DM9CP-integrin-RhoA pathway promotes the hemocyte phagocytosis in the immune priming of Crassostrea gigas

Abstract DM9 domain-containing proteins (DM9CPs) constitute a novel family of pattern recognition receptors (PRRs) recognizing specific ligands to initiate downstream immune responses. Their involvement in phagocytosis and immune priming is crucial for a comprehensive understanding of their regulatory mechanisms on immune responses. In the present study, the content of CgDM9CP-1 protein in the cell-free hemolymph of Crassostrea gigas was significantly upregulated at 3, 6, and 12 h after Vibrio splendidus stimulation, and CgDM9CP-1 was able to interact with CgIntegrin both in vitro and in vivo. After the expression of CgDM9CP-1 or CgIntegrin was knocked down by RNAi, the mRNA expression levels of CgRhoA and CgcathepsinL1, as well as the levels of reactive oxygen species (ROS) and nitric oxide (NO), and the phagocytic rate of hemocytes all decreased significantly. Moreover, the modification level of H3K4me3 in CgDM9CP-1 gene promoter increased significantly after the first V. splendidus stimulation, and the transcripts of CgDM9CP-1, CgIntegrin, CgRhoA and CgcathepsinL1, as well as the phagocytic rate of hemocytes all increased significantly after the secondary stimulation with V. splendidus. When the H3K4me3 modification level of the CgDM9CP-1 promoter was increased by the treatment with histone demethylases activator dimethyl 2-oxoglutarate (DM2O), the mRNA expression levels of CgDM9CP-1, the phagocytic rate and the ROS level of hemocytes decreased after the secondary V. splendidus stimulation, while the treatment with histone demethylases inhibitor dimethyl succinate (DMS) resulted in opposite effects. These results indicated the CgDM9CP-1-integrin-RhoA pathway regulated hemocyte phagocytosis in the immune priming of oyster.