[P1-2-02] Roles of macrophages during skeletal muscle regeneration
キーワード：macrophage、muscle regeneration、satellite cell
Purpose: Skeletal muscle regeneration relies on the proliferation and differentiation of satellite cells. During the regeneration process, infiltrating macrophages are essential to acute skeletal muscle injury repairs by phagocyting necrotic debris and secreting cytokines. However, the coordination between macrophages and satellite cells still remains poorly understood. In this study, we investigated the role of macrophages in the differentiation and kinetics of satellite cells.
Materials and methods: Muscle injury was induced by injecting 50 ul of 1.2% BaCl2 into mouse tibialis anterior muscle. Clodronate-liposomes and Csf1r neutralizing antibody AFS98 were intraperitoneally injected into those mice to deplete macrophage subpopulations. Besides that, we depleted macrophages by injecting tamoxifen into Csf1r CreERT2; Csf1r fl/fl mice. HE staining was used to assess the regeneration process and immunofluorescence was performed to analysis the cellular dynamic changes. The mobility was assessed by rotarod performance.
Results and conclusion: The number of macrophages reached a peak at 3 days post injury (dpi) and they mainly took part in the early stage of the regeneration process. At the first 2 dpi, F4/80(+)Csf1r(-) macrophages dominantly appeared, and F4/80(+)Csf1r(+) macrophages took a major part at 3dpi. Administrations of clodronate liposomes or AFS98 can effectively deplete F4/80(+)Csf1r(+) macrophages throughout the muscle regeneration process. The depletion of macrophages impaired the clearance of necrotic myofibers at 3 dpi and the regenerating myofiber formation at 5 dpi shown as the smaller cross-sectional area of regenerating muscle fibers. F4/80(+)Csf1r(+) macrophages were significantly decreased by injecting tamoxifen at 0 day, 1 dpi and 2 dpi in Csf1r cre; Csf1r fl/fl mice, whereas F4/80(+)Csf1r(-) macrophages were increased. Interestingly, calcium deposition and delayed muscle regeneration were observed in those mice at 5 dpi. These findings suggest that the increase in F4/80(+)Csf1r(-) macrophages are involved in the ectopic calcification. We found that the macrophage depletion impaired the proliferation and differentiation of satellite cells by assessing Ki67(+) pax7 (+) cells and myogenin (+) cells, respectively. The rotarod test, which assesses the recovery of grip strength, was markedly delayed in the hind limb of the macrophage-depleted mice, suggesting that macrophages promote the functional recovery of the injured muscles. Together, the time-dependent appearance of F4/80(+)Csf1r(+) macrophages govern successful skeletal muscle regeneration via the removal of necrotic fibers and regulations of satellite cell proliferation and differentiation.
Materials and methods: Muscle injury was induced by injecting 50 ul of 1.2% BaCl2 into mouse tibialis anterior muscle. Clodronate-liposomes and Csf1r neutralizing antibody AFS98 were intraperitoneally injected into those mice to deplete macrophage subpopulations. Besides that, we depleted macrophages by injecting tamoxifen into Csf1r CreERT2; Csf1r fl/fl mice. HE staining was used to assess the regeneration process and immunofluorescence was performed to analysis the cellular dynamic changes. The mobility was assessed by rotarod performance.
Results and conclusion: The number of macrophages reached a peak at 3 days post injury (dpi) and they mainly took part in the early stage of the regeneration process. At the first 2 dpi, F4/80(+)Csf1r(-) macrophages dominantly appeared, and F4/80(+)Csf1r(+) macrophages took a major part at 3dpi. Administrations of clodronate liposomes or AFS98 can effectively deplete F4/80(+)Csf1r(+) macrophages throughout the muscle regeneration process. The depletion of macrophages impaired the clearance of necrotic myofibers at 3 dpi and the regenerating myofiber formation at 5 dpi shown as the smaller cross-sectional area of regenerating muscle fibers. F4/80(+)Csf1r(+) macrophages were significantly decreased by injecting tamoxifen at 0 day, 1 dpi and 2 dpi in Csf1r cre; Csf1r fl/fl mice, whereas F4/80(+)Csf1r(-) macrophages were increased. Interestingly, calcium deposition and delayed muscle regeneration were observed in those mice at 5 dpi. These findings suggest that the increase in F4/80(+)Csf1r(-) macrophages are involved in the ectopic calcification. We found that the macrophage depletion impaired the proliferation and differentiation of satellite cells by assessing Ki67(+) pax7 (+) cells and myogenin (+) cells, respectively. The rotarod test, which assesses the recovery of grip strength, was markedly delayed in the hind limb of the macrophage-depleted mice, suggesting that macrophages promote the functional recovery of the injured muscles. Together, the time-dependent appearance of F4/80(+)Csf1r(+) macrophages govern successful skeletal muscle regeneration via the removal of necrotic fibers and regulations of satellite cell proliferation and differentiation.