Airborne particulate matter (PM2.5) collection
Atmospheric PM2.5 samples were obtained from January to October 2016 in Lanzhou, China, according to our previously described methods. PM samples were gathered on glass fiber filters by a flow air particle sampler (TH-150C, Wuhan Tianhong Instrument Factory, Wuhan, China) at a constant flow rate of 100 L/min. The filter membranes were then cut into 1 cm x 1 cm squares and extracted three times using an ultrasonic extractor at 100 W for 15 min in deionized water. Next, each sample suspension was filtered using 12 gauze layers, and dried by a vacuum freeze-drying machine (Labconco, Kansas, USA). PM2.5 samples were stored at ‒80℃, followed by resuspension of the resulting pellets in phosphate-buffered saline (PBS) before use.
C57BL/6 mice (3-4 weeks old) were purchased from the Shanghai Sippr-BK Laboratory Animal Co. Ltd. Mice were housed in clear cages loosely covered with air filters and containing a corncob pad as bedding. After a week of acclimatization, PM2.5 suspension was topically applied to the right corneas of mice.
Fluorescent mock PM2.5 particle tracing experiment
The fluorescent mock PM2.5 particles prepared from SiO2 (diameter: 10‒500 nm) were a gift from Prof. Yonghui Deng at Fudan University. Particles were re-dissolved in PBS and topically applied to the eyes of mice. Frozen sections were prepared, and particle distribution was observed under a confocal fluorescence microscope (Leica).
PM2.5 suspension topical exposure
Exposures were performed using a 1 mg/mL solution thrice daily (3 x 2 μL drops) over 3 months, with PBS applied topically to the contralateral eye as control. After 3 months, the mice were sacrificed, and outflow tissues were isolated and collected. All experiments complied with the Association for Research in Vision and Ophthalmology Statement on the use of animals in ophthalmic and vision research and were performed under the guidance of the Animal Care and Use Committee of Fudan University (Shanghai, China).
Mice IOP measurements
The IOP for both eyes was measured without anesthesia by rebound tonometry (TonoLab; ICare, Espoo, Finland). IOP was measured three times, and the average was used as the final value.
Mouse outflow tissue and HTM cells were lysed with a RIPA solution (Beyotime, Shanghai, China), and the protein concentration was determined by the BCA method (Beyotime, Shanghai, China). About 5‒20 μg of protein were loaded onto gels and separated by SDS-PAGE (10% or 12% acrylamide). Proteins were then transferred onto polyvinylidene fluoride membranes (PVDF, 0.45 μm; Millipore, Shanghai, China) by electrophoresis. Membranes were blocked with 5 % non-fat dry milk for 2 hours at room temperature. PVDF membranes were probed with a primary antibody (dilutions of the primary antibodies are presented in Table 1), followed by incubation with peroxidase-conjugated secondary antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.
HTM cell culture and PM2.5 treatment
HTM cells were purchased from ScienCell Research Laboratories (Shanghai, China). HTM cells were incubated in Trabecular Meshwork Cell Medium (ScienCell, Cat. No. 6591) containing 2% fetal bovine serum (FBS, Cat. No. 0010), 1% HTM growth supplement (TMCGS, Cat. No. 6592), and 1% penicillin/streptomycin (P/S, Cat. No. 0503) at 37°C and 5% CO2.
For the experiments, HTM cells were seeded at a concentration of 5 x 105 cells/well in 6-well plates and 5 x 103 or 5 x 106 cells/well in 96-well plates. After cell attachment, the culture medium was replaced with fresh medium containing a PM2.5 suspension or an equal volume of medium as a control. ROS scavenger acetylcysteine (N-Acetyl-L-cysteine, NAC, Sigma, Shanghai, China) 3 mM or caspase-1 inhibitor belnacasan (VX-765, Selleck, Shanghai, China) 100 uM were used. All experiments were performed at least three times.
Cell viability test
The viability of the PM2.5-treated HTM cells was tested using a cell counting kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, 100 µL of HTM suspensions (5000 cells/well) were added into the wells of a 96-well plate and incubated until cell adhesion. CCK-8 solution (1:10) was then added to each well of the plate 2 h before measurement. The optical density (OD) was measured at 450 nm using a microplate reader (Tecan, Männedorf, Switzerland), and cell viability was reported as a percentage of the optical density values from unexposed control cells (100%).
Contractility assay and treatments
Collagen gels were prepared in 96-well plates from a collagen solution (1.85 mg/ml, Cell Biolabs, Beijing, China) by following the manufacturer’s instructions. Briefly, HTM-containing medium (4 x 106 cells/mL) was added onto the collagen gel and incubated for 1 h at 37°C with 5% CO2. After collagen polymerization, culture medium was added to each collagen gel lattice. Following a 48-h incubation, the edge of the gel was gently detached using a pipette tip. The gel area was then imaged using a Fluorescent Stereomicroscope (Leica M165 FC) every hour for 15 h in order to determine the time required for cessation of the “natural contraction” of the gel by HTM cells. Drugs (NAC or VX-765) were added to the medium, and images were captured at 6, 24, and 48 h. The gel area was calculated using the Fluorescent Stereomicroscope.
Reactive oxygen species generation detection
The intracellular ROS levels were detected by the Reactive Oxygen Species Assay Kit (ROS Assay Kit), following the manufacturer’s instructions (Beyotime, Shanghai, China). HTM cells were washed with DMEM/F12 and were then treated with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, 1:1000, diluted by DMEM/F12) at 37°C for 20 min. After washing 3 times with the medium without FBS, the DCF fluorescence distribution of cells was detected. Rosup 50 μg/mL and 500 μg/mL were employed as the negative and positive control, respectively. The DCF fluorescence distribution of cells was observed under a fluorescence microscope (ZEISS, Shanghai, China).
RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
HTM cells were exposed to PM2.5 (100 μg/mL and 200 μg/mL) for 48 h, while control cells were treated with PBS. Total RNA was then extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA), and RNA concentration was measured by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). mRNA expression was measured using the SYBR Green quantitative real-time PCR kit (qRT-PCR, Takara, Osaka, Japan) according to the manufacturer’s instructions. Samples were amplified in a ViiA 7 Real-Time PCR System (Life Technologies, Pleasanton, CA), and mRNA expression was normalized to β-actin (the housekeeping gene). Expression was estimated using the comparative CT method (2-ΔΔCT) of relative quantification with the ViiA 7 Software (Life Technologies). Three independent experiments were conducted. The primer sequences used for the qRT-PCR are presented in Table 2.
Enzyme-linked immunosorbent assay (ELISA)
The IL-1β protein levels in the HTM cell culture medium were quantified using the human IL-1β ELISA kit (Abcam, Boston, MA) following the manufacturer’s instructions. The optical density (OD) was read at 450 nm using a microplate reader (Tecan, Männedorf, Switzerland).
Immunofluorescence analysis was performed using specific primary antibodies against NLRP3 (1:100, Abcam, Boston, USA) and caspase-1 (1:50, Proteintech, Shanghai, China). HTM cells grown on coverslips were fixed in 4% paraformaldehyde for 30 min at room temperature and were then washed three times with PBS. Cells were then treated with 0.1% Triton X-100 (Biotech Well, Shanghai, China) in PBS for 10 min and were once again washed three times. This was followed by blocking in PBS containing 0.5% bovine serum albumin (BSA, Roche, Shanghai, China) for 1 h at room temperature in a humidified chamber. The coverslips were then incubated with a primary antibody (dilutions of the primary antibodies are presented in Table 1) diluted in PBS containing 0.5% BSA overnight at 4°C in a humidified chamber. The cells were then washed three times with PBS and incubated with Alexa Fluor®555 anti-rabbit IgG (H+L) (1:200; donkey polyclonal; Beyotime, Shanghai, China) for 1 h at room temperature. After further washing with PBS, coverslips were stained with DAPI and stored at 4°C in the dark before being viewed under a confocal fluorescence microscope (Leica).
The results are presented as the mean ± standard deviation (SD) or mean ± standard error of mean (SEM). Data were analyzed using SPSS 21.0 (IBM, Chicago, IL, USA). For normally distributed data, the paired t-test, independent t-test, or the Student's t-test were used for two-level comparisons, while one-way analysis of variance (ANOVA) was used for ≥ 3-level comparison. The Mann-Whitney U test was used for two-level comparisons, while the Kruskal-Wallis H test was used for ≥ 3-level comparisons of non-normally distributed data. In all cases, differences were considered significant at p < 0.05.