miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques

Milessa Silva Afonso, Monika Sharma, Martin Schlegel, Coen Van Solingen, Graeme J. Koelwyn, Lianne C. Shanley, Lauren Beckett, Daniel Peled, Karishma Rahman, Chiara Giannarelli, Huilin Li, Emily J. Brown, Alireza Khodadadi-Jamayran, Edward A. Fisher, Kathryn J. Moore

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

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 Trem2hi (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 Trem2hi 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.

Original languageEnglish
Pages (from-to)1122-1138
Number of pages17
JournalCirculation Research
Volume128
Issue number8
DOIs
StatePublished - 16 Apr 2021

Keywords

  • adaptive immunity
  • atherosclerosis
  • inflammation
  • macrophages
  • microRNA

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