Temporal Single-Cell Sequencing Analysis Reveals That GPNMB-Expressing Macrophages Potentiate Muscle Regeneration

Macrophages play a crucial role in coordinating the skeletal muscle repair response, but their phenotypic diversity and the transition of specialized subsets to resolution-phase macrophages remain poorly understood. To address this issue, we induced injury and performed single-cell RNA sequencing on individual cells in skeletal muscle at different time points. Our analysis revealed a distinct macrophage subset that expressed high levels of Gpnmb and that coexpressed critical factors involved in macrophage-mediated muscle regeneration, including Igf1, Mertk, and Nr1h3. Gpnmb gene knockout inhibited macrophage-mediated efferocytosis and impaired skeletal muscle regeneration. Functional studies demonstrated that GPNMB acts directly on muscle cells in vitro and improves muscle regeneration in vivo. These findings provide a comprehensive transcriptomic atlas of macrophages during muscle injury, highlighting the key role of the GPNMB macrophage subset in regenerative processes. Targeting GPNMB signaling in macrophages could have therapeutic potential for restoring skeletal muscle integrity and homeostasis.


INTRODUCTION
Skeletal muscle is the most abundant human body tissue, comprising 30-45% of body weight, and the maintenance of its integrity and homeostasis is of critical importance.The complete regeneration of skeletal muscle after injury requires coordinated communication among several distinct cell types, including immune cells, muscle stem cells (MuSCs), bro-/adipogenic progenitors (FAPs), glial cells, and vascular cells 1,2 .Tight control of signal integration in the injury-induced immune response has been shown to promote regeneration in several tissues, such as the liver 3 , heart 4 , and skeletal muscle 5 .In the past, research on the role of macrophages in tissue regeneration has focused on their ability to phagocytose cellular debris.However, in ltrating macrophages can undergo a polarization shift toward an anti-in ammatory phenotype and exhibit various pro-regenerative functions.These functions include the remodeling of the extracellular matrix (ECM) 6 and the stimulation of MuSC proliferation 7 .
Disturbances in macrophage function lead to impaired muscle regeneration 8 , highlighting the importance of these cells in the regeneration process.It has been suggested that Ly6C lo macrophages contribute to skeletal muscle 9,10 and myocardial tissue regeneration 11 .The core genes expressed in Ly6C lo macrophages include secretory cytokines and growth factors such as insulin-like growth factor 1 (IGF1) 12 , the growth differentiation factors GDF3 13 , GDF15 14 , and transforming growth factor beta (TGFβ) 13,15 , which act in an anti-in ammatory manner and contribute to skeletal muscle regeneration.
Glycoprotein nonmetastatic melanoma protein B (GPNMB), which was initially identi ed as a regulator of tumor growth in melanoma with low metastatic potential 16 , is involved in the transendothelial migration of dendritic cells 17 .GPNMB also inhibits osteoclast differentiation by interacting with CD44 and inhibiting ERK activation 18 .In the brain, GPNMB is predominantly expressed in microglia, which serve as resident cells responsible for mediating in ammatory stimuli and neurodegeneration 19 .Furthermore, GPNMB expression is elevated in human liver samples from patients with hepatitis, cirrhosis, and paracetamol intoxication, all of which are associated with in ammatory diseases, compared to samples from healthy controls.Notably, the deletion of GPNMB in mice led to a signi cant increase in the levels of in ammatory cytokines in macrophages, suggesting that GPNMB may suppress the transition of macrophages toward a proin ammatory state 20 .However, the precise biological roles of GPNMB in macrophages during skeletal muscle regeneration remain to be elucidated.
In this study, we used a cardiotoxin (CTX)-induced skeletal muscle injury model, which induces myo ber necrosis and provides a highly reproducible framework.This model allowed us to visualize temporal changes in macrophage subsets and analyze their roles in muscle regeneration.We discovered that the expression of GPNMB was upregulated in a sustained manner, reaching its highest level on day 3, in a speci c macrophage subset that coexpresses critical factors involved in macrophage-mediated muscle regeneration.Furthermore, myeloid-speci c Gpnmb knockdown impaired the regeneration of skeletal muscle.The extrinsic administration of recombinant GPNMB to injured mice promoted myogenesis by activating myocyte differentiation.Overall, our study describes unrecognized macrophage subsets involved in muscle regeneration and demonstrated that GPNMB acts on myocytes in vitro and promotes muscle regeneration in vivo.

Animals
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University Hospital (license no.CMUIACUC-2023-061).Eight-to twelve-week-old female C57BL/6J mice were provided by the National Laboratory Animal Center (NLAC, Taiwan) and housed at the China Medical University Hospital Animal Center.Gpnmb −/− mice were generated in cooperation with the Transgenic Core Facility at the Academia Sinica, Taiwan.For this strain, exons 2-6 of the Gpnmb gene were deleted via CRISPR-Cas9 technology.The C57BL/6J wild-type strain was used as the control for animal experiments.

Cardiotoxin injections and cell isolation
A total of 40 µl of Naja pallida CTX (Merck KGaA), at a concentration of 10 µM in PBS, was injected into the TA muscle of anesthetized mice (i.m.).Furthermore, to investigate the potential impact of recombinant GPNMB (rGPNMB) on skeletal muscle regeneration, we simultaneously administered CTX at two concentrations (10 or 20 µg) of rGPNMB.Injured TA muscles were collected at the indicated time points after injury.To examine the regenerating myo bers, 10-mm cross sections were collected from the frozen TA muscles and stained with an embryonic myosin heavy chain (eMyHC, DSHB F1.652) antibody.The sections were imaged with a Nikon ECLIPSE Ti2 uorescence microscope.Quanti cation of eMyHC staining was performed with ImageJ.Individual bers were manually outlined to determine the crosssectional area.At least 50 bers per image and 3-5 images were analyzed at each indicated time point.We used a commercial murine skeletal muscle dissociation kit with a GentleMACS Octo Dissociator (Miltenyi Biotec).The TA muscles were excised and cut into small pieces following the manufacturer's protocol.The digested product was ltered through a 70-µm cell strainer using a plunger to disrupt the undigested tissue and washed with RPMI-1640 containing 1% P/S, 20 mM HEPES, and supplemented with 0.5% serum.After resuspension in 47% Percoll (Cytiva) and centrifugation at 1500 rpm for 10 min, the cells were collected and washed for ow cytometric analysis or single-cell RNA-seq.

Magnetic separation of mononuclear cells and ow cytometric analysis
We used MACS separators to enrich the mononuclear cells obtained from the TA muscle to increase the number of macrophages.The mononuclear cells were isolated using anti-mouse CD90.2 and B220 MicroBeads (Miltenyi Biotec) through negative sorting per the manufacturer's protocol to eliminate most T and B cells.Similarly, anti-mouse CD45 MicroBeads (Miltenyi Biotec) were used to sort the owthrough.

Cell culture
BMDCs from mice were cultured according to a previous protocol 21 .Brie y, bone marrow-derived cells from the femur and tibia were ushed out using Dulbecco's modi ed essential medium (DMEM) and ltered through a 70-µm cell strainer.Red blood cells were removed using ACK lysis buffer (Thermo Fisher).BMDCs were cultured in DMEM supplemented with 10% fetal bovine serum, antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), and 20 ng/ml macrophage colony-stimulating factor (M-CSF; PeproTech) for 7 days to allow differentiation into BMDMs.The BMDMs were polarized into M1 or M2 macrophages by using LPS (100 ng/ml, Sigma Aldrich) plus IFN-γ (45 ng/ml, PeproTech) or IL-4 (10 ng/ml, PeproTech), respectively.C2C12 myoblasts were purchased from ATCC and cultured in DMEM supplemented with 10% FBS until con uency.After reaching con uency, the myoblasts were differentiated in DMEM supplemented with 1% horse serum for 72 h, as previously described 22 .

Efferocytosis assay
An efferocytosis assay was designed to evaluate the capacity of macrophages to phagocytose apoptotic cells in the context of GPNMB de ciency.BMDMs were harvested from GPNMB-KO and WT mice.
Mononuclear cells were isolated from the TA muscle on day 3 post-CTX-induced injury.C2C12 myoblasts were labeled with carboxy uorescein succinimidyl ester (CFSE) to track their uptake by macrophages.
C2C12 cell apoptosis was induced using staurosporine (STS) treatment.BMDMs were cocultured with CFSE-labeled apoptotic C2C12 cells for 24 hours to allow for efferocytosis.After cocultivation, macrophages were stained with anti-F4/80 and anti-CD11b antibodies to identify the population of interest.Flow cytometry was subsequently utilized to quantify the percentage of macrophages that phagocytosed apoptotic C2C12 cells, as indicated by double positivity for F4/80, CD11b, and CFSE uorescence.The percentage of F4/80 + CD11b + macrophages containing CFSE + material was compared between the GPNMB-KO and WT groups to assess the impact of GPNMB on the efferocytosis of macrophages.

Single-cell RNA-seq (10x Genomics)
We isolated fresh cells and enriched them using MicroBeads to obtain high-quality single-cell RNA sequencing data, as previously described 23 .We then encapsulated these cells in droplet emulsions using a 10x Chromium Controller (10x Genomics) to achieve 10,000 cells per sample.The scRNA-seq libraries were prepared according to the manufacturer's protocol using the GenCode Single-Cell 3' Gel Bead and Library V3 kit.Subsequently, we pooled the libraries and sequenced them on a NovaSeq 6000 System (Illumina) following the manufacturer's instructions.

Single-cell RNA sequencing data processing
We obtained 31,395 single cells with a median of 80,046 reads per cell.Paired-end single-cell RNA sequencing (scRNA-seq) reads were demultiplexed, aligned to the mm10 reference genome, and processed for single-cell gene counting using Cell Ranger Software from 10X Genomics, Inc. (https://support.10xgenomics.com/single-cell-gene-expression/software).Downstream analysis of the combined sample gene counts was performed using Seurat, a scalable R-based package (version 4.3.0)designed for single-cell gene expression datasets.Gene counts were imported using the CreateSeuratObject function (min.cells= 25, min.features= 0), and low-quality cells were discarded using the following thresholds: a minimum of 500 and maximum of 5000 genes, a maximum of 10% of mitochondrial gene mapped reads, and a minimum of 1,000 and maximum of 40,000 UMIs.The total number of cells that passed quality control according to the abovementioned thresholds was 21,642.
SCTransform normalization, which uses regularized negative binomial regression for normalizing sparse single-cell data and variance stabilization, was performed for the ltered dataset using the SCTransform function in Seurat, regressing the percentage of mitochondrial genes per cell (vars.to.regress = "percent.mt").

Cell-cell communication analysis
Cellular communication networks were quantitatively inferred and analyzed using scRNA-seq data.The open-source R package CellChat was used to visualize the interactions among different cell groups 24 .Two hundred twenty-nine signaling pathway families were grouped as a library to analyze cell-cell communication.Circle, hierarchy, and river plots were generated according to the ligand-receptor interaction network.

Real-time qPCR
Total RNA was isolated from the TA muscle and C2C12 myoblasts with Direct-zol RNA Kits (Zymo Research), and mRNA levels for this study were determined by quantitative PCR on a CFX Opus 96 Real-Time PCR System (Bio-Rad, CA, USA).Primer 3 software was used across the intronic sequences to design all primers.

Dynamics of macrophage subset changes during skeletal muscle regeneration
To reveal the changes in macrophage heterogeneity and identify the speci c subsets in uencing skeletal muscle regeneration following injury, we intramuscularly injected CTX into the tibialis anterior (TA) of mice.The TA tissues were collected at the designated time points for multiple analyses (Fig. 1a).
Consistent with previous reports 25 , these in ltrated immune cells were mainly CD68 + monocytes/macrophages (Fig. 1b).Cyto uorometric analysis con rmed the robust in ltration of circulating monocytes and the generation of macrophage subsets in the regenerating muscle.These cells were initially proin ammatory Ly6C hi cells but transformed into anti-in ammatory Ly6C lo cells by day 4 (Fig. 1c-e).For unbiased analysis that integrates temporal gene pro ling of macrophages with analysis of their potentially varying roles in muscle regeneration, we enriched CD90.2 − B220 − CD45 + cell populations by using magnetic beads at the indicated time points during skeletal muscle regeneration.Subsequently, single-cell RNA-seq (scRNA-seq) analysis was performed using the 10X Genomics Chromium platform (Fig. S1a).After standard quality control and the removal of doublets, high-quality transcriptomes from 21,642 cells were revealed.We performed graph-based Leiden clustering and utilized uniform manifold approximation and projection (UMAP) embeddings for visualization.All clusters were annotated by utilizing the scMCA_MNN-muscle dataset and differentially expressed genes (DEGs).Fifteen cell clusters were revealed, including ve subsets of monocytes/macrophages (Mo/Mφ), ve subsets of neutrophils, and one subset each of dendritic cells, T cells, B cells, muscle progenitor cells, and stromal cells (Fig. S1b   and c).As expected, macrophages and neutrophils were the most abundant cell populations after, with proportions of 67.9% and 15.3%, respectively (Fig. S1c).

Identi cation of ve distinct macrophage subsets during skeletal muscle regeneration
We reclustered our scRNA-seq data of the macrophage subsets and identi ed ve groups of macrophage subsets (Fig. 1f) whose distributions changed dynamically at different time points (Fig. 1g).Pseudotime analysis was performed on all ve cell clusters along the injury-to-regeneration trajectory via Monocle (v3) to delineate the expression patterns of genes following muscle injury (Figs.1h and S2a).These dynamic changes suggest that the unique macrophage subset distributions may have critical biological functions at speci c time points.The ve distinct macrophage clusters (Mo/Mφ clusters: 1, 2, 3, 4, and 5) shared a common core of expressed macrophage markers, including Adgre1, Cd68, Csf1r, Fcgr1, Lgals3, and Lyz2 (Fig. S1d).However, they exhibited distinct transcriptional pro les: Cluster 1 exhibited increased expression of the M2 macrophage activation markers Arg1 and Mrc1; Cluster 2 exhibited increased expression of genes associated with tissue regeneration, such as Igf1 and Gdf15, which is in line with the recently described nding that GDF15 is a critical effector during skeletal regeneration 20 ; and Cluster 3 exhibited increased expression of genes associated with immune response activation and antigen presentation, including H2-Aa, H2-Ab1, and H2-Eb1.Moreover, the proportion of cells in Cluster 3 was notably greater than that of cells in other clusters in both the uninjured and regenerative stages; Cluster 4 exhibited high expression of genes involved in proin ammatory responses, such as I tm6, Gsr, and Hp; Cluster 5 exhibited increased expression of Acod1 (aconitate decarboxylase 1, also known as immunoresponsive gene 1), a key regulator of immunometabolism during infection and in ammation (Fig. S1e).During the acute in ammatory stage (days 1 and 2), most cells were cells from Clusters 1, 4, and 5, while there were only minor increases in the numbers of cells in Clusters 2 and 3.With the transition from the acute in ammatory stage to the regenerative stage (day 3), the proportions of cells in Clusters 1, 4, and 5 decreased, and there was a marked increase in the proportion of cells in Cluster 2, which then declined by day 7.In contrast, the proportion of cells in Cluster 3 gradually and consistently increased over 7 days, peaking at the regenerative stage.Thus, our ndings identi ed ve macrophage subsets with dynamic changes throughout the regenerative process.Although the Clusters 2 and 3 subsets were present in high proportions during the in ammatory-to-regenerative transition stage on day 3, Cluster 2 cells expressed relatively higher levels of genes involved in macrophage-mediated tissue regeneration than did Cluster 3 cells (Fig. S1e).For example, IGF1 is one of the best-characterized growth factors and has been shown to regulate muscle regeneration 26 .IGF1 binds its receptor IGF1R to phosphorylate the intracellular adapter protein insulin receptor substrate-1 (IRS-1), which in turn activates the PI3K/AKT pathway to facilitate skeletal muscle regeneration.Moreover, ablation of triggering receptor expressed on myeloid cells-2 (TREM2), a major macrophage sensor known for supporting immune cell responses, has been noted to impede hepatic reparative processes in response to metabolic disruptions 27,28 .In view of these ndings, it is conceivable that the macrophage Cluster 2 we identi ed could play a role in facilitating muscle regeneration.
A tissue-regenerative macrophage subset exhibiting a resident macrophage gene signature Resident and recruited macrophages play distinct roles in immune defense, with resident macrophages providing a constant level of immune surveillance, while recruited macrophages respond to acute infections or injuries.To visualize differential gene expression patterns, we summarized the origin of each monocyte/macrophage subset and examined the known marker genes associated with resident and recruited macrophages 29 .Resident macrophage signature genes, such as Axl, Cd74, and Cxcl16, were highly expressed in Clusters 2 and 3. Clusters 4 and 5 were characterized by the expression of the recruited macrophage markers Cxcr2, I tm1, and Sell.(Fig. S2a and b).However, it is noteworthy that Cluster 2 was conspicuously absent on day 0 (Fig. 1g), suggesting that Cluster 2 cells may not originate from resident macrophages.Our subsequent cell trajectory analysis demonstrated that Clusters 4 and 5 were the major in ltrating macrophage populations and likely contributed to Cluster 2 subset formation (Fig. S3a).Our ndings revealed that clusters 1, 4, and 5 consisted of in ammatory macrophages.In contrast, clusters 2 and 3 were tissue-resident macrophages in the in ammatory-to-regenerative transition stage during skeletal muscle regeneration.

Identi cation of GPNMB-expressing macrophages as critical effectors in skeletal muscle regeneration
A key analysis in the investigation of the molecular mechanisms underlying changes in the state of macrophages is the identi cation of differentially expressed genes along the pseudotime trajectory, i.e., that determined by trajectory inference 30 from single-cell RNA-sequencing data.This inferred trajectory highlights the key effectors within macrophage subsets that govern the biological processes of regeneration.By integrating the relative trajectory positions of the macrophage clusters with the distribution density of each identi ed group (Fig. 1h), Cluster 4 cells were identi ed as mainly present at the beginning of the trajectory; Cluster 3 cells were predominant at both ends of the trajectory, cells in Clusters 1 and 5 were identi ed at the early and middle positions, and Cluster 2 cells were identi ed at the end of the pseudotime axis.Notably, the positions of Cluster 3 cells at both ends of the pseudotime axis, together with their high proportions in uninjured muscle (Fig. 1g), suggest that Cluster 3 may represent a subset with steady-state characteristics.To further investigate the biological and functional roles of Cluster 2 macrophages during skeletal muscle regeneration, volcano plots were generated to visualize the DEGs in Cluster 2 cells vs. cells in Clusters 1, 3, 4, and 5.The top 10 up-and downregulated DEGs are labeled in the plots; among these DEGs, the Gpnmb gene was signi cantly upregulated, speci cally in Cluster 2 (Fig. S3b).To further investigate the importance of macrophages with high GPNMB expression in tissue regeneration, we analyzed signi cantly activated macrophage marker genes 31 using a pseudotime approach, focusing on critical factors involved in tissue regeneration and brosis.We found that Mertk, Igf1, and Nr1h3 exhibited the same expression pattern as Gpnmb (Fig. 2a); previous studies have highlighted the signi cance of these genes in tissue regeneration 26,[32][33][34] .Nuclear receptor subfamily 1 group H member 3 (Nr1h3) is a gene that encodes a transcription factor involved in lipid metabolism and cholesterol homeostasis.
In the late stage of tissue regeneration, macrophages adopt an anti-in ammatory phenotype that helps to suppress in ammatory responses and restore normal tissue structure and function.However, a dysregulated response can result in persistent in ammation and maladaptive regeneration, ultimately leading to tissue-destructive brosis.Previous studies have indicated that, in a chronic in ammatory environment, the GPNMB secreted by macrophages can stimulate excessive deposition of ECM, ultimately leading to pulmonary brosis 35 .After skeletal muscle injury, macrophages play a key role in clearing apoptotic cells and aiding tissue regeneration, a process that involves the conversion of in ltrating monocytes to macrophages with in ammatory and regenerative phenotypes.Our results revealed time-dependent gene expression changes in Gpnmb and Ly6c, revealing that the Gpnmb hi Ly6c lo macrophage population was predominantly enriched in Cluster 2 on day 3 (Fig. 2b).These ndings highlight the importance of identifying speci c GPNMB-expressing macrophage subsets during skeletal muscle regeneration.Based on the temporal dynamics of GPNMB expression and previous descriptions of tissue-resident macrophages 36,37 , we named this cell subset GPNMB hi Ly6C lo "regenerative macrophages".To validate the ndings from single-cell RNA-seq analysis, we employed ow cytometry to assess the abundance of the GPNMB hi Ly6C lo subset in the CD45 + CD11b + cell population in murine muscle postinjury and observed a peak on day 3, followed by a decrease to approximately 10.4%.Furthermore, our histological staining of tissue sections from various time points revealed cells with prominent GPNMB staining on day 3 (Fig. 2c).These ndings suggest that macrophages with high GPNMB expression exhibit characteristics reminiscent of M2 macrophages.To validate the ndings from single-cell RNA-seq analysis, we employed ow cytometry to identify the GPNMB hi Ly6C lo subset in the CD45 + CD11b + cell population in murine muscle postinjury and observed a peak in the proportion on day 3, followed by a decrease to approximately 10.4%.Furthermore, our histological staining of tissue sections at various time points revealed prominent GPNMB-positive cells on day 3 (Fig. 2d).These ndings suggest that macrophages with high GPNMB expression exhibit characteristics reminiscent of M2 macrophages.

CellChat identi es communication patterns and predicts the functions of macrophage subsets involved in skeletal muscle regeneration
Since a direct comparison of DEGs might not comprehensively capture the intricate signaling network, we conducted a thorough investigation of macrophage cellular communication dynamics.We performed CellChat 24 analysis at distinct time points and revealed regulatory cell-cell interactions.On day 3, we observed increased levels of tissue regeneration-related signaling factors, including IGF 12,38 , GAS 39,40 , GDF 14 , and nicotinamide phosphoribosyltransferase (NAMPT) 7 .On day 3, macrophages tended to send signals of IGF and GDF and receive signals of IGF, GAS, and GDF (Fig. S4a).In ammatory signals, such as IL-1, IL-2, and TNF in macrophages, were abundant on days 1 and 2 (Fig. S4b).In addition to the signaling pathway network as a time-resolved signature, we also predicted the putative interactions among ligand and receptor pairs (Fig. 2e and f).On day 3, three critical skeletal muscle regeneration signaling pathways, GAS6/AXL, GAS6/MERTK, and IGF1/IGF1R, were highly enriched.IGF1/IGF1R has been demonstrated to promote skeletal muscle regeneration 12,38 .The TYRO3, AXL, and MERTK (TAM) receptor tyrosine kinases and their cognate glycoprotein ligands growth arrest-speci c 6 (GAS6) and protein S (PROS1) are critical regulators of tissue homeostasis and in ammation 41 .Our results are consistent with the concept that TAM receptors are activated in macrophages in response to tissue injury 42 .The heightened activity of these three critical pathways con rms their roles in muscle regeneration and highlights their coordinated contribution to regenerative mechanisms.

GPNMB promotes M2 macrophage polarization via the upregulation of speci c transcription factors
To validate this hypothesis, we isolated murine bone marrow-derived cells (BMDCs) and induced their differentiation into M1 and M2 macrophages in vitro (Fig. 3a).mRNA and protein expression analyses revealed a signi cant increase in GPNMB expression in M2 macrophages (Fig. 3b and c).These results indicate that GPNMB is a marker of M2 macrophages involved in muscle tissue regeneration and led us to hypothesize that the overexpression of GPNMB promotes M2 macrophage polarization.We conducted GPNMB overexpression experiments to verify this hypothesis in murine bone marrow-derived macrophages (mBMDMs).Our ndings demonstrated that ectopic GPNMB expression increased the expression of M2 macrophage markers without affecting the expression of M1 macrophage markers (Fig. 3d).The overexpression of GPNMB was found to activate key transcription factors linked to M2 macrophage polarization, such as Irf4 and Pparg.In contrast, the expression of the critical regulators Irf5, Nfkb1, and Stat1 in M1 macrophages was unaffected by ectopic GPNMB expression (Fig. 3e), indicating that GPNMB may serve as part of the M2 gene expression program in macrophages.

GPNMB knockout impairs skeletal muscle regeneration
To investigate the role of GPNMB in muscle regeneration, we generated GPNMB-knockout mice and subjected them to CTX-induced muscle injury.The temporal evolution of muscle regeneration was monitored and compared with that of wild-type (WT) C57BL/6 controls (Fig. 4a).Western blot analysis of muscle tissue extracts con rmed the absence of the GPNMB protein in the KO group, con rming that the observed phenotypic differences were attributable to GPNMB de ciency (Fig. 4b).The gross morphological analysis of the TA muscles from the GPNMB-KO mice revealed marked impairment in muscle regeneration at days 4 and 7 postinjury (Fig. 4c).Compared with those from the WT mice, the injured muscles from the GPNMB-KO mice were visibly less striated.Histological evaluations conducted through hematoxylin and eosin (H&E) staining provided further insights into the compromised regenerative response in GPNMB-KO mice.Without GPNMB, the injured muscles exhibited exacerbated in ammatory responses and a notable delay in myo ber regeneration (Fig. 4d).The injured sites in the GPNMB-KO mice were characterized by increased cellular in ltration and a lack of newly formed myo bers.This phenotype starkly contrasted with the organized regenerative patterns observed in WT controls.Quantitative analyses were performed to measure the cross-sectional area (CSA) of muscle bers, a key indicator of regenerative progress.On day 4 postinjury, the mean CSA of regenerating myo bers in GPNMB-KO mice was signi cantly reduced, indicating a failure to initiate the regenerative process properly.This defect persisted through day 7, with KO mice displaying markedly smaller myo bers than WT mice (Fig. 4e).The statistical signi cance of these differences was con rmed, underscoring the necessity of GPNMB for e cient muscle regeneration.Our ndings unveil a previously unappreciated role for GPNMB in facilitating muscle tissue repair, suggesting that GPNMB may act as a modulator of the cellular and molecular events that orchestrate the complex process of muscle regeneration following acute injury.
Impaired macrophage efferocytosis and muscle regeneration following GPNMB knockout and MERTK inhibition GPNMB overexpression in primary murine macrophages led to the upregulation of the expression of efferocytosis-related genes, including Mertk and Axl, suggesting that GPNMB plays a regulatory role in the genetic orchestration of the efferocytosis process (Fig. 5a).We further investigated the functional role of GPNMB in macrophage-mediated efferocytosis.GPNMB-de cient macrophages exhibit a marked reduction in the phagocytosis of apoptotic cells.Fluorescence microscopy and ow cytometry analyses revealed that compared with their wild-type counterparts, mBMDMs from GPNMB-KO mice exhibited signi cantly decreased uptake of CFSE-labeled apoptotic C2C12 myoblasts, underscoring the importance of GPNMB in the clearance of apoptotic cells (Fig. 5b).To understand the broader implications of GPNMB de ciency for muscle regeneration, we further explored the impact of disruption of efferocytosis on muscle regeneration by employing a pharmacological approach to inhibit MERTK.The administration of a MERTK inhibitor resulted in signi cant de cits in muscle tissue architecture and repair, as evidenced by histopathological evaluations (Fig. 5c-f).Notably, administering the inhibitor led to a dose-dependent exacerbation of muscle repair impairment, particularly at higher dosages (Fig. 5c).Histological analysis of muscle regeneration revealed that MERTK inhibition, particularly at a higher dosage, severely compromised the muscle repair process on days 4 and 7 postinjury.These morphological defects were con rmed by measuring muscle ber CSA and length (Fig. 5d-f).These results underscore the importance of the GPNMB-MERTK axis in muscle regeneration and highlight a potential therapeutic target for promoting tissue repair following injury.

GPNMB stimulation promotes muscle regeneration and the myogenic differentiation of murine myoblasts
Previous research has indicated that the GPNMB expressed on macrophages undergoes enzymatic processing by disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) 43 to generate soluble GPNMB.Soluble GPNMB has critical functions; for example, it interacts with CD44 to promote cancer cell stemness and metastasis 44 .Via a similar pathway, soluble GPNMB promotes mesenchymal stromal cell survival, proliferation, and migration 45 .Moreover, the binding of soluble GPNMB to syndecan-4 impedes the extravasation of activated T cells into in amed skin 46 .To investigate the potential of GPNMB to promote myogenic differentiation of myoblasts, we performed myotube differentiation assays using C2C12 cells with or without the addition of recombinant GPNMB (rGPNMB).C2C12 cells treated with or without rGPNMB treatment were collected at several time points during the differentiation process, including 0 (before differentiation), 12, 24, and 72 hours.The expression of Myod and Myog gradually increased during myoblast differentiation, and the addition of rGPNMB signi cantly increased their expression.However, rGPNMB only temporarily elevated the expression of the early differentiation marker Myf2a at 12 hours and did not affect the expression of the myoblast proliferation marker Myf5 at any time point (Fig. 6a).Immuno uorescence staining revealed that treatment with rGPNMB resulted in increased MyHC expression (Fig. 6b) and the formation of larger myotubes (Fig. 6c) containing a greater number of nuclei (Fig. 6d) in C2C12 cells than in the rGPNMB withdrawal group.Furthermore, our in vitro data indicate that rGPNMB facilitates myoblast differentiation.In vivo, we examined the role of exogenous GPNMB in facilitating skeletal muscle regeneration by administering 10 or 20 µg of rGPNMB to injured skeletal muscle.The results showed that the delivery of rGPNMB restored muscle architecture at the injury site (Fig. 6e).Speci cally, on day 4, compared to injury alone, a single dose of rGPNMB led to a signi cant increase in the CSA of the TA muscle (225 ± 110.2 vs. 128 ± 43.2 µm 2 , p = 0.019, in the 10 µg group; 408 ± 139.5 vs. 128 ± 43.2 µm 2 , p < 0.0001, in the 20 µg group) Fig. 6f.Furthermore, on day 7, the effect of rGPNMB was sustained, as shown by the marked increase in the CSA of the TA muscle (728 ± 246.6 vs. 471 ± 195.3 µm 2 , p = 0.0013 in the 10 µg group; 1051 ± 340.9 vs. 471 ± 195.3 µm 2 , p < 0.0001 in the 20 µg group) Fig. 6g.

DISCUSSION
Skeletal muscle regeneration is a complex process orchestrated by various types of cells, including muscle stem cells, FAPs, and immune cells 1 .The contributions of macrophages to muscle regeneration promotion have been recognized for many years 9 .Recent studies have demonstrated that macrophagederived cells such as IGF1 12 , GDF15 14 , and NAMPT 7 are effectors of skeletal muscle regeneration.In this study, we provide the rst temporal single-cell transcriptional characterization of macrophage subsets during skeletal muscle regeneration and highlight the heterogeneity of the macrophage population and its importance in the dynamics of muscle regeneration.We identi ed ve macrophage clusters that differed in gene expression signatures and temporal dynamics during muscle regeneration.In particular, we identi ed a subset of macrophages characterized by elevated expression of GPNMB, IGF1, GDF15, and NAMPT.Furthermore, nutrient and oxygen availability, danger signals, antigens, or instructional signals from other cells trigger changes in key metabolic regulatory events in immune cells.Previous studies have linked macrophage activation status to metabolic remodeling 47 , such as the enhancement of FAO and OXPHOS in M2 macrophages, which are crucial for M2 activation 48 .We found that Cluster 1 macrophages exhibited signi cantly increased expression of M2 macrophage genes, including those involved in FAO, oxidative phosphorylation, and the TCA cycle.These results suggest that high GPNMB expression in Cluster 1 macrophages plays a role in facilitating metabolic reprogramming to support M2 activation.
GPNMB has recently been reported to regulate macrophage in ammatory responses by inhibiting NF-κB signaling through its interaction with CD44 20 .However, the function of GPNMB varies among different tissue cells.For instance, liver-derived GPNMB binds to the CD44 receptor on white adipose tissue, leading to an increase in lipogenesis via the CD44-PI3K-mTORC pathway and resulting in obesity and insulin resistance 49 .Furthermore, the soluble form of GPNMB has been demonstrated to promote the recruitment of mesenchymal stromal cells, thereby promoting cutaneous wound healing 18,50 .Additionally, growth factors such as IGF1 can promote skeletal muscle regeneration, and NAMPT can activate muscle stem cells through C-C motif chemokine receptor type 5. Here, we report a previously unrecognized subset of macrophages that contributes to muscle regeneration.Importantly, our CellChat results provided insights into the autocrine/paracrine mechanisms involving IGF1, GAS, GDF, and NAMPT signaling interactions among macrophages at different stages of muscle regeneration.
Macrophages secrete factors that facilitate tissue regeneration by promoting the proliferation, differentiation, and activation of various cell types, including stem and precursor cells.These cells adopt an anti-in ammatory phenotype during the later stages of tissue regeneration to suppress in ammatory responses and restore typical tissue structure.Dysregulation of this process can lead to persistent in ammation and maladaptive regeneration processes, ultimately resulting in tissue-destructive brosis 31 .Our RNA velocity analysis revealed the temporal dynamics of macrophages and identi ed Gpnmb, Mertk, Igf1, and Nr1h3 as pivotal indicators during skeletal muscle regeneration.These ndings highlight the importance of GPNMB in this regenerative process.In response to efferocytosis, MERTK is activated by the intracellular modi cation of membrane cholesterol, which yields steroid metabolites that activate Nr1h3, which binds directly to the MERTK promoter to promote transcription 51 .Previous studies have shown that GAS6 can bind to phosphatidylserine (PtdSer), which is externalized on apoptotic cell membranes 52 , and activate MERTK on macrophages 6 ; this provides positive feedback to further increase MERTK expression and ultimately shift macrophage polarization toward the M2 phenotype.Our results showed that the day 3 cell populations and Cluster 1 subset have the characteristics of both receivers and senders of signals in the GAS6 signaling pathway.Discrepancies in the expression patterns of proin ammatory and anti-in ammatory markers, as opposed to those of established genes, suggest the complexity of macrophage activation and the in uence of environmental factors, warranting further research.Importantly, we identi ed a speci c subset of high-GPNMB-expressing macrophages, termed GPNMB hi Ly6C lo regenerative macrophages, characterized by their time-dependent gene expression patterns and macrophage surface markers.Immunohistochemical staining revealed a high level of accumulation of these GPNMB hi Ly6C lo macrophages in the injured skeletal muscle area,supporting their involvement in tissue regeneration.In vitro examination of the expression of GPNMB in murine M1 and M2 macrophages revealed that GPNMB was signi cantly upregulated in M2 macrophages compared with M1 macrophages, suggesting that GPNMB plays a role in M2 macrophage polarization.A gain-offunction assay was conducted to further validate that GPNMB overexpression promotes M2 polarization, indicating that GPNMB may serve as a macrophage M2 marker.In summary, our study revealed the importance of GPNMB-expressing macrophages, along with the genes Mertk, Igf1, and Nr1h3, in tissue regeneration.Our study also revealed the signi cance of GPNMB-expressing macrophages and associated genes in tissue regeneration by revealing the gene expression timeline of regenerative macrophage subsets in injured muscle.
Muscle regeneration was indicated by signi cant increases in the CSA and multinuclear myo ber count in the TA muscle following the injection of rGPNMB.These results demonstrate the potential therapeutic role of GPNMB in promoting skeletal muscle regeneration.However, GPNMB may regulate multiple cell types involved in skeletal muscle regeneration in addition to in ltrating macrophages; these other cell types include FAPs and MuSCs.Further research is needed to elucidate the speci c interactions and contributions of GPNMB hi Ly6C lo /regenerative macrophages and other cell types to the overall process of skeletal muscle regeneration.The ndings presented in this single-cell study revealed the dynamics of macrophage subpopulation evolution during skeletal muscle regeneration and revealed the critical role of GPNMB hi Ly6C lo regenerative macrophages in this process.Our analyses suggested that these macrophages secrete soluble GPNMB into the microenvironment to promote the proliferation, differentiation, and maturation of MuSCs or myogenic progenitors.This research highlights the potential of GPNMB as a therapeutic target for promoting skeletal muscle regeneration and suggests a possible mechanism through which GPNMB exerts its effects.

CONFLICT OF INTEREST
The declare no competing interests. Figures

Figure 3 Ectopic
Figure 3 (e)Overexpression of GPNMB results in heightened expression of M2-related transcription factors Irf4 and Pparg, suggesting a potential pathway for GPNMB-mediated macrophage polarization.