Advertisement

Early Inflammation in Muscular Dystrophy Differs between Limb and Respiratory Muscles and Increases with Dystrophic Severity

  • Zachary M. Howard
    Affiliations
    Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio
    Search for articles by this author
  • Jeovanna Lowe
    Affiliations
    Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio
    Search for articles by this author
  • Anton J. Blatnik 3rd
    Affiliations
    Department of Biological Chemistry and Pharmacology, College of Medicine, The Ohio State University, Columbus, Ohio
    Search for articles by this author
  • Deztani Roberts
    Affiliations
    Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio
    Search for articles by this author
  • Arthur H.M. Burghes
    Affiliations
    Department of Biological Chemistry and Pharmacology, College of Medicine, The Ohio State University, Columbus, Ohio
    Search for articles by this author
  • Shyam S. Bansal
    Correspondence
    Shyam S. Bansal, Ph.D., Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, 607 Davis Heart and Lung Research Institute, 473 W. 12th Ave., Columbus, OH 43210.
    Affiliations
    Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio

    Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio
    Search for articles by this author
  • Jill A. Rafael-Fortney
    Correspondence
    Address correspondence to Jill A. Rafael-Fortney, Ph.D., Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, 390 Biomedical Research Tower, 460 W. 12th Ave., Columbus, OH 43210.
    Affiliations
    Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, Ohio

    Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio
    Search for articles by this author
Open ArchivePublished:January 23, 2021DOI:https://doi.org/10.1016/j.ajpath.2021.01.008
      Duchenne muscular dystrophy (DMD) is a genetic, degenerative, striated muscle disease exacerbated by chronic inflammation. Mdx mice in the genotypic DMD model poorly represent immune-mediated pathology observed in patients. Improved understanding of innate immunity in dystrophic muscles is required to develop specific anti-inflammatory treatments. Here, inflammation in mdx mice and the more fibrotic utrn+/-;mdx Het model was comprehensively investigated. Unbiased analysis showed that mdx and Het mice contain increased levels of numerous chemokines and cytokines, with further increased in Het mice. Chemokine and chemokine receptor gene expression levels were dramatically increased in 4-week–old dystrophic quadriceps muscles, and to a lesser extent in diaphragm during the early injury phase, and had a small but consistent increase at 8 and 20 weeks. An optimized direct immune cell isolation method prevented loss of up to 90% of macrophages with density-dependent centrifugation previously used for mdx flow cytometry. Het quadriceps contain higher proportions of neutrophils and infiltrating monocytes than mdx, and higher percentages of F4/80Hi, but lower percentages of F4/80Lo cells and patrolling monocytes compared with Het diaphragms. These differences may restrict regenerative potential of dystrophic diaphragms, increasing pathologic severity. Fibrotic and inflammatory gene expression levels are higher in myeloid cells isolated from Het compared with mdx quadriceps, supporting Het mice may represent an improved model for testing therapeutic manipulation of inflammation in DMD.
      Duchenne muscular dystrophy (DMD), a degenerative disease of striated muscles, is caused by loss-of-function mutations in the dystrophin gene. Dystrophin-deficient myofibers lead to chronic injuries that diminish regenerative potential of limb and respiratory skeletal muscles, resulting in progressive fibrosis and loss of function into early adulthood.
      • Hoffman E.P.
      • Brown Jr., R.H.
      • Kunkel L.M.
      • Dystrophin
      the protein product of the Duchenne muscular dystrophy locus.
      On injury, myofibers secrete cytokines and chemokines.
      • Miyatake S.
      • Shimizu-Motohashi Y.
      • Takeda S.
      • Aoki Y.
      Anti-inflammatory drugs for Duchenne muscular dystrophy: focus on skeletal muscle-releasing factors.
      ,
      • Rosenberg A.S.
      • Puig M.
      • Nagaraju K.
      • Hoffman E.P.
      • Villalta S.A.
      • Rao V.A.
      • Wakefield L.M.
      • Woodcock J.
      Immune-mediated pathology in Duchenne muscular dystrophy.
      These factors activate resident myoblasts, fibroblasts, endothelial cells, and immune cells in a paracrine manner, and amplify signals to also recruit immune cells from circulation.
      • De Rossi M.
      • Bernasconi P.
      • Baggi F.
      • de Waal Malefyt R.
      • Mantegazza R.
      Cytokines and chemokines are both expressed by human myoblasts: possible relevance for the immune pathogenesis of muscle inflammation.
      ,
      • Cannon J.G.
      • St Pierre B.A.
      Cytokines in exertion-induced skeletal muscle injury.
      Continuous, adjacent myofiber injuries produce a chronic inflammatory state in the muscle, which appears to peak at 4 weeks of age in the genotypic mdx mouse model of DMD and diminish to persistent, low-grade inflammation throughout the fibrotic phase.
      • Rosenberg A.S.
      • Puig M.
      • Nagaraju K.
      • Hoffman E.P.
      • Villalta S.A.
      • Rao V.A.
      • Wakefield L.M.
      • Woodcock J.
      Immune-mediated pathology in Duchenne muscular dystrophy.
      ,
      • Villalta S.A.
      • Nguyen H.X.
      • Deng B.
      • Gotoh T.
      • Tidball J.G.
      Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy.
      Although the mdx mouse is the genotypic model of DMD, the disease is more severe in humans. Mdx mice haplo-insufficient for the partially compensating protein utrophin (utrn+/-;mdx; Het mice) develop significantly more inflammation and fibrosis; however, the specific immune cell and cytokine signaling differences between mdx and Het mice have not been quantitatively compared.
      • Zhou L.
      • Rafael-Fortney J.A.
      • Huang P.
      • Zhao X.S.
      • Cheng G.
      • Zhou X.
      • Kaminski H.J.
      • Liu L.
      • Ransohoff R.M.
      Haploinsufficiency of utrophin gene worsens skeletal muscle inflammation and fibrosis in mdx mice.
      Identifying cytokine signaling and immune cells that underly increasing dystrophic severity is critical for developing new treatment strategies. Prednisone, an anti-inflammatory drug that is the standard of care for DMD patients, provides modest improvements in muscle strength.
      • Fenichel G.M.
      • Florence J.M.
      • Pestronk A.
      • Mendell J.R.
      • Moxley 3rd, R.T.
      • Griggs R.C.
      • Brooke M.H.
      • Miller J.P.
      • Robison J.
      • King W.
      • Signore L.
      • Pandya S.
      • Schierbecker J.
      • Wilson B.
      Long-term benefit from prednisone therapy in Duchenne muscular dystrophy.
      Severe side effects accompany the benefits of long-term prednisone usage and necessitate development of alternative immunomodulatory therapies.
      Innate myeloid and adaptive lymphoid immune responses are required for efficient tissue regeneration across various organs, injuries, and diseases.
      • Mescher A.L.
      • Neff A.W.
      • King M.W.
      Inflammation and immunity in organ regeneration.
      In normal skeletal muscle, similar to other highly regenerative organs, resident immune cells such as macrophages and dendritic cells contribute to the regenerative potential conferred by muscle stem cells.
      • Cordero-Espinoza L.
      • Huch M.
      The balancing act of the liver: tissue regeneration versus fibrosis.
      ,
      • Karin M.
      • Clevers H.
      Reparative inflammation takes charge of tissue regeneration.
      Resident macrophages in healthy mouse skeletal muscle are replaced continuously throughout the animal's lifespan by blood monocytes.
      • Brigitte M.
      • Schilte C.
      • Plonquet A.
      • Baba-Amer Y.
      • Henri A.
      • Charlier C.
      • Tajbakhsh S.
      • Albert M.
      • Gherardi R.K.
      • Chrétien F.
      Muscle resident macrophages control the immune cell reaction in a mouse model of notexin-induced myoinjury.
      Inflammation in dystrophic skeletal muscle is composed of myeloid and lymphoid cells, with myeloid cells found to be more abundant.
      • Spencer M.J.
      • Montecino-Rodriguez E.
      • Dorshkind K.
      • Tidball J.G.
      Helper (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin-deficient muscle.
      ,
      • Tidball J.G.
      • Welc S.S.
      • Wehling-Henricks M.
      Immunobiology of inherited muscular dystrophies.
      Neutrophils, monocytes, and macrophages are a heterogeneous group of myeloid immune cells that accelerate myofiber necrosis and fibrosis in DMD patients and mouse models.
      • Kranig S.A.
      • Tschada R.
      • Braun M.
      • Patry C.
      • Pöschl J.
      • Frommhold D.
      • Hudalla H.
      Dystrophin deficiency promotes leukocyte recruitment in mdx mice.
      Neutrophils and monocytes, the first responders to muscle damage, are required for the initial phases of regeneration, but can cause further tissue damage if their response is exacerbated.
      • Rizzo G.
      • Di Maggio R.
      • Benedetti A.
      • Morroni J.
      • Bouche M.
      • Lozanoska-Ochser B.
      Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair.
      • Mojumdar K.
      • Liang F.
      • Giordano C.
      • Lemaire C.
      • Danialou G.
      • Okazaki T.
      • Bourdon J.
      • Rafei M.
      • Galipeau J.
      • Divangahi M.
      • Petrof B.J.
      Inflammatory monocytes promote progression of Duchenne muscular dystrophy and can be therapeutically targeted via CCR2.
      • Hodgetts S.
      • Radley H.
      • Davies M.
      • Grounds M.D.
      Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNFalpha function with etanercept in mdx mice.
      Monocytes differentiate into macrophages, dendritic cells, or fibrocytes, depending on the genetic predisposition of that monocyte, as well as the cytokines and chemokines it encounters.
      • Mosser D.M.
      • Edwards J.P.
      Exploring the full spectrum of macrophage activation.
      • Yang J.
      • Zhang L.
      • Yu C.
      • Yang X.F.
      • Wang H.
      Monocyte and macrophage differentiation: circulation inflammatory monocyte as biomarker for inflammatory diseases.
      • Wang X.
      • Zhao W.
      • Ransohoff R.M.
      • Zhou L.
      Identification and function of fibrocytes in skeletal muscle injury repair and muscular dystrophy.
      Monocytes have also been shown to enter tissues and perform functions without immediately differentiating into one of the described cell types.
      • Jakubzick C.V.
      • Randolph G.J.
      • Henson P.M.
      Monocyte differentiation and antigen-presenting functions.
      The contributions of macrophages to dystrophic pathology are incredibly nuanced; subtypes of macrophages, including M1, M2a, M2b, and M2c, have varied, often conflicting functions and distributions that are temporally regulated.
      • Capote J.
      • Kramerova I.
      • Martinez L.
      • Vetrone S.
      • Barton E.R.
      • Sweeney H.L.
      • Miceli M.C.
      • Spencer M.J.
      Osteopontin ablation ameliorates muscular dystrophy by shifting macrophages to a pro-regenerative phenotype.
      • Acharyya S.
      • Villalta S.A.
      • Bakkar N.
      • Bupha-Intr T.
      • Janssen P.M.
      • Carathers M.
      • Li Z.W.
      • Beg A.A.
      • Ghosh S.
      • Sahenk Z.
      • Weinstein M.
      • Gardner K.L.
      • Rafael-Fortney J.A.
      • Karin M.
      • Tidball J.G.
      • Baldwin A.S.
      • Guttridge D.C.
      Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy.
      • Villalta S.A.
      • Deng B.
      • Rinaldi C.
      • Wehling-Henricks M.
      • Tidball J.G.
      IFN-γ promotes muscle damage in the mdx mouse model of Duchenne muscular dystrophy by suppressing M2 macrophage activation and inhibiting muscle cell proliferation.
      Myoblasts in the injury site are stimulated to replace or repair damaged myofibers by M1-like macrophages.
      • Bencze M.
      • Negroni E.
      • Vallese D.
      • Yacoub-Youssef H.
      • Chaouch S.
      • Wolff A.
      • Aamiri A.
      • Di Santo J.P.
      • Chazaud B.
      • Butler-Browne G.
      • Savino W.
      • Mouly V.
      • Riederer I.
      Proinflammatory macrophages enhance the regenerative capacity of human myoblasts by modifying their kinetics of proliferation and differentiation.
      As necrotic debris is cleared by immune cells and myoblasts differentiate, immunosuppressive M2-like CD206-positive macrophages that suppress the inflammation and conclude regeneration become more prevalent.
      • Saclier M.
      • Cuvellier S.
      • Magnan M.
      • Mounier R.
      • Chazaud B.
      Monocyte/macrophage interactions with myogenic precursor cells during skeletal muscle regeneration.
      • Saclier M.
      • Yacoub-Youssef H.
      • Mackey A.L.
      • Arnold L.
      • Ardjoune H.
      • Magnan M.
      • Sailhan F.
      • Chelly J.
      • Pavlath G.K.
      • Mounier R.
      • Kjaer M.
      • Chazaud B.
      Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration.
      • Desguerre I.
      • Mayer M.
      • Leturcq F.
      • Barbet J.P.
      • Gherardi R.K.
      • Christov C.
      Endomysial fibrosis in Duchenne muscular dystrophy: a marker of poor outcome associated with macrophage alternative activation.
      Macrophages present degraded self-antigens to infiltrating lymphocytes to assist in concluding regeneration. Advancements in understanding macrophage phenotypes, particularly in heart disease, have emphasized a reclassification of these populations using markers that are representative of cell function and origin, including major histocompatibility complex II (MHC II) and CCR2.
      • Epelman S.
      • Lavine K.J.
      • Beaudin A.E.
      • Sojka D.K.
      • Carrero J.A.
      • Calderon B.
      • Brija T.
      • Gautier E.L.
      • Ivanov S.
      • Satpathy A.T.
      • Schilling J.D.
      • Schwendener R.
      • Sergin I.
      • Razani B.
      • Forsberg E.C.
      • Yokoyama W.M.
      • Unanue E.R.
      • Colonna M.
      • Randolph G.J.
      • Mann D.L.
      Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
      • Bajpai G.
      • Bredemeyer A.
      • Li W.
      • Zaitsev K.
      • Koenig A.L.
      • Lokshina I.
      • Mohan J.
      • Ivey B.
      • Hsiao H.M.
      • Weinheimer C.
      • Kovacs A.
      • Epelman S.
      • Artyomov M.
      • Kreisel D.
      • Lavine K.J.
      Tissue resident CCR2- and CCR2+ cardiac macrophages differentially orchestrate monocyte recruitment and fate specification following myocardial injury.
      • Epelman S.
      • Lavine K.J.
      • Randolph G.J.
      Origin and functions of tissue macrophages.
      In this study, we use a comprehensive approach to define the inflammatory signatures underlying the differences in severity observed in the mdx and Het mouse models by comparing muscle cytokine protein levels and cytokine receptor gene expression levels together with flow cytometric analysis of immune cell populations. Flow cytometric analyses leverage prior immunohistochemistry and flow immune cell markers and incorporate functional markers and an unbiased isolation technique to provide a thorough understanding of early inflammation that leads to ultimate quantitative differences in fibrosis.

      Materials and Methods

      Mouse Breeding and Handling

      Animal protocols were approved by the Institutional Animal Care and Use Committee of The Ohio State University (Columbus, OH), which is in compliance with the laws of the United States of America and conforms to the NIH Guide for the Care and Use of Laboratory Animals
      Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council
      Guide for the Care and Use of Laboratory Animals: Eighth Edition.
      . Wild-type C57BL/10 (C57), mdx, and Het (mdx/utrn+/–) mice were bred and euthanized for tissue harvesting at 4, 8, and 20 weeks of age for all RNA, protein, and cell-based assays performed in this study. All mdx and Het mice were derived from the same colony and were on a C57BL/10 background. Het mice were genotyped, as previously described.
      • Rafael-Fortney J.A.
      • Chimanji N.S.
      • Schill K.E.
      • Martin C.D.
      • Murray J.D.
      • Ganguly R.
      • Stangland J.E.
      • Tran T.
      • Xu Y.
      • Canan B.D.
      • Mays T.A.
      • Delfín D.A.
      • Janssen P.M.
      • Raman S.V.
      Early treatment with lisinopril and spironolactone preserves cardiac and skeletal muscle in Duchenne muscular dystrophy mice.

      Tissue Homogenization and Mouse Cytokine Proteome Profiler

      Four-week–old C57BL/10 [n = 2 male (M) and 1 female (F)], mdx (n = 3M and 2F), and Het (n = 2M and 3F) LN2 flash-frozen quadriceps were homogenized in lysis buffer composed of 1% Triton X-100 (Sigma, St. Louis, MO; T9284) in Dulbecco’s phosphate-buffered saline (Thermo Fischer, Waltham, MA; 14190-144), containing 10 μg/mL aprotinin (Sigma; A1153), leupeptin (Sigma; L2884), and pepstatin A (Sigma; P5318) on ice. Following homogenization, samples were flash frozen and thawed on ice. Centrifugation was performed at 10,000 × g for 5 minutes (4°C), and supernatants from the same genotype were pooled for analysis. Protein concentrations were determined using the DC protein assay (Bio-Rad Laboratories, Hercules, CA; 5000166). Separate membranes from the Proteome Profiler Mouse Cytokine Array Kit A (R&D Systems, Minneapolis, MN; ARY006) were incubated with 5 mg of protein from each genotype. The assay was performed according to the manufacturer's instructions and detected using blue X-ray film (GeneMate Kaysville, UT; F90298X10). Relative cytokine levels were quantified using HL Image++ Quick Spots Tool version 25.0.0r (Western Vision Software, Salt Lake City, UT). Cytokines that were undetectable in C57 were not quantified and are not included in the data presented.

      Tissue RNA Extraction, Purification, and Real-Time Quantitative RT-PCR

      Four-week–old (n: C57 = 2M and 1F; mdx = 3M and 2F; Het = 2M and 3F), 8-week–old (n: C57 = 1M and 2F; mdx = 3M and 2F; Het = 4M and 1F), and 20-week–old (n: C57 = 1M and 2F; mdx = 3M and 2F; Het = 4M and 1F) C57, mdx, and Het LN2 flash-frozen quadriceps and diaphragms were homogenized in TRIzol reagent (Thermo Fisher;15596026) on ice. Extraction of RNA was performed according to the manufacturer's instructions and DNased using RQ1 DNase (Promega, Madison, WI; M6101). Purified RNA was reverse transcribed into cDNA using a high-capacity cDNA synthesis kit (Thermo Fisher; 4368814). Reaction efficiency was checked qualitatively utilizing RT-PCR for the endogenous control gene (Actb) before real-time quantitative RT-PCR. Real-time quantitative RT-PCR analysis was performed on cDNA derived from whole skeletal muscle. The list of transcripts and the corresponding primer sequences are listed in Table 1 (primers were acquired from Thermo Fisher).
      Table 1Primer Sequences and Predicted Sizes for Quantitative PCR Experiments
      GeneProtein productPrimer sequenceAmplicon, bp
      Actbβ-Actin (endogenous control)F: 5′-GATCAAGATCATTGCTCCTCCTG-3′183
      R: 5′-AGGGTGTAAAACGCAGCTCA-3′
      Ccl2Chemokine (C-C motif) ligand 2F: 5′-GCTGTAGTTTTTGTCACCAAGC-3′154
      R: 5′-GTGCTGAAGACCTTAGGGCA-3′
      Ccl4Chemokine (C-C motif) ligand 4F: 5′-AACCTAACCCCGAGCAACAC-3′111
      R: 5′-AGGGTCAGAGCCCATTGGTG-3′
      Ccr2CCR2F: 5′-AGGAGCCATACCTGTAAATGC-3′161
      R: 5′-GCCGTGGATGAACTGAGGTA-3′
      Ccr5CCR5F: 5′-TGAGACATCCGTTCCCCCTA-3′50
      R: 5′-AATCCATCCTGCAAGAGCCAGA-3′
      TnfTumor necrosis factor-αF: 5′-CCACGTCGTAGCAAACCACC-3′90
      R: 5′-TTGAGATCCATGCCGTTGGC-3′
      Il1bIL-1βF: 5′-TGCCACCTTTTGACAGTGATG-3′138
      R: 5′-TGATGTGCTGCTGCGAGATT-3′
      Il6IL-6F: 5′-GCCTTCTTGGGACTGATGCT-3′181
      R: 5′-TGCCATTGCACAACTCTTTTC-3′
      F, forward; R, reverse.

      Histology

      Four-week–old, 8-week–old, and 20-week–old mdx and Het quadriceps and diaphragms (n = 3M) were embedded in OCT compound, frozen in LN2-cooled isopentane, and sectioned on a cryostat (Bright, Huntingdon, UK). One 20-week–old C57 was used as a control. Frozen sections were hematoxylin and eosin-stained and imaged on a Nikon Eclipse 800 microscope using a Nikon DS-Ri2 Digital camera driven by Nikon Br Elements software.

      Skeletal Muscle Digestion, Generation of Single-Cell Suspensions, and Density-Dependent Centrifugation

      Four-week–old C57BL/10 (n = 6M), mdx (n = 3M and 3F), and Het (n = 3M and 3F) mice were euthanized via cervical dislocation, and quadriceps and diaphragms were harvested, rinsed in cold Dulbecco’s phosphate-buffered saline without CaCl2 or MgCl2 (Thermo Fisher; A1285601), and weighed. Tissues were finely minced with a razor blade and incubated in 10 mL/g digestion buffer (Dulbecco’s modified Eagle’s medium; Thermo Fisher; 21013024; 0.02% Collagenase P; Sigma; 11213857001; 0.1% RQ1 DNase) at 37°C for 30 minutes. The digested tissues were triturated using a 5-mL pipet coated in fetal bovine serum (R&D; S11150), and 3 mL of autoMACS Running Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany; 130091221) was added to quench collagenase P enzymatic activity. For the direct primary immune cell isolation method, 70- and 40-μm filtrations were performed to obtain single-cell suspensions and then cells were subsequently fixed in 1% paraformaldehyde (Sigma; P6148) in Dulbecco’s phosphate-buffered saline on ice for 10 minutes. For the density-dependent immune cell isolation method, filtered single-cell suspensions were carefully overlaid on 10 mL of either histopaque (Sigma; 10771) or lympholyte (Cedarlane, Burlington, NC; CL5031) and centrifuged at 400 × g for 30 minutes with no brake. Following separate interface and pellet collection, a wash step in autoMACS Running Buffer was performed before fixation. For the comparison between cell isolation methods, six pairs of mdx quadriceps (n = 3M and 3F) were pooled and separated into three aliquots for equal distribution of cells between the three different isolation techniques. For blood leukocyte isolation, 50 μL of blood obtained via submandibular blood collection was incubated with 2 mL of RBC Lysis Buffer (Thermo Fisher; 00433357) for 5 minutes and then fixed, as described earlier in this paragraph. Staining and analytical flow cytometry were completed within 24 to 96 hours of fixation for all experiments.

      Skeletal Muscle Single-Cell Suspension Staining and Flow Cytometry

      Following fixation of the single-cell suspensions, volumes of each sample were measured and adjusted to equal volumes with autoMACS Running Buffer. Flow cytometry antibodies used were as follows: CD45 (phycoerythrin-Cy7; Thermo Fisher; 25045182), CD11b (allophycocyanin; Biolegend, San Diego, CA; 101212), F4/80 (fluorescein isothiocyanate; Biolegend; 123108), CD68 (BV605; Biolegend; 137021), CD206 [peridinin-chlorophyll-protein (PerCP)-eFluo710; Thermo Fisher; 46206182], LY6C (eFluo450; Thermo Fisher; 48593282), MHC II (BV650; Biolegend; 107639), LY6G (allophycocyanin/FIRE750; Biolegend; 127652), and CCR2 (phycoerythrin; R&D; FAB5538P). An aliquot (100 μL) of each sample was taken in round-bottom 5-mL flow tubes, and extracellular staining was performed for 45 minutes on ice. Excess antibody was washed using autoMACS Running Buffer, followed by intracellular staining using 40 μL of 0.5% Tween-20 (Sigma; P1379) in Dulbecco’s phosphate-buffered saline as the permeabilizer. UltraComp eBeads (Thermo Fisher; 01222242) were used as single-color controls for compensation. Samples were washed again with autoMACS Running Buffer, and as an internal control 2000 (2 μL) AccuCount Blank Particles (Spherotech, Lake Forest, IL; ACBP10010) were added to each sample before flow cytometry to enable calculation of total cell counts in each sample. Immune cell populations were gated with the markers to distinguish the following populations: immune (CD45+), myeloid enriched (CD45+ CD11b+), neutrophils (CD45+ CD11b+ LY6G+), macrophages (CD45+ CD11b+ LY6G F4/80Hi/Lo CD68+), infiltrating monocytes (CD45+ CD11b+ LY6G F4/80Lo LY6CHi), and patrolling monocytes (CD45+ CD11b+ LY6G F4/80Lo LY6CLo). Experiments were conducted in the Analytical Cytometry Shared Resource at The Ohio State University using a Becton Dickinson LSRFortessa Flow Cytometrer (Becton Dickinson, Franklin Lakes, NJ), and all data were analyzed using FlowJo software Enzymatically-inactive Tissue-type Plasminogen Activator Reverses Disease Progression in the Dextran Sulfate Sodium Mouse Model of Inflammatory Bowel Disease version 10.7.1 (Becton Dickinson).

      Fluorescently Activated Cell Sorting, RNA Isolation, and Droplet Digital PCR

      Single-cell suspensions were prepared by the direct immune cell isolation method, as described above, from Het (n = 2M and 4F) and mdx (n = 3M and 3F) quadriceps. Freshly isolated unfixed cells were extracellularly stained with the following: CD45 (phycoerythrin-Cy7; Thermo Fisher; 25045182), CD11b (allophycocyanin; Biolegend; 101212), LY6G (allophycocyanin/FIRE750; Biolegend; 127652), F4/80 (PerCP/Cy5.5; Biolegend; 123128), CCR2 (fluorescein isothiocyanate; Biolegend; 150608), and LY6C (phycoerythrin; Thermo Fisher; 12593282), as described above. The cell populations isolated were as follows: infiltrating monocytes (CD45+ CD11b+ LY6G F4/80Lo LY6CHi), patrolling monocytes and LY6C−/Lo myeloid cells (CD45+ CD11b+ LY6G F4/80Lo LY6C−/Lo), F4/80Hi CCR2+ (CD45+ CD11b+ LY6G F4/80Hi CCR2+), and CCR2 myeloid cells (CD45+ CD11b+ LY6G F4/80Hi CCR2). UltraComp eBeads (Thermo Fisher; 01222242) were used as single-color controls for compensation in real time. The experiments were conducted in the Analytical Cytometry Shared Resource at The Ohio State University using the Becton Dickinson FACSAria III. Following fluorescently activated cell sorting, samples were pooled in duplicate and RNA was isolated from the cell populations using a NucleoSpin RNA XS kit (Takara, Mountain View, CA; 740902), according to the manufacturer's instructions. Purified RNA was reverse transcribed into cDNA, as described above. To absolutely quantify gene expression, digital droplet PCR was conducted using the synthesized cDNA. Droplet production and analysis were performed using the QX200 Droplet Digital PCR System (Bio-Rad). Targets were amplified and detected using commercially available primer/FAM-probe kits: Mrc1 (Bio-Rad; 5095688), Spp1 (Bio-Rad; 5108866), Nos2 (Bio-Rad; 5121694), Col1a (Bio-Rad; 5092596), and Timd4 (Bio-Rad; 5113448). Target copies per 20-μL reaction were calculated in QuantaSoft Analysis Pro software version 1.7 (Bio-Rad) and normalized to the RNA (ng) input into the reverse transcription reaction.

      Statistical Analysis

      All data are displayed as means ± SEM. For comparison of two groups, the two-tailed, unpaired t-test was utilized. Statistical comparisons between more than two groups were analyzed using one-way analysis of variance with either Tukey or Benjamini/Krieger/Yekutieli multiple-comparison post-hoc tests. Group variances were first compared using the Brown-Forsythe test. Tests were performed in GraphPad Prism software version 9.0.0 (GraphPad Prism Software, San Diego, CA). P ≤ 0.05 was considered significantly different.

      Results

      Het Quadriceps Muscles Contain Higher Cytokine Levels than Mdx Quadriceps Muscles during the Early Inflammatory Phase

      To evaluate differences in cytokine levels between wild-type C57BL/10 controls (C57), mdx, and Het skeletal muscles during the early inflammatory phase observed in muscular dystrophy, an unbiased mouse-specific cytokine array enabling the simultaneous detection of up to 40 cytokines was performed on pooled quadriceps lysates of each genotype (Figure 1A). After performing pixel densitometry, 21 cytokines were found to be up-regulated in both dystrophic genotypes relative to C57, with chemokine (C-C motif) ligand (CCL) 2, IL-1ra, and CCL12 representing the three highest fold changes in mdx compared with wild-type, with 33.65-fold, 22.53-fold, and 11.22-fold higher levels, respectively (Figure 1B). When comparing mdx with the more severe Het mouse model, increased levels of many chemokines, cytokines primarily responsible for mediating immune cell chemotaxis toward injury, including the following: CXCL13, CXCL10, CXCL1, CCL2, CCL12, CXCL9, CCL3, CCL4, and CCL5 (Figure 1, B and C) were observed. In addition to chemokines, IL-2 and triggering receptor expressed on myeloid cells 1 (TREM-1) were elevated, which are indicative of lymphoid and myeloid inflammation, respectively.
      • Lundberg I.
      • Brengman J.M.
      • Engel A.G.
      Analysis of cytokine expression in muscle in inflammatory myopathies, Duchenne dystrophy, and non-weak controls.
      ,
      • Campanholle G.
      • Mittelsteadt K.
      • Nakagawa S.
      • Kobayashi A.
      • Lin S.L.
      • Gharib S.A.
      • Heinecke J.W.
      • Hamerman J.A.
      • Altemeier W.A.
      • Duffield J.S.
      TLR-2/TLR-4 TREM-1 signaling pathway is dispensable in inflammatory myeloid cells during sterile kidney injury.
      Levels of anti-inflammatory IL-10 were correspondingly lower in Het relative to mdx quadriceps, further suggesting enhanced inflammation in Het mice when compared with mdx mice (Figure 1C).
      Figure thumbnail gr1
      Figure 1Unbiased analysis of cytokine protein levels from muscles of dystrophic mouse models. A: Proteome profiler dot blots incubated with pooled 4-week–old C57 wild-type, mdx, and Het quadriceps lysates. Technical duplicate dots correspond to the protein levels of a single cytokine, which are labeled with a coordinate [letter, number (L#)]. B: Cytokine pixel density ratios of mdx/C57, Het/C57, and Het/mdx measured from technical duplicate blots from pooled samples. Cytokines can be identified on the blot in A using the coordinates listed under L#. C: Mdx/C57 and Het/mdx chemokine and cytokine pixel density ratios displayed as a bar graph with a dotted horizontal line at y = 1.0. Chemokines and cytokines detected in the array are separated by a dashed vertical line. CCL, chemokine (C-C motif) ligand; M-CSF, macrophage colony stimulating factor; sICAM, soluble intercellular adhesion molecule; TIMP, tissue inhibitor of metalloproteinases; TNF-α, tumor necrosis factor-α; TREM, triggering receptor expressed on myeloid cells.

      Gene Expression for Cytokine and Chemokine Signaling Is Increased during the Early Inflammatory Phase and Diminishes in Both Dystrophic Models during Disease Progression

      After performing an unbiased cytokine assessment, gene expression for Ccl2, Ccl4, Ccr2, Ccr5, Tnf, Il1β, and Il6 was measured in C57, mdx, and Het quadriceps and diaphragms at 4, 8, and 20 weeks of age to evaluate temporal inflammation between the two dystrophic phenotypes (Figure 2). In 4-week–old mouse quadriceps, Ccl2, Ccl4, Ccr2, Ccr5, and Tnf gene expression was significantly up-regulated in both dystrophic models compared with C57 (one-way analysis of variance; Tukey; P ≤ 0.05) (Figure 2A). Il1b (11.05 ± 1.32 versus 1.00 ± 0.44; P = 0.007) and Il6 (1.89 ± 0.21 versus 0.53 ± 0.24; P = 0.047) expression levels were only significantly up-regulated in Het quadriceps compared with C57 (Figure 2A). Ccl2 (31.80 ± 3.80 versus 15.92 ± 1.87; P = 0.004) and Tnf (6.77 ± 0.59 versus 3.81 ± 0.47; P = 0.003) gene expression levels were significantly up-regulated in Het compared with mdx quadriceps (Figure 2A). In the 4-week–old mouse diaphragms, Ccl4 gene expression was significantly up-regulated in both dystrophic models compared with C57 (P ≤ 0.05) (Figure 2A). Only Ccl2 (18.92 ± 5.25 versus 1.11 ± 0.31; P = 0.023), Ccr2 (3.61 ± 0.17 versus 1.41 ± 0.21; P = 0.011), Ccr5 (5.03 ± 0.62 versus 1.88 ± 0.83; P = 0.003), Tnf (3.11 ± 0.17 versus 1.04 ± 0.02; P < 0.001), Il1b (5.17 ± 1.24 versus 0.94 ± 0.15; P = 0.026), and Il6 (2.39 ± 0.22 versus 0.74 ± 0.13; P = 0.008) expression levels were significantly up-regulated in Het diaphragms compared with C57 (Figure 2A). Ccr5 (5.03 ± 0.62 versus 2.76 ± 0.11; P = 0.011) and Tnf (3.11 ± 0.17 versus 1.80 ± 0.25; P = 0.001) were up-regulated in Het diaphragms compared with mdx (Figure 2A). The largest inflammatory gene expression differences between C57 and dystrophic muscle were at 4 weeks, which supports the peak of inflammation at this time point identified in previous studies (Figure 2A).
      • Villalta S.A.
      • Nguyen H.X.
      • Deng B.
      • Gotoh T.
      • Tidball J.G.
      Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy.
      ,
      • Wehling M.
      • Spencer M.J.
      • Tidball J.G.
      A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice.
      Figure thumbnail gr2
      Figure 2Temporal gene expression and histology comparing dystrophic mouse models A: Gene expression analysis of cytokines and chemokine receptors in C57, mdx, and Het quadriceps (QUADs) and diaphragms (DIAs) at 4, 8, and 20 week of age. Transcripts quantified by quantitative PCR from left to right are as follows: Ccl2, Ccl4, Ccr2, Ccr5, Tnf, Il1b, and Il6. Relative quantification was performed using β-actin (Actb) as an endogenous control. Statistical analysis was performed with one-way analysis of variance and the Tukey multiple-comparison post-hoc test. B: Hematoxylin and eosin staining of mdx and Het quadriceps and diaphragms at 4, 8, and 20 weeks of age, compared with C57 at 20 weeks of age. ∗P ≤ 0.05 versus C57; P ≤ 0.05 versus mdx. Scale bars = 100 μm (B).
      Similar patterns of significantly increased inflammatory gene expression in dystrophic compared with C57 muscles continue at 8-week–old and 20-week–old time points, although the magnitude of differences is lower at the later time points (Figure 2A). The only remaining significant increase in Het compared with mdx skeletal muscles at the later ages was Ccl4 quadriceps expression at 8 weeks (4.57 ± 0.29 versus 2.80 ± 0.24; P = 0.0032). Ccl4 is also more up-regulated in both dystrophic tissues at 20 weeks, which may indicate more lymphoid infiltration during the fibrotic phase of pathology (Figure 2A). These temporal changes in gene expression and differences between dystrophic models correlate with the more severe pathology observed in Het compared with mdx skeletal muscles (Figure 2B). Fibrosis becomes apparent by 20 weeks of age for both genotypes, with higher amounts visible in Het muscles, as previously quantified.
      • Zhou L.
      • Rafael-Fortney J.A.
      • Huang P.
      • Zhao X.S.
      • Cheng G.
      • Zhou X.
      • Kaminski H.J.
      • Liu L.
      • Ransohoff R.M.
      Haploinsufficiency of utrophin gene worsens skeletal muscle inflammation and fibrosis in mdx mice.

      Direct Primary Immune Cell Isolation Substantially Improves Yield of Dystrophic Skeletal Muscle Immune Cells Compared with Density-Dependent Centrifugation Methods

      Because we observed significant differences in several chemokines and cytokines that mediate immune cell influx into the tissues, we next measured immune cell infiltration into the single muscle tissues of mdx and Het mice and compared them with C57 mice. Several published studies use density-mediated cell separation techniques (lympholyte/histopaque) to remove tissue debris and enrich mononuclear cells.
      • Wang X.
      • Zhao W.
      • Ransohoff R.M.
      • Zhou L.
      Identification and function of fibrocytes in skeletal muscle injury repair and muscular dystrophy.
      ,
      • Hightower R.M.
      • Reid A.L.
      • Gibbs D.E.
      • Wang Y.
      • Widrick J.J.
      • Kunkel L.M.
      • Kastenschmidt J.M.
      • Villalta S.A.
      • van Groen T.
      • Chang H.
      • Gornisiewicz S.
      • Landesman Y.
      • Tamir S.
      • Alexander M.S.
      The SINE compound KPT-350 blocks dystrophic pathologies in DMD zebrafish and mice.
      ,
      • Kastenschmidt J.M.
      • Avetyan I.
      • Villalta S.A.
      Characterization of the inflammatory response in dystrophic muscle using flow cytometry.
      However, studies of immune cell isolation from other tissues support that, although density-mediated mononuclear cell isolation works well for peripheral blood mononuclear cells, altered cell size and granularity of tissue-infiltrated immune cells result in significant loss of leukocyte yield.
      • Covarrubias R.
      • Ismahil M.A.
      • Rokosh G.
      • Hamid T.
      • Accornero F.
      • Singh H.
      • Gumina R.J.
      • Prabhu S.D.
      • Bansal S.S.
      Optimized protocols for isolation, fixation, and flow cytometric characterization of leukocytes in ischemic hearts.
      These methods can potentially result in underrepresentation of specific immune cell populations isolated from skeletal muscle tissues. To achieve the goals of maximizing cell yield and minimizing cell manipulation, we compared previously used muscle isolation techniques with a direct immune cell isolation method recently described for heart.
      • Bansal S.S.
      • Ismahil M.A.
      • Goel M.
      • Zhou G.
      • Rokosh G.
      • Hamid T.
      • Prabhu S.D.
      Dysfunctional and proinflammatory regulatory T-lymphocytes are essential for adverse cardiac remodeling in ischemic cardiomyopathy.
      ,
      • Bansal S.S.
      • Ismahil M.A.
      • Goel M.
      • Patel B.
      • Hamid T.
      • Rokosh G.
      • Prabhu S.D.
      Activated T lymphocytes are essential drivers of pathological remodeling in ischemic heart failure.
      Immune cell numbers from mdx quadriceps muscles with and without density-mediated mononuclear cell isolation were compared using either histopaque or lympholyte on the same pooled samples. Myeloid cell populations (Supplemental Figure S1B) were found in the pellets generated from lympholyte and histopaque isolation (Figure 3, A and B ). Analysis of the pellets indicated 38.6% and 71.3% of CD45+ leukocytes were lost by isolating cells at the gradient interface of histopaque and lympholyte, respectively, compared with the direct isolation method confirmed via CD45+ back gating (Supplemental Figure S1A and Figure 3C). Further analysis of different immune cell populations showed 24% to 38% loss of neutrophils and monocytes (infiltrating and patrolling) in histopaque pellets and 60% to 70% loss in lympholyte pellets. More importantly, 80% to 90% of F4/80Hi CD68+ macrophages were found in both pellets, suggesting significant underrepresentation of this potent immune cell population by density-mediated immune cell isolation from tissue digests. Substantially more cells were lost using lympholyte than when using histopaque (Figure 3C).
      Figure thumbnail gr3
      Figure 3Comparison of flow cytometry analysis after direct versus density centrifugation methods of immune cell isolation from mdx quadriceps muscles. A and B: Representative flow cytometry scatterplots and gating of immune cell populations collected from the interface and the pellets after lympholyte (A) and histopaque (B) density-dependent centrifugation of single-cell suspensions obtained by digesting mdx muscles. C: Cell surface markers used for identifying total leukocytes and other innate immune cells of myeloid origin, including neutrophils, macrophages, and infiltrating (Inf.) and patrolling (Pat.) monocytes (left panel), and their relative frequencies in the histopaque and lympholyte pellets (right panel). FSC-W, forward scatter width; Histo., histopaque; Lympho., lympholyte.
      To define the mechanism underlying the loss of yield observed with density-mediated cell separation techniques, we show that leukocytes exhibit different sizes [forward scatter area (FSC-A)] and granularities [side scatter area (SSC-A)], depending on isolation from blood versus quadriceps muscles of Het mice. Leukocytes isolated from the blood were smaller, including the following: total immune cells (69,293.5 ± 660.0 versus 81,864.7 ± 979.0 FSC-A; t-test; P < 0.001) (Supplemental Figure S2A), myeloid cells (78,863.7 ± 767.0 versus 83,415.7 ± 1015.3 FSC-A; P = 0.005) (Supplemental Figure S2B), neutrophils (79,228.3 ± 931.3 versus 94,347.5 ± 1250.3 FSC-A; P < 0.001) (Supplemental Figure S2C), and monocytes (76,541.2 ± 1337.24 versus 92,455.3 ± 317.5 FSC-A; P < 0.001) (Supplemental Figure S2D). Blood immune cells were also less granulated: immune cells (6258.7 ± 278.7 versus 18,619.3 ± 785.4 SSC-A; P < 0.001), myeloid cells (10,642.5 ± 189.5 versus 19,139.5 ± 828.2 SSC-A; P < 0.001), neutrophils (12,023.8 ± 135.1 versus 15,637.7 ± 597.1 SSC-A; P < 0.001), and monocytes (6818.7 ± 234.3 versus 21,185.2 ± 951.7 SSC-A; P < 0.001). These results collectively showed that immune cells infiltrated into the muscles are larger and more granular compared with their counterparts in the circulation. Thus, lympholyte/histopaque-mediated cell enrichment is not as efficient of a technique to measure tissue-infiltrated immune cell populations and results in the biased enrichment of smaller and less granular cells at the interphase. The direct isolation method has the additional advantage of not exposing immune cells to the polysaccharide solution of density centrifugation methods. Therefore, all subsequent experiments use the direct isolation method.

      Mdx Quadriceps Muscles Contain a Greater Density of Myeloid Cells than Mdx Diaphragms

      To characterize different immune cell populations in C57 skeletal muscles and quantify differences in inflammation between mdx and C57 skeletal muscles, immune cell isolations using the direct method were performed separately using quadriceps and diaphragm muscles, from sex-matched mice of both genotypes. The representative gating strategy is shown in Supplemental Figure S1B, with representative gating dot plots for each tissue and genotype in Figure 4A. Flow cytometry isotype controls for markers lacking distinct populations are displayed in Supplemental Figure S1C. Quadriceps and diaphragms of C57 mice exhibited similar levels of CD45+ leukocytes (42.5 ± 9.1 versus 47.6 ± 14.7 cells/mg) at 4 weeks of age (Figure 4B). However, leukocyte numbers (per milligram of muscle tissue) were 100-fold and 10-fold higher in mdx quadriceps and diaphragms, respectively. Immune cell density differences between C57 and mdx skeletal muscle were assessed to demonstrate the severity of inflammation in dystrophic skeletal muscle. Mdx quadriceps contained significantly higher numbers of CD45+ immune (4740.4 ± 868.3 versus 42.5 ± 9.1 cells/mg; t-test; P = 0.0002) (Figure 4B), CD11b+ myeloid cells (4476.6 ± 808.1 versus 26.4 ± 6.6 cells/mg; P = 0.0002) (Figure 4C), and F4/80Hi macrophages (1730.6 ± 275.7 versus 3.5 ± 1.4 cells/mg; P = 0.00009) (Figure 4D) than C57 quadriceps. In addition, mdx diaphragms contained significantly fewer cells of CD45+, CD11b+, and F4/80Hi populations than mdx quadriceps [199.5 ± 71.8 cells/mg (P = 0.009), 172.7 ± 61.9 cells/mg (P = 0.008), and 20.5 ± 5.4 cells/mg (P = 0.003), respectively] (Figure 4, B–D). Mdx diaphragms also contained significantly higher F4/80Hi macrophage density than C57 diaphragms (20.5 ± 5.4 versus 0.6 ± 0.1 cells/mg; P = 0.021) (Figure 4D). In a parallel experiment, 8-week–old mdx skeletal muscle contained more immune cells than C57 skeletal muscle; however, the differences in immune cell density were substantially lower than at 4 weeks of age (data not shown). Combined with the gene expression analysis, inflammation appears to diminish by 8 weeks of age in mdx skeletal muscle.
      Figure thumbnail gr4
      Figure 4Comparison of immune cells in C57 and mdx quadriceps (QUADs) and diaphragm (DIA) muscles analyzed by flow cytometry using direct isolation method. A: Representative flow cytometry dot plots for CD45+ leukocytes, CD45+CD11b+ myeloid cells, CD45+CD11b+LY6GF4/80Lo monocytes, and CD45+CD11b+LY6GF4/80Hi macrophages in C57 and mdx QUAD and DIA skeletal muscles. B: Absolute quantification of the number of CD45+ leukocytes. C: CD45+CD11b+ myeloid cells. D: CD45+CD11b+LY6GF4/80Hi macrophages in C57 and mdx QUAD and DIA skeletal muscles. E: Marker analysis of CD45+ CD11b+ LY6G F4/80Hi CD68+ macrophages in C57 quadriceps. Statistical analysis was performed with t-test. ∗P ≤ 0.05 versus C57 QUAD; P ≤ 0.05 versus mdx DIA; P ≤ 0.05 versus C57 DIA. FSC, forward scatter; MHC, major histocompatibility complex.
      In C57 muscles, CD11b+ myeloid cells represented 60.2% of quadriceps and 42.4% of diaphragm CD45+ cells. Further analysis showed that most (≥98%) of CD11b+ cells were F4/80Hi macrophages and F4/80Lo LY6C+ monocytes. However, C57 diaphragms trend lower than quadriceps in F4/80Hi macrophage density (0.6 ± 0.1 versus 3.5 ± 1.4 cells/mg; P = 0.201) (Figure 4D). The low number of F4/80Hi macrophages in the diaphragm samples prevented comparison to C57 quadriceps for downstream markers. Only 16.9% of quadriceps CD11b+ F4/80Hi macrophages expressed CD68, suggesting that CD68, although specific, might not be a good marker to characterize muscle macrophages.
      F4/80Hi CD68+ macrophages from C57 quadriceps muscles were further characterized for CCR2, CD206, and MHC II marker expression (Figure 4E). We chose these phenotypic markers because CCR2 is a chemokine required for immune cell infiltration into the tissues, CD206+ M2-like macrophages are reparative and profibrotic, and high MHC II reflects antigen-presenting cells. Most (66.2%) of the CD45+ CD11b+ LY6G F4/80Hi CD68+ cells expressed the M2-like marker CD206, but a similar percentage (67.6%) of F4/80Hi CD68 cells also had CD206. This similar cell surface expression of CD206 in the CD68 population further supports the presence of macrophages without cell surface expression of CD68. More important, only 41.2% of F4/80Hi CD68+ macrophages expressed CCR2, whereas the majority did not, establishing the presence of two different subsets of macrophages in this population. Only 20.7% of F4/80Hi CD68+ macrophages expressed cell surface MHC II.

      Quadriceps from Het Mice Contain Significantly Higher Proportions of Neutrophils and Infiltrating Monocytes Compared with Mdx Mice

      Het skeletal muscle has greater levels of fibrosis than mdx skeletal muscle. We hypothesized that Het skeletal muscles would have increased or altered populations of infiltrated circulating leukocytes compared with mdx throughout the early phase of injury at 4 weeks of age. To identify differences in immune cell subsets, their frequencies were compared as a percentage of total CD45+ leukocytes. Representative dot plots for described significant differences are displayed in Figure 5A. LY6G+ neutrophils and LY6CHi infiltrating monocytes were significantly increased [t-test; 12.2% ± 2.0% versus 4.7% ± 0.6% (P = 0.004) and 25.7% ± 2.0% versus 15.8% ± 2.9% (P = 0.017), respectively] in the quadriceps of Het mice compared with the mdx mice (Figure 5C). These populations were also trending higher in Het diaphragms relative to mdx diaphragms, although the data were not significant because of the overall lower numbers of diaphragm inflammatory cells (neutrophils: P = 0.270; infiltrating monocytes: P = 0.070). Slightly higher total numbers of neutrophils and LY6CHi infiltrating monocytes were also present in the quadriceps and diaphragms of Het mice compared with mdx mice (Figure 5B). These changes, however, were not significant because of high variability observed in immune cell numbers for both genotypes. This variability also indicates that all the mice may not have an equivalent degree of injury at the chosen time point and may have slight differences in their time course of immune cell activation/infiltration. Because both neutrophils and LY6CHi monocytes are early responders of tissue injury, their higher frequencies in the Het mice reflect an ongoing early-phase inflammatory response. It is, therefore, possible that although mdx mice were at the peak of their inflammatory response at 4 weeks of age, Het mice were still undergoing early tissue remodeling and might exhibit peak inflammatory response at a different time scale given their more severe pathology.
      Figure thumbnail gr5
      Figure 5Flow cytometry analysis comparing myeloid populations in mdx and Het quadriceps (QUADs) and diaphragm (DIA) muscles. A: Representative flow cytometry dot plots for neutrophils, monocytes, F4/80Hi macrophages, and CD206+ F4/80Hi macrophages in the QUAD and DIA of mdx and Het mice. B: Bar graphs showing total cell counts of CD45+ leukocytes, CD11b+ myeloid cells, LY6G+ neutrophils (Nφ), F4/80Hi macrophages (MΦ), and F4/80Lo macrophages in the mdx and Het skeletal muscles. C: Cell populations quantified as a percentage of the total immune cell population (%CD45+). D: Distribution of CD206, CCR2, and major histocompatibility complex (MHC) II in F4/80Hi macrophages. Quantification for QUAD (left panel) and DIA (right panel) is shown. Statistical analysis was performed with t-test. ∗P ≤ 0.05 versus Het. Inf. Mono., infiltrating monocytes; Pat. Mono., patrolling monocytes.
      Because the inflammatory state in dystrophic muscle is severe, we predicted a large proportion of myeloid F4/80Hi/Lo cells would express extracellular CD68. However, no significant differences in the proportions of F4/80Hi/Lo CD68+ macrophages or LY6CLo patrolling monocytes were present between Het and mdx quadriceps or diaphragms. These cells compose a small percentage of the total CD45+ immune cell population in both genotypes and tissues. Quadriceps from both genotypes had 7% to 8% F4/80Hi CD68+ and approximately 2% F4/80Lo CD68+ cells and diaphragms had 3% to 4% F4/80Hi CD68+ and approximately 4% to 5% F4/80Lo CD68+ cells. More importantly, macrophages expressing high levels of F4/80 (7% to 8% of total CD45+ cells) were not different between both dystrophic genotypes in quadriceps. However, mdx diaphragm F4/80Hi macrophages were more M2 like, and 24.1% ± 3.7% of F4/80Hi macrophages expressed CD206, compared with only 10.5% ± 1.3% (P = 0.026) in Het mice (Figure 5D). The opposite is true for M1-like CD206 macrophages, which were present at much higher percentages in Het mice compared with mdx mice (75.9% and 89.5%, respectively), again suggesting an active proinflammatory phase in this genotype.

      Het Quadriceps and Diaphragms Display Tissue-Specific Differences in Myeloid Cell Composition

      Although all skeletal muscles have evolved the ability to regenerate, whether differences in the immune response between dystrophic limb and respiratory muscles would provide insight into why certain muscles have accelerated pathology in DMD was investigated. Because dystrophic diaphragms accumulate more fibrosis than quadriceps muscles and lose regenerative capacity earlier, diaphragm and quadriceps immune cell profiles from Het mice were compared. Het quadriceps show a nonsignificant trend for higher immune cell density than diaphragms for all the populations examined (Figure 6A). However, Het quadriceps have a significantly higher percentage of myeloid cells than diaphragms (94.8% ± 0.4% versus 90.2% ± 1.0%, t-test; P = 0.001). Quadriceps from Het mice also contained a significantly lower percentage of F4/80Lo CD68+ macrophages (1.9% ± 0.3% versus 5.4% ± 1.2%; P = 0.001) and patrolling monocytes (4.2% ± 0.3% versus 7.4% ± 0.6%; P = 0.008) than diaphragms (Figure 6B). When performing phenotypic analysis on both populations of macrophages, no differences in marker expression were found (Figure 6, C and D).
      Figure thumbnail gr6
      Figure 6Flow cytometry analysis comparing myeloid populations in Het quadriceps (QUADs) and diaphragm (DIA) muscles. Absolute cell density (A) and frequency (%CD45+) (B) of different immune cells in the QUAD and DIA of Het mice. Distribution of CD206, CCR2, and major histocompatibility complex (MHC) II in F4/80Hi (C) and F4/80Lo (D) macrophages (MΦ). F4/80Hi and F4/80Lo and cell density (E) and %CD45+ quantification (F) in Het skeletal muscle. Statistical analysis was performed with t-test. ∗P ≤ 0.05 versus Het DIA. Inf. Mono., infiltrating monocytes; Nφ, neutrophils; Pat. Mono., patrolling monocytes.
      Because extracellular CD68+ appears only to be present on a subset of macrophages, Het quadriceps and diaphragm F4/80Hi/Lo populations were examined in total CD11b+ cells without gating for CD68. Het quadriceps contained a significantly higher percentage of F4/80Hi macrophages (38.7% ± 3.1% versus 14.9% ± 2.1%; P = 0.002) and trended higher in cell numbers (P = 0.138) (Figure 6, E and F). Het quadriceps also contained a significantly lower percentage of F4/80Lo monocytes compared with Het diaphragms (43.4% ± 2.2% versus 58.3% ± 5.0%; P = 0.014).

      Het Quadriceps F4/80Hi CCR2+ Macrophages Are M2 Like, Profibrotic, and Proinflammatory

      CD206 was also assessed in each myeloid cell population from cytometry experiments to determine which populations were more M2 like. CD206 cell surface expression was enriched in CCR2+ (26.9%) and F4/80Hi (18.5%) macrophages, with much lower expression in the CCR2 (5.6%) and F4/80Lo (1.5%) populations. Enrichment of CD206 levels in F4/80Hi CCR2+ macrophages was also validated by t-distributed stochastic neighbor embedding dimensionality reduction (Figure 7).
      Figure thumbnail gr7
      Figure 7t-Distributed stochastic neighbor embedding (tSNE) dimensionality reduction of Het quadriceps CD45+ CD11b+ LY6G F4/80Hi cells (red) to show the distribution of CD45+ CD11b+ LY6G F4/80Hi CCR2+/- CD206+ cells (blue).
      To quantify gene expression in a cell-specific manner and confirm flow cytometry results, four myeloid cell populations were isolated using fluorescently activated cell sorting from mdx and Het quadriceps muscles, and gene expression was analyzed using droplet digital PCR. The four sorted populations included the following: infiltrating monocytes (CD45+ CD11b+ LY6G F4/80Lo LY6CHi), patrolling monocytes (CD45+ CD11b+ LY6G F4/80Lo LY6C−/Lo), F4/80Hi CCR2+ macrophages (CD45+ CD11b+ LY6G F4/80Hi CCR2+), and F4/80Hi CCR2 macrophages (CD45+ CD11b+ LY6G F4/80Hi CCR2). The gene expression of several M1-like and M2-like markers, including Mrc1 (CD206), Col1a (collagen I), Spp1 (osteopontin), Nos2 (inducible nitric oxide synthase), and Timd4, was compared between the described populations isolated from mdx and Het quadriceps (Table 2). Abundance of Mrc1, Timd4, and Nos2 transcripts were low in all cell populations, particularly in comparison to Col1a and Spp1 expression. Overall, F4/80Hi CCR2+ macrophages expressed the highest levels of all transcripts. Among the two genotypes, Het patrolling monocytes expressed significantly higher Mrc1 (3.67 ± 1.05 versus 0.07 ± 0.02 copies/ng RNA; t-test; P = 0.026), Col1a (738.67 ± 248.2 versus 41.33 ± 8.33 copies/ng RNA; P = 0.048), Spp1 (4188 ± 829.83 versus 45.69 ± 17.06 copies/ng RNA; P = 0.007), and Nos2 (1.64 ± 0.29 versus 0 copies/ng RNA; P = 0.004) than mdx-derived cells, whereas Het infiltrating monocytes expressed significantly higher Col1a (42.13 ± 9.67 versus 3.42 ± 1.54 copies/ng RNA; P = 0.016). In Het F4/80Hi CCR2+ macrophages, Nos2 expression was significantly up-regulated (17.11 ± 1.46 versus 5.87 ± 3.07 copies/ng RNA; P = 0.029). Mrc1 (P = 0.083) and Col1a (P = 0.104) trended higher in gene expression in Het F4/80Hi CCR2+ macrophages compared with mdx.
      Table 2Mdx and Het Comparison of Target Copies per Nanogram of RNA Isolated from Quadriceps CD45+ CD11b+ LY6G F4/80Lo LY6CHi Infiltrating Monocytes, CD45+ CD11b+ LY6G F4/80Lo LY6C−/Lo Patrolling Monocytes, and CD45+ CD11b+ LY6G F4/80Hi CCR2-/+ Macrophages, Including t-Test Significance Values
      TargetF4/80Lo LY6C−/LoF4/80Lo LY6CHiF4/80Hi CCR2F4/80Hi CCR2+
      MdxHetP valueMdxHetP valueMdxHetP valueMdxHetP value
      Mrc10.07 ± 0.023.67 ± 1.050.0260.02 ± 0.020.31 ± 0.200.2220.5 ± 0.310.19 ± 0.130.4081.23 ± 0.6613.1 ± 5.130.083
      Col1a41.33 ± 8.33738.67 ± 248.200.0483.42 ± 1.5442.13 ± 9.670.01626.22 ± 17.8924.67 ± 13.210.947228 ± 105.965565.33 ± 2543.170.104
      Spp145.69 ± 17.064188 ± 829.830.0073.78 ± 3.32212 ± 138.580.207110.76 ± 53.2781.33 ± 43.350.690316.44 ± 211.5712,225.33 ± 7850.590.204
      Nos20 ± 01.64 ± 0.290.0040 ± 00.36 ± 0.360.3730 ± 00 ± 0N.A.5.87 ± 3.0717.11 ± 1.460.029
      Timd40 ± 04.27 ± 4.270.3731.07 ± 1.074.93 ± 4.930.4860.93 ± 0.932 ± 20.6520 ± 05.87 ± 3.470.165
      Summary values are presented as means ± SEM copies/ng RNA. Targets quantified using droplet digital PCR include the following: Mrc1, Col1a, Spp1, Nos2, and Timd4.
      When comparing gene expression between F4/80Hi CCR2+ macrophages with the other three Het quadriceps populations (Table 3), F4/80Hi CCR2+ macrophages expressed significantly higher Mrc1 (one-way analysis of variance; Benjamini/Krieger/Yekutieli; P ≤ 0.05) than patrolling monocytes (13.1 ± 5.13 versus 3.67 ± 1.05; P = 0.034), infiltrating monocytes (0.31 ± 0.20 copies/ng RNA; P = 0.008), and F4/80Hi CCR2 macrophages (0.19 ± 0.13 copies/ng RNA; P = 0.008), confirming the flow cytometry data showing higher frequency of CD206+ cells in this population. In addition, Col1a expression was significantly higher in F4/80Hi CCR2+ macrophages relative to patrolling monocytes (5565.33 ± 2543.17 versus 738.67 ± 248.20; P = 0.039), infiltrating monocytes (42.13 ± 9.67 copies/ng RNA; P = 0.032), and F4/80Hi CCR2 macrophages (24.67 ± 13.21 copies/ng RNA; P = 0.032). A similar trend was seen in Spp1; however, there was no significant difference between mean target expression in the populations (P = 0.181). The F4/80Hi CCR2+ macrophages also expressed significantly higher Nos2 than all the other Het quadriceps-derived populations (17.11 ± 1.46 versus 1.64 ± 0.29, 0.36 ± 0.36, and 0 copies/ng RNA; P < 0.001). Phagocytic marker Timd4 expression was not different in any of the populations or genotypes (P = 0.903).
      Table 3Intragenotype Comparison of Gene Expression between the Described Populations Isolated from Het Quadriceps, including One-Way ANOVA and B.K.Y. Multicomparison Significance Values Comparing F4/80Hi CCR2+ Macrophages with the Other Isolated Cells
      PopulationMrc1Col1aSpp1Nos2Timd4
      HetANOVAB.K.Y.HetANOVAB.K.Y.HetANOVAB.K.Y.HetANOVAB.K.Y.HetANOVAB.K.Y.
      F4/80Lo LY6C–/Lo3.67 ± 1.050.0250.034738.67 ± 248.200.0420.0394188 ± 829.830.1810.1871.64 ± 0.29<0.001<0.0014.27 ± 4.270.9030.775
      F4/80Lo LY6CHi0.31 ± 0.200.00842.13 ± 9.670.032212 ± 138.580.0630.36 ± 0.36<0.0014.93 ± 4.930.867
      F4/80Hi CCR20.19 ± 0.130.00824.67 ± 13.210.03281.33 ± 43.350.0610 ± 0<0.0012 ± 20.495
      F4/80Hi CCR2+13.1 ± 5.135565.33 ± 2543.1712,225.33 ± 7850.5917.11 ± 1.465.87 ± 3.47
      Summary values are presented as means ± SEM copies/ng RNA.
      ∗F4/80Hi CCR2+ macrophages.
      ANOVA, analysis of variance; B.K.Y., Benjamini/Krieger/Yekutieli.

      Discussion

      In this study, a variety of techniques were utilized to characterize resident immune cells in healthy mouse skeletal muscle and quantify differences in skeletal muscle inflammation between two dystrophic mouse models, mdx and Het, with increasing severity. Presence of cytokines in C57 wild-type quadriceps indicates a continuous maintenance of resident immune cell populations in healthy skeletal muscle. Het skeletal muscle contained increased protein levels of numerous chemokines relative to mdx skeletal muscle, and gene expression cytokines and chemokine receptors identified a peak inflammatory phase at 4 weeks of age in both models and tissues. These data support what has previously been shown in the mdx model with an early peak in myofiber damage and inflammation, which appears to also occur in Het skeletal muscle.
      • Rosenberg A.S.
      • Puig M.
      • Nagaraju K.
      • Hoffman E.P.
      • Villalta S.A.
      • Rao V.A.
      • Wakefield L.M.
      • Woodcock J.
      Immune-mediated pathology in Duchenne muscular dystrophy.
      ,
      • Villalta S.A.
      • Nguyen H.X.
      • Deng B.
      • Gotoh T.
      • Tidball J.G.
      Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy.
      Increased Ccl2 and Tnf gene expression in Het relative to mdx quadriceps early in pathology corresponds to the increased recruitment of monocytes and neutrophils due to elevated damage, as well as an M1 bias in the macrophages present in the tissue observed in the flow cytometry results. Het diaphragms contained significantly higher expression of Ccr5 and Tnf compared with Het quadriceps, which may represent differences in monocyte or lymphocyte recruitment between limb and respiratory muscles. Infiltrating monocytes and neutrophils primarily follow CCL2 gradients via CCR2 to extravasate into damaged tissues, including skeletal muscle.
      • Warren G.L.
      • Hulderman T.
      • Mishra D.
      • Gao X.
      • Millecchia L.
      • O'Farrell L.
      • Kuziel W.A.
      • Simeonova P.P.
      Chemokine receptor CCR2 involvement in skeletal muscle regeneration.
      F4/80Hi macrophages in Het diaphragms are also more M1 like than in mdx diaphragms, which likely promotes myofiber cytotoxicity and may contribute to the higher levels of fibrosis in diaphragms.
      • Zhou L.
      • Rafael-Fortney J.A.
      • Huang P.
      • Zhao X.S.
      • Cheng G.
      • Zhou X.
      • Kaminski H.J.
      • Liu L.
      • Ransohoff R.M.
      Haploinsufficiency of utrophin gene worsens skeletal muscle inflammation and fibrosis in mdx mice.
      These results cumulatively indicate that Het skeletal muscle contains more inflammatory signaling than mdx skeletal muscle early in pathology, but like the mdx tissues, inflammation dampens as pathology progresses. Het skeletal muscle likely undergoes more necrosis during the early (4-week) phase of dystrophic mouse pathology because the sarcolemma is more vulnerable to contraction-induced damage without the partial compensation from reduced utrophin levels. Het skeletal muscle also displays more significant Il1b and Il6 up-regulation relative to C57, which indicates that Het shares more similarities with human DMD in terms of inflammatory markers.
      • Rosenberg A.S.
      • Puig M.
      • Nagaraju K.
      • Hoffman E.P.
      • Villalta S.A.
      • Rao V.A.
      • Wakefield L.M.
      • Woodcock J.
      Immune-mediated pathology in Duchenne muscular dystrophy.
      Immune cell isolation from dystrophic skeletal muscle has traditionally involved usage of density-dependent centrifugation to enrich for immune cells and remove tissue debris. Both histopaque and lympholyte are ficoll-like polysaccharide solutions optimized for isolating peripheral blood mononuclear cells from an interface,
      • Wang X.
      • Zhao W.
      • Ransohoff R.M.
      • Zhou L.
      Identification and function of fibrocytes in skeletal muscle injury repair and muscular dystrophy.
      ,
      • Hightower R.M.
      • Reid A.L.
      • Gibbs D.E.
      • Wang Y.
      • Widrick J.J.
      • Kunkel L.M.
      • Kastenschmidt J.M.
      • Villalta S.A.
      • van Groen T.
      • Chang H.
      • Gornisiewicz S.
      • Landesman Y.
      • Tamir S.
      • Alexander M.S.
      The SINE compound KPT-350 blocks dystrophic pathologies in DMD zebrafish and mice.
      ,
      • Kastenschmidt J.M.
      • Avetyan I.
      • Villalta S.A.
      Characterization of the inflammatory response in dystrophic muscle using flow cytometry.
      but their efficiency for isolating immune cells after extravasation into muscle tissues had not previously been quantified. Herein, we demonstrate increased size and granularity of immune cells from dystrophic skeletal muscle compared with blood and that muscle myeloid cells are lost in histopaque and lympholyte pellets. The direct isolation method and CD45+ back gating increase yield of total and specific cell populations and enable analysis of individual muscle types from one or two mice instead of previous pooling of multiple muscle types or of muscles from large numbers of animals.
      Monocytes express low levels of F4/80 in the blood, and macrophages derived from monocytes also tend to express low levels of F4/80.
      • Capote J.
      • Kramerova I.
      • Martinez L.
      • Vetrone S.
      • Barton E.R.
      • Sweeney H.L.
      • Miceli M.C.
      • Spencer M.J.
      Osteopontin ablation ameliorates muscular dystrophy by shifting macrophages to a pro-regenerative phenotype.
      Macrophages further along in differentiation are found to express higher levels of F4/80, but this process has not been demonstrated in skeletal muscle.
      • Lee Y.S.
      • Kim M.H.
      • Yi H.S.
      • Kim S.Y.
      • Kim H.H.
      • Kim J.H.
      • Yeon J.E.
      • Byun K.S.
      • Byun J.S.
      • Jeong W.I.
      CX(3)CR1 differentiates F4/80(low) monocytes into pro-inflammatory F4/80(high) macrophages in the liver.
      In dystrophic studies, F4/80Lo/Hi expression has been used to identify M1 and M2 macrophages, respectively, but our data support that expression of this marker may be more appropriate for visualizing the spectrum of states during the monocyte to macrophage differentiation process. It is possible that circulating cells present in capillaries supplying blood to the muscle-tissues were detected in our studies. However, this result is unlikely for two reasons. First, circulating monocytes do not have high expression of F4/80.
      • Lee Y.S.
      • Kim M.H.
      • Yi H.S.
      • Kim S.Y.
      • Kim H.H.
      • Kim J.H.
      • Yeon J.E.
      • Byun K.S.
      • Byun J.S.
      • Jeong W.I.
      CX(3)CR1 differentiates F4/80(low) monocytes into pro-inflammatory F4/80(high) macrophages in the liver.
      Second, CD11b+ myeloid cells represent only approximately 15% to 18% of total CD45+ leukocytes in the blood. In contrast, approximately 90% of CD45+ leukocytes were CD11b+ cells in our muscle digests, suggesting that the mdx/Het muscles were selectively enriched with myeloid cells and the inclusion of capillary entrapped leukocytes was minimal in our studies. We hypothesized that cell surface CD68 and F4/80 expression would enable us to discriminate activated macrophages from monocytes, dendritic cells, and granulocytes in flow cytometry experiments.
      Healthy mouse quadriceps, and to a lesser extent diaphragm, contain a population of CD45+ CD11b+ LY6G F4/80Hi cells that contain minimal extracellular CD68, but >60% of CD45+ CD45+ CD11b+ LY6G F4/80Hi CD68+ wild-type quadriceps macrophages express CD206. Because CD68 appears on the cell surface of only a small percentage of macrophages, it is possible that both intracellular and extracellular CD68 staining may more appropriately label all the macrophage populations. Resident skeletal muscle macrophages may suppress inflammation to a minimal level required for efficient tissue repair following injury. Alternative markers, such as CD64, shown to be expressed on macrophages, may be more informative for further differentiation between macrophages in future studies.
      • Tamoutounour S.
      • Henri S.
      • Lelouard H.
      • de Bovis B.
      • de Haar C.
      • van der Woude C.J.
      • Woltman A.M.
      • Reyal Y.
      • Bonnet D.
      • Sichien D.
      • Bain C.C.
      • Mowat A.M.
      • Reis e Sousa C.
      • Poulin L.F.
      • Malissen B.
      • Guilliams M.
      CD64 distinguishes macrophages from dendritic cells in the gut and reveals the Th1-inducing role of mesenteric lymph node macrophages during colitis.
      One of the primary causes of death in DMD patients is respiratory failure, resulting from diaphragm dysfunction. Quadriceps experience a larger range of contractile forces and are susceptible to external injuries that may require a more complex and sophisticated immunologic response to regulate regeneration and dampen detrimental inflammation. Het quadriceps were found to have a higher percentage of myeloid cells, but lower percentages of F4/80Lo macrophages and patrolling monocytes than Het diaphragms, with an even greater difference observed when not further gating for CD68. Because wild-type diaphragms do not have a significant F4/80Hi macrophage population to expand with chronic tissue injury, macrophages derived from circulating F4/80Lo monocytes may be more abundant in this tissue. Patrolling monocytes may play a role in early dystrophic fibrosis because these cells have been shown to be promote pathology in other diseases that end in tissue dysfunction.
      • Finsterbusch M.
      • Hall P.
      • Li A.
      • Devi S.
      • Westhorpe C.L.
      • Kitching A.R.
      • Hickey M.J.
      Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus.
      ,
      • Combadière C.
      • Potteaux S.
      • Rodero M.
      • Simon T.
      • Pezard A.
      • Esposito B.
      • Merval R.
      • Proudfoot A.
      • Tedgui A.
      • Mallat Z.
      Combined inhibition of CCL2, CX3CR1, and CCR5 abrogates Ly6C(hi) and Ly6C(lo) monocytosis and almost abolishes atherosclerosis in hypercholesterolemic mice.
      Interestingly, both F4/80Lo and F4/80Hi macrophage marker expression was not different between tissues, which may suggest that the downstream responses of the differentiated macrophages reach homeostasis, but the origins and amplitude of the myelogenous response are dysregulated.
      The roles of CCR2+ myeloid cells may be tissue-dependent. CCR2 levels in cardiac macrophages are associated with proinflammatory signaling, but macrophages expressing CCR2 and F4/80Hi are enriched for the M2-like marker CD206.
      • Bajpai G.
      • Bredemeyer A.
      • Li W.
      • Zaitsev K.
      • Koenig A.L.
      • Lokshina I.
      • Mohan J.
      • Ivey B.
      • Hsiao H.M.
      • Weinheimer C.
      • Kovacs A.
      • Epelman S.
      • Artyomov M.
      • Kreisel D.
      • Lavine K.J.
      Tissue resident CCR2- and CCR2+ cardiac macrophages differentially orchestrate monocyte recruitment and fate specification following myocardial injury.
      These cells also play a role in angiogenesis.
      • Ochoa O.
      • Sun D.
      • Reyes-Reyna S.M.
      • Waite L.L.
      • Michalek J.E.
      • McManus L.M.
      • Shireman P.K.
      Delayed angiogenesis and VEGF production in CCR2-/- mice during impaired skeletal muscle regeneration.
      Because Het myofibers undergo more damage due to the reduction of utrophin for mechanical reinforcement of the sarcolemma, myeloid cell up-regulation of Col1a and Spp1 early in pathology is likely required for efficient regeneration. Identical cell populations isolated from both genotypes are more transcriptionally active in Het muscle to mitigate more damage. Elevated COL1A and osteopontin in dystrophic muscle and serum have been shown to hasten development of fibrosis in response to severe myofiber damage.
      • Wang X.
      • Zhao W.
      • Ransohoff R.M.
      • Zhou L.
      Identification and function of fibrocytes in skeletal muscle injury repair and muscular dystrophy.
      ,
      • Capote J.
      • Kramerova I.
      • Martinez L.
      • Vetrone S.
      • Barton E.R.
      • Sweeney H.L.
      • Miceli M.C.
      • Spencer M.J.
      Osteopontin ablation ameliorates muscular dystrophy by shifting macrophages to a pro-regenerative phenotype.
      ,
      • Kuraoka M.
      • Kimura E.
      • Nagata T.
      • Okada T.
      • Aoki Y.
      • Tachimori H.
      • Yonemoto N.
      • Imamura M.
      • Takeda S.
      Serum osteopontin as a novel biomarker for muscle regeneration in Duchenne muscular dystrophy.
      In addition, Het F4/80Hi CCR2+ macrophages are enriched in expression of these targets relative to the other myeloid populations isolated from the same genotype. These macrophages paradoxically expressed the most Mrc1 and Nos2, which are markers for M2-like and M1-like macrophages, respectively. F4/80Hi CCR2+ macrophages are likely composed of different M2 macrophage subtypes, involved in both suppressing inflammation and promoting fibrosis through Col1a and Spp1 expression.
      • Braga T.T.
      • Agudelo J.S.
      • Camara N.O.
      Macrophages during the fibrotic process: M2 as friend and foe.
      Diaphragms, which contain a smaller quantity of F4/80Hi macrophages, may develop accelerated fibrosis through the increased infiltration of F4/80Lo monocyte-derived macrophages, as well as the activity of fibrocytes, which are likely present in the patrolling monocyte/LY6C−/Lo population based on their high Col1a expression relative to the infiltrating monocytes.
      • Wang X.
      • Zhao W.
      • Ransohoff R.M.
      • Zhou L.
      Identification and function of fibrocytes in skeletal muscle injury repair and muscular dystrophy.
      ,
      • Capote J.
      • Kramerova I.
      • Martinez L.
      • Vetrone S.
      • Barton E.R.
      • Sweeney H.L.
      • Miceli M.C.
      • Spencer M.J.
      Osteopontin ablation ameliorates muscular dystrophy by shifting macrophages to a pro-regenerative phenotype.
      Although it is unclear from the data whether the inflammatory differences are consequences or determinants of disease severity between the two models, the enhanced immune response in Het skeletal muscle is likely a necessary consequence of more muscle damage, albeit the increased infiltration may be more harmful than beneficial. Infiltration of CCR2+ LY6CHi monocytes and differentiation into various macrophage subtypes are required for efficient regeneration during acute or chronic skeletal muscle injury
      • Tidball J.G.
      • Villalta S.A.
      Regulatory interactions between muscle and the immune system during muscle regeneration.
      ,
      • Chazaud B.
      Inflammation and skeletal muscle regeneration: leave it to the macrophages!.
      ; however, infiltration of CCR2+ macrophages after cardiac injury promotes tissue fibrosis and dysfunction.
      • Bajpai G.
      • Bredemeyer A.
      • Li W.
      • Zaitsev K.
      • Koenig A.L.
      • Lokshina I.
      • Mohan J.
      • Ivey B.
      • Hsiao H.M.
      • Weinheimer C.
      • Kovacs A.
      • Epelman S.
      • Artyomov M.
      • Kreisel D.
      • Lavine K.J.
      Tissue resident CCR2- and CCR2+ cardiac macrophages differentially orchestrate monocyte recruitment and fate specification following myocardial injury.
      More infiltration of CCR2+ LY6CHi monocytes in Het skeletal muscle may increase the rate at which the regenerative potential of the tissue diminishes, occurring earlier in diaphragms than in quadriceps. Het quadriceps and diaphragms, during the early phase of murine dystrophic pathology, exhibit similar but different inflammatory responses that likely limit diaphragm regenerative potential, resulting in a more rapid accumulation of fibrosis. F4/80Hi macrophages may be important for mediating an efficient regenerative response in limb muscles, which are subject to mechanical and exercise-based injuries. The lack of environmental pressures on diaphragm muscle to evolve a more robust immune-mediated regenerative response may correlate with the lower resident F4/80Hi macrophage density observed and the capacity to mitigate myofiber damage.
      Early-onset inflammation in skeletal and cardiac muscle correlates with poor clinical outcomes in DMD, including loss of ambulation and heart failure.
      • Chen Y.W.
      • Nagaraju K.
      • Bakay M.
      • McIntyre O.
      • Rawat R.
      • Shi R.
      • Hoffman E.P.
      Early onset of inflammation and later involvement of TGFbeta in Duchenne muscular dystrophy.
      • Kim H.K.
      • Merrow A.C.
      • Shiraj S.
      • Wong B.L.
      • Horn P.S.
      • Laor T.
      Analysis of fatty infiltration and inflammation of the pelvic and thigh muscles in boys with Duchenne muscular dystrophy (DMD): grading of disease involvement on MR imaging and correlation with clinical assessments.
      • Mavrogeni S.
      • Papavasiliou A.
      • Spargias K.
      • Constandoulakis P.
      • Papadopoulos G.
      • Karanasios E.
      • Georgakopoulos D.
      • Kolovou G.
      • Demerouti E.
      • Polymeros S.
      • Kaklamanis L.
      • Magoutas A.
      • Papadopoulou E.
      • Markussis V.
      • Cokkinos D.V.
      Myocardial inflammation in Duchenne muscular dystrophy as a precipitating factor for heart failure: a prospective study.
      Inflammation-induced skeletal muscle fibrosis has even been shown in female carriers of DMD.
      • Preuße C.
      • von Moers A.
      • Kölbel H.
      • Pehl D.
      • Goebel H.H.
      • Schara U.
      • Stenzel W.
      Inflammation-induced fibrosis in skeletal muscle of female carriers of Duchenne muscular dystrophy.
      Future studies, beyond the scope of the current study, will be needed to more thoroughly characterize DMD myeloid cells. Understanding the tissue-specific, temporal functions of myeloid cells in dystrophic muscle will be important for developing targeted immunomodulatory therapies to improve quality of life and lifespan in DMD patients. Anti-inflammatory treatments synergistic with emerging genetic therapies are expected to provide the best possible outcomes for DMD patients.

      Acknowledgment

      We thank Bryan McElwain from the Flow Cytometry Shared Resource.

      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1Gating strategy, CD45+ back gating to identify CD45+ cells in muscle digests, and isotype controls A: CD45+ back gating to show specificity and exclusion of cellular debris for initial side scatter (SSC)-A versus forward scatter (FSC)-A gating in muscle digests. B: Representative gating strategy identifying different populations of immune cells derived from murine skeletal muscles: immune cells (CD45+), myeloid-enriched cells (CD45+ CD11b+), neutrophils (CD45+ CD11b+ LY6G+), macrophages (CD45+ CD11b+ LY6G F4/80Hi CD68+), inflammatory monocytes (CD45+ CD11b+ LY6G F4/80Lo LY6CHi), and patrolling monocytes (CD45+ CD11b+ LY6G F4/80Lo LY6CLo). Representative dot plots for analysis of CD206, CCR2, and major histocompatibility complex (MHC) II in F4/80Hi CD68+ macrophages C: Isotype controls corresponding to different flow cytometry target antibodies tested on dystrophic quadriceps and diaphragm suspensions. LY6C isotype gating is displayed as a contour plot for clarity of representation.
      Figure thumbnail figs2
      Supplemental Figure S2Differences in granularity and size of immune cells isolated from Het blood (BLD) and quadriceps (QUADs). Granularity [side scatter (SSC)-A] and size [forward scatter (FSC)-A] comparison between the CD45+ leukocytes (A), CD11b+ myeloid cells (B), LY6G+ neutrophils (C), and LY6C+ monocytes (D) isolated from Het blood (blue) and quadriceps (red).

      References

        • Hoffman E.P.
        • Brown Jr., R.H.
        • Kunkel L.M.
        • Dystrophin
        the protein product of the Duchenne muscular dystrophy locus.
        Cell. 1987; 51: 919-928
        • Miyatake S.
        • Shimizu-Motohashi Y.
        • Takeda S.
        • Aoki Y.
        Anti-inflammatory drugs for Duchenne muscular dystrophy: focus on skeletal muscle-releasing factors.
        Drug Des Devel Ther. 2016; 10: 2745-2758
        • Rosenberg A.S.
        • Puig M.
        • Nagaraju K.
        • Hoffman E.P.
        • Villalta S.A.
        • Rao V.A.
        • Wakefield L.M.
        • Woodcock J.
        Immune-mediated pathology in Duchenne muscular dystrophy.
        Sci Transl Med. 2015; 7: 299rv4
        • De Rossi M.
        • Bernasconi P.
        • Baggi F.
        • de Waal Malefyt R.
        • Mantegazza R.
        Cytokines and chemokines are both expressed by human myoblasts: possible relevance for the immune pathogenesis of muscle inflammation.
        Int Immunol. 2000; 12: 1329-1335
        • Cannon J.G.
        • St Pierre B.A.
        Cytokines in exertion-induced skeletal muscle injury.
        Mol Cell Biochem. 1998; 179: 159-167
        • Villalta S.A.
        • Nguyen H.X.
        • Deng B.
        • Gotoh T.
        • Tidball J.G.
        Shifts in macrophage phenotypes and macrophage competition for arginine metabolism affect the severity of muscle pathology in muscular dystrophy.
        Hum Mol Genet. 2009; 18: 482-496
        • Zhou L.
        • Rafael-Fortney J.A.
        • Huang P.
        • Zhao X.S.
        • Cheng G.
        • Zhou X.
        • Kaminski H.J.
        • Liu L.
        • Ransohoff R.M.
        Haploinsufficiency of utrophin gene worsens skeletal muscle inflammation and fibrosis in mdx mice.
        J Neurol Sci. 2008; 264: 106-111
        • Fenichel G.M.
        • Florence J.M.
        • Pestronk A.
        • Mendell J.R.
        • Moxley 3rd, R.T.
        • Griggs R.C.
        • Brooke M.H.
        • Miller J.P.
        • Robison J.
        • King W.
        • Signore L.
        • Pandya S.
        • Schierbecker J.
        • Wilson B.
        Long-term benefit from prednisone therapy in Duchenne muscular dystrophy.
        Neurology. 1991; 41: 1874-1877
        • Mescher A.L.
        • Neff A.W.
        • King M.W.
        Inflammation and immunity in organ regeneration.
        Dev Comp Immunol. 2017; 66: 98-110
        • Cordero-Espinoza L.
        • Huch M.
        The balancing act of the liver: tissue regeneration versus fibrosis.
        J Clin Invest. 2018; 128: 85-96
        • Karin M.
        • Clevers H.
        Reparative inflammation takes charge of tissue regeneration.
        Nature. 2016; 529: 307-315
        • Brigitte M.
        • Schilte C.
        • Plonquet A.
        • Baba-Amer Y.
        • Henri A.
        • Charlier C.
        • Tajbakhsh S.
        • Albert M.
        • Gherardi R.K.
        • Chrétien F.
        Muscle resident macrophages control the immune cell reaction in a mouse model of notexin-induced myoinjury.
        Arthritis Rheum. 2010; 62: 268-279
        • Spencer M.J.
        • Montecino-Rodriguez E.
        • Dorshkind K.
        • Tidball J.G.
        Helper (CD4(+)) and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin-deficient muscle.
        Clin Immunol. 2001; 98: 235-243
        • Tidball J.G.
        • Welc S.S.
        • Wehling-Henricks M.
        Immunobiology of inherited muscular dystrophies.
        Compr Physiol. 2018; 8: 1313-1356
        • Kranig S.A.
        • Tschada R.
        • Braun M.
        • Patry C.
        • Pöschl J.
        • Frommhold D.
        • Hudalla H.
        Dystrophin deficiency promotes leukocyte recruitment in mdx mice.
        Pediatr Res. 2019; 86: 188-194
        • Rizzo G.
        • Di Maggio R.
        • Benedetti A.
        • Morroni J.
        • Bouche M.
        • Lozanoska-Ochser B.
        Splenic Ly6Chi monocytes are critical players in dystrophic muscle injury and repair.
        JCI Insight. 2020; 5: e130807
        • Mojumdar K.
        • Liang F.
        • Giordano C.
        • Lemaire C.
        • Danialou G.
        • Okazaki T.
        • Bourdon J.
        • Rafei M.
        • Galipeau J.
        • Divangahi M.
        • Petrof B.J.
        Inflammatory monocytes promote progression of Duchenne muscular dystrophy and can be therapeutically targeted via CCR2.
        EMBO Mol Med. 2014; 6: 1476-1492
        • Hodgetts S.
        • Radley H.
        • Davies M.
        • Grounds M.D.
        Reduced necrosis of dystrophic muscle by depletion of host neutrophils, or blocking TNFalpha function with etanercept in mdx mice.
        Neuromuscul Disord. 2006; 16: 591-602
        • Mosser D.M.
        • Edwards J.P.
        Exploring the full spectrum of macrophage activation.
        Nat Rev Immunol. 2008; 8: 958-969
        • Yang J.
        • Zhang L.
        • Yu C.
        • Yang X.F.
        • Wang H.
        Monocyte and macrophage differentiation: circulation inflammatory monocyte as biomarker for inflammatory diseases.
        Biomark Res. 2014; 2: 1
        • Wang X.
        • Zhao W.
        • Ransohoff R.M.
        • Zhou L.
        Identification and function of fibrocytes in skeletal muscle injury repair and muscular dystrophy.
        J Immunol. 2016; 197: 4750-4761
        • Jakubzick C.V.
        • Randolph G.J.
        • Henson P.M.
        Monocyte differentiation and antigen-presenting functions.
        Nat Rev Immunol. 2017; 17: 349-362
        • Capote J.
        • Kramerova I.
        • Martinez L.
        • Vetrone S.
        • Barton E.R.
        • Sweeney H.L.
        • Miceli M.C.
        • Spencer M.J.
        Osteopontin ablation ameliorates muscular dystrophy by shifting macrophages to a pro-regenerative phenotype.
        J Cell Biol. 2016; 213: 275-288
        • Acharyya S.
        • Villalta S.A.
        • Bakkar N.
        • Bupha-Intr T.
        • Janssen P.M.
        • Carathers M.
        • Li Z.W.
        • Beg A.A.
        • Ghosh S.
        • Sahenk Z.
        • Weinstein M.
        • Gardner K.L.
        • Rafael-Fortney J.A.
        • Karin M.
        • Tidball J.G.
        • Baldwin A.S.
        • Guttridge D.C.
        Interplay of IKK/NF-kappaB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy.
        J Clin Invest. 2007; 117: 889-901
        • Villalta S.A.
        • Deng B.
        • Rinaldi C.
        • Wehling-Henricks M.
        • Tidball J.G.
        IFN-γ promotes muscle damage in the mdx mouse model of Duchenne muscular dystrophy by suppressing M2 macrophage activation and inhibiting muscle cell proliferation.
        J Immunol. 2011; 187: 5419-5428
        • Bencze M.
        • Negroni E.
        • Vallese D.
        • Yacoub-Youssef H.
        • Chaouch S.
        • Wolff A.
        • Aamiri A.
        • Di Santo J.P.
        • Chazaud B.
        • Butler-Browne G.
        • Savino W.
        • Mouly V.
        • Riederer I.
        Proinflammatory macrophages enhance the regenerative capacity of human myoblasts by modifying their kinetics of proliferation and differentiation.
        Mol Ther. 2012; 20: 2168-2179
        • Saclier M.
        • Cuvellier S.
        • Magnan M.
        • Mounier R.
        • Chazaud B.
        Monocyte/macrophage interactions with myogenic precursor cells during skeletal muscle regeneration.
        FEBS J. 2013; 280: 4118-4130
        • Saclier M.
        • Yacoub-Youssef H.
        • Mackey A.L.
        • Arnold L.
        • Ardjoune H.
        • Magnan M.
        • Sailhan F.
        • Chelly J.
        • Pavlath G.K.
        • Mounier R.
        • Kjaer M.
        • Chazaud B.
        Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration.
        Stem Cells. 2013; 31: 384-396
        • Desguerre I.
        • Mayer M.
        • Leturcq F.
        • Barbet J.P.
        • Gherardi R.K.
        • Christov C.
        Endomysial fibrosis in Duchenne muscular dystrophy: a marker of poor outcome associated with macrophage alternative activation.
        J Neuropathol Exp Neurol. 2009; 68: 762-773
        • Epelman S.
        • Lavine K.J.
        • Beaudin A.E.
        • Sojka D.K.
        • Carrero J.A.
        • Calderon B.
        • Brija T.
        • Gautier E.L.
        • Ivanov S.
        • Satpathy A.T.
        • Schilling J.D.
        • Schwendener R.
        • Sergin I.
        • Razani B.
        • Forsberg E.C.
        • Yokoyama W.M.
        • Unanue E.R.
        • Colonna M.
        • Randolph G.J.
        • Mann D.L.
        Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.
        Immunity. 2014; 40: 91-104
        • Bajpai G.
        • Bredemeyer A.
        • Li W.
        • Zaitsev K.
        • Koenig A.L.
        • Lokshina I.
        • Mohan J.
        • Ivey B.
        • Hsiao H.M.
        • Weinheimer C.
        • Kovacs A.
        • Epelman S.
        • Artyomov M.
        • Kreisel D.
        • Lavine K.J.
        Tissue resident CCR2- and CCR2+ cardiac macrophages differentially orchestrate monocyte recruitment and fate specification following myocardial injury.
        Circ Res. 2019; 124: 263-278
        • Epelman S.
        • Lavine K.J.
        • Randolph G.J.
        Origin and functions of tissue macrophages.
        Immunity. 2014; 41: 21-35
        • Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research Council
        Guide for the Care and Use of Laboratory Animals: Eighth Edition.
        National Academies Press, Washington, DC2011
        • Rafael-Fortney J.A.
        • Chimanji N.S.
        • Schill K.E.
        • Martin C.D.
        • Murray J.D.
        • Ganguly R.
        • Stangland J.E.
        • Tran T.
        • Xu Y.
        • Canan B.D.
        • Mays T.A.
        • Delfín D.A.
        • Janssen P.M.
        • Raman S.V.
        Early treatment with lisinopril and spironolactone preserves cardiac and skeletal muscle in Duchenne muscular dystrophy mice.
        Circulation. 2011; 124: 582-588
        • Lundberg I.
        • Brengman J.M.
        • Engel A.G.
        Analysis of cytokine expression in muscle in inflammatory myopathies, Duchenne dystrophy, and non-weak controls.
        J Neuroimmunol. 1995; 63: 9-16
        • Campanholle G.
        • Mittelsteadt K.
        • Nakagawa S.
        • Kobayashi A.
        • Lin S.L.
        • Gharib S.A.
        • Heinecke J.W.
        • Hamerman J.A.
        • Altemeier W.A.
        • Duffield J.S.
        TLR-2/TLR-4 TREM-1 signaling pathway is dispensable in inflammatory myeloid cells during sterile kidney injury.
        PLoS One. 2013; 8: e68640
        • Wehling M.
        • Spencer M.J.
        • Tidball J.G.
        A nitric oxide synthase transgene ameliorates muscular dystrophy in mdx mice.
        J Cell Biol. 2001; 155: 123-131
        • Hightower R.M.
        • Reid A.L.
        • Gibbs D.E.
        • Wang Y.
        • Widrick J.J.
        • Kunkel L.M.
        • Kastenschmidt J.M.
        • Villalta S.A.
        • van Groen T.
        • Chang H.
        • Gornisiewicz S.
        • Landesman Y.
        • Tamir S.
        • Alexander M.S.
        The SINE compound KPT-350 blocks dystrophic pathologies in DMD zebrafish and mice.
        Mol Ther. 2020; 28: 189-201
        • Kastenschmidt J.M.
        • Avetyan I.
        • Villalta S.A.
        Characterization of the inflammatory response in dystrophic muscle using flow cytometry.
        Methods Mol Biol. 2018; 1687: 43-56
        • Covarrubias R.
        • Ismahil M.A.
        • Rokosh G.
        • Hamid T.
        • Accornero F.
        • Singh H.
        • Gumina R.J.
        • Prabhu S.D.
        • Bansal S.S.
        Optimized protocols for isolation, fixation, and flow cytometric characterization of leukocytes in ischemic hearts.
        Am J Physiol Heart Circ Physiol. 2019; 317: H658-H666
        • Bansal S.S.
        • Ismahil M.A.
        • Goel M.
        • Zhou G.
        • Rokosh G.
        • Hamid T.
        • Prabhu S.D.
        Dysfunctional and proinflammatory regulatory T-lymphocytes are essential for adverse cardiac remodeling in ischemic cardiomyopathy.
        Circulation. 2019; 139: 206-221
        • Bansal S.S.
        • Ismahil M.A.
        • Goel M.
        • Patel B.
        • Hamid T.
        • Rokosh G.
        • Prabhu S.D.
        Activated T lymphocytes are essential drivers of pathological remodeling in ischemic heart failure.
        Circ Heart Fail. 2017; 10: e003688
        • Warren G.L.
        • Hulderman T.
        • Mishra D.
        • Gao X.
        • Millecchia L.
        • O'Farrell L.
        • Kuziel W.A.
        • Simeonova P.P.
        Chemokine receptor CCR2 involvement in skeletal muscle regeneration.
        FASEB J. 2005; 19: 413-415
        • Lee Y.S.
        • Kim M.H.
        • Yi H.S.
        • Kim S.Y.
        • Kim H.H.
        • Kim J.H.
        • Yeon J.E.
        • Byun K.S.
        • Byun J.S.
        • Jeong W.I.
        CX(3)CR1 differentiates F4/80(low) monocytes into pro-inflammatory F4/80(high) macrophages in the liver.
        Sci Rep. 2018; 8: 15076
        • Tamoutounour S.
        • Henri S.
        • Lelouard H.
        • de Bovis B.
        • de Haar C.
        • van der Woude C.J.
        • Woltman A.M.
        • Reyal Y.
        • Bonnet D.
        • Sichien D.
        • Bain C.C.
        • Mowat A.M.
        • Reis e Sousa C.
        • Poulin L.F.
        • Malissen B.
        • Guilliams M.
        CD64 distinguishes macrophages from dendritic cells in the gut and reveals the Th1-inducing role of mesenteric lymph node macrophages during colitis.
        Eur J Immunol. 2012; 42: 3150-3166
        • Finsterbusch M.
        • Hall P.
        • Li A.
        • Devi S.
        • Westhorpe C.L.
        • Kitching A.R.
        • Hickey M.J.
        Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus.
        Proc Natl Acad Sci U S A. 2016; 113: E5172-E5181
        • Combadière C.
        • Potteaux S.
        • Rodero M.
        • Simon T.
        • Pezard A.
        • Esposito B.
        • Merval R.
        • Proudfoot A.
        • Tedgui A.
        • Mallat Z.
        Combined inhibition of CCL2, CX3CR1, and CCR5 abrogates Ly6C(hi) and Ly6C(lo) monocytosis and almost abolishes atherosclerosis in hypercholesterolemic mice.
        Circulation. 2008; 117: 1649-1657
        • Ochoa O.
        • Sun D.
        • Reyes-Reyna S.M.
        • Waite L.L.
        • Michalek J.E.
        • McManus L.M.
        • Shireman P.K.
        Delayed angiogenesis and VEGF production in CCR2-/- mice during impaired skeletal muscle regeneration.
        Am J Physiol Regul Integr Comp Physiol. 2007; 293: R651-R661
        • Kuraoka M.
        • Kimura E.
        • Nagata T.
        • Okada T.
        • Aoki Y.
        • Tachimori H.
        • Yonemoto N.
        • Imamura M.
        • Takeda S.
        Serum osteopontin as a novel biomarker for muscle regeneration in Duchenne muscular dystrophy.
        Am J Pathol. 2016; 186: 1302-1312
        • Braga T.T.
        • Agudelo J.S.
        • Camara N.O.
        Macrophages during the fibrotic process: M2 as friend and foe.
        Front Immunol. 2015; 6: 602
        • Tidball J.G.
        • Villalta S.A.
        Regulatory interactions between muscle and the immune system during muscle regeneration.
        Am J Physiol Regul Integr Comp Physiol. 2010; 298: R1173-R1187
        • Chazaud B.
        Inflammation and skeletal muscle regeneration: leave it to the macrophages!.
        Trends Immunol. 2020; 41: 481-492
        • Chen Y.W.
        • Nagaraju K.
        • Bakay M.
        • McIntyre O.
        • Rawat R.
        • Shi R.
        • Hoffman E.P.
        Early onset of inflammation and later involvement of TGFbeta in Duchenne muscular dystrophy.
        Neurology. 2005; 65: 826-834
        • Kim H.K.
        • Merrow A.C.
        • Shiraj S.
        • Wong B.L.
        • Horn P.S.
        • Laor T.
        Analysis of fatty infiltration and inflammation of the pelvic and thigh muscles in boys with Duchenne muscular dystrophy (DMD): grading of disease involvement on MR imaging and correlation with clinical assessments.
        Pediatr Radiol. 2013; 43: 1327-1335
        • Mavrogeni S.
        • Papavasiliou A.
        • Spargias K.
        • Constandoulakis P.
        • Papadopoulos G.
        • Karanasios E.
        • Georgakopoulos D.
        • Kolovou G.
        • Demerouti E.
        • Polymeros S.
        • Kaklamanis L.
        • Magoutas A.
        • Papadopoulou E.
        • Markussis V.
        • Cokkinos D.V.
        Myocardial inflammation in Duchenne muscular dystrophy as a precipitating factor for heart failure: a prospective study.
        BMC Neurol. 2010; 10: 33
        • Preuße C.
        • von Moers A.
        • Kölbel H.
        • Pehl D.
        • Goebel H.H.
        • Schara U.
        • Stenzel W.
        Inflammation-induced fibrosis in skeletal muscle of female carriers of Duchenne muscular dystrophy.
        Neuromuscul Disord. 2019; 29: 487-496