Macrophage depletion overcomes human hematopoietic cell engraftment failure in zebrafish embryo.

Zebrafish is widely adopted as a grafting model for studying human development and diseases. Current zebrafish xenotransplantations are performed using embryo recipients, as the adaptive immune system, responsible for host versus graft rejection, only reaches maturity at juvenile stage. However, transplanted primary human hematopoietic stem/progenitor cells (HSC) rapidly disappear even in zebrafish embryos, suggesting that another barrier to transplantation exists before the onset of adaptive immunity. Here, using a labelled macrophage zebrafish line, we demonstrated that engraftment of human HSC induces a massive recruitment of macrophages which rapidly phagocyte transplanted cells. Macrophages depletion, by chemical or pharmacological treatments, significantly improved the uptake and survival of transplanted cells, demonstrating the crucial implication of these innate immune cells for the successful engraftment of human cells in zebrafish. Beyond identifying the reasons for human hematopoietic cell engraftment failure, this work images the fate of human cells in real time over several days in macrophage-depleted zebrafish embryos.

Quantitative imaging and semiotic phenotyping of mitochondrial network morphology in live human cells.

The importance of mitochondria in tissue homeostasis, stress responses and human diseases, combined to their ability to transition between various structural and functional states, makes them excellent organelles for monitoring cell health. There is therefore a need for technologies to accurately analyze and quantify changes in mitochondrial organization in a variety of cells and cellular contexts. Here we present an innovative computerized method that enables accurate, multiscale, fast and cost-effective analysis of mitochondrial shape and network architecture from confocal fluorescence images by providing more than thirty features. In order to facilitate interpretation of the quantitative results, we introduced two innovations: the use of Kiviat-graphs (herein named MitoSpider plots) to present highly multidimensional data and visualization of the various mito-cellular configurations in the form of morphospace diagrams (called MitoSigils). We tested our fully automated image analysis tool on rich datasets gathered from live normal human skin cells cultured under basal conditions or exposed to specific stress including UVB irradiation and pesticide exposure. We demonstrated the ability of our proprietary software (named MitoTouch) to sensitively discriminate between control and stressed dermal fibroblasts, and between normal fibroblasts and other cell types (including cancer tissue-derived fibroblasts and primary keratinocytes), showing that our automated analysis captures subtle differences in morphology. Based on this novel algorithm, we report the identification of a protective natural ingredient that mitigates the deleterious impact of hydrogen peroxide (H2O2) on mitochondrial organization. Hence we conceived a novel wet-plus-dry pipeline combining cell cultures, quantitative imaging and semiotic analysis for exhaustive analysis of mitochondrial morphology in living adherent cells. Our tool has potential for broader applications in other research areas such as cell biology and medicine, high-throughput drug screening as well as predictive and environmental toxicology.

Modelling 3D Tumour Microenvironment In Vivo: A Tool to Predict Cancer Fate.

Recently, many studies demonstrated the fundamental role of the tumour microenvironment (TME) in cancer progression. Here, we describe a method to visualize in 3D the behaviour of tumours in zebrafish embryos. We highlight two major actors of the TME, macrophages and vessels. This valuable tool is transposable to Patient-Derived Xenograft imaging in order to predict the fate of malignant tumours according to the dynamics of their TME.

Deficiency in hereditary hemorrhagic telangiectasia-associated Endoglin elicits hypoxia-driven heart failure in zebrafish.

Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease caused by mutations affecting components of bone morphogenetic protein (BMP)/transforming growth factor-ß (TGF-ß) signaling in endothelial cells. This disorder is characterized by arteriovenous malformations that are prone to rupture, and the ensuing hemorrhages are responsible for iron-deficiency anemia. Along with activin receptor-like kinase (ALK1), mutations in endoglin are associated with the vast majority of HHT cases. In this study, we characterized the zebrafish endoglin locus and demonstrated that it produces two phylogenetically conserved protein isoforms. Functional analysis of a CRISPR/Cas9 zebrafish endoglin mutant revealed that Endoglin deficiency is lethal during the course from juvenile stage to adulthood. Endoglin-deficient zebrafish develop cardiomegaly, resulting in heart failure and hypochromic anemia, which both stem from chronic hypoxia. endoglin mutant zebrafish display structural alterations of the developing gills and underlying vascular network that coincide with hypoxia. Finally, phenylhydrazine treatment demonstrated that lowering hematocrit/blood viscosity alleviates heart failure and enhances the survival of Endoglin-deficient fish. Overall, our data link Endoglin deficiency to heart failure and establish zebrafish as a valuable HHT model.

The ROSA-Like Prophage Colonizing Staphylococcus aureus Promotes Intracellular Survival, Biofilm Formation, and Virulence in a Chronic Wound Environment.

BACKGROUND: The transition from colonization to invasion is critical in diabetic foot ulcer (DFU). Staphylococcus aureus can colonize DFU, or invade the underlying tissues, causing serious infections. The ROSA-like prophage has previously been implicated in strain colonization characteristics of S aureus isolates in uninfected ulcers. METHODS: In this study, we investigated this prophage in the S aureus-colonizing strain using an in vitro chronic wound medium mimicking the chronic wound environment. RESULTS: Chronic wound medium reduced bacterial growth and increased biofilm formation and virulence in a zebrafish model. CONCLUSIONS: The ROSA-like prophage promoted intracellular survival of S aureus-colonizing strain in macrophages, keratinocytes, and osteoblasts.

DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma.

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP-AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro-inflammatory signaling. We show that the DNA-PK DNA repair complex (i) drives cGAS-independent IRF3-mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS-dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA-PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS-dependent signaling is acquired during tumorigenesis and that cGAS and DNA-PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity.

Bacteriophage Therapy for Staphylococcus Aureus Infections: A Review of Animal Models, Treatments, and Clinical Trials.

Staphylococcus aureus (S. aureus) is a common and virulent human pathogen causing several serious illnesses including skin abscesses, wound infections, endocarditis, osteomyelitis, pneumonia, and toxic shock syndrome. Antibiotics were first introduced in the 1940s, leading to the belief that bacterial illnesses would be eradicated. However, microorganisms, including S. aureus, began to develop antibiotic resistance from the increased use and abuse of antibiotics. Antibiotic resistance is now one of the most serious threats to global public health. Bacteria like methicillin-resistant Staphylococcus aureus (MRSA) remain a major problem despite several efforts to find new antibiotics. New treatment approaches are required, with bacteriophage treatment, a non-antibiotic strategy to treat bacterial infections, showing particular promise. The ability of S. aureus to resist a wide range of antibiotics makes it an ideal candidate for phage therapy studies. Bacteriophages have a relatively restricted range of action, enabling them to target pathogenic bacteria. Their usage, usually in the form of a cocktail of bacteriophages, allows for more focused treatment while also overcoming the emergence of resistance. However, many obstacles remain, particularly in terms of their effects in vivo, necessitating the development of animal models to assess the bacteriophage efficiency. Here, we provide a review of the animal models, the various clinical case treatments, and clinical trials for S. aureus phage therapy.

HRAS germline mutations impair LKB1/AMPK signaling and mitochondrial homeostasis in Costello syndrome models.

Germline mutations that activate genes in the canonical RAS/MAPK signaling pathway are responsible for rare human developmental disorders known as RASopathies. Here, we analyzed the molecular determinants of Costello syndrome (CS) using a mouse model expressing HRAS p.G12S, patient skin fibroblasts, hiPSC-derived human cardiomyocytes, a HRAS p.G12V zebrafish model, and human fibroblasts expressing lentiviral constructs carrying HRAS p.G12S or HRAS p.G12A mutations. The findings revealed alteration of mitochondrial proteostasis and defective oxidative phosphorylation in the heart and skeletal muscle of CS mice that were also found in the cell models of the disease. The underpinning mechanisms involved the inhibition of the AMPK signaling pathway by mutant forms of HRAS, leading to alteration of mitochondrial proteostasis and bioenergetics. Pharmacological activation of mitochondrial bioenergetics and quality control restored organelle function in HRAS p.G12A and p.G12S cell models, reduced left ventricle hypertrophy in CS mice, and diminished the occurrence of developmental defects in the CS zebrafish model. Collectively, these findings highlight the importance of mitochondrial proteostasis and bioenergetics in the pathophysiology of RASopathies and suggest that patients with CS may benefit from treatment with mitochondrial modulators.

Investigating Pathogenicity and Virulence of Staphylococcus pettenkoferi: An Emerging Pathogen.

Staphylococcus pettenkoferi is a coagulase-negative Staphylococcus identified in 2002 that has been implicated in human diseases as an opportunistic pathogenic bacterium. Its multiresistant character is becoming a major health problem, yet the pathogenicity of S. pettenkoferi is poorly characterized. In this study, the pathogenicity of a S. pettenkoferi clinical isolate from diabetic foot osteomyelitis was compared with a Staphylococcus aureus strain in various in vitro and in vivo experiments. Growth kinetics were compared against S. aureus, and bacteria survival was assessed in the RAW 264.7 murine macrophage cell line, the THP-1 human leukemia monocytic cell line, and the HaCaT human keratinocyte cell line. Ex vivo analysis was performed in whole blood survival assays and in vivo assays via the infection model of zebrafish embryos. Moreover, whole-genome analysis was performed. Our results show that S. pettenkoferi was able to survive in human blood, human keratinocytes, murine macrophages, and human macrophages. S. pettenkoferi demonstrated its virulence by causing substantial embryo mortality in the zebrafish model. Genomic analysis revealed virulence factors such as biofilm-encoding genes (e.g., icaABCD; rsbUVW) and regulator-encoding genes (e.g., agr, mgrA, sarA, saeS) well characterized in S. aureus. This study thus advances the knowledge of this under-investigated pathogen and validates the zebrafish infection model for this bacterium.

Mitochondrial morphology is associated with respiratory chain uncoupling in autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is associated with unique changes in mitochondrial metabolism, including elevated respiration rates and morphological alterations. We examined electron transport chain (ETC) complex activity in fibroblasts derived from 18 children with ASD as well as mitochondrial morphology measurements in fibroblasts derived from the ASD participants and four typically developing controls. In ASD participants, symptoms severity was measured by the Social Responsiveness Scale and Aberrant Behavior Checklist. Mixed-model regression demonstrated that alterations in mitochondrial morphology were associated with both ETC Complex I+III and IV activity as well as the difference between ETC Complex I+III and IV activity. The subgroup of ASD participants with relative elevation in Complex IV activity demonstrated more typical mitochondrial morphology and milder ASD related symptoms. This study is limited by sample size given the invasive nature of obtaining fibroblasts from children. Furthermore, since mitochondrial function is heterogenous across tissues, the result may be specific to fibroblast respiration. Previous studies have separately described elevated ETC Complex IV activity and changes in mitochondrial morphology in cells derived from children with ASD but this is the first study to link these two findings in mitochondrial metabolism. The association between a difference in ETC complex I+III and IV activity and normal morphology suggests that mitochondrial in individuals with ASD may require ETC uncoupling to function optimally. Further studies should assess the molecular mechanisms behind these unique metabolic changes.Trial registration: Protocols used in this study were registered in clinicaltrials.gov as NCT02000284 and NCT02003170.

Macrophage morphological plasticity and migration is Rac signalling and MMP9 dependant.

In vitro, depending on extracellular matrix (ECM) architecture, macrophages migrate either in amoeboid or mesenchymal mode; while the first is a general trait of leukocytes, the latter is associated with tissue remodelling via Matrix Metalloproteinases (MMPs). To assess whether these stereotyped migrations could be also observed in a physiological context, we used the zebrafish embryo and monitored macrophage morphology, behaviour and capacity to mobilise haematopoietic stem/progenitor cells (HSPCs), as a final functional readout. Morphometric analysis identified 4 different cell shapes. Live imaging revealed that macrophages successively adopt all four shapes as they migrate through ECM. Treatment with inhibitors of MMPs or Rac GTPase to abolish mesenchymal migration, suppresses both ECM degradation and HSPC mobilisation while differently affecting macrophage behaviour. This study depicts real time macrophage behaviour in a physiological context and reveals extreme reactivity of these cells constantly adapting and switching migratory shapes to achieve HSPCs proper mobilisation.

Modeling and live imaging of mechanical instabilities in the zebrafish aorta during hematopoiesis.

All blood cells originate from hematopoietic stem/progenitor cells (HSPCs). HSPCs are formed from endothelial cells (ECs) of the dorsal aorta (DA), via endothelial-to-hematopoietic transition (EHT). The zebrafish is a primary model organism to study the process in vivo. While the role of mechanical stress in controlling gene expression promoting cell differentiation is actively investigated, mechanisms driving shape changes of the DA and individual ECs remain poorly understood. We address this problem by developing a new DA micromechanical model and applying it to experimental data on zebrafish morphogenesis. The model considers the DA as an isotropic tubular membrane subjected to hydrostatic blood pressure and axial stress. The DA evolution is described as a movement in the dimensionless controlling parameters space: normalized hydrostatic pressure and axial stress. We argue that HSPC production is accompanied by two mechanical instabilities arising in the system due to the plane stress in the DA walls and show how a complex interplay between mechanical forces in the system drives the emerging morphological changes.

Phosphatidylinositol-3 kinase signaling controls survival and stemness of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos lacking functional pten, an important tumor suppressor with a central role in cell signaling. Through in vivo live imaging, we discovered that in pten mutant embryos a proportion of the HSPCs died upon emergence from the VDA, an effect rescued by inhibition of phosphatidylinositol-3 kinase (PI3K). Surprisingly, inhibition of PI3K in wild-type embryos also induced HSPC death. Surviving HSPCs colonized the caudal hematopoietic tissue (CHT) normally and committed to all blood lineages. Single-cell RNA sequencing indicated that inhibition of PI3K enhanced survival of multipotent progenitors, whereas the number of HSPCs with more stem-like properties was reduced. At the end of the definitive wave, loss of Pten caused a shift to more restricted progenitors at the expense of HSPCs. We conclude that PI3K signaling tightly controls HSPCs survival and both up- and downregulation of PI3K signaling reduces stemness of HSPCs.

Whole embryo culture, transcriptomics and RNA interference identify TBX1 and FGF11 as novel regulators of limb development in the mouse.

Identifying genes involved in vertebrate developmental processes and characterizing this involvement are daunting tasks, especially in the mouse where viviparity complicates investigations. Attempting to devise a streamlined approach for this type of study we focused on limb development. We cultured E10.5 and E12.5 embryos and performed transcriptional profiling to track molecular changes in the forelimb bud over a 6-hour time-window. The expression of certain genes was found to diverge rapidly from its normal path, possibly reflecting the activation of a stress-induced response. Others, however, maintained for up to 3 hours dynamic expression profiles similar to those seen in utero. Some of these resilient genes were known regulators of limb development. The implication of the others in this process was either unsuspected or unsubstantiated. The localized knockdown of two such genes, Fgf11 and Tbx1, hampered forelimb bud development, providing evidence of their implication. These results show that combining embryo culture, transcriptome analysis and RNA interference could speed up the identification of genes involved in a variety of developmental processes, and the validation of their implication.

Mechanical instabilities of aorta drive blood stem cell production: a live study.

During embryogenesis of all vertebrates, haematopoietic stem/progenitor cells (HSPCs) extrude from the aorta by a complex process named endothelial-to-haematopoietic transition (EHT). HSPCs will then colonize haematopoietic organs allowing haematopoiesis throughout adult life. The mechanism underlying EHT including the role of each aortic endothelial cell (EC) within the global aorta dynamics remains unknown. In the present study, we show for the first time that EHT involves the remodelling of individual cells within a collective migration of ECs which is tightly orchestrated, resulting in HSPCs extrusion in the sub-aortic space without compromising aorta integrity. By performing a cross-disciplinary study which combines high-resolution 4D imaging and theoretical analysis based on the concepts of classical mechanics, we propose that this complex developmental process is dependent on mechanical instabilities of the aorta preparing and facilitating the extrusion of HSPCs.

Correction to: TNF signaling and macrophages govern fin regeneration in zebrafish larvae.

Correction to: Cell Death Dis. 8, e2979 (2017); https://doi.org/10.1038/cddis.2017.374 ; published online 10th August 2017.

TNF signaling and macrophages govern fin regeneration in zebrafish larvae.

Macrophages are essential for appendage regeneration after amputation in regenerative species. The molecular mechanisms through which macrophages orchestrate blastema formation and regeneration are still unclear. Here, we use the genetically tractable and transparent zebrafish larvae to study the functions of polarized macrophage subsets during caudal fin regeneration. After caudal fin amputation, we show an early and transient accumulation of pro-inflammatory macrophages concomitant with the accumulation of non-inflammatory macrophages which, in contrast to pro-inflammatory macrophages, remain associated to the fin until the end of the regeneration. Chemical and genetic depletion of macrophages suggested that early recruited macrophages that express TNFα are critical for blastema formation. Combining parabiosis and morpholino knockdown strategies, we show that TNFα/TNFR1 signaling pathway is required for the fin regeneration. Our study reveals that TNFR1 has a necessary and direct role in blastema cell activation suggesting that macrophage subset balance provides the accurate TNFα signal to prime regeneration in zebrafish.

Identification of polarized macrophage subsets in zebrafish.

While the mammalian macrophage phenotypes have been intensively studied in vitro, the dynamic of their phenotypic polarization has never been investigated in live vertebrates. We used the zebrafish as a live model to identify and trail macrophage subtypes. We generated a transgenic line whose macrophages expressing tumour necrosis factor alpha (tnfa), a key feature of classically activated (M1) macrophages, express fluorescent proteins Tg(mpeg1:mCherryF/tnfa:eGFP-F). Using 4D-confocal microscopy, we showed that both aseptic wounding and Escherichia coli inoculation triggered macrophage recruitment, some of which started to express tnfa. RT-qPCR on Fluorescence Activated Cell Sorting (FACS)-sorted tnfa(+) and tnfa(-) macrophages showed that they, respectively, expressed M1 and alternatively activated (M2) mammalian markers. Fate tracing of tnfa(+) macrophages during the time-course of inflammation demonstrated that pro-inflammatory macrophages converted into M2-like phenotype during the resolution step. Our results reveal the diversity and plasticity of zebrafish macrophage subsets and underline the similarities with mammalian macrophages proposing a new system to study macrophage functional dynamic.