Papers - KONO Tomoya
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Inflammasomes in teleosts: Structures and mechanism that induce pyroptosis during bacterial infection Reviewed
Natsuki Morimoto, Tomoya Kono, Masahiro Sakai, Jun-ichi Hikima
International Journal of Molecular Sciences 22 ( 9 ) 4389 2021.4
Publishing type：Research paper (scientific journal)
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Natsuki Morimoto, Yo Okamura, Tomoya Kono, Masahiro Sakai, Jun-ichi Hikima
Developmental and Comparative Immunology In press 2021.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Developmental and Comparative Immunology
ASC is a component of the inflammasome playing crucial roles in the inflammatory response. In mammals, ASC induces pyroptosis and inflammatory cytokine production. In this study, three asc genes (asc1, asc2, and asc3) from the Japanese medaka (Oryzias latipes) were identified and characterized. These asc genes were tandem replicates on chromosome 16, and their exon-intron structures differed between them. All three ASCs conserved the pyrin and caspase-recruitment domains, which are important for inflammasome formation. In phylogenetic analysis, all ASCs clustered with those of other teleosts. The asc1 expression levels were significantly higher in several organs than those of asc2 and asc3, suggesting that asc1 may act as a dominant asc in the Japanese medaka. Expression of the three asc genes showed different patterns during Aeromonas hydrophila and Edwardsiella piscicida infections. Furthermore, their expression was adequately down-regulated in the medaka fin-derived cells stimulated with ATP for 12 h, while asc2 expression was statistically up-regulated after nigericin stimulation for 24 h. Moreover, the expression of asc2 and asc3 was significantly higher in the skin of ASC-1-knockout medaka than in that of the wild type medaka during A. hydrophila infection.
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Fish cytokines: current research and applications Invited Reviewed
Masahiro Sakai, Jun-ichi Hikima, Tomoya Kono
Fisheries Science 87 1 - 9 2020.12
Language：English Publishing type：Research paper (scientific journal)
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Morimoto N., Okamura Y., Maekawa S., Wang H.C., Aoki T., Kono T., Sakai M., Hikima J.i.
Fish and Shellfish Immunology 105 427 - 437 2020.10
Publishing type：Research paper (scientific journal) Publisher：Fish and Shellfish Immunology
Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is a component of inflammasome, which plays crucial roles in the inflammatory response. In mammals, ASC regulates caspase-1 activation, thereby inducing pyroptosis and producing activated inflammatory cytokines. In addition, ASC also interacts with receptor-interacting protein kinase 2 (RIPK2) and induces nuclear factor-κB (NF-κB) activation. However, the role of ASC remains poorly understood in fish. In this study, we focused on elucidating the role of ASC in fish that were infected with Aeromonas hydrophila using Japanese medaka (Oryzias latipes) as fish model, and ASC-knockout (KO) medaka was established using CRISPR-Cas9 system. ASC-KO and wild type (WT) medakas were infected with A. hydrophila, and mortality was observed. ASC-KO medaka demonstrated higher mortality than WT. Moreover, the expression of immune-related genes in the kidney and intestine of the ASC-KO and WT medakas challenged with A. hydrophila were analyzed. Following A. hydrophila infection, the kidney of ASC-KO medaka exhibited significantly lower expression of NF-κB regulated genes (e.g., IL-1β, IL-6, IL-8 and TNF-α) and RIPK2 gene than in WT kidney. Moreover, to investigate the immune response against A. hydrophila via ASC in the medaka, bacterial burden, superoxide anion production, and lactate dehydrogenase release in the kidney cells of ASC-KO medaka were measured. After infection, these responses in ASC-KO medaka were significantly decreased compared to those in WT. These results suggest that the medaka ASC plays a critical role against A. hydrophila infection by inducing inflammatory responses and cell death for bacterial clearance.
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Takahashi Y., Okamura Y., Morimoto N., Mihara K., Maekawa S., Wang H.C., Aoki T., Kono T., Sakai M., Hikima J.i.
Fish and Shellfish Immunology 103 143 - 149 2020.8
Publishing type：Research paper (scientific journal) Publisher：Fish and Shellfish Immunology
In mammals, interleukin (IL)-17A and IL-17F, mainly produced by Th17 cells, are hallmark inflammatory cytokines that play important roles in the intestinal mucosal immune response. In contrast, three mammalian IL-17A and IL-17F counterparts (IL-17A/F1-3) have been identified in teleosts, and most of their functions have been described in the lymphoid organs. However, their function in the intestinal mucosal immune response is poorly understood. In this study, a recombinant (r) tiger puffer fish fugu (Takifugu rubripes) IL-17A/F1 was produced and purified using a mammalian expression system, and was used to stimulate cells isolated from fugu head kidney and intestines. The gene expression levels of TNF-α, IL-1β, IL-6, and β-defensin-like protein-1 (BD-1) genes were evaluated at 0, 3, 6 and 12 h post-stimulation (hps). Phagocytic activity and superoxide anion production were evaluated at the same time points using an NBT assay. The rIL-17A/F1 protein was shown to induce the expression of pro-inflammatory cytokines and antimicrobial peptides in both head kidney and intestinal cells. Expression levels for IL-1β, TNF-α, and IL-6 were all up-regulated between 3 and 12 hps. In addition, stimulation with rIL-17A/F1 enhanced phagocytic activity at 24 hps. Superoxide anion production was increased at 48 hps in the head kidney cells and moderately increased at 48 hps in intestinal cells. This study suggests that fugu IL-17A/F1 plays an important role in promoting the innate immune response and may act as a bridge between innate and adaptive immunity in the head kidney and intestine.
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Diel variation of CC chemokine gene expression in the Japanese pufferfish Takifugu rubripes Reviewed
Yuri Tsutsui, Teika Onoue, Jun-ichi Hikima, Masahiro Sakai and Tomoya Kono
Marine Biotechnology 22 ( 5 ) 607 - 612 2020.8
Language：English Publishing type：Research paper (scientific journal) Publisher：Marine Biotechnology
CC chemokines are key molecules in the regulation of leukocyte trafficking to the site of injury, infection, or inflammation. In recent years, some mammalian chemokines have been shown to exhibit rhythmic expression, regulated by clock genes. However, the rhythmic expression of chemokines in teleost fish remains unknown. In the present study, the diel variation of teleost CC chemokine genes was investigated using the model fish, Fugu (Takifugu rubripes). Diel variation analysis revealed that clock (bmal1, clock1, per2, rorα, and rev-erbβ) and CC chemokine (ccl18l, ccl19, and ccl25l) genes show diel expression under 12:12 light-dark cycle (LD12:12) conditions. CC chemokine genes, which exhibit diel expression, contain RORE (ccl18l, ccl19, ccl25l) and/or E-box (ccl25l) motifs in their transcription regulatory region. Moreover, in vitro head kidney stimulation with lipopolysaccharide (LPS) at different zeitgeber times (ZT) under LD12:12 conditions affected the degree of ccl18l, ccl19, and ccl25l expression; high and low responsiveness to LPS stimulation at ZT12 and ZT0 (ccl25l), and ZT16 and ZT4 (ccl18l and ccl19), respectively, were observed. These results suggest that the expression of some fish CC chemokines is affected by the diel variation regulated by clock proteins, and that responsiveness against bacterial infection depends on the time zone.
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ASC-deficiency impairs host defense against Aeromonas hydrophila infection in Japanese medaka Oryzias latipes Reviewed
Natsuki Morimoto, Yo Okamura, Tsukasa Nakazaki, Shun Maekawa, Han-Ching Wang, Takashi Aoki, Tomoya Kono, Masahiro Sakai, Jun-ichi Hikima
Fish and Shellfish Immunology 105 427 - 437 2020.7
Language：English Publishing type：Research paper (scientific journal)
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Genki Taira, Teika Onoue, Jun-ichi Hikima, Masahiro Sakai and Tomoya Kono
Fish and Shellfish Immunology 105 438 - 445 2020.7
Language：English Publishing type：Research paper (scientific journal) Publisher：Fish and Shellfish Immunology
Currently, circadian regulation of immune molecules in lower vertebrates, particularly, diurnal oscillation in the immune status of a fish, is not well understood. In this study, the diurnal oscillation of toll-like receptor (Tlr) 9, which plays a role in pathogen recognition, was investigated in the Japanese medaka fish (Oryzias latipes). We confirmed the expression of tlr9 and clock genes (bmal1 and clock1) in the central and peripheral tissues of medaka. These genes were expressed in a diurnal manner in medaka acclimated to a 12-h:12-h light-dark (12:12 LD) cycle. In addition, increased tlr9 expression was detected in medaka embryo cells (OLHdrR-e3) overexpressing both bmal1 and clock1 genes; however, this result was not obtained when only one or neither of the genes was overexpressed. This suggests that the increase in expression was mediated by the Bmal1 and Clock1 proteins together. In vitro stimulation of the head kidney with CpG-oligodeoxynucleotides (CpG-ODNs) at different zeitgeber times (ZTs; ZT0 = light on, ZT12 = light off) affected the degree of tlr9 gene expression, showing high and low responsiveness to CpG-ODN stimulation at ZT6/10 and ZT18/22, respectively. Similarly, bacterial infection at different ZT points induced a difference in the expression of Tlr9 signaling pathway-related genes (tlr9 and myd88). These results suggested that fish tlr9 exhibits diurnal oscillation, which is regulated by clock proteins, and its responsiveness to immune-stimulation/pathogen infection depends on the time of the day.
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Immune responses in the Japanese pufferfish (Takifugu rubripes) head kidney and intestinal cells treated with recombinant interleukin-17A/F1 Reviewed
Yoshie Takahashi, Yo Okamura, Natsuki Morimoto, Koshin Mihara, Shun Maekawa, Hang-Ching Wang, Takashi Aoki, Tomoya Kono, Masahiro Sakai and Jun-ichi Hikima
Fish and Shellfish Immunology 103 143 - 149 2020.7
Language：English Publishing type：Research paper (scientific journal)
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Okamura Y., Morimoto N., Ikeda D., Mizusawa N., Watabe S., Miyanishi H., Saeki Y., Takeyama H., Aoki T., Kinoshita M., Kono T., Sakai M., Hikima J.I.
Frontiers in Immunology 11 2020.3
Publishing type：Research paper (scientific journal) Publisher：Frontiers in Immunology
In mammals, interleukin (IL)-17A and F are hallmark inflammatory cytokines that play key roles in protection against infection and intestinal mucosal immunity. In the gastrointestinal tract (GI), the induction of antimicrobial peptide (AMP) production via Paneth cells is a fundamental role of IL-17A and F in maintaining homeostasis of the GI microbiome and health. Although mammalian IL-17A and F homologs (referred to as IL-17A/F1-3) have been identified in several fish species, their function in the intestine is poorly understood. Additionally, the fish intestine lacks Paneth cells, and its GI structure is very different from that of mammals. Therefore, the GI microbiome modulatory mechanism via IL-17A/F genes has not been fully elucidated. In this study, Japanese medaka (Oryzias latipes) were used as a teleost model, and IL-17A/F1-knockout (IL-17A/F1-KO) medaka were established using the CRISPR/Cas9 genome editing technique. Furthermore, two IL-17A/F1-deficient medaka strains were generated, including one strain containing a 7-bp deletion (-7) and another with an 11-bp addition (+11). After establishing F2 homozygous KO medaka, transcriptome analysis (RNA-seq) was conducted to elucidate IL-17A/F1-dependent gene induction in the intestine. Results of RNA-seq and real-time PCR (qPCR) demonstrated down-regulation of immune-related genes, including interleukin-1β (IL-1β), complement 1q subunit C (C1qc), transferrin a (Tfa), and G-type lysozyme (LyzG), in IL-17A/F1-KO medaka. Interestingly, protein and lipid digestive enzyme genes, including phospholipase A2, group IB (pla2g1b), and elastase-1-like (CELA1), were also downregulated in the intestines of IL-17A/F1-KO medaka. Furthermore, to reveal the influence of these downregulated genes on the gut microbiome in IL-17A/F1-KO, 16S rRNA-based metagenomic sequencing analysis was conducted to analyze the microbiome constitution. Under a non-exposed state, the intestinal microbiome of IL-17A/F1-KO medaka differed at the phylum level from wild-type, with significantly higher levels of Verrucomicrobia and Planctomycetes. Additionally, at the operational taxonomic unit (OTU) level of the human and fish pathogens, the Enterobacteriaceae Plesiomonas shigelloides was the dominant species in IL-17A/F1-KO medaka. These findings suggest that IL-17A/F1 is involved in the maintenance of a healthy gut microbiome.
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Comparative analysis of plasmid DNAs from two strains of Photobacterium damselae subs. piscicida from Japan and the United States Reviewed
Akito Tsuchiya, Aki Nishihara, Ayumi Saeki, Yuki Teru, Takashi Aoki, Tomoya Kono, Masahiro Sakai and Jun-ichi Hikima
Fish Pathology In press 2020.3
Language：English Publishing type：Research paper (scientific journal)
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Interleukin-17AF1 deficiency reduces antimicrobial gene expression and contributes to microbiome alteration in intestines of Japanese medaka (Oryzias latipes) Reviewed
Yo Okamura, Natsuki Morimoto, Daisuke Ikeda, Nanami Mizusawa, Shugo Watabe, Hiroshi Miyanishi, Yuichi Saeki, Haruko Takeyama, Takashi Aoki, Masato Kinoshita, Tomoya Kono, Masahiro Sakai and Jun-ichi Hkima
Frontiers in Immunology In press 2020.3
Language：English Publishing type：Research paper (scientific journal)
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Yo Okamura, Natsuki Morimoto, Shuzo Sawada, Tomoya Kono, Jun-ichi Hikima and Masahiro Sakai
Comparative Biochemistry and Physiology, Part B In press 2020.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology
In mammals, interleukin 17 (IL-17), which is produced mainly by Th17 cells, is a hallmark inflammatory cytokine that plays key roles in the protection against infection and intestinal mucosal immunity. The mammalian IL-17 receptor family comprises five members (IL-17RA–E). Of these, IL-17RA is important in the control of the bacterial microbiota in mucosal tissues, particularly in the intestine, where it acts as a receptor for IL-17A and -F. In this study, the nucleotide sequence of IL-17RA1 cDNA from Japanese medaka (Oryzias latipes) of the Cab strain was determined and compared to two IL-17RA cDNAs (i.e., IL-17RA1 and IL-17RA2) of Japanese medaka Hd-rR strain downloaded from NCBI. Hd-rR 17RA1 and IL-17RA2 were located on chromosome 23 and chromosome 6, respectively, and phylogenetic tree analysis revealed that teleost IL-17RA1 and IL-17RA2 were separated in different clusters. Synteny analysis revealed that Japanese medaka IL-17RA1 and mammalian IL-17RA are conserved. IL-17RA1 expression levels in the gills, intestine, whole kidney, skin, and spleen were significantly higher than those of IL-17RA2, suggesting that IL-17RA1 is an important functional receptor in mucosal immunity. Interestingly, the expression levels of both IL-17RA genes were notably higher in the posterior than in the anterior intestinal tract section. Furthermore, despite its lower basal expression, IL-17RA2 expression was significantly increased at 72 h post Edwardsiella tarda infection.
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Tsuchiya Akito, Nishihara Aki, Saeki Ayumi, Teru Yuki, Aoki Takashi, Kono Tomoya, Sakai Masahiro, Hikima Jun-ichi
Fish Pathology 55 ( 1 ) 18 - 21 2020
Language：English Publishing type：Research paper (scientific journal) Publisher：The Japanese Society of Fish Pathology
Pseudotuberculosis pathogen, <i>Photobacterium damselae</i> subsp. <i>piscicida</i> (<i>Pdp</i>) shows some differences in virulence between Japanese and US strains. In this study, we analyzed plasmid DNAs derived from Japanese and US strains of <i>Pdp</i>, and found two homologous regions in these plasmids, pOT-51443-4/p91-197-1 and pOT-51443-1/p91-197-2. All genes in the p91-197-1 from US strain were observed in the pOT-51443-4 from Japanese strain. This event might have occurred during transmission of the US plasmid to Japanese strain. Inhibitor of vertebrate lysozyme (<i>ivy</i>), a known pathogenicity factor was encoded in the homologous region of pOT-51443-1/p91-197-2. Thus, the genomic region containing <i>ivy</i> may have been horizontally transmitted between the two <i>Pdp</i> strains.
DOI： 10.3147/JSFP.55.18
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Molecular characterization and expression of the teleost cytosolic DNA sensor genes cGAS, LSm14A, DHX9, and DHX36 in Japanese medaka, Oryzias latipes Reviewed
Murakami, S., Morimoto, N., Kono, T. Sakai, M., Hikima, J.
Developmental and Comparative Immunology In press 2019.10
Language：English Publishing type：Research paper (scientific journal)
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Circadian oscilattion of TNF-α gene expression regulated by clock gene, BMAL1 and CLOCK1 in the Japanese medaka (Oryzias latipes) Reviewed
Teika Onoue, Goshi Nishi, Jun-ichi Hikima, Masahiro Sakai and Tomoya Kono
International Immunopharmacology 70 362 - 371 2019.5
Language：English Publishing type：Research paper (scientific journal)
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Nonconservation of TLR5 activation site in Edwardsiella tarda flagellin decreases expression of interleukin-1β and NF-κB genes in Japanese flounder, Paralichthys Reviewed
Natsuki Morimoto, Masakazu Kondo, Tomoya Kono, Masahiro Sakai and Jun-ichi Hikima
Fish and Shellfish Immunology In press 2019.4
Language：English Publishing type：Research paper (scientific journal)
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Identification and immunoregulatory function of neuromedin U (NMU) in the Japanese pufferfish Takifugu rubripes Reviewed
Kono T., Ida T., Kawahara N., Watanabe F., Biswas G., Sato T., Mori K., Miyazato M.
Developmental and Comparative Immunology 73 246 - 256 2017.8
Language：English Publishing type：Research paper (scientific journal)
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Whole-genome sequence of Photobacterium damselae subsp. piscicida strain 91-197, isolated from hybrid striped bass (Morone sp.) in the United States Reviewed
Teru Y., Hikima J., Kono T., Sakai M., Takano T., Hawke J., Takeyama H., Aoki T.
Genome Announcements 5 ( 29 ) 2017.7
Language：English Publishing type：Research paper (scientific journal)
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Purification and characterization of bioactive peptides RYamide and CCHamide in the kuruma shrimp Marsupenaeus japonicus Reviewed
Mekata T., Kono T., Satoh J., Yoshida M., Mori K., Sato T., Miyazato M., Ida T.
General and Comparative Endocrinology 246 321 - 330 2017.5
Language：English Publishing type：Research paper (scientific journal)