Recombinant human tumor necrosis factor α tnf α

Human bronchial epithelial cells (BEAS-2B), human lung carcinoma cells (A549), and CF bronchi epithelial cells (IB3-1) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS). Recombinant human tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were obtained from R&D Systems (Minneapolis, MN) and reconstituted in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). For cytokines stimulation, TNF-α and IL-1β were used at 10 ng/mL each.

The Saos-2 cell line (ATCC^®HTB-85^TM) was cultured at 37°C in a 5% CO₂ humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM-Gibco) supplemented with 10% fetal calf serum (Dutscher) and 1% antibiotic solution PenStrep^®(Gibco) considered as standard medium (SM). Saos-2 cells were grown to 60–80% confluence in SM then rinsed with sterile Dulbecco’s Phosphate Buffered Saline (DPBS, Gibco) to eliminate antibiotics. Saos-2 cells were further incubated with DMEM and 10% fetal calf serum, without antibiotics, supplemented or not with recombinant human Tumor Necrosis Factor α (TNF α) at 20 ng/mL (R&D Systems) in 25 cm² flasks. After 72 h of incubation, collected supernatants of Saos-2 cells were applied to S. aureus cultures in minimal media (50% of supernatants and 50% of MM, named SN 50). In each condition, the initial quantity of bacteria was ∼10⁶ CFU/mL. Bacterial adhesion was evaluated after 24 h of contact in the static biofilm models (described above).

Cells were trypsinized, washed, and seeded at a density of 50–100 × 10³ cells/mL in multiwall plates. The reagents used to stimulate the HOKs were 1) 50 ng/mL recombinant human lipopolysaccharide-binding protein (LBP; R&D Systems, Inc., Minneapolis, MN, USA) combined with 5 µg/mL Escherichia coli ultrapure lipopolysaccharide (LPS-EB; Invivogen, San Diego, CA, USA); 2) 100 ng/mL recombinant human tumor necrosis factor-α (TNF-α); 3) 100 ng/mL recombinant-human interferon-γ (IFN-γ); both reagents were purchased from R&D Systems Inc. (Minneapolis, MN, USA); 4) 100 μM histamine ≥97.0% (Sigma-Aldrich, St. Louis, MO, USA). The cells were stimulated for 8 h, unless otherwise stated. To investigate the potential effect of the activation of H4R on hBD-2 signaling, the HOKs were incubated with 1 µM specific H4R-agonist HST-10 (N-(3-(1H-imidazol-4-yl) propyl)-2-cyclohexylacetamide) or 1 µM inverse agonist ST-1007 (both were kindly provided by Professor Holger Stark) for 8 h before adding treatment. Such ligands were previously utilized and showed effective influence on H4R signaling [17 (link),52 (link),53 (link)]. Cells were then stimulated with different inflammatory and bacterial mediators as above. Concentrations and time slots were optimized through pilot experiments.