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The Development of Macrophage-Mediated Cell Therapy to Improve Skeletal Muscle Function after Injury.

Rybalko V, Hsieh PL, Merscham-Banda M, Suggs LJ, Farrar RP - PLoS ONE (2015)

Bottom Line: A variety of MP phenotypes have been identified and characterized in vitro as well as in vivo.We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion.Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program.

View Article: PubMed Central - PubMed

Affiliation: Department of Kinesiology, The University of Texas at Austin, 1 University Station D3700, Austin, TX 78712, United States of America.

ABSTRACT
Skeletal muscle regeneration following acute injury is a multi-step process involving complex changes in tissue microenvironment. Macrophages (MPs) are one of the key cell types involved in orchestration and modulation of the repair process. Multiple studies highlight the essential role of MPs in the control of the myogenic program and inflammatory response during skeletal muscle regeneration. A variety of MP phenotypes have been identified and characterized in vitro as well as in vivo. As such, MPs hold great promise for cell-based therapies in the field of regenerative medicine. In this study we used bone-marrow derived in vitro LPS/IFN-y-induced M1 MPs to enhance functional muscle recovery after tourniquet-induced ischemia/reperfusion injury (TK-I/R). We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion. Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program. Furthermore, we show that delivery of M1 (LPS/IFN-γ) MPs early in regeneration accelerates myofiber repair, decreases fibrotic tissue deposition and increases whole muscle IGF-I expression.

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In vitro polarization of bone marrow-derived macrophages.BM MPs were either left untreated (M0) or treated with LPS/IFN-γ or TNF-α/IFN-γ for 42 hours to induce classical (M1) activation phenotype. Real-time PCR was performed to evaluate inflammatory (Tnfa, Nos2, Il1b) and anti-inflammatory (Arg1, PPARG, Igf1) gene expression of MPs polarized with LPS/IFN-γ or TNF-α/IFN-γ (10 ng/ml) relative to M0 MPs. β-actin was used as internal calibrator gene. Values expressed as mean ± SD.
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pone.0145550.g001: In vitro polarization of bone marrow-derived macrophages.BM MPs were either left untreated (M0) or treated with LPS/IFN-γ or TNF-α/IFN-γ for 42 hours to induce classical (M1) activation phenotype. Real-time PCR was performed to evaluate inflammatory (Tnfa, Nos2, Il1b) and anti-inflammatory (Arg1, PPARG, Igf1) gene expression of MPs polarized with LPS/IFN-γ or TNF-α/IFN-γ (10 ng/ml) relative to M0 MPs. β-actin was used as internal calibrator gene. Values expressed as mean ± SD.

Mentions: The purpose of this experiment was to compare the activation status of differentially polarized MPs treated with lipopolysaccharide (LPS)/Type-I interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α)/IFN-γ combinations. Classical (M1) activation was originally reported to require both IFN-γ and TNF-α, a “two signal model”. However, typical toll-like receptor ligand (TLR) ligand such as LPS can easily induce the transcription of TNF-α as well as IFN-β providing both signals and overcoming the “two signal” requirement [23, 43]. As anticipated, we saw higher inflammatory gene expression in M1 MPs treated with LPS/IFN-γ, probably due to synergistic signaling via TLR and IFN receptors (Fig 1). Relative expression of TNF-α (Tnfa), iNOS (Nos2) and IL-1β (Il1b) genes in M1 polarized MPs was considerably higher than that of unprimed controls (M0). As expected, there was downregulation of anti-inflammatory PPAR-γ (PPARG) and IGF-I (Igf1) gene expression in M1 polarized cells, while Arginase 1 (Arg1) expression was unaltered. For further studies, we used LPS/IFN-γ primed MPs, as signaling via TLR-4 receptor is important in recovery from ischemia/reperfusion injury [24].


The Development of Macrophage-Mediated Cell Therapy to Improve Skeletal Muscle Function after Injury.

Rybalko V, Hsieh PL, Merscham-Banda M, Suggs LJ, Farrar RP - PLoS ONE (2015)

In vitro polarization of bone marrow-derived macrophages.BM MPs were either left untreated (M0) or treated with LPS/IFN-γ or TNF-α/IFN-γ for 42 hours to induce classical (M1) activation phenotype. Real-time PCR was performed to evaluate inflammatory (Tnfa, Nos2, Il1b) and anti-inflammatory (Arg1, PPARG, Igf1) gene expression of MPs polarized with LPS/IFN-γ or TNF-α/IFN-γ (10 ng/ml) relative to M0 MPs. β-actin was used as internal calibrator gene. Values expressed as mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696731&req=5

pone.0145550.g001: In vitro polarization of bone marrow-derived macrophages.BM MPs were either left untreated (M0) or treated with LPS/IFN-γ or TNF-α/IFN-γ for 42 hours to induce classical (M1) activation phenotype. Real-time PCR was performed to evaluate inflammatory (Tnfa, Nos2, Il1b) and anti-inflammatory (Arg1, PPARG, Igf1) gene expression of MPs polarized with LPS/IFN-γ or TNF-α/IFN-γ (10 ng/ml) relative to M0 MPs. β-actin was used as internal calibrator gene. Values expressed as mean ± SD.
Mentions: The purpose of this experiment was to compare the activation status of differentially polarized MPs treated with lipopolysaccharide (LPS)/Type-I interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α)/IFN-γ combinations. Classical (M1) activation was originally reported to require both IFN-γ and TNF-α, a “two signal model”. However, typical toll-like receptor ligand (TLR) ligand such as LPS can easily induce the transcription of TNF-α as well as IFN-β providing both signals and overcoming the “two signal” requirement [23, 43]. As anticipated, we saw higher inflammatory gene expression in M1 MPs treated with LPS/IFN-γ, probably due to synergistic signaling via TLR and IFN receptors (Fig 1). Relative expression of TNF-α (Tnfa), iNOS (Nos2) and IL-1β (Il1b) genes in M1 polarized MPs was considerably higher than that of unprimed controls (M0). As expected, there was downregulation of anti-inflammatory PPAR-γ (PPARG) and IGF-I (Igf1) gene expression in M1 polarized cells, while Arginase 1 (Arg1) expression was unaltered. For further studies, we used LPS/IFN-γ primed MPs, as signaling via TLR-4 receptor is important in recovery from ischemia/reperfusion injury [24].

Bottom Line: A variety of MP phenotypes have been identified and characterized in vitro as well as in vivo.We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion.Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program.

View Article: PubMed Central - PubMed

Affiliation: Department of Kinesiology, The University of Texas at Austin, 1 University Station D3700, Austin, TX 78712, United States of America.

ABSTRACT
Skeletal muscle regeneration following acute injury is a multi-step process involving complex changes in tissue microenvironment. Macrophages (MPs) are one of the key cell types involved in orchestration and modulation of the repair process. Multiple studies highlight the essential role of MPs in the control of the myogenic program and inflammatory response during skeletal muscle regeneration. A variety of MP phenotypes have been identified and characterized in vitro as well as in vivo. As such, MPs hold great promise for cell-based therapies in the field of regenerative medicine. In this study we used bone-marrow derived in vitro LPS/IFN-y-induced M1 MPs to enhance functional muscle recovery after tourniquet-induced ischemia/reperfusion injury (TK-I/R). We detected a 15% improvement in specific tension and force normalized to mass after M1 (LPS/IFN-γ) MP transplantation 24 hours post-reperfusion. Interestingly, we found that M0 bone marrow-derived unpolarized MPs significantly impaired muscle function highlighting the complexity of temporally coordinated skeletal muscle regenerative program. Furthermore, we show that delivery of M1 (LPS/IFN-γ) MPs early in regeneration accelerates myofiber repair, decreases fibrotic tissue deposition and increases whole muscle IGF-I expression.

Show MeSH
Related in: MedlinePlus