Disulfide disruption reverses mucus dysfunction in allergic airway disease

Airway mucus is essential for lung defense, but excessive mucus in asthma obstructs airflow, leading to severe and potentially fatal outcomes. Current asthma treatments have minimal effects on mucus, and the lack of therapeutic options stems from a poor understanding of mucus function and dysfunction at a molecular level and in vivo. Biophysical properties of mucus are controlled by mucin glycoproteins that polymerize covalently via disulfide bonds. Once secreted, mucin glycopolymers can aggregate, form plugs, and block airflow. Here we show that reducing mucin disulfide bonds disrupts mucus in human asthmatics and reverses pathological effects of mucus hypersecretion in a mouse allergic asthma model. In mice, inhaled mucolytic treatment loosens mucus mesh, enhances mucociliary clearance, and abolishes airway hyperreactivity (AHR) to the bronchoprovocative agent methacholine. AHR reversal is directly related to reduced mucus plugging. These findings establish grounds for developing treatments to inhibit effects of mucus hypersecretion in asthma.


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No data were excluded.
Technical and biological replicates were studied on different days (>100 days). All attempts at replication were successful.
Mice, samples, and datasets were randomized for evaluations. For histological quantification randomized design-based stereology was performed.
Experimenters were blinded to genotypes and to treatment groups for all allergen vs control treatment groups in all mouse AHR and mucus clearance studies on days of experiments. MPT values were analyzed with blinding as to the treatment groups. Histological mage analysis was done blinded in all cases.
Knockout mouse samples were used for mouse-anti-MUC5AC (PMID: 23187315) and rabbit-anti-mouse Muc5b antibodies (PMID: 24317696). For human samples, molecular masses on agarose gels were indicative of very high molecular weight mucin polymers (>1 MDa). Anti human antibodies have also been validated by western blot and dot blot ELISA using MUC5AC and MUC5B knockout A549 cells. Data not shown. According to the vendor, 45M1 cross-reacts with Cat, Human, Mammal, Mouse, Non-human primate, Pig, Rabbit, Rat (>100 citations). H300 is no longer available commercially, but in practice we have found that it cross-reacts minimally with mouse, rat, and ferret tissues.
Male and female BALB/cJ wild type mice were purchased from the Jackson Labs (Bar Harbor, ME). Muc5ac /-mice were previously crossed onto a congenic BALB/cJ strain background. Animals were housed under specific pathogen-free conditions and used in allergic asthma studies beginning at ages 6-8 weeks.
The study did not involve wild animals.
The study did not involve samples collected in the field.
IRB and IACUC panels oversaw research at the University of Colorado, National Jewish Health, and Johns Hopkins.
Bronchoalveolar lavage samples from 4 Patients with stable asthma were used (2 M, 2 F). The patients were enrolled for a separate IRB approved study and anonymized samples were stored in a Universtiy of Colorado biobank. Subjects had no history of infection, steroid use, or asthma-related hospitalization within 4 wks of study Expectorated sputum was collected from 5 volunteers (3 M, 2 F) with CF as part of an unrelated study at Johns Hopkins. Discard material was used for the studies reported here. No patients were on CFTR modulator therapies, all CF patients had histories of positive bacterial cultures wihtin 1 year of study, 4 of 5 showed evidence of bacterial infection at time of study. CF mutations were: F508del/F508del, F508del/R1066C, F508del/W1282X, F508del/L88X, and F508del/exon 2 del.
All patients were adults, non-pregnant, non-smokers.