Engle, E. M. | bioRxiv Science

bioRxiv2026

An Inverse Transwell Assay for Airway Mucus Barrier Function Reveals both Virus- and Mucin-Specific Impacts on Infection

Respiratory viruses are a significant cause of morbidity and mortality world-wide and an important trigger of acute exacerbation in chronic lung disease. Secreted airway mucus - a front-line defense system against respiratory virus infection - is largely composed of glycosylated mucins that promote virus trapping via steric and adhesive interactions. Still, the degree to which mucus can trap specific viruses is unclear. Further, mucin expression is altered in chronic lung disease with undefined impacts on host susceptibility to infection. Here, we devised an inverse Transwell assay (ITA) to specifically probe the barrier function of mucus towards infection without the confounding effects of ongoing mucus secretion and transport on viral dynamics. Using the ITA, we assessed the barrier function of human airway epithelial (HAE) culture-derived mucus towards influenza (IAV), rhinovirus (RV), adenovirus, and parainfluenza virus. Results showed differences in mucus penetration efficiency between viruses, with IAV being the most inhibited relative to no mucus controls. Notably, IAV and RV penetration efficiency was similar between normal mucus and mucus sourced from an in-vitro model of asthmatic epithelium. To further explore the role of specific mucins during infection, we employed CRISPR/Cas9-modified HAE cultures lacking either MUC5B or MUC5AC expression. Direct infection in these cultures with IAV yielded higher viral titers compared to control HAE, suggesting both MUC5B and MUC5AC contribute to antiviral defense. Application of mucus harvested from specific mucin-knockout or control HAE in the ITA revealed that while RV penetration was similar across conditions, IAV was more efficient in breaching MUC5AC-depleted gels. Subsequent biophysical analysis of these mucus gels revealed a larger pore structure in the absence of MUC5AC. Together, these data indicate mucus-mediated restriction is virus dependent and highlight the contribution of MUC5AC to mucus structure and antiviral capabilities. Further, they establish the ITA as a tunable platform enabling investigation into mucus penetration by diverse viruses and the effects of altered mucus composition on barrier function. Clinical RelevanceMucus composition is altered in chronic lung disease states and during inflammation with unknown consequences on its barrier function towards respiratory virus infection. Using an inverse Transwell assay, we describe virus-specific kinetics through mucus representative of health and disease and identify a critical role for MUC5AC in defense towards influenza A virus. This work can inform future strategies to improve infection prevention or mucus targeted therapies, and may help explain differences in susceptibility to viral infections across the population.

microbiology↗

bioRxiv2024

Scalable Extraction of Airway Mucins from Porcine Trachea

Mucins are a major component of the innate defense system in the airways and their biological functions are important to consider in pulmonary disease research. However, the available mucus models for basic research relevant to the lung can be difficult to acquire in sufficient quantity to conduct such studies. Here, we present a new strategy to isolate airway mucins from pig trachea at the milligram to gram scale for use in pulmonary disease research. Using this protocol, we were able to isolate mucins with minimal DNA contamination consisting of [~]70% by weight protein. Compared to porcine gastric mucins extracted with the same procedure, the porcine tracheal extract possessed significantly greater O-linked glycoprotein (mucin) content. Particle tracking microrheology was used to evaluate the biophysical properties of porcine trachea mucins. We found porcine tracheal mucins formed a much tighter mesh network and possessed a significantly greater microviscosity compared to lab extracted porcine gastric mucins. In comparison to mucus harvested from human airway tissue cultures, we found porcine tracheal mucins also possessed a greater microviscosity suggesting these mucins can form into a gel-like material at physiological total solids concentrations. These studies establish an accessible means to isolate airway mucins from porcine trachea at large scale for use in pulmonary disease research.

biochemistry↗

bioRxiv2023

Mucus physically restricts influenza A viral particle access to the epithelium

Prior work suggests influenza A virus (IAV) crosses the airway mucus barrier in a sialic acid-dependent manner through the actions of the viral envelope glycoproteins, hemagglutinin and neuraminidase. However, host and viral factors that influence how efficiently mucus traps IAV remain poorly defined. In this work, we assessed how the physicochemical properties of mucus influence its ability to effectively capture IAV using fluorescence video microscopy and multiple particle tracking. We found an airway mucus gel layer must be produced with virus-sized pores to physically constrain IAV. While sialic acid binding by IAV may improve mucus trapping efficiency, sialic acid binding preference was found to have little impact on IAV mobility and the fraction of viral particles expected to penetrate the mucus barrier. Further, we demonstrate synthetic polymeric hydrogels engineered with mucus-like architecture are similarly protective against IAV infection despite their lack of sialic acid decoy receptors. Together, this work provides new insights on mucus barrier function toward IAV with important implications on innate host defense and interspecies transmission.

microbiology↗