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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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Phenotypic analysis of in vitro polarized BM MPs.MPs were either left untreated (M0) or stimulated with 10ng/ml LPS/IFN-γ and IL-4/IL-13for 42 hours to induce classical (M1) and alternative (M2) activation phenotypes respectively. Flow cytometry was used to evaluate the expression of CD206 and Ly-6C surface proteins. (A) Representative plots of surface protein after in vitro MP polarization, (B) Mean fluorescence intensity of CD206 expression on the surface of polarized MPs, (C) Representative plot of PKH2.6 label and F4/80expression by in vitro polarized macrophages prior to transplantation.
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pone.0145550.g002: Phenotypic analysis of in vitro polarized BM MPs.MPs were either left untreated (M0) or stimulated with 10ng/ml LPS/IFN-γ and IL-4/IL-13for 42 hours to induce classical (M1) and alternative (M2) activation phenotypes respectively. Flow cytometry was used to evaluate the expression of CD206 and Ly-6C surface proteins. (A) Representative plots of surface protein after in vitro MP polarization, (B) Mean fluorescence intensity of CD206 expression on the surface of polarized MPs, (C) Representative plot of PKH2.6 label and F4/80expression by in vitro polarized macrophages prior to transplantation.

Mentions: In the course of muscle regeneration MPs undergo phenotypic transition from an M1-like, inflammatory phenotype to the M2-like pro-regenerative state [32]. In general, M1 MPs are characterized by elevated expression of Ly-6C surface marker, while M2 MPs show increased expression of CD206, mannose receptor [32, 44]. We analyzed surface expression of both markers on BM-derived macrophages following in vitro polarization with LPS/IFN-γ (M1), IL-4/IL-13 (M2) compared to M0 untreated MPs (Fig 2A). Only small fraction of M1 (LPS/IFN-γ) polarized MPs (3–5%) upregulated Ly-6C expression. The mean fluorescence intensity of CD206 was different in all three treatment conditions (Fig 2B). M0 MPs showed intermediate levels of CD206 expression, M1 (LPS/IFN-γ) polarized macrophages exhibited the lowest expression levels as compared to M2 (IL-4/IL-13) polarized MPs. Unexpectedly, all three macrophage groups expressed differential levels of CD206 relative to isotype control (Fig 2B). For selected studies we additionally labelled in vitro polarized MPs with PKH 2.6 fluorescent membrane dye to allow for identification of transplanted MP populations after i.m. delivery (Fig 2C).


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)

Phenotypic analysis of in vitro polarized BM MPs.MPs were either left untreated (M0) or stimulated with 10ng/ml LPS/IFN-γ and IL-4/IL-13for 42 hours to induce classical (M1) and alternative (M2) activation phenotypes respectively. Flow cytometry was used to evaluate the expression of CD206 and Ly-6C surface proteins. (A) Representative plots of surface protein after in vitro MP polarization, (B) Mean fluorescence intensity of CD206 expression on the surface of polarized MPs, (C) Representative plot of PKH2.6 label and F4/80expression by in vitro polarized macrophages prior to transplantation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145550.g002: Phenotypic analysis of in vitro polarized BM MPs.MPs were either left untreated (M0) or stimulated with 10ng/ml LPS/IFN-γ and IL-4/IL-13for 42 hours to induce classical (M1) and alternative (M2) activation phenotypes respectively. Flow cytometry was used to evaluate the expression of CD206 and Ly-6C surface proteins. (A) Representative plots of surface protein after in vitro MP polarization, (B) Mean fluorescence intensity of CD206 expression on the surface of polarized MPs, (C) Representative plot of PKH2.6 label and F4/80expression by in vitro polarized macrophages prior to transplantation.
Mentions: In the course of muscle regeneration MPs undergo phenotypic transition from an M1-like, inflammatory phenotype to the M2-like pro-regenerative state [32]. In general, M1 MPs are characterized by elevated expression of Ly-6C surface marker, while M2 MPs show increased expression of CD206, mannose receptor [32, 44]. We analyzed surface expression of both markers on BM-derived macrophages following in vitro polarization with LPS/IFN-γ (M1), IL-4/IL-13 (M2) compared to M0 untreated MPs (Fig 2A). Only small fraction of M1 (LPS/IFN-γ) polarized MPs (3–5%) upregulated Ly-6C expression. The mean fluorescence intensity of CD206 was different in all three treatment conditions (Fig 2B). M0 MPs showed intermediate levels of CD206 expression, M1 (LPS/IFN-γ) polarized macrophages exhibited the lowest expression levels as compared to M2 (IL-4/IL-13) polarized MPs. Unexpectedly, all three macrophage groups expressed differential levels of CD206 relative to isotype control (Fig 2B). For selected studies we additionally labelled in vitro polarized MPs with PKH 2.6 fluorescent membrane dye to allow for identification of transplanted MP populations after i.m. delivery (Fig 2C).

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