Search

Edwardsiella tarda is an intracellular pathogenic bacteria that can imperil the health of farmed fish. However, the interactive networks of immune regulation and metabolic response in E. tarda-infected fish are still unclear. In this investigation, we aimed to explore immunometabolic interplay in crucian carp after E. tarda infection by utilizing multiomics analyses. Crucian carp (Carassius auratus) receiving E. tarda infection showed increased levels of tissue damage and oxidative injury in liver. Multiomics analyses suggested that carbon and amino acid metabolism may be considered as crucial metabolic pathways in liver of crucian carp following E. tarda infection, while spaglumic acid, isocitric acid and tetrahydrocortisone were the crucial liver biomarkers. After that, a potential antimicrobial peptide (AMP) sequence called apolipoprotein D (ApoD) was identified from omics study. Then, tissue-specific analysis indicated that liver CaApoD showed the highest expression among isolated tissues. After Aeromonas hydrophila stimulated, CaApoD expressions increased sharply in immune-related tissues. Moreover, CaApoD fusion protein could mediate the in vitro binding to A. hydrophila and E. tarda, attenuate bacterial growth as well as diminish bacterial biofilm forming activity. These findings may have a comprehensive implication for understanding immunometabolic response in crucian carp upon infection., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In the present study, an immunological arabinan, LCP70-2A, was isolated from Ligusticum chuanxiong for the first time. The absolute molecular weight of LCP70-2A was determined to be 6.46 × 10 4  g/mol using the HPSEC-MALLS-RID method. The absolute configuration of arabinose in LCP70-2A was determined to be L-configuration. Physicochemical characterization revealed that LCP70-2A was a homogeneous polysaccharide and had a backbone of (1 → 5)-linked α-L-Araf with terminal α-L-arabinose residues at position O-2 and O-3. Molecular conformation analysis showed that LCP70-2A was a branching polysaccharide with a compact coil chain conformation in 0.1 M NaCl solution. In addition, in vitro cell assays showed that LCP70-2A can activate macrophages by enhancing the phagocytosis and potentiating the secretion of immunoregulatory factors including NO, TNF-α, IL-6, and IL-1β. Furthermore, LCP70-2A was proved to promote the production of ROS and NO using the zebrafish model, suggesting that LCP70-2A can be further developed as a candidate supplement for immunological enhancement., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Zebrafish can efficiently regenerate complex tissue structures with a highly developed innate and adaptive immune system, which provides a model to investigate the roles of immune cells in tissue repair and regeneration. Two groups recently reported zebrafish mutants deficient in a forkhead box P3 (FOXP3) ortholog, which helped reveal the conserved immunosuppressive function of zebrafish FOXP3 in vivo. Zebrafish FOXP3 defines the development of a subset of T cell lineage with the conserved gene expression profile of mammalian regulatory T cells (Tregs). In damaged organs, zebrafish Tregs rapidly migrate to the injury site, where they promote the proliferation of regeneration precursor cells by producing tissue-specific regenerative factors through a distinct mechanism from the canonical anti-inflammatory pathway. These findings illuminate the potential for using zebrafish as an effective model in Treg research and demonstrate organ-specific roles for Tregs in maintaining proregenerative capacity that could potentially be harnessed for use in diverse regeneration therapies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The present study investigated dietary Enteromorpha prolifera polysaccharide (EPP) on the growth performance, body composition and non-specific immunity of crucian carp Carassius auratus. Crucian carps with body weight of 51.24 ± 4.08 g were randomly divided into five groups: one group was fed with basic diet, while the other four groups were fed with diets containing 20, 40, 60 and 80 g/kg EPP. After 60 days of feeding, dietary administration of 40 g/kg EPP increased the body weight gain rate, feed conversion ratio, specific growth rate, body crude protein content, intestine digestive enzyme activities, serum lysozyme activity, acid phosphatase activity, alkaline phosphatase activity, complement 3 level, superoxide dismutase activity and catalase activity and decreased body fat content and serum total cholesterol, triacylglycerol, alanine aminotransferase activity and glutamic oxaloacetic transaminase activity compared with those of the control group. All levels of EPP improved the resistance to Aeromonas hydrophilia compared with the control group. The results indicated that EPP could promote the growth of crucian carp and improve their disease resistance and may be used as a dietary supplement for crucian carp., (Copyright © 2020. Published by Elsevier B.V.)

Primarily used in genetic studies of development, the zebrafish (Danio rerio) has rapidly emerged as a promising animal model of human cancer. Cancer cell transplantation in zebrafish constitutes a key platform for clinical research since it allows to study cellular and molecular events involved in various aspects of tumorigenesis and to evaluate the efficacy of therapeutic molecules in vivo. Applied to patient-derived cells, the xenotransplantation approach in zebrafish allows to define the most appropriate therapeutic strategies for specific alterations found in patients in the context of personalized medicine. This review discusses the zebrafish transplantation model for the study of cancer development and drug discovery., (Copyright © 2019 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Triclosan (TCS, 5‑chloro‑2‑(2,4‑dichlorophenoxy) phenol) is becoming a major surface waters pollutant worldwide at concentrations ranging from ng L -1 to μg L -1 . Up to now, the adverse effects on aquatic organisms have been investigated at concentrations higher than the environmental ones, and the pathways underlying the observed toxicity are still not completely understood. Therefore, the aim of this study was to investigate the toxic effects of TCS at environmental concentrations on zebrafish embryos up to 120 hours post fertilization (hpf). The experimental design was planned considering both the quantity and the exposure time for the effects on the embryos, exposing them to two different concentrations (0.1 μg L -1 , 1 μg L -1 ) of TCS, for 24 h (from 96 to 120 hpf) and for 120 h (from 0 to 120 hpf). A suite of biomarkers was applied to measure the induction of embryos defence system, the possible increase of oxidative stress and the DNA damage. We measured the activity of glutathione‑S‑transferase (GST), P‑glycoprotein efflux and ethoxyresorufin‑o‑deethylase (EROD), the level of ROS, the oxidative damage through the Protein Carbonyl Content (PCC) and the activity of antioxidant enzymes. The genetic damage was evaluated through DNA Diffusion Assay, Micronucleus test (MN test), and Comet test. The results showed a clear response of embryos defence mechanism, through the induction of P-gp efflux functionality and the activity of detoxifying/antioxidant enzymes, preventing the onset of oxidative damage. Moreover, the significant increase of cell necrosis highlighted a strong cytotoxic potential for TCS. The overall results obtained with environmental concentrations and both exposure time, underline the critical risk associated to the presence of TCS in the aquatic environment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In a previous study we showed a clear relationship between immune system and behavior in zebrafish and we hypothesized that the immune system is capable of inducing behavioral changes. To further investigate this subject and to address our main question, here we induced an inflammatory response in one group of fish by the inoculation of formalin-inactivated Aeromonoas hydrophila bacterin and compared their social and exploratory behavior with control groups. After the behavioral tests, we also analyzed the expression of cytokines genes and markers of neuronal activity in fish brain. In the bacterin-inoculated fish, the locomotor activity, social preference and exploratory behavior towards a new object were reduced compared to the control fish while the expression of proinflammatory cytokines in the brain was upregulated. With this study we demonstrated for the first time that the immune system is capable of causing behavioral changes that are consistent with the sickness behavior observed in mammals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lead (Pb) poisoning in humans and fish represents a significant global problem. Bacillus subtilis (B. subtilis) is a widely used probiotic in aquaculture. Carassius auratus gibelio (C. gibelio) is one of the most important aquaculture species with great commercial value. The objective of this study was to evaluate the potential of B. subtilis in ameliorating lead-induced toxicity in C. gibelio. The fish were exposed for 60 days to waterborne Pb at 0, 0.05, 0.5 and 1 mg/L and/or dietary B. subtilis at 10 9 cfu/g. After 30 and 60 days, the fish were sampled and bioaccumulation, antioxidant activity and immune responses were assessed. The results revealed that B. subtilis confers significant protective effects against lead toxicity by preventing alterations in the levels of bioaccumulation, superoxide dismutase, catalase and glutathione. B. subtilis also assists in the recovery of blood δ-aminolevulinic acid dehydratase, lysozyme, and IgM levels while regulating the expression of immune-related genes including IL-10, lysozyme, TNF-α, IgM and Hsp70 after 60 days of lead exposure. Our results suggest that administration of B. subtilis (10 9 cfu/g) has the potential to combat lead toxicity in C. gibelio., (Copyright © 2018. Published by Elsevier Inc.)

Bergapten (5-methoxypsoralen), a coumarin-derivate compound isolated from Ficus hirta roots, was evaluated for its anti-inflammatory and proresolution activities in a tail-cutting-induced zebrafish larvae model. Bergapten was evaluated using a caudal fin-wounded transgenic zebrafish line "Tg(corola: eGFP)" to visualize the effects of the recruitment and clearance of neutrophils and macrophages at the injury site. We found that bergapten significantly suppressed the recruitment of neutrophils and macrophages toward the injury site, as well as promoted the clearance of neutrophils and macrophages from the wound site. We also investigated the reactive oxygen species (ROS) and nitric oxide (NO) level of bergapten in a tail-cutting-induced inflammation zebrafish model. The Results revealed that bergapten effectively inhibited the tail-cutting-induced production of ROS and NO in zebrafish larvae. This study reported for the first time the potential anti-inflammatory and proresolution activities of bergapten in an in vivo zebrafish model, suggesting that bergapten may be a potential candidate for inflammation therapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Regeneration is a post-embryonic developmental process that ensures complete morphological and functional restoration of lost body parts. The repair phase is a key step for the effectiveness of the subsequent regenerative process: in vertebrates, efficient re-epithelialisation, rapid inflammatory/immune response and post-injury tissue remodelling are fundamental aspects for the success of this phase, their impairment leading to an inhibition or total prevention of regeneration. Among deuterostomes, echinoderms display a unique combination of striking regenerative abilities and diversity of useful experimental models, although still largely unexplored. Therefore, the brittle star Amphiura filiformis and the starfish Echinaster sepositus were here used to comparatively investigate the main repair phase events after injury as well as the presence and expression of immune system and extracellular matrix (i.e. collagen) molecules using both microscopy and molecular tools. Our results showed that emergency reaction and re-epithelialisation are similar in both echinoderm models, being faster and more effective than in mammals. Moreover, in comparison to the latter, both echinoderms showed delayed and less abundant collagen deposition at the wound site (absence of fibrosis). The gene expression patterns of molecules related to the immune response, such as Ese-fib-like (starfishes) and Afi-ficolin (brittle stars), were described for the first time during echinoderm regeneration providing promising starting points to investigate the immune system role in these regeneration models. Overall, the similarities in repair events and timing within the echinoderms and the differences with what has been reported in mammals suggest that effective repair processes in echinoderms play an important role for their subsequent ability to regenerate. Targeted molecular and functional analyses will shed light on the evolution of these abilities in the deuterostomian lineage., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Transgenerational effects of environmental pollutants on humans and animals are complex. Thus, we used zebrafish to evaluate the effects of parental whole-life cycle exposure to bisphenol A and its analogs (bisphenol S and F) on offspring innate immunity. At adulthood, offspring were examined with/without continued chemicals treatment until 72h post-fertilization (hpf). To measure offspring immune function, larvae at 72 hpf were expose for 24h with/without the viral mimic polyinosinic-cytidylic acid (Poly I:C) or the bacterial mimic Pam3Cys-Ser-Lys4 (PAM3CSK4). Data show modified immunity in offspring. Specifically, lysozyme activity was significantly induced in F1 larvae and respiratory burst response and oxidative defense genes were inhibited. Genes of the innate immune system including Toll-like receptors and their downstream molecules and inflammatory cytokines were significantly down-regulated, whereas matrix metalloproteinases were up-regulated in larvae. In addition, recombination-activating genes in the immature adaptive immune system were significantly reduced. Thus, immune defense is diminished by exposing parental generations of zebrafish to environmentally relevant concentration of bisphenols and this suggests that fish chronically exposed to bisphenols in the wild may be vulnerable to pathogens., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Vitellogenin (Vtg) is a sensitive biomarker for environmental estrogens. In this study, an immunosensor for quantifying zebrafish Vtg was developed using the Octet system. First, Protein A sensors were immobilized with purified anti-lipovitellin (Lv) antibody that demonstrated specificity to Vtg. Then, antibody-coated biosensors were immersed into zebrafish Lv standards and diluted samples. The Octet system measured and recorded kinetic parameters between antigens and captured antibody within 5 min. Sample Vtg concentrations were automatically calculated by interpolating relative binding rates observed with each sample and the immobilized anti-Lv antibody into the developed standard curve. The sensor arrays exhibited a wide linear range from 78 to 5000 ng/mL, and the inter-assay coefficient of variation was 0.66-1.97%. Furthermore, the performance of the immunosensor in detecting Vtg was evaluated by quantifying Vtg induction in juvenile zebrafish exposed to 17β-estradiol (E 2 ). Compared with conventional immunoassay techniques, the Vtg immunosensor developed based on the Octet system was much simpler and less time-consuming, allowing rapid Vtg quantification within 15 min. Moreover, Protein A sensors could be reused many times to ensure that the assays have high reproducibility. Therefore, we suggest that immunosensors based on the Octet system are an easily operated detection method for ecotoxicological research., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Oxysterols are a family of cholesterol oxygenated derivatives with diverse roles in many biological activities and have recently been linked with the induction of a cellular antiviral state. The antiviral effects of 25-hydroxycholesterol (25HC) extend to several mammalian enveloped and non-enveloped viruses. It has been reported that the expression of the gene encoding cholesterol 25-hydroxylase (CH25H) is induced by interferons (IFNs). In this work, five ch25h genes were identified in the zebrafish (Danio rerio) genome. The ch25h genes showed different tissue expression patterns and differed in their expression after immune stimulation with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (PolyI:C) and Spring Viremia Carp Virus (SVCV). Only one of the 5 genes, ch25hb, was overexpressed after the administration of the treatments. Synteny and phylogenetic analyses revealed that ch25hb is the putative homolog of mammalian Ch25h in zebrafish, while the remaining zebrafish ch25h genes are products of duplications within the teleost lineage. Interestingly, its modulation was not mediated by type I IFNs, contrasting previous reports on mammalian orthologs. Nevertheless, in vivo overexpression of ch25hb in zebrafish larvae significantly reduced mortality after SVCV challenge. Viral replication was also negatively affected by 25HC administration to the zebrafish cell line ZF4. In conclusion, the interferon-independent antiviral role of 25HC was extended to a non-mammalian species for the first time, and dual activity that both protects the cells and interacts with the virus cannot be discarded., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Tributyltin (TBT) is a toxic compound released into aquatic ecosystems through antifouling paints. This study was designed to examine the effects of TBT on antioxidant ability and immune responses of zebrafish (Danio rerio). Three hundred sixty healthy zebrafish were randomly grouped into four groups and exposed to different doses of TBT (0, 1, 10 and 100ngL -1 ). At the end of 8 weeks, the fish were sampled, and antioxidant capability, immune parameters and immune-related genes were assessed. The results showed that with an increase in TBT dose, the concentration of malonaldehyde in the liver was significantly increased (p<0.05), whereas the activities of total superoxide dismutase, catalase and glutathione peroxidase were significantly decreased (p<0.05) compared to the control. The activity and expression of lysozyme and the content of immunoglobulin M were significantly decreased compared to those of the fish exposed to 0ngL -1 TBT (p<0.05). However, the expression of the HSP70, HSP90, tumor necrosis factor-α(TNF-α), interleukins (IL-1β, IL-6), and nuclear factor-kappa B p65 (NF-κ B p65) genes were all enhanced with an increase in TBT dose. The results indicated that TBT induced oxidative stress and had immunotoxic effects on zebrafish., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Synthetic glucocorticoids (GCs) are potential endocrine disrupting compounds that have been detected in the aquatic environment around the world in the low ng/L (nanomolar) range. GCs are used as immunosuppressants in medicine. It is of high interest whether clobetasol propionate (CP), a highly potent GC, suppresses the inflammatory response in fish after exposure to environmentally relevant concentrations. Bacterial lipopolysaccharide (LPS) challenge was used to induce inflammation and thus mimic pathogen infection. Zebrafish embryos were exposed to ≤1000nM CP from ~1h post fertilization (hpf) to 96 hpf, and CP uptake, survival after LPS challenge, and expression of inflammation-related genes were examined. Our initial experiments were carried out using 0.001% DMSO as a solvent vehicle, but we observed that DMSO interfered with the LPS challenge assay, and thus masked the effects of CP. Therefore, DMSO was not used in the subsequent experiments. The internal CP concentration was quantifiable after exposure to ≥10nM CP for 96h. The bioconcentration factor (BCF) of CP was determined to be between 16 and 33 in zebrafish embryos. CP-exposed embryos showed a significantly higher survival rate in the LPS challenge assay after exposure to ≥0.1nM in a dose dependent manner. This effect is an indication of immunosuppression. Furthermore, the regulation pattern of several genes related to LPS challenge in mammals supported our results, providing evidence that LPS-mediated inflammatory pathways are conserved from mammals to teleost fish. Anxa1b, a GC-action related anti-inflammatory gene, was significantly down-regulated after exposure to ≥0.05nM CP. Our results show for the first time that synthetic GCs can suppress the innate immune system of fish at environmentally relevant concentrations. This may reduce the chances of fish to survive in the environment, as their defense against pathogens is weakened., (Copyright © 2016. Published by Elsevier Inc.)

The objectives of this study were to determine the behavioral and molecular responses in the adult zebrafish (Danio rerio) central nervous system (CNS) following a dietary exposure to the pesticide dieldrin. Zebrafish were fed pellets spiked with 0.03, 0.15, or 1.8μg/g dieldrin for 21days. Behavioral analysis revealed no difference in exploratory behaviors or those related to anxiety. Transcriptional networks for T-cell aggregation and selection were decreased in expression suggesting an immunosuppressive effect of dieldrin, consistent with other studies investigating organochlorine pesticides. Processes related to oxidative phosphorylation were also differentially affected by dieldrin. Quantitative proteomics (iTRAQ) using a hybrid quadrupole-Orbitrap identified 226 proteins that were different following one or more doses. These proteins included ATP synthase subunits (mitochondrial) and hypoxia up-regulated protein 1 which were decreased and NADH dehydrogenases (mitochondrial) and signal recognition particle 9 which were up-regulated. Thus, proteins affected were functionally associated with the mitochondria and a protein network analysis implicated Parkinson's disease (PD) and Huntington's disease as diseases associated with altered proteins. Molecular networks related to mitochondrial dysfunction and T-cell regulation are hypothesized to underlie the association between dieldrin and PD. These data contribute to a comprehensive transcriptomic and proteomic biomarker framework for pesticide exposures and neurodegenerative diseases., Biological Significance: Dieldrin is a persistent organochlorine pesticide that has been associated with human neurodegenerative disease such as Parkinson's disease. Dieldrin is ranked 18th on the 2015 U.S. Agency for Toxic Substances and Disease Registry and continues to be a pesticide of concern for human health. Transcriptomics and quantitative proteomics (ITRAQ) were employed to characterize the molecular networks in the central nervous system that are altered with dietary exposure to dieldrin. We found that transcriptional and protein networks related to the immune system, mitochondria, and Parkinson's disease were preferentially affected by dieldrin. The study provides new insight into the mechanisms of dieldrin neurotoxicity that may explain, in part, the association between this pesticide and increased risks to neurodegeneration. These data contribute in a significant way to developing a molecular framework for pesticide induced neurotoxicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Vitellogenin (Vtg) in zebrafish (Danio rerio) is a recommended biomarker endpoint for detecting estrogenic activity of chemicals under the OECD test guidelines. The present paper reports the development of a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for the quantification of zebrafish Vtg based on monoclonal antibodies (MAbs) against lipovitellin (Lv), the major yolk protein derived from Vtg. The purified Lv was used to immunize mice and the spleen cells of mice were fused with myeloma cells. Two high-affinity MAbs (H3A8 and H4D9) were screened from hybridoma cells. Western blot analysis revealed that two MAbs were highly specific to zebrafish Vtg and recognized different antigenic epitopes because MAb H3A8 detected a main band of 143kDa in purified Vtg, while MAb H4D9 reacted with two clear bands of purified Vtg at 117 and 102kDa. Using MAb H3A8 as the coating antibody, HRP-labeled MAb H4D9 or HRP-labeled PAbs as the detecting antibody, two sandwich ELISAs for Vtg quantification were developed. The sandwich ELISA developed using HRP-labeled MAb H4D9 had a working range of 1.95-250ng/mL, with a detection limit of 0.78ng/mL, which was lower than that of the assay based on HRP-labeled PAbs. Parallelism between Lv standard curves and dilution curves of whole-body homogenates (WBH) from E 2 -treated male zebrafish confirmed the validity of the ELISAs for quantifying zebrafish Vtg. Finally, the usefulness of two assays for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to 17β-estradiol., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Macrophages are key components of the innate immune system and play pivotal roles in immune response, organ development, and tissue homeostasis. Studies in mice and zebrafish have shown that tissue-resident macrophages derived from different hematopoietic origins manifest distinct developmental kinetics and colonization potential, yet the genetic programs controlling the development of macrophages of different origins remain incompletely defined. In this study, we use zebrafish, where tissue-resident macrophages arise from the rostral blood island (RBI) and ventral wall of dorsal aorta (VDA), the zebrafish hematopoietic tissue equivalents to the mouse yolk sac and aorta-gonad-mesonephros for myelopoiesis, to address this issue. We show that RBI- and VDA-born macrophages are orchestrated by distinctive regulatory networks formed by the E-twenty-six (Ets) transcription factors Pu.1 and Spi-b, the zebrafish ortholog of mouse spleen focus forming virus proviral integration oncogene B (SPI-B), and the helix-turn-helix DNA-binding domain containing protein Irf8. Epistatic studies document that during RBI macrophage development, Pu.1 acts upstream of Spi-b, which, upon induction by Pu.1, partially compensates the function of Pu.1. In contrast, Pu.1 and Spi-b act in parallel and cooperatively to regulate the development of VDA-derived macrophages. Interestingly, these two distinct regulatory networks orchestrate the RBI- and VDA-born macrophage development largely by regulating a common downstream gene, Irf8. Our study indicates that macrophages derived from different origins are governed by distinct genetic networks formed by the same repertoire of myeloid-specific transcription factors., (© 2017 by The American Society of Hematology.)

Arsenic poisoning is a serious global issue. Apart from causing developmental and systemic toxicity, arsenic has recently been reported for its ability to hinder immune responses. The present study is designed to identify the global expression profile associated with arsenic-induced immune alterations at the organismic level. Adult zebrafish (Danio rerio) were exposed to 20, 40 and 80ppb of arsenic trioxide for 30days, sacrificed and global gene expression profile studied. Microarray data suggested 65 immune related genes were commonly affected in the three treatment regimens. The expression profile of key immune related genes (tlr1, nitr1f, nitr1c, crfb8, socs7, socs3b, abcb3/1, mch1uja, ifnγ1-2, cxcl12b and crlf1a) was validated by qPCR. Pathway analysis suggested the major involvement of JAK-STAT circuit in the process. The expression of these marker genes was also studied in arsenic exposed and bacteria (Aeromonas hydrophila) challenged zebrafish. Increase in bacterial colony forming units (CFU) coupled with gross histopathology of kidney in arsenic exposed-bacteria challenged fish suggested profound immuno-compromised condition. We propose that chronic arsenic exposure leads to hyperactivation of the immune system as a consequence when exposed to further stress (microbial) it induces immuno-suppression with pathological implications. The study provides a molecular snap shot for predicting arsenic immuno-toxicity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Zebrafish has been used for over a decade to study the mechanisms of a wide variety of inflammatory disorders and infections, with models ranging from bacterial, viral, to fungal pathogens. Zebrafish has been especially relevant to study the differentiation, specialization, and polarization of the two main innate immune cell types, the macrophages and the neutrophils. The optical accessibility and the early appearance of myeloid cells that can be tracked with fluorescent labels in zebrafish embryos and the ability to use genetics to selectively ablate or expand immune cell populations have permitted studying the interaction between infection, development, and metabolism. Additionally, zebrafish embryos are readily colonized by a commensal flora, which facilitated studies that emphasize the requirement for immune training by the natural microbiota to properly respond to pathogens. The remarkable conservation of core mechanisms required for the recognition of microbial and danger signals and for the activation of the immune defenses illustrates the high potential of the zebrafish model for biomedical research. This review will highlight recent insight that the developing zebrafish has contributed to our understanding of host responses to invading microbes and the involvement of the microbiome in several physiological processes. These studies are providing a mechanistic basis for developing novel therapeutic approaches to control infectious diseases., (© 2017 Elsevier Inc. All rights reserved.)

Immune deficiency or suppression in host animals is an essential precondition for the success of cancer cell xenotransplantation because the host immune system has a tendency to reject implanted cells. However, in such animals, the typical tumor microenvironment seen in cancer subjects does not form because of the lack of normal immunity. Here, we developed a novel zebrafish (Danio rerio) model based on 2 rounds of cancer cell xenotransplantation that achieved cancer-specific immunologic tolerance without immunosuppression. We irradiated human cancer cells (PC-3, K562 and HepG2) to abolish their proliferative abilities and implanted them into zebrafish larvae. These cells survived for 2 weeks in the developing host. Three months after the first implantation, the zebrafish were implanted with the same, but nonirradiated, cell lines. These cancer cells proliferated and exhibited metastasis without immune suppression. To reveal the transcriptional mechanism of this immune tolerance, we conducted dual RNA-seq of the tumor with its surrounding tissues and identified several regulatory zebrafish genes that are involved in immunity; the expression of plasminogen activator, urokinase, and forkhead box P3 was altered in response to immunologic tolerance. In conclusion, this xenograft method has potential as a platform for zebrafish-based anticancer drug discovery because it can closely mimic human clinical cancers without inducing immune suppression., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The present study examines the deleterious effect of biologically synthesized silver nanoparticles in adult zebrafish. Silver nanoparticles (AgNPs) used in the study were synthesized by treating AgNO3 with aqueous leaves extract of Malva crispa Linn., a medicinal herb as source of reductants. LC50 concentration of AgNPs at 96 h was observed as 142.2 μg/l. In order to explore the underlying toxicity mechanisms of AgNPs, half of the LC50 concentration (71.1 μg/l) was exposed to adult zebrafish for 14 days. Cytological changes and intrahepatic localization of AgNPs were observed in gills and liver tissues respectively, and the results concluded a possible sign for oxidative stress. In addition to oxidative stress the genotoxic effect was observed in peripheral blood cells like presence of micronuclei, nuclear abnormalities and also loss in cell contact with irregular shape was observed in liver parenchyma cells. Hence to confirm the oxidative stress and genotoxic effects the mRNA expression of stress related (MTF-1, HSP70) and immune response related (TLR4, NFKB, IL1B, CEBP, TRF, TLR22) genes were analyzed in liver tissues and the results clearly concluded that the plant extract mediated synthesis of AgNPs leads to oxidative stress and immunotoxicity in adult zebrafish., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Goldfish (Carassius auratus) vitellogenin (Vtg) is an efficient biomarker for estrogen contamination in aquatic environments. In this study, Vtg and lipovitellin (Lv) were purified from the plasma of 17β-estradiol (E2)-induced male goldfish and unfertilized eggs of females, and were used to generate polyclonal antibodies against Vtg (anti-Vtg) and Lv (anti-Lv), respectively. SDS-PAGE and Western blot were performed to confirm the specificity of the two antibodies and the immunological similarity between Vtg and Lv. As anti-Lv recognized more antigen epitopes than anti-Vtg, it was used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for goldfish Vtg with purified Lv as the standard. The detection limit of the assay was 1.82ng/mL, and the working range was 3.9-250ng/mL. The use of Lv instead of Vtg as the standard provided greater precision and strengthened the robustness of the sandwich ELISA. Western blot and the Lv-based ELISA were used to detect Vtg inductions in surface mucus and plasma of E2-induced goldfish. The surface mucus Vtg level in E2-induced males was significantly higher than that in the control males and E2-induced females, and was much closer to the plasma Vtg level in E2-induced males than that in E2-induced females. Therefore, the surface mucus Vtg level of male goldfish may be a reliable indicator of estrogenic activity in the aquatic environment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The tripartite motif (TRIM)-containing proteins exhibit various activities and play important roles in the immune system through regulating signaling pathways. Bloodthirsty gene is a multigene subset of TRIM genes. In this study we identified and characterized a new member of the bloodthirsty subset of TRIM genes, btr20, in zebrafish (Danio rerio). The gene is located on chromosome 19 and forms a cluster with btr18, btr21, btr22 and an E3 ubiquitin ligase TRIM39-like gene. Deduced btr20 represents a RBCC-B30.2 TRIM protein containing 544 amino acids. The mRNA expression level of btr20 was highest in intestine and gill, followed by in spleen and kidney. Challenge experiment with Aeromonas hydrophila strain NJ-1 showed that the levels of btr20 and NF-κB mRNA were remarkably upregulated in the four tissues mentioned above. btr20 was localized in the cytoplasm and formed aggregate in human embryonic kidney cell line 293T. In vitro self-ubiquitylation experiment demonstrated that btr20 has E3 ubiquitin ligase activity that can be self-ubiquitylated with most E2 enzymes, especially UbcH6. The results suggested that btr20 may involve in the anti-microbial activity in the immune system as an E3 ubiquitin ligase., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Goldfish (Carassius auratus) represents a good model to detect the estrogenic effects of chemicals, and vitellogenin (Vtg) is a vital indicator of estrogenic activity. The heterologous anti-carp Vtg antibody has previously been used for goldfish Vtg detection. Here, we report the preparation of an anti-goldfish Vtg antibody to improve the sensitivity and specificity of goldfish Vtg immunoassays. Vtg was purified from the plasma of 17β-estradiol (E2)-induced goldfish by gel filtration followed by anion-exchange chromatography. It was characterized as a phospholipoglycoprotein with an apparent molecular weight of ~460 kDa and separated into three major polypeptides corresponding to ~130, ~106, and ~81 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A polyclonal antibody against goldfish Vtg was raised in rabbits and found to be specific for goldfish Vtg through immunoelectrophoresis and Western blot. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of plasma Vtg, with a detection limit of 3.6 ng/mL and a detection range from 7.8 to 250 ng/mL. The intra- and inter-assay coefficients of variations were 2.4-6.8% and 6.7-10.8%, respectively. Additionally, we qualitatively and quantitatively detected the induction of Vtg in male fish exposed to 0.01, 0.01, and 1.00 mg/L monocrotophos pesticide by Western blot and ELISA. The homologous sandwich ELISA based on the anti-goldfish Vtg antibody could provide a valuable tool for the study of estrogenic effects of exogenous chemicals on goldfish., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid. While this approach relieved the cellular cloning bottleneck and had the desirable ability to screen antibody function prior to cloning, the small volume of hybridoma supernatant available for screening limited the number of antigens for pooled immunisation. Here, we report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening. This approach permits a significant increase in the number of antigens for parallel monoclonal antibody selection from a single animal. Finally, we show the successful use of a convenient small-scale transfection method to rapidly identify plasmids that encode functional cloned antibodies, addressing another bottleneck in this approach. In summary, we show that a hybrid approach of combining established hybridoma antibody technology with refined screening and antibody cloning methods can be used to select monoclonal antibodies of desired functional properties against many different antigens from a single immunised host., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Innate immunity constitutes the first line of the host defense after pathogen invasion. Viruses trigger the expression of interferons (IFNs). These master antiviral cytokines induce in turn a large number of interferon-stimulated genes, which possess diverse effector and regulatory functions. The IFN system is conserved in all tetrapods as well as in fishes, but not in tunicates or in the lancelet, suggesting that it originated in early vertebrates. Viral diseases are an important concern of fish aquaculture, which is why fish viruses and antiviral responses have been studied mostly in species of commercial value, such as salmonids. More recently, there has been an interest in the use of more tractable model fish species, notably the zebrafish. Progress in genomics now makes it possible to get a relatively complete image of the genes involved in innate antiviral responses in fish. In this review, by comparing the IFN system between teleosts and mammals, we will focus on its evolution in vertebrates., (© 2013 Elsevier Ltd. All rights reserved.)

Teleost fish are among the most ancient vertebrates possessing an adaptive immune system with B and T lymphocytes that produce memory responses to pathogens. Most bony fish, however, have only 2 types of B lymphocytes, in contrast to the 4 types available to mammals. To better understand the evolution of adaptive immunity, we generated transgenic zebrafish in which the major immunoglobulin M (IgM(+)) B-cell subset expresses green fluorescence protein (GFP) (IgM1:eGFP). We discovered that the earliest IgM(+) B cells appear between the dorsal aorta and posterior cardinal vein and also in the kidney around 20 days postfertilization. We also examined B-cell ontogeny in adult IgM1:eGFP;rag2:DsRed animals, where we defined pro-B, pre-B, and immature/mature B cells in the adult kidney. Sites of B-cell development that shift between the embryo and adult have previously been described in birds and mammals. Our results suggest that this developmental shift occurs in all jawed vertebrates. Finally, we used IgM1:eGFP and cd45DsRed;blimp1:eGFP zebrafish to characterize plasma B cells and investigate B-cell function. The IgM1:eGFP reporter fish are the first nonmammalian B-cell reporter animals to be described. They will be important for further investigation of immune cell evolution and development and host-pathogen interactions in zebrafish.

In the present study, we analyzed the oxidative stress related indices and immune related gene expression of zebrafish embryos after a short-term exposure to various concentrations of di-n-butyl phthalate (DBP), diethyl phthalate (DEP) and their mixture (DBP-DEP) from 4h post-fertilization (hpf) to 96hpf. Exposure to the chemicals was found to enhance the production of reactive oxygen species (ROS) and lipid peroxidation (LPO) in a concentration-dependent manner. Simultaneously, adaptive responses to DBP/DEP-induced oxidative stress were observed. The activity of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were all increased in a concentration-dependent manner. The transcription of innate immune related genes including interferon γ (IFNγ), interleukin-1β (IL1β), Myxovirus resistance (Mx), tumor necrosis factor α (TNFα), CC-chemokine, CXCL-clc, lysozyme (Lyz) and complement factor C3B (C3) were up-regulated upon DBP, DEP and their mixture exposure, suggesting the induction of immune response. In addition, co-exposure to DBP-DEP also induced antioxidant defense and immune response in zebrafish embryo. The results demonstrat that DBP/DEP exposure could induce the antioxidant and immune responses in zebrafish embryos., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We used the opportunities afforded by the zebrafish to determine upstream pathways regulating mast cell development in vivo and identify their cellular origin. Colocalization studies demonstrated zebrafish notch receptor expression in cells expressing carboxypeptidase A5 (cpa5), a zebrafish mast cell-specific marker. Inhibition of the Notch pathway resulted in decreased cpa5 expression in mindbomb mutants and wild-type embryos treated with the γ-secretase inhibitor, Compound E. A series of morpholino knockdown studies specifically identified notch1b and gata2 as the critical factors regulating mast cell fate. Moreover, hsp70::GAL4;UAS::nicd1a transgenic embryos overexpressing an activated form of notch1, nicd1a, displayed increased cpa5, gata2, and pu.1 expression. This increase in cpa5 expression could be reversed and reduced below baseline levels in a dose-dependent manner using Compound E. Finally, evidence that cpa5 expression colocalizes with lmo2 in the absence of hematopoietic stem cells revealed that definitive mast cells initially delineate from erythromyeloid progenitors. These studies identify a master role for Notch signaling in vertebrate mast cell development and establish developmental origins of this lineage. Moreover, these findings postulate targeting the Notch pathway as a therapeutic strategy in mast cell diseases.

Immunoglobulins (Ig) of Chondroichthyes have been extensively studied in sharks; in contrast, in skates investigations on Ig remain scarce and fragmentary despite the high occurrence of skates in all of the major oceans of the world. To focus on Rajidae Igμ, the most abundant heavy chain isotype, we have chosen the Antarctic species Bathyraja eatonii, Bathyraja albomaculata, Bathyraja brachyurops, and Amblyraja georgiana which live at high latitudes in the Southern Ocean, and at very low temperatures. We prepared mRNA from the spleen of individuals of each species and performed RT-PCR experiments using two oligonucleotides designed on the alignment of various elasmobranch Igμ heavy chain sequences available in GenBank. The PCR products, about 1400-nt long, were cloned and sequenced. Nucleotide sequence identities calculated for the constant region domains ranged from 88.5% to 97.5% between species, and from 91.1% to 99.7% within species. In a distance tree, including also Raja erinacea sequences, two major branches were obtained, one containing Arhynchobatinae sequences, the other one Rajinae sequences. Four presumptive D gene segments were identified in the region of the VH/D/JH recombination; two different D segments were often found in the same sequence. Moreover, 5-15 genomic fragments of different lengths, carrying the gene locus encoding Igμ chain were revealed by Southern blotting analysis. B. eatonii amino acid sequences were analyzed for the positional diversity by Shannon entropy analysis, showing CH4 as the most conserved domain, and CH3 as the most variable one. B. eatonii CDR3 region length varied between 11 and 15 amino acid residues; the mean length (13.4 aa) was greater than that of Leucoraja eglanteria sequences (7.7 aa). An alignment of representative sequences of Antarctic species and R. erinacea showed that more cysteine residues not involved in the intradomain disulfide bridges were present in Antarctic species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although platelets are the smallest cells in the blood, they are implied in various processes ranging from immunology and oncology to thrombosis and hemostasis. Many large-scale screening programs, genome-wide association, and "omics" studies have generated lists of genes and loci that are probably involved in the formation or physiology of platelets under normal and pathologic conditions. This creates an increasing demand for new and improved model systems that allow functional assessment of the corresponding gene products in vivo. Such animal models not only render invaluable insight in the platelet biology, but in addition, provide improved test systems for the validation of newly developed anti-thrombotics. This review summarizes the most important models to generate transgenic platelets and to study their influence on platelet physiology in vivo. Here we focus on the zebrafish morpholino oligonucleotide technology, the (platelet-specific) knockout mouse, and the transplantation of genetically modified human or murine platelet progenitor cells in myelo-conditioned mice. The various strengths and pitfalls of these animal models are illustrated by recent examples from the platelet field. Finally, we highlight the latest developments in genetic engineering techniques and their possible application in platelet research.

The evolutionarily conserved immune system of the zebrafish (Danio rerio), in combination with its genetic tractability, position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells (DCs), but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene, we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed double-transgenic animals demonstrated that kidney mhc2dab:GFP(hi); cd45:DsRed(hi) cells were exclusively mature monocytes/macrophages and DCs, as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but were most abundant in the intestine and spleen, where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.

Evaluating hematopoietic stem cell (HSC) function in vivo requires a long-term transplantation assay. Although zebrafish are a powerful model for discovering the genetics of hematopoiesis, hematopoietic transplantation approaches have been underdeveloped. Here we established a long-term reconstitution assay in adult zebrafish. Primary and secondary recipients showed multilineage engraftment at 3 months after transplantation. Limiting dilution data suggest that at least 1 in 65 000 zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We defined zebrafish haplotypes at the proposed major histocompatibility complex locus on chromosome 19 and tested functional significance through hematopoietic transplantation. Matching donors and recipients dramatically increased engraftment and percentage donor chimerism compared with unmatched fish. These data constitute the first functional test of zebrafish histocompatibility genes, enabling the development of matched hematopoietic transplantations. This lays the foundation for competitive transplantation experiments with mutant zebrafish HSCs and chemicals to test for effects on engraftment, thereby providing a model for human hematopoietic diseases and treatments not previously available.

In vertebrates, myeloid cells comprise polymorphonuclear and mononuclear lineages that arise from 2 successive waves of development: a transitory primitive wave giving rise to limited myeloid cells during embryonic stage and a definitive wave capable of producing myeloid cells throughout the fetal and adult life. One key unresolved question is what factors dictate polymorphonuclear versus mononuclear lineage fates during myelopoiesis. Here we show that during zebrafish embryogenesis interferon regulatory factor-8 (irf8) is expressed specifically in macrophages but not neutrophils. Suppression of Irf8 function in zebrafish causes a depletion of macrophages and an enhanced output of neutrophils but does not affect the overall number, proliferation, and survival of primitive myeloid cells. These data indicate that the skewed myeloid lineage development in Irf8 knockdown embryos results from a cell-fate switching. Such a conclusion is further supported by the observation showing that overexpression of Irf8 promotes macrophage formation at the expense of neutrophil development. Genetic epistasis analysis reveals that Irf8 acts downstream of Pu.1 but is insufficient to promote macrophage development in the absence of Pu.1. Our findings demonstrate that Irf8 is a critical determinant for neutrophil versus macrophage fate choice during zebrafish primitive myelopoiesis.

Eosinophils are granulocytic leukocytes implicated in numerous aspects of immunity and disease. The precise functions of eosinophils, however, remain enigmatic. Alternative models to study eosinophil biology may thus yield novel insights into their function. Eosinophilic cells have been observed in zebrafish but have not been thoroughly characterized. We used a gata2:eGFP transgenic animal to enable prospective isolation and characterization of zebrafish eosinophils, and demonstrate that all gata2(hi) cells in adult hematopoietic tissues are eosinophils. Although eosinophils are rare in most organs, they are readily isolated from whole kidney marrow and abundant within the peritoneal cavity. Molecular analyses demonstrate that zebrafish eosinophils express genes important for the activities of mammalian eosinophils. In addition, gata2(hi) cells degranulate in response to helminth extract. Chronic exposure to helminth- related allergens resulted in profound eosinophilia, demonstrating that eosinophil responses to allergens have been conserved over evolution. Importantly, infection of adult zebrafish with Pseudocapillaria tomentosa, a natural nematode pathogen of teleosts, caused marked increases in eosinophil number within the intestine. Together, these observations support a conserved role for eosinophils in the response to helminth antigens or infection and provide a new model to better understand how parasitic worms activate, co-opt, or evade the vertebrate immune response.

Previous studies have shown that vitellogenin (Vg) is an immune-relevant molecule, but its potential immunological role in vivo remains obscure. We demonstrated here that injection of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) into male Danio rerio rapidly induced a significant up-regulation of Vg at both transcriptional and translational levels, and that serum Vg produced was able to bind to both Escherichia coli and Staphylococcus aureus and to inhibit their growth in a dose-dependent manner. All these data suggest that serum Vg in zebrafish D. rerio is an acute phase protein with bacterial-binding and inhibiting activities. It also bolsters the notion that factors normally involved in control of female reproduction are linked with immunity in organisms that rely on Vg for oocyte development., (Copyright © 2009. Published by Elsevier GmbH.)

The Spi1/Pu.1 transcription factor plays a crucial role in myeloid cell development in vertebrates. Despite extensive studies of Spi1, the controlled gene group remains largely unknown. To identify genes dependent on Spi1, we used a microarray strategy using a knockdown approach in zebrafish embryos combined with fluorescence-activated cell sorting of myeloid cells from transgenic embryos. This approach of using knockdowns with specific green fluorescent protein-marked cell types was highly successful in identifying macrophagespecific genes in Spi1-directed innate immunity. We found a gene group downregulated on spi1 knockdown, which is also enriched in fluorescence-activated cell-sorted embryonic myeloid cells of a spi1:GFP transgenic line. This gene group, representing putative myeloidspecific Spi1 target genes, contained all 5 previously identified Spi1-dependent zebrafish genes as well as a large set of novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes cxcr3.2, mpeg1, ptpn6, and mfap4 were expressed specifically in early embryonic macrophages. In a functional approach, we demonstrated that gene cxcr3.2, coding for chemokine receptor 3.2, is involved in macrophage migration to the site of bacterial infection. Therefore, based on our combined transcriptome analyses, we discovered novel early macrophage-specific marker genes, including a signal transducer pivotal for macrophage migration in the innate immune response.

The amenability of the zebrafish to in vivo imaging and genetic analysis has fueled expanded use of this vertebrate model to investigate the molecular and cellular foundations of host-microbe relationships. Study of microbial encounters in zebrafish hosts has concentrated on developing embryonic and larval stages, when the advantages of the zebrafish model are maximized. A comprehensive understanding of these host-microbe interactions requires appreciation of the developmental context into which a microbe is introduced, as well as the effects of that microbial challenge on host ontogeny. In this review, we discuss how in vivo imaging and genetic analysis in zebrafish has advanced our knowledge of host-microbe interactions in the context of a developing vertebrate host. We focus on recent insights into immune cell ontogeny and function, commensal microbial relationships in the intestine, and microbial pathogenesis in zebrafish hosts., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The differentiation of endothelial cells is tightly connected with the formation of blood vessels during vertebrate development. The signaling pathways mediated by vascular endothelial growth factor (vegf) are required for these processes. Here we show that a proto-oncogene, meis1, plays important roles in the vascular development in zebrafish. Knockdown of meis1 by anti-sense meis1 morpholino (meis1 MO) led to the impairment of intersegmental vessel (ISV) formation. In meis1 morphants, the expression of an artery marker was reduced in dorsal aorta (DA), and the expression of vein markers was expanded in DA and posterior cardinal vein (PCV), suggesting the defects on artery development. Furthermore, the expression of vegf receptor, flk1, was significantly decreased in these embryos. Interestingly, flk1 MO-injected embryos exhibited similar defects as meis1 morphants. Thus, these results implicate that meis1 is a novel regulator involved in endothelial cell development, presumably affecting the vegf signaling pathway.

The infection of carp and other cyprinid fish with Trypanosoma danilewskyi was reported to cause significant morbidity and mortality in aquaculture. Tubulin is a component of parasite excretory/secretory (ES) products recognized by antibodies present in the serum of recovered hosts. To assess the role of parasite tubulin in the induction of a protective immune response in the goldfish, recombinant T. danilewskyi beta-tubulin was produced in Escherichia coli and used to immunize goldfish against challenge with live parasites. Affinity purified rabbit anti-recombinant tubulin IgG bound to both surface and internal structures of trypanosomes, and when added to parasite cultures caused a dose-dependent inhibition of their growth in vitro. Immunization of goldfish i.p. with either 40 microg or 80 microg of endotoxin-free beta-tubulin+Freund's complete adjuvant (FCA) caused a significant decrease in parasitemia during the establishment phase of the infection (days 3 and 7) and increased the time required to reach the maximal mean number of parasites compared to non-immunized sham-injected control fish. The serum from immune fish contained antibodies that recognized trypanosomes as determined by confocal immunofluorescence microscopy and specific antibodies that recognized recombinant tubulin as measured by ELISA. Thus, the immunization of goldfish with recombinant parasite beta-tubulin conferred partial antibody-mediated protection against a challenge infection with live trypanosomes. This is a first report that parasite tubulin is immunogenic in poikilothermic vertebrates.

The first leukocytes that arise in the development of vertebrate embryos are the primitive macrophages, which differentiate in the yolk sac and then quickly invade embryonic tissues. These macrophages have been considered to constitute a separate lineage, giving rise to no other cell type. Using an in vivo photoactivatable cell tracer in the transparent zebrafish (Danio rerio) embryo, we demonstrated that this lineage also gave rise to an equal or higher number of neutrophilic granulocytes. We were surprised to find that the differentiation of these primitive neutrophils occurs only after primitive myeloid progenitors have dispersed in the tissues. By 2 days after fertilization, these neutrophils have become the major leukocyte type found wandering in the epidermis and mesenchyme. Like the primitive macrophages, all primitive and larval neutrophils express PU.1 and L-plastin and they are highly attracted to local infections, yet only a small fraction of them phagocytose microbes, and to a much lesser extent per cell than the macrophages. They are also attracted to variously stressed or malformed tissues, suggesting a wider role than antimicrobial defense.

We have established an in vivo model for genetic analysis of the inflammatory response by generating a transgenic zebrafish line that expresses GFP under the neutrophil-specific myeloperoxidase promoter. We show that inflammation is induced after transection of the tail of zebrafish larvae and that this inflammation subsequently resolves over a similar time course to mammalian systems. Quantitative data can be generated from this model by counting of fluorescent cells or by digital image analysis. In addition, we show that the resolution of experimentally induced inflammation can be inhibited by the addition of a pancaspase inhibitor, zVD.fmk, demonstrating that experimental manipulation of the resolution of inflammation is possible in this model.

The discovery of antibiotics and other antimicrobial agents in the 1930s is arguably the most significant therapeutic advance in medical history. Penicillin and the sulfa drugs touched off the search for and discovery of countless derivative compounds and several new antibiotic classes. However, the pace of discovery has slowed down, and there is growing appreciation that much of the low-lying fruit accessible to traditional methods of antimicrobial discovery has been harvested. Combating emerging drug-resistant strains of infectious agents may require the adoption of fresh approaches to drug target validation, small-molecule discovery and safety assessment. The recent development of several infectious disease models in zebrafish raises the possibility of a new paradigm in antimicrobial discovery.

Goodpasture syndrome is an autoimmune vascular disease associated with kidney and lung failure, with pathogenic circulating autoantibodies targeted to a set of discontinuous epitope sequences within the noncollagenous domain-1 (NC1) of the alpha3 chain of type IV collagen (alpha3(IV)NC1), the Goodpasture autoantigen. We demonstrate that basement membrane extracted NC1 domain preparations from Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio do not bind Goodpasture autoantibodies, while Xenopus laevis, chicken, mouse and human alpha3(IV)NC1 domains bind autoantibodies. The alpha3(IV) chain is not present in C elegans and Drosophila melanogaster, but is first detected in the Danio rerio. Interestingly, native Danio rerio alpha3(IV)NC1 does not bind Goodpasture autoantibodies. Next, we cloned, sequenced, and generated recombinant Danio rerio alpha3(IV)NC1 domain. In contrast to recombinant human alpha3(IV)NC1 domain, there was complete absence of autoantibody binding to recombinant Danio rerio alpha3(IV)NC1. Three-dimensional molecular modeling from existing x-ray coordinates of human NC1 domain suggest that evolutionary alteration of electrostatic charge and polarity due to the emergence of critical serine, aspartic acid, and lysine residues, accompanied by the loss of asparagine and glutamine, contributes to the emergence of the 2 major Goodpasture epitopes on the human alpha3(IV)NC1 domain, as it evolved from the Danio rerio over 450 million years.