Cell culture
Buffy coat was collected from Karolinska University hospital, Sweden, and monocytes were isolated by negative selection kit (Stem cell technologies, UK). Isolated monocytes were stimulated with 50ng/ml of GM-CSF (Immunotools, Germany) for 5 days to differentiate into macrophages. Macrophage differentiation was confirmed by light microscopic visual observation and CD11B staining. The differentiated macrophages were trypsinized and seeded in 12 wells transwell inserts with 0.4µm pore size (Corning, Sigma Aldrich, Sweden). After overnight incubation, cell culture media was removed from the apical side to start culturing at air-liquid interface (ALI) and basal side was filled with the RPMI complete media with 10% FBS and 1% penicillin /Streptomycin.
ECIG exposure
Macrophages were exposed to ECIG smoke according to the earlier established protocol (14). According to the earlier study, we selected the ECIG flavor 2 (ripe strawberry, sweet apples and tart kiwi) which was more toxic than the flavor 1 (14). Shortly, macrophages in 12 well plates were placed in a glass jar with 3L desiccator volume, maintained at 37°C and humidity above 70% and allowed to equilibrate for 10–15 min. An air-tight pre-heated glass syringe was used to repeatedly collect 40 ml (representing one puff) of ECIG smoke and injected it into the desiccator. Ten puffs were injected to mimic one vaping session. The inlet tube contained multiple sidewise apertures for an even spread of the ECIG smoke within the desiccator. The macrophages cultured at ALI were exposed to ECIG smoke or filtered air for 15 min, where after they were transferred to a cell incubator (37°C, 60% humidity and 5% CO2) for 1 hour (h) until next exposure session. Macrophages were exposed total 3 times and 10 puffs in each exposure with 1 h between each exposure. Following completion of 3 exposures, the macrophages were incubated for various time points depending on the readout indicated below.
Cell viability and apoptosis assay
According to manufacturer instruction LDH assay (Thermofisher, Sweden) and annexin A5 assay (BD Bioscience, USA) were performed for cell viability and apoptosis respectively. Shortly, MQ was exposed to ECIG with or without nicotine and incubated for 18 hours. After the incubation, cell culture supernatant was used for LDH assay and cells were stained with annexin A5 and apoptosis was determined by flow cytometry.
ROS measurement
After the 3rd exposure to ECIG smoke, macrophages were incubated for 2 hours. After the incubation, basal media was removed, and both apical and basal side of the insert was washed three times with PBS. Five µM of Cell ROX reagent (Thermofisher, Sweden) in RPMI media was added both at the basal (500µl) and apical side (500µl) of the insert. After 30 minutes of incubation, cells were washed 3 times with PBS, trypsinized, resuspended in PBS and collected into flow cytometry tubes. In presence of reactive oxygen species (ROS), CellROX reagent generate fluorescence which is proportional to ROS level. The total ROS level was measured by flow cytometry, and median fluorescent intensity (MFI) was presented as level of ROS generation.
Malondialdehyde measurement
Macrophages were incubated for 18 hours after the 3rd exposure. According to the manufacturer instruction malondialdehyde (MDA) was measured from the cell lysate using MDA assay kit (Sigma Aldrich, Sweden). MDA-modified protein was investigated by FITC-labeled MDA antibodies (antibodies against MDA-modified protein, Abcam UK) using flow cytometry.
Phospholipid measurement
According to manufacturer protocol, levels of phospholipids in ECIG liquid and ECIG vapor condensate were measured by phospholipid assay kit (Abcam, UK)
Macrophage phenotypic markers
After the 3rd exposure to ECIG smoke, the macrophages were incubated for 18 hours. After the incubation, cells were trypsinized and stained with antibodies that are M1 markers (PerCp5.5-labeled CD86 and PE-labeled CD11C) and M2 marker (FITC-CD206 (BD bioscience, US). The expression of these cell surface markers were investigated by flow cytometry, and the expression level was presented as median fluorescence intensity.
RT-qPCR
After the 3rd exposure to ECIG smoke, the macrophages were incubated for 6 hours. After the incubation period, mRNA was extracted by RNA extraction mini kit (Qiagen, Germany). cDNA was synthesized from 300ng of RNA by cDNA synthesis kit (Applied biosystem, Germany) and 100 ng of cDNA was used for RT-qPCR. PCR amplification primers were selected according to earlier study (14). Housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The expression level of glutathione peroxidase (GPx), glutathione peroxidase (GPx4), IL-6, TNF-α, IL-12, IL-1β and IL-10 was calculated by delta-delta CT methods.
ELISA
After the 3rd exposure, cell culture media was collected after 18 hours and secreted cytokines including IL-6, TNF-α, IL1-β, IL-8, IL-12A and IL-10 levels were measured by ELISA duoset (Biotechne, UK). In addition, cell lysates were prepared after 6 hours of 3rd exposures and HSP60 was measured from the cell lysates using ELISA duoset (Biotechne, UK).
CD36 expression
Surface expression of lipid scavenger receptor CD36 (BD Bioscience, US) was measured by flow cytometry after 18 hours of ECIG exposure and expression levels were presented as median fluorescent intensity.
Lipid accumulation assay
Lipid accumulation was measured by flow cytometry and microscopy. After ECIG exposure, according to manufacturer protocol, cells were stained with lipid staining reagent BODIPY (Sigma Aldrich, Sweden) and lipid accumulation was investigated by flow cytometry and confocal microscopy. Median fluorescent intensity was presented as the level of expression from Flow cytometry measurement and microscopic image was presented after developed by image J software.
CD36 silencing
According to manufactural protocol, CD36 was silenced with ShRNA (Santa cruz, Germany). In brief, differentiated macrophages were cultured with serum free medium in 6 wells plate and transfected with CD36 shRNA or with a control shRNA. After 6 hours of transfection, 10% FBS was added to the culture medium. After 72 hours, more than 70% reduced expression of CD36 was confirmed at gene and protein level by qRT-PCR and flow cytometry, respectively.
TLR4 but not TLR2 expression
MQ were incubated for 18 hours after the 3rd exposure of ECIG. After the incubation, MQ were trypsinized and stained with TLR2 and TLR4 antibodies (BD Bioscience, US) and the surface expression of TLR2 and TLR4 was investigated by flow cytometry and the expression level was presented as median fluorescence intensity.
Phagocytosis
Phagocytosis of E. coli particles (FITC labeled) by MQ as investigated according to manufacturer protocol (Vybrant™ Phagocytosis Assay Kit, Thermo Fisher, Sweden). In short, MQ culture at ALI were exposed to ECIG smoke as described above. After overnight incubation, apical side of the insert was washed with RPMI media and E. coli particles in 100µl of media was added on the top of the MQ. After 3 hours of incubation, MQ was trypsinized, transferred into 96 wells plates and the plates were read at 485 excitation wavelengths by microplate reader (Bioteknik, US). Percentage of phagocytosis was calculated and compared between control and exposed condition. As described above, MQ were exposed to ECIG and incubated for 18 hours after the 3rd exposure and stained with CD35 (complement receptor1) and CD64 (Fc receptor) antibodies (BD bioscience, US) to detect surface expression. The expression of these cell surface markers was analyzed by flow cytometry, and the expression level was presented as median fluorescence intensity (MFI).
Statistical analysis
Each experiment was performed with MQ from 3 donors (N = 3), and technical replicates n = 2–3 from each donor. The results were expressed as median and interquartile ranges (25th–75th percentiles) followed by non-parametric statistical analysis. Within each group, the comparisons between control and ECIG exposure with or without nicotine were performed by Friedman test and followed by Wilcoxon signed-rank test. In all tests, difference with a P value ≤ 0.05 was as considered significant. Statistical significance in comparison to clean filtered air (sham) to all treatment condition expressed with * and between exposure with or without nicotine was expressed with #. All the data were analyzed using the GraphPad Prism 8.30 software.