Mir-33 Silencing Reprograms The Immune Cell Landscape In Atherosclerotic Plaques

Rationale:MicroRNA-33 (miR-33 [post-transcriptionally in the rationale]) post-transcriptionally represses genes involved in lipid metabolism and energy homeostasis. Targeted inhibition of miR-33 increases plasma HDL (high-density lipoprotein) cholesterol and promotes atherosclerosis regression, in part, by enhancing reverse cholesterol transport and dampening plaque inflammation. However, how miR-33 reshapes the immune microenvironment of plaques remains poorly understood.Objective:To define how miR-33 inhibition alters the dynamic balance and transcriptional landscape of immune cells in atherosclerotic plaques.Methods and Results:We used single-cell RNA-sequencing of aortic CD45(+) cells, combined with immunohistologic, morphometric, and flow cytometric analyses to define the changes in plaque immune cell composition, gene expression, and function following miR-33 inhibition. We report that anti-miR-33 treatment of Ldlr(-/-) mice with advanced atherosclerosis reduced plaque burden and altered the plaque immune cell landscape by shifting the balance of proatherosclerotic and antiatherosclerotic macrophage and T-cell subsets. By quantifying the kinetic processes that determine plaque macrophage burden, we found that anti-miR-33 reduced levels of circulating monocytes and splenic myeloid progenitors, decreased macrophage proliferation and retention, and promoted macrophage attrition by apoptosis and efferocytotic clearance. Single-cell RNA-sequencing of aortic arch plaques showed that anti-miR-33 reduced the frequency of MHCIIhi (major histocompatibility complex II) inflammatory and Trem2(hi) (Triggering Receptor Expressed On Myeloid Cells 2) metabolic macrophages, but not tissue-resident macrophages. Furthermore, anti-miR-33 led to derepression of distinct miR-33 target genes in the different macrophage subsets: in resident and Trem2(hi) macrophages, anti-miR-33 relieved repression of miR-33 target genes involved in lipid metabolism (eg, Abca1, Ncoa1, Ncoa2, Crot), whereas in MHCIIhi macrophages, anti-miR-33 upregulated target genes involved in chromatin remodeling and transcriptional regulation. Anti-miR-33 also reduced the accumulation of aortic CD8+ T cells and CD4+ T-helper 1 cells, and increased levels of FoxP3+ (Forkhead Box P3) regulatory T cells in plaques, consistent with an immune-dampening effect on plaque inflammation.Conclusions:Our results provide insight into the immune mechanisms and cellular players that execute anti-miR-33's atheroprotective actions in the plaque.