Journal: Frontiers in Immunology

Article Title: Efferocytosis-associated Mrc1 + Gas6 + macrophages are linked to abdominal aortic aneurysm progression through ERK-associated dysfunction

doi: 10.3389/fimmu.2026.1863507

Figure Lengend Snippet: ERK activation is associated with loss of Mrc1 + Gas6 + macrophage effectors and reduced macrophage efferocytosis-associated uptake. (A) Heatmap showing the average expression of representative M1- and M2-associated macrophage markers across Mφ1-Mφ4 subsets. The color scale indicates average expression. (B) Heatmap showing the average expression of efferocytosis-related molecules across macrophage subsets, including the bridging molecule Gas6, the TAM receptors Tyro3, Axl, and Mertk, and the CD147-associated molecules Bsg, Traf6, and Irf5. The color scale indicates average expression. (C) Feature plots showing the expression patterns of Gas6, Mertk, and Mrc1 (left), and Igf1, Entpd1, and Mrc1 (right) in re-clustered macrophages. The Mrc1 + Gas6 + macrophage subset is indicated in dashed line. (D) Dot plots showing the expression levels and proportions of Gas6, Mertk, Igf1, and Entpd1 across macrophage subsets in Sham, Elastase 7d, and Elastase 14d groups. Dot size indicates the fraction of cells expressing the indicated gene, and color indicates the average expression level. (E) Representative immunofluorescence images and quantification of MRC1, MERTK and CD68 staining in aortic tissues from Sham, and Elastase AAA mice (n = 6 biologically independent mice per group). Scale bar (low/high magnification): 100/20 μm. Line-scan analysis in Sham samples shows the colocalization pattern of CD68, MRC1, and MERTK along the selected line segment. The x-axis indicates distance along the selected line, and the y-axis indicates fluorescence intensity. (F) Gene set enrichment analysis (GSEA) showing enrichment of the ERK1_AND_ERK2_CASCADE signature in Mrc1 + Gas6 + macrophages from Elastase relative to Sham samples. (G) Representative western blot images and quantification of p-ERK and t-ERK protein levels in aortic tissues from Sham and Elastase-induced AAA mice. (n = 5 biologically independent mice per group). (H) qPCR analysis of Mertk, Igf1, Gas6, and Entpd1 mRNA levels in RAW264.7 macrophages treated with vehicle, TNF-α, U0126, or TNF-α plus U0126 (n = 3 biologically independent experiments). (I) Representative western blot images and quantification of p-ERK, t-ERK, MERTK, GAS6, IGF1, and ENTPD1 protein levels in RAW264.7 macrophages treated with vehicle, TNF-α, U0126, or TNF-α plus U0126. (n = 5 biologically independent experiments; quantitative comparisons between samples were run on the same gel). (J) Flow cytometric analysis of macrophage efferocytosis-associated uptake. RAW264.7 macrophages were co-cultured with apoptotic PKH26-labeled SMCs in the presence of vehicle, cytochalasin D, U0126, or the MERTK inhibitor UNC2025. Representative flow cytometry plots, PKH26 histograms, and quantification of the proportion of F4/80-positive PKH26-positive macrophages are shown (n = 5 biologically independent experiments). Data are presented as mean ± SEM (E, G–J) . Statistical significance was determined using two-tailed unpaired t test (E, G) or one-way ANOVA with Tukey’s post hoc test (H–J) . * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001; ns, not significant.

Article Snippet: For the phagocytosis-inhibition control, macrophages were pretreated with cytochalasin D (5 μM; MCE, Cat. No. HY-N6682) for 30 min before co-culture with PKH26-labeled apoptotic SMCs.

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot, Cell Culture, Labeling, Flow Cytometry, Two Tailed Test