Fig 1.
Optimum secretion of the IL-6 pro-inflammatory cytokine by P. aeruginosa-infected A549 cells in a cell-density and time-dependent kinetics.
Varying A549 cell numbers (104, 105 and 106 cells/well) plated in 24- and 12- well plates were incubated at 37°C in a 5% CO2 humidified atmosphere for 24 h. [A] mucoid and [B] non-mucoid P. aeruginosa strains (pre-determined optimum MOIs of 100) were then added and incubated at time-intervals of 1, 2, 3, 4 and 5 h. Cell-and bacteria-free culture supernatants were collected at each time-point to quantify IL-6 by specific ELISA.
Fig 2.
TP359 dose-dependently reduced the production levels of pro-inflammatory cytokines in P. aeruginosa-infected A549 cells.
A549 cells (1 × 106 cells/well/mL) were cultured in 12-well plates overnight followed by bacteria infection (MOI of 100) for 4 h. A549 cells infected with mucoid [A, C] or non-mucoid [B, D] strains of P. aeruginosa were then exposed to TP359 at various concentration (12.5, 25, 50 and 100 μg/mL) using three different strategies: [I] Exposing TP359 and bacteria simultaneously to A549 cells; [II] Exposing A549 cells to TP359 for 1 h, before bacterial infection; [III] Infecting cells with bacteria for 1 h, before exposing them to TP359. Positive control consisted of polymyxin (PB)-treated A549 cells infected with P. aeruginosa. Cell-and bacteria-free culture supernatants were collected to quantify cytokines by specific ELISAs. [A] and [B] are IL-6 concentrations, while [C] and [D] are TNFα concentrations, at each strategy for the mucoid- and non-mucoid P. aeruginosa-infected A549 cells. Each bar represents the mean ± SD of samples run in triplicates. The data are representative of three separate experiments. Statistical analysis was carried out using the one-way ANOVA. The number of asterisk was based on the degree of significance such as *P < 0.05, **P < 0.01 and ***P <0.001.
Fig 3.
TP359 dose-dependently reduced the production levels of the IL-8 pro-inflammatory cytokine in P. aeruginosa-infected A549 cells.
A549 cells (1 × 106 cells/well/mL) were cultured in 12-well plates overnight followed by bacteria infection (MOI of 100) for 4 h. A549 cells infected with either [A] mucoid and [B] non-mucoid strains of P. aeruginosa were then exposed to TP359 at 100 μg/mL. Positive control consisted of polymyxin (PB)-treated A549 cells infected with P. aeruginosa. Cell-and bacteria-free culture supernatants were collected to quantify IL-8 by specific ELISA. Each bar represents the mean ± SD of samples run in triplicates. The data are representative of three separate experiments. Statistical analysis was carried out using the one-way ANOVA. The number of asterisk was based on the degree of significance such as *P < 0.05, **P < 0.01 and ***P <0.001.
Fig 4.
The anti-inflammatory effect of TP359 is not entirely due to its anti-microbial properties.
A549 cells (1 × 106 cells/well/mL) were cultured in 12-well plates overnight. A549 cells were next infected with the mucoid and non-mucoid strains of P. aeruginosa (MOIs of 100) for 4 h in the presence and absence of TP359 (100 μg/mL) using three different strategies. [A] Infecting A549 cells with bacteria and TP359 simultaneously; [B] Exposing A549 cells to TP359 for 1 h, before bacterial infection; and [C] Infecting A549 cells with bacteria for 1 h, before exposing them to TP359. Culture supernatants were collected and colony count assay was done by serial dilution on Luria agar. Each bar represents the mean ± SD of samples run in triplicates. The data are representative of three separate experiments. Statistical analysis was carried out using the one-way ANOVA. The number of asterisk was based on the degree of significance such as *P < 0.05, **P < 0.01 and ***P <0.001.
Fig 5.
TP359 reduces the production of pro-inflammatory cytokines by A549 cells stimulated with FLA and LPS.
A549 cells (1 × 106/well) were cultured in 12-well plates overnight followed by stimulation with pre-determined optimum concentrations of FLA (100 ng/mL) or LPS (1 μg/mL) for 4 h in the presence or absence of TP359. Positive controls consisted of A549 cells exposed to live P. aeruginosa (MOI of 100) with or without treatment with polymyxin (PB) at 50 μg/mL. Unstimulated A549 cells served as the negative control. [A] and [B] are IL-6 and IL-8 concentration for FLA treated cells, respectively, while [C] and [D] are IL-6 and IL-8 concentration for LPS treated cells, respectively. Cell-free supernatants were collected and cytokine concentrations were measured by specific ELISAs. Each bar represents the mean ± SD of samples run in triplicates and each is representative of three independent experiments. Statistical analysis was carried out using the one-way ANOVA. Asterisk indicates significant difference *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 6.
Neutralization of TLR4 and TLR5 reduces the secretions of cytokines by A549 stimulated cells.
A549 cells (1 × 106 cells/well) were cultured in 12-well plates for 24 h, followed by exposure to optimum concentrations of anti-TLR4 (10 μg/mL), anti-TLR5 (10 μg/mL) and their isotype controls (10 μg/mL) for an additional 24 h. P. aeruginosa (MOI of 100), FLA (100 ng/mL) and LPS (1 μg/mL) were then added and incubated for an additional 4 h followed by collection of cell-and bacteria-free culture supernatants to quantify cytokines using specific ELISAs. Asterisk indicates significant difference *P < 0.05, **P < 0.01 and *** P < 0.001 and P values were calculated by use of one way ANOVA Each bar represents the mean ± SD of samples run in triplicates. The data are representative of two separate experiments. [A] and [B] are IL-6 concentrations in mucoid and non-mucoid P. aeruginosa-treated A549 cells respectively, [C] and [D] are IL-6 concentrations in FLA- and LPS-treated A549 cells respectively, [E] and [F] are IL-8 concentration in mucoid and non-mucoid P. aeruginosa treated A549 cells respectively. Each bar represents the mean ± SD of samples run in triplicates and the data are representative of three separate experiments. Statistical analysis was carried out using the one-way ANOVA. Asterisk indicates significant difference *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 7.
P. aeruginosa induces the secretion of pro-inflammatory cytokines via the p38 MAPK and JNK pathways.
A549 cells (1 × 106 cells/well) were cultured in 12-well plates for 24 h, and then exposed for 24 h to varying concentrations (2.5–20 μM) of pathway specific inhibitors: p38 MAPK (SB203580), ERK U0126), JNK (JNK-IN-8) and NF-κB (activation inhibitor IV). After 24 h incubation, bacterial strains of P. aeruginosa (MOIs of 100) were added and incubated for 4 h followed by collection of cell- and bacteria-free supernatants for quantification of cytokines using specific ELISAs. [A] and [C] are IL-8 and IL-6 concentrations, respectively in cultures with p38 and ERK inhibitors; [B] and [D] are IL-8 and IL-6 concentrations in supernatants with JNK and NF-kB inhibitors. Each bar represents the mean ± SD of samples run in triplicates with data being representative of three separate experiments. Statistical analysis was carried out using the one-way ANOVA. Asterisk indicates significant difference *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 8.
TP359 down-regulates the phosphorylation of p38 MAPK in A549 lung cells exposed to P. aeruginosa and FLA.
A549 (5 × 106 cells/well) were seeded in 60 mm × 15 mm cell-culture dishes and exposed to mucoid P. aeruginosa (5 × 108 CFU/mL), FLA (100 ng/mL) or LPS (1 μg/mL). Protein lysates were collected at different time-points (15, 30 and 60 mins) after cell exposure and subjected to SDS-PAGE as indicated in the materials and methods section. The presence of total p38 (internal control) and phosphorylated p38 (Phospho-p38) proteins was determined by western blotting as described in the materials and methods section. The 43 KDa phospho-p38 and total p38 proteins were determined from known positive controls and biotinylated ladder. Shown in blot [A] are bands of intensity for phospho-p38 at the different time-points for A549 cells exposed to P. aeruginosa: (1) Cells alone, 60 min; (2) Infected cells, 60 min; (3) Infected cells, 30 min; (4) Cells alone, 30 min; (5) Infected cells, 15 min; (6) Cells alone, 15 min. Shown in blot [B] are bands intensity for phospho-p38 and total p38 for A549 cells treated with bacteria, FLA or LPS in the presence and absence of TP359: (1) Cells alone, (2) Cells + TP359, (3) Cells + bacteria, (4) Cells + bacteria + TP359, (5) Cells + FLA, (6) Cells + FLA + TP359, (7) Cells + LPS, (8) Cells + LPS + TP359.
Fig 9.
Proposed mechanism of action of TLR5 recognition of flagellin and TP359 attenuation of inflammation in human A549 lung cells.
The TLR5 signaling cascade on the cell surface is stimulated by the binding of bacterial flagellum which is composed of flagellin. Flagellin-stimulated TLR5 recruits the adapter protein, MyD88, which activates MAP kinases, p38, JNK, and the transcription factor NF-κB. NF-κB further induces downstream pro-inflammatory pathways resulting in the secretion of pro-inflammatory cytokines such as IL-8, IL-6 and TNFα. TP359 attenuated inflammation in human A549 lung cells exposed to P. aeruginosa and flagellin by down-regulating the phosphorylation of the p38 MAPK pathway or blocking the binding of flagellin to TLR5 receptor, thereby inhibiting the expression of pro-inflammatory cytokines.