Expression profile of cytokine genes in Fugu monocytes stimulated with TLR agonists.

Monocytes are antigen-presenting cells (APCs) in mammals. Antigen presentation by monocytes via costimulatory molecules was recently confirmed in the Japanese pufferfish Fugu rubripes (also known as Takifugu rubripes) (Sugamata et al., 2009[1]). However, no detailed investigations examining how cytokine gene expression regulates the activation/differentiation of these cells have been conducted to date. Therefore, in this study, the expression of cytokine genes in Fugu monocytes stimulated with TLR agonists (LPS, polyI:C, and IMQ) was profiled. First, the morphological changes and phagocytic activity of stimulated monocytes were examined. At 5 days post-stimulation, the ratio of activated to inactivated monocytes increased (as determined by FCM analysis), and the phagocytic activity of the cells was higher (5-15%) than that of nonactivated cells. Analysis of cytokine gene expression in stimulated monocytes revealed that the genes encoding CSF-1b and IFN-γ are important cytokines in activation/differentiation of monocytes/macrophages, and that genes encoding inflammatory cytokines such as IL-1ß, IL-6, IL-18, and TNF-α are upregulated by stimulation. The present study revealed the types of cytokines expressed in Fugu monocytes stimulated with TLR agonists.

Cytokine Gene Expression in CD4 Positive Cells of the Japanese Pufferfish, Takifugu rubripes.

CD4(+) T (Th) cells are a central component of the adaptive immune response and are divided into distinct sets based on their specific cytokine production pattern. Several reports have suggested that fish possess Th subset activity similar to that of mammals. The aim of the present study was to isolate CD4(+) T cells from the blood of Japanese pufferfish, Fugu rubripes, and to characterize their cytokine expression profile. We produced a specific antibody against Fugu CD4 and performed cell sorting with the magnetic activated cell sorting system. Sorted Fugu CD4(+) cells were characterized by morphology and expression analysis of cell marker genes. Fugu CD4(+) cells expressed T-cell marker genes but not macrophage or B-cell marker genes. In addition, peripheral blood lymphocytes were stimulated with lipopolysaccharide (LPS), polycytidylic acid (polyI:C), concanavalin A (ConA) prior to sorting, and then Multiplex RT-PCR was used to examine the expression of Th cytokines by the stimulated Fugu CD4(+) cells. LPS and polyI:C stimulation upregulated the expression of Th1, Th17 and Treg cytokines and downregulated the expression of Th2 cytokines. ConA stimulation upregulated the expression of all Th cytokines. These results suggest that fish exhibit the same upregulation of Th-specific cytokine expression as in mammals.

Identification of IL-34 in teleost fish: differential expression of rainbow trout IL-34, MCSF1 and MCSF2, ligands of the MCSF receptor.

The mononuclear phagocyte system is composed of monocytes, macrophages and dendritic cells and has crucial roles in inflammation, autoimmunity, infection, cancer, organ transplantation and in maintaining organismal homeostasis. Interleukin-34 (IL-34) and macrophage colony stimulating factor (MCSF), both signalling through the MCSF receptor, regulate the mononuclear phagocyte system. A single IL-34 and MCSF gene are present in tetrapods. Two types of MCSF exist in teleost fish which is resulted from teleost-wide whole genome duplication. In this report, we first identified and sequence analysed six IL-34 genes in five teleost fish, rainbow trout, fugu, Atlantic salmon, catfish and zebrafish. The fish IL-34 molecules had a higher identity within fish group but low identities to IL-34s from birds (27.2-33.8%) and mammals (22.2-31.4%). However, they grouped with tetrapod IL-34 molecules in phylogenetic tree analysis, had a similar 7 exon/6 intron gene organisation, and genes in the IL-34 loci were syntenically conserved. In addition, the regions of the four main helices, along with a critical N-glycosylation site were well conserved. Taken together these data suggest that the teleost IL-34 genes described in this report are orthologues of tetrapod IL-34. Comparative expression study of the three trout MCSFR ligands revealed that IL-34, MCSF1 and MCSF2 are differentially expressed in tissues and cell lines. The expression of MCSF1 and MCSF2 showed great variance in different tissues and cell lines, suggesting a role in the differentiation and maintenance of specific macrophage lineages in specific locations. The relatively high levels of IL-34 expression across different tissues suggests a homeostatic role of IL-34 for the macrophage lineage in fish. One striking observation in the present study was the lack of induction of MCSF1 and MCSF2 expression but the quick induction of IL-34 expression by PAMPs and inflammatory cytokines in cell lines and primary head kidney macrophages in rainbow trout. In a parasitic proliferative kidney disease (PKD) model, the expression of IL-34 but not the dominant MCSF2 was affected by PKD, suggesting an involvement of macrophage function in this disease model. Thus IL-34 expression is sensitive to inflammatory stimuli and may regulate macrophage biology once up-regulated.

Establishment of a multiplex RT-PCR assay for the rapid detection of fish cytokines.

To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1ß, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-ß1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1ß, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.

Characterization and expression analysis of Th-POK from the Japanese pufferfish, Takifugu rubripes.

In fish, T cell lineage commitment has not been studied, although there are reports related to CD4 and CD8 positive cells. This study describes the cloning and analysis of a master regulator involved in this process, the Th-POK gene in Japanese pufferfish, Takifugu rubripes. The Fugu Th-POK cDNA was composed of 1901 bp, with a 75 bp 5'-UTR, a 131 bp 3'-UTR, and a 1692 bp open reading frame which translates into a peptide of 564 amino acid residues. The deduced Fugu Th-POK protein contained a BTB/POZ domain, Krüppel motif (H/C linker) and Krüppel-like zinc finger DNA binding domain with C2H2 structure. The homology analysis of Fugu Th-POK (ZBTB7B) with other known ZBTB7 members (ZBTB7A, 7C) showed low identity, and the phylogenetic tree analysis showed the Fugu Th-POK clustered with the mammalian Th-POK, away from other ZBTB7 members. The analysis of transcriptional control region of Th-POK gene suggested that the 5'-flanking region and intron 1 include numerous canonical binding motifs for transcription factors regulating T cell development. The genomic organization of the Fugu Th-POK gene was composed of three exons and two introns, and its structure was identical to that of its human counterpart. Comparison of the Fugu and human genomes showed that high levels of conserved synteny existed around the Th-POK gene. The high expression of the Fugu Th-POK gene in unstimulated tissues was seen in head kidney, muscle, skin and gills. Moreover, the expression of the Fugu Th-POK gene in thymic cells was increased by LPS, polyI:C and PHA stimulation.

The role of neuromedin U during inflammatory response in the common carp.

In the current study, we cloned and characterized the neuromedin U (NMU) gene from the common carp Cyprinus carpio L., and identified its participation in immune responses in the teleost. Five isoforms of the preproNMU genes were generated by alternative splicing and isolated from carp. The longest form of the carp preproNMU1 (isoform 1) cDNA was composed of 803 bp, and contained an 18 bp 5'-UTR, a 212 bp 3'-UTR and a 573 bp open reading frame, which translates into a peptide comprising 190 amino acid (aa) residues. The remaining carp preproNMU isoforms were composed of 175 (preproNMU2), 158 (preproNMU3), 150 (preproNMU4) and 133 (preproNMU5) aa residues. Isoforms 1-3 contained four processing signals (KR or RR), while isoforms 4 and 5 contained only two processing signals. High homology was demonstrated among fish and other vertebral NMU at the biologically active C-terminal region (aa position 175-182). Carp preproNMU transcript variants were identified in various tissues, and the expression pattern has been shown to change depending on feeding status. Moreover, it was shown that the expression of preproNMU3 and preproNMU5 was increased following treatment with bacterial or viral mimics. Finally, we investigated the functional aspect of carp NMU using a synthetic NMU peptide. The peptide was found to increase the expression of inflammation-related cytokine genes in intestinal cells within 1 h of treatment. In addition, the activation of phagocytic cells was also stimulated by the NMU peptide. The discovery of NMU in carp allows for a further understanding of immune regulation by biologically active substances.

Genomics of fish IL-17 ligand and receptors: a review.

Interleukin-17 (IL-17) is a cytokine family composed of six ligands (A-F). Especially, the IL-17A and IL-17F are best characterized cytokines of IL-17 family cytokine. These are produced by Th17 cells and induce the expression of many mediators of inflammation properties. In addition, the five member of IL-17 receptor family (RA-RE) have been identified in mammals. Although the research on fish IL-17 is a little to date, this review discusses some of the recent advances in research on IL-17 ligand and receptor genes in fish. IL-17 family member was chosen from the fish genome database, and its structure and phylogeny is analyzed in detail. Moreover, invertebrate IL-17 genes are also discussed, and the isolation and current status of fish IL-17 receptor genes are summarized. Comparative genomic analysis of the IL-17 family among mammals, teleost and invertebrates provided new insights. Novel IL-17 ligand (IL-17N) was identified from teleost, moreover it was suggested that IL-17N may be a teleost specific ligand by synteny and phylogenetic analysis. On the other hand, IL-17 receptors are well conserved between mammal and teleost, the five member of IL-17 receptor family: IL-17RA-RE were found on the teleost genome. In addition, the IL-17RA gene was duplicated in tandem on the stickleback and medaka genome. Knowledge about the IL-17 ligand/receptor in fish is very limited. Therefore this review will hopefully encourage future studies of IL-17 in fish.

Isolation of seven IL-17 family genes from the Japanese pufferfish Takifugu rubripes.

In humans, the IL-17 family is composed of six members (A-F). The A, E and F forms have been extensively studied in numerous mammalian species. However, there are few reports regarding IL-17 expression in teleost. In this study, IL-17 family genes were isolated from the Japanese pufferfish (Fugu) and their structure and expression profile were analyzed. Screening of the Fugu genome database revealed the existence of five scaffolds containing IL-17 family homologous genes. Scaffold_1 contained three IL-17 family homologues including IL-17A/F1, 2 and C2, and IL-17A/F1 and two located in tandem. This was similar to the IL-17A/F1 and two genes in zebrafish and to human IL-17A and F. Other scaffolds 38, 143 and 430, contained IL-17 family homologous genes that were identical to IL-17D, A/F3 and C1 in Fugu, respectively. Moreover, IL-17 family homologues on scaffold_264 included a novel type of IL-17 family genes in teleost. These isolates contained four cysteine residues that were involved in the formation of a typical cysteine knot consisting of two disulphide linkages. However, IL-17A/F2 did not demonstrate any conservation at the second and fourth cysteine residues. The tissue distribution of the Fugu IL-17 family genes was also found to differ. In particular, IL-17 family genes were highly expressed in the head kidney and gill. Moreover, expression of IL-17 family genes was significantly up-regulated in the lipopolysaccharide-stimulated head kidney. These results suggested that Fugu IL-17 family members were involved in inflammatory responses.

Characterization and expression analysis of type I interferon in common carp Cyprinus carpio L.

The type I interferon (I-IFN) gene has recently been cloned and sequenced in the common carp species Cyprinus carpio L. Carp I-IFN cDNA is composed of 675 base pairs and is translated into a protein of 186 amino acid residues. The carp I-IFN encodes a predicted signal peptide of 23 amino acid residues and contains the I-IFN family signature His(140)-Trp(158). Analysis of the homology between carp I-IFN and other known I-IFN and type II interferon (II-IFN) family members has revealed significant similarities to grass carp I-IFN. Phylogenetic analysis demonstrated that carp I-IFN clusters with I-IFN in teleosts, away from the other II-IFN family members. In addition, the gene structure for carp I-IFN is composed of 5 exons and 4 introns, a composition that is similar to that of the teleost I-IFN gene. RT-PCR analysis did not reveal gene expression in un-stimulated tissues including intestine, liver, gill, head kidney, muscle, spleen, mid-kidney and skin. However, I-IFN expression levels increased following stimulation with imiquimod in the head kidney cells. Furthermore, recombinant carp I-IFN protein (mature form) produced via the cell-free protein synthesis system stimulated the expression of the interferon-inducible Mx gene in the head kidney cells.

Immunostimulatory effects of natural human interferon-alpha (huIFN-alpha) on carps Cyprinus carpio L.

Human interferon-alpha (huIFN-alpha) is an important immunomodulatory substance used in the treatment and prevention of numerous infectious and immune-related diseases in animals. However, the immunostimulatory effects of huIFN-alpha in fish remain to be investigated. In the current study, the immune responses of the carp species Cyprinus carpio L. to treatment with huIFN-alpha were analyzed via measurement of superoxide anion production, phagocytic activity and the expression of cytokine genes including interleukin-1beta, tumor necrosis factor-alpha and interleukin 10. Low doses of huIFN-alpha were administered orally once a day for 3 days, and sampling was carried out at 1, 3 and 5 days post-treatment. Our results indicate that a low dose of huIFN-alpha significantly increased phagocytic activity and superoxide anion production in the carp kidney. The huIFN-alpha-treated fish also displayed a significant upregulation in cytokine gene expression. The current study demonstrates the stimulatory effects of huIFN-alpha on the carp immune system and highlights the immunomodulatory role of huIFN-alpha in fish.