Mucin Networks—A Mechanical Fuse that Protects Epithelial Cell Surfaces During Shear

Mucin dysfunction is implicated in many infectious and inflammatory diseases of the respiratory tract, the digestive tract, the reproductive tract, cancer, and the ocular surfaces. All moist epithelial surfaces are protected by a secreted layer of mucus, which is a low yield strength, high-water content, network of mucin glycoproteins. As a part of the innate immune system, these mucin networks provide hydration, protection, and help to exclude pathogens providing a reservoir for antimicrobial molecules. Although widely described as a “slippery” barrier on epithelial surfaces, the mechanics and self-healing properties of mucin network dynamics and their role in epithelial cell friction and shear are poorly understood. Experiments on self-mated epithelial cell surfaces at physiological contact pressures reveal shear stress limits of a mucin network, made of purified MUC2 and membrane mucins including MUC1 and MUC4¹. Micro-rheological measurements using magnetic tweezers provide insights into the network rheology, and molecular biology techniques reveal thresholds for the production of proinflammatory cytokines and apoptosis.<br/>In this work, hemi-spherical polyacrylamide hydrogel probes with a 2 mm radius of curvature and a shell thickness of 250 µm were used to maintain constant contact pressures ~ 1 kPa across the epithelial cell interfaces. The poly-acrylamide gels (water content ~ 87.5 %) were equilibrated in Keratinocyte Growth Media (KGM-Gold™). Bioconjugation of polyacrylamide with collagen type-I (0.4 mg/ml) was performed using 1mM Sulfo-SANPAH². Glass-bottomed culture-dishes were coated with fibronectin for cell adhesion. Human Corneal Epithelial Cells (hTCEpi) were cultured for ~ 2 days to form a monolayer with &gt;90% confluency on both the probes and the culture dishes. In situ biotribology experiments were performed at 37±0.2 °C, 5 % CO₂, and ~100 % relative humidity. A normal load of 400 µN was applied between the two surfaces. Friction force measurements were recorded on a custom fabricated biotribometer³ with a 3 mm stroke length, and a sliding speed of 1 mm/s. Contact pressure was calculated by from the measured contact area and applied force. All microscopy was performed in situ on a Nikon A1R confocal microscope. Purified MUC2 at 5 wt% in KGM-Gold™ was continuously flowed to the sample to induce transport conditions reducing inflammation and cell death. The magnetic tweezers were used for micro-rheology following the design of Lammerding⁴ with 6 µm superparamagnetic fluorescent bead.<br/>Experiments with the reciprocal sliding of two hTCEpi epithelial surfaces revealed the upregulation and production of proinflammatory cytokines at shear stresses ≥ 40 Pa, and almost complete apoptosis and cell removal at 100 Pa. Experiments on self-mated epithelial cells showed friction coefficients that monotonically increased to µ&gt;0.2 over 300 cycles without the addition of soluble MUC2. A 5 %w addition of MUC2 maintained low shear stress (t~40Pa) for over 300 cycles, and enhanced mucin protection. When transport conditions were induced, damage rate was significantly reduced, suggesting faster mucin-gel-self-healing phenomenon under this condition emphasizing the role of the self-healing time for appropriate mucin protection under shear. Magnetic-tweezers with microbeads revealed a definitive yield-stress and elasticity of the mucin network consistent with the measured shear stresses during sliding.<br/>In this model, the fragile mucin network reduced shear stress, friction, and friction-induced damage, and it was found to self-heal faster under transport conditions.<br/>¹Pedro DI, et al. Tribol Lett 69, 155 (2021) ²Wang YL and Pelham RJ Jr. (1998). Methods Enzymol. 298:489-496 ³Urueña JM, et al. Tribol Lett 66, 141 (2018) ⁴Rich JP, et al. (2011). Soft Matter 7:9933–9943