Influenza A virus diffusion through mucus gel networks

Abstract

Mucus in the lung plays an essential role as a barrier to infection by viral pathogens such as influenza A virus (IAV). Previous work determined mucin-associated sialic acid acts as a decoy receptor for IAV hemagglutinin (HA) binding and the sialic-acid cleaving enzyme, neuraminidase (NA), facilitates virus passage through mucus. However, it has yet to be fully addressed how the physical structure of the mucus gel influences its barrier function and its ability to trap viruses via glycan mediated interactions to prevent infection. To address this, IAV and nanoparticle diffusion in human airway mucus and mucin-based hydrogels is quantified using fluorescence video microscopy. We find the mobility of IAV in mucus is significantly influenced by the mesh structure of the gel and in contrast to prior reports, these effects likely influence virus passage through mucus gels to a greater extent than HA and NA activity. In addition, an analytical approach is developed to estimate the binding affinity of IAV to the mucus meshwork, yielding dissociation constants in the mM range, indicative of weak IAV-mucus binding. Our results provide important insights on how the adhesive and physical barrier properties of mucus influence the dissemination of IAV within the lung microenvironment.

Fig. 1. Characterization of fluorescent IAV.

a Fluorescence micrograph of purified IAV and 100 nm PS-NP (blue) stained with DiI (orange) and anti-HA antibody (green). DiI and anti-HA co-staining is denoted with white arrows. PS-NP indicated by white circles. Scale bar = 10 µm. b Measured hydrodynamic diameter for muco-inert PS-NP (dotted blue), unlabeled IAV(dashed orange), and DiI-labeled IAV (solid red).

Fig. 2. Diffusion of IAV and nanoparticles with similar diameter in human mucus.

a Representative trajectories of polyethylene glycol (PEG) coated 100 nm polystyrene nanoparticles (PS-NP) and IAV diffusion in mucus. Traces show 10 seconds of motion with a color scale to indicate time. The scale bar represents 0.2 µm. b Box-and-whisker plots of log-based 10 of MSD at τ = 1 s (log₁₀MSD) PS-NP (blue circles) and IAV (red triangles) in mucus samples collected from 10 individual patients are shown. The combined data set for all samples tested (C) is also shown. Patients are numbered in descending order according to the median MSD of PS-NP particles in each sample. c Estimated diffusion time calculated from the average effective diffusivity (D) for PS-NP (blue) and IAV (red) particles to diffuse through a 7 µm thick mucus layer. Dotted line at 30 min (0.5 hours) indicates cutoff to avoid removal due mucociliary clearance. Whiskers are drawn down to the 5th percentile, up to the 95th percentile, and the outliers are shown as points. Data sets (n = 10 patient samples) statistically analyzed with two-tailed Mann-Whitney test: ns = not significant; p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3. Quantifying IAV binding to human mucus.

a Representative PS-NP (blue) and IAV (red) trajectories centered around the average x and y value. IAV trajectory is offset by 0.5 µm in the x and y-direction. Average diameter of trajectories is indicated by black circles and labeled σ. b Radial position from the trajectory center (r) versus time for a single PS-NP (blue) and IAV (red) particle. c Histogram of sampled r for a single PS-NP (blue circles) and IAV (red triangles) particle. d Energy (U) versus radial position (r) for the average PS-NP energy of confinement for one video (〈U_3D,NP〉, blue circles), an individual IAV particle’s energy of confinement (U_3D,IAV, red triangles), and energy of IAV-mucus binding for the same IAV particle (U_B,IAV, green diamonds). Scatter plots of calculated (e) spring constant (k_s), and f dissociation constant (K_3D) for individual IAV particles. In e and f, combined data for all samples is denoted as (C). Line drawn at mean value. For f, IAV with a calculated K_3D ≥ 1000 mM are compiled with these high K_3D indicative of negligible IAV-mucus interactions. Patient numbers correspond with those in Fig. 2.

Fig. 4. The effect of neuraminidase (NA) inhibition on IAV diffusion through and adhesion to human mucus.

a Representative trajectories of PS-NP and IAV untreated and treated with NAI in human mucus. Scale bar = 0.2 µm. b Calculated pore size (ξ) in untreated (white) and NA inhibitor (NAI) treated (zanamivir; 10 µM final concentration; grey) based on PS-NP diffusion in human mucus. c Measured log₁₀MSD_1s for IAV diffusion in untreated and NAI treated human mucus. Box and whisker plots of d average trajectory diameter (σ) and e calculated dissociation constants (K_3D) with and without NAI treatment. IAV with a calculated K_3D ≥ 1000 mM are compiled with these high K_3D indicative of negligible IAV-mucus interactions. Whiskers are drawn down to the 5th percentile, up to the 95th percentile, and outliers are plotted as points. Data sets (n = 2 patient samples) statistically analyzed with two-tailed Mann-Whitney test: ns = not significant; p > 0.05, *p < 0.05, **p < 0.01. Patient numbers correspond with those in Figs. 2 and 3.

Fig. 5. Impact of reducing disulfide bonds within mucus gels on IAV diffusion and adhesion.

a Representative trajectories of PS-NP and IAV diffusion in mucus with and without DTT treatment. Scale bar = 0.2 µm. b Calculated mucus gel pore size (ξ) based on PS-NP diffusion in untreated (white) and DTT treated (grey) mucus (5 mM final concentration). c Measured log₁₀MSD_1s for IAV in untreated and DTT-treated mucus. Box and whisker plots of d average trajectory diameter (σ), and e calculated dissociation constant (K_3D) for individual IAV particles with and without DTT treatment. For e, IAV with a calculated K_3D ≥ 1000 mM are compiled with these high K_3D indicative of negligible IAV-mucus interactions. Whiskers are drawn down to the 5th percentile, up to the 95th percentile, and outliers are plotted as points. Data sets (n = 2 patient samples) statistically analyzed with two-tailed Mann-Whitney test: ns = not significant; p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Patient numbers correspond with those in Figs. 2–4.

Fig. 6. Modulation of crosslinking density effects IAV diffusion through synthetic mucus.

a Calculated mucus gel pore size (ξ) based on PS-NP diffusion in synthetic mucus. b Measured log₁₀MSD_1s for IAV in synthetic mucus. c Median gel pore size (ξ) calculated from the PS-NP compared to the median log₁₀ MSD value for the IAV particles in synthetic mucus of varying crosslinking densities, R² = 0.9435. d Calculated dissociation constant (K_3D) for individual IAV particles, the line at mean value for each data set. IAV with a calculated K_3D ≥ 1000 mM are compiled with these high K_3D indicative of negligible IAV-mucus interactions. Whiskers are drawn down to the 5th percentile, up to the 95th percentile, and outliers are plotted as points. Data sets (n = 3 synthetic hydrogels per group) statistically analyzed with Kruskal-Wallis test and Dunn’s test for multiple comparisons: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Color and symbol indicative of comparison group: 0%, light blue circles; 1%, orange squares; 2%, green triangles; 3%, red diamonds; 4%, dark blue hexagons.