Buffy coat was collected from Karolinska University hospital, Sweden, and monocytes were isolated from the buffy coats by a negative selection kit (Stem cell technologies, UK). Isolated monocytes were stimulated with 50 ng/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 0.4 µm pore size (Corning, Sigma Aldrich, Sweden) following earlier protocol for THP1 derived macrophages . After overnight incubation, cell culture media was removed from the apical side to start culturing at air–liquid interface (ALI) and the basal side was filled with the RPMI complete media with 10% FBS and 1% penicillin /Streptomycin.
MQ were exposed to ECIG-aerosol according to the earlier established protocol [17, 20]. According to the earlier study, we selected the ECIG flavor 2 (ripe strawberry, sweet apples and kiwi) which was more toxic than the flavor 1 . For this study, the difference between ECIG with nicotine (3 mg/ml) and without nicotine was just nicotine. The detail, characterization of these ECIG liquid was published in our earlier study .
Shortly, MQ 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-aerosol 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-aerosol within the desiccator. The macrophages cultured at ALI were exposed to ECIG-aerosol or filtered air for 15 min, where after they were transferred to a cell incubator (37 °C, 60% humidity and 5% CO2) for 1 h (h) until the next exposure session. MQ were exposed to ECIG-aerosol total 3 times and 10 puffs in each exposure with 1 h between each exposure. Following completion of 3 exposures, the MQ were incubated for various time points depending on the readout indicated below.
Cell viability and apoptosis assay
To evaluate whether ECIG-aerosol exposure induce apoptosis or cell death, LDH assay (Thermofisher, Sweden) and annexin V assay (BD Bioscience, USA) were performed following manufacturer protocol. Shortly, MQ were exposed to ECIG-aerosol with or without nicotine and incubated for 18 h. After the incubation, cell culture supernatant was used for LDH assay and cells were stained with annexin V for apoptosis measurement by flow cytometry.
Environmental insults including cigarette smoke induce oxidative stress and generate reactive oxygen species (ROS). Therefore, we evaluated the level of ROS in response to ECIG in the presence or absence of nicotine. After the 3rd exposure to ECIG-aerosol, MQ were incubated for 2 h. After the incubation, basal media was removed, and both apical and basal side of the insert were washed three times with PBS. Five µM of Cell ROX reagent (Thermofisher, Sweden) in RPMI media was added at both basal (500 µl) and apical side (500 µl) of the insert. After 30 min of incubation, cells were washed 3 times with PBS, trypsinized, resuspended in PBS and collected into flow cytometry tubes. In the presence of reactive oxygen species (ROS), CellROX reagents generate fluorescence which is proportional to ROS level. The total ROS level was determined by flow cytometry, and median fluorescence intensity (MFI) was presented as the level of ROS generation.
According to the manufacturer protocol (Abcam, UK), levels of phospholipids in ECIG liquid and ECIG vapor condensate were measured by phospholipid assay kit.
MQ were incubated for 18 h after the 3rd exposure to ECIG-aerosol. MQ were trypsinized and incubated with Fc blocker for 10 min on ice. After the incubation with Fc blocker, the MQ were incubated for 30 min on ice with the antibodies, specific to M1 MQ markers (PerCp5.5-labeled CD86 and PE-labeled CD11C), M2 MQ marker (FITC-labeled CD206), TLR2 (BV-421-labeled), TLR4 BV711-labeled) and lipid scavenger receptor ‘CD36’ (PE labeled-CD36) (BD bioscience, US). Cells were washed 3 times with PBS, resuspended in fresh PBS and the expression of these cell surface markers was investigated by flow cytometry using single or multi color fluorochrome. In the muti color assay, compensation was performed by compensation beads (BD bioscience, US) to avoid spectral overlap. The raw data from flow cytometry was analyzed by Flow Jo software and the expression level was presented as median fluorescence intensity.
According to the manufacturer instruction, Lipid peroxidation product malondialdehyde (MDA) was measured from the cell lysate using MDA assay kit (Sigma Aldrich, Sweden). In order to detect MDA-modified protein, MQ was incubated as above with FITC-labeled MDA antibodies (antibodies against MDA-modified protein, Abcam UK) and as above, the level of MDA-modified proteins was investigated by flow cytometry.
After the 3rd exposure to ECIG-aerosol, MQ were incubated for 6 h, and mRNA was extracted from the MQ by RNA extraction mini kit (Qiagen, Germany). cDNA was synthesized from 300 ng of RNA by cDNA synthesis kit (Applied biosystem, Germany) and 100 ng of cDNA was used in each reaction of RT-qPCR. PCR amplification primers were selected according to the earlier study . Housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The expression levels of oxidative stress related genes glutathione peroxidase (GPx), glutathione peroxidase (GPx4) and HOMOX1 as well as inflammatory cytokines genes IL-6, TNF-α, IL-12, IL-1β and IL-10 were calculated by delta-delta CT methods.
After the 3rd exposure, cell culture media was collected after 18 h of incubation. Secreted cytokines including IL-6, TNF-α, IL1-β, IL-8, IL-12A and IL-10 levels were measured from the culture supernatant by ELISA duoset (Biotechne, UK). In addition, cell lysates were prepared after 6 h of 3rd exposures and level of HSP60 was measured from the cell lysates using ELISA duoset (Biotechne, UK).
Lipid accumulation assay
Lipid accumulation was measured by flow cytometry and microscopy. After ECIG exposure, according to the manufacturer’s protocol, cells were stained with lipid staining reagent BODIPY (Sigma Aldrich, Sweden) and lipid accumulation was investigated by flow cytometry and confocal microscopy. Median fluorescence intensity was presented as the level of expression from Flow cytometry measurement and microscopic image was presented after developed by image J software.
According to the manufacturer protocol (Santa Cruz, Germany), CD36 was silenced with shRNA. In brief, differentiated MQ were cultured with serum free medium in 6 wells plate and transfected with CD36 shRNA or with a control shRNA. After 6 h of transfection, 10% FBS was added to the culture medium. After 72 h, more than 70% reduced expression of CD36 was confirmed at gene and protein level by qRT-PCR and flow cytometry, respectively.
According to the manufacturer’s protocol (Vybrant™ Phagocytosis Assay Kit, Thermo Fisher, Sweden), phagocytosis of E. coli particles (FITC labeled) by MQ was investigated. In short, MQ-cultured at ALI were exposed to ECIG-aerosol 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 h of incubation, MQ were 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 h 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 determined by flow cytometry, and the expression level was presented as median fluorescence intensity (MFI).
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 the Friedman test and followed by Wilcoxon signed-rank test. In all statistical tests, the difference with a P value ≤ 0.05 was considered statistically 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 #. Pearson two tailed analysis was performed to determine correlation between lipid accumulation and dependent variables and correlation matrix to evaluate the correlation between each variable. All the data were analyzed using the GraphPad Prism 8.30 software.