Dan-Dan Chen

IFN Regulatory Factor 10 Is a Negative Regulator of the IFN Responses in Fish

IFN regulatory factor (IRF) 10 belongs to the IRF family and exists exclusively in birds and fish. Most IRFs have been identified as critical regulators in the IFN responses in both fish and mammals; however, the role of IRF10 is unclear. In this study, we identified IRF10 in zebrafish (Danio rerio) and found that it serves as a negative regulator to balance the innate antiviral immune responses. Zebrafish IRF10 (DrIRF10) was induced by intracellular polyinosinic:polycytidylic acid in ZF4 (zebrafish embryo fibroblast-like) cells. DrIRF10 inhibited the activation of zebrafish IFN1 (DrIFN1) and DrIFN3 promoters in epithelioma papulosum cyprinid cells in the presence or absence of polyinosinic:polycytidylic acid stimulation through direct interaction with the IFN promoters, and this inhibition was also shown to block IFN signaling. Overexpression of DrIRF10 was able to abolish the induction of DrIFN1 and DrIFN3 mediated by the retinoic acid–inducible gene I–like receptors. In addition, functional domain analysis of DrIRF10 showed that either the DNA binding domain or the IRF association domain is sufficient for its inhibitory activity for IFN signaling. Lastly, overexpression of DrIRF10 decreased the transcription level of several IFN-stimulated genes, resulting in the susceptibility of host cells to spring viremia of carp virus infection. Collectively, these data suggest that DrIRF10 inhibits the expression of DrIFN1 and DrIFN3 to avoid an excessive immune response, a unique regulation mechanism of the IFN responses in lower vertebrates.

Distinctive Structural Hallmarks and Biological Activities of the Multiple Cathelicidin Antimicrobial Peptides in a Primitive Teleost Fish

Cathelicidin antimicrobial peptides (CAMPs) represent a crucial component of the innate immune system in vertebrates. Although widely studied in mammals, little is known about the structure and function of fish CAMPs. Further to the previous findings, two more cathelicidin genes and multiple transcripts from rainbow trout were identified in the present study. Interestingly, we found that trout have evolved energy-saving forms of cathelicidins with the total deletion of the characteristic cathelin-like domain. Sequence analysis revealed that salmonid CAMPs have formed a special class of antimicrobial peptides in vertebrates with three distinctive hallmarks: the N terminus is intensified by positive charges, the central region consists of repetitive motifs based on RPGGGS, and the C terminus is lowly charged. Immunofluorescence localization of trout CAMPs demonstrated that these peptides expressed mainly at the mucosal layer of gut. Meanwhile, signals around sinusoids were also detected in head kidney. Moreover, the biological activities of trout CAMPs were proved to be mediated by the N terminus. Additionally, the repetitive motifs characteristically existing in Salmonidae increased the structural flexibilities of peptides and further increased the antibacterial and IL-8–stimulating activities. Unlike most α helical and cytotoxic mammalian CAMPs, trout CAMPs, mainly consisting of β-sheet and random coil, exhibited no cytotoxic activities. The distinctive structural features of trout CAMPs provide new insights into the understanding of the evolution of CAMPs in vertebrates. Moreover, the high bacterial membrane selectivity of trout CAMPs will help to design excellent peptide antibiotics.

Spring Viremia of Carp Virus N Protein Suppresses Fish IFNφ1 Production by Targeting the Mitochondrial Antiviral Signaling Protein

For a virus to replicate efficiently, it must try and inhibit host IFN expression because IFN is an important host defense at early stages after viral infection. For aquatic viruses, the mechanisms used to escape the hosts IFN system are still unclear. In this study, we show that the N protein of spring viremia of carp virus (SVCV) inhibits zebrafish IFNφ1 production by degrading the mitochondrial antiviral signaling protein (MAVS). First, the upregulation of IFNφ1 promoter activity stimulated by polyinosinic:polycytidylic acid, retinoic acid–inducible gene I (RIG-I) or MAVS was suppressed by the SVCV infection. However, the upregulation by the downstream factor of the RIG-I–like receptor signaling pathway, TANK-binding kinase 1, was not affected. Notably, at the protein level, MAVS decreased remarkably when cells were infected with SVCV. Second, consistent with the result of the SVCV infection, overexpression of the N protein of SVCV blocked the IFNφ1 transcription activated by MAVS and downregulated MAVS expression at the protein level but not at the mRNA level. Further analysis demonstrated that the N protein targeted MAVS for K48-linked ubiquitination, which promoted the degradation of MAVS. These data indicated that fish MAVS could be degraded by the N protein of SVCV through the ubiquitin-proteasome pathway. To our knowledge, this is the first article of a fish RIG-I–like receptor pathway interfered by an aquatic virus in an ubiquitin-proteasome manner, suggesting that immune evasion of a virus also exists in lower vertebrates.

Fish gut-liver immunity during homeostasis or inflammation revealed by integrative transcriptome and proteome studies

The gut-associated lymphoid tissue, connected with liver via bile and blood, constructs a local immune environment of both defense and tolerance. The gut-liver immunity has been well-studied in mammals, yet in fish remains largely unknown, even though enteritis as well as liver and gallbladder syndrome emerged as a limitation in aquaculture. In this study, we performed integrative bioinformatic analysis for both transcriptomic (gut and liver) and proteomic (intestinal mucus and bile) data, in both healthy and infected tilapias. We found more categories of immune transcripts in gut than liver, as well as more adaptive immune in gut meanwhile more innate in liver. Interestingly reduced differential immune transcripts between gut and liver upon inflammation were also revealed. In addition, more immune proteins in bile than intestinal mucus were identified. And bile probably providing immune effectors to intestinal mucus upon inflammation was deduced. Specifically, many key immune transcripts in gut or liver as well as key immune proteins in mucus or bile were demonstrated. Accordingly, we proposed a hypothesized profile of fish gut-liver immunity, during either homeostasis or inflammation. Current data suggested that fish gut and liver may collaborate immunologically while keep homeostasis using own strategies, including potential unique mechanisms.

The P Protein of Spring Viremia of Carp Virus Negatively Regulates the Fish Interferon Response by Inhibiting the Kinase Activity of TANK-Binding Kinase 1

ABSTRACT Spring viremia of carp virus (SVCV) is an efficient pathogen causing high mortality in the common carp. Fish interferon (IFN) is a powerful cytokine enabling host cells to establish an antiviral response; therefore, the strategies that SVCV uses to avoid the cellular IFN response were investigated. Here, we report that the SVCV P protein is phosphorylated by cellular TANK-binding kinase 1 (TBK1), which decreases IFN regulatory factor 3 (IRF3) phosphorylation and suppresses IFN production. First, overexpression of P protein inhibited the IFN promoter activation induced by SVCV and the IFN activity activated by the mitochondrial antiviral signaling protein (MAVS) although TBK1 activity was not blocked by P protein. Second, P protein colocalized and interacted with TBK1. Dominant negative experiments suggested that the TBK1 N-terminal kinase domain interacted with P protein and was essential for P protein and IRF3 phosphorylation. Finally, P protein overexpression reduced the IRF3 phosphorylation activated by TBK1 and reduced host cellular ifn transcription. Collectively, our data demonstrated that the SVCV P protein is a decoy substrate for the host phosphokinase TBK1, preventing IFN production and facilitating SVCV replication. IMPORTANCE TBK1 is a pivotal phosphokinase that activates host IFN production to defend against viral infection; thus, it is a potential target for viruses to negatively regulate IFN response and facilitate viral evasion. We report that the SVCV P protein functions as a decoy substrate for cellular TBK1, leading to the reduction of IRF3 phosphorylation and suppression of IFN expression. These findings reveal a novel immune evasion mechanism of SVCV.

Identification and characterization of Bacillus subtilis from grass carp (Ctenopharynodon idellus) for use as probiotic additives in aquatic feed

Bacillus subtilis is widely used as probiotic species in aquaculture for water quality control, growth promoting, or immunity enhancing. The aim of this study is to find novel B. subtilis strains from fish as potential probiotics for aquaculture. Eleven B. subtilis isolates derived from the intestinal tract of grass carp were identified by gene sequencing and biochemical tests. These isolates were classified into 4 groups, and the representatives (GC-5, GC-6, GC-21 and GC-22) of each group were further investigated for antibiotic susceptibility, sporulation rate, biofilm formation, activity against pathogenic bacteria, resistance to stress conditions of intestinal tract (high percentage of bile and low pH) and high temperature, which are important for probiotics to be used as feed additives. Additionally, the adhesion properties of the 4 characterized strains were assessed using Caco-2 cell and gut mucus models. The results showed that the 4 strains differed in their capacities to adhere to intestinal epithelial cells and mucus. Furthermore, the strains GC-21 and GC-22 up-regulated the expression levels of IL-10 and TGF-β but down-regulated IL-1β, suggesting their potential anti-inflammatory abilities. Based on physiological properties of the 4 characterized B. subtilis strains, one or more strains may have potential to be used as probiotics in aquaculture.

B Cell Functions Can Be Modulated by Antimicrobial Peptides in Rainbow Trout Oncorhynchus mykiss: Novel Insights into the Innate Nature of B Cells in Fish

B cells in fish were recently proven to have potent innate immune activities like macrophages. This inspired us to further explore the innate nature of B cells in fish. Moreover, antimicrobial peptides (AMPs) are representative molecules of innate immunity, and they can modulate the functions of macrophages. These make fish an appropriate model to study the interactions between B cells and AMPs. Interestingly, the results in this study revealed that the IgM+ and IgT+ B cells of rainbow trout could express multiple AMP genes, including four cathelicidin genes and one β-defensin gene. The expression levels of the cathelicidin genes were obviously higher than that of the β-defensin gene. Further studies revealed that intracellular, extracellular, in vitro, and in vivo stimulations could significantly increase the expression of the cathelicidin genes in trout IgM+ and IgT+ B cells but not the expression of the β-defensin gene, indicating that cathelicidin peptides are the main innate immune effectors of trout B cells. More interestingly, we found that cathelicidin peptides could significantly enhance the phagocytic, intracellular bactericidal, and reactive oxygen species activities of trout IgM+ and IgT+ B cells, a phenomenon previously reported only in macrophages, and these activities might also be mediated by the P2X7 receptor. These results collectively suggest that B cells play multiple roles in the innate immunity of fish, and they provide new evidence for understanding the close relationship between B cells and macrophages in vertebrates.

Molecular characterization and expression analysis of three subclasses of IgT in rainbow trout (Oncorhynchus mykiss).

ABSTRACT As the teleost specific immunoglobulin, IgT plays important roles in systemic and mucosal immunity. In the current study, in rainbow trout, we have cloned the heavy chain (Ig&tgr;) genes of a secretory form of IgT2 as well as the membrane and secretory forms of a third IgT subclass, termed IgT3. Conserved cysteine and tryptophan residues that are crucial for the folding of the immunoglobulin domain as well as hydrophobic and hydrophilic residues within CART motif were identified in all IgT subclasses. Through analysis of the rainbow trout genome assembly, Ig&tgr;3 gene was found localized upstream of Ig&tgr;1 gene, while Ig&tgr;2 gene situated on another scaffold. At the transcriptional level, Ig&tgr;1 was mainly expressed in both systemic and mucosal lymphoid tissues, while Ig&tgr;2 was largely expressed in systemic lymphoid organs. After LPS and poly (I:C) treatment, Ig&tgr;1 and Ig&tgr;2 genes exhibited different expression profiles. Interestingly the transcriptional level of Ig&tgr;3 was negligible, although its protein product could be identified in trout serum. Importantly, a previously reported monoclonal antibody directed against trout IgT1 was able to recognize IgT2 and IgT3. These data demonstrate that there exist three subclasses of IgT in rainbow trout, and that their heavy chain genes display different expression patterns during stimulation. Overall, our data reflect the diversity and complexity of immunoglobulin in trout, thus provide a better understanding of the IgT system in the immune response of teleost fish. HIGHLIGHTSThe heavy chain gene of a third IgT subclass (Ig&tgr;3) is characterized in rainbow trout.Trout Ig&tgr; genes are located probably at two Ig loci.Trout Ig&tgr; genes respond differentially to stimulants.The anti‐trout IgT1 mAb recognizes all of the three IgT subclasses.

Fish IRF6 is a positive regulator of IFN expression and involved in both of the MyD88 and TBK1 pathways

Interferon (IFN) regulatory factors (IRF) are the crucial transcription factors for IFN expression, leading host cell response to viral infection. In mammals, only IRF6 is unaffected by IFN expression in the IRF family; however, in fish, a lower vertebrate, whether IRF6 is related to IFN regulation is unclear. In this study, we identified that zebrafish IRF6 was a positive regulator of IFN transcription and could be phosphorylated by both MyD88 and TBK1. First, the transcript level of cellular irf6 was upregulated by treatment with poly I:C (a mimic of viral RNAs), indicating IRF6 might be involved in the process of host cell response to viruses. Overexpression of IRF6 could upregulate IFN promoter activity significantly, meaning IRF6 is a positive regulator of IFN transcription. Subsequently, at the protein regulation level and in the interaction relationship, IRF6 was phosphorylated by and associated with both MyD88 and TBK1. In addition, overexpression of IRF6 activated the transcription of isg15, rig-i and mavs of host cells; meanwhile, the transcripts of p, m and n genes of SVCV were significantly declined in IRF6-overexpressing cells. Taken together, our data demonstrate that fish IRF6 is distinguished from the homolog of mammals by being a positive regulator of IFN transcription and phosphorylated by MyD88 and TBK1, suggesting that differences in the IRF6 regulation pattern exist between lower and higher vertebrates.

Preferential combination between the light and heavy chain isotypes of fish immunoglobulins

Immunoglobulin light chain (IgL) is necessary for the assembly of an Ig molecule, which plays important roles in the immune response. IgL genes were identified in various teleost species, but the basic functions of different IgL isotypes and the preferential combination between IgL and IgH (Ig heavy chain) isotypes remain unclear. In the current study, by EST database searching and cDNA cloning in rainbow trout, 8 IgL sequences were obtained, which could be classified into the IgLκF, IgLκG, IgLσ and IgLλ isotypes, respectively. Trout IgL isotypes were highly expressed in the immune-related tissues, and participated in the immune responses in spleen and gut by stimulation with LPS and poly (I:C). The results of FACS and LC-MS/MS indicated that the IgLκG and IgLσ isotypes preferentially bonded with the heavy chains of IgM and IgT, respectively, in trout B cells and serum. In addition, the genomic organization of trout IgL isotypes and the utilization of recombination signal sequences were studied.