Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation (Hartstichting) Aim Epigenetic processes are essential modulators of macrophage inflammatory responses. We postulate that interference in the epigenetic machinery of macrophages might offer novel approaches to combat atherosclerosis. Here, we investigate the repressive histone modification H3K27Me3 deposited by the polycomb repressive complex 2 (PRC2) with its catalytic component EZH2. We studied the therapeutic potential of macrophage EZH2 inhibition in the context of atherosclerosis. Methods Human monocyte-derived macrophages and murine peritoneal and bone-marrow derived macrophages were treated with the EZH2-specific inhibitor GSK126 and subsequently activated with LPS to mimic TLR4-inflammatory responses. The impact of Ezh2 inhibition on macrophage differentiation and activation compared to vehicle (DMSO) was assessed by RNA-seq, ChIP-seq, flow cytometry, western blot, and ELISA. To study its impact on atherosclerosis, female Ldlr-/- mice on a high-cholesterol diet (9weeks) were treated the last four weeks with GSK126 or control. Results Ezh2 inhibition by GSK126 in vitro lowered global H3K27Me3 levels without altering macrophage viability and differentiation, showcasing effective EZH2 inhibition. RNA-seq revealed that of more than one-third of the LPS-induced genes were significantly downregulated by EZH2 inhibition. Subsequent pathway analysis identified cytokine and interferon signaling, co-stimulation, and cell migration as the top down-regulated pathways (padj<0.05). Furthermore, ChIP-seq revealed considerably altered regulation of H3K27Me3 deposition. Indeed, we confirmed that gene and cytokine expression of the inflammatory mediators IL-6, IL-12, and TNF were reduced. Furthermore, membrane marker expression of co-stimulatory CD40, CD80, and CD86 were significantly decreased. In murine atherosclerosis, we observed a reduction in the Virmani plaque severity score and are currently assessing lesion size and composition. Conclusion Overall, we show that EZH2 inhibition reduces inflammatory responses in human and murine macrophages in vitro. We are currently assessing the impact of EZH2 inhibition on lesion size and composition in murine atherosclerosis. Additionally, we are performing ex vivo experiments on human endarterectomy plaques to assess the therapeutic potential of EZH2 inhibition on human atherosclerosis.

中文翻译：

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EZH2 抑制可减少动脉粥样硬化中的巨噬细胞炎症反应


资助致谢 资助来源类型：基金会。主要资金来源：荷兰心脏基金会 (Hartstichting) 目标 表观遗传过程是巨噬细胞炎症反应的重要调节剂。我们假设，干扰巨噬细胞的表观遗传机制可能会提供对抗动脉粥样硬化的新方法。在这里，我们研究了多梳抑制复合物 2 (PRC2) 及其催化组分 EZH2 沉积的抑制性组蛋白修饰 H3K27Me3。我们研究了巨噬细胞 EZH2 抑制在动脉粥样硬化背景下的治疗潜力。方法用 EZH2 特异性抑制剂 GSK126 处理人单核细胞来源的巨噬细胞以及小鼠腹膜和骨髓来源的巨噬细胞，然后用 LPS 激活以模拟 TLR4 炎症反应。通过 RNA-seq、ChIP-seq、流式细胞术、蛋白质印迹和 ELISA 评估与载体 (DMSO) 相比，Ezh2 抑制对巨噬细胞分化和激活的影响。为了研究其对动脉粥样硬化的影响，在最后 4 周用 GSK126 或对照组治疗高胆固醇饮食（9 周）的雌性 Ldlr-/- 小鼠。结果 GSK126 体外抑制 Ezh2 可降低整体 H3K27Me3 水平，而不改变巨噬细胞活力和分化，显示出有效的 EZH2 抑制作用。 RNA-seq 显示，超过三分之一的 LPS 诱导基因因 EZH2 抑制而显着下调。随后的通路分析确定细胞因子和干扰素信号传导、共刺激和细胞迁移是最下调的通路（padj<0.05）。此外，ChIP-seq 揭示了 H3K27Me3 沉积的调控发生了显着改变。 事实上，我们证实炎症介质 IL-6、IL-12 和 TNF 的基因和细胞因子表达降低。此外，共刺激CD40、CD80和CD86的膜标志物表达显着降低。在小鼠动脉粥样硬化中，我们观察到 Virmani 斑块严重程度评分降低，目前正在评估病变大小和成分。结论 总体而言，我们表明 EZH2 抑制可减少体外人和小鼠巨噬细胞的炎症反应。我们目前正在评估 EZH2 抑制对小鼠动脉粥样硬化病变大小和成分的影响。此外，我们正在对人动脉内膜切除术斑块进行离体实验，以评估 EZH2 抑制对人动脉​​粥样硬化的治疗潜力。