Macrophages are Required for Recovery from Physiological Muscle Stress in the mdx Mouse Model of Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a lethal genetic disease associated with muscle degeneration and chronic inflammation. Current therapies are limited to corticosteroids which resolve inflammation at the expense of detrimental side effects, and exon skipping which is applicable only to a small subset of patients. Cardiosphere‐derived cells (CDCs) are cardiac progenitor/stromal cells with anti‐inflammatory, anti‐fibrotic, and regenerative properties. Recently, CDCs have been shown to reverse many of the abnormalities of heart and skeletal muscle in mdx mice, motivating the HOPE‐Duchenne clinical trial (Aminzadeh M, Rogers R et al., Stem Cell Reports 2018). Several lines of evidence now support the idea that CDCs exert their bioactivity by secreting nanoparticles called exosomes (CDC‐exos), which mediate cell‐cell transfer of genetic information. A single intravenous dose of CDC‐exos dramatically improves contractile function of the mdx mouse soleus, and restores muscle architecture. Given the anti‐inflammatory effects of CDC‐exos, RNA‐sequencing unexpectedly revealed a striking upregulation of inflammatory pathways in the solei of CDC‐exo treated mdx mice, as confirmed by confocal imaging of tissue macrophages using CD68 immunohistochemistry. Thus, we investigated the role of macrophages in mdx pathology. Clodronate liposomes were used to deplete monocytes and macrophages in vivo, as confirmed in blood by flow cytometry and in tissue by immunohistochemistry. Macrophage‐depleted and control mdx mice were subjected to a single bout of downhill running to induce muscle stress. Maximal exercise capacity was recorded prior to, and every other day for two weeks, following downhill running. Macrophage‐depleted mdx mice failed to recover during the two‐week period, whereas control mdx mice had recovered by the third day. Further, macrophage depletion shifted the force‐frequency relationship to higher frequencies and reduced maximal force developed by the soleus by 24%. Cultured macrophages from mdx bone marrow (BMDMs) express 4‐fold higher levels of the pro‐inflammatory cytokine TNF‐α than those from age‐matched wild‐type mice. In vitro conditioning of mdx BMDMs with CDC‐exos reduced the expression of TNF‐α, suggesting that CDC‐exos may skew mdx macrophage polarization toward a tissue healing phenotype. This interpretation is consistent with the finding that CDC‐exos polarize macrophages to a phenotype with enhanced efferocytosis capacity, minimizing scar formation after acute myocardial infarction. Thus, macrophages are required for recovery from physiological muscle stress in mdx mice, and CDC‐exos decrease pro‐inflammatory gene expression in macrophages. The unexpected requirement for macrophages in mdx skeletal muscle recovery from injury suggests that a macrophage population promotes tissue regeneration. We are currently testing the hypothesis that CDC‐exos may modulate the pro‐inflammatory phenotype of recruited macrophages to stimulate myogenesis and support mdx muscle regeneration.Support or Funding InformationCoalition DuchenneNIH T32 HL116273This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.