Concentration of the antibacterial precursor thiocyanate in cystic fibrosis airway secretions

Abstract

A recently discovered enzyme system produces antibacterial hypothiocyanite (OSCN(-)) in the airway lumen by oxidizing the secreted precursor thiocyanate (SCN(-)). Airway epithelial cultures have been shown to secrete SCN(-) in a CFTR-dependent manner. Thus, reduced SCN(-) availability in the airway might contribute to the pathogenesis of cystic fibrosis (CF), a disease caused by mutations in the CFTR gene and characterized by an airway host defense defect. We tested this hypothesis by analyzing the SCN(-) concentration in the nasal airway surface liquid (ASL) of CF patients and non-CF subjects and in the tracheobronchial ASL of CFTR-ΔF508 homozygous pigs and control littermates. In the nasal ASL, the SCN(-) concentration was ~30-fold higher than in serum independent of the CFTR mutation status of the human subject. In the tracheobronchial ASL of CF pigs, the SCN(-) concentration was somewhat reduced. Among human subjects, SCN(-) concentrations in the ASL varied from person to person independent of CFTR expression, and CF patients with high SCN(-) levels had better lung function than those with low SCN(-) levels. Thus, although CFTR can contribute to SCN(-) transport, it is not indispensable for the high SCN(-) concentration in ASL. The correlation between lung function and SCN(-) concentration in CF patients may reflect a beneficial role for SCN(-).

Fig. 1. Anion composition analysis of nasal ASL in CF and non-CF subjects

(A) A representative chromatogram of human ASL sample. ASL was collected from the nasal mucosa and diluted 50-fold prior to chromatography. The F⁻ (I), Cl⁻ (II), NO₃⁻ (III), HPO₄²⁻ (IV), SO₄²⁻ (V), and SCN⁻ (VI) peaks are indicated with Roman numerals. The identity of the SCN⁻ peak was verified by oxidizing the SCN⁻ content of diluted ASL using an excess of LPO and H₂O₂ (inset). The detector response is shown in arbitrary units (AU). (B) SCN⁻ concentrations in the nasal ASL of CF and non-CF subjects, as determined by ion-exchange chromatography (n=23 CF, 21 non-CF; Mann-Whitney test, p=0.89). (C) Correlation between SCN⁻ concentrations in serum and nasal ASL of non-CF (▲) and CF (□) subjects. Each data point represents one subject. Solid and dotted lines indicate the best-fit linear regression lines to non-CF and CF data, respectively (non-CF: r=0.60, **p=0.0084; CF: r=0.97, ***p<0.0001). The slopes of the two regression lines are not significantly different (F-test, p=0.31).

Fig. 2. SCN⁻ secretion in the lower airways of CF and control pigs

(A and B) Representative chromatograms of pig ASL samples. ASL samples were collected from the trachea and bronchi of (A) an untreated pig (representative of 4 newborn animals) and (B) a pig intravenously injected with 8 mg NaSCN/kg body weight (representative of 3 animals). The F⁻ (I), Cl⁻ (II), NO₃⁻ (III), HPO₄²⁻ (IV), SO₄²⁻ (V), and SCN⁻ (VI) peaks are indicated with Roman numerals. Note that SCN⁻ was not detected in the ASL of the untreated pig. The detector response is shown in arbitrary units (AU). (C) Relationship between SCN⁻ concentrations in the serum and tracheobronchial ASL of homozygous CFTR-ΔF508 (open symbols) and wild-type (closed symbols) pigs 2 hours after intravenous injection of NaSCN at the following doses (in mg/kg body weight): 0.25 (up triangle), 1 (circle), 2 (square), 4 (down triangle), and 8 (diamond). Each data point represents one animal. Dotted (CF) and solid (wild-type, WT) lines were generated by fitting the CF and non-CF data to the Michaelis-Menten equation. The two curves are significantly different (F-test, *p=0.019).

Fig. 3. The relationship between nasal ASL SCN⁻ concentration and FEV₁ in CF patients

Each data point represents the ASL SCN⁻ concentration and FEV1 of one subject. The Pearson’s correlation test indicates association between the ASL SCN⁻ concentration and FEV1 (r=0.7457, ***p<0.0001).

Fig. 4. SCN⁻ concentration dependence of OSCN⁻ production and bacterial killing by airway epithelia

(A) OSCN⁻ production by primary cultures of human airway epithelia in the presence of indicated SCN⁻ concentrations, ATP (100 μM), and LPO (7 μg/ml) (n=3 for each data point). (B) Bacterial survival on the apical side of airway epithelia as a function of SCN⁻ concentrations. The apical surface of human airway epithelial cultures was inoculated with 3,000 CFU S. aureus in the absence (squares) or presence of apically added H₂O₂ scavengers (5 mM glutathione, circles; 1,000 U/mL catalase, triangles). The apical buffer of all epithelial cultures also contained the indicated SCN⁻ concentrations plus ATP (100 μM) and LPO (7 μg/ml); the number of surviving bacteria was evaluated after a 3-hour incubation using a quantitative bacterial culture method (n=5 for each data point).