Characterization of the structural properties of mucus model systems and human mucus in health and disease

Mucus plays an important role in the pathophysiology of diseases, such as cystic fibrosis and Crohn’s disease. To develop effective treatment options for mucus-related diseases, validated testing procedures that account for variability within patient groups are required. To address this need, this thesis aims to understand mucus function and dysfunction by characterizing the structural properties of mucus model systems and human mucus in healthy and diseased states. To achieve this, experimental methods were optimized before their application to native human mucus samples. Selecting appropriate model systems was essential, and these systems underwent comprehensive characterization to assess their suitability and properties for the intended studies. Different methods such as macrorheology, supported by microrheology, magnetic microwire rheology (MMWR), and scanning electron microscopy (SEM) were applied to assess the viscoelastic properties of mucus on both macro- and microscopic scales, as well as evaluating the internal mucus network structure. The mucus model systems included bovine submaxillary mucin (BSM) and mucus from primary human nasal epithelial cells (phNECs) cultured on an air-liquid interface (ALI). Human mucus samples consisted of native sputum from healthy individuals and patients with cystic fibrosis (CF), as well as native mucus from the terminal ileum of both healthy individuals and patients with Crohn’s disease (CD). Significantly higher viscoelastic moduli for CF sputum compared to healthy sputum were identified by macrorheological measurements of sputum from patients with CF and healthy individuals utilizing a protocol with optimized environmental conditions. The rheological study of increasing concentrations of BSM showed a notable increase in viscoelasticity with increasing mucin concentrations, especially under varying ionic conditions, which was accurately represented by a fractional Kelvin-Voigt model. A comprehensive proteomic analysis revealed MUC19 as the dominant mucin in BSM. The concentration dependence of the BSM viscoelastic properties was also confirmed using microrheology through fluorescence correlation spectroscopy (FCS). By applying the generalized Stokes-Einstein relation and a shell model of local viscosities surrounding the tracer particles, the FCS microrheology data were successfully compared to bulk rheological measurements. The investigation of the viscoelastic behavior of the thin mucus layer from donor-derived phNECs was non-destructively achieved through in situ measurements using MMWR. A substantial increase in the viscosity of the mucus compared to the untreated samples was observed upon the addition of a defined concentration of calcium and exposure to a mildly acidic pH. The viscoelastic properties of native human mucus from the terminal ileum of healthy individuals and patients with CD were investigated using the aforementioned macrorheological protocol. The results revealed significantly higher viscoelastic moduli and reduced mesh size in the mucus samples from patients with CD compared to those from healthy individuals. This work provides valuable insights into the physicochemical properties of human mucus from various sources. The results offer a scientific foundation for better understanding the studied diseases in order to develop effective patient therapies. The experimental methods were evaluated for their suitability by using mucus model systems, and the characteristics of these systems were critically assessed in terms of their relevance and reliability as model systems. Now, the validated methods can be more extensively used to explore human mucus samples.