Myeloid Ezh2 Deficiency Limits Atherosclerosis Development

Abstract

Macrophages define a key component of immune cells present in atherosclerotic lesions and are central regulators of the disease. Since epigenetic processes are important in controlling macrophage function, interfering with epigenetic pathways in macrophages might be a novel approach to combat atherosclerosis. Histone H3K27 trimethylation is a repressive histone mark catalyzed by polycomb repressive complex with EZH2 as the catalytic subunit. EZH2 is described to increase macrophage inflammatory responses by supressing the suppressor of cytokine signaling, Socs3. We previously showed that myeloid deletion of Kdm6b, an enzymes that in contrast to EZH2 removes repressive histone H3K27me3 marks, results in advanced atherosclerosis. Because of its opposing function and importance of EZH2 in macrophage inflammatory responses, we here studied the role of myeloid EZH2 in atherosclerosis. A myeloid-specific Ezh2 deficient mouse strain (Ezh2^del) was generated (LysM-cre+ x Ezh2^fl/fl) and bone marrow from Ezh2^del or Ezh2^wt mice was transplanted to Ldlr^-/- mice which were fed a high fat diet for 9 weeks to study atherosclerosis. Atherosclerotic lesion size was significantly decreased in Ezh2^del transplanted mice compared to control. The percentage of macrophages in the atherosclerotic lesion was similar, however neutrophil numbers were lower in Ezh2^del transplanted mice. Correspondingly, the migratory capacity of neutrophils was decreased in Ezh2^del mice. Moreover, peritoneal Ezh2^del foam cells showed a reduction in the inflammatory response with reduced production of nitric oxide, IL-6 and IL-12. In Conclusion, myeloid Ezh2 deficiency impairs neutrophil migration and reduces macrophage foam cell inflammatory responses, both contributing to reduced atherosclerosis.

Keywords: H3K27; PRC2; atherosclerosis; epigenetic; histone modification; macrophage; polycomb.

Figure 1

Bone marrow of Ezh2^wt and Ezh2^del mice was effectively transplanted to Ldlr^-/- mice. (A) Relative normalized Ezh2 and Ezh1 mRNA expression in Ezh2^wt and Ezh2^del BMDMs (B) EZH2 protein expression on whole cell lysates (left) and H3K27Me3 levels on histone extracts (right) of BMDMs. α-tubulin and H3 were used as loading control (C) H3K27 methyltransferase activity in nuclear lysates of BMDMs. (D) Chimerism determination by qPCR for the Ldlr in the DNA of blood of Ezh2^wt (black bars) and Ezh2^del (white bars) transplanted mice. (E) Mouse weight in grams at the start of the diet (week 0) and after 8 weeks of HFD (week 8). (F) Cholesterol and (G) triglyceride levels in the plasma at the start and after 8 weeks of HFD. Percentage of (H) blood and (I) spleen leukocyte subsets assessed by flow cytometry. B cells (CD45⁺, CD19⁺), T cells (CD45⁺, CD3⁺), Neutrophils (CD45⁺, CD11b⁺, Ly6G⁺) and monocytes (CD45⁺, CD11b⁺, Ly6G^-). N=15 each group. Data represent mean ± SEM.

Figure 2

Atherosclerotic lesions size is reduced in Ezh2^del transplanted mice. (A) Representative Toluidin Blue staining of the aortic root of Ezh2^wt and Ezh2^del transplanted mice. (B) Aortic lesion area presented as the sum of the three valves per mice. (C) Representative Sirius Red staining to measure collagen content. (D) Collagen content as percentage of total lesion area. (E) Representative MOMA-2 staining for macrophages. (F) Macrophage area as percentage of total lesion area. (G) Necrotic core area indicated in red on the toluidine blue staining. (H) Necrotic core as percentage of total lesion area. (I) Ly6G (1A8) neutrophil staining in red and nuclei in blue (DAPI). (J) Neutrophil counts, corrected for total lesion area. Data represent mean ± SEM of 15 mice. *P < 0.05.

Figure 3

EZH2 regulates neutrophil migration. (A) Neutrophils were isolated from bone marrow of Ezh2^wt or Ezh2^del mice. Neutrophils were fluorescently labeled and chemotaxis was measured in response to supernatants of unstimulated or LPS activated Ezh2^wt or Ezh2^del BMDMs. (B) Neutrophil migration as the increase of RFU in time in relative fluorescence units (delta RFU). Presented is one out of three experiment of 2 pooled Ezh2^wt and 2 pooled Ezh2^del mice plated in duplo. Left is Ezh2^wt neutrophil migration toward supernatant of Ezh2^wt or Ezh2^del BMDMs and right is Ezh2^wt or Ezh2^del neutrophil migration toward wildtype supernantants. (C) Cxcl1 and Cxcl2 mRNA expression in the aortic arch of Ezh2^wt or Ezh2^del transplanted mice. Statistics are performed on duplicates with a two-way anova with bonferroni correction. Data represent mean ± SEM. *P < 0.05. Panel (A) was made with use of smart servier medical art, lincesed under a Creative Common Attribution 3.0 Unported License. https://smart.servier.com/.

Figure 4

EZH2 affects inflammatory responses in peritoneal foam cells. (A) Cytokine production by Ezh2^wt (black bars) and Ezh2^del peritoneal foam cells after stimulation for 24 h with LPS or LPS+IFNγ. (B) mRNA expression in Ezh2^wt and Ezh2^del peritoneal foam cells after 24 h of IL-4 stimulation. Foam cells of five individual mice per group were pooled and plated in triplicate. Data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.