Mucus as a Physicochemical Barrier to Influenza A Diffusion and Infection.

Introduction: The mucosal barrier consists of a viscous mucus gel layer that serves as the first line of defense against inhaled pathogens, such as Influenza A Virus (IAV). This barrier is composed of mucin glycoproteins that function to trap inhaled substances for removal. Despite this barrier, respiratory viral infections continue to cause a large number of deaths each year.(1) IAV infects the airway epithelium by initiating receptor-mediated endocytosis through the glycan receptor, sialic acid (Sia).(2) Sia is present not only in the epithelium but also in secreted mucins and as such IAV must navigate between binding to Sia present in mucins and to Sia present on epithelial cells.(2) IAV has two envelope proteins that function to interact with Sia and allow for continual binding and release: hemagglutinin (HA) and neuraminidase (NA). HA binds to Sia for eventual cell entry; whereas NA cleaves Sia to allow for the release of IAV-Sia associations and continual diffusion of IAV.(2) Historically, IAV trapping in the mucosal barrier has been attributed to Sia-IAV associations allowing time for mucosal ciliary clearance of the particulate.(2) This study illuminates the contribution of steric hindrance, resulting from disulfide bond formation and entanglement within the mucus, as a second factor hindering IAV penetration through the mucosal barrier.(3) This study reveals the dual IAV trapping mechanisms employed by the mucosal network resulting from both the physically restrictive network and Sia concentration.(3)

1. CDC. Respiratory Virus Updates (2023).
2. De Vries et al., Trends in Microbiology (2019).
3. Kaler et. al., BioRxiv (2023).

Materials and

Methods: Three human airway epithelial cell lines were cultured under an air-liquid interface until full epithelial cell differentiation: Normal Human Bronchial Epithelial cells (NHBE), Human Lung Adenocarcinoma Epithelial cells (Calu-3), and Immortalized Human Bronchial Epithelial cells (BCi-NS1.1). Mucus was collected and concentrated from fully differentiated cultures for characterization of disulfide bond concentration, mucin content, and sialic acid concentration. To understand how these mucosal properties impact IAV diffusion, A/Udorn/307/72, an H3N2 IAV virus, (gifted by Dr. Margaret A. Scull, University of Maryland), and size control muco-inert nanoparticles (NP) were added to each mucus source and tracked using video microscopy for later multiple particle tracking (MPT). These tracked trajectories were utilized to calculate the mean square displacement of each particle as well as the mucosal pore size. To explore the role of sialic acid on IAV diffusion, an exogenous neuraminidase from Arthobacter Ureafaciens was preincubated with NHBE mucus to deplete sialic acid prior to analyzing the diffusion of Udorn and NPs through MPT. To isolate the contribution of the physical architecture of the mucosal network on IAV penetration and infection, a glycan-free artificial polyethylene glycol hydrogel that emulates the physical architecture of native mucus was utilized (2% polyethylene glycol orthopyridyl disulfide or PEG-OPSS; and 2% polyethylene glycol thiol or PEG-SH). IAV diffusion in this synthetic gel was analyzed through MPT and the protective properties of this gel was measured through an IAV infection study in which this gel was applied apically on cultures in place of mucus prior to IAV challenging.

Results, Conclusions, and Discussions: Historically, mucosal trapping of IAV has been primarily attributed to the amount of sialic acid present that can act as a “decoy” receptor to IAV. There has been minimal discussion and exploration of other factors contributing to IAV trapping. Our work has revealed that inhibition of IAV diffusion as well as infection is caused by the duality of both chemical associations between IAV and the environment as well as steric hindrance provided by the mucosal network itself. Of the three mucus sources, the NHBE-derived mucus contained the highest concentration of Sia, and thus provided an environment with highest potential for IAV-Sia associations (Fig 1B). Despite the high Sia content present in the NHBE-derived mucus, the BCi-derived mucus provided an environment in which IAV had the lowest diffusion among the three mucosal sources (Fig. 1A, Fig. 1B & Fig. 1C). This mucosal source contained the highest percentage of pores on the size scale of IAV providing an environment in which IAV can become trapped in a restrictive environment (Fig. 1C & Fig. 1D). This revealed the importance of the size of the physical mucosal network on hindering IAV diffusion. It was further revealed that IAV has increased diffusion in sialic acid-depleted mucus resulting from the lack of IAV-Sia association, affirming the importance of IAV-Sia associations in addition to the physical mucosal network in dictating IAV diffusion. The protective abilities of mucus acting as only a physical barrier to infection was demonstrated to be comparable to the protective abilities of native mucus in an IAV infection study using transplanted glycan-free synthetic PEG gel (Fig. 1E). Overall, this study examined the impact of the physicochemical properties of three separate mucosal sources on IAV diffusion and revealed the importance of both the mucosal architecture as well as sialic acid content providing a barrier to diffusion and infection.

Acknowledgements (Optional): This project was funded by the Burroughs Welcome Fund CASI, the Cystic Fibrosis Foundation (BOBOLT23H0), the National Institutes of Health (R21 AI142050, R01 HL160540, R01 HL151840, & T32 AI089621), and the National Science Foundation (CBET 2129624).