6 well culture plate

Human Embryonic Kidney 293T cells (HEK cells) were cultured in DMEM/F12 medium with L‐glutamine supplemented with 10% FCS, 1% NEAA, and 1% Pen/Strep (all Gibco). Cells were plated in 6‐well culture plates (Greiner) at equal densities 1 day prior to transfection at 37°C, 5% CO₂.

HaCaT immortalized human keratinocytes were obtained from Cell Lines Service and cultured in DMEM medium (Lonza, Cat No.: 12-604F) supplemented with 10% FBS (Gibco Thermo Fisher, Waltham, MA, USA). Cultures were maintained in 6-well culture plates (Greiner, Frickenhausen, Germany) at 37 °C in a humidified incubator with 5% CO ${}_2$ atmosphere. Once confluent, cell monolayers were washed in phosphate-buffered saline (PBS) twice, briefly incubated in trypsin-EDTA (Lonza), then resuspended in culture medium and transferred to the sample holder device at 1200 cells/mm ${}^2$ density. Cells attached to the fibronectin-coated electrospun membrane and formed a confluent monolayer. Cultures were maintained for various durations up to 3 weeks while the sample holder devices were kept in standard 24-well tissue culture plates (Greiner) and medium was refreshed every 3 days.

Tracheas of B. pseudohinzii-negative C57BL/6 J mice were explanted aseptically, surrounding tissue was removed and tracheas were cut into approximately 2 mm long pieces consisting of 2–3 cartilage rings. For electron microscopy, the rings were opened by cutting the trachealis muscle, washed in sterile PBS (Thermo Fisher Scientific) supplemented with penicillin (100 U/ml; PAA, Etobicoke, Canada) and streptomycin (0.1 mg/ml; PAA) and transferred into 6-well culture plates (Greiner bio–one, Kremsmünster, Austria). The rings were then submerged in phenol red–free MEM (Thermo Fisher Scientific) supplemented with penicillin, streptomycin and L-glutamine (2 mM) (Thermo Fischer Scientific) and cultured at 37 °C and 5% CO₂ for 24 h. After 24 h, viability of explants was validated by visualizing the beating of ciliated cells. Vital explants were then transferred into fresh plates and washed with subsequent addition of B. pseudohinzii cultures (strain 3227). Throughout, explants were kept in 1 ml of medium.
For measurements of PTS and CBF of in vitro-infected tracheal explants, each trachea was divided into 2 halves. They were transferred into a delta T-dish, washed with PBS, bacteria were added and the samples were incubated for 4 h at 37 °C. PTS and CBF were examined as described below. Throughout, explants were kept in 1 ml of medium.

Human monocytic leukemia THP-1 (ATCC: TIB-202, Rockville, MA) and mouse macrophage cell line RAW264.7 (ATCC: TIB-71) were maintained in RPMI 1640 and DMEM media, respectively, supplemented with 10% fetal bovine serum (FBS), penicillin G (100 IU/ml), streptomycin (100 μg/ml), and L-glutamine (2 mM) and incubated at 37°C. TDM cells were obtained by treating THP-1 monocytes with 25 ng/ml PMA for 48 h in 6-well culture plates (Greiner, Germany) with 2 ml cell suspension (5 × 10⁵ cells) in each well. Differentiated cells were washed and incubated for another 24 h in the culture medium to return to the resting state of macrophages (M0). For chronic iron overload experiments, THP-1, TDM and RAW264.7 cells were persistently maintained and subcultured in media containing 100 μM FeSO₄.

A human hepatocellular liver carcinoma (HepG2) cell line was used as an in vitro model of liver cancer, because HepG2 cells show similar cell morphology as hepatocytes [20 (link)] and are commonly used to test immunomodulatory effects of molecules on cancer cells [15 (link)]. The HepG2 cell line was maintained in complete media composed of RPMI 1640 (Lonza, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Lonza, USA) and 5% penicillin-streptomycin (Lonza, USA), incubated in 5% CO₂ at 37°C. For MTT assays, cells were seeded at 2 × 10⁴ cells/well in 96-well plates (Greiner Bio-One, Germany). For RNA and protein analysis experiments, cells were seeded at 3 × 10⁵ cells/well in 6-well culture plates (Greiner Bio-One, Germany). After seeding, HepG2 cells were cultured overnight prior to stimulation. For morphological changes and cell death features, cells were examined using an inverted microscope (Olympus 1X70, USA). The viable cell count was determined by trypan blue staining using a hemocytometer (Hausser Scientific, USA). HepG2 cells and PBMCs were counted before seeding, in order to fix the count and ratio of PBMCs: HepG2 cells.

Murine bone marrow cells were obtained from femur and tibia bone marrow. Cells were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Invitrogen, Grand Island, NY, USA) containing 10% heat-inactivated fetal bovine serum, 100 mg/ml of streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 100 mg/ml of penicillin (Sigma-Aldrich), and 40 ng/ml of recombinant macrophage colony-stimulating factor (Peprotech, Rocky Hill, NJ, USA) for 5 d at 37°C. Bone marrow-derived macrophages (BMDMs) were harvested and seeded at 5×10⁵ cells/well in 6-well culture plates (Greiner Bio-One, Monroe, NC, USA). After overnight incubation with 10 ng/ml IFN-γ(R&D Systems, Minneapolis, MN, USA), the wells were washed and challenged with stimulators as follows: Pam3CSK4 (Toll-like receptor [TLR]2 ligand) 50 to 500 ng/ml, LPS (TLR4 ligand) 100 ng/ml, and ODN1585 (TLR9 ligand) 500 nM (Pam3CSK4 and ODN1585 from InvivoGen [San Diego, CA, USA] and LPS from Sigma-Aldrich). The cells were stimulated for 24 h before the supernatants were collected and stored at -80°C until assayed.