Modeling airway dysfunction in asthma using synthetic mucus biomaterials

Abstract

As asthma worsens, occlusion of airways with mucus significantly contributes to airflow obstruction and reduced lung function. Recent evidence from clinical studies has shown mucus obtained from adults and children with asthma possesses altered mucin composition. However, how these changes alter the functional properties of the mucus gel is not yet fully understood. To study this, we have engineered a synthetic mucus biomaterial to closely mimic the properties of native mucus in health and disease. We demonstrate this model possesses comparable biophysical and transport properties to native mucus ex vivo collected from human subjects and in vitro isolated from human airway epithelial (HAE) tissue cultures. We found by systematically varying mucin composition that mucus gel viscoelasticity is enhanced when predominantly composed of mucin 5AC (MUC5AC), as is observed in asthma. As a result, asthma-like synthetic mucus gels are more slowly transported on the surface of HAE tissue cultures and at a similar rate to native mucus produced by HAE cultures stimulated with the type 2 cytokine IL-13, known to contribute to airway inflammation and MUC5AC hypersecretion in asthma. We also discovered the barrier function of asthma-like synthetic mucus towards influenza A virus was impaired as evidenced by the increased frequency of infection in MUC5AC-rich hydrogel coated HAE cultures. Together, this work establishes a biomaterial-based approach to understand airway dysfunction in asthma and related muco-obstructive lung diseases.

Tear Film Stability as a Function of Tunable Mucin Concentration Attached to Supported Lipid Bilayers

“…3 We note that in our study we use bovine submaxillary mucins (BSM) as a commercially available and economical reagent, though the same chemistry can be readily applied to other mucins. BSM is commonly used as a source of the secretory mucin MUC5B, 17 but it exhibits high degrees of O-glycosylation and a comparable molecular weight to MUC1 and MUC4. 3 Our platform is also adaptable to combinations of several mucins by exposure to a mixture during the mucin incubation step.…”

MUC5B mobilizes and MUC5AC spatially aligns mucociliary transport on human airway epithelium

“…Samples were loaded into Amicon Ultra 100 kDa filters (Millipore-Sigma) and centrifuged at 14,000 xg for 20 minutes to remove excess PBS for mucin isolation. Mucus samples were stored at −80 °C for long-term storage (> 2 weeks) or stored at 4 °C ( 26 ). Pooled mucus samples were used for western blot analysis, solids concentration analysis, biophysical characterization, and transport experiments.…”

“…Images were collected at a frame rate of 0.5 Hz for 20 seconds on the plane of the mucus gel. The microsphere tracking data analysis is based on an image processing algorithm that was custom written in MATLAB ( 26 , 39 ). Briefly, the analysis software computes the xy-plane trajectories of each fluorescent microsphere in each frame.…”

“…Reconstituted mucus samples from KO culture washings were used for transplantation studies. 20 μL of mucus was applied to the apical surface of ALI cultures that had been washed with PBS for 30 minutes ( 26 , 40 ). 2 μm red-fluorescent polystyrene microspheres were applied immediately after transplantation of exogenous mucus.…”

“…The sgRNAs were cloned into a GFP-expressing lentiviral vector that also encodes the Cas9 nuclease. After lentivirus production, we transduced BCi-NS1.1 cells, an immortalized HAE cell line, which has previously been used for manipulating mucin expression (25)(26)(27). Subsequently, transduced cells were expanded, and sorted for GFP Using mucus samples collected from apical washes, we confirmed KO expression on western blot analysis demonstrating loss of MUC5B and MUC5AC expression in KO cultures using respective sgRNA (Fig 1D).…”