Dicer prevents activation of the type I interferon pathway in lipid-loaded macrophages

Abstract

Abstract Background The expression of the endonuclease Dicer in macrophages decreases atherosclerosis and necrotic core formation by producing microRNAs such as miR-10a. This effect of Dicer is associated with enhanced mitochondrial respiration in lipid-loaded macrophages. However, the mechanism by which Dicer-dependent production of miRNAs in lipid-loaded macrophages regulates mitochondrial function is unclear. Purpose We aimed to determine the effect of Dicer on lipid-loaded macrophages in the context of atherosclerosis. Methods Mice with a myeloid cell-specific knockout of Dicer (Lys-Cre/Dicerflox/flox/Apoe−/− mice [M-Dicer−/−]) and control mice (Lys-Cre/DicerWT/WT/Apoe−/− mice [M-Dicer+/+]) were fed a high-fat diet (HFD) for 24 weeks. The oxygen consumption rate (OCR) in aortic arch plaques was studied ex vivo by Seahorse Flux XF 24 Analyzer. Bone marrow-derived macrophages (BMDMs) were stimulated with oxLDL (100 μg/mL) for 72 h. Lipid-loaded macrophages were used for proteomic analysis by mass spectrometry. RIP-prime- seq was performed in tAgo2 immunoprecipitates (IP) from Dicer+/+ and Dicer−/− BMDMs. The miRNA expression profile was determined in lipid-loaded BMDMs by NanoString technology. Stat1 phosphorylation was determined in lipid-loaded macrophages by Jess automated western blot system (ProteinSimple). Result The OCR in aortic arch tissue with plaques was higher than in those without plaques in M-Dicer+/+ mice after 24 weeks of HFD feeding. Dicer knockout in macrophages decreased the OCR in aortic arch tissues with plaques but not in aortic arch tissues without plaques (n=3–4, p<0.05). The proteomic analysis of oxLDL-treated BMDMs indicated that Dicer knockout activated the type I interferon signaling pathway by up-regulating the expression of STAT1/2 and interferon-stimulated genes, such as ISG15. Proteins related to mitochondrial DNA were upregulated (e.g., Dnmt3a) or downregulated (e.g., Tfam) by Dicer knockout (n=5, adj.p<0.05). RIP-prime-seq from BMDMs showed that 1376 transcripts were significantly enriched in the tAgo IP from Dicer+/+ compared with that from Dicer−/− BMDMs (n=3–5, adj.p<0.05) including Dnmt3a and Stat2. STAT1 phosphorylation was increased in Dicer−/− compared with Dicer+/+ BMDMs. Among the 299 miRNAs downregulated by Dicer knockout in lipid-loaded macrophages (n=6, adj.p<0.05), miR-29 and miR-30 have highly conserved binding sites (predicted by TargetScan) for Dnmt3a. Conclusion Our results indicate that Dicer expression in lipid-loaded macrophages limits Stat1/2-driven type I interferon response due to mitochondrial damage. This effect may be mediated by the suppression of Dnmt3a by miRNAs such as miR-29 and miR-30. This suggests that targeting Dnmt3a-mediated mitochondrial damage in lipid-loaded macrophages by miRNAs may be therapeutic strategy to limit atherosclerosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG