Ezh2 Shapes T Cell Plasticity to Drive Atherosclerosis.

Abstract

The activation and polarization of T cells play a crucial role in atherosclerosis and dictate athero-inflammation. The epigenetic enzyme EZH2 (enhancer of zeste homolog 2) mediates the H3K27me3 (trimethylation of histone H3 lysine 27) 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 <i>Apoe</i> (apolipoprotein E)-deficient mice with CD (cluster of differentiation) 4⁺ or CD8⁺ T cell-specific <i>Ezh2</i> deletion (Ezh2^cd4-knockout [KO], Ezh2^cd8-KO) were analyzed to unravel the role of T cell Ezh2 in atherosclerosis and T cell-associated immune status. <i>EZH2</i> 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. Ezh2^cd4-KO, but not Ezh2^cd8-KO, mice showed reduced atherosclerosis with fewer 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 Ezh2^cd4-KO mice revealed that absence of Ezh2 results in a type 2 immune response with increased Il-4 (interleukin 4) gene and protein expression in the aorta and lymphoid organs. In vitro, <i>Ezh2</i>-deficient T cells polarized macrophages toward an anti-inflammatory phenotype. Single-cell RNA-sequencing of splenic T cells revealed that <i>Ezh2</i> deficiency reduced naive, Ccl5⁺ (C-C motif chemokine ligand 5) 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 toward Th2 (type 2 T helper) effector CD4⁺ T cells in Ezh2^cd4-KO mice and confirmed a profound increase in splenic iNKT cells with increased expression of Plzf (promyelocytic leukemia zinc finger), which is the characteristic marker of the iNKT2 subset. Likewise, <i>Zbtb16</i> ([zinc finger and BTB domain containing 16], the Plzf-encoding gene) transcripts were elevated in the aorta of Ezh2^cd4-KO mice, suggesting an accumulation of iNKT2 cells in the plaque. H3K27me3-chromatin immunoprecipitation followed by quantitative polymerase chain reaction showed that T cell-Ezh2 regulates the transcription of the <i>Il-4</i> and <i>Zbtb16</i> genes. Our study uncovers the importance of T cell EZH2 in human and mouse atherosclerosis. Inhibition of Ezh2 in CD4⁺ T cells drives type 2 immune responses, resulting in an accumulation of iNKT2 and Th2 cells, memory T cells and anti-inflammatory macrophages that limit the progression of atherosclerosis.