Inhibition of inflammatory CCR2 signaling promotes aged muscle regeneration and strength recovery after injury

Abstract

Muscle regeneration depends on a robust albeit transient inflammatory response. Persistent inflammation is a feature of age-related regenerative deficits, yet the underlying mechanisms are poorly understood. Here, we find inflammatory-related CC-chemokine-receptor 2 (Ccr2) expression in non-hematopoietic myogenic progenitors (MPs) during regeneration. After injury, the expression of Ccr2 in MPs corresponds to the levels of its ligands, the chemokines Ccl2, 7, and 8. We find stimulation of Ccr2-activity inhibits MP fusion and contribution to myofibers. This occurs in association with increases in MAPKp38δ/γ signaling, MyoD phosphorylation, and repression of the terminal myogenic commitment factor Myogenin. High levels of Ccr2-chemokines are a feature of regenerating aged muscle. Correspondingly, deletion of Ccr2 in MPs is necessary for proper fusion into regenerating aged muscle. Finally, opportune Ccr2 inhibition after injury enhances aged regeneration and functional recovery. These results demonstrate that inflammatory-induced activation of Ccr2 signaling in myogenic cells contributes to aged muscle regenerative decline.

Fig. 1. Ccr2 expression in activated SCs and MPs from regenerating muscle.

a, b Representative FACS histograms of Ccr2 reporter fluorescence (eGFP) in a hematopoietic cells (CD45+), and b SCs and MPs (Lin- β1Int + VCAM+) from adult (2 months old) mouse skeletal muscles uninjured (U) and injured muscles, 2- and 5-days post injury (2dpi and 5dpi) (n = 3 mice per condition). c Mean fluorescence intensity of eGFP (Ccr2) in SCs and MPs from adult uninjured and injured limb muscles, 2- and 5-days post injury (2dpi and 5dpi) (n = 3 mice per condition). d Expression of Ccr2 in SCs and MPs from adult uninjured and injured limb muscles, 2- and 5-days post injury (2dpi and 5dpi) (n = 3 mice per condition). e Levels of inflammatory Ccr2 chemokine ligands (Ccl2, Ccl7 and Ccl8) from adult uninjured, 2dpi and 5dpi muscles with multiplex Luminex assay (n = 6 mice per condition). Flow cytometry data are reported as mean ± s.e.m. from at least 500000 events per acquisition. Cells were gated for single cells and live cells (DAPI) prior to specific cell lineage markers (Supplementary Fig. S1) and GFP gating. mRNA levels are reported as fold-change ± s.d. relative to Gapdh and B2m and normalized to experimental control. Luminex data are reported as protein concentration from muscle lysates (mean ± s.e.m., ng/mL). Non-significant (n.s.) P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, non-parametric one-way ANOVA followed by Tukey’s multiple comparisons test. * is relative to uninjured except if otherwise indicated (bar).

Fig. 2. Ccr2 chemokines prevent SC-derived MP terminal commitment and fusion.

a Representative images from immunofluorescence staining of SC cultures derived from adult (2 months old) wildtype (Ccr2^+/+) and Ccr2-null mutant (Ccr2^−/−) mice. Cells were purified by MACS and, 5000 SCs were plated and cultured for 96 h in growing media prior to low serum condition media (differentiation media; DM) supplemented with vehicle (V), Ccr2-ligands (Ccl^2/7/8) or Ccl^2/7/8 and Ccr2 small inhibitor (Ccr2i) for 24 hours. Red, Pax7 (SC); Green, MyoG (Differentiating MP); Blue, DAPI (Nuclei). Experiments were performed at least three times. b, c Percentage of b MyoG+ and c Pax7+ cells of total cells (DAPI+) per culture of SCs derived from adult Ccr2^+/+ (n = 12) and Ccr2^−/− (n = 3) mice. Each dot represents the percentage of positive cells averaged from duplicate cultures from one mouse (n = 3–12 mice). d Representative images from immunofluorescence staining of SC cultures derived from adult wildtype Ccr2^+/+ and Ccr2^−/− mice. Cells were purified by MACS and 10000 cultured for differentiation and myotube formation in DM supplemented with vehicle, Ccl^2/7/8 or Ccl^2/7/8 and Ccr2i for 24 hours. Green, Myosin (Myotubes); Blue, DAPI (Nuclei). Experiments were performed at least three times. e, f MP cultures e fusion index and f cell density (DAPI + /um²). Fusion index was measured by number of fused myonuclei within myotubes relative to total number of cells (DAPI) per culture of SCs derived from adult Ccr2 + /+ (n = 12) and Ccr2−/− (n = 3) mice. Each dot is the averaged data from duplicate cultures from one mouse. Culture data are percentage mean ± s.e.m. For each experiment, cells were derived from one mouse and cultured into at least two wells per condition and >300 cells were counted. Scale bars, (a) 20 μm, (d) 50 μm. Non-significant (n.s.) P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, non-parametric two-way ANOVA followed by Tukey’s multiple comparisons test. Stats are relative to Vehicle except if otherwise indicated (bar).

Fig. 3. Ccr2 represses Myogenin expression through p38δγ signaling.

a Myogenin mRNA expression in SCs derived myotubes from adult (2 months old) wildtype (Ccr2^+/+) (n = 6) and Ccr2-null mutants (Ccr2^−/−) (n = 3) mice treated with vehicle (V), Ccr2-ligands (Ccl^2/7/8) or Ccl^2/7/8 and Ccr2 small inhibitor (Ccr2i) for 24 h. b, c Representative immunoblotting from treated SC derived myotubes (b) and c quantification of phospho-p38δγ normalized to total p38, and of phospho-MyoD normalized to total protein (n = 3 mice). d Immunofluorescence staining of adult SC derived cultures treated with siRNAs against Mapk12/13 or siGLO (control) after 72 h then directed toward differentiation at 96 h in DM supplemented with vehicle or Ccl^2/7/8 for 24 h. Pax7 (Red); MyoG (Green); DAPI (Blue). Experiments were performed at least three times. e, f Percentage of e MyoG+ MPs and f Pax7+ SCs normalized to total number of cells (DAPI+). Each dot represents the percentage of positive cells averaged from duplicate cultures from one mouse (n = 3 mice). RTqPCR analysis was performed in triplicate from at least two animal per experiment and condition. mRNA levels are reported as fold-change ± s.d. relative to Gapdh and B2m and normalized to experimental control. Immunoblots were performed from lysates derived from at least two animal per condition from at least three experiments. Data are mean ± s.e.m. For each experiment we plated cells from one mouse into at least two wells per condition and used at least three mice per group and counted >400 cells. siRNA experiments were pre-validated using two different siRNAs for each gene target and efficient siRNAs were controlled for off-target effects toward other p38MAPK gene members (Supplementary Fig. S5e). Scale bars, d 25 μm. n.s. not significant, *P < 0.05, **P < 0.01, ***P < 0.001, non-parametric one-way ANOVA followed by Tukey’s multiple comparisons test. Asterisk is relative to vehicle from sample condition except if otherwise indicated by a line.

Fig. 4. Prolonged Ccr2 signaling during adult muscle regeneration negatively affects myogenesis.

a Strategy to extend Ccr2-ligands (Ccl^2/7/8) presence in regenerating muscles. Adult (2 months old) mice were injured with intramuscular (IM) injection of barium chloride, followed by IM injection of recombinant chemokines or saline (vehicle) and harvested for analysis at 5dpi. b Immunofluorescence staining of the injured muscles from adult mice treated with vehicle (n = 5) or Ccl^2/7/8 (n = 5). Laminin (red); MyoG (green); DAPI (blue). c Percentage of MyoG+ MPs per area of regeneration (regen. area; μm²) from adult injured muscles at 5dpi following treatment (n = 5 mice per condition). d vMyogenin transcript levels in freshly isolated SCs and MPs from adult injured muscles (5dpi) following chemokines treatment (n = 3 mice per condition). Data are reported as mean ±  s.e.m. Scale bars, 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, non-parametric one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 5. Elevated Ccr2 chemokines in aged regenerating muscle restrains MP contribution.

a Experimental strategy for freshly isolated SC long-term transplant. Adult SCs derived from Ccr2^+/+; Pax7^CreER/+; Rosa26^nTnG/+ (SC^{Ccr2+/+; P7nTnG}; n = 4) or Ccr2^−/−; Pax7^CreER/+; Rosa26^nTnG/+ (SC^{Ccr2−/−; P7nTnG}; n = 4) are indelibly labelled for nuclear GFP (nGFP) allowing to track engraftment and contribution to regeneration. 3000 freshly isolated nGFP SCs were transplanted into an adult or aged regenerating muscle at 4dpi, and contribution analyzed at 35dpi. b, c Representative images (b) and c quantification of long-term transplanted adult (n = 5 mice per condition) and aged (n = 5 mice per condition) muscles. d Experimental strategy for freshly isolated SC culture to obtain fusion-ready MPs prior to transplant and in vivo fusion assays. Adult SCs derived from Ccr2^+/+; Pax7^CreER/+; Rosa26^nTnG/+ (SC^{Ccr2+/+; P7nTnG}; n = 4) or Ccr2^−/−; Pax7^CreER/+; Rosa26^nTnG/+ (SC^{Ccr2−/−; P7nTnG}; n = 4) were isolated and cultured on recombinant extra cellular matrix gels to obtain fusion-competent MPs. 15000 fusion-competent MPs were injected into adult or aged regenerating muscle (4dpi) that were analyzed at 10dpi for fusion competency. e, f Representative images (e) and f quantification of regenerating adult (n = 5 mice per condition) and aged (n = 5 mice per condition) muscles 6 days post-transplant (10dpi) for in vivo fusion assay. Successful engraftment was assessed by quantifying ratio of nGFP+ centrally nucleated fiber (CNF; regenerating) relative to total centrally nucleated fibers in the area of transplant. Area of transplant was determined by staining serial 10 μm cryosections through 2500 μm of the transplanted muscles. Analysis of the engraftment efficiency was confined to the area of transplant and acquired at 20X to keep the area consistent. Scale bars, 25 μm. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 6. Timely inhibition of Ccr2 promotes aged muscle regeneration and strength recovery.

a Schematic for acute muscle injury followed by single intramuscular injection of Ccr2 inhibitor (Ccr2i) or Saline (Vehicle) at 4 days post injury (4dpi). At 5dpi muscles were collected to assess MyoG+ MPs. b, c Immunofluorescence of MyoG+ MPs in adult (4 months old; n = 6 for vehicle and n = 6 for Ccr2i) and aged (24 months old; n = 4 per condition) 5dpi muscles (b) and c number of MyoG+ cells per area of regeneration (regen. area; um²). Laminin (Red); MyoG (Green); DAPI (Blue). d Schematic depicting the strategy to assess cross sectional area (CSA; um²) of regenerated fibers (centrally nucleated fibers; CNF) in adult and aged 10dpi muscles that received a single intramuscular injection of Ccr2i or Vehicle at 4dpi. e, f Representative images (e) and f quantification of regenerated fibers CSA in adult (n = 5 per condition) and aged (n = 5 per condition) mice at 10dpi. Laminin (Red); DAPI (Blue). g, Representative force curves from 10dpi and uninjured, non-injected contralateral (CL) EDL muscles from adult or aged mice treated with Vehicle or Ccr2i at 4dpi. h, i Quantification of the h absolute force recovery and i specific force recovery in regenerated muscles from adult (n = 3 mice) and aged (n = 8 mice per condition). Data are reported as mean ± s.e.m. Scale bars, b 100 μm, e 25 μm. non-significant (n.s.) P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001, non-parametric one-way or two-way ANOVA followed by Tukey’s multiple comparisons test.