EZH2 inhibition reduces macrophage inflammatory responses in atherosclerosis

Background and Aims: 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 repressor 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 EZH2-specific inhibitor GSK126 and subsequently activated with LPS to mimic TLR4-inflammatory responses. The impact of GSK126 on macrophage differentiation and activation compared to vehicle (DMSO) was assessed by RNA-seq, flow cytometry, western blot, ELISA, RT-qPCR, and ChIP-seq. Results: GSK126 treatment lowered global H3K27Me3 levels without altering macrophage viability and differentiation, showing effective EZH2 inhibition. RNA-seq revealed that more than one-third of the LPS-induced genes were significantly downregulated by GSK126 treatment. Subsequent pathway analysis identified cytokine and interferon signaling, co-stimulation, and cell migration as the top down-regulated pathways (padj<0.05). Indeed, we confirmed that gene expression and cytokine secretion 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. Conclusions: Overall, we show that EZH2 inhibition reduces inflammatory responses in human and murine macrophages. We are currently analyzing ChIP-seq data to identify direct targets of EZH2. Furthermore, we are performing ex vivo experiments on human endarterectomy plaques and an in vivo murine atherosclerosis study to assess the therapeutic potential of EZH2 inhibition on atherosclerosis development and progression.