Effect of TLR4 and 5 expression on KC and IL-6 synthesis by lung epithelial cells challenged with <i>P. aeruginosa</i>.

<p>Lung epithelial cells (EC) collected from WT and TLR4,5^−/− mice were infected for 4 h with increasing concentrations (number of bacteria/well) of <i>P. aeruginosa</i> wild-type strain PAK. <i>NS</i>, non-stimulated cells cultured with medium only. LPS (1 µg/mL), and flagellin (20 ng/mL) were used as control agonists to stimulate the cells. Values represent means±SEM of three to five experiments performed in triplicate. *, <i>p</i><0.05 when compared with the corresponding WT values.</p

Effect of TLR2,4 and flagellin expression on KC, TNF-α and IL-6 synthesis by alveolar macrophages challenged with <i>P. aeruginosa</i>.

<p>Alveolar macrophages (AM) collected from WT and TLR2,4^–/– mice were infected for 4 h with increasing concentrations (number of bacteria/well) of a mutant <i>P. aeruginosa</i> devoid of flagellin production, Δ<i>fliC</i>. <i>NS</i>, non-stimulated cells cultured with medium only. LPS (1 µg/mL) and flagellin (20 ng/mL) were used as control agonists to stimulate the cells. Values represent means±SEM of three to five experiments performed in triplicate. *, <i>p</i><0.05 when compared with the corresponding WT values.</p

Effect of MyD88 expression on KC, TNF-α and IL-6 synthesis by alveolar macrophages challenged with <i>P. aeruginosa</i>.

<p>Alveolar macrophages (AM) collected from WT and MyD88^–/– mice were infected for 4 h with increasing concentrations (number of bacteria/well) of <i>P. aeruginosa</i> wild-type strain PAK. <i>NS</i>, non-stimulated cells cultured with medium only. LPS (1 µg/mL) and flagellin (20 ng/mL) were used as control agonists to stimulate the cells. Values represent means±SEM of three to five experiments performed in triplicate. *, <i>p</i><0.05 when compared with the corresponding WT values.</p

Effect of MyD88 expression on KC and IL-6 synthesis by lung epithelial cells challenged with <i>P. aeruginosa</i>.

<p>Lung epithelial cells (EC) collected from WT and MyD88^–/– mice were infected for 4 h with increasing concentrations (number of bacteria/well) of <i>P. aeruginosa</i> wild-type strain PAK. <i>NS</i>, non-stimulated cells cultured with medium only. LPS (1 µg/mL) and flagellin (20 ng/mL) were used as control agonists to stimulate the cells. Values represent means±SEM of three to five experiments performed in triplicate. *, <i>p</i><0.05 when compared with the corresponding WT values.</p

Effect of TLR2,4 and flagellin expression on KC and IL-6 synthesis by lung epithelial cells challenged with <i>P. aeruginosa</i>.

<p>Lung epithelial cells (EC) collected from mice were infected for 4 h with increasing concentrations (number of bacteria/well) of <i>P. aeruginosa</i>. Different combinations were used, <i>i.e.</i>, WT and TLR2,4^−/− mice cells stimulated with the wild-type bacteria strain PAK (7a); WT mice cells stimulated with the wild type bacteria strain PAK and its mutant Δ<i>fliC</i> (7b); WT and TLR2,4^−/− mice cells stimulated with the Δ<i>fliC</i> mutant (7c). <i>NS</i>, non-stimulated cells cultured with medium only. LPS (1 µg/mL), and flagellin (20 ng/mL) were used as control agonists to stimulate the cells. Values represent means±SEM of three to five experiments performed in triplicate. *, <i>p</i><0.05 when compared with either the corresponding WT mice or wild-type PAK strain values.</p

Effect of flagellin expression on KC, TNF-α and IL-6 synthesis by alveolar macrophages challenged with <i>P. aeruginosa</i>.

<p>Alveolar macrophages (AM) collected from WT mice were infected for 4 h with increasing concentrations (number of bacteria/well) of <i>P. aeruginosa</i> wild-type strain PAK or with a mutant devoid of flagellin production, Δ<i>fliC</i>. <i>NS</i>, non-stimulated cells cultured with medium only. LPS (1 µg/mL) and flagellin (20 ng/mL) were used as control agonists to stimulate the cells. Values represent means±SEM of three to five experiments performed in triplicate. *, <i>p</i><0.05 when Δ<i>fliC</i> values are compared with the corresponding PAK values.</p

Effect of TLR2 and 4 expression on KC, TNF-α and IL-6 synthesis by alveolar macrophages challenged with <i>P. aeruginosa</i>.

<p>Alveolar macrophages (AM) collected from wild-type (WT) and TLR2,4^–/– mice were infected for 4 h with increasing concentrations (number of bacteria/well) of <i>P. aeruginosa</i> wild-type strain PAK. <i>NS</i>, non-stimulated cells cultured with medium only. LPS (1 µg/mL) and flagellin (20 ng/mL) were used as control agonists to stimulate the cells. Values represent means±SEM of three to five experiments performed in triplicate. *, <i>p</i><0.05 when compared with the corresponding WT values.</p

Lack of flagellin reduces <i>P. aeruginosa</i> supernatant-induced MUC5AC expression by NCI-H292 cells.

<p>NCI-H292 cells were exposed for 3, 6, 10 or 24 hrs to supernatants from either PAK or ΔFliC at 1∶8 dilution (A, b) or at the indicated dilution (B). The Insert (A, a) indicates immunoblotting analysis which shows the presence of flagellin in PAK (P) and its absence in ΔFliC (ΔF) supernatants. A 20 ng of purified flagellin from PAK (Flag) was used as a positive control and the bacteria culture medium (LB) was used as a negative control. Total mRNA was extracted and subjected to RT-qPCR (A, B). The level of MUC5AC protein produced in NCI-H292 supernatant was measured by ELISA, as indicated in <i>Material and Methods</i> (C). In (D) NCI-H292 cells were pre-treated with TLR4i (5 µM) one hour before incubation with bacterial supernatants from either ΔFliC or PAK and treated again during stimulation. The results show the percentage of inhibition of MUC5AC mRNA expression in TLR4i treated NCI-H292 cell compared to untreated NCI-H292 cells. E) Shows the LPS levels in ΔFliC vs. PAK bacterial supernatant after 24 h of culture. C  =  control (untreated cells), LB  =  Luria Bertoni medium 1∶8 dilution. Values represent means ± SM of 3 independent experiments. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 <i>vs</i> Control (C); &&& <i>P</i><0.001, && <i>P</i><0.01 <i>vs.</i> corresponding PAK-treated controls or LB.</p

Impact of TLR5 and NAIP silencing on MUC5AC expression.

<p>NCI-H292 cells were transfected with TLR5 or Naip siRNA and then treated with or without purified flagellin (Flag, 1 µg/ml) for 24 h. Scrambled siRNA was used as a negative control for transfection. (A) Shows the effect of siRNA transfection on flagellin-induced MUC5AC expression. (B) Shows TLR5, Naip and Ipaf expression in NCI-H292 analyzed by RT-PCR. (C) and (D) show the efficiency of siRNA transfection on TLR5 and Naip expressions compared to siRNA controls (C). Data are expressed as the means ± SM. P<0,05. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 <i>vs</i> Controls, 3 independent experiments.</p

Deletion of flagellin abrogates <i>P. aeruginosa</i>-induced MUC2 expression in NCI-H292 cells.

<p>Cells were exposed to various MOI of living bacteria (A, C) or bacterial supernatants (B and D) as indicated in <i>Material</i> and <i>Methods</i>. Total mRNA was prepared and subjected to RT-qPCR to measure MUC2 (A, B) and MUC5B (C, D) levels. (E) NCI-H292 cells were pre-treated with TLR4i (5 µM) one hour and then incubated with bacteria supernatants. The result shows the percentage of inhibition of MUC2 mRNA expression following TLR4i treatment as compared to untreated control cells. Values represent means ± SM of 3 independent experiments. ns  =  no significant. C  =  control cells. * <i>P</i><0.05 infected <i>vs.</i> no infected cells:</p