Lusheng Xin

Identification and functional analysis of a novel IFN-like protein (CgIFNLP) in Crassostrea gigas

Interferons (IFNs) belong to class II helical cytokines family with pleiotropic biological activities, which have been demonstrated to play crucial roles in innate and adaptive immunity in vertebrates. In the present study, a novel IFN-like protein (designed CgIFNLP) was identified from oyster Crassostrea gigas, which contained an interferon domain from 14 to 97 amino acids showing low sequence similarities with vertebrates IFNs, but shared a similar three-dimensional structure with class II helical cytokines. The mRNA transcripts of CgIFNLP was detected in all the tested tissues including gonad, adductor muscle, hemocytes, mantle, gills, and hepatopancreas, with the highest expression level in gills (39-fold, P < 0.05). Moreover, the expression level of CgIFNLP mRNA in hemocytes increased significantly at 12 h (8.35-fold, P < 0.01) and 24 h (4.95-fold, P < 0.01) after poly (I: C) stimulation. After the treatments by recombinant CgIFNLP protein (rCgIFNLP) at different concentrations, the apoptosis and phagocytosis rates of oyster hemocytes increased obviously. The proliferation rate of L929 did not change obviously after incubation with rCgIFNLP for 72 h, but the proliferation rate of A549 abated significantly at 36 h and 48 h after incubation with rCgIFNLP. The results collectively suggested that the IFN-like molecule existed in oyster and it tended to present conserved functions rather than conserved amino acid sequence in comparison with vertebrate IFNs.

CgIL17-5, an ancient inflammatory cytokine in Crassostrea gigas exhibiting the heterogeneity functions compared with vertebrate interleukin17 molecules.

Interleukin 17 (IL17) is a proinflammatory cytokine that plays an important role in immune response. Recently, five novel IL17 homologs have been identified by screening and analyzing the genome of pacific oyster Crassostrea gigas. In the present study, the functions of CgIL17-5 were investigated by examining the distribution of its mRNA and protein, ligands binding and modulation in immune response. The mRNA expression levels of CgIL17-5 in hemocytes of oysters post twice challenges of Vibrio splendidus were all significantly up-regulated (P < 0.01), while the secondary pathogen infection attenuated the expression level of CgIL17-5 mRNA compared with the primary challenge. CgIL17-5 was found to be located on oyster hemocyte membranes through fluorescence confocal assay. The luciferase reporter assays showed that CgIL17-5 could activate the transfactors NF-κB, CREB and ATF-1, and involve in their signal pathways in HEK293T cells. Meanwhile, CgIL17-5 could augment the IL6 synthesis in HuVEC cells, playing the similar roles as human IL17 in inflammatory response. Additionally, the recombinant CgIL17-5 (rCgIL17-5) could directly bind peptidoglycan (PGN), lipopolysaccharide (LPS), poly (I:C) and β-1,3-glucan, with the highest affinity to PGN, and significantly inhibit the growth of Micrococcus luteus and Escherichia coli. All the results collectively suggested that CgIL17-5, as an ancient inflammatory cytokine, could not only activate signal transduction for the release of other cytokines, but also mediate the clearance of extracellular bacteria in oysters.

A cytokine-like factor astakine accelerates the hemocyte production in Pacific oyster Crassostrea gigas

Astakine has been reported to be a hematopoietic growth factor of prokineticin homolog firstly found in arthropods freshwater crayfish Pacifastacus leniusculus. In the present study, an astakine homologous gene was identified from Pacific oyster Crassostrea gigas (designated CgAstakine). The full length cDNA of CgAstakine encoded a polypeptide of 103 amino acids containing a prokineticin (PK) domain homologous to that in astakine from freshwater crayfish P. leniusculus. The deduced amino acid sequence of CgAstakine shared higher similarity with those of other invertebrate astakines than prokineticins from vertebrates. The mRNA of CgAstakine was highly expressed in hepatopancreas and adductor muscle of oyster, while the CgAstakine protein was mainly distributed in hepatopancreas, gill and hemocytes. The mRNA expression of CgAstakine in hemocytes was significantly increased (p < 0.01) and maintained at a high level from 3 h to 9 h after Vibrio anguillarum challenge. After the oyster hemocytes were incubated with 5 μg/mL recombinant CgAstakine protein (rCgAstakine) for 24 h in vitro, the proliferation of hemocytes was significantly increased to 1.89 fold of that in control group (p < 0.05). Moreover, the total count of oyster hemocytes was significantly upregulated (2.45 fold of that in control group, p < 0.05) at 12 h after the oysters were received an injection of rCgAstakine (0.5 μg/g). These results collectively indicated that CgAstakine could modulate the hemocytes proliferation both in vitro and in vivo, and probably involved in the hematopoietic process fighting against the invasion of foreign pathogens.

The inhibitory role of γ-aminobutyric acid (GABA) on immunomodulation of Pacific oyster Crassostrea gigas.

γ-aminobutyric acid (GABA) is an inhibitory neurotransmitter to suppress the immune-mediated pro-inflammatory reactions, and it has been used in the treatment of many inflammation-related diseases in vertebrates, while its immunomodulatory role in invertebrates has never been reported. In the present study, GABA was found to exist in the hemolymph of Pacific oyster Crassostrea gigas, and its concentration decreased slightly from 8.00 ± 0.37 μmol L(-1) at normal condition to 7.73 ± 0.15 μmol L(-1) at 6 h after LPS stimulation, and then increased to 9.34 ± 0.15 μmol L(-1), 8.86 ± 0.68 μmol L(-1) at 12 h and 48 h, respectively. After LPS stimulation, the mRNA expressions of pro-inflammatory cytokines (CgIL-17 and CgTNF) and immune effectors (CgSOD and CgBPI), and the protein expression of NOS increased significantly, and these increased trends were remarkably inhibited by GABA stimulation. At the same time, the phagocytosis rate and apoptosis rate of immunocytes also increased obviously after LPS stimulation, whereas the increase was repressed with the addition of GABA. The results collectively demonstrated that GABA was an indispensable inhibitory agent for both humoral and cellular immune response, which mainly functioned at the late phase of immune response to avoid the excess immune reactions and maintain the immune homeostasis.

Comparative study of three C1q domain containing proteins from pacific oyster Crassostrea gigas

ABSTRACT C1q domain containing proteins (C1qDCs) are a family of proteins containing a globular head C1q domain (ghC1q) in C‐terminus, which serve as pattern recognition receptors (PRRs) and mediate a series of immune responses. In the present study, three C1qDC proteins from pacific oyster Crassostrea gigas (CgC1qDC‐2, CgC1qDC‐3, CgC1qDC‐4) were characterized and comparatively investigated to understand their roles in the immune response. All the three recombinant CgC1qDC proteins (rCgC1qDCs) could bind lipopolysaccharide (LPS) significantly but they could not bind lipoteichoic acid (LTA), &bgr;‐1,3‐glucan (GLU), mannan (MAN), and polyinosinic‐polycytidylic acid (Poly I:C). Correspondingly, they all exhibited higher binding activities towards Gram‐negative bacteria Vibrio anguillarum and V. splendidus. Moreover, they could enhance the phagocytosis of oyster hemocytes, and the enhancements towards Gram‐negative bacteria were significantly higher than that towards Gram‐positive bacteria (p < 0.01). The LPS binding affinity of rCgC1qDC‐3 (KD = 8.74 Symbol 10−7 M) was higher than that of rCgC1qDC‐2 (KD = 7.76 Symbol 10−5 M) and rCgC1qDC‐4 (KD = 1.09 Symbol 10−5 M). Meanwhile, rCgC1qDC‐3 exhibited significantly higher enhancement on phagocytosis of oyster hemocytes towards Gram‐negative bacteria than that of rCgC1qDC‐2 and rCgC1qDC‐4 (p < 0.05). After the secondary challenge with V. splendidus, the up‐regulations of CgC1qDC‐2 and CgC1qDC‐4 mRNA in hemocytes occurred at 6 h, while that of CgC1qDC‐3 was observed at 3 h and lasted for 24 h. And CgC1qDC‐3 responded with high mRNA level for tested 24 h upon the secondary challenge with V. anguillarum as well. These results collectively suggested that three CgC1qDCs could serve as PRRs to specifically recognize certain Gram‐negative bacteria and opsonins to enhance phagocytosis. CgC1qDC‐3, with higher binding affinity to LPS, stronger opsonization and more rapid and persistent mRNA expression response upon the secondary challenge with homologous Vibrios, might exert efficient functions in the immune responses against invading pathogens. Symbol. No Caption available. Symbol. No Caption available. Symbol. No Caption available. HighlightsThree C1qDC proteins from C. gigas could specifically recognize LPS and Gram‐negative bacteria.Three C1qDC proteins could enhance cell phagocytosis towards bacteria especially towards Gram‐negative bacteria.CgC1qDC‐3 with higher LPS binding affinity exhibited stronger ability of enhancing phagocytosis.CgC1qDC‐3 mRNA could more rapidly and persistently up‐regulate when the oysters reencountered with homologous Vibrios.

The modulation role of serotonin in Pacific oyster Crassostrea gigas in response to air exposure

ABSTRACT Serotonin, also known as 5‐hydroxytryptamine (5‐HT), is a critical neurotransmitter in the neuroendocrine‐immune regulatory network and involved in regulation of the stress response in vertebrates and invertebrates. In the present study, serotonin was found to be widely distributed in the tissues of Pacific oyster Crassostrea gigas, including haemolymph, gonad, visceral ganglion, mantle, gill, labial palps and hepatopancreas, and its concentration increased significantly in haemolymph and mantle after the oysters were exposed to air for 1 d. The apoptosis rate of haemocytes was significantly declined after the oysters received an injection of extra serotonin, while the activity of superoxide dismutase (SOD) in haemolymph increased significantly. After the stimulation of serotonin during air exposure, the apoptosis rate of oyster haemocytes and the concentration of H2O2 in haemolymph were significantly decreased, while the SOD activity was significantly elevated. Furthermore, the survival rate of oysters from 4th to 6th d after injection of serotonin was higher than that of FSSW group and air exposure group. The results clearly indicated that serotonin could modulate apoptotic effect and redox during air exposure to protect oysters from stress. HighlightsNeurotransmitter 5‐HT existed extensively in the tissues of oyster C. gigas.The level of 5‐HT in haemolymph and mantle increased significantly against air exposure stress.5‐HT could reduce apoptotic effects of haemocytes and modulate redox in haemolymph after air exposure.5‐HT could decrease the mortality of oysters during air exposure, and protect them from stress.

A norepinephrine-responsive miRNA directly promotes CgHSP90AA1 expression in oyster haemocytes during desiccation

ABSTRACT Oyster Crassostrea gigas is one model mollusc inhabiting in the intertidal zone and is frequently stressed by desiccation. The adaptation mechanism of oyster to environmental stress involves multiple levels, and miRNA is one of the most important regulators in post‐transcriptional level. In the present study, an oyster norepinephrine‐responsive miRNA cgi‐miR‐365 was proved to contribute to the host adaptation against desiccation by directly promoting the expression of CgHSP90AA1. Briefly, a significant increase of cgi‐miR‐365 was observed from the first day after aerial exposure and the up‐regulation was vigorously repressed when oysters were injected with adrenoceptors antagonists. A total of 15 genes involved in biological regulation, metabolic process and response to stimulus were predicted to be modulated by cgi‐miR‐365. Among these genes, CgHSP90AA1 was up‐regulated significantly during desiccation and could be down‐regulated after simultaneous injection of adrenoceptors antagonists. The interaction between cgi‐miR‐365 and CgHSP90AA1 was subsequently verified in vitro, and a significant promotion of CgHSP90AA1 transcripts was observed after overexpressing cgi‐miR‐365 in either in vitro luciferase reporter assay or primarily cultured haemocytes. Meanwhile, CgHSP90AA1 transcripts decreased in vivo when cgi‐miR‐365 was repressed by its inhibitor during desiccation. Collectively, it was suggested that cgi‐miR‐365 could be induced by norepinephrine during desiccation and promote CgHSP90AA1 expression directly after binding to its 3′‐UTR, which would provide new evidence in miRNA‐mediated adaptation mechanism in oysters against intertidal stress. HIGHLIGHTScgi‐miR‐365 was found fast and strongly induced in NE‐dependent way after desiccation stress.Fifteen genes involved in biological regulation and metabolic process were targeted by cgi‐miR‐365.CgHSP90AA1, as one putative target of cgi‐miR‐365, was also found induced after desiccation by NE.Interaction between cgi‐miR‐365 and CgHSP90AA1 was verified by in vitro and in vivo.CgHSP90AA1 mRNA could be promoted directly by cgi‐miR‐365 during desiccation.