Investigation Of Neutrophil Extracellular Traps Formation And Neutrophils' Gene Expression Against Bacillus Calmette-Gue'rin Infection

Objectives: To investigate the neutrophil extracellular traps formation under BCG stimulation,the capture and bactericidal effect of NETs on BCG,and also explore the molecular mechanism of the formation of NETs.By using gene chip technology we screened differentially expressed genes when neutrophil under BCG stimulation and to explore the role of neutrophils in BCG-mediated cell immune response.Methods: Neutrophils were isolated from heparinized venous blood of healthy volunteers.(1)Fluorescent staining for neutrophils after stimulation with BCG,using confocal microscope to observe the formation of NETs.The scanning electron microscopy was used to observe NETs formation and how NETs capture BCG,using cytochalasin D inhibit neutrophil phagocytosis and degranulation,when measuring the efficiency of NETs of sterilization,neutrophils were pretreated with DNase observed no NETs formation.(2)Using ROS assay kit to detect the ROS release from neutrophils in different groups.The neutrophils after pretreatment of various protease inhibitors,was observed NETs formation with a fluorescent staining method under BCG stimulation and with IPP software to count the percentage of the formed NETs.(3)Using gene chip to screen neutrophil differentially genes expressed after BCG stimulation,and to make the gene functional analysis.Results:(1)BCG can stimulate the neutrophils to form NETs,the main ingredient of NETs is DNA,histones,elastase,etc;DNase can inhibite the formation of NETs and degradate the NETs structure;the NETs also have capture and killing effect on BCG.(2)NADPH oxidase inhibitor DPI and Acetylcysteine pretreated neutrophils in BCG stimulation was not observed the formation of NETs nor produce ROS;similarly with PI3 K inhibitors,PKC inhibitors,MEK inhibitors of neutral pretreatment granulocytes in BCG stimulation was not observed the formation NETs and ROS production,on the contrary,with p38 MAPK inhibitor and JNK inhibitor pretreated neutrophils in BCG stimulation still can be observed NETs formation and the production of ROS.(3)the stimulation of neutrophils with BCG compared to without BCG showed significant change gene expression,indicating that the BCG plays a significant impact on the neutrophil function;in the BCG-mediated cell immune response,neutrophils plays an important role in Cytokine-cytokine receptor interaction signaling pathway,Chemokine signaling pathway signaling and Focal adhesion signaling pathway.Conclusion: BCG can induce neutrophils to form NETs,NETs formation can also capture and sterilization BCG;the ROS is necessary for BCG-induced neutrophil NETs formation;PI3K signaling pathway regulates cell autophagy play an important role in NETs formation;in the process of BCG-induced neutrophil to form NETs,the PKC and MEK may in the upstream signaling pathway of ROS production,however,the p38 MAPK and JNK may in the downstream signaling pathway.Gene chip technology screening differences in gene expression in neutrophils under stimulation of BCG.