FTR33, a member of fish-specific TRIM (finTRIM) subfamily, regulates negatively type I IFN antiviral immunity in zebrafish.

In mammals, the tripartite motif (TRIM) proteins have been identified as critical factors involved in various cellular processes, including antiviral immunity. In teleost fish, a subfamily of fish-specific TRIM (finTRIM, FTR) has emerged in genus- or species-specific duplication. In this study, a finTRIM gene, called ftr33, was identified in zebrafish (Danio rerio), and phylogenic analysis revealed that FTR33 is closely related with zebrafish FTR14. The FTR33 protein contains all conservative domains reported in other finTRIMs. The ftr33 has a constitutive expression in embryos and in tissues/organs of adult fish, and its expression can be induced following spring viremia of carp virus (SVCV) infection and interferon (IFN) stimulation. The overexpression of FTR33 significantly downregulated the expression of type I IFNs and IFN-stimulated genes (ISGs) both in vitro and in vivo, respectively, leading to the increased replication of SVCV. It was also found that FTR33 interacted with melanoma differentiation associated gene 5 (MDA5) or mitochondrial anti-viral signaling protein (MAVS) to weaken the promoter activity of type I IFN. It is thus concluded that the FTR33, as an ISG, in zebrafish can negatively regulate IFN-mediated antiviral response.

Identification and functional characterization of CL-11 in black rockfish (Sebastes schlegelii).

CL-11 (Collectin-11, also known as Collectin kidney-1 or CL-K1) is a member of collectin family that works as a pattern recognition molecule (PRM) and participating in lectin-complement pathway in host defense against pathogens. We identified the CL-11 homologue SsCL-11 in black rockfish (Sebastes schlegelii) and investigated the functional characteristics in this study. The SsCL-11 has conserved protein modules, i.e. an N-terminal hydrophobic region, a collagen-like region, an α-helical neck region and a carbohydrate recognition domain (CRD). SsCL-11 has varying degrees of expressions in difference tissues, among which the highest expression is observed in liver. It also shows induced expressions in immune-related tissues following Aeromonas salmonicida (A. salmonicida) infection. In addition, SsCL-11 exhibits binding abilities to different kinds of carbohydrates, pathogen-associated molecular patterns (PAMPs) and bacteria. It exhibits comparatively strong binding to l-fucose, d-mannose, and d-glucose, which is consistent with the functional EPN motif in its CRD. SsCL-11 also shows agglutinating effects on various bacteria in the presence of Ca2+. Furthermore, SsCL-11 is confirmed to be a secretory lectin and can form multimers. These findings collectively demonstrate that SsCL-11 can function as a recognition molecule in pathogen resistance in black rockfish, which will promote our understanding of immunological roles of fish collectins.

Molecular characterization, antibacterial activity and mechanism analyzation of three different piscidins from black rockfish, Sebastes schlegelii.

Sebastes schlegelii (black rockfish) is a popular and economically important fish species in aquaculture. However, disease outbreaks have hindered the development of its cultivation. Antimicrobial peptides (AMPs) are a group of important components in fish innate immune system, that are active in the first line of defense against pathogens. The piscidin family, which are a group of fish-specific AMPs, have been isolated in a part of teleost but still poorly understood in S. schlegelii. In this study, three piscidin genes (Ss-piscidin1, 2, 3) are identified in S. schlegelii and their antibacterial activities and related mechanisms are analyzed. Three Ss-piscidins have conserved signal peptides but highly variable mature peptides and prodomains, and their mature regions all have predicted amphipathic and α-helical structures. Phylogenetic analysis shows that three Ss-piscidins cluster with different fish piscidin sequences into three sister clades, which correspond to three groups of fish piscidin family, respectively. Ss-piscidins have constitutive expressions in different tissues of healthy fish and enhanced expressions after Aeromonas salmonicida challenge. All three piscidins exhibit antibacterial activities, and are able to enhance bacterial membrane permeability and change bacterial morphology to different degrees, with a positive correlation observed among these activities. This suggests that three peptides exert their antibacterial activity through a "membrane-attack" mechanism. Moreover, hemolytic activities of three piscidins are also analyzed, and Ss-piscidin1, with low hemolytic ability and high antibacterial activity, is considered to be a possible candidate template for design of AMP drugs. Results in this study can promote a better understanding of immune responses in black rockfish and facilitate the future development of strategies in fish disease control in aquaculture.

Functional characterization of four TIR domain-containing adaptors, MyD88, TRIF, MAL, and SARM in mandarin fish Siniperca chuatsi.

Toll/interleukin-1 receptor (TIR) domain-containing adaptors, serve as pivotal signal transduction molecules in Toll-like receptor (TLR) signalling pathway to mediate downstream signalling cascades. In this study, four TIR-domain containing adaptors, MyD88, TRIF, MAL and SARM, were identified in mandarin fish Siniperca chuatsi, and they all contain TIR domains, of which MyD88 and SARM had high sequence homology with their vertebrate homologues. The expression analysis at mRNA level indicated that these genes were ubiquitously distributed in different tissues, being high in immune- and mucosa-related tissues such as head-kidney and intestine. The transcripts of these adaptor genes were up-regulated by poly(I:C) and LPS stimulation in isolated head-kidney lymphocytes (HKLs) of mandarin fish. Fluorescence microscopy revealed that all these molecules were localized in cytoplasm, and further investigations showed that the over-expression of MyD88, TRIF and MAL activated the NF-κB, ISRE or type Ι IFN promoters and inhibited SVCV replication, whereas their antiviral effects were significantly impaired when co-transfected with SARM. It was also confirmed by co-immunoprecipitation (Co-IP) that SARM interacts separately with MyD88, TRIF and MAL, and MAL interacts with MyD88. However, the regulatory mechanisms of these adaptors involved in signalling pathways of different TLRs should be of interest for further research.

Identification and expression analysis of sixteen Toll-like receptor genes, TLR1, TLR2a, TLR2b, TLR3, TLR5M, TLR5S, TLR7-9, TLR13a-c, TLR14, TLR21-23 in mandarin fish Siniperca chuatsi.

Toll-like receptors (TLRs), as a family of pattern recognition receptors (PRRs), possess specific pathogen-related molecular pattern (PAMP) recognition spectrum in inducing immune responses. In this study, sixteen TLRs were identified and characterized in mandarin fish (Siniperca chuatsi). All these TLRs consist of leucine-rich repeats (LRRs), a transmembrane domain and a Toll/interleukin-I receptor (TIR) domain, with the exception of TLR5S which lacks TIR domain, and they can be clustered into five branches, i.e. TLR1 subfamily, TLR3 subfamily, TLR5 subfamily, TLR7 subfamily and TLR11 subfamily in phylogenetic tree. These TLR genes were expressed in all tested tissues and had high expression levels in immune-related tissues such as head-kidney and spleen or mucosa-related tissues such as intestine and pyloric caecum. The transcripts of TLR2a, TLR2b, TLR3, TLR13a, TLR14, TLR22 and TLR23 were all significantly up-regulated after stimulation with poly(I:C); TLR1, TLR2a, TLR2b, TLR3, TLR5M, TLR5S, TLR13a and TLR13b transcripts were all significantly up-regulated after stimulation with PGN; and TLR2a, TLR2b, TLR5M, TLR5S, TLR7, TLR8, TLR9, TLR13c, TLR14 and TLR22 transcripts were all significantly up-regulated after stimulation with LPS in isolated head kidney lymphocytes of mandarin fish. The findings in this study may provide a valuable basis for functional study on TLR genes in mandarin fish.

Specific bioactivity of IL-22 in intestinal cells as revealed by the expression of IL-22RA1 in Mandarin fish, Siniperca chuatsi.

IL-22, a multifunctional cytokine, acts as an important regulator in host immunity in mammals. IL-22 homologues have been characterized in several species of fish, with its expression found in multiple tissues/cells in fish, but its target cells have not been fully analyzed. In the present research, different organ/tissue isolated cells were examined for the expression of IL-22 and the induced IL-22 responses in mandarin fish. The mandarin fish IL-22 was found to be expressed in all these tested cells with high basal expression in intestinal cells. The HKLs showed low basal expression but significant increase in expression of IL-22 after LPS treatment or bacterial infection. Only intestinal cells showed response to IL-22 by enhanced expression of hepcidin, LEAP2 and IL-22BP, with unresponsiveness observed in other tested cells, which indicated the cell-specificity of IL-22 bioactivity in mandarin fish. One of the heterodimeric receptor components for IL-22, the IL-22RA1, was cloned in mandarin fish, with four tandem fibronectin type III (FNIII) domains identified in its extracellular part. IL-22RA1 exhibited an intestinal cell-specific expression pattern, although another receptor component of IL-22, IL-10R2, displayed constitutive expressions in all these tested cells. The present study reveals that the mandarin fish IL-22 exhibits its bioactivity in a cell-specific manner in intestinal cells, which is reflected in the restrictive expression of its receptor unit, IL-22RA1.

In Primitive Zebrafish, MHC Class II Expression Is Regulated by IFN-γ, IRF1, and Two Forms of CIITA.

Mammalian CIITA isoforms are tightly regulated by independent promoters. These promotors are induced by IFN-γ through JAK-STAT signaling pathway. The induction of CIITA controls the expression of MHC class II (MHCII) and Ag presentation to the adaptive immune system. In the current study, to our knowledge, we first identified two independent promoters, p1 and p2, in the zebrafish (Danio rerio) that control the expression of the two variants of CIITA, CIITA variant 1 (CIITAv1), and CIITA variant 2 (CIITAv2), respectively. Moreover, although IRF1 in an IFN-γ signaling pathway induced CIITAv2, which has two ISRE motifs in its promoter, CIITAv1 expression was not induced by this signal. Further, the transcription of MHCII DAB was controlled by IRF1 via two distinct mechanisms: 1) the transcription of MHCII DAB was controlled by IRF1 indirectly through the two ISREs in p2; and 2) directly via the ISRE in MHCII DAB promoter. We also found that IRF1 associated with CIITAv1 and CIITAv2 via protein-protein interactions to synergistically drive the transcription of MHCII DAB. The IFN-γ-IRF1-CIITA-MHCII signaling cascade was functional in early life stages of CIITA-/- and IRF1-/- zebrafish. Our findings imply that the immune system develops early in fishes and that the IFN-γ signaling cascade-induced CIITA and MHCII DAB is conserved in teleost fishes and mammals.

Myxovirus resistance (Mx) gene and its differential expression regulated by three type I and two type II IFNs in mandarin fish, Siniperca chuatsi.

Interferons (IFNs) can induce the expression of IFN-stimulated genes (ISGs), such as myxovirus resistance (Mx) protein, to inhibit virus replication. In this study, the expression of Mx gene in mandarin fish, and the IFN-sensitive response elements (ISREs) and gamma-interferon activated sites (GASs) in the promoter of Mx gene were analyzed in relation to the stimulation of three distinct type I IFNs, IFNc, IFNd and IFNh, and two type II IFNs, IFN-γ and IFN-γ related molecule (IFN-γrel). A single Mx gene was found in mandarin fish, and its expression was highly and constitutively observed in all organs/tissues examined. The Mx gene was significantly induced in vivo for 120 h following infectious spleen and kidney necrosis virus (ISKNV) infection. Furthermore, the overexpression and recombinant of IFNh, IFNc, as well as IFN-γ can significantly induce Mx expression in MFF-1 cells at transcript and protein levels, although all the three type I IFNs and the two type II IFNs can activate the Mx promoter. In addition, ISRE1 which is the proximal one among the three predicted ISREs seems to be the important ISRE for the higher and efficient activation of the Mx promoter. However, the possible interaction between the GASs and type II IFN signalling molecules require further study.

Functional characterization of IL-10 and its receptor subunits in a perciform fish, the mandarin fish, Siniperca chuatsi.

Interleukin (IL)-10 is an immune-regulatory cytokine with multiple functions. In the current study, IL-10 and its two receptors, IL-10R1 and IL-10R2 were identified in mandarin fish, Siniperca chuatsi. The inhibitory effect of mandarin fish IL-10 was investigated on pro-inflammatory cytokine expression and the ligand-receptor relationship. This IL-10 possesses conserved cysteine residues, predicted α-helices and a typical IL-10 family signature motif, similar to its mammalian orthologue, and IL-10R1 harbours predicted JAK1 and STAT3 binding sites in the intracellular region. The fish IL-10 and IL-10R1 exhibit high expression levels in several immune-related organs/tissues, such as spleen, trunk kidney and head kidney, and IL-10R2 possesses a constitutive expression pattern. The expression of IL-10 shows significant increase in spleen from infectious spleen and kidney necrosis virus (ISKNV) infected mandarin fish, where the two receptors also exhibit different levels of induced expression. Mandarin fish IL-10 also exhibits significant response to the stimulation of LPS, PHA and PMA, with the two receptors exhibiting an interesting decrease in expression following the treatment of PMA. The pro-inflammatory cytokines, IL-6, IL-1ß, IL-8, TNF-α, show diminished up-regulation in LPS-stimulated splenocytes pre-incubated with IL-10, indicating the anti-inflammatory roles of mandarin fish IL-10. In EPC cells transfected with different combinations of receptors, IL-10 can enhance the expression of suppressor of cytokine signalling 3 (SOCS3) only when IL-10R1 and IL-10R2 are both expressed, suggesting the participation of the two receptors in signal transduction of mandarin fish IL-10. Similar results are observed with the usage of chimeric receptors, IL-10R1/CRFB1 and IL-10R2/CRFB5. Overall, mandarin fish IL-10 shares conserved ligand-receptor system and the prototypical inhibitory activities on pro-inflammatory cytokine expression with mammalian IL-10, implying the evolutionary conservation of this cytokine.

Receptor complex and signalling pathway of the two type II IFNs, IFN-γ and IFN-γrel in mandarin fish or the so-called Chinese perch Siniperca chuatsi.

IFN-γ, as the sole member of mammalian type II IFN, is a multifunctional cytokine which exerts its effects through two distinct IFN-γ receptors, IFNGR1 and IFNGR2. However, in teleost fish, another IFN-γ homologous gene, namely IFN-γ related gene (IFN-γrel), has been identified. Although IFN-γ and IFN-γrel genes have been described in some fish species, many important aspects remain poorly understood in relation with their signalling and function. In the present study, IFN-γ and IFN-γrel, as well as their receptors, cytokine receptor family B (CRFB) 17, CRFB13, two of which are homologous to IFNGR1 in mammals, and CRFB6, homolomous to IFNGR2, have been characterized in mandarin fish, Siniperca chuatsi. It was revealed that the two type IFN members exhibit antiviral activity, and IFN-γ transduces downstream signalling through CRFB13 and CRFB6, while IFN-γrel interacts with CRFB17 to activate downstream signalling. Moreover, IFN-γ and IFN-γrel have been shown to exert antiviral biological activity in a STAT1-dependent manner. Intracellular domain analysis of CRFB17 and CRFB13 demonstrated that the Y386 tyrosine residue of CRFB13 is required for the activation of the IFN-γ-mediated biologic response, and the Y324 and Y370 residues in CRFB17 are required to activate IFN-γrel signalling.

Functional characterization of interleukin (IL)-22 and its inhibitor, IL-22 binding protein (IL-22BP) in Mandarin fish, Siniperca chuatsi.

As an important immune regulatory molecule, interleukin (IL)-22 has been reported in several species of fish, but its soluble receptor, IL-22 binding protein (IL-22BP), discovered as a natural antagonist of IL-22 in mammals, has not been functionally characterized in fish to date. In the present study, IL-22 and IL-22BP genes were cloned in mandarin fish Siniperca chuatsi. They all exhibited a high basal expression level in mucosa-enriched tissues, implying their possible roles in mucosal immunity. The IL-22 was found to show a potent response to LPS stimulation, acting as an inducer of antimicrobial peptide (AMP) genes, such as hepcidin and Liver-expressed antimicrobial peptide-2 (LEAP-2) in intestinal cells. IL-22BP, via co-incubation with IL-22, inhibited completely the induction of downstream genes by IL-22. Through a yeast two-hybrid assay, the interaction between IL-22BP and IL-22 was confirmed, which may account for the inhibitory effect of IL-22BP. Moreover, two hot spot residues for IL-22 binding, as reported in mammalian IL-22BP, were found to be conserved both in sequence location and function in mandarin fish IL-22BP, indicating that the interaction mode between IL-22 and IL-22BP may be also conserved in fish and mammals. In conclusion, the mandarin fish IL-22 and IL-22BP are conserved in their interaction and function with their mammalian orthologues, and these findings provide basis for future research on IL-22-IL-22BP axis in fish immunity.

Functional, signalling and transcriptional differences of three distinct type I IFNs in a perciform fish, the mandarin fish Siniperca chuatsi.

Teleost fish are unique in having type I and type II interferons (IFNs) only, and the type I IFNs are classified into Group one and Group two based on the presence of two or four cysteines respectively, and are further classified into seven subgroups. In the present study, three distinct type I IFNs, IFNc, IFNd and IFNh, have been identified in the genome sequences of a perciform fish, the mandarin fish Siniperca chuatsi. These IFNs are induced following the stimulation of Polyinosinic polycytidylic acid (poly(I:C)) and Resiquimod (R848) either in vivo or in vitro. But, the infectious spleen and kidney necrosis virus (ISKNV) infection caused a delayed response of IFNs, which may be resulted from the viral inhibition of type I IFN production and related signalling. The three receptor subunits, cytokine receptor family B 1 (CRFB1), CRFB2 and CRFB5 are also expressed in a similar manner as observed for the IFNs, and IFNc, IFNd and IFNh use preferentially the receptor complex, CRFB2 and CRFB5, CRFB1 and CRFB5, CRFB1 and CRFB5 respectively for their effective signalling in the induction of IFN-stimulated genes (ISGs). Moreover, the IFNs are able to induce their own expression, and also the IRF3 and IRF7 expression, leading to the amplification of IFN cascade. It is further revealed that these three IFNs are transcribed differently by IRF7 and IRF3. The composition, function, signalling and transcription of type I IFNs have been investigated in detail in a teleost fish.

Composition and transcription of all interferon regulatory factors (IRFs), IRF1‒11 in a perciform fish, the mandarin fish, Siniperca chuatsi.

Interferon regulatory factors (IRFs) are a family of mediators in various biological processes including immune modulation of interferon (IFN) and proinflammatory cytokine expression. However, the data on the complete composition of IRFs is rather limited in teleost fish. In the present study, all IRF members, i.e. IRF1‒11 with two IRF4, IRF4a and IRF4b have been characterised in an aquaculture species of fish, the mandarin fish, Siniperca chuatsi, in addition to the previous report of IRF1, IRF2, IRF3 and IRF7 from the fish. These IRFs are constitutively expressed in various organs/tissues of the fish, and their expression can be induced following the stimulation of polyinosinic:polycytidylic acid (poly(I:C)) and the infection of infectious spleen and kidney necrosis virus (ISKNV), a viral pathogen of mandarin fish in aquaculture. The ISKNV infection induced the significant increase in the expression of some IRF genes, i.e. IRF2, IRF4a, IRF7, IRF9, IRF10 at 24 or 36 h post-infection (hpi) in spleen and head-kidney, and the significant increase of some other IRF genes, e.g. IRF1, IRF3, IRF4b, IRF5, IRF6, IRF8 at later stage of infection from 72, or 96, or even 120 hpi, which may imply the inhibitory effect of ISKNV on fish immune response. It is considered that the present study provides the first detailed analysis on all IRF members in an aquaculture species of fish, and can be served as the base for further investigation on the role of IRFs in teleost fish.

[Study on the composition and pharmacological function change of rhizoma polygonati before and after processing].

OBJECTIVE: To discuss the processing mechanism of Rhizoma Polygonati (RP) through studying the correlation between the change of composition and pharmacological function in raw and processed RP. METHODS: The extraction of petroleum ether, methylene dichloride, ethyl acetate and 1-butanol of the raw and processed RP were compared by HPLC. The compounds changed in processed RP in the methylene dichloride extraction were further identified with reference substances. The immune function of methylene dichloride extraction of raw and processed RP were compared. RESULTS: The changed compound in concentration was determined to be 5-Hydroxymethylfurfural. After processed, the concentration of 5-Hydroxymethylfurfural sharply increased. The carbon clearance index (P < 0.01) and coefficient of phagocytosis (P < 0.05) were increased remarkably by processed RP comparing to those of the normal saline and raw RP. CONCLUSION: The increase of immune function of processed RP may be related to increasing of concentration of 5-Hydroxymethylfurfural. The results provide a better understanding of RP processing.