| Objective Klebsiella pneumoniae can cause a wide range of infections,is an important factor of hospital-acquired and community-acquired pneumonia.The emergence of hypervirulent K.pneumoniae poses a serious clinical therapeutic challenge and is associated with a high mortality.The goal of this work was to investigate the influence of K.pneumoniae infection on host cells and innate immune system,particularly pyroptosis,apoptosis,and autophagy in the context of host-pathogen interactions,to better understanding the pathogenic mechanism of K.pneumoniae.Methods In this study,classical K.pneumoniae(cKp)and hypervirulent K.pneumoniae(hv Kp)isolates were identified by string test and PCR,and then the two strains were used to infect RAW264.7 cells and C57BL/6 mice for in vitro and in vivo investigation.Phagocytosis of macrophages infected with K.pneumoniae was assessed by flow cytometry;Calcein-AM/PI double staining and Lactate dehydrogenase(LDH)release test were performed to determine the viability of macrophages;The inflammatory response was evaluated by measuring the pro-inflammatory cytokines and ROS production;Western blot was used to determine the expression levels of pyroptosis-related proteins,flow cytometry was used to detect the apoptosis of macrophages,and immunofluorescence analysis was used to detect the expression levels of autophagy protein LC3.In addition,mouse pneumonia models were constructed by intratracheal instillation of K.pneumoniae for in vivo validation experiments.Survival rates of mice were measured continuously for 7 days and recorded every 24 h;Lung histopathological changes in mice were assessed by H&E staining;The occurrence of pyroptosis,apoptosis and autophagy in vivo was verified by quantitative real-time PCR and immunohistochemistry assay.Results 1.The average phagocytosis rate of macrophage was 28.2% in the c Kp group and only 12.6% in the hv Kp group,the difference was statistically significant(P < 0.05).2.Calcein-AM/PI staining analysis revealed that K.pneumoniae infection caused more dead cells,and the number of dead cells in hv Kp group was much higher than that in c Kp group;LDH release test demonstrated that K.pneumoniae infection promoted LDH release from macrophages and the LDH release was higher in the hv Kp group(P < 0.05),which indicated that hv Kp infection could cause more severe cytotoxicity.3.All mice in hv Kp group died at 4days post-infection,while 75% of mice in cKp group remained alive during the entire period of observation.Compared with c Kp strain,hv Kp strain caused a much higher mortality of mice(P < 0.05).4.H&E staining showed that hv Kp strain caused more severe lung injury than c Kp strain.5.The expression of proinflammatory cytokines IL-1β,IL-6,TNF-α as well as inflammatory mediator ROS was increased following K.pneumoniae infection,and the expression levels were much higher in hv Kp group than in c Kp group(P < 0.05).6.The expression levels of pyroptosis-related proteins NLRP3,ASC,GSDMD,and Caspase-1 p20 were significantly higher in the hv Kp group than in the c Kp group(P < 0.05).7.K.pneumoniae infection promoted apoptosis,the protein levels of cleaved-Caspase-3 were significant higher in the hv Kp group than in the c Kp group(P < 0.05).8.c Kp strain dramatically elevated the expression levels of LC3 and beclin-1,while hv Kp group manifested relatively lower levels(P < 0.05).Conclusion 1.hv Kp strain was much more resistant to macrophage-mediated phagocytosis compared with c Kp strain.2.hv Kp infection could cause more severe cellular damage and lung tissue damage compared to c Kp infection.3.K.pneumoniae infection induced pyroptosis in vitro and in vivo,and the degree of pyroptosis was much stronger in hv Kp group.4.K.pneumoniae infection triggered apoptosis both in vitro and in vivo,and the pro-apoptotic effect in hv Kp group was more pronounced.5.c Kp infection induced autophagy,while hv Kp inhibited autophagy,at least in part.6.The inhibition of autophagy may be partly responsible for the high virulence and high pathogenicity of hvKp. |
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