| Neuroendocrine-immune(NEI)regulatory network is consisted of multidirectional interactions among the immune,endocrine and nervous systems,and it plays vital roles in homeostasis maintenance of organism.Recent studies in vertebrates suggest that neurotransmitters are not only synthesized and released from neurocytes to regulate immune response,but also released from immunocytes and functioned on the immune cells in an autocrine or paracrine manner.The research on the roles of neurotransmitters synthesized from immune cells in the origin and development of immune system,and immune response of immune cells have become the focus in recent years.At present,some neurotransmitters and their synthetic related genes have also been found in invertebrates,but the researches on the distribution and immune response pattern of their in invertebrate immunocytes are still poorly understood.In the present study,the expression profiles of neurotransmitters synthetic related genes and their receptors in haemocytes of the pacific oyster Crassostrea gigas,response patterns of various neurotransmitters after immune stimulations and their mediated immune responses were investigated by multiple approaches including RNAseq,molecular biology,cell biology and immunology.Multiple neurotransmitter synthetic pathways were revealed in oyster haemocytes,and these neurotransmitters were found to respond to immune stimulation differentially and palyed multiple immunomodulatory roles,which provided valuable reference for further understanding the neuroendocrine-immune system and its involvements in maintaining immune homeostasis in invertebrates.The main results obtained from this study are listed as follows:1.There were multiple neurotransmitters synthetic pathways in oyster haemocytes,and those neurotransmitter-related genes could respond dynamically to LPS stimulation.The genes expressed in oyster haemocytes accounted for 59%of the total number of genomic genes,of which 809 genes were significantly higher expressed in haemocytes and mainly enriched in neural-endocrine-immune-related pathways.There were 147 genes specifically expressed in haemocytes including 23 receptors for neurotransmitters,neuropeptide or immune recognition which played important roles in the signal sensor and pathogen recognition in haemocytes.The synthetic related genes for monoamine neurotransmitters(dopamine,DA,5-hydroxytryptamine,5-HT,and norepinephrine,NE),choline neurotransmitter(acetylcholine,ACh),neuropeptide neurotransmitter(FMRFamide)and amino acid neurotransmitters(glutamate,Glu and gamma aminobutyric acid,GABA)were all expressed in haemocytes.After LPS stimulation,neurotransmitter Glu synthetic related genes,GABA and ACh synthetic related genes,and DA and 5-HT synthetic related genes increased significantly at early and middle stages(6,12 h),late stage(24 h),and middle and late stages(12,24 h),respectively.2.The highly expressed synthases and receptors of neurotransmitters in oyster haemocytes had conservative structures and functions,as well as distinct distributions.The highly expressed NE synthase CgDBH-1 and its receptor Cga2AR,FMRFamide precursor CgFMRFamide and its receptor CgFMRFR-1,and acetylcholine synthase CgChAT and its receptor CgnAChR in oyster haemocytes were screened according to transcriptome and bioinformatics analysis.And sequence and functional characteristics of these molecules indicated that they were evolutionarily conservative.CgDBH-1,with 569 amino acids,contained nine conserved cysteine residues and six conserved dopamine β-hydroxylase catalytic activity sites,and possessed enzyme activity on synthesizing NE.Cga2AR,with 399 amino acids,had the highest similarity to α2 type AR sequences from human and mouse,and was closely clustered with α2 ARs from other invertebrates.CgFMRFamide contained 432 amino acids and could be processed into nineteen FMRFamide peptides.CgFMRFR-1,with 363 amino acids,contained seven transmembrane domains.CgChAT,with 523 amino acids,had the ability to synthesize ACh.CgnAChR,with 276 amino acids,contained a LBD domain.CgDBH1,Cga2AR,CgFMRFR-1 and CgnAChR were all specifically highly expressed in oyster haemocytes.CgDBH-1 and Cga2AR,CgFMRFR-1,and CgChAT and CgnAChR were mainly distributed in granulocytes,semi-granulocytes and agranulocytes,respectively.3.Binding NE to the receptor Cgα2AR mediated the expressions of cytokines by regulating cAMP concentration in haemocytes.Both of NE synthase CgDBH-1 and receptor Cgα2AR exhibited a tendency of upregulation at 12 h after LPS stimulation.NE was found to regulate the concentration of intracellular cAMP,but had no effect on the concentration of intracellular Ca2+through binding to Cga2AR.In vivo RNA interference and receptor antagonist treatment experiments both proved that Cga2AR could up-regulate the expressions of CgIL17-5,CgTNF-1 and CgTNF-2 in oyster haemoctyes.4.FMRFamide could activate p38 MAP kinase pathway,and regulate the expressions of immune effectors and apoptosis-related genes in oyster haemocytes.Both of FMRFamide precursor CgFMRFamide and its receptor CgFMRFR-1 could respond to immune stimulation and air exposure.The expressions of CgFMRFamide and CgFMRFR-1 were significantly up-regulated at 12 h and 6-12 h after LPS stimulation,respectively.The highest expression levels of CgFMRFamide and CgFMRFR-1 were observed at 3 d and 6 d after air exposure,respectively.After the concentration of FMRFamide in oysters was increased by exogenous injection,an increased phosphorylation of p38 MAP kinase in haemocytes was detected,and the expressions of pro-inflammatory cytokine CgIL17-5,as well as the apoptosis-related genes of CgCaspase-1 and CgCaspase-3 in haemocytes were promptly increased,while the concentration of nitric oxide(NO)in haemolymph supernatant was significantly down-regulated.Meanwhile,glucose concentration in haemolymph supernatant was increased significantly after FMRFamide injection.On the contrary,the expression of CgFMRFamide in oyster haemocytes was decreased significantly after glucose injection.5.The ACh system in oyster haemocytes was involved in regulating the immune priming.Both of the expressions of ACh synthase CgChAT and its receptor CgnAChR in oyster haemocytes were significantly decreased at 12 h,but significantly increased at 48 h after LPS stimulation,indicated that ACh system played a vital role at the late stage of immune response.The expressions of CgChAT and CgnAChR in oyster haemocytes,and the concentration of ACh in haemolymph supernatant were all significantly up-regulated at 6 h after two consecutive stimulations with Vibrio splendidus,suggested that ACh system was involved in the immune adaption of oyster.In conclusion,there were multiple neurotransmitters synthetic pathways in oyster haemocytes.The expression profiles of different neurotransmitters’s synthases and receptors showed immune cell type specific charactristics,and the synthases and receptors of those neurotransmitters also had varied response patterns at different stages of immune response.The neurotransmitters of NE and FMRFamide responding at the early and middle stages of immune response could enhance immune defense to eliminate the pathogen quickly and maintain the homeostasis.The neurotransmitters of ACh responding at the late stage of immune response might prevent the body from damage caused by excessive immunity and achieve the recovery of homeostasis by inhibiting the immune response.The results in the present study not only enriched a new role for invertebrate immunocytes on autonomously synthesizing neurotransmitters,but also improved the understanding of the relationship between nervous system and immune system in evolution.Meanwhile,it was of great significance to uncover the maintenance mechanism of immune homeostasis in invertebrates from the perspective of neuroendocrine-immune regulatory system. |
