MicroRNA-33–dependent regulation of macrophage metabolism directs immune cell polarization in atherosclerosis
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
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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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 6

Anti–miR-33–treated macrophages induce differentiation of Tregs.

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Anti–miR-33–treated macrophages induce differentiation of Tregs. (A) Nai...
(A) Naive CD4+ T cells were cultured in the presence or absence of retinoic acid for 6 days with macrophages transfected with control anti-miR or anti-miR33 and (B) subsequently analyzed by flow cytometry for nuclear FoxP3 expression to determine Treg differentiation. Data are representative of 2 independent experiments. (C) Percentage of naive CD4+ T cells differentiated into Tregs in coculture with WT and Mir33–/– macrophages in the presence or absence of LE540 to block RA signaling. Data are representative of 2 independent experiments. (D) Real-time PCR analysis of mRNA from aortic arches of Western diet–fed Ldlr–/– mice treated with control anti-miR or anti–miR-33 (8 weeks) for markers of T cell subsets and associated cytokines. Data represent the mean ± SEM of n = 6 mice/group. (E) Immunofluorescent staining for Foxp3 in plaques of Western diet–fed Ldlr–/– mice treated with control anti-miR or anti–miR-33 (8 weeks). Dotted line indicates the outline of the intima. L, lumen. FoxP3+ cells accumulate in both the intima (arrowheads) and adventitia (arrows) of plaques of anti–miR-33–treated mice. Data represent the mean ± SEM of n = 6 mice/group. Scale bar: 100 μM. (F) Quantification of FoxP3 staining is shown in D. n = 5 mice/group. (B–D and F) All values are mean ± SEM. Statistical comparisons were made using 2-tailed Student’s t test. *P ≤ 0.05; **P ≤ 0.005, compared with controls. (G) Model of molecular mechanisms involved in miR-33 regulation of macrophage polarization and Treg differentiation. When miR-33 levels are high, AMPK, CPT1A, CROT, and HADHB are repressed, leading to reduced FAO and increased glycolysis, which polarizes macrophages to the inflammatory M1 phenotype. Conversely, when miR-33 levels are low, AMPK, CPT1A, CROT, and HADHB are no longer repressed, leading to increased FAO and polarization of macrophages to the M2 phenotype. M2 macrophages suppress inflammation through their production of antiinflammatory cytokines (e.g., IL-10) and retinoic acid, which can foster the differentiation of iTregs.