Fine ambient particulate matter (PM2.5)
Fine ambient particulate matter (PM2.5), a Standard Reference Material® (SRM 2786) with a mean diameter of approximately 2.8 μm, was purchased from the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). Its certificate of analysis is available online (https://tsapps.nist.gov/srmext/certificates/2786.pdf). In this study, PM2.5 was prepared at a concentration of 5 mg/mL in physiological saline and diluted with physiological saline if necessary. The PM2.5 was vortexed thoroughly before each experiment.
Chemicals and reagents
Primary antibodies including anti-IL-1β (D6D6T) (cat. no. 31202, 1:500), anti-cleaved IL-1β (E7V2A) (cat. no. 63124, 1:1000), anti-NLRP3 (D4D8T) (cat. no. 15101, 1:1000), anti-TLR2 (E1J2W) (cat. no. 13744, 1:1000), anti-Histone H3 (cat. no. 9715, 1:2000), and anti-β-actin (E4D9Z) (cat. no. 58169, 1:2000) antibodies, and secondary antibodies including HRP-conjugated goat anti-rabbit IgG (cat. no. 7074, 1:2000) and horse anti-mouse IgG (cat. no. 7076, 1:2000) were purchased from the Cell Signaling Technology (Beverly, MA, USA). Anti-NF-κB p65 (F-6) (cat. no. sc-8008, 1:200) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture and PM2.5 and/or high glucose treatment
Immortalized mouse alveolar macrophages MH-S were purchased from American Type Culture Collection (ATCC, cat. no. CRL-2019, Manassas, VA, USA) and cultured in an incubator with a humidified atmosphere of 5% CO2 at 37 °C. The complete cell culture medium contained RPMI 1640 medium (cat. no. 10–043-CV, without glucose, Corning, Manassas, VA, USA) supplemented with 10% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin (Corning), 0.05 mM 2-mercaptoethanol (Gibco, Grand Island, NY, USA), and 5 mM glucose (Acros Organics, NJ, USA).
To determine the mRNA or protein expression levels of pro-inflammatory cytokines (IL-1β and IL-18), components of NLRP3 inflammasome (NLRP3 and caspase-1), Toll-like receptors (TLR2 and TLR4), NF-κB p65 (nuclear and cytoplasmic), MMPs (MMP-2 and MMP-9), and TIMPs (TIMP-1 and TIMP-2) in MH-S macrophages after PM2.5 exposure, the cells were treated with 25 or 50 μg/mL of PM2.5 for 1, 3, 6, 12, or 24 h (time-response study), or with 25, 50, and 100 μg/mL of PM2.5 for 6 h (dose–response study). To determine whether the effects of PM2.5 were enhanced at a high glucose setting, the MH-S cells were cultured in complete medium containing 30 mM glucose. The control cells were cultured in complete medium only. Mannitol was used as an osmolality control. 3 × 106 cells in 20 mL medium were seeded in each 75 cm2 flask (day 0). The medium was refreshed on day 4 and day 7. On day 9, the cells were split, and 3 × 106 cells were seeded for another cycle. After 2 cycles (18 days), the cells were collected and 1 × 106 cells in 2 mL medium were seeded in each well of 6-well plates. After overnight culture, the medium was refreshed, and the cells were treated with 25 or 50 μg/mL of PM2.5 (20 μL of 2.5 or 5 mg/mL of PM2.5) for 3 h (for IL-1β, NLRP3, or TLR2 mRNA), 6 h (for IL-1β, NLRP3, or TLR2 protein by Western blot), or 24 h (for MMPs and TIMPs). The cells treated with physiological saline (20 μL) were used as a control.
Cytotoxicity of PM2.5 and/or glucose on mouse alveolar macrophages MH-S
The cells were seeded in 96-well plates. After overnight culture, the cells were treated with 0, 25, 50, 100, 200, and 400 μg/mL of PM2.5 or 5, 10, 20, 30, 40, and 50 mM of glucose for 24 h. The cytotoxicity of PM2.5 or glucose in MH-S cells was determined by two different methods: CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS assay) (Promega, Madison, WI, USA) and alamarBlue™ assay (Invitrogen, Eugene, OR, USA) according to the manufacturer’s instructions. To observe whether there was cytotoxicity with PM2.5 treatment in a high glucose environment, the cells were pretreated with 30 mM glucose for 18 days followed by 25 or 50 μg/mL of PM2.5 treatment for another 24 h. Mannitol was used as an osmolality control.
Total RNA isolation and RT-qPCR
Total RNA was isolated from the cells by using TRIzol® reagent (Sigma-Aldrich, St. Louis, MO, USA), and RT-qPCR was performed as described previously [18, 19]. 2 µg total RNA was reverse-transcribed into cDNA by using M-MLV reverse transcriptase (Promega, Madison, WI, USA). qPCR was performed by using iTaq™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA) on a BioRad iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). The PCR reaction was performed as follows: 40 cycles at 94 °C for 10 s, at 58 °C for 45 s, and at 72 °C for 45 s. Data were quantified by using the 2−ΔΔCt (Livak) method . β-actin was used as an internal control. All the primers used were listed in Table 1.
Protein extraction and Western blot
Total protein was isolated from the cells by RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) while nuclear and cytoplasmic proteins were extracted by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagent (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Western blot was performed as described previously [18, 21]. The expression of β-actin was used as an internal reference for cytoplasmic or total protein, and histone H3 was for nuclear protein. Immunoreactive bands were quantified by using NIH ImageJ software (http://imagej.nih.gov/ij/). Uncropped versions of Western blots were shown in Additional file 5.
Measurement of ROS
ROS generation in mouse alveolar macrophages MH-S after PM2.5 and/or high glucose exposure was determined by using 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCF-DA, Molecular Probes, Eugene, OR, USA). The cells seeded in 96-well plates were pretreated with 5 µM of H2DCF-DA for 2 h before the cells were exposed to 0, 6.3, 12.5, 25, and 50 μg/mL of PM2.5 for 12 h, or to 50 μg/mL of PM2.5 for 3, 6, 12, and 24 h. To determine the effects of PM2.5 on ROS generation in MH-S cells with high glucose pretreatment, the cells were pretreated with 30 mM of glucose for 18 days. Then the cells were seeded and pretreated with 5 µM of H2DCF-DA for 2 h, followed by treatment with 50 μg/mL of PM2.5 for another 12 h. The cells treated with physiological saline were used as the control, and mannitol was used as an osmolality control. The DCF fluorescence was measured by using a Synergy HT microreader (BioTek, Winooski, VT, USA) at λex485/ λem528.
To observe the effects of ROS scavenges or inhibitors on ROS generation in MH-S cells after PM2.5 exposure, the following reagents were used: (1) 100 μM of Trolox (Sigma-Aldrich, St. Louis, MO, USA), a water-soluble analog of vitamin E which has an antioxidant effect; (2) 10 μM of diphenyleneiodonium chloride (DPI) (Alexis, San Diego, CA, USA), a specific inhibitor of NADPH oxidase; and (3) 300 U/mL of superoxide dismutase (SOD) (Sigma-Aldrich), an enzyme that catalyzes the dismutation of the superoxide radical into oxygen and hydrogen peroxide. The cells were pre-treated with ROS scavenges or inhibitors for 2 h and 5 μM of H2-DCFDA for another 2 h, followed by 25 or 50 μg/mL of PM2.5 treatment for 12 h. The fluorescence values were measured as described above.
MitoSOX™ Red Mitochondrial Superoxide Indicator (Invitrogen, Eugene, OR, USA) was used to detect mitochondrial ROS generation in MHS cells after PM2.5 exposure according to the manufacturer’s instructions. Briefly, the cells were pretreated with 5 µM of MitoSOX™ for one hour before 0, 6.3, 12.5, 25, and 50 μg/mL of PM2.5 exposure for another 12 h. The fluorescence at λex530/ λem590 was recorded by a Synergy HT microreader (BioTek, Winooski, VT, USA).
Measurement of cytokines and MMP-2/9 by ELISA
The levels of IL-1β in the cell culture media were determined by Mouse IL-1beta ELISA kit (cat. no. BMS6002, Invitrogen by Thermo Fisher Scientific, Vienna, Austria) while the MMP-2 and MMP-9 protein levels were analyzed by Mouse MMP-2 or MMP-9 PicoKine™ ELISA Kit (cat. no. EK0460 or EK0466, Boster Biological Technology, Pleasanton, CA, USA) according to the manufacturer’s instructions.
Gelatin zymography assay
The enzymatic activities of MMP-2 and MMP-9 were determined by gelatin zymography assay as described in our previous studies [19, 22]. Briefly, the cells were cultured in FBS-free media for 24 h prior to PM2.5 exposure. At the end of exposure, the cell culture media were collected, and an equal volume of media was loaded in each lane of 10% SDS-PAGE copolymerized with 0.5 mg/mL gelatin, which was used as the substrate under nonreducing conditions. After washing 4 times (15 min each) with renaturing buffer [50 mM Tris–HCl buffer (pH 7.5), 2.5% Triton X-100] at room temperature, the gels were incubated in calcium assay buffer [50 mM Tris–HCl buffer (pH 7.5), 0.2 M NaCl, 7.55 mM CaCl2, 1 μM ZnCl2, and 1% Triton X-100] at 37 °C overnight. After briefly washing with ddH2O, the gel was stained with 0.1% Coomassie Brilliant Blue R-250 (Bio-Rad, Hercules, CA, USA) at room temperature for one hour, then destained with 10% acetic acid until the clear bands were observed.
Isolation of alveolar macrophages from mice
Animal use was reviewed and approved by the University of Louisville Institutional Animal Care and Use Committee. Wild-type C57BL/6J (JAX stock no. 000664), NLRP3 knockout (KO) (B6.129S6-Nlrp3tm1Bhk/J, JAX stock no. 021302), and IL-1R1 KO (B6.129S7-Il1r1tm1Imx/J, JAX stock no. 003245) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA), and both strains of homozygous KO mice are viable, fertile, and bred in our university animal facility. The mice were housed in an air-conditioned room (temperature of 20 ± 2 °C, relative humidity of 60 ± 10%) with a 12 h light and 12 h dark cycle environment and with free access to food and water.
Primary alveolar macrophages were isolated from C57BL/6J, NLRP3 KO, and IL-1R1 KO mice by bronchoalveolar lavage (BAL) as described previously [23, 24]. 0.8 mL of ice-cold FBS- and glucose-free RPMI 1640 medium supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin, and 0.4 mM EDTA was used to lavage the lungs. Each mouse was lavaged six times, and approximately 1 × 105 macrophages were obtained from each mouse. The lavage fluid from the same strain of mice was combined and centrifugated at 200 × g and 4 °C for 10 min. After centrifugation, the cells were resuspended at 2 × 105 or 6 × 105 cells/mL in RPMI 1640 medium supplemented with 10% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin, 0.05 mM 2-mercaptoethanol, and 5 mM glucose. Then, 1 × 105 (for IL-1β and NLRP3 mRNA) or 3 × 105 (for MMP-2/9 protein and activity) cells were seeded into each well of a 24-well plate. The cells were treated with 50 μg/mL of PM2.5 for 3 h (for IL-1β and NLRP3 mRNA) or 24 h (for MMP-2/9 protein and activity) with/without 30 mM glucose pretreatment for 24 h. After treatment, the cells or the cell culture media were collected. If the cell culture media would be used for the determination of MMP-2 and MMP-9 protein levels by ELISA or their activities by gelatin zymography assay, FBS-free medium was used to culture the cells. The above procedures were repeated three times.
Data were expressed as the mean ± SEM, and the differences were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett (for comparison with the control) or Bonferroni (for all pairwise comparisons) post-hoc test or two-way ANOVA followed by the Holm-Sidak test by using SigmaPlot 13.0 software (Systat Software, Inc., San Jose, CA, USA). A difference was considered statistically significant when a p-value was less than 0.05.