Role of mir-147 and let-7b in efferocytosis

Abstract Background MicroRNAs generated by Dicer regulate macrophage polarization, such as the increase of miR-147 in pro-inflammatory macrophages and let-7b in anti-inflammatory macrophages. In atherosclerosis, defective efferocytosis accelerates apoptotic cell (AC) accumulation and plaque progression, emphasizing the importance of macrophage efferocytic capacity in disease outcomes. Macrophages polarized into anti-inflammatory (e.g., IL-4–induced M2) or pro-inflammatory (LPS/IFNγ-induced M1) phenotypes seem to affect their efferocytic activity. However, it remains unclear whether microRNAs regulate the efferocytic function of different macrophage subtypes. Purpose We investigated whether miRNAs regulate efferocytosis in different macrophage states and hypothesized that miR-147 selectively enhances efferocytosis in pro-inflammatory (M1) macrophages. Methods Bone marrow-derived macrophages (BMDMs) expressing membrane-bound EGFP (mEGFP) were either left untreated (M0) or treated with LPS and IFN-γ (M1) to induce a pro-inflammatory state, or with IL-4 (M2) to promote anti-inflammatory polarization. Annexin V-labeled, staurosporine-induced apoptotic BMDMs were added to wild-type or Dicer, Mirlet7b, or Mir147 knockout mEGFP+ macrophages. Efferocytosis was monitored over 2 hours using 4D confocal live-cell imaging, with manual counting of total uptake, partial uptake, and continual uptake in macrophages. The volumes of engulfed and degraded AC were automatically measured using the 3D analysis module of the LAS X software. To identify the underlying molecular mechanisms, liquid chromatography-tandem mass spectrometry was performed to compare the proteomes of Mir147+/+ and Mir147-/- BMDMs. The results were analyzed by LIMMA R test (adj. P0.05, absolute fold change1.3). Results The percentage of efferocytosing M1 macrophages was higher than that of M0 or M2 macrophages, with M2 exceeding M0 macrophages. Additionally, the volume of engulfed ACs and the fraction of macrophages engulfing more than one AC were greater in M1 than in M0 or M2 macrophages. Although M2 macrophages showed a lower efferocytosis rate, they exhibited stronger AC degradation capabilities compared with M1 macrophages. Knocking out Mirlet7b in M0 and M1 macrophages reduced the volume of engulfed ACs. In contrast, knocking out Dicer or Mir147 affected efferocytosis only in M1 macrophages. In Mir147−/− M1 macrophages, out of the 43 upregulated and 77 downregulated proteins compared to Mir147+/+ M1 macrophages, three are known to be involved in efferocytosis, including TLR7, CD93, and Rac2, indicating they may contribute to miR-147-mediated efferocytosis. Conclusions LPS/IFNγ-stimulated macrophages have the highest efferocytic ability, with miR-147 specifically enhancing the uptake of apoptotic cells in this state. Let-7b broadly promotes efferocytosis across macrophage states, suggesting regulation beyond subtype specificity.