Pyroptosis, apoptosis, and autophagy are involved in infection induced by two clinical Klebsiella pneumoniae isolates with different virulence

Abstract

Klebsiella pneumoniae can cause widespread infections and is an important factor of hospital- 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, particularly pyroptosis, apoptosis, and autophagy in the context of host-pathogen interactions to better understand the pathogenic mechanism of K. pneumoniae. Two clinical K. pneumoniae isolates, one classical K. pneumoniae isolate and one hypervirulent K. pneumoniae isolate, were used to infect RAW264.7 cells to establish an in vitro infection model. We first examined the phagocytosis of macrophages infected with K. pneumoniae. Lactate dehydrogenase (LDH) release test, and calcein-AM/PI double staining was conducted to determine the viability of macrophages. The inflammatory response was evaluated by measuring the pro-inflammatory cytokines and reactive oxygen species (ROS) production. The occurrence of pyroptosis, apoptosis, and autophagy was assessed by detecting the mRNA and protein levels of the corresponding biochemical markers. In addition, mouse pneumonia models were constructed by intratracheal instillation of K. pneumoniae for in vivo validation experiments. As for results, hypervirulent K. pneumoniae was much more resistant to macrophage-mediated phagocytosis but caused more severe cellular damage and lung tissues damage compared with classical K. pneumoniae. Moreover, we found increased expression of NLRP3, ASC, caspase-1, and GSDMD associated with pyroptosis in macrophages and lung tissues, and the levels were much higher following hypervirulent K. pneumoniae challenge. Both strains induced apoptosis in vitro and in vivo; the higher apoptosis proportion was observed in infection caused by hypervirulent K. pneumoniae. Furthermore, classical K. pneumoniae strongly triggered autophagy, while hypervirulent K. pneumoniae weakly activated this process. These findings provide novel insights into the pathogenesis of K. pneumoniae and may form the foundation for the future design of treatments for K. pneumoniae infection.

Keywords: Klebsiella pneumoniae; apoptosis; autophagy; inflammation; macrophage; pyroptosis.

Figure 1

Effects of cKp and hvKp infection on phagocytic capability and cell viability of macrophages. (A) The histograms of flow cytometric analysis of phagocytosis in RAW264.7 cells treated by cKp and hvKp. (B) Phagocytic ability (PA) values of RAW264.7 cells. (C) Phagocytic index (PI) values of RAW264.7 cells. (D) Bacterial load of macrophages was detected by colony forming units counting. (E) Calcein−AM/PI double staining assay was performed for living cells (green fluorescence) and dead cells (red fluorescence) (scale bar, 50 μm). (F) The levels of LDH release in RAW264.7 cells. All experiments were independently repeated three times. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. cKp, classical Klebsiella pneumoniae; hvKp, hypervirulent Klebsiella pneumoniae.

Figure 2

Effects of cKp and hvKp infection on the production of pro-inflammatory cytokines and ROS in macrophages. (A) Relative mRNA expression of IL-1β, IL-6, and TNF-α in RAW264.7 cells by qRT-PCR. (B) The protein concentrations of IL-1β, IL-6, and TNF-α in cell supernatant by ELISA. (C) Fluorescence images of ROS accumulation in RAW264.7 cells (scale bar, 50 μm). (D) The quantitative analysis of ROS production in RAW264.7 cells by flow cytometry. All experiments were independently repeated three times. Data are presented as mean ± SD. ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001. cKp, classical Klebsiella pneumoniae; hvKp, hypervirulent Klebsiella pneumoniae.

Figure 3

Effects of cKp and hvKp infection on pyroptosis, apoptosis, and autophagy of macrophages. (A) The protein expressions of NLRP3, GSDMD-N, and caspase-1 p20 were detected by Western blot and the bands densities were analyzed using Image J. (B) The apoptosis of RAW264.7 cells were determined by flow cytometry. FSC/SSC panels were flow gating strategy for apoptosis analysis, Q1-LR (Annexin V+/PI−) was considered as early apoptosis and Q1-UR (Annexin V+/PI+) was considered as late apoptosis in the pseudo color plot. (C) The protein levels of LC3 were detected by Western blot and analyzed using Image J. (D) The LC3 puncta were detected by immunofluorescence staining (scale bar, 25 μm). All experiments were independently repeated three times. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. cKp, classical Klebsiella pneumoniae; hvKp, hypervirulent Klebsiella pneumoniae.

Figure 4

Effects of cKp and hvKp infection on survival and lung injury in mice. (A) Survival rates of mice were recorded from day 0 to day 7. (B) The pathological changes in lung tissues were determined by H&E staining (×200). (C) The mRNA levels of IL-1β and TNF-α in lung tissues were detected by qRT-PCR. (D) The macrophages marker F4/80 was detected by immunohistochemical staining (×200). (E) Statistical analysis of F4/80 positive cells. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. cKp, classical Klebsiella pneumoniae; hvKp, hypervirulent Klebsiella pneumoniae.

Figure 5

Effects of cKp and hvKp infection on cell death pathways in mice. (A) The relative mRNA levels of NLRP3, caspase-1, GSDMD, caspase-3, bcl-2/bax, LC3, and beclin-1 were detected by qRT-PCR. (B) Immunohistochemical staining was used to detect the protein expressions of NLRP3, ASC, caspase-3, and LC3 (×200). (C) Statistical analyses were performed by ImageJ. Data are presented as mean ± SD. ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001. cKp, classical Klebsiella pneumoniae; hvKp, hypervirulent Klebsiella pneumoniae.