Cell cultures and exposure

THP-1 monocytes (EEAC sigma) were seeded at a density of 5 × 10⁵ cells/mL in 1640 RPMI cell culture medium with L-glutamine (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) supplemented with sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), hepes (Sigma-Aldrich, St. Louis, MO, USA), gentamicin (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) and 10% foetal calf serum (FCS; Biochrom, Berlin, Germany), and maintained at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were passaged every 2–3 days to maintain proper cell density. Prior to the experiments, the cells were seeded on 6-well Corning® Costar® cell culture plates (Merck, Darmstadt, Germany) at a density of 5 × 10⁵ cells/mL in 2 mL cell culture medium. To initiate differentiation into macrophage-like cells, 64 nM phorbol myrisate acetate (PMA; Merck, Darmstadt, Germany) was added to each well followed by 48 h incubation at 37 °C in an atmosphere containing 5% CO₂. PMA-differentiated THP-1 cells are referred to as THP-1 macrophages in the main body of the text. Prior to exposure, each well was washed once with 1 mL PBS before adding 1 mL of serum-free RPMI and incubating the plate for an additional 24 h.

Human bronchial epithelial cells (HBEC3-KT) were cultured in LHC-9 medium (Lonza, Basel, Switzerland) in collagen-coated T75 flasks and maintained at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were passaged twice every week to ensure appropriate culture conditions. For experimental procedures, cells were seeded on collagen-coated 6-well cell culture plates at a concentration of 220,000 cells per well in 1 mL LHC-9 medium. The LHC-9 medium was replaced after 24 h. Then, 24 h prior to exposure, each well was washed once with 1 mL PBS before replacing the medium with 1 mL of serum-free DMEM (Gibco, Thermo Fischer Scientific, Waltham, MA, USA), supplemented with penicillin-streptomycin (Lonza, Basel, Switzerland), ampicillin (New York, NY, USA) and amphotericin B (Sigma, St. Louis, MO, USA).

HBEC3-KT cells were seeded in 6-well cell culture plates in LHC-9 medium at a concentration of 220,000 cells per well and incubated for 48 h at 37 °C in a humidified atmosphere containing 5% CO₂. THP-1 cells were cultured in a T175 cell culture flask and differentiated with PMA as outlined above. The differentiated THP-1 macrophages were then loosened by accutase (A6964; Sigma Aldrich, Merck, Darmstadt, Germany) treatment and resuspended in equal amounts of serum-free RPMI and DMEM at a concentration of 100,000 cells/mL. Next, the HBEC3-KT cell cultures were washed once with 1 mL PBS before adding 1 mL of THP-1 suspension. The co-cultures were then incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂. For experiments with conditioned medium, HBEC3-KT and THP-1 cells were cultured and seeded at the same densities and under the same conditions as outlined above. However, THP-1 macrophages were seeded on new 6-well cell culture plates after being loosened with accutase, while the medium of the HBEC3-KT cells was changed to equal amounts of serum-free RPMI and DMEM.

Particle stocks were prepared in serum-free cell culture medium. To ensure even suspension of the particles, the stock solutions were sonicated for 5 min on ice using a Vibra-Cell™ probe sonicator (Sonics & Materials Inc., Newtown, CT, USA). The primary stock concentration of 2 mg/mL was further diluted in the wells to yield the final concentrations of 50, 100, 200, 300 and 400 μg/mL. HBEC3-KT cells, THP-1 macrophages and HBEC3-KT/THP-1 co-culture were exposed in 1 mL serum-free DMEM, RPMI and DMEM/RPMI, respectively. For experiments assessing the role of NLRP3, cells were incubated with medium containing 0.001, 0.01, 0.1, 1 and 10 μM of the NLRP3 inhibitor MCC950 (Invivogen, San Diego, CA, USA) for 30 min prior to particle exposure. MCC950 was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, United States), according to the manufacturer’s instructions. The level of DMSO was kept below 0.05% of the total well volume and was equalized between wells. Following exposure, the cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 12 or 24 h, depending on the experiment. Next, the cell culture supernatants were transferred to Eppendorf tubes and centrifuged at 290 x g for 10 min to remove cells and debris. The supernatant was then centrifuged for an additional 10 min at 1200 x g to remove particles, before further analyses. For experiments with conditioned medium as described in the "HBEC3-KT/THP-1 co-culture" section, the particle-free supernatants were used to expose HBEC3-KT cells and THP-1 macrophages for 24 h under the same conditions as for particle exposure. Samples from finished experiments were stored at − 80 °C awaiting analysis.