Molecular Characterization and Expression Analysis of ATP-Gated P2X7 Receptor Involved in Japanese Flounder (Paralichthys olivaceus) Innate Immune Response

ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60–88% sequence similarity and 45–78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1β and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.     

Molecular cloning and characterization of Toll-like receptor 14 in Japanese flounder,Paralichthys olivaceus

Toll-like receptors (TLRs) are essential for activation of the innate immune system in response to invading pathogens. TLR14, which is unique to fish, has been identified in several fish species, but its function is unclear. In this study, Japanese flounder (Paralichthys olivaceus) TLR14 gene (JfTLR14) was cloned and its expression profiles were analyzed after infection with viral hemorrhagic septicemia virus, gram-positive Streptococcus iniae and gram-negative Edwardsiella tarda. The coding region of JfTLR14 cDNA was 2,607 bp, encoding 878 amino acid residues. JfTLR14 was highly expressed in head kidney of healthy flounder. In response to infection with VHSV and S. iniae, the JfTLR14 gene was up-regulated at only 1 day post-infection (dpi). However, E. tarda infection increased JfTLR14 gene expression from 1 to 6 dpi. These results imply that JfTLR14 participates more in the immune response against E. tarda infection than in the immune responses to other pathogen infections.     

Molecular characterization and gene expression of a CXC chemokine gene from Japanese flounder Paralichthys olivaceus

Chemokines are small, secreted cytokine peptides that have the ability to recruit a wide range of immune cells to sites of infection and disease. A novel CXC chemokine was obtained from Japanese flounder Paralichthys olivaceus. This chemokine cDNA contains an open reading frame of 333 nucleotides encoding 111 amino acid residues containing four conserved cysteine residues. The gene is composed of four exons and three introns as are those of mammalian and fish CXC chemokines. Results of homology and phylogenetic analysis revealed that the Japanese flounder CXC chemokine is closest to CXCL13 subgroup. The gene was expressed in immune-related organs, including head kidney, trunk kidney, spleen and peripheral blood leukocytes (PBLs). Japanese flounder CXC chemokine gene expression was observed at 3 and 6h after induction by LPS, but not at 3 and 6h after induction by poly I:C. These results suggest that the Japanese flounder CXC chemokine is probably associated with inflammatory as well as homeostatic functions.     

The use of innate immune responses as biomarkers in a programme of integrated biological effects monitoring on flounder (Platichthys flesus) from the southern North Sea

Immunological biomarkers that reflect the effects of exposure to environmental contaminants in coastal marine habitats were sought in European flounder (Platichthys flesus) from five locations in the German Bight with different anthropogenic impacts. During a 2-year period of sampling, innate immune responses were monitored from a total of 331 individual flounder of a body length of 18 to 25 cm. From the fish, plasma lysozyme, phagocytosis and respiratory burst activity of head kidney leucocytes were analysed and implemented as part of an integrated biological effects monitoring programme. As the measurements of the parameters applied here varied within wide ranges at some locations, spatial differences could not always be established, but some general trends could be drawn: plasma lysozyme activity was decreased in flounder contaminated with DDT adducts and some PCBs, while cellular functions such as phagocytosis and respiratory burst were stimulated by some chlorinated hydrocarbons. Correlation analysis also revealed connections not only between the parameters applied here and some contaminants but also with some biochemical parameters used as biomarkers in pollution monitoring: in flounder with decreased integrity of hepatocyte lysosomal membranes, immune functions also were impaired, and plasma lysozyme as well as phagocytosis activity of head kidney cells were impaired when the activity of cytochrome P450 1A was induced. The data presented here indicate that innate immune responses may be useful parameters to monitor cellular functions in a battery of biomarkers of different levels of biological organisation. Communicated by H. v. Westernhagen, A. Diamant     

胞外ATP对壳聚糖诱导的ROS和PAL活性变化的影响

以烟草BY-2悬浮细胞为材料,探讨了胞外ATP对壳聚糖引起的活性氧(reactive oxygen species,ROS)水平和苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)活性变化的影响。结果表明,5~20μg·mL^-1壳聚糖处理导致了烟草悬浮细胞细胞内ROS水平逐渐增加;壳聚糖也导致了PAL活性的增加,其活性在15μg·mL^-1壳聚糖处理下达到峰值,此后有所降低。10~40μmol·L^-1外源ATP处理未引起烟草悬浮细胞内ROS水平和PAL活性的显著变化。细胞外ATP水平则随壳聚糖浓度的增加而逐渐下降。本文进一步分析了细胞外ATP对壳聚糖引起的ROS水平和PAL活性变化的影响。结果显示,外源施加20μmol·L^-1ATP可以有效降低壳聚糖诱导的烟草悬浮细胞ROS水平上升,同时外源ATP也明显减缓了壳聚糖所诱导的PAL活性的上升。上述结果表明,细胞外ATP水平能够影响壳聚糖引起的ROS水平和PAL活性的变化。     

Structural characterisation and expression analysis of toll-like receptor 2 gene from catfish

Toll-like receptors (TLRs) are important components of innate immunity. They were found to recognise specific structures on pathogens termed pathogen-associated molecular patterns (PAMPs) and utilise conserved signaling pathways to activate pro-inflammatory cytokines and type-1 interferons. In spite of much understanding gained from the mammalian systems, many fish TLRs are unknown. Recent studies in Japanese flounder as well as in zebrafish suggested that the ligand binding and activation of inflammatory responses in fish may be different from and more complex than those found in mammals. In channel catfish, the major aquaculture species in the United States, only partial sequences of TLR3 and TLR5 were reported. As a part of efforts to characterise the innate immune components in channel catfish, here we cloned and sequenced both the cDNA and the gene for TLR2, a receptor believed mostly responsible for recognition of lipopeptides on the surface of most Gram-positive bacteria. However, expression analysis after infection with a Gram-negative bacterium, Edwardsiella ictaluri indicated that TLR2 was modestly down-regulated in the head kidney tissue of blue catfish, and with a similar pattern in the head kidney of channel catfish though the down-regulation in channel catfish was not statistically significant. In the spleen, an insignificant down-regulation was initially observed early after infection, with an increase of TLR expression later after infection. These results suggest the involvement of TLR2 in the responses after the bacterial infection. As LPS is believed to be the major PAMP for Gram-negative bacteria, additional research is warranted to determine the functions and mechanisms of TLR2 in infections of Gram-negative bacteria.     

Measuring some flounder (Platichthys flesus L.) innate immune responses to be incorporated in effect biomonitoring concepts

For an implementation of innate immune responses of flounder (Platichthys flesus) in an integrated biological effect monitoring concept, leucocytes were isolated from peripheral blood, head kidney and spleen, and analysed for their capacity to mount a respiratory burst response upon phorbol ester stimulation. Responding cells were identified by reduced nitro-blue-tetrazolium salt deposits and by dihydro-rhodamine fluorescence in light microscope and flow cytometric analysis. Responding cells were found in head kidney derived cell suspensions rather than in peripheral blood or spleen. Parallel cytometric and microscopic analysis indicated that responding cells had a granulocyte or monocyte morphology, were alpha-naphtyl-esterase or myeloperoxidase positive and in flow cytometry exhibited a characteristic forward and side scatter (FSC/SSC) pattern. These cells represented 30–40% of head kidney derived cell suspensions and only 4–5 % of peripheral blood and spleen. In order to reduce sampling effort in field studies, leucocyte cell suspensions derived from flounder head kidney could be used in respiratory burst assays without further enrichment protocols. This paper combines, for the first time, conventional and cytometric analysis of phagocytes derived from flounder peripheral blood and head kidney. Communicated by H. v. Westernhagen, A. Diamant     

MKP-1 Is Necessary for T Cell Activation and Function

MAPKs are evolutionarily conserved immune regulators. MAPK phosphatases (MKPs) that negatively regulate MAPK activities have recently emerged as critical players in both innate and adaptive immune responses. MKP-1, also known as DUSP1, was previously shown to negatively regulate innate immunity by inhibiting pro-inflammatory cytokine production. Here, we found that MKP-1 is necessary in T cell activation and function. MKP-1 deficiency in T cells impaired the activation, proliferation, and function of T cells in vitro, associated with enhanced activation of JNK and reduced NFATc1 translocation into the nucleus. Consistently, MKP-1^−/− mice were defective in anti-influenza immunity in vivo and resistant to experimental autoimmune encephalomyelitis. Our results thus demonstrate that MKP-1 is a critical positive regulator of T cell activation and function and may be targeted in treatment of autoimmune diseases.     

Cloning and characterization of the IkappaBalpha gene from Japanese flounder, Paralichthys olivaceus

We identified and characterized the Japanese flounder (Paralichthys olivaceus) inhibitor kappa B alpha (JFIKBA) cDNA. The JFIKBA cDNA contains an open reading frame of 960bp encoding 320 amino acid residues. JFIKBA contains 6 ankyrin repeats in the central coding region. Expression studies by RT-PCR showed constitutive expression of the JFIKBA gene in several Japanese flounder tissues (brain, muscle, gill, heart, kidney, liver, spleen and intestine). Moreover, expression of JFIKBA mRNA was induced in kidney by LPS stimulation. To investigate the role of JFIKBA, we constructed a recombinant plasmid expressing the JFIKBA coding region under the control of the cytomegalovirus (CMV) promoter. Over-expression of the JFIKBA gene in the Japanese flounder cultured cell line derived from kidney, suppressed the expression of the TNF alpha gene with lipopolysaccharide stimulation. These results indicated that JFIKBA has an important role in the innate immune system, especially in the signaling of the cytokine network.     

Extracellular ATP: a potential regulator of plant cell death

Adenosine 5′‐triphosphate (ATP) has been regarded as an intracellular energy currency molecule for many years. In recent decades, it has been determined that ATP is released into the extracellular milieu by animal, plant and microbial cells. In animal cells, this extracellular ATP (eATP) functions as a signalling compound to mediate many cellular processes through its interaction with membrane‐associated receptor proteins. It has also been reported that eATP is a signalling molecule required for the regulation of plant growth, development and responses to environmental stimuli. Recently, the first plant receptor for eATP was identified in Arabidopsis thaliana. Interestingly, some studies have shown that eATP is of particular importance in the control of plant cell death. In this review article, we summarize and discuss the theoretical and experimental advances that have been made with regard to the roles and mechanisms of eATP in plant cell death. We also make an attempt to address some speculative aspects to help develop and expand future research in this area.     

Role of extracellular nucleotides in the immune response against intracellular bacteria and protozoan parasites

Extracellular nucleotides are danger signals involved in recognition and control of intracellular pathogens. They are an important component of the innate immune response against intracellular pathogens, inducing the recruitment of inflammatory cells, stimulating secretion of cytokines, and producing inflammatory mediators such as reactive oxygen species (ROS) and nitric oxide (NO). In the case of extracellular ATP, some of the immune responses are mediated through activation of the NLRP3 inflammasome and secretion of the cytokine, interleukin-1β (IL-1β), through a mechanism dependent on ligation of the P2X7 receptor. Here we review the role of extracellular nucleotides as sensors of intracellular bacteria and protozoan parasites, and discuss how these pathogens manipulate purinergic signaling to diminish the immune response against infection.     

A pore-forming protein,perforin, from a non-mammalian organism,Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.     

TLR9 and RIG-I Signaling in Human Endocervical Epithelial Cells Modulates Inflammatory Responses of Macrophages and Dendritic Cells In Vitro

The innate immune system has evolved to recognize invading pathogens through pattern recognition receptors (PRRs).Among PRRs, Toll like receptors (TLRs 3, 7/8,9) and RIG-I like receptors (RLRs) have been shown to recognize viral components. Mucosal immune responses to viral infections require coordinated actions from epithelial as well as immune cells. In this respect, endocervical epithelial cells (EEC''s) play an important role in initiating innate immune responses via PRRs. It is unknown whether EEC''s can alter immune responses of macrophages and dendritic cells (DC''s) like its counterparts in intestinal and respiratory systems. In this study, we show that endocervical epithelial cells (End1/E6E7) express two key receptors, TLR9 and RIG-I involved in anti-viral immunity. Stimulation of End1/E6E7 cells lead to the activation of NF-κB and increased secretion of pro-inflammatory cytokines, IL-6 and IL-8. Polarized End1/E6E7 cells responded to apical stimulation with ligands of TLR9 and RIG-I, CpG-ODN and Poly(I:C)LL respectively, without compromising End1/E6E7 cell integrity. At steady state, spent medium from End1/E6E7 cells significantly reduced secretion of pro-inflammatory cytokines from LPS treated human primary monocyte derived macrophages (MDMs) and DC:T cell co-cultures. Spent medium from End1/E6E7 cells stimulated with ligands of TLR9/RIG-I restored secretion of pro-inflammatory cytokines as well as enhanced phagocytosis and chemotaxis of monocytic U937 cells. Spent medium from CpG-ODN and Poly(I:C)LL stimulated End1/E6E7 cells showed significant increased secretion of IL-12p70 from DC:T cell co-cultures. The anti-inflammatory effect of spent media of End1/E6E7 cell was observed to be TGF-β dependent. In summary, the results of our study indicate that EEC''s play an indispensable role in modulating anti-viral immune responses at the female lower genital tract.     

Use of zinc chloride as alternative stimulant for in vitro study of nitric oxide production pathway in avian splenocyte culture

Nitric Oxide (NO) plays an important role for regulation of cellular and vascular response of inflammation and initiates killing mechanism in the host-defense reactions. NO production is regulated through inducible nitric oxide synthase (iNOS) pathway in response to infections and injurious agents besides pro-inflammatory cytokines secreted by the host. Evaluation of NO pathway is one of the major targets which can evaluate various immunomodulators for their therapeutic interaction on innate immune system. Lipopolysaccharide (LPS) and concavalin A (ConA) are used as standard mitogen for peripheral blood mononuclear cells and splenocyte of mammalian and avian cell culture. During the present investigation ZnCl₂ has been explored as standard mitogen and result was comparable with standard mitogen. The result has been evaluated on the basis of relative mRNA expression of iNOS and interferon gamma and nitrite assay. Observation indicated significantly higher expression of both biomolecules in comparison to control, LPS, ConA treated group. This study indicated that, ZnCl₂ can also be used as standard stimulant for molecular mining of innate immunity.     

Differential induction of innate memory in porcine monocytes by β-glucan or bacillus Calmette-Guerin

Innate immunomodulation via induction of innate memory is one mechanism to alter the host’s innate immune response to reduce or prevent disease. Microbial products modulate innate responses with immediate and lasting effects. Innate memory is characterized by enhanced (training) or depressed (tolerance) innate immune responses, including pro-inflammatory cytokine production, to secondary exposure following a priming event. To investigate the ability of β-glucans and bacillus Calmette-Guerin to induce innate training or tolerance in pig cells, porcine monocytes were cultured with priming agonist (β-glucans or bacillus Calmette-Guerin) then re-stimulated 5 d later with a heterologous microbial agonist to determine induction of innate memory. Priming with β-glucan from Saccharomyces cerevisiae depressed IL-1β and TNF-α cytokine responses to re-stimulation with LPS, indicative of a tolerized state. However, bacillus Calmette-Guerin priming induced a trained state in porcine monocytes, as LPS re-stimulation enhanced IL-1β and TNF-α gene expression and protein production. We present the first evidence of innate memory in pig monocytes, with bacillus Calmette-Guerin (training) or Saccharomyces cerevisiae β-glucan (tolerance). Induction of a trained or tolerized state in vitro is a first step to identify agonists to alter the innate immune system at the animal level with the intent of enhancing disease resistance.     

Investigating wastewater treatment plant effluent and pharmaceutical exposure on innate cytokine expression of darters (Etheostoma spp.) in the Grand River watershed

Fish live in continuous contact with various stressors and antigenic material present within their environments. The impact of stressors associated with wastewater-exposed environments on fish has become of particular interest in toxicology studies. The objectives of this study were to examine potential effects of wastewater treatment plant (WWTP) effluent-associated stressors on innate cytokine expression within the gills of darter species (Etheostoma spp.), using both field and laboratory approaches. Male and female darters (rainbow, greenside, fantail, and johnny darters) were collected upstream and downstream of the Waterloo WWTP in the Grand River, Ontario. Gill samples were collected from fish in the field and from a second subset of fish brought back to the laboratory. Laboratory fish were acutely exposed (96-h) to an environmentally relevant concentration of venlafaxine (1.0 μg/L), a commonly prescribed antidepressant. To assess the impacts of these stressors on the innate immunity of darters, the expression of key innate cytokines was examined. Minor significant effects on innate cytokine expression were observed between upstream and downstream fish. Moderate effects on cytokine expression were observed in venlafaxine-exposed fish compared to their control counterparts however, changes were not indicative of a biologically significant immune response occurring due to the exposure. Although the results of this study did not display extensive impacts of effluent and pharmaceutical exposure on innate cytokine expression within the gills, they provide a novel avenue of study, illustrating the importance of examining potential impacts that effluent-associated stressors can have on fundamental immune responses of native fish species.