Infection of zebrafish embryos with intracellular bacterial pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions (1). The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage (2, 3). The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle (4-6). In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection (7). A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization (8). Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system

Pioneer neutrophils release chromatin within in vivo swarms

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

Sipuleucel-T in patients with metastatic castration-resistant prostate cancer: an insight for oncologists

Sipuleucel-T represents a novel immunotherapeutic compound designed to stimulate an immune response against castration-resistant prostate cancer (CRPC). Sipuleucel-T is an autologous active cellular immunotherapy product, which includes autologous dendritic cells pulsed ex vivo with PAP2024, a recombinant fusion protein made of prostatic acid phosphatase and granulocyte-macrophage colony-stimulating factor. Despite the lack of prostate-specific antigen and objective response, a recent phase III randomized trial demonstrated a significant improvement in overall survival in asymptomatic and minimally symptomatic CRPC patients. This review summarizes the clinical development of Sipuleucel-T in CRPC that led to the regulatory approval of this compound in the USA

Characterisation of duplicate zinc finger like 2 erythroid precursor genes in zebrafish

In separate expression pattern and micro-array screens the zinc finger containing factor, znfl2, has been previously implicated in hematopoiesis. Here we analysed znfl2 expression in detail and performed genetic epistatic analysis in a series of hematopoietic mutants and transient gain-of-function models. znfl2 expression in the hematopoietic intermediate mesoderm and derived erythrocytes required early genes cloche and spadetail, but not gata1. Expression was up-regulated in scl gain-of-function embryos, identifying znfl2 as an early erythroid factor that is regulated upstream or independently of gata1. Furthermore, we identified a duplicate znfl2 gene in the genome (znfl2b) which was expressed in early mesendoderm and weakly in the lateral plate mesoderm, overlapping in expression with znfl2. The production of loss-of-function models for znfl2, znfl2b and znfl2/znfl2b together suggested that these erythrocyte specific zinc finger genes are dispensible for erythropoiesis

Zebrafish gcm2 is required for gill filament budding from pharyngeal ectoderm

The pharyngeal arches give rise to multiple organs critical for diverse processes, including the thymus, thyroid and parathyroids. Several molecular regulators of thymus and thyroid organogenesis are strikingly conserved between mammals and zebrafish. However, land animals have parathyroids whereas fish have gills. The murine transcription factor Glial cells missing 2 (Gcm2) is expressed specifically in the parathyroid primordium in the endodermal epithelium of the third pharyngeal pouch, and in both mice and humans is required for normal development of parathyroid glands. The molecular regulation of fish gill organogenesis remains to be described. We report the expression of gcm2 in the zebrafish pharyngeal epithelium and a requirement for Hox group 3 paralogs for gcm2 expression. Strikingly, zebrafish gcm2 is expressed in the ectodermal portion of the pharyngeal epithelium and is required for the development of the gill filament buds, precursors of fish-specific gill filaments. This study identifies yet another role for a GCM gene in embryonic development and indicates a role for gcm2 during the evolution of divergent pharyngeal morphologies

Specification of the primitive myeloid precursor pool requires signaling through Alk8 in zebrafish

In the zebrafish embryo, primitive hematopoiesis initiates in two spatially distinct regions. Rostrally, the cells of the anterior lateral plate mesoderm (ALPM) give rise exclusively to cells of the myeloid lineage in a pu.1-dependent manner [1-5]. Caudally, in the posterior lateral plate mesoderm (PLPM), the expression of gata1 defines a precursor pool that gives rise predominantly to the embryonic erythrocytes [6]. The transcription factor scl acts upstream of both gata1 and pu.1 in these precursor pools, activating a series of conserved transcription factors that cell-autonomously specify either myeloid or erythroid fates [1, 4, 7, 8]. However, the mechanisms underlying the spatial separation of the hematopoietic precursor pools and the induction of differential gene expression within these pools are not well understood. We show here that the Bmp receptor lost-a-fin/alk8 is required for rostral pu.1 expression and myelopoiesis, identifying an early genetic event that distinguishes between the induction of anterior and posterior hematopoiesis. Introducing a constitutively active version of the Alk8 receptor led to increased pu.1 expression, but the role of alk8 was independent of the scl-dependent cell-fate pathway. Furthermore, the role of Alk8 in myelopoiesis was genetically separable from its earlier role in dorsal-ventral embryonic patterning