Papers - KONO Tomoya
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Yamamoto J., Deguchi H., Sumiyoshi T., Nakagami K., Saito A., Miyanishi H., Kondo M., Kono T., Sakai M., Kinoshita M., Hikima J.I.
Marine Biotechnology 26 ( 4 ) 658 - 671 2024.8
Publishing type：Research paper (scientific journal) Publisher：Marine Biotechnology
Intracellular bacteria such as those belonging to the genus Edwardsiella can survive and proliferate within macrophages. However, the detailed mechanisms underlying the host macrophage immune response and pathogen evasion strategies remain unknown. To advance the field of host macrophage research, we successfully established transgenic (Tg) Japanese medaka Oryzias latipes that possesses fluorescently visualized macrophages. As a macrophage marker, the macrophage-expressed gene 1.1 (mpeg1.1) was selected because of its predominant expression across various tissues in medaka. To validate the macrophage characteristics of the fluorescently labeled cells, May-Grünwald Giemsa staining and peroxidase staining were conducted. The labeled cells exhibited morphological features consistent with those of monocyte/macrophage-like cells and tested negative for peroxidase activity. Through co-localization studies, the fluorescently labeled cells co-localized with E. piscicida in the intestines and kidneys of infected medaka larvae, confirming the ingestion of bacteria through phagocytosis. In addition, the labeled cells expressed macrophage markers but lacked a neutrophil marker. These results suggested that the fluorescently labeled cells of Tg[mpeg1.1:mCherry/mAG] medaka were monocytes/macrophages, which will be useful for future studies aimed at understanding the mechanisms of macrophage-mediated bacterial infections.
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Accumulation and Phagocytosis of Fluorescently Visualized Macrophages Against Edwardsiella piscicida Infection in Established mpeg1.1‑Transgenic Japanese Medaka Oryzias latipes Reviewed
Juna Yamamoto, Hana Deguchi, Takechiyo Sumiyoshi, Kentaro Nakagami, Akatsuki Saito, Hiroshi Miyanishi, Masakazu Kondo, Tomoya Kono, Masahiro Sakai1, Masato Kinoshita, Jun‑ichi Hikima
Marine Biotechnology 26 658 - 671 2024.6
Language：English Publishing type：Research paper (scientific journal)
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Comprehensive analysis of diel rhythmic expression of the medaka tlr gene family Reviewed
Takahiko Hata, Suzuka Setoguchi, Natuki Morimoto, Hidetoshi Shimawaki, Jun-ichi Hikima, Masahiro Sakai, Tomoya Kono
Developmental and Comparative Immunology in press 2024.5
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
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Comprehensive analysis of diel rhythmic expression of the medaka toll-like receptor gene family Reviewed
Hata T., Shimawaki H., Setoguchi S., Morimoto N., Hikima J.i., Sakai M., Kono T.
Developmental and Comparative Immunology 154 2024.5
Publishing type：Research paper (scientific journal) Publisher：Developmental and Comparative Immunology
Several immune-related genes, including Toll-like receptors (TLR), are associated with circadian rhythms in mammals. However, information on the circadian rhythmic expression of TLRs in fish is limited. In this study, we aimed to analyze the regulation of diel oscillations in the expression of TLR genes in Japanese medaka (Oryzias latipes). The expression analysis revealed diel expression patterns of tlr1, tlr5m, tlr21, and clock genes (bmal1 and clock1) under a 12 h light:12 h dark cycle. The clock gene response element (E-box) was identified in the transcriptional regulatory regions of tlr1, tlr5m, and tlr21. Moreover, overexpressed bmal1 and clock1 enhanced expression levels of tlr1, tlr5m, and tlr21 in medaka embryo (OLHdrR-e3) cells. The expression of tlr1, tlr5m, and tlr21 was significantly decreased in OLHdrR-e3 after generating a bmal1 knockdown using a morpholino oligo. These results indicate the regulation of the diel rhythmic expression of several fish TLRs by clock genes.
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Susceptibility f Japanese Medaka to Edwardsiella piscicida and E. anguillarum isolated from aquacultured fish. Reviewed
Aki Nishihara, Takechiyo Sumiyoshi, Koushirou Arakawa, Natsuki Morimoto, Shinya Yasumoto, Masakazu Kondo, Tomoya Kono, Masahiro Sakai, Jun-ichi Hikima
Fish Pathology 58 ( 4 ) 175 - 179 2023.12
Language：English Publishing type：Research paper (scientific journal)
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Watanabe M., Okamura Y., Kono T., Sakai M., Hikima J.i.
Developmental and Comparative Immunology 148 2023.11
Publishing type：Research paper (scientific journal) Publisher：Developmental and Comparative Immunology
The cytokine interleukin (IL)-22 has been identified in several fish species; however, its functional significance in the gills of these fish species remains unclear. In this study, we analyzed the expression of proinflammatory cytokines, antimicrobial peptides, and IL-22 binding protein in the gills of wild-type and IL-22-knockout (IL-22 KO) medaka under dextran sulfate sodium-induced inflammation. We also produced medaka recombinant IL-22 (rIL-22) and analyzed the expression of immune-related genes in rIL-22-stimulated primary cell cultures from gills. The il1b, il6, tnfa, and hamp genes were significantly upregulated in wild-type gills upon dextran sulfate sodium stimulation compared with the naïve state but not in IL-22 KO gills. il22bp transcripts were barely detectable in the IL-22 KO medaka gills. However, the expression of il1b, il6, hamp, and il22bp was upregulated in rIL-22-stimulated gill cell culture. These results suggest IL-22 could be involved in immune responses through inflammatory cytokine and antimicrobial peptide production in fish gills.
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Tsuchiya A., Okamura Y., Kono T., Hikima J.i., Sakai M.
Fisheries Science 89 ( 4 ) 449 - 462 2023.7
Publishing type：Research paper (scientific journal) Publisher：Fisheries Science
The progress of virology research in shrimp has been hampered by a lack of immortalized shrimp cell lines, and even long-term and stable primary cell cultures have not been perfectly achieved. Here, transcriptomic analysis of primary lymphoid cells in kuruma shrimp Marsupenaeus japonicus was performed to understand gene expression changes and stressors under existing culture conditions. In our optimized culture, the primary cells showed dead cell increases on days 3–4 and 6–7 after seeding. Next, we performed transcriptomic analysis at four time points (days 1, 3, 4, and 6 after tissue seeding), and RNA sequencing revealed that detected genes could be divided into three groups according to their expression changes: (1) downregulated on days 3–6, (2) upregulated on days 3–4, and (3) upregulated on day 6. Group 1 included platelet-derived growth factor receptor alpha and vascular endothelial growth factor 3 (MjVEGF3). Consistently, quantitative real-time polymerase chain reaction (qPCR) results showed a significant decrease in MjVEGF3 and its receptor (MjVEGFR) expression. These results suggest the importance of supplementation to recover VEGF signaling in the proper time-dependent manner for long and stable culture of shrimp lymphoid organ cells.
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Evolutional perspective and functional characteristics of interleukin-17 in teleosts Reviewed
Yo Okamura, Tomoya Kono, Masahiro Sakai (Review article)
Fish and Shellfish Immunology 132 108496 2023.1
Language：English Publishing type：Research paper (scientific journal) Publisher：Fish and Shellfish Immunology
Interleukin (IL)-17 is a proinflammatory cytokine and plays essential roles in adaptive and innate immune responses against bacterial and fungal infections. Especially in mammalian mucosal tissues, it is well known that innate immune responses via IL-17A and IL-17F, such as the production of antimicrobial peptides, are very important for microbiota control. In contrast, interesting insights into the functions of IL-17 have recently been reported in several teleost species, although little research has been conducted on teleost IL-17. In the present review, we focused on current insights on teleost IL-17 and speculated on the different or consensus parts of teleost IL-17 signaling compared to that of mammals. This review focuses on the role of teleost IL-17 in intestinal immunity. We expect that this review will encourage a further understanding of the roles and importance of IL-17 signaling in teleosts.
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Tomoya Takeuchi, Takahiko Hata, Hiroshi Miyanishi, Takumi Yuasa, Suzuka Setoguchi, Ayaka Takeda, Natsuki Morimoto, Jun-ichi Hikima, Masahiro Sakai and Tomoya Kono
Fish and Shellfish Immunology 127 ( 2022 ) 238 - 246 2022.8
Authorship：Corresponding author Publishing type：Research paper (scientific journal) Publisher：Fish and Shellfish Immunology
In recent years, studies on circadian control in immunity have been actively conducted in mammals, but little is known about circadian rhythms in the field of fish immunology. In this study, we aimed to analyse the regulation of the diel oscillation of inflammatory cytokine interleukin-1β (il1b) gene expression by core components of the circadian clock in Japanese medaka (Oryzias latipes). The expression of il1b and clock genes (bmal1 and clock1) in medaka acclimated to a 12:12 light (L): dark (D) cycle showed diel rhythm. Additionally, higher expression of il1b was detected in medaka embryo cells (OLHdrR-e3) overexpressing bmal1 and clock1. A significant decrease in il1b expression was observed in OLHdrR-e3 cells after bmal1 knockdown using morpholino oligos. These changes may be mediated by transcriptional regulation via clock proteins, which target the E-box sequence in the cis-element of il1b as identified using luciferase reporter assays. Moreover, LPS stimulation and pathogenic bacterial infection at different zeitgeber time (ZT) under LD12:12 conditions affected the degree of il1b expression, which showed high and low responsiveness to both immuno-stimulations at ZT2 and ZT14, respectively. These results suggested that fish IL-1β exhibited diel oscillation regulated by clock proteins, and its responsiveness to immune-stimulation depends on the time of day.
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Inhibition of lysozyme lytic activity by Ivy derived from Photobacterium damselae subsp. piscicida Reviewed
Aki Nishihara, Natsuki Morimoto, Takechiyo Sumiyoshi, Shinya Yasumoto, Masakazu Kondo, Tomoya Kono, Masahiro Sakai, Jun-ichi Hikima
Fish and Shellfish Immunology 124 280 - 288 2022.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Fish and Shellfish Immunology
A pseudotuberculosis pathogen, Photobacterium damselae subsp. piscicida (Pdp), has caused enormous economic damage to yellowtail aquaculture in Japan. The Ivy gene has been discovered in plasmid of Pdp, and it has been proposed that it may help bacteria evade lysozyme-mediated lysis during interaction with an animal host. However, the lysozyme-inhibiting activity of Pdp-derived Ivy (Ivy-Pdp) is unknown, and it is unclear whether it acts as a virulence factor for host biophylaxis. In this study, the inhibitory effect of Ivy-Pdp on lysozyme was evaluated by expressing and purifying the recombinant Ivy-Pdp protein (rIvy-Pdp). The rIvy-Pdp protein inhibited hen egg white lysozyme activity in an rIvy-Pdp-concentration-dependent manner, and its inhibitory effect was similar under different temperature and pH conditions. The serum and skin mucus of the yellowtail (which is the host species of Pdp), Japanese flounder, and Nile tilapia showed bacteriolytic activity. In contrast, the addition of rIvy-Pdp inhibited the lytic activity in the serum of these fish species. In particular, it significantly inhibited lytic activity in the serum and skin mucus of Nile tilapia. On the basis of these results, we suggest that Ivy-Pdp is a temperature- and pH-stable lysozyme inhibitor. Additionally, Ivy-Pdp inhibited the lytic activity of lysozyme, which is involved in host biophylaxis. In summary, we inferred that Ivy-Pdp is an important factor that diminishes the sterilization ability of C-type lysozyme when Pdp infects the host.
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Okamura Y., Kinoshita M., Kono T., Sakai M., Hikima J.i.
Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics 40 2021.12
Publishing type：Research paper (scientific journal) Publisher：Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics
The mutual relationship between the intestinal immune system and the gut microbiota has received a great deal of attention. In mammals, interleukin-17A and F (IL-17A/F) are inflammatory cytokines and key regulators of the gut microbiota. However, in teleosts, the function of IL-17A/F in controlling the gut microbiota is poorly understood. We attempted to elucidate the importance of teleost IL-17 signaling in controlling gut microbiota. We previously established a knockout (KO) of IL-17 receptor A (RA) 1, a receptor for IL-17A/F, in the Japanese medaka (Oryzias latipes) using the CRISPR-Cas9 system and performed 16S rRNA-based metagenomic analyses using the anterior and posterior sections of the intestinal tract. The number of observed OTUs in the anterior intestine was significantly decreased in IL-17RA1 KO medaka compared to that in the wild-type (WT). Furthermore, β-diversity analysis (weighted UniFrac) revealed considerably different bacterial composition in the anterior intestine of IL-17RA1 KO compared to WT, with similar findings in α-diversity. Notably, the pathogen Plesiomonas shigelloides was significantly increased in the posterior intestine of IL-17RA1 KO medaka. These findings indicate that signaling via IL-17RA1 is required to maintain a healthy gut microbiota in teleosts and mammals. The involvement of IL-17RA1 in controlling the gut microbiota has been demonstrated, resulting in microbiome dysbiosis in IL-17RA1 KO medaka.
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Okamura Y., Miyanishi H., Kinoshita M., Kono T., Sakai M., Hikima J.i.
Scientific Reports 11 ( 1 ) 2021.12
Publishing type：Research paper (scientific journal) Publisher：Scientific Reports
In the intestine, the host must be able to control the gut microbiota and efficiently absorb transiently supplied metabolites, at the risk of enormous infection. In mammals, the inflammatory cytokine interleukin (IL)-17A/F is one of the key mediators in the intestinal immune system. However, many functions of IL-17 in vertebrate intestines remain unclarified. In this study, we established a gene-knockout (KO) model of IL-17 receptor A1 (IL-17RA1, an IL-17A/F receptor) in Japanese medaka (Oryzias latipes) using genome editing technique, and the phenotypes were compared to wild type (WT) based on transcriptome analyses. Upon hatching, homozygous IL-17RA1-KO medaka mutants showed no significant morphological abnormality. However, after 4 months, significant weight decreases and reduced survival rates were observed in IL-17RA1-KO medaka. Comparison of gene-expression patterns in WT and IL-17RA1-KO medaka revealed that various metabolism- and immune-related genes were significantly down-regulated in IL-17RA1-KO medaka intestine, particularly genes related to mevalonate metabolism (mvda, acat2, hmgcs1, and hmgcra) and genes related to IL-17 signaling (such as il17c, il17a/f1, and rorc) were found to be decreased. Conversely, expression of genes related to cardiovascular system development, including fli1a, sox7, and notch1b in the anterior intestine, and that of genes related to oxidation–reduction processes including ugp2a, aoc1, and nos1 in posterior intestine was up-regulated in IL-17RA1-KO medaka. These findings show that IL-17RA regulated immune- and various metabolism-related genes in the intestine for maintaining the health of Japanese medaka.
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Yo Okamura, Hiroshi Miyanishi, Tomoya Kono, Masahiro Sakai, Jun-ichi Hikima
Fish and Shellfish Immunology Reports 2 100028 2021.12
Publishing type：Research paper (scientific journal) Publisher：Fish and Shellfish Immunology Reports
Phospholipase A2 (PLA2), a phospholipid hydrolase, has recently attracted attention owing to its broad functionality. Immunological evidence has revealed increased susceptibility to infectious diseases and immunodeficiency in knockout (KO) mice of several pla2 genes. However, no progress has been made in terms of immunological research on any pla2 gene in fish. In this study, we focused on the intestinal immune responses of fish PLA2s. The full-length open reading frames of pla2g1b, pla2g3, pla2g10, pla2g12b1, pla2g12b2, and pla2g15 cDNAs were cloned in Japanese medaka (Orizias latipes), and their gene expressions were quantified by real-time PCR (qPCR) and in situ hybridization (ISH). Characterization of pla2 genes revealed a functional domain and three-dimensional structure similar to the mammalian counterparts. In addition, expression of pla2g1b, pla2g12b1, and pla2g12b2 was extremely high in Japanese medaka intestines. ISH detected strong expression of pla2g1b mRNAs in the basal muscle layer, and pla2g12b1 and pla2g12b2 mRNAs were detected in the epithelial cells. In the medaka exposed to Edwardsiella piscicida, pla2g12b1, pla2g12b2 and pla2g15 were significantly induced in the anterior and posterior intestines, and pla2g1b was upregulated in the anterior intestine. Furthermore, pla2g1b, pla2g3, pla2g10, and pla2g12b2 were significantly downregulated in the IL-17A/F1 KO medaka compared to those in wild-type medaka. These results suggest that these PLA2s are involved in intestinal immunity in teleosts.
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Nanaki Harada, Yo Okamura, Tomoya Kono, Masahiro Sakai, Jun-ichi Hikima
Developmental and Comparative Immunology 124 ( 104179 ) 2021.11
Publishing type：Research paper (scientific journal) Publisher：Developmental and Comparative Immunology
In mammals, interleukin (IL)-17 receptor C (IL-17RC) and IL-17RA mediate IL-17A and IL-17F signaling to produce mucin, antimicrobial peptides, and maintain healthy intestinal flora. However, IL-17RC signaling in fish remains unclear. In this study, three il17rc transcripts (il17rca1, il17rca2, and il17rcb) from the Japanese medaka (Oryzias latipes) were cloned; il17rca1 and il17rca2 mRNAs were alternatively spliced from il17rca pre-mRNA as transcript variants. The il17rca and il17rcb genes were located on chromosomes 7 and 5, respectively. Teleost clades containing medaka il17rca and il17rcb clustered separately from the tetrapod clade. In adult tissues, il17rca1 expression was significantly higher than il17rca2 and il17rcb. Conversely, il17rcb expression was significantly higher in embryos and larvae. These expression patterns changed following infection with Edwardsiella piscicida and Aeromonas hydrophila. Furthermore, an immunoprecipitation assay using recombinant IL-17RCs and rIL-17A/Fs suggested that, in teleosts, three ligands could function in signaling through two IL-17RCs.
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Takahashi Y., Okamura Y., Harada N., Watanabe M., Miyanishi H., Kono T., Sakai M., Hikima J.I.
Frontiers in Immunology 12 2021.10
Publishing type：Research paper (scientific journal) Publisher：Frontiers in Immunology
Mucosal tissue forms the first line of defense against pathogenic microorganisms. Cellular damage in the mucosal epithelium may induce the interleukin (IL)-22-related activation of many immune cells, which are essential for maintaining the mucosal epithelial barrier. A previous study on mucosal immunity elucidated that mammalian IL-22 contributes to mucus and antimicrobial peptides (AMPs) production and anti-apoptotic function. IL-22 has been identified in several teleost species and is also induced in response to bacterial infections. However, the roles of IL-22 in teleost immunity and mucus homeostasis are poorly understood. In this study, Japanese medaka (Oryzias latipes) was used as a model fish. The medaka il22, il22 receptor A1 (il22ra1), and il22 binding protein (il22bp) were cloned and characterized. The expression of medaka il22, il22ra1, and il22bp in various tissues was measured using qPCR. These genes were expressed at high levels in the mucosal tissues of the intestines, gills, and skin. The localization of il22 and il22bp mRNA in the gills and intestines was confirmed by in situ hybridizations. Herein, we established IL-22-knockout (KO) medaka using the CRISPR/Cas9 system. In the IL-22-KO medaka, a 4-bp deletion caused a frameshift in il22. To investigate the genes subject to IL-22-dependent regulation, we compared the transcripts of larval medaka between wild-type (WT) and IL-22-KO medaka using RNA-seq and qPCR analyses. The comparison was performed not only in the naïve state but also in the dextran sulfate sodium (DSS)-exposed state. At the transcriptional level, 368 genes, including immune genes, such as those encoding AMPs and cytokines, were significantly downregulated in IL-22-KO medaka compared that in WT medaka in naïve states. Gene ontology analysis revealed that upon DSS stimulation, genes associated with cell death, acute inflammatory response, cell proliferation, and others were upregulated in WT medaka. Furthermore, in DSS-stimulated IL-22-KO medaka, wound healing was delayed, the number of apoptotic cells increased, and the number of goblet cells in the intestinal epithelium decreased. These results suggested that in medaka, IL-22 is important for maintaining intestinal homeostasis, and the disruption of the IL-22 pathway is associated with the exacerbation of inflammatory pathology, as observed for mammalian IL-22.
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Interleukin-22 deficiency contributes to the dextran 1 sulfate sodium (DSS)-induced inflammation in Japanese medaka (Olyzias latipes) Reviewed
Yoshie Takahashi, Yo Okamura, Nanaki Harada, Hiroshi Miyanishi, Tomoya Kono, Masahiro Sakai, Jun-ichi Hikima
Frontiers in Immunology 12 688036 2021.10
Publishing type：Research paper (scientific journal)
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Deficiency of interleukin-17 receptor A1 induces microbiome dysbiosis in intestine of Japanese medaka, Oryzias latipes Reviewed
Yo Okamura, Masato Kinoshita, Tomoya Kono, Masahiro Sakai, Jun-ichi Hikima*
Comparative Biochemistry and Physiology - Part D: Genomics and Proteomics 40 ( 100885 ) 2021.7
Publishing type：Research paper (scientific journal)
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IL-17RA1 gene knockout causes weight loss and reduction of intestinal metabolism-related genes in Japanese medaka, Oryzias latipes Reviewed
Yo Okamura, Hiroshi Miyanishi, Masato Kinoshita, Tomoya Kono, Masahiro Sakai, Jun-ichi Hikima
Scientific Reports 11 ( 12099 ) 2021.6
Publishing type：Research paper (scientific journal)
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Study on fish cytokines regulating innate immune responses Invited
KONO TOMOYA
NIPPON SUISAN GAKKAISHI 87 ( 3 ) 213 - 216 2021.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society of Fisheries Science
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Morimoto N., Kono T., Sakai M., Hikima J.I.
International Journal of Molecular Sciences 22 ( 9 ) 2021.5
Publishing type：Research paper (scientific journal) Publisher：International Journal of Molecular Sciences
Pattern recognition receptors (PRRs) play a crucial role in inducing inflammatory responses; they recognize pathogen‐associated molecular patterns, damage‐associated molecular patterns, and environmental factors. Nucleotide‐binding oligomerization domain‐leucine‐rich repeat‐containing receptors (NLRs) are part of the PRR family; they form a large multiple‐protein complex called the inflammasome in the cytosol. In mammals, the inflammasome consists of an NLR, used as a sensor molecule, apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) as an adaptor protein, and pro‐caspase1 (Casp1). Inflammasome activation induces Casp1 activation, promoting the maturation of proinflammatory cytokines, such as interleukin (IL)‐1β and IL‐18, and the induction of inflammatory cell death called pyroptosis via gasdermin D cleavage in mammals. Inflammasome activation and pyroptosis in mammals play important roles in protecting the host from pathogen infection. Recently, numerous inflammasome-related genes in teleosts have been identified, and their conservation and/or differentiation between their expression in mammals and teleosts have also been elucidated. In this review, we summarize the current knowledge of the molecular structure and machinery of the inflammasomes and the ASC‐spec to induce pyroptosis; moreover, we explore the protective role of the inflammasome against pathogenic infection in teleosts.
DOI： 10.3390/ijms22094389