MicroRNA-33–dependent regulation of macrophage metabolism directs immune cell polarization in atherosclerosis
Mireille Ouimet, … , P’ng Loke, Kathryn J. Moore
Mireille Ouimet, … , P’ng Loke, Kathryn J. Moore
Published December 1, 2015; First published October 26, 2015
Citation Information: J Clin Invest. 2015;125(12):4334-4348. https://doi.org/10.1172/JCI81676.
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Research Article Vascular biology

MicroRNA-33–dependent regulation of macrophage metabolism directs immune cell polarization in atherosclerosis

Abstract

Cellular metabolism is increasingly recognized as a controller of immune cell fate and function. MicroRNA-33 (miR-33) regulates cellular lipid metabolism and represses genes involved in cholesterol efflux, HDL biogenesis, and fatty acid oxidation. Here, we determined that miR-33–mediated disruption of the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Macrophage-specific Mir33 deletion increased oxidative respiration, enhanced spare respiratory capacity, and induced an M2 macrophage polarization–associated gene profile. Furthermore, miR-33–mediated M2 polarization required miR-33 targeting of the energy sensor AMP-activated protein kinase (AMPK), but not cholesterol efflux. Notably, miR-33 inhibition increased macrophage expression of the retinoic acid–producing enzyme aldehyde dehydrogenase family 1, subfamily A2 (ALDH1A2) and retinal dehydrogenase activity both in vitro and in a mouse model. Consistent with the ability of retinoic acid to foster inducible Tregs, miR-33–depleted macrophages had an enhanced capacity to induce forkhead box P3 (FOXP3) expression in naive CD4+ T cells. Finally, treatment of hypercholesterolemic mice with miR-33 inhibitors for 8 weeks resulted in accumulation of inflammation-suppressing M2 macrophages and FOXP3+ Tregs in plaques and reduced atherosclerosis progression. Collectively, these results reveal that miR-33 regulates macrophage inflammation and demonstrate that miR-33 antagonism is atheroprotective, in part, by reducing plaque inflammation by promoting M2 macrophage polarization and Treg induction.

Authors

Mireille Ouimet, Hasini N. Ediriweera, U. Mahesh Gundra, Frederick J. Sheedy, Bhama Ramkhelawon, Susan B. Hutchison, Kaitlyn Rinehold, Coen van Solingen, Morgan D. Fullerton, Katharine Cecchini, Katey J. Rayner, Gregory R. Steinberg, Phillip D. Zamore, Edward A. Fisher, P’ng Loke, Kathryn J. Moore

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Figure 1

miR-33 regulates macrophage polarization.

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miR-33 regulates macrophage polarization. (A) Relative expression levels...
(A) Relative expression levels of miR-33 in BMDMs activated to M1 or M2 by treatment with LPS and IFN-γ, or IL-4, for 24 hours, respectively. Data are representative of 3 independent experiments. (B and C) Real-time PCR analysis of markers of classical M1 and alternative M2 activation in peritoneal macrophages transfected with (B) control (ctrl) or miR-33 mimics or (C) control or miR-33 inhibitors for 48 hours. Data are representative of 4 independent experiments. (D) 3′ UTR luciferase reporter activity of mouse Arg1 in HEK293 cells transfected with miR-33 or control mimics. Data are representative of 3 independent experiments. (E) Real-time PCR analysis of M1 and M2 markers in BMDMs isolated from WT or Abca1–/– mice and transfected with control anti-miR or anti–miR-33 for 48 hours. Data represent the mean ± SEM of 3 experiments. (F) ECAR of BMDMs cultured for 24 hours with IFN-γ and LPS (M1) or with IL-4 (M2), followed by sequential treatment (arrows) with glucose, oligomycin (Oligo), and 2-DG. (G) OCR of M1 and M2 macrophages prepared as in E and sequentially treated with oligomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone plus antimycin (Rtn/AA). (H and I) Maximal ECAR and OCR of macrophages treated with (H) control or miR-33 mimic or (I) control anti-miR or anti–miR-33 at 48 hours after transfection, measured in real time and presented as change in mpH per unit time (ECAR) and pMoles per unit time (OCR). Data are representative of 3 independent experiments (F–I). (J) 14C-oleic acid oxidation in macrophages treated with control or miR-33 inhibitors in the presence or absence of the FAO inhibitor etomoxir. Veh, vehicle. Data are representative of 2 independent experiments. Statistical comparisons were made using 2-tailed Student’s t test (A–C and H–J) or ANOVA (E). *P ≤ 0.05; **P ≤ 0.005, compared with controls. All values are mean ± SEM.