Norepinephrine regulates TNF expression via the A1AR-p38 MAPK-Relish pathway in granulocytes of oyster Crassostrea gigas.

Norepinephrine (NE) is involved in regulating cytokine expression and phagocytosis of immune cells in the innate immunity of vertebrates. In the present study, the modulation mechanism of NE on the biosynthesis of TNFs in oyster granulocytes was explored. The transcripts of CgTNF-1, CgTNF-2 and CgTNF-3 were highly expressed in granulocytes, and they were significantly up-regulated after LPS stimulation, while down-regulated after NE treatment. The phagocytic rate and apoptosis index of oyster granulocytes were also triggered by LPS stimulation and suppressed by NE treatment. The mRNA expressions of CgMAPK14 and CgRelish were significantly induced after NE treatment, and the translocation of CgRelish from cytoplasm to nucleus was observed. The concentration of intracellular Ca2+ in granulocytes was significantly up-regulated upon NE incubation, and this trend reverted after the treatment with DOX (specific antagonist for NE receptor, CgA1AR-1). No obvious significance was observed in intracellular cAMP concentrations in the PBS, NE and NE + DOX groups. Once CgA1AR-1 was blocked by DOX, the mRNA expressions of CgMAPK14 and CgRelish were significantly inhibited, and the translocation of CgRelish from cytoplasm to nucleus was also dramatically suppressed, while the mRNA expression of CgTNF-1 and the apoptosis index increased significantly to the same level with those in LPS group, respectively. These results collectively suggested that NE modulated TNF expression in oyster granulocyte through A1AR-p38 MAPK-Relish signaling pathway.

A novel surface marker CD49d promotes TNF expression in oyster agranulocytes by mediating the MAPK pathway.

CD49d, encoded by the gene Integrin α4, is a significant member of cell adhesion receptors, which is widely expressed in various immune cells to trigger immune responses against invading pathogens. In the present study, the expression of CgCD49d and its regulatory role in TNF expression were investigated in the Pacific oyster Crassostrea gigas. There were five Int-alpha domains, an Integrin_alpha2 region and a unique FG-GAP repeat region inserted identified in CgCD49d. CgCD49d transcript was specifically expressed in haemocytes, and its mRNA expression level in haemocytes increased after LPS and Vibrio splendidus stimulation. After CgCD49d was blocked by using its antibody, the phosphorylation level of CgJNK in the MAPK signaling pathway and CgTNF transcripts decreased significantly post V. splendidus stimulation. After phosphorylation level of CgJNK was inhibited by using its inhibitor, the nuclear translocation of CgRel was restrained and CgTNF transcripts also decreased significantly post V. splendidus stimulation. Furthermore, CgCD49d was found to be mainly expressed in the agranulocyte subpopulation, and Alexa Fluor 488-conjugated CgCD49d antibody labeled agranulocytes with a circle of green fluorescence signals on CgCD49d+ agranulocyte surface under Confocal microscopy, which accounted for 24.9 ± 4.53% of total haemocytes. Collectively, these results suggested that CgCD49d promoted TNF expression in oyster haemocytes against bacterial invasion by mediating MAPK pathway, and it could be used as a surface marker to type and sort a subset of agranulocyte subpopulation among haemocytes.

CgSHIP2 negatively regulates the mRNA expressions of CgIL-17s in response to Vibrio splendidus stimulation in Crassostrea gigas.

SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.

CgADAR1 involved in regulating the synthesis of interferon-like protein in Crassostrea gigas.

Adenosine deaminases acting on RNA 1 (ADAR1) is a dsRNA adenosine (A)-to-inosine (I) editing enzyme that regulates the innate immune response against virus invasion. In the present study, a novel CgADAR1 was identified from the oyster Crassostrea gigas. The open reading frame (ORF) of CgADAR1 was of 3444 bp encoding a peptide of 1147 amino acid residues with two Zα domains, one dsRNA binding motif (DSRM) and one RNA adenosine deaminase domain (ADEAMc). The mRNA transcripts of CgADAR1 were detected in all the examined tissues, with higher expression levels in mantle and gill, which were 7.11-fold and 4.90-fold (p < 0.05) of that in labial palp, respectively. The mRNA transcripts of CgADAR1 in haemocytes were significantly induced at 24 h and 36 h after Poly (A: U) stimulation, which were 6.03-fold (p < 0.01) and 1.37-fold (p < 0.001) of that in control group, respectively. At 48 h after Poly (A:U) stimulation, the mRNA expression of CgRIG-Ⅰ, CgIRF8 and CgIFNLP significantly increased, which were 4.36-fold (p < 0.001), 1.82-fold (p < 0.05) and 1.92-fold (p < 0.05) of that in control group. After CgADAR1 expression was inhibited by RNA interference (RNAi), the mRNA expression levels of CgMDA5, CgRIG-Ⅰ, CgTBK1, CgIRF8 and CgIFNLP were significantly increased, which were 11.88-fold, 11.51-fold, 2.22-fold, 2.85-fold and 2.52-fold of that in control group (p < 0.001), and the phosphorylation level of CgTBK1 was also significantly increased. These results suggested that CgADAR1 played a regulation role in the early stages of viral infection by inhibiting the synthesis of interferon-like protein.

Polymorphisms in the cysteine dioxygenase gene and their association with taurine content in the Pacific oyster Crassostrea gigas.

The Pacific oyster Crassostrea gigas is rich in taurine, which is crucial for its adaptation to the fluctuating intertidal environment and presents significant potential in improving taurine nutrition and boosting immunity in humans. Cysteine dioxygenase (CDO) is a key enzyme involved in the initial step of taurine biosynthesis and plays a crucial role in regulating taurine content in the body. In the present study, polymorphisms of CDO gene in C. gigas (CgCDO) and their association with taurine content were evaluated in 198 individuals. A total of 24 single nucleotide polymorphism (SNP) loci were identified in the exonic region of CgCDO gene by direct sequencing. Among these SNPs, c.279G>A and c.287C>A were found to be significantly associated with taurine content, with the GG and AA genotype at the two loci exhibiting enhanced taurine accumulation (p < 0.05). Haplotype analysis revealed that the 279GG/287AA haplotype had the highest taurine content of 29.24 mg/g, while the 279AA/287CC haplotype showed the lowest taurine content of 21.19 mg/g. These results indicated that the SNPs of CgCDO gene could influence the taurine content in C. gigas and have potential applications in the selective breeding of high-taurine varieties.

An LPS-induced TNF-α factor involved in immune response of oyster Crassostrea gigas by regulating haemocytes apoptosis.

LPS induced TNF-α Factor (LITAF) is a transcription factor widely involving in activation of Tumor Necrosis Factor (TNF) and other cytokines in the inflammatory response. In the present study, a homologue of LITAF with a conserved LITAF domain was identified from the Pacific oyster Crassostrea gigas. The transcripts of CgLITAF were detected in all examined tissues with highest expression in hepatopancrease. The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgLITAF protein in haemocytes. While the mRNA level of CgLITAF changed slightly after LPS stimulation. When the siRNA of CgLITAF was injected to inhibit its expression, the apoptotic level of haemocytes decreased observably after LPS stimulation. Consistently, the transcripts of CgTNF3 and CgTNF4 (LOC105343080, LOC105341146), the apoptotic-related molecules including CgBax, CgCytochrome c, CgCaspase9 and CgCaspase3, were significantly suppressed in the CgLITAF-RNAi oysters. While the mRNA expression level of CgBcl was enhanced significantly in the CgLITAF-RNAi oysters. These results indicated that CgLITAF promoted haemocyte apoptosis by regulating the expression of apoptotic-related factors, suggesting its important role in the immune response of oysters.

Synergistic modulation of neuroendocrine-inflammation pathway by microRNAs facilitates intertidal adaptation of molluscs.

Neuroendocrine-immune system is an evolution-conserved regulatory network in maintaining the homeostasis of animals. While knowledge on the roles of neuroendocrine-immune system in the disease and stress responses of organisms is growing, the ecological roles of neuroendocrine-immune system, especially how it shapes the unique lifestyle of organisms remain insufficiently investigated. As an endemic and dominant mollusc in intertidal region, oysters have evolved with a primitive neuroendocrine-immune system and with a sessile lifestyle. Recently, a novel neuroendocrine-immune pathway, Ca2+/calmodulin (CaM)-nitrite oxide synthase (NOS)/nitrite oxide (NO)-tumor necrosis factor (TNF) pathway, is identified in oysters and found altered dynamically during aerial exposure, one common but challenging stresses for intertidal organisms and a decisive factor shaping their habitat. Since the pathway proves fatal in prolonged aerial exposure, we hypothesized that the activation/deactivation of pathway could be strictly modulated in adaptation to the sessile lifestyle of oysters. Here, a synergistic modulation on the Ca2+/CaM-NOS/NO-TNF pathway by four members of miR-92 family and two oyster-specific miRNAs was identified, which further hallmarks the resilience and survival strategy of oysters to aerial exposure. Briefly, these six miRNAs were down-regulating CgCaM24243 post-transcriptionally and deactivating the pathway during the early-stage of stress. However, a robust recession of these miRNAs occurred at the late-stage of stress, resulting in the reactivation of pathway and overwhelming accumulation of cytokines. These results demonstrated a complicated interaction between miRNAs and ancient neuroendocrine-immune system, which facilitates the environmental adaptation of intertidal oysters and provides novel insight on the function and evolution of neuroendocrine-immune system in ecological context.

A circadian clock protein cryptochrome inhibits the expression of inflammatory cytokines in Chinese mitten crab (Eriocheir sinensis).

Cryptochrome (Cry), as important flavoprotein, plays a key role in regulating the innate immune response, such as the release of inflammatory cytokines. In the present study, a cryptochrome homologue (EsCry) was identified from Chinese mitten crab Eriocheir sinensis, which contained a typical DNA photolyase domain, a FAD binding domain. The transcripts of EsCry were highly expressed at 11:00, and lowest at 3:00 within one day, while those of Interleukin enhancer binding factor (EsILF), Lipopolysaccharide-induced TNF-alpha factor (EsLITAF), Tumor necrosis factor (EsTNF) and Interleukin-16 (EsIL-16) showed a rhythm expression pattern contrary to EsCry. After EsCry was knocked down by dsEsCry injection, mRNA transcripts of Timeless (EsTim), Cycle (EsCyc), Circadian locomotor output cycles kaput (EsClock), Period (EsPer), and EsLITAF, EsTNF, EsILF, EsIL-16, as well as phosphorylation level of Dorsal significantly up-regulated. The transcripts of EsLITAF, EsTNF, EsILF, and EsIL-16 in EsCry-RNAi crabs significantly down-regulated after injection of NF-κB inhibitor. The interactions of EsCyc and EsCry, EsCyc and Dorsal were observed in vitro. These results indicated that EsCry negatively regulated the expression of the cytokine TNF and IL-16 via inhibiting their transcription factor LITAF and ILF through NF-κB signaling pathway, which provide evidences to better understand the circadian regulation mechanism of cytokine production in crabs.

A bone morphogenetic protein regulates the shell formation of Crassostrea gigas under ocean acidification.

Bone morphogenetic proteins (BMPs) are key factors controlling osteoblast differentiation, which have been proved to be involved in the hard tissue formation of marine mollusks. In the present study, a member of BMPs gene (CgBMP7) was identified from Pacific oyster Crassostrea gigas (C. gigas) with the aim to understand its possible role in the regulation of shell formation under ocean acidification (OA) conditions. The open reading frame (ORF) of CgBMP7 was of 1254 bp encoding a polypeptide of 417 amino acids. The deduced amino acid sequence of CgBMP7 was comprised of one signal peptide, one prodomain and one TGF-ß domain, which shared 21.69%-61.10% identities with those from other species. The mRNA transcript of CgBMP7 was ubiquitously expressed in all the tested tissues of adult oysters with a higher expression level in mantle, notably highest in the middle fold (MF) of the three folds of mantle. The expression level of bone morphogenetic protein type I receptor (CgBMPR1B) mRNA was also highest in the MF and up-regulated dramatically post recombinant BMP7 protein (rCgBMP7) stimulation. After the blockage of BMPR1B with inhibitor LDN193189 (LDN), the mRNA expression level and phosphorylation level of CgSmad1/5/8 in mantle were decreased, and the mRNA expression levels of CgCaM and Cgengrailed-1 were down-regulated significantly. During the oysters were exposed to acidified seawater for weeks, the expression levels of CgBMP7, CgBMPR1B and CgSmad1/5/8 in the MF decreased significantly (p < 0.01) at the 4th week, and CgCaM and Cgengrailed-1 also exhibited the same variable expression patterns as CgBMP7. In addition, the growth of shell in the treatment group (pH 7.8) was slower than that in the control group (pH 8.1). These results collectively indicated that BMP7 was able to trigger the BMPR-Smad signaling pathway and involved in controlling the formation of oyster calcified shell under OA conditions.

The involvement of AMP-activated protein kinase α in regulating glycolysis in Yesso scallop Patinopecten yessoensis under high temperature stress.

AMP-activated protein kinase α subunit (AMPKα), the central regulatory molecule of energy metabolism, plays an important role in maintaining energy homeostasis and helping cells to resist the influence of various adverse factors. In the present study, an AMPKα was identified from Yesso scallop Patinopecten yessoensis (PyAMPKα). The open reading frame (ORF) of PyAMPKα was of 1599 bp encoding a putative polypeptide of 533 amino acid residues with a typical KD domain, a α-AID domain and a α-CTD domain. The deduced amino acid sequence of PyAMPKα shared 59.89-74.78% identities with AMPKαs from other species. The mRNA transcripts of PyAMPKα were found to be expressed in haemocytes and all the examined tissues, including gill, mantle, gonad, adductor muscle and hepatopancreas, with the highest expression level in adductor muscle. PyAMPKα was mainly located in cytoplasm of scallop haemocytes. At 3 h after high temperature stress treatment (25 °C), the mRNA transcripts of PyAMPKα, the phosphorylation level of PyAMPKα at Thr170 and the lactic acid (LD) content in adductor muscle all increased significantly, while the glycogen content decreased significantly. The activity of pyruvate kinase (PyPK) and the relative mRNA expression level of phosphofructokinase (PyPFK) were significantly up-regulated at 3 h after high temperature stress treatment (25 °C). Furthermore, the PyAMPKα activator AICAR could effectively upregulate the phosphorylation level of PyAMPKα, and increase activities of PyPFK and pyruvate kinase (PyPK). Meanwhile the glycogen content also declined under AICAR treatment. These results collectively suggested that PyAMPKα was involved in the high temperature stress response of scallops by enhancing glycolysis pathway of glycogen. These results would be helpful for understanding the functions of PyAMPKα in maintaining energy homeostasis under high temperature stress in scallops.

A autophagy related-like protein 16-1 promotes the formation of autophagosomes and autolysosomes in antibacterial immune response of Pacific oyster Crassostrea gigas.

Autophagy related 16-like (ATG16L) protein is a core autophagy protein, which promotes the extension of autophagosome membrane through microtubule-associated protein light chain 3 (LC3). In the present study, an ATG16L was identified from oyster Crassostrea gigas (defined as CgATG16L1). The full-length cDNA of CgATG16L1 was of 3184 bp with an open reading frame of 1650 bp that encoded a polypeptide of 549 amino acids. There was an ATG5-interacting motif (AFIM) domain, a coiled-coil (CC) domain and seven tryptophan-aspartic acid 40 (WD40) repeats in CgATG16L1. ATG16L1 mRNA was expressed in all the examined tissues with the highest expression in haemolymph (11.22-fold of that in hepatopancreas, p < 0.05). The mRNA expressions of CgATG16L1 in haemocytes increased significantly at 3, 6, 12, 24 and 72 h after lipopolysaccharide (LPS) stimulation, which were 81.15-fold, 24.95-fold, 6.02-fold, 3.90-fold and 5.97-fold (p < 0.05) of that in control group, respectively. The green positive signals of CgATG16L1 protein and the red positive signals of CgLC3 protein were dotted in the cytoplasm of agranulocytes, semi-granulocytes and granulocytes. The co-localization of CgATG16L1 and CgLC3 was observed in haemocytes after Vibrio splendidus stimulation. In CgATG16L1-RNAi oysters, the number of autophagosomes and autolysosomes in haemocytes was reduced. All these results suggested that CgATG16L1 participated in the bacteria-induced autophagy process in the haemocytes of oyster response to bacteria invasion.

Species-specific CgCaspase-Cg-5 in the pacific oyster induces haemocyte apoptosis by regulating the mRNA expression of apoptosis-related genes in the early stage of immune response.

Caspases are cysteinyl aspartate-specific proteinases, playing critical roles in apoptotic pathway to induce apoptosis and inflammatory response. In this study, the expanded repertoire of Caspases was revealed in the Pacific oyster Crassostrea gigas, and a total of 30 Caspases were identified from the genomic and stress-induced transcriptomic databases of the Pacific oyster. They were clustered into CgCaspase-2/9, CgCaspase-8/10, CgCaspase-3/6/7, CgCaspase-Cg, and CgCaspase-L. CgCaspase-Cg subgroup was found to be specifically expanded after a positive selection in oyster with average Ka/Ks of 0.50. The mRNA expression of CgCaspase-Cg-5 was found to be obviously induced against various bacterial and viral stimulations or environmental stresses. The relative expression level of CgCaspase-Cg-5 in haemocytes increased and reached the peak at 6 h after Vibrio splendidus stimulation, which was 5.57-fold of that in the control group (p < 0.01). In the oysters whose CgCaspase-Cg-5 expression was knocked down, the mRNA expression of apoptosis-related genes including CgBcl2, CgBax, CgCaspase3 and CgCaspase9 changed significantly at 12 h after V. splendidus stimulation. The expression of CgBax, CgCaspase3 and CgCaspase9 decreased, which was 0.64-fold (p < 0.05), 0.53-fold (p < 0.05) and 0.62-fold (p < 0.01), while the expression of CgBcl2 increased, which was 2.81-fold (p < 0.01) of that in the EGFP-dsRNA group, respectively. Meanwhile, the apoptotic rate of haemocytes (1.90 ± 0.71%) significantly decreased compared to that in the EGFP-dsRNA group (5.40 ± 0.72%) (p < 0.05), and the histological damages of widened cell spacing, gill filament swelling and loose cytoplasm were observed in the CgCaspase-Cg-5-knockdown oysters after V. splendidus stimulation. Collectively, CgCaspase-Cg subgroup was specifically expanded in oyster and some bivalve species, and species-specific CgCaspase-Cg-5 regulated the mRNA expression of the apoptosis-related genes to induce haemocyte apoptosis in the early stage of immune response. This provided insight into the evolutionary and functional characteristics of Caspase repertoire in the Pacific oyster and highlighted the important role of CgCaspase-Cg-5 in the response to pathogen infection and environmental stresses.

A MASP-like functions as PRR to regulate the mRNA expressions of inflammatory factors in the Pacific oyster Crassostrea gigas.

Mannose-binding lectin-associated serine protease (MASP) is a type of central serine protease in the complement lectin pathway. In the present study, a MASP-like was identified from the Pacific oyster Crassostrea gigas, defined as CgMASPL-2. The cDNA sequence of CgMASPL-2 was of 3399 bp with an open reading frame of 2757 bp and encoded a polypeptide of 918 amino acids containing three CUB domains, an EGF domain, two IG domains, and a Tryp_SPC domain. In the phylogenetic tree, CgMASPL-2 was firstly clustered with Mytilus californianus McMASP-2-like, and then assigned into the invertebrate branch. CgMASPL-2 shared similar domains with M. californianus McMASP-2-like and Littorina littorea LlMReM1. CgMASPL-2 mRNA was expressed in all the tested tissues with the highest expression in haemolymph. CgMASPL-2 protein was mainly distributed in the cytoplasm of haemocytes. The mRNA expression of CgMASPL-2 increased significantly in haemocytes after Vibrio splendidus stimulation. The recombinant 3 × CUB-EGF domains of CgMASPL-2 displayed binding activities to diverse polysaccharides (lipopolysaccharide, peptidoglycan and mannose) and microbes (Staphylococcus aureus, Micrococcus luteus, Pichia pastoris, Vibrio anguillarum, V. splendidus and Escherichia coli). In anti-CgMASPL-2 treated oysters, the mRNA expressions of CgIL17-1 and CgIL17-2 in haemocytes decreased significantly after V. splendidus stimulation. The results indicated that CgMASPL-2 could directly sense microbes and regulate the mRNA expressions of inflammatory factors.

Syk regulates the haemocyte autophagy through inducing the mRNA expressions of autophagy-related genes and the cleavage of CgLC3 in oyster antibacterial immunity.

Spleen tyrosine kinase (Syk) is reported to be involved in activating the autophagy. Recently, a homologue of Syk was identified from Pacific oyster Crassostrea gigas (defined as CgSyk). In the present study, the molecular characteristics of CgSyk and its regulation mechanism in autophagy were investigated in oyster C. gigas. The full-length cDNA of CgSyk was of 4566 bp with an open reading frame (ORF) of 1989 bp. CgSyk encoded a polypeptide of 662 amino acids, containing two Src homology 2 (SH2) domains and one tyrosine kinase catalytic (TyrKc) domain. The deduced amino acid sequence of CgSyk shared low similarity with the previously identified Syks from other species. In the phylogenetic tree, CgSyk was first clustered with Crassostrea virginica CvSyk, and then classified into a branch of invertebrate Syks. In CgSyk-RNAi oysters, the mRNA expressions of CgLC3, CgP62, CgBeclin-1 and CgATG5 in haemocytes decreased significantly at 12 h after Vibrio splendidus stimulation. At the same time, the abundance of CgLC3Ⅱ in haemocytes, and the autophagy rate of haemocytes in CgSyk-RNAi oysters decreased significantly at 12 h after V. splendidus stimulation. All the results collectively suggested that CgSyk regulated the autophagy through inducing the mRNA expressions of autophagy-related genes and the cleavage of CgLC3 to defend against bacterial invasion in oysters.

An immunoglobulin superfamily member (CgIgIT2) functions as immune inhibitory receptor to inhibit the inflammatory cytokine expressions in Crassostrea gigas.

Immune inhibitory receptors are increasingly acknowledged as potent regulators of immune response, which inhibit the overactivation of immune system and play an important role in maintaining immune homeostasis. In the present study, a novel immunoglobulin superfamily member (CgIgIT2) was identified from the Pacific oyster, Crassostrea gigas. The protein sequence of CgIgIT2 contained one signal peptide, four Ig domains, one fibronectin type III domain, one transmembrane domain, and a cytoplasmic tail with two intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and one immunoreceptor tyrosine-based switch motif (ITSM). The mRNA transcripts of CgIgIT2 were widely expressed in all the tested tissues, including haemolymph, gill, mantle, adductor muscle, labial palp, gonad and hepatopancreas, with the highest expression in haemolymph. The mRNA expressions of CgIgIT2 in haemocytes increased significantly at 24, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgIgIT2 protein were mainly detected in granulocytes of haemocytes, which were 1.27-fold and 2.15-fold (p < 0.05) higher than that of semi-granulocytes and agranulocytes, respectively. And CgIgIT2 was mainly located in the membrane and cytoplasm of haemocytes. The recombinant protein of CgIgIT2-4 × Ig (rCgIgIT2-4 × Ig) exhibited binding activity towards multiple pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS), peptidoglycan (PGN), mannose (MAN) and polyinosinic-polycytidylic acid (Poly (I: C)) with the highest affinity for LPS. rCgIgIT2-4 × Ig could also bind Gram-negative bacteria (V. splendidus, V. anguillarum, Escherichia coli), Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis), and fungi (Pichia pastoris). In the blocking assay with anti-CgIgIT2 antibody, the mRNA expressions of interleukins (CgIL17-1, CgIL17-3 and CgIL17-6) and tumor necrosis factors (CgTNF-1 and CgTNF-2) in haemocytes all increased significantly at 12 h after V. splendidus stimulation. These results suggested that CgIgIT2 could function as an inhibitor receptor to bind different PAMPs and microbes, as well as inhibit the mRNA expressions of multiple inflammatory cytokines in oysters.

An IRF2BP member (CgIRF2BP) involved in negative regulation of CgIFNLP expression in oyster Crassostrea gigas.

The IRF2BP family of transcription regulators act as corepressor molecules by inhibiting both enhancer-activated and basal transcription involving in many biological contexts. In the present study, an IRF2BP homologue (CgIRF2BP) was identified from oyster C. gigas. Its open reading frame is of 1809 bp encoding a polypeptide of 602 amino acids, which contains an IRF-2BP1_2 domain and a RING domain. The mRNA transcripts of CgIRF2BP were detected in all tested tissues with highest level in haemocytes (28.99-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the expression level of CgIRF2BP was significantly down-regulated at 3 h (0.50-fold of that in control group, p < 0.001) and gradually increased from 6 h to 48 h (2.69-fold of that in control group, p < 0.01). The recombinant protein of CgIRF2BP (rCgIRF2BP) showed high affinity to both rCgIRF1 and rCgIRF8 with Kd value of 1.02 × 10-7 and 2.09 × 10-7, respectively. In CgIRF2BP-RNAi oysters, the mRNA expression of CgIFNLP, CgMx1, CgViperin and CgIFI44L were significantly increased after poly (I:C) stimulation, which were 2.88 (p < 0.01), 1.83 (p < 0.05), 2.47 (p < 0.05), and 1.99-fold (p < 0.01) of that in EGFP group, respectively. These findings suggested that CgIRF2BP negatively regulated CgIFNLP expression by binding with CgIRF1 and CgIRF8.

An ATP-binding cassette transporter G2 (CgABCG2) regulates the haemocyte proliferation by modulating the G1/S phase transition of cell cycle in oyster Crassostrea gigas.

ATP-binding cassette transporter G2 (ABCG2) is a half-transporter of the G subfamily in ATP-binding cassette transporters (ABC transporter), which is involved in the regulation of multidrug-resistant, cell cycle, and cell proliferation. In the present study, a homologue of ABCG2 (named as CgABCG2) with the conserved AAA domain and ABC2 membrane domain was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgABCG2 was of 1956 bp encoding a predicted polypeptide of 652 amino acids, which shared 56.7%-65.7% sequence similarities with previously identified ABCG2s from other animals. The mRNA transcripts of CgABCG2 were detected in all the tested tissues with higher expression levels in gonad and haemocytes (19.31-fold and 11.23-fold of that in adductor muscle respectively, p < 0.05). CgABCG2 was mainly distributed on the cell membrane of the haemocytes with a partial distribution in the cytoplasm and nucleus. After Vibrio splendidus stimulation, the mRNA expression level of CgABCG2 in haemocytes was significantly up-regulated at 3 h and 6 h, which was 5.22-fold and 8.60-fold (p < 0.05) of that in control, respectively. After the expression of CgABCG2 was interfered by RNAi, the number of cells with EdU positive signals was reduced in both haemocytes and the potential hematopoietic sites. And the mRNA expression level of CgPCNA, CgGATA3, CgRunx, CgSCL and CgC-kit decreased significantly (p < 0.05), which were about 0.66-, 0.37-, 0.32-, 0.50-, and 0.50-fold of that in the negative control group, respectively. While the mRNA expression level of CgCDK2 increased significantly (1.84-fold to that in control, p < 0.05) and that of stem cell-related factor CgSOX2 did not change significantly in the si-CgABCG2 oysters. Moreover, the cell cycle of haemocytes was detected by flow cytometry, which was arrested at G0/G1 phase in the si-CgABCG2 oysters. All the results collectively suggested that CgABCG2 might involve the proliferation of haemocytes by regulating the expression of haematopoiesis related transcription factors and the G1/S phase transition of the cell cycle in oyster C. gigas.

CgTNF-2 promotes the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in the Pacific oyster Crassostrea gigas.

A TNF-α family member, CgTNF-2, was previously identified from the oyster Crassostrea gigas to involve in the antibacterial response. In the present study, the role of CgTNF-2 in mediating the proliferation of haemocytes was further explored. The mRNA expression of CgTNF-2 in granulocytes was significantly higher than that in semi-granulocytes and agranulocytes, and the percentages of CgTNF-2 antibody labeled cells in agranulocytes, semi-granulocytes and granulocytes were 19.15%, 40.25% and 94.07%, respectively. After the treatment with rCgTNF-2, the percentage of EdU+ cells in haemocytes increased significantly (1.77-fold, p < 0.05) at 6 h compared with that in rGST-treated group, and the mRNA expressions of CgRunx, CgCyclin A, CgCDK2 and CgCDC45 in haemocytes all increased significantly (p < 0.05), which were 1.94-fold, 2.13-fold, 1.97-fold, 1.76-fold of that in rGST-treated group, respectively. Meanwhile, the protein abundance of CgRunx and CgCyclin A in the haemocytes of oysters in the rCgTNF-2-treated group increased, and the percentage of PI+ haemocytes in S phase also increased significantly (2.19-fold, p < 0.05) compared with that in rGST-treated group. These results collectively confirmed that CgTNF-2 was highly expressed in granulocytes and involved in the proliferation of haemocytes by regulating the expressions of CgRunx and cell cycle related genes in C. gigas.

CgCaspase-3 activates the translocation of CgGSDME in haemocytes of Pacific oyster Crassostrea gigas.

Cysteinyl aspartate specific proteinase-3 (Caspase-3) is an important protein involved in the apoptosis and gasdermin E (GSDME)-mediated cell pyroptosis pathways in vertebrates. A Caspase-3 homologue (designated as CgCaspase-3) was previously identified as an immune receptor specific for lipopolysaccharide (LPS) to regulate apoptosis in the Pacific oyster Crassostrea gigas. In the present study, the binding activity of CgCaspase-3 to different pathogen associated molecular patterns (PAMPs) and its effects on CgGSDME translocation in haemocytes were further investigated in C. gigas. The mRNA expression of CgCaspase-3 could be detected in all the tested tissues, including hepatopancreas, labial palp, adductor muscle, gonad, gill, mantle and haemocytes, and it was highly expressed in labial palp, gonad, haemocytes, and adductor muscle. The mRNA expression of CgCaspase-3 in haemocytes increased significantly at 3, 24, 48 and 72 h after LPS stimulation, and it increased significantly at 6, 12, 24 and 48 h after Vibrio splendidus stimulation. The recombinant CgCaspase-3 displayed binding activity towards LPS, mannose (MAN), peptidoglycan (PGN), and polyinosinic-polycytidylic acid potassium salt (Poly (I:C)). The positive signals of CgGSDME on haemocyte membrane became stronger at 3 h after V. splendidus stimulation, compared with that of Seawater group, and the co-localization of CgCaspase-3 and CgGSDME was observed in the haemocyte membrane. After the injection of dsCgCaspase-3, the positive signals of CgGSDME on haemocyte membrane became weaker compared with that of EGFP-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgCaspase-3 was able to bind diverse PAMPs and activate the translocation of CgGSDME in haemocytes of oyster response against pathogen invasion.

The involvement of an interferon-induced protein 44-like (CgIFI44L) in the antiviral immune response of Crassostrea gigas.

Interferon-stimulated genes (ISGs) encoding proteins are the essential executors of interferon (IFN) mediated antiviral defense. In the present study, an ISG member, interferon-induced protein 44-like (IFI44L) gene (designed as CgIFI44L-1) was identified from the Pacific oyster Crassostrea gigas. The ORF of CgIFI44L-1 cDNA was of 1437 bp encoding a polypeptide of 479 amino acids with a TLDc domain and an MMR_HSR1 domain. The mRNA transcripts of CgIFI44L-1 were detected in all the tested tissues with highest level in haemocytes, which was 15.78-fold of that in gonad (p < 0.001). Among the haemocytes, the CgIFI44L-1 protein was detected to be highly expressed in granulocytes with dominant distribution in cytoplasm. The mRNA expression level of CgIFI44L-1 in haemocytes was significantly induced by poly (I:C) stimulation, and the expression level peaked at 24 h, which was 24.24-fold (p < 0.0001) of that in control group. After the treatment with the recombinant protein of an oyster IFN-like protein (rCgIFNLP), the mRNA expression level of CgIFI44L-1 was significantly enhanced at 6 h, 12 h and 24 h, which was 2.67-fold (p < 0.001), 5.44-fold (p < 0.001) and 5.16-fold (p < 0.001) of that in control group, respectively. When the expressions of CgSTAT and CgIFNLP were knocked down by RNA interference (RNAi), the mRNA transcripts of CgIFI44L-1 were significantly down-regulated after poly (I:C) stimulation, which was 0.09-fold (p < 0.001) and 0.06-fold (p < 0.001) of those in EGFP group, respectively. These results suggested that CgIFI44L-1 was a conserved ISG in oyster, which was regulated by CgIFNLP and CgSTAT, and involved in the oyster antiviral immune response.