RESUMEN
Fish rely, to a high degree, on the innate immune system to protect them against the constant exposure to potential pathogenic invasion from the surrounding water during homeostasis and injury. Zebrafish larvae have emerged as an outstanding model organism for immunity. The cellular component of zebrafish innate immunity is similar to the mammalian innate immune system and has a high degree of sophistication due to the needs of living in an aquatic environment from early embryonic stages of life. Innate immune cells (leukocytes), including neutrophils and macrophages, have major roles in protecting zebrafish against pathogens, as well as being essential for proper wound healing and regeneration. Zebrafish larvae are visually transparent, with unprecedented in vivo microscopy opportunities that, in combination with transgenic immune reporter lines, have permitted visualisation of the functions of these cells when zebrafish are exposed to bacterial, viral and parasitic infections, as well as during injury and healing. Recent findings indicate that leukocytes are even more complex than previously anticipated and are essential for inflammation, infection control, and subsequent wound healing and regeneration.
Asunto(s)
Neutrófilos , Pez Cebra , Animales , Macrófagos , Inmunidad Innata , Animales Modificados Genéticamente , Larva , MamíferosRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1007597.].
RESUMEN
Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis.
Asunto(s)
Cryptococcus neoformans/metabolismo , Dinoprostona/análogos & derivados , Eicosanoides/metabolismo , Animales , Animales Modificados Genéticamente , Técnicas de Cultivo de Célula , Criptococosis/metabolismo , Cryptococcus neoformans/crecimiento & desarrollo , Cryptococcus neoformans/patogenicidad , Dinoprostona/metabolismo , Dinoprostona/fisiología , Modelos Animales de Enfermedad , Eicosanoides/inmunología , Interacciones Huésped-Patógeno/fisiología , Humanos , Macrófagos/microbiología , PPAR gamma/metabolismo , Virulencia/fisiología , Pez Cebra/microbiologíaRESUMEN
Senescence drives the onset and severity of multiple ageing-associated diseases and frailty. As a result, there has been an increased interest in mechanistic studies and in the search for compounds targeting senescent cells, known as senolytics. Mammalian models are commonly used to test senolytics and generate functional and toxicity data at the level of organs and systems, yet this is expensive and time consuming. Zebrafish share high homology in genes associated with human ageing and disease. They can be genetically modified relatively easily. In larvae, most organs develop within 5 days of fertilisation and are transparent, which allows tracking of fluorescent cells in vivo in real time, testing drug off-target toxicity and assessment of cellular and phenotypic changes. Here, we have generated a transgenic zebrafish line that expresses green fluorescent protein (GFP) under the promoter of a key senescence marker, p21. We show an increase in p21:GFP+ cells in larvae following exposure to ionising radiation and with natural ageing. p21:GFP+ cells display other markers of senescence, including senescence-associated ß-galactosidase and IL6. The observed increase in senescent cells following irradiation is associated with a reduction in the thickness of muscle fibres and mobility, two important ageing phenotypes. We also show that quercetin and dasatinib, two senolytics currently in clinical trials, reduce the number of p21:GFP+ cells, in a rapid 5-day assay. This model provides an important tool to study senescence in a living organism, allowing the rapid selection of senolytics before moving to more expensive and time-consuming mammalian systems.
Asunto(s)
Senescencia Celular , Pez Cebra , Animales , Envejecimiento/genética , Proteínas Fluorescentes Verdes/genética , Mamíferos , Senoterapéuticos , Pez Cebra/genéticaRESUMEN
Little is known about the distal excretory component of the urinary tract in Danio rerio (zebrafish). This component is affected by many human diseases and disorders of development. Here, we have undertaken multi-level analyses to determine the structure and composition of the distal urinary tract in the zebrafish. In silico searches identified uroplakin 1a (ukp1a), uroplakin 2 (upk2) and uroplakin 3b (upk3b) genes in the zebrafish genome (orthologues to genes that encode urothelium-specific proteins in humans). In situ hybridization demonstrated ukp1a expression in the zebrafish pronephros and cloaca from 96â h post-fertilization. Haematoxylin and Eosin staining of adult zebrafish demonstrated two mesonephric ducts uniting into a urinary bladder that leads to a distinct urethral opening. Immunohistochemistry identified Uroplakin 1a, Uroplakin 2 and GATA3 expression in zebrafish urinary bladder cell layers that match human urothelial expression. Fluorescent dye injections demonstrated zebrafish urinary bladder function, including urine storage and intermittent micturition, and a urethral orifice separate from the larger anal canal and rectum. Our findings reveal homology between the urinary tracts of zebrafish and humans, and offer the former as a model system to study disease.
Asunto(s)
Glicoproteínas de Membrana , Pez Cebra , Animales , Humanos , Adulto , Pez Cebra/metabolismo , Glicoproteínas de Membrana/metabolismo , Uroplaquina Ia/metabolismo , Uroplaquina II/metabolismo , Vejiga Urinaria/metabolismoRESUMEN
Neutrophils are rapidly recruited to inflammatory sites where their coordinated migration forms clusters, a process termed neutrophil swarming. The factors that modulate early stages of neutrophil swarming are not fully understood, requiring the development of new in vivo models. Using transgenic zebrafish larvae to study endogenous neutrophil migration in a tissue damage model, we demonstrate that neutrophil swarming is a conserved process in zebrafish immunity, sharing essential features with mammalian systems. We show that neutrophil swarms initially develop around an individual pioneer neutrophil. We observed the violent release of extracellular cytoplasmic and nuclear fragments by the pioneer and early swarming neutrophils. By combining in vitro and in vivo approaches to study essential components of neutrophil extracellular traps (NETs), we provide in-depth characterisation and high-resolution imaging of the composition and morphology of these release events. Using a photoconversion approach to track neutrophils within developing swarms, we identify that the fate of swarm-initiating pioneer neutrophils involves extracellular chromatin release and that the key NET components gasdermin, neutrophil elastase, and myeloperoxidase are required for the swarming process. Together our findings demonstrate that release of cellular components by pioneer neutrophils is an initial step in neutrophil swarming at sites of tissue injury.
Asunto(s)
Cromatina/metabolismo , Trampas Extracelulares/metabolismo , Neutrófilos/inmunología , Heridas y Lesiones/patología , Animales , Agregación Celular/fisiología , Elastasa de Leucocito/metabolismo , Neutrófilos/patología , Peroxidasa/metabolismo , Heridas y Lesiones/inmunología , Pez CebraRESUMEN
Inflammation-related progressive lung destruction is the leading causes of premature death in cystic fibrosis (CF), a genetic disorder caused by a defective cystic fibrosis transmembrane conductance regulator (CFTR). However, therapeutic targeting of inflammation has been hampered by a lack of understanding of the links between a dysfunctional CFTR and the deleterious innate immune response in CF. Herein, we used a CFTR-depleted zebrafish larva, as an innovative in vivo vertebrate model, to understand how CFTR dysfunction leads to abnormal inflammatory status in CF. We show that impaired CFTR-mediated inflammation correlates with an exuberant neutrophilic response after injury: CF zebrafish exhibit enhanced and sustained accumulation of neutrophils at wounds. Excessive epithelial oxidative responses drive enhanced neutrophil recruitment towards wounds. Persistence of neutrophils at inflamed sites is associated with impaired reverse migration of neutrophils and reduction in neutrophil apoptosis. As a consequence, the increased number of neutrophils at wound sites causes tissue damage and abnormal tissue repair. Importantly, the molecule Tanshinone IIA successfully accelerates inflammation resolution and improves tissue repair in CF animal. Our findings bring important new understanding of the mechanisms underlying the inflammatory pathology in CF, which could be addressed therapeutically to prevent inflammatory lung damage in CF patients with potential improvements in disease outcomes.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Inmunidad Innata/inmunología , Inflamación/inmunología , Infiltración Neutrófila/inmunología , Cicatrización de Heridas/inmunología , Proteínas de Pez Cebra/inmunología , Animales , Animales Modificados Genéticamente , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Neutrophilic inflammation is central to disease pathogenesis, for example, in chronic obstructive pulmonary disease, yet the mechanisms that retain neutrophils within tissues remain poorly understood. With emerging evidence that axon guidance factors can regulate myeloid recruitment and that neutrophils can regulate expression of a class 3 semaphorin, SEMA3F, we investigated the role of SEMA3F in inflammatory cell retention within inflamed tissues. We observed that neutrophils upregulate SEMA3F in response to proinflammatory mediators and following neutrophil recruitment to the inflamed lung. In both zebrafish tail injury and murine acute lung injury models of neutrophilic inflammation, overexpression of SEMA3F delayed inflammation resolution with slower neutrophil migratory speeds and retention of neutrophils within the tissues. Conversely, constitutive loss of sema3f accelerated egress of neutrophils from the tail injury site in fish, whereas neutrophil-specific deletion of Sema3f in mice resulted in more rapid neutrophil transit through the airways, and significantly reduced time to resolution of the neutrophilic response. Study of filamentous-actin (F-actin) subsequently showed that SEMA3F-mediated retention is associated with F-actin disassembly. In conclusion, SEMA3F signaling actively regulates neutrophil retention within the injured tissues with consequences for neutrophil clearance and inflammation resolution.
Asunto(s)
Movimiento Celular/inmunología , Proteínas de la Membrana/inmunología , Proteínas del Tejido Nervioso/inmunología , Neutrófilos/inmunología , Transducción de Señal/inmunología , Proteínas de Pez Cebra/inmunología , Pez Cebra/inmunología , Animales , Humanos , Inflamación/inmunología , Inflamación/patología , Ratones , Neutrófilos/patología , Regulación hacia Arriba/inmunologíaRESUMEN
The inappropriate retention of neutrophils at inflammatory sites is a major driver of the excessive tissue damage characteristic of respiratory inflammatory diseases including COPD, ARDS, and cystic fibrosis. The molecular programmes which orchestrate neutrophil recruitment to inflammatory sites through chemotactic guidance have been well-studied. However, how neutrophil sensitivity to these cues is modulated during inflammation resolution is not understood. The identification of neutrophil reverse migration as a mechanism of inflammation resolution and the ability to modulate this therapeutically has identified a new target to treat inflammatory disease. Here we investigate the role of the CXCL12/CXCR4 signaling axis in modulating neutrophil retention at inflammatory sites. We used an in vivo tissue injury model to study neutrophilic inflammation using transgenic zebrafish larvae. Expression of cxcl12a and cxcr4b during the tissue damage response was assessed using in situ hybridization and analysis of RNA sequencing data. CRISPR/Cas9 was used to knockdown cxcl12a and cxcr4b in zebrafish larvae. The CXCR4 antagonist AMD3100 was used to block the Cxcl12/Cxcr4 signaling axis pharmacologically. We identified that cxcr4b and cxcl12a are expressed at the wound site in zebrafish larvae during the inflammatory response. Following tail-fin transection, removal of neutrophils from inflammatory sites is significantly increased in cxcr4b and cxcl12a CRISPR knockdown larvae. Pharmacological inhibition of the Cxcl12/Cxcr4 signaling axis accelerated resolution of the neutrophil component of inflammation, an effect caused by an increase in neutrophil reverse migration. The findings of this study suggest that CXCR4/CXCL12 signaling may play an important role in neutrophil retention at inflammatory sites, identifying a potential new target for the therapeutic removal of neutrophils from the lung in chronic inflammatory disease.
Asunto(s)
Movimiento Celular/inmunología , Quimiocina CXCL12/inmunología , Neutrófilos/inmunología , Receptores CXCR4/inmunología , Transducción de Señal/inmunología , Proteínas de Pez Cebra/inmunología , Pez Cebra/inmunología , Animales , Movimiento Celular/genética , Quimiocina CXCL12/genética , Técnicas de Silenciamiento del Gen , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Neutrófilos/patología , Receptores CXCR4/genética , Transducción de Señal/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Neutrophils are the first immune cells recruited to a site of injury or infection, where they perform many functions. Having completed their role, neutrophils must be removed from the inflammatory site-either by apoptosis and efferocytosis or by reverse migration away from the wound-for restoration of normal tissue homeostasis. Disruption of these tightly controlled physiological processes of neutrophil removal can lead to a range of inflammatory diseases. We used an in vivo zebrafish model to understand the role of lipid mediator production in neutrophil removal. Following tailfin amputation in the absence of macrophages, neutrophillic inflammation does not resolve, due to loss of macrophage-dependent handling of eicosanoid prostaglandin E2 (PGE2) that drives neutrophil removal via promotion of reverse migration. Knockdown of endogenous PGE synthase gene reveals PGE2 as essential for neutrophil inflammation resolution. Furthermore, PGE2 is able to signal through EP4 receptors during injury, causing an increase in Alox12 production and switching toward anti-inflammatory eicosanoid signaling. Our data confirm regulation of neutrophil migration by PGE2 and LXA4 (lipoxin A4) in an in vivo model of inflammation resolution. This pathway may contain therapeutic targets for driving inflammation resolution in chronic inflammatory disease.
Asunto(s)
Dinoprostona/metabolismo , Inflamación/patología , Neutrófilos/fisiología , Heridas y Lesiones/fisiopatología , Animales , Animales Modificados Genéticamente , Movimiento Celular , Dinoprostona/farmacología , Modelos Animales de Enfermedad , Inflamación/metabolismo , Lipoxinas/metabolismo , Lipoxinas/farmacología , Lipooxigenasas/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Neutrófilos/efectos de los fármacos , Fenotipo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Pez Cebra/genética , Pez Cebra/fisiologíaRESUMEN
There remains a need to identify novel pro-resolution drugs for treatment of inflammatory disease. To date, there are no neutrophil-specific anti-inflammatory treatments in clinical use, perhaps due to our lack of understanding of how drugs access this complex cell type. Here we present the first comprehensive description and expression of both major classes of drug transporters, SLC and ABC, in resting human blood neutrophils. Moreover, we have studied the expression of these carriers in the tractable model system, the zebrafish (Danio rerio), additionally examining the evolutionary relationship between drug transporters in zebrafish and humans. We anticipate that this will be a valuable resource to the field of inflammation biology and will be an important asset in future anti-inflammatory drug design.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Neutrófilos/metabolismo , Análisis de Secuencia de ARN/métodos , Proteínas Transportadoras de Solutos/genética , Pez Cebra/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Evolución Molecular , Regulación de la Expresión Génica , Humanos , Anotación de Secuencia Molecular , Familia de Multigenes , Filogenia , Proteínas Transportadoras de Solutos/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Familial amyotrophic lateral sclerosis (FALS) is caused, in 20% of cases, by mutations in the Cu/Zn superoxide dismutase gene (SOD1). Although motor neuron injury occurs through a toxic gain of function, the precise mechanism(s) remains unclear. Using an established NSC34 cellular model for SOD1-associated FALS, we investigated the effects of mutant SOD1 specifically in cells modelling the vulnerable cell population, the motor neurons, without contamination from non-neuronal cells present in CNS. Using gene expression profiling, 268 transcripts were differentially expressed in the presence of mutant human G93A SOD1. Of these, 197 were decreased, demonstrating that the presence of mutant SOD1 leads to a marked degree of transcriptional repression. Amongst these were a group of antioxidant response element (ARE) genes encoding phase II detoxifying enzymes and antioxidant response proteins (so-called 'programmed cell life' genes), the expression of which is regulated by the transcription factor NRF2. We provide evidence that dysregulation of Nrf2 and the ARE, coupled with reduced pentose phosphate pathway activity and decreased generation of NADPH, represent significant and hitherto unrecognized components of the toxic gain of function of mutant SOD1. Other genes of interest significantly altered in the presence of mutant SOD1 include several previously implicated in neurodegeneration, as well as genes involved in protein degradation, the immune response, cell death/survival and the heat shock response. Preliminary studies on isolated motor neurons from SOD1-associated motor neuron disease cases suggest key genes are also differently expressed in the human disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Neuronas Motoras/metabolismo , Mutación , Superóxido Dismutasa/genética , Transactivadores/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Antioxidantes , Apoptosis/genética , Línea Celular , Supervivencia Celular/genética , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Neuronas Motoras/patología , Factor 2 Relacionado con NF-E2 , Degeneración Nerviosa/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Elementos de Respuesta , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available 'cromones' are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease.
Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/farmacología , Cumarinas/química , Cumarinas/farmacología , Evaluación Preclínica de Medicamentos , Pez Cebra/metabolismo , Animales , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Cromolin Sódico/química , Cromolin Sódico/farmacología , Furocumarinas/química , Furocumarinas/farmacología , Inflamación/patología , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
Diseases of failed inflammation resolution are common and largely incurable. Therapeutic induction of inflammation resolution is an attractive strategy to bring about healing without increasing susceptibility to infection. However, therapeutic targeting of inflammation resolution has been hampered by a lack of understanding of the underlying molecular controls. To address this drug development challenge, we developed an in vivo screen for proresolution therapeutics in a transgenic zebrafish model. Inflammation induced by sterile tissue injury was assessed for accelerated resolution in the presence of a library of known compounds. Of the molecules with proresolution activity, tanshinone IIA, derived from a Chinese medicinal herb, potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Tanshinone IIA blocked proinflammatory signals in vivo, and its effects are conserved in human neutrophils, supporting a potential role in treating human inflammation and providing compelling evidence of the translational potential of this screening strategy.
Asunto(s)
Abietanos/farmacología , Antiinflamatorios/farmacología , Movimiento Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Inflamación/tratamiento farmacológico , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Pez Cebra , Animales , Animales Modificados Genéticamente , Apoptosis/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Larva , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Investigación Biomédica Traslacional , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/inmunología , Pez Cebra/metabolismoRESUMEN
Many key components of innate immunity to infection are shared between Drosophila and humans. However, the fly Toll ligand Spaetzle is not thought to have a vertebrate equivalent. We have found that the structurally related cystine-knot protein, nerve growth factor ß (NGFß), plays an unexpected Spaetzle-like role in immunity to Staphylococcus aureus infection in chordates. Deleterious mutations of either human NGFß or its high-affinity receptor tropomyosin-related kinase receptor A (TRKA) were associated with severe S. aureus infections. NGFß was released by macrophages in response to S. aureus exoproteins through activation of the NOD-like receptors NLRP3 and NLRP4 and enhanced phagocytosis and superoxide-dependent killing, stimulated proinflammatory cytokine production, and promoted calcium-dependent neutrophil recruitment. TrkA knockdown in zebrafish increased susceptibility to S. aureus infection, confirming an evolutionarily conserved role for NGFß-TRKA signaling in pathogen-specific host immunity.
Asunto(s)
Factor de Crecimiento Nervioso/inmunología , Receptor trkA/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/inmunología , Evolución Molecular , Técnicas de Silenciamiento del Gen , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Macrófagos/inmunología , Factor de Crecimiento Nervioso/genética , Fagocitosis/genética , Fagocitosis/inmunología , Receptor trkA/genética , Infecciones Estafilocócicas/genética , Pez Cebra/genética , Pez Cebra/inmunologíaRESUMEN
To understand inflammation and immunity, we need to understand the biology of the neutrophil. Whereas these cells can readily be extracted from peripheral blood, their short lifespan makes genetic manipulations impractical. Murine knockout models have been highly informative, and new imaging techniques are allowing neutrophils to be seen during inflammation in vivo for the first time. However, there is a place for a new model of neutrophil biology, which readily permits imaging of individual neutrophils during inflammation in vivo, combined with the ease of genetic and chemical manipulation. The zebrafish has long been the model of choice for the developmental biology community, and the availability of genomic resources and tools for gene manipulation makes this an attractive model. Zebrafish innate immunity shares many features with mammalian systems, including neutrophils with morphological, biochemical, and functional features, also shared with mammalian neutrophils. Transgenic zebrafish with neutrophils specifically labeled with fluorescent proteins have been generated, and this advance has led to the adoption of zebrafish, alongside existing models, by a number of groups around the world. The use of these models has underpinned a number of key advances in the field, including the identification of a tissue gradient of hydrogen peroxide for neutrophil recruitment following tissue injury and direct evidence for reverse migration as a regulatable mechanism of inflammation resolution. In this review, we discuss the importance of zebrafish models in neutrophil biology and describe how the understanding of neutrophil biology has been advanced by the use of these models.
Asunto(s)
Modelos Animales , Neutrófilos/inmunología , Pez Cebra/inmunología , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Humanos , Inmunidad/inmunologíaRESUMEN
Neutrophil function is thought to be regulated, in large part, by limitation of lifespan by apoptosis. A number of studies suggest that circulating neutrophils have a half-life of approximately 6 hours, although contradictory evidence exists. Measuring tissue neutrophil lifespan, however, is more problematic. It is thought that tissue neutrophils survive longer, perhaps with a half-life in the order of 3-5 days, but this has never been directly measured. Zebrafish are an emerging model organism, with several advantages for the study of vertebrate immunity. In zebrafish, neutrophils constitutively assume tissue locations allowing their direct study in vivo. Using a transgenic approach, neutrophils were labelled with a photoconvertible pigment, Kaede. Photoconversion parameters were optimised and the stability of the Kaede confirmed. Individual neutrophils were photoconverted by scanning a confocal 405 nm laser specifically over each cell and their survival monitored for 48 hours, revealing an in vivo half-life for zebrafish tissue neutrophils of around 120 hours (117.7 hrs, 95% CI 95.67-157.8). Laser energy did not extend neutrophil lifespan, and we conclude that this represents a lower bound for the lifespan of a resting tissue neutrophil in the developing zebrafish larva. This is the first direct measurement of the lifespan of an in vivo tissue neutrophil.
RESUMEN
A key feature of inflammatory cells is the ability to migrate to a site of injury or infection quickly and efficiently. Infectious agents can then be taken up by these inflammatory cells, preventing established infection. Inflammatory cell migration is driven by a complex interaction between inflammatory cells and their environment. In order to maintain health, inflammation needs to resolve, allowing the surrounding tissues to recover and heal. These processes are not fully understood and have been difficult to study in cell culture due to the complex interactions between cell types. We therefore use a range of techniques in near-transparent zebrafish (Danio rerio) larvae to study these migration events in a whole-organism, in vivo model. Using a transgenic zebrafish line that specifically marks neutrophils with green fluorescent protein, Tg(mpx:GFP)i114, we are able to follow neutrophil behaviour at a single cell level. Using these methods, the cellular processes involved in all phases of inflammation can be studied and better understood.
Asunto(s)
Rastreo Celular/métodos , Quimiotaxis de Leucocito , Neutrófilos/fisiología , Pez Cebra/fisiología , Aletas de Animales/inmunología , Aletas de Animales/patología , Animales , Animales Modificados Genéticamente , Proteínas Fluorescentes Verdes/biosíntesis , Larva/genética , Larva/inmunología , Larva/fisiología , Microscopía Confocal , Microscopía Fluorescente , Proteínas Recombinantes/biosíntesis , Programas Informáticos , Imagen de Lapso de Tiempo , Pez Cebra/genética , Pez Cebra/inmunologíaRESUMEN
Zebrafish are a unique model for pharmacological manipulation of physiological processes such as inflammation; they are small and permeable to many small molecular compounds, and being transparent, they permit the visualization and quantitation of the inflammatory response by observation of transgenically labeled inflammatory cell populations. Using a transgenic line specifically labeling neutrophils in vivo (mpx:GFP), we studied the effects of a range of pharmacological agents on the resolution of inflammation in vivo. These agents were selected for their ability to modulate neutrophil function and lifespan in human neutrophils in vitro. Agents delaying neutrophil apoptosis (LPS, dbcAMP, and several caspase inhibitors) all lead to a delay in resolution of neutrophilic inflammation. Reciprocally, pyocyanin and roscovitine (inducers of neutrophil apoptosis) lead to reduced neutrophil numbers. The occurrence of apoptosis was observed by time-lapse analysis and confirmed by dual staining for neutrophil-specific mpx activity (TSA staining) and an apoptotic marker (TUNEL). During inflammation, macrophages follow neutrophils into the inflamed site, and TUNEL/TSA dual-positive material can be demonstrated within macrophages, consistent with their uptake of apoptotic neutrophils. This model has several advantages over mammalian models and lends itself to the study of pharmaceutical agents modulating inflammation.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inflamación/tratamiento farmacológico , Neutrófilos/metabolismo , Purinas/farmacología , Piocianina/farmacología , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Inflamación/metabolismo , Inflamación/patología , Neutrófilos/patología , Roscovitina , Pez Cebra/genéticaRESUMEN
Neutrophilic inflammation is responsible for much of the tissue damage seen in many lung diseases. For resolution of inflammation to occur, neutrophils must die by apoptosis, allowing their recognition and removal by macrophages. The molecular events controlling this important regulatory step are poorly understood, in large part due to the genetic intractability of the human neutrophil granulocyte. The authors have established a model of inflammation in the Zebrafish, which shares many features of the innate immune system with those of humans. Injury to the Zebrafish tailfin induces a reproducible and quantifiable inflammatory response, which resolves with kinetics similar to mammalian models of neutrophilic inflammation, including pulmonary inflammation. Pharmacological modulation of neutrophil apoptosis can modulate the outcome of experimentally induced inflammation. In addition, the authors have generated a construct that expresses green fluorescent protein under the myeloperoxidase promoter, allowing in vivo visualization of neutrophils during experimentally induced inflammation. The authors are also performing an unbiased forward genetic screen for mutants with defective resolution of inflammation, and to date have identified a number of putative mutants. Further study and characterization of these mutants is underway. The authors have thus established an important experimental link between apoptosis and resolution of inflammation in an in vivo system, and defined an important new model for the study of inflammation resolution. The authors hope that these tools will permit detailed study of the genetic controls of the resolution of inflammation, and provide insights with potential clinical utility.