Exosomes derived from inflammatory myoblasts promote M1 polarization and break the balance of myoblast proliferation/differentiation

Abstract

BACKGROUND

Acute muscle injuries are one of the most common injuries in sports. Severely injured muscles are prone to re-injury due to fibrotic scar formation caused by prolonged inflammation. How to regulate inflammation and suppress fibrosis is the focus of promoting muscle healing. Recent studies have found that myoblasts and macrophages play important roles in the inflammatory phase following muscle injury; however, the crosstalk between these two types of cells in the inflammatory environment, particularly the exosome-related mechanisms, had not been well studied.

AIM

To evaluate the effects of exosomes from inflammatory C2C12 myoblasts (IF-C2C12-Exos) on macrophage polarization and myoblast proliferation/differentiation.

METHODS

A model of inflammation was established in vitro by lipopolysaccharide stimulation of myoblasts. C2C12-Exos were isolated and purified from the supernatant of myoblasts by gradient centrifugation. Multiple methods were used to identify the exosomes. Gradient concentrations of IF-C2C12-Exos were added to normal macrophages and myoblasts. PKH67 fluorescence tracing was used to identify the interaction between exosomes and cells. Microscopic morphology, Giemsa stain, and immunofluorescence were carried out for histological analysis. Additionally, ELISA assays, flow cytometry, and western blot were conducted to analyze molecular changes. Moreover, myogenic proliferation was assessed by the BrdU test, scratch assay, and CCK-8 assay.

RESULTS

We found that the PKH-67-marked C2C12-Exos can be endocytosed by both macrophages and myoblasts. IF-C2C12-Exos induced M1 macrophage polarization and suppressed the M2 phenotype in vitro. In addition, these exosomes also stimulated the inflammatory reactions of macrophages. Furthermore, we demonstrated that IF-C2C12-Exos disrupted the balance of myoblast proliferation/differentiation, leading to enhanced proliferation and suppressed fibrogenic/myogenic differentiation.

CONCLUSION

IF-C2C12-Exos can induce M1 polarization, resulting in a sustained and aggravated inflammatory environment that impairs myoblast differentiation, and leads to enhanced myogenic proliferation. These results demonstrate why prolonged inflammation occurs after acute muscle injury and provide a new target for the regulation of muscle regeneration.

Key Words: C2C12 myoblast; Exosomes; Macrophage polarization; Inflammation; Differentiation; Proliferation

Core Tip: For successful muscle regeneration, macrophage polarization and myogenesis should be supported by an appropriate combination of cells and their signals. As the communication between myoblasts and macrophages within the inflammatory environment is unknown, we aimed to evaluate the effects of IF-C2C12-Exos on macrophage polarization and myoblast proliferation/differentiation. We found that IF-C2C12-Exos could induce M1 polarization, resulting in a sustained and exacerbated inflammatory environment, impaired myoblast fibrogenic/myogenic differentiation, and led to abnormal myogenic proliferation. These results indicate a potential mechanism for the development of long-term inflammation following acute muscle injury, but further preclinical evaluations targeting IF-C2C12-Exos in animal models are necessary.

INTRODUCTION

More than half of sports injuries in athletes have been reported to be related to muscle damage[1]. In addition to promoting muscle regeneration, preventing scar formation is a key factor in the healing of injured muscle[2-4], as localized fibrotic tissue can lead to susceptibility to re-injury of injured muscles[5,6].

Skeletal muscle fibrosis is related to excessive local inflammation and myoblast fibrogenic differentiation in the early stage after injury[7-11]. During the general inflammatory process following injury, M1 macrophages (classically activated) are recruited and lead to further muscle damage[12], which are then substituted by M2 macrophages (alternatively activated) to promote muscle regeneration and differentiation[13]. Sometimes, M2 polarization is suppressed by the excessive inflammatory conditions that follow acute muscle injury[14,15]. Given the timing of the appearance of M1 coupled with that of myoblasts within 1-3 d after muscle injury[12,16], we suppose that myoblasts may be able to influence the shift from M1 macrophages to M2 subtype. Periodontal ligament stem cells and adipocytes within the inflammatory environment have been reported to inhibit the M2 polarization of macrophages through exosomes[17,18]. Many species of living cells such as myoblasts secrete exosomes that can be transported and regulate the essential cell activities such as proliferation and differentiation of neighboring cells[19-22]. Therefore, we speculated that in the prolonged inflammatory micro-environment of injured muscle, myoblasts might promote M1 macrophage polarization and suppress M2 phenotype through exosomes.

Additionally, Guescini et al[23] reported that exosomes derived from myotubes can influence the balance of proliferation and differentiation of normal myoblasts after H₂O₂ administration, suggesting that the C2C12-Exos could also regulate muscle healing through self-control[23]. In our previous studies, we found that myoblasts could be converted to myofibroblasts after muscle injury due to its intracellular signals including lncRNA-MFAT1/H19, miR-122-5p/25-3p, and transforming growth factor-β (TGF-β)/SMAD[24-26], which may contribute to muscle fibrosis. Therefore, it is worth investigating whether C2C12-Exos could influence the fibrogenic/myogenic differentiation and proliferation of normal myoblasts.

In this study, we isolated C2C12-Exos from myoblasts within an inflammatory environment, tested their functions, and investigated the potential underlying mechanisms of the effects of C2C12-Exos on the immunomodulation of macrophages and myoblasts proliferation/differentiation in vitro.

MATERIALS AND METHODS

Cell culture

C2C12 myoblasts (murine cell line), purchased from ScienCell Research Laboratories, were maintained in high glucose Dulbecco's modified eagle medium (DMEM) (HyClone) with 10% fetal bovine serum (FBS) and 0.5 mL of penicillin/streptomycin solution (#0503; ScienCell Research Laboratories) in a humidified incubator at 37°C and 5% CO₂ atmosphere. Muscle differentiation was induced using 2% horse serum (ThermoFisher) for C2C12. Lipopolysaccharide (LPS), at a concentration of 1000 ng/mL was used to induce an inflammatory environment for C2C12. The medium was then washed three times to remove all the LPS and a fresh exosome-depleted medium was added. C2C12 conditioned media (C2C12-CM) were collected using a 1 mL pipette and added into 50 mL tubes after 24 h incubation. The media were then kept at -80°C before use. Exosomes from C2C12 myoblasts (C2C12-Exos) were extracted by the following steps.

RAW264.7 cells (mouse leukemia cells of monocyte-macrophage), purchased from the American Type Culture Collection, were cultured in DMEM containing 10% FBS and 0.5 mL of penicillin/streptomycin solution in a humidified incubator at 37°C and 5% CO₂ atmosphere.

Isolation and identification of C2C12-Exos

The extraction procedure for C2C12-Exos was based on a previous method[27]. 50 mL conditioned culture medium containing 5 mL exosome-free FBS (Exosome-depleted FBS, Gibco), and 0.5 mL of penicillin/streptomycin solution were used to culture C2C12s for 48 h. After the C2C12s had grown to more than 90% confluence, the cells were treated with LPS for 1 d. Then, fresh culture medium was added, and the cells were kept quiescent for 24 h, and then all the supernatant was collected. After that, all media were subjected to sequential centrifugation (Optima XPN-100 ultracentrifuge; Beckman Coulter SW 41 Ti rotor) at 10000 ×g for 35 min (to remove the cell debris) and then at 100000 ×g for 70 min. After this step, at 100000 ×g for 70 min the precipitate was washed twice with phosphate-buffered saline (PBS). The C2C12-Exos were resuspended in PBS and stored at -80°C prior to processing.

To observe the morphology of exosomes, C2C12-Exos were assessed directly under transmission electron microscopy (Tecnai G2 Spirit, Tecnai) and scanning electron microscopy (SEM, MIRA3 FEG-SEM, TESCAN). The SEM procedures were based on a previous study[28]. Generally, 100 μL of the exosome suspension was frozen overnight in the refrigerator and then transferred to a vacuum dryer for lyophilization. An appropriate amount of freezing glue was applied to the sample table and then the exosome lyophilized powder was spread onto the sample table. The samples were then coated with gold with an ion sputterer and observed under the microscope.

To assess the absolute size distribution of C2C12-Exos, they were analyzed using a NanoSight NS300 (Malvern, United Kingdom). The particles were automatically tracked and sized using nanoparticle tracking analysis (NTA) based on Brownian motion and diffusion coefficients.

To identify the surface markers of C2C12-Exos, exosomes were assessed by flow cytometry analysis using a commercially Exo-Flow capture kit, including CD9, CD47, CD63, and CD81 flow antibodies (System Biosciences, CA, United States). These procedures were per the published studies[29].

Additionally, the C2C12-Exos were identified by Western blotting with anti-CD63, anti-CD9, anti-Alix, and anti-HSP60 antibodies (purchased from Abcam or Affinity) (Table 1).

Table 1 Primary antibodies used in the experiment.

Antibody	Source	Catalog No.	Type	Dilution	M.W. (kD)
CD63	Affinity	AF5117	Rabbit mAb	1:1000(W.B.)	47
CD9	Affinity	AF5139	Rabbit mAb	1:2000(W.B.)	23
Alix	Affinity	ab275377AF0184	Rabbit mAb	1:2000(W.B.)	95
HSP60	Affinity	AF0199	Rabbit mAb	1:2000(W.B.)	60
iNOS	Affinity		Rabbit mAb	1:1000(W.B.)	130
ARG1	Affinity	DF6657	Rabbit mAb	1:1000(W.B.)	35
CD86	Abcam	ab220188	Rabbit mAb	1:1000(W.B.)	38
				1:100(IF)
CD86 PE	eBioscience	12-0862-81	Rat mAb	0.125 μg/test	(Flow Cyt)
CD206	Affinity	DF4149	Rabbit mAb	1:1000(W.B.)	120
CD206	Abcam	ab64693	Rabbit mAb	1:500(IF)
	eBioscience	17-1631-80	Rat mAb	0.25 μg/test	(Flow Cyt)
CD163APC	Abcam	ab51263	Mouse mAb	1:1000(W.B.)	227
MYHC				1:500(IF)
MyoD1	Affinity	AF7733	Rabbit mAb	1:1000(W.B.)	60
MyoD1	Proteintech	18943-1-AP	Rabbit PAb	1:200(IF)
MyoG	Abcam	ab1835	Mouse mAb	1:1000(W.B.)	25
Collage 1	Affinity	AF7001	Rabbit mAb	1:1000(W.B.)	140
α-SMA	Affinity	AF1032	Rabbit mAb	1:1000(W.B.)	42
Tubulin	Affinity	T0023	Mouse mAb	1:1000(W.B.)	55
GAPDH	Affinity	AF7021	Rabbit mAb	1:1000(W.B.)	37
F4/80 APC	eBioscience	47-4801-80	Rat mAb	0.125 μg/test	(Flow Cyt)

α-SMA: α-smooth muscle actin.

Cell intervention

C2C12 and RAW264.7 cells were incubated at 1 × 10⁷ cells in 10 cm plates and divided into groups according to different IF-C2C12-Exos concentrations or conditioned medium. After 24 or 48 h of incubation, macrophages were collected for flow cytometry/western blot or fixation to assess histological changes. After 48 h of treatment with high concentrations of IF-C2C12-Exos, macrophages were collected in conditioned medium. In this study, we defined this as M1CM, meaning that the macrophages in the medium were more of the M1 subtype rather than M2 or M0 macrophages. In addition, C2C12 cells were incubated with exosomes for 24 h for further experiments, including proliferation and differentiation (Figure 1).

Figure 1 Experimental design for both the in vitro and in vivo studies. IF-C2C12-Exos, Myoblast-derived exosomes within the inflammatory environment; Molecular evaluation included Western Blot, ELISA, immunofluorescence, and flow cytometry. Histological evaluation included immunofluorescence and electron microscopy. PBS: Phosphate-buffered saline.

Exosome labeling with PKH67

Isolated C2C12-Exos were labeled with PKH67 (a Green Fluorescent Labeling Kit (Sigma, Aldrich, MINI67-1KT)), and the procedures followed the manufacturer’s protocol. C2C12-Exos or PBS were stained with PKH67 dye in 500 μL of Diluent C solution for 5 min at room temperature. After that, 1 mL 1% BSA was used to stop the labeling process. Then the re-purified exosomes underwent ultracentrifugation with a PBS rinse for 30 min. The labeled PBS or C2C12-Exos were co-incubated with C2C12 cells and macrophages (M0) for 12 h at 37°C, in a 5% CO₂ cell incubator. After incubation, the culture medium was discarded and the cells were washed with PBS three times. Cells were fixed with 4% PFA and nuclei were counterstained with DAPI for 5 min. The uptake of labeled exosomes by C2C12 and macrophages was observed by a fluorescence microscope (ECHO Revolve, United States). Six random images of cells were taken, and PKH-67 positive cells were counted. The total number of cells was calculated using the DAPI staining method. The positive rate of PKH-67 = PKH-67 positive cells/total number of cells.

Histological analysis

Macrophage differentiation and morphology: M0 macrophages were incubated with different concentrations of IF-C2C12-Exos for 2 d (1 × 10⁹, 1 × 10¹⁰, 2 × 10¹⁰/medium). Macrophage cultures were then viewed directly under a microscope (ECHO Revolve, United States).

Immunofluorescence staining: To examine the expression and location of target proteins, cells were immunofluorescently stained as previously described[30]. The primary antibodies used were anti-CD86, anti-CD206, anti-MYHC, anti-iNOS, and anti-MyoD1. DAPI was used to locate the nuclei of the cells. Images were taken using fluorescence microscopy (ECHO Revolve, United States).

Giemsa stain: C2C12 cells were washed with PBS, fixed with 4% PFA for 10 min, and rinsed three times with fresh PBS for 5 min each time. The fixed cells were then incubated with Giemsa stain (Phygene, China) for 50 min. Cells were rinsed with tap water for 3 min and then photographed using an inverted microscope. Three images were taken randomly at 200 × magnification for each well. The numbers of total and fused cell nuclei were counted. Cell counting was performed using ImageJ. The fusion rate was defined as the percentage of total nuclei being in myotubes/total nuclei of C2C12 cells. The diameter and length of the myotube were analyzed by Image-Pro Plus 7.0.

Flow cytometry for M1/M2

Flow cytometry was performed using anti-CD86-PE, anti-CD163-APC, and anti-F4/80-FITC (Thermo/eBio). The percentage of CD86 +/CD163 +/F4/80 + cell population (macrophages) was evaluated using Cytomics™ FC 500 (Beckman Coulter). In detail, different groups of macrophages were collected with flow cytometry staining buffer (eBioscience). Then, 2 μL antibody was added to each 100 μL of cell suspension for 60 min at 4°C in the dark. Then, 5 mL staining buffer was put into each tube and the cell suspension was centrifuged for 5 min (500 ×g, 4℃). The washing procedures were repeated three times. Finally, the cell precipitation was re-suspended in 200 μL PBS for flow cytometry analysis.

Western blot

Protein was extracted and analyzed using an established method[31]. Briefly, total protein from C2C12 was collected using RIPA lysis buffer (R0010; Solarbio, Beijing, China) with phenylmethanesulfonyl fluoride (PMSF; Solarbio, Beijing, China). Protein concentrations were measured using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Next, 10 μg protein samples from each group were separated by 10% SDS-PAGE. Afterward, they were transferred to nitrocellulose membranes. 5% non-fat milk dissolved in Tris-buffered saline containing Tween-20 was utilized to block the blots before applying primary antibodies overnight at 4℃. Anti-CD206, anti-Arginase 1, anti-iNOS, anti-CD86, anti-MYHC, anti-MyoD1, anti-MyoG, anti-Col 1, anti-α-SM, anti-β-Tubulin, and anti-GAPDH antibodies were applied as primary antibodies (Table 1). Each group contained 4 protein samples for calculation (n = 4).

C2C12 proliferation and migration

BrdU test: The proliferation of C2C12 cells was determined using the 5-Bromo-2’-deoxyuridine (BrdU) incorporation assay kit (Cell Signaling Technology, MA, United States) according to the manufacturer’s instructions. Briefly, cells were seeded into a 6-well plate with 1 × 10⁶ cells per well. BrdU solution was added into each well 3 h prior to IF-C2C12-Exos treatment. Cell proliferation was evaluated using the average number of BrdU + cells per view.

Scratch assay: To assess the cell migration properties of C2C12, an in vitro scratch assay was performed by scratching a straight line in the middle of cells cultured for 24 h. Then, different concentrations of IF-C2C12-Exos were applied for 24 h and observed under an inverted microscope. Cell migration ability was evaluated by the percentage of wound-healing rate (distance migrated/ original wound distance × 100%).

CCK-8 assay: The Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, Shanghai, China) assay was used to assess the viability of C2C12 cells after IF-C2C12-Exos treatment. The general procedure followed that outlined in a previous study[32]. Briefly, primary cells were seeded into a 96-well plate (Thermo Fisher Scientific, MA, United States) with 1 × 10³ cells per well and treated with IF-C2C12-Exos for 24 h. Next, CCK-8 solution (10 μL) was applied to each well, and the plate was incubated for 2 h and the absorbance of each well was measured at 450 nm using a Microplate Reader (Bio-Rad, Hercules, CA, United States). Cell viability was evaluated by average absorbance of the IF-C2C12-Exos treatment group/absorbance of control group × 100%.

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) kits, including interleukin (IL)-6, IL-1β, TGF-β, and tumor necrosis factor-α (TNF-α), were purchased from Laizee (LEM060-2, LEM012-2, LEM822-2, LEM810-2). The protocol followed previous studies[33]. Cell supernatants were collected from each group and the kits were then utilized following the manufacturer's instructions. In detail, 100 μL of samples and standard samples were added to the corresponding well, the plates were sealed and incubated at room temperature for 2 h. The samples were then discarded and the plates were washed 5 times with 300 μL of wash and drained on paper. Then 100 μL avidin HRP solution was added to each well, the plate was sealed, and incubated at room temperature for 30 min. The washing procedure was repeated. Next, 100 μL TMB solution was added to each well, the plate was sealed, and incubated for 15 min at room temperature. Finally, 50 μL of terminator solution was added to terminate the reaction and the plate was analyzed at 450 nm.

Statistical analysis

All experiments were performed at least three times. Data were analyzed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, United States) and presented as the mean ± SD. Significance was typically analyzed by Student's t-test, or one-way ANOVA followed by post hoc LSD test. P < 0.05 was regarded as significant. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001 and ^dP < 0.0001.

RESULTS

C2C12-CM regulated macrophage polarization

We first investigated the effects of conditioned medium on macrophage polarization in normal myocytes or myoblasts in an inflammatory environment. LPS at 500 ng/mL and IL-4 at 20 ng/mL were utilized as a positive control, while the fresh exosome-depleted medium served as a negative control. We found that IF-CM and LPS induced M1 polarization (significantly more CD86+ cells compared with the control group), while NC-CM and IL-4 promoted M2 polarization (markedly more CD163+ cells compared with the control group) (Supplementary Figure 1). These results suggested that there was crosstalk between myoblasts and macrophages through their secretion. Therefore, we performed further experiments.

M1CM hindered myoblast myogenic differentiation

IF-C2C12 stimulated macrophages towards the M1 subtype for two days. Fresh medium was then added to the macrophages and 24 h later conditioned medium was collected to treat normal C2C12 (Supplementary Figure 2A). Giemsa stain and immunofluorescence results showed that the myotubes after M1CM administration were smaller and fewer than those of controls, which suggested that the myoblast myogenic differentiation ability was weakened by M1CM (Supplementary Figure 2B and C).

Identification and characterization of C2C12-Exos after LPS stimulation

Under transmission and SEM, C2C12-Exos appeared spherical or globular in shape (Figure 2A and B). Flow cytometry results demonstrated that exosomal markers (CD9, CD47, CD63, and CD81) were highly expressed in C2C12-Exos (Figure 2C). The NTA experiment evaluated the size of C2C12-Exos. The diameters ranged from 50-130 nm, which are consistent with the data from previous studies (Figure 2D). Western blot results showed that exosomal marker proteins (CD9, CD63, Alix, and HSP60) were highly expressed in C2C12-Exos, while these proteins were expressed in the exosome-depleted fractions (Figure 2E).

Figure 2 Purification, isolation, and characterization of C2C12-Exos. A: Flowchart of C2C12-Exos purification based on differential ultra-centrifugation. Lipopolysaccharide, 1000 ng/mL for 1 d; B: The morphology of C2C12-Exos was observed by transmission electron microscopy (left) and scanning electron microscopy (right). The red arrows indicate representative exosomes; C: Representative flow cytometry plots showing the phenotypes of exosome markers, including CD9, CD47, CD63, and CD81; D: The particle size distribution of C2C12-Exos was analyzed by the qNano platform; E: Western blotting showed the presence of exosomal markers, including CD63, HSP60, Alix, and CD9. The four lanes represent different exosomal proteins and deproteinized supernatants extracted from the two independent conditioned media. LPS: Lipopolysaccharide.

Exosomes tracing in vitro

To investigate how C2C12-Exos communicated with C2C12 and macrophages, C2C12-Exos were added to cultured C2C12 and RAW264.7 cells. PKH-67 (green) labeled C2C12-Exos were internalized into C2C12/macrophages and were identified in the cytoplasm after 12 h of co-culture. The Dye-only group showed no PKH67 signals (Figure 3A). The PKH-67 positivity rate was significantly higher in macrophages and C2C12 than in the Dye-only group (Figure 3B and C).

Figure 3 C2C12-Exos were taken up by C2C12 and RAW264.7 cells in vitro. A: Representative images of the uptake of PKH67-labeled exosomes (green) by RAW264.7 cells or C2C12 cells (DAPI blue) and fluorescence uptake by negative control samples. Dye-only, only PKH67 incubated with RAW264.7 cells and C2C12 cells; scale bar = 25 μm; B and C: The percentage of PKH67 positive cells in RAW264.7 or C2C12 is presented. ^dP < 0.0001, n = 6.

IF-C2C12-Exos induced M1 macrophage polarization in vitro

To examine the effects of IF-C2C12-Exos on macrophages, different concentrations of IF-C2C12-Exos were added to the culture system. After 48 h, as the concentration increased, the macrophages became more elongated and predominantly spindle-shaped compared with their original circular shape (Figure 4A). In addition, immunofluorescence staining showed an increase in CD86-positive cells and a decrease in CD206-positive cells as the concentration of IF-C2C12-Exos increased (Figure 4B). Moreover, flow cytometry results showed a significant increase in the proportion of M1 macrophages and a significant decrease in the proportion of M2 macrophages in the IF-C2C12-Exos group (Figure 4C-E). These data demonstrate that IF-C2C12-Exos induce the polarization of macrophages towards M1 in vitro.

Figure 4 IF-C2C12-Exos induced M1 macrophage polarization in vitro. A: Images of macrophages cultured with different concentrations of C2C12-Exos for 2 d (1 × 10⁹, 1 × 10¹⁰, 2 × 10¹⁰/medium). Scale bar = 200 μm or 100 μm; B: Immunofluorescence localization of CD86 (red), marker of M1 and CD206 (green), marker of M2 after culture with different concentrations of C2C12-Exos for 2 d. Scale bar = 90 μm. Arrows indicate CD206 or CD86 positive cells; C: Representative flow cytometry plots showing the percentages of M1 (CD86 + CD4/80+) and M2 (CD163 + CD4/80+) phenotype in macrophages after culture with different concentrations of C2C12-Exos for 24 h; D: Quantification of flow cytometry data (n = 3); E: M2 macrophages percentage. Data are presented as mean ± SD. ^aP < 0.05; ^bP < 0.01. NS: Not significant.

IF-C2C12-Exos induced inflammatory reactions of macrophages in vitro

To further investigate the effects of IF-C2C12-Exos on macrophages, inflammatory-related factors were evaluated. The results of western blot demonstrated that IF-C2C12-Exos significantly up-regulated the protein expression (iNOS and CD86) and down-regulated that of CD206 and Arg1 (Figure 5A-D). In addition, immunofluorescence staining results showed that the relative expression and area of iNOS gradually increased with increasing concentrations of IF-C2C12-Exos (Figure 5E). Furthermore, ELISA results showed that the concentrations of cytokine IL-6, TNF-α, and IL-1β in the culture supernatants significantly increased, while that of TGF-β did not show obvious changes after IF-C2C12-Exos treatment for 24 h (Figure 5F). Taken together, these data suggested that IF-C2C12-Exos can induce inflammatory reactions of macrophages in vitro.

Figure 5 IF-C2C12-Exos induced inflammatory reactions of macrophages in vitro. A-D: iNOS, CD86, CD206, and Arg1 protein levels in macrophages were determined by western blot after culturing with different concentrations of C2C12-Exos for 24 h (1 × 10¹⁰, 2 × 10¹⁰/medium). (n = 4). Data are expressed as the mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001; E: Immunofluorescence localization and relative expression of iNOS, a marker of inflammatory level, in macrophage medium after culture with different concentrations of C2C12-Exos for 24 h. Scale bar = 90 μm; F: The concentration of cytokine interleukin (IL)-6, transforming growth factor-β, tumor necrosis factor-α, and IL-1β in supernatants of macrophage cells after culture with IF-C2C12-Exos or phosphate-buffered saline for 24 h measured by ELISA (n = 3). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. PBS: Phosphate-buffered saline. IL: Interleukin; TGF-β: Transforming growth factor-β; TNF-α: Tumor necrosis factor-α; NS: Not significant.

IF-C2C12-Exos promoted C2C12 proliferation in vitro

To evaluate the effects of IF-C2C12-Exos on normal myoblasts, we first examined the proliferative capacity of the cells. The BrdU assay results showed that treatment with high concentrations of IF-C2C12-Exos (1 × 10¹⁰, 2 × 10¹⁰/medium) significantly increased the mean number of BrdU-positive cells, indicating that these C2C12 cells gained higher proliferation capacity (Figure 6A and C). Next, the wound-healing rate in the IF-C2C12-Exos group was significantly higher than that of the control group, which suggested that the migration capacity of C2C12 was enhanced by the IF-C2C12-Exos (Figure 6B and D). Furthermore, the results of the CCK-8 assay revealed a significant increase in the viability of C2C12 cells in the 2 × 10¹⁰/medium concentration group after IF-C2C12-Exos treatment, while the low concentration group (1 × 10⁹, 1 × 10¹⁰) showed a small but insignificant increase in viability (Figure 6E). Overall, these data demonstrate that IF-C2C12-Exos can stimulate normal C2C12 proliferation in vitro.

Figure 6 IF-C2C12-Exos stimulated C2C12 proliferation in vitro. A: Cell proliferation ability of C2C12 was determined using the BrdU incorporation assay after 24 h IF-C2C12-Exos incubation. Green signal = BrdU. Scale bar = 180 μm; B: The cell migration ability of C2C12 was determined by the wound-healing assay after 24 h IF-C2C12-Exos incubation; C and D: Quantification of BrdU and wound-healing data (n = 3). Data are presented as mean ± SD. ^aP < 0.05, ^bP < 0.01; E: Cell proliferation ability of C2C12 was further determined using the CCK-8 assay. Data are presented as mean ± SD. ^aP < 0.05.

IF-C2C12-Exos impaired C2C12 differentiation in vitro

The fusion index of control myoblasts in differentiation and those treated with IF-C2C12-Exos were slightly different at the early stages (2 d) under all conditions tested (Figure 7A and B). More interestingly, after 4 d of differentiation, the fusion indices of the IF-C2C12-Exos treatment groups were significantly lower than that of the control group (14%-16% vs 32%) (Figure 8A and B).

Figure 7 IF-C2C12-Exos impaired early C2C12 muscle differentiation in vitro. A: Immunofluorescence was used to detect relative expression and distribution of MYHC and Myod1 on day 2 after different treatments. Green, red, and blue signals represent MYHC, Myod1, and nucleus, respectively. Scale bar = 90 μm; B, D and E: Quantification of myotube length, diameter, and fusion rate. Data are presented as mean ± SD. n = 12 or 3. ^aP < 0.05; C: Representative images of myotube after culture with different concentrations of IF-C2C12-Exosomes for 24 h by Giemsa staining (2 d 2% horse-serum incubation). Scale bar = 100 μm.

Figure 8 IF-C2C12 Exos impaired C2C12 muscle differentiation in vitro. A: Immunofluorescence was used to detect relative expression and distribution of MYHC and Myod1 on day 4 after different treatments. Green, red, and blue signals represent MYHC, Myod1, and nucleus, respectively. Scale bar = 90 μm; B, D and E: Quantification of myotube length, diameter, and fusion rate. Data are presented as mean ± SD. n = 12 or 3. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001; C: Representative images of myotube after culture with different concentrations of IF-C2C12-Exosomes for 24 h by Giemsa staining (4 d 2% horse-serum incubation). Scale bar = 100 μmT.

In addition, our data also showed that myotube diameter and length were significantly affected by IF-C2C12-Exos treatments, with IF-C2C12-Exos inducing a significant decrease in myotube size at both early and late stages of muscle differentiation. As a result, there was a significant difference in the diameter and length of control and IF-C2C12-Exos myotubes, and this difference increased at higher IF-C2C12-Exos concentrations (Figures 7C-E and 8C-E). Finally, modulation of the myoblast fibrogenic/myogenic differentiation process in response to IF-C2C12-Exos treatment was investigated by western blot analysis. MyoG, MyoD1, MYHC, α-smooth muscle actin (SMA), and Col 1 protein expression levels showed a significant downregulation after 24 h of IF-C2C12-Exos treatment (Figure 9). Altogether, these data suggest that IF-C2C12-Exos impairs C2C12 muscle differentiation in vitro and triggers a shift in the balance of proliferation/differentiation towards proliferation.

Figure 9 IF-C2C12 Exos down-regulated C2C12 fibrogenic/myogenic differentiation-related proteins in vitro. A-D: MYHC, MYOD1, and MYOG protein levels in C2C12 medium were determined by western blot after incubation with different concentrations of C2C12-Exos for 24 h (1 × 10⁹, 1 × 10¹⁰, 2 × 10¹⁰/medium) (n = 4); E-G: Col 1 and α-smooth muscle actin. Data are presented as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. α-SMA: α-smooth muscle actin.

DISCUSSION

This is the first study to demonstrate that myoblasts within the inflammatory environment crosstalk with surrounding macrophages. In this study, IF-C2C12-Exos was shown to promote M1 polarization of M0 macrophages and to influence the balance of myoblast proliferation/differentiation in vitro.

Following acute mechanical injury, skeletal muscle develops significant inflammation[34,35]. If the M1 stage of macrophages persists after acute muscle injury, it will result in a prolonged inflammatory environment in the damaged area[14,36]. However, the underlying mechanism by which the M1 phenotype remains at the early and mid-stage of injury is less well understood. The similar appearance time of myoblasts and M1 macrophages after skeletal muscle injury may suggest a crosstalk between themselves[12,16]. CD86 and CD163 expression levels can reflect the polarization stage of macrophages[37]. In this study, the exosomes from inflammatory C2C12 myoblasts was found to induce higher levels of CD86 expression (M1 marker) than that of CD163 expression (M2 marker) in macrophages. Furthermore, we found that the inflammatory reactions in macrophages were also aggravated by IF-C2C12-Exos. A previous study[23] reported that exosomes from H₂O₂ treated myotubes could stimulate RAW264.7 macrophages to express higher levels of IL-6, which is consistent with our findings. However, we found other inflammatory factors, such as IL-1β, TNF-α, and iNOS, were also upregulated in macrophage cultures following administration of high concentrations of IF-C2C12-Exos. This suggests that IF-C2C12-Exos may regulate macrophages to exert higher level inflammatory responses than those of H₂O₂-myotube-Exos. Interestingly, IF-C2C12-Exos reduced M2 macrophage expression, while IF-C2C12-CM did not exert that effect but also induced M1 polarization. Many studies have proved that macrophage polarization was regulated by surrounding environmental factors including cytokines and exosomes[38,39]. These results suggested that IF-C2C12-Exos can regulate the polarization of macrophages and maintain a prolonged inflammatory environment, while myoblasts in the inflammatory environment can continue to secrete IF-C2C12-Exos. This local positive feedback intercellular mechanism was observed in Xu et al[20]'s study, whereby C2C12-Exos promoted pre-osteoblasts differentiation to osteoblasts[20].

In this study, IF-C2C12-Exos were found to impair C2C12 differentiation and promote proliferation in vitro. In detail, decreased levels of MyoD, MyoG, and MYHC protein levels[25,26,40] suggesting that IF-C2C12-Exos reduced the myogenic differentiation ability of myoblasts. Induction experiments of myogenic differentiation also provided direct evidence for this result. In addition, the BrdU, CCK-8, and scratch assays[41] showed that a higher ability of myoblast proliferation was induced by IF-C2C12-Exos. This result is consistent with previous literature where exosomes from C2C12 myotube after H₂O₂ or TNF-α/interferon-γ treatment enhanced proliferation but impaired myogenic differentiation[23,42].

Significant and prolonged inflammation after acute muscle injury can result in muscle fibrosis[10,11,14,36]. Additionally, promoting M1 macrophages to M2 during the inflammatory phase after muscle injury prevents muscle fibrosis[27,43,44]. Interestingly, myocyte IF-C2C12-Exos treatment resulted in a significant decrease in protein levels of the fibrosis markers (Col 1, α-SMA), implying that the fibrogenic capacity of normal myoblasts was also suppressed[45]. We speculate that IF-C2C12-Exos are only secreted by myoblasts in the acute inflammatory stage (1-5 d after injury). They may disappear after day 5 due to inflammatory dissipation, meanwhile, M2 macrophages secrete a large amount of TGF-β, which activates myoblasts into fibroblasts, leading to ECM production[24,26,46]. However, due to prolonged inflammation caused by M1 macrophages, M2 polarization is incomplete and tissue remodeling is maladaptive, which leads to subsequent fibrosis[27,47]. Our results suggest that myoblasts passed information to surrounding myoblasts, telling them to grow faster but not differentiate under inflammatory conditions. This effect would have favored muscle regeneration, as large numbers of myoblasts are required to support muscle repair[48,49]. However, abnormal proliferation with down-regulated myogenic differentiation indeed hinders the muscle healing process after the acute inflammatory stage[50,51]. Additionally, the main effects of IF-C2C12-Exos are not to induce myoblast-derived fibrosis but to induce proliferation. A previous study proved that myoblasts are key in muscle fibrosis[52], and another study found that only promotion of myoblast proliferation cannot prevent fibrosis[6]. Therefore, we speculate that the IF-C2C12-Exos actually prepare the conditions for later fibrotic differentiation (accumulation of undifferentiated myogenic cells).

Together, the above results suggest that (1) IF-C2C12-Exos can induce macrophages towards M1 polarization, leading to a prolonged and aggravated inflammatory environment. In turn, the inflammatory factors stimulate myoblasts to produce more IF-C2C12-Exos, which forms a vicious circle; and (2) IF-C2C12-Exos can impair fibrogenic/myogenic differentiation, and lead to proliferation (Figure 10).

Figure 10  Graphic abstract. IF-C2C12-Exos promoted M1 macrophage polarization and myoblast proliferation, and inhibited myoblast fibrogenic/myogenic differentiation. IL: Interleukin; TNF-α: Tumor necrosis factor-α.

There are several limitations in this study that should be noted. First, we only utilized an in vitro model to study the effects of IF-C2C12-Exos on macrophages and myoblasts. However, muscle injury is a complex physiological and pathological issue with complex in vivo factors[53]. Further studies should consider validating the role of exosomes in animal models. Second, we only added IF-C2C12-Exos once; thus, multiple administrations should be considered, and the optimal concentration of exosomes should be studied in future research. Third, our study did not involve an in-depth mechanistic study. For example, myotubes were identified to promote myoblast fusion through exosomal miRNA[54]. Therefore, understanding the potential underlying mechanisms of how IF-C2C12-Exos can control macrophage polarization and influence the balance of myoblast proliferation/differentiation would be our future goal.

Given the pathophysiological significance of the findings of this study, further studies are needed to elucidate mechanisms responsible for these effects which deserve investigation. It is hoped that further studies will identify specific targets involved in muscle regeneration and fibrosis, such as lncRNAs and miRNAs.

CONCLUSION

Overall, this study demonstrates that IF-C2C12-Exos can promote M1 polarization and impair myoblasts differentiation. Normal or inflammatory myoblasts play a pivotal role, in fact, they release distinct Exos carrying a complex range of signals directed to the surrounding cells. Signals associated with Exos, by virtue of their diversity and specificity, may contribute to a fine reprogramming of the muscle regeneration process in a cooperative manner.

ARTICLE HIGHLIGHTS

Research background

More than half of sports injuries in athletes have been reported to be related to muscle damage. Severely injured muscles are prone to re-injury due to fibrotic scar formation caused by prolonged inflammation. How to regulate inflammation and suppress fibrosis is the focus of promoting muscle healing.

Research motivation

Recent studies have found that myoblasts and macrophages play important roles in the inflammatory phase following muscle injury; however, the crosstalk between these two types of cells in the inflammatory environment, particularly the exosome-related mechanisms, has not been well studied.

Research objectives

This study aimed to evaluate the effects of exosomes from inflammatory C2C12 myoblasts (IF-C2C12-Exos) on macrophage polarization and myoblast proliferation/ differentiation.

Research methods

A model of inflammation was established in vitro by lipopolysaccharide stimulation of myoblasts. Multiple methods were used to isolate and identify the exosomes. Gradient concentrations of IF-C2C12-Exos were added to normal macrophages and myoblasts. PKH67 fluorescence tracing, microscopic morphology, Giemsa staining, immunofluorescence, ELISA assays, flow cytometry, western blot, BrdU test, scratch assay, and CCK-8 assay were conducted to determine the mechanism of IF-C2C12-Exos.

Research results

We found that the PKH-67-marked C2C12-Exos can be endocytosed by both macrophages and myoblasts. IF-C2C12-Exos induced M1 macrophage polarization and suppressed the M2 phenotype in vitro. These exosomes also stimulated the inflammatory reactions of macrophages. Furthermore, we demonstrated that IF-C2C12-Exos disrupted the balance of myoblast proliferation/differentiation, leading to enhanced proliferation and suppressed fibrogenic/myogenic differentiation.

Research conclusions

IF-C2C12-Exos can induce M1 polarization, resulting in a sustained and aggravated inflammatory environment that impairs myoblast differentiation, and leads to enhanced myogenic proliferation. These results demonstrate why prolonged inflammation occurs after acute muscle injury and provide a new target for the regulation of muscle regeneration.

Research perspectives

Given the pathophysiological significance of the findings of this study, further studies are needed to elucidate the mechanisms responsible for these effects which deserve investigation. It is hoped that further studies will identify specific targets involved in muscle regeneration and fibrosis, such as lncRNAs and miRNAs.

ACKNOWLEDGEMENTS

We are grateful to the Shanghai Institute of Nutrition and Health for providing the experimental platform.