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Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti‐AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane‐bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one‐hundred nanomolar range and optimal binding under reducing conditions. 3D‐modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD‐dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes. [ABSTRACT FROM AUTHOR]

Simple Summary: Inflammation is a central part of the body's response to harm that can be elicited by microbes, environmental factors, or internal injuries. This complex physiological process has evolved to protect the body and eliminate the threat. To do so, it relies on immune cells and molecular mediators that act in concert to provide protection and tissue repair. If return to homeostasis fails, prolonged inflammation can cause tissue damage and chronic diseases. It is therefore essential to understand in detail the mechanisms at play during the inflammatory response, in order to modulate them in a pathological context. Such studies strongly benefit from in vivo models, as these can capture the complexity of inflammation as a whole. Zebrafish has proven to be a valuable animal model to study innate immune responses as its immune system shows high similarity with the human one and it offers numerous advantages such as its genetics and transparency. We here review the current knowledge on the molecular actors of inflammation in zebrafish, highlighting how the tools developed to study them have helped gain insights into the mechanisms of inflammation. This will allow to design more refined models of inflammation, mimicking human diseases, for drug screening in zebrafish. Inflammation is a hallmark of the physiological response to aggressions. It is orchestrated by a plethora of molecules that detect the danger, signal intracellularly, and activate immune mechanisms to fight the threat. Understanding these processes at a level that allows to modulate their fate in a pathological context strongly relies on in vivo studies, as these can capture the complexity of the whole process and integrate the intricate interplay between the cellular and molecular actors of inflammation. Over the years, zebrafish has proven to be a well-recognized model to study immune responses linked to human physiopathology. We here provide a systematic review of the molecular effectors of inflammation known in this vertebrate and recapitulate their modes of action, as inferred from sterile or infection-based inflammatory models. We present a comprehensive analysis of their sequence, expression, and tissue distribution and summarize the tools that have been developed to study their function. We further highlight how these tools helped gain insights into the mechanisms of immune cell activation, induction, or resolution of inflammation, by uncovering downstream receptors and signaling pathways. These progresses pave the way for more refined models of inflammation, mimicking human diseases and enabling drug development using zebrafish models. [ABSTRACT FROM AUTHOR]

Author(s): Olivier Jaillon [1]; Jean-Marc Aury [1]; Frédéric Brunet [2]; Jean-Louis Petit [1]; Nicole Stange-Thomann [3]; Evan Mauceli [3]; Laurence Bouneau [1]; Cécile Fischer [1]; Catherine Ozouf-Costaz [4]; Alain Bernot [...]

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. [ABSTRACT FROM AUTHOR]

In humans, several members of the CEACAM receptor family have been shown to interact with intestinal pathogens in an inflammatory context. While CEACAMs have long been thought to be only present in mammals, recent studies have identified ceacam genes in other vertebrates, including teleosts. The function of these related genes remains however largely unknown. To gain insight into the function of CEACAM proteins in fish, we undertook the study of a putative member of the family, CEACAMz1, identified in Danio rerio. Sequence analysis of the ceacamz1 gene product predicted a GPI-anchored extracellular protein containing eleven immunoglobulin domains but revealed no evident orthology with human CEACAMs. Using a combination of RT-PCR analyses and in situ hybridization experiments, as well as a fluorescent reporter line, we showed that CEACAMz1 is first expressed in discrete cells on the ventral skin of zebrafish larvae and later on in the developing gills. This distribution remains constant until juvenile stage is reached, at which point CEACAMz1 is almost exclusively expressed in gills. We further observed that at late larval stages, CEACAMz1-expressing cells mostly localize on the afferent side of the branchial filaments and possibly in the inter-lamellar space. Using immunolabelling and 3D-reconstructions, we showed that CEACAMz1 is expressed in cells from the uppermost layer of skin epidermis. These cells are embedded within the keratinocytes pavement and we unambiguously identified them as proton-pump rich ionocytes (HR cells). As the expression of ceacamz1 is turned on concomitantly to that of other known markers of HR cells, we propose that ceacamz1 may serve as a novel marker of mature HR cells from the zebrafish epidermis. [ABSTRACT FROM AUTHOR]

Immediately after a wound, macrophages are activated and change their phenotypes in reaction to danger signals released from the damaged tissues. The cues that contribute to macrophage activation after wounding in vivo are still poorly understood. Calcium signaling and Reactive Oxygen Species (ROS), mainly hydrogen peroxide, are conserved early wound signals that emanate from the wound and guide neutrophils within tissues up to the wound. However, the role of these signals in the recruitment and the activation of macrophages is elusive. Here we used the transparent zebrafish larva as a tractable vertebrate system to decipher the signaling cascade necessary for macrophage recruitment and activation after the injury of the caudal fin fold. By using transgenic reporter lines to track pro-inflammatory activated macrophages combined with high-resolutive microscopy, we tested the role of Ca²⁺ and ROS signaling in macrophage activation. By inhibiting intracellular Ca²⁺ released from the ER stores, we showed that macrophage recruitment and activation towards pro-inflammatory phenotypes are impaired. By contrast, ROS are only necessary for macrophage activation independently on calcium. Using genetic depletion of neutrophils, we showed that neutrophils are not essential for macrophage recruitment and activation. Finally, we identified Src family kinases, Lyn and Yrk and NF-κB as key regulators of macrophage activation in vivo , with Lyn and ROS presumably acting in the same signaling pathway. This study describes a molecular mechanism by which early wound signals drive macrophage polarization and suggests unique therapeutic targets to control macrophage activity during diseases. [ABSTRACT FROM AUTHOR]

Abstract Background The high degree of sequence conservation between coding regions in fish and mammals can be exploited to identify genes in mammalian genomes by comparison with the sequence of similar genes in fish. Conversely, experimentally characterized mammalian genes may be used to annotate fish genomes. However, gene families that escape this principle include the rapidly diverging cytokines that regulate the immune system, and their receptors. A classic example is the class II helical cytokines (HCII) including type I, type II and lambda interferons, IL10 related cytokines (IL10, IL19, IL20, IL22, IL24 and IL26) and their receptors (HCRII). Despite the report of a near complete pufferfish (Takifugu rubripes) genome sequence, these genes remain undescribed in fish. Results We have used an original strategy based both on conserved amino acid sequence and gene structure to identify HCII and HCRII in the genome of another pufferfish, Tetraodon nigroviridis that is amenable to laboratory experiments. The 15 genes that were identified are highly divergent and include a single interferon molecule, three IL10 related cytokines and their potential receptors together with two Tissue Factor (TF). Some of these genes form tandem clusters on the Tetraodon genome. Their expression pattern was determined in different tissues. Most importantly, Tetraodon interferon was identified and we show that the recombinant protein can induce antiviral MX gene expression in Tetraodon primary kidney cells. Similar results were obtained in Zebrafish which has 7 MX genes. Conclusion We propose a scheme for the evolution of HCII and their receptors during the radiation of bony vertebrates and suggest that the diversification that played an important role in the fine-tuning of the ancestral mechanism for host defense against infections probably followed different pathways in amniotes and fish.

Background and Purpose: Specialized pro-resolving mediators (SPMs) are a family of lipids controlling the resolution of inflammation and playing a role in many processes including organ protection and tissue repair. While SPMs are potent bioactive molecules in vivo, their role in epimorphic regeneration of organs in vertebrates has not been tested. Using the zebrafish larva as a robust regenerative vertebrate system, we studied the role of the SPM neuroprotectin/protectin D1 (PD1) during the caudal fin fold regeneration.Experimental Approach: Regeneration of the fin fold was analysed when exposed to a synthetic PD1. The effect of PD1 on immune cell recruitment and activation was further investigated using live imaging combined with fluorescent reporter lines. Using genetic and pharmacological approaches, we dissected the role of neutrophils and macrophages on driving the pro-regenerative effect of PD1.Key Results: We showed that PD1 improves fin fold regeneration. Acting in a narrow time window during regeneration, PD1 accelerates the resolution of inflammation without affecting the initial kinetic of neutrophil recruitment but instead, promotes their reverse migration potential. In addition, PD1 induces macrophage polarization switch towards non-inflammatory states in both zebrafish and mammalian system. Finally, macrophages but not neutrophils are essential for PD1-mediated regeneration.Conclusion and Implications: These results reveal the pro-regenerative action of PD1 and its role in regulating neutrophil and macrophage response in vertebrates. These findings strongly support the development of pro-resolving mediators as natural therapeutic candidates for degenerative disorders and the use of the zebrafish as a tool to investigate pro-regenerative drugs. [ABSTRACT FROM AUTHOR]

Understanding the roles of neutrophils and macrophages in fighting bacterial infections is a critical issue in human pathologies. Although phagocytic killing has been extensively studied, little is known about how bacteria are eliminated extracellularly in live vertebrates. We have recently developed an infection model in the zebrafish embryo in which leukocytes cannot reach the injected bacteria. When Escherichia coli bacteria are injected within the notochord, both neutrophils and macrophages are massively recruited during several days, but do not infiltrate the infected tissue presumably because of its tough collagen sheath. Nevertheless, the bacteria are killed during the first 24 hours, and we report here that neutrophils, but not macrophages are involved in the control of the infection. Using genetic and chemical approaches, we show that even in absence of phagocytosis, the bactericidal action relies on NADPH oxidase-dependent production of superoxide in neutrophils. We thus reveal a host effector mechanism mediated by neutrophils that eliminates bacteria that cannot be reached by phagocytes and that is independent of macrophages, NO synthase or myeloperoxidase. [ABSTRACT FROM AUTHOR]

Zebrafish gastrulation and particularly epiboly that involves coordinated movements of several cell layers is a dynamic process for which regulators remain to be identified. We show here that Flotillin 1 and 2, ubiquitous and highly conserved proteins, are required for epiboly. Flotillins knockdown compromised embryo survival, strongly delayed epiboly and impaired deep cell radial intercalation and directed collective migration without affecting enveloping layer cell movement. At the molecular level, we identified that Flotillins are required for the formation of E-cadherin-mediated cell-cell junctions. These results provide the first in vivo evidence that Flotillins regulate E-cadherin-mediated cell-cell junctions to allow epiboly progression. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus is considered the most common respiratory pathogen among the rapidly growing non-tuberculous mycobacteria. Infections with M. abscessus are increasingly found in patients with chronic lung diseases, especially cystic fibrosis, and are often refractory to antibiotic therapy. M. abscessus has two morphotypes with distinct effects on host cells and biological responses. The smooth (S) variant is recognized as the initial airway colonizer while the rough (R) is known to be a potent inflammatory inducer associated with invasive disease, but the underlying immunopathological mechanisms of the infection remain unsolved. We conducted a comparative stepwise dissection of the inflammatory response in S and R pathogenesis by monitoring infected transparent zebrafish embryos. Loss of TNFR1 function resulted in increased mortality with both variants, and was associated with unrestricted intramacrophage bacterial growth and decreased bactericidal activity. The use of transgenic zebrafish lines harboring fluorescent macrophages and neutrophils revealed that neutrophils, like macrophages, interact with M. abscessus at the initial infection sites. Impaired TNF signaling disrupted the IL8-dependent neutrophil mobilization, and the defect in neutrophil trafficking led to the formation of aberrant granulomas, extensive mycobacterial cording, unrestricted bacterial growth and subsequent larval death. Our findings emphasize the central role of neutrophils for the establishment and maintenance of the protective M. abscessus granulomas. These results also suggest that the TNF/IL8 inflammatory axis is necessary for protective immunity against M. abscessus and may be of clinical relevance to explain why immunosuppressive TNF therapy leads to the exacerbation of M. abscessus infections. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus (Mabs) is a rapidly growing Mycobacterium and an emerging pathogen in humans. Transitioning from a smooth (S) high-glycopeptidolipid (GPL) producer to a rough (R) low-GPL producer is associated with increased virulence in zebrafish, which involves the formation of massive serpentine cords, abscesses, and rapid larval death. Generating a cord-deficient Mabs mutant would allow us to address the contribution of cording in the physiopathological signs of the R variant. Herein, a deletion mutant of MAB_4780, encoding a dehydratase, distinct from the β-hydroxyacyl-ACP dehydratase HadABC complex, was constructed in the R morphotype. This mutant exhibited an alteration of the mycolic acid composition and a pronounced defect in cording. This correlated with an extremely attenuated phenotype not only in wild-type but also in immunocompromised zebrafish embryos lacking either macrophages or neutrophils. The abolition of granuloma formation in embryos infected with the dehydratase mutant was associated with a failure to replicate in macrophages, presumably due to limited inhibition of the phagolysosomal fusion. Overall, these results indicate that MAB_4780 is required for Mabs to successfully establish acute and lethal infections. Therefore, targeting MAB_4780 may represent an attractive antivirulence strategy to control Mabs infections, refractory to most standard chemotherapeutic interventions. The combination of a dehydratase assay with a highresolution crystal structure of MAB_4780 opens the way to identify such specific inhibitors. [ABSTRACT FROM AUTHOR]

The article presents the study that identifies the macrophages vital in the diverse abilities of zebrafish using the polarization process in France.

Chikungunya Virus (CHIKV), a re-emerging arbovirus that may cause severe disease, constitutes an important public health problem. Herein we describe a novel CHIKV infection model in zebrafish, where viral spread was live-imaged in the whole body up to cellular resolution. Infected cells emerged in various organs in one principal wave with a median appearance time of ∼14 hours post infection. Timing of infected cell death was organ dependent, leading to a shift of CHIKV localization towards the brain. As in mammals, CHIKV infection triggered a strong type-I interferon (IFN) response, critical for survival. IFN was mainly expressed by neutrophils and hepatocytes. Cell type specific ablation experiments further demonstrated that neutrophils play a crucial, unexpected role in CHIKV containment. Altogether, our results show that the zebrafish represents a novel valuable model to dynamically visualize replication, pathogenesis and host responses to a human virus. [ABSTRACT FROM AUTHOR]

The embryonic development of the posterior lateral line of zebrafish involves the migration from head to tail of a primordium comprising approximately 100 cells, and the deposition at regular intervals of presumptive mechanosensory organs (neuromasts). Migration depends on the presence of chemokine SDF1 along the pathway, and on the asymmetrical distribution of chemokine receptors CXCR4 and CXCR7 in the primordium. Primordium polarization depends on Wnt signaling in the leading region. Here, we examine the role of a major effector of Wnt signaling, lef1, in this system. We show that, although its inactivation has no overt effect on the expression of cxcr4b and cxcr7b, lef1 contributes to their control. We also show that cell proliferation, which ensures constant primordium size despite successive rounds of cell deposition, is reduced upon lef1 inactivation. Because of this defect, the primordium runs short of cells and vanishes before the line has been completed. We conclude that lef1-mediated Wnt signaling is involved in various aspects of primordium migration, although part of this implication is masked by a high level of developmental redundancy. Developmental Dynamics 239:3163-3171, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Background: The high degree of sequence conservation between coding regions in fish and mammals can be exploited to identify genes in mammalian genomes by comparison with the sequence of similar genes in fish. Conversely, experimentally characterized mammalian genes may be used to annotate fish genomes. However, gene families that escape this principle include the rapidly diverging cytokines that regulate the immune system, and their receptors. A classic example is the class II helical cytokines (HCII) including type I, type II and lambda interferons, IL10 related cytokines (IL10, IL19, IL20, IL22, IL24 and IL26) and their receptors (HCRII). Despite the report of a near complete pufferfish (Takifugu rubripes) genome sequence, these genes remain undescribed in fish. Results: We have used an original strategy based both on conserved amino acid sequence and gene structure to identify HCII and HCRII in the genome of another pufferfish, Tetraodon nigroviridis that is amenable to laboratory experiments. The 15 genes that were identified are highly divergent and include a single interferon molecule, three IL10 related cytokines and their potential receptors together with two Tissue Factor (TF). Some of these genes form tandem clusters on the Tetraodon genome. Their expression pattern was determined in different tissues. Most importantly, Tetraodon interferon was identified and we show that the recombinant protein can induce antiviral MX gene expression in Tetraodon primary kidney cells. Similar results were obtained in Zebrafish which has 7 MX genes. Conclusion: We propose a scheme for the evolution of HCII and their receptors during the radiation of bony vertebrates and suggest that the diversification that played an important role in the fine-tuning of the ancestral mechanism for host defense against infections probably followed different pathways in amniotes and fish. [ABSTRACT FROM AUTHOR]