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.

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.

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.

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.

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.

Antibody-mediated trapping in biological hydrogels is governed by sugar-sugar hydrogen bonds.

N-glycans on IgG and IgM antibodies (Ab) facilitate Ab-mediated crosslinking of viruses and nanoparticles to the major structural elements of mucus and basement membranes. Nevertheless, the chemical moieties in these biological hydrogel matrices to which Ab can bind remain poorly understood. To gain insights into the chemistries that support Ab-matrix interactions, we systematically evaluated IgG- and IgM-mediated trapping of nanoparticles in different polysaccharide-based biogels with unique chemical features. In agarose, composed of alternating d-galactose and 3,6-anhydro-l-galactopyranose (i.e. hydroxyl groups only), anti-PEG IgM but not anti-PEG IgG trapped PEGylated nanoparticles. In alginate, comprised of homopolymeric blocks of mannuronate and guluronate (i.e. both hydroxyl and carboxyl groups), both IgG and IgM trapped PEGylated nanoparticles. In contrast, chitosan, comprised primarily of glucosamine (i.e. both hydroxyl and primary amine groups), did not facilitate either IgG- or IgM-mediated trapping. IgG-mediated trapping in alginate was abrogated upon removal of IgG N-glycans, whereas IgM-mediated trapping was eliminated in agarose but not alginate upon desialylation. These results led us to propose a model in which hydrogen bonding between carboxyl and hydroxyl groups of glycans on both Ab and matrix facilitates Ab-mediated trapping of pathogens in biogels. Our work here offers a blueprint for designing de novo hydrogels that could harness Ab-matrix interactions for various biomedical and biological applications. STATEMENT OF SIGNIFICANCE: Here, we interrogated the molecular mechanism of antibody-mediated trapping to address what are the chemical moieties on biogels that are essential for facilitating trapping in biogels. We systematically evaluated the potencies of IgG and IgM to trap nanoparticles in different polysaccharide-based biogels with unique and highly defined chemical moieties: hydroxyl groups (agarose), amine groups (chitosan), and carboxyl groups (alginate). We discovered that only hydroxyl/carboxyl hydrogen bonds (and stronger) are sufficiently strong enough to facilitate antibody-mediated trapping; weaker hydroxyl/hydroxyl bonds or hydroxyl/amine bonds fail to adequately slow particles. Our findings presents the first blueprint for how to engineer de novo biogels that are capable of harnessing antibodies to immobilize foreign entities in the biogels, for applications ranging from infectious disease to contraception to purification processes.

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.

Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice.

The gain-of-function MUC5B promoter variant rs35705950 is the dominant risk factor for developing idiopathic pulmonary fibrosis (IPF). Here we show in humans that MUC5B, a mucin thought to be restricted to conducting airways, is co-expressed with surfactant protein C (SFTPC) in type 2 alveolar epithelia and in epithelial cells lining honeycomb cysts, indicating that cell types involved in lung fibrosis in distal airspace express MUC5B. In mice, we demonstrate that Muc5b concentration in bronchoalveolar epithelia is related to impaired mucociliary clearance (MCC) and to the extent and persistence of bleomycin-induced lung fibrosis. We also establish the ability of the mucolytic agent P-2119 to restore MCC and to suppress bleomycin-induced lung fibrosis in the setting of Muc5b overexpression. Our findings suggest that mucociliary dysfunction might play a causative role in bleomycin-induced pulmonary fibrosis in mice overexpressing Muc5b, and that MUC5B in distal airspaces is a potential therapeutic target in humans with IPF.

Pathological mucus and impaired mucus clearance in cystic fibrosis patients result from increased concentration, not altered pH.

Cystic fibrosis (CF) is a recessive genetic disease that is characterised by airway mucus plugging and reduced mucus clearance. There are currently alternative hypotheses that attempt to describe the abnormally viscous and elastic mucus that is a hallmark of CF airways disease, including: 1) loss of CF transmembrane regulator (CFTR)-dependent airway surface volume (water) secretion, producing mucus hyperconcentration-dependent increased viscosity, and 2) impaired bicarbonate secretion by CFTR, producing acidification of airway surfaces and increased mucus viscosity.A series of experiments was conducted to determine the contributions of mucus concentration versus pH to the rheological properties of airway mucus across length scales from the nanoscopic to macroscopic.For length scales greater than the nanoscopic, i.e. those relevant to mucociliary clearance, the effect of mucus concentration dominated over the effect of airway acidification.Mucus hydration and chemical reduction of disulfide bonds that connect mucin monomers are more promising therapeutic approaches than alkalisation.