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.

Starvation Affects the Muscular Morphology, Antioxidant Enzyme Activity, Expression of Lipid Metabolism-Related Genes, and Transcriptomic Profile of Javelin Goby (Synechogobius hasta).

Fish in natural and cultivated environments can be challenged by starvation. However, inducing starvation in a controlled manner cannot only reduce feed consumption but also reduces aquatic eutrophication and even improves farmed fish quality. This study investigated the effects of starvation on the muscular function, morphology, and regulatory signaling in javelin goby (Synechogobius hasta) by evaluating the biochemical, histological, antioxidant, and transcriptional changes in the musculature of S. hasta subjected to 3, 7, and 14 days fasting. The muscle glycogen and triglyceride levels in S. hasta were gradually reduced under starvation, reaching their lowest at the end of the trial (P < 0.05). The levels of glutathione and superoxide dismutase were significantly elevated after 3-7 days of starvation (P < 0.05), but later returned to the level of the control group. The muscle of starved S. hasta developed structural abnormalities in some areas after 7 days of food deprivation, and more vacuolation and more atrophic myofibers were observed in 14-day fasted fish. The transcript levels of stearoyl-CoA desaturase 1 (scd1), the key gene involved in the biosynthesis of monounsaturated fatty acids, were markedly lower in the groups starved for 7 or more days (P < 0.05). However, the relative expressions of genes associated with lipolysis were decreased in the fasting experiment (P < 0.05). Similar declines in the transcriptional response to starvation were found in muscle fatp1 and ppar γ abundance (P < 0.05). Furthermore, the de novo transcriptome of muscle tissue from the control, 3-day and 14-day starved S. hasta generated 79,255 unigenes. The numbers of differentially expressed genes (DEGs) identified by pairwise comparisons among three groups were 3276, 7354, and 542, respectively. The enrichment analysis revealed that the DEGs were primarily involved in metabolism-related pathways, including ribosome, TCA pathway, and pyruvate metabolism. Moreover, the qRT-PCR results of 12 DEGs validated the expression trends observed in the RNA-seq data. Taken together, these findings demonstrated the specific phenotypical and molecular responses of muscular function and morphology in starved S. hasta, which may offer preliminary reference data for optimizing operational strategies incorporating fasting/refeeding cycles in aquaculture.

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.

cDNA sequence and tissues expression analysis of lipoprotein lipase from common carp (Cyprinus carpio Var. Jian).

A full-length cDNA coding lipoprotein lipase (LPL) was cloned from liver of adult common carp (Cyprinus carpio Var. Jian) by RT-PCR and rapid amplification of cDNA ends (RACE) approaches. The cDNA obtained was 2,411 bp long with a 1,524 bp open reading frame (ORF) encoding 507 amino acids. This amino acid sequence contains two structural regions: N-terminus (24-354 residues) and C-terminus (355-507 residues). Before N-terminus, 1-23 residues is signal peptide, 6-23 residues is transmembrance helix. At N-terminus, some conversed functional sites were found, including two N-linked glycosylation sites Asn(41) and Asn(88); one catalytic triad Ser(174), Asp(198) and His(283); one conserved heparin-binding site Arg(321) to Arg(324) (RKNR); eight cysteines residues Cys(69) and Cys(82), Cys(258) and Cys(281), Cys(306) and Cys(325), Cys(317) and Cys(320) which are involved in four disulfide bridges; one polypeptide "lid" that participates in substrate specificity. At C-terminus, Asn(401) is another N-linked glycosylation site, and Trp(434) and Trp(435) (WW) is lipid-binding site. The amino acid sequence has a high similarity, and shows similar structural features to LPL of other species. Tissue distribution of LPL mRNA in liver, head kidney, mesenteric adipose tissue, heart and white muscle of common carp was analyzed by semi-quantitative RT-PCR method using beta-actin gene as internal control. The result showed that the expressions of LPL mRNA were detected in all examined tissues of common carp. The expression levels of LPL in the mesenteric adipose tissue was highest among these tissues, following in liver and head kidney, and the lowest expression was found in heart and white muscle.