Effects of hypoxia\u2013reoxygenation stimuli on renal redox status and nuclear factor erythroid 2-related factor 2 pathway in sickle cell SADmice

Hypoxia\u2013reoxygenation (H/R) stress is known to increase oxidative stress in transgenic sickle mice and can cause organ failure. Here we described the effects of H/R on nuclear factor erythroid 2-related factor 2 (Nrf2) as a putative regulator of redox status in the kidneys of SAD mice investigating Nrf2-regulated antioxidant enzymes. Transgenic SAD mice and healthy C57Bl/6J mice were exposed to 4 h of hypoxia followed by various times of reoxygenation at ambient air (2 or 6 h). Regardless of the conditions (i.e. normoxia or H/R), SAD mice expressed higher renal oxidative stress levels. Nuclear Nrf2 protein expression decreased after 2 h post-hypoxia only in the medulla region of the kidney and only in SAD mice. Simultaneously, haem oxygenase transcripts were affected by H/R stimulus with a significant enhancement after 2 h post-hypoxia. Similarly, hypoxia inducible factor-1 staining increased after 2 h post-hypoxia in SAD mice in both cortex and medulla areas. Our data confirm that the kidneys are organs that are particularly sensitive toH/R stimuli in sickle cell SAD mice. Also, these results suggest an effect of the duration of recovery period (short vs. long) and specific responses according to kidney areas, medulla vs. cortex, on Nrf2 expression in response to H/R stimuli in SAD mice

H3K18 lactylation marks tissue-specific active enhancers

Background: Histone lactylation has been recently described as a novel histone post-translational modification linking cellular metabolism to epigenetic regulation. Results: Given the expected relevance of this modification and current limited knowledge of its function, we generate genome-wide datasets of H3K18la distribution in various in vitro and in vivo samples, including mouse embryonic stem cells, macrophages, adipocytes, and mouse and human skeletal muscle. We compare them to profiles of well-established histone modifications and gene expression patterns. Supervised and unsupervised bioinformatics analysis shows that global H3K18la distribution resembles H3K27ac, although we also find notable differences. H3K18la marks active CpG island-containing promoters of highly expressed genes across most tissues assessed, including many housekeeping genes, and positively correlates with H3K27ac and H3K4me3 as well as with gene expression. In addition, H3K18la is enriched at active enhancers that lie in proximity to genes that are functionally important for the respective tissue. Conclusions: Overall, our data suggests that H3K18la is not only a marker for active promoters, but also a mark of tissue specific active enhancers. Keywords: Adipocyte; CUT&Tag; ChromHMM; Embryonic stem cell; Enhancer; Epigenetics; H3K18la; Histone post-translational modification; Lactate; Lactylation; Macrophage; Muscle; Promoter

The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1$^{-/-}$ mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1$^{-/-}$ mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation

Interactions entre le Récepteur aux glucocorticoïdes et l'AMP-activated protein kinase dans les macrophages au cours de la régénération du muscle strié squelettique

Skeletal muscle regenerates ad integrum after a sterile acute injury thanks to satellite cells (muscle stem cells). Inflammation, and notably macrophages, plays important roles during this process. Just after injury, monocytes infiltrate the tissue from the blood and convert into pro-inflammatory damaged associated macrophages. These macrophages phagocyte muscle debris and promote the proliferation of muscle stem cells. Then, macrophages switch their phenotype toward an anti-inflammatory restorative profile and promote muscle stem differentiation, fusion and myofiber growth. This sequence of macrophage profile is essential for an efficient skeletal muscle regeneration. The lab has shown that this phenotype switch is dependent of AMP kinase (AMPK)a1, a major energetic sensor in the cell controlling cellular metabolism. Besides, glucocorticoids have been used for decades for their anti-inflammatory effects on inflammation. Their actions are mediated by the Glucocorticoid Receptor which induces or represses gene expression by direct or indirect DNA-binding. As AMPKa1 and glucocorticoids induce similar anti-inflammatory effects on macrophages, we hypothesized that these 2 pathways could be interconnected in macrophages to allow the resolution of inflammation and muscle repair. Data from an in vitro model of skeletal muscle injury using bone marrow derived macrophages showed that: i) glucocorticoids induce AMPK phosphorylation; ii) AMPKa1 is required for the functional acquisition of the anti-inflammatory phenotype induced by glucocorticoids. Indeed, AMPKa1-deficient macrophages did not switch their phenotype and did not sustain myogenesis. In vivo experiments using LysMCre/+;AMPKa1fl/fl mice in which AMPKa1 is depleted only in myeloid cells, showed that macrophagic AMPK drove the beneficial effects of glucocorticoids during skeletal muscle regeneration. Inversely, in absence of AMPK in macrophages, glucocorticoids induced a delayed muscle regeneration and a modification in myofiber maturation, assessed by the alteration of myosin heavy chain expression. Altogether, these data show that glucocorticoids need AMPKa1 in macrophages for the resolution of inflammation and an efficient skeletal muscle regenerationLe muscle strié squelettique régénère ad integrum après une lésion aigüe stérile grâce aux cellules satellites qui sont les cellules souches du muscle strié squelettique. L'inflammation, et notamment les macrophages, joue un rôle important durant ce processus. En effet, après une lésion, les monocytes sanguins infiltrent le tissu et deviennent des macrophages avec un phénotype pro-inflammatoire associé à la lésion. Ces macrophages phagocytent les débris cellulaires et promeuvent la prolifération des cellules souches musculaires. Ensuite, les macrophages changent leur phénotype vers un phénotype anti-inflammatoire associé à la restauration du tissu. Ils promeuvent la différenciation, puis la fusion des cellules souches musculaires et la croissance des myofibres. Cette séquence de phénotypes inflammatoires est essentielle pour une régénération musculaire efficace. Le laboratoire a montré que ce changement de phénotype est dépendant d'un senseur énergétique majeur de la cellule qui contrôle le métabolisme cellulaire, l'AMP kinase (AMPK)al. Par ailleurs, les glucocorticoïdes sont utilisés depuis des décennies pour leurs effets anti-inflammatoires sur l'inflammation. Leur action est médiée par le Récepteur aux Glucocorticoïdes qui induit ou réprime l'expression de gènes par interaction directe ou indirecte à l'ADN. Comme l'AMPKal et les glucocorticoïdes induisent des effets anti-inflammatoires similaires sur les macrophages, nous avons posé l'hypothèse que ces 2 voies de signalisation pourraient être interconnectées dans les macrophages afin de permettre leur changement de phénotype et la régénération musculaire. Les données issues d'un modèle in vitro de lésion musculaire utilisant des macrophages dérivés de la moelle osseuse de souris ont montré que : i) les glucocorticoïdes induisaient la phosphorylation de l'AMPKal ; ii) l'AMPKal était requise pour l'acquisition fonctionnelle du statut anti-inflammatoire des macrophages induit par les glucocorticoïdes puisque des macrophages déficients pour l'AMPKal ne modifiaient pas leur phénotype et ne stimulaient pas la myogenèse. Les expériences in vivo utilisant des souris LysMCre/+;AMPKalfl/fl dans lesquelles l'AMPKal est invalidée uniquement dans les cellules myéloïdes ont montré que l'AMPKal dans les macrophages régulait les effets bénéfiques des glucocorticoïdes au cours de la régénération du muscle strié squelettique. En effet, en absence d'AMPKal dans les macrophages, les glucocorticoïdes induisaient un retard de régénération et une modification de la maturation des fibres attestée par une modification de l'expression des isoformes des chaînes lourdes de myosines. En conclusion, ces données montrent que l'AMPKal est requise pour le changement de phénotype des macrophages induit par les glucocorticoïdes et une régénération musculaire efficac

Histone lactylation in macrophages is predictive for gene expression changes during ischemia induced-muscle regeneration

Objectives: We have previously shown that lactate is an essential metabolite for macrophage polarisation during ischemia-induced muscle regeneration. Recent in vitro work has implicated histone lactylation, a direct derivative of lactate, in macrophage polarisation. Here, we explore the in vivo relevance of histone lactylation for macrophage polarisation after muscle injury. Methods: To evaluate macrophage dynamics during muscle regeneration, we subjected mice to ischemia-induced muscle damage by ligating the femoral artery. Muscle samples were harvested at 1, 2, 4, and 7 days post injury (dpi). CD45+CD11b+F4/80+CD64+ macrophages were isolated and processed for RNA sequencing, Western Blotting, and CUT&Tag-sequencing to investigate gene expression, histone lactylation levels, and histone lactylation genomic localisation and enrichment, respectively. Results: We show that, over time, macrophages in the injured muscle undergo extensive gene expression changes, which are similar in nature and in timing to those seen after other types of muscle-injuries. We find that the macrophage histone lactylome is modified between 2 and 4 dpi, which is a crucial window for macrophage polarisation. Absolute histone lactylation levels increase, and, although subtly, the genomic enrichment of H3K18la changes. Overall, we find that histone lactylation is important at both promoter and enhancer elements. Lastly, H3K18la genomic profile changes from 2 to 4 dpi were predictive for gene expression changes later in time, rather than being a reflection of prior gene expression changes. Conclusions: Our results suggest that histone lactylation dynamics are functionally important for the function of macrophages during muscle regeneration

Effects of hypoxia–reoxygenation stimuli on renal redox status and nuclear factor erythroid 2‐related factor 2 pathway in sickle cell SAD mice

International audienc

Open-CSAM, a new tool for semi-automated analysis of myofiber cross-sectional area in regenerating adult skeletal muscle

Abstract Adult skeletal muscle is capable of complete regeneration after an acute injury. The main parameter studied to assess muscle regeneration efficacy is the cross-sectional area (CSA) of the myofibers as myofiber size correlates with muscle force. CSA analysis can be time-consuming and may trigger variability in the results when performed manually. This is why programs were developed to completely automate the analysis of the CSA, such as SMASH, MyoVision, or MuscleJ softwares. Although these softwares are efficient to measure CSA on normal or hypertrophic/atrophic muscle, they fail to efficiently measure CSA on regenerating muscles. We developed Open-CSAM, an ImageJ macro, to perform a high throughput semi-automated analysis of CSA on skeletal muscle from various experimental conditions. The macro allows the experimenter to adjust the analysis and correct the mistakes done by the automation, which is not possible with fully automated programs. We showed that Open-CSAM was more accurate to measure CSA in regenerating and dystrophic muscles as compared with SMASH, MyoVision, and MuscleJ softwares and that the inter-experimenter variability was negligible. We also showed that, to obtain a representative CSA measurement, it was necessary to analyze the whole muscle section and not randomly selected pictures, a process that was easily and accurately be performed using Open-CSAM. To conclude, we show here an easy and experimenter-controlled tool to measure CSA in muscles from any experimental condition, including regenerating muscle

Macrophagic AMPKα1 orchestrates regenerative inflammation induced by glucocorticoids

International audienceAbstract 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

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

H3K18 lactylation marks tissue-specific active enhancers

Background: Histone lactylation has been recently described as a novel histone post-translational modification linking cellular metabolism to epigenetic regulation. Results: Given the expected relevance of this modification and current limited knowledge of its function, we generate genome-wide datasets of H3K18la distribution in various in vitro and in vivo samples, including mouse embryonic stem cells, macrophages, adipocytes, and mouse and human skeletal muscle. We compare them to profiles of well-established histone modifications and gene expression patterns. Supervised and unsupervised bioinformatics analysis shows that global H3K18la distribution resembles H3K27ac, although we also find notable differences. H3K18la marks active CpG island-containing promoters of highly expressed genes across most tissues assessed, including many housekeeping genes, and positively correlates with H3K27ac and H3K4me3 as well as with gene expression. In addition, H3K18la is enriched at active enhancers that lie in proximity to genes that are functionally important for the respective tissue. Conclusions: Overall, our data suggests that H3K18la is not only a marker for active promoters, but also a mark of tissue specific active enhancers