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.

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.

The origins and non-canonical functions of macrophages in development and regeneration.

The discovery of new non-canonical (i.e. non-innate immune) functions of macrophages has been a recurring theme over the past 20 years. Indeed, it has emerged that macrophages can influence the development, homeostasis, maintenance and regeneration of many tissues and organs, including skeletal muscle, cardiac muscle, the brain and the liver, in part by acting directly on tissue-resident stem cells. In addition, macrophages play crucial roles in diseases such as obesity-associated diabetes or cancers. Increased knowledge of their regulatory roles within each tissue will therefore help us to better understand the full extent of their functions and could highlight new mechanisms modulating disease pathogenesis. In this Review, we discuss recent studies that have elucidated the developmental origins of various macrophage populations and summarize our knowledge of the non-canonical functions of macrophages in development, regeneration and tissue repair.

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.

AMPK in skeletal muscle function and metabolism.

Skeletal muscle possesses a remarkable ability to adapt to various physiologic conditions. AMPK is a sensor of intracellular energy status that maintains energy stores by fine-tuning anabolic and catabolic pathways. AMPK's role as an energy sensor is particularly critical in tissues displaying highly changeable energy turnover. Due to the drastic changes in energy demand that occur between the resting and exercising state, skeletal muscle is one such tissue. Here, we review the complex regulation of AMPK in skeletal muscle and its consequences on metabolism ( e.g., substrate uptake, oxidation, and storage as well as mitochondrial function of skeletal muscle fibers). We focus on the role of AMPK in skeletal muscle during exercise and in exercise recovery. We also address adaptations to exercise training, including skeletal muscle plasticity, highlighting novel concepts and future perspectives that need to be investigated. Furthermore, we discuss the possible role of AMPK as a therapeutic target as well as different AMPK activators and their potential for future drug development.-Kjøbsted, R., Hingst, J. R., Fentz, J., Foretz, M., Sanz, M.-N., Pehmøller, C., Shum, M., Marette, A., Mounier, R., Treebak, J. T., Wojtaszewski, J. F. P., Viollet, B., Lantier, L. AMPK in skeletal muscle function and metabolism.

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.

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.

Myeloid HIF-1 is protective in Helicobacter pylori-mediated gastritis.

Helicobacter pylori infection triggers chronic inflammation of the gastric mucosa that may progress to gastric cancer. The hypoxia-inducible factors (HIFs) are the central mediators of cellular adaptation to low oxygen levels (hypoxia), but they have emerged recently as major transcriptional regulators of immunity and inflammation. No studies have investigated whether H. pylori affects HIF signaling in immune cells and a potential role for HIF in H. pylori-mediated gastritis. HIF-1 and HIF-2 expression was examined in human H. pylori-positive gastritis biopsies. Subsequent experiments were performed in naive and polarized bone marrow-derived macrophages from wild-type (WT) and myeloid HIF-1α-null mice (HIF-1(Δmyel)). WT and HIF-1(Δmyel) mice were inoculated with H. pylori by oral gavage and sacrificed 6 mo postinfection. HIF-1 was specifically expressed in macrophages of human H. pylori-positive gastritis biopsies. Macrophage HIF-1 strongly contributed to the induction of proinflammatory genes (IL-6, IL-1ß) and inducible NO synthase in response to H. pylori. HIF-2 expression and markers of M2 macrophage differentiation were decreased in response to H. pylori. HIF-1(Δmyel) mice inoculated with H. pylori for 6 mo presented with a similar bacterial colonization than WT mice but, surprisingly, a global increase of inflammation, leading to a worsening of the gastritis, measured by an increased epithelial cell proliferation. In conclusion, myeloid HIF-1 is protective in H. pylori-mediated gastritis, pointing to the complex counterbalancing roles of innate immune and inflammatory phenotypes in driving this pathology.

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.

Nanotubular crosstalk with distressed cardiomyocytes stimulates the paracrine repair function of mesenchymal stem cells.

Mesenchymal stem cells (MSC) are known to repair broken heart tissues primarily through a paracrine fashion while emerging evidence indicate that MSC can communicate with cardiomyocytes (CM) through tunneling nanotubes (TNT). Nevertheless, no link has been so far established between these two processes. Here, we addressed whether cell-to-cell communication processes between MSC and suffering cardiomyocytes and more particularly those involving TNT control the MSC paracrine regenerative function. In the attempt to mimic in vitro an injured heart microenvironment, we developed a species mismatch coculture system consisting of terminally differentiated CM from mouse in a distressed state and human multipotent adipose derived stem cells (hMADS). In this setting, we found that crosstalk between hMADS and CM through TNT altered the secretion by hMADS of cardioprotective soluble factors such as VEGF, HGF, SDF-1α, and MCP-3 and thereby maximized the capacity of stem cells to promote angiogenesis and chemotaxis of bone marrow multipotent cells. Additionally, engraftment experiments into mouse infarcted hearts revealed that in vitro preconditioning of hMADS with cardiomyocytes increased the cell therapy efficacy of naïve stem cells. In particular, in comparison with hearts treated with stem cells alone, those treated with cocultured ones exhibited greater cardiac function recovery associated with higher angiogenesis and homing of bone marrow progenitor cells at the infarction site. In conclusion, our findings established the first relationship between the paracrine regenerative action of MSC and the nanotubular crosstalk with CM and emphasize that ex vivo manipulation of these communication processes might be of interest for optimizing current cardiac cell therapies.

AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity.

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that plays a central role in skeletal muscle metabolism. We used skeletal muscle-specific AMPKα1α2 double-knockout (mdKO) mice to provide direct genetic evidence of the physiological importance of AMPK in regulating muscle exercise capacity, mitochondrial function, and contraction-stimulated glucose uptake. Exercise performance was significantly reduced in the mdKO mice, with a reduction in maximal force production and fatigue resistance. An increase in the proportion of myofibers with centralized nuclei was noted, as well as an elevated expression of interleukin 6 (IL-6) mRNA, possibly consistent with mild skeletal muscle injury. Notably, we found that AMPKα1 and AMPKα2 isoforms are dispensable for contraction-induced skeletal muscle glucose transport, except for male soleus muscle. However, the lack of skeletal muscle AMPK diminished maximal ADP-stimulated mitochondrial respiration, showing an impairment at complex I. This effect was not accompanied by changes in mitochondrial number, indicating that AMPK regulates muscle metabolic adaptation through the regulation of muscle mitochondrial oxidative capacity and mitochondrial substrate utilization but not baseline mitochondrial muscle content. Together, these results demonstrate that skeletal muscle AMPK has an unexpected role in the regulation of mitochondrial oxidative phosphorylation that contributes to the energy demands of the exercising muscle.-Lantier, L., Fentz, J., Mounier, R., Leclerc, J., Treebak, J. T., Pehmøller, C., Sanz, N., Sakakibara, I., Saint-Amand, E., Rimbaud, S., Maire, P., Marette, A., Ventura-Clapier, R., Ferry, A., Wojtaszewski, J. F. P., Foretz, M., Viollet, B. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity.

Tissue LyC6- macrophages are generated in the absence of circulating LyC6- monocytes and Nur77 in a model of muscle regeneration.

There are several open questions regarding the origin, development, and differentiation of subpopulations of monocytes, macrophages (MFs), and dendritic cells. It is a particularly intriguing question how circulating monocyte subsets develop and contribute to the generation of steady-state and inflammatory tissue MF pools and which transcriptional mechanisms contribute to these processes. In this study, we took advantage of a genetic model in which LyC6(-) circulating monocyte development is severely diminished due to the lack of the nuclear receptor, NUR77. We show that, in a mouse model of skeletal muscle injury and regeneration, the accumulation of leukocytes and the generation of LyC6(+) and LyC6(-) MF pools are intact in the absence of circulating LyC6(-) blood monocytes. These data suggest that NUR77, which is required for LyC6(-) blood monocyte development, is expressed but not critically required for LyC6(+) to LyC6(-) tissue MF specification. Moreover, these observations support a model according to which tissue macrophage subtype specification is distinct from that of circulating monocytes. Lastly, our data show that in the used sterile inflammation model tissue LyC6(-) MFs are derived from LyC6(+) cells.

Differentially activated macrophages orchestrate myogenic precursor cell fate during human skeletal muscle regeneration.

Macrophages (MPs) exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for adult skeletal muscle repair, notably by acting on myogenic precursor cells. However, these interactions have not been fully characterized. Here, we explored both in vitro and in vivo, in human, the interactions of differentially activated MPs with myogenic precursor cells (MPCs) during adult myogenesis and skeletal muscle regeneration. We showed in vitro that through the differential secretion of cytokines and growth factors, proinflammatory MPs inhibited MPC fusion while anti-inflammatory MPs strongly promoted MPC differentiation by increasing their commitment into differentiated myocytes and the formation of mature myotubes. Furthermore, the in vivo time course of expression of myogenic and MP markers was studied in regenerating human healthy muscle after damage. We observed that regenerating areas containing proliferating MPCs were preferentially associated with MPs expressing proinflammatory markers. In the same muscle, regenerating areas containing differentiating myogenin-positive MPCs were preferentially coupled to MPs harboring anti-inflammatory markers. These data demonstrate for the first time in human that MPs sequentially orchestrate adult myogenesis during regeneration of damaged skeletal muscle. These results support the emerging concept that inflammation, through MP activation, controls stem cell fate and coordinates tissue repair.

Measurement of Myonuclear Accretion In Vitro and In Vivo.

Adult skeletal muscle stem cells (MuSC) are the regenerative precursors of myofibers and also have an important role in myofiber growth, adaptation, and maintenance by fusing to the myofibers-a process referred to as "myonuclear accretion." Due to a focus on MuSC function during regeneration, myofibers remain a largely overlooked component of the MuSC niche influencing MuSC fate. Here, we describe a method to directly measure the rate of myonuclear accretion in vitro and in vivo using ethynyl-2'-deoxyuridine (EdU)-based tracing of MuSC progeny. This method supports the dissection of MuSC intrinsic and myofiber-derived factors influencing myonuclear accretion as an alternative fate of MuSCs supporting myofiber homeostasis and plasticity.

Impaired skeletal muscle regeneration in diabetes: From cellular and molecular mechanisms to novel treatments.

Diabetes represents a major public health concern with a considerable impact on human life and healthcare expenditures. It is now well established that diabetes is characterized by a severe skeletal muscle pathology that limits functional capacity and quality of life. Increasing evidence indicates that diabetes is also one of the most prevalent disorders characterized by impaired skeletal muscle regeneration, yet underlying mechanisms and therapeutic treatments remain poorly established. In this review, we describe the cellular and molecular alterations currently known to occur during skeletal muscle regeneration in people with diabetes and animal models of diabetes, including its associated comorbidities, e.g., obesity, hyperinsulinemia, and insulin resistance. We describe the role of myogenic and non-myogenic cell types on muscle regeneration in conditions with or without diabetes. Therapies for skeletal muscle regeneration and gaps in our knowledge are also discussed, while proposing future directions for the field.

The AMPK-related kinase NUAK1 controls cortical axons branching by locally modulating mitochondrial metabolic functions.

The cellular mechanisms underlying axonal morphogenesis are essential to the formation of functional neuronal networks. We previously identified the autism-linked kinase NUAK1 as a central regulator of axon branching through the control of mitochondria trafficking. However, (1) the relationship between mitochondrial position, function and axon branching and (2) the downstream effectors whereby NUAK1 regulates axon branching remain unknown. Here, we report that mitochondria recruitment to synaptic boutons supports collateral branches stabilization rather than formation in mouse cortical neurons. NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration, and upregulation of mitochondrial function is sufficient to rescue axonal branching in NUAK1 null neurons in vitro and in vivo. Finally, we found that NUAK1 regulates axon branching through the mitochondria-targeted microprotein BRAWNIN. Our results demonstrate that NUAK1 exerts a dual function during axon branching through its ability to control mitochondrial distribution and metabolic activity.

Co-cultures of Macrophages with Muscle Stem Cells with Fibroadipogenic Precursor Cells from Regenerating Skeletal Muscle.

Adult muscle stem cells rebuild myofibers after damage. Although they are highly powerful to implement the adult myogenic program, they need environmental cues provided by surrounding cells for efficient and complete regeneration. Muscle stem cell environment includes fibroadipogenic precursors, vascular cells, and macrophages. A way to decipher the complexity of the interactions muscle stem cells establish with their neighborhood is to co-culture cells freshly isolated from the muscle and assess the impact of one cell type on the behavior/fate of the other cell type. Here, we present a protocol allowing the isolation of primary muscle stem cells, macrophages, and fibroadipogenic precursors by Fluorescence Activated Cell Sorting (FACS) or Magnetic Cell Separation (MACS), together with co-culture methods using a specific setup for a short time window to keep as much as possible the in vivo properties of the isolated cells.

AMPKα2 is a skeletal muscle stem cell intrinsic regulator of myonuclear accretion.

Due to the post-mitotic nature of skeletal muscle fibers, adult muscle maintenance relies on dedicated muscle stem cells (MuSCs). In most physiological contexts, MuSCs support myofiber homeostasis by contributing to myonuclear accretion, which requires a coordination of cell-type specific events between the myofiber and MuSCs. Here, we addressed the role of the kinase AMPKα2 in the coordination of these events supporting myonuclear accretion. We demonstrate that AMPKα2 deletion impairs skeletal muscle regeneration. Through in vitro assessments of MuSC myogenic fate and EdU-based cell tracing, we reveal a MuSC-specific role of AMPKα2 in the regulation of myonuclear accretion, which is mediated by phosphorylation of the non-metabolic substrate BAIAP2. Similar cell tracing in vivo shows that AMPKα2 knockout mice have a lower rate of myonuclear accretion during regeneration, and that MuSC-specific AMPKα2 deletion decreases myonuclear accretion in response to myofiber contraction. Together, this demonstrates that AMPKα2 is a MuSC-intrinsic regulator of myonuclear accretion.

A new model of experimental fibrosis in hindlimb skeletal muscle of adult mdx mouse mimicking muscular dystrophy.

INTRODUCTION: Duchenne Muscular Dystrophy (DMD) is characterized by the lack of dystrophin that leads to severe myofiber degeneration. We have shown that endomysial fibrosis is correlated with age at ambulation loss in DMD patients. However, the dystrophin-deficient mdx mouse does not have fibrotic lesions in adult limb muscles. Here, we describe a model of chronic mechanical muscle injury that triggers chronic lesions in mdx hindlimb muscle. METHODS: Micromechanical injuries were performed daily in tibialis anterior muscles for 2 weeks. RESULTS: Endomysial fibrosis appeared beginning 1 week post-injury, remained stable for 3 months and was associated with loss of specific maximal force. Fibrosis was associated with an increased expression of factors involved in fibrogenesis including α-smooth muscle actin, connective tissue growth factor, and lysyl oxidase, which colocalized with collagen deposits. CONCLUSIONS: This induced fibrotic dystrophic model may be useful to study mechanisms of fibrosis in dystrophinopathies and to evaluate antifibrotic treatments.