New biophysical and immunoregulatory mechanisms in neutrophil extracellular trap mediated lung dysfunction in cystic fibrosis

PROJECT SUMMARY Mucus acts as a defensive barrier in the airways by trapping inhaled particles within a mucin gel network and clearing them from the airway via mucociliary transport performed by the underlying airway epithelium. Muco- obstructive airway diseases including cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease are caused by the buildup of thick mucus with an aberrant composition that is not able to be dynamically cleared, resulting in occluded airways. Mucus accumulation also leads to chronic bacterial infection in the airways, especially by Pseudomonas aeruginosa in CF patients. One abnormal component found in excess within the mucus of patients with muco-obstructive diseases, most prominently in CF mucus, is neutrophil extracellular traps (NETs). NETs are web-like complexes comprised mainly of decondensed chromatin intermixed with neutrophilic granular proteins that are secreted extracellularly to capture and kill bacteria in a process known as NETosis. DNase is currently used by CF patients to degrade the chromatin structure of NETs in the mucus but often does not fully restore mucociliary transport in patients, indicating the granular components of NETs are also likely involved in mucus dysfunction. In our previous research, we used a synthetic biomaterial model of the chromatin structure of NETs to evaluate the effects on mucus biophysical properties and mucociliary transport velocity. Building upon this, we propose to use both biomaterial and human airway tissue culture models to pursue the following objectives: 1) determine how various granular proteins within NETs differentially affect mucus biophysical properties and transportability across the airway epithelium, and 2) determine how the alterations to mucin composition and glycosylation in CF mucus contribute to the increased NETosis observed. For the first objective, we will employ similar synthetic NET biomaterial models, but incorporate neutrophilic granular proteins into the formulation to evaluate their specific contributions in enhancing mucus viscosity and decreasing mucociliary transport in CF airways. We will also account for the effects of inhaled DNase therapy used by CF patients to determine if granular proteins continue to cause mucociliary transport dysfunction after degradation of the chromatin scaffold of NETs. In the second objective, we will manipulate the expression of secreted mucins and mucin glycosylation patterns of human airway tissue to understand how mucins and their glycans modulate the activation of NETosis in neutrophils. We will account for the effects of P. aeruginosa bacteria on mucin glycosylation to determine how NETosis is altered during infection in CF patients. This research will identify novel anti-NET mucosal drug targets to prevent NETosis and neutralize the effects of NETs in the airway mucus barrier. Ultimately, we believe this will lead to improved treatment of CF and other related muco-obstructive lung diseases.