BCG induced neutrophil extracellular traps formation and its regulatory mechanism

Abstract Background Intravesical BCG is one of the most effective immunotherapies for bladder cancer. Our previous study showed that BCG could induce the formation of neutrophil extracellular traps (NETs), which play an important role in bladder tumor treatment. To identify how BCG induced NETs formation, in this study, we examined NETs formation induced by BCG in vitro and in mouse model, then analyzed the effects of NETs on BCG, and the relevant regulatory mechanism.Methods The formation of NETs was visualized by Confocal Laser Scanning Microscope (CLSM) and Scanning Electron Microscopy (SEM). NETs quantitation was evaluated by the strength of extracellular DNA. ROS and NETs formation were examined by co-culturing with the inhibitors of ROS, NADPH oxidase and relevant pathways respectively. FITC–labeled BCG was used to observe the capturing by NETs. Finally, NETs formation was observed in mouse urine and subcutaneous tumor after BCG perfusion.Results BCG time-dependently induced in vitro NETs formation, as well as in urine and subcutaneous tumor of mouse, which could be inhibited by pretreatment with DNase I and protease. Interestingly, BCG was trapped but not killed in vitro by NETs, which was different from the effect on S. aureus. Moreover, ROS was required for BCG-induced NETs formation, which was regulated by star pathways, such as MEK, p38, PI3K and PKC pathways.Conclusions By exploring how BCG induce the formation of NETs and regulatory mechanism, we concluded a novel immune reaction of neutrophils in the early stages of BCG treatment.