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The imidazoquinoline compound R848 is a specific agonist of toll-like receptor (TLR) 7/TLR8 that has been used as an immunostimulant in humans against viral diseases. Although R848-induced immune response has been reported in teleost fish, the relevant mechanism is not clear. In this study, we investigated the antiviral potential and the signaling pathway of R848 in a model of Japanese flounder ( Paralichthys olivaceus ). We found that R848 was able to inhibit the replication of megalocytivirus, stimulated the proliferation of peripheral blood leukocytes (PBL), enhanced the expression of immune genes, and reduced apoptosis of PBL. When endosomal acidification was blocked by chloroquine (CQ), R848-mediated antiviral activity and immune response were significantly reduced. Likewise, inhibition of Myd88 activation markedly impaired the pro-proliferation and anti-apoptosis effect of R848. Cellular study showed that cultured founder cells treated with R848 exhibited augmented NF-κB activity, which, however, was dramatically reduced in the presence of CQ and Myd88 inhibitor. Furthermore, when NF-κB was inactivated, the effect of R848 on cell proliferation and apoptosis was significantly decreased. Taken together, these results indicate that R848 is an immunostimulant with antiviral property in a teleost species, and that the immune response of R848 is mediated by, most likely, TLR7/TLR8 signaling pathway, in which Myd88 and NK-κB play an essential role. [ABSTRACT FROM AUTHOR]

Abstract: Tissue factor pathway inhibitor 1 (TFPI-1) is a Kunitz-type serine protease inhibitor that regulates the activation of tissue factor-induced coagulation. In teleosts, TFPI-1-like sequences have been found to exist in two species (Danio rerio and Cyprinus carpio); however, the potential function of fish TFPI-1 has not been investigated. In this study, we identified and analyzed a TFPI-1 homologue, SoTFPI-1, from red drum (Sciaenops ocellatus). The deduced amino acid sequence of SoTFPI-1 is 284 residues in length and contains three Kunitz domains, an acidic N-terminus, and a basic C-terminus. SoTFPI-1 shares 49.5% and 46.9% overall sequence identities with the TFPI-1 of D. rerio and C. carpio, respectively. Quantitative real time RT-PCR analysis showed that constitutive SoTFPI-1 expression occurred, in increasing order, in kidney, brain, liver, gill, blood, spleen, muscle, and heart. Bacterial infection and lipopolysaccharide exposure upregulated SoTFPI-1 expression in kidney in time-dependent manners. Recombinant SoTFPI-1 (rSoTFPI-1) purified from Escherichia coli exhibits not only serine protease inhibitor activity but also bactericidal activity in a manner that is independent of any host factors. A synthetic peptide, TO17, corresponding to the C-terminal basic region of SoTFPI-1 also possesses antibacterial effect that is more potent than that of the full-length rSoTFPI-1. Taken together, these results demonstrate that (i) SoTFPI-1 is a biologically active serine protease inhibitor endowed with bactericidal property; (ii) provide the first indication that teleost TFPI-1 is likely to be involved in anti-microbial infection and thus is linked to innate immune defense. [Copyright &y& Elsevier]

Anti-lipopolysaccharide factor (ALF) is a type of antimicrobial peptides (AMPs) with a vital role in antimicrobial defense. Although a large amount of ALFs have been identified from neritic and fresh water crustacean species, no functional investigation of ALFs from deep-sea animals have been documented. In the present study, we characterized the immune function of an ALF molecule (named RspALF1) from the shrimp Rimicaris sp. residing in the deep-sea hydrothermal vent in Desmos, Manus Basin. RspALF1 shares 51.5%–62.4% overall sequence identities with known shrimp ALFs and contains the conserved LPS binding domain (LBD). Both recombinant RspALF1 (rRspALF1) and the LBD-derived peptide (ALF1P1) bound to the cell wall components of Gram-negative and Gram-positive bacteria and killed a wide range of bacteria, especially those from deep-sea hydrothermal field, by damaging bacterial cellular structures. The bactericidal activities of rRspALF1 and ALF1P1 were optimal and stably maintained from 4 °C to 37 °C, which is comparable to the ambient temperature range of the habitat of Rimicaris sp. In addition to bacteria, rRspALF1 and ALF1P1 also exhibited anti-fungal activity. rRspALF1 and ALF1P1 exhibited high killing efficiencies, which, in terms of MIC values, were ranged between 0.25 μM and 4 μM for bacteria and 4 μM–8 μM for fungi. When introduced in vivo , both rRspALF1 and ALF1P1 effectively inhibited bacterial infection in shrimp and reduced the dissemination of bacterial and viral pathogens in fish. Together, these results provide the first insight into the biological property of deep-sea ALF and indicate that RspALF1 very likely plays a significant role in immune defense by functioning as a highly effective antimicrobial with a broad target range. [ABSTRACT FROM AUTHOR]

In mammals, CD83 is a surface marker on mature dendritic cells and vital to lymphocyte activation. In teleost, studies on the function of CD83 are very limited. In this study, we examined the potential involvement of turbot ( Scophthalmus maximus ) CD83, SmCD83, in vaccine-induced immunity. For this purpose, turbot were immunized with pORF75, a DNA vaccine against megalocytivirus, in the presence or absence of pSmCD83, a plasmid that constitutively expresses SmCD83. Immune response and protection analysis showed that the presence of pSmCD83 significantly (i) enhanced the activation of head kidney macrophages (HKM) and immune gene expression, (ii) inhibited viral replication in fish tissues following megalocytivirus challenge and increased the survival of the vaccinated fish, and (iii) stimulated production of specific serum antibody and the cytotoxicity of peripheral blood leukocytes. To further examine the effect of SmCD83, pORF75 was administered into turbot in which SmCD83 was knocked down. Subsequent analysis showed that in fish with SmCD83 knockdown, vaccine-induced HKM activation and antibody production were severely reduced, and, consistently, the protectivity of pORF75 was drastically decreased. Taken together, these results indicate for the first time that teleost CD83 is required for the induction of protective immune response by DNA vaccine. [ABSTRACT FROM AUTHOR]

Highlights: [•] Three CpG ODNs that inhibited megalocytivirus replication in flounder were selected. [•] ODN C7 was able to promote leukocyte and phagocyte activation. [•] Knockdown of toll-like receptor 9 expression blocked C7-mediated immune response. [•] C7 exhibited adjuvant property and augmented the protection of a DNA vaccine. [•] C7 enhanced the expression of vaccine-induced genes of innate and adaptive immunity. [ABSTRACT FROM AUTHOR]

Highlights: [•] SmLMWPTP is a low molecular weight protein tyrosine phosphatase from turbot. [•] rSmLMWPTP exhibited optimum tyrosine phosphatase activity at pH 5 and 50°C. [•] Peripheral blood leukocytes (PBL) infected by pathogen expressed and secreted SmLMWPTP. [•] rSmLMWPTP reduced the biological activity, gene expression, and phagocytosis of PBL. [•] Mutation of the P-loop sequence abolished the regulatory effect of SmLMWPTP on PBL. [ABSTRACT FROM AUTHOR]

Abstract: IκBα is a member of the NF-κB inhibitor family that inhibits NF-κB activity by sequestering NF-κB in an inactive form in the cytosol. Unlike mammalian IκBα, which has been extensively studied, very little is known about the function of fish IκBα. In this study, we identified and analyzed an IκBα homologue, CsIκBα from half-smooth tongue sole (Cynoglossus semilaevis), a marine flatfish with important economic value. The deduced amino acid sequence of CsIκBα contains 308 residues and shares 58–82% overall sequence identities with the IκBα of a number of teleosts. In silico analysis identified in CsIκBα conserved domains that in mammals are known to be involved in phosphorylation, ubiquitination, and degradation of IκBα. Quantitative real time RT-PCR detected constitutive expression of CsIκBα in gut, spleen, liver, gill, heart, brain, muscle, and kidney. Experimental challenge with a bacterial pathogen-induced significant inductions of CsIκBα expression in head and trunk kidney, which, however, were transient and much lower in magnitude than that of interleukin-1β. To examine the effect of unregulated overexpression of CsIκBα in a live fish model, tongue sole were administered via intramuscular injection with plasmid pCNCsIkBa, which constitutively expresses CsIκBα. PCR, RT-PCR, and immunohistochemistry analysis showed that pCNCsIkBa was able to translocate to internal tissues, where transcription and translation of the recombinant CsIκBα took place. Compared to control fish, fish administered with pCNCsIkBa were impaired in the ability to block bacterial dissemination and survival in kidney and exhibited significantly reduced expression of multiple immune genes. These results suggest the possible existence in tongue sole of a NF-κB–IκBα signaling pathway that is negatively regulated by CsIκBα and required for effective defense against bacterial infection. [Copyright &y& Elsevier]

Highlights: [•] CsEBI3 expression was detected in multiple tissues and induced by pathogen infection. [•] CsEBI3 was secreted by peripheral blood leukocytes (PBL) upon bacterial stimulation. [•] rCsEBI3 enhanced the immune response and bacterial resistance of PBL. [•] Antibody blocking of CsEBI3 on PBL reduced cell resistance to bacterial invasion. [•] The immunoregulatory function of rCsEBI3 depends on the FN3 domain of the protein. [Copyright &y& Elsevier]

Highlights: [•] SoMIF is a macrophage migration inhibitory factor from red drum. [•] SoMIF was expressed in multiple tissues and induced by bacterial and viral infection. [•] SoMIF was secreted by head kidney (HK) leukocytes. [•] rSoMIF inhibited the migration of HK monocytes and lymphocytes. [•] Fish received rSoMIF showed enhanced monocyte activation and bacterial clearance. [Copyright &y& Elsevier]

Abstract: The immunoglobulin superfamily (IgSF) is a large group of cell surface proteins that include various immunoregulatory receptors such as novel immune type receptors (NITRs), which are a family of diversified proteins found exclusively in bony fish. In this study, we identified and analyzed an IgSF protein, SoIgSF1, from red drum (Sciaenops ocellatus). SoIgSF1 is composed of 225 amino acid residues and moderately related to teleost NITRs. In silico analysis indicated that SoIgSF1 is a type I transmembrane glycoprotein and contains an N-terminal signal peptide sequence, a single extracellular immunoglobulin V domain, a transmembrane region, and a cytoplasmic region. However, unlike most NITRs, the cytoplasmic region of SoIgSF1 exhibits no consensus inhibitory or stimulatory signaling sequences but has two tyrosine-containing motifs that conform to the right-half sequence of the immunoreceptor tyrosine-based inhibitory motif (ITIM). Quantitative real time RT-PCR analysis showed that SoIgSF1 expression occurred mainly in immune organs and was drastically induced by viral and bacterial infection. Immunofluorescence microscopy indicated that viral infection of head kidney (HK) leukocytes induced surface expression of SoIgSF1, which was able to interact with antibodies against recombinant SoIgSF1. Antibody cross-linking of SoIgSF1 on HK leukocytes inhibited the expression of immune relevant genes and promoted viral and bacterial infection. Taken together, these results indicate that SoIgSF1, though lacking canonical intracellular signaling motifs, is involved in regulation of host immune response during pathogen infection possibly by functioning as a negative signaling receptor through a novel mechanism. [Copyright &y& Elsevier]