Innate Immune Response In Fish Research Articles

Single-nuclei RNA-seq reveals skin cell responses to Aeromonas hydrophila infection in Chinese longsnout catfish Leiocassis longirostris.

As the primary natural barrier that protects against adverse environmental conditions, the skin plays a crucial role in the innate immune response of fish, particularly in relation to bacterial infections. However, due to the diverse functionality and intricate anatomical and cellular composition of the skin, deciphering the immune response of the host is a challenging task. In this study, single nuclei RNA-sequencing (snRNA-seq) was performed on skin biopsies obtained from Chinese longsnout catfish (Leiocassis longirostris), comparing Aeromonas hydrophila-infected subjects to healthy control subjects. A total of 19,581 single nuclei cells were sequenced using 10x Genomics (10,400 in the control group and 9,181 in the treated group). Based on expressed unique transcriptional profiles, 33 cell clusters were identified and classified into 12 cell types including keratinocyte (KC), fibroblast (FB), endothelial cells (EC), secretory cells (SC), immune cells, smooth muscle cells (SMC), and other cells such as pericyte (PC), brush cell (BC), red blood cell (RBC), neuroendocrine cell (NDC), neuron cells (NC), and melanocyte (MC). Among these, three clusters of KCs, namely, KC1, KC2, and KC5 exhibited significant expansion after A. hydrophila infection. Analysis of pathway enrichment revealed that KC1 was primarily involved in environmental signal transduction, KC2 was primarily involved in endocrine function, and KC5 was primarily involved in metabolism. Finally, our findings suggest that neutrophils may play a crucial role in combating A. hydrophila infections. In summary, this study not only provides the first detailed comprehensive map of all cell types present in the skin of teleost fish but also sheds light on the immune response mechanism of the skin following A. hydrophila infection in Chinese longsnout catfish.

Identification and functional regulation of three alternative splicing isoforms of the fthl27 gene in miiuy croaker, Miichthys miiuy

Alternative splicing is an important basic mechanism for eukaryotes to control gene expression. Different forms of alternative splicing may lead to the production of protein subtypes with different functions, leading to the expansion of protein diversity in organisms, affecting cell production and metabolism, and is even related to the occurrence of many diseases. Many studies have shown that ferritin is usually associated with inflammation, vascular proliferation, and tumors, which is the focus of immunological research. It not only plays a role in iron metabolism and storage in the body, but also plays an important regulatory role in pathways related to immune and inflammatory regulation. However, there are few studies on alternative splicing events of the ferritin gene nowadays. Therefore, this study identified three different splicing isoforms in its ferritin gene fthl27 of Miichthys miiuy through Sanger sequencing, qRT-PCR, and other experimental techniques, and we found that three different splicing isoforms of the ferritin gene fthl27 in M. Miiuy cells showed an upregulation trend after being stimulated by Lipopolysaccharide (LPS) and poly (I: C). The experiment also found that the three isoforms may have different regulatory effects on the expression of inflammatory factors and antiviral immune factors, playing an important role in the innate immune response of fish.

Inclusion effect of jack mackerel, Trachurus japonicus, meal in the diet of rockfish, Sebastes schlegeli, on growth, feed utilization, biochemical composition, and innate immune responses

AbstractDietary inclusion effects of various levels of jack mackerel meal (JMM) on growth, feed utilization, biochemical composition, and innate immune response of rockfish were determined. The control (Con) diet included 550 g/kg fish meal (FM). Ten, 30, 50, 100, 200, 400, 600, and 1000 g/kg of JMM were included in the Con diet, referred to as the JMM10, JMM30, JMM50, JMM100, JMM200, JMM400, JMM600, and JMM1000 diets, respectively. Eight hundred and ten fish (initial mean weight of 2.3 g) were distributed into 27 flow‐through tanks. The experimental diets were assigned to triplicate groups of fish. Fish were hand‐fed for 8 weeks. Weight gain of fish fed the JMM400 diet was statistically better than that of fish fed the Con, JMM10, JMM30, JMM50, JMM100, and JMM200 diets. Feed consumption of fish fed the JMM400 diet was statistically higher than that of fish fed the Con, JMM10, JMM30, and JMM50 diets. Dietary inclusion of JMM did not influence feed utilization, biochemical composition, and innate immune responses of fish. In conclusion, inclusion of JMM up to 400 g/kg of FM in diet improved growth and feed consumption of rockfish, and dietary optimum inclusion levels of JMM were estimated to be 396.7 and 403.1 g/kg based on regression analysis of weight gain and feed consumption, respectively.

Identification and characterization of two novel antimicrobial peptides from Japanese sea bass (Lateolabrax japonicus) with antimicrobial activity and MO/MФ activation capability

Piscidins participate in the innate immune response of fish, which aims to eliminate recognized foreign microbes and restore the homeostasis of immune system. We characterized two piscidin-like antimicrobial peptides (LjPL-3 and LjPL-2) isolated from Japanese sea bass (Lateolabrax japonicus). LjPL-3 and LjPL-2 showed different expression patterns in tissues. After Vibrio harveyi infection, the mRNA expression of LjPL-3 and LjPL-2 was upregulated in the liver, spleen, head kidney, and trunk kidney. The synthetic mature peptides LjPL-3 and LjPL-2 exhibited different antimicrobial spectra. Furthermore, LjPL-3 and LjPL-2 treatments decreased inflammatory cytokine production while promoting chemotaxis and phagocytosis in monocytes/macrophages (MO/MФ). LjPL-2, but not LjPL-3, displayed bacterial killing capability in MO/MФ. LjPL-3 and LjPL-2 administration increased Japanese sea bass survival after V. harveyi challenge, which was accompanied by a decline in bacterial burden. These data suggested that LjPL-3 and LjPL-2 participate in immune response through direct bacterial killing and MO/MФ activation.

Evolution of LPS recognition and signaling: The bony fish perspective

Fish are the most diverse and successful group of vertebrate animals, with about 30,000 species. The study of fish immunity is of great importance for understanding the evolution of vertebrate immunity, as they are the first animals to show both innate and adaptive immune responses. Although fish immunity is similar to that of mammals, there are obvious differences, such as their dependence of ambient temperature, their poor antibody response, and lack of antibody switching and lymph nodes. In addition, several important differences have also been found between the innate immune responses of fish and mammals. Among these, we will discuss in this review the high resistance of fish to the toxic effects of lipopolysaccharide (LPS) which can be explained by the absence of a Toll-like receptor 4 (Tlr4) ortholog in most fish species or by the inability of the Tlr4/Md2 (Myeloid differentiation 2) complex to recognize LPS, together with the presence of a negative regulator of the LPS signaling complex formed by the TLR-like molecule Rp105 (Radioprotective 105) and Md1. Taken together, these data support the idea that, although TLR4 and RP105 arose from a common ancestor to fish and tetrapods, the TLR4/MD2 receptor complex for LPS recognition arose after their divergence about 450 million years ago.

Effect of dietary Spirulina ( Arthrospira platensis ) on the growth performance, immune‐related gene expression and resistance to Vibrio anguillarum in European seabass ( Dicentrarchus labrax )

This research was conducted to reveal the effects of dietary Spirulina (Arthrospira platensis) meal on the growth performance, immune responses and resistance against Vibrio anguillarum in European seabass (Dicentrarchus labrax). In the study, the fish (5.74 ± 0.02 g) were fed with control (C) and three different levels (1%, 2.5% and 5%) of Spirulina meal-containing diets (SP1, SP2.5 and SP5) for 60 days. The adaptive and innate immune responses of fish were determined every 30th day of the study. The results showed that respiratory burst activity (RBA) was significantly elevated on the SP2.5 and SP5 groups on the 30th day of the study. Lysozyme activity (LYS) was increased on the SP1 group on the 30th day and the SP5 group on the 60th day (p < 0.05). Myeloperoxidase activity (MPO) was raised on the 60th day of the SP2.5 and SP5 groups compared with the C (p < 0.05). IL-1β gene expression increased in the kidney and intestine of fish on the 30th day and in the spleen on the 60th day. IL-10 gene expression in the intestine of fish fed the Spirulina-containing diet was elevated at each sampling time relative to the C group (p < 0.05). Elevated IL-6 activity was determined in the kidney and the intestine on all Spirulina-fed groups compared with control at both sampling times. Similar to IL-6, IL-8 gene expressions were elevated in all Spirulina-fed groups (p < 0.05). TNF-α was up-regulated in the kidney and intestine of all Spirulina-fed groups compared with the C group on the 30th day of the study (p < 0.05). TGF-β gene expression was increased in the kidney and intestine of all Spirulina groups compared with the C group on all sampling times. COX-2 gene expression in the kidneys and intestines of fish fed with Spirulina diets was significantly elevated compared with the C diet at days 60th and 30th respectively. Hepcidin gene expression of all groups was elevated in all tissue samples on the 30th day of the study. The survival rate of fish infected with V. anguillarum was higher in all Spirulina-fed groups than in the C group. The feed conversion rate and protein efficiency ratio of fish fed with a 1% Spirulina diet was found to be better than those fed with other diets. These results show that up to the 5% inclusion of Spirulina meal in European seabass diets promotes some growth parameters and supports immunity in fish.

Oral delivery of a dsRNA-Phytoglycogen nanoparticle complex enhances both local and systemic innate immune responses in rainbow trout

Salmonids are one of the most farmed fish species worldwide. These aquatic vertebrates rely heavily on their innate immune responses as the first line of defense to defend themselves against invading pathogens. Although commercial vaccines are available against some viral and bacterial pathogens affecting salmonids, their protective efficacy varies. Using a prophylactic inducer of local and systemic innate immune responses to limit infection could have significant implications in salmonid aquaculture. A potent inducer of innate immune responses in fish is double-stranded RNA (dsRNA), a molecule that all viruses make during their replicative cycle. Polyinosinic: polycytidylic acid (polyI:C) is a synthetic dsRNA commonly used to induce type I interferons (IFNs), interferon stimulated genes (ISGs) as well as an antiviral state in vertebrate species. Based on in vitro data it was hypothesized that both local and systemic innate immune responses, in salmonids, would be enhanced by orally delivering high molecular weight polyI:C (HMW polyI:C) using cationic phytoglycogen nanoparticles (NPs) as a delivery method. The present study investigates this hypothesis using two feed delivery methods. In the first in vivo study, to ensure an equal distribution of dose, individual rainbow trout (Oncorhynchus mykiss) were orally gavaged with feed moistened with a solution containing HMW-NP (polyI:C complexed with cationic phytoglycogen nanoparticles) or HMW polyI:C alone. In a second in vivo experiment, to better mimic a more realistic feeding scenario, rainbow trout were fed feed pellets to which HMW, or HMW-NP was added. The expression of IFN1 and ISGs (vig-3, Mx1) were quantified using real-time PCR in the intestine (local response) and head kidney (systemic response). The results of these studies indicate that HMW-NP induced a higher level of IFN1 and ISG expression in the intestine and head kidney compared to the HMW fed fish. The results of this study could lead to new advances in therapeutics for the aquaculture industry by utilizing the innate immune response against invading pathogens using an orally delivered stimulant.

Effect of dietary Ficus carica polysaccharides on the growth performance, innate immune response and survival of crucian carp against Aeromonas hydrophila infection

Ficus carica polysaccharides (FCPS), one of the most effective and important compo-nents in Ficus carica L., had been considered to be a beneficial immunostimulant and may be used in immunotherapy for animals and human. However, studies were little about the effect of FCPS used as immunomodulatory and the suitable dosage in fish. The present study investigated the effect of four different dietary levels of FCPS (0.1%, 0.2%, 0.4%, 0.8%) on the growth performance, innate immune responses and survival of crucian carp against Aeromonas hydrophila infection. The results showed that compared with control group, dietary FCPS had positive effects the growth performance (final weight, feed conversion ratio and survival rate) of crucian carp. FCPS induced significant higher (p < 0.05) leukocyte phagocytosis activity, serum bactericidal activity, lysozyme activity, com-plement C3, SOD activity and total protein level in the serum of crucian carp. Moreover, innate immune response of fish in FCPS groups increased first and then decreased with increasing dietary FCPS from 0.1% to 0.8%, and reached up to the peak in 0.4% dietary FCPS groups. Besides, the cumulative mortalities in FCPS groups were remarkably lower than that of control group when challenged with A. hydrophila, the relative percent survivals were 22.67%, 55.56%, 62.22% and 17.78% in 0.1% group, 0.2% group, 0.4% group and 0.8% group, respectively. These results suggested that dietary FCPS could improve the growth performance, innate immune response and disease resistance against A. hydrophila in fish, and the suitable dietary dose of FCPS was 0.4% in crucian carp.

TLR1 in Nile tilapia: The conserved receptor cannot interact with MyD88 and TIRAP but can activate NF-κB in vitro

Toll-like receptors (TLRs) play a critical role in the innate immune response of fish. In this study, we isolated the cDNA sequence of Nile tilapia TLR1 (OnTLR1). The deduced OnTLR1 protein contains a signal peptide, 7 leucine-rich repeats (LRRs), a C-terminal LRR (LRR-CT), a transmembrane region and a highly conserved TIR domain. In healthy Nile tilapia, the OnTLR1 transcript was broadly expressed in all examined tissues, with the highest expression levels in the spleen. After infection with Streptococcus agalactiae, the OnTLR1 transcripts were upregulated in the gill and kidney. After stimulation with polyinosinic-polycytidylic acid (poly(I:C)), the expression levels of OnTLR1 were significantly downregulated in the intestine, whereas OnTLR1 transcripts were significantly upregulated in the kidney. After challenge with lipopolysaccharide (LPS), the expression levels of OnTLR1 were significantly upregulated in the spleen and kidney. The subcellular localization showed that OnTLR1 was expressed in the cytoplasm. TLR1 significantly increased MyD88-dependent NF-κB activity. However, the results of a pull-down assay showed that OnTLR1 did not interact with MyD88 or TIRAP. Binding assays revealed the specificity of OnTLR1 for pathogen-associated molecular patterns (PAMPs) and bacteria that included S. agalactiae, Aeromonas hydrophila and poly(I:C) and LPS. Taken together, these findings suggest that OnTLR1, as a pattern recognition receptor (PRR), might play an important role in the immune response to pathogen invasion.

Molecular characterization and expressional analysis of two poly (ADP-ribose) polymerase (PARP) domain-containing Gig2 isoforms in rockfish (Sebastes schlegelii) and their antiviral activity against viral hemorrhagic septicemia virus

Remedies toward sustainable aquaculture rely upon research that unveils the molecular mechanisms behind host immunity and their interactions with pathogens. Antiviral defense is a major innate immune response in fish. The antiviral protein GCHV-induced gene-2 (Gig2), a member of the interferon-stimulated gene (ISG), was identified and characterized from rockfish (Sebastes schlegelii). Gig2 exists in two isoforms, namely, SsGig2-I1 and SsGig2-I2, in rockfish with lengths of 163 and 223 bp, respectively. Bioinformatic analysis indicated the availability of poly (ADP-ribose) polymerase domain in both proteins, and 51.3% identity and 71.3% similarity between both isoforms were observed. The basal expression pattern revealed the highest tissue-specific expression in rockfish gills for both isoforms. The immune challenge experiment disclosed a distinctive and strong expression of each transcript in the presence of poly I:C. Both isoforms are localized in the endoplasmic reticulum. Interferon (IFN) pathway gene analysis revealed no significant upregulation of IFN related genes. Viral hemorrhagic septicemia virus (VHSV) gene expression analysis revealed strong downregulation of viral transcripts after 48 h of infection in the presence of Gig2 isoforms. Collectively, these results indicate the protective role of Gig2 in rockfish against VHSV infection and help broaden our understanding of the innate immunity of fish.

Grass carp (Ctenopharyngodon idella) interferon regulatory factor 8 down-regulates interferon1 expression via interaction with interferon regulatory factor 2 in vitro

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a negative regulatory factor of interferon (IFN) and plays an important role in cell differentiation and innate immunity in mammals. In recent years, some irf8 homologous genes have been cloned and confirmed to take part in innate immune response in fish, but the mechanism still remains unclear. In this paper, a grass carp (Ctenopharyngodon idella) irf8 gene (Ciirf8) was cloned and characterized. The deduced protein (CiIRF8) possesses a highly conserved N-terminal DNA binding domain but a less well-conserved C-terminal IRF association domain (IAD). Ciirf8 was widely expressed in all tested tissues of grass carp and up-regulated following poly(I:C) stimulation. Ciirf8 expression was also up-regulated in CIK cells upon treatment with poly(I:C). To explore the molecular mechanism of how fish IRF8 regulates ifn1 expression, the similarities and differences of grass carp IRF8 and IRF2 were compared and contrasted. Subcellular localization analysis showed that CiIRF8 is located both in the cytoplasm and nucleus; however, CiIRF2 is only located in the nucleus. The nuclear-cytoplasmic translocation of CiIRF8 was observed in CIK cells under stimulation with poly(I:C). The interaction of CiIRF8 and CiIRF2 was further confirmed by a co-immunoprecipitation assay in the nucleus. Dual-luciferase reporter assays showed that the promoter activity of Ciifn1 was significantly inhibited by co-transfection with CiIRF2 and CiIRF8. The transcription inhibition of Ciifn1 was alleviated by competitive binding of CiIRF2 and CiIRF8 to CiIRF1. In conclusion, CiIRF8 down-regulates Ciifn1 expression via interaction with CiIRF2 in cells.

Polyunsaturated Fatty Acids Influence LPS-Induced Inflammation of Fish Macrophages Through Differential Modulation of Pathogen Recognition and p38 MAPK/NF-κB Signaling.

Polyunsaturated fatty acids (PUFAs) not only serve as essential nutrients but also function as modulators of the immune response in marine fish. However, their immunomodulatory mechanism is poorly understood given that the underlying regulation of the innate immune response in fish has not been fully elucidated. Hence, study of the innate immunity of fish could help elucidate the mechanism by which PUFAs affect the fish immune response. Here, we used combined transcriptome analysis and in vitro experimentation to study the mechanism of LPS-induced inflammation. Transcriptome profiling indicated that LPS elicited strong pro-inflammatory responses featuring high expression levels of pathogen recognition receptors (PRRs) and cytokines along with the activation of NF-κB and MAPK signaling pathways. The transcription factor p65 alone could increase the transcription of IL1β by binding to the promoter of IL1β, and this promoting effect disappeared after mutation or deletion of its binding sites. We then examined the effects of PUFAs on the levels of gene expression and the abundance of proteins of critical kinases associated with LPS-induced inflammation. We found that LA exerts pro-inflammatory response while ALA, EPA, and DHA induced anti-inflammatory effects by modulating the expression of PRRs, phosphorylation of IKK and p38, and the nuclear translocation of p65. Overall, this study advances our understanding of the regulatory mechanisms by which PUFAs regulate LPS-induced inflammation in a non-model fish species.

Beyond 2020

Beyond 2020

Grass carps co-exposed to environmentally relevant concentrations of cypermethrin and sulfamethoxazole bear immunodeficiency and are vulnerable to subsequent Aeromonas hydrophila infection

The aquatic ecosystem is seriously damaged because of the heavy use of pesticides and antibiotics. Fish is the indispensable link between environmental pollution and human health. However, the toxic effects of environment-related concentrations of pesticides and antibiotics in fish have not been thoroughly studied. In this study, grass carps exposed to cypermethrin (CMN, 0.651 μg/L) or/and sulfamethoxazole (SMZ, 0.3 μg/L) for 42 days caused oxidative stress, apoptosis and immunodeficiency in the spleen of grass carps. CMN or/and SMZ exposure led to oxidative damage (consumption of antioxidant enzymes (superoxide dismutase and catalase)) and lipid peroxidation (accumulation of malondialdehyde), induced apoptosis (increases in TUNEL index, Bax/bcl-2, p53, puma and Caspase family expression). In addition, the levels of immunoglobulin M (IgM), complement 3 (C3) were significantly decreased in all treatment groups, which trend was also found in C-reactive protein in CMN and MIX group, and lysozyme in MIX group. Transcription of almost all genes involved in the Toll-like receptors (TLR) signaling pathway was up-regulated under CMN or/and SMZ exposure. However, when subsequently attacked by Aeromonas hydrophila for 2 days, the TLR pathway was inhibited in spleens of all treatment groups accompanied by higher mortality. Overall, the environmentally relevant concentration of CMN and SMZ damages the immune system, triggering oxidative stress and apoptosis in carps. And by affecting the conduction of TLR signaling pathway, CMN or/and SMZ exposure inhibits the innate immune response of fish and reducing their disease resistance. This study highlights the importance of rational and regulated use of these pesticides and antibiotics.

Grass carp (Ctenopharyngodon idella) GPATCH3 initiates IFN 1 expression via the activation of STING-IRF7 signal axis

GPATCH3, a protein with G-patch domain, is known to participate in innate immune response and organ development in mammals. However, there are few reports on GPATCH3 in fish. Here the cDNA sequence of GPATCH3 was cloned from Ctenopharyngodon idella (CiGPATCH3, MN149902) and was determined its character. A cDNA sequence of CiGPATCH3 is 1646 bp and contains an ORF of 1221 bp translating a protein of 407 amino acids. Phylogenetic analysis uncovered that CiGPATCH3 possesses a relatively high degree of homology with Cyprinus carpio GPATCH3. The mRNA level of CiGPATCH3 was increased following the intracellular stimulation of poly (I:C) into CIK cells. In vivo, over-expression of CiGPATCH3 can significantly up-regulate IFN 1 and ISG15 expression at mRNA and protein levels. To investigate the molecular mechanism by which GPATCH3 initiates the innate immune response in fish, co-IP experiments were performed to analyze the substrates of CiGPATCH3. The results showed that CiGPATCH3 directly interacted with CiSTING, but not with CiIRF3, CiIRF7, CiTBK1 or CiIPS-1. As compared with the single transfection of CO cells with either CiGPATCH3 or CiSTING, the expression of IFN 1 was more significantly up-regulated in cells under treatment with dual transfection of CiGPATCH3 and CiSTING. Knockdown of CiGPATCH3 inhibited STING-mediated IFN 1 expression in fish cells. Over-expression of CiGPATCH3 and CiSTING facilitated the phosphorylation and cytoplasmic-to-nuclear translocation of CiIRF7. These results explicitly showed that CiGPATCH3 up-regulates IFN 1 and ISG15 expression via the activation of STING-IRF7 signal axis in vivo.

In vivo effects on the immune function of fathead minnow (Pimephales promelas) following ingestion and intraperitoneal injection of polystyrene nanoplastics

Nanoplastics (1–100 nm) are potentially the most hazardous litter in the environment. Recent scientific studies have documented their toxic effects at the cellular and molecular levels, but knowledge underlying mechanisms of their toxicity is still scarce. Nanoplastics are known for their ability to induce immune and inflammatory responses as well as generating reactive oxygen species. While some studies have addressed the immunotoxicity of nanoplastics in vitro and on in vivo in fish after intraperitoneal injection, no information is available on adult fish after ingestion of a contaminated prey. The present study is the first to attempt to address the immunotoxicity of nanoplastics in adult fish after trophic transfer. Pimephales promelas is a well-established bioindicator species to study the immunotoxicity of nanoparticles and the innate immune responses of fish. This study aims to assess the in vivo innate immune response of adult P. promelas following exposure to polystyrene nanoplastics by measuring the gene expression of ncf, nox2, mst1 and c3; these genes are related with the immune function of neutrophils, macrophages and complement in fish. Two target organs (liver and head kidney) and two routes of exposure (IP- injection and ingestion) were analyzed. After 48 h of exposure, polystyrene nanoplastics were encountered in the liver and kidney of both IP-injection and ingestion exposed fish, and significantly affected the innate immune system of P. promelas by downregulating the gene expression ncf, mst1, and c3 in liver and kidney. Significant difference between treatments was only observed for the gene expression of nfc in liver. Results of this study indicate that polystyrene nanoplastics can exhibit immunotoxicity in fish through an environmentally relevant route of exposure, interfering with the synthesis and function of neutrophils, macrophages, and complement of P. promelas in their principal hematopoietic tissues, which may potentially compromise its ability to survive in nature.

Extracellular ATP is a potent signaling molecule in the activation of the Japanese flounder (Paralichthys olivaceus) innate immune responses.

Innate immunity is the first line of defense against pathogen infections. Extracellular ATP (eATP) is one of the most studied danger-associated molecular pattern molecules that can activate host innate immune responses through binding with and activating purinergic receptors on the plasma membrane. The detailed actions of eATP on fish innate immunity, however, remain poorly understood. In this study, we investigated bacterial pathogen-induced ATP release in head kidney cells of the Japanese flounder Paralichthys olivaceus. We also examined the actions of eATP on pro-inflammatory cytokine and immune-related gene expression, the activity of induced NO synthase (iNOS), and the production of reactive oxygen species (ROS) and NO in Japanese flounder immune cells. We demonstrate that ATP is dynamically released from Japanese flounder head kidney cells into the extracellular milieu during immune challenge by formalin-inactivated Edwardsiella tarda and Vibrio anguillarum. In addition, we show that eATP administration results in profound up-regulation of pro-inflammatory cytokine gene expression, iNOS activity, and inflammatory mediator production, including ROS and NO, in Japanese flounder immune cells. Altogether, our findings demonstrate that eATP is a potent signaling molecule for the activation of innate immune responses in fish.

Glutaredoxin 2 from big belly seahorse (Hippocampus abdominalis) and its potential involvement in cellular redox homeostasis and host immune responses

Glutaredoxins are oxidoreductases present in almost all living organisms. They belong to the thioredoxin superfamily and share the thioredoxin structure and catalytic motif. Glutaredoxin 2 has been identified as a mitochondrial protein in vertebrates. In this study, the sequence of Glutaredoxin 2 from Hippocampus abdominalis (HaGrx2) was analyzed by molecular, transcriptional, and functional assays. In-silico analysis revealed that HaGrx2 shows the highest homology with Hippocampus comes, while distinctly cluster with fish Grx2 orthologs. Tissue distribution analysis showed that HaGrx2 is ubiquitously expressed in all tissues tested, and the highest expression was observed in the brain and skin. Significant HaGrx2 transcript modulation was identified in blood and liver upon injecting bacterial and Pathogen Associated Molecular Patterns. The redox activity of HaGrx2 was revealed by Dehydroascorbic reduction and insulin disulfide reduction activity assays. Further, the deglutathionylation activity of 1 nM HaGrx2 was found to be equivalent to that of 0.84 nM HaGrx1. HaGrx2 exhibited antiapoptotic activity against H2O2-induced oxidative stress in FHM cells. Altogether, the results of this study suggest that HaGrx2 plays a role in redox homeostasis and innate immune responses in fish.

Transcriptome analysis in the skin of Carassius auratus challenged with Aeromonas hydrophila

Skin plays an important role in the innate immune responses of fish, particularly towards bacterial infection. To understand the molecular mechanism of mucosal immunity of fish during bacterial challenge, a de novo transcriptome assembly of crucian carp Carassius auratus skin upon Aeromonas hydrophila infection was performed, the latter with Illumina Hiseq 2000 platform. A total of 118111 unigenes were generated and of these, 9693 and 8580 genes were differentially expressed at 6 and 12 h post-infection, respectively. The validity of the transcriptome results of eleven representative genes was verified by quantitative real-time PCR (qRT-PCR) analysis. A comparison with the transcriptome profiling of zebrafish skin to A. hydrophila with regards to the mucosal immune responses revealed similarities in the complement system, chemokines, heat shock proteins and the acute-phase response. GO and KEGG enrichment pathway analyses displayed the significant immune responses included TLR, MAPK, JAK-STAT, phagosome and three infection-related pathways (ie., Salmonella, Vibrio cholerae and pathogenic Escherichia coli) in skin. To our knowledge, this study is the first to describe the transcriptome analysis of C. auratus skin during A. hydrophila infection. The outcome of this study contributed to the understanding of the mucosal defense mechanisms in cyprinid species.

Induction of IL-22 protein and IL-22-producing cells in rainbow trout Oncorhynchus mykiss

IL-22 is a critical cytokine which is involved in modulating tissue responses during inflammation, and is produced mainly by T cells and innate leucocytes. In mammals, IL-22 is a key component in mucosal defences, tissue repair, epithelial cell survival and proliferation. In teleosts, IL-22 has been cloned and studied in several species, and the transcript is highly expressed in mucosal tissues and induced by pathogen associated molecular patterns (PAMPs), suggesting IL-22 also functions as an important component of the innate immune response in fish. To investigate these immune responses further, we have validated and characterised two monoclonal antibodies (mAbs) which were raised against two different peptide immunogens of salmonid IL-22. Our results show that both mAbs specifically react to their own peptide immunogens and recombinant IL-22, and are able to detect the induction of native protein expression after stimulation. In flow cytometry, an increase in IL-22 positive cells was detected after stimulation in vitro with cytokines and PAMPs and in vivo after bacterial challenge. The immunohistochemistry results showed that IL-22 is highly upregulated in the gills after challenge, both in cells within the gill filaments and in the interbranchial lymphoid tissue. Such results suggest IL-22 may have a role in triggering local antimicrobial defences in fish that may facilitate efficient microbial clearance. Hence monitoring IL-22 producing cells/protein secretion may provide an alternative mean to assess the effectiveness of mucosal vaccines.