Zhaoqun Liu

The enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas

Enkephalinergic neuroendocrine-immune regulatory system is one of the most important neuroendocrine-immune systems in both vertebrates and invertebrates for its significant role in the immune regulation. In the present study, the early onset of enkephalinergic nervous system and its immunomodulation on the developing immune system during the ontogenesis of oyster Crassostrea gigas were investigated to illustrate the function of neural regulation on the innate immune system in oyster larvae. [Met(5)]-enkephalin (Met-ENK) was firstly observed on the marginal of the dorsal half of D-hinged larvae. Six immune-related molecules, including four PRRs (CgCTL-1, CgCTL-2, CgCTL-4, CgNatterin-3) and two immune effectors (CgTNF-1 and CgEcSOD) were detected in the early developmental stages of trochophore, D-hinged and umbo larvae of oyster. After incubated with [Met(5)]-enkephalin, the mRNA expression level of all the PRRs changed significantly (p < 0.05). In trochophore larvae, the expression level of CgNatterin-3 decreased dramatically (p < 0.05) at 6 h, and the expression level of CgCTL-4 was significantly down-regulated at 3 h and 6 h (p < 0.05), respectively. In D-hinged and umbo larvae, only CgCTL-1 was significantly down-regulated and the differences were significant at 3 h and 6 h (p < 0.05), while the expression level of CgCTL-2 and CgCTL-4 increased significantly at 3 h after treatment (p < 0.05). Moreover, the expression levels of immune effectors were up-regulated significantly at 3 h and 6 h in trochophore larvae (p < 0.05). The expression level of CgTNF-1 in both blank and experiment groups was up-regulated but there was no significant difference in D-hinged larvae stage. On the contrary, the expression level of CgEcSOD in D-hinged larvae decreased dramatically at 3 h and 6 h after [Met(5)]-enkephalin incubation (p < 0.05). In umbo larvae, the expression level of CgTNF-1 and CgEcSOD in the experiment group increased significantly at 6 h after [Met(5)]-enkephalin treatment (p < 0.05), while no significant difference was found in the blank group. In addition, the anti-bacterial activities of the total protein extract from trochophore, D-hinged and umbo larvae increased significantly (p < 0.05) at both 3 h and 6 h after [Met(5)]-enkephalin incubation compared to that in the blank group, and PO activities of both D-hinged and umbo larvae total protein extract increased significantly (p < 0.05) while no significant difference was observed in trochophore larvae. The PO activities of the total protein extract in all the experiment groups decreased after the treatment with [Met(5)]-enkephalin for 6 h, but no significant difference was observed when compared to the blank group. Furthermore, after incubation for 6 h, the concentration of both CgTNF-1 and CgIL17-5 increased dramatically compared to that in the blank group (p < 0.05). These results together indicated that the enkephalinergic nervous system of oyster was firstly appeared in D-hinged larvae, while the primitive immune defense system existed in the region of prototroch in trochophore larvae and developed maturely after D-hinged larvae. The developing immune system could be regulated by the neurotransmitter [Met(5)]-enkephalin released by the neuroendocrine system in oyster C. gigas.

The immunomodulation mediated by a delta-opioid receptor for [Met(5)]-enkephalin in oyster Crassostrea gigas

Opioid receptors (OR) are a group of G protein-coupled receptors with opioids as ligands, which play an important role in triggering the second messengers to modulate immune response in vertebrate immunocytes. In the present study, the full length cDNA of a homologue of δ-opioid receptor (DOR) for [Met(5)]-enkaphalin was cloned from oyster Crassostrea gigas (designated as CgDOR), which was 1104 bp encoding a peptide of 367 amino acids containing a conserved 7tm_1 domain. After the stimulation of [Met(5)]-enkephalin, the concentration of second messengers Ca(2+) and cAMP in the HEK293T cells decreased significantly (p <0.05) with the expression of CgDOR. However, this trend was reverted with the addition of DOR antagonist BNTX. The CgDOR transcripts were ubiquitously detected in the tested tissues including haemocytes, gonad, mantle, kidney, gill, adductor muscle and hepatopancreas, with the highest expression level in the hepatopancreas. After LPS stimulation, the expression level of CgDOR mRNA began to increase (4.05-fold, p <0.05) at 6 h, and reached the highest level (5.00-fold, p <0.05) at 12 h. Haemocyte phagocytic and antibacterial activities increased significantly after [Met(5)]-enkephalin stimulation, whereas the increase was repressed with the addition of DOR antagonist BNTX. These results collectively suggested that CgDOR for [Met(5)]-enkephalin could modulate the haemocyte phagocytic and antibacterial functions through the second messengers Ca(2+) and cAMP, which might be requisite for pathogen elimination and homeostasis maintenance in oyster.

The simple neuroendocrine-immune regulatory network in oyster Crassostrea gigas mediates complex functions

The neuroendocrine-immune (NEI) regulatory network is a complex system, which plays an indispensable role in the immunity of the host. In the present study, the bioinformatical analysis of the transcriptomic data from oyster Crassostrea gigas and further biological validation revealed that oyster TNF (CgTNF-1 CGI_10018786) could activate the transcription factors NF-κB and HSF (heat shock transcription factor) through MAPK signaling pathway, and then regulate apoptosis, redox reaction, neuro-regulation and protein folding in oyster haemocytes. The activated immune cells then released neurotransmitters including acetylcholine, norepinephrine and [Met5]-enkephalin to regulate the immune response by arising the expression of three TNF (CGI_10005109, CGI_10005110 and CGI_10006440) and translocating two NF-κB (Cgp65, CGI_10018142 and CgRel, CGI_10021567) between the cytoplasm and nuclei of haemocytes. Neurotransmitters exhibited the immunomodulation effects by influencing apoptosis and phagocytosis of oyster haemocytes. Acetylcholine and norepinephrine could down-regulate the immune response, while [Met5]-enkephalin up-regulate the immune response. These results suggested that the simple neuroendocrine-immune regulatory network in oyster might be activated by oyster TNF and then regulate the immune response by virtue of neurotransmitters, cytokines and transcription factors.

The granulocytes are the main immunocompetent hemocytes in Crassostrea gigas

ABSTRACT Hemocytes comprise diverse cell types with morphological and functional heterogeneity and play indispensable roles in immunological homeostasis of invertebrates. The morphological classification of different hemocytes in mollusk has been studied since the 1970s, yet the involvement of the different sub‐populations in immune functions is far from clear. In the present study, three types of hemocytes were morphologically identified and separated as agranulocytes, semi‐granulocytes and granulocytes by flow cytometry and Percoll® density gradient centrifugation. The granulocytes were characterized functionally as the main phagocytic and encapsulating population, while semi‐granulocytes and agranulocytes exhibited low or no such capacities, respectively. Meanwhile, the lysosome activity and the productions of ROS and NO were all mainly concentrated in granulocytes under both normal and immune‐activated situations. Further, the mRNA transcripts of some immune related genes, including CgTLR, CgClathrin, CgATPeV, CgLysozyme, CgDefensin and CgIL‐17, were mainly expressed in granulocytes, lower in semi‐granulocytes and agranulocytes. These results collectively suggested that the granulocytes were the main immunocompetent hemocytes in oyster C. gigas, and a differentiation relationship among these three sub‐population hemocytes was inferred based on the gradual changes in morphological, functional and molecular features. HighlightsAgranulocytes, semi‐granulocytes and granulocytes were identified and separated.Phagocytosis and encapsulation were primarily executed by granulocytes, with little by others.Lysosome activity and production of ROS and NO were mostly concentrated in granulocytes.Immune genes CgTLR, CgLysozyme, CgDefensin and CgIL‐17, were mainly expressed in granulocytes.

The RNA-seq analysis suggests a potential multi-component complement system in oyster Crassostrea gigas.

Abstract The complement system is one of the major effector mechanisms of immune system, playing essential roles in both the innate and adaptive immune responses. In the present study, the counterparts of vertebrate complement components were identified by screening the sequenced genome of Crassostrea gigas, resulting in the identification of 792 gene models containing complement‐related domains. The transcriptome of haemocytes at 6, 12 and 24 h post lipopolysaccharides (LPS) stimulation showed differential expression of 77 C1q domain containing proteins, 53 C‐type lectins and 42 fibrinogen‐related proteins. mRNAs encoding 18 serine protease domain‐containing (SPC) proteins, 4 MACPF‐domain containing proteins and 11 C3 receptor‐like proteins were up‐regulated upon LPS stimulation, and CgC3 mRNA was significantly increased at 12 h. The presence of CgC3 was confirmed in cell free plasma and was present in three subunit chains as expected for the processed mature protein. The complement related PRRs with coiled coil regions and SPC proteins with CUB domains may function in the activation of CgC3, whereas, the C3‐like receptors with integrin‐&agr;/&bgr; domain mediated the phagocytosis of C3‐labled pathogens. These PRRs appear to serve as opsonins to promote phagocytosis of opsonized pathogens. The overall results suggested the existence of a potential multi‐component complement system in C. gigas. HighlightsThe counterparts of vertebrate complement components were identified from the genome of Crassostrea gigas.The transcriptome of haemocytes post LPS stimulation showed hundreds of differential expression genes of potential complement components.The presence of CgC3 was confirmed in cell free plasma and was present in three subunit chains.Complement related PRRs with coiled coil regions and SPC proteins with CUB domains may function in activation of CgC3.C3‐like receptors with integrin‐&agr;/&bgr; domain might mediate phagocytosis of C3‐labled pathogens.

The neuroendocrine immunomodulatory axis-like pathway mediated by circulating haemocytes in pacific oyster Crassostrea gigas

The neuroendocrine-immune (NEI) regulatory network is a complex system, which plays an indispensable role in the immunity of host. In this study, a neuroendocrine immunomodulatory axis (NIA)-like pathway mediated by the nervous system and haemocytes was characterized in the oyster Crassostrea gigas. Once invaded pathogen was recognized by the host, the nervous system would temporally release neurotransmitters to modulate the immune response. Instead of acting passively, oyster haemocytes were able to mediate neuronal immunomodulation promptly by controlling the expression of specific neurotransmitter receptors on cell surface and modulating their binding sensitivities, thus regulating intracellular concentration of Ca2+. This neural immunomodulation mediated by the nervous system and haemocytes could influence cellular immunity in oyster by affecting mRNA expression level of TNF genes, and humoral immunity by affecting the activities of key immune-related enzymes. In summary, though simple in structure, the ‘nervous-haemocyte’ NIA-like pathway regulates both cellular and humoral immunity in oyster, meaning a world to the effective immune regulation of the NEI network.

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.

Soluble adenylyl cyclase mediates mitochondrial pathway of apoptosis and ATP metabolism in oyster Crassostrea gigas exposed to elevated CO2

Abstract Ocean acidification (OA) has deleterious impacts on immune response and energy homeostasis status of Mollusca. In the present study, the apoptosis ratio of hemocytes and the adenosine triphosphate (ATP) allocation in gill tissues were determined after Pacific oysters Crassostrea gigas were exposed to elevated CO2 environment (pH = 7.50) for 16 days.The apoptosis ratio in CO2 exposure group (35.2%) was significantly higher (p < 0.05) than that in the control group, and the increased apoptosis ratio induced by elevated CO2 could be significantly inhibited (p < 0.05) by KH7, a specific inhibitor of a bicarbonate sensor soluble adenylyl cyclase (sAC). After CO2 exposure, sAC in oyster (CgsAC) was found to be clustered with mitochondria in the cytoplasm, and the pro‐caspase‐3 was cleaved into two small fragments. Moreover, the activities of caspase‐3 and caspase‐9 also increased post CO2 exposure and these increases could be inhibited by KH7. However, the activities of caspase‐8 did not change significantly compared with that in the control group. After CO2 exposure, the ATP content in the gill increased significantly (p < 0.05) and such increase could also be inhibited by KH7. The ATP content in purified gill mitochondria decreased significantly (p < 0.05) after CO2 exposure, which was also inhibited by KH7. These results implied that the elevated CO2 could activate the mitochondria‐CgsAC pathway of apoptosis and ATP metabolism in oyster, and this pathway played essential roles in maintaining the homeostasis and the balance of energy metabolism in response to OA. HighlightsCgsAC mediates the increase of apoptosis rate caused by CO2 exposure.The signal of CgsAC was clustered with mitochondria after CO2 exposure.CgsAC mediates the increase of Caspase‐3 and caspase‐9 activity caused by CO2 exposure.CgsAC mediates the increase of ATP in gill tissue caused by CO2 exposure.CgsAC mediates the decrease of ATP in gill mitochondria caused by CO2 exposure.

Transcriptomic analysis of oyster Crassostrea gigas larvae illustrates the response patterns regulated by catecholaminergic system upon acute heat and bacterial stress

&NA; Bacterial infection and heat stress, as two major environmental threats of marine molluscs, could affect larval development and dramatically promote mortality of oysters. In the present study, next‐generation sequencing, together with determinations of mRNA expression and measurements of enzyme activities, were employed to understand the response patterns of oyster larvae under acute heat and bacterial stress. After RNA‐seq, a total of 9472 differentially expressed genes including 4895 significantly up‐regulated ones and 4577 significantly down‐regulated ones were obtained from 12 transcriptome libraries. GO overrepresentation analysis of the up‐regulated genes revealed that the neuroendocrine immunomodulation pathway was activated after acute heat and bacterial stimulation, in which the catecholaminergic regulation played an important role. GO overrepresentation analysis of the down‐regulated genes suggested that the immune capacity of Crassostrea gigas larvae was suppressed under stress, which was further validated since superoxide dismutase (SOD) and phenoloxidase (PO) activities in the total protein extract of larvae decreased dramatically after stress. Moreover, the shell formation of trochophore was inhibited and severe mortality was caused after acute heat and bacterial stress. These results collectively indicated that acute heat and bacterial stress could significantly inhibit larval development and suppress immune response of oyster C. gigas larvae. And the neuroendocrine immunomodulation, especially the catecholaminergic regulation, played an indispensable role in the stress response of molluscan larvae. HighlightsThe catecholaminergic system was activated after acute heat and bacterial stress in oyster C. gigas larvae.Acute heat and bacterial stress could inhibit shell formation and cause severe mortality of C. gigas larvae.SOD and PO activities in the total protein extract of larvae decreased dramatically after stress.The immune capacity of C. gigas larvae was suppressed under acute heat and bacterial stress.

A novel junctional adhesion molecule A (CgJAM-A-L) from oyster (Crassostrea gigas) functions as pattern recognition receptor and opsonin.

Junctional adhesion molecule (JAM), a subfamily of immunoglobulin superfamily (IgSF) with a couple of immunoglobulin domains, can act as regulator in homeostasis and inflammation of vertebrates. In the present study, a structural homolog of JAM-A (designated CgJAM-A-L) was screened out from oyster, Crassostrea gigas, through a search of JAM-A D1 domain (N-terminal Ig domain in JAM-A). The cDNA of CgJAM-A-L was of 1188 bp encoding a predicted polypeptide of 395 amino acids. The immunoreactive area of CgJAM-A-L mainly distributed over the plasma membrane of hemocytes. After Vibro splendidus or tumor necrosis factor (CgTNF-1) stimulation, the mRNA transcripts of CgJAM-A-L in hemocytes increased significantly by 4.46-fold and 9.00-fold (p < 0.01) of those in control group, respectively. The recombinant CgJAM-A-L protein (rCgJAM-A-L) could bind multiple PAMPs including lipopolysaccharides (LPS), peptidoglycan (PGN), lipoteichoic acid (LTA), mannose (MAN), β-glucan (GLU) and poly(I:C), and various microorganisms including Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Vibro anguillarum, V. splendidus, Pastoris pastoris and Yarrowia lipolytica. The phagocytic rates of oyster hemocytes towards Gram-negative bacteria V. anguillarum and yeast P. pastoris were significantly enhanced after the incubation of rCgJAM-A-L, and even increased more significantly after the pre-incubation of rCgJAM-A-L with microbes (p < 0.01). The results collectively indicated that CgJAM-A-L functioned as an important pattern recognition receptor (PRR) and opsonin in the immune defense against invading pathogen in oyster. Moreover, as the most primitive specie with homolog of JAMs, the information of CgJAM-A-L in oyster would provide useful clues for the evolutionary study of JAMs and immunoglobulins.