Oxidation increases mucin polymer cross-links to stiffen airway mucus gels

Abstract

Airway mucus in cystic fibrosis (CF) is highly elastic, but the mechanism behind this pathology is unclear. We hypothesized that the biophysical properties of CF mucus are altered because of neutrophilic oxidative stress. Using confocal imaging, rheology, and biochemical measures of inflammation and oxidation, we found that CF airway mucus gels have a molecular architecture characterized by a core of mucin covered by a web of DNA and a rheological profile characterized by high elasticity that can be normalized by chemical reduction. We also found that high levels of reactive oxygen species in CF mucus correlated positively and significantly with high concentrations of the oxidized products of cysteine (disulfide cross-links). To directly determine whether oxidation can cross-link mucins to increase mucus elasticity, we exposed induced sputum from healthy subjects to oxidizing stimuli and found a marked and thiol-dependent increase in sputum elasticity. Targeting mucin disulfide cross-links using current thiol-amino structures such as N-acetylcysteine (NAC) requires high drug concentrations to have mucolytic effects. We therefore synthesized a thiol-carbohydrate structure (methyl 6-thio-6-deoxy-α-D-galactopyranoside) and found that it had stronger reducing activity than NAC and more potent and fast-acting mucolytic activity in CF sputum. Thus, oxidation arising from airway inflammation or environmental exposure contributes to pathologic mucus gel formation in the lung, which suggests that it can be targeted by thiol-modified carbohydrates.

Fig. 1. Mucin and DNA biopolymers in mucus and effect of mucolytics

(A) Confocal image of sputum from healthy and CF subjects. Mucins (red) are present in healthy and CF sputum, whereas DNA (green) is more abundant in CF. Abs, antibodies. (B) 3D rendering of XY images of mucins. (C) Z-stack images of mucins. (D) Relationship between mucin and DNA polymers in CF mucus gel. (E) 3D rendering shows a dense mucin core with overlying DNA polymers. (F) DNA web with mucin core subtracted. (G) Typical frequency sweep of the elastic (G′) and viscous modulus (G″) of healthy (open symbols) and CF sputum (closed symbols), showing that G′ predominates over the G″ across a broad range of frequencies, indicating a viscoelastic gel. (H) Summary of average G′ and G″ (at a frequency of 1.0 Hz) in induced sputum from 15 healthy and 14 CF subjects. (I) Effects of treatment with mucolytics on G′ of induced sputum from 10 CF patients over a 4-hour test period. rhDNase (0.1 mg/ml) significantly reduced G′, but NAC (61 mM) had a larger effect (74 ± 10% reduction versus 34 ± 16%, P = 0.04). (J) Effects of treatment with mucolytics on the G″ of induced sputum from 10 patients with CF. rhDNase had little effect on G″, whereas NAC had a significant effect. The normalized ratio of elastic modulus refers to the posttreatment G′ divided by pretreatment G′. The effects of mucolytics on G′ and G″ are compared to normal saline. Scale bars, 20 mm (unless otherwise indicated). Data in (H) to (J) are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2. Measurements of oxidative stress in CF mucus

(A) Neutrophil cell number is significantly higher in induced sputum from patients with CF (n = 13) than in induced sputum from healthy subjects (n = 14). (B) MPO concentration measurements are higher in induced sputum from CF patients (n = 13) than in induced sputum from healthy controls (n = 15). (C) MPO activity measurements are higher in induced sputum from CF patients (n = 13) than in induced sputum from healthy controls (n = 15). (D) ROS, as detected by conversion of carboxy-H2DCFDA to its green fluorescent form, are markedly higher in spontaneously expectorated supernatant from CF patients (n = 5) than in induced sputum from healthy controls (n = 5). Hydrogen peroxide contributes significantly to the ROS signal, as evidenced by the reduction in ROS with catalase. The elimination of the ROS signal by HCl is included as a negative control of the enzyme-based assay. (E) Oxidized protein products measured using MS are higher in spontaneously expectorated supernatant from CF patients (n = 5) than in induced sputum from healthy controls (n = 5). (F) A fluorescent assay using dithiothreitol (DTT) and monobromobimane (mBBr) shows that the amount of total accessible cysteines is not significantly different between the spontaneously expectorated CF sputum (n = 5) and the induced healthy sputum (n = 5). (G) With iodoacetamide pre-treatment, the same assay shows that the concentration of cystines (disulfide bonds) is markedly higher in the CF sputum (n= 5) than in the healthy sputum (n = 5). (H) The concentration of disulfide bonds correlates positively with ROS. r_s = 0.77, P = 0.013 [data from (D) and (F)]. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3. Oxidative stress causing excessive disulfide bond formation in mucus

(A) Addition of DMSO [20% (v/v)] to PGM (5 mg/ml) rapidly increases its elastic modulus. An increase in the concentration of PGM from 5 to >20 mg/ml was required to have the same effect on G. (B) The effect of DMSO on the viscous modulus (G″) of PGM is smaller than that on G′ (C) Addition of DMSO [20% (v/v)] to induced sputum from healthy subjects causes a doubling in elastic modulus, an effect caused by disulfide bridge formation, because it is prevented by addition of iodoacetamide (50 mM). The area under the curve (AUC) of G′ for DMSO/phosphate-buffered saline (PBS) was significantly larger than that for DMSO/Iodoacetamide (5679 ± 974 versus 3619 ±589, P = 0.01). (D) DMSO had little effect on the viscous modulus of the induced sputum. (E) Schematic showing a cone and plate rheometer modified to permit exposure of mucus to oxygen or nitrogen in a closed system that controls humidity, temperature, and gas concentration. (F) Exposure of induced sputum samples from healthy subjects (n = 5) to 100% oxygen caused a time-dependent significant increase in elastic modulus, whereas exposure to nitrogen gas has no significant effect. The AUC of G′ for oxygen was significantly larger than that for nitrogen (31804 ±5507 versus 15391 ± 1539, P = 0.04). (G) Schematic representation for how healthy mucus can transition to pathologic mucus when oxidation promotes mucin chain extension through end-to-end disulfides and side-to-side cross-links of internal cysteines.

Fig. 4. A thiol-modified galactose is a novel and potent reducing agent with superior mucolytic activity

(A) Chemical structure of NAC. (B) Chemical structure of TDG. (C) The ORP of TDG is lower than that of NAC and of the parent sugar (MDG). (D) Effects of high concentrations (61 mM) of TDG, NAC, and MDG on the elastic properties of CF sputum (n = 5 donors) over a 12-min test period. (E) The mucolytic effect of TDG at 2 min is significantly larger than NAC and MDG; the mucolytic effects of TDG and NAC at 12 min are similar. (F) Effects of low concentrations (10 mM) of TDG, MDG, and NAC on the elastic properties of CF sputum (n = 5 donors) over a 12-min test period. (G) The mucolytic effects of TDG at 2 and 12 min are significantly larger than those of NAC and MDG. Data in (C) to (G) are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.