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 induces the formation of neutrophil extracellular traps (NETs), which play an important role in bladder tumor treatment. To identify how BCG induced NETs formation, we examined NETs formation induced by BCG in vitro and in a mouse model, then analyzed the effects of NETs on BCG and the relevant regulatory mechanism. Results: We observed the NETs at different time points by Confocal Laser Scanning Microscope (CLSM) and Scanning Electron Microscopy (SEM), the results found that BCG induced in vitro NETs formation in a time-dependent fashion, which was inhibited by pretreatment with DNase I and protease. FITC-labeled BCG was used to observe capturing by NETs. Interestingly, BCG was trapped but not killed in vitro by NETs, which was different from the effect on Staphylococcus aureus. Moreover, ROS was required for BCG-induced NETs formation, which was regulated by MEK, p38, PI3K, and PKC pathways. Finally, NETs formation was observed in mouse urine and subcutaneous tumors after BCG perfusion. Conclusions: By exploring how BCG induced the formation of NETs and the regulatory mechanism, we conclude that a novel immune reaction involving neutrophils exists in the early stages of BCG treatment.