Aerobic glycolysis of bronchial epithelial cells rewires Mycoplasma pneumoniae pneumonia and promotes bacterial elimination

Abstract

The immune response to Mycoplasma pneumoniae infection plays a key role in clinical symptoms. Previous investigations focused on the pro-inflammatory effects of leukocytes and the pivotal role of epithelial cell metabolic status in finely modulating the inflammatory response have been neglected. Herein, we examined how glycolysis in airway epithelial cells is affected by M. pneumoniae infection in an in vitro model. Additionally, we investigated the contribution of ATP to pulmonary inflammation. Metabolic analysis revealed a marked metabolic shift in bronchial epithelial cells during M. pneumoniae infection, characterized by increased glucose uptake, enhanced aerobic glycolysis, and augmented ATP synthesis. Notably, these metabolic alterations are orchestrated by adaptor proteins, MyD88 and TRAM. The resulting synthesized ATP is released into the extracellular milieu via vesicular exocytosis and pannexin protein channels, leading to a substantial increase in extracellular ATP levels. The conditioned medium supernatant from M. pneumoniae-infected epithelial cells enhances the secretion of both interleukin (IL)-1β and IL-18 by peripheral blood mononuclear cells, partially mediated by the P2X7 purine receptor (P2X7R). In vivo experiments confirm that addition of a conditioned medium exacerbates pulmonary inflammation, which can be attenuated by pre-treatment with a P2X7R inhibitor. Collectively, these findings highlight the significance of airway epithelial aerobic glycolysis in enhancing the pulmonary inflammatory response and aiding pathogen clearance.

Keywords: Mycoplasma pneumoniae; aerobic glycolysis; bronchial epithelial cells; extracellular ATP; peripheral blood mononuclear cells; purine receptor.

Fig 1

Overview of differentially expressed genes in M. pneumoniae- infected BEAS-2B cells. The human bronchial epithelial cell line BEAS-2B was subjected to M. pneumoniae infection for 16 h. The upregulated genes were subjected to gene ontology (GO) enrichment analysis (A), and the top 20 pathways with the highest KEGG pathway enrichment levels were displayed according to the amount and enrichment level of total differentially expressed genes annotated in the control vs M. pneumoniae comparison (B). TNF, tumor necrosis factor.

Fig 2

M. pneumoniae-induced aerobic glycolysis in bronchial epithelial cells. HBE cells were cultured in 96-well plates and infected with M. pneumoniae at an MOI of 100 for 24 h. Thereafter, the ECAR and OCR were detected using fluorescence luminescence. (A and B) HBE cells were infected with various MOIs of M. pneumoniae for 24 h, and the expressions of HK1, PFKM, and pyruvate kinase M2 (PKM2) mRNA (C–E) or protein (F) were measured using quantitative PCR and western blot analysis. Data shown are one experiment representative of three independent experiments with three duplicate samples for each group. *P <0.05, as compared with the untreated group (C–E).

Fig 3

M. pneumoniae enhances glucose uptake in bronchial epithelial cells. HBE cells were seeded in 24-well plates and infected with various MOIs of M. pneumoniae for 24 h or with MOI 100 for 0, 8, 12 and 24 h. Glucose and lactate concentrations were detected using the glucose oxidase method and enzyme colorimetry, respectively (A–D); (E) HBE cells were pre-treated with the glycolysis inhibitor 2-DG (1 and 5 mM) for 3 h, and subsequently infected with 100 MOI of M. pneumoniae for 24 h. The concentrations of lactate in the supernatant was detected; (F–H) HBE cells were infected with M. pneumoniae for various time intervals. GLUT1 expression was detected using quantitative PCR or western blot analysis. Data represent at least three independent experiments with similar results. *P <0.05, as compared with the control (A, C, F, and G) or specified groups (B, D, and E).

Fig 4

Metabolic shift in response to transfection of dominant negative MyD88 and TRAM. BEAS-2B cells were transfected with the dominant negative MyD88 or TRAM plasmid before infection with M. pneumoniae (MOI = 100) for 24 h. ECAR (A and C), OCR (B and D), glucose uptake (E), lactate production (F), and GLUT1 expression (G) were measured. Data shown are one experiment representative of three independent experiments, and n = 3 per group for each experiment. *P <0.05, compared with the indicated groups.

Fig 5

M. pneumoniae induces the bronchial epithelial cell secretion of eATP. HBE cells were seeded on 24-well plates and infected with varying doses of M. pneumoniae for specified time intervals. The concentration of eATP was measured using luminescence ( A and B ). (C–G ) Cells were pre-treated with glycolysis inhibitor (5 mM 2-DG) or ATP channel inhibitors (10- and 50 -µM CBX, 10- and 50 -µM 18α-GA, 20- and 100 -µM N-ethylmaleimide (NEM), and 50- and 100 -µM FFA), and subsequently infected with 100 MOI of M. pneumoniae for 4 h . eATP concentration was measured using luminescence. Data shown are one experiment representative of three independent experiments, and n = 3 per group for each experiment. *P <0.05, as compared with the uninfected ( A and B ) or indicated groups ( C–G ). ns, no significant difference.

Fig 6

eATP enhances the pro-inflammatory effect of PBMCs via the P2 X7 receptor. PBMCs were infected with M. pneumoniae (MOI = 100) for 4 h and stimulated with 1:1 diluted CM from infected bronchial epithelial cells for 24 h , and the concentrations of IL-1β (A) and IL-18 (B) were measured using enzyme-linked immunosorbent assay (ELISA). ( C and D ) PBMCs were infected with M. pneumoniae for 4 h and treated with the P2 X7 receptor inhibitor A740003 (10 µM ) or oxATP (300  µM ) before CM stimulation, and the concentrations of cytokines were detected using ELISA. (E) HBE cells were infected with M. pneumoniae at an MOI of 100 for 24 h , and the expression of P2X7 mRNA was detected using quantitative PCR. (F) PBMCs were infected with M. pneumoniae for 4 h and subsequently co-incubated with CM collected from HBE cells pre-treated with 2-DG before M. pneumoniae infection. IL-1β and IL-18 levels in the supernatant were measured 24 h later using ELISA. Data are derived from the results of one experiment representative of three independent experiments performed with n = 3 for each group. *P <0.05 as compared with the indicated groups ( A–D and F ) or uninfected group (E). CTRL, control.

Fig 7

Histological assessment and pathogen burden after eATP treatment. C57BL/6 mice were intratracheally infected with 10⁷ M. pneumoniae for 2 h. Thereafter, 40 µL of CM (or 200-µmol ATP) was nasally instilled (once daily for 3 days) or mice were intraperitoneally administered oxATP (6mg/kg) (30 min prior to CM administration). The mice were euthanized, and their lung tissues were subsequently analyzed using histological techniques (A); the BALF was examined for IL-1β and IL-18 concentrations (B).(C and D) M. pneumoniae burden in lung and CFUs in BALF were quantified using quantitative PCR (qPCR) for the Mp P1-adhesin gene relative to GAPDH and cultured on solid pleuropneumonia-like organism (PPLO) agar plates, respectively. (E and F) Mice were nasally instilled 10⁷ M. pneumoniae plus 0.25-µmol 2-DG. The tissue was obtained for qPCR or cultured on PPLO agar 72 h after infection. Data are derived from the results of one experiment representative of three independent experiments performed with n = 4–6 mice per group. *P < 0.05, as compared with indicated groups.