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

Because Pa induced CHAC1 expression in non-CF cells, we investigated which Pa virulence factors were involved. We studied the effects of two very well-known virulence factors, LPS and flagellin from Pa, on hAECBs from CF and non-CF patients and on NCI-H292 cells, which expressed CHAC1 mRNA at sufficient levels for further knockdown experiments in comparison to BEAS-2B cells (Supplementary Figure 2A) . Notably, 6 h of stimulation with LPS and flagellin induced CHAC1 expression in hAECBs from non-CF patients, similar to whole bacteria at a MOI of 0.25 for 6 h. This induction by LPS and flagellin and by living bacteria was not found in hAECBs from CF patients (Figure 1C) . We observed similar effects of LPS and flagellin on CHAC1 expression in NCI-H292 cells (Figure 2A) . To validate the role of flagellin in CHAC1 expression, we used a mutant of the Pa PAK strain, FliC, in which flagellin was deleted. We observed a significantly lower increase in CHAC1 mRNA expression when NCI-H292 cells were stimulated with the FliC strain than with the WT PAK strain (Figure 2B) . We also tested a double mutant of the Pa PAK strain with both type II and III secretion systems deleted ( pscF xcpQ). This mutant induced CHAC1 mRNA expression, similar to the WT PAK strain (Figure 2B) . The difference in CHAC1 expression observed with the FliC strain was not due to different MOI since equal numbers of Pa colonies were found on LB agar (Supplementary Figure 3A) . Subsequently, in order to determine whether the observed CHAC1 upregulation was restricted to the laboratory PAK strain, we used Pa strains freshly isolated from different patients with CF. We observed that clinical isolates of Pa were also able to induce CHAC1 mRNA expression in NCI-H292 cells (Figure 2C ) with an effect similar to PAK. This induction is highly variable between the clinical strains used and may reflect difference of virulence of each strain, since the number of the bacteria used for stimulation is similar (Supplementary Figure 3B) . CHAC1 induction by PAK strain is also highly variable (Figure 2C) , and is likely due to the variability in the inoculum used (Supplementary Figure 3B) . We also observed that the induction of CHAC1 mRNA expression by PAK was higher in NCI-H292 cells than in hAECBs (Figures 2B,C vs. Figure 1C ). Finally, we tested two other bacterial species known to colonize the airways of patients with CF, i.e., S. aureus and S. maltophilia. Using a similar MOI, we observed reduced CHAC1 mRNA induction in NCI-H292 cells infected with S. aureus and S. maltophilia compared with that in the cells infected with Pa. Concomitantly, induction of IL-8 mRNA expression was reduced ( Figure 2D ). The differences in CHAC1 and IL-8 mRNA expression observed with S. aureus and S. maltophilia were not caused by different MOI because equal numbers of colonies were found on LB agar (Supplementary Figure 3C) .