DCs unchained: Whetting dendritic-cell appetite boosts wound healing.

Apoptotic-cell uptake (efferocytosis) by dendritic cells (DCs) has been mainly linked to their antigen presentation property. In a recent issue of Nature, Maschalidi et al. identified a break to efferocytosis in DCs, the inhibition of which improves skin debris cleansing after a wound, accelerating healing.

Macrophage PPARγ, a Lipid Activated Transcription Factor Controls the Growth Factor GDF3 and Skeletal Muscle Regeneration.

Tissue regeneration requires inflammatory and reparatory activity of macrophages. Macrophages detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of effector functions of macrophages coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of macrophages involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARγ, is required for proper skeletal muscle regeneration, acting in repair macrophages. PPARγ controls the expression of the transforming growth factor-ß (TGF-ß) family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell fusion. This work establishes PPARγ as a required metabolic sensor and transcriptional regulator of repair macrophages. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair.

Macrophagic AMPKα1 orchestrates regenerative inflammation induced by glucocorticoids.

Macrophages are key cells after tissue damage since they mediate both acute inflammatory phase and regenerative inflammation by shifting from pro-inflammatory to restorative cells. Glucocorticoids (GCs) are the most potent anti-inflammatory hormone in clinical use, still their actions on macrophages are not fully understood. We show that the metabolic sensor AMP-activated protein kinase (AMPK) is required for GCs to induce restorative macrophages. GC Dexamethasone activates AMPK in macrophages and GC receptor (GR) phosphorylation is decreased in AMPK-deficient macrophages. Loss of AMPK in macrophages abrogates the GC-induced acquisition of their repair phenotype and impairs GC-induced resolution of inflammation in vivo during post-injury muscle regeneration and acute lung injury. Mechanistically, two categories of genes are impacted by GC treatment in macrophages. Firstly, canonical cytokine regulation by GCs is not affected by AMPK loss. Secondly, AMPK-dependent GC-induced genes required for the phenotypic transition of macrophages are co-regulated by the transcription factor FOXO3, an AMPK substrate. Thus, beyond cytokine regulation, GR requires AMPK-FOXO3 for immunomodulatory actions in macrophages, linking their metabolic status to transcriptional control in regenerative inflammation.

Kinetics of skeletal muscle regeneration after mild and severe muscle damage induced by electrically-evoked lengthening contractions.

Post-injury skeletal muscle regeneration requires interactions between myogenic and non-myogenic cells. Our knowledge on the regeneration process is mainly based on models using toxic, chemical, or physical (e.g., based on either muscle freezing or crushing) injury. Strikingly, the time course and magnitude of changes in the number of cells involved in muscle regeneration have been poorly described in relation to mild and severe muscle damage induced by electrically-evoked lengthening contractions. We investigated for the first time the kinetics and magnitude of changes in mononuclear cells in relation to the extent of muscle damage. Mild and severe injury were induced in vivo in the mouse gastrocnemius muscle by 1 and 30 electrically-evoked lengthening contractions, respectively. Several days after muscle damage, functional analysis of maximal torque production and histological investigations were performed to assess the related cellular changes. Torque recovery was faster after mild injury than after severe muscle damage. More necrotic and regenerating myofibers were observed after severe muscle damage as compared with mild injury, illustrating an association between functional and histological alterations. The kinetics of changes in muscle stem cells (total, proliferating, and differentiating), endothelial cells, fibro-adipogenic progenitors (FAPs), and macrophages in the regenerating muscle was similar in mild and severe models. However, the magnitude of changes in the number of differentiating muscle stem cells, hematopoietic cells, among which macrophages, and FAPs was higher in severe muscle damage. Collectively, our results show that the amount of myogenic and non-myogenic cells varies according to the extent of skeletal muscle injury to ensure efficient skeletal muscle regeneration while the kinetics of changes is independent of muscle tissue alterations. The possibility to experimentally modulate the extent of muscle damage will be useful to further investigate the cellular and molecular events involved in muscle regeneration.

Inflammation during post-injury skeletal muscle regeneration.

The adult skeletal muscle fully regenerates after injury thanks to the properties of muscle stem cells that follow the adult myogenic program to replace damaged myofibers. Muscle regeneration also relies upon the coordinated actions of several other cell types, among which immune cells. Leukocytes infiltrate the damaged muscle soon after injury and support the regeneration process in a variety of ways, from the activation of muscle stem cells to the maturation of newly formed myofibers. Leukocytes also interact with other cell types such as fibroadipogenic precursors and endothelial cells. This review presents the interactions that leukocytes develop with the cells present in their vicinity and the impact they have on skeletal muscle regeneration.

Interplay between myofibers and pro-inflammatory macrophages controls muscle damage in mdx mice.

Duchenne muscular dystrophy is a genetic muscle disease characterized by chronic inflammation and fibrosis mediated by a pro-fibrotic macrophage population expressing pro-inflammatory markers. Our aim was to characterize cellular events leading to the alteration of macrophage properties and to modulate macrophage inflammatory status using the gaseous mediator hydrogen sulfide (H2S). Using co-culture experiments, we first showed that myofibers derived from mdx mice strongly skewed the polarization of resting macrophages towards a pro-inflammatory phenotype. Treatment of mdx mice with NaHS, an H2S donor, reduced the number of pro-inflammatory macrophages in skeletal muscle, which was associated with a decreased number of nuclei per fiber, as well as reduced myofiber branching and fibrosis. Finally, we established the metabolic sensor AMP-activated protein kinase (AMPK) as a critical NaHS target in muscle macrophages. These results identify an interplay between myofibers and macrophages where dystrophic myofibers contribute to the maintenance of a highly inflammatory environment sustaining a pro-inflammatory macrophage status, which in turn favors myofiber damage, myofiber branching and establishment of fibrosis. Our results also highlight the use of H2S donors as a potential therapeutic strategy to improve the dystrophic muscle phenotype by dampening chronic inflammation. This article has an associated First Person interview with the first author of the paper.

Inflammation and Skeletal Muscle Regeneration: Leave It to the Macrophages!

Inflammation is usually considered as harmful; however, it is also necessary for tissue recovery after injury. Macrophages exert immune and nonimmune functions throughout this process. During skeletal muscle regeneration, they mount an inflammatory response while exerting trophic roles on muscle and mesenchymal stem cells. Proinflammatory macrophages shift to being anti-inflammatory, triggering the resolution of inflammation. Studies have highlighted that during this shift, a crosstalk ensues, integrating cues for resolution, efferocytosis, cellular metabolism, and signaling pathways. During the restorative phase, macrophages dampen inflammation while promoting stem cell differentiation, angiogenesis, and matrix remodeling. Since blunting the inflammatory phase can be detrimental for muscle regeneration, we suggest that rather than fighting inflammation, it should be allowed to operate and resolve, thus allowing for tissue recovery.

Immunometabolism-fit: How exercise and training can modify T cell and macrophage metabolism in health and disease.

BACKGROUND: The term immunometabolism describes cellular and molecular metabolic processes that control the immune system and the associated immune responses. Acute exercise and regular physical activity have a substantial influence on the metabolism and the immune system, so that both processes are closely associated and influence each other bidirectionally. SCOPE OF REVIEW: We limit the review here to focus on metabolic phenotypes and metabolic plasticity of T cells and macrophages to describe the complex role of acute exercise stress and regular physical activity on these cell types. The metabolic and immunological consequences of the social problem of inactivity and how, conversely, an active lifestyle can break this vicious circle, are then described. Finally, these aspects are evaluated against the background of an aging society. MAJOR CONCLUSIONS: T cells and macrophages show high sensitivity to changes in their metabolic environment, which indirectly or directly affects their central functions. Physical activity and sedentary behaviour have an important influence on metabolic status, thereby modifying immune cell phenotypes and influencing immunological plasticity. A detailed understanding of the interactions between acute and chronic physical activity, sedentary behaviour, and the metabolic status of immune cells, can help to target the dysregulated immune system of people who live in a much too inactive society.

Benefits and pathologies associated with the inflammatory response.

Adult skeletal muscle regenerates completely after a damage, thanks to the satellite cells, or muscle stem cells (MuSCs), that implement the adult myogenic program. This program is sustained by both robust intrinsic mechanisms and extrinsic cues coming from the close neighborhood of MuSCs during muscle regeneration. Among the various cell types present in the regenerating muscle, immune cells, and particularly macrophages, exert numerous functions and provide sequential transient niches to support the myogenic program. The adequate orchestration of the delivery of these cues ensures efficient muscle regeneration and full functional recovery. The situation is very different in muscular dystrophies where asynchronous and permanent microinjuries occur, triggering contradictory regenerating cues at the same time in a specific area, that lead to chronic inflammation and fibrogenesis. Here we review the beneficial effects that leukocytes, and particularly macrophages, exert on their neighboring cells during skeletal muscle regeneration after an acute injury. Then, the more complicated (and less beneficial) roles of leukocytes during muscular dystrophies are presented. Finally, we discuss how the inflammatory compartment may be a target to improve muscle regeneration in both acute muscle injury and muscle diseases.

Sex differences in semitendinosus muscle fiber-type composition.

Sex differences in muscle fiber-type composition have been documented in several muscle groups while the hamstring muscle fiber-type composition has been poorly characterized. This study aimed to compare the semitendinosus muscle composition between men and women. Biopsy samples were obtained from the semitendinosus muscle of twelve men and twelve women during an anterior cruciate ligament reconstruction. SDH and ATPase activities as well as the size and the proportion of muscle fibers expressing myosin heavy chain (MyHC) isoforms were used to compare muscle composition between men and women. The proportion of SDH-positive muscle fibers was significantly lower (37.4 ± 11.2% vs. 49.3 ± 10.6%, p < 0.05), and the percentage of fast muscle fibers (i.e., based on ATPase activity) was significantly higher (65.8 ± 10.1% vs. 54.8 ± 8.3%, p < 0.05) in men versus women. Likewise, men muscles exhibited a lower percentage of the area that was occupied by MyHC-I labeling (35.6 ± 10.1% vs. 48.7 ± 8.9%; p < 0.05) and a higher percentage of the area that was occupied by MyHC-IIA (38.3 ± 6.7% vs. 32.5 ± 6.5%; p < 0.05) and MyHC-IIX labeling (26.1 ± 9.6% vs. 18.8 ± 8.5%; p = 0.06) as compared with women muscles. The cross-sectional area of MyHC-I, MyHC-IIA, and MyHC-IIX muscle fibers was 31%, 43%, and 50% larger in men as compared with women, respectively. We identified sex differences in semitendinosus muscle composition as illustrated by a faster phenotype and larger muscle size in men as compared with women. This sexual dimorphism might have functional consequences.

AMPKα1-LDH pathway regulates muscle stem cell self-renewal by controlling metabolic homeostasis.

Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self-renewal) is crucial for tissue repair. Here, we showed that AMP-activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self-renewal. AMPKα1-/- MuSCs displayed a high self-renewal rate, which impairs muscle regeneration. AMPKα1-/- MuSCs showed a Warburg-like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non-limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1-/- phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1-/- MuSC self-renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.

Recombinant HvRNASET2 protein induces marked connective tissue remodelling in the invertebrate model Hirudo verbana.

The RNASET2 ribonuclease, belonging to the highly conserved RH/T2/s RNase gene family, has been recently shown to modulate inflammatory processes in both vertebrates and invertebrates. Indeed, the RNASET2 protein acts as a chemoattractor for macrophages in both in vitro and in vivo experimental settings and its expression significantly increases following bacterial infections. Moreover, we recently observed that injection of human recombinant RNASET2 protein in the body wall of the medicinal leech (a consolidated invertebrate model for both immune response and tissue regeneration) not only induced immune cell recruitment but also apparently triggered massive connective tissue remodelling as well. Based on these data, we evaluate here a possible role of leech recombinant RNASET2 protein (rHvRNASET2) in connective tissue remodelling by characterizing the cell types involved in this process through histochemical, morphological and immunofluorescent assays. Moreover, a time-course expression analysis of newly synthesized pro-collagen1α1 (COL1α1) and basic FGF receptor (bFGFR, a known fibroblast marker) following rHvRNASET2 injection in the leech body wall further supported the occurrence of rHvRNASET2-mediated matrix remodelling. Human MRC-5 fibroblast cells were also investigated in order to evaluate their pattern of collagen neosynthesis driven by rHvRNASET2 injection.Taken together, the data reported in this work provide compelling evidence in support of a pleiotropic role for RNASET2 in orchestrating an evolutionarily conserved crosstalk between inflammatory response and regenerative process, based on macrophage recruitment and fibroblast activation, coupled to a massive extracellular reorganization.

Interferon-signature in idiopathic inflammatory myopathies.

PURPOSE OF REVIEW: The present review describes the interferon (IFN)-signature currently emerging as a tool for the diagnosis of idiopathic inflammatory myopathies (IIMs), and aims at presenting the interests and limitations of this recent tool for the clinics and the research. RECENT FINDINGS: Recent in-vivo and in-vitro transcriptomic studies have evidenced the involvement of IFNs in the pathogenesis of IIMs. A correlation between the IFN-signature and the clinical severity of IIMs has been established. Moreover, studies pointed out differences in the IFN-signature regarding the IIM subgroup (dermatomyositis, polymyositis, inclusion body myositis, anti-synthetase syndrome, immuno-mediated necrotizing myopathies), raising the hypothesis of several pathogenic processes in IIMs. SUMMARY: IIM pathogenesis remains partially understood. IFN-signature represents one of the main recent advances in the field. IFN-signature was identified thanks to transcriptomic analyses of tissues or cells from IIM patients (muscle, skin, blood cells, muscle cells) and should allow to establish new diagnosis and better monitoring of IIM patients. It also provides a tool for investigation of IIM pathogenesis. Nevertheless, IFN-signature still requires accurate definition in order to standardize its use, notably in the clinical practice.

Cell sorting of various cell types from mouse and human skeletal muscle.

Muscle stem cells or satellite cells are required for skeletal muscle regeneration. It has been shown that the satellite cell microenvironment, including neighboring cells such as endothelial cells, macrophages or fibroblasts are essential for complete and efficient regeneration. A deficient behavior of these cells compromises regeneration. Therefore, there is a strong interest in understanding the cellular and molecular interactions at work between these cell types during muscle regeneration. Fluorescence-activated cell sorting allows to isolate these four cell types at different time points of regeneration, for further high throughput or behavioral experiments. We present here a method for the concomitant isolation of 4 cell types present in the regenerating skeletal muscle: muscle stem cells, endothelial cells, fibro-adipogenic precursor cells and macrophages.

Highly Dynamic Transcriptional Signature of Distinct Macrophage Subsets during Sterile Inflammation, Resolution, and Tissue Repair.

Macrophage gene expression determines phagocyte responses and effector functions. Macrophage plasticity has been mainly addressed in in vitro models that do not account for the environmental complexity observed in vivo. In this study, we show that microarray gene expression profiling revealed a highly dynamic landscape of transcriptomic changes of Ly6C(pos)CX3CR1(lo) and Ly6C(neg)CX3CR1(hi) macrophage populations during skeletal muscle regeneration after a sterile damage. Systematic gene expression analysis revealed that the time elapsed, much more than Ly6C status, was correlated with the largest differential gene expression, indicating that the time course of inflammation was the predominant driving force of macrophage gene expression. Moreover, Ly6C(pos)/Ly6C(neg) subsets could not have been aligned to canonical M1/M2 profiles. Instead, a combination of analyses suggested the existence of four main features of muscle-derived macrophages specifying important steps of regeneration: 1) infiltrating Ly6C(pos) macrophages expressed acute-phase proteins and exhibited an inflammatory profile independent of IFN-γ, making them damage-associated macrophages; 2) metabolic changes of macrophages, characterized by a decreased glycolysis and an increased tricarboxylic acid cycle/oxidative pathway, preceded the switch to and sustained their anti-inflammatory profile; 3) Ly6C(neg) macrophages, originating from skewed Ly6C(pos) cells, actively proliferated; and 4) later on, restorative Ly6C(neg) macrophages were characterized by a novel profile, indicative of secretion of molecules involved in intercellular communications, notably matrix-related molecules. These results show the highly dynamic nature of the macrophage response at the molecular level after an acute tissue injury and subsequent repair, and associate a specific signature of macrophages to predictive specialized functions of macrophages at each step of tissue injury/repair.

Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration.

KEY POINTS: Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross-talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell-culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell-cell cross-talk during physiological and pathological muscle remodelling. ABSTRACT: Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross-sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co-cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable membrane. MPC proliferation, differentiation and fusion were assessed from cells stained for BrdU, desmin and myogenin. On biopsy cross-sections, fibroblast number was seen to increase, along with myogenic cell number, by d7 and increase further by d30, where fibroblasts were observed to be preferentially located immediately surrounding regenerating muscle fibres. In vitro, the presence of fibroblasts in direct contact with MPCs was found to moderately stimulate MPC proliferation and strongly stimulate both MPC differentiation and MPC fusion. It thus appears, in humans, that fibroblasts exert a strong positive regulatory influence on MPC activity, in line with observations during in vivo skeletal muscle regeneration.

Myeloid HIFs are dispensable for resolution of inflammation during skeletal muscle regeneration.

Besides their role in cellular responses to hypoxia, hypoxia-inducible factors (HIFs) are involved in innate immunity and also have anti-inflammatory (M2) functions, such as resolution of inflammation preceding healing. Whereas the first steps of the inflammatory response are associated with proinflammatory (M1) macrophages (MPs), resolution of inflammation is associated with anti-inflammatory MPs exhibiting an M2 phenotype. This M1 to M2 sequence is observed during postinjury muscle regeneration, which provides an excellent paradigm to study the resolution of sterile inflammation. In this study, using in vitro and in vivo approaches in murine models, we demonstrated that deletion of hif1a or hif2a in MPs has no impact on the acquisition of an M2 phenotype. Furthermore, using a multiscale methodological approach, we showed that muscles did not require macrophagic hif1a or hif2a to regenerate. These results indicate that macrophagic HIFs do not play a crucial role during skeletal muscle regeneration induced by sterile tissue damage.

Palmitoleate Reverses High Fat-induced Proinflammatory Macrophage Polarization via AMP-activated Protein Kinase (AMPK).

A rise in tissue-embedded macrophages displaying "M1-like" proinflammatory polarization is a hallmark of metabolic inflammation during a high fat diet or obesity. Here we show that bone marrow-derived macrophages (BMDM) from high fat-fed mice retain a memory of their dietary environment in vivo (displaying the elevated proinflammatory genes Cxcl1, Il6, Tnf, Nos2) despite 7-day differentiation and proliferation ex vivo. Notably, 6-h incubation with palmitoleate (PO) reversed the proinflammatory gene expression and cytokine secretion seen in BMDM from high fat-fed mice. BMDM from low fat-fed mice exposed to palmitate (PA) for 18 h ex vivo also showed elevated expression of proinflammatory genes (Cxcl1, Il6, Tnf, Nos2, and Il12b) associated with M1 polarization. Conversely, PO treatment increased anti-inflammatory genes (Mrc1, Tgfb1, Il10, Mgl2) and oxidative metabolism, characteristic of M2 macrophages. Therefore, saturated and unsaturated fatty acids bring about opposite macrophage polarization states. Coincubation of BMDM with both fatty acids counteracted the PA-induced Nos2 expression in a PO dose-dependent fashion. PO also prevented PA-induced IκBα degradation, RelA nuclear translocation, NO production, and cytokine secretion. Mechanistically, PO exerted its anti-inflammatory function through AMP-activated protein kinase as AMP kinase knockout or inhibition by Compound C offset the PO-dependent prevention of PA-induced inflammation. These results demonstrate a nutritional memory of BMDM ex vivo, highlight the plasticity of BMDM polarization in response to saturated and unsaturated fatty acids, and identify the potential to reverse diet- and saturated fat-induced M1-like polarization by administering palmitoleate. These findings could have applicability to reverse obesity-linked inflammation in metabolically relevant tissues.

Skeletal Muscle Microvasculature: A Highly Dynamic Lifeline.

Skeletal muscle is highly irrigated by blood vessels. Beyond oxygen and nutrient supply, new vessel functions have been identified. This review presents vessel microanatomy and functions at tissue, cellular, and molecular levels. Mechanisms of vessel plasticity are described during skeletal muscle development and acute regeneration, and in physiological and pathological contexts.