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

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.

Less Severe Sepsis in Cecal Ligation and Puncture Models with and without Lipopolysaccharide in Mice with Conditional Ezh2-Deleted Macrophages (LysM-Cre System)

“…However, there was no difference in most of the sepsis severity biomarkers between Ezh2 null mice with LPS-CLP and those with CLP alone, except the higher histology score in LPS-CLP Ezh2 null mice (Figure 7C-L). The data implied no or less immune exhaustion after LPS tolerance in Ezh2 null mice, as mentioned in previous publications [39,58]. Between Ezh2 null versus Ezh2 control mice with CLP-induced sepsis hyperinflammation (CLP) and LPS tolerance with subsequent sepsis (LPS-CLP), sepsis severity was more severe in Ezh2 control mice as indicated by survival analysis, organ injury (kidney and liver), cell-free DNA, endotoxemia, bacteremia, and serum cytokines (TNF-α and IL-6, but not IL-10) (Figure 7C-L), supporting a beneficial impact of Ezh2 deletion in macrophages during sepsis in both conditions.…”

“…Indeed, the increased mortality in patients with high Ezh2 and H3K27 is mentioned [70,71] and inhibition of Ezh2 [72] might be beneficial. M1 macrophage polarization (IL-1β and iNOS) along with pro-inflammatory molecules (TNF-α, IL-6, and NF-κB) were less prominent in LPS-activated Ezh2 null macrophages than the control (Figure 6), possibly correlated with more profound suppressor of cytokine signaling 3 (Socs3; an anti-inflammatory molecule) as mentioned in our previous publication [39]. Perhaps Ezh2 more potently inhibits some molecules than others.…”

“…However, the expression of genes for proinflammatory molecule nuclear factor kappa B (NF-kB) was similarly higher than in the control group (N/N) (Figure 6J). On the other hand, there was a less prominent M1 polarization (pro-inflammatory macrophages), as indicated by IL-1β and iNOS, together with less pro-inflammatory responses (TNF-α, IL-6, and NF-κB) in Ezh2 null macrophages compared with control cells after a single LPS stimulation (N/LPS) (Figure 6C-J) supporting a pro-inflammatory effect of Ezh2 on macrophages [39]. In LPS tolerance (LPS/LPS), there was a non-difference in supernatant IL-1β, macrophage polarization, and cytokine genes between Ezh2 null macrophages and control cells, despite lower NF-κB expression in Ezh2 null macrophages (Figure 6B-J).…”

“…Ezh2 causes histone methylation that block transcription of both pro-inflammatory genes (cytokines) and anti-inflammatory genes (Sosc3) and the impact of Ezh2 might be different among various genes and cell types. Indeed, in mice with conditional Ezh2 deletion by the LysM-Cre system, Ezh2 deletion only in the myeloid cells (monocytes, macrophages, and neutrophils) demonstrates less severe responses against a single LPS injection, but not LPS tolerance (two LPS injections), and bone marrow-derived macrophages from these mice indicated less potent LPS responses (lower supernatant cytokines) and less severe LPS tolerance (higher supernatant cytokines) compared with the control cells [39]. Although Ezh2 impacts on sepsis are still inconclusive, Ezh2 is one of the upregulated genes in LPS-tolerant macrophages [52] which is improved by the Ezh2 inhibitor (enhanced TNF-α expression) [53] as an interesting control of macrophage through epigenetic manipulation [13,54].…”

“…Interestingly, the tolerance against LPS, especially in monocytes or macrophages, is possibly due to the epigenetic modifications, chromatin remodeling, and interferences in cell energy status [34][35][36], among which epigenetic alteration is the most extensively studied [37,38]. Epigenetics is the phenotypic alterations without the changes in the DNA sequence for the switch "on" and "off" of DNA transcription through (i) the modifications of DNA and/ or histone through several enzymes [3,39], and (ii) noncoding RNA (microRNA) [2]. Among all, the methylation at histone 3 lysine 27 (H3K27) is one of the most common epigenetic processes through histone changes for several cell activities, including after LPS activation [40].…”