LPS tolerance. Thus, we then determined whether loss of EZH2 or EED affect the LPS tolerance by using Ezh2 or Eed deficient macrophages as models. We determined the impact of Ezh2 KO or Eed KO on LPS tolerized BMDMs using the protocol shown in Fig. 2A. Both WT and Ezh2 KO macrophages exhibited comparable levels of TNF-α and IL-6 during the LPS priming (Fig. 2B and C). In contrast, significantly decreased levels of TNF-α and IL-6 were observed in LPS-primed Eed KO macrophages, compared with littermate WT control (Fig. 2D and E).

Fig. 2
figure 2

Pro-inflammatory cytokine productions of LPS-stimulated BMDMs. (A) Schematic method for unstimulated, LPS-primed, LPS-tolerant BMDMs. (B-F) TNF-α and IL-6 productions of (B-D) Ezh2 KO, (E-F) Eed KO and its littermate WT macrophages stimulated as described. Data are representative of at least 3 independent experiments. *, indicate p < 0.05 using unpaired t-test

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In LPS-tolerized macrophages, either WT macrophages (both Ezh2fl/fl and Eedfl/fl) or Ezh2/Eed KO macrophages showed significantly dampened TNF-α and IL-6 levels, compared to the LPS-primed cells, confirming the tolerant phenotype with reduced production of pro-inflammatory cytokines after successive LPS stimulation. Interestingly, only Eed KO macrophages produced significantly lower levels of these two cytokines than the control WT while no difference was observed in LPS tolerized Ezh2 KO macrophages (Fig. 2D and E). Therefore, loss of EED further reduced the level of pro-inflammatory cytokines during LPS tolerance and, thus the involvement of EED in LPS tolerance was further explored.

The effect of Eed KO on LPS tolerance was investigated for expression of some key genes that were previously characterized in LPS tolerance model using the scheme depicted in Fig. 3A [9]. As shown in Fig. 3B, the levels of cytokine genes and tolerizable genes, i.e. Tnf, Il6, Il1b and Il10 transcripts in LPS tolerized Eed KO BMDMs all showed the reducing trends, but the differences did not reach statistical significance (Fig. 3B). In contrast, the levels of the two representative non-tolerizable genes, Marco and Saa3, were increased in Eed KO BMDMs (Fig. 3C). We also examined the level of proinflammatory cytokine genes (Tnf, Il6) in LPS tolerized Ezh2 KO macrophages; however, we did not observe any effect of Ezh2 deficiency on LPS tolerance (Supplementary Figure S4). The results suggested that EED plays a more pronounced role in tolerance than EZH2. Taken together, these findings indicated that EED, but not EZH2, plays a role in regulating expression of tolerized and non-tolerizable genes in LPS tolerance. Loss of Eed in macrophages decreased expression of some tolerizable genes while increased the expression of some non-tolerizable genes.

Fig. 3
figure 3

Expression of pro-inflammatory cytokines, anti-inflammatory cytokines and representative tolerizable genes in LPS tolerized Eed KO BMDMs. (A) Schematic method for unstimulated, LPS-primed, LPS-tolerant BMDMs for RNA isolation. (B) Expression of cytokine encoding genes: Tnf, Il6, Il1b, and Il10 transcripts and (C) non-tolerizable genes: Marco and Saa3 were measured using qRT-PCR. Data are representative of 3 biological replicates. *, indicate p < 0.05 using unpaired t-test

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TLR4 signaling pathways were not altered in LPS tolerized E ed KO BMDMs

LPS signals via Toll-like receptor 4 (TLR-4) which in turn regulates the expression of critical pro-inflammatory genes essential for inflammatory immune responses [42]. We assessed whether the impaired production of pro-inflammatory cytokines in LPS-tolerized Eed KO macrophages was the result of the compromised TLR4 signal transduction. LPS tolerized BMDMs were subjected to Western blot to evaluate the downstream TLR4 signaling pathways, including phospho-p65 NF-κB, phospho-p44/42 ERK, phospho-p38 MAPK and phospho-SAPK/JNK. In both WT and Eed KO macrophages, the rise of phosphorylated p65-NF-κB, MAPK, and SAPK/JNK were observed over time while the level of phosphorylated ERK was significantly reduced at the beginning of LPS re-stimulation (Supplementary Figure S5A and B). No marked differences in the activation kinetics of these signaling pathways were observed with or without EED, suggesting that loss of EED did not impinge upon the immediate downstream signaling of TLR4.

Loss of EED reduced the glycolytic function and the level of Hif1a expression in LPS tolerized macrophages

Metabolic flux plays essential roles in the outcome of macrophage response to external stimuli and activation via TLR4 leads to a switch from oxidative phosphorylation toward glycolysis in inflammatory macrophages whereas a switch from aerobic glycolysis to fatty acid oxidation results in anti-inflammatory response during tolerance [43,44,45]. It has been shown that pro-inflammatory (M1) macrophage primarily exhibit glycolytic metabolism [46]. The LPS-activated macrophages shift their cellular metabolism to the glycolytic pathway, allowing them to quickly generate the energy needed to produce inflammatory substances [47, 48]. In addition, LPS tolerance of human monocytes showed defect lactate production while inhibition of glycolysis reduced TNF-α further in LPS tolerized cells [49].

Collectively, understanding glycolytic activity during LPS tolerance can provide insights into how LPS-tolerized Eed KO macrophages utilize and generate energy in response to LPS. Accordingly, we measured the glycolytic capacity of Eed KO macrophages after LPS priming and tolerance for 24 h, respectively, using Seahorse Glycolytic assay to measure ECAR generated in the medium, resulting from the extrusion of protons during glycolysis breakdown. As shown in Fig. 4, Eed KO BMDMs exhibited significantly lower glycolytic activities as measured by ECAR, compared to the WT control. This result suggested that low glycolytic activity in Eed KO macrophages leads to decreased cytokine productions.

Fig. 4
figure 4

Glycolytic activity measurement of LPS tolerized BMDMs. WT and Eed KO BMDMs were primed and challenged with primary and secondary LPS as described earlier. (A). Glycolytic utilization and capability of Eed KO and WT tolerant macrophages were measured by Seahorse Glycolytic assay. (B). The hypoxia-inducible factor-1α (Hif1a), was quantified in unstimulated, LPS-primed, and tolerant cells by qRT-PCR. Data are analyzed in 3 biological replicates. *, indicate p < 0.05 using unpaired t-test

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Furthermore, the interplay between the transcription factor hypoxia-inducible factor-1α (Hif-1α), a key regulator of hypoxia-induced gene expression, and LPS tolerance has been identified in monocytes and macrophages during sepsis [45, 50, 51]. We wondered whether Hif-1α in LPS-tolerized macrophages was altered in Eed KO macrophages. LPS priming increased the level of Hif1a over the unstimulated cells in similar fashion in both WT and Eed KO macrophages (Fig. 4B). When the expression levels of Hif1a in tolerized macrophages were quantified, further increased Hif1a transcript was detected in WT control cells, compared to its LPS-primed condition, consistent with its roles in LPS tolerance. In contrast, failure to increase Hif1a expression was noticed in the Eed KO BMDMs, suggesting a defect in regulatory mechanism of LPS tolerance in the absence of EED. Taken together, loss of Eed resulted in reduced glycolytic capacity and failure to increase Hif1a expression in LPS tolerized macrophages.

Transcriptomic changes were observed in E ed KO BMDMs

Although many studies have evaluated the regulation of the PRC2 complex, its precise function in macrophages remains unclear. The role of PRC2 in innate immune cells, particularly in macrophages, has been focused mainly on EZH2 function [39]. More importantly, the function of EED in macrophages has not been documented. In addition, the loss of Eed exhibited a more dramatic reduction of H3K27me3 than Ezh2-deficient ES cells, leading to our interest in EED. To identify genes affected by Eed deficiency in macrophages, we first evaluated the whole transcriptome of unstimulated BMDMs in Eed KO macrophages and the control WT. In the absence of EED, BMDMs upregulated expression of 78 genes (log2 FC > 0.75, FDR < 0.05) while 7 genes were downregulated (log2 FC < -0.75, FDR < 0.05) (Supplementary Tables 3 and 4, Supplementary Figure S6). The GSEA analysis revealed that the TGF-β signaling and Wnt/β-catenin signalling hallmarks were enriched in Eed KO macrophages. Ctsk, Lyz1, and Lyz2 encoding for proteins that are involved in macrophage effector functions were among the downregulated genes.

We further identified differentially expressed genes in the absence of EED upon LPS priming. A total of 199 and 33 genes were up-regulated and down-regulated, respectively (log2 FC > 0.75 or < -0.75, FDR < 0.05) (Supplementary Tables 5 and 6, Supplementary Figure S7). The higher numbers of upregulated genes found in Eed KO BMDMs strongly supported that EED/PRC2 deposits the repressive marks on the histone tails that suppress gene expression. Wnt/β-catenin signaling and the inflammatory response hallmarks were the topmost enriched hallmarks among the differentially expressed genes in Eed KO BMDMs.

In LPS tolerized macrophages, we observed significant numbers of upregulated genes in Eed KO BMDMs than the downregulated ones (Fig. 5). A total of 142 genes were upregulated and listed in the Supplementary Table 7. They were depicted in the heatmap and the volcano plot in Fig. 5A and B (log2FC > 0.75, p < 0.05). Higher level of expressions of the non-tolerizable genes such as Slc13a3, Lox, Ass1, and Ccl8 in the Eed KO BMDMs were also found from the RNA-seq data set. The impact of EED loss on associated pathways in LPS-tolerized macrophages was analyzed using gene set enrichment analysis (GSEA). The GSEA analysis revealed that the Hypoxia, TGF-β signaling and Wnt/β-catenin signaling hallmarks were shown to be positively enriched in Eed KO macrophages (p < 0.05). Furthermore, GSEA showed that the loss of EED was negatively correlated with the IFNγ response hallmark (p < 0.05) (Fig. 5D).

Fig. 5
figure 5

Transcriptomics profiles of LPS tolerized Eed KO and WT macrophages BMDMs were primed and tolerized as previously mentioned. RNA-Seq was performed and analyzed as described in the “Material and methods”. (A). Heatmap (B). Volcano plot depicted up-regulated genes and differentially expressed genes (DEGs) in Eed KO tolerant macrophages, respectively. DEGs were filtered using |log2FC| > 0.75, FDR < 0.05. (C). The expression of Runx3, was confirmed by qRT-PCR in unstimulated and tolerant macrophages from both Eed KO and WT BMDMs. (D). GSEA showed the top positive and negative enrichment hallmarks that correlate with the loss of EED in tolerant macrophages and the indicated hallmarks, respectively (n = 3, p < 0.05) (E-F). Heatmap showing the expression patterns of histone methyltransferase (E) and demethylase (F) encoding genes from RNA-Seq data for Eed KO and WT under tolerance conditions

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One of the notable upregulated genes is Runt-related transcription factor 3 (Runx3), encoding the RUNX3 transcription factor known to function in activation or repressing specific gene expression during cellular development and differentiation, including T lymphocytes [52]. The upregulation of Runx3 was validated by qPCR that confirmed the findings from RNA-seq data (Fig. 5C).

Because PRC2/EED plays a crucial role in histone modification, the loss of H3K27me3 might impact the expression of genes encoding other histone modifying enzymes, i.e. histone methyltransferases (HMT) and demethylases (HDM). Therefore, the transcripts of indicated histone modifying enzymes were analyzed and shown in Fig. 5E-F. Most HMT and HDM exhibited increased expression in Eed KO LPS tolerized macrophages. The expression profiles of histone acetyltransferase (HAT) and histone deacetylase (HDAC) were also analyzed by comparing Eed KO BMDMs to the WT control in unstimulated and LPS tolerized macrophages. In LPS tolerized BMDMs, Ep300, Crebbp, Kat2a and Kat7 of HATs are upregulated in the absence of EED. For HDACs, the expression of most HDAC encoding genes, except for Hdac3, 4 and 7, were slightly downregulated. The combination of increasing HAT and reducing HDAC expression may contribute to increased histone acetylation in Eed KO BMDMs.

Apart from the up-regulated genes, we observed only a small number of down-regulated genes in Eed KO LPS tolerized macrophages. Total of 20 genes including Eed (log2FC < -0.75, FDR < 0.05) exhibited decreased transcripts in Eed KO LPS tolerized macrophages, including Ctsk and Ctsl encoding cathepsins, as listed in Supplementary Table 8. Among these genes, only one gene, Myadm, was categorized as tolerizable gene. However, the pro-inflammatory genes, Tnf, Il6, and Il1b were found to be stable in our RNA-Seq data (Supplementary Fig S8). This result suggested that the mRNA profiles provide a momentary snapshot of gene expression and may not directly reflect the final protein products, particularly secreted cytokines.

Silencing Runx3 in E e d KO BMDMs rescued the dampened LPS tolerized gene expression

Because Runx3 was highly upregulated in the absence of EED, the silencing approach was taken to reduce the expression of Runx3 in BMDMs using the protocol as shown in Fig. 6A. shRNA specific to Runx3 successfully reduced the expression of Runx3 in both WT and Eed KO BMDMs (Fig. 6B). The effect of silencing Runx3 was evaluated in LPS tolerance. As shown in Fig. 6C-E, pro-inflammatory cytokine genes were increased in WT BMDMs when Runx3 was silenced in the LPS tolerance setting. More importantly, in Eed KO BMDMs, the dampened expression of Il6 and Il1b was rescued by silencing Runx3. As shown above, Eed KO BMDMs failed to upregulate Hif1a during LPS tolerance. We thus hypothesized that Runx3 and HIF-1a may cross-regulate and play a role in hypo-responsiveness of Eed KO BMDMs under tolerance. AS shown in Fig. 6F, Runx3 silencing in Eed KO restored Hif1a expression under LPS tolerance. In addition, since the TGF-β signaling hallmark was positively enriched in the LPS tolerized Eed KO BMDMs, we tested whether Runx3 mediated the induction of TGF-β signaling cascades. Upon Runx3 silencing in Eed BMDMs, the significant decrease in Tgfb expression was detected, comparing to its mock control, suggesting that RUNX3 and TGF-β may cooperate in regulating LPS tolerance (Fig. 6G).

Fig. 6
figure 6

Runx3 knock down in BMDMs and its effect in LPS tolerized BMDMs. (A). Schematic methodology for sh-RNA transfection and LPS tolerance. mRNA expression level of (B). Runx3, (C). Tnf, (D). Il6, (E). Il1b, (F). Hif1aand (G) Tgfb in Runx3 knock down BMDMs from Eed KO and WT under LPS tolerance was measured by qRT-PCR. (H). Schematic methodology for LPS treatment and ChIP assay. (I). ChIP-qPCR of EED occupancy at Runx3, Tnf-α, and Il-6 promoters from Eed KO and WT tolerant macrophages. The promoter enrichment quantification was normalized to a 5% input. The assay was conducted in 3 biological replicates. *, **, ***, ****; indicate p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively, using unpaired t-test or Two-Way ANOVA according to the assay

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To test whether EED directly interacts with the promoter of Runx3, ChIP-qPCR was performed and as shown in Fig. 6H, significant reduction in the enrichment of EED was detected in the Runx3 promoter when EED was deleted, while the enrichment levels in the promoter of Tnf and Il6 were not different (Fig. 6I). These results strongly implied that EED directly interacts with the regulatory regions of Runx3 gene in LPS tolerized macrophages, possibly depositing the H3K27me3 silencing marks and suppressing its expression through epigenetic mechanism, which in turn regulates LPS tolerance.

Changes in the H3K27me3 profiles were observed in LPS tolerized E e d KO BMDMs

To obtain a global view of H3K27me3 profiles in LPS tolerized macrophages in the absence of EED, CUT&Tag and sequencing was carried out. As shown in Fig. 7A, three clusters of H3K27me3 profiles were identified. Cluster 1 represented the class of loci in which the level of H3K27me3 was most affected by Eed KO. The enrichments were drastically reduced in LPS tolerized Eed KO BMDMs. Cluster 2 showed a class of loci where the intermediate effect of decreasing H3K27me3 was observed in the Eed KO BMDMs. Cluster 3 represented elements with no difference in the H3K27me3 levels with or without EED. The up-regulated genes from RNA-seq data which showed overlapping profiles with genes in cluster I were evaluated and presented in a heatmap (Fig. 7B). These genes are potential direct targets of PRC2/EED-mediated gene silencing via H3K27me3 deposition, including Runx3. From the RNA seq data, we identified 20 downregulated genes in Eed KO LPS tolerized macrophages and 16 of which were listed in cluster 3. Furthermore, Il6 and Il1b were also identified in cluster 3. The results imply that EED/PRC2 may indirectly regulate genes that were downregulated during LPS tolerance.

Fig. 7
figure 7

Profiling of H3K27me3 of LPS tolerized Eed KO and WT BMDMs. (A) Heatmap showing the genomic regions with binding profiles of H3K27me3 in Eed KO and its littermate WT macrophages at 10 kb flanking the transcriptional start site of genes under LPS tolerance. (B) Heatmap showing up-regulated genes overlapped with gene under cluster I. (C) Genome tracks showing H3K27me3 enrichment at the promotor regions of representative of up- regulated genes among Eed KO and littermate WT BMDM under LPS tolerized condition. (D) Enriched motifs with sequences in cluster I genes that uniquely overlap exons (motif score > 5, E-value < 10 using the binomial test). Logos were drawn using the top 4-8nucleotides from the top enriched motif. (E) Enrichment of H3K27me3 at the Ets1 and Gabpa promotor, genes in ETS family. The experiment was conducted in 2 biological replicates. (F) Heatmap showing expression profiles of ETS family from RNA-Seq dataset. The assay was conducted in 3 biological replicates. The statistical analysis was conducted using unpaired t-test

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The enrichment of H3K27me3 at the regulatory regions of some of the up-regulated genes identified from the RNA-seq data set were depicted in the genome track as shown in Fig. 7C. We observed the decreased enrichment of the H3K27me3 around the regulatory regions of Runx3, Tnc, Mmp2, and Serpine2, indicating potential direct regulatory role of PRC2/EED against these genes. In contrast, EED/PRC2 may also play an indirect role in transcription regulation of other genes because many up-regulated genes from our RNA-seq data set showed no alteration in the H3K27me3 levels at their promotor loci. Thus, it is possible that EED/PCR2 might epigenetically regulate the expression of other transcription factors that affect LPS tolerance, as exemplified by Runx3.

To identify the transcription factor binding motifs that are enriched in the gene promoters in the absence of EED in LPS tolerized macrophages, motif finding on the cluster I loci was performed by MEME-ChIP. Ets transcription factors family binding motif was found as the top motif among the cluster I genes, suggesting their involvements in LPS tolerance (Fig. 7D). H3K27me3 enrichment in some members of Ets transcription factors family gene, Ets1 and Gabpa, was found to be reduced (Fig. 7E) and the upregulation of ETS family genes were also observed in our RNA-seq data, implying that increased ETS family proteins may bind to their target genes and regulates their expression (Fig. 7F). However, this analysis was based on the bioinformatic predictions, and additional experiments are required to evaluate the impact of the ETS family on LPS re-stimulation.

Discussion

Polycomb-group proteins negatively regulate gene expression via post translational modification of histones where PRC1 and PRC2 function through H2AK119 ubiquitination and H3K27 methylation, respectively. Previous studies reported that the PRC2 complex via EZH2 subunit represses the expression of anti-inflammatory protein SOCS3 and reduces inflammatory response in macrophages [20, 53]. This finding is in line with the report that Ezh2 deficiency in myeloid lineage cells reduces atherosclerosis development [54]. Because our previous study on an unbiased screening of epigenetic modifying enzyme inhibitors demonstrated that PRC2 inhibitor altered innate immune memory in macrophages (LPS tolerance and trained immunity), the current study aimed at addressing how PRC2 regulates such memory responses in the form of endotoxin tolerance [24].

Deletion of either Eed or Ezh2 in macrophages showed a similar global reduction of H3K27me3, whereas increased H3K27ac, an active enhancer mark, was observed. This result correlated with the findings that indicated a direct competition between PRC2 and HAT p300/CBP in the modification of H3K27 [55]. Several HAT enzymes encoding genes were upregulated in Eed KO macrophages, including Ep300 and Crebbp. H3K27ac is found to be dynamically changed by LPS tolerance. H3K27ac marks were gained in a subset of more than 500 enhancer sites and lost at a subset of more than 600 sites [56, 57]. Changes in repressive and active histone marks may coordinately affect gene expression during LPS tolerance response in macrophages.

Compared to Ezh2 KO cells, Eed KO cells showed more robust changes in the level of histone modifications and other responses to LPS stimulation/tolerance. The difference in the phenotypes observed between Ezh2 and Eed KO macrophages may be because of the functional redundancy between EZH1 and EZH2 in the context of PRC2 [58]. In fact, the deletion of Ezh2 did not affect the mRNA level of Ezh1. We could not rule out the possibility that EED may function in regulating LPS tolerance independent of its role in the context of PRC2. EED is known as a epigenetic reader that is associated with gene repression but it was reported to function in cooperation with BRD4 to activate gene expression as well [59].

During LPS tolerance, not all genes are equally repressed, and it is now recognized that the transcription of a subset of genes are induced during tolerized response. These genes have functions in anti-microbial effector activities and are postulated to be beneficial for the host during systemic infection [9, 56]. In the absence of EED, LPS tolerized macrophages showed further depressed pro-inflammatory cytokine production while increased the expression of non-tolerizable genes. LPS tolerance is accompanied by increased levels of negative-feedback regulators of TLR4 signaling, including A20 and IRAK-M [56]. In the absence of EED, the levels of these regulators did not robustly change, indicating that other mechanisms downstream of TLR4 signaling may be responsible for the altered LPS tolerance in Eed KO macrophages.

Based on the RNA-seq and GSEA analysis, the TGF-β signaling, Wnt/β-catenin signaling and hypoxia hall marks were enriched in Eed KO LPS tolerized macrophages. Endotoxin tolerance is characterized by down-regulation of pro-inflammatory cytokines but up-regulation of anti-inflammatory cytokines such as IL-10 and TGF-β [60]. TGF-β has been shown to play a key role in suppressing inflammation. TGF-β /SMAD4 axis is crucial for maximal endotoxin tolerance which is mediated via increasing the expression of phosphatase SHIP1 [61]. Moreover, restimulation of LPS to human peripheral blood mononuclear cells (PBMCs) resulted in hyporesponsive phenotype associated with an induction of transforming growth factor beta-induced (TGFBI) expression when compared to healthy unstimulated control [62].

Regarding upregulated genes we identified, cyclin-dependent kinase inhibitor 2 A encoding gene, Cdkn2a was upregulated about 3.22 folds (log2FC = 1.69). This gene is associated with cellular senescence and known to be regulated by PRC2 [63]. The senescent macrophages increased Cdkn2a (p16INK4a) transcripts in DNA repair deficient mice and downregulated glycolysis, which leads to an energy-depleted state that weaken the macrophage function [64, 65]. It has reported that chronic inflammation facilitates senescence which lead to impair immune function [66]. This phenomenon is consistent with our finding in which Eed KO LPS tolerant macrophages expressed higher level of Cdkn2a and lower glycolytic activity followed which culminated in hypo-responsiveness.

Metabolic reprogramming emerges as one of the key regulators of immune cell responses to stimuli. While glycolysis is necessary for pro-inflammatory response during acute LPS priming, LPS tolerized macrophages failed to undergo glycolytic switch and are unable to actively produce pro-inflammatory cytokines [67]. In cancer cells, PRC2/EZH2 facilitates glucose metabolisms by directly influencing the expression of genes in the glycolysis pathway and fuels tumor growth [68]. Signaling via TLR4 is known to regulate the metabolic reprograming and the use of glycolysis in polarization of M1 macrophages. Although no drastic changes in TLR4 signaling pathways (MAPK and NF-kB) in Eed KO macrophages, other signaling pathways such as PI3K/AKT may be affected or PRC2/EED may directly regulate genes in the metabolic pathways as seen in the cancer cells [68, 69]. In addition, the HIF1-a is also shown to induce metabolic reprogramming in inflammatory macrophages [70]. Recent evidence also supports that hypoxic condition induces transcriptional reprograming toward inflammatory macrophages in HIF-1a and NF-kB [71]. Our results indicated that EED directly controls the cellular metabolic shift during LPS tolerance and in the absence of EED, cells failed to use glycolytic pathway for energy needs which resulted in further dampening of LPS tolerance.

The enrichment of H3K27me3 was significantly reduced in regulatory elements of a group of genes in Eed KO macrophages. Indeed, upregulated genes from the RNA-seq showed correlation with decreased H3K27me3 enrichment, indicating a direct regulatory role of EED/PRC2 as repressor of these genes. To further investigate how deletion of EED affects gene expression under LPS tolerance, the binding motif of ETS family transcription factors were found to be enriched among genes decreased in H3K27me3 occupancy (cluster I). Previous report has also shown that ETS family members are most associated with endotoxin tolerance such as ETS variant4 (ETV4), GA binding protein transcription factor a subunit (GABPA), and Sp1 transcription factor (SP1) [72].

Among upregulated genes in the RNA-seq data set, we focused our attention on Runx3. RUNX3 belongs to the runt domain-containing family of transcription factors that either activate or suppress transcription of the target genes which is well characterized in T cells and cancer cells [52, 73]. However, the roles in innate immune cells are only limitedly documented. One report showed that RUNX3 promotes the differentiation of anti-inflammatory mononuclear phagocytes and prevents spontaneous colitis [74]. In addition, it was shown that TGF-β promotes tolerance in human monocytes through the regulation of Runx3 which mediates the up-regulation of miR-146b [75]. Runx3 is also directly induced by TGF-β signaling [76]. It has been demonstrated that RUNX3 directly interacts with SMAD1, SMAD2, SMAD3, and SMAD5 through the MH2 domain, functioning as a transcriptional activator [77]. Interestingly, it was reported that suppressing TGF-β signaling or silencing Smad2 can recover LPS tolerance, indicating a link between TGF-β/RUNX3 and LPS tolerance [78]. Our ChIP-qPCR assay together with our CUT&Tag data revealed that EED is associated with Runx3 promoter and deletion of Eed increased Runx3 mRNA in unstimulated and LPS tolerized macrophages. EED through PRC2 may suppress Runx3 transcription by depositing repressive histone marks. We further provided evidence that silencing Runx3 during LPS tolerance significantly enhances proinflammatory response.

We thus proposed a model in which EED/PRC2 directly and indirectly regulates expression of tolerizable and non-tolerizable genes during LPS tolerance (Fig. 8). For a subset of non-tolerizable genes, EED/PRC2 may act directly to suppress the transcription by depositing the repressive epigenetic marks and in the absence of EED, H3K27me3 disappears from the cis-regulatory elements and the mRNA level increases in LPS tolerance. For tolerizable genes, in which expression is repressed upon successive LPS stimulation, EED/PRC2 may indirectly affect the expression of the subsets of these genes. One of the mechanisms may be via regulation of TGF-β/Runx3 axis. EED deficiency results in expression of Runx3 that cooperatively functions to dampen inflammatory response with TGF-β.

Fig. 8
figure 8

A proposed model of how EED may regulate LPS tolerance in macrophages. See text for details. Created with BioRender.com

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Conclusions

We provided strong evidence linking the PRC2 component, EED, with LPS tolerance via epigenetic regulation. PRC2 via EED directly and indirectly targets genes that regulate tolerizable and non-tolerizable genes during LPS tolerance. Lacking EED affects metabolic switch, the transcription factor RUNX3 and TGF-β signaling hallmarks in LPS tolerized macrophages. Targeting these components may be useful for designing treatment of immune paralysis in sepsis patients.

Data availability

The RNA-Seq and CUT&Tag-Seq datasets generated or analyzed for this study were deposited in a public database and can be found under the GEO accession number GSE234655 and GSE249512, respectively.

Abbreviations

PRC2:

Polycomb repressive complex 2

H3K27me3:

Histone H3 lysine 27 trimethylation

H3K27ac:

Histone H3 lysine 27 acetylation

H2AK119:

Histone 2 A lysine119

LPS:

Lipopolysaccharide

EED:

Embryonic ectoderm development

EZH1/EZH2:

EZH1/EZH2 Enhancer of Zeste Homolog 1 and 2

PCR1:

Polycomb repressive complex 1

BMDMs:

Bone marrow-derived macrophages

WT:

Wild-type

KO:

Knock-out

TNF-α:

Tumor necrosis factor

IL-6:

Interleukin-6

il-1ß:

Interleukin-1 beta

il-10:

Interleukin-10

TGF-β:

Transforming growth factor beta

Runx3:

Runt-related transcription factor 3

JAK/STAT:

Janus kinase/signal transducers and activators of transcription

TLR4:

Toll like receptor 4

NF-κB:

The nuclear factor kappa B

MAPK:

Mitogen-activated protein kinase

SAPK:

Stress-activated protein kinases

JNK:

Jun amino-terminal kinases

ERK:

Extracellular-signal-regulated kinases

ECAR:

Extracellular acidification rate

Hif-1α:

Hypoxia-inducible factor-1α

GSEA:

Gene Set Enrichment Analysis

HMT:

Histone methyltransferases

HDM:

Histone demethylases

HAT:

Histone acetyltransferase

HDAC:

Histone deacetylase

CUT&Tag:

Cleavage Under Targets and Tagmentation

ChIP:

Chromatin immunoprecipitation

ELISA:

Enzyme-Linked Immunosorbent Assay

DMEM:

Dulbecco’s modified Eagle’s medium

PBMCs:

Peripheral blood mononuclear cells

TGFBI:

Transforming growth factor beta-induced

SOCS3:

Suppressor of cytokine signaling 3

BRD4:

Bromodomain-containing protein 4

IRAK-M:

Interleukin-1 receptor associated kinase

SMAD4:

Mothers against decapentaplegic homolog 4

ETS:

Erythroblast Transformation Specific

TMB:

Tetramethylbenzidine

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Acknowledgements

The authors thank Dr. Nayuta Yakushiji-Kaminatsui at RIKEN IMS for technical assistance on CUT&Tag analysis.

Funding

This work was supported in part by and by the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (B16F640117), Thailand Science Research and Innovation (TSRI) Fund B05F640209 and Thailand Science Research and Innovation Fund Chulalongkorn University (FF67) to T.P. A.B. is supported by the Second Century Fund (C2F) postdoctoral fellowship program, Chulalongkorn University. S.B. is supported by the 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship, Chulalongkorn University.

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  1. Atsadang Boonmee

    Present address: Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand

Authors and Affiliations

  1. Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand

    Atsadang Boonmee, Benjawan Wongprom, Thitiporn Pattarakankul, Kittitach Sri-Ngern-Ngam & Tanapat Palaga

  2. Center of Excellence in Immunology and Immune-mediated Diseases, Chulalongkorn University, Bangkok, 10330, Thailand

    Atsadang Boonmee, Salisa Benjaskulluecha, Patipark Kueanjinda, Benjawan Wongprom, Kittitach Sri-Ngern-Ngam & Tanapat Palaga

  3. Inter-disciplinary Graduate Program in Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok, 10330, Thailand

    Salisa Benjaskulluecha

  4. Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, 10330, Thailand

    Patipark Kueanjinda

  5. Center of Excellence in Materials and Bio-Interfaces, Chulalongkorn University, Bangkok, 10330, Thailand

    Thitiporn Pattarakankul

  6. Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Science, Chulalongkorn University, Bangkok, 10330, Thailand

    Supawadee Umthong

  7. Laboratory for Developmental Genetics, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, 230-0045, Japan

    Junichiro Takano & Haruhiko Koseki

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Contributions

T.P. and A.B. designed and conceptualized the overall experiments. A. B., S. B., P. K., B. W., J. T., K.S., S. U. and T. P. conducted the experiments and analyzed the data. T. P. and H.K. supervised all research and acquired funding. A.B. and T.P. prepared the manuscript for submission. All authors have revised the manuscript.

Corresponding author

Correspondence to Tanapat Palaga.

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Ethics approval

No human samples or subjects were used in this study. 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) and Chulalongkorn University Laboratory Animal Center (CULAC, approval protocol 2073015. All experiments were performed according to the guidelines issued by the IACUC. This study is reported in accordance with the ARRIVE guidelines.

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Additional file 1: Tables 1, 2, 3, 4, 5, 6, 7, 8 and 9: RNA-Seq data, lists of genes, and list of primers

13072_2024_562_MOESM2_ESM.xlsx

Additional file 2: Supplementary Figure S1: Global repressive and active histone profiles of Eed KO and Ezh2 KO macrophages compared to their littermate WT. Supplementary Figure S2: BMDMs from Eed and Ezh2 KO mice. Supplementary Figure S3: EED226-treated macrophage phenotypes. Supplementary Figure S4: Transcript levels of Tnf and Il6 measured in Ezh2 KO macrophages. Supplementary Figure S5: Downstream TLR4 signaling pathway activity in LPS tolerized BMDMs (WT and Eed KO). Supplementary Figure S6: Transcriptomics profiles of unstimulated Eed KO and WT macrophages. Supplementary Figure S7: Transcriptomics profiles of LPS-primed Eed KO and WT macrophages. Supplementary Figure S8: Log2 TPM (Transcripts Per Million) values of pro-inflammatory cytokine genes derived from RNA-Seq data

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Boonmee, A., Benjaskulluecha, S., Kueanjinda, P. et al. A polycomb group protein EED epigenetically regulates responses in lipopolysaccharide tolerized macrophages. Epigenetics & Chromatin 17, 36 (2024). https://doi.org/10.1186/s13072-024-00562-6

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Keywords

Epigenetics & Chromatin

ISSN: 1756-8935