miR-31 dysregulation in cystic fibrosis airways contributes to increased pulmonary cathepsin S production

Abstract

Rationale: Cathepsin S (CTSS) activity is increased in bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF). This activity contributes to lung inflammation via degradation of antimicrobial proteins, such as lactoferrin and members of the β-defensin family.

Objectives: In this study, we investigated the hypothesis that airway epithelial cells are a source of CTSS, and mechanisms underlying CTSS expression in the CF lung.

Methods: Protease activity was determined using fluorogenic activity assays. Protein and mRNA expression were analyzed by ELISA, Western blotting, and reverse-transcriptase polymerase chain reaction.

Measurements and main results: In contrast to neutrophil elastase, CTSS activity was detectable in 100% of CF BAL fluid samples from patients without Pseudomonas aeruginosa infection. In this study, we identified epithelial cells as a source of pulmonary CTSS activity with the demonstration that CF airway epithelial cells express and secrete significantly more CTSS than non-CF control cells in the absence of proinflammatory stimulation. Furthermore, levels of the transcription factor IRF-1 correlated with increased levels of its target gene CTSS. We discovered that miR-31, which is decreased in the CF airways, regulates IRF-1 in CF epithelial cells. Treating CF bronchial epithelial cells with a miR-31 mimic decreased IRF-1 protein levels with concomitant knockdown of CTSS expression and secretion.

Conclusions: The miR-31/IRF-1/CTSS pathway may play a functional role in the pathogenesis of CF lung disease and may open up new avenues for exploration in the search for an effective therapeutic target.

Keywords: cystic fibrosis; epithelium; microRNA; protease.

Figure 1.

Increased cathepsin S (CTSS) levels and activity in cystic fibrosis (CF) bronchoalveolar lavage (BAL) fluid correlate negatively with lung function. (A) CTSS activity in BAL fluid from a cohort of pediatric Pseudomonas-infected (Ps+; n = 15) and noninfected (Ps−; n = 11) patients with CF (Group 1) was determined using the fluorogenic FR-AMC substrate (pH 7.5). Results were expressed as the change (Δ) in relative fluorescence units (ΔRFU) over time. (B) Total CTSS levels (tCTSS) in Ps− and Ps+ CF BAL fluid were quantified by ELISA. There was no significant difference between Ps− and Ps+ CF BAL fluid for either parameter measured. The correlation between CF BAL fluid (C) CTSS activity and (D) tCTSS levels and FEV₁ (n = 22) was determined using Spearman correlation. The correlation between CF BAL fluid CTSS activity and (E) neutrophils and (F) macrophages were determined using Spearman correlation.

Figure 2.

Profile of cathepsin S (CTSS) and neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid from non–cystic fibrosis (CF) and CF children who tested negative for Pseudomonas. (A) CTSS and (B) NE activity were quantified in BAL fluid from a cohort of preschool, Pseudomonas-negative non-CF (n = 9) and CF children (n = 43; Group 2). CTSS and NE activities were detected using the fluorogenic substrates FR-AMC (pH 7.5) and AAPV-AMC, respectively. Results were expressed as the change (Δ) in relative fluorescence units (ΔRFU) over time. CTSS activity was detectable in all 43 CF samples analyzed. However, in contrast, NE activity was undetectable in most samples (>60%). The correlation between CF BAL fluid CTSS activity and (C) neutrophils and (D) macrophages were determined using Spearman correlation. *P < 0.05.

Figure 3.

Basal cathepsin S (CTSS) secretion from unstimulated human cystic fibrosis (CF) and non-CF pulmonary epithelial cells. CTSS activity in cell-free supernatants of non-CF and CF (A) tracheal (human tracheal epithelial cell line 9HTEo− [HTE], CF tracheal epithelial cell line CFTE29o− [CFTE]) and (B) bronchial epithelial (human bronchial epithelial cell line 16HBE14o− [HBE], CF bronchial epithelial cell line CFBE41o− [CFBE]) cell lines, (C) primary human bronchial epithelial cell (PBEC, n = 4) apical washes, and (D) PBEC basolateral media was analyzed using FR-AMC (pH 7.5). Results were expressed as the change (Δ) in relative fluorescence units (ΔRFU) over time. (E–H) Total CTSS (tCTSS) levels in cell line supernatants (E and F), PBEC apical washes (G), and PBEC basolateral media (H) were quantified by ELISA. **P < 0.01, ***P < 0.001.

Figure 4.

Increased basal cathepsin S (CTSS) mRNA expression in cystic fibrosis (CF) epithelial cells. (A) CTSS and glyceraldehyde phosphate dehydrogenase (GAPDH) expression were detected in tracheal (human tracheal epithelial cell line 9HTEo− [HTE], CF tracheal epithelial cell line CFTE29o− [CFTE]) and bronchial epithelial (human bronchial epithelial cell line 16HBE14o− [HBE], CF bronchial epithelial cell line CFBE41o− [CFBE]) cell lines and (B) primary human bronchial epithelial cells (PBEC, n = 4) by reverse-transcriptase polymerase chain reaction. (C) Densitometry of PBEC CTSS relative to GAPDH. *P < 0.05.

Figure 5.

IRF-1 levels in non–cystic fibrosis (CF) and CF epithelial cells. (A) Western blotting of IRF-1 and glyceraldehyde phosphate dehydrogenase (GAPDH) in tracheal (human tracheal epithelial cell line 9HTEo− [HTE], CF tracheal epithelial cell line CFTE29o− [CFTE]) and bronchial (human bronchial epithelial cell line 16HBE14o− [HBE], CF bronchial epithelial cell line CFBE41o− [CFBE]) epithelial cell lines and (B) primary cultures of well-differentiated human bronchial epithelial cells (PBECs) (n = 4) whole-cell lysates. (C) Densitometry of PBEC IRF-1 relative to GAPDH. **P < 0.01.

Figure 6.

IRF-1 knockdown decreases cathepsin S (CTSS) expression and secretion from cystic fibrosis epithelial cells. The effects of IRF-1 knockdown on CTSS levels in cystic fibrosis bronchial epithelial cells (CFBEs) were investigated using IRF-1 siRNA. CFBEs were transfected with 100 nM IRF-1 siRNA (IRF-1), scrambled control (Scr), or mock transfected (Con) for 48 hours. After a further 24-hour incubation in fresh media, (A) knockdown of IRF-1 levels was confirmed by Western blotting and (B) CTSS mRNA levels were assessed by reverse-transcriptase polymerase chain reaction. (C) Extracellular CTSS activity was determined using the FR-AMC (pH 7.5) substrate and (D) extracellular tCTSS levels were quantified by ELISA. The data are the mean ± SEM of n = 3 and are expressed as % of mock transfected control cells (C and D). **P < 0.01, ***P < 0.001. GAPDH = glyceraldehyde phosphate dehydrogenase; tCTSS = total CTSS levels.

Figure 7.

miR-31 expression is decreased in cystic fibrosis (CF) epithelial cells. miR-31 levels in (A) bronchial brushings (n = 5), (B) primary cultures of well-differentiated human bronchial epithelial cells (n = 4) and (C) tracheal and (D) bronchial epithelial cell lines were quantified by quantitative reverse-transcriptase polymerase chain reaction and normalized to miR-16. *P < 0.05, **P < 0.01, ***P < 0.001. CFBE = CF bronchial epithelial cell line CFBE41o−; CFTE = CF tracheal epithelial cell line CFTE29o−; HBE = human bronchial epithelial cell line 16HBE14o−; HTE = human tracheal epithelial cell line 9HTEo−.

Figure 8.

miR-31 dysregulation in cystic fibrosis epithelial cells leads to increased cathepsin S (CTSS) expression via IRF-1. The effects of pre–miR-31 overexpression on IRF-1 and CTSS levels were determined by transfecting cystic fibrosis bronchial epithelial cells (CFBEs) with 30 nM pre–miR-31 (PM-31), negative control (PM-Neg), or mock transfected (Con) for 48 hours. (A) IRF-1 levels in whole-cell lysates were analyzed by Western blotting. (B) CTSS mRNA levels were assessed by reverse-transcriptase polymerase chain reaction. (C) Extracellular CTSS was quantified by ELISA. The data are the mean ± SEM of n = 3 and are expressed as % of mock transfected control cells. ***P < 0.001. GAPDH = glyceraldehyde phosphate dehydrogenase; tCTSS = total CTSS levels.