Qing Chu

The genome of the miiuy croaker reveals well-developed innate immune and sensory systems

The miiuy croaker, Miichthys miiuy, is a representative Sciaenidae known for its exceptionally large otoliths. This species mainly inhabits turbid aquatic environments with mud to sandy mud bottoms. However, the characteristics of the immune system of this organism and its specific aquatic environment adaptations are poorly understood. Thus, we present a high-quality draft genome of miiuy croaker. The expansions of several gene families which are critical for the fish innate immune system were identified. Compared with the genomes of other fishes, some changes have occurred in the miiuy croaker sensory system including modification of vision and expansion of taste and olfaction receptors. These changes allow miiuy croaker to adapt to the environment during the long-term natural selection. The genome of miiuy croaker may elucidate its relatively well-developed immune defense and provide an adaptation model of the species thriving in turbid deep aquatic environments.

Discovery of toll-like receptor 13 exists in the teleost fish: Miiuy croaker (Perciformes, Sciaenidae).

Toll-like receptors (TLRs) play an indispensable role in the immune response for pathogen recognition and triggering not only innate immunity but also adaptive immunity. Here we report the TLR13 homologue, one member of TLRs, in Perciformes (especially Sciaenidae). And we used the miiuy croaker as represented species for further functional experiments. Former study reported the TLR13 only expressed in murine, and we are the first to report the teleost TLR13 (mmiTLR13). MmiTLR13 expressed highly in immune defense related tissues, such as the liver, spleen, and kidney, and Vibrio anguillarum or poly(I:C) infection showed the immune response of mmiTLR13. Further luciferase reporter assays showed the ability for activation of ISRE luciferase reporter, but it failed to active NF-κB. And further gene silence by short hairpin RNA (shRNA) confirmed the results. Immunofluorescence of mmiTLR13 presents the cytoplasmic distribution in Hela cell. In addition, the Toll/interleukin 1 receptor (TIR) domain of mammal TLR5 exhibits high identity with TLR13, which indicated the high homology between TLR5 and TLR13. These findings will lay the fundamental cornerstone for further research of teleost TLR13 and expand the horizon for better understand the teleost TLRs system.

Transcriptome comparative analysis revealed poly(I:C) activated RIG-I/MDA5-mediated signaling pathway in miiuy croaker.

Miiuy croker (Miichthys miiuy) as an important economical aquaculture species has challenged many more diseases caused by various pathogens recently. To better explore the immune response to virus, we have analyzed the transcriptome profiling of miiuy croaker challenged with poly(I:C) synthetic analog of virus dsRNA. We have obtained differentially expressed genes (DEGs) with up/down-relevant from comparison of the Ctrl and Poly transcriptome libraries. Through GO and KEGG enrichment analysis, immune-relevant DEGs whose expression are significantly rise or fall after challenged have been identified and classified. In order to detailedly analysis host immune response patterns for dsRNA virus, we have performed a map based on RIG-I/MDA5-mediated and TLR3-mediated signaling pathway which both induced type I IFNs response. In this pathway, both MDA5 and LGP2 are important RLRs in host surveillance against infection of dsRNA viruses and induce type I IFNs response which subsequently form a transcription factor complex ISGF3 that promote downstream genes referred to as ISGs to inhibits virus replication.

MicroRNA-3570 Modulates the NF-κB Pathway in Teleost Fish by Targeting MyD88

The inflammatory response, a protective process to clear detrimental stimuli, constitutes the defense against infectious pathogens. However, excessive inflammation disrupts immune homeostasis, which may induce autoimmune and inflammatory diseases. In this study, we report that microRNA (miR)-3570 plays a negative role in the bacteria-induced inflammatory response of miiuy croaker. Upregulation of miR-3570 by Vibrio anguillarum and LPS inhibits LPS-induced inflammatory cytokine production, thus avoiding an excessive inflammation response. Evidence showed that miR-3570 targets MyD88 and posttranscriptionally downregulates its expression. Overexpression of miR-3570 in macrophages suppresses the expression of MyD88, as well as its downstream signaling of IL-1R–associated kinases 1 and 4 and TNFR-associated factor 6. These results suggest that miR-3570 plays a regulatory in the bacteria-induced inflammatory response through the MyD88-mediated NF-κB signaling pathway by targeting MyD88.

Up-regulated of miR-8159-5p and miR-217-5p by LPS stimulation negatively co-regulate TLR1 in miiuy croaker.

ABSTRACT Toll‐like receptors (TLRs) are a group of pattern‐recognition receptors which play vital roles in ligand recognition and activation of the innate immune response. As an important member of TLRs family, TLR1 is mainly responsible for PAMPs from bacteria and play a pivotal role in sensing microbial products. Recent studies revealed that TLR1 could perceive LPS stimulation and transfer signals to activate the NF‐&kgr;B pathway, whereas ligands and signaling pathway of TLR1 are still unclear in fish. Growing evidence has shown that miRNAs (microRNAs) play as negative regulators in controlling the diverse of biophysical and biochemical processes at the post‐transcriptional level. In this study, we used a combination of bioinformatics and experimental techniques to exhibit that both miR‐8159‐5p and miR‐217‐5p were the direct negative regulators of TLR1 in miiuy croaker. Furthermore, dual‐luciferase reporter assays showed that combining miR‐8159‐5p and miR‐217‐5p exhibited a greater negative regulatory effect on TLR1 than only miR‐8159‐5p or miR‐217‐5p. Additionally, we also demonstrated that the expression of both the two miRNAs could be up‐regulated by LPS stimulation in either LPS‐stimulation spleen tissue or LPS‐treated cultured macrophage, which indicating that miR‐8159‐5p and miR‐217‐5p could be induced by LPS and may be as the negative regulators of TLR1 involved in the immune response to LPS stimulation. These results would enhance our understanding of the miRNA regulation in fish TLR signaling pathways. HighlightsTLR1 could response to LPS stimulation in miiuy croaker.miR‐8159‐5p and miR‐217‐5p negatively co‐regulated TLR1.Expression profiles of two miRNAs responding to LPS was conducted in vitro and in vivo.

A genome-wide survey of expansive NLR-C subfamily in miiuy croaker and characterization of the NLR-B30.2 genes

NOD-like receptors (NLRs) are essential intracellular pattern-recognition receptors that respond to pathogens and regulate innate immunity. NLRs include three distinct subfamilies: NLR-A, NLR-B and NLR-C, thereinto, NLR-C as a large subfamily is unique to bony fish and little research about it has been done. In the current study, we identified the members of NLR-B and NLR-C subfamilies containing 2 and 48 genes respectively in miiuy croaker. Compared with other teleosts except for zebrafish, NLR-C subfamily genes occurred expansion in miiuy croaker. The gene expansions of NLR-C subfamily may illustrate adaptive genome evolution in response to specific aquatic environments. Structural analysis showed that the N-terminus of NLR-C subfamily receptors has different characteristics of the domains including RING domain, FISNA domain or PYRIN domain. Interestingly, the C-terminus of 18 NLR-C subfamily members contains an extra B30.2 domain (named NLR-B30.2 genes) which plays an important role in antiviral immune recognition. Simultaneously, molecular evolutionary analysis indicated that the positively sites in miiuy croaker are mainly located in NACHT domain which was the vital region for signal transduction in immune response. Significantly, pathogens challenge in spleen and macrophages demonstrated that NLR-B30.2 genes exhibited more sensitive response to virus than bacteria, suggesting these genes play enhanced roles in innate antiviral immunity, which may represent a new family used for antiviral infection.

Comparative genomic evidence for duplication of TLR1 subfamily and miiuy croaker TLR1 perceives LPS stimulation via MyD88 and TIRAP

Being indispensable pattern recognition receptors in innate immune responses in host protection, Toll-like receptors (TLRs) play an important role in pathogen recognition. Fish TLRs exhibit high variety and distinct features, although little is known about their function on ligand recognition and signaling pathway in fish. This paper reports the evolutionary spectrum of the TLR1 subfamily (referred to as TLR1, TLR6, and TLR10) as determined using the comparative genomic approach. We hypothesized that the TLR1 subfamily underwent two rounds of gene duplication events; the first duplication occurred prior to the divergence of amphibians, and the second one occurred prior to the divergence of eutherians. To further study the function of fish TLR1, we identified miiuy croaker (Miichthys miiuy) TLR1 (mmiTLR1) and determined its potential ability to perceive Vibrio anguillarum and lipopolysaccharide stimulation. Data further suggested that mmiTLR1 is dependent on TIRAP and MyD88 for signal transmission. In addition, immunocytochemistry showed the speculative interaction between MyD88 and mmiTLR1 TIR domain. Overall, we systematically and comprehensively analyzed evolution of TLR1 subfamily and the function of mmiTLR1, which will provide the basis for future scientific research on fish TLRs.

The evolution and functional characterization of miiuy croaker cytosolic gene LGP2 involved in immune response

The laboratory of genetics and physiology 2 (LGP2) is a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors), which may participate in the immune regulation process. The role of LGP2 on modulating signaling was ambiguous, some researchers suggested that the regulation mechanism of LGP2 to melanoma differentiation-associated gene 5 (MDA5) and retinoic acid inducible gene-I (RIG-I) were different. In this study, the bioinformatics and functions of LGP2 from miiuy croaker (mmLGP2) were characterized. Comparative genomic analysis showed that the evolution of LGP2 in mammals was more conserved than it in fish. LGP2 contains three structural domains: ResIII, HelicaseC and RD, and ResIII structural domain of LGP2 was extremely conservative. The mmLGP2 was ubiquitously expressed in the tested miiuy croaker tissues and the expressions were significantly upregulated after stimulation with poly(I:C), indicating that LGP2 might participate in the immune response, especially antiviral immunity. Furthermore, immunofluorescence of miiuy croaker LGP2 presents in the cytoplasm in Hela cells. The overexpression of mmLGP2 can activate ISRE, but cannot activate NF-κB luciferase reporter, implying that mmLGP2 might act as a positive regulator in immune responses through activating ISRE to induce the expression of IFN. The research of mmLGP2 will enrich the information of fish LGP2, and the functional experiments will be helpful for the future research about fish immune systems.

IRF9 as a negative regulator involved in TRIF-mediated NF-κB pathway in a teleost fish, Miichthys miiuy

&NA; Proinflammatory cytokines and type I IFNs were produced by TLR signaling and these responses are crucial for host defensive responses against pathogens. In order to avoid harmful and inappropriate inflammatory responses, there are multiple mechanisms to negatively regulate TLR signaling. In this paper, we have firstly studied IRF9 functions as a negative regulator involved in TRIF‐mediated NF‐&kgr;B pathway. Moreover, we found inhibitory effect of IRF9 primary depends on DBD domain. Interestingly, we also demonstrated that else mutants of IRF9, except for IRF9‐&Dgr;DBD, have different inhibitory effects upon TRIF‐mediated NF‐&kgr;B pathway. This study provides a novel evidence about the negatively regulation of innate immune signaling pathway in teleost fish. In addition, this finding provides new insights into the regulatory mechanism in mammals.

Genome-guided transcriptome analysis of miiuy croaker provides insights into pattern recognition receptors and cytokines in response to Vibrio anguillarum

&NA; Miiuy croaker (Miichthys miiuy), as an economically important marine fish is affected by numerous bacterial diseases. Infection by bacterial pathogen Vibrio anguillarum causes high mortality and great economic loss in aquaculture. To understand the immune response of the miiuy croaker to V. anguillarum infection, we analyzed the transcriptomic profile of V. anguillarum‐challenged M. miiuy, and two cDNA libraries, namely, the normal library (Ctrl) and V. anguillarum challenged library (Van) were constructed. Combined with the whole genome‐guided assembly, total 47,971 unigenes were acquired. Moreover, 2482 significantly differentially expressed unigenes were identified based on the expression patterns. Immune‐related differentially expressed genes (DEGs) that were significantly up‐ or down‐regulated after V. anguillarum injection were identified via enrichment analysis using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. To further understand the expression profiles that underlie the response to V. anguillarum infection, we investigated the DEGs that are related to pattern‐recognition receptors and cytokines. The expression patterns of relevant DEGs were validated by qRT‐PCR. Additionally, the expression profiles of DEGs that are related to pattern‐recognition receptors and cytokines were further measured in miiuy croaker macrophages. This study is the first to identify and characterize the transcriptome of miiuy croaker in response to V. anguillarum infection. The results expand the general understanding of the immune defense mechanisms of miiuy croaker and provide helpful information for counteracting V. anguillarum infection. Highlights2482 DEGs were identified within 1313 up‐regulated unigenes.Immune‐related DEGs were identified and classified.DEGs related to PPRs and cytokines were analyzed in detail.