Kidney Leukocytes Research Articles

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.

Effect of symbiotic supplemented diet on innate-adaptive immune response, cytokine gene regulation and antioxidant property in Labeo rohita against Aeromonas hydrophila

Administration of probiotic, prebiotic or symbiotic supplemented diets boosts the antioxidant property, pro and/or anti-inflammatory cytokine gene transcription, innate-adaptive immunity, growth rate and feed digestibility with very low or no mortality in healthy and infected (both groups) in Labeo rohita against Aeromonas hydrophila is reported. The probiotic diet increased the white blood cell (WBC) count and globulin (GB) level significantly on or after 6th week whereas with the symbiotic diet the increase was noted two weeks earlier in both groups; the total protein (TP) level also increased significantly when fed with probiotic diet on weeks 6 and 8, whereas with symbiotic diet the significant increase manifested earlier at 4th week itself. The serum phagocytic activity (PA), respiratory burst activity (RBA), complement C3 (CC3) level, alternative complement pathway (ACP), lysozyme activity (LA), and immunoglobulin M (IgM) levels in head kidney (HK) leucocytes increased significantly (P < 0.05) in both groups fed with probiotic diet on weeks 6 and 8; with symbiotic diet from weeks 2–8; but with prebiotic diet only on 8th week. With probiotic diet the superoxide dismutase (SOD) and catalase (CAT) activities increased significantly (P < 0.05) on weeks 6 and 8; with symbiotic diet from weeks 4–8 but the prebiotics diet only on 8th week. However, glutathione peroxidase (GPx) activity increased significantly (P < 0.05) with probiotic diet on weeks 6 and 8 and with symbiotic diet from weeks 4–8. When healthy fish fed with any supplementation diet for a period of 30 days there was no mortality while 5%, 10%, and 10% mortality was observed in infected group fed with symbiotic, probiotic, and prebiotic supplementation diets. In head kidney (HK) leucocytes, the IL-1β, IL-8, TNF-α, and NF-κB gene transcriptions were significantly up-regulation in both groups when fed with probiotic diet on weeks 6 and 8, symbiotic diet from weeks 4–8 while the prebiotic diet only on 8th week. The iNOS expression was up-regulation significantly in both groups fed with probiotic and symbiotic diets on weeks 6 and 8; however, with any diet, the relative IL-10 and TGF-β gene expressions were down-regulated. The present study suggested that dietary administration of symbiotic diet elicited earlier antioxidant activity, innate-adaptive immune response, immune related cytokine gene modulation, and disease protection earlier i.e. on 4th week than with probiotic or prebiotic diets in L. rohita against A. hydrophila.

Production and functional insights into the potential regulation of three isoforms of grass carp p40 subunit in inflammation

The p40 subunit is known as a component of Interleukin (IL)-12 and IL-23. In mammals, p40 can be secreted as a monomer or homodimer and acts independently to mediate cellular responses. Recently, three p40 paralogues were isolated and identified from grass carp and other fish species, but whether they exist independently as well as their functional consequences and significance remain unclear. In the present study, using grass carp as the model, we for the first time demonstrated the existence of natural fish p40a, p40b and p40c (gcp40a, gcp40b and gcp40c) mainly as a monomer in culture supernatant of head kidney leukocytes (HKLs). Particularly, their excessive secretion induced by various immune stimuli suggests possible involvement of free p40s in fish immune responses. To define their functions, recombinant grass carp p40a/b/c (rgcp40a, rgcp40b and rgcp40c) were prepared by Pichia pastoris expression system, and they possessed the activities to enhance the secretion of pro-inflammatory cytokines including Il-1β and tumor necrosis factor-α (Tnf-α) in grass carp HKLs. These pro-inflammatory properties of p40 isoforms prompted us to investigate their roles during the inflammatory process. In line with this, in vivo study revealed the pathogenic effect of rgcp40a on intestinal inflammation, whereas gcp40a polyclonal antibodies remarkably ameliorated Aeromonas hydrophila-induced intestinal histopathological changes. Taken together, our results uncover the biological significance of free p40s in teleost, and provide new clue for targeting fish intestinal inflammation.

In vitro immunomodulatory activities of peptides derived from Salmo salar NK-lysin and cathelicidin in fish cells

Antimicrobial peptides (AMPs) are amphipathic peptides, which play an important role in innate defence. These peptides are gene-encoded and either constitutively expressed and/or upregulated during an infection. NK-lysins are AMPs with a three-dimensional globular structure. They are larger molecules, which comprise 74–78 amino acid residues and six conserved cysteine residues forming three disulphide bonds. Cathelicidins are a family of antimicrobial peptides that act as important components of the innate immune system with a broad spectrum of antimicrobial activity and immunomodulatory properties. Although they are widely studied in mammals, little is known about their immunomodulatory function. In the present study, we identified and characterized for the first time four NK-lysin-like transcripts from Atlantic salmon (Salmo salar) based on EST reported sequences. In vitro, NK-lysin derived peptides were able to induce the expression of IL-1β and IL-8 in Salmo salar head kidney leukocytes. We also tested Salmo salar cathelicidin 1 derived peptide in a similar assay, showing its ability to induce the expression of IFN-γ. These results indicate that NK-lysin and cathelicidin 1 derived peptides are able to modulated immune response, suggesting their potential use to enhance immune response in fish.

Lyn is involved in host defense against S. agalactiae infection and BCR signaling in Nile tilapia (Oreochromis niloticus)

Lyn, a member of Src protein kinase family, plays a crucial role in immune reactions against pathogenic infection. In this study, Lyn from Nile tilapia (Oreochromis niloticus) (OnLyn) was identified and characterized at expression pattern against bacterial infection, and regulation function in BCR signaling. The open reading frame of OnLyn contained 1536 bp of nucleotide sequence encoded a protein of 511 amino acids. The OnLyn protein was highly conversed to other species Lyn, including SH3, SH2 and a catalytic Tyr kinase (TyrKc) domain. Transcriptional expression analysis revealed that OnLyn was detected in all examined tissues and was highly expressed in the head kidney. The up-regulation OnLyn expression was observed in the head kidney and spleen following challenge with Streptococcus agalactiae (S. agalactiae) in vivo, and was also displayed in head kidney leukocytes challenge with S. agalactiae and LPS in vitro. In addition, after induction with mouse anti-OnIgM mAb in vitro, the OnLyn expression and phosphorylation of OnLyn (Y507) were significantly up-regulated in the head kidney leukocytes. Moreover, after treatment with AZD0530 and mouse anti-OnIgM monoclonal antibody, the down-regulation of cytoplasmic free-Ca2+ concentration was detected in the head kidney leukocytes in vitro. Taken together, the findings of this study revealed that OnLyn might play potential roles in BCR signaling and get involved in host defense against bacterial infection in Nile tilapia.

Innate-immune responses of tilapia (Oreochromis mossambicus) exposure to acute cold stress.

In this present study, Oreochromis mossambicus tilapia were transferred to cold water at 12°C for various time intervals (1, 4, 8, 24, and 48 hr) and its innate immune response was analyzed by studying cellular and humoral parameters. In vivo, alternative complement pathway activity in blood plasma was rapidly increased at 1 hr of cold water (12°C) exposure. Lysozyme activity and cortisol levels of plasma were increased at 4 and 1 hr, respectively. Surprisingly, only plasma cortisol levels remained unchanged through 24 hr of cold water transfer. Phagocytic ability, phagocytic capacity, and respiratory burst (RB) activity of head kidney (HK) leukocytes and splenocytes showed no any significant changes. In peripheral blood leukocytes, phagocytic capacity, and RB activity were increased at 24 hr of cold water exposure. The expressions of genes involved innate immunity in splenocytes and HK leukocytes of tilapia cold water exposure were analyzed, messenger RNA (mRNA) expressions of HSP70, HSP90, and immunoglobulin M failed to change upon exposure to cold stress. Major histocompatibility complex-I and II mRNAs were significantly increased in tilapia splenocytes at 1 hr of cold water transferred. Whereas myxovirus (Mx) expression was increased in splenocytes and HK leukocytes of tilapia after 1 hr of cold water exposed. Our result reveals that the exposure of tilapia to acute cold stress condition significantly enhances plasma acid phosphatase activity and both phagocytic capacity and RB activity. Furthermore, cold stress significantly stimulates Mx gene expression in splenocytes and HK leukocytes.

Effects of aloe-emodin on innate immunity, antioxidant and immune cytokines mechanisms in the head kidney leucocytes of Labeo rohita against Aphanomyces invadans

The effect of aloe-emodin incorporated diets on innate immune response, disease resistance, pro and/or anti-inflammatory cytokine gene transcription in Labeo rohita against Aphanomyces invadans is reported for the first time. In healthy and infected groups fed with 5 mg aloe-emodin enriched diet the white blood cell (WBC) count increased significantly (p > 0.05) after 6th week. In both groups fed with any enriched diet the biochemical parameters such as albumin, globulin, and albumin/globulin ratio did not vary significantly; however with 5 mg aloe-emodin diet the albumin and globulin levels increased significantly (p > 0.05) after 6th week. The serum phagocytic activity (PA), respiratory burst activity (RBA), serum complement C3 (CC3), and lysozyme activity (LA) did not increase with any diet between weeks 2 and 4, whereas with 5 mg aloe-emodin diet increased significantly in both groups after 6th week. The pro-inflammatory cytokines such as IL-1β, IL-8, TNF-α, and iNOS significantly modulated the expression in both groups on being fed with 5 mg aloe-emodin incorporation diet on 8th week. Healthy fish fed with any aloe-emodin diet did not suffer mortality. However, the infected fish fed with 1, 5, and 10 mg kg−1 aloe-emodin diets registered 5%, 10%, and 15% mortality. The present study indicates that healthy and infected L. rohita exhibited enhanced innate immune response, disease resistance, pro and/or anti-inflamatory cytokine gene transcription levels against A. invadans.

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.

In vitro effects of the neuroactive substances serotonin and γ-aminobutyric acid on leucocytes from sticklebacks (Gasterosteus aculeatus)

The majority of parasites have evolved strategies to evade the immune responses of their hosts. Neuroactive substances produced by cestodes are possible candidate molecules for regulating host immune responses. The neurons of helminths can synthesize a wide range of molecules that are identical to the ones functioning in their host organisms, and host lymphocytes have receptors for these neuroactive substances. We hypothesized that in teleost fish, antihelminthic immune responses are regulated via 5-hydroxytryptamine (5-HT, or serotonin) and γ-aminobutyric acid (GABA). In the present study, we investigated the in vitro influence of serotonin, GABA and Schistocephalus solidus (helminth) antigens on basic characteristics of the three-spined stickleback Schistocephalus solidus cellular immune response. Head kidney leucocytes (HKLs) were analysed by flow cytometry for cell viability and the frequency of leucocyte subsets (the granulocyte-to-lymphocyte ratio) and by a chemiluminescence assay for the production of reactive oxygen species (ROS). In short-term (2-h) HKL cultures, 5-HT did not change the total numbers of live HKLs, but the production of ROS decreased significantly with all 5-HT concentrations. In long-term (96-h) cultures, high 5-HT concentrations induced a decrease in leucocyte viability. This coincided with elevated ROS production in cultures with all 5-HT concentrations. In short-term (2-h) HKL cultures, GABA did not change the total numbers of live HKLs, but the production of ROS decreased significantly with high (100 nmol L−1) GABA concentrations. In long-term (96-h) cultures, high and medium concentrations of GABA (100 nmol L−1 and 10 nmol L−1) elevated the numbers of live HKLs compared to controls. The granulocyte-to-lymphocyte ratios generally increased upon exposure to GABA at all concentrations. All concentrations of GABA alone elevated the ROS production of HKLs compared to controls. In the present work, we showed that the neuroactive substances serotonin and GABA regulate the teleost immune system. Our study supports the hypothesis that these substances might be immunomodulators in tapeworm–fish parasite-host interactions.

Immunohistochemical identification of the bacterial antigen of Aeromonas hydrophila in the kidneys and spleen of carps and evaluation of the activity of kidney phagocytes after intraperitoneal and immersion immunization

The aim of this study was to evaluate the activity of kidney phagocytes after immunization of fish with inactivated bacteria of Aeromonas hydrophila and to determine the location and permanence of the bacterial antigen in the lymphoid organs, the kidney and the spleen, of carps immunized intraperitoneally (group I) and by immersion (group II) at 12oC. We examined the absorption of bacteria by kidney leucocytes (PK%), the killing capacity of phagocytes (IB) and chemiluminescent activity (CL). Evaluations were performed 12 h, as well as 3, 7, 14 and 28 days after immunization. At all these time points, the values of the parameters examined were higher in fish immunized intraperitoneally than in those immunized by immersion. The absorption of bacterial cells by kidney phagocytes was particularly high after 14 days after immunization in fish group I whereas their killing and chemiluminescent activity was particularly high in 7 days after immunization in group I. Immunohistochemical examination revealed an extensive distribution and large amounts of the antigen in the interstitial part and melanomacrophage centers of the kidneys and spleen after the intraperitoneal administration of the antigen, By contrast, small amounts of the antigen were found in these organs after it had been administered by immersion.

Generation of virus-specific CD8+ T cells by vaccination with inactivated virus in the intestine of ginbuna crucian carp

Although a previous study using ginbuna crucian carp suggested that cell-mediated immunity can be induced by the oral administration of inactivated viruses, which are exogenous antigens, there is no direct evidence that CD8+ cytotoxic T cells (CTLs) in teleost fish are generated by vaccination with exogenous antigens. In the present study, we investigated whether antigen-specific CD8+ CTLs in ginbuna crucian carp can be elicited by intestinal immunization with an exogenous antigen without any adjuvant. The IFNγ-1 and T-bet mRNA expressions were up-regulated in intestinal leukocytes following the administration of formalin-inactivated crucian hematopoietic necrosis virus (FI-CHNV), whereas the down-regulation of these genes was observed in kidney leukocytes. Furthermore, an increase in the percentage of proliferating CD8+ cells was detected in the posterior portion of the hindgut, suggesting that the virus-specific CTLs are locally generated in this site. In addition, cell-mediated cytotoxicity against CHNV-infected syngeneic cells and the in vivo inhibition of viral replication were induced by immunization with FI-CHNV. Unexpectedly, intraperitoneal immunization with FI-CHNV induced a type I helper T cell (Th1)-response in the intestine, but not in the kidney; however, its effect was slightly lower than that reported after intestinal immunization. These findings suggest that the posterior portion of the intestine is an important site for generating virus-specific CTLs by vaccination with the inactivated vaccine.

Molecular characterization of grass carp interleukin-6 receptor and the agonistic activity of its soluble form in head kidney leucocytes

Interleukin (IL)-6 receptor (IL-6R) can specifically bind to IL-6 and the complex subsequently recruits a transmembrane signal transducer, gp130, to trigger the intracellular signal transduction. IL-6R exists in two forms, a transmembrane IL-6R and a soluble IL-6R (sIL-6R), leading to different signal transduction mechanisms as classic signaling and trans-signaling, respectively. There is now a general consensus that these two modes of signal transduction can mediate anti-inflammatory and pro-inflammatory activities of IL-6. The study on Il-6r is limited although Il-6 has been well studied in teleost. In the present study, a cDNA encoding grass carp Il-6r (gcIl-6r) was isolated. An in-silico analysis showed that gcIl-6r shared the same functional domains and conserved gene synteny at its loci with mouse homologue, and its amino acid sequence was conserved in fish species. A tissue distribution assay demonstrated that gcil6r mRNA was expressed with high levels in immune tissues including spleen and head kidney, and its expression was induced by LPS and Poly I:C in grass carp head kidney leucocytes (HKLs). An in vitro binding assay showed that recombinant soluble gcIl-6r (rgcsIl-6r) could specifically bind to recombinant gcIl-6 (rgcIl-6) protein. Moreover, rgcIl-6 stimulated suppressor of cytokine signaling 3 (socs3)'s mRNA expression in grass carp HKLs and it combined with rgcsIl-6r increased socs3 mRNA expression in CIK cells with gp130 but without Il-6r expression. In HKLs, rgcIl-6 stimulated the mRNA levels of both pro-inflammatory (tnfa and il1b) and anti-inflammatory (il10) cytokines, and rgcsIl-6r could augment these stimulatory effects of gcIl-6. Taken these data together, gcsIl-6r can mediate the immuno-regulatory functions of gcIl-6 and has an agonistic property in these actions of Il-6 in grass carp.

Identification and functional characterization of grass carp (Ctenopharyngodon idella) tumor necrosis factor receptor 2 and its soluble form with potentiality for targeting inflammation

Tumor necrosis factor-alpha (TNF-α) signals through two distinct cell surface receptors, TNFR1 and TNFR2 in mammals. In the present study, grass carp Tnfr2 (gcTnfr2) was isolated and characterized. Sequence alignment and phylogenetic analysis suggested that gcTnfr2 was a homolog of goldfish and zebrafish Tnfr2. Tissue distribution assay showed gctnfr2 transcripts were expressed in all examined tissues similar to gctnfr1. To functionally characterize the newly cloned molecule, gcTnfr2 was overexpressed in COS7 cell lines and it showed the ability to mediate the recombinant grass carp Tnf (rgcTnf)-α-triggered NF-κΒ activity and gcil1b promoter activity, clarifying its role in mediating Tnf-α signaling. The recombinant soluble form of gcTnfr2 (rgcsTnfr2) was prepared and it was able to interact with rgcTnf-α with higher affinity than that of rgcsTnfr1. Moreover, grass carp soluble Tnfr2 (gcsTnfr2) were detected in the culture medium of grass carp head kidney leukocytes (HKLs) and heat-inactivated A. hydrophila challenge significantly induced its production, indicating involvement of gcsTnfr2 in inflammation response. In agreement with this notion, rgcsTnfr2 effectively antagonized the effect of rgcTnf-α on il1b mRNA expression in HKLs, suggesting anti-Tnf-α property of gcsTnfr2. To strengthen the anti-inflammatory role of soluble Tnfr2, bacteria were injected intraperitoneally in grass carp followed by rgcsTnfr2. Hematoxylin-eosin (HE) staining of head kidney, spleen and intestine showed that rgcsTnfr2 could significantly improve infection-induced histopathological changes. These results functionally identified gcTnfr2 and its soluble form, particularly highlighting the role of gcsTnfr2 against Tnf-α-triggered inflammatory signaling. In this line, rgcsTnfr2 displayed anti-inflammatory potentiality during infection, thereby providing a powerful mediator of inflammation control in fish.

Characterization and functional analysis of grouper (Epinephelus coioides) MEK1 and MEK2

MEK dual-specificity protein kinases are a group of mitogen-activated protein kinase kinases, which act as an integration point by transferring extracellular signals to the nucleus. To investigate the function of MEK in teleost fish, we cloned MEK1 and MEK2 cDNA sequences from the orange-spotted grouper (Epinephelus coioides). EcMEK1 and EcMEK2 shared 80% amino acid identity with each other. EcMEK1 had 89–99% amino acid identity with teleosts or mammals, whereas EcMEK2 shared 85–97% amino acid identity. The exon structures of the grouper MEK1/2 genes were conserved with zebrafish and human MEK1/2. Tissue distribution analysis showed that EcMEK1 and EcMEK2 had a similar expression pattern in grouper tissues and was mainly transcribe in systemic immune organs. Both EcMEK1 and EcMEK2 were distributed throughout the cytoplasm of transfected GS or HEK293T cells. Overexpression of EcMEK1 or EcMEK2 activated Activator protein 1 dependent luciferase. The phosphorylation levels of EcMEK1/2 and EcERK1/2 were significantly increased in head kidney leukocytes by stimulation with PMA treatment. The grouper MEK1/2-ERK1/2 axis was activated in Cryptocaryon irritans infection and showed an enhanced phosphorylation after immunization.

Characterization of Bacillus subtilis from gastrointestinal tract of hybrid Hulong grouper (Epinephelus fuscoguttatus × E. lanceolatus) and its effects as probiotic additives

Probiotics are widely used for the improvement of animals' growth and health. However, few marine aquatic probiotics are applied and licensed in China. In this study, a Bacillus spp. strain was isolated from the Hulong grouper gastrointestinal tract, which was identified as a new strain of Bacillus subtilis and was named as 7k. B. subtilis 7k showed desirable capability of sporulation and resistance to heat, simulated gastric juice and simulated duodenum juice, indicating its potential as probiotics. Seven antimicrobial chemicals were found in the secretion of the B. subtilis 7k. B. subtilis 7k addition in diet promoted the growth rate of Hulong groupers. Moreover, B. subtilis 7k can inhibit infection by iridovirus, making B. subtilis 7k a suitable kind of probiotic for maintaining fishes’ health. Our results also revealed that B. subtilis 7k induced non-specific immune response in Hulong grouper under virus infection. Hulong grouper fed by diets containing B. subtilis 7k at 108 and 1010 cfu g−1 for 4–8 weeks were significantly strengthened in serum lysozyme activity, serum alternative complement activity (ACH50), serum bactericidal activity, respiratory burst, superoxide dismutase activity (SOD), and phagocytic activity of head kidney leucocytes when compared with those fed by control diets. In conclusion, B. subtilis 7k was isolated and characterized to be a kind of process enduring, growth stimulating, immunity enhancing and health promoting probiotic using in grouper culture.

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.

Dietary administration of probiotic Paenibacillus ehimensis NPUST1 with bacteriocin-like activity improves growth performance and immunity against Aeromonas hydrophila and Streptococcus iniae in Nile tilapia (Oreochromis niloticus)

Bacteria-induced diseases are a major cause of mortality in aquaculture. Probiotics have commonly been used to replace antibiotics for prophylactic biocontrol in aquaculture. In the present study, Paenibacillus ehimensis NPUST1 was isolated from a tilapia culture pond. This probiotic has bacteriocin-like activities against Aeromonas hydrophila and was characterized by biochemical analysis and 16S rDNA sequencing. The physiochemical properties of a crude extract of the bacteriocin-like substance revealed low pH and high thermal tolerance. The substance exhibited broad-spectrum antimicrobial activity against diverse aquatic pathogens, food spoilage, clinical pathogens, and plant pathogens. The effect of dietary supplementation with P. ehimensis NPUST1 was evaluated in regard to the growth of Nile tilapia (Oreochromis niloticus) and immunity against pathogenic infection. The results showed significantly increased weight gain (WG), feed conversion ratio (FCR), and feed efficiency (FE) in Nile tilapia fed P. ehimensis NPUST1 for 2 months compared with fish fed a control diet. When challenged with A. hydrophila and S. iniae, the fish fed P. ehimensis NPUST1 also exhibited a higher survival rate than fish fed the control diet. The immune parameters revealed that the P. ehimensis NPUST1-fed fish had significantly higher phagocytic activity, respiratory burst, and superoxide dismutase (SOD) of the head kidney leukocytes, as well as higher serum lysozyme activity and expression of cytokines TNF-α and IL-1β than the fish fed the control diet. These results indicate that dietary supplementation with P. ehimensis NPUST1 improved the growth performance, immunity, and disease resistance in Nile tilapia.

Complement 1q-binding protein from Nile tilapia (Oreochromis niloticus): Molecular characterization, expression pattern upon bacterial infection and its binding properties

Complement 1q-binding protein (gC1qR), a multi-functional cellular protein, is able to bind the globular head region of C1q molecule (gC1q), playing an important role in regulation of immune response and host defense against bacterial infection. In this study, the full length of a gC1qR ortholog (OngC1qR) was identified and characterized from Nile tilapia (Oreochromis niloticus). The cDNA of OngC1qR ORFs consisted of 870 bp of nucleotide sequence encoding a 289 amino acid residue polypeptide. The deduced amino acid sequence is highly homologous to teleost, containing a MAM33p domain, two potential glycosylation sites, and a putative cell adhesion site EGD (Glu-Gly-Asp). Spatial mRNA expression analysis revealed that the OngC1qR was expressed ubiquitously in all examined tissues, with a high expression in the liver. The OngC1qR expression was significantly up-regulated in liver, spleen and head kidney following in vivo challenges with Streptococcus agalactiae. A high up-regulation was also detected in head kidney leukocytes in vitro stimulation with Streptococcus agalactiae. In addition, immunofluorescence detection illustrated that the OngC1qR was located at the cell surface of leukocytes. Recombinant OngC1qR protein was able to not only bind LPS, LTA, S. agalactiae and Aeromonas hydrophila, but also possess binding capability with the ligand OnC1qs. Taken together, the results indicated that OngC1qR might be involved in host defense against bacterial infection in Nile tilapia.

Interleukin-6 gets involved in response to bacterial infection and promotes antibody production in Nile tilapia (Oreochromis niloticus)

Interleukin 6 (IL-6), a pleiotropic cytokine, plays an important role in humoral immune response, not only inducing the differentiation of B cells into plasma cells, but also promoting antibody-secreting cells (ASCs) to produce antibodies. In this study, Nile tilapia (Oreochromis niloticus) IL-6 (OnIL-6) was identified and characterized at expression level in response to bacterial infection and promotion of antibody production. The open reading frame of OnIL-6 ORF is consisted of 663 bp encoding a polypeptide of 220 amino acids. The deduced OnIL-6 protein contained an IL-6/G-CSF family signature, two conserved cysteine, and four α-helix bundles, which was highly homologous to other species. Spatial mRNA expression analysis revealed that the highest expression of OnIL-6 was observed in the thymus. After in vivo challenges of lipopolysaccharide (LPS) and Streptococcus agalactia (S. agalactiae), OnIL-6 expressions were significantly up-regulated in head kidney and spleen. The similar up-regulation of OnIL-6 was observed in the head kidney and spleen leukocytes in vitro stimulation with LPS and S. agalactiae. In addition, inducement with the recombinant OnIL-6 ((r)OnIL-6) in vitro caused significant increases in expressions of both sIgM and mIgM. Moreover, the (r)OnIL-6 stimulation enhanced the secretion of sIgM (more especially in P50 plasma-like B cells) and the production of mIgM in P60 and P70 B cell subsets (resting B cells, activated B cells and plasmablast-like B cells) in vitro. Taken together, this study indicated that OnIL-6 might be involved in host defense against bacterial infection and promote the production of antibody in Nile tilapia.

Pseudomonas aeruginosa FARP72 Offers Protection Against Aeromonas hydrophila Infection in Labeo rohita.

Use of probiotics as the biocontrol agent for disease prevention in aquaculture is gaining importance as an alternative to the indiscriminate use of antibiotics and other chemotherapeutics. In view of this trend, the probiotic properties of a potent antagonistic bacterium, Pseudomonas aeruginosa FARP72, was characterized in terms of safety, antagonistic activities, in vitro immunomodulation, and in vivo disease resistance. Immunomodulatory activity was ascertained by measuring the production of intracellular superoxide anion, nitric oxide, total leukocyte peroxidase content, and the leukocyte proliferation in head kidney leukocytes. The bacterium isolated from the skin mucus of freshwater catfish Clarias batrachus was harmless to Labeo rohita. It showed inhibitory activity against Aeromonas caviae, A. hydrophila, Edwardsiella tarda, Pseudomonas putida, and Streptococcus agalactiae as revealed by cross and parallel streaking methods. Significantly higher superoxide anion and nitric oxide production, peroxidase content, and proliferative responses of leucocytes delineated the strains' ability to interact with immune cells to activate the immune system in vitro. Significant growth inhibition of A. hydrophila from 1.55 × 105CFU/mL was observed when co-cultured with P. aeruginosa FARP72 in phosphate-buffered saline (PBS) at levels ranging from 2.61 × 107 to 2.61 × 109CFU/mL in 10days. P. aeruginosa FARP72 increased the survival rate of rohu fingerlings against pathogenic A. hydrophila challenge in biocontrol study in vivo as determined by cohabitation challenge. These results suggest that P. aeruginosa FARP72 is a potential probiotic strain and can be used in aquaculture to improve the health status and disease resistance of fish.