Fish Immune Cells Research Articles

Rodlet Cells Provide First Line of Defense against Swimbladder Nematode and Intestinal Coccidian in Anguilla anguilla

A subpopulation of 97 European eels, Anguilla anguilla, was obtained from a local consortium of Lake Trasimeno. The fish were examined for parasites in the swimbladder (SB) and intestine; the SBs of 66 (68%) of the 97 eels contained 480 adult specimens of the nematode Anguillicoloides crassus (Kuwahara, Niimi and Itagaki, 1974) and the intensity of infection ranged from 1 to 18 adult worms per SB (7.27 ± 0.43, mean ± SE). In heavily infected SB, the wall was thicker; upon excision, black-brownish adult nematodes were noticed inside the lumen. The infected SBs showed a papillose aspect of the epithelium and frequent erosion, inflammation, hemorrhages, and dilation of blood vessels. In parasitized SBs, mast cells and macrophages were encountered in the mucosal layer; in several heavily infected SB, rodlet cells were the sole fish immune cells noticed in the epithelium and in close contact with the A. crassus. With reference to the eel intestinal epithelium, in fifteen eels, two developmental stages of coccidian were close to clusters of rodlet cells, the coccidian induced erosion of the epithelium. The results of a panel of 12 antibodies in the European eel infected tissues will be presented.

Potential Applications of Fluorescence-Activated Cell Sorting (FACS) and Droplet-Based Microfluidics in Promoting the Discovery of Specific Antibodies for Characterizations of Fish Immune Cells.

Akin to their mammalian counterparts, teleost fish possess a complex assortment of highly specialized immune cells that are capable of unleashing potent innate immune responses to eradicate or mitigate incoming pathogens, and also differentiate into memory lymphocytes to provide long-term protection. Investigations into specific roles and functions of fish immune cells depend on the precise separation of each cell type. Commonly used techniques, for example, density gradient centrifugation, rely on immune cells to have differing sizes or densities and thus fail to separate between similar cell types (e.g. T and B lymphocytes). Furthermore, a continuously growing database of teleost genomic information has revealed an inventory of cellular markers, indicating the possible presence of immune cell subsets in teleost fish. This further complicates the interpretation of results if subsets of immune cells are not properly separated. Consequently, monoclonal antibodies (mAbs) against specific cellular markers are required to precisely identify and separate novel subsets of immune cells in fish. In the field of fish immunology, mAbs are largely generated using the hybridoma technology, resulting in the development of mAbs against specific cellular markers in different fish species. Nevertheless, this technology suffers from being labour-intensive, time-consuming and most importantly, the inevitable loss of diversities of antibodies during the fusion of antibody-expressing B lymphocytes and myeloma cells. In light of this, the focus of this review is to discuss the potential applications of fluorescence-activated cell sorting and droplet-based microfluidics, two emerging technologies capable of screening and identifying antigen-specific B lymphocytes in a high-throughput manner, in promoting the development of valuable reagents for fish immunology studies. Our main goal is to encourage the incorporation of alternative technologies into the field of fish immunology to promote the production of specific antibodies in a high-throughput and cost-effective way, which could better allow for the precise separation of fish immune cells and also facilitate the identification of novel immune cell subsets in teleost fish.

A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies

In this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

Molecular, Cellular and Functional Analysis of TRγ Chain along the European Sea Bass Dicentrarchus labrax Development.

In jawed vertebrates, adaptive immune responses are enabled by T cells. Two lineages were characterized based on their T cell receptor (TcR) heterodimers, namely αβ or γδ peptide chains, which display an Ig domain-type sequence that is somatically rearranged. γδ T cells have been less extensively characterized than αβ and teleost fish, in particular, suffer from a severe scarcity of data. In this paper, we worked on the well-known model, the European sea bass Dicentrarchus labrax, to broaden the understanding of teleost γδ-T cells. The T cell receptor chain (TR) γ transcript was expressed at a later developmental stage than TRβ, suggesting a layered appearance of fish immune cells, and the thymus displayed statistically-significant higher mRNA levels than any other organ or lymphoid tissue investigated. The polyclonal antibody developed against the TRγ allowed the localization of TRγ-expressing cells in lymphoid organs along the ontogeny. Cell positivity was investigated through flow cytometry and the highest percentage was found in peripheral blood leukocytes, followed by thymus, gut, gills, spleen and head kidney. Numerous TRγ-expressing cells were localized in the gut mucosa, and the immunogold labelling revealed ultrastructural features that are typical of T cells. At last, microalgae-based diet formulations significantly modulated the abundance of TRγ+ cells in the posterior intestine, hinting at a putative involvement in nutritional immunity. From a comparative immunological perspective, our results contribute to the comprehension of the diversity and functionalities of γδ T cells during the development of a commercially relevant marine teleost model.

Autophagy promotes innate defense against Aeromonas hydrophila in grass carp (Ctenopharyngodon idella) monocytes/macrophages

Aeromonas hydrophila (A. hydrophila) is a gram-negative opportunistic pathogen widely distributed in the aquatic environment, which can infect humans, birds, amphibians, reptiles, and fishes. However, the interaction between A. hydrophila and host cells has not been well characterized. In the present study, we investigated the role of autophagy in grass carp (Ctenopharyngodon idella) monocytes/macrophages following infection with A. hydrophila. Our results found that A. hydrophila challenge significantly induced autophagy, as shown by the increase of microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) proteins and LC3 puncta in the cells. Moreover, by using confocal microscopy, the A. hydrophila-elicited autophagy was further confirmed by the colocalization of the A. hydrophila with LC3-positive autophagosome in the cells. In return, the effect of the activated autophagy on the survival of A. hydrophila was examined in the same cell model, showing that rapamycin-induced autophagy was effective in restricting the survival of A. hydrophila. In contrast, autophagy blocked by 3-methyladenine (3-MA) enhanced the amount of A. hydrophila in the cells. All these findings for the first time revealed the role of autophagy in the process of A. hydrophila invading fish immune cells.

Fluorescent Microplastic Uptake by Immune Cells of Atlantic Salmon (Salmo salar L.)

The ubiquitous presence of microplastics and their marine ecotoxicity are major public concerns. Microplastics are ingested accidentally by the marine fauna or are taken up indirectly through the food chain. These particles can accumulate in cells and tissues and affect the normal biological functions of organisms, including their defense mechanisms. There is limited information available about the response of immune cells to microplastics; the degree of uptake by the cells, the response of different organs or the impact of environmental concentrations of microplastic are matters that remain unclear. Moreover, very little is known about the toxicity of different polymer types. This study aimed to shed light on the physical impact of small microplastics (1–5 μm) on cells from Atlantic salmon. Immune cells from intestine, blood, and head kidney were exposed to green fluorescent polyethylene microplastic (PE-MP), yellow fluorescent polystyrene microplastic (PS-MP) and both. High (50 mg/L), medium (5 mg/L), and low (0.05 mg/L) concentrations were tested for 1, 24, 48, and 72 h to study cell mortality and microplastic uptake. Quantitative data of microplastic uptake by fish immune cells were obtained for the first time by imaging flow cytometry. Salmon immune cells showed a relatively low ability to phagocytose microplastics. Less than 6% of the cells ingested the particles after 48 h of exposure to high concentrations. Cells also phagocytosed microplastics at low concentrations although at low rates (<0.1%). PE-MPs was phagocytosed by higher percentage of cells compared to PS-MPs and the former bioaccumulated in time while the latter decreased over time. However, each cell generally phagocytosed more PS-MPs particles than PE-MPs. Cells from different tissues showed different responses to the microplastic polymers. In conclusion, this study shows that immune cells of Atlantic salmon can phagocytose microplastics, and the impact is dependent on the microplastic type. PE-MPs, the most abundant polymer in the oceans and a widely used plastic in salmon aquaculture, was more easily taken up than PS-MPs. Furthermore, the study demonstrates how imaging flow cytometry can be applied in microplastics research.

Characterization of miRNAs in Extracellular Vesicles Released From Atlantic Salmon Monocyte-Like and Macrophage-Like Cells.

Cell-derived extracellular vesicles (EVs) participate in cell-cell communication via transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily “monocyte-like” at Day 1 to primarily “macrophage-like” at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.

Regulation of Il-10 gene expression by Il-6 via Stat3 in grass carp head kidney leucocytes.

Interleukin (IL)-10 is a critical anti-inflammatory and late cytokine being produced after the proinflammatory mediators while IL-6 is a promptly synthesized cytokine in response to inflammation in mammals. This chronological expression of interleukin (Il)-6 and Il-10 was also found in grass carp head kidney leucocytes (HKLs) treated by heat-killed Aeromonas hydrophila, supporting the possible interplay between grass carp (gc)Il-6 and gcIl-10 in HKLs. Our further findings were in agreement with this hypothesis that recombinant gcIl-6 (rgcIl-6) promptly and transiently increased gcil10 mRNA levels in grass carp HKLs. Moreover, rgcIl-6 enhanced its own mRNA level and this self-enhancement of gcil6 mRNA level could be partially blocked by rgcIl-10. These results collectively suggest that gcIl-10 production stimulated by gcIl-6 may provide a negative feedback to gcIl-6 production. Interestingly, rgcIl-6 significantly decreased gcil10 mRNA levels in grass carp HKLs after the treatment for 12 and 24h in contrast to its enhancement of gcil10 levels after the treatment for 3h. Involvement of Stat3 but not MEK, p38 MAPK or JNK pathway in the increase of gcil10 mRNA levels by rgcIl-6 was revealed by using the signaling pathway inhibitors. This was supported by the fact that rgcIl-6 stimulated Stat3 phosphorylation in grass carp HKLs. Furthermore, rgcIl-6 had no effect on the stability of gcil10 mRNA after the treatment for 3 to 36h while it increased gcil10 promoter activity after the treatment for 24h. Taken these data together, gcIl-6 can stimulate Il-10 production at early stage but subsequently inhibit il10 mRNA expression in grass carp HKLs, shedding light on the dynamic regulation of il10 mRNA expression by Il-6 in fish immune cells.

Novel functions of grass carp three p40 isoforms as modulators of Th17 signature cytokine expression in head kidney leukocytes

Interleukin (IL)-12p40, a component of IL-12 and IL-23, can be secreted as monomer and homodimer in mammals. Our previous study has proved the existence of natural three p40 isoforms and their proinflammatory properties in grass carp. In the present study, we unexpectedly found that recombinant grass carp p40a/b/c (rgcp40a, rgcp40b and rgcp40c) were able to enhance the mRNA levels of grass carp il-17a/f1 (gcil-17a/f1) in a dose- and time-dependent manner in head kidney leukocytes (HKLs). In agreement with these findings, the enzyme-linked immunosorbent assay (ELISA) showed that rgcp40a, rgcp40b and rgcp40c markedly stimulated gcIl-17a/f1 secretion from the HKLs. Together with their stimulatory effects on grass carp gcil-22 and gcil-26 expression, our data suggested their potential to mediate Th17-like response in grass carp. To support this notion, we investigated the underlying mechanisms for the regulation of rgcp40 isoforms on gcil-17a/f1 expression, and found that three rgcp40 isoforms significantly induced the activation of Erk, Jnk and Stat3 pathways in a time-dependent oscillation in the same cell model. Moreover, three rgcp40 isoforms-induced gcil-17a/f1 mRNA expression was suppressed by the inhibition on Erk, Jnk and Stat3 pathways, suggesting the signaling pathways in the p40 isoforms-mediating il-17a/f1 transcription. These studies for the first time proved the involvement of three gcp40 isoforms in mediating Th17 signature cytokine expression in fish immune cells, therefore providing new insights into the roles of p40 in teleost immunity.

Functional characterization of purinergic receptor P2Y14 in the Japanese flounder (Paralichthys olivaceus) head kidney macrophages

Extracellular nucleotides and nucleotide sugars are important danger-associated signaling molecules that play critical roles in regulation of immune responses in mammals through activation of purinergic receptors located on the cell surface. However, the immunological role of extracellular UDP-glucose-activated P2Y14 receptor (P2Y14R) in fish still remains unknown. In this study, we identified and characterized a P2Y14R paralog in the Japanese flounder (Paralichthys olivaceus). The mRNA transcripts of P2Y14R are detected in all examined Japanese flounder tissues. Compared with the UDP-activated P2Y6 receptor, however, P2Y14R gene is highly expressed in Japanese flounder head kidney macrophages (HKMs). In addition, P2Y14R is significantly upregulated following inflammatory stimulation with LPS and poly (I:C) in the HKMs, suggesting a role of P2Y14R in response to inflammation in fish. Furthermore, activation of P2Y14 receptor with its potent and selective agonist MRS 2905 resulted in a decreased expression of LPS-induced pro-inflammatory cytokine IL-1beta gene in the HKMs. In contrast, inhibition of P2Y14 receptor activity or down-regulation of the endogenous expression of P2Y14R by small interfering RNA significantly upregulates the LPS-induced pro-inflammatory cytokine IL-1beta gene expression in the HKMs, demonstrating that P2Y14R is involved in inflammation regulation in fish. Moreover, stimulation of the Japanese flounder HKMs with UDP-glucose evoked a rapid increase of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent manner, indicating the involvement of P2Y14R in activation of ERK1/2 signaling in fish immune cells. Taken together, we demonstrated that the inducible P2Y14R plays an important role in regulation of fish innate immunity.

Optimization of macrophage isolation from the Persian sturgeon and the Caspian kutum fish: a comparative study.

The aim of this research was a comparative study on the isolation and culture of head kidney macrophages derived from Acipenser persicous and Rutilus frisii kutum as teleost and chondrostei species of fish. The macrophages were isolated by density gradient sedimentation, followed by adherence to a plastic surface. They exhibited strong phagocytic activity against bacteria. The effect of cell density, incubation time, FBS percentage, pH and temperatures on the cell number and viability were determined and compared. Also, the effect of light/dark regimen on viability, adherence, release of reactive oxygen species (ROS) and nitric oxide (NO) in the macrophages was determined. The results showed that the Caspian kutum macrophages were more sensitive to FBS percentage and cell density whereas the Persian sturgeon macrophages were more sensitive to pH of the cell culture media. The adherence and viability of the macrophages from both fish species firstly increased (P < 0.05) after exposure to a light/dark regimen, but then significantly decreased as did ROS and NO productions. For the first time, this study has determined the optimal conditions for primary culture of macrophages derived from sturgeons, and shows the unique effect of light on the biology of fish immune cells.

In vitro investigations on the effects of semi-synthetic, sulphated carbohydrates on the immune status of cultured common carp (Cyprinus carpio) leucocytes

The rapid emergence of drug resistance, unfavourable immunosuppression and mounting evidence to suggest the deleterious accumulation of drug breakdown residues within animal tissues has driven a strong desire to move away from these current methods of disease control. Some natural products such as β-glucan, which are extracted from, for example, plants and fungi, are able to modulate the immune system and increase protection against diseases. However, these products are heterogeneous and their effects can be variable thus limiting their applicability and reliability. Carbohydrates were modified via chemical sulphation and these semi-synthetic, sulphated carbohydrates analysed for their immunological activity utilising carp pronephric cells and a carp leucocyte cell line (CLC). A sulphated β(1,4)-glucan, methyl hydroxyethyl cellulose sulphate (MHCS), demonstrated a stimulatory effect on fish immune cells. MHCS induced a range of bioactive effects in carp leucocyte cells whilst not affecting cell viability when cells were exposed for 24 h at concentrations of 1–150 μgml−1. MHCS stimulated the innate immune system where a significant increase in respiratory burst activity was observed at concentrations 25–250 μgml−1 in comparison to control (sterile water), cellulose ether, MacroGard® and zymosan. Also, under in mock bacterial and viral infection conditions i.e. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (Poly(I:C)), MHCS enhanced the immune responses of pronephric cells by stimulating the respiratory burst activity at concentrations 50 and 150 μgml−1. MHCS also enhanced the expression of cytokines including interleukin 1 beta (IL1β), tumor necrosis factor alpha 1 and 2 (TNFα 1,2), interferons alpha 2 (IFN α2) and inducible nitric oxide synthase (iNOS) in carp pronephric cells. It is proposed that this new semi-synthetic carbohydrate is a potential candidate for the development of a new generation of immunostimulants and adjuvants for use in vaccination strategies in aquaculture.

Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types.

The immune system of vertebrate species consists of many different cell types that have distinct functional roles and are subject to different evolutionary pressures. Here, we first analyzed conservation of genes specific for all major immune cell types in human and mouse. Our results revealed higher gene turnover and faster evolution of trans-membrane proteins in NK cells compared with other immune cell types, and especially T cells, but similar conservation of nuclear and cytoplasmic protein coding genes. To validate these findings in a distant vertebrate species, we used single-cell RNA sequencing of lck:GFP cells in zebrafish and obtained the first transcriptome of specific immune cell types in a nonmammalian species. Unsupervised clustering and single-cell TCR locus reconstruction identified three cell populations, T cells, a novel type of NK-like cells, and a smaller population of myeloid-like cells. Differential expression analysis uncovered new immune-cell–specific genes, including novel immunoglobulin-like receptors, and neofunctionalization of recently duplicated paralogs. Evolutionary analyses confirmed the higher gene turnover of trans-membrane proteins in NK cells compared with T cells in fish species, suggesting that this is a general property of immune cell types across all vertebrates.

Lipopolysaccharide-induced autophagy participates in the control of pro-inflammatory cytokine release in grass carp head kidney leukocytes

Microtubule-associated protein 1 light chain 3B (LC3B) is a known marker of autophagy in mammals. In the present study, we isolated and identified grass carp LC3B (gcLC3B) cDNA and found its inductive expression in response to bacterial infection in vivo. To assess the occurrence of autophagy in immune response, the role of gcLC3B as an autophagy marker in grass carp was characterized. Accordingly, grass carp kidney cells (CIKs) with stable expression of GFP-gcLC3B were established and GFP-gcLC3B puncta were counted under a confocal fluorescence microscope. Results showed that starvation, a conventional inducer of autophagy, significantly enhanced GFP-gcLC3B puncta number, indicating the existence of gcLC3B-linked autophagy in fish cells. Moreover, a commercial antibody recognizing gcLC3B and 3-methyladenine (3-MA) were validated in grass carp CIKs, and used to evaluate autophagy in grass carp head kidney leukocytes (HKLs) in response to LPS. Western blotting assay showed that LPS significantly induced the conversion of gcLC3B protein, providing the evidence for autophagy induced by LPS in fish immune cells. Importantly, autophagy inhibition by 3-MA enhanced grass carp IL-1β and TNF-α secretion, indicating the involvement of autophagy in pro-inflammatory cytokine production. Besides, 3-MA could amplify LPS-induced IL-1β and TNF-α release, implying that autophagic induction may drive a mechanism for controlling inflammatory response in fish. Thus, our data highlight the role of autophagy in fish immunity and provide new insight into the mechanism for the regulation of inflammation in fish.

Effects of diazinon on the lymphocytic cholinergic system of Nile tilapia fish (Oreochromis niloticus)

Fish rearing under intensive farming conditions can be easily disturbed by pesticides, substances that have immunotoxic properties and may predispose to infections. Organophosphorus pesticides (OPs) are widely used in agricultural activities; however, the mechanism of immunotoxicity of these substances is unclear. The aim of this study was to evaluate the effect of diazinon pesticides (OPs) on the cholinergic system of immune cells as a possible target of OP immunotoxicity. We evaluated ACh levels and cholinergic (nicotinic and muscarinic) receptor concentration. Additionally, AChE activity was evaluated in mononuclear cells of Nile tilapia (Oreochromis niloticus), a freshwater fish mostly cultivated in tropical regions around the world. The obtained results indicate that acute exposure to diazinon induces an increase in ACh concentration and a decrease in nAChR and mAChR concentrations and AChE activity in fish immune cells, This suggests that the non-neuronal lymphocytic cholinergic system may be the main target in the mechanism of OP immunotoxicity. This study contributes to the understanding of the mechanisms of immunotoxicity of pollutants and may help to take actions for animal health improvement.

Effects of cylindrospermopsin on the phagocytic cells of the common carp (Cyprinus carpio L.).

Cylindrospermopsin is a cyanotoxin with cytotoxic activity. It is released into water during and after cyanobacterial water blooms and thus poses a threat to the health of fish. There is very little information available concerning the effects of the toxin on fish immune cells. In this study, we assessed the potential impact of cylindrospermopsin on the basic functions of phagocytic cells from common carp (Cyprinus carpio L.), including phagocytosis, reactive oxygen and nitrogen species production, and the structure of microfilaments and selected cytokine expression. Phagocytic cells, isolated from fish head kidneys, were exposed to the toxin at concentrations of 0.05, 0.1, 0.5 or 1 µg ml(-1), for up to 24 h. Cytotoxicity, detected by lactate dehydrogenase release, was observed at the highest studied concentration. A decrease in phagocytic activity and changes in actin cytoskeletal structures were observed after the cell exposure to the toxin at 0.5 and 1 µg ml(-1). Moreover, at all tested concentrations, cylindrospermopsin increased the production of reactive oxygen and nitrogen species. It also evidently influenced the expression of genes of proinflammatory cytokines interleukin-1β and tumour necrosis factor-α and, to a minor extent, anti-inflammatory transforming growth factor-β, but had no effects on interleukin-10. The results indicated that the cyanotoxin cylindrospermopsin is able to modify basic features of carp phagocytic cells, which might result in adverse consequences for fish health.

Cellular activation, expression analysis and functional characterization of grass carp IκBα: Evidence for its involvement in fish NF-κB signaling pathway

IκBα is a well-known member of the inhibitors of kappa B (IκB) family that controls NF-κB signaling by blocking NF-κB translocation from cytoplasm to nucleus. In the present study, an IκBα homologue was identified from grass carp (gcIκBα), showing the structural characteristics of IκB family. Moreover, mRNA expression of this molecule in grass carp periphery blood lymphocytes (PBLs) was enhanced significantly by both LPS and PHA in a time- and dose-dependent manner, indicating the involvement of gcIκBα in fish immune response. Further analysis demonstrated that LPS but not PHA induced gcIκBα phosphorylation and protein degradation in PBLs, implying different signaling pathways mediated by LPS and PHA in gcIκBα expression regulation in grass carp PBLs. In particular, the time-dependent oscillation of gcIκBα phosphorylation and total protein levels induced by LPS is in accordance with the characteristics of mammalian IκBα phosphorylation followed by protein degradation during NF-κB activation. In support of this notion, overexpression of gcIκBα was able to block both basal and LPS-stimulated NF-κB activity in grass carp kidney cell line, indicating the negatively regulatory role of gcIκBα in NF-κB activity as seen in mammals. Therefore, our results not only reveal a dynamic variation of NF-κB activity based on the activation and expression of IκBα for the first time, but also provide the direct evidence for the involvement of IκBα in NF-κB signaling in fish immune cells.

The in vitro effect of kynurenic acid on the rainbow trout (Oncorhynchus mykiss) leukocyte and splenocyte activity.

Kynurenic acid (KYNA), an endogenous neuroprotectant formed along the kynurenine pathway of tryptophan degradation, is a selective ligand of the GPR35 receptor, which can be found on the surface of various populations of human immune cells. In infections and inflammations, KYNA produces an anti-inflammatory effect through this receptor, by depressing the synthesis of reactive oxygen species and pro-inflammatory cytokines. However, it is still unrecognized whether receptors for kynurenic acid are also localized on immune cells of poikilothermic animals, or whether KYNA is able to affect these cells. The objective of this study has been to determine the effect of different concentrations of kynurenic acid (12.5 microM to 10 mM) on the viability and mitogenic response of lymphocytes and on the activity of phagocytic cells isolated from blood and the spleen of rainbow trout. The results imply low toxicity of kynurenic acid towards fish immune cells, and the proliferative effect observed at the two lowest concentrations of KYNA (12.5 microM and 25 microM) seems indicative of endogenous kynurenic acid being capable of activating fish lymphocytes. Non-toxic, micromole concentrations of KYNA, however, had no influence on the mitogenic response of lymphocytes nor on the activity of phagocytes in rainbow trout under in vitro conditions. There is some likelihood that such an effect could be observed at lower, nanomole concentrations of KYNA.

Protective role of oligomeric proanthocyanidin complex against hazardous nodularin-induced oxidative toxicity in Carassius auratus lymphocytes

Nodularin (NOD) is a hazardous material widely detected in water blooms. Fish immune cells are extremely vulnerable to NOD-induced oxidative stress. Oligomeric proanthocyanidin complex (OPC), extracted from grapeseed, was used as an antioxidant to eliminate reactive oxygen species and prevent apoptotic effects. Carassius auratus lymphocytes were treated with different concentrations (0, 10, 100, and 1000μg/L) of OPC and a constant dose (100μg/L) of NOD for 12h in vitro. OPC inhibited mitosis by decreasing intracellular levels of oxidative stress, regulating antioxidant enzymes (CAT, SOD, GPx, GR, and GST), mediating bcl-2 family proteins, and deactivating caspase-3. Glutathione (GSH) levels in group V (NOD 100μg/L; OPC 1000μg/L) showed a twofold increase compared with corresponding levels in group II (NOD 100μg/L). Structure parameters of NOD and NOD-GSH were calculated using SYBYL 7.1 software. ClogP and HINK logP values of NOD-GSH decreased by 10.4- and 2.3-fold, respectively, compared with corresponding values of NOD. OPC-stimulated GSH can lower the lipophilicity and polarity of NOD. OPC, as a protective agent, can alleviate NOD-induced toxicity in C. auratus lymphocytes by regulating oxidative stress and inducing NOD-GSH detoxification.

Effects of cylindrospermopsin on a common carp leucocyte cell line

Cylindrospermopsin (CYN) is a cytotoxin produced by different cyanobacterial species, increasingly detected in water reservoirs worldwide. There is very little information available concerning the effects of the toxin on fish immune cells. The aim of the study was to elucidate the potential impact of cylindrospermopsin on the selected parameters of a common carp (Cyprinus carpio L.) leucocyte cell line (CLC). The cells were incubated with the cyanotoxin at concentrations of 10, 1 or 0.1 µg ml(-1) for up to 48 h. Cell viability and proliferation, apoptosis/necrosis induction, cell morphology and phagocytic activity were determined. The two higher toxin concentrations occurred to be evidently cytotoxic in a time-dependent manner and influenced all studied parameters. The lowest used concentration had no effects on cell viability and cell number; however, a strong reduction of bacteria uptake after 24-h exposure was detected. The obtained results indicate that cylindrospermopsin may interfere with the basic functions of fish phagocytic cells and as a consequence influence the fish immunity.