Absence of CCR2 results in an inflammaging environment in young mice with age-independent impairments in muscle regeneration

Abstract

Skeletal muscle regeneration requires coordination between dynamic cellular populations and tissue microenvironments. Macrophages, recruited via CCR2, are essential for regeneration; however, the contribution of macrophages and the role of CCR2 on nonhematopoietic cells has not been defined. In addition, aging and sex interactions in regeneration and sarcopenia are unclear. Muscle regeneration was measured in young (3-6 mo), middle (11-15 mo), old (24-32 mo) male and female CCR2^-/- mice. Whereas age-related muscle atrophy/sarcopenia was present, regenerated myofiber cross-sectional area (CSA) in CCR2^-/- mice was comparably impaired across all ages and sexes, with increased adipocyte area compared with wild-type (WT) mice. CCR2^-/- mice myofibers achieved approximately one third of baseline CSA even 84 d after injury. Regenerated CSA and clearance of necrotic tissue were dependent on bone marrow-derived cellular expression of CCR2. Myogenic progenitor cells isolated from WT and CCR2^-/- mice exhibited comparable proliferation and differentiation capacity. The most striking cellular anomaly in injured muscle of CCR2^-/- mice was markedly decreased macrophages, with a predominance of Ly6C^- anti-inflammatory monocytes/macrophages. Ablation of proinflammatory TLR signaling did not affect muscle regeneration or resolution of necrosis. Of interest, many proinflammatory, proangiogenic, and chemotactic cytokines were markedly elevated in injured muscle of CCR2^-/- relative to WT mice despite impairments in macrophage recruitment. Collectively, these results suggest that CCR2 on bone marrow-derived cells, likely macrophages, were essential to muscle regeneration independent of TLR signaling, aging, and sex. Decreased proinflammatory monocytes/macrophages actually promoted a proinflammatory microenvironment, which suggests that inflammaging was present in young CCR2^-/- mice.

Keywords: TLRs; monocytes/macrophages; myogenic progenitor cells; sarcopenia.

Figure 1.. Effects of sex and age on baseline myofiber size, regenerated myofiber size, and fat area in CCR2^−/− mice.

CSA of myocytes and area (%) of adipocytes in TA muscle of male and female mice of different ages—young (3–6 mo), middle (11–15 mo), and old (24–32 mo)—obtained at baseline (no injury, day 0) and at various times (d) after injury. (A) Myofiber CSA at baseline and in regenerated myofibers after CTX muscle injury. Regenerated myofibers were decreased (P < 0.001) at all postinjury time points compared with baseline. (B) Intramuscular fat area at baseline and in injured/regenerated muscle. Fat area was increased (P ≤ 0.02) at all postinjury time points compared with baseline. Data are presented as means ± sem; n = 3–18 mice/group/time point. ^P ≤ 0.05 compared with young mice for each sex at the corresponding time point; ⁺P ≤ 0.03 compared with middle-age mice for each sex at the corresponding time point; ^#P ≤ 0.03 compared with male mice at the corresponding age group and time point.

Figure 2.. Effects of BM donor genotype on myofiber regeneration and fat area in young, male chimera mice.

(A) Regenerated myofiber CSA was decreased (P < 0.001) at all postinjury time points compared with baseline; range of 2736–3174 µm². (B) Intramuscular fat area in injured/regenerated muscle; baseline fat area range of 0.00–0.01%. Data are presented as means ± sem; n = 5–11 mice/group/time point; *P ≤ 0.03 compared with WT→WT; ^#P ≤ 0.04 compared with CCR2^−/−→CCR2^−/−.

Figure 3.. Similar in vitro proliferation and differentiation between MPCs isolated from young, male WT and CCR2^−/− mice.

For proliferation, MPCs were subcultured after the third passage and maintained in MPC growth media on type I collagen–coated dishes. (A) WT and CCR2^−/− MPC proliferation. (B and C) Distribution of cells in different phases of the cell cycle was established by flow cytometry in WT (B) and CCR2^−/− (C) MPCs. For differentiation, MPCs were subcultured after the third passage and maintained in MPC differentiation media on entactin-collagen IV-laminin–coated dishes. (D and E) Differentiation potential (D) and fusion index (E) were determined on the basis of nuclei in myosin heavy chain (MHC)–positive cells and myotubes. Data are presented as means ± sem; n = 3 different MPC primary cultures/mouse strain used at passage 3. *P ≤ 0.04 compared with 24-h time point for each strain.

Figure 4.. Inflammatory cell recruitment in young, male WT and CCR2^−/− mice after CTX injury.

Cells isolated from CTX-injured muscle were analyzed by flow cytometry at the indicated times. (A–F) Total cells (A), neutrophils (CD11b⁺Ly6G⁺) (B), monocytes (CD11b⁺(CD90/B220/CD49/NK1.1/Ly6G)⁻(F4/80/I-Ab/CD11c)⁻Ly6C^+/−) (C), macrophages (CD11b⁺F4/80⁺) (D), monocyte subsets (E), and macrophage subsets (F). Data are presented as means ± sem; n = 3 mice/strain/time point. ^#P ≤ 0.05 WT vs. CCR2^−/− mice at the corresponding time point; ^†P ≤ 0.003 Ly6C⁺ monocyte subset WT vs. CCR2^−/− mice at the corresponding time point; ^§P ≤ 0.01 Ly6C⁻ monocyte subset WT vs. CCR2^−/− mice at the corresponding time point; *P ≤ 0.03 for Ly6C⁺CD301⁻ macrophage subset WT vs. CCR2^−/− mice at the corresponding time point; ^‡P ≤ 0.02 for Ly6C⁻CD301⁻ macrophage subset WT vs. CCR2^−/− mice at the corresponding time point.

Figure 5.. Increased proinflammatory cytokines/chemokines in young, male CCR2^−/− mice compared with WT mice after CTX injury.

(A–H) Measurement of tissue cytokines/chemokines normalized to total protein in the anterior compartment of WT and CCR2^−/− mice after CTX-induced injury for TNF-α (Α), IL-12p70 (B), GM-CSF (C), MIP-1β (D), MCP-1 (E), MCP-3 (F), MCP-5 (G), and MIP-2 (H). Data are presented as means ± sem; n = 4–8 mice/strain/time point. ^#P ≤ 0.02 WT vs. CCR2^−/− mice at the corresponding time point.

Figure 6.. Diverse cytokine/chemokine elevations in young, male CCR2^−/− mice compared with WT mice after CTX injury.

(A–H) Measurement of tissue cytokines/chemokines normalized to total protein in the anterior compartment of WT and CCR2^−/− mice after CTX-induced injury for KC/GRO (A), IL-3 (B), IL-4 (C), IL-7 (D), SCF (E), SDF-1 (F), oncostatin (OSM) (G), and TIMP-1 (H). Data are presented as means ± sem; n = 4–8 mice/strain/time point. ^#P ≤ 0.04 WT vs. CCR2^−/− mice at the corresponding time point.