A host-directed oxadiazole compound potentiates antituberculosis treatment via zinc poisoning in human macrophages and in a mouse model of infection.

Antituberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multiresistant strains of Mycobacterium tuberculosis (MTB) are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high-content imaging, we identified novel 1,2,4-oxadiazole-based compounds, which allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, MTB became even more sensitive to antituberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus.

Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.

MuscleJ2: a rebuilding of MuscleJ with new features for high-content analysis of skeletal muscle immunofluorescence slides.

Histological analysis of skeletal muscle is of major interest for understanding its behavior in different pathophysiological conditions, such as the response to different environments or myopathies. In this context, many software programs have been developed to perform automated high-content analysis. We created MuscleJ, a macro that runs in ImageJ/Fiji on batches of images. MuscleJ is a multianalysis tool that initially allows the analysis of muscle fibers, capillaries, and satellite cells. Since its creation, it has been used in many studies, and we have further developed the software and added new features, which are presented in this article. We converted the macro into a Java-language plugin with an improved user interface. MuscleJ2 provides quantitative analysis of fibrosis, vascularization, and cell phenotype in whole muscle sections. It also performs analysis of the peri-myonuclei, the individual capillaries, and any staining in the muscle fibers, providing accurate quantification within regional sublocalizations of the fiber. A multicartography option allows users to visualize multiple results simultaneously. The plugin is freely available to the muscle science community.

Impact of the gut microbiome on nicotine's motivational effects and glial cells in the ventral tegmental area in male mice.

A link between gut dysbiosis and the pathogenesis of brain disorders has been identified. A role for gut bacteria in drug reward and addiction has been suggested but very few studies have investigated their impact on brain and behavioral responses to addictive drugs so far. In particular, their influence on nicotine's addiction-like processes remains unknown. In addition, evidence shows that glial cells shape the neuronal activity of the mesolimbic system but their regulation, within this system, by the gut microbiome is not established. We demonstrate that a lack of gut microbiota in male mice potentiates the nicotine-induced activation of sub-regions of the mesolimbic system. We further show that gut microbiota depletion enhances the response to nicotine of dopaminergic neurons of the posterior ventral tegmental area (pVTA), and alters nicotine's rewarding and aversive effects in an intra-VTA self-administration procedure. These effects were not associated with gross behavioral alterations and the nicotine withdrawal syndrome was not impacted. We further show that depletion of the gut microbiome modulates the glial cells of the mesolimbic system. Notably, it increases the number of astrocytes selectively in the pVTA, and the expression of postsynaptic density protein 95 in both VTA sub-regions, without altering the density of the astrocytic glutamatergic transporter GLT1. Finally, we identify several sub-populations of microglia in the VTA that differ between its anterior and posterior sub-parts, and show that they are re-organized in conditions of gut microbiota depletion. The present study paves the way for refining our understanding of the pathophysiology of nicotine addiction.

The FDA-approved drug Auranofin has a dual inhibitory effect on SARS-CoV-2 entry and NF-κB signaling.

Patients with severe COVID-19 show an altered immune response that fails to control the viral spread and suffer from exacerbated inflammatory response, which eventually can lead to death. A major challenge is to develop an effective treatment for COVID-19. NF-κB is a major player in innate immunity and inflammatory process. By a high-throughput screening approach, we identified FDA-approved compounds that inhibit the NF-κB pathway and thus dampen inflammation. Among these, we show that Auranofin prevents post-translational modifications of NF-κB effectors and their recruitment into activating complexes in response to SARS-CoV-2 infection or cytokine stimulation. In addition, we demonstrate that Auranofin counteracts several steps of SARS-CoV-2 infection. First, it inhibits a raft-dependent endocytic pathway involved in SARS-CoV-2 entry into host cells; Second, Auranofin alters the ACE2 mobility at the plasma membrane. Overall, Auranofin should prevent SARS-CoV-2 infection and inflammatory damages, offering new opportunities as a repurposable drug candidate to treat COVID-19.

A Role for Taok2 in Listeria monocytogenes Vacuolar Escape.

The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.

Antibody Neutralization of HIV-1 Crossing the Blood-Brain Barrier.

HIV-1 can cross the blood-brain barrier (BBB) to penetrate the brain and infect target cells, causing neurocognitive disorders as a result of neuroinflammation and brain damage. Here, we examined whether antibodies targeting the HIV-1 envelope glycoproteins interfere with the transcytosis of virions across the human BBB endothelium. We found that although the viral envelope spike gp160 is required for optimal endothelial cell endocytosis, no anti-gp160 antibodies blocked the BBB transcytosis of HIV-1 in vitro Instead, both free viruses and those in complex with antibodies transited across endothelial cells in the BBB model, as observed by confocal microscopy. HIV-1 infectious capacity was considerably altered by the transcytosis process but still detectable, even in the presence of nonneutralizing antibodies. Only virions bound by neutralizing antibodies lacked posttranscytosis infectivity. Overall, our data support the role of neutralizing antibodies in protecting susceptible brain cells from HIV-1 infection despite their inability to inhibit viral BBB endocytic transport.IMPORTANCE HIV-1 can cross the blood-brain barrier (BBB) to penetrate the brain and infect target cells, causing neurocognitive disorders as a result of neuroinflammation and brain damage. The HIV-1 envelope spike gp160 is partially required for viral transcytosis across the BBB endothelium. But do antibodies developing in infected individuals and targeting the HIV-1 gp160 glycoproteins block HIV-1 transcytosis through the BBB? We addressed this issue and discovered that anti-gp160 antibodies do not block HIV-1 transport; instead, free viruses and those in complex with antibodies can transit across BBB endothelial cells. Importantly, we found that only neutralizing antibodies could inhibit posttranscytosis viral infectivity, highlighting their ability to protect susceptible brain cells from HIV-1 infection.

Hepatic stellate cell hypertrophy is associated with metabolic liver fibrosis.

Hepatic fibrosis is a major consequence of chronic liver disease such as non-alcoholic steatohepatitis which is undergoing a dramatic evolution given the obesity progression worldwide, and has no treatment to date. Hepatic stellate cells (HSCs) play a key role in the fibrosis process, because in chronic liver damage, they transdifferentiate from a "quiescent" to an "activated" phenotype responsible for most the collagen deposition in liver tissue. Here, using a diet-induced liver fibrosis murine model (choline-deficient amino acid-defined, high fat diet), we characterized a specific population of HSCs organized as clusters presenting simultaneously hypertrophy of retinoid droplets, quiescent and activated HSC markers. We showed that hypertrophied HSCs co-localized with fibrosis areas in space and time. Importantly, we reported the existence of this phenotype and its association with collagen deposition in three other mouse fibrosis models, including CCl4-induced fibrosis model. Moreover, we have also shown its relevance in human liver fibrosis associated with different etiologies (obesity, non-alcoholic steatohepatitis, viral hepatitis C and alcoholism). In particular, we have demonstrated a significant positive correlation between the stage of liver fibrosis and HSC hypertrophy in a cohort of obese patients with hepatic fibrosis. These results lead us to conclude that hypertrophied HSCs are closely associated with hepatic fibrosis in a metabolic disease context and may represent a new marker of metabolic liver disease progression.

Muscle Injury Induces Postoperative Cognitive Dysfunction.

Postoperative cognitive dysfunction (POCD) is a major complication affecting patients of any age undergoing surgery. This syndrome impacts everyday life up to months after hospital discharge, and its pathophysiology still remains unclear. Translational research focusing on POCD is based on a wide variety of rodent models, such as the murine tibial fracture, whose severity can limit mouse locomotion and proper behavioral assessment. Besides, influence of skeletal muscle injury, a lesion encountered in a wide range of surgeries, has not been explored in POCD occurrence. We propose a physical model of muscle injury in CX3CR1GFP/+ mice (displaying green fluorescent microglial cells) to study POCD, with morphological, behavioral and molecular approaches. We highlighted: alteration of short- and long-term memory after muscle regeneration, wide microglial reactivity in the brain, including hippocampus area, 24 hours after muscle injury, and an alteration of central brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) balance, 28 days after muscle injury. Our results suggest for the first time that muscle injury can have early as well as late impacts on the brain. Our CX3CR1GFP/+ model can also facilitate microglial investigation, more specifically their pivotal role in neuroinflammation and synaptic plasticity, in the pathophysiology of POCD.

USF1 defect drives p53 degradation during Helicobacter pylori infection and accelerates gastric carcinogenesis.

OBJECTIVE: Helicobacter pylori (Hp) is a major risk factor for gastric cancer (GC). Hp promotes DNA damage and proteasomal degradation of p53, the guardian of genome stability. Hp reduces the expression of the transcription factor USF1 shown to stabilise p53 in response to genotoxic stress. We investigated whether Hp-mediated USF1 deregulation impacts p53-response and consequently genetic instability. We also explored in vivo the role of USF1 in gastric carcinogenesis. DESIGN: Human gastric epithelial cell lines were infected with Hp7.13, exposed or not to a DNA-damaging agent camptothecin (CPT), to mimic a genetic instability context. We quantified the expression of USF1, p53 and their target genes, we determined their subcellular localisation by immunofluorescence and examined USF1/p53 interaction. Usf1-/- and INS-GAS mice were used to strengthen the findings in vivo and patient data examined for clinical relevance. RESULTS: In vivo we revealed the dominant role of USF1 in protecting gastric cells against Hp-induced carcinogenesis and its impact on p53 levels. In vitro, Hp delocalises USF1 into foci close to cell membranes. Hp prevents USF1/p53 nuclear built up and relocates these complexes in the cytoplasm, thereby impairing their transcriptional function. Hp also inhibits CPT-induced USF1/p53 nuclear complexes, exacerbating CPT-dependent DNA damaging effects. CONCLUSION: Our data reveal that the depletion of USF1 and its de-localisation in the vicinity of cell membranes are essential events associated to the genotoxic activity of Hp infection, thus promoting gastric carcinogenesis. These findings are also of clinical relevance, supporting USF1 expression as a potential marker of GC susceptibility.

Microglial production of quinolinic acid as a target and a biomarker of the antidepressant effect of ketamine.

Major depressive disorder is a complex multifactorial condition with a so far poorly characterized underlying pathophysiology. Consequently, the available treatments are far from satisfactory as it is estimated that up to 30% of patients are resistant to conventional treatment. Recent comprehensive evidence has been accumulated which suggests that inflammation may be implied in the etiology of this disease. Here we investigated ketamine as an innovative treatment strategy due to its immune-modulating capacities. In a murine model of LPS-induced depressive-like behavior we demonstrated that a single dose of ketamine restores the LPS-induced depressive-like alterations. These behavioral effects are associated with i/ a reversal of anxiety and reduced self-care, ii/ a decrease in parenchymal cytokine production, iii/ a modulation of the microglial reactivity and iv/ a decrease in microglial quinolinic acid production that is correlated with plasmatic peripheral production. In a translational approach, we show that kynurenic acid to quinolinic acid ratio is a predictor of ketamine response in treatment-resistant depressed patients and that the reduction in quinolinic acid after a ketamine infusion is a predictor of the reduction in MADRS score. Our results suggest that microglia is a key therapeutic target and that quinolinic acid is a biomarker of ketamine response in major depressive disorder.

Next-Generation Phenotypic Screening in Early Drug Discovery for Infectious Diseases.

Cell-based phenotypic screening has proven to be valuable, notably in recapitulating relevant biological conditions, for example, the host cell/pathogen niche. However, the corresponding methodological complexity is not readily compatible with high-throughput pipelines, and fails to inform either molecular target or mechanism of action, which frustrates conventional drug-discovery roadmaps. We review the state-of-the-art and emerging technologies that suggest new strategies for harnessing value from the complexity of phenotypic screening and augmenting powerful utility for translational drug discovery. Advances in cellular, molecular, and bioinformatics technologies are converging at a cutting edge where the complexity of phenotypic screening may no longer be considered a hinderance but rather a catalyst to chemotherapeutic discovery for infectious diseases.

Imaging of Red-Shifted Light From Bioluminescent Tumors Using Fluorescence by Unbound Excitation From Luminescence.

Early detection of tumors is today a major challenge and requires sensitive imaging methodologies coupled with new efficient probes. In vivo optical bioluminescence imaging has been widely used in the field of preclinical oncology to visualize tumors and several cancer cell lines have been genetically modified to provide bioluminescence signals. However, the light emitted by the majority of commonly used luciferases is usually in the blue part of the visible spectrum, where tissue absorption is still very high, making deep tissue imaging non-optimal, and calling for optimized optical imaging methodologies. We have previously shown that red-shifting of bioluminescence signal by Fluorescence Unbound Excitation from Luminescence (FUEL) is a mean to increase bioluminescence signal sensitivity detection in vivo. Here, we applied FUEL to tumor detection in two different subcutaneous tumor models: the auto-luminescent human embryonic kidney (HEK293) cell line and the murine B16-F10 melanoma cell line previously transfected with a plasmid encoding the Luc2 firefly luciferase. Tumor size and bioluminescence were measured over time and tumor vascularization characterized. We then locally injected near infrared emitting Quantum Dots (NIR QDs) in the tumor site and observed a red-shifting of bioluminescence signal by (FUEL) indicating that FUEL could be used to allow deeper tumor detection in mice.

Tri-mannose grafting of chitosan nanocarriers remodels the macrophage response to bacterial infection.

BACKGROUND: Infectious diseases are still a leading cause of death and, with the emergence of drug resistance, pose a great threat to human health. New drugs and strategies are thus urgently needed to improve treatment efficacy and limit drug-associated side effects. Nanotechnology-based drug delivery systems are promising approaches, offering hope in the fight against drug resistant bacteria. However, how nanocarriers influence the response of innate immune cells to bacterial infection is mostly unknown. RESULTS: Here, we used Mycobacterium tuberculosis as a model of bacterial infection to examine the impact of mannose functionalization of chitosan nanocarriers (CS-NCs) on the human macrophage response. Both ungrafted and grafted CS-NCs were similarly internalized by macrophages, via an actin cytoskeleton-dependent process. Although tri-mannose ligands did not modify the capacity of CS-NCs to escape lysosomal degradation, they profoundly remodeled the response of M. tuberculosis-infected macrophages. mRNA sequencing showed nearly 900 genes to be differentially expressed due to tri-mannose grafting. Unexpectedly, the set of modulated genes was enriched for pathways involved in cell metabolism, particularly oxidative phosphorylation and sugar metabolism. CONCLUSIONS: The ability to modulate cell metabolism by grafting ligands at the surface of nanoparticles may thus be a promising strategy to reprogram immune cells and improve the efficacy of encapsulated drugs.

Altered spinogenesis in iPSC-derived cortical neurons from patients with autism carrying de novo SHANK3 mutations.

The synaptic protein SHANK3 encodes a multidomain scaffold protein expressed at the postsynaptic density of neuronal excitatory synapses. We previously identified de novo SHANK3 mutations in patients with autism spectrum disorders (ASD) and showed that SHANK3 represents one of the major genes for ASD. Here, we analyzed the pyramidal cortical neurons derived from induced pluripotent stem cells from four patients with ASD carrying SHANK3 de novo truncating mutations. At 40-45 days after the differentiation of neural stem cells, dendritic spines from pyramidal neurons presented variable morphologies: filopodia, thin, stubby and muschroom, as measured in 3D using GFP labeling and immunofluorescence. As compared to three controls, we observed a significant decrease in SHANK3 mRNA levels (less than 50% of controls) in correlation with a significant reduction in dendritic spine densities and whole spine and spine head volumes. These results, obtained through the analysis of de novo SHANK3 mutations in the patients' genomic background, provide further support for the presence of synaptic abnormalities in a subset of patients with ASD.

MuscleJ: a high-content analysis method to study skeletal muscle with a new Fiji tool.

BACKGROUND: Skeletal muscle has the capacity to adapt to environmental changes and regenerate upon injury. To study these processes, most experimental methods use quantification of parameters obtained from images of immunostained skeletal muscle. Muscle cross-sectional area, fiber typing, localization of nuclei within the muscle fiber, the number of vessels, and fiber-associated stem cells are used to assess muscle physiology. Manual quantification of these parameters is time consuming and only poorly reproducible. While current state-of-the-art software tools are unable to analyze all these parameters simultaneously, we have developed MuscleJ, a new bioinformatics tool to do so. METHODS: Running on the popular open source Fiji software platform, MuscleJ simultaneously analyzes parameters from immunofluorescent staining, imaged by different acquisition systems in a completely automated manner. RESULTS: After segmentation of muscle fibers, up to three other channels can be analyzed simultaneously. Dialog boxes make MuscleJ easy-to-use for biologists. In addition, we have implemented color in situ cartographies of results, allowing the user to directly visualize results on reconstituted muscle sections. CONCLUSION: We report here that MuscleJ results were comparable to manual observations made by five experts. MuscleJ markedly enhances statistical analysis by allowing reliable comparison of skeletal muscle physiology-pathology results obtained from different laboratories using different acquisition systems. Providing fast robust multi-parameter analyses of skeletal muscle physiology-pathology, MuscleJ is available as a free tool for the skeletal muscle community.

Fluorescence imaging host pathogen interactions: fifteen years benefit of hindsight .

We consider in review current state-of-the-art fluorescence microscopy for investigating the host-pathogen interface. Our perspective is honed from years with literally thousands of microbiologists using the variety of imaging technologies available within our dedicated BSL2/BSL3 optical imaging research service facilities at the Institut Pasteur Paris founded from scratch in 2001. During fifteen years learning from the success and failures of introducing different fluorescence imaging technologies, methods, and technical development strategies we provide here a synopsis review of our experience to date and a synthesis of how we see the future in perspective for fluorescence imaging at the host-pathogen interface.

Rev-erb-α regulates atrophy-related genes to control skeletal muscle mass.

The nuclear receptor Rev-erb-α modulates hepatic lipid and glucose metabolism, adipogenesis and thermogenesis. We have previously demonstrated that Rev-erb-α is also an important regulator of skeletal muscle mitochondrial biogenesis and function, and autophagy. As such, Rev-erb-α over-expression in skeletal muscle or its pharmacological activation improved mitochondrial respiration and enhanced exercise capacity. Here, in gain- and loss-of function studies, we show that Rev-erb-α also controls muscle mass. Rev-erb-α-deficiency in skeletal muscle leads to increased expression of the atrophy-related genes (atrogenes), associated with reduced muscle mass and decreased fiber size. By contrast, in vivo and in vitro Rev-erb-α over-expression results in reduced atrogenes expression and increased fiber size. Finally, Rev-erb-α pharmacological activation blocks dexamethasone-induced upregulation of atrogenes and muscle atrophy. This study identifies Rev-erb-α as a promising pharmacological target to preserve muscle mass.

Assessing Vacuolar Escape of Listeria Monocytogenes.

Listeria monocytogenes is a bacterial pathogen which invades and multiplies within non-professional phagocytes. Signaling cascades involved in cellular entry have been extensively analyzed, but the events leading to vacuolar escape remain less clear. In this chapter, we detail a microscopy FRET-based assay which allows quantitatively measuring L. monocytogenes infection and escape from its internalization vacuole, as well as a correlative light/electron microscopy method to investigate the morphological features of the vacuolar compartments containing L. monocytogenes.

Phenotypic clustering: a novel method for microglial morphology analysis.

BACKGROUND: Microglial cells are tissue-resident macrophages of the central nervous system. They are extremely dynamic, sensitive to their microenvironment and present a characteristic complex and heterogeneous morphology and distribution within the brain tissue. Many experimental clues highlight a strong link between their morphology and their function in response to aggression. However, due to their complex "dendritic-like" aspect that constitutes the major pool of murine microglial cells and their dense network, precise and powerful morphological studies are not easy to realize and complicate correlation with molecular or clinical parameters. METHODS: Using the knock-in mouse model CX3CR1(GFP/+), we developed a 3D automated confocal tissue imaging system coupled with morphological modelling of many thousands of microglial cells revealing precise and quantitative assessment of major cell features: cell density, cell body area, cytoplasm area and number of primary, secondary and tertiary processes. We determined two morphological criteria that are the complexity index (CI) and the covered environment area (CEA) allowing an innovative approach lying in (i) an accurate and objective study of morphological changes in healthy or pathological condition, (ii) an in situ mapping of the microglial distribution in different neuroanatomical regions and (iii) a study of the clustering of numerous cells, allowing us to discriminate different sub-populations. RESULTS: Our results on more than 20,000 cells by condition confirm at baseline a regional heterogeneity of the microglial distribution and phenotype that persists after induction of neuroinflammation by systemic injection of lipopolysaccharide (LPS). Using clustering analysis, we highlight that, at resting state, microglial cells are distributed in four microglial sub-populations defined by their CI and CEA with a regional pattern and a specific behaviour after challenge. CONCLUSIONS: Our results counteract the classical view of a homogenous regional resting state of the microglial cells within the brain. Microglial cells are distributed in different defined sub-populations that present specific behaviour after pathological challenge, allowing postulating for a cellular and functional specialization. Moreover, this new experimental approach will provide a support not only to neuropathological diagnosis but also to study microglial function in various disease models while reducing the number of animals needed to approach the international ethical statements.