IFN-γ enhances protective efficacy against Nocardia seriolae infection in largemouth bass (Micropterus salmoides).

Introduction: Nocardia seriolae adversely impacts a diverse range of fish species, exhibiting significant pathogenic characteristics that substantially impede the progress of aquaculture. N. seriolae infects in fish has a long incubation period, and clinical symptoms are not obvious in the early stages. There is presently no viable and eco-friendly approach to combat the spread of the disease. According to reports, N. seriolae primarily targets macrophages in tissues after infecting fish and can proliferate massively, leading to the death of fish. Interferon-gamma (IFN-γ) is a crucial molecule that regulates macrophage activation, but little is known about its role in the N. seriolae prevention. Methods: IFN-γ was first defined as largemouth bass (Micropterus salmoides, MsIFN-γ), which has a highly conserved IFN-γ characteristic sequence through homology analysis. The recombinant proteins (rMsIFN-γ) were obtained in Escherichia coli (E. coli) strain BL21 (DE3). The inflammatory response-inducing ability of rMsIFN-γ was assessed in vitro using monocytes/macrophages. Meanwhile, the protective effect of MsIFN-γ in vivo was evaluated by N. seriolae infection largemouth bass model. Results: In the inflammatory response of the monocytes/macrophages activated by rMsIFN-γ, various cytokines were significantly increased. Interestingly, interleukin 1ß (IL-1ß) and tumor necrosis factor alpha (TNF-a) increased by 183- and 12-fold, respectively, after rMsIFN-γ stimulation. rMsIFN-γ improved survival by 42.1% compared with the control. The bacterial load in the liver, spleen and head kidney significantly decreased. rMsIFN-γ was also shown to better induce increased expression of IL-1ß, TNF-α, hepcidin-1(Hep-1), major histocompatibility complex I (MHCI), and MHC II in head kidney, spleen and liver. The histopathological examination demonstrated the transformation of granuloma status from an early necrotic foci to fibrosis in the infection period. Unexpectedly, the development of granulomas was successfully slowed in the rMsIFN-γ group. Discussion: This work paves the way for further research into IFN-γ of largemouth bass and identifies a potential therapeutic target for the prevention of N. seriolae.

Chitooligosaccharide boosts the immunity of immunosuppressed blunt snout bream against bacterial infections.

The immunosuppression hazard of fish brought by intensive aquaculture needs to be addressed urgently, while chitooligosaccharide (COS) shows the potential application in the prevention the immunosuppression of fish due to its superior biological properties. In this study, COS reversed the cortisol-induced immunosuppression of macrophages and improved the immune activity of macrophages in vitro, promoting the expression of inflammatory genes (TNF-α, IL-1ß, iNOS) and NO production, and increasing the phagocytic activity of macrophages. In vivo, the oral COS was absorbed directly through the intestine, significantly ameliorating the innate immunity of cortisol-induced immunosuppression of blunt snout bream (Megalobrama amblycephala). Such as facilitated the gene expression of inflammatory cytokines (TNF-α, IL-1ß, IL-6) and pattern recognition receptors (TLR4, MR) and potentiated bacterial clearance, resulting in an effective improvement in survival and tissue damage. Altogether, this study demonstrates that COS offers potential strategies in the application of immunosuppression prevention and control in fish.

Blunt snout bream (Megalobrama amblycephala) MaCSF-1 contributes to proliferation, phagocytosis and immunoregulation of macrophages via MaCSF-1R.

CSF-1 and CSF-1R have been well demonstrated in humans, regulating the differentiation, proliferation and survival of the mononuclear phagocyte system. However, the functional study on MaCSF-1 and MaCSF-1R from blunt snout bream (Megalobrama amblycephala) is still unknown. In the present study, we cloned and functionally characterized MaCSF-1 and MaCSF-1R. Multiple sequence alignment and phylogenetic tree analysis showed that both MaCSF-1 and MaCSF-1R were mostly close to the grass carp counterparts. Tissue distribution analysis showed that both MaCSF-1 and MaCSF-1R were widely distributed in all examined tissues, dominantly distributed in spleen, blood and head kidney tissues. Furthermore, confocal microscopy assay and flow cytometry assay showed that MaCSF-1R was the marker on the surface of macrophages. Recombinant MaCSF-1 promoted macrophage proliferation, phagocytosis and the production of IL-10. Through the pull-down experiments and indirect immunofluorescence experiments, the interaction between MaCSF-1 and MaCSF-1R was confirmed. To explore the relationship between MaCSF-1 and its receptor, MaCSF-1R and MaCSF-1R antibody was prepared. Then the MaCSF-1R blockage assay indicated that the role of MaCSF-1 on the macrophages proliferation and phagocytosis was weakened, leading the reduction of IL-10 expression level. In conclusion, MaCSF-1R is the marker on the surface of macrophage membrane; and MaCSF-1 promotes macrophage proliferation, phagocytosis, and significantly increased the expression levels of IL-10 depended on the interacting with MaCSF-1R. This study provides basal data for the biological function of MaCSF-1 and MaCSF-1R, and is valuable for the exploration of MaCSF-1 and MaCSF-1R molecular interactions.

Hepcidin Protects Yellow Catfish (Pelteobagrus fulvidraco) against Aeromonas veronii-Induced Ascites Disease by Regulating Iron Metabolism.

Aeromonas veronii (A. veronii) is one of the main pathogens causing bacterial diseases in aquaculture. Although previous studies have shown that hepcidin as an antimicrobial peptide can promote fish resistance to pathogenic bacterial infections, but the mechanisms remain unclear. Here, we expressed and purified recombinant yellow catfish (Pelteobagrus fulvidraco) hepcidin protein (rPfHep). rPfHep can up-regulate the expression of ferritin and enhance the antibacterial activity in primary hepatocytes of yellow catfish. We employed berberine hydrochloride (BBR) and Fursultiamine (FSL) as agonists and antagonists for hepcidin, respectively. The results indicated that agonist BBR can inhibit the proliferation of pathogenic bacteria, and the antagonist FSL shows the opposite effect. After gavage administration, rPfHep and the agonist BBR can enhance the accumulation of iron in liver, which may hinder the iron transport and limit the amount of iron available to pathogenic bacteria. Moreover, rPfHep and the agonist BBR can also reduce the mortality rate, bacterial load and histological lesions in yellow catfish infected with A. veronii. Therefore, hepcidin is an important mediator of iron metabolism, and it can be used as a candidate target for prevent bacterial infections in yellow catfish. Hepcidin and BBR have potential application value in preventing anti-bacterial infection.

Hamp Type-1 Promotes Antimicrobial Defense via Direct Microbial Killing and Regulating Iron Metabolism in Grass Carp (Ctenopharyngodon idella).

Hepcidin is an antimicrobial peptide and regulator of iron homeostasis which has two isoforms in most fishes and some mammals. Previous studies have reported that the two hepcidin isoforms have different roles. Hamp type-1 plays a regulatory role in iron metabolism and hamp type-2 mostly performs an antimicrobial role. In this study, we found that Ctenopharyngodon idella (C. idella) have only one hepcidin isoform (hamp type-1), which showed both broad-spectrum antibacterial and iron regulatory functions. C. idella hepcidin mature peptide (hepcidin-25) and truncated peptide (hepcidin-20) exhibited bactericidal activities against both Gram-positive and Gram-negative bacteria in a dose-dependent manner in part through membrane rupture and binding to bacterial genomic DNA. The data from challenge tests demonstrated that the administration of hepcidin-25 significantly reduced mortality rates of C. idella by A. hydrophila infection, probably due to direct bactericidal activities of the peptide and a reduction of iron content in the fish serum. In addition, a comparison between hepcidin-20 and -25 suggests that the N terminal 5 amino acids play a critical role in reducing iron content in fish serum. Our findings revealed an important role of hamp type-1 in maintaining iron homeostasis and fighting against bacterial infections, suggesting the hepcidin has implications for the prevention and control of bacterial infection in aquaculture.

Immunomodulatory Effects and Induction of Apoptosis by Different Molecular Weight Chitosan Oligosaccharides in Head Kidney Macrophages From Blunt Snout Bream (Megalobrama amblycephala).

Prophylactic administration of immunopotentiators has been tested and practiced as one of the most promising disease prevention methods in aquaculture. Chitosan oligosaccharide (COS), as an ideal immunopotentiator, is mainly used as feed additives in aquaculture, and the antimicrobial and immune enhancement effects are highly correlated with molecular weight (MW), but little is known about the mechanisms in teleost. Here, we isolated and purified macrophages in head kidney from blunt snout bream (Megalobrama amblycephala), stimulated them with three different MW (~500 Da, ~1000 Da and 2000~3000 Da) COSs, performed RNA-sequencing, global transcriptional analyses, and verification by quantitative real-time PCR (qRT-PCR) and immunofluorescent staining methods. Differential expression gene (DEG) analysis indicated that gene expression patterns are different and the proportion of unique genes are relatively high in different treatment groups. Biological process and gene set enrichment analysis (GSEA) demonstrated that all three COSs activate resting macrophages, but the degrees are different. Weighted gene co-expression network analysis (WGCNA) reflected gene modules correlated to MW, the module hub genes and top GO terms showed the activation of macrophage was positively correlated with the MW, and larger MW COS activated cell death associated GO terms. Further use of the screening and enrichment functions of STRING and Pfam databases discovered that apoptosis-related pathways and protein families were activated, such as the P53 pathway and caspase protein family. qRT-PCR results showed that as the stimulation time extends, the innate immune-related and P53 pathways are gradually activated, and the degree of activation is positively correlated with the stimulation time. In addition, apoptosis was detected by immunofluorescent staining in three groups. Therefore, the use of COS has two sides-it can activate the immune system against pathogen invasion, but with the increase in stimulation time and MW, macrophage apoptosis is induced, which may be caused by abnormal replication of DNA and excessive inflammation. This study provides a theoretical basis for the rational use of COS as an immunopotentiator in aquaculture.

Transcriptome Analysis Provides Insights into the Markers of Resting and LPS-Activated Macrophages in Grass Carp (Ctenopharyngodon idella).

Macrophages are very versatile immune cells, with the characteristics of a proinflammatory phenotype in response to pathogen-associated molecular patterns. However, the specific activation marker genes of macrophages have not been systematically investigated in teleosts. In this work, leukocytes (WBC) were isolated using the Percoll gradient method. Macrophages were enriched by the adherent culture of WBC, then stimulated with lipopolysaccharide (LPS). Macrophages were identified by morphological features, functional activity and authorized cytokine expression. Subsequently, we collected samples, constructed and sequenced transcriptomic libraries including WBC, resting macrophage (Mø) and activated macrophage (M(LPS)) groups. We gained a total of 20.36 Gb of clean data including 149.24 million reads with an average length of 146 bp. Transcriptome analysis showed 708 differential genes between WBC and Mø, 83 differentially expressed genes between Mø and M(LPS). Combined with RT-qPCR, we proposed that four novel cell surface marker genes (CD22-like, CD63, CD48 and CD276) and two chemokines (CXCL-like and CCL39.3) would be emerging potential marker genes of macrophage in grass carp. Furthermore, CD69, CD180, CD27, XCL32a.2 and CXCL8a genes can be used as marker genes to confirm whether macrophages are activated. Transcriptome profiling reveals novel molecules associated with macrophages in C. Idella, which may represent a potential target for macrophages activation.

Hepcidin protects grass carp (Ctenopharyngodon idellus) against Flavobacterium columnare infection via regulating iron distribution and immune gene expression.

Columnaris disease (CD) caused by Flavobacterium columnare (F. columnare) is lack of knowledge on effective treatment measures. Bacterial pathogens require iron as an essential nutrient to infect the host. While hepcidin acts as a master regulator in iron metabolism, its contribution to host defense is emerging as complex and multifaceted. In vitro, recombinant Ctenopharyngodon idellus (C. idellus) hepcidin (CiHep) and synthetic CiHep both showed the ability to increase the expression of hepcidin and ferritin in C. idellus kidney cells, especially the recombinant CiHep. In vivo, recombinant CiHep improved the survival rate of C. idellus challenged with F. columnare. In addition, the fish fed diet containing recombinant CiHep (group H-1) had a higher survival rate than other pretreatment groups. The study showed that recombinant CiHep regulated iron metabolism causing iron redistribution, decreasing serum iron levels and increasing iron accumulation in the hepatopancreas. Moreover, the expression of iron-related genes was upregulated in various degrees at a different time except for group H-1. Immune-related genes were also evaluated, showing higher expression in the groups pretreated with CiHep at an early stage of infection. Of note, a clear upregulation of more immune genes occurred in the groups pretreated with recombinant CiHep than that pretreated with synthetic CiHep in the late stage of infection. In conclusion, the recombinant CiHep has a protective effect on the host response to bacterial pathogens. We speculate that hepcidin protects C. idellus against F. columnare infection via regulating the iron distribution and immune gene expression.

Chemotactic effect of ß-defensin 1 on macrophages in Megalobrama amblycephala.

Besides their function as a physical barrier against pathogens, ß-defensins possess the ability to induce direct or indirect chemotaxis in leukocytes of mammals. However little is known about the ability of defensins to guide the migration of macrophages in fish. The objective of our study was to investigate whether ß-defensin 1 (maBD1) can recruit leukocytes (specifically macrophages) in vivo and in vitro in a farmed cyprinid fish Megalobrama amblycephala. The M. amblycephala ß-defensin 1 (maBD1) gene was amplified from the head-kidney transcriptome. Synthetic maBD1 polypeptide (as well as its N-terminus half, but not the C-terminus half) was capable of inducing the migration of leukocytes (specifically macrophages) at concentrations from 26.0 µg/mL to 52.0 µg/mL in head kidney tissue in vitro. When injected intraperitoneally in vivo, the number of leukocytes in the peritoneal cavity was in positive correlation with the maBD1 concentration. maBD1 also induced the expression of two proinflammatory cytokines (IL-1beta and TNF-alpha) in spleen, head and body kidney, and hepatopancreas. These results strongly indicate that BD1 has a chemoattractant capacity for macrophages, as well as the ability to modulate the expression of proinflammatory cytokines in fish.

Molecular cloning and functional characterization of cyclophilin A in yellow catfish (Pelteobagrus fulvidraco).

Cyclophilin A (CypA) is a ubiquitously expressed protein which involves in diverse pathological conditions including infection and inflammation. In this report, a CypA gene (designated as YC-CypA) was cloned from yellow catfish (Pelteobagrus fulvidraco) which is an important cultured fish species in Asian countries. The open reading frame (ORF) of YC-CypA encoded a polypeptide of 164 amino acids with calculated molecular weight of 17.70 kDa. The deduced amino acid sequences of the YC-CypA shared highly conserved structures with CypAs from the other species, indicating that YC-CypA should be a new member of the CypA family. Full-length YC-CypA protein was expressed in Escherichia coli and specific polyclonal antibody against YC-CypA was generated. The YC-CypA protein showed chemotactic activity by transwell migration assay. The mRNA and protein of YC-CypA could be detected in all examined tissues with relatively higher mRNA level in spleen and higher protein level in head kidney, respectively. The temporal expression patterns of YC-CypA, IL-1ß and TNF-α mRNAs were analyzed in the liver, spleen and head kidney post of Edwardsiella ictaluri infection. By immunohistochemistry assay, slight enhancement of YC-CypA protein was observed in the liver, spleen, body kidney and head kidney of yellow catfish infected with E. ictaluri. In conclusion, YC-CypA of yellow catfish showed chemotactic activity in vitro and might have been involved in cytokines secretion in yellow catfish during the infection of E. ictaluri.

Oligochitosan stimulated phagocytic activity of macrophages from blunt snout bream (Megalobrama amblycephala) associated with respiratory burst coupled with nitric oxide production.

The immunostimulating effects of oligochitosan have been proven in several fish, however, the mechanisms underlying the stimulation are not characterized. In the present study, the effects of oligochitosan were investigated using macrophages isolated from blunt snout bream (Megalobrama amblycephala). The results showed that the phagocytic activity of the macrophages was enhanced by the addition of oligochitosan in vitro and in vivo. The two of the most important antimicrobial pathways of macrophages, NADPH oxidase and iNOS pathways were included for further studies. The amounts of superoxide anion and the mRNAs of the five subunits of NADPH oxidase genes were significantly enhanced in the oligochitosan-treated macrophages and macrophages isolated from fish fed with feed containing oligochitosan. In addition, the NO production, iNOS activity and iNOS gene expression were all significantly increased in the presence of oligochitosan. Furthermore, the mRNA levels of the TNF-α and IL-1ß were also significantly increased in the macrophages derived from fish fed with oligochitosan. In conclusion, the stimulation effects of oligochitosan on the phagocytic activity of the fish macrophages were associated with respiratory burst coupled with nitric oxide production.

Molecular cloning and expression analysis of mannose receptor in blunt snout bream (Megalobrama amblycephala).

Mannose receptor (MR) plays a significant role in innate immune responses to pathogens in vertebrates. Here we characterized the first teleost MR from Megalobrama amblycephala, named maMR and its expression patterns were investigated. The full-length maMR consists of 5,295 bp encoding a putative protein of 1,433 amino acids. The predicted amino acid sequences showed that maMR contained a signal peptide, a cysteine-rich domain, a single fibronectin type II domain, eight tandemly arranged C-type lectin-like domains, a transmembrane domain and a C-terminal cytoplasmic domain. Phylogenetic analysis revealed the highest similarity of maMR with Danio rerio MR predicted by computational analysis. The maMR-mRNAs were ubiquitously transcribed in different tissues, However the highest transcripts were observed in head kidney. Transcripts of maMR significantly increased at the late stages of embryo and continued to be at the high levels after hatching. The maMR transcripts were significantly increased in M. amblycephala after stimulation with killed Aeromonas hydrophila.

In vivo studies on the immunotoxic effects of microcystins on rabbit.

Microcystins (MCs) are the toxic molecules produced by common cyanobacterium in freshwater blooms. Their toxicities raise severe health issues in livestock and human beings. In current study, the immunotoxic effects of MC-LR were investigated in rabbit through evaluating the dynamics of white blood cell (WBC) numbers and cytokine production such as interleukin-3 (IL-3), IL-4, IL-6, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α). MCs at the high dose (50 µg MC-LReq kg(-1) ) significantly induced increase in the WBC number but decrease in the Th1 (IFN-γ, TNF-α) and Th2 (IL-3, IL-4, IL-6) production. In the low dose group(12.5 µg MC-LReq kg(-1) ), the number of WBC and the production of IFN-γ, IFN-α, IL-4, IL-3, and IL-6 increased gradually in first 12 h, reach the peaks at 12 h, and dropped after 24 h. Significantly positive correlations were found between the cytokines production of IL-4 and IL-6, IFN-γ and IFN-α, or IL-4 and IFN-γ. In conclusion, MC-LR is able to disturb the rabbit immune system and there exists time-dose response relationship in the MC-LR-eliciting perturbation, which probably give a better insight into investigating the immunotoxicity mechanisms of MCs in vivo. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

Anemia induced by repeated exposure to cyanobacterial extracts with explorations of underlying mechanisms.

Hematological abnormalities or derangements have been demonstrated in patients suffering form microcystins (MCs) in hemodialysis unit in Caruaru, Brazil, 1996. While experimental study on hematological effect of microcystins has been rare and the underlying mechanisms are still puzzling. In the present study, microcystins were repeatedly intraperitoneally injected with a dose of 6 µg/kg/day in rabbits (Oryctolagus cuniculus) for 14 days, and the prolonged effects of extracted microcystins on hematotoxicology were investigated. Significant decreases were observed in the hematological indices red blood cell counts, hematocrit, hemoglobin, and platelet count, while an obvious anemia occurred in rabbits after 14-day exposure. Moreover, red blood cell volume distribution width, mean corpuscular volume, and mean corpuscular hemoglobin did not vary significantly, indicating that rabbits suffered from normocytic anemia. In bone marrow, on the 14th day after toxin exposure, the frequency of micronucleus increased significantly, and the viability of bone marrow cells decreased markedly compared with the control. Serum erythropoietin levels declined on the 7th and 14th day, which suggested that the ability to regulate differentiation and maturation of erythrocytes was impaired. These results indicate that repeated exposure of microcystins can result in normocyte anemia, and the bone marrow injures and the sharp decreases of erythropoietin levels were responsible for the anemia.

Effective production and purification of the glycosylated TSOL18 antigen, which is protective against pig cysticercosis.

Cysticercosis caused by Taenia solium metacestodes is a worldwide public health problem. Important progress in the development of effective and practical vaccines against this disease has been made. In this study, the promising T. solium oncospheral vaccine candidate named TSOL18 antigen was produced in a 5-liter fermentor. During the process of fermentation, the pH of the culture was always kept below 5.0, and in order to prevent foaming, an antifoam agent was added. In addition, the oxygen content of the culture was constantly kept at >50% in our experiment. A high level of the glycosylated protein (2.5 g/liter) was obtained, and the protein was easily purified by gel chromatography. Vaccination trials showed that the recombinant TSOL18 antigen induced 94 and 100% reductions in metacestode burdens in vaccinated pigs, obviously higher than the 89% reduction in pigs immunized with cysticercus crude extracts in trial 1. These are very promising results in the development of an efficient tool to control cysticercosis in Asia.

Induction of protection against porcine cysticercosis in growing pigs by DNA vaccination.

A DNA vaccine, pcDNA3-B, was developed by using the nucleotide sequence of Taenia solium B antigen and cloning into pcDNA3.1 plasmid. The growing pigs were vaccinated by one intramuscular infection of 200 or 1000 microg pcDNA3-B. The immunization with 1000 microg of pcDNA3-B showed 92.6% protection when the pigs were challenged by T. solium eggs and four of the five pigs vaccinated had no viable cysts. The results provide encouraging information on the use of pcDNA3-B vaccination for the prevention of cysticercosis.