Antibacterial Defense of Human Airway Epithelial Cells from Chronic Obstructive Pulmonary Disease Patients Induced by Acute Exposure to Nontypeable Haemophilus influenzae: Modulation by Cigarette Smoke

Abstract

Antimicrobial proteins and peptides (AMPs) are a central component of the antibacterial activity of airway epithelial cells. It has been proposed that a decrease in antibacterial lung defense contributes to an increased susceptibility to microbial infection in smokers and patients with chronic obstructive pulmonary disease (COPD). However, whether reduced AMP expression in the epithelium contributes to this lower defense is largely unknown. We investigated the bacterial killing activity and expression of AMPs by air-liquid interface-cultured primary bronchial epithelial cells from COPD patients and non-COPD (ex-)smokers that were stimulated with nontypeable Haemophilus influenzae (NTHi). In addition, the effect of cigarette smoke on AMP expression and the activation of signaling pathways was determined. COPD cell cultures displayed reduced antibacterial activity, whereas smoke exposure suppressed the NTHi-induced expression of AMPs and further increased IL-8 expression in COPD and non-COPD cultures. Moreover, smoke exposure impaired NTHi-induced activation of NF-κB, but not MAP-kinase signaling. Our findings demonstrate that the antibacterial activity of cultured airway epithelial cells induced by acute bacterial exposure was reduced in COPD and suppressed by cigarette smoke, whereas inflammatory responses persisted. These findings help to explain the imbalance between protective antibacterial and destructive inflammatory innate immune responses in COPD.

Keywords: Antimicrobial peptides; Chronic obstructive pulmonary disease; Cigarette smoke; Epithelium; Host defense.

Fig. 1

Impaired bacterial killing by COPD ALI-PBEC. a Schematic representation of the bacterial killing assay. ALI-PBEC cultures were stimulated with 0.5 × 10⁹ CFU/mL UV-inactivated NTHi or PBS as the negative control for 6 h, washed at the apical surface, and incubated for 42 h. Next, streptavidin-coated glass coverslips linked to biotin-bound NTHi were placed on the apical surface of ALI-PBEC. Bacterial killing was determined by counting individual live and dead bacteria. b Bacterial killing was assessed for cultured ALI-PBEC from COPD patients (gray boxplots, n = 5 patients) and non-COPD smokers (white boxplots, n = 5 patients), either unstimulated or stimulated with 0.5 × 10⁹ CFU/mL UV-inactivated NTHi. Data are shown as the percentage of dead bacteria. The killing assay was performed in triplicates. COPD and non-COPD comparison results are depicted as boxplots with whiskers from minimum to maximum or bars (means ± SEM). The analysis of differences was conducted with a 2-way ANOVA and Bonferroni post hoc test. * p < 0.05.

Fig. 2

AMP expression by COPD ALI-PBEC is lower compared to non-COPD. COPD (gray boxplots, n = 12 patients) and non-COPD (white boxplots, n = 8 patients) ALI-PBEC were stimulated with different concentrations of UV-inactivated NTHi for 24 h. mRNA expression of the AMPs DEFB4/hBD-2 (a), S100A7 (b), LCN2 (c), and CCL20 (d) was assessed by qPCR. Stimulations were performed in duplicate. Data are shown as the fold change in mRNA compared to untreated cells. Assessment of hBD-2 (e) and CCL20 (f) protein secretion in the apical surface liquid (ASL) and basal medium (BM) of COPD (gray boxplots, n = 9) and non-COPD (white boxplots, n = 7–8) ALI-PBEC stimulated with 1 × 10⁹ CFU/mL UV-inactivated NTHi for 24 h. Stimulations were performed in duplicate. Results are shown as boxplots with whiskers from minimum to maximum or bars (means ± SEM). The analysis of differences was conducted with a 2-way ANOVA and Bonferroni post hoc test. * p < 0.05, ** p < 0.01.

Fig. 3

Differences in early- and late-induced transcriptional responses between COPD and non-COPD ALI-PBEC. Time course of NTHi-induced mRNA expression in COPD (gray bars, n = 12) and non-COPD AL-PBEC (white bars, n = 9). COPD and non-COPD ALI-PBEC were stimulated with 1 × 10⁹ CFU/mL UV-inactivated NTHi for 3, 12, and 24 h, afterwards mRNA expression of DEFB4/hBD-2 (a), S100A7 (b), LCN2 (c), and CCL20 (d) was examined by qPCR. Stimulations were performed in duplicate. Data are shown as the fold change compared to unstimulated cells. All results are depicted as the mean ± SEM. The analysis of differences was conducted with an unpaired t test. * p < 0.05.

Fig. 4

Expression of epithelial differentiation markers and barrier function in COPD and non-COPD ALI-PBEC. Baseline mRNA expression of the cell differentiation markers SCGB1A1 (club cell; a), FOXJ1 (ciliated cell; b), MUC5AC (goblet cell; c), and TP63 (basal cell; d) was determined in differentiated ALI-PBEC from COPD (gray boxplots, n = 11 patients) and non-COPD (white boxplots, n = 8 patients) ALI-PBEC. Data are shown as normalized values. e The epithelial barrier integrity of COPD (gray boxplots, n = 9 patients) and non-COPD (white boxplots, n = 7 patients) ALI-PBEC was determined by measuring the TEER values. ALL results are shown as boxplots with whiskers from minimum to maximum or bars (means ± SEM). The analysis of differences was conducted with a 2-way ANOVA and Bonferroni post hoc test.

Fig. 5

CS differentially modulates the innate immune gene expression. ALI-PBEC (n = 7) were exposed to AIR or CS and subsequently stimulated with 1 × 10⁹ CFU/mL UV-inactivated NTHi for 3, 12, and 24 h. mRNA expression of DEFB4 (a), S100A7 (b), LCN2 (c), CCL20 (d), IL8 (e), and IL6 (f) was measured by qPCR. Stimulations were performed in duplicate. Data are shown as the fold change in mRNA compared to untreated cells. Assessment of hBD-2 (g) secretion in the apical surface liquid and CCL20 (h) and IL-8 (i) secretion in the basal medium of AIR/CS-exposed ALI-PBEC (n = 7) stimulated with 1 × 10⁹ CFU/mL UV-inactivated NTHi. Stimulations were performed in duplicate. All results are shown as the mean ± SEM. The analysis of differences was conducted with a 1-way ANOVA and Bonferroni post hoc test (a–f), and paired t test (g–i). * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 6

Suppression of AMPs by CS in both COPD and non-COPD ALI-PBEC. COPD (gray bars, n = 12 patients) and non-COPD (white bars, n = 8 patients) ALI-PBEC were exposure to AIR or CS and subsequently stimulated with 1 × 10⁹ CFU/mL UV-inactivated NTHi for 3 h. mRNA expression of DEFB4 (a), S100A7 (b), LCN2 (c), CCL20 (d), and IL8 (e) was determined by qPCR. Stimulations were performed in duplicate. Data are shown as the fold change in mRNA compared to untreated cells and depicted as the mean ± SEM. The analysis of differences was conducted with a paired t test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Fig. 7

CS impairs NTHi-induced NF-κB but not MAPK signal transduction in ALI-PBEC. a ALI-PBEC (n = 4–8) were exposed to AIR/CS and stimulated with 1 × 10⁹ CFU/mL UV-inactivated NTHi for 30 min. NF-κB activation was assessed by measuring the phosphorylation of IKKα/β and degradation of IκB-α. MAPK signaling was assessed by determining the phosphorylation of ERK1/2 and p38. b–e Analysis of the data by densitometry. Results are shown as the mean ± SEM. Analysis of differences was conducted with a paired t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig. 8

CS impairs NF-κB transcriptional activity by ALI-PBEC. ALI-PBEC were left untreated or exposed to AIR or CS and stimulated with UV-inactivated NTHi for 1 h. a Cellular localization of the NF-κB subunits p50 and p65 was determined by immunofluorescence microscopy. The data shown represent n = 3 independent donors. b ALI-PBEC (n = 4) were exposed to AIR or CS and stimulated with 1 × 10⁹ CFU/mL UV-inactivated NTHi for 1 h. Protein expression of p50 and p65 was measured in isolated nuclear extracts. c The data were quantified by densitometry. ALI-PBEC were unstimulated or exposed to AIR or CS and stimulated with 1 × 10⁹ CFU/ml UV-inactivated NTHi for 1 h. ALI-PBEC (n = 4) were exposed to AIR or CS and subsequently stimulated with 1 × 10⁹ CFU/mL UV-inactivated NTHi for 5, 15, 30, 60, and 120 min. mRNA expression of early induced NF-κB target genes NFKBIA, ZC3H12A, and NFKBIZ (d), and early induced MAPK-target genes JUN, FOS, and FOSL1 (e), was determined by qPCR. Data are shown as the fold change in mRNA compared to AIR-exposed cells. All results are shown as the mean ± SEM. Analysis of differences was conducted with a paired t test (c) and 2-way ANOVA with Bonferroni post hoc test (d, e). Significant differences compared to AIR: * p < 0.05, ** p < 0.01, *** p < 0.001. Significant differences compared to CS+NTHi: ^##p < 0.01, ^###p < 0.001.