(408) Neutrophil Extracellular Traps (NETs) Must Spread and Overlap to Adversely Affect Mucus Rheology.

Background: Hyperconcentrated mucus associated with cystic fibrosis (CF) results from the combination of dehydration of the airway surface liquid (ASL) and mucin hypersecretion. The problems associated with hyperconcentrated mucus, increased infections and inflammation as well as decreased mucociliary clearance, are compounded by the accumulation of DNA within the mucus layer. DNA is a stiff polymer that can entangle with and stiffen the mucin-laden mucus network. The majority of DNA in CF mucus comes from neutrophils, recruited to fight persistent infections, which have undergone programmed necrosis, termed NETosis, to form neutrophil extracellular traps (NETs). We hypothesize that in order for NETs to adversely affect both mucus rheology and clearance, the NETs must spread through the mucus gel matrix such that the released DNA overlaps with adjacent NETs.

Methods: Mucus was harvested from human bronchial epithelium (HBE) cell cultures and prepared to concentrations that mimic healthy (1% organic solids, os), mild (3% os), moderate (5% os), and severe (7% os) CF airway disease. Neutrophils were isolated from HL-60 cells or whole blood, added to mucus, and stimulated with PLA to undergo NETosis. The spreading rates of NETs over a 72-hour period was measured by fluorescence microscopy. The macro- and microrheology properties of mucus with and without NETs was measured over a 72-hour period by cone and plate and particle tracking microrheology (PTMR) respectively.

Results: The spreading rate of NETs is inversely proportional to the concentration of mucus, i.e., NETs spread faster in 1% os, healthy-like mucus, than in high concentrations of mucus associated with CF. Further, at 24 hrs., the complex viscosity (η*) of 1% mucus was increased roughly two-fold while at the 48hr time point, η* was increased 10-fold. This increase is characteristic of the previously reported difference in rheology for mucus harvested from culture compared to CF sputum(1). The increased η* in mild- and severe-like concentration of HBE mucus took > 24 hours to increase 10-fold.

Conclusions: The overlapping of NETs results in significant increase in η* of mucus. The rate at which NETs overlap is dependent on the concentration of mucus, suggesting that for NETs to readily overlap in hyperconcentrated CF mucus, a greater number of neutrophils must be stimulated to undergo NETosis. These results indicate that a threshold concentration of DNA is required for the use of DNA-cleaving agents as therapy to be effective.

Acknowledgements: We are grateful to the Cystic Fibrosis Foundation for their support of this work (HILL19G0 and HILL20Y2-OUT) as well as the NC TraCS Institute (550KR282124).

References: [1] Hill DB, Button B. Establishment of Respiratory Air-Liquid Interface Cultures and Their Use in Studying Mucin Production, Secretion, and Function. Methods Mol Biol. 2012;842:245-58.