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. 2023 Mar 10;24(6):5363.
doi: 10.3390/ijms24065363.

The Regulatory Roles of Ezh2 in Response to Lipopolysaccharide (LPS) in Macrophages and Mice with Conditional Ezh2 Deletion with LysM-Cre System

Areerat Kunanopparat  1   2 , Asada Leelahavanichkul  2   3   4 , Peerapat Visitchanakun  2 , Patipark Kueanjinda  2 , Pornpimol Phuengmaung  2 , Kritsanawan Sae-Khow  2 , Atsadang Boonmee  1   5 , Salisa Benjaskulluecha  1   5 , Tanapat Palaga  1   5 , Nattiya Hirankarn  1   2
Affiliations

The Regulatory Roles of Ezh2 in Response to Lipopolysaccharide (LPS) in Macrophages and Mice with Conditional Ezh2 Deletion with LysM-Cre System

Areerat Kunanopparat et al. Int J Mol Sci. .

Abstract

The responses of macrophages to lipopolysaccharide (LPS) might determine the direction of clinical manifestations of sepsis, which is the immune response against severe infection. Meanwhile, the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, might interfere with LPS response. Transcriptomic analysis on LPS-activated wild-type macrophages demonstrated an alteration of several epigenetic enzymes. Although the Ezh2-silencing macrophages (RAW264.7), using small interfering RNA (siRNA), indicated a non-different response to the control cells after a single LPS stimulation, the Ezh2-reducing cells demonstrated a less severe LPS tolerance, after two LPS stimulations, as determined by the higher supernatant TNF-α. With a single LPS stimulation, Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) macrophages demonstrated lower supernatant TNF-α than Ezh2 control (Ezh2fl/fl; LysM-Cre-/-), perhaps due to an upregulation of Socs3, which is a suppressor of cytokine signaling 3, due to the loss of the Ezh2 gene. In LPS tolerance, Ezh2 null macrophages indicated higher supernatant TNF-α and IL-6 than the control, supporting an impact of the loss of the Ezh2 inhibitory gene. In parallel, Ezh2 null mice demonstrated lower serum TNF-α and IL-6 than the control mice after an LPS injection, indicating a less severe LPS-induced hyper-inflammation in Ezh2 null mice. On the other hand, there were similar serum cytokines after LPS tolerance and the non-reduction of serum cytokines after the second dose of LPS, indicating less severe LPS tolerance in Ezh2 null mice compared with control mice. In conclusion, an absence of Ezh2 in macrophages resulted in less severe LPS-induced inflammation, as indicated by low serum cytokines, with less severe LPS tolerance, as demonstrated by higher cytokine production, partly through the upregulated Socs3.

Keywords: Ezh2; epigenetics; lipopolysaccharide; macrophages; sepsis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The transcriptome profiles and the log2 of the transcript count per million (TPM) of genes in bone-marrow-derived macrophages from wild-type mice 24 h after incubation by lipopolysaccharide (LPS) or media control (untreated) as indicated by the heatmap (A) and the bar plot showing some epigenetic-associated genes in several groups with statistically significant difference (or with a tendency of the difference), including (i) lysine deacetylase (eraser); Hdac1 (histone deacetylase 1), Sirt6 (sirtuin 6), Kdm3a (lysine demethylase 3A), Kdm6b (lysine demethylase 6b) (B), (ii) lysine methyltransferase (writer); Ezh1 (histone-lysine N-methyltransferase-1), Ezh2 (histone-lysine N-methyltransferase-2), and Kmt5a (lysine methyltransferase 5A) (C), (iii) serine-threonine/tyrosine kinase (writer); Aurkb (aurora kinase B) and Aurkc (aurora kinase C) (D), and (iv) lysine ubiquitin ligase (writer); Rnf2 (ring finger protein 2) (E), are demonstrated. Macrophages were isolated from 3 different mice for the triplicate analysis. Mean ± SEM is presented with Student’s t-test analysis (*, p < 0.05).
Figure 2
Figure 2
The schema of the experiments in a murine macrophage cell line (RAW264.7) with the silencing of Ezh2 gene using small interfering RNA (Ezh2 siRNA) or the control siRNA (non-targeting pool siRNA; non-siRNA) and activated by lipopolysaccharide (LPS) in a single protocol (N/LPS) which began with the culture media followed by LPS 24 h later or LPS tolerance (LPS/LPS) by two LPS stimulations, or control (N/N) using the culture media incubation twice (A). The characteristics of these macrophages with different protocols as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10) (B–D), expression of pro-inflammatory genes of M1 polarization (iNOS and IL-1β) (E,F), and anti-inflammatory genes of M2 polarization (Fizz-1, Arg-1, and TGF-β) (G–I) are demonstrated. Triplicated independent experiments were performed. Mean ± SEM is presented with the one-way ANOVA followed by Tukey’s analysis (*, p < 0.05 vs. non-siRNA N/N and #, p < 0.05).
Figure 3
Figure 3
The schema of the experiments in bone-marrow-derived macrophages from Ezh2 control (Ezhfl/fl; LysM-Cre−/−) or Ezh2 null (Ezhfl/fl; LysM-Crecre/−) mice after activation by lipopolysaccharide (LPS) in a single protocol (N/LPS), which began with the culture media followed by LPS 24 h later, or LPS tolerance (LPS/LPS) through two LPS stimulations, or control (N/N) using the culture media incubation twice (A). The characteristics of these macrophages with different protocols as indicated by supernatant cytokines (TNF-α, IL-6, and IL-10) (B–D) are also demonstrated. Triplicate independent experiments were performed. Mean ± SEM is presented with the one-way ANOVA followed by Tukey’s analysis (*, p < 0.05 vs. WT N/N and #, p < 0.05).
Figure 4
Figure 4
Characteristics of bone-marrow-derived macrophages (BMDM) from Ezh2 control (Ezhfl/fl; LysM-Cre−/−) or Ezh2 null (Ezhfl/fl; LysM-Cre cre/−) mice 24 h after stimulation by lipopolysaccharide (LPS) tolerance (twice LPS stimulation; LPS/LPS) or a single LPS stimulation (N/LPS; started with phosphate buffer solution (PBS) followed by LPS) as indicated by the expression of several genes, including Ezh1, Ezh2, and Socs3 (A–C) and the energy status of cells (extracellular flux analysis) (D,E). Independent triplicate experiments were performed. Mean ± SEM is presented with the one-way ANOVA followed by Tukey’s analysis (*, p < 0.05 and #, p < 0.05 vs. untreated).
Figure 5
Figure 5
Schematic workflow (A) demonstrates the experimental groups, including lipopolysaccharide (LPS) tolerance; starting with LPS intraperitoneal (ip) injection (0.8 mg/kg) followed by LPS (4 mg/kg) (LPS tolerance; LPS/LPS), a single LPS stimulation; starting with phosphate buffer solution (PBS) followed by LPS (4 mg/kg) (a single LPS; N/LPS), in Ezh2 control (Ezhfl/fl; LysM-Cre−/−) or Ezh2 null (Ezhfl/fl; LysM-Crecre/−) mice as indicated by serum cytokines (TNF-α, IL-6, and IL-10) (B–D) (n = 7/group or time-point). Mean ± SEM is presented with the one-way ANOVA followed by Tukey’s analysis (*, p < 0.05 vs. others and #, p < 0.05).
Figure 6
Figure 6
The proposed working hypothesis demonstrates the impact of Ezh2 (Enhancer of zeste homolog 2) in response against lipopolysaccharide (LPS) of macrophages. In normal macrophages (left side), LPS recognition by Toll-like receptor 4 (TLR-4) initiates several downstream signals, including nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) [85] (number 1). The presence of Ezh2 causes the methylation on the 27th lysine of the histone (H3K27Me) resulting in the blockage of NF-κB and STAT3, which reduces cytokine production and suppressor of cytokine signaling 3 (Socs3; the inhibitor of NF-κB signaling [35,49]), respectively (number 2). The downregulated Sosc3 causes an increase in cytokines, highlighting the impact of the Ezh2 gene on the induction of LPS pro-inflammatory responses (number 3). Without Ezh2 (right side), both NF-κB and STAT3 effectively transcribe DNA into RNA of cytokines and Sosc3 (number 2); the upregulated Socs3 inhibits the synthesis of pro-inflammatory cytokines (TNF-α and IL-6), but not anti-inflammatory cytokines (IL-10) (number 3). More experiments are needed for a solid conclusion. Picture created by BioRender.com.
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