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1.
Fish Shellfish Immunol ; 150: 109650, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38788912

ABSTRACT

Nectins are adhesion molecules that play a crucial role in the organization of epithelial and endothelial junctions and function as receptors for the entry of herpes simplex virus. However, the role of Nectin4 remains poorly understood in fish. In this study, nectin4 gene was cloned from medaka (OlNectin4). OlNectin4 was located on chromosome 18 and contained 11 exons, with a total genome length of 25754 bp, coding sequences of 1689 bp, coding 562 amino acids and a molecular weight of 65.5 kDa. OlNectin4 contained four regions, including an Immunoglobulin region, an Immunoglobulin C-2 Type region, a Transmembrane region and a Coiled coil region. OlNectin4 shared 47.18 % and 25.00 % identity to Paralichthys olivaceus and Mus musculus, respectively. In adult medaka, the transcript of nectin4 was predominantly detected in gill. During red spotted grouper nervous necrosis virus (RGNNV) infection, overexpression of OlNectin4 in GE cells significantly increased viral gene transcriptions. Meanwhile, Two mutants named OlNectin4△4 (+4 bp) and OlNectin4△7 (-7 bp) medaka were established using CRISPR-Cas9 system. Nectin4-KO medaka had higher mortality than WT after infected with RGNNV. Moreover, the expression of RGNNV RNA2 gene in different tissues of the Nectin4-KO were higher than WT medaka after challenged with RGNNV. The brain and eye of Nectin4-KO medaka which RGNNV mainly enriched, exhibited significantly higher expression of interferon signaling genes than in WT. Taken together, the OlNectin4 plays a complex role against RGNNV infection by inducing interferon responses for viral clearance.


Subject(s)
Fish Diseases , Fish Proteins , Nectins , Nodaviridae , Oryzias , RNA Virus Infections , Animals , Oryzias/genetics , Oryzias/immunology , Nodaviridae/physiology , RNA Virus Infections/veterinary , RNA Virus Infections/immunology , Nectins/genetics , Nectins/immunology , Fish Proteins/genetics , Fish Proteins/immunology , Fish Proteins/chemistry , Fish Diseases/immunology , Fish Diseases/virology , Phylogeny , Amino Acid Sequence , Immunity, Innate/genetics , Sequence Alignment/veterinary , Gene Expression Regulation/immunology , Gene Expression Profiling/veterinary
2.
Front Immunol ; 13: 812899, 2022.
Article in English | MEDLINE | ID: mdl-35185906

ABSTRACT

Organized intestinal mucosal immune response appears to be restricted to tetrapods. In teleost fish, there is no evidence for the existence of a particular intestinal region that facilitates the interaction of antigen-presenting cells (APCs) and T cells, such as secondary lymphoid organs. Indeed, despite their importance in the defense against pathogens, the location and manner of APC-T cell interaction within the fish gut is unknown. Here, using non-invasive live imaging of newly developed transgenic reporter lines, we addressed the spatial organization and behavior of APCs and T cells in the intestine of medaka fish both during homeostasis and inflammation. We report that Ccr9a+ T cells are recruited to a band in the lamina propria next to the muscularis mucosa in which Ccl25-expressing cells are present. Ccr9a+ T cells contact APCs for several minutes, in a process mediated by connexin 43. This type of interaction was observed in homeostasis and inflammation, with the interaction being longer and more frequent during inflammation. Thus, our results demonstrate that the mucosal immune response in the intestine of medaka is organized and endowed with a specific region with specialized microenvironment and function.


Subject(s)
Antigen-Presenting Cells/immunology , Intestinal Mucosa/immunology , T-Lymphocytes/immunology , Animals , Chemokines, CC/metabolism , Oryzias/immunology , Receptors, CCR/metabolism
3.
Eur J Immunol ; 52(2): 261-269, 2022 02.
Article in English | MEDLINE | ID: mdl-34731490

ABSTRACT

The differentiation of T cells from lymphoid progenitors in the thymus follows sequential developmental stages that constantly require interaction with thymic epithelial cells. Several distinct aspects of early T cell development depend on the activation of Notch receptors on thymocytes, while the selection of thymocytes at later stages are believed to be Notch independent. Using reverse genetic approaches and whole-thymus live imaging in an in vivo teleost model, the medaka, we report that Notch1 signals is required for proliferation and specification of developing T cells as well as involved in their selection in the thymus. We reveal that Notch1 controls the migratory behavior of thymocytes through controlling the chemokine receptor Ccr9b and thereby influence the T cell receptor (TCR) activation. Hence, we propose that, in lower vertebrates, the function of Notch signaling extends to all stages of T cell development, except when thymocytes undergo TCRß rearrangement.


Subject(s)
Cell Movement , Fish Proteins/immunology , Oryzias , Receptor, Notch1/deficiency , Signal Transduction , T-Lymphocytes/immunology , Thymus Gland/immunology , Animals , Cell Movement/genetics , Cell Movement/immunology , Fish Proteins/deficiency , Oryzias/genetics , Oryzias/immunology , Receptor, Notch1/immunology , Signal Transduction/genetics , Signal Transduction/immunology
4.
Dev Comp Immunol ; 124: 104179, 2021 11.
Article in English | MEDLINE | ID: mdl-34171369

ABSTRACT

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.


Subject(s)
Interleukin-17/metabolism , Oryzias/immunology , Receptors, Interleukin-17/metabolism , Aeromonas hydrophila/physiology , Alternative Splicing , Animals , Chromosome Mapping , Edwardsiella/physiology , Fish Proteins/genetics , Fish Proteins/metabolism , Gene Expression/immunology , Ligands , Oryzias/genetics , Oryzias/microbiology , Phylogeny , Receptors, Interleukin-17/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Synteny , Tissue Distribution/immunology
5.
Dev Comp Immunol ; 115: 103894, 2021 02.
Article in English | MEDLINE | ID: mdl-33080274

ABSTRACT

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.


Subject(s)
Fish Diseases/immunology , Fish Proteins/genetics , Gene Duplication/immunology , Oryzias/immunology , Aeromonas hydrophila/immunology , Animals , Animals, Genetically Modified , Cell Line , Edwardsiella/immunology , Fish Diseases/genetics , Fish Diseases/microbiology , Fish Proteins/metabolism , Gene Expression Regulation/immunology , Gene Knockout Techniques , Inflammasomes/genetics , Oryzias/genetics , Oryzias/microbiology , Phylogeny , Pyroptosis
6.
Fish Shellfish Immunol ; 105: 438-445, 2020 Oct.
Article in English | MEDLINE | ID: mdl-32653586

ABSTRACT

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.


Subject(s)
ARNTL Transcription Factors/genetics , Circadian Clocks/genetics , DNA-Binding Proteins/genetics , Fish Proteins/genetics , Gene Expression Regulation , Oryzias/genetics , Toll-Like Receptor 9/genetics , Zebrafish Proteins/genetics , ARNTL Transcription Factors/immunology , ARNTL Transcription Factors/metabolism , Animals , Circadian Clocks/immunology , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Fish Proteins/immunology , Fish Proteins/metabolism , Oryzias/immunology , Toll-Like Receptor 9/immunology , Toll-Like Receptor 9/metabolism , Zebrafish Proteins/immunology , Zebrafish Proteins/metabolism
7.
Front Immunol ; 11: 425, 2020.
Article in English | MEDLINE | ID: mdl-32256492

ABSTRACT

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.


Subject(s)
Fish Proteins/immunology , Gastrointestinal Microbiome/immunology , Immunity, Mucosal/immunology , Interleukin-17/immunology , Oryzias/immunology , Animals , Gene Expression , Interleukin-17/deficiency
8.
Int J Mol Sci ; 20(17)2019 Aug 26.
Article in English | MEDLINE | ID: mdl-31454991

ABSTRACT

Over the past two decades, studies have demonstrated that several features of T-cell and thymic development are conserved from teleosts to mammals. In particular, works using zebrafish (Danio rerio) and medaka (Oryzias latipes) have shed light on the cellular and molecular mechanisms underlying these biological processes. In particular, the ease of noninvasive in vivo imaging of these species enables direct visualization of all events associated with these processes, which are, in mice, technically very demanding. In this review, we focus on defining the similarities and differences between zebrafish and medaka in T-cell development and thymus organogenesis; and highlight their advantages as two complementary model systems for T-cell immunobiology and modeling of human diseases.


Subject(s)
Oryzias/embryology , Oryzias/immunology , T-Lymphocytes/cytology , T-Lymphocytes/physiology , Thymus Gland/embryology , Zebrafish/embryology , Zebrafish/immunology , Animals , Biomarkers , Cell Differentiation , Genetic Testing , Humans , Molecular Imaging , Organogenesis , Species Specificity
9.
Dev Comp Immunol ; 99: 103402, 2019 10.
Article in English | MEDLINE | ID: mdl-31141705

ABSTRACT

Numerous cytosolic DNA sensors (CDSs), which are very important for recognizing cytosolic dsDNA derived from intracellular viruses and bacteria, exist in mammals. However, teleost CDSs are poorly understood. In this study, four CDSs, including the cyclic GMP-AMP synthase (cGAS), Sm-like protein 14 homolog A (LSm14A), DEAH-box helicase (DHX) 9, and DHX36 genes were identified in Japanese medaka, Oryzias latipes, and their expression patterns were elucidated. The expression of these genes was upregulated in the intestines and kidney of CpG-ODN-stimulated medaka. The cGAS and LSm14A genes were significantly induced in the intestines, kidney, and spleen of formalin-killed Edwardsiella tarda-treated medaka; the DHX9 and DHX36 genes were not. cGAS gene expression was induced only in the intestines of live E. tarda-treated medaka. These results suggest that the transcription of four CDS genes of medaka responds to dsDNA stimulation, and cGAS is probably more important for the immune response against E. tarda infection.


Subject(s)
DNA-Binding Proteins/genetics , Fish Proteins/genetics , Gene Expression Regulation , Oryzias/genetics , Receptors, Pattern Recognition/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Edwardsiella tarda/immunology , Fish Proteins/immunology , Fish Proteins/metabolism , Gene Expression Regulation/immunology , Oligodeoxyribonucleotides/immunology , Oryzias/classification , Oryzias/immunology , Phylogeny , Receptors, Pattern Recognition/immunology , Receptors, Pattern Recognition/metabolism , Tissue Distribution
10.
Environ Pollut ; 251: 502-509, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31103010

ABSTRACT

Benzo[a]pyrene (BaP), a common environmental pollutant, can modulate the immune-associated signal pathway NF-κB, which is one of the critical signal pathways involved in various immune responses. BaP exposure usually generates reactive oxygen species (ROS), but whether ROS are predominantly involved in the modulation mechanism of the NF-κB pathway has not been clearly understood. In this study, an in vivo examination of Oryzias melastigma demonstrated that BaP exposure led to a down-regulation of the NF-κB pathway and increased levels of ROS. Conversely, in vitro results using the medaka liver cell line DIT-29 and a widely applied H2O2 method showed the opposite: up-regulation of the NF-κB pathway. However, the down-regulation of NF-κB upon BaP exposure in vitro was inhibited by the addition of a ROS inhibitor, indicating ROS are involved in the modulation of NF-κB. The discrepancy between in vivo and in vitro results of ROS impacts on NF-κB activation might be related to the concentration and persistence of ROS. Using a modified luminol detection system, BaP was found to generate sustained physiological concentrations of ROS for 24 h, while an H2O2 bolus generated ROS for less than 30 min. Furthermore, a steady-state sub-micromolar H2O2 system (H2O2ss) was developed in parallel as a positive control of ROS, by which H2O2 could be maintained for 24 h. Comparative evaluation using H2O2, H2O2ss and BaP exposures on the medaka cell line with pGL4.32 demonstrated that the persistent physiological concentrations of ROS generated upon BaP exposure or treatment with H2O2ss inhibited the NF-κB pathway, but direct H2O2 exposure had the opposite effect. Moreover, a western-blot assay and EMSA detection further confirmed the modulation of the NF-κB pathway in DIT-29. Taken together, this study shows that BaP exposure inhibits the NF-κB pathway by generating sustained physiological concentrations of ROS.


Subject(s)
Benzo(a)pyrene/toxicity , Environmental Pollutants/toxicity , NF-kappa B/metabolism , Oryzias/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Animals , Antioxidants/metabolism , Cell Line , Down-Regulation , Hydrogen Peroxide/pharmacology , Liver/drug effects , Liver/enzymology , Male , Oryzias/immunology
11.
Gen Comp Endocrinol ; 277: 30-37, 2019 06 01.
Article in English | MEDLINE | ID: mdl-30395804

ABSTRACT

Hepatitis A virus cellular receptor2 (Havcr2) also named T-cell immunoglobulin and mucin domain containing-3 (Tim-3) was initially described as a T helper 1-specific cell surface protein, a member of Tim family implicated in the regulating process of adaptive and innate immune responses. Here, medaka (Oryzias latipes) Havcr2 (OlHavcr2) was isolated and characterized. Unlike other Havcr2 proteins, OlHavcr2 possesses two Ig-like domains but lacks cytoplasmic and transmembrane domains. RT-PCR results revealed that OlHavcr2 mRNA was expressed strongly in the liver, moderately in the intestine, heart and ovary, and weakly in the muscle, gill, brain, eye, spleen, and testis. OlHavcr2 expression begun from gastrula stage and was maintained until hatching. The signal of OlHavcr2 was mainly identified in the blood system in the yolk sac by in situ hybridization. These results indicated that OlHavcr2 is expressed ubiquitously in adult tissues, and is a zygotic gene expressed from gastrula onwards in embryogenesis. OlHavcr2 may play a significant role in the blood system of medaka. In the immune organs, OlHavcr2 expression was affected by the immune stimulants, lipopolysaccharide and poly I:C, suggesting that OlHavcr2 was involved in innate immunity and adaptive immunity in medaka.


Subject(s)
Fish Proteins/genetics , Gene Expression Profiling , Oryzias/genetics , Aging/genetics , Animals , Embryonic Development/genetics , Female , Fish Proteins/chemistry , Fish Proteins/metabolism , Gene Expression Regulation, Developmental , Male , Oryzias/immunology , Phylogeny
12.
Article in English | MEDLINE | ID: mdl-30292753

ABSTRACT

Bcl6B, also known as BAZF, plays important roles in the immune response, repression of cancers, and maintenance of spermatogonial stem cells in mammals. In this study, the homologous gene bcl6b and its 5 alternative splicing variants, namely bcl6bX1 to bcl6bX5, were isolated from medaka fish, Oryzias latipes. Medaka bcl6b possesses conserved domains such as BTB domain, RD2 domain and four zinc fingers. Medaka bcl6bX1 to bcl6bX3 possess all three previously mentioned domains with minor differences in sequences. Medaka bcl6bX4 possesses only the BTB domain due to premature stopping, and bcl6bX5 possesses both the BTB domain and zinc fingers without the RD2 domain. Medaka bcl6b was expressed in the tissues including the brain, heart, gill, muscle, spleen, kidney, intestine, ovary and testes of adult fish. Medaka bcl6b was expressed in the embryos from very early stage, and could be detected clearly in the developing eyes by RT-PCR and in situ hybridization. Medaka bcl6b could respond to the stimuli of polyI:C and LPS in the kidney and spleen. Medaka bcl6bX1 to bcl6bX3 were the majority of the variants expressed in the adult tissues and the embryos, and were the major response to the stimulation of polyI:C and LPS in the spleen. These results suggested that bcl6b, including its isoforms, could function in various tissues and embryogenesis. Moreover, bcl6b might be a factor for immune response in medaka.


Subject(s)
Alternative Splicing , Embryonic Development , Fish Proteins/metabolism , Gene Expression Regulation, Developmental , Oryzias/physiology , Repressor Proteins/metabolism , Alternative Splicing/drug effects , Amino Acid Sequence , Animals , Conserved Sequence , Embryo, Nonmammalian/immunology , Embryo, Nonmammalian/physiology , Eye/embryology , Eye/metabolism , Fish Proteins/chemistry , Fish Proteins/genetics , Gene Expression Regulation, Developmental/drug effects , Immunity, Innate/drug effects , Interferon Inducers/pharmacology , Kidney/drug effects , Kidney/growth & development , Kidney/immunology , Kidney/metabolism , Lipopolysaccharides/pharmacology , Organ Specificity , Oryzias/embryology , Oryzias/growth & development , Oryzias/immunology , Poly I-C/pharmacology , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Random Allocation , Repressor Proteins/chemistry , Repressor Proteins/genetics , Sequence Alignment , Spleen/drug effects , Spleen/growth & development , Spleen/immunology , Spleen/metabolism
13.
Aquat Toxicol ; 203: 95-106, 2018 Oct.
Article in English | MEDLINE | ID: mdl-30099325

ABSTRACT

Estrogenic endocrine disrupting chemicals (EEDCs) are present ubiquitously in sediments and aquatic ecosystems worldwide. The detrimental impact of EEDCs on the reproduction of wildlife is widely recognized. Increasing evidence shows the immunosuppressive effects of EEDCs in vertebrates. Yet, no studies have considered concomitantly EEDC-induced impacts on reproductive impairment and immune suppression in vivo, which are deemed essential for risk assessment and environmental monitoring. In this study, EE2 was used as a representative EEDC, for parallel evaluation of EEDC-induced immune suppression (immune marker gene expression, leukocyte numbers, host resistance assay, and immune competence index) and reproductive impairment (estrogen responsive gene expression, fecundity, fertilization success, hatching success, and reproductive competence index) in an established fish model (marine medaka Oryzias melastigma), considering sex-specific induction and adaptation and recovery responses under different EE2 exposure scenarios. The findings in marine medaka reveal distinct sex differences in the EE2-mediated biological responses. For female fish, low concentration of exogenous EE2 (33 ng/L) could induce hormesis (immune enhancement), enable adaptation (restored reproduction) and even boost fish resistance to bacterial challenge after abatement of EE2. However, a prolonged exposure to high levels of EE2 (113 ng/L) not only impaired F0 immune function, but also perturbed females recovering from reproductive impairment, resulting in a persistent impact on the F1 generation output. Thus, for female fish, the exposure concentration of EE2 is more critical than the dose of EE2 in determining the impacts of EE2 on immune function and reproduction. Conversely, male fish are far more sensitive than females to the presence of low levels of exogenous EE2 in water and the EE2-mediated biological impacts are clearly dose-dependent. It is also evident in male fish that direct contact of EE2 is essential to sustain impairments of immune competence and reproductive output as well as deregulation of immune function genes in vivo. The immunomodulatory pathways altered by EE2 were deciphered for male and female fish, separately. Downregulation of hepatic tlr3 and c3 (in female) and tlr3, tlr5 and c3 (in male) may be indicative of impaired fish immune competence. Taken together, impaired immune competence in the EE2-exposed fish poses an immediate thread on the survival of F0 population. Impaired reproduction in the EE2-exposed fish can directly affect F1 output. Parallel evaluation of immune competence and reproduction are important considerations when assessing the risk of sublethal levels of EE2/EEDCs in aquatic environments.


Subject(s)
Ethinyl Estradiol/toxicity , Immunologic Factors/toxicity , Oryzias/physiology , Reproduction/drug effects , Sex Characteristics , Animals , Biomarkers/metabolism , Environmental Monitoring , Female , Fertility/drug effects , Gene Expression Regulation/drug effects , Leukocytes/drug effects , Leukocytes/metabolism , Liver/drug effects , Liver/metabolism , Male , Oryzias/immunology , Water Pollutants, Chemical/toxicity
14.
Fish Shellfish Immunol ; 82: 421-431, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30125706

ABSTRACT

17α-ethinylestradiol (EE2) exerts endocrine disrupting effect and immunotoxic effect on marine animals, including modulation of hepcidin expression. The antimicrobial peptide hepcidin displays a crucial role in innate immunity in fish against invading pathogens. It is known that the transcription of hepcidin in mammals is individually regulated by many stimuli, including inflammation, iron overload, anemia or hypoxia, through several distinct molecular pathways. The canonical mechanism for endocrine disrupting effects is mediated by an estrogen receptor (ER) and estrogen responsive element (ERE), whereas the underlying mechanism for immunotoxic effect is still unclear. In this study, a hepcidin from Oryzias melastigma (OM-hep1) was found to be down-regulated upon EE2 exposure and was associated with ERα. Unlike the revealed signaling pathways for hepcidin regulation in mammals, it was revealed by promoter activity analysis that the OM-hep1 transcription was not associated with canonical immune-associated and hormone-associated regulatory elements, known as the nuclear factor κB (NF-κB), signal transducer and activator of transcription 3 (STAT3), ERE and estrogen-related receptor responsive element (ERRE). Further analysis through a series of base mutations revealed a short fragment from -315 to -289 bp on the OM-hep1 promoter with high activity. This fragment was composed of a putative ERE-like element (23 bases) plus an adjacent down-streamed four bases motif GTGT. Replacement of either of the core bases (GGTCA) of ERE-like or GTGT motif showed non-activity and non-response to EE2 exposure, thus a new hepcidin-associated element named as HepERE was revealed. Evidences from electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) assay demonstrated that the EE2-mediated down-regulation of OM-hep1 expression was associated with ERα binding to HepERE but not classical ERE. Taken together, a novel signaling pathway was revealed and the regulatory mechanism associated with the ERα and HepERE element on immunomodulation of OM-hep1 expression upon EE2 exposure was first reported here.


Subject(s)
Endocrine Disruptors/toxicity , Ethinyl Estradiol/toxicity , Fish Proteins/genetics , Gene Expression Regulation , Hepcidins/genetics , Oryzias/genetics , Water Pollutants, Chemical/toxicity , Animals , Base Sequence , Fish Proteins/metabolism , Hepcidins/metabolism , Immunity, Innate , Male , Oryzias/immunology , Oryzias/metabolism , Regulatory Elements, Transcriptional
15.
Fish Shellfish Immunol ; 70: 260-269, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28882797

ABSTRACT

Growing evidence suggests that the immune system of teleost is vulnerable to xenoestrogens, which are ubiquitous in the marine environment. This study detected and identified the major circulatory immune proteins deregulated by 17α-ethinylestradiol (EE2), which may be linked to fish susceptibility to pathogens in the marine medaka, Oryzias melastigma. Fish immune competence was determined using a host resistance assay to pathogenic bacteria Edwardsiella tarda. Females were consistently more susceptible to infection-induced mortality than males. Exposure to EE2 could narrow the sex gap of mortality by increasing infection-induced death in male fish. Proteomic analysis revealed that the major plasma immune proteins of adult fish were highly sexually dimorphic. EE2 induced pronounced sex-specific changes in the plasma proteome, with the male plasma composition clearly becoming "feminised". Male plasma was found to contain a higher level of fibrinogens, WAP63 and ependymin-2-like protein, which are involved in coagulation, inflammation and regeneration. For the first time, we demonstrated that expression of C1q subunit B (C1Q), an initiating factor of the classical complement pathway, was higher in males and was suppressed in both sexes in response to EE2 and bacterial challenge. Moreover, cleavage and post-translational modification of C3, the central component of the complement system, could be altered by EE2 treatment in males (C3dg down; C3g up). Multiple regression analysis indicated that C1Q is possibly an indicator of fish survival, which warrants further confirmation. The findings support the potential application of plasma immune proteins for prognosis/diagnosis of fish immune competence. Moreover, this study provides the first biochemical basis of the sex-differences in fish immunity and how these differences might be modified by xenoestrogens.


Subject(s)
Complement System Proteins/genetics , Complement System Proteins/immunology , Estrogens/metabolism , Fish Diseases/immunology , Immunity, Innate/genetics , Oryzias/genetics , Oryzias/immunology , Animals , Blood Proteins/genetics , Blood Proteins/metabolism , Edwardsiella tarda/physiology , Enterobacteriaceae Infections/immunology , Ethinyl Estradiol/metabolism , Female , Fish Proteins/genetics , Fish Proteins/immunology , Male , Proteomics
16.
Fish Shellfish Immunol ; 67: 561-570, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28600196

ABSTRACT

Antimicrobial peptides (AMPs) represent an efficient part of innate immunity and are found in a variety of life. Among them Histone 2A (H2A), as a promising class of AMPs, attracts great attention, but the in vivo mechanism of H2A derived AMP is still less known. Based on the acquisition of Sphistin, a synthetic 38-amino acid H2A derived peptide from Scylla paramamosain, as reported in our previous study, was truncated into three short fragments (Sph12-38, Sph20-38 and Sph30-38) and further investigated for its possible functional domains. The antimicrobial activities of these analogs against different Gram-positive bacteria, Gram-negative bacteria and fungi were illustrated. Among the analogs, Sph12-38 showed a stronger activity with a much lower minimum inhibitory concentration (3 µM) against Staphylococcus aureus, Corynebacterium glutamicum, Micrococcus lysodeikticus Fleming, Bacillus subtilis, Pseudomonas fluorescens, Aeromonas hydrophila and A. sobria in comparison with the reported Sphistin. A leakage of intracellular content was described in E. coli treated with Sph12-38. Unlike Sphistin which mainly disrupts the membrane integrity, Sph12-38 could also combine the A. sobria genomic DNA with a minimum concentration of 6 µM and was located intracellularly in cells observed under confocal laser scanning microscope imaging. In comparison with the control group of Oryzias melastigma injected with A. sobria alone, the group treated with a mixture of Sph12-38 and A. sobria showed a higher survival rate 7 days post-injection. Furthermore, in a pretreatment assay at 6 h, a higher survival rate was observed in the group injected with the mixture of Sph12-38 and A. sobria. Taken together, the synthetic peptide of Sph12-38 had a potent antimicrobial activity against bacteria. However, Sph12-38 had no cytotoxicity towards the hemolymph of S. paramamosain. Our study suggested that, as with Sph12-38, the H2A derived peptides were more likely prone to exert their activities in vivo through the truncated fragments while defending against different species of pathogens.


Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Fungi/drug effects , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Immunity, Innate , Oryzias/immunology , Animals , Fungi/physiology , Gram-Negative Bacteria/physiology , Gram-Positive Bacteria/physiology , Microbial Sensitivity Tests
17.
Article in English | MEDLINE | ID: mdl-28347744

ABSTRACT

Environmentally induced alterations of the immune system during sensitive developmental stages may manifest as abnormalities in immune organ configuration and/or immune cell differentiation. These not only render the early life stages more vulnerable to pathogens, but may also affect the adult immune competence. Knowledge of these sensitive periods in fish would provide an important prognostic/diagnostic tool for aquatic risk assessment of immunotoxicants. The marine medaka Oryzias melastigma is an emerging seawater fish model for immunotoxicology. Here, the presence and onset of four potentially sensitive periods during the development of innate and adaptive cellular immune defence were revealed in O. melastigma: 1.) initiation of phagocyte differentiation, 2.) migration and expansion of lymphoid progenitor cells, 3.) colonization of immune organs through lymphocyte progenitors and 4.) establishment of immune competence in the thymus. By using an established bacterial resistance assay for O. melastigma, larval immune competence (from newly hatched 1dph to 14dph) was found concomitantly increased with advanced thymus development and the presence of mature T-lymphocytes. A comparison between the marine O. melastigma and the freshwater counterpart Oryzias latipes disclosed a disparity in the T-lymphocyte maturation pattern, resulting in differences in the length of T-lymphocyte maturation. The results shed light on a potential difference between seawater and freshwater medaka in their sensitivity to environmental immunotoxicants. Further, medaka immune system development was compared and contrasted to economically important fish. The present study has provided a strong scientific basis for advanced investigation of critical windows for immune system development in fish.


Subject(s)
Embryo, Nonmammalian/immunology , Immunity, Cellular , Immunity, Innate , Immunocompetence , Larva/immunology , Morphogenesis , Oryzias/immunology , Animals , Aquaculture , Bacterial Load , Cell Differentiation , Edwardsiella tarda/growth & development , Edwardsiella tarda/immunology , Edwardsiella tarda/isolation & purification , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/microbiology , Embryonic Development , Gene Expression Regulation, Developmental , Head Kidney/cytology , Head Kidney/growth & development , Head Kidney/immunology , Head Kidney/microbiology , In Situ Hybridization/veterinary , Larva/cytology , Larva/growth & development , Larva/microbiology , Lymphoid Progenitor Cells/cytology , Lymphoid Progenitor Cells/immunology , Lymphoid Progenitor Cells/microbiology , Oryzias/embryology , Oryzias/growth & development , Oryzias/microbiology , Phagocytes/cytology , Phagocytes/immunology , Phagocytes/microbiology , Species Specificity , Spleen/cytology , Spleen/growth & development , Spleen/immunology , Spleen/microbiology , Survival Analysis , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/microbiology , Thymus Gland/cytology , Thymus Gland/growth & development , Thymus Gland/immunology , Thymus Gland/microbiology
18.
Dev Comp Immunol ; 68: 12-20, 2017 03.
Article in English | MEDLINE | ID: mdl-27825821

ABSTRACT

In fish, as well as vertebrates, type I interferons (IFNs) are important cytokines that help to provide innate, antiviral immunity. Although low amounts of IFN are constitutively secreted under normal physiological conditions, long-term and excessive IFN stimulation leads to reduced sensitivity to the IFN signal. This provides a negative feedback mechanism that prevents inappropriate responses and autoimmunity. At present, however, neither IFN desensitization nor the normal physiological role of constitutive IFN are well characterized in fish. The objective here was therefore to produce and characterize a transgenic medaka fish (Oryzias latipes), designated IFNd-Tg, that constitutively overexpressed the IFNd gene. A dual promoter expression vector was constructed for overexpression of IFNd under an EF1α promoter and a DsRed reporter gene under control of a γF-crystaline promoter. The phenotype of the IFNd-Tg fish had a lower response to poly(I:C) and increased susceptibility to nervous necrosis virus (NNV) infection compared to wild-type (WT). Furthermore, transduction of IFN signals for STAT1b, STAT2 and IRF9 were down-regulated in the IFNd-Tg fish, and expression levels of RLR signal molecules (MDA5, MITA, IRF1 and IRF3) were lower than in WT. The constitutive overexpression of IFNd resulted in desensitization of IFN-stimulation, apparently due to downregulation of IFN signal transduction, and this caused increased susceptibility to NNV.


Subject(s)
Fish Diseases/immunology , Fish Proteins/metabolism , Interferon Type I/metabolism , Nodaviridae/immunology , Oryzias/immunology , RNA Virus Infections/immunology , Animals , Animals, Genetically Modified , Cells, Cultured , Disease Susceptibility , Fish Proteins/genetics , Immunity, Innate , Interferon Regulatory Factors/metabolism , Interferon Type I/genetics , Oryzias/genetics , Poly I-C/immunology , STAT1 Transcription Factor/metabolism , STAT2 Transcription Factor/metabolism , Signal Transduction/genetics , Transgenes/genetics , Vertebrates/genetics
19.
Environ Sci Pollut Res Int ; 24(36): 27687-27701, 2017 Dec.
Article in English | MEDLINE | ID: mdl-27473621

ABSTRACT

Many anthropogenic pollutants in coastal marine environments can induce immune impairments in wild fish and reduce their survival fitness. There is a pressing need to establish sensitive and high throughput in vivo tools to systematically evaluate the immunosuppressive effects of contaminants in marine teleosts. This study reviewed a battery of in vivo immune function detection technologies established for different biological hierarchies at molecular (immune function pathways and genes by next generation sequencing (NGS)), cellular (leukocytes profiles by flow cytometry), tissues/organ system (whole adult histo-array), and organism (host resistance assays (HRAs)) levels, to assess the immune competence of marine medaka Oryzias melastigma. This approach enables a holistic assessment of fish immune competence under different chemical exposure or environmental scenarios. The data obtained will also be useful to unravel the underlying immunotoxic mechanisms. Intriguingly, NGS analysis of hepatic immune gene expression profiles (male > female) are in support of the bacterial HRA findings, in which infection-induced mortality was consistently higher in females than in males. As such, reproductive stages and gender-specific responses must be taken into consideration when assessing the risk of immunotoxicants in the aquatic environment. The distinct phenotypic sexual dimorphism and short generation time (3 months) of marine medaka offer additional advantages for sex-related immunotoxicological investigation.


Subject(s)
Immunotoxins/toxicity , Oryzias/immunology , Water Pollutants, Chemical/toxicity , Animals , Female , High-Throughput Nucleotide Sequencing , Liver/metabolism , Male , Oryzias/genetics , Sex Characteristics , Transcriptome
20.
Fish Physiol Biochem ; 43(1): 27-37, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27431678

ABSTRACT

Here, we investigated the effect of dietary resveratrol (20, 40, and 80 µg/g BW/day) on cell-mediated immunity (activity of spleen phagocytes and proliferative response of lymphocytes) and reproductive parameters (egg and sperm quality, i.e. fecundity-total number of eggs produced by individual fish, fertility, embryo survival, and hatching rate) in medaka. Fish fed feed with resveratrol at 40 and 80 µg/g BW/day had significantly higher metabolic activity and intracellular phagocyte killing activity than control. The proliferative lymphocyte activity of the fish from R80 group was greater by more than 20 % in comparison with the control group (P < 0.05). The percentage of macrophages (MO) and their mean fluorescence intensities (MFI) in R40 and R80 groups were significantly higher compared to C and R20 groups (P < 0.05). The differences in MO and MFI values ranged from 52.5 % (±1.5; R0 group) to 65.8 % (±1.6; R80 group) and from 23.2 (±1.4; R0 group) to 38.2 (±2.4; R80 group), respectively. Moreover, resveratrol at 80 µg/g BW/day decreased liver COX activity, i.e. 5.4 in R80 group and 7.9 in R0 group (P < 0.05). The motility parameters of the sperm obtained from the males fed feed supplemented with resveratrol at 80 µg/g BW/day exhibited the highest values except the linearity, which was lower as compared to the control (P < 0.05). The results indicate that diet supplemented with resveratrol at a dosage of 40 µg/g BW/day improves phagocyte killing ability and lymphocyte proliferation in broodstock and accelerates offspring hatch. Also, the results suggest that COX activity influences sperm and oocyte quality in fish; the presence of a COX inhibitor in the dose of 40 µg/g BW/day decreased the embryo survival.


Subject(s)
Diet/veterinary , Immunologic Factors/pharmacology , Oryzias/immunology , Oryzias/physiology , Reproduction/drug effects , Stilbenes/pharmacology , Animals , Cell Proliferation/drug effects , Female , Immunity, Cellular/drug effects , Liver/drug effects , Liver/enzymology , Lymphocytes/drug effects , Lymphocytes/physiology , Macrophages/drug effects , Macrophages/immunology , Male , Phagocytosis/drug effects , Prostaglandin-Endoperoxide Synthases/metabolism , Resveratrol , Sperm Motility/drug effects , Spleen/cytology
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