Recessively Inherited Deficiency of Secreted WFDC2 (HE4) Causes Nasal Polyposis and Bronchiectasis.

RATIONALE: Bronchiectasis is a pathological dilatation of the bronchi in the respiratory airways associated with environmental or genetic causes (e.g., cystic fibrosis, primary ciliary dyskinesia and primary immunodeficiency disorders), but most cases remain idiopathic. OBJECTIVES: To identify novel genetic defects in unsolved cases of bronchiectasis presenting with severe rhinosinusitis, nasal polyposis, and pulmonary Pseudomonas aeruginosa infection. METHODS: DNA was analyzed by next-generation or targeted Sanger sequencing. RNA was analyzed by quantitative PCR and single-cell RNA sequencing. Patient-derived, cells, cell cultures and secretions (mucus, saliva, seminal fluid) were analyzed by Western blotting and immunofluorescence microscopy, and mucociliary activity was measured. Blood serum was analyzed by electrochemiluminescence immunoassay. Protein structure and proteomic analyses were used to assess the impact of a disease-causing founder variant. MEASUREMENTS AND MAIN RESULTS: We identified bi-allelic pathogenic variants in WFDC2 in 11 individuals from 10 unrelated families originating from the United States, Europe, Asia, and Africa. Expression of WFDC2 was detected predominantly in secretory cells of control airway epithelium and also in submucosal glands. We demonstrate that WFDC2 is below the limit of detection in blood serum and hardly detectable in samples of saliva, seminal fluid, and airway surface liquid from WFDC2-deficient individuals. Computer simulations and deglycosylation assays indicate that the disease-causing founder variant p.Cys49Arg structurally hampers glycosylation and thus secretion of mature WFDC2. CONCLUSIONS: WFDC2 dysfunction defines a novel molecular etiology of bronchiectasis characterized by the deficiency of a secreted component of the airways. A commercially available blood test combined with genetic testing allows its diagnosis. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

Mucus polymer concentration and in vivo adaptation converge to define the antibiotic response of Pseudomonas aeruginosa during chronic lung infection.

The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa, which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic tolerance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro. We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa. Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. IMPORTANCE: Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro, is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.

Combination treatment to improve mucociliary transport of Pseudomonas aeruginosa biofilms.

People with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grown Pseudomonas aeruginosa biofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models.

BPIFB1 loss alters airway mucus properties and diminishes mucociliary clearance.

Airway mucociliary clearance (MCC) is required for host defense and is often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the gene Bpifb1/Lplunc1 as a regulator of airway MUC5B protein levels using genetic approaches. Here, we show that BPIFB1 is required for effective MCC in vivo using Bpifb1 knockout (KO) mice. Reduced MCC in Bpifb1 KO mice occurred in the absence of defects in epithelial ion transport or reduced ciliary beat frequency. Loss of BPIFB1 in vivo and in vitro altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and the protein mesh of secreted mucus in human airway epithelia cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.NEW & NOTEWORTHY BPIFB1, also known as LPLUNC1, was found to regulate mucociliary clearance (MCC), a key aspect of host defense in the airway. Loss of this protein was also associated with altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC.

Combination Treatment to Improve Mucociliary Transport of Pseudomonas aeruginosa Biofilms.

People with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grown Pseudomonas aeruginosa biofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models.

Delivery Characterization of SPL84 Inhaled Antisense Oligonucleotide Drug for 3849 + 10 kb C- > T Cystic Fibrosis Patients.

Recent advances in the therapeutic potential of RNA-related treatments, specifically for antisense oligonucleotide (ASO)-based drugs, have led to increased numbers of ASO regulatory approvals. In this study, we focus on SPL84, an inhaled ASO-based drug, developed for the treatment of the pulmonary disease cystic fibrosis (CF). Pulmonary drug delivery is challenging, due to a variety of biological, physical, chemical, and structural barriers, especially when targeting the cell nucleus. The distribution of SPL84 throughout the lungs, penetration into the epithelial cells and nucleus, and structural stability are critical parameters that will impact drug efficacy in a clinical setting. In this study, we demonstrate broad distribution, as well as cell and nucleus penetration of SPL84 in mouse and monkey lungs. In vivo and in vitro studies confirmed the stability of our inhaled drug in CF patient-derived mucus and in lung lysosomal extracts. The mobility of SPL84 through hyperconcentrated mucus was also demonstrated. Our results, supported by a promising preclinical pharmacological effect of full restoration of cystic fibrosis transmembrane conductance regulator channel activity, emphasize the high potential of SPL84 as an effective drug for the treatment of CF patients. In addition, successfully tackling the lung distribution of SPL84 offers immense opportunities for further development of SpliSense's inhaled ASO-based drugs for unmet needs in pulmonary diseases.

Normalizing salt content by mixing native human airway mucus samples normalizes sample rheology.

Across the globe, millions of people are affected by muco-obstructive pulmonary diseases like cystic fibrosis, asthma, and chronic obstructive pulmonary disease. In MOPDs, the airway mucus becomes hyperconcentrated, increasing viscoelasticity and impairing mucus clearance. Research focused on treatment of MOPDs requires relevant sources of airway mucus both as a control sample type and as a basis for manipulation to study the effects of additional hyperconcentration, inflammatory milieu, and biofilm growth on the biochemical and biophysical properties of mucus. Endotracheal tube mucus has been identified as a prospective source of native airway mucus given its several advantages over sputum and airway cell culture mucus such as ease of access and in vivo production that includes surface airway and submucosal gland secretions. Still, many ETT samples suffer from altered tonicity and composition from either dehydration, salivary dilution, or other contamination. Herein, the biochemical compositions of ETT mucus from healthy human subjects were determined. Samples were characterized in terms of tonicity, pooled, and restored to normal tonicity. Salt-normalized ETT mucus exhibited similar concentration-dependent rheologic properties as originally isotonic mucus. This rheology agreed across spatial scales and with previous reports of the biophysics of ETT mucus. This work affirms previous reports of the importance of salt concentration on mucus rheology and presents methodology to increase yield native airway mucus samples for laboratory use and manipulation.

Altering the viscoelastic properties of mucus-grown Pseudomonas aeruginosa biofilms affects antibiotic susceptibility.

The viscoelastic properties of biofilms are correlated with their susceptibility to mechanical and chemical stress, and the airway environment in muco-obstructive pulmonary diseases (MOPD) facilitates robust biofilm formation. Hyperconcentrated, viscoelastic mucus promotes chronic inflammation and infection, resulting in increased mucin and DNA concentrations. The viscoelastic properties of biofilms are regulated by biopolymers, including polysaccharides and DNA, and influence responses to antibiotics and phagocytosis. We hypothesize that targeted modulation of biofilm rheology will compromise structural integrity and increase antibiotic susceptibility and mucociliary transport. We evaluate biofilm rheology on the macro, micro, and nano scale as a function of treatment with a reducing agent, a biopolymer, and/or tobramycin to define the relationship between the viscoelastic properties of biofilms and susceptibility. Disruption of the biofilm architecture is associated with altered macroscopic and microscopic moduli, rapid vector permeability, increased antibiotic susceptibility, and improved mucociliary transport, suggesting that biofilm modulating therapeutics will improve the treatment of chronic respiratory infections in MOPD.

Mucus polymer concentration and in vivo adaptation converge to define the antibiotic response of Pseudomonas aeruginosa during chronic lung infection.

The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa , which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic recalcitrance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro . We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa . Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. Importance: Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro , is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.

Effects of Mucin and DNA Concentrations in Airway Mucus on Pseudomonas aeruginosa Biofilm Recalcitrance.

The pathological properties of airway mucus in cystic fibrosis (CF) are dictated by mucus concentration and composition, with mucins and DNA being responsible for mucus viscoelastic properties. As CF pulmonary disease progresses, the concentrations of mucins and DNA increase and are associated with increased mucus viscoelasticity and decreased transport. Similarly, the biophysical properties of bacterial biofilms are heavily influenced by the composition of their extracellular polymeric substances (EPS). While the roles of polymer concentration and composition in mucus and biofilm mechanical properties have been evaluated independently, the relationship between mucus concentration and composition and the biophysical properties of biofilms grown therein remains unknown. Pseudomonas aeruginosa biofilms were grown in airway mucus as a function of overall concentration and DNA concentration to mimic healthy, and CF pathophysiology and biophysical properties were evaluated with macro- and microrheology. Biofilms were also characterized after exposure to DNase or DTT to examine the effects of DNA and mucin degradation, respectively. Identifying critical targets in biofilms for disrupting mechanical stability in highly concentrated mucus may lead to the development of efficacious biofilm therapies and ultimately improve CF patient outcomes. Overall mucus concentration was the predominant contributor to biofilm viscoelasticity and both DNA degradation and mucin reduction resulted in compromised biofilm mechanical strength. IMPORTANCE Pathological mucus in cystic fibrosis (CF) is highly concentrated and insufficiently cleared from the airway, causing chronic inflammation and infection. Pseudomonas aeruginosa establishes chronic infection in the form of biofilms within mucus, and this study determined that biofilms formed in more concentrated mucus were more robust and less susceptible to mechanical and chemical challenges compared to biofilms grown in lower concentrated mucus. Neither DNA degradation nor disulfide bond reduction was sufficient to fully degrade biofilms. Mucus rehydration should remain a priority for treating CF pulmonary disease with concomitant multimechanistic biofilm degradation agents and antibiotics to clear chronic infection.

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer.

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., viral/bacterial infections), mucus biophysical properties are altered by goblet cell hypersecretion, airway dehydration, oxidative stress, and the presence of extracellular DNA. Previous studies showed that sputum viscoelasticity correlated with pulmonary function and that treatments affecting sputum rheology (e.g., mucolytics) can result in remarkable clinical benefits. In general, rheological measurements of non-Newtonian fluids employ elaborate, time-consuming approaches (e.g., parallel/cone-plate rheometers and/or microbead particle tracking) that require extensive training to perform the assay and interpret the data. This study tested the reliability, reproducibility, and sensitivity of Rheomuco, a user-friendly benchtop device that is designed to perform rapid measurements using dynamic oscillation with a shear-strain sweep to provide linear viscoelastic moduli (G', G", G*, and tan δ) and gel point characteristics (γc and σc) for clinical samples within 5 min. Device performance was validated using different concentrations of a mucus simulant, 8 MDa polyethylene oxide (PEO), and against traditional bulk rheology measurements. A clinical isolate harvested from an intubated patient with status asthmaticus (SA) was then assessed in triplicate measurements and the coefficient of variation between measurements is <10%. Ex vivo use of a potent mucus reducing agent, TCEP, on SA mucus resulted in a five-fold decrease in elastic modulus and a change toward a more "liquid-like" behavior overall (e.g., higher tan δ). Together, these results demonstrate that the tested benchtop rheometer can make reliable measures of mucus viscoelasticity in clinical and research settings. In summary, the described protocol could be used to explore the effects of mucoactive drugs (e.g., rhDNase, N-acetyl cysteine) onsite to adapt treatment on a case-by-case basis, or in preclinical studies of novel compounds.

Mucus concentration-dependent biophysical abnormalities unify submucosal gland and superficial airway dysfunction in cystic fibrosis.

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway model of CF revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Using proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, CF sputum proteomics analyses revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and in SMGs of the CF lung.

Mucus and mucus flake composition and abundance reflect inflammatory and infection status in cystic fibrosis.

BACKGROUND: Mucus hyperconcentration in cystic fibrosis (CF) lung disease is marked by increases in both mucin and DNA concentration. Additionally, it has been shown that half of the mucins present in bronchial alveolar lavage fluid (BALF) from preschool-aged CF patients are present in as non-swellable mucus flakes. This motivates us to examine the utility of mucus flakes, as well as mucin and DNA concentrations in BALF as markers of infection and inflammation in CF airway disease. METHODS: In this study, we examined the mucin and DNA concentration, as well as mucus flake abundance, composition, and biophysical properties in BALF from three groups; healthy adult controls, and two CF cohorts, one preschool aged and the other school aged. BALFs were characterized via refractometry, PicoGreen, immunofluorescence microscopy, particle tracking microrheology, and fluorescence image tiling. RESULTS: Mucin and DNA BALF concentrations increased progressively from healthy young adult controls to preschool-aged people and school-aged people with CF. Notably, mucin concentrations were increased in bronchoalveolar lavage fluid (BALF) from preschool-aged patients with CF prior to decreased pulmonary function. Infrequent small mucus flakes were identified in normal subjects. A progressive increase in the abundance of mucus flakes in preschool and school-aged CF patients was observed. Compositionally, MUC5B dominated flakes from normal subjects, whereas an increase in MUC5AC was observed in people with CF, reflected in a reduced flaked MUC5B/MUC5AC mucin ratio. CONCLUSION: These findings suggest mucus composition and flake properties are useful markers of inflammatory and infection-based changes in CF airways.

Treatment of cystic fibrosis airway cells with CFTR modulators reverses aberrant mucus properties via hydration.

QUESTION: Cystic fibrosis (CF) is characterised by the accumulation of viscous adherent mucus in the lungs. While several hypotheses invoke a direct relationship with cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction (i.e. acidic airway surface liquid (ASL) pH, low bicarbonate (HCO3 -) concentration, airway dehydration), the dominant biochemical alteration of CF mucus remains unknown. MATERIALS/METHODS: We characterised a novel cell line (CFTR-KO Calu3 cells) and the responses of human bronchial epithelial (HBE) cells from subjects with G551D or F508del mutations to ivacaftor and elexacaftor-tezacaftor-ivacaftor. A spectrum of assays such as short-circuit currents, quantitative PCR, ASL pH, Western blotting, light scattering/refractometry (size-exclusion chromatography with inline multi-angle light scattering), scanning electron microscopy, percentage solids and particle tracking were performed to determine the impact of CFTR function on mucus properties. RESULTS: Loss of CFTR function in Calu3 cells resulted in ASL pH acidification and mucus hyperconcentration (dehydration). Modulation of CFTR in CF HBE cells did not affect ASL pH or mucin mRNA expression, but decreased mucus concentration, relaxed mucus network ultrastructure and improved mucus transport. In contrast with modulator-treated cells, a large fraction of airway mucins remained attached to naïve CF cells following short apical washes, as revealed by the use of reducing agents to remove residual mucus from the cell surfaces. Extended hydration, but not buffers alkalised with sodium hydroxide or HCO3 -, normalised mucus recovery to modulator-treated cell levels. CONCLUSION: These results indicate that airway dehydration, not acidic pH and/or low [HCO3 -], is responsible for abnormal mucus properties in CF airways and CFTR modulation predominantly restores normal mucin entanglement.

Induction of ciliary orientation by matrix patterning and characterization of mucociliary transport.

Impaired mucociliary clearance (MCC) is a key feature of many airway diseases, including asthma, bronchiectasis, chronic obstructive pulmonary disease, cystic fibrosis, and primary ciliary dyskinesia. To improve MCC and develop new treatments for these diseases requires a thorough understanding of how mucus concentration, mucus composition, and ciliary activity affect MCC, and how different therapeutics impact this process. Although differentiated cultures of human airway epithelial cells are useful for investigations of MCC, the extent of ciliary coordination in these cultures varies, and the mechanisms controlling ciliary orientation are not completely understood. By introducing a pattern of ridges and grooves into the underlying collagen substrate, we demonstrate for the first time, to our knowledge, that changes in the extracellular matrix can induce ciliary alignment. Remarkably, 90% of human airway epithelial cultures achieved continuous directional mucociliary transport (MCT) when grown on the patterned substrate. These cultures maintain transport for months, allowing carefully controlled investigations of MCC over a wide range of normal and pathological conditions. To characterize the system, we measured the transport of bovine submaxillary gland mucin (BSM) under several conditions. Transport of 5% BSM was significantly reduced compared with that of 2% BSM, and treatment of 5% BSM with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) reduced viscosity and increased the rate of MCT by approximately twofold. Addition of a small amount of high-molecular-weight DNA increased mucus viscosity and reduced MCT by ∼75%, demonstrating that the composition of mucus, as well as the concentration, can have significant effects on MCT. Our results demonstrate that a simple patterning of the collagen substrate results in highly coordinated ciliated cultures that develop directional MCT, and can be used to investigate the mechanisms controlling the regulation of ciliary orientation. Furthermore, the results demonstrate that this method provides an improved system for studying the effects of mucus composition and therapeutic agents on MCC.

Molecular Dynamics Simulations to Explore the Structure and Rheological Properties of Normal and Hyperconcentrated Airway Mucus.

We develop the first molecular dynamics model of airway mucus based on the detailed physical properties and chemical structure of the predominant gel-forming mucin MUC5B. Our airway mucus model leverages the LAMMPS open-source code [https://lammps.sandia.gov], based on the statistical physics of polymers, from single molecules to networks. On top of the LAMMPS platform, the chemical structure of MUC5B is used to superimpose proximity-based, non-covalent, transient interactions within and between the specific domains of MUC5B polymers. We explore feasible ranges of hydrophobic and electrostatic interaction strengths between MUC5B domains with 9 nanometer spatial and 1 nanosecond temporal resolution. Our goal here is to propose and test a mechanistic hypothesis for a striking clinical observation with respect to airway mucus: a 10-fold increase in non-swellable, dense structures called flakes during progression of cystic fibrosis disease. Among the myriad possible effects that might promote self-organization of MUC5B networks into flake structures, we hypothesize and confirm that the clinically confirmed increase in mucin concentration, from 1.5 to 5 mg/mL, alone is sufficient to drive the structure changes observed with scanning electron microscopy images from experimental samples. We post-process the LAMMPS simulated datasets at 1.5 and 5 mg/mL, both to image the structure transition and compare with scanning electron micrographs and to show that the 3.33-fold increase in concentration induces closer proximity of interacting electrostatic and hydrophobic domains, thereby amplifying the proximity-based strength of the interactions.

Pseudomonas aeruginosa Biofilm Eradication via Nitric Oxide-Releasing Cyclodextrins.

Pseudomonas aeruginosa is the main contributor to the morbidity and mortality of cystic fibrosis (CF) patients. Chronic respiratory infections are rarely eradicated due to protection from CF mucus and the biofilm matrix. The composition of the biofilm matrix determines its viscoelastic properties and affects antibiotic efficacy. Nitric oxide (NO) can both disrupt the physical structure of the biofilm and eradicate interior colonies. The effects of a CF-like growth environment on P. aeruginosa biofilm susceptibility to NO were investigated using parallel plate macrorheology and particle tracking microrheology. Biofilms grown in the presence of mucins and DNA contained greater concentrations of DNA in the matrix and exhibited concomitantly larger viscoelastic moduli compared to those grown in tryptic soy broth. Greater viscoelastic moduli correlated with increased tolerance to tobramycin and colistin. Remarkably, NO-releasing cyclodextrins eradicated all biofilms at the same concentration. The capacity of NO-releasing cyclodextrins to eradicate P. aeruginosa biofilms irrespective of matrix composition suggests that NO-based therapies may be superior antibiofilm treatments compared to conventional antibiotics.

Endotracheal tube mucus as a source of airway mucus for rheological study.

Muco-obstructive lung diseases (MOLDs), like cystic fibrosis and chronic obstructive pulmonary disease, affect a spectrum of subjects globally. In MOLDs, the airway mucus becomes hyperconcentrated, increasing osmotic and viscoelastic moduli and impairing mucus clearance. MOLD research requires relevant sources of healthy airway mucus for experimental manipulation and analysis. Mucus collected from endotracheal tubes (ETT) may represent such a source with benefits, e.g., in vivo production, over canonical sample types such as sputum or human bronchial epithelial (HBE) mucus. Ionic and biochemical compositions of ETT mucus from healthy human subjects were characterized and a stock of pooled ETT samples generated. Pooled ETT mucus exhibited concentration-dependent rheologic properties that agreed across spatial scales with reported individual ETT samples and HBE mucus. We suggest that the practical benefits compared with other sample types make ETT mucus potentially useful for MOLD research.

Mucus accumulation in the lungs precedes structural changes and infection in children with cystic fibrosis.

Although destructive airway disease is evident in young children with cystic fibrosis (CF), little is known about the nature of the early CF lung environment triggering the disease. To elucidate early CF pulmonary pathophysiology, we performed mucus, inflammation, metabolomic, and microbiome analyses on bronchoalveolar lavage fluid (BALF) from 46 preschool children with CF enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program and 16 non-CF disease controls. Total airway mucins were elevated in CF compared to non-CF BALF irrespective of infection, and higher densities of mucus flakes containing mucin 5B and mucin 5AC were observed in samples from CF patients. Total mucins and mucus flakes correlated with inflammation, hypoxia, and oxidative stress. Many CF BALFs appeared sterile by culture and molecular analyses, whereas other samples exhibiting bacterial taxa associated with the oral cavity. Children without computed tomography-defined structural lung disease exhibited elevated BALF mucus flakes and neutrophils, but little/no bacterial infection. Although CF mucus flakes appeared "permanent" because they did not dissolve in dilute BALF matrix, they could be solubilized by a previously unidentified reducing agent (P2062), but not N-acetylcysteine or deoxyribonuclease. These findings indicate that early CF lung disease is characterized by an increased mucus burden and inflammatory markers without infection or structural lung disease and suggest that mucolytic and anti-inflammatory agents should be explored as preventive therapy.

An Improved Inhaled Mucolytic to Treat Airway Muco-obstructive Diseases.

RATIONALE: Airways obstruction with thick, adherent mucus is a pathophysiologic and clinical feature of muco-obstructive respiratory diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis (CF). Mucins, the dominant biopolymer in mucus, organize into complex polymeric networks via the formation of covalent disulfide bonds, which govern the viscoelastic properties of the mucus gel. For decades, inhaled N-acetylcysteine (NAC) has been used as a mucolytic to reduce mucin disulfide bonds with little, if any, therapeutic effects. Improvement of mucolytic therapy requires the identification of NAC deficiencies and the development of compounds that overcome them. OBJECTIVES: Elucidate the pharmacological limitations of NAC and test a novel mucin-reducing agent, P3001, in preclinical settings. METHODS: The study used biochemical (e.g., Western blotting, mass spectrometry) and biophysical assays (e.g., microrheology/macrorheology, spinnability, mucus velocity measurements) to test compound efficacy and toxicity in in vitro and in vivo models and patient sputa. MEASUREMENTS AND MAIN RESULTS: Dithiothreitol and P3001 were directly compared with NAC in vitro and both exhibited superior reducing activities. In vivo, P3001 significantly decreased lung mucus burden in ßENaC-overexpressing mice, whereas NAC did not (n = 6-24 mice per group). In NAC-treated CF subjects (n = 5), aerosolized NAC was rapidly cleared from the lungs and did not alter sputum biophysical properties. In contrast, P3001 acted faster and at lower concentrations than did NAC, and it was more effective than DNase in CF sputum ex vivo. CONCLUSIONS: These results suggest that reducing the viscoelasticity of airway mucus is an achievable therapeutic goal with P3001 class mucolytic agents.