Identification of distinct LRC- and Fc receptor complex-like chromosomal regions in fish supports that teleost leukocyte immune-type receptors are distant relatives of mammalian Fc receptor-like molecules.

Leukocyte immune-type receptors (LITRs) are a large family of immunoregulatory receptor-types originally identified in the channel catfish (Ictalurus punctatus (Ip)LITRs). Phylogenetic analyses of LITRs show that they share distant evolutionary relationships with important mammalian immunoregulatory receptors belonging to the Fc receptors family and the leukocyte receptor complex (LRC), but their syntenic relationships with these immunoglobulin superfamily members have not been investigated. To further examine the possible evolutionary connections between teleost LITRs and various mammalian immunoregulatory receptor-types, we surveyed the genomic databases of representative vertebrate taxa and our results show that teleost LITRs generally exist in large genomic clusters, which are linked to vangl2, arhgef11, and slam family genes, features that are also shared by amphibian and mammalian Fc receptor-like molecules (FCRLs). Moreover, detailed phylogenetic comparisons between the individual Ig-like domains of LITRs and mammalian FCRLs shows that these receptors share related Ig-like domains indicative of their common ancestry. However, contrary to our previous reports, no supportive evidence for phylogenetic relationships between the Ig-like domains of LITRs with the Ig-like domains of LRC-encoded mammalian immunoregulatory receptors was found. We also identified an LRC-like region in the zebrafish genome, but no expanded litr-related genes were located in this region. Similarly, no lilr-related genes were found in spotted gar, a representative basal ray-finned fish. Finally, two distantly related fcrls and an LRC-like gene were identified in the elephant shark genome, suggesting that the loss of an immunoregulatory receptor-containing LRC region may be unique to ray-finned fish.

Identification of goldfish (Carassius auratus L.) leukocyte immune-type receptors shows alternative splicing as a potential mechanism for receptor diversification.

Leukocyte immune-type receptors (LITRs) are a multigene family of teleost immunoregulatory proteins that share structural, phylogenetic, and likely functional relationships with several innate immune receptor proteins in other vertebrates, including mammals. Originally discovered in channel catfish (Ictalurus punctatus), representative IpLITR-types have been shown to regulate diverse innate immune cell effector responses including phagocytosis, degranulation, and cytokine secretion. To date, IpLITRs have been primarily characterized using mammalian cell line expression systems, therefore many unanswered questions remain regarding their actual regulatory roles in fish immunity. In the present study, we report on the preliminary molecular characterization of five goldfish (Carassius auratus) CaLITR-types and the identification of several putative splice variants of these receptors cloned from various goldfish tissues and primary myeloid cell cultures. In general, CaLITR mRNA transcripts were detected in all goldfish tissues tested, and also in primary kidney macrophage and neutrophil cultures. Specifically, CaLITR1 is a functionally ambiguous receptor with no charged amino acids in its transmembrane (TM) segment and is devoid of tyrosine-based signaling motifs in its short cytoplasmic tail (CYT) region. CaLITR2 is a putative activating receptor-type that contains immunotyrosine-based activation motifs (ITAMs) within its long CYT region, and CaLITR3 has a positively charged TM segment, suggesting that it may recruit intracellular stimulatory adaptor signaling molecules. CaLITR4 and CaLITR5 appear to have diverse signaling capabilities since they contain various immunoregulatory signaling motifs within their CYT regions including putative Nck and STAT recruitment motifs as well as ITAM-like and ITIM sequences. We also identified putative CaLITR splice variants with altered extracellular Ig-like domain compositions and variable CYT regions. Interestingly, this suggests that alternative splicing-mediated diversification of CaLITRs can generate receptor forms with possible variable binding and/or intracellular signaling abilities. Overall, these findings reveal new information about the teleost LITRs and sets the stage for exploring how alternative splicing leads to the functional diversification of this complex multigene immunoregulatory receptor family.

Inorganic fraction of oil sands process-affected water induces mammalian macrophage stress gene expression and acutely modulates immune cell functional markers at both the gene and protein levels.

The focus of the present study was to examine the acute immunotoxic properties of oil sands process-affected waters (OSPW) using the RAW 264.7 macrophage cell line. Specifically, we used a quantitative PCR assay to monitor changes in the expression of stress, cytokine, and antimicrobial enzyme genes in activated macrophages following acute (i.e. < 24 h) exposure of the cells to whole OSPW and its fractions. Overall, our data shows that OSPW inorganic fraction (IF) significantly induces the expression of genes associated with oxidative stress and DNA damage and that the OSPW-IF also significantly augmented cytokine gene expression. These effects are similar to what was observed following whole OSPW exposures, which contrasts the minimal effects observed when cells were treated with equivalent doses of the OSPW organic fraction (OF). Surprisingly, OSPW-IF had reciprocal effects on gene and protein expression levels of two key macrophage enzymes (e.g. inducible nitric oxide (iNOS) synthase and arginase), which indicates that components within OSPW-IF have the unique ability to alter the overall functional states of macrophage by polarizing them towards an alternatively activated status; concomitant with the reciprocal depression of iNOS levels and enhanced expression and activity of arginase. Collectively, these findings show that at sub-lethal exposure doses, the inorganic constituents of OSPW have significant immunotoxicological properties that could potentially affect innate cellular defense responses of exposed animals.

Separation of oil sands process water organics and inorganics and examination of their acute toxicity using standard in-vitro bioassays.

Oil sands process water (OSPW) contains complex components of inorganics and organics. This is the first study that separated OSPW inorganic and organic fractions and examined their relative acute toxicity when compared with the original whole OSPW using an in-vitro cell-based bio-indicator system. The separation of OSPW inorganic and organic fractions would be conducive to the understanding of the toxic contribution of organic and inorganic fractions as well as the identification and treatment of organic fractions. In this research, we demonstrated that the highest organic fraction extraction was obtained using HLB solid phase extraction with 95.4 ±â€¯0.7% of dissolved organic carbon (DOC) and 90.0 ±â€¯5.3% of naphthenic acid (NA) recovered from OSPW, which were higher than those obtained using the traditional dichloromethane liquid-liquid extraction (48.8 ±â€¯0.2% of DOC and 81.0 ±â€¯2.6% of NA recovery) or other SPE cartridges tested. We also reported the first isolation method for OSPW inorganic fraction by removing 96.1 ±â€¯0.2% of DOC in OSPW using granular activated carbon. The difference of other parameters such as pH, alkalinity, conductivity, and concentration of detected ions between OSPW and isolated inorganic fraction was negligible. The acute toxicity of whole OSPW, separated OSPW inorganic fraction and organic fraction, and the reconstituted fractions were assessed using in-vitro bioassays with RAW 264.7 mouse macrophage cell lines. OSPW organic fraction demonstrated significant cytotoxicity at 14 mg/L O2-NA and affected the cellular metabolic activity at 10 mg/L of O2-NAs. No significant cytotoxicity or effect on cellular metabolic activity was observed for whole OSPW, OSPW inorganic fraction and the reconstituted fractions. Overall, this study provides the procedure for the isolation of the major components of OSPW (i.e., organics and inorganics), which allows the assessment of their relative toxicological effects using a standard in-vitro bioassay and would allow more accurate characterization and treatment study for each fraction in OSPW.

Exposure to Organic Fraction Extracted from Oil Sands Process-Affected Water Has Negligible Impact on Pregnancy and Lactation of Mice.

Dissolved organic compounds are major contaminants in oil sands process-affected water (OSPW), of which naphthenic acids (NAs) are one of the main persistent toxicants. In the present study, we explore the toxic effects of the organic fraction extracted from OSPW (OSPW-OF) in mice during pregnancy and lactation. Here, we report that acute oral exposure of female Balb/c mice during gestation, and subchronic exposure throughout gestation and lactation to OSPW-OF (containing naturally occurring levels of NAs found in tailings ponds), had negligible effects on their reproductive performance. Specifically, mating behavior, pregnancy success, embryonic implantation, gestation period, litter size, and offspring viability were not affected by OSPW-OF containing up to 55 mg/L NAs. OSPW-OF exposure also did not affect plasma concentrations of pregnancy-associated hormones or pro- and anti-inflammatory cytokines, and it had minimal effects on liver stress gene expression. This study presents the first comprehensive in vivo analysis of mammalian toxicity associated with OSPW-OF exposure. Overall, our results suggest that the risk of acute and subchronic toxicity to mice exposed to OSPW-OF at environmentally relevant concentrations of NAs in contaminated drinking water is likely negligible.

Trypanosoma carassii infection in goldfish (Carassius auratus L.): changes in the expression of erythropoiesis and anemia regulatory genes.

Trypanosoma carassii is a flagellated bloodstream parasite of cyprinid fish with pathogenesis manifesting primarily as anemia in experimentally infected fish. This anemia is characterized by decreases in the number of circulating red blood cells (RBCs) during peak parasitemia. We examined changes in the key blood metrics and expression of genes known to be important in the regulation of erythropoiesis. Increasing parasitemia was strongly correlated with an overall decrease in the total number of circulating RBCs. Gene expression of key erythropoiesis regulators (EPO, EPOR, GATA1, Lmo2, and HIFα) and proinflammatory cytokines (IFNγ and TNFα) were measured and their expressions differed from those in fish made anemic by injections of phenylhydrazine (PHZ). Significant upregulation of pro-erythropoietic genes was observed in PHZ-induced anemia, but not during peak parasitic infection. Previously, we reported on functional characterization of goldfish erythropoietin (rgEPO) and its ability to induce survival and differentiation of erythroid progenitor cells in vitro. Treatment of goldfish during the infection with rgEPO reduced the severity of anemia but failed to fully prevent the onset of the anemic state in infected fish. Proinflammatory cytokines have been implicated in the suppression of erythropoiesis during trypanosomiasis, specifically the cytokines TNFα, IFNγ, and IL-1ß. Analysis of key proinflammatory cytokines revealed that mRNA levels of IFNγ and TNFα were upregulated in response to infection, but only TNFα increased in response to PHZ treatment. Synergistic activity of the proinflammatory cytokines may be required to sustain prolonged anemia. These findings provide insight into the relationship between T. carassii and host anemia and suggest that T. carassii may directly or indirectly suppress host erythropoiesis.

Teleost contributions to the understanding of mycobacterial diseases.

Few pathogens have shaped human medicine as the mycobacteria. From understanding biological phenomena driving disease spread, to mechanisms of host-pathogen interactions and antibiotic resistance, the Mycobacterium genus continues to challenge and offer insights into the basis of health and disease. Teleost fish models of mycobacterial infections have progressed significantly over the past three decades, now supplying a range of unique tools and new opportunities to define the strategies employed by these Gram-positive bacteria to overcome host defenses, as well as those host antimicrobial pathways that can be used to limit its growth and spread. Herein, we take a comparative perspective and provide an update on the contributions of teleost models to our understanding of mycobacterial diseases.

Teleost antimicrobial peptide hepcidin contributes to host defense of goldfish (Carassius auratus L.) against Trypanosoma carassii.

Hepcidin is an antimicrobial peptide and an iron regulatory protein that prevents the release of excess iron in the blood. There is evidence suggesting that teleost hepcidin is a major player in antimicrobial defense against various bacteria species, but little is known regarding the effects of teleost hepcidin in protozoan parasitic infections. We examined the role of hepcidin during the course of infection of goldfish with Trypanosoma carassii. Quantitative real-time PCR was used to determine the expression of hepcidin in goldfish immune organs during the course of T. carassii infection. During the acute phase of the T. carassii infection, the mRNA levels of hepcidin were up-regulated in liver and kidney. In contrast, an up-regulation of hepcidin mRNA expression in spleen was observed during the chronic phase of the infection. Furthermore, a synthetic goldfish hepcidin peptide induced trypanosome lysis in vitro, and parasite surface disruption was confirmed by scanning electron microscopy (SEM) analysis. These results suggest that, in addition to well-characterized direct antibacterial activities, teleost hepcidin also exhibits trypanocidal activity.

Mechanisms of Fish Macrophage Antimicrobial Immunity.

Overcrowding conditions and temperatures shifts regularly manifest in large-scale infections of farmed fish, resulting in economic losses for the global aquaculture industries. Increased understanding of the functional mechanisms of fish antimicrobial host defenses is an important step forward in prevention of pathogen-induced morbidity and mortality in aquaculture setting. Like other vertebrates, macrophage-lineage cells are integral to fish immune responses and for this reason, much of the recent fish immunology research has focused on fish macrophage biology. These studies have revealed notable similarities as well as striking differences in the molecular strategies by which fish and higher vertebrates control their respective macrophage polarization and functionality. In this review, we address the current understanding of the biological mechanisms of teleost macrophage functional heterogeneity and immunity, focusing on the key cytokine regulators that control fish macrophage development and their antimicrobial armamentarium.

Characterization and functional assessment of the NLRC3-like molecule of the goldfish (Carassius auratus L.).

The NLRC3-like (NLRC3L) molecule from the goldfish transcriptome database was identified and characterized. Quantitative gene expression analysis revealed the highest mRNA levels of NLRC3L were in the spleen and intestine, with lower mRNA levels observed in muscle and liver. Goldfish NLRC3L was differentially expressed in goldfish immune cell populations with highest mRNA levels measured in PBLs and macrophages. We generated a recombinant form of the molecule (rgfNLRC3L) and an anti-CT-NLRC3L IgG. Treatment of goldfish primary kidney macrophages in vitro with ATP, LPS and heat-killed Aeromonas salmonicida up-regulated the NLRC3L mRNA and protein. Confocal microscopy and co-immunoprecipitation assays indicated that goldfish rgfNLRC3L interacted with apoptosis-associated spec-like protein (ASC) in eukaryotic cells, indicating that NLRC3L may participate in the regulation of the inflammasome responses. The dual-luciferase reporter assay showed that NLRC3L over-expression did not cause the activation of NF-κB, but that it cooperated with RIP2 to down-regulate NF-κB activation. Our results indicate that the NLRC3L may function as a regulator of NLR pathways in teleosts.

Comparison of the Acute Immunotoxicity of Nonfractionated and Fractionated Oil Sands Process-Affected Water Using Mammalian Macrophages.

OSPW is a complex mixture of inorganic and organic substances and its principal toxic components have yet to be fully characterized. Previously, we showed in vitro that the oil sands process-affected water (OSPW) organic fraction (OF) caused a concentration-dependent immunotoxicity in mammals. In the present study we further explore the immunotoxicological properties of OSPW in mammals using a series of in vitro bioassays. Specifically, using the RAW 264.7 mouse macrophage cell line we show that whole OSPW containing naphthenic acid (NA) concentrations ranging from 12 to 18 mg/L, significantly inhibited cell proliferation, reduced cell viability, and was directly cytotoxic, whereas the exposure of cells to equivalent doses of the OSPW-OF had no measurable effects. Whole OSPW exposures also caused morphological changes in RAW 264.7 cells, and at sublethal doses (i.e., 10 mg/L) it induced the early expression of the stress genes hmox1 and gadd45. In addition, at NA concentrations of 10 mg/L, whole OSPW but not the OSPW-OF had significant effects on pro-inflammatory cytokine mRNA levels and cytokine protein secretion activities. Finally, whole OSPW also impaired the ability of RAW 264.7 cells to perform phagocytosis. Overall, we demonstrate that exposure to whole OSPW (at NA doses ranging from 10 to 20 mg/L), but not the OSPW-OF caused both cytotoxic and immunomodulatory changes in mouse macrophages. This suggests that the complex mixture of inorganic and organic components found in whole OSPW are acutely toxic at much lower doses than we previously reported for the OSPW-OF (i.e., 50 mg/L) due to unknown additive and/or synergistic interactions that likely occur between the various components present in whole OSPW.

The toxicity of oil sands process-affected water (OSPW): A critical review.

Large volumes of oil sands process-affected water (OSPW) are produced by the surface-mining oil sands industry in Alberta. Both laboratory and field studies have demonstrated that the exposure to OSPW leads to many physiological changes in a variety of organisms. Adverse effects include compromised immunological function, developmental delays, impaired reproduction, disrupted endocrine system, and higher prevalence of tissue-specific pathological manifestations. The composition of OSPW varies with several factors such as ore sources, mining process, and tailings management practices. Differences in water characteristics have confounded interpretation or comparison of OSPW toxicity across studies. Research on individual fractions extracted from OSPW has helped identify some target pollutants. Naphthenic acids (NAs) are considered as the major toxic components in OSPW, exhibiting toxic effects through multiple modes of action including narcosis and endocrine disruption. Other pollutants, like polycyclic aromatic hydrocarbons (PAHs), metals, and ions may also contribute to the overall OSPW toxicity. Studies have been conducted on OSPW as a whole complex effluent mixture, with consideration of the presence of unidentified components, and the interactions (potential synergistic or antagonistic reactions) among chemicals. This review summarizes the toxicological data derived from in vitro and in vivo exposure studies using different OSPW types, and different taxa of organisms. In general, toxicity of OSPW was found to be dependent on the OSPW type and concentration, duration of exposures (acute versus sub chronic), and organism studied.

Recombinant IL-4/13A and IL-4/13B induce arginase activity and down-regulate nitric oxide response of primary goldfish (Carassius auratus L.) macrophages.

We report on the expression analysis and functional characterization of IL-4/13A and IL-4/13B in goldfish. Quantitative analysis indicated the highest expression in the heart, spleen, brain, and kidney, with comparable expression patterns for both IL-4/13A and IL-4/13B. The mRNA levels of IL-4/13A and IL-4/13B in the immune cells examined were highest in macrophage and monocytes. Assessment of spleen mRNA following infection with Trypanosoma carassii, a prominent protozoan pathogen of fish, revealed decrease in IL-4/13B and arginase expression 14 days post infection, followed by an increase in IL-4/13B and arginase-2 at 28 days post infection. Recombinant forms of IL-4/13A and IL-4/13B induced an increase in arginase activity in macrophages in a dose-dependent manner. Recombinant IL-4/13A and IL-4/13B also induced significant increase in mRNA levels of arginase -2 in macrophages at 6, 12, 18 and 24 h after treatment. Furthermore, treatment with both IL-4/13 recombinants interfered with the IFNγ-induced nitric oxide response of macrophages. Our results suggest a conserved role of IL-4/IL-13 in induction of alternative activation phenotype in teleost macrophages.

Functional characterization of apoptosis-associated speck-like protein (ASC) of the goldfish (Carassius auratus L.).

Quantitative expression analysis of goldfish ASC indicated the highest and lowest mRNA levels in spleen and muscle, respectively. The ASC was differentially expressed in normal goldfish tissues and different immune cell populations. The highest ASC mRNA levels were observed in the spleen and macrophages. We generated a recombinant form of the molecule (rgfASC) and an anti-ASC IgG antibody, and report that treatment of goldfish macrophages with nigericin, an inducer of inflammasome pathway, up-regulated the expression of ASC at both mRNA and protein levels. rgfASC aggregated to form multimers in cross-linking assays, and formed speck-like structures visualized by confocal microscopy. Co-immunoprecipitation assays showed that rgfASC interacted with caspase-1 and receptor-interacting serine/threonine kinase 2 (RIP2). The dual luciferase reporter assay showed that ASC over-expression did not cause the activation of NF-κB directly, but down-regulated RIP2 ability to activate NF-κB. Goldfish ASC was found to interact with both Nod-like receptor and inflammasome signaling pathway molecules, suggesting multifunctional roles for ASC in regulation of different NLR signaling pathways and eventual proinflammatory cytokine production by activated macrophages.

Pilot-scale UV/H2O2 advanced oxidation process for municipal reuse water: Assessing micropollutant degradation and estrogenic impacts on goldfish (Carassius auratus L.).

Low concentrations (ng/L-µg/L) of emerging micropollutant contaminants in municipal wastewater treatment plant effluents affect the possibility to reuse these waters. Many of those micropollutants elicit endocrine disrupting effects in aquatic organisms resulting in an alteration of the endocrine system. A potential candidate for tertiary municipal wastewater treatment of these micropollutants is ultraviolet (UV)/hydrogen peroxide (H2O2) as an advanced oxidation process (AOP) which was currently applied to treat the secondary effluent of the Gold Bar Wastewater Treatment Plant (GBWWTP) in Edmonton, AB, Canada. A new approach is presented to predict the fluence-based degradation rate constants (kf') of environmentally occurring micropollutants including carbamazepine [(0.87-1.39) × 10(-3) cm(2)/mJ] and 2,4-Dichlorophenoxyacetic acid (2,4-D) [(0.60-0.91) × 10(-3) cm(2)/mJ for 2,4-D] in a medium pressure (MP) UV/H2O2 system based on a previous bench-scale investigation. Rather than using removal rates, this approach can be used to estimate the performance of the MP UV/H2O2 process for degrading trace contaminants of concern found in municipal wastewater. In addition to the ability to track contaminant removal/degradation, evaluation of the MP UV/H2O2 process was also accomplished by identifying critical ecotoxicological endpoints (i.e., estrogenicity) of the treated wastewater. Using quantitative PCR, mRNA levels of estrogen-responsive (ER) genes ERα1, ERα2, ERß1, ERß2 and NPR as well as two aromatase encoding genes (CYP19a and CYP19b) in goldfish (Carassius auratus L.) were measured during exposure to the GBWWTP effluent before and after MP UV/H2O2 treatment (a fluence of 1000 mJ/cm(2) and 20 mg/L of H2O2) in spring, summer and fall. Elevated expression of estrogen-responsive genes in goldfish exposed to UV/H2O2 treated effluent (a 7-day exposure) suggested that the UV/H2O2 process may induce acute estrogenic disruption to goldfish principally because of the possible formation of various oxidation by-products. However, prolonged exposure of goldfish (60 days) in UV/H2O2 treated effluent showed a restoration trend of ER gene expressions, especially in the summer. Collectively, our findings provide valuable indications regarding the long-term in vivo assessment of the MP UV/H2O2 process for removing/degrading endocrine disrupting compounds detected in the municipal wastewater effluents.

Comparison of UV/hydrogen peroxide, potassium ferrate(VI), and ozone in oxidizing the organic fraction of oil sands process-affected water (OSPW).

The efficiency of three different oxidation processes, UV/H2O2 oxidation, ferrate(VI) oxidation, and ozonation with and without hydroxyl radical (OH) scavenger tert-butyl alcohol (TBA) on the removal of organic compounds from oil sands process-affected water (OSPW) was investigated and compared. The removal of aromatics and naphthenic acids (NAs) was explored by synchronous fluorescence spectra (SFS), ion mobility spectra (IMS), proton and carbon nuclear magnetic resonance ((1)H and (13)C NMR), and ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC TOF-MS). UV/H2O2 oxidation occurred through radical reaction and photolysis, transforming one-ring, two-ring, and three-ring fluorescing aromatics simultaneously and achieving 42.4% of classical NAs removal at 2.0 mM H2O2 and 950 mJ/cm(2) UV dose provided with medium pressure mercury lamp. Ferrate(VI) oxidation exhibited high selectivity, preferentially removing two-ring and three-ring fluorescing aromatics, sulfur-containing NAs (NAs + S), and NAs with high carbon and high hydrogen deficiency. At 2.0 mM Fe(VI), 46.7% of classical NAs was removed. Ozonation achieved almost complete removal of fluorescing aromatics, NAs + S, and classical NAs (NAs with two oxygen atoms) at the dose of 2.0 mM O3. Both molecular ozone reaction and OH reaction were important pathways in transforming the organics in OSPW as supported by ozonation performance with and without TBA. (1)H NMR analyses further confirmed the removal of aromatics and NAs both qualitatively and quantitatively. All the three oxidation processes reduced the acute toxicity towards Vibrio fischeri and on goldfish primary kidney macrophages (PKMs), with ozonation being the most efficient.

Goldfish (Carassius auratus L.) as a model system to study the growth factors, receptors and transcription factors that govern myelopoiesis in fish.

The process of myeloid cell development (myelopoiesis) in fish has mainly been studied in three cyprinid species: zebrafish (Danio rerio), ginbuna carp (Carassius auratus langsdorfii) and goldfish (C. auratus, L.). Our studies on goldfish myelopoiesis have utilized in vitro generated primary kidney macrophage (PKM) cultures and isolated primary kidney neutrophils (PKNs) cultured overnight to study the process of macrophage (monopoiesis) and neutrophil (granulopoiesis) development and the key growth factors, receptors, and transcription factors that govern this process in vitro. The PKM culture system is unique in that all three subpopulations of macrophage development, namely progenitor cells, monocytes, and mature macrophages, are simultaneously present in culture unlike mammalian systems, allowing for the elucidation of the complex mixture of cytokines that regulate progressive and selective macrophage development from progenitor cells to fully functional mature macrophages in vitro. Furthermore, we have been able to extend our investigations to include the development of erythrocytes (erythropoiesis) and thrombocytes (thrombopoiesis) through studies focusing on the progenitor cell population isolated from the goldfish kidney. Herein, we review the in vitro goldfish model systems focusing on the characteristics of cell sub-populations, growth factors and their receptors, and transcription factors that regulate goldfish myelopoiesis.

Biology of Bony Fish Macrophages.

Macrophages are found across all vertebrate species, reside in virtually all animal tissues, and play critical roles in host protection and homeostasis. Various mechanisms determine and regulate the highly plastic functional phenotypes of macrophages, including antimicrobial host defenses (pro-inflammatory, M1-type), and resolution and repair functions (anti-inflammatory/regulatory, M2-type). The study of inflammatory macrophages in immune defense of teleosts has garnered much attention, and antimicrobial mechanisms of these cells have been extensively studied in various fish models. Intriguingly, both similarities and differences have been documented for the regulation of lower vertebrate macrophage antimicrobial defenses, as compared to what has been described in mammals. Advances in our understanding of the teleost macrophage M2 phenotypes likewise suggest functional conservation through similar and distinct regulatory strategies, compared to their mammalian counterparts. In this review, we discuss the current understanding of the molecular mechanisms governing teleost macrophage functional heterogeneity, including monopoetic development, classical macrophage inflammatory and antimicrobial responses as well as alternative macrophage polarization towards tissues repair and resolution of inflammation.

Molecular and functional characterization of goldfish (Carassius auratus L.) Serum Amyloid A.

Quantitative expression analysis of goldfish SAA revealed the highest mRNA levels in the kidney, spleen and intestine with lower mRNA levels in muscle and liver. Goldfish SAA was differentially expressed in goldfish immune cells with highest mRNA levels observed in neutrophils. To functionally assess goldfish SAA, recombinant protein (rgSAA) was generated by prokaryotic expression and functionally characterized. Monocytes and macrophages treated with rgSAA exhibited differential gene expression of pro-inflammatory and anti-inflammatory cytokines. rgSAA induced gene expression of both pro-inflammatory (TNFα1, TNFα2) and anti-inflammatory cytokines (IL-10, TGFß) in monocytes. rgSAA induced IL-1ß1 and SAA gene expression in macrophages. rgSAA was chemotactic to macrophages and neutrophils, but not monocytes. rgSAA did not affect respiratory burst induced by heat-killed Aeromonas salmonicida. rgSAA treatment of macrophages down-regulated their production of nitric oxide. rgSAA exhibited antibacterial properties against Escherichia coli in a concentration dependent manner.