Biodiesel exhaust particle (BDEP) and petro-diesel exhaust particle (DEP) generation and characterization
BDEP and DEP were generated during combustion in a diesel engine in a laboratory at Umea University, Sweden, which has extensive experience in diesel exhaust research. Pure rapeseed methyl ester (RME) is a major biodiesel fuel in Europe and was compared with standard petrodiesel fuel (SD10), The DEP and BDEP particles are well characterized and used in previous studies28,29. The particle emission from the biodiesel and petro-diesel combustion differed qualitatively and quantitatively. PM and CO emission was reduced in biodiesel while emission of NOx was increased in biodiesel emission compared to petro-diesel. The particle size distribution in the biodiesel exhaust was shifted towards smaller particles as compared to petro-diesel exhaust, with a bimodal distribution indicating a larger fraction of nanoparticles. Furthermore, the concentration of polycyclic-aromatic hydrocarbons (PAHs) was lower in the biodiesel particles29.
Cell culture
Human pulmonary bronchial epithelial cell (PBEC)
Human primary bronchial epithelial cells (PBEC) were obtained from bronchial healthy tissues of donors who underwent lobectomy. The study protocol was approved by the Ethics Committee of Karolinska Institutet, Stockholm, Sweden. All the donors gave their informed written consent. The harvested PBEC were stored in liquid nitrogen and have been used in several studies17, with confirmed cellular characteristic. In the current study, cell passage number 3 and 4 was used. The air- liquid interface model was established according to earlier protocol17. In short, 1.5 × 105 PBEC/transwell inserts 0.9 cm2 with 0.4 μm pore size, (BD Falcon™) were cultured under submerged conditions in PneumaCult™-Ex plus medium with supplement (Stemcell technologies, UK), 10% growth factor and supplemented with 0.1% hydrocortisone (Stemcell technologies, UK) and 1% penicillin/streptomycin (life technologies) The culture medium was replaced every 48 hours and at day 7 when cells had reached ≥ 90% confluency, the cells were cultured at air-liquid interface and expansion medium was replaced with PneumaCult™-ALI maintenance medium with 0.5% 10X supplement (Stemcell technologies, UK), 1% hydrocortisone, 2 mg/ml heparin (Stemcell technologies, UK) and 1% antibiotic and added to the basal chamber only. The standard cell culture condition 37°C and 5% CO2 was maintained during culture period. After 3 weeks of culturing, the cells have differentiated, cell morphology has changed and the cell mucosa models included basal cells, ciliated cells, mucus producing cells and Club cells17.
THP-1 derived macrophage (MQ) and MQ-ALI monocultures
The THP-1 cell line was purchased from the American Type Culture Collection (TIB-202™, ATCC, Rockville, MD, USA), and grown with RPMI complete media (Gibco Life technologies, UK) supplemented with 10% FBS and 1% penicillin streptomycin (Gibco Life technologies, UK). THP-1 cells passage 28, 29 and 30 were differentiated into macrophage-like cells by stimulation of 10ng/ml of PMA (Sigma Aldrich, US) for 48 hours. Cells were trypsinized and purity of the macrophages was determined by microscopic observation and CD11b staining followed by analyzing by FACS. Differentiated MQ at a concentration of 1.5 × 105 cells/transwell inserts in 200ul of RPMI media were added to the apical side of the insert. After 3 hours of incubation and adherence of the macrophages, the media was carefully removed before the exposure to diesel or biodiesel.
PBEC-ALI and MQ co-culture
MQ and PBEC were co-cultured according to earlier established protocol5. In short, PBEC-ALI cultures were developed as described above. Differentiated MQ (1.5 × 104 cells/transwell cells) re-suspended in 200µl of RPMI media was added to the apical side of the insert and on top of the differentiated PBEC-ALI culture and incubated for 3 h. After the adherence of the MQ, the cell culture media form the apical side was removed carefully, followed by the addition of BDEP/DEP (see below). Cell culture temperature and presence of CO2 was maintained as above.
Exposure of PBEC-ALI, MQ-ALI and PBEC-ALI/MQ to DEP or BDEP
DEP or BDEP at two different concentrations (18µg/cm2 and 36µg/cm2) in a small volume (40 ul in ALI or RPMI media) was added on the top (apical side) of the cells cultured at ALI conditions and incubated for different time points depending on the experimental outcomes. As control (Sham exposure) cell models were exposed to the same volume of fresh media.
Reactive oxygen species (ROS)measurement
PBEC-ALI or MQ-ALI models were exposed to BDEP or DEP at the above-mentioned concentration and was incubated for 2 hours. After the incubation, basal media was removed, and apical side of the insert was washed three times with PBS. Cell ROX reagent (Thermofisher, Sweden) at a concentration of 5uM at ALI or RPMI media was added both to the basal and apical side of the insert and incubated for 30 minutes. Cells were washed 3 times, trypsinized, washed, resuspended in 400µl of PBS and collected into flow cytometry tube. The ROS level was measured by flow cytometry, analyzed according to manual and median fluorescent intensity were presented as level of ROS.
Cell viability
Following exposure to either BDEP or DEP, PBEC and MQ or the combination of both cell types were incubated for 18 hours. Apoptosis induction and cell viability were measured by annexin A5 and propidium iodide staining, respectively.
Glutathione measurement
Glutathione protects cells from oxidative damage by neutralizing reactive oxygen species, where glutathione peroxidase (GPx) catalyzes the reaction and increases the level of oxidized glutathione (GSSG). PBEC or MQ were exposed to DEP or BDEP as described above for 6 hours. The cells were washed and lyzed with lysis buffer, centrifuged and the supernatant was used for GSH measurement by the colorimetric assay kit (Sigma Aldrich,USA).
ELISA
The PBEC, MQ or PBEC with MQ cells culture media was collected after 18 hours of the exposures and secreted cytokines including Interleukin -6 (IL-6), tumor necrosis factor alpha (TNF-α), interleukin -1beta (IL-1β), interleukin -13 (IL-13), chemokine (C-X-C motif) ligand 8 (CXCL8), as well as secreted proteins including uteroglobin, MMP9 and PGE2 level were measured by ELISA using duoset (Bio-techne, UK). In addition, to measure intracellular level of heat shock protein 60 (HSP60) and COX-2, cell lysates were prepared after 6 hours and 18 hours of exposures and HSP60 and COX-2 respectively were measured by ELISA duoset (Bio-techne, UK).
RT-qPCR
The cells exposed to DEP or BDEP were collected after 6 hours, and mRNA were extracted by mini kit (Qiagen, Germany). cDNA was synthesized from 300ng of RNA by cDNA synthesis kit (Applied biosystem, Germany). 1ul of cDNA was used in 20 ul reaction mixture of RT-PCR. Housekeeping gene GAPDH was used as an internal control. The expression levels of GPx, GSS, IL-6, TNF-α, IL-12, IL-10 and IL-4 were calculated as delta-delta CT methods.
Macrophage polarization markers CD86 and CD206
MQ in presence or absence of PBEC was exposed to DEP or BDEP for 18 hours. After the incubation, cells were trypsinized and stained with M1 marker CD86, and M2 marker CD206 antibodies (BD bioscience, USA,). Cells were stained with CD11b (Bd Bioscience, USA) as a macrophage marker to distinguish from PBEC. The expression of these cell surface markers was analyzed by flow cytometry, and the expression level were presented as median fluorescence intensity.
Histone phosphorylation and DNA damage assay
Histone phosphorylation is an indication of DNA damage. Histone phosphorylation was investigated by flow cytometry kit (BD Bioscience, US). Level of histone phosphorylation was presented as median fluorescence intensity (MFI). DNA damage was investigated by of 8-hydroxy-2deoxyguanoisne release in cell culture supernatant. The level of 8-hydroxy-2-deoxyguanosine was measured by competitive ELISA (Biotechne UK).
Prostaglandin E2 inhibition
To investigate the role of COX-2 on PGE2 and inflammatory cytokines release, PBEC was exposed to BDEP or DEP in presence or absence of COX-2 inhibitor (Valdecoxib, Biotenchne UK). In short, according to manufacturer instruction, cells were incubated with 5nM valdecoxib for 45 min prior exposure to BDEP or DEP.
Phagocytosis assay
Phagocytosis of E. coli particles (FITC labeled) by MQ was investigated according to manufacturer protocol (Vybrant™ Phagocytosis Assay Kit, Thermo fisher, Sweden). In short, MQ-ALI models were exposed to BDEP or DEP 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-ALI models. After 3 hours of incubation, cells were washed, and fixed with 3.7% formaldehyde. The membrane of the insert was cut and placed on a microscopic glass slide, stained with DAPI with anti-fade mounting media (2 scientific, US) and covered with a glass cover slip. The microscopic images were taken by Zeiss LSM 900 confocal microscope (Zeiss, Germany) and the images were analyzed by image J software. Furthermore, quantitative phagocytosis analysis was performed by fluorometric microplate reader. In short, MQ-ALI or PBEC/MQ-ALI models were exposed to BDEP or DEP and incubated with E. coli particles as described above. After 3 hours of incubation, MQ were trypsinized, transferred into 96 wells plates and the plates were read at 488 excitation wavelengths by microplate reader (Bioteknik, US. According to manufacturer instructions, phagocytic activity was calculated, and percentage of phagocytosis was compared between control and BDEP/DEP exposed condition. As described above, MQ-ALI models were exposed to BDEP or DEP for 18 hours and stained with CD35, CD36 and CD64 antibodies (BD bioscience, US) to detect surface expression of these proteins. The expression of these cell surface markers was analyzed by flow cytometry, and the expression level was presented as median fluorescence intensity.
Statistical analysis
Each experiment was performed with PBEC from 3 (N=3) donors, and technical replicates were 6 (n=6), and experiments with THP1 derived MQ was performed 3 times (N=3) with a minimum of 3 technical replicates (n=3). 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 BDEP/DEP exposure at different doses were assessed by Friedman test and followed by Wilcoxon signed rank t test. In all tests, difference with a P value below 0.05 was considered significant. Statistical significance in comparison to control (sham) to all treatment condition expressed with * and between different treatment condition was expressed with #, Ф, ¤ or ±. All the data were analyzed using the GraphPad Prism 8.30 software.