Head Kidney Cells Research Articles

Development of monoclonal antibody against IgM of large yellow croaker (Larimichthys crocea) and characterization of IgM+ B cells

In the present study, a monoclonal antibody (mAb) against large yellow croaker IgM was produced by immunizing mice with purified large yellow croaker serum IgM. Western blotting showed that this mAb could specifically react with the heavy chain of large yellow croaker serum IgM. Indirect immunofluorescence assay (IFA) analysis suggested that the resulting mouse anti-IgM mAb could recognize membrane-bound IgM (mIgM) molecules of large yellow croaker. This mouse anti-IgM mAb also can be used for sorting of large yellow croaker IgM+ B cells through the magnetic-activated cell sorting (MACS) method, which was further confirmed by RT-PCR analysis of specific marker genes for B cells. Flow cytometry analysis showed that the percentages of IgM+ B cells in head kidney, spleen and peripheral blood lymphocytes were 29.00 ± 1.58%, 33.00 ± 1.64%, and 16.50 ± 2.39%, respectively. Additionally, the phagocytosis rates of IgM+ B cells for 0.5 μm beads in head kidney, spleen and peripheral blood were calculated to be 7.56 ± 0.58%, 4.053 ± 0.62% and 23.17 ± 2.26%, respectively, while only 2.36 ± 0.23%, 1.16 ± 0.44% and 6.41 ± 0.45 of IgM+ B cells in these three tissues ingested 1 μm beads. Taken together, our data demonstrated that the mouse anti-IgM mAb produced in this study could be used as a tool to characterize IgM+ B cells and to study functions of IgM in large yellow croaker.

Identification and characterization of fish-specific IL-23 isoforms and their roles in fish immunity

Abstract IL-23 is well known as a regulator in inflammation response and host defense by promoting the development of Th17 cells. In fish, three genes coding p40 (p40a, p40b and p40c) are originated from fish-specific genome duplication, thereby generating three IL-23 isoforms (IL-23a, IL-23b and IL-23c). Considering the functional diversity of fish IL-23, it is possible that the fish-specific immunoregulatory mechanism of IL-23 may have existed. In this study, we isolated and identified the subunits of IL-23 from grass carp. In response to bacterial infection in vivo, the mRNA levels of gcp19 were upregulated in headkidney, liver and gill, while those of p40a were significantly induced in the selected tissues except liver. Surprisingly, the levels of gcp40b and gcp40c did not show marked change in tissues. What’s more, LPS, PHA stimulation in vitro, the mRNA levels of these subunits also showed a significant increase in periphery blood lymphocytes. Meanwhile, the result of Co-IP showed p19 could couple with each of the three p40 to form IL-23 and be secreted into the supernatants, verifying the existence of three IL-23 isoforms in grass carp. In order to assess their functions in fish immunity, we constructed plasmids containing gcp19 CDS combined with gcp40a/b/c CDS by a linker sequence and over-expressed them in CHO cells. The positive stable transfectants were selected, and the recombinant proteins were produced and purified from the culture medium. At present, we have gained these grass carp IL-23 proteins. Their bioactivities were investigated in grass carp headkidney cells, showing same or different functions in the immune regulation of grass carp. Finally, our work has provided a basis for elucidating the roles of IL-23 isoforms in fish immunity.

Identification and bioactivity of a granulocyte colony-stimulating factor b homologue from large yellow croaker (Larimichthys crocea)

Granulocyte colony-stimulating factor (GCSF) is a pleiotropic cytokine that plays a key role in regulation of hematopoiesis, innate and adaptive immune responses in mammals. However, bioactivity of GCSF in teleost fish remains largely unknown. In this study, a GCSFb homologue from large yellow croaker (Larimichthys crocea) (LcGCSFb) was cloned by RACE-PCR techniques. The open reading frame (ORF) of LcGCSFb is 603 bp long and encoded a protein precursor of 200 amino acids (aa), with a 19-aa signal peptide and a 181-aa mature peptide. In healthy fish, the LcGCSFb was constitutively expressed in all examined tissues, with the highest levels in mucous tissues, such as gills, intestine, and stomach. Its transcripts in head kidney, spleen, gills, intestine and stomach were significantly induced by Vibrio alginolyticus challenge. LcGCSFb transcripts were also detected in primary head kidney leukocytes (PKL), primary head kidney macrophages (PKM), primary head kidney granulocytes (PKG) and head kidney cell line (LYCK), and markedly upregulated by inactivated V. alginolyticus. These data suggested that LcGCSFb may play a role in immune response against bacterial infection. In vivo administration of recombinant LcGCSFb protein (rLcGCSFb) significantly upregulated the expression levels of the inflammatory cytokines (IL-6 and TNFα), and transcription factor C/EBPβ, which is required for proliferation of neutrophils. Furthermore, rLcGCSFb showed an ability to strengthen the phagocytosis of PKL in vitro. Taken together, LcGCSFb may be involved in antibacterial immunity via promoting the inflammatory response and the phagocytic activity of leukocytes. To our knowledge, this is the first report on immunoregulatory roles of GCSF in teleost.

Sequence characterization and expression pattern analysis of six kinds of IL-17 family genes in the Asian swamp eel (Monopterus albus)

Interleukin-17 (IL-17) is an important cytokine that plays a critical role in the inflammatory response and host defense against extracellular pathogens. In the present study, six novel IL-17 family genes (MaIL-17) were identified by analyzing Asian swamp eel (Monopterus albus) genome. Sequence analysis revealed that the MaIL-17 family genes shared similar features, comprising a signal peptide, an IL-17 superfamily region, and four conserved cysteines. Phylogenetic analysis showed that the MaIL-17 genes were clustered together with their corresponding IL-17 genes from other species. The similarity and identity of all IL-17 family genes indicated that the MaIL-17 genes are conserved among teleosts, while Ma-IL-17D is more conserved than the other Ma-IL-17s. Except for MaIL-17A/F3 and MaIL-17D, all MaIL-17s shared the same genomic structure as the genes from other fish, namely three exons and two introns. The MaIL-17s showed conserved synteny among fish, and we found that the MaIL-17D locus has a more conserved syntenic relationship with the loci from other fish and humans. These results demonstrated that MaIL-17D and human IL-17D might have evolved from a common ancestral gene and subsequently diverged. The analysis of swamp eel reference genes revealed that EEF1A1 (encoding eukaryotic translation elongation factor 1 alpha 1) was an ideal reference gene for accurate real-time qRT-PCR normalization in the swamp eel. The MaIL-17 genes are widely distributed throughout tissues, suggesting that MaIL-17s carry out their biological functions in immune and non-immune tissues compartments. The transcript of Ma-IL17s exhibited different fold changes in head kidney cells in response to Aeromonas veronii phorbol 12-myristate 13-acetate (PMA) and polyinosinic:polycytidylic acid (poly I:C) challenge, showing that MaIL-17A/F1 has stronger antiviral activities compared with other MaIL-17 family genes, and that MaIL-17A/F3 and MaIL-17A/F2 possess stronger effects against extracellular pathogens compared with the others; however, MaIL-17C2 and MaIL-17D may play vital roles during pathogen infection. The differential immune responses of these genes to Aeromonas veronii, PMA and poly I:C implied distinct mechanisms of host defense against extracellular pathogens.

Immuno-stimulatory effects of sulfated polysaccharides isolated from Codium fragile in olive flounder, Paralichthys olivaceus

Sulfated polysaccharides isolated from Codium fragile have been previously demonstrated to possess immune-stimulating effects on murine cell lines and the fraction F2 (F2) isolated by ion exchange chromatography was the most effective. In this study, the effects of the fraction F2 were evaluated on the expressions of immune genes including IL-1β, TNF-α, IL-8, IFN-γ and lysozyme in vitro and in vivo as well as lysozyme and complement activities in serum of olive flounder, Paralichthys olivaceus. In vitro, these gene expressions were up-regulated by F2 in head kidney cells. In vivo, IL-1β and IL-8 gene expressions were up-regulated in peritoneal cells, head kidney, liver, gill and spleen, while TNF-α, IFN-γ and lysozyme gene expressions were mostly up-regulated but varied depending on tissue types or time points. Indeed, lysozyme and complement activities in serum were increased. Overall, these results indicate that the sulfated polysaccharides from C. fragile have immuno-stimulatory effects on olive flounder and may be used to enhance immunity during aquaculture.

Profiling the Atlantic Salmon IgM+ B Cell Surface Proteome: Novel Information on Teleost Fish B Cell Protein Repertoire and Identification of Potential B Cell Markers.

Fish immunology research is at a pivotal point with the increasing availability of functional immunoassays and major advances in omics approaches. However, studies on fish B cells and their distinct subsets remain a challenge due to the limited availability of differentially expressed surface markers. To address this constraint, cell surface proteome of Atlantic salmon IgM+ B cells were analyzed by mass spectrometry and compared to surface proteins detected from two adherent salmon head kidney cell lines, ASK and SSP-9. Out of 21 cluster of differentiation (CD) molecules identified on salmon IgM+ B cells, CD22 and CD79A were shortlisted as potential markers based on the reported B cell-specific surface expression of their mammalian homologs. Subsequent RT-qPCR analyses of flow cytometry-sorted subpopulations from head kidney leukocytes confirmed that both cd22 and cd79a genes were highly expressed in IgM+ lymphoid cells but were observed in barely detectable levels in IgM− non-lymphoid suspension and adherent cells. Similarly, significantly high cd22 and cd79a mRNA levels were observed in IgM+ or IgT+ lymphoid cells from the spleen and peritoneal cavity, but not in their corresponding IgM− IgT− non-lymphoid fractions. This suggests that the B cell restrictive expression of CD22 and CD79A extend down to the transcription level, which was consistent across different lymphoid compartments and immunoglobulin isotypes, thus strongly supporting the potential of CD22 and CD79A as pan-B cell markers for salmon. In addition, this study provides novel information on the salmon B cell surface protein repertoire, as well as insights on B cell evolution. Further investigation of the identified salmon CD molecules, including development of immunological tools for detection, will help advance our understanding of the dynamics of salmon B cell responses such as during infection, vaccination, or immunostimulation.

Cloning and expressional analysis of secretory and membrane-bound IgM in rock bream (Oplegnathus fasciatus) under megalocytivirus infection and vaccination

In this study, for better understanding the humoral immunity of rock bream (Oplegnathus fasciatus), 2 transcripts of immunoglobulin M (IgM) heavy chain gene including membrane bound (m-IgM) and secretory (s-IgM) forms were sequenced and analyzed their tissue distribution and differential expression in rock bream under rock bream iridovirus (RBIV) infection and vaccination since RBIV has caused mass mortality in rock bream aquaculture in Korea. Consequently, s-IgM cDNA was 1902 bp in length encoding a leader region, a variable region, four constant regions (CH1, CH2, CH3, CH4) and a C-terminal region while m-IgM cDNA was 1689 bp in length encoding shorter three constant regions (CH1, CH2, CH3) and two transmembrane regions. The predicted s-IgM and m-IgM represent a high structural similarity to other species including human. In tissue distribution analysis in healthy fish, the highest expression of s-IgM was observed in head kidney followed by body kidney, spleen, and mid gut whereas m-IgM expression was the highest in blood followed by head kidney and spleen. In vitro, s-IgM expression was up-regulated by LPS in head kidney and spleen cells at 24 h with no change of m-IgM expression. In vivo upon vaccination, s-IgM expression was up-regulated in liver and blood but not in head kidney while m-IgM expression was only up-regulated in head kidney. After challenge with RBIV, s-IgM expression level was higher in vaccinated fish than in unvaccinated fish and m-IgM expression was up-regulated in head kidney of vaccinated group. In conclusion, differential expression of m-IgM and s-IgM may indicate their differential functions to produce the most effective IgM during adaptive immune response. Although it is not able to assess specific IgM at protein level due to a lack of antibody against rock bream IgM, the present study on s-IgM and m-IgM gene expressions upon infection and vaccination will be useful in developing efficient vaccines in the future.

Surplus arginine reduced lipopolysaccharide induced transcription of proinflammatory genes in Atlantic salmon head kidney cells

In aquaculture production, studies of salmon health and interaction between pathogens and nutrition are of high importance. This study aimed to compare genes and pathways involved in salmon head kidney cells and liver cells, isolated from the same fish, towards polyinosinic acid: polycytidylic acid (poly I:C) and lipopolysaccharide (LPS), with and without addition of surplus arginine. Selected transcriptional responses of genes involved in inflammation, polyamine synthesis, oxidation and apoptosis were elucidated.For the genes related to inflammation, viperin, Mx and Toll like receptor 3 (TLR3), transcription were significantly upregulated by poly I:C in head kidney cells, while viperin was upregulated in liver cells. Surplus arginine did not affect poly I:C induced responses with the exception of reducing poly I:C induced Mx transcription in head kidney cells. Gene transcription of Interleukin 1β (IL-1β), Interleukin-8 (IL-8) and cyclooxygenase 2 (Cox2) were elevated during LPS treatment in all liver and head kidney cell cultures. In addition, LPS induced significantly, CD83 transcription in liver cells and TNF-α transcription in head kidney cells. Surplus arginine significantly reduced IL-8, Cox2 and TNF-α transcription in head kidney cells. LPS upregulated arginase in head kidney cells while poly I:C upregulated S-adenosyl methionine decarboxylase (SAMdc) transcription in liver cells. This suggests that LPS and poly I:C modulates genes involved in polyamine synthesis. In addition, in head kidney cells, surplus arginine, when cultured together with LPS, increased the transcription of ornithine decarboxylase (ODC) the limiting enzyme of polyamine synthesis. The genes involved with oxidation and apoptosis were not affect by any of the treatments in liver cells, while LPS decreased caspase 3 transcription in head kidney cells. In liver cells, protein expression of catalase was reduced by surplus arginine alone and when challenged with poly I:C. Both liver cells and head kidney cells isolated from the same individual fish responded to LPS and poly I:C, depending on the gene analyzed. Additionally, arginine could modulate transcription of pro-inflammatory genes induced by LPS in salmon immune cells, thus affecting salmon immunity.

Immune-enhancing Activity of Potential Probiotic Strains of Lactobacillus Plantarum in the Common Carp (Cyprinus Carpio) Fingerling.

IntroductionImmune-potentiating functions of Lactobacillus plantarum strains in the common carp were evaluated.Material and MethodsFourteen days of feeding fish dry diet supplemented with the bacteria provided parameters of nonspecific humoral immunity (lysozyme, ceruloplasmin, γ-globulin, total protein levels, and serum bactericidal activity) and cellular immunity (pinocytosis, respiratory burst activity, and potential killing activity of organ phagocytes), as well as the proliferative response of organ lymphocytes stimulated with mitogens. The resistance of fish to infection with Aeromonas hydrophila was also determined.ResultsDietary supplementation with L. plantarum had a substantial influence on the activity of organ phagocytes, especially the potential killing activity of head kidney cells. A significant increase in the proliferative activity of LPS-stimulated B lymphocytes and in the levels of γ-globulins and total protein was observed. The supplemented diet conveyed higher resistance than the control diet as the cumulative fish mortalities after infection with A. hydrophila were 65% and 85%, respectively.ConclusionThe results indicate that dietary supplementation with L. plantarum stimulates the antibacterial resistance of common carp and may reinforce defence against bacterial infections, but further studies need to be conducted.

Cellular Composition and Proliferation Levels in the Hematopoietic Tissue of Black Scorpionfish (Scorpaena porcus L.) Head Kidney and Spleen During the Spawning and Wintering Periods.

To identify cells and analyze proliferative activity of hematopoietic tissue, black scorpionfish head kidney and spleen cells were characterized by light microscopy and flow cytometry. Hematopoiesis of black scorpionfish head kidney was formed by the following series: erythropoietic, granulopoietic, lymphopoietic, and thrombopoietic. Flow cytometric analysis allowed dividing blood cells in hematopoietic organs into subpopulations differing by size, granularity, and proliferative activity. Three distinct subpopulations were observed during the wintering period. The number of small low-granulated cells, identified as lymphocytes and thrombocytes, was 41% ± 4% in both wintering and spawning fish. Proliferating subpopulation of blast (high-granulated) cells amounted to about 36% of the total cell count with 50% ± 5% of proliferating cells; the largest low-granulated cells (10% of total cells) comprised maturing white blood cells, monocytes, and macrophages. The spawning period was accompanied with an increase of maturing neutrophils and enhancement of blast cell proliferation. In the spleen three distinct subpopulations were observed. The subpopulation of small low-granulated cells comprised lymphocytes and thrombocytes similar to the head kidney and amounted 33% ± 4%. Other cells with larger diameter were identified as red blood cells. No proliferation was observed during the wintering period in the spleen, however, spawning induced cell proliferation of erythroblasts (small granulated cells) with the number of dividing cells 84% ± 1%. Anat Rec, 302:1136-1143, 2019. © 2018 Wiley Periodicals, Inc.

Characterization and function of a group II type I interferon in the perciform fish, large yellow croaker (Larimichthys crocea)

Teleost fish possess two groups of type I interferons (IFNs) with two (group I IFNs) or four (group II IFNs) conserved cysteines, which are further classified into seven subgroups. In our previous study, two group I type I IFNs, LcIFNd and LcIFNh (a new subgroup member), were identified in the perciform fish, large yellow croaker (Larimichthys crocea). Here, we identified a group II type I IFN, LcIFNc, in this species. The deduced LcIFNc contained six cysteines, four of which are highly conserved (C1: C28, C2:C53, C3: C130, and C4:C159) in the fish group II type I IFNs, and a typical type I IFN signature motif was also found in it. Phylogenetic analysis indicated that LcIFNc belongs to the IFNc subgroup of fish group II type I IFNs. LcIFNc was constitutively expressed in all examined tissues, and was rapidly up-regulated in spleen and head kidney by poly(I:C) and Aeromonas hydrophila. Recombinant LcIFNc protein (rLcIFNc) could increase the expression of antiviral genes, Mx1, PKR and ISG15, in large yellow croaker peripheral blood leukocytes (PBLs). The rLcIFNc also exhibited obvious antiviral activity based on less cytopathic effect (CPE) and decreased expression levels of several viral genes in the rLcIFNc-treated grouper spleen (GS) cells following Singapore grouper iridovirus (SGIV) infection. Additionally, rLcIFNc was able to induce the expression of LcIFNc, as well as LcIFNd and LcIFNh in the PBLs and primary head kidney cells (HKCs) from large yellow croaker. These results therefore indicated that LcIFNc not only had antiviral activity, but also mediated the regulation of type I IFN response.

Molecular characterization of a new fish specific chemokine CXCL_F6 in large yellow croaker (Larimichthys crocea) and its role in inflammatory response

Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in regulating cell migration and activation. Currently, five subgroups of fish specific CXC chemokines, named CXCL_F1-CXCL_F5, have been identified in teleost fish. However, understanding of the functions of these fish specific CXC chemokines is still limited. Here, a new member of fish specific CXC chemokines, LcCXCL_F6, was cloned from large yellow croaker Larimichthys crocea. Its open reading frame (ORF) is 369 nucleotides long, encoding a peptide of 122 amino acids (aa). The deduced LcCXCL_F6 protein contains a 19-aa signal peptide and a 103-aa mature polypeptide, which has four conserved cysteine residues (C28, C30, C56, and C72), as found in other known CXC chemokines. Phylogenetic analysis showed LcCXCL_F6 formed a separate clade with sequences from other fish species, tentatively named CXCL_F6, distinct from the clades formed by fish CXCL_F1-5 and mammalian CXC chemokines. The LcCXCL_F6 transcripts were constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney tissues by poly (I:C) and Vibrio alginolyticus. Its transcripts were also detected in primary head kidney leukocytes (HKLs), peripheral blood leucocytes (PBLs), and large yellow croaker head kidney (LYCK) cell line, and significantly up-regulated by poly(I:C), lipopolysaccharide (LPS), and peptidoglycan (PGN) in HKLs. Recombinant LcCXCL_F6 protein (rLcCXCL_F6) could not only chemotactically attract monocytes/macrophages and lymphocytes from PBLs, but also enhance NO release and expression of proinflammatory cytokines (TNF-α, IL-1β, and CXCL8) in monocytes/macrophages. These results indicate that LcCXCL_F6 plays a role in mediating the inflammatory response.

Expression analysis of Pannexin1 channel gene in response to immune challenges and its role in infection-induced ATP release in tilapia (Oreochromis niloticus)

ATP released from immune cells plays an important role in activation of host innate immunity. However, the molecular mechanisms for pathogen infection-induced ATP release in fish remains unclear. Pannexin1 (Panx1) is a recently identified ATP release channel important for controlling immune responses. The immune relevance of Panx1 in fish, however, is still poorly understood. In this study, we characterized a Panx1 gene homologue (termed tPanx1) from Nile tilapia (Oreochromis niloticus) and analyzed its expression in response to different immune challenges. We also investigated the role of tPanx1 channel in bacterial infection-induced ATP release. Real-time quantitative PCR analysis revealed that tPanx1 gene is expressed in all tested tissues with predominant expression in intestine. Immune challenges with lipopolysaccharide, polyinosinic-polycytidylic acid and zymosan led to increased gene expression of tPanx1 in tilapia head kidney cells and peripheral blood leucocytes. In addition, tPanx1 gene was up-regulated in hepatopancreas, muscle, spleen, gill, head kidney and blood after Aeromonas hydrophila infection. Furthermore, pharmacological inhibition of tPanx1 channel activity with Panx1 channel inhibitor, carbenoxolone, significantly attenuated A. hydrophila infection-induced ATP release in tilapia head kidney cells. Taken together, our findings suggested that tPanx1 is an important immune response gene involved in bacterial infection-induced ATP release in tilapia O. niloticus.

Molecular and biological characterization of gamma-interferon-inducible lysosomal thiol reductase in silver carp (Hypophthalmichthys molitrix)

Gamma-interferon-inducible lysosomal thiol reductase (GILT) plays an important role in the processing of major histocompatibility complex (MHC) class II-restricted antigens by catalyzing disulfide bonds reduction. Herein, a GILT homolog (ScGILT) was identified from silver carp. Its open reading frame covers 771 base pairs, encoding a protein of 256 amino acids that possesses GILT signature sequence CQHGX2ECX2NX4C, active-site CXXC motif, and two potential N-linked glycosylation sites. The predicted tertiary structures of ScGILT and other GILTs were quite similar in shape and positional arrangement of the key motifs. ScGILT mRNA was constitutively expressed in all detected tissues, with high-level expression in fish immune organs, spleen and head kidney. After stimulation with lipopolysaccharide, the expression of ScGILT mRNA significantly increased in spleen and head kidney cells, and ScGILT protein translocated to late endosomes and lysosomes in HeLa cells. Recombinant ScGILT fused with a His6 tag was expressed and purified, and could reduce the interchain disulfide bonds of IgG at pH 4.5. These results suggested that ScGILT was capable of catalyzing disulfide bonds reduction, and then might play an important role in the processing of MHC class II-restricted antigens in silver carp.

Leukocyte immune-type receptor expressing cells increase in response to Edwardsiella ictaluri infection in Channel Catfish, Ictalurus punctatus

Abstract Unique to teleosts, leukocyte immune-type receptors (LITRs) represent a large polygenic, polymorphic family of immunoregulatory receptors. Monoclonal antibodies (mAb), CC34 and CC41, were developed against a catfish clonal CTL line and a catfish NK cell line, respectively. Both recognize members of the LITR family. One subset of LITRs, defined by reactivity with mAb CC41, is a distinct marker of a cytotoxic cell population, which expands during the antiviral response to Channel Catfish Virus (CCV). It is hypothesized that these mAb CC41-reactive LITRs function as NK-like receptors and recognize stress induced ligands on target cells. Edwardsiella ictaluri, an intracellular pathogen, primarily establishes infection in pronephros (head kidney) monocytes and may elicit a strong cytotoxic response. To explore this, small fish (<1kg) were infected with E. ictaluri via bath immersion (1×107cfu/ml). Following infection, whole blood was collected and analyzed daily by flow cytometry for mAb CC34 and CC41 staining. In parallel, an in vitro model was developed to monitor the number of CC34 and CC41-reactive cells in response to E. ictaluri infection. Briefly, head kidney monocytes and peripheral blood lymphocytes (PBL) were isolated from catfish of either susceptible or resistant background. Head kidney cells were infected with E. ictaluri (MOI 10) and then mixed with PBL from the same individual catfish and analyzed daily by flow cytometry. There was an increase in staining with both mAbs CC34 and CC41 by day 3 after infection both in vivo and in vitro. This study highlights the utilization of these mAb reagents to monitor cytotoxic cell response to E. ictaluri infection.

The effect of intermittent hypoxia under different temperature on the immunomodulation in Streptococcus agalactiae vaccinated Nile tilapia (Oreochromis niloticus)

Dissolved oxygen (DO) and temperature are the potential immunomodulators in fish and play the important roles in regulating immunity. We studied the effect of intermittent hypoxia under different temperature on the immunomodulation in vaccinated Nile tilapia (Oreochromis niloticus). The expression of immune-related genes, enzymatic activities, histology, cumulative mortality, and S. agalactiae clearance were assessed. Study conditions were intermittently hypoxic (4.0 ± 1.0 mg/L DO) at 30 ± 0.5 °C or 35 ± 0.5 °C. Interleukin-1beta (IL-1β), tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) mRNA expression in spleen and head kidney were significantly lower in vaccinated hypoxic fish compared to the vaccinated normoxic fish. Levels of heat shock protein 70 (HSP70) in tissues showed an opposite tendency. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities were significantly lower in vaccinated hypoxic fish. Malondialdehyde levels were significantly greater under hypoxic conditions. In vitro studies evaluated the effects of intermittent hypoxia at different temperatures on cells of vaccinated O. niloticus. Phagocytic activity of peripheral blood leucocytes (PBLs) and intracellular reactive oxygen species (ROS) production in head kidney cells were significantly decreased by intermittent hypoxia at either 30 °C or 35 °C, while nitric oxide levels in tissues cells increased significantly under hypoxic conditions. These changes were well reflected by the further suppression modulation on S. agalactiae clearance in vaccinated O. niloticus and higher cumulative mortality by intermittent hypoxia. Taken together, intermittent hypoxia at either 30 °C or 35 °C could suppress immunomodulation in vaccinated Nile tilapia.

Comparative studies on the cytotoxic effects induced by nodularin in primary carp leukocytes and the cells of the fish CLC line

Nodularin, one of the toxins produced by cyanobacteria commonly occurring in brackish waters, is a cyclic pentapeptide, that may exert adverse effects on animal and human health. The aim of the present study was to evaluate the cytotoxicity of nodularin on different fish cellular models. The leukocytes isolated from carp head kidney and common carp (Cyprinus carpio L.) leukocyte cell line (CLC) were incubated with the cyanotoxin at the concentrations of 0.001, 0.01, 0.05 or 0.1 μg/mL. Treatment of cells with the higher concentrations of the toxin resulted in a dramatic decrease in cell viability. Simultaneously, we observed an increase in the percentage of necrotic cells. Nodularin also induced apoptosis as demonstrated by increased caspase-3/7 activity, and DNA fragmentation. We found, that primary cell cultures were more sensitive to the toxin than CLC cells. Our study also demonstrated significant increases in intracellular ROS production and decreases in GSH levels, observed in both primary leukocytes and CLC cells exposed to nodularin at 0.01–0.1 μg/mL. These cells exhibited similar GSH/GSSG ratios and similar level of oxidized glutathione.

The discovery and comparative expression analysis of three distinct type I interferons in the perciform fish, meagre (Argyrosomus regius)

Type I interferons (IFN) play an important role in anti-viral responses. In teleost fish multiple genes exist, that are classified by group/subgroup. That multiple subgroups are present in Acanthopterygian fish has only become apparent recently, and 3 subgroups are now known to be expressed, including a new subgroup termed IFNh. However, the potential to express multiple IFN subgroups and their interplay is not well defined. Hence this study aims to clarify the situation and undertook the first in-depth analysis into the nature and expression of IFNc, IFNd and IFNh in the perciform fish, meagre. Constitutive expression was analysed initially during larval development and in adult tissues (gills, mid-gut, head kidney, spleen). During early ontogeny IFNc was the highest expressed IFN, and this was also the case in adult tissues with the exception of gills where IFNd was highest. However, comparison between tissues for individual isoforms showed that spleen had high transcript levels of all three IFNs, IFNd/IFNh were also highly expressed in gills. The expression of each sub-group was increased significantly in the four tissues following injection of poly I:C, however, this increase was only seen in the mid-gut for IFNh. Following in vitro stimulation with poly I:C again all three isoforms were upregulated, although with differences in kinetics and the cell source used. For example, early induction was seen for IFNc/IFNh in gill cells, IFNd/IFNh in splenocytes and all three isoforms in head kidney cells. Induction was sustained in splenocytes and head kidney cells, but in gut cells only a late induction was seen. These results demonstrate a complex pattern of regulation between the different IFN isoforms present in meagre and highlights potential sub-functionalisation of these IFN subgroups during perciform anti-viral responses.

Molecular characterization and functional activity of CXCL8_L3 in large yellow croaker Larimichthys crocea

CXCL8, also called interleukin-8, is a typical CXC chemokine that plays a key role in promoting inflammation. Phylogenetically, fish CXCL8 chemokines can be divided into three subgroups, CXCL8_L1, CXCL8_L2, and CXCL8_L3, of which CXCL8_L3 is a new subgroup. The CXCL8_L3 gene sequences have been reported in many fish species, but their function remains unknown. Here, a CXCL8_L3 (LycCXCL8_L3) gene was cloned from large yellow croaker Larimichthys crocea. Its open reading frame (ORF) was 309 nucleotides long, encoding a peptide of 102 amino acids. The deduced LycCXCL8_L3 protein contains an 18-aa signal peptide and an 84-aa mature polypeptide, which has four conserved cysteine residues (C30, C32, C57, and C73) as found in other known CXCL8 chemokines. Phylogenetic analysis showed LycCXCL8_L3 formed a major clade with CXCL8_L3 sequences from other fish species. The LycCXCL8_L3 transcript was constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney tissues by inactivated trivalent bacterial vaccine. The LycCXCL8_L3 transcript was also detected in peripheral blood leukocytes (PBLs), primary head kidney macrophages (PKM), and large yellow croaker head kidney cell line (LYCK), with the highest levels in PKM. Recombinant LycCXCL8_L3 (rLycCXCL8_L3) protein could not only chemotactically attract lymphocytes and eosinophils in PBLs, but also enhance the respiratory burst activity of PKM. These results indicate that LycCXCL8_L3 may play an important role in the inflammatory response of large yellow croaker. To our knowledge, this is the first report on functional study of the CXCL8_L3 in fish.

Procedures for leukocytes isolation from lymphoid tissues and consequences on immune endpoints used to evaluate fish immune status: A case study on roach (Rutilus rutilus)

The effects of two protocols (density gradient versus hypotonic lysis) used for leukocyte isolation from three major lymphoid tissue of fish (head-kidney, spleen and blood) were examined on some cell functional activities (tissue leucocytes distributions, phagocytosis, basal and burst oxidative activities) classically used to estimate the fish immune status. Experiments were conducted on roach (Rutilus rutilus), a cyprinid fish model often studied in different eco-physiological contexts (aquaculture, ecotoxicology …). All of immune endpoints were assessed either immediately after cell isolation or after a 12 h of incubation in order to observe if a post-isolation incubation may influence the leukocytes activities.Compared to the density gradient, hypotonic lysis is associated with granulocytes enrichments of cell suspensions. This is particularly true for leukocyte suspensions isolated from head kidney where granulocytes are naturally abundant. However, important variabilities in leukocyte distributions were observed in head kidney and spleen cells samples obtained by the use of hypotonic lysis for two incubation conditions used (no incubation or 12 h of incubation at 4 °C). The density gradient protocol leads to a transitory increase in basal ROS production in spleen lymphocytes and macrophages The blood leukocytes isolated by this same method exhibit high basal oxidative activities after 12 h of incubation at 4 °C and for the three leukocyte types (lymphocytes, monocytes and granulocytes). The hypotonic lysis is associated with an increase in PMA-induced ROS production especially in head kidney leukocytes. The increases in cell oxidative activities are consistent with increases in granulocyte proportions observed in leukocyte suspensions obtained by hypotonic lysis. Finally, the two protocols have no effect on leukocyte mortality and phagocytic activity.Within limits of our experimental conditions, the spleen is the organ whose leukocyte oxidative activities (stimulated or not) are only slightly influenced by the methods used for leukocyte isolation. This is also the case for the anterior kidney, but for this tissue, it is necessary to incubate the isolated cells for 12 h at 4 °C before functional analyses.Each of the two methodologies used has advantages and disadvantages. The hypotonic lysis allows to isolate a greater variety of leukocytes types whereas the density gradient used ensures a better stability of cells distributions over time. However, for the same fish species and for the same tissue, the method used to isolate leukocytes influences results and must be taken into consideration during acquired data analysis for evaluation of fish immune status.