The Neuroendocrine Immunomodulatory Network In Oyster Crassostrea Gigas

The neuroendocrine-immune(NEI)regulatory network consists of nervous system,endocrine system and immune system,which carries a reciprocal regulation to maintain homeostasis in the host with the involvement of signaling molecules,such as neurotransmitters,hormones and cytokines.By far,most of the research conducted in vertebrates focus on neuropsychiatric disorders,developmental programming,disease pathogenesis,and comparative approaches,while little is known about the NEI network in invertebrates,especially in molluscs.In the present study,the structural basis,molecular composition,regulation patterns,as well as the physiological significances of the NEI network in molluscs have been explored by using multiple approaches including RNA-seq,bioinformatical analysis and biological validation.The nervous system of oyster is composed of two cerebral ganglia(CG)under the palps,one visceral ganglia(VG)next to the adductor muscle,and the nerve cords and nerve fibers which connect them together.One opioid receptor for enkephalin(ENK)(CgDOR),one muscarinic acetylcholine(ACh)receptor(CgmAChR-1)and one norepinephrine(NE)receptor(CgA1AR-1)were identified and cloned from oyster haemocytes.The ORF(open reading frame)of CgDOR was 1104 bp,encoding a protein of 367 amino acids.Three conserved cysteine residues(Cys95,Cys125 and Cys174)were identified by the alignment of CgDOR with other two vertebrate DORs,and the conserved Cys95 and Cys174 were predicted to form an important disulphide bridge by PROSITE.The ORF of CgmAChR-1 was 1983 bp and the polypeptide sequence contained 660 amino acids with a predicted molecular mass of 74.08 kDa.According to the phylogenic tree,CgmAChR-1 was closely matched with m5 muscarinic receptors found in invertebrates.Also,the nucleotide sequence of CgA1AR-1 was 1149 bp,and the polypeptide sequence contained 382 amino acids with the molecular mass predicted as 43.18 kDa.According to the phylogenic tree,CgA1AR-1 was closely matched with A1 ARs previously identified in vertebrates.The mRNA transcripts of all these three receptors were detected in oyster tissues such as haemocytes,gonad,gill,mantle,adductor muscle,kidney and hepatopancreas.The neuroendocrine system of oyster could be activated by CgTNF-1 through the activation of MAPK signal pathway and the transcription factor NF-κB and HSF,regulating the apoptosis,redox reaction,neuro-regulation and protein folding in oyster haemocytes.Besides,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.Moreover,the neuroendocrine system could also be triggered by LPS,resulting in the synthesis and release of neurotransmitters in both individual oyster and primarily cultured haemocytes.Oyster haemocytes were able to work as just immune cells and form the simple “nervous-haemocyte” neuroendocrine immunomodulatory axis-like pathway to mediate neuronal immunomodulation promptly by controlling the expression of specific neurotransmitter receptors on cell surface and modulating their binding sensitivities.Meanwhile,oyster haemocytes could also serve as a new source of cholinergic and adrenergic neurotransmitters to execute a negative regulation on innate immune response with similar autocrine/paracrine self-regulatory mechanisms identified in vertebrate lymphocytes.Moreover,oyster neurons might be activated after LPS or heat stress and then release neurotransmitters to modulate the immune response by promoting the expression of TNF and translocation of NF-κB in cytoplasm and nuclei of haemocytes.And,the immunomodulation of neurotransmitters ACh and ENK could induce differentially expression of lots of proteins and affect both humoral(SOD and CAT)and cellular(haemocyte apoptosis and phagocytosis)immunity through p53 signaling pathway,EGF-R-ErbB signaling pathway,and FcγR signaling pathway.All these results suggested that an analogous NEI network was also exist in molluscs,which was simple-in-structure but complex-in-function.This network could be activated by both cytokines and environmental stressors.The activated neuroendocrine system then release neurotransmitters,such as ACh,NE and ENK to modulate cellular and humoral immune responses through the EkbB,p53 and EGF signaling pathways,regulating the expression of cytokines and transcription factors including TNF-α,NF-κB and AP-1.In particular,oyster haemocytes could work as immune cells by mediating the immunomodulation of neurotransmitters released from the neuroendocrine system,and as a new sort of neuroendocrine cells by producing neurotransmitters and preforming autocrine/paracrine self-regulatory processes.Results in the present study would contribute to a better understanding of the NEI network lower forms of live,and shed light on the study of the evolution of NEI network.