| In recent years,there has been increasing evidence that invertebrates have adaptations similar to vertebrate innate immunity,known as"immune priming".Immune priming has been reported in annelids,mollusks,arthropods,and echinoderms,etc.,but the mechanism of its occurrence and regulation has not been thoroughly studied.Previous studies in the laboratory found that energy metabolism,epigenetics,phagocytosis and inflammation may play an important role in immune priming of Crassostrea gigas.In this study,C.gigas was taken as the research object to explore the regulation of Cg Clec-HTM/Cg Ig R-Cg Syk-Cg BCL10 signaling pathway on glycolysis pathway,histone methylation,cell phagocytosis and inflammatory response in immune priming,and the following results were obtained.1.Cg BCL10 can activate MAPK-NF-κB/Rel signaling pathway to promote the production of inflammatory factors.In the present study,a BCL10 homologue was identified from C.gigas(designed as Cg BCL10).The full length c DNA of Cg BCL10 was of 897 bp with an open reading frame of 522bp encoding a polypeptide of 174 amino acids containing a classical CARD domain.The deduced amino acid sequence of Cg BCL10 shared low similarity with the previously identified BCL10s from other species.In the phylogenetic tree,Cg BCL10 was firstly clustered with Cv BCL10 from Crassostrea virginica and then assigned into the branch of invertebrate BCL10s.The m RNA transcripts of Cg BCL10 were highly expressed in gonad,gill,adductor muscle,and haemocytes.After Vibrio splendidus stimulation,the m RNA expression level of Cg BCL10 in haemocytes increased significantly(p<0.05)at 24,72 and 96 h.In Cg BCL10-RNAi oysters,the phosphorylation level of mitogen-activated protein kinases(MAPKs),nuclear translocation of NF-κB/Rel and activator protein-1(AP-1)in haemocytes were inhibited,and the m RNA expressions of inflammatory cytokines including Cg IL17s and Cg TNF all decreased significantly(p<0.05)at12 h after V.splendidus stimulation.These results suggested that Cg BCL10 regulated the expression of inflammatory cytokines by activating MAPK kinase,and nuclear translocation of NF-κB/Rel and AP-1 to defense pathogen.2.In immune priming,Cg Clec-HTM/Cg Ig R-Cg Syk-Cg BCL10 can activate m TOR signaling pathway to regulate anaerobic glycolysis pathway.The expression of Cg Clec-HTM,Cg Ig R,Cg Syk and Cg BCL10 genes was interfered by RNAi technology,and C.gigas was given the first immune stimulationt with V.splendidus.Then the phosphorylation level of m TOR and anaerobic glycolysis were detected.After the first V.splendidus stimulation for 6 h and 7 d,the phosphorylation of m TOR and the m RNA expression of Cg Glu T and Cg GK in haemocytes,the contents of glucose,NAD~+/NADH,lactate and other key components of anaerobic glycolysis pathway in gill were significantly decreased(p<0.05).The changes of anaerobic glycolysis were detected by the second immune stimulation after the first stimulation for 7 d.After the second V.splendidus stimulation for 6 h,the m RNA expression levels of Cg Glu T and Cg GK in haemocytes,the contents of glucose,NAD+/NADH,lactste and other key components of anaerobic glycolysis pathway in gill were significantly decreased(p<0.05).These results suggest that Cg Clec-HTM/Cg Ig R-Cg Syk-Cg BCL10 signaling pathway can activate m TOR signaling pathway to regulate anaerobic glycolysis pathway,which provides energy for immune response in the immune priming of C.gigas.3.Cg Clec-HTM/Cg Ig R-Cg Syk-Cg BCL10 can mediate histone methylation and participate in the regulation of immune priming.The expression of Cg Clec-HTM,Cg Ig R,Cg Syk and Cg BCL10 genes was interfered by RNAi technology,and C.gigas was given the immune stimulationt with V.splendidus.Then the levels of histone monomethylation and trimethylation in haemocytes were detected by western blot.After the first V.splendidus stimulation for 6 h and 7 d,the immunoimprinting bands of the experimental group were thinner and lighter than the control group after quantitative analysis of tubulin,indicating that the levels of histone monomethylation and trimethylation were reduced.These results suggest that Cg Clec-HTM/Cg Ig R-Cg Syk-Cg BCL10 signaling pathway can mediate histone methylation and participate in the regulation of immune priming.4.Cg Clec-HTM/Cg Ig R-Cg Syk-Cg BCL10 can regulate the phagocytosis of cells in immune priming.The expression of Cg Clec-HTM,Cg Ig R,Cg Syk and Cg BCL10 genes was interfered by RNAi technology,and C.gigas was given the first immune stimulationt with V.splendidus.Then the changes of phagocytosis were detected.After the first V.splendidus stimulation for 6 h and 7d,the m RNA levels of Cg Clathrin,Cg ATPe V and Cg Lysozyme in haemocytes,the content of lysozyme in gill were significantly decreased(p<0.05).The changes of phagocytosis were detected by the second immune stimulation after the first stimulation for 7 d.After the second V.splendidus stimulation for 6 h,the m RNA levels of Cg Clathrin,Cg ATPe V and Cg Lysozyme in haemocytes,the content of lysozyme in gill were significantly decreased(p<0.05),and the phagocytic activity of hemocytes all decreased significantly(p<0.05).These results suggest that Cg Clec-HTM/Cg Ig R-Cg Syk-Cg BCL10 signaling pathway can promote cell phagocytosis in immune priming.5.Cg Clec-HTM/Cg Ig R-Cg Syk-Cg BCL10 can regulate the inflammatory response in immune priming.The expression of Cg Clec-HTM,Cg Ig R,Cg Syk and Cg BCL10 genes was interfered by RNAi technology,and C.gigas was given the first immune stimulationt with V.splendidus.Then the phosphorylation level of MAPKs and the expression of cytokines were detected.After the first V.splendidus stimulation for 6 h and 7 d,the phosphorylation levels of Cg ERK,Cg JNK and Cg P38in haemocytes,the m RNA expression levels of inflammatory factors IL17s and TNF were significantly decreased(p<0.05).The expression of cytokines was detected by the second immune stimulation after the first stimulation for 7 d.After the second V.splendidus stimulation for 6 h,the m RNA levels of inflammatory factors IL17s and TNF were significantly decreased(p<0.05).These results suggest that Cg Clec-HTM/Cg Ig R-Cg Syk-Cg BCL10 signaling pathway can activate MAPK signaling pathway to promote inflammatory response in immune priming.In conclusion,in this study,a Cg Clec-HTM/Cg Ig R-Cg Syk-Cg BCL10 signaling pathway was identified in immune priming of C.gigas.On the one hand,this pathway mediated histone methylation through activation of m TOR-anaerobic glycolysis pathway and MAPK-NF-κB signaling pathway in immune priming.In addition,it can regulate the phagocytosis of cells and the inflammatory response in vivo,and then participate in cellular and humoral immunity in immune priming.Relevant research results deepened the understanding of the immune priming mechanism of C.gigas,provided new clues for studying the evolution of animal immune adaptability,and provided new ideas for the prevention and control of important aquaculture animal diseases in China. |
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