Campygen atmosphere generation systems

H. pylori strain ATCC43504 was obtained from American Type Culture Collection (Manassas, Va, USA) and cultured as previously described.^{23 (link)} The bacteria were incubated on trypticase soy (TS) agar plates supplemented with 5% sheep blood at 37°C for 3 days under microaerophilic conditions (CampyGen Atmosphere Generation Systems, Thermo Scientific, Vienna, Austria). Then, the bacteria were collected in clean TS broth, subjected to centrifugation at 3000 g for 5 minutes, and resuspended in the broth at 10^{9 (link)} colony-forming units (CFUs)/mL. Cultures incubated for 72 hours on plates were used in the following experiments.

All H. pylori strains (H. pylori wild-type P12 (wt) and P12 ΔcagPAI) were cultured under microaerophilic conditions (CampyGen Atmosphere Generation Systems, Thermo Scientific, Vienna Austria) and at 37 °C on GC agar plates (10% horse serum). The mutant strain P12 ΔcagPAI is kanamycin resistant, and thus selective GC agar plates supplemented with kanamycin (8 µg/mL) were used to cultivate P12 ΔcagPAI. For infection of cDC2s, H. pylori was harvested in PBS and added to the cells at a multiplicity of infection (MOI) of 5 for the indicated time points. For TLR inhibition experiments, primary DCs were treated with α-TLR2 (PAb-hTLR2, InvivoGen, Toulouse, France), α-TLR4 (W7C11, α-hTLR4-IgG, InvivoGen, Toulouse, France), or α-TLR10 (3C10C5, Abcam, Cambridge, UK) at a concentration of 1 µg/mL or 10 µg/mL 20 min prior to infection.

H. pylori wild-type P12 (wt) and P12 ΔcagPAI were cultured at 37 °C on GC agar plates (10% horse serum) under microaerophilic conditions (CampyGen Atmosphere Generation Systems, Thermo Scientific). For P12 ΔcagPAI, selective GC agar plates supplemented with kanamycin (8 μg/ml) were used. For infection, H. pylori was harvested and added to the DCs at a multiplicity of infection (MOI) of 0.2–20 or MOI = 5, respectively, for the indicated time points. For TLR inhibition experiments, DCs were treated with α-TLR2 (PAb-hTLR2, InvivoGen) and α-TLR4 (W7C11, α-hTLR4-IgG, InvivoGen) (1 μg/ml each) 20 min prior to infection. As a control, DCs were stimulated with 1 ng/ml LPS (E. coli). For the MAPK inhibition experiments, cells were treated with specific inhibitors of MEK1/2 (U0126, InvivoGen) or p38 (SB203, InvivoGen) at a concentration of 10 μM 1 h prior to infection. JAK kinase inhibition was performed by addition of a specific inhibitor (CAS 457081–03-7, Calbiochem, 1 μg/ml) 20 min prior to infection. TNFα was neutralized by using a human neutralizing antibody (D1B4, Cell Signaling) at a concentration of 100 ng/ml.