Evolutional perspective and functional characteristics of interleukin-17 in teleosts.

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.

ASC-deficiency impairs host defense against Aeromonas hydrophila infection in Japanese medaka, Oryzias latipes.

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-1ß, 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.

Interleukin-17A/F1 from Japanese pufferfish (Takifugu rubripes) stimulates the immune response in head kidney and intestinal cells.

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-1ß, IL-6, and ß-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-1ß, 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.

Molecular characterization and gene expression analysis of hypoxia-inducible factor and its inhibitory factors in kuruma shrimp Marsupenaeus japonicus.

In shrimp aquaculture, overcrowded farming causes fluctuations in dissolved oxygen concentrations. Low-oxygen conditions (hypoxia) affect shrimp growth. Hypoxia-inducible factor (HIF) is a transcriptional factor in the basic helix-loop-helix/PAS family and is activated in response to hypoxic stress. However, little is known about HIF and other inhibitors of the HIF pathway in crustaceans. In this study, we cloned MjHIF-1α, an inhibitory factor, MjFIH-1 (factor inhibiting HIF-1α), and MjVHL (Von Hippel-Lindau tumor suppressor) from kuruma shrimp (Marsupenaeus japonicus). MjVHL is the first crustacean VHL ortholog to be cloned. MjHIF-1α, MjFIH-1, and MjVHL exhibit significant sequence similarity and share key functional domains with previously described vertebrate and invertebrate genes. As a result of gene expression analysis in various tissues, MjHIF-1α and MjVHL were more highly expressed in the intestine than in any other organ tissues. In hypoxia experiments, HIF-induced expression levels of MjHIF-1α in the hypoxic group increased significantly for 24 h after initiating hypoxia stimulation and expression of MjVHL decreased significantly for 6 h after hypoxia stimulation (P < 0.05).

Enhancement of immune response and resistance against Vibrio parahaemolyticus in kuruma shrimp (Marsupenaeus japonicus) by dietary supplementation of ß-1,4-mannobiose.

Supplementation of prebiotic carbohydrates can act as a potent immunomodulator and have the efficacy to induce immune-related genes which are involved in host defense. Pure ß-1,4-mannobiose (MNB) showed activation of prophenoloxidase system of shrimp hemocytes in vitro. The resistance of kuruma shrimp Marsupenaeus japonicus against Vibrio parahaemolyticus was examined after the shrimp were fed with 0 (control), 0.02, 0.2, and 2% MNB supplemented diets. The results showed significantly higher survival rates in MNB supplemented shrimp than those of the control one from 2 to 12 days post challenge. In another experiment, the hemocyte count, ROS production, phagocytic, phenoloxidase and bactericidal activities, and expression of immune-related genes were investigated in the control and MNB supplemented groups at day 1, 4, 6, 8 and 11 of the feeding. These immune parameters were significantly enhanced in MNB supplemented groups. Furthermore, the gene expression analysis showed that transcripts of lysozyme, crustin, penaeidin and TNF were significantly up-regulated in hemolymph, lymphoid organs and intestines of MNB treated shrimp. Overall, the results provided evidence that MNB supplementation could improve the immune response and increase shrimp resistance against V. parahaemolyticus infection.

Characterization of xanthine dehydrogenase and aldehyde oxidase of Marsupenaeus japonicus and their response to microbial pathogen.

Reactive oxygen species (ROS) play key roles in many physiological processes. In particular, the sterilization mechanism of bacteria using ROS in macrophages is a very important function for biological defense. Xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX), members of the molybdo-flavoenzyme subfamily, are known to generate ROS. Although these enzymes occur in many vertebrates, some insects, and plants, little research has been conducted on XDHs and AOXs in crustaceans. Here, we cloned the entire cDNA sequences of XDH (MjXDH: 4328 bp) and AOX (MjAOX: 4425 bp) from Marsupenaeus japonicus (kuruma shrimp) using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). Quantitative real-time RT-PCR transcriptional analysis revealed that MjXDH mRNA is highly expressed in heart and stomach tissues, whereas MjAOX mRNA is highly expressed in the lymphoid organ and intestinal tissues. Furthermore, expression of MjAOX was determined to be up-regulated in the lymphoid organ in response to Vibrio penaeicida at 48 and 72 h after injection; in contrast, hydrogen peroxide (H2O2) concentrations increased significantly at 6, 12, 48, and 72 h after injection with white spot syndrome virus (WSSV) and at 72 h after injection with V. penaeicida. To the best of our knowledge, this study is the first to have identified and cloned XDH and AOX from a crustacean species.

Interleukin-22 induces immune-related gene expression in the gills of Japanese medaka Oryzias latipes.

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.

Identification and expression of phospholipase A2 genes related to transcriptional control in the interleukin-17A/F1 pathway in the intestines of Japanese medaka Oryzias latipes.

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.

Identification of two interleukin 17 receptor C (IL-17RC) genes and their binding activities to three IL-17A/F ligands in the Japanese medaka, Oryzias latipes.

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.

Deficiency of interleukin-17 receptor A1 induces microbiota disruption in the intestine of Japanese medaka, Oryzias latipes.

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, ß-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.

Characterization and expression analysis of tandemly-replicated asc genes in the Japanese medaka, Oryzias latipes.

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.

A defective interleukin-17 receptor A1 causes weight loss and intestinal metabolism-related gene downregulation in Japanese medaka, Oryzias latipes.

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.

Interleukin-22 Deficiency Contributes to Dextran Sulfate Sodium-Induced Inflammation in Japanese Medaka, Oryzias latipes.

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.

Molecular characterization and expression of two interleukin-17 receptor A genes on different chromosomes in Japanese medaka, Oryzias latipes.

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.

Interleukin-17A/F1 Deficiency Reduces Antimicrobial Gene Expression and Contributes to Microbiome Alterations in Intestines of Japanese medaka (Oryzias latipes).

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-1ß (IL-1ß), 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.