Inductive immune responses in the Japanese pufferfish (Takifugu rubripes) treated with recombinant IFN-γ, IFN-γrel, IL-4/13A and IL-4/13B.

Immune regulation in response to recombinant cytokines (Th1- and Th2-types) is poorly understood in fish. Therefore, we synthesized and purified the Japanese pufferfish, (Takifugu rubripes) recombinant cytokines, namely interferon-gamma (IFN-γ), IFN-γrel, interleukin (IL)-4/13A and IL-4/13B, and then administered by injection to examine the immune regulatory effects on phagocytic activity, superoxide anion production and lysozyme activity, and cytokine gene expressions in treated fish head kidney (HK) at 1, 3 and 5 days post injection (dpi). Pufferfish that received rIFN-γ injection had an elevated phagocytic activity at 1 and 3 dpi, whereas rIFN-γrel and rIL-4/13A treated fish showed an increased phagocytic activity only at 1 and 3 dpi, respectively. Superoxide anion production increased only at 1 dpi in rIFN-γ, rIFN-γrel and rIL-4/13A injected pufferfish. Lysozyme showed a high activity at 1 dpi in rIFN-γ injected fish, at 5 dpi in rIL-4/13A injected fish and all through the time course in rIL-4/13B injected fish. rIFN-γ stimulation caused an elevated IL-1ß and tumor necrosis factor-alpha (TNF-α) transcriptions, whereas stimulation with rIFN-γrel resulted in IFN-γ gene induction and a long term increase in IL-6 and IL-12p40 gene expressions. Injection of rIL-4/13A induced transcription of IL-6, IL-12p40 and TNF-α, while rIL-4/13B treatment enhanced IL-6, IL-12p35 and IL-12p40 gene expressions. Our results suggest that each recombinant cytokine has a role in activation of immune cells. More precisely, rIFN-γ may be potentially the most effective immune inducer among the tested cytokines. Therefore, use of recombinant Th1 and Th2 cytokines as immune enhancers could be an efficient technique for disease prevention in fish.

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.

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.