Preliminary Study On Induction And Antibacterial Effects Of Neutrophil Extracellular Traps In Grass Carp

Neutrophil extracellular traps(NETs)are a reticular fibrous structure in which neutrophils release a DNA skeleton attached to a large number of antimicrobial proteins after exposure to exogenous microorganisms or certain chemical stimuli.Based on its special network structure,NETs can capture a variety of pathogenic microorganisms to prevent their diffusion in the tissue,and use local high concentration of antimicrobial particle protein to kill it,can effectively resist the invasion of pathogenic microorganisms,the NETs production process is known as "NETosis".This paper,we conducted a related study on microbial-induced NETs by using kidney-derived neutrophils from PMA fish.First,the neutrophil was isolated by the kit and examined by optical and electron microscopy for morphological identification and in vitro culture activity.Then,the neutrophil NETs of grass carp was induced and verified by PMA and two strains of bacteria,and the factors affecting NETs production and related factors were explored.Finally,the antibacterial effect of the two strains was detected.The results showed that we isolated purified and highly active neutrophils,and the results showed that neutrophils in grass carp could produce a large number of NETs structures under the stimulation of phorbol 12-myristate 13-acetate,and A.veronii highly virulent(GZ09007)and lowly virulent(FS12001).And we can see that the NETs of PMA induced neutrophil release is based on the nucleus DNA.The amount of NETs induced by both strains decreased significantly after treatment of neutrophils with inhibitors of ROS and NO,indicating that ROS and NO play an important role in the formation of NETs;But when the neutrophils were induced by two strains,the amount of ROS and NO detected did not increase significantly compared with the control group,presumably because only a small amount of ROS and NO involvement was required during NETs formation.Using GZ09007 and FS12001 to stimulate neutrophils,it was found that the production of NETs increased gradually within 5 h,and the NETs produced by FS12001 induction was always higher than that of GZ09007.The results of colony cfu counting showed that the bactericidal efficiency of NETs on FS12001 was significantly higher than that of GZ09007;when capturing the killing GZ09007,the NETs and phagocytosis have a good synergistic effect,but the two have less synergistic effect when capturing the killing FS12001.It is suggested that this may be related to the difference of virulence factors and pathogenicity between the two strains.To sum up,neutrophils from kidney source of grass carp can produce antibacterial NETs,under the guidance of bacteria,and the amount of production NETs a certain time is positively correlated with time,and the production of NETs requires a small amount of ROS and NO participation;the production of NETs is closely related to the bactericidal efficiency and inducer.The NETs produced by different strains is different,and the bactericidal efficiency is different.the synergistic effect of NETs bactericidal action and phagocytosis has strain differences.these results help to further understand the mechanism of fish NETosis and its interaction with pathogenic microorganisms.