M4-like Macrophages Drive Skeletal Muscle Fibrosis and Suppress Myogenesis via the MMP12/SPP1/NF-κB Axis
- 1Department of Orthopedics, The Second Affiliated Hospital of Chongqing Medical University, Yuzhong, Chongqing 400010, China
Abstract
Acute skeletal muscle repair depends on a finely tuned balance between myogenic regeneration and fibrotic remodeling. Macrophages are essential to this process, and specific subsets have been linked to fibrosis in some organs. The M4-like macrophage, a distinct polarization subset implicated in fibrotic diseases, has not been well characterized in the context of injured muscle, particularly regarding its impact on fibroblast activation and myogenic suppression. The study aims to explore the effects of M4-like macrophages in the repair of muscle injury and possible mechanism. We analyzed a murine single-cell RNA-sequencing dataset from cardiotoxin (CTX)-injured skeletal muscle to identify injury-expanded macrophage subsets and their molecular programs. Immunohistochemistry and immunofluorescence staining of muscle sections were used to assess CXCL4 expression and platelet (CD41+) infiltration in injured versus uninjured tissues. CXCL4-induced M4-like macrophages were established as an in vitro model to examine their effects on L929 fibroblasts and C2C12 myoblasts. The MMP12–SPP1 interaction was predicted by AlphaFold3 and validated by co-immunoprecipitation and immunofluorescence colocalization. Loss-of-function and rescue experiments targeting MMP12 and SPP1 were performed to determine their causal roles in fibroblast activation and myogenic suppression. Transcriptomic and biochemical analyses were used to define downstream macrophage-intrinsic NF-κB/TNF-α signaling. Finally, AAV-mediated macrophage-targeted SPP1 knockdown and pharmacological NF-κB inhibition were evaluated in the CTX injury model. Using a BALB/c mouse model of cardiotoxin (CTX)-induced acute skeletal muscle injury and a publicly available murine scRNA-seq dataset from CTX-injured skeletal muscle, we observed an injury-induced MMP12/SPP1-positive macrophage subset with an M4-like transcriptional signature. Mechanistically, co-immunoprecipitation assays and immunofluorescence colocalization supported an association between MMP12 and SPP1. MMP12 knockdown attenuated the pro-fibrotic and anti-myogenic effects of M4-like macrophages, whereas recombinant SPP1 largely restored these phenotypes. Furthermore, transcriptomic and biochemical analyses revealed that autocrine SPP1 activated NF-κB signaling in M4-like macrophages, and increased TNF-α secretion. Pharmacological inhibition of NF-κB signaling with QNZ reduced fibroblast activation and restored the expression of myogenic markers. However, QNZ is not TNF-α specific and may affect multiple cell types. In vivo, AAV-mediated macrophage-targeted SPP1 knockdown driven by the F4/80 promoter, as well as QNZ treatment in the CTX-induced muscle injury model, markedly decreased collagen deposition, enhanced myogenic regeneration, and improved functional recovery. These findings identify an MMP12/SPP1/NF-κB signaling pathway in M4-like macrophages, with TNF-α as a potential downstream mediator, that may contribute to an imbalance between regeneration and fibrosis and may represent a therapeutic target for enhancing skeletal muscle repair after acute injury.
Related
- From Fixed Lineage to Dynamic States: Fibro-Adipogenic Progenitors in Skeletal Muscle Regeneration, Fibrosis, and Fibrofatty Degeneration
- RUNX1‐Mediated Hypoxic Signaling Impairs Myogenesis and Promotes Fibrosis in Myopathies
- Pseudomonas aeruginosa Cystic Fibrosis Isolates Induce Rapid, Type III Secretion-Dependent, but ExoU-Independent, Oncosis of Macrophages and Polymorphonuclear Neutrophils
- Distinct muscle stem cell fates correlated with hyperplasia and hypertrophy during skeletal muscle growth in rainbow trout
- Biological Accessibility and Analytical Recovery in Myofibrillar Protein Loss During Skeletal Muscle Atrophy
BibTeX
@article{yao2026m4like,
title = {{M4-like Macrophages Drive Skeletal Muscle Fibrosis and Suppress Myogenesis via the MMP12/SPP1/NF-κB Axis}},
author = {Senwen Yao and Zhenyuan Lu and Guosheng Zhao and Yang Liu and Shan Wu and Jiaxuan Zhang and Renjie Dong and Yangming Hu and Ye Zhang and Yang Wang},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27209369},
url = {https://www.mdpi.com/1422-0067/27/20/9369},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - M4-like Macrophages Drive Skeletal Muscle Fibrosis and Suppress Myogenesis via the MMP12/SPP1/NF-κB Axis
AU - Senwen Yao
AU - Zhenyuan Lu
AU - Guosheng Zhao
AU - Yang Liu
AU - Shan Wu
AU - Jiaxuan Zhang
AU - Renjie Dong
AU - Yangming Hu
AU - Ye Zhang
AU - Yang Wang
PY - 2026
DA - 2026/10/21
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27209369
UR - https://www.mdpi.com/1422-0067/27/20/9369
AB - Acute skeletal muscle repair depends on a finely tuned balance between myogenic regeneration and fibrotic remodeling. Macrophages are essential to this process, and specific subsets have been linked to fibrosis in some organs. The M4-like macrophage, a distinct polarization subset implicated in fibrotic diseases, has not been well characterized in the context of injured muscle, particularly regarding its impact on fibroblast activation and myogenic suppression. The study aims to explore the effects of M4-like macrophages in the repair of muscle injury and possible mechanism. We analyzed a murine single-cell RNA-sequencing dataset from cardiotoxin (CTX)-injured skeletal muscle to identify injury-expanded macrophage subsets and their molecular programs. Immunohistochemistry and immunofluorescence staining of muscle sections were used to assess CXCL4 expression and platelet (CD41+) infiltration in injured versus uninjured tissues. CXCL4-induced M4-like macrophages were established as an in vitro model to examine their effects on L929 fibroblasts and C2C12 myoblasts. The MMP12–SPP1 interaction was predicted by AlphaFold3 and validated by co-immunoprecipitation and immunofluorescence colocalization. Loss-of-function and rescue experiments targeting MMP12 and SPP1 were performed to determine their causal roles in fibroblast activation and myogenic suppression. Transcriptomic and biochemical analyses were used to define downstream macrophage-intrinsic NF-κB/TNF-α signaling. Finally, AAV-mediated macrophage-targeted SPP1 knockdown and pharmacological NF-κB inhibition were evaluated in the CTX injury model. Using a BALB/c mouse model of cardiotoxin (CTX)-induced acute skeletal muscle injury and a publicly available murine scRNA-seq dataset from CTX-injured skeletal muscle, we observed an injury-induced MMP12/SPP1-positive macrophage subset with an M4-like transcriptional signature. Mechanistically, co-immunoprecipitation assays and immunofluorescence colocalization supported an association between MMP12 and SPP1. MMP12 knockdown attenuated the pro-fibrotic and anti-myogenic effects of M4-like macrophages, whereas recombinant SPP1 largely restored these phenotypes. Furthermore, transcriptomic and biochemical analyses revealed that autocrine SPP1 activated NF-κB signaling in M4-like macrophages, and increased TNF-α secretion. Pharmacological inhibition of NF-κB signaling with QNZ reduced fibroblast activation and restored the expression of myogenic markers. However, QNZ is not TNF-α specific and may affect multiple cell types. In vivo, AAV-mediated macrophage-targeted SPP1 knockdown driven by the F4/80 promoter, as well as QNZ treatment in the CTX-induced muscle injury model, markedly decreased collagen deposition, enhanced myogenic regeneration, and improved functional recovery. These findings identify an MMP12/SPP1/NF-κB signaling pathway in M4-like macrophages, with TNF-α as a potential downstream mediator, that may contribute to an imbalance between regeneration and fibrosis and may represent a therapeutic target for enhancing skeletal muscle repair after acute injury.
SN - 1422-0067
ER -