Engineered Models of Neutrophil Extracellular Trap Mediated Dysfunction in Cystic Fibrosis Airways

Introduction::
One of the most important aspects in developing new inhaled therapeutics for cystic fibrosis (CF) is to accurately model the airway mucus barrier properties. Traditionally, human airway epithelial (HAE) cells cultured at air-liquid interface have been used for in vitro studies. However, one of the key aspects currently missing from in vitro models of the CF airway is the presence of neutrophil extracellular traps (NETs) in the mucus. NETs are webbed complexes composed of a decondensed chromatin scaffold decorated with antimicrobial granular proteins.^1,2 Neutrophils are the most abundant immune cells in the CF airway, responding to bacteria that grow rampantly in the mucus.³ NET release is an innate immune response to trap and kill pathogens.⁴ However, the excess release of NETs in the airway is extremely detrimental, causing increased mucus viscoelasticity, often promoting bacterial biofilm formation, and slowing mucus clearance by cilia on the surface of airway epithelial cells, known as mucociliary transport.⁴ In CF patients, declining lung function is highly correlated with increasing NET concentrations in the mucus.⁵ Despite the pivotal role of NETs in CF pathogenesis and mucosal inflammation, current models of the airway mucus barrier do not account for their presence. Therefore, we are developing systems to model the effects of NETs in mucus both ex vivo using a synthetic mucus biomaterial and in vitro using HAE cells cultured at air-liquid interface. These models will be useful in applications such as assessing the efficacy of new inhaled therapeutics and studying mucosal host-pathogen interactions.




Materials and Methods::
We adapted a previous protocol to create a biomaterial mimicking the chromatin scaffold of NETs, called “microwebs”.⁶ Microwebs are synthesized by combining DNA and histones, then sonicating to create a suspension.⁶Using microwebs avoids the loss of proteins that can occur while purifying NETs.⁶ Microwebs have the added advantage of using readily available materials and confers flexibility to adjust the concentrations used in the models to mimic a wide range of CF mucus conditions. We used these microwebs in a novel application to recapitulate the effects of NETs in the airway mucosal environment. We devised a method of adding microwebs into a hydrogel composed of mucins, the gel-forming proteins in mucus. The mucin-microweb hydrogels were characterized using macrorheology and microrheoloy. Microrheology involves tracking the diffusion of nanoparticles within hydrogels to obtain the log₁₀ of the mean squared displacement at 1 second (log₁₀[MSD_1s]). A lower value of log₁₀[MSD_1s] indicates that nanoparticles are confined by a denser hydrogel network. Hydrogels were also treated with DNase to determine how microweb degradation affects the material.









We also created an in vitro model of the CF airway using HAE cells cultured at air-liquid interface by incorporating microwebs into the apical mucosal barrier. We measured the velocity of mucociliary transport by tracking the movement of fluorescent microspheres on the surface of the cultures. The cultures were treated with DNase to the observe effects on mucociliary transport. Finally, we are currently studying the interactions of the common CF bacteria Pseudomonas aeruginosa with our models incorporating microwebs.




Results, Conclusions, and Discussions::
The addition of microwebs to the synthetic mucus hydrogel increased the elastic and viscous moduli compared to mucin-only hydrogels with the same percentage of solids. We found that the log₁₀[MSD_1s] was significantly reduced in the mucin-microweb and mucin-DNA hydrogels compared to the mucin-histone and mucin-only hydrogels (figure 1a, b). Therefore, DNA is the component of microwebs (and NETs) contributing the most to microstructural alterations. When treated with DNase, the log₁₀[MSD_1s] of the mucin-microwebs hydrogel increased significantly.









Next, we constructed the in vitro model incorporating microwebs into the mucus layer of the HAE cells and assessed the impacts on mucociliary transport. We found that the velocity of mucociliary transport was significantly reduced with the addition of microwebs to the mucus layer of airway epithelial cells (figure 1c, d). Transport was restored with the addition of DNase to the mucus containing the microwebs. The results of the bacterial studies are in progress.









NETs are extremely important within the CF airway environment and should be accounted for in preclinical models for inhaled drug delivery studies and studies of host-pathogen interactions. We have engineered a novel in vitro HAE cell model and ex vivo synthetic mucus hydrogel model capable of recapitulating the biophysical effects of NETs on mucus viscoelasticity and microstructure, and mucociliary transport. We also demonstrated the ability of each of the models to respond to DNase treatment applied at a physiologically relevant concentration. DNase is inhaled by over 91% of CF patients, making this a clinically relevant aspect of our characterization.⁷ The results of the bacterial studies are currently in progress.




Acknowledgements (Optional): : This work is supported by the Cystic Fibrosis Foundation Student Traineeship Award BOBOLT23H0.

References (Optional): :
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