Temporal single-cell sequencing analysis reveals that GPNMB-expressing macrophages potentiate muscle regeneration
Posted on: 19 October 2025 , updated on: 21 October 2025
Preprint posted on 28 March 2024
A glycoprotein nonmetastatic melanoma protein B (GPNMB) expressing macrophage subset emerged as a regulator of macrophage-mediated muscle regeneration, following muscle injury!
Selected by Hafsa Zahid
Categories: cell biology, immunology
Updated 20 October 2025 with a postLight by Hafsa Zahid
This study was published in Nature Experimental and Molecular Medicine with minimal updates and revisions. Most of the updates were done in the placement of subfigures in the figure panels and re-writing some parts of the discussion section.
- In the original pre-print, Figure 1h showed the pseudo time analysis for five monocyte–macrophage subsets, all mapped together in a single plot, which made it difficult to understand for the reader. In the peer-reviewed version, the same pseudo time analysis is now depicted in five separate plots for each monocyte-macrophage subset, hence the data visualization is improved. Along with this, the placements of western blot plots in figure panels 4 and 5 were also updated to ensure a better flow of information for the readers.
- The pre-print also underwent some changes in discussion section and thereby also addressed one initial question raised in my pre-light post about whether cluster2 is M2 skeletal muscle-resident macrophage subset or an infiltrating subset giving rise to M2 subset. In the updated discussion section, the authors highlighted how the cluster2 subset is an infiltrating macrophage subset which partially adopts a tissue-resident anti-inflammatory profile (M2), hence aiding in skeletal muscle regeneration. Moreover, this also adds another interesting avenue to the study as it shows that infiltrating macrophages can acquire tissue-resident macrophage phenotype upon exposure to environmental cues at the site of muscle injury.
Overall, this study is indeed a fascinating one as it shows that macrophage phenotypic states are more fluid and more complex than previously thought. Moreover, the differences in figure placements between the pre-print and the published version of the study show that data visualization is indeed a core factor in helping the readers contextualize the findings.
Background
Temporal resolution of skeletal muscle tissue debris upon muscle injury involves a complex cellular crosstalk between immune cells, muscle stem cells (MuSCs), Fibroadipogenic progenitors (FAPs), vascular cells and glial cells (1). While optimal functioning of each of these cell types is a pre-requisite for effective muscle regeneration, recently macrophages have emerged as key regulators of skeletal muscle repair response. However, their phenotypic diversity and transitions between pro- and anti-inflammatory subsets remains elusive. While a variety of cytokines and growth factors have been previously implicated in controlling macrophage phenotype and function, Chen and colleagues identify glycoprotein nonmetastatic melanoma protein B (GPNMB) signalling as one of the major cellular networks controlling macrophage-mediated muscle regeneration. GPNMB has been previously identified as a regulator of melanoma growth and its metastatic potential (2). Furthermore, an elevated inflammatory cytokine profile in macrophages has been observed previously in mice harbouring GPNMB deletion, hinting towards a possible role of GPNMB in governing pro-inflammatory to anti-inflammatory microenvironment transition (3). Of note, the precise function of GPNMB signalling mediated by macrophages during skeletal muscle regeneration is only elucidated recently by Chen and colleagues in this study.
Injury Model
Cardiotoxin (CTX)-induced skeletal muscle injury in C57BL/6 WT and GPNMB KO mice.
Key Findings
- Single-cell sequencing analysis identified five distinct macrophage subsets during skeletal muscle repair, following injury (Figure 1). Furthermore, Pseudo-time mapping of the identified macrophage subsets along the injury-to-regeneration trajectory revealed that each subset not only has a temporal association, but a unique gene expression profile, with Cluster 2 being of high interest as it appears at a transition point governing pro-to anti-inflammatory regeneration axis. Differential gene expression (DEGS) analysis revealed that Cluster 2 not only distinctively express GPNMB, but also co-express crucial factors involved in tissue regeneration and fibrosis e.g., Mertk, Igf1 and Nr1h3.
- In vitro functional assays using bone-marrow derived cells (BMDCs) reveal a significant increase in GPNMB expression in anti-inflammatory macrophage subset (M2) as compared to the pro-inflammatory subset (M1). Moreover, overexpression of GPNMB in BMDCs showed an upregulation of M2-associated genes i.e., Arg1, Mrc1, IL-4, Mertk, Axl and Igf1r.
- GPNMB-KO mice exhibit myofibers with smaller cross-sectional area (CSA) at day 4 and day 7, following CTX injury, hence pointing towards an overall impaired muscle regeneration. Flow cytometry experiment utilizing CSFE-labelled apoptotic bodies revealed that GPNMB-KO macrophages have a substantially reduced capacity to phagocytose apoptotic cells. Moreover, investigation of cross-talk between GPNMB and Mertk expression revealed that in vivo inhibition of Mertk following CTX-injury, exhibits a similar phenotype as observed in GPNMB KO mice.
- Lastly, myotube differentiation assays using C2C12 myoblast cell line, with or without the addition of recombinant GPNMB (rGPNMB), showed that GPNMB stimulation promotes myogenic differentiation. Moreover, in vivo administration of rGPNMB rescues the fibrotic phenotype following CTX injury, by increasing the myofiber cross-sectional area at 4- and 7-days post-injury.
Figure 1: Representation of five transcriptionally unique macrophage subsets identified via scRNA sequencing, with special focus on GPNMB-expressing macrophages as a key contributor in aiding skeletal muscle regeneration.
Why I think this work is important?
This work is interesting as it provides an extensive temporal record of gene expression changes in macrophages at a single cell level following CTX injury. This dataset can help us understand macrophages beyond the classical terminology of pro versus anti inflammation and shows that macrophage phenotypic states are more fluid than previously thought. Moreover, delineating the mechanisms (e.g., GPNMB signalling) underlying these phenotypic switches can help develop effective therapies to counteract fibrosis.
Future directions and questions for the authors
- The traditional definition of M1 and M2 macrophage subsets states that M1 is classically activated pro-inflammatory macrophage subset and M2 is alternatively activated anti-inflammatory subset. In the study, that a higher GPNMB expression is observed in fully differentiated M2 macrophages derived from BMDCs, so should the cluster 2 identified via scRNA be regarded as a tissue-resident M2 or an infiltrating population transitioning to M2?
- Quantitative PCR and Western blots from BMDCs experiment revealed a considerable upregulation of GPNMB in M0 macrophages when compared with M1, what does this hint towards? Can one extrapolate that in order for M0 to give rise to M1 macrophages, the GPNMB signalling needs to be switched off? If that’s the case, then it could be a nice idea to look into why this signalling needs to be turned off for M1 (I reckon it has to do something with metabolism).
- Does the increased CSA of myofibers following in vivo administration of rGPNMB, reflects a healthy homeostatic outcome i.e., when compared to a WT homeostatic muscle (without injury)? For example, maximal isometric force measurements in rGPNMB administered mice would help in assessing whether force production is improved and is closer to a homeostatic muscle.
References
- Wosczyna, M. N., & Rando, T. A. (2018). A muscle stem cell support group: coordinated cellular responses in muscle regeneration. Developmental cell, 46(2), 135-143.
- Weterman, M. A., Ajubi, N., van Dinter, I. M., Degen, W. G., van Muijen, G. N., Ruiter, D. J., & Bloemers, H. P. (1995). nmb, a novel gene, is expressed in low‐metastatic human melanoma cell lines and xenografts. International journal of cancer, 60(1), 73-81.
- Prabata, A., Ikeda, K., Rahardini, E. P., Hirata, K. I., & Emoto, N. (2021). GPNMB plays a protective role against obesity-related metabolic disorders by reducing macrophage inflammatory capacity. Journal of Biological Chemistry, 297(5), 101232.
doi: https://doi.org/10.1242/prelights.41735
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