Let-7b limits the survival of anti-inflammatory macrophages through non-canonical targeting of afadin

Dicer-mediated microRNA (miRNA) biogenesis is essential for macrophage polarization, shaping inflammatory and anti-inflammatory responses relevant to cardiovascular disease. These miRNAs act through the Ago2-containing RNA-induced silencing complex (RISC), leading to target cleavage or translational repression. Dicer promotes mitochondrial respiration in IL-4–stimulated macrophages through let-7b, independent of other let-7 family members, yet the regulatory role and targeting mechanisms of let-7b across polarization states remain unclear. This study examines let-7b's target landscape and its functional effects under pro-inflammatory (IFNγ/LPS) and anti-inflammatory (IL-4) conditions to clarify its context-dependent mechanisms. Bone marrow–derived macrophages (BMDMs) from let-7b knockout and wild-type mice on an Apoe-deficient background were polarized with IFNγ/LPS (10 ng/ml) or IL-4 (5 ng/ml). Functional effects of let-7b loss were assessed by measuring apoptosis, proliferation, iron storage, mitochondrial activity, and lipid droplet formation using live cell fluorescence microscopy. Proteomic profiling was performed on wild-type and let-7b knockout BMDMs. Ago2 immunoprecipitation (IP) was conducted using BMDMs from Apoe-/- mice expressing GFP-tagged AGO2. AGO2 complexes were isolated with GFP-Trap agarose beads (Chromotek). Let-7b target enrichment in RISC was evaluated by prime-RNA sequencing of RNA before (BIP) and after (AIP) IP. A dual-luciferase reporter assay confirmed let-7b binding sites. IL-4–stimulated macrophages showed higher cell turnover, phagocytic activity, iron content, mitochondrial activity, and ROS levels but accumulated fewer lipid droplets than LPS/IFNγ–stimulated macrophages. Let-7b knockout increased iron content and ROS in both cell types, enhanced mitochondrial activity in LPS/IFNγ-stimulated macrophages, and decreased it in IL-4–treated cells. Let-7b Knockout also increased lipid droplets in LPS/IFNγ-stimulated macrophages while reducing apoptosis and promoting proliferation in IL-4–stimulated macrophages. Most proteins altered by let-7b loss differed between polarization states. In M1 macrophages, let-7b deficiency strongly upregulated Arfrp1 and Cox7a1, both linked to lipid-droplet formation. In M2 macrophages, Afdn (Afadin) was the most strongly upregulated protein and was enriched in the RISC complex. Although Afdn lacks a canonical let-7b binding site, a non-canonical site with GU-wobble interactions in the seed was identified. Blocking this interaction increased Afdn expression and confirmed its role in the M2-specific apoptosis phenotype. let-7b limits lipid-droplet formation in inflammatory macrophages, while in anti-inflammatory macrophages it decreases cell survival by targeting Afdn. These findings underscore a polarization-state–specific role of let-7b and suggest a potential strategy for therapeutically modifying macrophage polarization in atherosclerosis.