| The increasing evidence has revealed that there is some degree of specific acquiredimmunity in invertebrates, which is defined as immune priming, but the underlyingmechanism is not fully understood yet. In the present study, we analyzed themolecular and cellular characteristics of immune priming phenomena in Pacific oysterCrassostrea gigas, ascertained the relationships between the signaling pathway andmolecules related to phagocytosis and immune priming, and then identified the rolesof oyster hemocytes and plasma in the immune priming.The oyster stimulated primarily by heat-killed Vibrio splendidus was observed torespond more strongly at cellular level to the secondary challenge of live V.splendidus. The total hemocyte counts (THC) and the number of new generatedcirculating hemocytes increased significantly and slightly at24h after the primarystimulation of heat-killed V. splendidus respectively, while the phagocytic rate peakedat12h. After the secondary challenge with live V. splendidus, the THC had a moresharp increase and peaked fast at6h, and the number of new generated circulatinghemocytes increased significantly at6h and the proliferative rate exceeded about4times. Furthermore, the phagocytic rate increased more significantly at12h comparedwith that of the primary stimulation. Meanwhile, the enhanced hemocyte phagocytosisafter the secondary challenge was highly specific for V. splendidus and they coulddistinguish Vibrio anguillarum, Vibrio coralliilyticus, Yarrowia lipolytica, andMicrococcus luteus efficiently.The relative mRNA expression levels of CgRunx1and CgBMP7probablyinvolved in the oyster hemopoiesis had a higher peak after the successive challengewith V. splendidus than that of the primary sitmulation of V. splendidus, but theexpression level of CgGATA3mRNA did not change significantly. The expressionlevels of phagocytic receptor CgIntegrin and the key signaling pathway moleculesCgPI3K, CgRho J, CgMAPKK, CgRab32, CgNADPH oxidase in the hemocytes ofpre-stimulated oysters were higher than that after the secondary stimulation of V.splendidus, and the expression level of CgEcSOD was lower than that after theprimary stimulation. However, there were no significant change for the expression level of CgPKC, CgMyosin, CgActin, and CgToll6genes. CgIntegrin was observedto locate on the membrane of oyster hemocytes. The blocking experiment withpolyclonal antibody proved that CgIntegrin was mainly involved in the phagocytosisof hemocytes against V. splendidus. CgToll6was located on the membrane and in thecytoplasm of oyster hemocytes, and it could bind LPS and PGN, but not Mannan.Accordingly, CgToll6displayed a very strong binding activity to V. splendidus, V.anguillarum, S. aureus, M. luteus and P. pastoris, while weakly to Y. lipolytica. Thenatural CgEcSOD in oyster plasma had a strong binding activity to LPS, PGN andPloy I:C dependent on the dose of plasma. Accordingly, CgEcSOD could bind V.splendidus, V. anguillarum, S. aureus, M. luteus, P. pastoris and Y. lipolytica with aCu2+dose-dependent manner.The oyster transferred with the primed plasma (the plasma of oyster stimulated4days by killed V. splendidus) displayed the significantly raised proliferative rate ofhemocytes at6h and declined apoptotic rate from6h to24h, but their THC wassignificantly decreased at6h and48h and the phagocytic rate just began to enhanceat late stage after infection of V. splendidus. It was presented in the oyster transferredwith the primed hemocytes (the hemocytes of oyster stimulated4days by killed V.splendidus) that the proliferative rate of hemocytes raised significantly at6h, and theapoptotic rate declined significantly at24h and rose at48h after infection of V.splendidus The THC increased significantly at24h and decreased at48h, while thephagocytic rate of hemocytes declined significantly at12h and began to enhance at48h against the infection of V. splendidus. The co-transfer of primed hemocytes andplasma (haemolymph of oyster stimulated4days by killed V. splendidus) aroused thesignificantly reduced apoptotic rate at6and24h and the significantly declinedhemocyte proliferative rate after infection of V. splendidus, while the THC andphagocytic rate were significantly increased.In summary, the primary stimulation of heat-killed V. splendidus could induceimmune priming in oyster with rapidly promoted regeneration of circulatinghemocytes and the specifically enhanced phagocytosis to respond the secondarychallenge against V. splendidus. The recepoter CgIntegrin and the downstreamsignaling pathways participated in the specifically enhanced phagocytosis in theimmune priming response of oyster hemocytes. The pattern recognition receptorCgToll6could not be directly involved in the immune priming of oyster, but the high-abundant plasma protein CgEcSOD could mediate the immune priming. Ahandful of specific hemocytes might play vital roles in the forming and maintenanceimmune priming of oyster. The results provided further information for the knowledgeof immune priming and laid a solid foundation for unveiling the cellular andmolecular mechanisms of immune priming in invertebrates as well. |
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