Hiding in the yolk: A unique feature of Legionella pneumophila infection of zebrafish.

The zebrafish has become a powerful model organism to study host-pathogen interactions. Here, we developed a zebrafish model to dissect the innate immune response to Legionella pneumophila during infection. We show that L. pneumophila cause zebrafish larvae death in a dose dependent manner. Additionally, we show that macrophages are the first line of defence and cooperate with neutrophils to clear the infection. Immunocompromised humans have an increased propensity to develop pneumonia, similarly, when either macrophages or neutrophils are depleted, these "immunocompromised" larvae become lethally sensitive to L. pneumophila. Also, as observed in human infections, the adaptor signalling molecule Myd88 is not required to control disease in the larvae. Furthermore, proinflammatory cytokine genes il1ß and tnf-α were upregulated during infection, recapitulating key immune responses seen in human infection. Strikingly, we uncovered a previously undescribed infection phenotype in zebrafish larvae, whereby bloodborne, wild type L. pneumophila invade and grow in the larval yolk region, a phenotype not observed with a type IV secretion system deficient mutant that cannot translocate effectors into its host cell. Thus, zebrafish larva represents an innovative L. pneumophila infection model that mimics important aspects of the human immune response to L. pneumophila infection and will allow the elucidation of mechanisms by which type IV secretion effectors allow L. pneumophila to cross host cell membranes and obtain nutrients from nutrient rich environments.

CSF-contacting neurons respond to Streptococcus pneumoniae and promote host survival during central nervous system infection.

The pathogenic bacterium Streptococcus pneumoniae (S. pneumoniae) can invade the cerebrospinal fluid (CSF) and cause meningitis with devastating consequences. Whether and how sensory cells in the central nervous system (CNS) become activated during bacterial infection, as recently reported for the peripheral nervous system, is not known. We find that CSF infection by S. pneumoniae in larval zebrafish leads to changes in posture and behavior that are reminiscent of pneumococcal meningitis, including dorsal arching and epileptic-like seizures. We show that during infection, invasion of the CSF by S. pneumoniae massively activates in vivo sensory neurons contacting the CSF, referred to as "CSF-cNs" and previously shown to detect spinal curvature and to control posture, locomotion, and spine morphogenesis. We find that CSF-cNs express orphan bitter taste receptors and respond in vitro to bacterial supernatant and metabolites via massive calcium transients, similar to the ones observed in vivo during infection. Upon infection, CSF-cNs also upregulate the expression of numerous cytokines and complement components involved in innate immunity. Accordingly, we demonstrate, using cell-specific ablation and blockade of neurotransmission, that CSF-cN neurosecretion enhances survival of the host during S. pneumoniae infection. Finally, we show that CSF-cNs respond to various pathogenic bacteria causing meningitis in humans, as well as to the supernatant of cells infected by a neurotropic virus. Altogether, our work uncovers that central sensory neurons in the spinal cord, previously involved in postural control and morphogenesis, contribute as well to host survival by responding to the invasion of the CSF by pathogenic bacteria during meningitis.

Latest trends in bioimaging and building a proactive network of early-career young scientists around bioimaging in Europe.

Biological research is in constant need of new methodological developments to assess organization and functions at various scales ranging from whole organisms to interactions between proteins. One of the main ways to evidence and quantify biological phenomena is imaging. Fluorescence microscopy and label-free microscopy are in particular highly active fields of research due to their compatibility with living samples as well as their versatility. The Imabio Young Scientists Network (YSN) is a group of young scientists (PhD students, postdocs and engineers) who are excited about bioimaging and aim to create a proactive network of researchers with the same interest. YSN is endorsed by the bioimaging network GDR Imabio in France, where the initiative was started in 2019. Since then, we aim to organize the Imabio YSN conference every year to expand the network to other European countries, establish new collaborations and ignite new scientific ideas. From 6-8 July 2022, the YSN including researchers from the domains of life sciences, chemistry, physics and computational sciences met at the Third Imabio YSN Conference 2022 in Lyon to discuss the latest bioimaging technologies and biological discoveries. In this Meeting Review, we describe the essence of the scientific debates, highlight remarkable talks, and focus on the Career Development session, which is unique to the YSN conference, providing a career perspective to young scientists and help to answer all their questions at this career stage. This conference was a truly interdisciplinary reunion of scientists who are eager to push the frontiers of bioimaging in order to understand the complexity of biological systems.

Exploring Zebrafish Larvae as a COVID-19 Model: Probable Abortive SARS-CoV-2 Replication in the Swim Bladder.

Animal models are essential to understanding COVID-19 pathophysiology and for preclinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap, and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, or bloodstream, resulted in a rapid decrease of SARS-CoV-2 RNA in wild-type larvae. However, when the virus was inoculated in the swim bladder, viral RNA stabilized after 24 h. By immunohistochemistry, epithelial cells containing SARS-CoV-2 nucleoprotein were observed in the swim bladder wall. Our data suggest an abortive infection of the swim bladder. In some animals, several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. A mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro. In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung.

A User-Friendly Approach for Routine Histopathological and Morphometric Analysis of Skeletal Muscle Using CellProfiler Software.

Adult skeletal muscle is capable of active and efficient differentiation in the event of injury in both physiological and pathological conditions, such as in Duchenne muscular dystrophy (DMD). DMD is characterized by different features, such as continuous cycles of degeneration/regeneration, fiber heterogeneity, chronic inflammation and fibrosis. A well-defined and standardized approach for histological and morphometric analysis of muscle samples is necessary in order to measure and quantify specific regenerative parameters in myopathies. Indeed, non-automatic methods are time-consuming and prone to error. Here, we describe a simple automatized computational approach to quantify muscle parameters with specific pipelines to be run by CellProfiler software in an open-source and well-defined fashion. Our pipelines consist of running image-processing modules in CellProfiler with the aim of quantifying different histopathological muscle hallmarks in mdx mice compared to their wild-type littermates. Specifically, we quantified the minimum Feret diameter, centrally nucleated fibers and the number of macrophages, starting from multiple images. Finally, for extracellular matrix quantification, we used Sirius red staining. Collectively, we developed reliable and easy-to-use pipelines that automatically measure parameters of muscle histology, useful for research in myobiology. These findings should simplify and shorten the time needed for the quantification of muscle histological properties, avoiding challenging manual procedures.

IFN-Stimulated Genes in Zebrafish and Humans Define an Ancient Arsenal of Antiviral Immunity.

The evolution of the IFN system, the major innate antiviral mechanism of vertebrates, remains poorly understood. According to the detection of type I IFN genes in cartilaginous fish genomes, the system appeared 500 My ago. However, the IFN system integrates many other components, most of which are encoded by IFN-stimulated genes (ISGs). To shed light on its evolution, we have used deep RNA sequencing to generate a comprehensive list of ISGs of zebrafish, taking advantage of the high-quality genome annotation in this species. We analyzed larvae after inoculation of recombinant zebrafish type I IFN, or infection with chikungunya virus, a potent IFN inducer. We identified more than 400 zebrafish ISGs, defined as being either directly induced by IFN or induced by the virus in an IFNR-dependent manner. Their human orthologs were highly enriched in ISGs, particularly for highly inducible genes. We identified 72 orthology groups containing ISGs in both zebrafish and humans, revealing a core ancestral ISG repertoire that includes most of the known signaling components of the IFN system. Many downstream effectors were also already present 450 My ago in the common ancestor of tetrapods and bony fish and diversified as multigene families independently in the two lineages. A large proportion of the ISG repertoire is lineage specific; around 40% of protein-coding zebrafish ISGs had no human ortholog. We identified 14 fish-specific gene families containing multiple ISGs, including finTRIMs. This work illuminates the evolution of the IFN system and provides a rich resource to explore new antiviral mechanisms.