CgDM9CP-5-Integrin-MAPK Pathway Regulates the Production of CgIL-17s and Cgdefensins in the Pacific Oyster, Crassostrea gigas.

DM9 domain containing protein (DM9CP) is a family of newly identified recognition receptors exiting in most organisms except plants and mammals. In the current study, to our knowledge, a novel DM9CP-5 (CgDM9CP-5) with two tandem DM9 repeats and high expression level in gill was identified from the Pacific oyster, Crassostrea gigas. The deduced amino acid sequence of CgDM9CP-5 shared 62.1% identity with CgDM9CP-1 from C. gigas, and 47.8% identity with OeFAMeT from Ostrea edulis. The recombinant CgDM9CP-5 (rCgDM9CP-5) was able to bind d-mannose, LPS, peptidoglycan, and polyinosinic-polycytidylic acid, as well as fungi Pichia pastoris, Gram-negative bacteria Escherichia coli and Vibrio splendidus, and Gram-positive bacteria Staphylococcus aureus. The mRNA transcript of CgDM9CP-5 was highly expressed in gill, and its protein was mainly distributed in gill mucus. After the stimulations with V. splendidus and mannose, mRNA expression of CgDM9CP-5 in oyster gill was significantly upregulated and reached the peak level at 6 and 24 h, which was 13.58-fold (p < 0.05) and 14.01-fold (p < 0.05) of that in the control group, respectively. CgDM9CP-5 was able to bind CgIntegrin both in vivo and in vitro. After CgDM9CP-5 or CgIntegrin was knocked down by RNA interference, the phosphorylation levels of JNK and P38 in the MAPK pathway decreased, and the expression levels of CgIL-17s (CgIL-17-3, -4, -5, and -6), Cg-Defh1, Cg-Defh2, and CgMolluscidin were significantly downregulated. These results suggested that there was a pathway of DM9CP-5-Integrin-MAPK mediated by CgDM9CP-5 to regulate the release of proinflammatory factors and defensins in C. gigas.

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.

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.

CgRab1 regulates Cgcathepsin L1 expression and participates in the phagocytosis of haemocytes in oyster Crassostrea gigas.

Rab protein plays an important role in a variety of cellular activities, especially the fusion process of the inner membrane during endocytosis. In the present study, a Rab1 protein (CgRab1) was identified from the Pacific oyster Crassostrea gigas. The full-length cDNA sequence of CgRab1 was of 2248 bp with an open reading frame of 618 bp, encoding a polypeptide of 205 amino acids containing a Rab domain. The deduced amino acid sequence of CgRab1 shared 94.2% and 89.3% identity with Rab1 from Pomacea canaliculata and Homo sapiens respectively. In the phylogenetic tree, CgRab1 was firstly clustered with the Rab1s from Aplysia californica and Pomacea canaliculata to form a sister group with Rab1 from invertebrates. The recombinant CgRab1 protein (rCgRab1) was able to bind GTP. The mRNA transcripts of CgRab1 were highly expressed in oyster haemocytes, and its expression level in oyster haemocytes was significantly up-regulated at 24 h after the stimulations with Vibro splendidus, which was 2.43-fold (p < 0.01) of that in the control group. After the expression of CgRab1 was knocked down (0.38-fold of that in EGFP-RNAi experimental group) by RNAi，the protein expression of Cgcathepsin L1 were reduced (0.63-fold, p < 0.01) compared with that in EGFP-RNAi experimental group. The phagocytic rate and phagocytic index of haemocytes in CgRab1-RNAi oysters decreased after V. splendidus stimulation, which was 0.50-fold (p < 0.01) and 0.58-fold (p < 0.01) of that in EGFP-RNAi experimental group. These results indicated that CgRab1 was involved in the process of haemocytes phagocytosis by regulating the expression of Cgcathepsin L1 in oyster C. gigas.

The characterization of an agranulocyte-specific marker (CgCD9) in the Pacific oyster Crassostrea gigas.

The agranulocytes in the Pacific oyster Crassostrea gigas are a group of haemocytes that are significantly different from semi-granulocytes and granulocytes on the morphology. Agranulocytes are the smallest haemocytes characterized by a spherical shape, the largest ratio of nucleus to cytoplasm, and no granules in the cytoplasm. The lack of unique cell surface markers impedes the isolation of agranulocytes from total haemocytes. Previous transcriptome sequencing analysis of three subpopulations of haemocytes revealed that a homologue of CD9 (designed as CgCD9) was highly expressed in agranulocytes of oyster C. gigas (data not shown). In the present study, CgCD9 was identified to share a similarity of 60% with other vertebrates CD9s, and it harbored a typical four transmembrane domain and a conserved Cys-Cys-Gly (CCG) motif. The mRNA transcript of CgCD9 was found to be highly expressed in agranulocytes, which was 6.63-fold (p < 0.05) and 3.68-fold (p < 0.05) of that in granulocytes and semi-granulocytes, respectively. A specific monoclonal antibody of CgCD9 (named 3D8) was successfully prepared by traditional hybridoma technology, and a single positive band at 25.2 kDa was detected in the haemocyte proteins by Western Blotting, indicating that this monoclonal antibody exhibited high specificity and sensitivity to CgCD9 protein. The ELISA positive value of 3D8 monoclonal antibody to recognize agranulocytes, semi-granulocytes and granulocytes was 17.35, 4.48 and 1.55, respectively, indicating that monoclonal antibody was specific to agranulocytes. Immunocytochemistry assay revealed that CgCD9 was specifically distributed on the membrane of agranulocytes. Using immunomagnetic beads coated with 3D8 monoclonal antibody, CgCD9+cells with a purity of 94.53 ± 5.60% were successfully isolated with a smaller diameter, a larger N:C ratio and no granules in cytoplasm, and could be primary culture in the modified L-15 medium in vitro. Collectively, these results suggested that CgCD9 was a specific cell surface marker for agranulocytes, which offered a tool for high-purity capture of agranulocytes from total haemocytes in C. gigas.

CgATP synthase ß subunit involved in the regulation of haemocytes proliferation as a CgAstakine receptor in Crassostrea gigas.

Astakine is considered as an endogenous cytokine-like factor of prokineticin homologue in invertebrate. Recently, an astakine homologue (CgAstakine) has been identified and characterized in oyster Crassostrea gigas. In the present study, a CgATP synthase ß subunit was identified as the receptor of CgAstakine in C. gigas. There was an ATP-synt_ab_N domain and an AAA domain in the CgATP synthase ß subunit protein. The mRNA transcripts of CgATP synthase ß subunit were detected in all tested tissues, with the highest expression level in hepatopancreas and gills, which was 109.11-fold (p < 0.01) and 97.21-fold (p < 0.01) of that in labial palps, respectively. After rCgAstakine stimulation, the mRNA transcripts of CgATP synthase ß subunit in agranulocytes and semi-granulocytes were significantly increased at 24 h (2.44-fold, and 9.01-fold of that in control group, p < 0.01), and those in granulocytes were significantly increased at 6 h (1.83-fold, p < 0.01), 12 h (1.92-fold, p < 0.01) and 24 h (3.47-fold, p < 0.01). The expression level of CgATP synthase ß subunit protein in agranulocytes and granulocytes was also significantly increased after rCgAstakine stimulation, which was 1.64-fold (p < 0.05) and 1.85-fold (p < 0.05) of that in control group, respectively, while there were no significant changes in semi-granulocytes. The immunofluorescence assay showed that CgATP synthase ß subunit positive signals were mainly located on the membrane of haemocytes. The number of haemocytes with EdU positive signals was significantly increased after rCgAstakine stimulation (2.04-fold of seawater group, p < 0.01), while significantly decreased after the RNA interference (RNAi) of CgATP synthase ß subunit, which was 0.28-fold of that in NC group (p < 0.01). Bio-layer interferometry (BLI) assay confirmed in vitro interaction between rCgAstakine and rCgATP synthase ß subunit. There results suggested that CgATP synthase ß subunit acts as the receptor of CgAstakine and plays important roles in CgAstakine induced renewal of haemocytes in C. gigas.

A new member of the runt domain family from Pacific oyster Crassostrea gigas (CgRunx) potentially involved in immune response and larvae hematopoiesis.

The Runx family is a kind of heteromeric transcription factors, which is defined by the presence of a runt domain. As transcriptional regulator during development and cell fate specification, Runx is best known for its critical roles in hematopoiesis. In the present study, a Runx transcription factor (designed as CgRunx) was identified and characterized from the oyster Crassostrea gigas. The complete coding sequence of CgRunx was of 1638 bp encoding a predicted polypeptide of 545 amino acids with one conserved runt domain, which shared high similarity with other reported Runx proteins. CgRunx was highly expressed in hemocytes, gill and mantle both at the protein and nucleic acid levels. CgRunx protein was localized specifically in the cell nuclei of hemocytes, and distributed at the tubule lumen of gill filament. During the larval developmental stages, the mRNA transcripts of CgRunx gradually increased after fertilization, reached to a relative high level at the 8 cell embryos and the blastula stage of 2-4 hpf (hours post fertilization) (about 40-fold), and peaked at early trochophore larvae (10 hpf) (about 60-fold). Whole-mount immunofluorescence assay further revealed that the abundant immunofluorescence signals of CgRunx distributed through the whole embryo at blastula stage (5 hpf), and progressively reduced with the development to a ring structure around the dorsal region in trochophore larvae (10 hpf). Scattered positive immunoreactivity signals finally appeared in the velum region of D-veliger larvae. After LPS and Vibrio splendidus stimulations, the expression levels of CgRunx mRNA in hemocytes were up-regulated significantly compared with that in the control (0 h), which were 2.98- and 2.46-fold (p < 0.05), 2.67- and 1.5-fold (p < 0.05), 2.36- and 1.38-fold (p < 0.05) at 3 h, 6 h and 12 h, respectively. These results collectively suggested that CgRunx involved in immune response and might participate in larvae hematopoiesis in oyster.

CgAATase with specific expression pattern can be used as a potential surface marker for oyster granulocytes.

Granulocytes are known as the main immunocompetent hemocytes that play important roles in the immune defense of oyster Crassostrea gigas. In the present study, an alcohol acyltransferase (designed as CgAATase) with specific expression pattern was identified from oyster C. gigas, and it could be employed as a potential marker for the isolation of oyster granulocytes. The open reading frame (ORF) of CgAATase was of 1431 bp, encoding a peptide of 476 amino acids with a typically conserved AATase domain. The mRNA transcripts of CgAATase were highest expressed in hemocytes, lower expressed in hepatopancreas, mantle, gonad, gill, ganglion, adductor muscle, and labial palp. The mRNA expression level of CgAATase in hemocytes was significantly up-regulated at 3-12 h and reached the highest level (27.40-fold compared to control group, p < 0.05) at 6 h after Vibrio splendidus stimulation. The total hemocytes were sorted as granulocytes, semi-granulocytes and agranulocytes by Percoll® density gradient centrifugation. CgAATase transcripts were dominantly observed in granulocytes, which was 8.26-fold (p < 0.05) and 2.80-fold (p < 0.05) of that in agranulocytes and semi-granulocytes, respectively. The monoclonal antibody against CgAATase was produced and employed for the isolation of granulocytes with the immunomagnetic bead. CgAATase protein was mainly detected on the cytomembrane of granulocytes. About 85.7 ±â€¯4.60% of the granulocytes were positive for CgAATase and they could be successfully separated by flow cytometry with immunomagnetic bead coated with anti-CgAATase monoclonal antibody, and 97.7 ±â€¯1.01% of the rest hemocytes (agranulocytes and semi-granulocytes) were negative for CgAATase. The isolated primary granulocytes could maintain cell activity for more than one week in vitro culture that exhibited numerous filopodia. These results collectively suggested that CgAATase was a potential marker of oyster granulocytes, and the granulocytes could be effectively isolated from total circulating hemocytes by immunomagnetic bead coated with the anti-CgAATase monoclonal antibody.

E3 Ubiquitin Ligase CHIP Inhibits Haemocyte Proliferation and Differentiation via the Ubiquitination of Runx in the Pacific Oyster.

Mollusca first evolve primitive immune cells (namely, haemocytes), which assemble a notable complex innate immune system, which are continuously produced through proliferation and differentiation and infused in the haemolymph. As a typical E3 ligase, CHIP is critical for immune cell turnover and homeostasis in vertebrates. In this study, a CHIP homolog (CgCHIP) with a high expression in haemocytes was identified in oysters to investigate its role in the proliferation and differentiation of ancient innate immune cells. CgCHIP exhibited a widespread distribution across all haemocyte subpopulations, and the knockdown of CgCHIP altered the composition of haemocytes as examined by flow cytometry. Mechanistically screened with bioinformatics and immunoprecipitation, a key haematopoietic transcription factor CgRunx was identified as a substrate of CgCHIP. Moreover, amino acids in the interacted intervals of CgCHIP and CgRunx were determined by molecular docking. Experimental evidence from an in vitro culture model of an agranulocyte subpopulation and an in vivo oyster model revealed that the knockdown of CgCHIP and CgRunx had opposing effects on agranulocyte (precursor cells) differentiation and granulocyte (effector cells) proliferation. In summary, CgCHIP negatively regulated agranulocyte differentiation and granulocyte proliferation by mediating the ubiquitination and degradation of CgRunx in oysters. These results offer insight into the involvement of ubiquitylation in controlling haemocyte turnover in primitive invertebrates.

A transcription factor ATF3 involves in the phagocytosis of granulocytes in oyster Crassostrea gigas.

Phagocytosis is a major cellular mechanism for mollusk granulocytes to eliminate nonself substances and dead cells, and thus to preserve the immune homeostasis. The knowledge of the regulatory mechanisms controlling phagocytic capacity is vital to understanding the immune system. In the present study, an ATF3 homolog (CgATF3) with a typical bZIP domain was identified in the Pacific oyster Crassostrea gigas. Its highly conserved bZIP domain consisted of two structural features, a basic region for DNA binding and a leucine zipper region for dimerization. Its transcript was found to be abundantly expressed in haemocytes, which was induced by Vibrio splendidus stimulation and recombinant CgTNF-2 treatment, along with an increase of its protein content in the nucleus. Moreover, CgATF3 showed a consistent and specific high expression in granulocytes, and CgATF3+ granulocytes were characterized morphologically by the largest diameter, smaller nucleus to cytoplasmic ratio, and abundant cytoplasmic granules, and functionally by a higher capacity for phagocytosis. When CgATF3 expression was inhibited by RNAi, the expression levels of CgRab1, CgRab33 and CgCathepsin L1, as well as the phagocytic rate and index of granulocytes all decreased after V. splendidus stimulation. These results together demonstrated the involvement of CgATF3 in regulating the expressions of Rabs and Cathepsin L1, as well as the phagocytosis of granulocytes in oyster C. gigas.