MUC5B mobilizes and MUC5AC spatially aligns mucociliary transport on human airway epithelium

Abstract

Secreted mucus is a frontline defense against respiratory infection, enabling the capture and swift removal of infectious or irritating agents from the lungs. Airway mucus is composed of two mucins: mucin 5B (MUC5B) and 5AC (MUC5AC). Together, they form a hydrogel that can be actively transported by cilia along the airway surface. In chronic respiratory diseases, abnormal expression of these mucins is directly implicated in dysfunctional mucus clearance. Yet, the role of each mucin in supporting normal mucus transport remains unclear. Here, we generate human airway epithelial tissue cultures deficient in either MUC5B or MUC5AC to understand their individual contributions to mucus transport. We find that MUC5B and MUC5AC deficiency results in impaired and discoordinated mucociliary transport, respectively, demonstrating the importance of each mucin to airway clearance.

Fig. 1.. Generation and validation of MUC5B/AC-KO cultures.

(A) Overview of the MUC5B/MUC5AC gene-targeting approach via lentivirus-mediated delivery of sgRNA and CRISPR-Cas9. Transduced and sorted cells were differentiated at air-liquid interface (ALI). Once fully differentiated, the mucus gel was collected by apically washing cultures. HEK293T, human embryonic kidney–293T; FACS, fluorescence-activated cell sorting; EGFP, green fluorescent protein. (B) TEER measurements in fully differentiated MUC5B/MUC5AC KO cultures. KO1 corresponds to BCi-NS1.1 cells transduced with sgRNA1, and KO2 corresponds to cells transduced with sgRNA2. (n = 4 biological replicates). No statistically significant differences between groups as assessed by one-way analysis of variance (ANOVA). (C) Immunofluorescence staining for MUC5B (green) and MUC5AC (orange) in MUC5B/AC-KO cultures. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Representative two-dimensional (2D) projections from z-stack images for each secreted mucin are shown. Cultures were washed with 10 mM dithiothreitol before fixing and staining for intracellular mucins. Scale bar, 10 μm. (D) Western blot analysis of mucus gels collected from apical washes of fully differentiated control and KO cultures. Samples were separated by electrophoresis (4 to 20% tris-glycine gel, reducing conditions) and detected by immunoblot for MUC5B and MUC5AC.

Fig. 2.. Viscoelastic properties of mucus produced from MUC5B/AC-KO cultures.

(A) Elastic and viscous moduli (G′ and G″) at ω = 1 rad/s measured using macrorheology. Moduli show G′-dominant behavior, indicative of a viscoelastic gel. (B) Complex viscosity measured using macrorheology. (C) Solids concentration (%, w/v) in pooled mucus samples from MUC5B/AC-KO and control cultures. (A to C) Individual data points represent each independent measurement. (D) Airway mucus acts as a vehicle for pathogen clearance, providing the lungs with a robust defense mechanism. Mucus also has a microstructural network that can immobilize diffusing particles. (E) Diffusion of 100-nm polyethylene glycol (PEG)–coated nanoparticles (PEG-NP) in mucus samples from KO cultures as measured by log₁₀ MSD at time lag of 1 s (MSD_1s). Whiskers are drawn down to the 5th percentile, up to the 95th percentile, and the outliers are shown as individual points. (F) Estimated pore sizes of mucus samples. (G to J) Frequency distribution of log₁₀(MSD_1s) for 100 nm PEG-NP diffusion in mucus harvested from control cultures compared to (G) MUC5B-KO1, (H) MUC5B-KO2, (I) MUC5AC-KO1, and (J) MUC5AC-KO2 cultures. (n = 3 biological replicates). Significance was determined by one-way ANOVA (A to C) and Kruskal-Wallis test with Dunn’s correction (E and F). ****P < 0.0001.

Fig. 3.. MCC dynamics of MUC5B/AC-KO cultures.

(A) Mucociliary transport rate is measured by apically applying 2-μm microspheres to fully differentiated cultures and measuring particle displacement over time. (B) Images of microsphere trajectories over time. Trajectories show 20 s of motion with a color scale that indicates elapsed time. Scale bar, 100 μm. (C) Mucociliary transport rates of MUC5B/AC-KO cultures. (D) Alignment in microsphere trajectory angle was calculated as Δθ = θ_Average – θ_i, where θ_Average is the average flow direction and θ_i is individual microsphere trajectory angle. (E to G) Frequency distribution of Δθ in (E) control, (F) MUC5AC-KO1, and (G) MUC5AC-KO2 cultures. Percentages refer to percent of particles falling within 10° to −10° of Δθ, which is indicated by the arrows. Transport rates and directionality were determined from 7 cultures (n = 7). Individual data points represent median transport rate of microspheres determined from three fields per culture. (H) Cilia beat frequencies of MUC5B/AC KO cultures. Beat frequency was determined from six cultures (n = 6). Individual data points represent average beat frequency determined from nine fields per culture. (I) Immunofluorescence staining for acetylated α-tubulin in MUC5B/AC-KO cultures. Micrographs show 2D projections of acquired z-stack images. Scale bar, 10 μm. (J) Quantification of +tubulin % area. Significance was determined by one-way ANOVA (C, H, and J). **P < 0.05 and ****P < 0.0001.

Fig. 4.. MCC dynamics after MUC5B and MUC5AC supplementation in KO cultures.

(A) Exogenous mucus collected from KO cultures was transplanted onto fully differentiated KO cultures to supplement with additional and/or absent MUC5B/AC. After transplantation, cultures were incubated for 12 hours to allow equilibration. (B) Mucociliary transport rates after transplanting MUC5AC and MUC5B on MUC5B-KO1 culture. Transport rates of transplanted mucus were determined from three cultures (n = 3). (C) Images of microsphere trajectories on MUC5B-KO1 cultures with transplanted mucus. Scale bar, 100 μm. (D) Frequency distribution of Δθ on MUC5B-KO1 cultures supplemented with MUC5B. (E) Mucociliary transport rates after transplanting MUC5AC and MUC5B on MUC5AC-KO2 cultures. (F) Images of microsphere trajectories on MUC5AC-KO2 cultures with transplanted mucus. Scale bars, 100 μm. (G) Frequency distribution of Δθ on MUC5AC-KO2 cultures supplemented with MUC5AC. (C and F) Trajectories show 20 s of motion with a color scale that indicates elapsed time. Individual data points represent median transport rate of microspheres determined from three fields per culture. Percentages refer to percent of particles falling within 10° to −10° of Δθ, which is indicated by the arrows. Significance was determined by one-way ANOVA (B and E). **P < 0.05.