αtlr2

For antibody neutralization, mice were administered via the intraperitoneal injection αTLR2 (InvivoGen), αTLR4 (eBiosciences), αTLR5 (InvivoGen), or αIL6 (eBiosciences) neutralizing antibodies (100 μg/mouse) or pertinent isotype control, as indicated, three days prior to, and concomitant with, consortia inoculation. For pharmacological inhibition of canonical NF-κB, mice were administered BOT-64 (30 mg/kg/dose) via intraperitoneal injection concomitant with consortia inoculation. For pharmacological inhibition of P38, mice were administered SB203580 (2 mg/kg/dose) via intraperitoneal injection concomitant with consortia inoculation.

Cell lines were obtained from ECACC (SK-GT 4, OE33, FLO-1); ATCC (GO (CP-B)); and Invivogen (THP1-XBlueTM-CD14 (NF-kB/AP-1- Reporter Monocytes)). Cell lines were maintained in culture for no more than 20 passages. Cells were not authenticated in the past year. Cell lines from ECACC were tested for mycoplasma contamination twice per year using PCR as described by Young et al. [19 (link)]. SK-GT 4, OE33, FLO-1 and THP1-XBlue-CD14 were cultured in RPMI 1640 GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA, Catalog no. 61870036) with 10% foetal bovine serum (Labtech, Sorisole, Italy, Catalog no. FCS-SA/500) and penicillin–streptomycin (100 U/mL, 100 µg/mL, Catalog No. 15140148). GO were cultured in BEGM™ Bronchial Epithelial Cell Growth Medium Bullet kit (Lonza, Basel, Switzerland, Catalog no. CC-3170). Where indicated, cells were pre-incubated (1 h) with the TLR2 neutralising antibody (αTLR2 (0.4–40 μg/mL), T2.5, Invivogen) prior to stimulation. Cells were stimulated using Pam3CSK4 (P3C; 0.05 μg/mL, Invivogen, San Diego, CA, USA, tlrl-pms), Pam2CSK4 (P2C; 0.05 μg/mL, Invivogen, tlrl-pm2s-1), recombinant HMGB1 expressed from HEK 293 (stock conc. 50 µg/mL; Sigma-Aldrich, St. Louis, MO, USA; SRP6265), and LPS from Escherichia coli 0111:B4 (1 μg/mL, Sigma-Aldrich, L2630), or with 50% conditioned media (CM), generated as described below.