Impact of neutrophil serine proteases on mucus properties and function in in muco-obstructive lung diseases

Principal Investigator

Scientific interest within the context of the graduate college:

Our airways are covered by a thin mucus layer that plays a pivotal role in maintaining lung health and homeostasis. In healthy, this mucus layer entraps constantly inhaled pathogens, pollutants and irritants that are then removed from the lungs by beating cilia on airway surfaces that facilitate mucocilary clearance, which constitutes an important innate defense mechanism of the lung. In chronic muco-obstructive lung diseases such as cystic fibrosis (CF) and bronchiectasis the viscoelastic properties of airway mucus are characteristically altered.^1,2 Increased viscosity and elasticity of the mucus in these diseases leads to impaired mucociliary clearance, which in turn leads to airway mucus plugging, chronic infection with Pseudomonas aeruginosa and other bacterial pathogens, and chronic neutrophilic inflammation. Neutrophils express a group of proteases called neutrophil serine proteases (NSP), including neutrophil elastase (NE), protease 3 (PR3) and cathepsin G (CG). It is well established that increased activity of these NSPs leads to the degradation of endogenous anti-proteases, which in turn causes a protease/anti-protease imbalance that plays a central role in the pathogenesis of progressive structural lung damage in CF and bronchiectasis.^3,4 Preliminary data from our group suggest that these proteases may also change the viscoelastic properties and function of the mucus by cleavage of the mucins (MUC5B and MUC5AC) that form the mucus layer. However, a systemic evaluation of the effects of NSPs on mucus properties has not been performed and whether proteolytic degradation of mucus is beneficial or detrimental in muco-obstructive lung diseases remains unknown. Finding answers to these questions is also relevant in the context of current development of novel therapies for CF and bronchiectasis that inhibits NSP activity⁵ and may thereby also have an effect on mucus properties and mucociliary clearance.

Project description:

The overall aim of this project is to characterize the impact of NSPs on mucus properties and function in muco-obstructive lung diseases such as in CF and bronchiectasis. Elevated levels of NSP activity are characteristically found in expectorated sputum from patients with CF and patients with bronchiectasis,^1,5 and are associating with increased mucus viscoelasticity and increased proinflammatory cytokines. Three NSPs have been identified, NE, PR3 and CG that have been implicated to play a crucial role in the pathogenesis of muco-obstructive lung diseases.^6,7 To study the role of the three NSPs in mucin processing and modulation of mucus properties and function in health and disease, we will perform ex vivo studies of native sputum samples from healthy people and patients with CF or bronchiectasis and in vitro studies of the native mucus layer on patient-derived airway epithelial cultures. Specifically, sputum collected from CF or bronchiectasis and healthy people will be treated with individual NSPs (NE, PR3 or CG) at various pathophysiologically relevant concentrations and the impact on mucus processing and viscoelastic properties will be determined biochemically by Western blotting and mass spectrometry and functionally by rheological measurements using a cone and plate rheometer. Since NSP activity is commonly elevated in expectorated patient sputum, these measurements will also be performed in samples that will be treated with protease inhibitors. To gain mechanistic insight, these studies in patient sputum will be complemented by studies of highly differentiated patient-derived primary airway epithelial cell cultures that allow to study the native mucus layer under near physiological condition.⁸ For this purpose, airway cultures will be treated with the different NSPs added at different concentrations to the mucus layer and effects on viscoelastic properties will be determined using several sophisticated imaging-based techniques such as FRAP (fluorescent recovery after photobleaching)⁹ and magnetic microwire rheometry (MMWR)¹⁰ that have been established in our laboratory. By stimulating primary airway cultures with proinflammatory mediators in the presence of NSPs, we can recapitulate the chronic muco-inflammatory environment characteristic of CF and bronchiectasis in vitro. These functional studies of the properties of the native mucus layer will be accompanied by biochemical studies of abundancy and processing of the airway mucins MUC5AC and MUC5B by Western blotting and mass spectrometry, as described for patient sputum samples above, and inflammatory mediators such as proinflammatory cytokines secreted by epithelial cells will be assessed by bead-based multiplexed immunoassay. Thereby, this MD thesis will apply a spectrum of molecular, cellular, and state-of-the-art imaging techniques to patient-derived sputum and airway cultures to tackle pertinent questions related to the pathogenesis and therapy of muco-obstructive lung diseases such as in CF and bronchiectasis, which have emerged as the third leading chronic lung disease worldwide with limited therapeutic options.  

Application details

References