Myeloid cell-mediated targeting of LIF to dystrophic muscle causes transient increases in muscle fiber lesions by disrupting the recruitment and dispersion of macrophages in muscle.

Leukemia inhibitory factor (LIF) can influence development by increasing cell proliferation and inhibiting differentiation. Because of its potency for expanding stem cell populations, delivery of exogenous LIF to diseased tissue could have therapeutic value. However, systemic elevations of LIF can have negative, off-target effects. We tested whether inflammatory cells expressing a LIF transgene under control of a leukocyte-specific, CD11b promoter provide a strategy to target LIF to sites of damage in the mdx mouse model of Duchenne muscular dystrophy, leading to increased numbers of muscle stem cells and improved muscle regeneration. However, transgene expression in inflammatory cells did not increase muscle growth or increase numbers of stem cells required for regeneration. Instead, transgene expression disrupted the normal dispersion of macrophages in dystrophic muscles, leading to transient increases in muscle damage in foci where macrophages were highly concentrated during early stages of pathology. The defect in inflammatory cell dispersion reflected impaired chemotaxis of macrophages to C-C motif chemokine ligand-2 and local increases of LIF production that produced large aggregations of cytolytic macrophages. Transgene expression also induced a shift in macrophage phenotype away from a CD206+, M2-biased phenotype that supports regeneration. However, at later stages of the disease when macrophage numbers declined, they dispersed in the muscle, leading to reductions in muscle fiber damage, compared to non-transgenic mdx mice. Together, the findings show that macrophage-mediated delivery of transgenic LIF exerts differential effects on macrophage dispersion and muscle damage depending on the stage of dystrophic pathology.

Differential Effects of Myeloid Cell PPARδ and IL-10 in Regulating Macrophage Recruitment, Phenotype, and Regeneration following Acute Muscle Injury.

Changes in macrophage phenotype in injured muscle profoundly influence regeneration. In particular, the shift of macrophages from a proinflammatory (M1 biased) phenotype to a proregenerative (M2 biased) phenotype characterized by expression of CD206 and CD163 is essential for normal repair. According to the current canonical mechanism regulating for M1/M2 phenotype transition, signaling through PPARδ is necessary for obtaining the M2-biased phenotype. Our findings confirm that the murine myeloid cell-targeted deletion of Ppard reduces expression in vitro of genes that are activated in M2-biased macrophages; however, the mutation in mice in vivo increased numbers of CD206+ M2-biased macrophages and did not reduce the expression of phenotypic markers of M2-biased macrophages in regenerating muscle. Nevertheless, the mutation impaired CCL2-mediated chemotaxis of macrophages and slowed revascularization of injured muscle. In contrast, null mutation of IL-10 diminished M2-biased macrophages but produced no defects in muscle revascularization. Our results provide two significant findings. First, they illustrate that mechanisms that regulate macrophage phenotype transitions in vitro are not always predictive of mechanisms that are most important in vivo. Second, they show that mechanisms that regulate macrophage phenotype transitions differ in different in vivo environments.

Macrophages escape Klotho gene silencing in the mdx mouse model of Duchenne muscular dystrophy and promote muscle growth and increase satellite cell numbers through a Klotho-mediated pathway.

Duchenne muscular dystrophy (DMD) is a muscle wasting disease in which inflammation influences the severity of pathology. We found that the onset of muscle inflammation in the mdx mouse model of DMD coincides with large increases in expression of pro-inflammatory cytokines [tumor necrosis factor-α (TNFα); interferon gamma (IFNγ)] and dramatic reductions of the pro-myogenic protein Klotho in muscle cells and large increases of Klotho in pro-regenerative, CD206+ macrophages. Furthermore, TNFα and IFNγ treatments reduced Klotho in muscle cells and increased Klotho in macrophages. Because CD206+/Klotho+ macrophages were concentrated at sites of muscle regeneration, we tested whether macrophage-derived Klotho promotes myogenesis. Klotho transgenic macrophages had a pro-proliferative influence on muscle cells that was ablated by neutralizing antibodies to Klotho and conditioned media from Klotho mutant macrophages did not increase muscle cell proliferation in vitro. In addition, transplantation of bone marrow cells from Klotho transgenic mice into mdx recipients increased numbers of myogenic cells and increased the size of muscle fibers. Klotho also acted directly on macrophages, stimulating their secretion of TNFα. Because TNFα is a muscle mitogen, we tested whether the pro-proliferative effects of Klotho on muscle cells were mediated by TNFα and found that increased proliferation caused by Klotho was reduced by anti-TNFα. Collectively, these data show that pro-inflammatory cytokines contribute to silencing of Klotho in dystrophic muscle, but increase Klotho expression by macrophages. Our findings also show that macrophage-derived Klotho can promote muscle regeneration by expanding populations of muscle stem cells and increasing muscle fiber growth in dystrophic muscle.

Aging of the immune system causes reductions in muscle stem cell populations, promotes their shift to a fibrogenic phenotype, and modulates sarcopenia.

Aging is associated with diminished muscle mass, reductions in muscle stem cell functions, and increased muscle fibrosis. The immune system, especially macrophages, can have important roles in modulating muscle growth and regeneration, suggesting that the immune system may also have significant influences on muscle aging. Moreover, the immune system experiences changes in function during senescence, suggesting that regulatory interaction between muscle cells and the immune system may also change during aging. In this study, we performed bone marrow transplantations between age-mismatched donor and recipient mice to test the influence of the age of the immune system on muscle aging. Transplantation of young bone marrow cells into old recipients prevented sarcopenia and prevented age-related change in muscle fiber phenotype. Transplantation of old bone marrow cells into young animals reduced satellite cell numbers and promoted satellite cells to switch toward a fibrogenic phenotype. We also demonstrated that conditioned media from young, but not old, bone marrow cells promoted myoblast proliferation in vitro, and we found that factors released by young bone marrow cells were more supportive of myotube differentiation in vitro. Together, our results demonstrate that aging of bone marrow cells promotes the age-related reduction of satellite cell number and function and contributes to sarcopenia.-Wang, Y., Wehling-Henricks, M., Welc, S. S., Fisher, A. L., Zuo, Q., Tidball, J. G. Aging of the immune system causes reductions in muscle stem cell populations, promotes their shift to a fibrogenic phenotype, and modulates sarcopenia.

Modulation of Klotho expression in injured muscle perturbs Wnt signalling and influences the rate of muscle growth.

NEW FINDINGS: What is the central question of this study? Does modulating the expression of Klotho affect myogenesis following acute injury of healthy, non-senescent muscle? What is the main finding and its importance? Klotho can accelerate muscle growth following acute injury of healthy, adult mice, which supports the possibility that increased delivery of Klotho could have therapeutic value for improving repair of damaged muscle. ABSTRACT: Skeletal muscle injuries activate a complex programme of myogenesis that can restore normal muscle structure. We tested whether modulating the expression of klotho influenced the response of mouse muscles to acute injury. Our findings show that klotho expression in muscle declines at 3 days post-injury. That reduction in klotho expression coincided with elevated expression of targets of Wnt signalling (Ccnd1; Myc) and increased MyoD+ muscle cell numbers, reflecting the onset of myogenic cell differentiation. klotho expression subsequently increased at 7 days post-injury with elevated expression occurring primarily in inflammatory lesions, which was accompanied by reduced expression of Wnt target genes (Ccnd1: 91%; Myc: 96%). Introduction of a klotho transgene maintained high levels of klotho expression over the course of muscle repair and attenuated the increases in Ccnd1 and Myc expression that occurred at 3 days post-injury. Correspondingly, transgene expression reduced Wnt signalling in Pax7+ cells, reflected by reductions in Pax7+ cells expressing active ß-catenin, and reduced the numbers of MyoD+ cells at 3 days post-injury. At 21 days post-injury, muscles in klotho transgenic mice showed increased Pax7+ and decreased myogenin+ cell densities and large increases in myofibre size. Likewise, treating myogenic cells in vitro with Klotho reduced Myod expression but did not affect Pax7 expression. Muscle inflammation was only slightly modulated by increased klotho expression, initially reducing the expression of M2-biased macrophage markers Cd163 and Cd206 at 3 days post-injury and later increasing the expression of pan-macrophage marker F480 and Cd68 at 21 days post-injury. Collectively, our study shows that Klotho modulates myogenesis and that increased expression accelerates muscle growth after injury.

Klotho gene silencing promotes pathology in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease involving progressive loss of muscle regenerative capacity and increased fibrosis. We tested whether epigenetic silencing of the klotho gene occurs in the mdx mouse model of DMD and whether klotho silencing is an important feature of the disease. Our findings show that klotho undergoes muscle-specific silencing at the acute onset of mdx pathology. Klotho experiences increased methylation of CpG sites in its promoter region, which is associated with gene silencing, and increases in a repressive histone mark, H3K9me2. Expression of a klotho transgene in mdx mice restored their longevity, reduced muscle wasting, improved function and greatly increased the pool of muscle-resident stem cells required for regeneration. Reductions of fibrosis in late, progressive stages of the mdx pathology achieved by transgene expression were paralleled by reduced expression of Wnt target genes (axin-2), transforming growth factor-beta (TGF-ß1) and collagens types 1 and 3, indicating that Klotho inhibition of the profibrotic Wnt/TGFß axis underlies its anti-fibrotic effect in aging, dystrophic muscle. Thus, epigenetic silencing of klotho during muscular dystrophy contributes substantially to lost regenerative capacity and increased fibrosis of dystrophic muscle during late progressive stages of the disease.

Myeloid cells are capable of synthesizing aldosterone to exacerbate damage in muscular dystrophy.

FDA-approved mineralocorticoid receptor (MR) antagonists are used to treat heart failure. We have recently demonstrated efficacy of MR antagonists for skeletal muscles in addition to heart in Duchenne muscular dystrophy mouse models and that mineralocorticoid receptors are present and functional in skeletal muscles. The goal of this study was to elucidate the underlying mechanisms of MR antagonist efficacy on dystrophic skeletal muscles. We demonstrate for the first time that infiltrating myeloid cells clustered in damaged areas of dystrophic skeletal muscles have the capacity to produce the natural ligand of MR, aldosterone, which in excess is known to exacerbate tissue damage. Aldosterone synthase protein levels are increased in leukocytes isolated from dystrophic muscles compared with controls and local aldosterone levels in dystrophic skeletal muscles are increased, despite normal circulating levels. All genes encoding enzymes in the pathway for aldosterone synthesis are expressed in muscle-derived leukocytes. 11ß-HSD2, the enzyme that inactivates glucocorticoids to increase MR selectivity for aldosterone, is also increased in dystrophic muscle tissues. These results, together with the demonstrated preclinical efficacy of antagonists, suggest MR activation is in excess of physiological need and likely contributes to the pathology of muscular dystrophy. This study provides new mechanistic insight into the known contribution of myeloid cells to muscular dystrophy pathology. This first report of myeloid cells having the capacity to produce aldosterone may have implications for a wide variety of acute injuries and chronic diseases with inflammation where MR antagonists may be therapeutic.

Protection of intestinal injury during heat stroke in mice by interleukin-6 pretreatment.

KEY POINTS: Heat stroke afflicts thousands of humans each year, worldwide. The immune system responds to hyperthermia exposure resulting in heat stroke by producing an array of immunological proteins, such as interleukin-6 (IL-6). However, the physiological functions of IL-6 and other cytokines in hyperthermia are poorly understood. We hypothesized that IL-6 plays a protective role in conditions of heat stroke. To test this, we gave small IL-6 supplements to mice prior to exposing them to hot environments sufficient to induce conditions of heat stroke. Pretreatment with IL-6 resulted in improved ability to withstand heat exposure in anaesthetized mice, it protected the intestine from injury, reducing the permeability of the intestinal barrier, and it attenuated the release of other cytokines involved in inflammation. The results support the hypothesis that IL-6 is a 'physiological stress hormone' that plays an important role in survival during acute life-threatening conditions such as heat stroke. ABSTRACT: The role of interleukin-6 (IL-6) in hyperthermia and heat stroke is poorly understood. Plasma IL-6 is elevated following hyperthermia in animals and humans, and IL-6 knockout mice are more intolerant of severe hyperthermia. We evaluated the effect of IL-6 supplementation on organ injury following severe hyperthermia exposure in anaesthetized mice. Two hours prior to hyperthermia, mice were treated with 0.6 µg intraperitoneal IL-6, or identical volumes of saline in controls. Mice were anaesthetized, gavaged with FITC-dextran for measures of gastrointestinal permeability, and exposed to incremental (0.5°C every 30 min) increases in temperature. Heating stopped when maximum core temperature (Tc) of 42.4°C was attained (Tc,max). The mice recovered at room temperature (≈22°C) for 30 or 120 min, at which time plasma and tissues were collected. IL-6-treated mice, on average, required ≈25 min longer to attain Tc,max . Injury and swelling of the villi in the duodenum was present in untreated mice after 30 min of recovery. These changes were blocked by IL-6 treatment. IL-6 also reduced gastrointestinal permeability, assayed by the accumulation of FITC-dextran in plasma. Plasma cytokines were also attenuated in IL-6-treated animals, including significant reductions in TNFα, MCP-1 (CXCL2), RANTES (CCL5) and KC (CCL5). The results demonstrate that IL-6 has a protective influence on the pattern of physiological responses to severe hyperthermia, suggesting that early endogenous expression of IL-6 may provide a protection from the development of organ damage and inflammation.

Functional cardiac consequences of ß-adrenergic stress-induced injury in the mdx mouse model of Duchenne muscular dystrophy.

Cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD), however, in the mdx mouse model of DMD, the cardiac phenotype differs from that seen in DMD-associated cardiomyopathy. Although some have used pharmacologic stress to enhance the cardiac phenotype in the mdx model, many methods lead to high mortality, variable cardiac outcomes, and do not recapitulate the structural and functional cardiac changes seen in human disease. Here, we describe a simple and effective method to enhance the cardiac phenotype model in mdx mice using advanced 2D and 4D high-frequency ultrasound to monitor cardiac dysfunction progression in vivo. For our study, mdx and wild-type (WT) mice received daily low-dose (2 mg/kg/day) isoproterenol injections for 10 days. Histopathologic assessment showed that isoproterenol treatment increased myocyte injury, elevated serum cardiac troponin I levels, and enhanced fibrosis in mdx mice. Ultrasound revealed reduced ventricular function, decreased wall thickness, increased volumes, and diminished cardiac reserve in mdx mice compared to wild-type. Our findings highlight the utility of low-dose isoproterenol in mdx mice as a valuable model for exploring therapies targeting DMD-associated cardiac complications.

Both enantiomers of ß-aminoisobutyric acid BAIBA regulate Fgf23 via MRGPRD receptor by activating distinct signaling pathways in osteocytes.

With exercise, muscle and bone produce factors with beneficial effects on brain, fat, and other organs. Exercise in mice increased fibroblast growth factor 23 (FGF23), urine phosphate, and the muscle metabolite L-ß-aminoisobutyric acid (L-BAIBA), suggesting that L-BAIBA may play a role in phosphate metabolism. Here, we show that L-BAIBA increases in serum with exercise and elevates Fgf23 in osteocytes. The D enantiomer, described to be elevated with exercise in humans, can also induce Fgf23 but through a delayed, indirect process via sclerostin. The two enantiomers both signal through the same receptor, Mas-related G-protein-coupled receptor type D, but activate distinct signaling pathways; L-BAIBA increases Fgf23 through Gαs/cAMP/PKA/CBP/ß-catenin and Gαq/PKC/CREB, whereas D-BAIBA increases Fgf23 indirectly through sclerostin via Gαi/NF-κB. In vivo, both enantiomers increased Fgf23 in bone in parallel with elevated urinary phosphate excretion. Thus, exercise-induced increases in BAIBA and FGF23 work together to maintain phosphate homeostasis.

Skeletal muscle interleukin-6 regulation in hyperthermia.

We previously reported that IL-6 production is acutely elevated in skeletal muscles exposed to ≥41°C, but the regulatory pathways are poorly understood. The present study characterizes the heat-induced transcriptional control of IL-6 in C2C12 muscle fibers. Hyperthermia exposure (42°C for 1 h) induced transcription from an IL-6 promoter-luciferase reporter plasmid. Heat shock factor-1 (HSF-1), a principal mediator of the heat shock response, was then tested for its role in IL-6 regulation. Overexpression of a constitutively active HSF-1 construct increased basal (37°C) promoter activity, whereas overexpression of a dominant negative HSF-1 reduced IL-6 promoter activity during basal and hyperthermia conditions. Since hyperthermia also induces stress-activated protein kinase (SAPK) signaling, we tested whether mutation of a transcription site downstream of SAPK, (i.e., activator protein-1, AP-1) influences IL-6 transcription in hyperthermia. The mutation had no effect on baseline reporter activity but completely inhibited heat-induced activity. We then tested whether pharmacologically induced states of protein stress, characteristic of cellular responses to hyperthermia and known to induce SAPKs and HSF-1, would induce IL-6 production in the absence of heat. The proteasome was inhibited with MG-132 in one set of experiments, and the unfolded protein response was stimulated with dithiothreitol, thapsigargin, tunicamycin, or castanospermine in other experiments. All treatments stimulated IL-6 protein secretion in the absence of hyperthermia. These studies demonstrate that IL-6 regulation in hyperthermia is directly controlled by HSF-1 and AP-1 signaling and that the IL-6 response in C2C12 myotubes is sensitive to categories of protein stress that reflect accumulation of damaged or unfolded proteins.

The regulation of interleukin-6 implicates skeletal muscle as an integrative stress sensor and endocrine organ.

Skeletal muscle has been identified as an endocrine organ owing to its capacity to produce and secrete a variety of cytokines (myokines) and other proteins. To date, myokines have primarily been studied in response to exercise or metabolic challenges; however, numerous observations suggest that skeletal muscle may also release myokines in response to certain categories of internal or external stress exposure. Internal stress signals include oxidative or nitrosative stress, damaged or unfolded proteins, hyperthermia or energy imbalance. External stress signals, which act as indicators of organismal stress or injury in other cells, employ mediators such as catecholamines, endotoxin, alarmins, ATP and pro-inflammatory cytokines, such as tumour necrosis factor-α and interleukin-1ß. External stress signals generally induce cellular responses through membrane receptor systems. In this review, we focus on the regulation of interleukin-6 (IL-6) as a prototypical stress response myokine and highlight evidence that IL-6 gene regulation in muscle is inherently organized to respond to a wide variety of internal and external stressors. Given that IL-6 can initiate protective, anti-inflammatory or restorative processes throughout the organism during life-threatening conditions, we present the argument that skeletal muscle has a physiological function as a sensor and responder to stress. Furthermore, we hypothesize that it may comprise a fundamental component of the organism's acute stress response.

Hyperthermia increases interleukin-6 in mouse skeletal muscle.

Skeletal muscles produce and contribute to circulating levels of IL-6 during exercise. However, when core temperature is reduced, the response is attenuated. Therefore, we hypothesized that hyperthermia may be an important and independent stimulus for muscle IL-6. In cultured C2C12 myotubes, hyperthermia (42°C) increased IL-6 gene expression 14-fold after 1 h and 35-fold after 5 h of 37°C recovery; whereas exposure to 41°C resulted in a 2.6-fold elevation at 1 h. IL-6 protein was secreted and significantly elevated in the cell supernatant. Similar but reduced responses to heat were seen in C2C12 myoblasts. Isolated soleus muscles from mice, exposed ex vivo to 41°C for 1 h, yielded similar IL-6 gene responses (>3-fold) but without a significant effect on protein release. When whole animals were exposed to passive hyperthermia, such that core temperature increased to 42.4°C, IL-6 mRNA in soleus increased 5.4-fold compared with time matched controls. Interestingly, TNF-α gene expression was routinely suppressed at all levels of hyperthermia (40.5-42°C) in the isolated models, but TNF-α was elevated (4.2-fold) in the soleus taken from intact mice exposed, in vivo, to hyperthermia. Muscle HSP72 mRNA increased as a function of the level of hyperthermia, and IL-6 mRNA responses increased proportionally with HSP72. In cultured C2C12 myotubes, when heat shock factor was pharmacologically blocked with KNK437, both HSP72 and IL-6 mRNA elevations, induced by heat, were suppressed. These findings implicate skeletal muscle as a "heat stress sensor" at physiologically relevant hyperthermia, responding with a programmed cytokine expression pattern characterized by elevated IL-6.

Myeloid cell-specific mutation of Spi1 selectively reduces M2-biased macrophage numbers in skeletal muscle, reduces age-related muscle fibrosis and prevents sarcopenia.

Intramuscular macrophages play key regulatory roles in determining the response of skeletal muscle to injury and disease. Recent investigations showed that the numbers and phenotype of intramuscular macrophages change during aging, suggesting that those changes could influence the aging process. We tested that hypothesis by generating a mouse model that harbors a myeloid cell-specific mutation of Spi1, which is a transcription factor that is essential for myeloid cell development. The mutation reduced the numbers of macrophages biased to the CD163+/CD206+ M2 phenotype in muscles of aging mice without affecting the numbers of CD68-expressing macrophages and reduced the expression of transcripts associated with the M2-biased phenotype. The mutation did not affect the colony-forming ability or the frequency of specific subpopulations of bone marrow hematopoietic cells and did not affect myeloid/lymphoid cell ratios in peripheral blood leukocyte populations. Cellularity of most myeloid lineage cells was not influenced by the mutation. The Spi1 mutation in bone marrow-derived macrophages in vitro also did not affect expression of transcripts that indicate the M2-biased phenotype. Thus, myeloid cell-targeted mutation of Spi1 influences macrophage phenotype in muscle but did not affect earlier stages of differentiation of cells in the macrophage lineage. The mutation reduced age-related muscle fibrosis, which is consistent with the reduction of M2-biased macrophages, and reduced expression of the pro-fibrotic enzyme arginase. Most importantly, the mutation prevented sarcopenia. Together, our observations indicate that intramuscular, M2-biased macrophages play significant roles in promoting detrimental, age-related changes in muscle.

Muscle-Specific Ablation of Glucose Transporter 1 (GLUT1) Does Not Impair Basal or Overload-Stimulated Skeletal Muscle Glucose Uptake.

Glucose transporter 1 (GLUT1) is believed to solely mediate basal (insulin-independent) glucose uptake in skeletal muscle; yet recent work has demonstrated that mechanical overload, a model of resistance exercise training, increases muscle GLUT1 levels. The primary objective of this study was to determine if GLUT1 is necessary for basal or overload-stimulated muscle glucose uptake. Muscle-specific GLUT1 knockout (mGLUT1KO) mice were generated and examined for changes in body weight, body composition, metabolism, systemic glucose regulation, muscle glucose transporters, and muscle [3H]-2-deoxyglucose uptake ± the GLUT1 inhibitor BAY-876. [3H]-hexose uptake ± BAY-876 was also examined in HEK293 cells-expressing GLUT1-6 or GLUT10. mGLUT1KO mice exhibited no impairments in body weight, lean mass, whole body metabolism, glucose tolerance, basal or overload-stimulated muscle glucose uptake. There was no compensation by the insulin-responsive GLUT4. In mGLUT1KO mouse muscles, overload stimulated higher expression of mechanosensitive GLUT6, but not GLUT3 or GLUT10. In control and mGLUT1KO mouse muscles, 0.05 µM BAY-876 impaired overload-stimulated, but not basal glucose uptake. In the GLUT-HEK293 cells, BAY-876 inhibited glucose uptake via GLUT1, GLUT3, GLUT4, GLUT6, and GLUT10. Collectively, these findings demonstrate that GLUT1 does not mediate basal muscle glucose uptake and suggest that a novel glucose transport mechanism mediates overload-stimulated glucose uptake.

Aging of the immune system and impaired muscle regeneration: A failure of immunomodulation of adult myogenesis.

Skeletal muscle regeneration that follows acute injury is strongly influenced by interactions with immune cells that invade and proliferate in the damaged tissue. Discoveries over the past 20 years have identified many of the key mechanisms through which myeloid cells, especially macrophages, regulate muscle regeneration. In addition, lymphoid cells that include CD8+ T-cells and regulatory T-cells also significantly affect the course of muscle regeneration. During aging, the regenerative capacity of skeletal muscle declines, which can contribute to progressive loss of muscle mass and function. Those age-related reductions in muscle regeneration are accompanied by systemic, age-related changes in the immune system, that affect many of the myeloid and lymphoid cell populations that can influence muscle regeneration. In this review, we present recent discoveries that indicate that aging of the immune system contributes to the diminished regenerative capacity of aging muscle. Intrinsic, age-related changes in immune cells modify their expression of factors that affect the function of a population of muscle stem cells, called satellite cells, that are necessary for normal muscle regeneration. For example, age-related reductions in the expression of growth differentiation factor-3 (GDF3) or CXCL10 by macrophages negatively affect adult myogenesis, by disrupting regulatory interactions between macrophages and satellite cells. Those changes contribute to a reduction in the numbers and myogenic capacity of satellite cells in old muscle, which reduces their ability to restore damaged muscle. In addition, aging produces changes in the expression of molecules that regulate the inflammatory response to injured muscle, which also contributes to age-related defects in muscle regeneration. For example, age-related increases in the production of osteopontin by macrophages disrupts the normal inflammatory response to muscle injury, resulting in regenerative defects. These nascent findings represent the beginning of a newly-developing field of investigation into mechanisms through which aging of the immune system affects muscle regeneration.

Targeting a therapeutic LIF transgene to muscle via the immune system ameliorates muscular dystrophy.

Many potentially therapeutic molecules have been identified for treating Duchenne muscular dystrophy. However, targeting those molecules only to sites of active pathology is an obstacle to their clinical use. Because dystrophic muscles become extensively inflamed, we tested whether expressing a therapeutic transgene in leukocyte progenitors that invade muscle would provide selective, timely delivery to diseased muscle. We designed a transgene in which leukemia inhibitory factor (LIF) is under control of a leukocyte-specific promoter and transplanted transgenic cells into dystrophic mice. Transplantation diminishes pathology, reduces Th2 cytokines in muscle and biases macrophages away from a CD163+/CD206+ phenotype that promotes fibrosis. Transgenic cells also abrogate TGFß signaling, reduce fibro/adipogenic progenitor cells and reduce fibrogenesis of muscle cells. These findings indicate that leukocytes expressing a LIF transgene reduce fibrosis by suppressing type 2 immunity and highlight a novel application by which immune cells can be genetically modified as potential therapeutics to treat muscle disease.

Immunobiology of Inherited Muscular Dystrophies.

The immune response to acute muscle damage is important for normal repair. However, in chronic diseases such as many muscular dystrophies, the immune response can amplify pathology and play a major role in determining disease severity. Muscular dystrophies are inheritable diseases that vary tremendously in severity, but share the progressive loss of muscle mass and function that can be debilitating and lethal. Mutations in diverse genes cause muscular dystrophy, including genes that encode proteins that maintain membrane strength, participate in membrane repair, or are components of the extracellular matrix or the nuclear envelope. In this article, we explore the hypothesis that an important feature of many muscular dystrophies is an immune response adapted to acute, infrequent muscle damage that is misapplied in the context of chronic injury. We discuss the involvement of the immune system in the most common muscular dystrophy, Duchenne muscular dystrophy, and show that the immune system influences muscle death and fibrosis as disease progresses. We then present information on immune cell function in other muscular dystrophies and show that for many muscular dystrophies, release of cytosolic proteins into the extracellular space may provide an initial signal, leading to an immune response that is typically dominated by macrophages, neutrophils, helper T-lymphocytes, and cytotoxic T-lymphocytes. Although those features are similar in many muscular dystrophies, each muscular dystrophy shows distinguishing features in the magnitude and type of inflammatory response. These differences indicate that there are disease-specific immunomodulatory molecules that determine response to muscle cell damage caused by diverse genetic mutations. © 2018 American Physiological Society. Compr Physiol 8:1313-1356, 2018.

Myeloid cell-derived tumor necrosis factor-alpha promotes sarcopenia and regulates muscle cell fusion with aging muscle fibers.

Sarcopenia is age-related muscle wasting that lacks effective therapeutic interventions. We found that systemic ablation of tumor necrosis factor-α (TNF-α) prevented sarcopenia and prevented age-related change in muscle fiber phenotype. Furthermore, TNF-α ablation reduced the number of satellite cells in aging muscle and promoted muscle cell fusion in vivo and in vitro. Because CD68+ macrophages are important sources of TNF-α and the number of CD68+ macrophages increases in aging muscle, we tested whether macrophage-derived TNF-α affects myogenesis. Media conditioned by TNF-α-null macrophages increased muscle cell fusion in vitro, compared to media conditioned by wild-type macrophages. In addition, transplantation of bone marrow cells from wild-type mice into TNF-α-null recipients increased satellite cell numbers and reduced numbers of centrally nucleated myofibers, indicating that myeloid cell-secreted TNF-α reduces muscle cell fusion. Transplanting bone marrow cells from wild-type mice into TNF-α-null recipients also increased sarcopenia, although transplantation did not restore the age-related change in muscle fiber phenotype. Collectively, we show that myeloid cell-derived TNF-α contributes to muscle aging by affecting sarcopenia and muscle cell fusion with aging muscle fibers. Our findings also show that TNF-α that is intrinsic to muscle and TNF-α secreted by immune cells work together to influence muscle aging.