CHAC1 Is Differentially Expressed in Normal and Cystic Fibrosis Bronchial Epithelial Cells and Regulates the Inflammatory Response Induced by Pseudomonas aeruginosa

Perra, Léa; Balloy, Viviane; Foussignière, Tobias; Moissenet, Didier; Petat, Hortense; Mungrue, Imran N.; Touqui, Lhousseine; Corvol, Harriet; Chignard, Michel; Guillot, Loic

The Pa WT PAK strain, the mutant FliC with deletion of flagellin and the double mutant pscF xcpQ with deletion of type II and III secretion systems were used and grown as previously (20, 21) . The OD measured at 600 nm was adjusted to give the desired MOI. MOI were verified by serial 10fold dilutions of the bacterial suspensions and plating on LB agar (Supplementary Figure 3) . Cells seeded in 6-well (hAECBs) or 12-well plates (NCI-H292) were infected with Pa in their culture medium without antibiotics. After each stimulation, supernatants were collected and centrifuged at 3,000 × g for 15 min to remove the bacteria. Clinical strains of Pa were isolated according to the standards procedures of the microbiological department of St-Antoine Hospital, Paris, France. Sputum from patients was diluted (v/v) with prediluted Digest-Eur (Eurobio, Courtaboeuf, France), vortexed, and incubated for 15 min at room temperature. A 10-µL aliquot was plated on different selective media for 5 days at 37 • C under aerobic conditions. Pa was isolated from Drigalski's agar plates, and identification was confirmed by mass spectrometry (Maldi-Biotyper, Brucker, France). Stenotrophomonas maltophilia was freshly isolated from a CF patient, and its identification was confirmed by mass spectrometry. S. maltophilia and Staphyloccocus aureus [Newman strain (22) ] were cultured using the same protocol for Pa as described above.