Dy201

N9 cells were seeded at the concentration of 9 × 10⁴ cells/well into a 12-well plate and then the day after were treated as described above. Levels of TNF-α released into the culture medium were quantified after 3, 6, and 24 h by using the corresponding quantification enzyme-linked immunosorbent assay (ELISA) kits (88-7324, Thermo Fisher Scientific, Monza, Italy) according to the manufacturer’s instructions. Levels of IL-6 and IL-1β released into the culture medium were quantified after 24 h by using the corresponding quantification enzyme-linked immunosorbent assay (ELISA) kits (DY406, DY401, R&D systems, Minneapolis, MN, USA). PMA-THP-1 cells were seeded at a concentration of 8 × 10⁴ cells/per well into a 96-well plate and differentiated as described above. Cells were then treated with 100 nM PEA or RePEA as indicated above. Levels of TNF-α, IL-6, and IL-1β released into the culture medium were quantified after 24 h by using the corresponding quantification enzyme-linked immunosorbent assay (ELISA) kits (DY210, DY206, DY201, R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The data were expressed as pg/mL following interpolation on the basis of a standard curve. The experiment was done in triplicate and repeated for three independent measurements.

Within 30 min of blood sampling, 50 μL of whole-blood was LPS stimulated in triplicates after having been diluted 1:10 in RPMI medium (LONZA, BE12-167F) supplemented with LPS (Sigma-Aldrich, L2645-1MG) in a final concentration of 1 μg mL⁻¹. Samples were incubated for ~24 h at 37 °C and 5% CO₂ in order to determine ex vivo cytokine production. After incubation supernatants were harvested and stored at −80 °C until ELISA measurements of IL-1β, IL-6, TNF-α and IFN-γ (R&D Systems, DY201, DY206, and DY210, respectively)^{73 (link)}. The CV% was 3.6–5.2%.

The IL-1β levels in the cellular supernatants and peritoneal lavages were measured using an enzyme-linked immunosorbent assay (ELISA) kit (DY201 or DY401, R&D SYSTEMS). LDH secretion was analyzed using an assay kit (BCT-LDHP, BIOMAX, Seoul, Republic of Korea). For the ROS measurement, LPS-primed BMDMs (1.25 × 10⁵ cells/well in a 96-well-black plate) were treated with dihydrorhodamine 123 (DHR123, CAYMAN CHEMICAL, Ann Arbor, MI, USA) in the presence of rotenone (160 μM) and JC for 6 h. The caspase-1 activity was determined using mouse recombinant caspase-1 (one unit per reaction, BIOVISION, Milpitas, CA, USA) and a caspase-1 fluorometric assay kit (BIOVISION) in the presence of JC or the pan-caspase inhibitor (Z-VAD-FMK, 10 μg/mL; R&D SYSTEMS). The recombinant caspase-1 was co-incubated JC or Z-VAD-FMK in the reaction buffer containing YVAD-AFC for 1 h at 37 °C according to the manufacturer's protocol. The plates were analyzed using a multi-microplate spectrophotometer (Synergy H1 Hybrid Multi-Mode Reader, BIOTEK, Winooski, VT, USA).

The release of IL-1β, IL-8, TNF-α, and TGF-β1 was quantified using cell-free supernatants from the BL compartment after 4, 24, or 48 h of co-culture. The ELISA was run as described by Kinsner et al. (2006) (link) using commercially available antibody pairs (R&D Systems, Cat.: DY210, DY201, DY208, DY240; Abingdon, Oxfordshire, UK). Briefly, the primary antibodies were incubated overnight at room temperature (RT) in coating buffer (0.1 M NaHCO₃ in MilliQ H₂O) on high protein-binding 96-well plates (Thermo Fisher Scientific). After washing (PBS + 0.05% Tween20) and blocking (3% BSA/PBS) for 1 h at RT, the cytokine standards and supernatant samples were added and incubated for 2 h at RT, followed by incubation for 45 min with biotinylated secondary antibodies. After 30 min incubation with streptavidin-peroxidase (Biotrend Chemikalien; Cologne, NRW, Germany), 100 μL 3,3′,5,5′-Tetramethylbenzidine (Sigma-Aldrich) was added and the reaction was stopped with sulfuric acid (1 M) after 5 (IL-8) or 15 min (TNF-α, TGF-β1, and IL-1β). The absorbance was read spectrophotometrically (Enspire, Perkin Elmer; Milano, Lombardy, Italy) at 450 nm.

Lentivirus was produced as previously described [28 (link)]. One million THP-1 cells MEFV KO were infected using 3 ml of virus and 7 μg of polybrene. Plates were centrifuged at 800 g for 3 h at 32°C and then incubated at 37°C. Cells were washed in Dulbecco’s PBS (DPBS, Gibco) 24 h after infection and seeded 48 h after infection with or without Pam3CSK4 (500 ng/ml, InvivoGen, San Diego, CA, USA, 112208–00-1) and native Clostridioides difficile Toxin B protein at various concentrations (TcdB, ab124001, Cambridge, UK). Cells were lysed and immunoblotted for protein expression. IL-1β and IL-18 were measured in culture supernatants by ELISA 24 h after seeding (R&D Systems, DY201 and DY318-05).