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A nine-week feeding trial was conducted to comprehensively investigate the effects of different levels of dietary lipid on intestinal physiology of juvenile turbot. Three diets with different lipid levels (8%, 12% and 16%) were formulated, which were designated as the low-lipid group (LL), medium-lipid group (ML) and high-lipid group (HL), respectively. Each diet was fed to six replicate tanks, and each tank was stocked with 35 fish. The results revealed that medium dietary lipid (12%) increased the activities of intestinal digestive enzymes and brush border enzymes. Excessive dietary lipid (16%) decreased the intestinal antioxidative enzyme levels and increased the lipid peroxidation pressure. In addition, HL stimulated the occurrence of intestinal inflammation and significantly up-regulated the mRNA expression level of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β). Dietary LL and HL induced the apoptosis of intestinal epithelial cells. Sequencing of bacterial 16 s rRNA V4 region indicated that the abundance and diversity of intestinal microflora in fish fed with medium lipid diet (12%) were significantly higher than those in other groups, indicating the intestinal microflora ecology in group ML was more balanced. MetaStat analysis indicated that both low- and high-lipid diets significantly reduced the relative abundance of intestinal beneficial bacteria. In conclusion, results of this study demonstrated the sensitivity of intestinal health and microbiota to dietary lipid levels. From the perspective of microecological balance, medium dietary lipid (12%) was more conducive to maintaining the intestinal microflora stability of turbot., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Interleukin-17 (IL-17) is a cytokine secreted by a variety of immune cells that plays an important role in host defense against pathogens. IL-17 usually activates downstream immune signaling pathway by binding to heterodimeric or homodimeric complex formed by IL-17 receptors (IL-17R). Describing the characteristics, tissue distribution of IL-17 and IL-17 receptor family members and their expression after pathogen infection will provide a reference for host defense against disease of turbot. In this study, six IL-17 family members and nine IL-17 receptor family members were identified by analyzing the turbot (Scophthalmus maximus) genome. Different from other vertebrates, most members of the IL-17 receptor family own two copies. Protein structure analysis showed that the six IL-17 family members contained typical "IL-17" domains, and the nine IL-17 receptor family members contained typical "SEFIR domain" or "IL17_R_N domain". Syntenic analysis revealed that all IL-17s and IL-17Rs were chromosomally conserved compared with other fish. The phylogenetic analysis further confirmed the evolutionary conservatism of different copies of IL-17C and IL-17Rs. Tissue distribution results showed that IL-17 and IL-17R genes were highly expressed in immune-related tissues. The expression of IL-17C and its receptor in the mucosal immune tissues after infection with V. anguillarum were analyzed subsequently, which were significantly increased in the skin. The results are consistent with previous studies showing that IL-17 and IL-17 receptor play an important role in promoting innate immune response., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In mammals, Class II, major histocompatibility complex (MHC II) transactivator (CIITA) recognizes microbial pathogens and triggers immune responses. In Chinese tongue sole Cynoglossus semilaevis, Cs-CIITA was prevalently expressed in various tissues. Cs-CIITA, Cs-MHC IIA and Cs-MHC IIB were expressed significantly higher in skin in susceptible families infected with Vibrio harveyi, while higher expression of Cs-CIITA and Cs-MHC IIB was examined in liver in resistant families. In addition, the three genes were up-regulated in gill, skin, intestine, liver, spleen and kidney at 48 h or 72 h after V. harveyi infection. Furthermore, the three genes were co-expressed in the epithelial mucous cells of gill, skin, and intestine. Knockdown of Cs-CIITA regulates the expression of other inflammation-related genes, including CD40, IL-1β, IL-8, RelB, NFκB, and Myd88. These results suggest that CIITA functions in the inflammatory responses of C. semilaevis against V. harveyi, via MHC II transcriptional regulation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Host protective inflammatory caspase activity must be tightly regulated to prevent pathogens infection, however, the inflammatory caspase-engaged inflammasome activation in teleost fish remains largely unknown. In this study, we reveal a bifurcated evolutionary role of the inflammatory caspase in mediating both non-canonical and canonical inflammasome pathways in teleost fish. Through characterization of a unique inflammatory SmCaspase from the teleost Scophthalmus maximus (turbot), we found it can directly recognize cytosolic lipopolysaccharide (LPS) via its N-terminal CARD domain, resulting in caspase-5-like proteolytic enzyme activity-mediated pyroptosis in Turbot Muscle Fibroblasts. Interestingly, we also found that this inflammatory caspase can be recruited to SmNLRP3-SmASC to form the NLRP3 inflammasome complex, engaging the SmIL-1β release in Head Kidney-derived Macrophages. Consequently, the SmCaspase activation can recognize and cleave the SmGSDMEb to release its N-terminal domain, mediating both pyroptosis and bactericidal activities. Furthermore, the SmCaspase-SmGSDMEb axis-gated pyroptosis governs the bacterial clearance and epithelial desquamation in fish gill filaments in vivo. To our knowledge, this study is the first to identify an inflammatory caspase acting as a central coordinator in NLRP3 inflammasome, as well as a cytosolic LPS receptor; thus uncovering a previously unrecognized function of inflammatory caspase in turbot innate immunity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Long noncoding RNAs (lncRNAs) play a multifaceted role in transcriptional regulation and are important regulators of immune function. Scarce information is available regarding lncRNAs in fish. Peripheral blood mononuclear cells participate in the immune response of fish and aid resistance to infection with pathogenic microorganisms. Chitosan oligosaccharide can improve cellular and humoral immunity to enhance disease resistance in fish. In this study, we obtained peripheral blood leukocytes from half-smooth tongue sole and studied the effect of chitosan oligosaccharide on the lncRNA-mRNA expression profile of these cells using high-throughput sequencing and bioinformatics techniques. A total of 609 differentially expressed mRNAs and 50 differentially expressed lncRNAs were identified. The GO term enrichment analysis of the differentially expressed genes was annotated by 220 GO terms, 137 biological processes, 18 cellular components, and 65 molecular functions. Sixteen KEGG pathways, including immune signaling pathways, metabolism, and genetic information processing, were significantly enriched in differentially expressed genes. Thirty-six differentially expressed lncRNAs and 32 differentially expressed mRNAs produced a coexpression network containing 90 relationship pairs. The prediction of lncRNA target genes revealed 244 lncRNAs that potentially cis-regulated 294 differentially expressed mRNAs. qPCR verified that the expression levels of 17 differentially expressed lncRNAs and 15 differentially expressed mRNAs were consistent with the RNA-Seq results. Among them, 6 lncRNAs and 7 mRNAs were differentially expressed genes obtained from the prediction and analysis of lncRNA target genes, and 8 lncRNAs and 4 mRNAs were differentially expressed genes that participated in the construction of the coexpression network. In peripheral blood leukocytes after chitosan oligosaccharide treatment, as well as in peripheral blood and spleen after Vibrio anguillarum stimulation, lncRNAs and mRNAs showed significant differential expression. The results indicated that they may be related to the immune response, providing novel reference information for further research on the role of lncRNAs in immune regulation in half-smooth tongue sole., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The cargo of exosomes contains proteins with various functions, which might be promising biomarkers for disease diagnosis and prognosis. To explore the impact of the Vibrio harveyi pathogen on Cynoglossus semilaevis from a different perspective and develop promising biomarkers for infection, the exosomes from epidermal mucus of healthy controls（EC）and sick fish（ES）were extracted and identified, coupled with proteomic screening through iTRAQ followed with LC-MS/MS. 1531 credible proteins were obtained relating to structural, metabolic and immunological functions. 359 different expressed proteins (DEPs) (FC > 2 or FC < 0.5) were found, with 161 up-regulated and 198 down-regulated in ES. Based on the database of C. semilaevis on Uniprot, 71 proteins were characterized as concrete names, including 19 up-regulated proteins and 52 down-regulated proteins, and were selected as subjects for further studies. Ferritin, Toll-like receptor 5S protein and Calcium-transporting ATPase were upregulated, while Histone H2B and Eukaryotic translation initiation factor 5A were downregulated, consistent with the expression levels of related mRNAs in skin tissue verified by qRT-PCR. The integrated analysis between miRomics and proteomics also provided possible regulatory relationships mediated by mucous exosomes during infection. The signature proteins in mucosal exosomes could make sense in the explanation of the infection defending mechanism and the development of biomarkers which can differentiate diseased and healthy C. semilaevis individuals., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The NOD-like receptor X1 (NLRX1) is a member of highly conserved nucleotide-binding domain (NBD)- and leucine-rich-repeat (LRR)-containing family (known as NLR), that localizes to the mitochondrial outer membrane and regulate the innate immunity by interacting with mitochondrial antiviral-signaling protein (MAVS). As one of cytoplasmic PRRs, NLRX1 plays key roles for pathogen recognition, autophagy and regulating of subsequent immune signaling pathways. In this study, we identified the nlrx1 in turbot as well as its expression profiles in mucosal surfaces following bacterial infection. In our results, the full-length nlrx1 transcript consists of an open reading frame (ORF) of 4,886 bp encoding the putative peptide of 966 amino acids. The phylogenetic analysis revealed the SmNlrx1 showed the closest relationship to Cynoglossus semilaevis. In addition, the Nlrx1 mRNA expression could be detected in all the examined tissues, with the most abundant expression level in head kidney, and the lowest expression level in liver. Moreover, Nlrx1 showed similar expression patterns following Vibrio anguillarum and Streptococcus iniae infection, that were both significantly up-regulated following challenge, especially post S. iniae challenge. Finally, fluorescence microscopy unveiled that the SmNlrx1 localized to mitochondria in HEK293T by N-terminal mitochondrial targeting sequence. Characterization of Nlrx1 might have an important implication in bioenergetic adaptation during metabolic stress, oncogenic transformation and innate immunity and will probably contribute to the development of novel intervention strategies for farming turbot., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Interleukin (IL)-10, an immune-regulatory cytokine, exerts various biological functions through interaction with IL-10 receptors. In teleost, very limited functional studies on IL-10 receptors have been documented. In this study, we reported the expression patterns of IL-10 receptor 1 (CsIL-10R1) and receptor 2 (CsIL-10R2) of tongue sole (Cynoglossus semilaevis) and examined their biological properties. The expression of CsIL-10R1 and CsIL-10R2 occurred in multiple tissues and were regulated by bacterial challenge. In vitro binding studies showed that recombinant extracellular region of CsIL-10R1 (rCsIL-10R1ex) rather than rCsIL-10R2ex could bind with rCsIL-10. Cellular study showed that both CsIL-10R1 and CsIL-10R2 were expressed on peripheral blood leukocytes (PBLs), and blockade of CsIL-10R1 or CsIL-10R2 by antibody could reduce inhibitory effect of CsIL-10 on ROS production of PBLs. When injected in vivo, anti-rCsIL-10R1 or anti-rCsIL-10R2 antibody dramatically promoted the expression of proinflammatory cytokines and suppressed bacterial dissemination in tongue sole tissues. Consistently, the overexpression of CsIL-10R1 or CsIL-10R2 significantly enhanced bacterial dissemination, and the overexpression of CsIL-10R1M bearing STAT3 site mutation reduced bacterial dissemination. Overall, these results demonstrate for the first time teleost IL-10 receptors play a negative role in antibacterial immunity and add insight into the function of CsIL-10 receptors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This report describes the cloning, characterization and gene expression pattern of two Th17 cytokines, interleukin (IL)-17A/F and -22, in turbot Scophthalmus maximus. The turbot IL-17A/F cDNA contains a 516 bp open reading frame encoding a deduced IL-17A/F protein of 171 amino acid (aa) residues, containing a predicted signal peptide of 31 aa. Turbot IL-22 had a 564 bp ORF coding for a 187 aa protein with a 33 aa signal peptide. The turbot IL-22 protein contained a typical IL-10 family signature. Both cytokines had highest expression levels in the intestine followed by head kidney and gills. Stimulation with the Gram negative bacterium Aeromonas salmonicida was able to modulate IL-17A/F and IL-22 expression in head kidney, spleen and liver but not the intestine. PMA and PHA were also able to induce the expression of both cytokines, suggesting that, as expected, T-cells are likely the main producers of these molecules in turbot as in mammals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Chemokines are small cytokines that, based on their structural differences, are classified into four groups, one of which is called CXC chemokines. In this study, we identified a CXC chemokine, CsCXCe1, from half-smooth tongue sole (Cynoglossus semilaevis) and analyzed its function. The deduced amino acid sequence of CsCXCe1 contains 115 residues and is phylogenetically distinct from known CXC chemokines. CsCXCe1 possesses the conserved RCXC motif in the form of RCWC but lacks the ELR sequence that is found in some CXC chemokines. Expression of CsCXCe1 as determined by quantitative real time RT-PCR occurred abundantly in immune organs and was upregulated by bacterial and viral infection in time dependent manners. Purified recombinant CsCXCe1 (rCsCXCe1) exhibited comparable chemotactic activities against tongue sole and turbot (Scophthalmus maximus) peripheral blood leukocytes (PBL). Microscopic analysis identified lymphocytes as the major cellular population in PBL that responds to rCsCXCe1. Mutational study showed that when the two cysteine residues in the RCWC motif of CsCXCe1 were substituted by serine, the chemoattractive activity of CsCXCe1 was completely lost. Further study showed that treatment of PBL with rCsCXCe1 (i) stimulated cellular proliferation and respiratory burst activity, (ii) upregulated the expression of a wide spectrum of immune relevant genes, and (iii) enhanced cellular resistance against bacterial infection. Taken together, these results indicate that CsCXCe1 is likely a new type of CXC chemokine that exerts chemotactic and immunostimulatory effects on PBL., (Copyright © 2011 Elsevier Ltd. All rights reserved.)