Ezh2 shapes T cell plasticity to drive atherosclerosis
The activation and polarization of T cells play a crucial role in atherosclerosis and dictate athero-inflammation. The epigenetic enzyme enhancer of zeste homolog 2 (EZH2) mediates the tri-methylation of lysine27 on histone3 (H3K27me3) and is pivotal in controlling T cell responses. To detail the role of T cell-EZH2 in atherosclerosis, we used human carotid endarterectomy specimens to reveal plaque expression and geography of EZH2. Atherosclerosis-prone Apoe-deficient mice with CD4+ and/or CD8+ T cell-specific Ezh2 deletion (Ezh2cd4-KO, Ezh2cd8-KO) were analyzed to unravel T cell-Ezh2's role in atherosclerosis and T cell-associated immune status.EZH2 expression is elevated in advanced human atherosclerotic plaques and primarily expressed in the T cell nucleus, suggesting the importance of canonical EZH2 function in atherosclerosis. Ezh2cd4-KO, but not Ezh2cd8-KO mice showed reduced atherosclerosis with less advanced plaques, which contained less collagen and macrophages, indicating that Ezh2 in CD4+ T cells drives atherosclerosis. In-depth analysis of CD4+ T cells of Ezh2cd4-KO mice revealed that absence of Ezh2 results in a type II immune response with increased Il-4 gene and protein expression in the aorta and lymphoid organs. In vitro, Ezh2-deficient T cells polarized macrophages towards an anti-inflammatory phenotype. scRNAseq of splenic T cells revealed that Ezh2-deficiency reduced naive, Ccl5+ and regulatory T cell populations and increased the frequencies of memory T cells and invariant natural killer T (iNKT) cells. Flow cytometric analysis identified a shift towards T helper 2 (Th2) effector CD4+ T cells in Ezh2cd4-KO mice and confirmed a profound increase in splenic iNKT cells with high expression of Plzf, the characteristic marker of the iNKT2 subset. Likewise, Zbtb16 (Plzf-encoding gene) transcripts were elevated in the aorta of Ezh2cd4-KO mice, suggesting an accumulation of iNKT2 cells in the plaque. H3K27me3-chromatin immunoprecipitation followed by qPCR showed that T cell-Ezh2 regulates the transcription of the Il-4 and Zbtb16 genes. Our study uncovers the importance of T cell-EZH2 in human and mouse atherosclerosis. Inhibition of Ezh2 in CD4+ T cells drives type II immune responses, resulting in an accumulation of iNKT2 and Th2 cells, memory T cells and anti-inflammatory macrophages that limit the progression of atherosclerosis. We performed scRNAseq of splenic T cells isolated from Ezh2cd4-KO and respective controls fed with Western type diet, to study T cells during atherosclerosis progression. Overall design: Following Western-type diet, spleens were isolated from Ezh2cd4-KO mice and controls after cervical dislocation. Living splenic CD45+CD3+ T cells (B220-Gr1-Ter119-) were sorted using a FACSAria III (BD Biosciences) and analyzed using scRNA-Seq.