Synergistic induction of apoptosis by LL-37 and P. aeruginosa is isolate-specific and independent of type III secretion system and pilus expression. Human bronchial epithelial cells (16HBE14o⁻) were assessed for mitochondrial membrane depolarization using Mitocapture dye, as described in Materials and Methods, after incubation for 1 hour with a range of concentrations of LL-37, in Ultroser G serum–substitute supplemented media, in the presence and absence of (A) log-phase clinical P. aeruginosa isolate J1386 (MOI 10:1), (B) log-phase P. aeruginosa PA01exsA∷Ω or isogenic PAO1 control strain (MOI 10:1), and (C) log-phase pilA P. aeruginosa mutant or isogenic PAO1 control strain (MOI 10:1). Data represent mean values ± SEM, for n = 3 independent experiments for each condition. Two-way ANOVAs were performed to evaluate significance, with Bonferroni post hoc tests comparing (A) LL-37/P. aeruginosa to LL-37 alone, and (B) LL-37/P. aeruginosa mutant to LL-37/isogenic controls. *P ≤ 0.05,***P ≤ 0.001.

Synergistic induction of apoptosis by LL-37 and P. aeruginosa requires epithelial-cell internalization of bacteria. Human bronchial epithelial cells (16HBE14o⁻) were incubated for 60 minutes in Ultroser G serum–substitute supplemented media, in the presence and absence of (MOI 10:1) log-phase P. aeruginosa strains PA01, ΔmexAB-oprM mutant (A–C), isogenic PAO1 control strain (B), or ΔmexAB-oprM mutant added concurrently with sterile conditioned supernatant collected from 16HBE14o⁻ cells infected with PA01 (C). (A) Invasion of epithelial cells by bacteria was determined by gentamicin exclusion, quantifying the number of viable CFUs surviving extracellular gentamicin treatment (50 μg/ml). Data are plotted as mean values ± SEM, for n = 3 independent experiments plated in duplicate for each condition. (B, C) Infected epithelial cells were concurrently incubated with a range of concentrations of LL-37, and mitochondrial membrane depolarization was determined using Mitocapture dye, as described in Materials and Methods. Data represent mean values ± SEM, for n = 3 independent experiments for each condition. Two-way ANOVAs were performed to evaluate significance, with Bonferroni post hoc tests comparing (B) LL-37/ΔmexAB-oprM mutant to LL-37/isogenic controls, and (C) LL-37/ΔmexAB-oprM mutant to LL-37/ΔmexAB-oprM mutant in PAO1-conditioned supernatant. **P ≤ 0.01,***P ≤ 0.001.

Synergistic induction of apoptosis by LL-37 and P. aeruginosa requires specific bacteria–epithelial cell interactions with whole, live bacteria. (A) P. aeruginosa PA01 was cultured to log-phase, then exposed to LL-37 over a range of concentrations for 1 hour at 37°C in Ultroser G serum–substitute supplemented media. Serial dilutions were performed, incubated on LB agar plates in triplicate, and cultured for 16 hours before colony-forming units were counted. Data represent mean values ± SEM, for n = 3 independent experiments for each condition. (B) Human bronchial epithelial cells (16HBE14o⁻) were assessed for mitochondrial membrane depolarization using Mitocapture dye, as described in Materials and Methods, after incubation for 1 hour with a range of concentrations of LL-37, in serum-substitute supplemented media, in the presence and absence of live log-phase P. aeruginosa PA01 (MOI 10:1), heat-killed or UV-killed PA01 (MOI 10:1), P. aeruginosa PA01 LPS (1 μg/ml), P. aeruginosa PA01 conditioned medium, or live P. aeruginosa PA01 (MOI 10:1) separated from the cells via a semipermeable polyester membrane with 0.4-μm pore size. Data represent mean values ± SEM, for n = 3 independent experiments for each condition. Two-way ANOVAs were performed to evaluate significance, with Bonferroni post hoc tests comparing LL-37 alone to LL-37/stimuli. ***P ≤ 0.001.

LL-37 and P. aeruginosa synergistically induce DNA fragmentation and caspase activation in airway epithelial cells. Human bronchial epithelial cell line 16HBE14o⁻ (A, C, D) or primary human bronchial epithelial cells (B) were incubated for 6 hours (A, B) or 5 hours (C, D) over a range of LL-37 concentrations (or scrambled LL-37 [sLL-37] at 50 μg/ml) in Ultroser G serum–substitute supplemented media, in the presence and absence of log-phase P. aeruginosa PA01 (MOI 10:1) added concurrently. (A, B) Cells were treated as described, with or without preincubation for 1 hour with the polycaspase inhibitor Z-VAD-FMK (50 μM), and were then fixed. Apoptosis was assessed by TUNEL assay. Four random fields of view, each containing more than 100 cells, were counted for each sample. and the number of TUNEL-positive cells was expressed as a percentage of the number of DAPI-positive nuclei. Data represent mean values ± SEM, for n ≥ 3 independent experiments for each condition. Two-way ANOVA with Bonferroni post hoc test was used to compare LL-37/P. aeruginosa–treated samples with LL-37 only–treated samples at corresponding concentrations, or LL-37/P. aeruginosa/Z-VAD-FMK–treated samples with LL-37/P. aeruginosa–treated samples at corresponding concentrations. *P ≤ 0.05, **P ≤ 0.01. (C, D) Whole-cell protein lysates were prepared and analyzed by SDS-PAGE and Western immunoblotting. Immunoblots were performed using antibodies specific for cleaved caspase-3, XIAP, cleaved caspase-9, or actin. Images shown are representative of n ≥ 3 independent experiments.

LL-37–induced mitochondrial depolarization and DNA fragmentation involve Bax-dependent mechanisms. Human bronchial epithelial cells (16HBE14o⁻) were incubated for 1 hour (A) or 6 hours (B) over a range of LL-37 concentrations in Ultroser G serum–substitute supplemented media, in the presence and absence of log-phase P. aeruginosa PA01 (MOI 10:1) added concurrently, with or without preincubation for 1 hour with Bax-inhibiting peptide V5 (BIP-V5; 100 μM). (A) Mitochondrial membrane depolarization was determined using Mitocapture dye, quantifying the percentage of apoptotic cells displaying diffuse green fluorescence (cells with depolarized mitochondria), compared with healthy cells displaying punctuate red fluorescence (cells with polarized mitochondrial membranes). Four random fields of view were counted for each sample (minimum of 300 cells per sample), and the number of apoptotic cells was expressed as a percentage of total number of cells. Data were corrected for a background level of approximately 10% positive cells in control untreated samples, and plotted as mean values ± SEM, for n = 3 independent experiments for each condition. A two-way ANOVA with Bonferroni post hoc test was used to compare LL-37–only treated samples with LL-37/BIP-V5–treated samples, or LL-37/P. aeruginosa–treated samples with LL-37/P. aeruginosa/BIP-V5–treated samples at corresponding concentrations. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (B) Cells were fixed and apoptosis was assessed by TUNEL assay. Four random fields of view, each containing more than 100 cells, were counted for each sample, and the number of TUNEL-positive cells was expressed as a percentage of the number of DAPI-positive nuclei. Data represent mean values ± SEM, for n = 3 independent experiments for each condition. Two-way ANOVA with Bonferroni post hoc test was used to compare LL-37 only–treated samples with LL-37/BIP-V5–treated samples, or LL-37/P. aeruginosa–treated samples with LL-37/P. aeruginosa/BIP-V5–treated samples at corresponding concentrations **P ≤ 0.01, ***P ≤ 0.001.

Pseudomonas aeruginosa infection of airway epithelial cells synergistically enhances LL-37–mediated mitochondrial depolarization and cytochrome c release. Human bronchial epithelial cells (16HBE14o⁻) were incubated with a range of LL-37 concentrations (or scrambled LL-37 [sLL-37] at 50 μg/ml) in Ultroser G serum–substitute supplemented media, in the presence and absence of log-phase P. aeruginosa PA01 (MOI 10:1). Bacteria and LL-37 were added concurrently and incubated for 60 minutes (A) or 90 minutes (C), or epithelial cells were preinfected with bacteria for 60 minutes, washed, and exposed to LL-37 for 60 minutes (B). (A, B) Mitochondrial membrane depolarization was determined using Mitocapture dye, quantifying the percentage of apoptotic cells displaying diffuse green fluorescence (cells with depolarized mitochondria), compared with healthy cells displaying punctuate red fluorescence (cells with polarized mitochondrial membranes). Four random fields of view were counted for each sample (minimum of 300 cells per sample), and number of apoptotic cells was expressed as a percentage of the total number of cells. Data were corrected for a background level of approximately 10% positive cells in control untreated samples, and plotted as mean values ± SEM, for n = 6 (A) or n = 3 (B) independent experiments for each condition. Two-way ANOVA with Bonferroni post hoc test was performed to determine significance. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. (C) Cellular localization of cytochrome c was assessed by ELISA analysis of mitochondrial fractions after subcellular fractionation. Data represent the mean percentage of cytochrome c present in this fraction as a proportion of total cytochrome c detected in each sample ± SEM for n = 3 independent experiments, measured in duplicate for each condition. Two-way ANOVA was performed with Bonferroni post hoc test to compare each treatment to appropriate LL-37–free negative control sample. **P ≤ 0.01, ***P ≤ 0.001.