Dynamics of macrophage polarization support Salmonella persistence in a whole living organism.

Numerous intracellular bacterial pathogens interfere with macrophage function, including macrophage polarization, to establish a niche and persist. However, the spatiotemporal dynamics of macrophage polarization during infection within host remain to be investigated. Here, we implement a model of persistent Salmonella Typhimurium infection in zebrafish, which allows visualization of polarized macrophages and bacteria in real time at high resolution. While macrophages polarize toward M1-like phenotype to control early infection, during later stages, Salmonella persists inside non-inflammatory clustered macrophages. Transcriptomic profiling of macrophages showed a highly dynamic signature during infection characterized by a switch from pro-inflammatory to anti-inflammatory/pro-regenerative status and revealed a shift in adhesion program. In agreement with this specific adhesion signature, macrophage trajectory tracking identifies motionless macrophages as a permissive niche for persistent Salmonella. Our results demonstrate that zebrafish model provides a unique platform to explore, in a whole organism, the versatile nature of macrophage functional programs during bacterial acute and persistent infections.

Macrophages undergo a behavioural switch during wound healing in zebrafish.

In response to wound signals, macrophages are immediately recruited to the injury where they acquire distinct phenotypes and functions, playing crucial roles both in host defense and healing process. Although macrophage phenotypes have been intensively studied during wound healing, mostly using markers and expression profiles, the impact of the wound environment on macrophage shape and behaviour, and the underlying mechanisms deserve more in-depth investigation. Here, we sought to characterize the dynamics of macrophage recruitment and behaviour during aseptic wounding of the caudal fin fold of the zebrafish larva. Using a photo-conversion approach, we demonstrated that macrophages are recruited to the wounded fin fold as a single wave where they switch their phenotype. Intravital imaging of macrophage shape and trajectories revealed that wound-macrophages display a highly stereotypical set of behaviours and change their shape from amoeboid to elongated shape as wound healing proceeds. Using a pharmacological inhibitor of 15-lipoxygenase and protectin D1, a specialized pro-resolving lipid, we investigated the role of polyunsaturated fatty acid metabolism in macrophage behaviour. While inhibition of 15-lipoxygenase using PD146176 or Nordihydroguaiaretic acid (NDGA) decreases the switch from amoeboid to elongated shape, protectin D1 accelerates macrophage reverse migration and favours elongated morphologies. Altogether, our findings suggest that individual macrophages at the wound switch their phenotype leading to important changes in behaviour and shape to adapt to changing environment, and highlight the crucial role of lipid metabolism in the control of macrophage behaviour plasticity during inflammation in vivo.

NRG1/ErbB signalling controls the dialogue between macrophages and neural crest-derived cells during zebrafish fin regeneration.

Fish species, such as zebrafish (Danio rerio), can regenerate their appendages after amputation through the formation of a heterogeneous cellular structure named blastema. Here, by combining live imaging of triple transgenic zebrafish embryos and single-cell RNA sequencing we established a detailed cell atlas of the regenerating caudal fin in zebrafish larvae. We confirmed the presence of macrophage subsets that govern zebrafish fin regeneration, and identified a foxd3-positive cell population within the regenerating fin. Genetic depletion of these foxd3-positive neural crest-derived cells (NCdC) showed that they are involved in blastema formation and caudal fin regeneration. Finally, chemical inhibition and transcriptomic analysis demonstrated that these foxd3-positive cells regulate macrophage recruitment and polarization through the NRG1/ErbB pathway. Here, we show the diversity of the cells required for blastema formation, identify a discrete foxd3-positive NCdC population, and reveal the critical function of the NRG1/ErbB pathway in controlling the dialogue between macrophages and NCdC.

Occurrence of foamy macrophages during the innate response of zebrafish to trypanosome infections.

A tightly regulated innate immune response to trypanosome infections is critical to strike a balance between parasite control and inflammation-associated pathology. In this study, we make use of the recently established Trypanosoma carassii infection model in larval zebrafish to study the early response of macrophages and neutrophils to trypanosome infections in vivo. We consistently identified high- and low-infected individuals and were able to simultaneously characterise their differential innate response. Not only did macrophage and neutrophil number and distribution differ between the two groups, but also macrophage morphology and activation state. Exclusive to high-infected zebrafish, was the occurrence of foamy macrophages characterised by a strong pro-inflammatory profile and potentially associated with an exacerbated immune response as well as susceptibility to the infection. To our knowledge, this is the first report of the occurrence of foamy macrophages during an extracellular trypanosome infection.

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.

Damage-Induced Calcium Signaling and Reactive Oxygen Species Mediate Macrophage Activation in Zebrafish.

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 Ca²âº and ROS signaling in macrophage activation. By inhibiting intracellular Ca²âº 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.

Pseudomonas aeruginosa OprF plays a role in resistance to macrophage clearance during acute infection.

While considered an extracellular pathogen, Pseudomonas aeruginosa has been reported to be engulfed by macrophages in cellular and animal models. However, the role of macrophages in P. aeruginosa clearance in vivo remains poorly studied. The major outer membrane porin OprF has been recently shown to be involved in P. aeruginosa fate within cultured macrophages and analysis of an oprF mutant may thus provide insights to better understand the relevance of this intramacrophage stage during infection. In the present study, we investigated for the first time the virulence of a P. aeruginosa oprF mutant in a vertebrate model that harbors functional macrophages, the zebrafish (Danio rerio) embryo, which offers powerful tools to address macrophage-pathogen interactions. We established that P. aeruginosa oprF mutant is attenuated in zebrafish embryos in a macrophage-dependent manner. Visualization and quantification of P. aeruginosa bacteria phagocytosed by macrophages after injection into closed cavities suggested that the attenuated phenotype of oprF mutant is not linked to higher macrophage recruitment nor better phagocytosis than wild-type strain. Using cultured macrophages, we showed an intramacrophage survival defect of P. aeruginosa oprF mutant, which is correlated with elevated association of bacteria with acidic compartments. Notably, treatment of embryos with bafilomycin, an inhibitor of acidification, increased the sensibility of embryos towards both wild-type and oprF mutant, and partially suppressed the attenuation of oprF mutant. Taken together, this work supports zebrafish embryo as state-of-the-art model to address in vivo the relevance of P. aeruginosa intramacrophage stage. Our results highlight the contribution of macrophages in the clearance of P. aeruginosa during acute infection and suggest that OprF protects P. aeruginosa against macrophage clearance by avoiding bacterial elimination in acidified phagosomes.

Pro-resolving mediator protectin D1 promotes epimorphic regeneration by controlling immune cell function in vertebrates.

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.

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.

Mycobacterium abscessus-Induced Granuloma Formation Is Strictly Dependent on TNF Signaling and Neutrophil Trafficking.

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.

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.

Transient infection of the zebrafish notochord with E. coli induces chronic inflammation.

Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils) during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation.

Temporally regulated traffic of HuR and its associated ARE-containing mRNAs from the chromatoid body to polysomes during mouse spermatogenesis.

BACKGROUND: In mammals, a temporal disconnection between mRNA transcription and protein synthesis occurs during late steps of germ cell differentiation, in contrast to most somatic tissues where transcription and translation are closely linked. Indeed, during late stages of spermatogenesis, protein synthesis relies on the appropriate storage of translationally inactive mRNAs in transcriptionally silent spermatids. The factors and cellular compartments regulating mRNA storage and the timing of their translation are still poorly understood. The chromatoid body (CB), that shares components with the P. bodies found in somatic cells, has recently been proposed to be a site of mRNA processing. Here, we describe a new component of the CB, the RNA binding protein HuR, known in somatic cells to control the stability/translation of AU-rich containing mRNAs (ARE-mRNAs). METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of cell imagery and sucrose gradient fractionation, we show that HuR localization is highly dynamic during spermatid differentiation. First, in early round spermatids, HuR colocalizes with the Mouse Vasa Homolog, MVH, a marker of the CB. As spermatids differentiate, HuR exits the CB and concomitantly associates with polysomes. Using computational analyses, we identified two testis ARE-containing mRNAs, Brd2 and GCNF that are bound by HuR and MVH. We show that these target ARE-mRNAs follow HuR trafficking, accumulating successively in the CB, where they are translationally silent, and in polysomes during spermatid differentiation. CONCLUSIONS/SIGNIFICANCE: Our results reveal a temporal regulation of HuR trafficking together with its target mRNAs from the CB to polysomes as spermatids differentiate. They strongly suggest that through the transport of ARE-mRNAs from the CB to polysomes, HuR controls the appropriate timing of ARE-mRNA translation. HuR might represent a major post-transcriptional regulator, by promoting mRNA storage and then translation, during male germ cell differentiation.

[Aberrant regulation of mRNA 3' untranslated region in cancers and inflammation]. / Contribution de la régulation post-transcriptionnelle à l'émergence de maladies: l'enfance de l'ARE.

Almost 10% of mammalian coding mRNAs contain in their 3' untranslated region a sequence rich in adenine and uridine residues known as AU-rich element (ARE). Many of them encode oncogenes (for instance c-Myc and c-Fos), cell cycle regulators (cyclin D1, A1, B1), cytokines (TNFalpha, IL2) and growth factors (GM-CSF) which are overexpressed in cancer or inflammatory diseases due to increased mRNA stability and/or translation. AREs are recognized by a group of proteins, collectively called AUBPs which display various functions. For instance, HuR/ELAV is mainly known to protect ARE-containing mRNAs from degradation, while AUF1, TTP and KSRP act to destabilize their bound target mRNAs and TIA/TIAR to inhibit their translation. Alterations in ARE sequences or AUBP abundance, cellular localization or activity due to post-translational modifications such as phosphorylation can promote or enhance malignancy or perturb immune homeostasis. Here, c-myc and TNFalpha are chosen as examples to illustrate how altered 3' UTR gene regulation impacts on pathologies.