Intelectin enhances the phagocytosis of macrophages via CDC42-WASF2-ARPC2 signaling axis in Megalobrama amblycephala.

Intelectin has been identified in various vertebrates and plays an important role in the host immune system. In our previous studies, recombinant Megalobrama amblycephala intelectin (rMaINTL) protein with excellent bacterial binding and agglutination activities enhances the phagocytic and killing activities of macrophages in M. amblycephala; however, the underlying regulatory mechanisms remain unclear. The present study showed that treatment with Aeromonas hydrophila and LPS induced the expression of rMaINTL in macrophages, and its level and distribution in macrophages or kidney tissue markedly increased after incubation or injection with rMaINTL. The cellular structure of macrophages was significantly affected after incubation with rMaINTL, resulting in an increased surface area and pseudopodia extension, which might contribute to enhancing the phagocytic ability of macrophages. Then, digital gene expression profiling analysis of the kidneys from rMaINTL-treated juvenile M. amblycephala identified some phagocytosis-related signaling factors that were enriched in pathways involved in the regulation of the actin cytoskeleton. In addition, qRT-PCR and western blotting verified that rMaINTL upregulated the expression of CDC42, WASF2, and ARPC2 in vitro and in vivo; however, the expression of these proteins was inhibited by a CDC42 inhibitor in macrophages. Moreover, CDC42 mediated the promotion of rMaINTL on actin polymerization by increasing the F-actin/G-actin ratio, which led to the extension of pseudopodia and remodeling of the macrophage cytoskeleton. Furthermore, the enhancement of macrophage phagocytosis by rMaINTL was blocked by the CDC42 inhibitor. These results suggested that rMaINTL induced the expression of CDC42 as well as the downstream signaling molecules WASF2 and ARPC2, thereby facilitating actin polymerization to promote cytoskeletal remodeling and phagocytosis. Overall, MaINTL enhanced the phagocytosis activity of macrophages in M. amblycephala via activation of the CDC42-WASF2-ARPC2 signaling axis.

Molecular characterization and expression patterns of CXCL8 gene from blunt snout bream (Megalobrama amblycephala) and its chemotactic effects on macrophages and neutrophils.

CXCL8 is a typical CXC-type chemokine, which mediates the migration of immune cells from blood vessels to the site of inflammation or injury to clear pathogenic microorganisms and repair damaged tissues. In this study, Megalobrama amblycephala CXCL8 (MaCXCL8) gene was identified and characterized. Sequence analysis showed that the deduced MaCXCL8 protein possessed the typical structure of CXCL8 from other species, with the characteristic CXC cysteine residues in the N-terminal and accompanied by a DLR motif (Asp-Leu-Arg motif). Phylogenetic analysis revealed that MaCXCL8 was homologous to that of Ctenopharyngodon idella and other cyprinid fishes. MaCXCL8 gene was expressed in all detected healthy tissues, with the highest expression levels in the spleen, and its expression was significantly up-regulated upon the challenge of Aeromonas hydrophila and Lipopolysaccharide (LPS) both in juvenile M. amblycephala tissues and primary macrophages. The immunohistochemical assay showed that MaCXCL8 was mainly distributed in the nucleus and cytoplasm, and its expression levels increased observably with the prolongation of bacterial infection. In addition, recombinant MaCXCL8 protein exhibited significant chemotactic effects on neutrophils and macrophages. In conclusion, MaCXCL8 is involved in the immune response of M. amblycephala, and these findings will be helpful to understand the biological roles of MaCXCL8 and provide a theoretical basis for the prevention and control of fish bacterial diseases.

Comparative Expression Profiling Reveals the Regulatory Effects of Dietary Mannan Oligosaccharides on the Intestinal Immune Response of Juvenile Megalobrama amblycephala against Aeromonas hydrophila Infection.

Mannan oligosaccharides (MOS) are functional oligosaccharides with beneficial effects on the non-specific immunity of Megalobrama amblycephala, but systematic studies on the immunomodulatory mechanisms of MOS are still lacking. To investigate the protective mechanisms of three different levels of dietary MOS supplementation on the intestinal immunity of juvenile M. amblycephala, comparative digital gene expression (DGE) profiling was performed. In this study, 622 differentially expressed genes (DEGs) were identified, while the similar expression tendency of 34 genes by qRT-PCR validated the accuracy of the DGE analyses. Gene Ontology (GO) enrichment revealed that the DEGs were mainly enriched in two functional categories of biological process and molecular function. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEGs were mainly related to complement and coagulation cascades, coagulation cascades, platelet activation, natural killer cell mediated cytotoxicity, Fc gamma R-mediated phagocytosis and antigen processing and presentation. In addition, the pro-inflammatory, apoptosis and tight junction-related genes were more significantly up-regulated upon infection in the dietary MOS groups to enhance host immune functions and maintain the stability of the intestinal barrier. These results will be helpful to clarify the regulatory mechanism of MOS on the intestinal immunity of M. amblycephala and lay the theoretical foundation for the prevention and protection of fish bacterial diseases.

Dietary Mannan Oligosaccharides Enhance the Non-Specific Immunity, Intestinal Health, and Resistance Capacity of Juvenile Blunt Snout Bream (Megalobrama amblycephala) Against Aeromonas hydrophila.

Mannan oligosaccharides (MOS) have been studied and applied as a feed additive, whereas their regulation on the growth performance and immunity of aquatic animals lacks consensus. Furthermore, their immunoprotective effects on the freshwater fish Megalobrama amblycephala have not been sufficiently studied. Thus, we investigated the effects of dietary MOS of 0, 200, and 400 mg/kg on the growth performance, non-specific immunity, intestinal health, and resistance to Aeromonas hydrophila infection in juvenile M. amblycephala. The results showed that the weight gain rate of juvenile M. amblycephala was not significantly different after 8 weeks of feeding, whereas the feed conversion ratio decreased in the MOS group of 400 mg/kg. Moreover, dietary MOS increased the survival rate of juvenile M. amblycephala upon infection, which may be attributed to enhanced host immunity. For instance, dietary MOS increase host bactericidal and antioxidative abilities by regulating the activities of hepatic antimicrobial and antioxidant enzymes. In addition, MOS supplementation increased the number of intestinal goblet cells, and the intestine was protected from necrosis of the intestinal folds and disruption of the microvilli and junctional complexes, thus maintaining the stability of the intestinal epithelial barrier. The expression levels of M. amblycephala immune and tight junction-related genes increased after feeding dietary MOS for 8 weeks. However, the upregulated expression of immune and tight junction-related genes in the MOS supplemental groups was not as notable as that in the control group postinfection. Therefore, MOS supplementation might suppress the damage caused by excessive intestinal inflammation. Furthermore, dietary MOS affected the richness and composition of the gut microbiota, which improved the gut health of juvenile M. amblycephala by increasing the relative abundance of beneficial gut microbiota. Briefly, dietary MOS exhibited significant immune protective effects to juvenile M. amblycephala, which is a functional feed additive and immunostimulant.

Identification, expression patterns, evolutionary characteristics and recombinant protein activities analysis of CD209 gene from Megalobrama amblycephala.

CD209 is a type II transmembrane protein in the C-type lectin family, which is involved in the regulation of innate and adaptive immune system. Although it has been widely studied in mammals, but little has been reported about fish CD209 genes. In the present study, Megalobrama amblycephala CD209 (MaCD209) gene was cloned and characterized, its expression patterns, evolutionary characteristics, agglutinative and bacteriostatic activities were analyzed. These results showed that the open reading frame (ORF) of MaCD209 gene was 795 bp, encoding 264 aa, and the calculated molecular weight of the encoded protein was 29.7 kDa. MaCD209 was predicted to contain 2 N-glycosylation sites, 1 functional domain (C-LECT-DC-SIGN-like) and 1 transmembrane domain. Multiple sequence alignment showed that the amino acid sequence of MaCD209 was highly homologous with that of partial fishes, especially the highly conserved C-LECT-DC-SIGN-like domain and functional sites of CD209. Phylogenetic analysis showed that the CD209 genes from M. amblycephala and other cypriniformes fishes were clustered into one group, which was reliable and could be used for evolutionary analysis. Then, adaptive evolutionary analysis of teleost CD209 was conducted, and several positively selected sites were identified using site and branch-site models. Quantitative real-time PCR analysis showed that MaCD209 gene was highly expressed in the liver and heart. Moreover, the expression of MaCD209 was significantly induced upon Aeromonas hydrophila infection, with the peak levels at 4 h or 12 h post infection. The immunohistochemical analysis also revealed increased distribution of MaCD209 protein post bacterial infection. In addition, recombinant MaCD209 (rMaCD209) protein was prepared using a pET32a expression system, which showed excellent bacterial binding and agglutinative activities in a Ca2+-independent manner. However, rMaCD209 could only inhibit the proliferation of Escherichia coli rather than A. hydrophila. In conclusion, this study identified the MaCD209 gene, detected its expression and evolutionary characteristics, and evaluated the biological activities of rMaCD209 protein, which would provide a theoretical basis for understanding the evolution and functions of fish CD209 genes.

Novel insights into the immune regulatory effects of Megalobrama amblycephala intelectin on the phagocytosis and killing activity of macrophages.

Previous studies have found that the expression level of Megalobrama amblycephala intelectin (MaINTL) increased significantly post Aeromonas hydrophila infection, and recombinant MaINTL (rMaINTL) protein could activate macrophages and enhance the phagocytosis and killing activity of macrophages. In order to reveal the immune regulatory mechanisms of MaINTL, primary M. amblycephala macrophages were treated with endotoxin-removed rMaINTL and GST-tag proteins, then total RNA were extracted and used for comparative Digital Gene Expression Profiling (DGE). 1247 differentially expressed genes were identified by comparing rMaINTL and GST-tag treated macrophage groups, including 482 up-regulated unigenes and 765 down-regulated unigenes. In addition, eleven randomly selected differentially expressed genes were verified by qRT-PCR, and most of them shared the similar expression patterns as that of DGE results. GO enrichment revealed that the differentially expressed genes were mainly concentrated in the membrane part and cytoskeleton of cellular component, the binding and signal transducer activity of molecular function, the cellular process, regulation of biological process, signaling and localization of biological process, most of which might related with the phagocytosis and killing activity of macrophages. KEGG analysis revealed the activation and involvement of differentially expressed genes in immune related pathways, such as Tumor necrosis factor (TNF) signaling pathway, Interleukin 17 (IL-17) signaling pathway, Toll-like receptor signaling pathway, and NOD like receptor signaling pathway, etc. In these pathways, TNF-ɑ, Activator protein-1 (AP-1), Myeloid differentiation primary response protein MyD88 (MyD88), NF-kappa-B inhibitor alpha (ikBɑ) and other key signaling factors were significantly up-regulated. These results will be helpful to clarify the immune regulatory mechanisms of fish intelectin on macrophages, thus providing a theoretical basis for the prevention and control of fish bacterial diseases.