Resting natural killer cell homeostasis relies on tryptophan/NAD+^{+} metabolism and HIF-1α

Natural killer (NK) cells are forced to cope with different oxygen environments even under resting conditions. The adaptation to low oxygen is regulated by oxygen-sensitive transcription factors, the hypoxia-inducible factors (HIFs). The function of HIFs for NK cell activation and metabolic rewiring remains controversial. Activated NK cells are predominantly glycolytic, but the metabolic programs that ensure the maintenance of resting NK cells are enigmatic. By combining in situ metabolomic and transcriptomic analyses in resting murine NK cells, our study defines HIF-1α as a regulator of tryptophan metabolism and cellular nicotinamide adenine dinucleotide (NAD$^{+}$ ) levels. The HIF-1α/NAD$^{+}$ axis prevents ROS production during oxidative phosphorylation (OxPhos) and thereby blocks DNA damage and NK cell apoptosis under steady-state conditions. In contrast, in activated NK cells under hypoxia, HIF-1α is required for glycolysis, and forced HIF-1α expression boosts glycolysis and NK cell performance in vitro and in vivo. Our data highlight two distinct pathways by which HIF-1α interferes with NK cell metabolism. While HIF-1α-driven glycolysis is essential for NK cell activation, resting NK cell homeostasis relies on HIF-1α-dependent tryptophan/NAD$^{+}$ metabolism

Vaccination-based immunotherapy to target profibrotic cells in liver and lung

Fibrosis is the final path of nearly every form of chronic disease, regardless of the pathogenesis. Upon chronic injury, activated, fibrogenic fibroblasts deposit excess extracellular matrix, and severe tissue fibrosis can occur in virtually any organ. However, antifibrotic therapies that target fibrogenic cells, while sparing homeostatic fibroblasts in healthy tissues, are limited. We tested whether specific immunization against endogenous proteins, strongly expressed in fibrogenic cells but highly restricted in quiescent fibroblasts, can elicit an antigen-specific cytotoxic T cell response to ameliorate organ fibrosis. In silico epitope prediction revealed that activation of the genes Adam12 and Gli1 in profibrotic cells and the resulting “self-peptides” can be exploited for T cell vaccines to ablate fibrogenic cells. We demonstrate the efficacy of a vaccination approach to mount CD8+ T cell responses that reduce fibroblasts and fibrosis in the liver and lungs in mice. These results provide proof of principle for vaccination-based immunotherapies to treat fibrosis

Functional analysis of Ki-67 protein in the cell cycle

Ki-67 maintient l'hétérochromatine constitutive lors de la prolifération cellulaire. La protéine Ki-67 est fortement exprimée dans le noyau des cellules de mammifères en prolifération. Sa présence est devenue un marqueur de référence en histopathologie diagnostic dans le cancer avec 330 millions de références sur des sites Internet et plus de 18 000 articles indexés avec le mot clé Ki-67 dans PubMed. Malgré son utilisation comme référence incontestable de la prolifération cellulaire, les rôles fonctionnels, les régulations et les mécanismes moléculaires où Ki-67 est impliquée demeurent obscurs. Depuis l'identification de Ki-67 en 1983, les différentes études avec l'utilisation d'outils moléculaires (anticorps, oligonucléotide antisens, shRNA, siRNA) qui inhibent l'expression de Ki-67 dans des cellules cancéreuses de tous types ont mis en évidence une forte régression à leur prolifération. La localisation prédominate de Ki-67 est dans le nucléole, son inactivation photodynamique abroge la transcription de l'ARN ribosomal qui est requis pour la prolifération cellulaire. Le consensus est que Ki-67 favorise la prolifération cellulaire, mais à ce jour aucune étude n'a mis en évidence ni un mécanisme d'action, ni un rôle essentiel de Ki-67 dans la prolifération cellulaire in vivo. Dans notre travail, nous abordons la question du rôle de Ki-67 dans les cellules humaines cancéreuses non transformées en culture ainsi qu'in vivo chez la souris. Nous avons montré que Ki-67 n'est pas nécessaire pour la prolifération cellulaire. En revanche, Ki-67 est indispensable pour maintenir la structure de l'hétérochromatine constitutive. Nous avons mis en évidence des nouveaux mécanismes de régulation de l'expression de Ki-67 au cours du cycle cellulaire, sa transcription étant contrôlé par CDK4/6 via la phosphorylation de la protéine rétinoblastome, et sa dégradation en G1 tardive via APC/C-Cdh1. Après extinction de l'expression de Ki-67 par ARNi ou par ablation du gène de Ki-67 par la nouvelle approche TALEN, nous n'avons observé aucun effet sur la synthèse de l'ARN ribosomique, sur le déroulement normal du cycle cellulaire, sur le développement embryonnaire ou la fertilité de la souris. Par contre, son extinction inhibe la progression des tumeurs dans des modèles de Xénogreffes, et induit un remodelage du paysage de l'expression de certain gènes. Notre étude par protéomique des protéines interragissant avec Ki-67 a identifié des protéines nucléolaires à l'interface entre l'hétérochromatine périnucléolaires et les composants granulaires nucléolaires. Ces complexes protéiques empêchent la dispersion de l'hétérochromatine constitutive au cours du cycle cellulaire. Au vu de nos résultats, nous concluons que dans les cellules non-proliférantes l'anémie de Ki-67 est associée à la diffusion de l'hétérochromatine constitutive. Ki-67 est indispensable à la maintenance de cette hétérochromatine qui serait essentielle pour le développement des tumeurs.Ki-67 links constitutive heterochromatin maintenance to cell proliferation.Ubiquitous nuclear expression of Ki-67 in proliferating mammalian cells has led to its use as a benchmark diagnostic marker for cell proliferation, especially in cancer histopathology. Its importance is reflected by over 330 million hits when searching using the keyword “Ki-67” on Google, and over 18,000 papers on PubMed. In spite of its use as a surrogate marker for cell proliferation, the mechanisms of regulation of Ki-67 expression and its physiological functions in cell proliferation remain obscure. Early functional studies found that inhibition of Ki-67 expression by injection of antisense oligonucleotides or inactivating antibodies into cultured cancer cells inhibited cell proliferation. This is in agreement with later results obtained by peptide-nucleic acid, antisense oligonucleotide, siRNA or shRNA experiments in various cancer cell lines. Photodynamic inactivation of Ki-67 abrogates ribosomal RNA transcription, consistent with its predominantly nucleolar localisation and apparent requirement for cell proliferation. However, a recent study in HeLa cells found only minor effects on the cell cycle distribution upon Ki-67 knockdown. Nevertheless, Ki-67 is required for localising several nucleolar proteins to the mitotic chromosome periphery, potentially providing a mechanism for nucleolar assembly, as previously suggested by segregating nucleolar components upon cell division and chromosome segregation. Therefore, although the consensus is that Ki-67 promotes cell proliferation, this has not been clearly demonstrated, and no studies have ascertained requirements for Ki-67 in vivo.In this work, we address these questions in non-transformed human cells and cancer cells in culture and in vivo in mice. We have shown that Ki-67 is dispensable for cell proliferation but is required to maintain constitutive heterochromatin. We found that Ki-67 expression is cell cycle-dependent due to dynamic control by CDK4/6 and Cdh1. However, silencing of Ki-67 by RNAi or a TALEN-mediated Ki-67 gene ablation had no effect on ribosomal RNA synthesis, cell cycling, mouse development or fertility, but prevented tumour progression and led to remodelling of the gene expression landscape in cycling cells. Interaction proteomics and functional assays revealed that Ki-67 defines the boundary between perinucleolar heterochromatin and the nucleolar granular components, and prevents dispersal of constitutive heterochromatin during cell cycling. Conversely, Ki-67 downregulation in non-proliferating cells is associated with constitutive heterochromatin dispersal. The results suggest that Ki-67 mediates organisation of heterochromatin, and allows efficient tumour progression

MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells

Summary: Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. DNA adenine methyltransferase identification (DamID) has emerged as a comprehensive method to map genome-wide occupancy of proteins of interest. A caveat of DamID is the specificity of Dam methyltransferase for GATC motifs that are not homogenously distributed in the genome. Here, we developed an optimized method named MadID, using proximity labeling of DNA by the methyltransferase M.EcoGII. M.EcoGII mediates N6-adenosine methylation in any DNA sequence context, resulting in deeper and unbiased coverage of the genome. We demonstrate, using m6A-specific immunoprecipitation and deep sequencing, that MadID is a robust method to identify protein-DNA interactions at the whole-genome level. Using MadID, we revealed contact sites between human telomeres, repetitive sequences devoid of GATC sites, and the nuclear envelope. Overall, MadID opens the way to identification of binding sites in genomic regions that were largely inaccessible. : Mapping the binding sites of DNA- or chromatin-interacting proteins is essential to understanding biological processes. Sobecki et al. developed an optimized method named MadID based on proximity labeling of DNA by the bacterial methyltransferase M.EcoGII. MadID results in deep and unbiased coverage for genome-wide mapping studies. Keywords: MadID, M.EcoGII, m6A, LADs, telomeres, nuclear envelope, proximity labeling, methylatio

Revisited Synthesis of Aryl-H-phosphinates

International audienceA systematic study of the reaction conditions for the preparation of pure aryl-H-phosphinate esters, originally developed by Sander and optimized by Petneházy, is reported. The influence of the reaction concentration has been investigated for the formation of phosphonite intermediates via direct addition of triethyl phosphite to the appropriate Grignard reagent. Subsequent hydrolysis of the phosphonites under acidic conditions gives various aryl-Hphosphinates in high yields and purities

Cell-Cycle Regulation Accounts for Variability in Ki-67 Expression Levels

International audienc

NK cells in hypoxic skin mediate a trade-off between wound healing and antibacterial defence

During skin injury, immune response and repair mechanisms have to be coordinated for rapid skin regeneration and the prevention of microbial infections. Natural Killer (NK) cells infiltrate hypoxic skin lesions and Hypoxia-inducible transcription factors (HIFs) mediate adaptation to low oxygen. We demonstrate that mice lacking the Hypoxia-inducible factor (HIF)-1α isoform in NK cells show impaired release of the cytokines Interferon (IFN)-γ and Granulocyte Macrophage - Colony Stimulating Factor (GM-CSF) as part of a blunted immune response. This accelerates skin angiogenesis and wound healing. Despite rapid wound closure, bactericidal activity and the ability to restrict systemic bacterial infection are impaired. Conversely, forced activation of the HIF pathway supports cytokine release and NK cell-mediated antibacterial defence including direct killing of bacteria by NK cells despite delayed wound closure. Our results identify, HIF-1α in NK cells as a nexus that balances antimicrobial defence versus global repair in the skin

Loss of HIF-1α in natural killer cells inhibits tumour growth by stimulating non-productive angiogenesis

Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels

NK cells in hypoxic skin mediate a trade-off between wound healing and antibacterial defence

International audienceAbstract During skin injury, immune response and repair mechanisms have to be coordinated for rapid skin regeneration and the prevention of microbial infections. Natural Killer (NK) cells infiltrate hypoxic skin lesions and Hypoxia-inducible transcription factors (HIFs) mediate adaptation to low oxygen. We demonstrate that mice lacking the Hypoxia-inducible factor (HIF)-1α isoform in NK cells show impaired release of the cytokines Interferon (IFN)-γ and Granulocyte Macrophage - Colony Stimulating Factor (GM-CSF) as part of a blunted immune response. This accelerates skin angiogenesis and wound healing. Despite rapid wound closure, bactericidal activity and the ability to restrict systemic bacterial infection are impaired. Conversely, forced activation of the HIF pathway supports cytokine release and NK cell-mediated antibacterial defence including direct killing of bacteria by NK cells despite delayed wound closure. Our results identify, HIF-1α in NK cells as a nexus that balances antimicrobial defence versus global repair in the skin

The transcription factor HIF-1α mediates plasticity of NKp46+ innate lymphoid cells in the gut.

Gut innate lymphoid cells (ILCs) show remarkable phenotypic diversity, yet microenvironmental factors that drive this plasticity are incompletely understood. The balance between NKp46+, IL-22-producing, group 3 ILCs (ILC3s) and interferon (IFN)-γ-producing group 1 ILCs (ILC1s) contributes to gut homeostasis. The gut mucosa is characterized by physiological hypoxia, and adaptation to low oxygen is mediated by hypoxia-inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not well understood. Mice lacking the HIF-1α isoform in NKp46+ ILCs show a decrease in IFN-γ-expressing, T-bet+, NKp46+ ILC1s and a concomitant increase in IL-22-expressing, RORγt+, NKp46+ ILC3s in the gut mucosa. Single-cell RNA sequencing revealed HIF-1α as a driver of ILC phenotypes, where HIF-1α promotes the ILC1 phenotype by direct up-regulation of T-bet. Loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the expression of IL-22-inducible genes, and confers protection against intestinal damage. Taken together, our results suggest that HIF-1α shapes the ILC phenotype in the gut