(447) Mucociliary Interactions and the Vertical Clearance Assay 2.0

Background: The accumulation of hyperconcentrated mucus along the airways of cystic fibrosis (CF) patients results in failed mucociliary clearance (MCC), which promotes infection and inflammation and indicates the progression of CF lung disease. While considerable efforts in the community focus more on developing therapies to increase MCC by decreasing mucus rheology, little is known about the underlying mucociliary adhesions that facilitate or impede MCC under healthy or pathological conditions. Here, we present an assay for measuring the effectiveness of mucus transport against gravity when particulate-sized microspheres, surface-coated with constituent mucins, polyethylene glycol (PEG), lectins, or lectin-like proteins. To further elucidate the effect of specific adhesive interactions with beating cilia, we study the effect of interfering agents, which in the future could include candidate muco-corrective therapies. Combining these studies with our previous efforts to quantify adhesive forces of the mucus-mucus interaction reveals more of the role adhesion plays in MCC.

Methods: We covalently bound human bronchial epithelial (HBE) mucus to the surfaces of fluorescent, magnetic 24-micron diameter beads. For control conditions, we used similar attachment methods to couple beads to PEG or lectins with known adhesion constants, which specifically target common glycans found on mucins, including GalNAc, GlcNAc, and sialic acid. These beads are then distributed across confluent, fully ciliated HBE cell cultures and their clearance/transport monitored with our tilting microscope set at 0° or against gravity at 30° (0.5 G). For adhesion measurements, magnetic forces are applied to the identical bead-types until particles detach.

Results: In the adhesion assay, PEG-coated beads, serving as a negative control, showed the lowest detachment force (0.1 nN) from a mucus-coated substrate. When checked in the vertical clearance assay, the same PEG beads show negligible transport at either angle. In contrast, beads functionalized with HBE mucus required more force to detach the bead (~ 10 nN) in the adhesion assay and cleared with ease against a 30° tilt (0.5 G) in the vertical clearance assay, their velocities ranging from 3 to 25 µm/s.

Conclusions: These data show mucus-mucus adhesive interactions may facilitate or even govern the success of mucociliary transport. Further studies on isolated mucins and candidate cross-linking mucociliary proteins are needed to understand more precisely the nature of the interactions described here. Next steps include adding free glycans to the beads after and prior to dosing onto cell culture to mimic both rescue and prophylactic treatments, respectively.

Acknowledgements: We are grateful to CFF for their support of this research (SUPERF20G0).

References: