Depletion of CD206+ M2-like macrophages induces fibro-adipogenic progenitors activation and muscle regeneration

Abstract

Muscle regeneration requires the coordination of muscle stem cells, mesenchymal fibro-adipogenic progenitors (FAPs), and macrophages. How macrophages regulate the paracrine secretion of FAPs during the recovery process remains elusive. Herein, we systemically investigated the communication between CD206⁺ M2-like macrophages and FAPs during the recovery process using a transgenic mouse model. Depletion of CD206⁺ M2-like macrophages or deletion of CD206⁺ M2-like macrophages-specific TGF-β1 gene induces myogenesis and muscle regeneration. We show that depletion of CD206⁺ M2-like macrophages activates FAPs and activated FAPs secrete follistatin, a promyogenic factor, thereby boosting the recovery process. Conversely, deletion of the FAP-specific follistatin gene results in impaired muscle stem cell function, enhanced fibrosis, and delayed muscle regeneration. Mechanistically, CD206⁺ M2-like macrophages inhibit the secretion of FAP-derived follistatin via TGF-β signaling. Here we show that CD206⁺ M2-like macrophages constitute a microenvironment for FAPs and may regulate the myogenic potential of muscle stem/satellite cells.

Fig. 1. Depletion of CD206⁺ M2-like MΦ promotes muscle regeneration.

a Hematoxylin and eosin (H&E)-stained TA tissue paraffin sections of muscle obtained from male C57BL/6J mice following acute injury by cardiotoxin (CTX)-administration. Muscles were harvested at 0, 4, 7, and 14 dpi. n = 2 (D14), and 4 (D0, 4, 7). Scale bar, 200 μm. Asterisks (*) denote necrotic area. b Representative flow cytometry analysis of M2-like MΦ (CD45⁺CD11b⁺F4/80⁺CD206⁺) at 4, 7, and 14 dpi (n = 4 mice per group). c Quantification of CD206⁺ M2-like MΦ (n = 4 mice per group). d Representative flow cytometry analysis of M2-like MΦ (CD45⁺CD11b⁺F4/80⁺CD206⁺) and quantification (e) in muscle of WT and Tg mice harvested at 7 dpi (n = 3 mice per group). f Relative mRNA expression of Mrc1 (CD206) gene in muscle of Tg mice, compared with their littermate WT controls, harvested at 7 dpi (n = 3 mice per group). g Histological sections of Gc muscle from Tg mice and WT control mice, harvested at 7 dpi, stained with H&E (n = 3 mice per group) or WGA and DAPI (n = 4 mice per group). Scale bar, 200 μm (H&E) and 75 μm (WGA). h, i Images stained with DAPI and WGA were used to determine the cross-sectional areas (CSA) (in μm²) of centrally nucleated (CN) fibers (h) and % of CN fibers (i) of the Gc muscle of Tg and WT mice (n = 4 mice per group). j Relative mRNA expression of myogenesis-related marker genes in muscle of Tg mice, compared with their littermate WT control mice. n = 4 (WT), and 5 (Tg) mice. Source data are provided as a Source Data file. The data are shown as the means ± SEM. *p < 0.05, and **p < 0.01 were considered significant as determined using the one-way ANOVA followed by Tukey’s post hoc tests (c), and two-tailed Student t-tests (e–j). WT wild-type, Tg transgenic, mRNA messenger RNA, AU arbitrary units, % percentage, WGA wheat germ agglutinin, and D day.

Fig. 2. Depletion of CD206⁺ M2-like MΦ enhances expression of myogenesis-related marker genes and FAPs-related marker genes.

a Total RNA sequencing (RNA-Seq) analysis of tibialis anterior (TA) of Tg mice and control WT following acute injury. Heatmap of RNA-Seq expression data showing the most significantly altered FAPs-related marker genes in Tg mice, compared with littermate control WT mice (n = 2 mice per group). b Volcano plot analysis highlights the most significant gene alterations in Tg mice versus control WT mice based on false discovery rate threshold (red) [FDR q value < 0.05 and −0.58 > Log₂ fold change (FC) > 0.58] and P value threshold (green) [P value < 0.05 and −0.58 > Log₂ fold change (FC) > 0.58]. Highlighted in gray are the filtered non-significant changes, as indicated. The red dots represent genes that were significantly upregulated/downregulated in the muscle of Tg mice. The Y-axis denotes the −Log₁₀ P values, while the X-axis shows the Log₂ fold change values. The volcano plot was generated using RStudio (n = 2 mice per group). c High resolution confocal imaging of frozen TA sections from Tg mice and WT control mice, stained with anti-PDGFRα and anti-Fst antibodies. Scale bar, 25 μm. Arrows indicate co-localization of PDGFRα and Fst (n = 3 mice per group). Data are representative of at least three independent experiments. d Representative flow cytometry analysis of lineage negative Sca-1/PDGFRα double-positive (FAPs) cells in WT and Tg mice (n = 3 mice per group). Gating strategy is given in Supplementary Fig. 3b. e Relative mRNA expressions of the FACS-isolated FAPs fraction in muscle harvested from WT and Tg mice (n = 3 mice per group). Source data are provided as a Source Data file. The data are shown as the means ± SEM. *p < 0.05, compared with their littermates as determined using the two-tailed Student t-test. f Heatmap representation of differential expression analysis of activated FAPs-related marker genes in isolated FAPs of WT versus Tg mice (n = 2 mice per group).

Fig. 3. Depletion of CD206⁺ M2-like MΦ specifically enhances FAP-derived Fst.

a Histological sections of Gc muscle from FAP-derived Fst KO and Fst^f/f control mice, harvested at 7 dpi, stained with H&E (upper) or WGA and DAPI (lower) (n = 3 mice per group). Scale bar, 200 μm (H&E) and 100 μm (WGA). b Images stained with DAPI and WGA were used to quantify centrally nucleated (CN) fibers of muscle from FAP-derived Fst KO and Fst^f/f control mice. Quantification of CN myofibers (cross-sectional areas (in µm²) of the Gc muscle) (n = 3 mice per group). c Relative mRNA expression of myogenesis-related marker genes in muscle of FAP-derived Fst KO mice, compared with their littermate controls. n = 4 (Fst^f/f), and 3 (Fst KO) mice. d Relative mRNA expression of activated FAP-related marker genes in FACS-isolated FAPs of FAP-derived Fst KO mice, compared with their littermate control Fst^f/f mice (n = 3 mice per group). e Representative images of frozen sections of muscle from Fst KO mice and their littermate control Fst^f/f mice, stained with anti-laminin, and anti-MyoD antibodies. Scale bar, 50 μm. Quantification is given in right panel (n = 3 mice per group). f Representative images of sirius red-stained sections of TA from tamoxifen-treated FAP-derived Fst KO mice compared to tamoxifen-treated Fst^f/f control mice. Quantification is given in right panel (n = 3 mice per group). Scale bars, 200 μm. Fibrotic area (red) was analyzed by ImageJ software. Data are representative of three independent experiments. g Relative mRNA expression of fibrosis-related marker genes in the muscle of Fst KO mice, compared with their littermate controls (n = 3 mice per group). Source data are provided as a Source Data file. The data are shown as the means ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered significant as determined using the two-tailed Student t-test. Fst follistatin, f/f flox/flox, and KO knockout.

Fig. 4. Depletion of CD206⁺ M2-like MΦ reduces TGF-β signaling and improves muscle recovery.

a Representative images of paraffin sections of muscle from Tg mice and their littermate control WT mice following acute injury (7 dpi). Scale bar, 200 μm, 20×. Quantification of TGF-β1⁺, and p27⁺cells/field is given in right panel (n = 5 mice per group). b Histological sections of gastrocnemius muscle from CD206⁺ M2-like MΦ-derived TGF-β1 KO and TGF-β1^f/f mice, harvested at 7 dpi, stained with H&E (upper) or WGA and DAPI (lower) (n = 4 mice per group). Scale bar, 200 μm (H&E) and 100 μm (WGA). c Quantification of CN myofibers of the gastrocnemius muscle (n = 4 mice per group). d Relative mRNA expression of myogenesis-related marker genes in the muscle of TGF-β1 KO mice compared to TGF-β1^f/f littermate control mice (n = 6 mice per group). e Relative mRNA expression of activated FAP-related marker genes in FACS-isolated FAPs of TGF-β1 KO mice compared to TGF-β1^f/f littermate control mice (n = 3 mice per group). f Representative images of sirius red-stained sections of TA from tamoxifen-treated TGF-β1 KO mice compared to tamoxifen-treated TGF-β1^f/f control mice. Quantification is given in right panel (n = 3 mice per group). Scale bars, 200 μm. Fibrotic area (red) was analyzed by ImageJ software. Data are representative of at least two independent experiments. g Relative mRNA expression of fibrosis-related marker genes in the muscle of TGF-β1 KO mice, compared with their littermate TGF-β1^f/f control mice (n = 3 mice per group). The data are shown as the means ± SEM. *p < 0.05, and **p < 0.01 were considered significant as determined using the two-tailed Student t-test. Source data are provided as a Source Data file. CN centrally nucleated, and TGF-β transforming growth factor-beta.

Fig. 5. Impact of TGF-β signaling on C2C12 myoblast differentiation.

a Relative mRNA expression levels of myogenesis-related marker genes in C2C12 myoblast co-cultured with M2-induced BMDM treated with recombinant TGF-β1 and TGF-β receptor I/II inhibitor (Ly21) (n = 4 wells per group). Data are shown as the means ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc tests. *p < 0.05 was considered as significant. b Relative mRNA expression levels of myogenesis-related marker genes. M2-induced BMDM inhibitory effect on primary myoblast differentiation was released by the depletion of CD206⁺ M2-like MΦ upon diphtheria toxin (DT) treatment to the BMDM obtained from Tg mice (n = 3 wells per group). Data are shown as the means ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc tests. *p < 0.05, and **p < 0.01 were considered as significant. c Representative images of primary myoblast co-cultured with M2-induced BMDM with or without DT, stained with anti-MyoG antibody (n = 3 wells per group). d Representative images of C2C12 myoblast co-cultured with M2-induced BMDM (obtained from uninjured Tg mice) with or without DT, stained with embryonic myosin heavy chain (eMyH3) antibody, and DAPI (n = 4 wells per group). The images were taken using Keyence microscope. Scale bar 200 μm. e, f Relative myotube area and fusion index were quantified (n = 4 wells per group). Data are expressed as mean ± SEM. Statistical significance for e was determined by one-way ANOVA followed by Tukey’s post hoc tests, and for f was determined using the two-tailed Student t-test (*p < 0.05, and ns non-significant). AU arbitrary units, Con control, BMDM bone marrow-derived macrophages, and Ly21 Ly2109761 (TGF-β receptor I/II inhibitor).