Mycoplasma Pneumoniae Induces Bronchial Epithelial Cells Aerobic Glycolysis To Enhance PBMCs Secretion Of IL-1β

Inflammatory damage caused by Mycoplasma pneumoniae infection is the most important pathogenic mechanism.The majority of the current work is focused on the traditional immune signal transduction pathway,and the regulation of cellular metabolism on inflammatory response has been overlooked.This study aimed to investigate the impact of M.pneumoniae infection on aerobic glycolysis in bronchial epithelial cells,and to figure out the mechanism of M.pneumoniae induced extracellular ATP(e ATP)secretion through aerobic glycolysis pathway to regulate the secretion of IL-1β by human peripheral blood mononuclear cells(PBMCs).Firstly,BEAS-2B cells were cultured in vitro and were infected with M.pneumoniae of different multiplicity of infection(MOI).The differential gene expression before and after infection was analyzed by RNA-Seq.Secondly,the influences of M.pneumoniae infection on aerobic glycolysis were ascertained by cell energy metabolism analysis,enzymology experiment,RT-q PCR and western blot;The content of e ATP before and after M.pneumoniae infection was measured by luminescence method,and inhibitors were used to elucidate the mechanism of e ATP secretion.Finally,the PBMCs were co-incubated with conditioned medium(CM)from M.pneumoniae-infected BEAS-2B,then the concentration of IL-1β in the supernatant,as well as the M.pneumoniae burdens were evaluated by ELISA and RT-q PCR,respectively.ATPase and purine receptor inhibitors were used to explore how e ATP promotes the secretion of IL-1β from PBMCs and its influence on M.pneumoniae burdens.The results showed that after BEAS-2B cells infected with M.pneumoniae,1165 genes were up-regulated and 824 genes were down-regulated,and the up-regulated genes were associated with the cellular metabolic pathway.After M.pneumoniae infection,the cell oxygen consumption rate(OCR)decreased,whereas the extracellular acidification rate(ECAR)increased.Meanwhile,the expression of hexokinase(HK),6-phosphofructokinase(PFK)and pyruvate kinase(PK)increased with the increase of MOI;M.pneumoniae infection could increased the expression of glucose transporter 1(GLUT1),as well as the lactate concentration in a dose-and time-dependent manner,while the concentration of glucose was decreased in a dose-and time-dependent manner;M.pneumoniae infection could also increased the secretion of e ATP,which decreased after treatment with the glycolysis inhibitor 2DG,the selective pannexin/connexin inhibitor CBX,and the vesicular exocytosis inhibitor NEM.Furthermore,after PBMCs infected with M.pneumoniae,co-incubation of CM from M.pneumoniae-infected BEAS-2B cells with PBMCs can significantly increased IL-1β secretion,whilst abrogate the M.pneumoniae load in the co-culture system;After CM pretreated with ATPase or PBMCs pretreated with P2X7 inhibitor,the levels of IL-1β in the supernatant were significantly decreased,while the M.pneumoniae load was higher than that of untreated group.The above results indicate that M.pneumoniae induces ATP synthesis in BEAS-2B cells through aerobic glycolysis pathway,which results in the accumulation of ATP in the extracellular space through vesicle exocytosis and pannexin protein channel.The e ATP ultimatly promotes PBMCs to secret IL-1β,as well as eliminate M.pneumoniae through P2X7 receptor.