Scientific rigor and reproducibility
We have applied robust unbiased experimental design and data analysis approaches throughout the study. We have validated the methods and ensured reproducibility with repeated experiments. All methods are presented in detail with transparency. Results were reported and interpreted without bias. Laboratory grade biological and chemical resources were purchased from commercial sources. Our methodologies, data, and results adhered to strict NIH standards of reproducibility and scientific rigor.
Ethics statement
Institutional biosafety and animal protocol approval.
Experiments in this study were performed according to the standards and guidelines approved by The University of Rochester Institutional Biosafety Committee (Study approval #Rahman/102054/09-167/07-186; identification code: 07-186; date of approval: 01/05/2019 and 02/03/2020). Validated cell lines, human bronchial epithelial cell lines (16-HBE and BEAS2B) and human monocytic leukemia derived cell line (MONO-MAC-6), were procured from ATCC, USA. Ethical approval was not necessary for the utilized cell lines.
All mouse housing, handling, exposure, and procedure protocols used in this study were approved by the University Committee on Animal Research (UCAR) Committee of the University of Rochester, Rochester, NY (UCAR protocol 102204/UCAR-2007-070E, date of approval: 01/05/2019 and 02/03/2020).
Human plasma samples used for lipidomics analysis were from the study conducted at the University of Rochester Medical Center (Rochester, NY, USA) (Institutional Review Board approval RSRB00064337) [10].
Aerosol exposure setup
An Ooze slim twist vaping pen set at 3.8V was connected to the Scireq Inexpose pump (Montreal, Canada). For in vitro exposures, cell culture plates were placed inside an Enzyscreen chamber (Enzyscreen BV, Netherlands) and exposed to two 70 mL puffs of the aerosol under air-liquid interface conditions. The cells were allowed to incubate in the vapor for 10-minutes.
For in vivo exposures, wild type mice with C57BL/6 background were exposed to 1 hr MCT, VEA, and cartridge aerosols with 70 mL puffs, two puffs/min using the Scireq Inexpose system (Montreal, Canada) [11].
Vitamin E acetate (VEA) 50% w/v preparation
A refillable vape oil cartridge was filled with either MCT oil (GreenIVe, Amazon) or tocopherol acetate (Sigma Cat# PHR1030-500MG) was mixed with MCT oil to make a 50% w/v or 1.06 M VEA.
E-cigarette cartridges
For the cell and mouse exposures in this study, e-cig cartridges used in our previous study characterizing constituents of EVALI were used [9].
Physicochemical characteristics of MCT and VEA
Respirable particle concentration and distribution:
Dusttrack II aerosol monitor 8530 (TSI, MN) was used to measure respirable particles with aerodynamic diameter 1.0, 2.5, 4.0, and 10.0 µm by taking readings immediately after a single puff was released to a chamber with dimensions 8”x6”x5.25” (Enzyscreen, Netherlands). Data collection duration is one minute with a 10s moving average. These fine particle measurements were obtained for air, MCT, VEA, and e-cig cartridge aerosols.
Measurement of total volatile organic compounds (VOC) levels in aerosols
To measure the VOC levels inside the Enzyscreen chamber, the exhaust tubing was connected to a 50 mL conical tube. A photoionization detector probe 985 (TSI, MN) was placed inside the conical tube, and the total VOC levels in air, MCT, VEA, and cartridge aerosols were recorded.
Acellular ROS Assay
Cell-free ROS generated by MCT, VEA, and e-cig cartridge aerosols were determined using 2’7’dichlorofluorescien diacetate (H2-DCF-DA) fluorogenic probe (Sigma-Aldrich Cat# 287810). H2-DCF-DA [5 mM] with NaOH [0.01N] was reacted for 30 minutes to prepare the dye. After incubation, 25 mM phosphate buffer was added to stop the reaction followed by the addition of 2 mL HRP. A set of hydrogen peroxide standards ranging from 0 to 50 µM was prepared from a 1M H2O2 stock. Subsequently, two puffs of MCT, VEA, and e-cig cartridges were bubbled through an impinger containing 10 mL of the dye connected to Scireq Inexpose pump. After aerosolization, the samples were incubated for 15 minutes at 37 °C in a water bath. Subsequently, the absorbance/emission was read at 485/535 nm with Turner Quantech fluorometer (FM109535, Barnstead international).
Cellular ROS Assay
2’7’dichlorofluorescien diacetate (H2-DCF-DA) fluorogenic probe (Sigma-Aldrich Cat# 287810) dye was prepared as described. 15,000 BEAS-2B cells/well were cultured in a 96-well plate in complete medium to 80% confluency. After serum depriving the cells at 1% FBS for 12 hours, 20 µM dye was added to each well of the 96-well plate and was incubated for 30 minutes. After incubation, the cells were treated with 0.25% (v/v) MCT, and 0.025% (w/v) or 50 μM VEA, and a mixture of e-cig cartridge liquids at 0.0001% (v/v). The absorbance/emission was read at 485/535 nm at 1.5 hours, 4 hours, and 6 hours using microplate spectrophotometer (Cytation 5, Biotek).
Cell Culture
Bronchial epithelial cells, BEAS-2B, were cultured in DMEM F-12 50/50 base media and supplemented with 5% FBS, 1% Pen/Strep, and HEPES. Cells were plated at 300,00 cells/well in 6 well plates in complete medium. At 80% confluency, the cells were serum-deprived in 1% FBS. Similarly, epithelial cells, 16-HBE, were cultured in DMEM base media and supplemented with 1% Pen/Strep, 10% FBS, and 1ml Amphotericin B. 16-HBE cells were plated and serum-deprived in 1% FBS. Monocytes, Mono-Mac-6 (MM6), were cultured in 24-well culture plates in RPMI 1640 media and supplemented with 1% Pen/Strep, 1mM Sodium Pyruvate, 2mM L-glutamine, non-essential amino acid, Transferrin, Oxaloacetic acid, Polymixin B, and Bovine Insulin. At 80% confluency, MM6 cells were serum-deprived in 1% FBS 12-hours before treatment.
Aerosol exposures and treatments to cells
Epithelial cells (BEAS-2B and 16-HBE) were exposed to MCT, VEA (50% w/v), and e-cig cartridges, as described in the exposure setup section. Twenty-four hours post-exposure, the conditioned media, and the cell pellets were collected for further analysis.
Monocytes, MM6 cells were treated with %0.25 (v/v) MCT, 0.025% (w/v) or 50 μM, and 0.0001% cartridges in 1% FBS media. Twenty-four hours later, conditioned media and cell pellets were collected.
Cytotoxicity Assay
To assess the induced cytotoxicity by MCT and VEA treatments, 20 µL of cells were mixed with 20µL of Acridine Orange/Propidium Iodide. Then, 20 µl of the mixture was added to the Nexcelom automated cell counter slide, and viability and the live and total cell counts were performed using a Nexcelom 2000 Cellometer (Nexcelom Bioscience, Lawrence MA).
Cytokine ELISA
Conditioned media collected from each well was used to determine inflammatory cytokine release. Interleukin 6 (IL-6) was measured using an IL-6 ELISA kit following manufacturer’s instruction (Invitrogen, Catalog # CHC1263). Similarly, an interleukin-8 (IL-8) ELISA kit (Invitrogen, Catalog # CHC1303) to quantify secreted IL-8 levels in conditioned media. The cartridge aerosol exposed data points were then pooled, and the average was compared with air, MCT, and VEA.
Trans-epithelial electrical resistance (TEER) measurement
16-HBE cells (20,000 cells/well) were cultured on 24-well transwell inserts (Corning cat# 3470) until the cells reached a complete monolayer. Cells were then serum-deprived at 1% FBS 12-hours before exposure. Cells were exposed to MCT, VEA, and cartridge aerosols using the previously described aerosol exposure setup. 24-hours later, the barrier function was evaluated by recording the transepithelial voltage and resistance by EVOM2 (WPI instruments, FL). Each well was measured three times, and the average unit area resistance was calculated. Cartridge aerosol exposed data points were then pooled, and the average was compared with the unexposed air group.
In vivo mouse exposures
Approximately four month old male and female mice with C57BL/6 background were exposed to medium-chain triglyceride oil (MCT), vitamin E acetate (50% w/v in MCT), and e-cig cartridge aerosols one-hour per day for three consecutive days, as described in the aerosol exposure setup section [11]. For cartridge aerosols, six different cartridges were aerosolized for 10-minute cycles. Immediately following the last exposure, mice were euthanized and the tissues were collected.
Mouse arterial oxygen saturation
Immediately prior to mouse euthanasia, arterial oxygen saturation was measured in mice by MouseOX plus device (STARR life science, PA). Data collected over approximately ~8 minutes and any unstable data points were removed, including the first 60s.
Bronchoalveolar lavage (BALF) collection
Upon anesthesia 0.6 mL of 0.9% NaCl saline solution was instilled three times (1.8 mLs cumulative volume) into the trachea and the recovered BALF centrifuged at 1000 RPM for 7 minutes. The acellular fraction of the BALF was stored at -80°C for cytokine analysis by Luminex assay. The pelleted cells were then used for flow cytometry analysis to obtain differential cell counts.
Luminex assay
To determine inflammatory mediators released due to MCT ,VEA, and e-cig cartridge aerosol exposures , 50 μL of the BALF sample was used with BioRad 23-plex-Group I kit (BioRad Cat# M60009RDPD) according to the manufacturer’s instructions. Briefly, capture antibody coupled magnetic beads were added to the plate, followed by the addition of samples and the standards. After incubating, detection antibody and streptavidin-PE were added. The appropriate number of washing steps and incubation steps were followed as instructed. After resuspending the sample in 125 μL of assay buffer, the plate was read on a FLEXMAP 3D system (Luminex). The concentrations of each analyte were compared to the unexposed air group and the analytes that showed significant differences were reported.
Flow cytometry analysis
Collected cells from the BALF recovery were counted by AO/PI assay to obtain total cell counts. The cells were then blocked with anti-CD16/32 (Fc block) for 10 minutes. Followed by a PBS wash step, cells were stained with CD45, F4/80, Ly6B.2, CD4, and CD8 cell surface markers in staining buffer to identify approximate counts of cell populations. After 30 minutes of incubation in the dark at 4°C the cells were washed twice in PBS and resuspended in 100 μL buffer. Appropriate FMO controls and compensation beads were used for compensation. Sample acquisition was performed using Guava easycyte 8 flow cytometer (Luminex). Data analysis was performed using GuavaSoft 3.3.
Western blot analysis
Snap-frozen mouse lung tissues were homogenized in RIPA buffer with protease inhibitor cocktail and 25µg of protein from air, VEA, MCT, and cartridge aerosol exposed mice (N=6 per group for male (n=3) and female (n=3) were extracted and quantified using BCA protein assay. Extracted proteins were separated on 7.5% SDS-PAGE gels, which were then transferred onto nitrocellulose membranes. The membrane was probed with SP-A antibody (ab115791), ACE2 antibody (ab108252), Furin (ab183495), and TMPRSS2 (ab92323). Β-actin (ab20272) was used as a loading control for normalization.
Lipidomics analysis
Mouse BALF samples (200 μL) and human plasma from one of our e-cig studies were analyzed by Cayman chemicals, MI, for eicosanoids/oxylipins and short-chain-fatty-acids by LC-MS/MS. Heat maps were generated depicting the changes in analytes. Analytes showing distinct differences were then graphed as box-whisker plots. Untargeted lipidomic profiling was performed by normalizing peak areas with internal standards. Air control samples were compared with VEA and cartridge exposed samples, and only the statistically significant analytes were included in Table 1.
Oil-Red-O staining
Oil-Red-O staining was performed on lung OCT-cryosections and immobilized MM6 cells using BioVision Cat# K580-24 according to manufacturer’s instructions. In brief, the oil-red-o stock was made in 20 mL 100% isopropanol. The slides were incubated in 60% isopropanol for 5 minutes, followed by evenly covering with Oil-Red-O working solution. The slides were then placed on a shaker and incubated for 20 minutes. After rinsing the slides with dH2O, hematoxylin was added and incubated for 1 minute. After rinsing the slides with dH2O five times, the slides were viewed under the microscope. Lipid-laden-indices were calculated in representative images of MM6 stained with Oil-Red-O.
Lipid-laden-index (LLI) scoring
The method for LLI scoring of the Oil-Red-O stained monocytes was adapted from Kazachkov et al. [12]. In two representative images, the total macrophages in each field were counted. Then, macrophages with <50% of the cytoplasm opacified by the stain were assigned a score of “1”. Macrophages with >50% of the cytoplasm opacified by the stain were assigned a scored of “2”. The LLI was then calculated as follows: LLI=((% 1+ macrophages)×1)+((% 2+ macrophages)×2).
Statistical Analysis
Statistical analysis of data was done by One-Way ANOVA with Tukey’s multiple comparison test for multiple sample groups with one variable and by Two-Way ANOVA with Tukey’s multiple comparison test for multiple sample groups with two variables using GraphPad Prism 8.0. Data are reported by mean ± SEM and statistical significance was reported as *p< 0.05, **p<0.01, and ***p<0.001.