PM preparation

SRM 1648a (standard reference material) was supplied by the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA). SRM 1648a is a conglomeration of fine and ultrafine particles (with high levels of transition metals (TM): iron (Fe) and zinc (Zn)) with a mean particle diameter 5.85 µm. According to the certificate of analysis it contains also organic compounds, including polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and chlorinated pesticides [56]. Our own additional analysis also documented in the NIST particles high content of LPS (Lipopolysaccharide/endotoxin) [57]. SRM 1648a was also treated for 120 min with low-temperature (cold) plasma using a Plasma Zepto system (Diener Electronic GmbH, Germany) at the highest power (100 W) to remove organic compounds from the reference material (hereinafter referred to as LAP). The carbon, hydrogen, nitrogen, and sulfur contents were evaluated by elemental analysis (Elementar, Vario Micro Cube, Germany). The carbon and organic carbon contents in the NIST and LAP samples were determined using a total organic carbon analyzer (Shimadzu, TOC-V series with a Total Nitrogen accessory, Japan). Additionally, a general physicochemical analysis of PM was performed by our partners from the Department of Inorganic Chemistry, Jagiellonian University, Cracow, Poland. NIST contains ca. 13% carbon, including 10.5% organic carbon, and less than 2% organic carbon, after the removal of organic compounds from the reference material (LAP) [58]. Similarly, the LPS content was significantly lower than in the NIST particles [57]. For in vitro culture, PM preparations were weighed on a high-precision microbalance and freshly suspended in RPMI (Roswell Park Memorial Institute) 1640 medium (Corning, Manassas, VA, USA) under sterile conditions. The final concentrations of PM (1, 10, and 100 µg/mL) in monocyte cultures were established experimentally, based on our [57] and other in vitro studies [59,60,61].

Monocytes isolation and cell culture

CPDA-treated blood samples from healthy donors were purchased from the Regional Center of Blood Donation and Blood Therapy in Krakow, Poland. Peripheral blood mononuclear cells (PBMCs) were isolated using standard Pancoll human (Panbiotech, Aidenbach, Germany) density gradient centrifugation. Monocytes were separated from PBMCs by counter-current centrifugal elutriation (JE-6B elutriation system equipped with a 5-mL Sanderson separation chamber; Beckmann-Coulter, Palo Alto, CA, USA), as described previously [62]. The cells were washed and resuspended in RPMI 1640 medium (Corning) and kept in an ice bath until use. The purity of isolated monocytes was confirmed by flow cytometry using an anti-CD14 antibody (BD Biosciences Pharmingen, San Diego, CA, USA) and did not drop below 90%.

Monocytes (5 × 105/mL) were cultured in Ultra Low Attachment tubes (Corning) in RPMI 1640 medium (Corning) supplemented with 2 mM L-glutamine, phenol red, 5–10% heat-inactivated fetal bovine serum (FBS, EURx, Gdańsk, Poland) and 25 µg/mL gentamycin (Sigma, St. Louis, MO, USA) (complete medium), with or without NIST and LAP at three concentrations (1, 10, and 100 µg/mL). Cells were kept at 37 °C in a humidified atmosphere with 5% CO2 and collected after 15 min, 2 and 4 h of culture. The time points of the analysis were experimentally established during the cell culture. For some experiments, a population of lymphocytes containing approximately 80% CD3-positive cells was isolated by counter-current centrifugal elutriation from PBMCs.

Additionally, in some experiments, monocytes were simulated with 10 µM CCCP (15 min, carbonyl cyanide m-chlorophenylhydrazone, Sigma), an uncoupler of oxidative phosphorylation; 200 µM TBHP (tert-butyl hydroperoxide, Invitrogen, Carlsbad, CA, USA); 100 ng/mL of LPS (lipopolysaccharide) from Salmonella abortus equi (Sigma); or 400 U/mL of human recombinant IFN-γ (Sigma) with 100 ng/mL of LPS from Salmonella abortus equi (Sigma) (positive controls). As a negative control, a 1 h cell pre-incubation with the inhibitor of mitochondrial ROS production (1.5 mM; Mito-TEMPO ((2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride), Sigma) or Caspase-1 inhibitor (50 µM; Ac-yvad-cmk; InvivoGen, San Diego, CA, USA), was done prior to PM exposure. In the experiments assessing the role of phagocytosis in PM internalisation, monocytes were preincubated (1 h) with cytochalasin D (0.1 μM; Sigma) or were incubated with PM on ice. Additionally, polyclonal mouse anti-human TLR4 antibody and relevant isotype control (both 5 μg/mL; InvivoGen) were used to block LPS binding. To block NLRP3 activation, a selective inhibitor – N-((1,2,3,5,6,7-heksahydro-s-indacen-4-ylcarbamoyl)-4-(2-hydroxy-2-propanyl)-2-furansulfonamide) (MCC950; 10 μM; Sigma) was used prior to PM stimulation.

Antigen pulse and T-cell proliferation

T cell proliferation in response to PPD (purified protein derivative of tuberculin) antigen (Statens Serum Institute, Copenhagen, Denmark) stimulation was analyzed, as described previously [63]. Briefly, monocytes at the density of 4 × 105/mL suspended in DMEM (Dulbecco's Modification of Eagle's Medium) medium (Corning), containing phenol red and supplemented with 2 mM L-glutamine, 10% human AB serum (Sigma), and 25 µg/mL gentamycin (Sigma) – complete DMEM medium were exposed (15 min) in Ultra Low Attachment tubes (Corning) to NIST or LAP used in three concentrations: 1 µg/mL, 10 µg/mL, and 100 µg/mL. Thereafter, control samples (monocytes not exposed to PM) and monocytes exposed to PM were divided into two parts: one part was pulsed with a “recall” PPD at a concentration of 100 µg/mL, while the second part of monocytes was incubated with no antigen (additional control). For these experiments only blood donors responding for PPD were selected. After a 2 h antigen pulse (37 °C, humidified atmosphere with 5% CO2), monocytes were washed with complete DMEM medium. Four groups of monocytes (control and control PPD-pulsed, PM, and PM PPD-pulsed) at a density of 1 × 104/mL were cultured for 7 days (37 °C, 5% CO2 in a humidified atmosphere) in complete DMEM medium (0.2 mL) in flat-bottom 96-well plates (Corning) with 1 × 105/mL of autologous T lymphocytes. 3H-thymidine (Hartmann Analytic GmbH, Braunschweig, Germany) (1 µCi per well) was added for overnight incubation. Subsequently, cultures were harvested, and cell radioactivity was measured using a Beckman LS1801 Scintillation Beta Counter (Beckman-Coulter, Brea, CA, USA). Results were expressed as counts per minute (cpm), and proliferation index was calculated as a ratio of cpm from T cell cultures with antigen-pulsed monocytes to cpm from T cell cultures with monocytes without antigen pulse.

Cell morphology

To estimate the morphological changes in monocytes after 15 min, 2 and 4 h exposure to PM, 2 × 105/mL cells per glass slide were spun in a Cytospin 2 centrifuge (Shandon, England) for 5 min, fixed in methanol, and stained with May-Grünwald-Giemsa dye (Sigma). Cells that were not exposed to PM were used as controls. Monocyte morphology was assessed by light microscopy (Olympus BX53, Olympus Corporation, Tokyo, Japan), using 100x/1.40 magnification (Oil objective, Olympus). Images were processed using CellSens Dimension v.1.16 software (Olympus).

Monocyte viability

Monocyte viability was assessed by flow cytometry using Annexin-V Apoptosis Detection Kit I (BD Pharmingen), according to the manufacturer’s instructions. Briefly, monocytes after 15 min, 2 and 4 h of culture with or without PM were harvested, washed in PBS (Corning), resuspended in binding buffer, and stained with Annexin V-FITC (15 min at room temperature in the dark). The stained cells were examined using flow cytometry (FACSCalibur, BD Biosciences). Typically, 10,000 monocytes were acquired for analysis. Additionally, monocyte viability and Annexin-V binding was determined after Mito-TEMPO (Sigma) or Ac-yvad-cmk (InvivoGen) pre-treatment prior to 15 min of PM exposure and after 15 min of incubation with CCCP (positive control; Sigma). Moreover, in some experiments, monocytes after 2 h exposure to PM were re-challenged with the same dose of PM for an additional 15 min or 2 h. Thereafter, cell viability was determined (as described above). The results are expressed as a percentage of Annexin V-FITC-positive cells.

ROS production

Monocytes (5 × 104/mL) in 50 µl of complete medium with or without PM and 100 µl of 2 mM luminol (5 amino-2,3 dihydro-1,4 phthalazinedione) (Sigma) in Krebs–Ringer buffer (1.12 mM Mg2 + ; 0.54 mM Ca2 +) and 11 mM glucose (Sigma) were suspended in 96-well White Opaque Tissue Culture Plates (Falcon, Corning, NY, USA). The plates were placed in the measuring chamber of a Victor2 plate reader (EG&G WALLAC, Turku, Finland) at 37 °C, and ROS formation was analyzed using luminol-dependent chemiluminescence measurements. The results were expressed as cumulative counts of responses recorded for 100 min from 60 cycles. Additionally, detection of ROS in monocytes was performed by flow cytometry using a FACSCanto X flow cytometer (BD Biosciences) after cell staining with 1 µM CellROX Green reagent (Invitrogen). The results are expressed as a frequency of CellROX Green positive cells.

Mitochondrial membrane potential (ΨMMP)

To determine changes in the mitochondrial membrane potential (ΔΨMMP) of monocytes after exposure to PM, cells were stained with MitoScreen JC-1 dye (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide; BD Pharmingen) for 15 min at 37 °C, according to the manufacturer’s instructions. Thereafter, cells were washed twice in warm washing buffer and ΨMMP was analyzed by flow cytometry (FACSCalibur, BD Biosciences) after 15 min of monocyte exposure to PM. Data from 10,000 monocyte events were recorded and analyzed using CellQuest v.3.1 software (BD Biosciences). The relative changes in ΨMMP were determined as the ratio of aggregates (high fluorescence intensity) to monomers (low fluorescence intensity).

Cellular respiration

For the assessment of mitochondrial respiration, monocytes were seeded at 150,000 cells/well in XFe 8 well cell culture microplates (Agilent Technologies, Palo Alto, CA, USA), covered with a combination of laminin, fibronectin, and poly-l-lysine (all from Sigma) in 80 µl of complete medium. Cells were kept in microplates at 37 °C and 5% CO2 in a humidified atmosphere overnight to allow them adherence to the plastic. Thereafter, 60 µl of supernatant was discarded and 180 μl of the Seahorse media (XF-based minimal DMEM; Agilent Technologies) supplemented with 5.5 mM glucose (Sigma), 1 mM Sodium Pyruvate (Gibco, Gaithersburg, MD, USA), and 2 mM L-glutamine (Gibco) were carefully added to avoid disturbing the cell layer. The XFe 8 well cell culture microplates were incubated at 37 °C in a non-CO2 incubator for 1 h. The assay sensor cartridge was hydrated overnight with 200 μl of water for injection at 37 °C (in a non-CO2 incubator). Thereafter, water was discarded and replaced with 200 µl of XF Calibrant Media (Agilent Technologies) for at least 3 h. Before the measurement of OCR (oxygen consumption rate), PM was added directly to XFe 8 well cell culture microplate with monocytes, Oligomycin (1 µM; Agilent Technologies) was added to Port A of the assay cartridge, FCCP (2 µM; carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, Agilent Technologies) was added to Port B and Rotenone/antimycin A (0.5 µM; Agilent Technologies) were added to Port C (final concentrations). Optimization of cell density and Oligomycin and FCCP concentrations were experimentally established. Key parameters of mitochondrial function, OCR, basal, maximal, and non-mitochondrial respiration, ATP production, proton leak, and spare capacity were measured using a Seahorse XFp Extracellular Flux Analyzer (Agilent Technologies) using Seahorse XFp Cell Mito Stress Test (Agilent Technologies), according to the manufacturer’s instructions, and analyzed by Wave Desktop and Controller 2.6 Software (Agilent Technologies).

Western blotting

For Western blot analysis, monocytes were harvested after a 15 min exposure to PM, washed in PBS (Corning), and lysed in Mammalian Protein Extraction Reagent (Sigma) containing protease inhibitor (Sigma). Cell-free supernatants were collected for protein analysis. Equal amounts of proteins were loaded onto 10% polyacrylamide gels, resolved by SDS-PAGE, and transferred to PVDF membranes. PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa, (catalog number: 26620; Thermo Fisher Scientific Inc., Waltham, MA, USA), was used as size standards in SDS-PAGE and Western blotting. Next, membranes were incubated for 1 h with 5% BSA (bovine serum albumin; Sigma) in tris-buffered saline with 0.05% Tween 20 (Sigma) to block non-specific binding and washed before the overnight incubation with primary rabbit monoclonal antibodies: anti-Apaf-1 (clone D7G4; 1:1000), anti-NLRP3 (clone D4D8T; 1:1000), anti-Gasdermin E (clone E2X7E; 1:1000) and anti-Gasdermin D (clone E8G3F; 1:1000, all from Cell Signaling Technology, Danvers, MA, USA) at 4 °C, followed by 1 h incubation at RT with secondary anti-rabbit IgG HRP-conjugated antibody (1:5000; Cell Signaling Technology). Blotting with rabbit anti-GAPDH monoclonal antibody (D16H11) (1:5000; Cell Signaling Technology) was performed to confirm the equivalent protein loading. Protein expression was detected using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific Inc.) and a ChemiDoc Imaging System (Bio-Rad, Warsaw, Poland).

Caspase-1, Caspase-3, and Caspase-9 activity

Caspase-1 activity was detected after 15 min, 2 and 4 h of monocyte incubation with or without PM using a Caspase 1 (active) Staining Kit (Abcam, Cambridge, MA, USA), according to the manufacturer’s instructions. Cells were harvested and stained with FAM-YVAD-FMK in the dark at 37 °C in a humidified atmosphere with 5% CO2 for 1 h. The stained cells were immediately examined for green fluorescence of FAM-YVAD-FMK by flow cytometry (FACSCanto X, BD Biosciences).

Caspase-3 activity was determined at the same time points of monocyte exposure to PM. Cells were harvested and washed in PBS (Corning) with 2% FBS (EURx), fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences), according to manufacturer’s instructions. Then, the cells were washed twice in Perm/Wash solution (BD Biosciences) and stained (30 min at 4 °C, dark condition) for active Caspase-3 using PE-conjugated monoclonal antibody (BD Pharmingen). After staining cells were washed twice in PBS with 2% FBS and suspended in PBS for flow cytometry analysis (FACSCanto X, BD Biosciences).

In some experiments after 2 h of monocyte exposure to NIST or LAP, cells were additionally re-challenged with the same dose of PM for next 15 min or 2 h and stained for Caspase-1 and Caspase-3 activity (as above).

Caspase-9 activity was assessed after 15 min, 2 and 4 h of monocyte incubation with or without PM using an active Caspase-9 FITC Staining Kit (Abcam). For this purpose, cells were harvested and stained with FITC-LEHD-FMK in the dark at 37 °C in a humidified atmosphere with 5% CO2 for 45 min. The stained cells were immediately examined using a FACSCanto X flow cytometer (BD Biosciences) and analyzed for green fluorescence of FITC-LEHD-FMK by CellQuest v.3.1 software (BD Biosciences).

Immunostaining for intracellular IL-1β

Monocytes (3 × 105/mL) were cultured in Ultra Low Attachment tubes (Corning) in complete medium with or without PM, at 37 °C in a humidified 5% CO2 atmosphere. For intracellular detection of IL-1β, 2 µM Golgi Stop (containing monensin; BD Biosciences) was added at the beginning of culture. After 15 min and 4 h of the culture, cells were harvested, washed in PBS with 2% FBS, fixed and permeabilized with Cytofix/Cytoperm solution. Next, cells were washed twice in Perm/Wash solution and stained (30 min at 4 °C, dark) for intracellular cytokine using a PE-labelled mouse anti-human IL-1β monoclonal antibody (BD Pharmingen). The IL-1β producing monocytes were analyzed using a FACSCanto X flow cytometer with BD FACSDiva v. 8.0.1 software. In some experiments, monocytes after the first exposure to PM for indicated time were re-challenged with the same doses of PM for additional 2 h. Thereafter, the level of IL-1β positive cells was determined (as above).

Cytokine detection by CBA

Monocytes (3 × 105/mL) were cultured at 37 °C in a humidified, 5% CO2 atmosphere in Ultra Low Attachment tubes (Corning) in complete medium with or without PM in three concentrations (1 µg/mL, 10 µg/mL and 100 µg/mL). Supernatants from monocyte cultures were collected after 4 h and the concentration of cytokines (IL-1β, IL-6, IL-8 and TNF-α) was determined by Cytokine Bead Array (CBA, Human Inflammatory Cytokine Kit; BD Biosciences), according to manufacturer’s instructions. Typically, data from 3,600 beads were acquired by FACSCanto X flow cytometer and analyzed using FCAP Array v. 3.0 software (BD Biosciences).

Statistics

The normal distribution of the data was verified using the Shapiro–Wilk test. Statistical analysis was performed using the Kruskal–Wallis test for data without normal distribution and ANOVA for data with normal distribution using PRISM GraphPad 6.01 software (GraphPad Software Inc., San Diego, CA, USA). Data are presented as median with interquartile range. Statistically significant differences were considered at the following p-values: p < 0.05, p < 0.01, p < 0.001, and p < 0.0001.