(319f) Native Airway Mucus Rheology in Health and Patients with Cystic Fibrosis Having Positive or Negative Microbial Culture

In health, airway mucus is composed primarily of long, polymeric mucin proteins as well as some globular proteins and cell debris. Airway dehydration in cystic fibrosis (CF) gives rise to hyperconcentrated, viscoelastic mucus that is difficult for the mucociliary escalator to clear. This unclearable mucus traps pathogens in the lungs leading to inflammation and airway destruction. Because disease onset occurs first in the distal airways, sampling of airway mucus from infant and adolescent patient lungs often relies on the collection of Bronchioalveolar Lavage Fluid (BALF). While the lavage inherently dilutes the airway surface milieu, we show that roughly half (53%) of the mucus-forming mucin biopolymers in CF are present in the airways in insoluble “flakes” or “rafts” that are observable via microscopy. Because such a proportion of airway mucins are insoluble, we hypothesize that the characterization of these flakes will yield important advances in understanding the etiology of CF lung disease. Flakes in BALF present a number of research obstacles mostly arising from the difficulties associated with characterizing the biophysics of heterogeneous samples. To address this, we employ a combination of particle tracking microrheology (PTMR) and Gaussian Mixture Modeling (GMM) in order to isolate and study the biophysical properties and biomedical implications of these flakes.

We collected BALF from 29 patients with CF (age 10.5±4.4 years) and 6 healthy adults. We added 1µm fluorescent polystyrene beads to the samples to probe flake rheology via PTMR. The local complex viscosity (η*, Pa∙s) of BALF samples was computed from mean squared displacement of each individual bead and the generalized Stokes-Einstein equation. Bulk rheology was assessed in the CF samples using a cone and plate rheometer. Samples were scanned for flakes and videos of bead thermal motion in and near the mucus flakes were recorded at 60fps for 30s. Rheological signals were clustered into “watery” and mucus components via GMM.

Comparison between η* measurements from bulk rheology and the ensemble mean of all beads in each CF patient sample revealed a moderate correlation (r=0.42, p<0.05). However, comparison of only the mucus component η* from PTMR enhanced the correlation (r=0.60, p<0.01), indicating that bulk rheology is more reflective of the thickest components of a heterogeneous sample. Comparing CF samples to healthy adults, weighted η* (i.e. the sum of the mean η* in the water and mucus components multiplied by their mixing proportion, p_m) was increased in CF (log₁₀(η*)=-0.96) vs. non-CF (-2.5, p<0.01), but the mean of the mucus component was similar. The span of η* in the mucus component was nearly 3 logs larger in CF samples, and mucin concentration was increased 3,000-fold in CF BALF relative to control samples. Furthermore, the GMM mixing proportion (p_m) of mucus was increased in CF (p_m=0.83) vs. non-CF (p_m=0.27) samples (p<0.01). Interestingly, neither hyperconcentration nor hypersecretion was observed in the subset (n=5) of patients with CF whose BALF was negative for microbial culture. Culture negative samples had weighted log₁₀(η*) = -2.8 and p_m = 0.23. The culture positive values of weighted log₁₀(η*) = -0.75 and p_m = 0.85 were significantly increased versus the healthy controls (p<0.01, both) and the culture negative patients with CF (p<0.01, both).

Through a combination of PTMR and Gaussian Mixture Modeling we find that while mucus flakes arise in health and in CF, hyperconcentration only occurs in CF mucus flakes, and orders of magnitude more mucus is found in CF samples. Importantly, increased rheological signal is only found in CF samples that were positive for microbial culture, indicating that infection may be linked to hyperconcentration and hypersecretion.