Recombinant human interleukin 1β il 1β

Monocyte-derived DCs harvested at day 6 were treated with LPS at 50 ng/ml (Sigma-Aldrich) or a cytokine cocktail composed of recombinant human IL-6 (Peprotech), recombinant human interleukin-1β (IL-1β) (Peprotech), and recombinant bovine interferon-γ (IFN-γ) (Biorad), each of them at 50 ng/ml, in moDC medium for 14 h at 38.5°C with 5% CO₂. Treated cells were gently harvested with rubber scrapers in cold PBS with 2 mM EDTA. For stimulations with bacteria, moDC were harvested at day 9 and infected with a S. aureus strain (#1403) isolated from a bovine clinical mastitis at a multiplicity of infection (MOI) of 50 in moDC medium for 2 h under standard conditions. Afterwards, the cells were washed with DPBS and processed for reverse transcription-quantitative PCR (RT-qPCR) as follows.

Test compounds and Bay 11-7082 (Calbiochem, San Diego, CA, USA), used as a pharmacological control, were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Co) so that its final concentration in the culture medium did not exceed 0.1% (v/v). This vehicle was used as control. Lipopolysaccharides from Escherichia coli 026:B6 (LPS; Sigma-Aldrich Co.) were dissolved in phosphate buffered saline (PBS). Recombinant human interleukin-1β (IL-1β; Peprotech, Rocky Hill, NJ, USA) was dissolved in PBS containing 0.1% Bovine Serum Albumin. Test compounds or the vehicle were added to macrophage cell cultures or human chondrocytes 1 h before the pro-inflammatory stimulus, 1 µg/mL LPS or 10 ng/mL IL-1β respectively, and maintained for the rest of the experimental period, except for experiments in Fig. 4 (details in the Results section and figure legend). The concentrations of each compound and the experimental treatment periods are indicated in figure legends.

Frozen PBMCs isolated from peripheral venous blood were thawed and differentiated to PBMDMs, as described earlier. Transduced PBMDMs were cultured in 24-well plates (OptiPlate-24, White Opaque 24-well Microplate) in 0.4 mL medium containing 1 mM luciferin. Cells were stimulated with 200 ng/mL LPS and luminescence detected over time in a CO₂ Lumistar Omega luminometer. Post-assay, pro-viral copies were measured by qPCR, using a Lenti-X™ Provirus Quantitation Kit (Clontech; Oxford, UK). For in vitro stimulation, other ligands used included human recombinant Interleukin-1β [IL-1β](PeproTech), Flagellin FliC from Salmonella typhimurium (NovusBio; Littleton CO, USA), muramyl-dipeptide [MDP] (InvivoGen; Toulouse, France), and LPS extracted using modified phenol/water method (27 (link)) from IBD mucosa-associated E. coli isolates, LF82 and LF10 (28 (link), 29 (link)).