Macrophages (MPs) are important for skeletal muscle regeneration in vivo and may exert beneficial effects on myogenic cell growth through mitogenic and antiapoptotic activities in vitro. However, MPs are highly versatile and may exert various, and even opposite, functions depending on their activation state. We studied monocyte (MO)/MP phenotypes and functions during skeletal muscle repair. Selective labeling of circulating MOs by latex beads in CX3CR1GFP/+ mice showed that injured muscle recruited only CX3CR1lo/Ly-6C+ MOs from blood that exhibited a nondividing, F4/80lo, proinflammatory profile. Then, within muscle, these cells switched their phenotype to become proliferating antiinflammatory CX3CR1hi/Ly-6C− cells that further differentiated into F4/80hi MPs. In vitro, phagocytosis of muscle cell debris induced a switch of proinflammatory MPs toward an antiinflammatory phenotype releasing transforming growth factor β1. In co-cultures, inflammatory MPs stimulated myogenic cell proliferation, whereas antiinflammatory MPs exhibited differentiating activity, assessed by both myogenin expression and fusion into myotubes. Finally, depletion of circulating MOs in CD11b–diphtheria toxin receptor mice at the time of injury totally prevented muscle regeneration, whereas depletion of intramuscular F4/80hi MPs at later stages reduced the diameter of regenerating fibers. In conclusion, injured skeletal muscle recruits MOs exhibiting inflammatory profiles that operate phagocytosis and rapidly convert to antiinflammatory MPs that stimulate myogenesis and fiber growth.

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Adult skeletal muscle has the unique capacity to regenerate. Muscle regeneration is always associated with inflammation and notably macrophages (MPs), which play dual role. Soon after injury, inflammatory monocyte‐derived macrophages (M1 phenotype) stimulate myogenic cell proliferation. After phagocytosis of muscle debris, MPs switch their phenotype to acquire an anti‐inflammatory profile (M2) and stimulate myogenic cell differentiation and myofibre growth. Here, we explored the role of AMPK in the resolution of inflammation during muscle repair. AMPKα1 KO muscle shows both a delay and an impairment of post‐injury regeneration. These deficiencies are also observed in LysM‐CRE;AMPKfl/fl muscle, confirming the MP specificity of AMPK requirement. In vitro, AMPKα1 KO MPs hardly acquire a M2 profile upon cytokine stimulation. Their phagocytic activity is also altered. In vivo analysis of MP subpopulations (using the AMPKα1−/−;CX3CR1GFP/+ mouse) during muscle repair shows that the number of intramuscular MPs exhibiting the M2 phenotype is reduced in the AMPKα1 KO compared to the WT mouse. Accordingly, leukocytes from AMPKα1 KO muscle do not increase their expression of markers associated with the resolution of inflammation during muscle regeneration. These results strongly support that AMPKα1 is one key regulator of MP switch at time of resolution of inflammation and is essential for a proper muscle repair.

Macrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/ polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here, we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory MPs inhibited MPC fusion while anti-inflammatory MPs strongly promoted MPC differentiation by increasing their commitment into differentiated myocytes and the formation of mature myotubes. Furthermore, the in vivo time course of expression of myogenic and MP markers was studied in regenerating human healthy muscle after damage. We observed that regenerating areas containing proliferating MPCs were preferentially associated with MPs expressing proinflammatory markers. In the same muscle, regenerating areas containing differentiating myogenin-positive MPCs were preferentially coupled to MPs harboring anti-inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates tissue repair. STEM CELLS 2013;31:384-396 Disclosure of potential conflicts of interest is found at the end of this article.

Highlights d In DMD muscle, Ly6C pos macrophages produce latent TGF-b1 due to high LTBP4 synthesis d AMPK activation downregulates LTBP4 expression and TGF-b1 production by macrophages d Metformin treatment of DMD mice decreases fibrosis and improves muscle function d Fibroblast-derived enzymes activate latent TGF-b1, which acts on fibroblasts

Macrophages are necessary for skeletal muscle regeneration after injury. Muscle recruits inflammatory monocytes/macrophages that switch toward an anti-inflammatory profile upon phagocytosis of debris. In vitro, proinflammatory macrophages stimulate myoblast proliferation, whereas anti-inflammatory macrophages stimulate their differentiation. Thus, macrophages are involved in both phases of skeletal muscle regeneration: first, inflammation and cleansing of necrosis, and then myogenic differentiation and tissue repair.

The mechanisms underlying stromal cell supportive functions are incompletely understood but probably implicate a mixture of cytokines, matrix components and cell adhesion molecules. Skeletal muscle uses recruited macrophages to support post-injury regeneration. We and others have previously shown that macrophages secrete mitogenic factors for myogenic cells. Here, we focused on macrophage-elicited survival signals. We demonstrated that: (1) macrophage influx is temporally correlated with the disappearance of TUNEL-positive apoptotic myogenic cells during post-injury muscle regeneration in mice; (2) direct cell-cell contacts between human macrophages and myogenic cells rescue myogenic cells from apoptosis, as assessed by decreased annexin V labelling and caspase-3 activity, and by increased DIOC-6 staining, Bcl-2 expression and phosphorylation of Akt and ERK1/2 survival pathways; (3) four pro-survival cell-cell adhesion molecular systems detected by DNA macroarray are expressed by macrophages and myogenic cells in vitro and in vivo - VCAM-1-VLA-4, ICAM-1-LFA-1, PECAM-1-PECAM-1 and CX3CL1-CX3CR1; (4) macrophages deliver anti-apoptotic signals through all four adhesion systems, as assessed by functional analyses with blocking antibodies; and (5) macrophages more strongly rescue differentiated myotubes, which must achieve adhesion-induced stabilisation of their structure to survive. Macrophages could secure these cells until they establish final association with the matrix.

BackgroundIt has been shown previously that it is possible to obtain growth of Plasmodium falciparum in human erythrocytes grafted in mice lacking adaptive immune responses by controlling, to a certain extent, innate defences with liposomes containing clodronate (clo-lip). However, the reproducibility of those models is limited, with only a proportion of animals supporting longstanding parasitemia, due to strong inflammation induced by P. falciparum. Optimisation of the model is much needed for the study of new anti-malarial drugs, drug combinations, and candidate vaccines.Materials/MethodsWe investigated the possibility of improving previous models by employing the intravenous route (IV) for delivery of both human erythrocytes (huRBC) and P. falciparum, instead of the intraperitoneal route (IP), by testing various immunosuppressive drugs that might help to control innate mouse defences, and by exploring the potential benefits of using immunodeficient mice with additional genetic defects, such as those with IL-2Rγ deficiency (NSG mice).ResultsWe demonstrate here the role of aging, of inosine and of the IL-2 receptor γ mutation in controlling P. falciparum induced inflammation. IV delivery of huRBC and P. falciparum in clo-lip treated NSG mice led to successful infection in 100% of inoculated mice, rapid rise of parasitemia to high levels (up to 40%), long-lasting parasitemia, and consistent results from mouse-to-mouse. Characteristics were closer to human infection than in previous models, with evidence of synchronisation, partial sequestration, and receptivity to various P. falciparum strains without preliminary adaptation. However, results show that a major IL-12p70 inflammatory response remains prevalent.ConclusionThe combination of the NSG mouse, clodronate loaded liposomes, and IV delivery of huRBC has produced a reliable and more relevant model that better meets the needs of Malaria research.