Role of major histocompatibility complex II antigen-presentation pathway genes in orange-spotted grouper infected with Cryptocaryon irritans.

Cryptocaryon irritans, a pathogen model for fish mucosal immunity, causes skin mucosal and systematic humoral immune response. Where and how MHC II antigen presentation occurs in fish infected with C. irritans remain unknown. In this study, the full-length cDNA of the grouper cysteine protease CTSS was cloned. The expression distributions of six genes (CTSB, CTSL, CTSS, GILT, MHC IIA and MHC IIB) involved in MHC II antigen presentation pathway were tested. These genes were highly expressed in systematic immune tissues and skin and gill mucosal-associated immune tissues. All six genes were upregulated in skin at most time points. Five genes expected CTSS was upregulated in spleen at most time points. CTSB, CTSL and MHC IIA were upregulated in the gill and head kidney at some time points. These results indicate that the presentation of MHC II antigen intensively occurred in local infected skin and gill. Spleen, not head kidney, had the most extensive systematic antigen presentation. In skin, six genes most likely peaked at day 2, earlier than in spleen (5-7 days), marking an earlier skin antibody peak than any recorded in serum previously. This significant and earlier mucosal antigen presentation indicates that specific immune response occurs in local mucosal tissues.

Immunomodulatory Effects of N-Acetyl Chitooligosaccharides on RAW264.7 Macrophages.

The ongoing development of new production methods may lead to the commercialization of N-acetyl chitooligosaccharides (NACOS), such as chitosan oligosaccharides (COS). The bioactivity of NACOS, although not well detailed, differs from that of COS, as they have more acetyl groups than COS. We used two enzymatically produced NACOS with different molecular compositions and six NACOS (NACOS1-6) with a single degree of polymerization to verify their immunomodulatory effects on the RAW264.7 macrophage cell line. We aimed to identify any differences between COS and various NACOS with a single degree of polymerization. The results showed that NACOS had similar immune enhancement effects on RAW264.7 cells as COS, including the generation of reactive oxygen species (ROS), phagocytotic activity, and the production of pro-inflammation cytokines (IL-1ß, IL-6, and TNF-α). However, unlike COS and lipopolysaccharide (LPS), NACOS1 and NACOS6 significantly inhibited nitric oxide (NO) production. Besides their immune enhancement effects, NACOS also significantly inhibited the LPS-induced RAW264.7 inflammatory response with some differences between various polymerization degrees. We confirmed that the NF-κB pathway is associated with the immunomodulatory effects of NACOS on RAW264.7 cells. This study could inform the application of NACOS with varying different degrees of polymerization in human health.

Distribution of Mpeg1+ cells in healthy grouper (Epinephelus coioides) and after Cryptocaryon irritans infection.

Cryptocaryon irritans is an extremely harmful ciliated obligate parasite that is responsible for large economic losses in aquaculture. C. irritans infection can cause an insect-resistant immune response in fish, and many immune cells can be observed in the local infection site. However, it is unclear whether macrophages are involved in the host defense against C. irritans infection. The Mpeg1 protein can form pores and destroy the cell membrane of invading pathogens, and is also used as a macrophage-specific marker in mammals. Therefore, a polyclonal antibody against grouper recombinant Mpeg1a was produced to mark macrophages in this study, which could recognize both isoforms of Mpeg1 (Mpeg1a/b). Immunofluorescence revealed that EcMpeg1 positive cells were mostly distributed in the head kidney and spleen in healthy grouper. Immunofluorescence and immunohistochemistry showed that the number of EcMpeg1 positive cells increased in the gills after infection with C. irritans, implying that EcMpeg1 positive cells may be involved in the process of grouper resistance against C. irritans infection.

Identification and characterization of c-raf from orange-spotted grouper (Epinephelus coioides).

C-Raf proto-oncogene serine/threonine kinase is a mitogen-activated protein kinase (MAP) kinase kinase, which can initiate a mitogen-activated protein kinase (MAPK) cascade by phosphorylating the dual-specific MAP kinase kinases (MEK1/2), and in turn activate the extracellular signal-regulated kinases (ERK1/2). To study the function of c-Raf in teleost fish, a c-Raf cDNA sequence from orange-spotted grouper (Epinephelus coioides) was cloned. Ecc-Raf shared 81%-99% amino acid identity with other vertebrate c-Raf molecules, and shared the highest amino acid identity (99%) with Lates calcarifer c-Raf. Genomic structure analysis revealed that grouper c-Raf shared a conserved exon structure with other vertebrates. Tissue distribution showed that Ecc-Raf was mainly transcribed in systemic immune organs. Ecc-Raf was distributed throughout the cytoplasm of transfected GS cells and the overexpression of Ecc-Raf only slightly enhanced the activation of Activator protein 1. The phosphorylation levels of Ecc-Raf can be induced by PMA and H2O2 treatment, in contrast to DMSO or untreated HKLs. Moreover, the phosphorylation level of the Raf-MEK-ERK axis was downregulated after 24 h of SGIV infection. On the other hand, the total level and phosphorylation level of c-Raf significantly increased post C. irritans infection and showed an enhanced level post immunization. The results of this study suggested that the Raf-MEK-ERK cascade was involved in the response to viral or parasitic infections.

Molecular characteristics and function study of TNF receptor-associated factor 5 from grouper (Epinephelus coioides).

Tumor necrosis factor receptor-associated factor 5 (TRAF5) is a key adapter molecule that participates in numerous signaling pathways. The function of TRAF5 in fish is largely unknown. In the present study, a TRAF5 cDNA sequence (EcTRAF5) was identified in grouper (Epinephelus coioides). Similar to its mammalian counterpart, EcTRAF5 contained an N-terminal RING finger domain, a zinc finger domain, a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. The EcTRAF5 protein shared relatively low sequence identity with that of other species, but clustered with TRAF5 sequences from other fish. Real-time PCR analysis revealed that EcTRAF5 mRNA was broadly expressed in numerous tissues, with relatively high expression in skin, hindgut, and head kidney. Additionally, the expression of EcTRAF5 was up-regulated in gills and head kidney after infection with Cryptocaryon irritans. Intracellular localization analysis demonstrated that the full-length EcTRAF5 protein was uniformly distributed in the cytoplasm; while a deletion mutant of the coiled-coil domain of EcTRAF5 was observed uniformly distributed in the cytoplasm and the nucleus. After exogenous expression in HEK293T cells, TRAF5 significantly activated NF-κB. The deletion of the EcTRAF5 RING domain or of the zinc finger domain dramatically impaired its ability to activate NF-κB, implying that the RING domain and the zinc finger domain are required for EcTRAF5 signaling.

Molecular characteristics and functional study of tumor necrosis factor receptor-associated factor 2 from the orange-spotted grouper (Epinephelus coioides).

In mammals, tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial intracellular adaptor protein, which performs a vital role in numerous signaling pathways that activate NF-κB, MAPKs, and IRFs. In the present study, three TRAF2 sequences were identified from the orange-spotted grouper (Epinephelus coioides), and named EcTRAF2-1, EcTRAF2-2, and EcTRAF2-3. These sequences contained conserved structure features that were similar to those of mammals. EcTRAF2-1 shared relatively low sequence identity with the other two EcTRAF2s. In healthy E. coioides, EcTRAF2s were widely expressed in all tissues tested, but with distinct expression profiles. After infection with Cryptocaryon irritans, EcTRAF2s was markedly upregulated in the gill and head kidney at most time points, implying that EcTRAF2s may be involved in host defense against C. irritans infection. In HEK293T cells, EcTRAF2s were scattered in the cytoplasm. EcTRAF2-1 and EcTRAF2-2 increased the activity of NF-κB, while EcTRAF2-3 reduced NF-κB activation mediated by EcTRAF2-1 implying that EcTRAF2-3 might be a negative regulator of EcTRAF2-1.

Identification and characterization of myeloperoxidase in orange-spotted grouper (Epinephelus coioides).

Cryptocaryon irritans is an important protozoan ciliate, which has led to heavy economic losses in marine aquaculture. Previous studies have indicated that C. irritans infection could induce the migration of neutrophils to infection sites. Myeloperoxidase (MPO) mainly exists in the cytoplasmic granules of the neutrophil and performs its function by a unique enzymatic capacity to produce hypohalous acid and other toxic oxidants. To determine the involvement of MPO and neutrophils against C. irritans infection in the host, we amplified MPO cDNA (EcMPO) from orange-spotted grouper (Epinephelus coioides). The open reading frame (ORF) of EcMPO encodes a putative polypeptide of 770 amino acids and has typical structural characteristics of mammalian MPO, including a signal peptide, a propeptide, a light chain, a heavy chain, and a peroxidase domain. Bioinformatics analysis has demonstrated that the most important functional sites in mammalian MPO were also conserved in grouper and other piscine MPO, implying the functional conservation of this protein during evolution. A rabbit anti-MPO recombinant protein polyclonal antibody was produced, which could recognize the native MPO protein. The expression of EcMPO was higher in the lympho-hematopoietic organs, such as head kidney, trunk kidney, spleen, but lower in muscle, heart, and brain. After infection with C. irritans, the EcMPO transcript was significantly up-regulated at specific time points in the infection sites (skin and gill) and systemic immune organs (head kidney and spleen); The number of EcMPO positive cells first increased and then decreased in the gill, but was still higher than the control after 7 days. These results demonstrated that EcMPO and its positive cells may be involved in anti-C. irritans infection in the grouper, which is attributed to the innate immune mechanisms of the host against parasite infection.

Identification and expression analysis of three XCR1-like receptors from Epinephelus coioides after Cryptocaryon irritans infection.

The unique receptor XCR1 of the XC subfamily of chemokines is specially expressed in CD8α-like dendritic cells. This receptor has one ligand in mice (XCL1) and two ligands in humans (XCL1 and XCL2). In mammals, the XCR1-XCL1 complex performs a vital role in regulating the localization and function of T cells, dendritic cells, and other cell types. In this study, three XCR1-like receptors (EcXCR1, EcXCR1L, and EcCCR12) were identified from a transcriptome database of orange-spotted grouper. The open reading frames (ORFs) of EcXCR1, EcXCR1L, and EcCCR12 predictably encode 337, 348, and 358 amino acids, respectively. All receptors are seven trans-membrane proteins, and contain conserved functional regions, and conserved sites, that are crucial for the role of chemokine receptors in mammals. Conserved features include four cysteine residues in the extracellular regions, a "DRY" motif in the second intracellular loop, and common characteristics at the N-terminus that are important for ligand interaction. In healthy grouper, EcXCR1, EcXCR1L, and EcCCR12 were broadly expressed in all the tissues tested. EcXCR1 was expressed at high levels in the liver, and EcXCR1L, and EcCCR12 in the thymus. After grouper infection with Cryptocaryon irritans, EcXCR1 and EcCCR12 were up-regulated in the skin and the spleen, and EcCCR12 in the skin, gill, and spleen. EcXCR1L expression changed only slightly. These results imply that EcXCR1 and EcCCR12 may be involved in host defense against parasite infection. A polyclonal antibody was produced against EcCCR12, and used to detect EcCCR12-positive cells in peripheral blood. These results will contribute considerably to elucidate the biological role of piscine XCR1-like receptors and their ligands system in the future.

Grouper (Epinephelus coioides) IL-34/MCSF2 and MCSFR1/MCSFR2 were involved in mononuclear phagocytes activation against Cryptocaryon irritans infection.

MCSF and its well-known receptor MCSFR had been well studied in humans, regulating the differentiation, proliferation, and survival of the mononuclear phagocyte system. IL-34, which is an alternative ligand of MCSF receptor, was recently identified as a novel cytokine and functionally overlaps with MCSF. However, the functional study of these receptors and their ligands in fish are largely unknown. In the present study, the cDNA of two potential grouper MCSFR ligands have been cloned, EcIL-34 (657 bp) and EcMCSF2 (804 bp), as well as an additional copy of grouper MCSFR, EcMCSFR2 (3141 bp). Sequence analysis showed that these three molecules had higher identities with other fish counterparts compared to mammals and their conserved structures and important functional residues were also analyzed. Tissue distribution analysis showed that EcIL-34 is dominant in brain, gill and spleen compared to EcMCSF2, which is dominant in head kidney, trunk kidney, skin, heart and muscle. EcMCSFR1 was dominant in the most tissues except head kidney and liver compared to EcMCSFR2. The different tissue distribution patterns of these two grouper MCSF receptors and their two ligands indicate the different mononuclear phagocyte differentiation and activation modes in different tissues. In Cryptocaryon irritans infected grouper, EcIL-34 and EcMCSFR2 were the most strongly up-regulated ligand and receptor in the infected sites, gill and skin. Their up-regulation confirmed the proliferation and activation of phagocytes in C. irritans infected sites, which would improve the antigen presentation and elicit the host local specific immune response. In C. irritans infected grouper head kidney, both ligands EcIL-34 and EcMCSF2 (especially EcMCSF2) were up-regulated, but both receptors EcMCSFR1 and EcMCSFR2 were down-regulated, which indicated that the phagocytes differentiation and proliferation may have occurred in this hemopoietic organ, and after that they migrated to the infected cites. The down-regulation of EcIL-34 and EcMCSF2 and no significant change of EcMCSFR1 and EcMCSFR2 in most time point of grouper spleen showed it was less involved in phagocytes response to C. irritans infection.

TAK1-binding proteins (TAB1 and TAB2) in grass carp (Ctenopharyngodon idella): identification, characterization, and expression analysis after infection with Ichthyophthirius multifiliis.

Transforming growth factor-ß activated kinase-1 (TAK1) is a key regulatory molecule in toll-like receptor (TLR), interleukin-1 (IL-1), and tumor necrosis factor (TNF) signaling pathways. The activation of TAK1 is specifically regulated by two TAK1-binding proteins, TAB1 and TAB2. However, the roles of TAB1 and TAB2 in fish have not been reported to date. In the present study, TAB1 (CiTAB1) and TAB2 (CiTAB2) in grass carp (Ctenopharyngodon idella) were identified and characterized, and their expression profiles were analyzed after fish were infected with the pathogenic ciliate Ichthyophthirius multifiliis. The full-length CiTAB1 cDNA is 1949 bp long with an open reading frame (ORF) of 1497 bp that encodes a putative protein of 498 amino acids containing a typical PP2Cc domain. The full-length CiTAB2 cDNA is 2967 bp long and contains an ORF of 2178 bp encoding a putative protein of 725 amino acids. Protein structure analysis revealed that CiTAB2 consists of three main structural domains: an N-terminal CUE domain, a coiled-coil domain, and a C-terminal ZnF domain. Multiple sequence alignment showed that CiTAB1 and CiTAB2 share high sequence identity with other known TAB1 and TAB2 proteins, and several conserved phosphorylation sites and an O-GlcNAc site were deduced in CiTAB1. Phylogenetic tree analysis demonstrated that CiTAB1 and CiTAB2 have the closest evolutionary relationship with TAB1 and TAB2 of Danio rerio, respectively. CiTAB1 and CiTAB2 were both widely expressed in all examined tissues with the highest levels in the heart and liver, respectively. After infection with I. multifiliis, the expressions of CiTAB1 and CiTAB2 were both significantly up-regulated in all tested tissues at most time points, which indicates that these proteins may be involved in the host immune response against I. multifiliis infection.

Molecular characterization and functional analysis of TRAF6 in orange-spotted grouper (Epinephelus coioides).

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial signal transducer in both the TNFR superfamily and Toll-like receptor/interleukin 1R family. Although significant progress has been made in clarifying the role of TRAF6 in mammals, the function of TRAF6 in fish is still poorly understood. In this study, we cloned the orange-spotted grouper (Epinephelus coioides) TRAF6 (EcTRAF6) cDNA, with an open reading frame of 1713bp encoding 570 amino acids. Sequence analysis indicated that EcTRAF6 contains the four characteristic domains conserved in the TRAF family, including an N-terminal RING finger, two zinc fingers, a coiled-coil domain, and a C-terminal MATH domain. Homology alignment and phylogenetic analysis demonstrated that EcTRAF6 shares high sequence identity with TRAF6 of other fish species. The EcTRAF6 gene contains seven exons and six introns, which is similar to the organization in ayu, but not in the common carp, human, or mouse (six exons and five introns). EcTRAF6 transcripts were broadly expressed in all tissues tested, and increased after infection with Cryptocaryon irritans. Intracellular localization showed EcTRAF6 was distributed mainly in the cytoplasm. Over-expression of wild type (WT) EcTRAF6, truncated forms of EcTRAF6, including ΔZinc finger 2 and ΔMATH, and a mutant of C78A activated NF-κB strongly in HEK293T cells; whereas truncations, including ΔRING, ΔZinc finger 1 and Δcoiled-coiled, and a mutant of K132R induced the activity of NF-κB slightly compared to WT EcTRAF6, implying the latter has a more crucial role in downstream signal transduction. Together, these results suggested EcTRAF6 functions like that of mammals to activate NF-κB, and it might have an important role in host defense against parasitic infections.

Cloning and expression analysis of grouper (Epinephelus coioides) M-CSFR gene post Cryptocaryon irritans infection and distribution of M-CSFR(+) cells.

The M-CSF/M-CSFR system plays a central role in the cell survival, proliferation, differentiation and maturation of the monocyte/macrophage lineage. In present study, we cloned the sequence of the M-CSFR cDNA from the orange-spotted grouper (Epinephelus coioides). Sequence analysis reveals that ten cysteines in the extracellular immunoglobulin-like (Ig-like) domains of EcM-CSFR are conserved in fish and mammals, its nine possible N-glycosylation sites are conserved in fish but not mammals, 7 of 8 identified mammal M-CSFR intracellular autophosphorylation tyrosine sites was found in EcM-CSFR. Real-time PCR showed that the constitutive expression level of EcM-CSFR was the highest in the spleen, less in the gill, kidney, head kidney and liver, least in the blood, skin, gut and thymus. A rabbit anti-EcM-CSFR polyclonal antibody against the recombinant EcM-CSFR extracellular domain was developed and it was efficient in labeling the monocytes and macrophages isolated from the head kidney. Immunochemistry analysis showed that M-CSFR(+) cells located in all tested paraffin-embedded tissues and M-CSFR(+) cell centres with the characteristic of melano-macrophage centres(MMCs) was found in the spleen, head kidney, kidney, gut and liver. All these results indicate the widespread distribution of macrophages in grouper tissues and its importance in fish immune system. In Crytocaryon irritans infected grouper, EcM-CSFR was transient up-regulated and rapidly down-regulated in skin, gill, head kidney and spleen. The possible activation mechanism of macrophage via EcM-CSFR signal transduction in the fish anti-C. irritans infection was discussed.

Grass carp (Ctenopharyngodon idella) TRAF6 and TAK1: molecular cloning and expression analysis after Ichthyophthirius multifiliis infection.

Ichthyophthirius multifiliis, a pathogenic ciliate parasite, infects almost all freshwater fish species and causes significant economic losses. Tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-ß-activated kinase 1 (TAK1) are two important signaling molecules involved in toll-like receptor (TLR) signal transduction. To date, the roles of TRAF6 and TAK1 in host defense against fish parasites are still poorly understood. In the present study, TRAF6 (CiTRAF6) and TAK1 (CiTAK1) were identified from grass carp (Ctenopharyngodon idella). The full-length cDNA sequence of CiTRAF6 (2250 bp) includes an open reading frame (ORF) of 1629 bp, which shows a high similarity to that of Cyprinus carpio TRAF6 and encodes a putative protein of 542 amino acids containing one RING domain, two zinc fingers, one coiled-coil region, and one MATH domain. The full-length CiTAK1 cDNA sequence is 2768 bp and includes an ORF of 1626 bp that encodes a putative protein of 541 amino acids containing a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. Phylogenetic analysis showed that CiTRAF6 and CiTAK1 were clustered with TRAF6 and TAK1 of other teleosts, respectively. CiTRAF6 and CiTAK1 were both constitutively expressed in all examined tissues but with varied expression levels. The highest expressions of CiTRAF6 and CiTAK1 were in the head kidney and spleen, respectively. The expression profiles of CiTRAF6 and CiTAK1 were detected in grass carp after I. multifiliis infection. Expressions of both genes were significantly up-regulated in the skin, gill, head kidney, and spleen at most time points after infection, indicating that CiTRAF6 and CiTAK1 may play essential roles in grass carp defense against I. multifiliis.

Molecular cloning of orange-spotted grouper (Epinephelus coioides) TLR21 and expression analysis post Cryptocaryon irritans infection.

TLR21, a non-mammalian Toll like receptor, was recently identified in chicken as a pattern recognition receptor of unmethyl-CpG ODN, functionally similar to that of mammalian TLR9. Its role in fish immune defense and whether it is involved in anti-parasite immunity has not yet been proven. In this study, we identified a cDNA sequence encoding orange-spotted grouper Toll-like receptor 21 (EcTLR21), the open reading frame (ORF) was 2937 bp encoding a putative polypeptide of 979 amino acid residues. Some conserved motifs in mammalian TLR9 were also conserved in grouper and other fish species' TLR9 and TLR21. Tissue distribution analysis indicated that EcTLR21 is broadly expressed in all the tissue we tested except muscle. High expression levels were found in the head kidney, trunk kidney, spleen and heart. Post Cryptocaryon irritans infection, TLR21 and TLR9 transcripts were induced at the local infection sites (skin and gill), while suppressed in systemic immune organs (spleen and head kidney), indicating that these two receptors may play a role in host anti-parasitic immune responses.