Pericytes in the myovascular niche promote post-natal myofiber growth and satellite cell quiescence.

The satellite cells, which serve as adult muscle stem cells, are both located beneath myofiber basement membranes and closely associated with capillary endothelial cells. We observed that 90% of capillaries were associated with pericytes in adult mouse and human muscle. During post-natal growth, newly formed vessels with their neuroglial 2 proteoglycan (NG2)-positive pericytes became progressively associated with the post-natal muscle stem cells, as myofibers increased in size and satellite cells entered into quiescence. In vitro, human muscle-derived pericytes promoted myogenic cell differentiation through insulin-like growth factor 1 (IGF1) and myogenic cell quiescence through angiopoietin 1 (ANGPT1). Diphtheria toxin-induced ablation of muscle pericytes in growing mice led both to myofiber hypotrophy and to impaired establishment of stem cells quiescence. Similar effects were observed following conditional in vivo deletion of pericyte Igf1 and Angpt1 genes, respectively. Our data therefore demonstrate that, by promoting post-natal myogenesis and stem cell quiescence, pericytes play a key role in the microvascular niche of satellite cells.

Characterizing Acute-Onset Small Fiber Neuropathy.

BACKGROUND AND OBJECTIVES: Immune-mediated small fiber neuropathy (SFN) is increasingly recognized. Acute-onset SFN (AOSFN) remains poorly described. Herein, we report a series of AOSFN cases in which immune origins are debatable. METHODS: We included consecutive patients with probable or definite AOSFN. Diagnosis of SFN was based on the NEURODIAB criteria. Acute onset was considered when the maximum intensity and extension of both symptoms and signs were reached within 28 days. We performed the following investigations: clinical examination, neurophysiologic assessment encompassing a nerve conduction study to rule out large fiber neuropathy, laser-evoked potentials (LEPs), warm detection thresholds (WDTs), electrochemical skin conductance (ESC), epidermal nerve fiber density (ENF), and patient serum reactivity against mouse sciatic nerve teased fibers, mouse dorsal root ganglion (DRG) sections, and cultured DRG. The serum reactivity of healthy subjects (n = 10) and diseased controls (n = 12) was also analyzed. Data on baseline characteristics, biological investigations, and disease course were collected. RESULTS: Twenty patients presenting AOSFN were identified (60% women; median age: 44.2 years [interquartile range: 35.7-56.2]). SFN was definite in 18 patients (90%) and probable in 2 patients. A precipitating event was present in 16 patients (80%). The median duration of the progression phase was 14 days [5-28]. Pain was present in 17 patients (85%). Twelve patients (60%) reported autonomic involvement. The clinical pattern was predominantly non-length-dependent (85%). Diagnosis was confirmed by abnormal LEPs (60%), ENF (55%), WDT (39%), or ESC (31%). CSF analysis was normal in 5 of 5 patients. Antifibroblast growth factor 3 antibodies were positive in 4 of 18 patients (22%) and anticontactin-associated protein-2 antibodies in one patient. In vitro studies showed IgG immunoreactivity against nerve tissue in 14 patients (70%), but not in healthy subjects or diseased controls. Patient serum antibodies bound to unmyelinated fibers, Schwann cells, juxtaparanodes, paranodes, or DRG. Patients' condition improved after a short course of oral corticosteroids (3/3). Thirteen patients (65%) showed partial or complete recovery. Others displayed relapses or a chronic course. DISCUSSION: AOSFN primarily presents as an acute, non-length-dependent, symmetric painful neuropathy with a variable disease course. An immune-mediated origin has been suggested based on in vitro immunohistochemical studies.

Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis.

Macrophages (MPs) are important for skeletal muscle regeneration in vivo and may exert beneficial effects on myogenic cell growth through mitogenic and antiapoptotic activities in vitro. However, MPs are highly versatile and may exert various, and even opposite, functions depending on their activation state. We studied monocyte (MO)/MP phenotypes and functions during skeletal muscle repair. Selective labeling of circulating MOs by latex beads in CX3CR1(GFP/+) mice showed that injured muscle recruited only CX3CR1(lo)/Ly-6C(+) MOs from blood that exhibited a nondividing, F4/80(lo), proinflammatory profile. Then, within muscle, these cells switched their phenotype to become proliferating antiinflammatory CX3CR1(hi)/Ly-6C(-) cells that further differentiated into F4/80(hi) MPs. In vitro, phagocytosis of muscle cell debris induced a switch of proinflammatory MPs toward an antiinflammatory phenotype releasing transforming growth factor beta1. In co-cultures, inflammatory MPs stimulated myogenic cell proliferation, whereas antiinflammatory MPs exhibited differentiating activity, assessed by both myogenin expression and fusion into myotubes. Finally, depletion of circulating MOs in CD11b-diphtheria toxin receptor mice at the time of injury totally prevented muscle regeneration, whereas depletion of intramuscular F4/80(hi) MPs at later stages reduced the diameter of regenerating fibers. In conclusion, injured skeletal muscle recruits MOs exhibiting inflammatory profiles that operate phagocytosis and rapidly convert to antiinflammatory MPs that stimulate myogenesis and fiber growth.

Myoinjury transiently activates muscle antigen-specific CD8+ T cells in lymph nodes in a mouse model.

OBJECTIVE: To investigate the influence of myoinjury on antigen presentation to T cells in draining lymph nodes (LNs). METHODS: Muscle crush was performed in mice injected with exogenous ovalbumin (OVA) and in transgenic SM-OVA mice expressing OVA as a muscle-specific self antigen. Antigen exposure and the resulting stimulation of T cell proliferation in draining LNs was assessed by transferring carboxyfluorescein succinimidyl ester (CFSE)-labeled OVA-specific CD8+ and CD4+ T cells from OT-I and OT-II mice and by measuring the dilution of CFSE, which directly reflects their proliferation. The role of monocyte-derived dendritic cells (DCs) in T cell priming was assessed using pharmacologic blockade of DC migration. Immunofluorescence was used to detect CD8+ T cells, inflammatory monocyte-derived DCs, and type I major histocompatibility complex (MHC)-expressing myofibers in crushed muscle, and to assess expression of perforin, interferon-γ (IFNγ), interleukin-2 (IL-2), IL-10, and transforming growth factor ß1 (TGFß1). RESULTS: OVA injection into intact muscle induced strong proliferation of CD4+ and CD8+ T cells, indicating efficient exposure of soluble antigens in draining LNs. OVA-specific CD8+ T cell proliferation in draining LNs of SM-OVA mice required myoinjury and was unaffected by pharmacologic inhibition of monocyte-derived DC migration. On day 7 postinjury, activated CD8+ T cells expressing perforin, IFNγ and IL-2 were transiently detected in crushed muscle, and these cells were in close contact with class I MHC-positive regenerating myofibers. Beginning on day 7, the immunosuppressive cytokines IL-10 and TGFß1 were conspicuously expressed by CD11b+ cells, and CD8+ T cells rapidly disappeared from the healing muscle. CONCLUSION: Myofiber damage induces an episode of muscle antigen-specific CD8+ T cell proliferation in draining LNs. Activated CD8+ T cells transiently infiltrate the injured muscle, with prompt control by immunosuppressive cues. Inadequate control might favor sustained autoimmune myositis.

Obesity impairs skeletal muscle repair through NID-1 mediated extracellular matrix remodeling by mesenchymal progenitors.

Obesity triggers skeletal muscle physio-pathological alterations. However, the crosstalk between adipose tissue and myogenic cells remains poorly understood during obesity. We identified NID-1 among the adipose tissue secreted factors impairing myogenic potential of human myoblasts and murine muscle stem cells in vitro. Mice under High Fat Diet (HFD) displayed increased NID-1 expression in the skeletal muscle endomysium associated with intramuscular fat adipose tissue expansion and compromised muscle stem cell function. We show that NID-1 is highly secreted by skeletal muscle fibro-adipogenic/mesenchymal progenitors (FAPs) during obesity. We demonstrate that increased muscle NID-1 impairs muscle stem cells proliferation and primes the fibrogenic differentiation of FAPs, giving rise to an excessive deposition of extracellular matrix. Finally, we propose a model in which obesity leads to skeletal muscle extracellular matrix remodeling by FAPs, mediating the alteration of myogenic function by adipose tissue and highlighting the key role of NID-1 in the crosstalk between adipose tissue and skeletal muscle.

Optimized Flow Cytometry Strategy for Phenotyping Intramuscular Leukocytes: Application to the Evaluation of Myopathological Processes.

Phenotyping intramuscular immune cells is essential for the characterization of dysimmune/inflammatory myopathies (DIM). Flow cytometry (FC) is the most reliable technique for analyzing leukocyte subpopulations and evaluating their activation levels. We developed a purely mechanical protocol for extracting cells from muscle tissue allowing us to preserve cell surface epitopes and determined its applicability to experimental pathology in mice and myopathological diagnosis in human. Skeletal muscle regeneration in mice was associated with a transient enrichment of macrophages (CD11bhighGr-1+), myeloid dendritic cells (CD3-C8+CD11bhigh), CD8+ T cells (CD3+C8+), and NK cells (CD3- CD11bhighNKp46+). In murine models of inherited muscle dystrophies, leukocytes represented 23%-84% of intramuscular mononuclear cells, with a percentage of CD8+ T cells (4%-17%) mirroring that of all CD45+ cells, while MDCs remained a minority. In human 16 samples (DIM: n = 9; nonimmune conditions: n = 7), DIM was associated with intramuscular recruitment of CD8+ T cells, but not CD4+ T cells and NK cells. FC allowed concomitant quantification of HLA-DR, CD25, CD38, and CD57 activation/differentiation biomarkers and showed increased activation levels of CD4+ and CD8+ T cells in DIM. In conclusion, FC is an appropriate method for quantifying intramuscular leukocyte subpopulations and analyzing their activation states.

Immunolabelling Myofiber Degeneration in Muscle Biopsies.

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Platelet-Rich Plasma Improves the Wound Healing Potential of Mesenchymal Stem Cells through Paracrine and Metabolism Alterations.

Chronic and acute nonhealing wounds represent a major public health problem, and replacement of cutaneous lesions by the newly regenerated skin is challenging. Mesenchymal stem cells (MSC) and platelet-rich plasma (PRP) were separately tested in the attempt to regenerate the lost skin. However, these treatments often remained inefficient to achieve complete wound healing. Additional studies suggested that PRP could be used in combination with MSC to improve the cell therapy efficacy for tissue repair. However, systematic studies related to the effects of PRP on MSC properties and their ability to rebuild skin barrier are lacking. We evaluated in a mouse exhibiting 4 full-thickness wounds, the skin repair ability of a treatment combining human adipose-derived MSC and human PRP by comparison to treatment with saline solution, PRP alone, or MSC alone. Wound healing in these animals was measured at day 3, day 7, and day 10. In addition, we examined in vitro and in vivo whether PRP alters in MSC their proangiogenic properties, their survival, and their proliferation. We showed that PRP improved the efficacy of engrafted MSC to replace lost skin in mice by accelerating the wound healing processes and ameliorating the elasticity of the newly regenerated skin. In addition, we found that PRP treatment stimulated in vitro, in a dose-dependent manner, the proangiogenic potential of MSC through enhanced secretion of soluble factors like VEGF and SDF-1. Moreover, PRP treatment ameliorated the survival and activated the proliferation of in vitro cultured MSC and that these effects were accompanied by an alteration of the MSC energetic metabolism including oxygen consumption rate and mitochondrial ATP production. Similar observations were found in vivo following combined administration of PRP and MSC into mouse wounds. In conclusion, our study strengthens that the use of PRP in combination with MSC might be a safe alternative to aid wound healing.

Novel Missense CAPN3 Mutation Responsible for Adult-Onset Limb Girdle Muscular Dystrophy with Calves Hypertrophy.

CAPN3 gene encodes for calpain-3; this protein is a calcium-dependent intracellular protease. Deficiency of this enzyme leads to weakness of the proximal limb muscles and pelvic and shoulder girdles, the so-called limb-girdle muscular dystrophy type 2A (LGMD2A). Here, we reported the case of a Tunisian patient with LGMD2A associated with a novel missense mutation (c.T1681C/p.Y561H). A 61-year-old man, with consanguineous parents, was referred for gait difficulties and slowly progressive proximal weakness of the four limbs associated with moderate hypertrophy of the calves but his facial muscles were unaffected. Electromyography showed that the profile was myopathic pattern and creatine kinase (CK) level was high. Muscle biopsy processing included routine histological, immunohistochemical, and Western Blot reactions, using a panel of antibodies directed against dystrophin, dysferlin, calpain-3, sarcoglycan α, ß, γ, and δ. For mutation analysis, we designed an NGS-based screening. Immunological analyses demonstrated a total deficiency in calpain-3 and δ-sarcoglycan, and a reduced expression of dysferlin. The genetic study yielded a homozygous missense mutation (c.T1681C) of the 13th exon of the CAPN3 gene. The mutation found in our patient (c.T1681C/p.Y561H) has not been previously reported. It is responsible for complete calpain-3 and δ-sarcoglycan deficiency and reduced dysferlin expression. The genetic study is mandatory in such cases with multiple-protein deficiency and ambiguous results of immune-histology and Western Blot studies.

Distinct interferon signatures stratify inflammatory and dysimmune myopathies.

Objective: The role of interferons (IFN) in the pathophysiology of primary inflammatory and dysimmune myopathies (IDM) is increasingly investigated, notably because specific neutralisation approaches may constitute promising therapeutic tracks. In present work we analysed the muscular expression of specific IFNα/ß and IFNγ-stimulated genes in patients with various types of IDM. Methods: 39 patients with IDM with inclusion body myositis (IBM, n=9), dermatomyositis (DM, n=10), necrotising autoimmune myopathies (NAM, n=10) and antisynthetase myositis (ASM, n=10), and 10 controls were included. Quantification of expression levels of IFNγ, ISG15, an IFNα/ß-inducible gene and of six IFNγ-inducible genes (GBP2, HLA-DOB, HLA-DPB, CIITA, HLA-DRB and HLA-DMB) was performed on muscle biopsy samples. Results: DM usually associated with strong type I IFNα/ß signature, IBM and ASM with prominent type II IFNγ signature and NAM with neither type I nor type II IFN signature. Immunofluorescence study in ASM and IBM showed myofibre expression of major histocompatibility class 2 (MHC-2) and CIITA, confirming the induction of the IFNγ pathway. Furthermore, MHC-2-positive myofibres were observed in close proximity to CD8+ T cells which produce high levels of IFNγ. Conclusion: Distinct IFN signatures allow a more distinct segregation of IDMs and myofibre MHC-2 expression is a reliable biomarker of type II IFN signature.

[The effect of interferon-gamma on skeletal muscle cell biology]. / 15es JSFM : Prix Master 2017* - Effet de l'interféron-gamma sur la biologie des cellules musculaires squelettiques.

Dysimmune and inflammatory myopathies (DIMs) affect around 14/100,000 people worldwide. Based on immupour nopathological criteria, DIMs are divided in four groups: (1) polymyositis (PM)/inclusion body myositis (IBM), (2) dermatomyositis (DM), (3) immune-mediated necrotizing myopathies (IMNM) and (iv) overlapping myositis including anti-synthetase syndrome (ASS). ASS and PM/IBM are characterized by the activation of inflammation with lymphocytic infiltrations. Recently, we showed that an expression of the major histocompatibility complex class 2 (MHC2) was present in myofibers from ASS and IBM muscle biopsies. Interestingly, MHC2 expression is known to be stimulated by Interferon-gamma (IFNγ) in myogenic cells. LTCD8 cells, which are well-known producers of IFNγ, are commonly found in close vicinity to MHC2 positive myofibers. This inflammatory cytokine also inhibits myogenic differentiation in vitro by CIITA-myogenin interaction. The mechanisms involved in the lymphocyte-driven muscle toxicity in DIMs are unclear. The objectives of this project are to characterize IFNγ effects on the biology of human myogenic cells by morphological, molecular and cellular approaches. Then, we aim to investigate the role of IFNγ in these myopathies and its impact during muscular regeneration. In vitro preliminary studies have been performed using human and mouse myoblasts treated or not with IFNγ. Our results should lead to a better understanding of the role of IFNγ in the pathophysiology of DIMs, and would hopefully help identify new therapeutic targets.

Non-linear dose-response of aluminium hydroxide adjuvant particles: Selective low dose neurotoxicity.

Aluminium (Al) oxyhydroxide (Alhydrogel®), the main adjuvant licensed for human and animal vaccines, consists of primary nanoparticles that spontaneously agglomerate. Concerns about its safety emerged following recognition of its unexpectedly long-lasting biopersistence within immune cells in some individuals, and reports of chronic fatigue syndrome, cognitive dysfunction, myalgia, dysautonomia and autoimmune/inflammatory features temporally linked to multiple Al-containing vaccine administrations. Mouse experiments have documented its capture and slow transportation by monocyte-lineage cells from the injected muscle to lymphoid organs and eventually the brain. The present study aimed at evaluating mouse brain function and Al concentration 180days after injection of various doses of Alhydrogel® (200, 400 and 800µg Al/kg of body weight) in the tibialis anterior muscle in adult female CD1 mice. Cognitive and motor performances were assessed by 8 validated tests, microglial activation by Iba-1 immunohistochemistry, and Al level by graphite furnace atomic absorption spectroscopy. An unusual neuro-toxicological pattern limited to a low dose of Alhydrogel® was observed. Neurobehavioural changes, including decreased activity levels and altered anxiety-like behaviour, were observed compared to controls in animals exposed to 200µg Al/kg but not at 400 and 800µg Al/kg. Consistently, microglial number appeared increased in the ventral forebrain of the 200µg Al/kg group. Cerebral Al levels were selectively increased in animals exposed to the lowest dose, while muscle granulomas had almost completely disappeared at 6 months in these animals. We conclude that Alhydrogel® injected at low dose in mouse muscle may selectively induce long-term Al cerebral accumulation and neurotoxic effects. To explain this unexpected result, an avenue that could be explored in the future relates to the adjuvant size since the injected suspensions corresponding to the lowest dose, but not to the highest doses, exclusively contained small agglomerates in the bacteria-size range known to favour capture and, presumably, transportation by monocyte-lineage cells. In any event, the view that Alhydrogel® neurotoxicity obeys "the dose makes the poison" rule of classical chemical toxicity appears overly simplistic.

Muscle resident macrophages control the immune cell reaction in a mouse model of notexin-induced myoinjury.

OBJECTIVE: Skeletal muscle may be the site of a variety of poorly understood immune reactions, particularly after myofiber injury, which is typically observed in inflammatory myopathies. This study was undertaken to explore both the cell dynamics and functions of resident macrophages and dendritic cells (DCs) in damaged muscle, using a mouse model of notexin-induced myoinjury to study innate immune cell reactions. METHODS: The myeloid cell reaction to notexin-induced myoinjury was analyzed by microscopy and flow cytometry. Bone marrow (BM) transplantation studies were used to discriminate resident from exudate monocyte/macrophages. Functional tests included cytokine screening and an alloantigenic mixed leukocyte reaction to assess the antigen-presenting cell (APC) function. Selective resident macrophage depletion was obtained by injection of diphtheria toxin (DT) into CD11b-DT receptor-transgenic mice transplanted with DT-insensitive BM. RESULTS: The connective tissue surrounding mouse muscle/fascicle tissue (the epimysium/perimysium) after deep muscle injury displayed a resident macrophage population of CD11b+F4/80+CD11c-Ly-6C-CX3CR1- cells, which concentrated first in the epimysium. These resident macrophages were being used by leukocytes as a centripetal migration pathway, and were found to selectively release 2 chemokines, cytokine-induced neutrophil chemoattractant and monocyte chemoattractant protein 1, and to crucially contribute to massive recruitment of neutrophils and monocytes from the blood. Early epimysial inflammation consisted of a predominance of Ly-6C(high)CX3CR1(low)CD11c- cells that were progressively substituted by Ly-6C(low)CX3CR1(high) cells displaying an intermediate, rather than high, level of CD11c expression. These CD11c(intermediate) cells were derived from circulating CCR2+ monocytes, functionally behaved as immature APCs in the absence of alloantigenic challenge, and migrated to draining lymph nodes while acquiring the phenotype of mature DCs (CD11c+Ia+CD80+ cells, corresponding to an inflammatory DC phenotype). CONCLUSION: The results in this mouse model show that resident macrophages in the muscle epimysium/perimysium orchestrate the innate immune response to myoinjury, which is linked to adaptive immunity through the formation of inflammatory DCs.