Characterization of the Inflammatory Response in Dystrophic Muscle Using Flow Cytometry

  • Jenna M. Kastenschmidt
  • Ileen Avetyan
  • S. A. Villalta
Part of the Methods in Molecular Biology book series (MIMB, volume 1687)


Although mutations of the dystrophin gene are the causative defect in Duchenne muscular dystrophy (DMD) patients, secondary disease processes such as inflammation contribute greatly to the pathogenesis of DMD. Genetic and histological studies have shown that distinct facets of the immune system promote muscle degeneration or regeneration during muscular dystrophy through mechanisms that are only beginning to be defined. Although histological methods have allowed the enumeration and localization of immune cells within dystrophic muscle, they are limited in their ability to assess the full spectrum of phenotypic states of an immune cell population and its functional characteristics. This chapter highlights flow cytometry methods for the isolation and functional study of immune cell populations from muscle of the mdx mouse model of DMD. We include a detailed description of preparing single-cell suspensions of dystrophic muscle that maintain the integrity of cell-surface markers used to identify macrophages, eosinophils, group 2 innate lymphoid cells, and regulatory T cells. This method complements the battery of histological assays that are currently used to study the role of inflammation in muscular dystrophy, and provides a platform capable of being integrated with multiple downstream methodologies for the mechanistic study of immunity in muscle degenerative diseases.

Key words

Muscular dystrophy mdx Flow cytometry FACS Muscle inflammation Tregs ILC2 Macrophages Eosinophils Immune system Inflammatory cells 



We thank Chairut Vareechon for critical reviewing of this chapter, and for his insightful comments.


  1. 1.
    Hoffman EP, Brown RHJ, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919–928CrossRefPubMedGoogle Scholar
  2. 2.
    Koenig M et al (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50:509–517CrossRefPubMedGoogle Scholar
  3. 3.
    Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney HL (1993) Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci U S A 90:3710–3714CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Rosenberg AS et al (2015) Immune-mediated pathology in Duchenne muscular dystrophy. Sci Transl Med 7:299rv4CrossRefPubMedGoogle Scholar
  5. 5.
    Manzur AY, Kuntzer T, Pike M, Swan A (2008) Glucocorticoid corticosteroids for Duchenne muscular dystrophy. Cochrane Database Syst Rev CD003725Google Scholar
  6. 6.
    Merlini L et al (2003) Early prednisone treatment in Duchenne muscular dystrophy. Muscle Nerve 27:222–227CrossRefPubMedGoogle Scholar
  7. 7.
    Bulfield G, Siller WG, Wight PA, Moore KJ (1984) X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci U S A 81:1189–1192CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Spencer MJ, Tidball JG (2001) Do immune cells promote the pathology of dystrophin-deficient myopathies? Neuromuscul Disord 11:556–564CrossRefPubMedGoogle Scholar
  9. 9.
    Wehling M, Spencer MJ, Tidball JG (2001) A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice. J Cell Biol 155:123–131CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Spencer MJ, Montecino-Rodriguez E, Dorshkind K, Tidball JG (2001) Helper (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin-deficient muscle. Clin Immunol 98:235–243CrossRefPubMedGoogle Scholar
  11. 11.
    McDouall RM, Dunn MJ, Dubowitz V (1990) Nature of the mononuclear infiltrate and the mechanism of muscle damage in juvenile dermatomyositis and Duchenne muscular dystrophy. J Neurol Sci 99:199–217CrossRefPubMedGoogle Scholar
  12. 12.
    Pescatori M et al (2007) Gene expression profiling in the early phases of DMD: a constant molecular signature characterizes DMD muscle from early postnatal life throughout disease progression. FASEB J 21:1210–1226CrossRefPubMedGoogle Scholar
  13. 13.
    Haslett JN et al (2002) Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle. Proc Natl Acad Sci U S A 99:15000–15005CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Flanigan KM et al (2013) Anti-dystrophin T cell responses in duchenne muscular dystrophy: prevalence and a glucocorticoid treatment effect. Hum Gene Ther 24:797. doi: 10.1089/hgtb.2013.092 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Mendell JR et al (2010) Dystrophin immunity in Duchenne’s muscular dystrophy. N Engl J Med 363:1429–1437CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Tidball JG (2005) Inflammatory processes in muscle injury and repair. Am J Physiol Regul Integr Comp Physiol 288:R345–R353CrossRefPubMedGoogle Scholar
  17. 17.
    Arnold L et al (2007) Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J Exp Med 204:1057–1069CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Varga T et al (2016) Highly dynamic transcriptional signature of distinct macrophage subsets during sterile inflammation, resolution, and tissue repair. J Immunol 196:4771–4782CrossRefPubMedGoogle Scholar
  19. 19.
    Villalta SA, Nguyen HX, Deng B, Gotoh T, Tidball JG (2009) Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy. Hum Mol Genet 18:482–496CrossRefPubMedGoogle Scholar
  20. 20.
    Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8:958–969CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3:23–35CrossRefPubMedGoogle Scholar
  22. 22.
    Mosser DM, Zhang X (2008) Interleukin-10: new perspectives on an old cytokine. Immunol Rev 226:205–218CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Makita N, Hizukuri Y, Yamashiro K, Murakawa M, Hayashi Y (2015) IL-10 enhances the phenotype of M2 macrophages induced by IL-4 and confers the ability to increase eosinophil migration. Int Immunol 27:131–141CrossRefPubMedGoogle Scholar
  24. 24.
    Villalta SA et al (2014) Regulatory T cells suppress muscle inflammation and injury in muscular dystrophy. Sci Transl Med 6:258ra142CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Tang Q, Bluestone JA (2008) The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat Immunol 9:239–244CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Panduro M, Benoist C, Mathis D (2016) Tissue tregs. Annu Rev Immunol 34:609–633CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Fontenot JD, Gavin MA, Rudensky AY (2003) Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330–336CrossRefPubMedGoogle Scholar
  28. 28.
    Hori S, Nomura T, Sakaguchi S (2003) Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057–1061CrossRefPubMedGoogle Scholar
  29. 29.
    Burzyn D et al (2013) A special population of regulatory T cells potentiates muscle repair. Cell 155:1282–1295CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Villalta SA et al (2011) Interleukin-10 reduces the pathology of mdx muscular dystrophy by deactivating M1 macrophages and modulating macrophage phenotype. Hum Mol Genet 20:790–805CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Jenna M. Kastenschmidt
    • 1
    • 2
  • Ileen Avetyan
    • 1
    • 2
  • S. A. Villalta
    • 1
    • 2
  1. 1.Department of Physiology and BiophysicsUniversity of California, IrvineIrvineUSA
  2. 2.Institute for ImmunologyUniversity of California, IrvineIrvineUSA

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