Immunomodulatory Role Of Neuropeptides AST-B,sNPF And Their Receptor In The Mud Crab,Scylla Paramamosain

Based on molecular,cellular and physiological studies in animal models,it has been demonstrated that the functions of the nervous,endocrine and immune systems are interdependent and that these systems are regulated in both directions by the release of signaling molecules such as neuropeptides,cytokines and hormones,resulting in formation of the neuroendocrine-immune regulatory system(NEI system).The NEI system currently plays an integral role in optimizing the immune response and maintaining homeostasis,and neuropeptides as an important part of this system have attracted more and more attention.B-allatostatin(AST-B)and short neuropeptide F(sNPF)are neuropeptides with pleiotropic physiological effects in arthropods,have been identified in many crustaceans,however,research on their physiological functions in crustaceans is still very scarce.The mud crab Scylla paramamosain is a marine crustacean with high economic value in China.It possesses complete neural and endocrine organs,which can be used as an ideal model to study the NEI system.In this study,the tissue distribution of two neuropeptides,AST-B and sNPF,and their respective receptors,AST-BR and sNPF-R,were analyzed in the mud crab,and their expression patterns following immune stimulation were examined.Subsequently,their neuroendocrine-immunomodulatory effects mediated in the mud crab were investigated by in vitro and in vivo experiments.The results of the study are as follows:1)This study investigated the immunomodulatory function of AST-B in hemocytes of the mud crab.Tissue distribution studies showed that Sp-AST-B is mainly expressed in the nervous system,while its receptor Sp-AST-BR can be expressed in all tested tissues including hemocytes.In situ hybridization analysis further showed that Sp-AST-B was widely localized in a variety of neurons in the thoracic ganglion and SpAST-BR was localized in granular cells of the hemocyte subpopulation.Immune stimulation experiments showed that the pathogenic mimics lipopolysaccharide(LPS)and polyriboinosinic polyribocytidylic acid(Poly(I:C))could significantly increase the expression levels of Sp-AST-B in the thoracic ganglion,while the expression levels of Sp-AST-BR in hemocytes was also significantly changed.Subsequently,we screened the concentration range for the action of Sp-AST-B peptide by in vitro experiments.In primary hemocyte culture experiments,it was shown that the administration of Sp-ASTB peptide significantly increased the expression levels of Sp-AST-BR,NF-κB signaling pathway factors(Sp-Dorsal and Sp-Relish),pro-inflammatory cytokines(Sp-IL-16),prophenoloxidase(Sp-PO),C-type lectin(Sp-C-lectin)and antimicrobial peptides(SpLyz,Sp-Crustin,Sp-ALF1,Sp-ALF5,Sp-Hyastatin)in hemocytes.In addition,in vitro experiments showed that injection of Sp-AST-B peptide also increased the expression levels of the above-mentioned receptors and immune effector molecules in hemocytes,and increased nitric oxide(NO)concentrations and phagocytic activity.dsRNA interference experiments further showed that knockdown of Sp-AST-B in the nervous system significantly suppressed NO concentrations,phagocytic activity,the expression levels of receptor and immune effector molecules,and led to a significant impairment of the ability to inhibit bacterial proliferation in the mud crab.The above results indicated that AST-B and its receptor were involved in the humoral and cellular immune processes of the mud crab hemocytes,and play an important role in resistance to bacterial infection.2)This study investigated the immunomodulatory function of AST-B in the hepatopancreas of the mud crab.In situ hybridization analysis showed that Sp-AST-BR was mainly localized in the fibroblasts of the hepatopancreas.We also found that SpAST-BR expression levels were significantly upregulated in the hepatopancreas following LPS or Poly(I:C)immune stimulation.The results of in vitro experiments showed that the administration of Sp-AST-B peptide significantly increased the expression levels of Sp-AST-BR,NF-κB signaling pathway factors(Sp-Dorsal and SpRelish),pro-inflammatory cytokines(Sp-IL-16)and antimicrobial peptides(Sp-Lyz,SpCrustin,Sp-ALF1,Sp-ALF2,Sp-Hyastatin)in the hepatopancreas.In addition,administration of Sp-AST-B peptide significantly increased NO concentrations in hepatopancreas cultured medium in vitro and enhanced its ability to clear bacteria.In vitro experiments also demonstrated that overexpression and knockdown of Sp-AST-B regulated the expression levels of Sp-AST-BR and immune effector molecules.The above results indicated that AST-B could trigger downstream signaling pathways via its receptor,and then participate in the regulation of immune response in the hepatopancreas.3)This study investigated the immunomodulatory function of sNPF in hemocytes of the mud crab.Tissue distribution studies have shown that Sp-sNPF and Sp-sNPF-R are commonly expressed in neural tissue and other tissues including hemocytes.In situ hybridization analysis further showed that Sp-sNPF was widely localized in cerebral ganglion,and both Sp-sNPF and Sp-sNPF-R were specifically localized in hemocytes.Furthermore,Sp-sNPF expression in cerebral ganglion could be significantly activated upon LPS and Poly(I:C)immune stimulation,and immune stimulation also significantly changed the expression levels of hemocyte-derived Sp-sNPF and SpsNPF-R.Subsequently,we analyzed the concentration range of Sp-sNPF peptide action by in vivo experiments.In vitro experiments also showed that the administration of SpsNPF peptide enhanced the phagocytosis of primary hemocyte culture,and the enhanced effect could be blocked by sNPF-R dsRNA and adenylate cyclase inhibitor SQ 22536.The administration of Sp-sNPF peptide also significantly increased the contents of the signaling molecules adenylate cyclase(AC),cyclic adenosine monophosphate(cAMP)and protein kinase A(PKA)in hemocytes.In addition,the administration of Sp-sNPF peptide also significantly increased the expression levels of Sp-sNPF-R,NF-κB signaling pathway factors(Sp-Dorsal and Sp-Relish),proinflammatory cytokine(Sp-IL-16),prophenoloxidase(Sp-PO),C-type lectin(Sp-Clectin)and antimicrobial peptides(Sp-Lyz,Sp-Crustin,Sp-ALF1,Sp-ALF2,SpHyastatin)in hemocytes.Finally,in vivo experiments showed that injection of Sp-sNPF peptide significantly increased the expression levels of receptor and immune effector molecules in hemocytes of the mud crab,and enhanced NO concentration and phagocytic activity.The above results indicated that sNPF could mediate phagocytosis of hemocytes via its receptor sNPF-R-coupled AC-cAMP-PKA pathway,and was involved in the process of innate immune response in the mud crab.4)This study investigated the immunomodulatory function of sNPF in the hepatopancreas of the mud crab.In situ hybridization showed that Sp-sNPF-R was mainly localized in the fibroblasts of the hepatopancreas,and LPS and Poly(I:C)immune stimuli significantly increased Sp-sNPF-R expression in the hepatopancreas.Both in vitro and in vivo experiments showed that injection and administration of SpsNPF peptide significantly increased the expression levels of Sp-sNPF-R,NF-κB signaling pathway factors(Sp-Dorsal and Sp-Relish),pro-inflammatory cytokines(SpIL-16)and antimicrobial peptides(Sp-Lyz,Sp-Crustin,Sp-ALF1,Sp-ALF2,SpHyastatin)in the hepatopancreas.In addition,the administration of Sp-sNPF peptide significantly increased the concentration of NO in hepatopancreas cultured medium in vitro and enhanced its bacterial clearance ability.The above results indicated that sNPF and its receptors were involved in the regulation of immune responses in the hepatopancreas and further improve the understanding of the NEI system in the mud crab.In summary,we found for the first time that the neuropeptides AST-B and sNPF were involved in the regulation of immune function and explored their possible immune regulatory pathways at the molecular,cellular,organ and individual levels.At the same time,we have also established the basic NEI system in mud crab and revealed the neuropeptide-mediated immune regulatory network,which could provide new strategies for disease prevention and control in aquaculture.