Immune response mechanisms underlying bacterial infection in Aquarana catesbeiana erythrocytes

Erythrocytes are the most abundant cell type in the blood circulation and are predominantly responsible for oxygen transport. Recent studies have revealed a crucial role of erythrocytes in the immune response. However, the immune function of Bullfrog (Aquarana catesbeiana) erythrocytes is still murky. In this study, we systematically investigated the immune response mechanisms underlying bacterial infection in bullfrog erythrocytes. Firstly, the phagocytic and adhesion assays showed that the bullfrog erythrocytes exhibited extensive phagocytosis of beads with diameters of 0.1-1.0 mu m. Scanning electron microscopy (SEM) showed several beads adhering to the surface of bullfrog erythrocytes, transmission electron microscopy (TEM) further revealed that multiple beads were present in the bullfrog erythrocytes. Followed, the flow cytometry analysis data revealed that the bullfrog erythrocytes had a wide range of phagocytic and adhesion activities toward different bacteria (Staphylococcus aureus, Elizabethkingia miricola, and Aeromonas hydrophila). The reactive oxygen species (ROS) detection assay suggested that the bacterial stimulation significantly increased the ROS content in erythrocytes, and showed an extensive bactericidal effect. In addition, the stimulation of bacteria modulated the antioxidant enzyme activities and mRNA expression levels of cytokines in bullfrog erythrocytes. The transcriptome sequencing was performed to further explore the molecular regulatory mechanisms of antibacterial immunity in bullfrog erythrocytes after we injected the Escherchia coil into the blood circulation. The in vivo observation experiment showed that the erythrocytes had a certain phagocytic activity toward E. coil and formed a "rosette" structure. Furthermore, the RNA-seq analyses showed that the infection of Elizabethkingia miricola activated numerous immune-related pathways and various programmed cell death pathways in the bullfrog erythrocytes. In summary, bullfrog erythrocytes exhibited phagocytic activity against pathogens and bactericidal effects through the release of ROS, activated the expression of immune response genes, and also induced multiple forms of cell death.