Table 1.
Bacterial strains.
Fig 1.
IL-8 response in AECs stimulated with PAF or SAF alone, or PAF+SAF co-stimulation.
A) Time-dependent IL-8 response to PAF and SAF. Beas-2B cells were stimulated with PAF (2.5% v/v) and/or SAF (10% v/v) for the indicated duration. B) Dose-dependent IL-8 response to PAF+SAF co-stimulation. Beas-2B cells were stimulated for 6h with PAF (2.5% v/v) and SAF at the following concentration: + = 2.5% v/v; ++ = 5% v/v; +++ = 10% v/v. C) Time-dependent IL-8 response to PAF+SAF co-stimulation. Beas-2B cells were stimulated with PAF (2.5% v/v) and/or SAF (10% v/v) for the indicated duration. D) IL-8 mRNA response to PAF, SAF and PAF+SAF co-stimulation. Beas-2B cells were stimulated with PAF (2.5% v/v) and/or SAF (10% v/v) for 1h. Relative mRNA expression of IL-8 / GAPDH are expressed as fold increase. In A, B and C, extracellular IL-8 levels were measured in the AEC conditioned supernatant by ELISA after stimulation. Stimulation with LB medium was used as control. Results represent the mean (±SEM) of n≥3 independent biological replicates. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared to PAF alone at the similar time point using 2-way ANOVA (A and C) and 1-way ANOVA (B and D), followed by multiple comparisons Bonferroni correction test.
Fig 2.
Effects of SAF on TLR agonist-mediated IL-8 responses.
Beas-2B cells were stimulated with the TLR agonists Pam3CysSK4 (TLR 1/2), LPS (TLR4) and flagellin (TLR5) alone or in co-stimulation with SAF (10% v/v) for 6h. Extracellular IL-8 levels were measured in the AEC conditioned supernatant by ELISA after stimulation. Results are shown as mean (±SEM) of four independent biological replicates. ***P<0.001, ****P<0.0001 compared to stimulation with the agonist alone at the same concentration using an unpaired two-tailed student’s t-test.
Fig 3.
Effects of SAF on IKKαβ, IκB and p38 MAPK.
Beas-2B cells were stimulated with Pam3CysSK4 (10 μg/mL) and/or SAF (10% v/V) for 45 min, and levels of p-IKKαβ (A), IκB (B) and P-p38MAPK (C) were measured in Beas-2B cell by immunoblots. Quantitative analysis of the band signals are shown in the top panels as the mean (±SEM) of three independent biological replicates. Representative blots are shown in the bottom panels. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 compared to the Pam3CysSk4 alone using 1-way ANOVA, followed by multiple comparisons Bonferroni correction test.
Fig 4.
SAF represses PAF and TLR1/2 dependent NF-κB activity.
Beas-2B cells stably expressing a NF-κB promoter-reporter (pGL4.28NF-κB) were stimulated with PAF (2.5% v/v), Pam3CysSK4 (10 μg/ml) and/or with SAF (10% v/V) for 3h. Where indicated, Beas-2B cells were pre-treated with the IKKβ inhibitor Bi605906 (7.5 μg/mL) 1h prior to stimulation with Pam3CysSK4. Following stimulation, cells were lysed, and the relative luminescence (RLU) was measured in the supernatant using a luciferase assay to determine NF-κB activity. LB media was used as a control. Results are shown as mean (±SEM) of n≥3 independent biological replicates. ****P<0.0001 compared to PAF stimulation alone using 1-way ANOVA, followed by multiple comparisons Bonferroni correction test.
Fig 5.
Relative mRNA expression of IL-8, CXCL2 and ATF3 in response to Pam3CysSK4 alone or in co-stimulation with SAF.
Beas-2B cells were stimulated with Pam3CysSK4 (10 μg/mL) and/or with SAF (10% v/v) for 3h. All results are shown as mean (±SEM) of three independent biological replicates. *P<0.05, **P<0.01, and ***P<0.001 compared to Pam3CysSK4 alone using an unpaired two-tailed student’s t-test.
Fig 6.
IL-8 response in AEC co-stimulated with PAF and UV killed SA, or SAF from Δhla and Δhlb mutants.
A) IL-8 response to UV-killed SA. Beas-2B cells were stimulated with PAF (2.5% v/v) and/or a suspension of UV-killed whole SA bacteria (108 CFU/mL) for 6h. B) IL-8 response to Δhla and Δhlb SAF. Beas-2B cells were stimulated with PAF (2.5% v/v) and/or SAF (10% v/v) for 6h. PAF and SAF were prepared from bacterial cultures grown in TSB medium for 24h. WT = 8325–4 SA wild-type parental strain; Δhla and Δhlb are its isogenic mutants. In A and B, extracellular IL-8 levels were measured in the AEC supernatant by ELISA after stimulation. Results are shown as mean (±SEM) of four independent biological replicates. *P<0.05, ***P<0.001 compared to PAF alone and to SAF WT, using 1-way ANOVA, followed by multiple comparisons Bonferroni correction test.
Fig 7.
AEC IL-8 responses to co-stimulation with different SA and PA strains.
A). IL-8 responses to co-stimulation of PAF+SAF from different SA strains. Beas-2B cells were stimulated with SA filtrates (10% v/v) alone or co-stimulated with PAO1 PAF (2.5% v/v) for 6 h. B). IL-8 responses to co-stimulation of PAF+SAF from different PA strains. Beas-2B cells were stimulated with filtrates from different PA strains (2.5% v/v) alone, or co-stimulated with ATCC29213 SAF (10% v/v) for 6 h. Extracellular IL-8 levels were measured in the AEC supernatant by ELISA after stimulation. All results are shown as mean (±SEM) of n≥4 independent biological replicates. *P<0.05, **P<0.01, ***P<0.001 compared to PAF alone using an unpaired two-tailed student’s t-test.
Fig 8.
IL-8 responses in CFBE41o- cells stimulated with PAF and SAF.
The CFBE41o- airway epithelial cells (CFTR ΔF508 homozygotes) were stimulated for 6h with PAF (2.5% v/v) and/or SAF at the following volumes. + = 2.5% v/v; ++ = 5% v/v;. Extracellular IL-8 levels were measured in the AEC supernatant by ELISA after stimulation. Results are shown as mean (±SEM) of four independent biological replicates. *P<0.05, **P<0.01 compared to PAF alone using 2-way ANOVA, followed by multiple comparisons Bonferroni correction test.