Preparation of bacteria

K. pneumoniae was obtained from the Biological Resource center of Tianjin First Central Hospital and grown as described previously (Anand et al. 2020). K. pneumoniae was cultured in the microbial culture room and grown in Luria Bertani (LB) broth with constant shaking at 37 °C or solid media containing LB and 1.5% agar in an incubator maintained at 5% CO2, pH 7.0–7.4, and 37 °C. The bacteria grown overnight in the exponential phase were harvested by centrifugation at 2700× g for 20 min and resuspended in PBS.

Culturing of lung cell lines

HLF-1 (human lung fibroblast) cells and BEAS-2B cells (normal human bronchial epithelial cells) were purchased from Zishi Biotechnology (Shanghai, China) and cultured in Dulbecco’s modified eagle medium (DMEM, Cat No. 11054001, Thermo Fisher, Carthage, USA) containing 10% fetal bovine serum (FBS, Cat No. 12664025C, Thermo Fisher), 1% penicillin–streptomycin (Cat No. 15140163, Thermo Fisher, Carthage, USA), and 1% 2 mM L-glutamine (Cat No. 25030081, Thermo Fisher, Carthage, USA) at 37 °C and 5% CO2.

Culturing of three-dimensional (3D) lung spheroids

3D lung spheroids were purchased from Innovation Biotechnology (Tianjin, China). Culture of 3D lung spheroids was used previously published method (Kumari et al. 2020). Briefly, a mixture of 3000 HLF-1 cells and 3000 BEAS-2B cells were plated on 96-well ultra-low attachment plates (Corning) in complete media (DMEM media containing 10% FBS, 1% penicillin–streptomycin, and 1% 2 mM L-glutamine) supplemented with 4% Matrigel™ (Corning, New York, USA). Media was refreshed every 4 days by carefully aspirating 50% of the well volume and replacing with fresh complete media containing 1% Matrigel™. The 3D spheroids were re-fed with fresh media every 2 or 3 days.

Bacterial infection

HLF-1 or BEAS-2B cells or 3D lung spheroids grown in DMEM medium at 5% CO2 and 37 °C were serum-starved for 18 h and infected with the bacterial inoculum prepared in DMEM. Briefly, 80–90% confluent HLF-1 or BEAS-2B cells were seeded in 24-well plates at a density of 2 × 105 cells per well or 3D lung spheroids were seeded in a 4% Matrigel coated 24-well plate at a density of 50 spheroids per well and infected with K. pneumoniae at a multiplicity of infection (MOI) ranging from 5:1 to 10:1. The cells or 3D lung spheroids were then centrifuged for 4 min at 200× g at 22 °C and incubated between 2 and 24 h at 37 °C and 5% CO2 in a humidified incubator. HLF-1 or BEAS-2B cells infected with K. pneumoniae or 3D lung spheroids were treated with 1 μM melatonin for 24 h.

CCK-8 assay of cell viability

Lung cell viability was analyzed using the CCK8 assay kit (Beyotime, Cat. No. C0037, Shanghai, China) according to the manufacturer’s instructions. Briefly, 1 × 104 HLF-1 or BEAS-2B cells were seeded in 96-well culture plates, infected with K. pneumoniae, and treated with or without 1 μM melatonin for specified periods of time. The cells were then incubated for 4 h with 100 μL CCK-8 and the absorbance was measured at 450 nm. Cell viability (%) = OD (treated cells)/OD (control cells) × 100.

Western blotting

Protein lysates were prepared from HLF-1 and BEAS-2B cells using RIPA (catalog#P0013B, Beyotime Biotechnology, Shanghai, China). Equal amounts of protein lysates (30 µg/lane) were separated on a 12% polyacrylamide gel, transferred to PVDF membranes, and blocked with 5% FBS at room temperature for 1 h. The blots were then incubated overnight at 4 °C with the primary rabbit AMPK alpha 1 polyclonal antibody (1:1000, Cat. No. ab3759, Abcam, Cambridge, UK), anti-IL-6 antibody (1:1000, Cat. No. Ab233706, Cambridge, UK), anti-CXCL1 antibody (1:1000, Cat. No. ab124731, Cambridge, UK), anti-CXCL2 antibody (1:1000, Cat. No. ab275879, Cambridge, UK), and anti-GAPDH antibody (1:1000, Cat. No. ab8245, Abcam, Cambridge, UK). The membranes were washed with TBST for 10 min and incubated with the anti-rabbit secondary antibody (1:1000, Cat. No. ab288151, Abcam, Cambridge, UK) for 30 min at room temperature. Finally, membranes were developed using the enhanced chemiluminescence (ECL) agent (Cat. No. P0018M, Beyotime Biotechnology, Shanghai, US). The protein bands were then developed and visualized using ChemiDoc XRS + System (BIO-RAD, Cat. No. 1708265, California, US).

Reverse transcription-polymerase chain reaction (RT-qPCR)

Total RNA was extracted from lung cells using the RNeasy plus Mini Kit (Qiagen, Maryland, USA). Reverse transcription (RT) was performed with 500 ng of total RNA using the BeyoRT™ III cDNA synthesis kit (Cat. No. D7178M, Beyotime, Shanghai, China). qPCR was performed with the cDNA samples using the TB Green qPCR Kit (Takara) in the 7500 Fast Real-Time PCR System (Applied Biosystem, CA, USA). The qPCR reaction conditions included initial denaturation at 94 °C for 5 min followed by 40 cycles of amplification at 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s. The reaction was stopped at 25 °C for 5 min. Relative gene expression levels were analyzed using the 2−ΔΔCt method. GAPDH was used as an endogenous control. RT-PCR primers used in this study are listed in Table 1.

Table 1 QPCR primers used in the studyFlow cytometry

For analysis of apoptosis rates, after treatments, 3D lung spheroids were harvested to 15 centrifuge tubes, followed by spinning down at 100 g for 5 min. Then, supernatant was discarded, followed by adding 5 mL TrypLE™ Express Enzyme (1×, phenol red, Thermo Fisher Scientific GmbH, Waltham, Massachusetts, US) and incubating in water bath (°C) for 10 min. Then, dissociation was terminated by adding 5 mL FBS. Then, single cells were washed with PBS for three times, followed by being resuspended in 1× annexin binding buffer (10× , 0.1M hepes (pH 7.4), 1.4M NaCl, 25 mM CaCl2) and incubated for 5 min at room temperature. Afterwards, cells were pelleted and stained with annexin-V-FITC (Miltenyi Biotech, Bergisch Gladbach, Germany) and propidium iodide (PI) solution (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer’s manual. They were incubated for 15 min at room temperature before an additional 300 μl Annexin V Binding Buffer was added to each sample. Gating parameters were determined using Veh/PBS-treated cells as a negative control. Unstained cells were run to account for auto fluorescence. Stained cells were analyzed by flow cytometry using a FACSLyric system (BD Biosciences, Franklin Lakes, New Jersey, US). For instrument setup, the excitation wavelength was set to 488 nm, and emission wavelength was set to 530 for annexin-V, and the excitation wavelength was set to 488 nm, and emission wavelength was set to 585 for PI.

Statistical analysis

GraphPad Prism (version 9, GraphPad Software, San Diego, CA, US) was used for statistical analysis. Data are shown as mean ± SEM. One-way analysis of variance (ANOVA) was used to compare the data between multiple groups. P < 0.05 was considered statistically significant.