New Insights into IgZ as a Maternal Transfer Ig Contributing to the Early Defense of Fish against Pathogen Infection.

IgZ or its equivalent IgT is a newly discovered teleost specific Ig class that is highly specialized in mucosal immunity. However, whether this IgZ/IgT class participates in other biological processes remains unclear. In this study, we unexpectedly discovered that IgZ is highly expressed in zebrafish ovary, accumulates in unfertilized eggs, and is transmitted to offspring from eggs to zygotes. Maternally transferred IgZ in zygotes is found at the outer and inner layers of chorion, perivitelline space, periphery of embryo body, and yolk, providing different lines of defense against pathogen infection. A considerable number of IgZ+ B cells are found in ovarian connective tissues distributed between eggs. Moreover, pIgR, the transporter of IgZ, is also expressed in the ovary and colocalizes with IgZ in the zona radiata of eggs. Thus, IgZ is possibly secreted by ovarian IgZ+ B cells and transported to eggs through association with pIgR in a paracrine manner. Maternal IgZ in zygotes showed a broad bacteriostatic activity to different microbes examined, and this reactivity can be manipulated by orchestrating desired bacteria in water where parent fish live or immunizing the parent fish through vaccination. These observations suggest that maternal IgZ may represent a group of polyclonal Abs, providing protection against various environmental microbes encountered by a parent fish that were potentially high risk to offspring. To our knowledge, our findings provide novel insights into a previously unrecognized functional role of IgZ/IgT Ig in the maternal transfer of immunity in fish, greatly enriching current knowledge about this ancient Ig class.

Identification of the 14-3-3 ß/α-A protein as a novel maternal peptidoglycan-binding protein that protects embryos of zebrafish against bacterial infections.

14-3-3 proteins are widespread in animals, but their functions and mechanisms remain poorly defined. Here we clearly demonstrate that 14-3-3 ß/α-A is a newly identified PGN-binding protein present abundantly in the eggs/embryos of zebrafish. We also show that recombinant 14-3-3 ß/α-A acts as a pattern recognition receptor capable of identifying the bacterial signature molecule PGN, binding the bacteria, and functions as an antibacterial effector molecule directly killing the bacteria. Importantly, microinjection of r14-3-3 ß/α-A into early embryos significantly enhanced the resistance of the embryos against pathogenic A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-14-3-3 ß/α-A antibody. Collectively, these results indicate that 14-3-3 ß/α-A is a maternal PGN-binding protein that can protect the early embryos of zebrafish against pathogenic attacks, a novel role assigned to 14-3-3 ß/α-A proteins. This work also provides new insights into 14-3-3 proteins that are widely distributed in various animals.

The Zebrafish Perivitelline Fluid Provides Maternally-Inherited Defensive Immunity.

In the teleost egg, the embryo is immersed in an extraembryonic fluid that fills the space between the embryo and the chorion and partially isolates it from the external environment, called the perivitelline fluid (PVF). The exact composition of the PVF remains unknown in vertebrate animals. The PVF allows the embryo to avoid dehydration, to maintain a safe osmotic balance and provides mechanical protection; however, its potential defensive properties against bacterial pathogens has not been reported. In this work, we determined the global proteomic profile of PVF in zebrafish eggs and embryos, and the maternal or zygotic origin of the identified proteins was studied. In silico analysis of PVF protein composition revealed an enrichment of protein classes associated with non-specific humoral innate immunity. We found lectins, protease inhibitors, transferrin, and glucosidases present from early embryogenesis until hatching. Finally, in vitro and in vivo experiments done with this fluid demonstrated that the PVF possessed a strong agglutinating capacity on bacterial cells and protected the embryos when challenged with the pathogenic bacteria Edwardsiella tarda. Our results suggest that the PVF is a primitive inherited immune extraembryonic system that protects the embryos from external biological threats prior to hatching.

Effects of thermal stress-induced lead (Pb) toxicity on apoptotic cell death, inflammatory response, oxidative defense, and DNA methylation in zebrafish (Danio rerio) embryos.

Lead (Pb) is a toxic environmental pollutant that is frequently present in effluents from urban, mining, and industrial sources. The combinatorial effects of heavy metal exposure and temperature in aquatic organisms have received considerable attention as heat stress occurs simultaneously in conjunction with several contaminants in a natural environment. In this study, we examined the potential effects of Pb exposure in conditions of thermal stress (34 °C) in zebrafish (Danio rerio) embryos. Thermal stress at 34 °C induced a dramatic decrease in the survival rate, although exposure to Pb at 26 °C decreased the survival rate of the embryos. Malformations, such as the curved body shape, were increased in response to exposure to a combination of Pb and heat stress. The combination of Pb and heat stress also caused a decrease in the heart rate. Moreover, Pb and high-temperature exposure induced the upregulation of SOD, CAT, TNF-α, IL-1ß, p53, and BAX transcripts, and downregulation of Dnmt1 and Dnmt3b transcripts. Thermal stress enhanced transcriptional responses of eight indicator genes following Pb toxicity. The induction of cell death in response to combined exposures was also confirmed in the body of zebrafish by fluorescence intensity image analysis. These data indicated that thermal stress enhanced the poisonous effects of Pb exposure on antioxidant defense, inflammation, and apoptotic mechanisms. Transcriptional inhibition of DNA methylation-related genes might serve as a crucial factor contributing to the possibility of epigenetic adaptation by altering combined stress. We suggest that a careful evaluation of the potential effects of climate change (especially temperature) should be considered when investigating the toxic levels of metal pollution, such as Pb, in an aquatic environment.

Graphene oxide nanoparticles induce hepatic dysfunction through the regulation of innate immune signaling in zebrafish (Danio rerio).

Graphene oxide (GO) is an increasingly important nanomaterial that exhibits great promise in the area of bionanotechnology and nanobiomedicine. However, the toxic effects of GO on the vertebrate developmental system are still poorly understood. Here, we aimed to investigate the toxic effects and molecular mechanisms of GO exposure in larval and adult zebrafish. The results showed that the major hepatotoxic phenotype induced by GO in zebrafish embryos was a significant decrease in liver area and a dose-dependent decrease in the hepatocytes. Moreover, the number of macrophages and neutrophils in zebrafish embryos were reduced but the expressions of pro-inflammatory cytokines were increased after GO treatment. High through-put RNA-Seq identified 314 differentially expressed genes (DEGs) in GO-induced zebrafish embryos including 192 up-regulated and 122 down-regulated. KEGG and GO functional analysis revealed that steroid hormone biosynthesis, lipoprotein metabolic process, and PPAR signaling pathway were significantly enriched. Most of the lipid metabolism genes were down-regulated while majority of the immune genes were up-regulated after GO treatment. Moreover, GO induced NF-κB p65 into the nucleus and increased the protein levels of NF-κB p65, JAK2, STAT3, and Bcl2 in adult zebrafish liver. In addition, pharmacological experiments showed that inhibition of ROS and blocking the MAPK signaling could rescue the hepatotoxic phenotypes induced by GO exposure. On the contrary, pharmacological activation of PPAR-α expression have increased the hepatotoxic effects in GO-induced larval and adult zebrafish. Taken together, these informations demonstrated that GO induced hepatic dysfunction mainly through the ROS and PPAR-α mediated innate immune signaling in zebrafish.

The immunotoxicity and neurobehavioral toxicity of zebrafish induced by famoxadone-cymoxanil.

As a new protective and therapeutic fungicide, studies on famoxadone-cymoxanil are rare, and its toxicity to aquatic organisms has not been reported. In the present study, zabrafish embryos were exposed to several concentrations of famoxadone-cymoxanil at 10 hpf. Then, the changes of their shape, heart rate, development and function of innate and adaptive immune cells, oxidative stress, apoptosis, the expression of apoptosis-related genes and immune-related genes, the locomotor behavior were observed and detected in acute toxicity of famoxadone-cymoxanil. Our studies showed that, after exposure to famoxadone-cymoxanil, zebrafish embryos had decreased heart rate, shortened body length, swollen yolk sac. Secondly, the number of innate and adaptive immune cells was significantly reduced; and neutrophil migration and retention at the injury area were inhibited, indicating the developmental toxicity and immunotoxicity of famoxadone-cymoxanil on the zebrafish. We also found that the oxidative stress related indicators of embryos were changed significantly, and apoptosis were substantially increased. Further investigation of changes of some key genes in TLR signaling including TLR4, MYD88 and NF-κB p65 revealed that the mRNA expression of these genes was up-regulated. Meanwhile, the mRNA expression of some proinflammatory cytokines such as TNF-α, IFN-γ, IL6 and IL-1ß was also up-regulated. In addition, the activity, the total distance, time and average speed were decreased along with the increase of exposure concentration. The absolute turn angle, sinuosity and the enzymatic activity of acetylcholinesterase (AChE) were also increased. These results suggested that famoxadone-cymoxanil can induce developmental toxicity, immunotoxicity and neurobehavioral toxicity in zebrafish larvae.

Prokaryotic ribosomal RNA stimulates zebrafish embryonic innate immune system.

OBJECTIVES: Cell-culture studies reported that prokaryotic RNA molecules among the various microbe-associated molecular patterns (MAMPs) were uniquely present in live bacteria and were categorized as viability-associated MAMPs. They also reported that specific nucleotide modifications are instrumental in the discrimination between self and nonself RNAs. The aim of this study was to characterize the in vivo immune induction potential of prokaryotic and eukaryotic ribosomal RNAs (rRNAs) using zebrafish embryos as novel whole animal model system. Additionally, we aimed to test the possible role of rRNA modifications in immune recognition. RESULTS: We used three immune markers to evaluate the induction potential of prokaryotic rRNA derived from Escherichia coli and eukaryotic rRNAs from chicken (nonself) and zebrafish (self). Lipopolysaccharide (LPS) of Pseudomonas aeruginosa served as a positive control. E. coli rRNA had an induction potential equivalent to that of LPS. The zebrafish innate immune system could discriminate between self and nonself rRNAs. Between the nonself rRNAs, E. coli rRNA was more immunogenic than chicken rRNA. The in vitro transcript of zebrafish 18S rRNA gene without the nucleotide modifications was not recognized by its own immune system. Our data suggested that prokaryotic rRNA is immunostimulatory in vivo and could be useful as an adjuvant.

Effects of norfloxacin nicotinate on the early life stage of zebrafish (Danio rerio): Developmental toxicity, oxidative stress and immunotoxicity.

Norfloxacin nicotinate (NOR-N), an adduct of norfloxacin (NOR) and nicotinic acid, has been widely used for replacing NOR in animal husbandry and fishery industry. Nowadays, increasing evidences showed that NOR could pose toxic effects on fish and other aquatic organisms, but as its adduct, whether NOR-N could cause adverse effects on aquatic organisms is still unclear. To evaluate the toxic effects of NOR-N on the early life stage of zebrafish, we determined the changes in embryonic development (hatching rate, body length, malformation rate and mortality), antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx)) activities, malondialdehyde (MDA) content and gene expression levels related to antioxidant enzymes (Cu/Zn-sod, Mn-sod, CAT and Gpx) and innate immune system (tumor necrosis factor α (TNFα), interferon (IFN), Interleukin-1 beta (IL-1ß), IL-8, CXCL-clc, CC-chemokine, lysozyme (Lzy) and complement factors (C3)) after embryonic exposure to NOR-N till 96 hpf. The results showed that NOR-N exposure could decreased the hatching rate and body length, and increased abnormality and mortality as concentration-dependent during embryonic development process. NOR-N induced oxidative stress in zebrafish larvae through increasing the contents of MDA and the activities of SOD, CAT and Gpx, as well as the mRNA levels of genes related to these antioxidant enzymes. Moreover, the expression of TNFα, IFN, IL-1ß, IL-8, CXCL-clc, CC-chemokine, Lzy and C3 genes were significantly up-regulated after exposure to high concentration (5 and/or 25 mg/L) of NOR-N till 96 hpf, indicating that the innate immune system in zebrafish larvae was disturbed by NOR-N. Overall, our results suggested that NOR-N caused development toxicity, oxidative stress and immunotoxicity on the early life stage of zebrafish. Thus, widespread application of NOR-N might pose potential ecotoxicological risk on aquatic ecosystems.

Effects of spinetoram on the developmental toxicity and immunotoxicity of zebrafish.

Our study investigated the effects of spinetoram on the developmental toxicity and immunotoxicity of zebrafish. 10 h post-fertilization (hpf) zebrafish embryos were exposed to several concentrations of spinetoram (0, 5.0 mg/L, 7.5 mg/L, 10 mg/L) for up to 96 hpf, and their mortality, heart rate, number of innate and adaptive immune cells, oxidative stress, apoptosis and gene expression were detected. Studies indicated that the spinetoram exposed zebrafish embryos showed yolk sac edema, slow growth, decreased heart rate, decreased number of immune cells, delayed thymic development and cell apoptosis. In addition, there were also significant changes in oxidative stress related indicators in zebrafish, the content of ROS and MDA and the activity of CAT and SOD increased with the increase of spinetoram concentration. Moreover, we detected the expression of TLR4 related genes including TLR4, MYD88 and NF-κB p65 which were significantly up-regulated in the treated groups. Meanwhile, we also found that pro-inflammatory factors IL-6, IL-8, IFN-γ and CXCL-c1c were up-regulated, but anti-inflammatory factor IL-10 was down-regulated in the treated groups. Briefly, our results show that spinetoram induces the developmental toxicity and immunotoxicity of zebrafish to a certain extent, providing basis for the further research on the molecular mechanism of spinetoram exposure to aquatic ecosystems.

Bifenthrin induces developmental immunotoxicity and vascular malformation during zebrafish embryogenesis.

Bifenthrin is a synthesized pyrethroid insecticide which is frequently used in the farmland to eradicate insects. Bifenthrin mainly disrupts sodium ion channel inducing neurotoxicity in the target insects. It also exerts toxic effects such as hormone dysregulation, hepatotoxicity and immunotoxicity in other vertebrates. However, there is no evidence of the acute-toxicity associated embryogenesis and organogenesis of bifenthrin in zebrafish. Here we first demonstrated that bifenthrin induced acute-toxicity accompanying inflammatory response and physiological degradations resulting in loss of embryogenesis and vascular development in zebrafish embryos. We found that bifenthrin increased intestinal ROS accumulation and the inflammatory genes including tnfa, il6, il8 and ptgs2b, thereby increasing embryo mortality. Moreover, bifenthrin disrupted angiogenesis by down-regulation of VEGF receptors in embryos. Not only in the zebrafish, bifenthrin also decreased cell viability and hampered vascular formation of HUVECs. Collectively, bifenthrin induced developmental toxicity, inflammatory cell death and anti-angiogenesis during embryogenesis.

Chemical constituents from Alismatis Rhizoma and their anti-inflammatory activities in vitro and in vivo.

Six new compounds, including a new compound with an unusual 2, 4, 6-cycloheptatrien ketone skeleton (1), two new diphenylpropanoid ethers (2, 3), a new protostane-type triterpenoid (4), two new norsesquiterpene (5a, 5b), and two new natural products (6, 7), together with eleven known compounds (8-18) were isolated from the aqueous extract of Alismatis Rhizoma (AR). Their structures were elucidated by a combination of 1D and 2D NMR (1H and 13C NMR, COSY, HSQC, HMBC, and NOESY), HRESIMS spectroscopic data, experimental and calculated electronic circular dichroism (ECD) spectra. Some of the compounds were evaluated for their inhibitory effects on nitric oxide (NO) production in LPS-induced RAW 264.7 cells. Two protostane-type triterpenoids, compounds 4 and 17, exhibited potent inhibitory activities with the IC50 values of 39.3 and 63.9 µM compared with indomethacin. In the meanwhile, their anti-inflammatory effects were also confirmed by acute inflammation model induced by CuSO4 in zebrafish.

The role of infectious hematopoietic necrosis virus (IHNV) proteins in recruiting the ESCRT pathway through three ways in the host cells of fish during IHNV budding.

In cytokinetic abscission, phagophore formation, and enveloped virus budding are mediated by the endosomal sorting complex required for transport (ESCRT). Many retroviruses and RNA viruses encode "late-domain" motifs that can interact with the components of the ESCRT pathway to mediate the viral assembly and budding. However, the rhabdovirus in fish has been rarely investigated. In this study, inhibition the protein expression of the ESCRT components reduces the extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in IHNV release. The respective interactions of IHNV proteins including M, G, L protein with Nedd4, Tsg101, and Alix suggest the underlying molecular mechanism by which IHNV gets access to the ESCRT pathway. These results are the first observation that rhabdovirus in fish gains access to the ESCRT pathway through three ways of interactions between viral proteins and host proteins. In addition, the results show that IHNV is released from host cells through the ESCRT pathway. Taken together, our study provides a theoretical basis for studying the budding mechanism of IHNV.

Exposure to diclofop-methyl induces immunotoxicity and behavioral abnormalities in zebrafish embryos.

Diclofop-methyl (DM) is widely used in agriculture and may lead to serious toxicity. However, a limited number of studies have been performed to evaluate the toxicity of DM in the immune and nervous systems of animals. Here, we utilized a good vertebrate model, zebrafish, to evaluate the toxicity of DM during the developmental process. Exposure of zebrafish embryos to 0.1, 0.3 and 0.5 mg/l DM from 6 h post fertilization (hpf) to 72 hpf induced developmental abnormalities, such as shorter body lengths and yolk sac edemas. The number of immune cells in zebrafish larvae was significantly reduced, but the inflammatory response was not influenced by DM treatment. The expression of immune-related genes were downregulated and the levels of oxidative stress were upregulated by DM exposure. Moreover, locomotor behaviors were inhibited by DM exposure. Therefore, our results suggest that DM has the potential to induce immunotoxicity and cause behavioral changes in zebrafish larvae. This study provides new evidence of the influence of DM exposure on aquatic ecosystems.

The biodistribution and immuno-responses of differently shaped non-modified gold particles in zebrafish embryos.

Important questions raised in (nano)ecotoxicology are whether biodistribution of nanoparticles (NPs) is affected by particle shape and to what extent local adverse responses are subsequently initiated. For nanomedicine, these same questions become important when the labeled NPs lose the labeling. In this study, we investigated the biodistribution patterns of gold nanoparticles (AuNPs) as well as immune-related local and systemic sublethal markers of exposure and behavioral assessment. Hatched zebrafish embryos were exposed to four differently shaped non-coated AuNPs with comparable sizes: nanospheres, nanorods, nano-urchins, and nano-bipyramids. Shape-dependent trafficking of the particles resulted in a different distribution of the particles over the target organs. The differences across the distribution patterns indicate that the particles behave slightly different, although they eventually reach the same target organs - yet in different ratios. Mainly local induction of the immune system was observed, whereas systemic immune responses were not clearly visible. Macrophages were found to take AuNPs from the body fluid, be transferred into the veins and transported to digestive organs for clearance. No significant behavioral toxicological responses in zebrafish embryos were observed after exposure. The trafficking of the particles in the macrophages indicates that the particles are removed via the mononuclear phagocytic system. The different ratios in which the particles are distributed over the target organs indicate that the shape influences their behavior and eventually possibly the toxicity of the particles. The observed shape-dependent biodistribution patterns might be beneficial for shape-specific targeting in nanomedicine and stress the importance of incorporating shape-features in nanosafety assessment.

Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model.

Cystic fibrosis (CF) is a hereditary disease due to mutations in the CFTR gene and causes mortality in humans mainly due to respiratory infections caused by Pseudomonas aeruginosa. In a previous work we used phage therapy, which is a treatment with a mix of phages, to actively counteract acute P. aeruginosa infections in mice and Galleria mellonella larvae. In this work we apply phage therapy to the treatment of P. aeruginosa PAO1 infections in a CF zebrafish model. The structure of the CFTR channel is evolutionary conserved between fish and mammals and cftr-loss-of-function zebrafish embryos show a phenotype that recapitulates the human disease, in particular with destruction of the pancreas. We show that phage therapy is able to decrease lethality, bacterial burden, and the pro-inflammatory response caused by PAO1 infection. In addition, phage administration relieves the constitutive inflammatory state of CF embryos. To our knowledge, this is the first time that phage therapy is used to cure P. aeruginosa infections in a CF animal model. We also find that the curative effect against PAO1 infections is improved by combining phages and antibiotic treatments, opening a useful therapeutic approach that could reduce antibiotic doses and time of administration.

Environmental concentrations of triclosan activate cellular defence mechanism and generate cytotoxicity on zebrafish (Danio rerio) embryos.

Triclosan (TCS, 5­chloro­2­(2,4­dichlorophenoxy) 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 glutathione­S­transferase (GST), P­glycoprotein efflux and ethoxyresorufin­o­deethylase (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.

Expression of the alternative splicing variants of bcl6b in medaka Oryzias latipes.

Bcl6B, also known as BAZF, plays important roles in the immune response, repression of cancers, and maintenance of spermatogonial stem cells in mammals. In this study, the homologous gene bcl6b and its 5 alternative splicing variants, namely bcl6bX1 to bcl6bX5, were isolated from medaka fish, Oryzias latipes. Medaka bcl6b possesses conserved domains such as BTB domain, RD2 domain and four zinc fingers. Medaka bcl6bX1 to bcl6bX3 possess all three previously mentioned domains with minor differences in sequences. Medaka bcl6bX4 possesses only the BTB domain due to premature stopping, and bcl6bX5 possesses both the BTB domain and zinc fingers without the RD2 domain. Medaka bcl6b was expressed in the tissues including the brain, heart, gill, muscle, spleen, kidney, intestine, ovary and testes of adult fish. Medaka bcl6b was expressed in the embryos from very early stage, and could be detected clearly in the developing eyes by RT-PCR and in situ hybridization. Medaka bcl6b could respond to the stimuli of polyI:C and LPS in the kidney and spleen. Medaka bcl6bX1 to bcl6bX3 were the majority of the variants expressed in the adult tissues and the embryos, and were the major response to the stimulation of polyI:C and LPS in the spleen. These results suggested that bcl6b, including its isoforms, could function in various tissues and embryogenesis. Moreover, bcl6b might be a factor for immune response in medaka.

Developmental neurotoxicity and immunotoxicity induced by graphene oxide in zebrafish embryos.

Graphene oxide (GO) has emerged as the worldwide promising candidate for biomedical application, such as for drug delivery, bio-sensing and anti-cancer therapy. This study was focused on the zebrafish and RAW264.7 cell line as in vivo and in vitro models to assess the potential developmental neurotoxicity and immunotoxicity of GO. No obvious acute developmental toxicity was observed upon treatments with 0.01, 0.1, and 1 µg/mL GO for five consecutive days. However, decreased hatching rate, increased malformation rate, heart beat rate and hypoactivity of locomotor behavior were detected when exposed to 10 µg/mL GO. Also, RT-PCR analysis revealed that expressions of genes related to the nervous system were up-regulated. The potential risk of GO for developmental neurotoxicity may be ascribed to the high level of oxidative stress induced by high concentration of GO. Most importantly, the mRNA levels of immune response associated genes, such as interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ) were significantly increased under environmental concentration exposure. The activation of pro-inflammatory immune response was also observed in macrophage cell line. Taken together, our results demonstrated that immunotoxicity is a sensitive indicator for assessment of bio-compatibility of GO.

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy.

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

Palmitate induces nitric oxide production and inflammatory cytokine expression in zebrafish.

Inflammation markers in zebrafish embryos reflect a toxic response that is common to other animal models and humans. Free fatty acids (FFAs) are known to cause damage in various tissues by inducing inflammation. In this study, we investigated whether a FFA (palmitate) induces inflammation in zebrafish embryos. Nitrous oxide (NO) production and cyclooxygenase-2 (COX-2) mRNA expression were increased in palmitate-treated zebrafish embryos in a dose-dependent manner. mRNA expression of pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF- α), were also increased. Additionally, the mRNA expression of p65 nuclear factor-kB and I-kB-α were significantly increased after palmitate-treatment. Increased reactive oxygen species (ROS) expression was observed in palmitate-treated zebrafish embryos as well as pericardial edema. Additionally, mRNA expression of pro-inflammatory cytokines were increased in zebrafish liver and pancreas fed with palmitate-contained diet. Taken together, these results indicated that palmitate increases pro-inflammatory mediators in zebrafish embryos, suggesting that zebrafish could be an alternative animal model for inflammatory disease including diabetes.