The RNA modification N 6 -methyladenosine restrains SIRPα to license myeloid efferocytosis in atherosclerosis
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Background
Macrophages participate in atherosclerotic plaque development, progression and resolution, integrating lipid and inflammatory cues to balance tissue damage and repair. Despite considerable understanding of macrophage function in atherosclerosis, the underlying regulatory mechanisms governing these responses continue to evolve. Emerging evidence implicates epitranscriptomic gene regulation by the RNA modification N 6 -methyladenosine (m 6 A) in control of context specific immune responses, but its role in atherosclerosis is not well-defined.
Methods
We integrated m 6 A quantitative trait loci (m 6 A QTLs) with Coronary Artery Disease (CAD) GWAS summary statistics. Using Oxford Nanopore direct RNA sequencing and MeRIP- seq, we constructed a high-resolution map of m 6 A modifications in macrophages. To determine the functional consequences of myeloid m 6 A loss in-vivo , we generated a myeloid-specific loss- of-function mouse model of m 6 A ( Mettl14 f/f LysM Cre ) and performed atherosclerosis studies using bone marrow transplantation into irradiated Ldlr --/- mice. Atherosclerosis lesions were analyzed using light microscopy and immunofluorescence imaging, with complementary in-vitro mechanistic studies performed in murine bone marrow derived macrophages (BMDMs).
Results
m 6 A quantitative trait loci (m 6 A -QTLs) were enriched in CAD GWAS loci relevant to immune and lipid function, implicating m 6 A regulation in human CAD genetics. Using Oxford Nanopore direct RNA sequencing in conjunction with public MeRIP-sequencing data, we generated a single-base resolution, stoichiometric map of m 6 A modification in macrophages, nominating genes central to macrophage biology in atherosclerosis as high-confidence m 6 A targets. Myeloid specific deletion of the m 6 A “writer” complex subunit Mettl14 drove the development of larger, more necrotic and unstable atherosclerotic plaques on an Ldlr --/- background compared with Mettl14 f/f controls, establishing an atheroprotective role for myeloid m 6 A. Loss of myeloid Mettl14 impaired macrophage efferocytosis in atherosclerotic plaques, in a dexamethasone-induced apoptosis in the thymus and in-vitro . Molecularly, m 6 A methylation of the “don’t eat me” antiphagocytic receptor SIRPα RNA suppressed its protein expression.
Conclusions
These findings identify the myeloid m 6 A - SIRPα axis as a novel homeostatic regulator of efferocytosis and establish epitranscriptomic remodeling as a crucial layer of macrophage regulation in atherosclerosis.
