Characterization of WS2 nanosheets
WS2 nanosheets (XF143) prepared by lithium intercalation were purchased from Nanjing XFNANO Materials Tech Co., Ltd., China. To remove small WS2 nanosheets, the WS2 nanosheet dispersion was centrifuged at 1500 × g for 5 min, and then the supernatant was removed. The pellet was used in subsequent experiments. WS2 nanosheet morphology was examined using atomic force microscopy (AFM, Dimension Icon, Bruker, USA) and high-resolution transmission electron microscopy (HRTEM, JEM-2800, JEOL, Japan). X-ray photoelectron spectroscopy (XPS) was performed on a Thermo Scientific ESCALAB 250Xi spectrometer equipped with a monochromatic Al-Kα X-ray source (1486.6 eV). UV − vis absorption spectra were recorded on a T90 spectrophotometer (Purkinje General, China). The hydrodynamic size and surface charge of WS2 nanosheet dispersions in deionized water, phosphate-buffered saline (PBS) and cell culture medium were measured using a ZetaSizer Nano-ZS instrument (Malvern Instruments, UK).
Cell Culture And Coculture System
A549 and THP-1 cells were obtained from the Shanghai Cell Bank of Type Culture Collection of China. Both cell lines were grown in Roswell Park Memorial Institute medium (RPMI 1640, Genview, China) supplemented with 10% fetal bovine serum (FBS, AusGeneX, Australia) and 100 units/mL penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2. THP-1 monocytes were differentiated into macrophages by incubation with 160 nM phorbol 12-myristate 13-acetate (PMA, MedChem Express, China) for 24 h.
To study the factors in WS2-treated A549 cells that were involved in the bystander effect in untreated THP-1 cells, a modified transwell insert coculture system was designed and utilized (Fig. 1a). Transwell inserts were ligated to dialysis membranes (with an MWCO of 1000 kDa, approximately 100 nm) and placed in a 24-well plate or 6-well plate. WS2 nanosheet-pretreated A549 cells were placed in the upper chamber, and untreated THP-1 cells were seeded in the lower chamber. The insert had a porous membrane with a pore size of 0.4 µm to allow the passage of molecules but not cells. Dialysis membranes between the chambers were applied to prevent the diffusion of WS2 nanosheets from the upper chamber to the lower chamber.
For the coculture assay, A549 and THP-1 cells were seeded and cultured on 24-well (6-well) Millicell hanging cell culture inserts (Millipore, Germany) and 24-well (6-well) plates, respectively. After 24 h of incubation, the THP-1 cell medium containing PMA was replaced with fresh medium, and the A549 cell medium was replaced with fresh medium containing WS2 nanosheets or other inhibitors. Then, cell culture inserts with A549 cells were transferred to plates containing differentiated THP-1 macrophages. After 24 h of coincubation, the upper chamber was removed, and the cells in the lower chamber were used for further analysis.
Medium Transfer Assay
A medium transfer assay was further used to study bystander effects (Fig. 1b). A549 cells were seeded in 24-well plates overnight and then exposed to WS2 nanosheets for 24 h. After exposure, the medium of A549 cells was harvested and filtered through a sterile 0.1-µm filter (Millipore, Ireland) to ensure that no cells and no nanosheets remained in the medium. Then, the conditioned medium was transferred to 24-well plates where differentiated THP-1 macrophages were previously seeded. THP-1 cells were treated with the conditioned medium for another 24 h and then analyzed.
Cell viability, cellular reactive oxygen species and mitochondrial membrane potential assay
THP-1 monocytes were seeded at 1.6 × 105 cells/well in 24-well plates and cocultured with A549 cells or cultured with conditioned medium as described above. After incubation, the cell viability of differentiated THP-1 macrophages was determined using a cell counting kit-8 (CCK-8, Bimake, USA). The optical density (OD) at 450 nm was measured by a microplate reader (Synergy H4, Bio-Tek, USA). The cell viability of the treatment group was expressed as the percentage of live cells in the treatment group relative to that of live cells in the control group.
The generation of cellular ROS was determined by the change in fluorescence of an ROS probe, 2’,7’-dichlorofluorescin diacetate (DCFH-DA, MedChem Express, China). The fluorescence intensities of DCFH-DA were measured by a microplate reader (Synergy H4, Bio-Tek, USA). The excitation/emission wavelengths for DCFH-DA was 485/530. The fluorescence of DCFH-DA was normalized to the cell viability of macrophages and presented as a percentage relative to the control group.
Mitochondrial membrane potential was analyzed by using the probe 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazolo carbocyanine iodide (JC-1, MedChem Express, China). After staining, the red fluorescence (excitation/emission wavelengths,530/590 nm) and green fluorescence (excitation/emission wavelengths, 485/530 nm) of macrophages were analyzed by a microplate reader (Synergy H4, Bio-Tek, USA). Mitochondrial membrane potential was determined by the ratio of the red to green fluorescence intensity and presented as a percentage relative to the control group. A decrease in the ratio indicated mitochondrial depolarization.
A cytokinesis-blocked micronucleus assay was employed to detect genotoxicity in bystander effects. When THP-1 cells were cocultured with WS2 nanosheet-pretreated A549 cells or were exposed to the medium derived from WS2 nanosheet-pretreated A549 cells, the culture medium contained 1 µg/mL cytochalasin-B (Meilune Biological Technology, China), an inhibitor of cytokinesis. After culturing for 24 h, the cells were fixed with methanol:acetic acid (3:1, v/v) for 30 min. After air-drying, the cells were stained with 4'-6-diamidino-2- phenylindole (DAPI, Beyotime Biotechnology, China). The nuclei with micronuclei were counted using fluorescence microscopy (IX71, Olympus, Japan).
Intracellular And Extracellular Nitric Oxide Detection
A549 cells were seeded onto 96-well plates at 1.0 × 104 cells per well overnight. WS2 nanosheets and related inhibitors were resuspended in culture media and then added to 96-well plates. Intracellular NO was analyzed using the NO-specific fluorescent probe 3-amino,4-aminomethyl-2',7'-difluorescein, diacetate (DAF-FM DA, Beyotime Biotechnology, China). After exposure to WS2 nanosheets for 24 h, the medium was removed, and the cells were incubated with DAF-FM DA (5 µM) for 20 min. The fluorescence intensities of DAF-FM were measured by a microplate reader (Synergy H4, Bio-Tek, USA). The excitation/emission wavelengths used for DAF-FM were 495/515 nm. The fluorescence intensities of DAF-FM were presented as a percentage relative to the control group. The NO levels in the culture medium supernatant were measured by a Griess reagent kit (Beyotime Biotechnology, China). After exposure, 50 µL of culture medium supernatant was mixed with 50 µL of Griess Reagent I and 50 µL of Griess Reagent II. After incubation for 10 min at room temperature, the absorbance at 540 nm was measured by a microplate reader (Synergy H4, Bio-Tek, USA). The NO levels of each sample were calculated using a standard curve.
To analyze transforming growth factor (TGF)-β1 secretion, A549 cells were seeded onto 96-well plates at 1.0 × 104 cells per well overnight. WS2 nanosheets and related inhibitors were resuspended in culture media and then added to each well. After incubation for 24 h, the supernatant was collected, and the concentrations of TGF-β1 in culture medium were assayed with ELISA kits, according to the manufacturer’s instructions (Dakewe, China). To analyze TNF-α and IL-6 secretion, THP-1 cells were seeded onto 96-well plates at 4.0 × 104 cells per well and differentiated into macrophages by incubation with PMA for 24 h. THP-1 cells were treated with conditioned medium derived from WS2 nanosheet-pretreated A549 cells with or without inhibitors for another 24 h. Then, the conditioned medium was removed, and the cells were washed with PBS and cultured for 6 h in fresh medium with or without lipopolysaccharide (LPS) (200 ng/mL). The supernatants were collected, and cytokines were analyzed with ELISA kits according to the manufacturer’s instructions (LiankeBio, China).
A wound-healing assay was performed to analyze the migration of differentiated THP-1 macrophages. THP-1 cells were seeded onto 24-well plates at 1.0 × 106 cells per well and differentiated into macrophages by incubation with PMA for 24 h. A scratch wound in the monolayer cells was made by a 200 µL pipette tip. Cells were washed three times with warm PBS and exposed to conditioned medium derived from WS2 nanosheet-pretreated A549 cells. The cell migration into the scratch area at 0 h, 24 h, and 48 h was photographed using microscopy (IX71, Olympus, Japan).
Quantitative Real-time Polymerase Chain Reaction (qpcr)
The genes related to the polarization state of differentiated THP-1 macrophages modulated by paracrine factors produced by A549 cells exposed to WS2 nanosheets were quantitatively analyzed by qPCR. For the M1 macrophage control, THP-1 cells were treated with 200 ng/mL LPS and 50 ng/mL recombinant interferon-gamma (IFN-γ) for 24 h. For the M2 macrophage control, THP-1 cells were treated with 100 ng/mL IL-4 for 24 h. After the macrophages were cocultured with A549 cells exposed to WS2 nanosheets for 24 h, the total RNA of macrophages was extracted using a total RNA Extraction Kit (Solarbio, China), and cDNA was generated using All-in-One cDNA Synthesis SuperMix (Bimake, USA) by the S1000 Thermal Cycler system (Bio-Rad, USA). qPCR was performed using the Bio-Rad IQ5 system (Bio-Rad, USA) with 2 × SYBR Green qPCR Master Mix (Bimake, USA). The PCR conditions were as follows: 95 °C for 10 min, then 45 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The sequences of specific primers are shown in Table S1. To quantify gene expression changes, the 2−ΔΔCT method was used with glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as a housekeeping gene. The expression level of each gene in the treatment groups was subsequently normalized to that in the control group and presented as relative fold expression.
The polarization of differentiated THP-1 macrophages was further analyzed by immunofluorescent staining. After coculture with A549 cells exposed to WS2 nanosheets for 24 h, the differentiated THP-1 macrophages were fixed with 4% (w/v) paraformaldehyde for 20 min at room temperature and thoroughly washed with PBS. The treated macrophages were permeabilized with 0.5% (v/v) Triton X-100 for 30 min at room temperature. Then, the permeabilized macrophages were incubated in a blocking buffer containing 5% (w/v) goat serum (Beyotime Biotechnology, China) for 40 min at room temperature. The treated macrophages were incubated in blocking buffer containing primary antibodies against iNOS (M1-specific indicator, 1:200, Santa Cruz, USA) and Arg-1 (M2-specific indicator, 1:200, Bioss, China) at 4 °C for 16 h and thoroughly washed with PBS containing 0.1% Tween 20. The cells were then incubated in blocking buffer containing secondary antibodies (goat anti-rabbit IgG/Alexa Fluor 555 and goat anti-mouse IgG/Alexa Fluor 488, 1:350, Bioss, China) for 2 h at room temperature. The cells were subsequently washed with PBS containing 0.1% Tween 20. The nuclei of macrophages were stained with DAPI. Images were observed with CLSM (Leica, Germany).
When THP-1 cells were cocultured with WS2 nanosheet-pretreated A549 cells, the culture medium contained 10 µg/mL green fluorescent polystyrene microspheres (Aladdin, China). After culturing for 24 h, the cells were washed three times with PBS and collected. The fluorescence emission was measured by using flow cytometry (Accuri C6, Becton-Dickinson, USA).
Differentiated THP-1 macrophages were seeded on cover slips in 24-well plates. After coculture with A549 cells exposed to WS2 nanosheets for 24 h, the THP-1 cells were fixed with 4% (w/v) paraformaldehyde for 15 min on ice and thoroughly washed with PBS. The cells were then permeabilized using 0.5% (v/v) Triton X-100 for 10 min at room temperature. YF555-phalloidin (US Everbright Inc., China) and DAPI were used to stain actin and cell nuclei, respectively. Images were acquired using confocal laser scanning microscopy (CLSM, Leica, Germany).
THP-1 cells were seeded onto 6-well plates at 8.0 × 105 cells per well and differentiated into macrophages by incubation with PMA for 24 h. THP-1 cells were treated with conditioned medium derived from WS2 nanosheet-pretreated A549 cells for another 24 h. Then, the conditioned medium was removed, and the cells were washed with PBS and cultured for 6 h in fresh medium with or without LPS (200 ng/mL). At the end of the exposure period, the cells were washed with PBS. Total protein, cytoplasmic protein and nuclear protein were extracted using radioimmunoprecipitation assay (RIPA) lysate buffer and nucleoprotein extraction kit (Solarbio, China), respectively. The protein concentrations were measured using a BCA kit (Beyotime Biotechnology, China). Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Anti-IκBα, anti-NFκB p65, anti-β-actin and anti-poly (ADPribose) polymerase (PARP) antibodies were used according to the manufacturer’s instructions (Bioss, China). β-Actin was used as a loading control for total proteins and cytoplasmic protein, and PARP was used as a loading control for nuclear proteins. The density of bands was quantified using ImageJ software (National Institutes of Health, USA).
After coculture with A549 cells exposed to WS2 nanosheets for 24 h, the cell culture medium of differentiated THP-1 macrophages was removed, and the cells were washed twice with 1 mL of prewarmed PBS. Then, 1 mL of ice-cold 80:20 (v/v) methanol/water was immediately added, and the cells were scraped and collected in a 2 mL Eppendorf tube. The wells were washed again with an additional 1 mL of a methanol/water solution and combined with the previous solution. The metabolites were extracted using ice bath ultrasound (400 W, 30 min) followed by centrifugation (12000 × g, 5 min, 4 °C). The supernatant was filtered through a 0.22 µm organic membrane filter, removed via nitrogen blowing, and lyophilized. Methoxamine hydrochloride (20 mg/mL, 50 µL) and N-methyl-N-(trimethylsilyl) trifluoroacetamide (80 µL) were added as derivatives. After derivatization, the samples (1 µL) were injected and analyzed using gas chromatography-mass spectrometry (GC-MS, 6890N/5973, Agilent, USA). The metabolites were identified using full-scan monitoring with a detection slope of m/z 50–650, based on the National Institute of Standards and Technology (NIST) Mass Spectral Library in ChemStation software.
Statistical analysis was performed using SPSS 22.0 software (IBM, USA). Differences between two groups were analyzed using Student's t-test, and differences among three or more groups were analyzed using one-way analysis of variance (ANOVA) with Tukey's test. Before ANOVA, the Kolmogorov − Smirnov (KS) test (a significance level of 0.05) was performed to assess the normal distribution. P values less than 0.05 were considered statistically significant. Metabolic pathway analysis was performed using MetaboAnalyst version 4.0 (http://www.metaboanalyst.ca).