Preliminary Study On Cholinergic Neuro-immune Regulatory Ystem In Scallop Chlamvs Farreri

The neuroendocrine immune system mediated by acetylcholine (ACh) plays acrucial role in the immunomodulation process of vertebrate. The knowledge aboutcholinergic neuro-immune regulatory is mainly from vertebrates, while there is norelative report in invertebrate. In this study, the components and activation ofcholinergic neuro-immune system, and its immunomodulation in immune responsewere investigated to preliminarily understand the molecular mechanism of cholinergicneuro-immune regulatory network in scallop Chlamys farreri.In the present study, an acetylcholinesterase (CfAChE) gene and two nicotinicacetylcholine receptor alpha subunit (CfnAChR1and CfnAChR2) genes werecloned from scallop Chlamys farreri, which encoded522,443and384amino acidsrespectively. The CfAChE shared the conserved classic active site triad and peripheralanionic site with all the other AChEs. The CfnAChR1and CfnAChR2had theconserved extracellular ligand binding domain and transmembrane domain, whichwas similar to nAChR7from vertebrates. The mRNA transcripts of CfAChE,CfnAChR1CfnAChR2were expressed constitutively in the haemocytes, adductormuscle, gills, kidney, mantle, gonad and hepatopancreas. The ACh and ChAT weredetected in all the tested tissues by using immunofluorescence assay with anti-ChATand anti-ACh antibodies. And nAChR7, CfnAChR1and CfnAChR2were alsodetected in all the tested tissues except mantle. The recombinant CfAChE in Pichiapastoris GS115could catalyse the reaction from acetylcholine (ACh) to choline andacetic acid effectively. The results indicated a cholinergic nervous system probablyexisted in scallop C. farreri.After the lipopolysaccharide (LPS) stimulation, the ACh concentration in hemolymph of scallop increased significantly and reached the highest level (14.36×10-6M,P <0.05) at6h. The CfAChE mRNA transcripts level was up-regulatedobviously between12h and24h (P <0.05). The CfnAChR1mRNA transcripts levelin haemocytes was10.8and13.9folds of that in the blank group at3h and24hresepectively. When scallops were treated with tumor necrosis factor-alpha (TNF-),the ACh concentration in hemolymph reached the highest level (15.51×10-6M, P <0.05) at3h. The CfAChE and CfnAChR1expression level in haemocytes weresignificantly higher than that in the blank and control groups (P <0.05). Nosignificant change of CfnAChR2expression level was observed in haemocytesduring LPS and TNF-stimulations. The mRNA expression level of immune-relatedenzymes, including superoxide dismutase (SOD), catalase (CAT) and lysozyme (LYZ),reached the peak at3h,6h and12h respectively after LPS challenge (P <0.05),while their up-regulation level was significantly lower than that in the AChCl+LPStreatment group. When the haemocytes were incubated with anti-CfnAChR1andanti-CfnAChR2antibodies, nicotine and LPS, the nicotine could inhibited thehaemocytes releasing TNF-to the cell culture medium, while blocking theCfnAChR1receptor could eliminate the nicotine inhibition.During the embryo development stages of scallop, the mRNA transcripts ofCfAChE, CfnAChR1and CfnAChR2could be detected from the fertilized eggs toeyebot larval stage through real-time PCR assay. And the cholinergic neuron ofscallop could be detected in trochophore, D-hinged larval and umbo-veliger stages. Inthe trochophore and D-hinged larval stages, the CfAChE and CfnAChR1mRNAtranscripts increased significantly after the Vibrio anguillarum challenge, while theCfnAChR2expression level did not change in trochophore and increasedsignificantly in D-hinged larval. After the different concentration of nicotine treatment,the CfSOD mRNA transcripts was inhibited significantly during trochophore andD-hinged larvals, and the CfCAT and CfLYZ mRNA expression level decreased introchophore and increased in D-hinged larval stages significantly. The differentconcentration of MG treatment could increase the CfSOD, CfCAT and CfLYZ mRNAexpression level in trochophore and D-hinged larvals obviously. All the results suggested the existence of the complete cholinergicimmunomodulation system in scallop C. farreri, and this system could be activated byimmune response against LPS snd TNF-stimulations. The activated cholinergicnervous system regulated the ACh concentration and the mRNA expression involvedin the cholinergic signal pathway. Then the increased ACh could modulate theimmune response of scallop ACh receptors located in the immunocytes.