Biology subjects

Boboltz, A.

Publications and source records attributed to Boboltz, A..

10 recordsLinked to original sources

bioRxiv2026

MUC5B and MUC5AC function in combination to regulate mucociliary transport on human airway epithelium

Muco-obstructive lung diseases are characterized by impaired airway clearance and altered mucin composition. MUC5B and MUC5AC are the primary gel-forming mucins in airway mucus, yet how their relative abundance influences the physical and functional properties of mucus remains poorly understood. Here, we investigated how compositional variations in MUC5B and MUC5AC within mucus impact mucociliary transport. Mucus enriched in either MUC5B or MUC5AC was generated using human airway epithelial models depleted for each mucin via CRISPR/Cas9-targeted knockout. Defined mixtures of these mucins were generated at physiologically relevant ratios, for assessment of their microrheological properties and mucociliary transport behavior in differentiated primary human airway epithelial tissue cultures. We found that increasing MUC5AC content reduced network pore size and increased microviscosity, concomitant with reduced mucociliary transport, demonstrating that mucin composition alters the biophysical and functional properties of the mucus barrier. In normal airway tissue cultures with MUC5B-predominant mucus, overlay of MUC5AC on the apical surface impaired mucociliary transport, whereas MUC5B had minimal effect. Conversely, MUC5B supplementation uniquely improved mucociliary transport in IL-13 stimulated cultures exhibiting mucostasis, whereas additional MUC5AC did not alter transport. Together, these findings demonstrate that the ratio of MUC5B to MUC5AC can shape mucus organization at the microscale, which in turn governs mucociliary transport at the tissue scale.

cell biology↗

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↗

bioRxiv2026

Airway mucins function as endogenous inhibitors of neutrophil extracellular traps

Neutrophils recruited to the airways are important for innate lung defense and can release neutrophil extracellular traps (NETs) to capture and eliminate microbes. While NETs are not abundant in healthy airways, uncontrolled NETosis is a known pathological feature and contributor to both chronic and acute respiratory diseases. Prior studies have shown that mucin glycoproteins secreted in the oral cavity and cervicovaginal tract can modulate NETosis, but it remains unknown whether mucins secreted in the respiratory tract influence NET formation. In these studies, we discovered that human airway mucus strongly inhibits NETosis in primary human neutrophils in a sialic acid dependent manner. In comparison, mucus produced by human airway epithelial cells genetically engineered to lack either MUC5B or MUC5AC secreted airway mucins showed a reduced ability to suppress NETosis. To assess how the lung microenvironment in obstructive lung diseases may influence mucus-dependent NET formation, we engineered a synthetic, mucin-laden hydrogel model with physical properties resembling that of mucus in a healthy lung and a disease-affected lung. When neutrophils were cultured on these gel substrates, we found that increasing gel stiffness led to a significantly greater extent of NETosis. Together these data demonstrate a new functional role of airway mucus in modulating neutrophil homeostasis in the respiratory tract and provide evidence that mucus dysfunction in disease can impair its ability to regulate NETosis.

immunology↗

bioRxiv2026

Gene regulatory networks define human airway epithelial cell types and their distinct responses to type I interferon

The human airway epithelium (HAE) is composed of diverse cell types that coordinate essential functions and host defenses. Among these defenses, interferons (IFNs) are central to antiviral programs. However, the gene regulatory networks (GRNs) governing HAE cellular identities and their IFN responses are incompletely defined. At single-cell resolution, we characterized the transcriptomes and accessible chromatin landscapes of HAE cell types, at steady state and following IFN{beta} stimulation. The resulting scRNA-seq and snATAC-seq data informed genome-scale GRN construction and inference of transcriptional circuits underlying cell identities. In response to IFN, we identified a shared transcriptional program across HAE cell types, and an expanded set of interferon-responsive genes exhibiting cell type-associated expression patterns. Cell type-associated transcription factors and chromatin accessibility contribute to distinct IFN-responsive gene expression programs. Together, these data provide a blueprint for molecular regulation of complex HAE responses and a foundation for therapeutic strategies to enhance host antiviral defense.

systems biology↗

bioRxiv2025

Myeloperoxidase impairs mucociliary transport on human airway epithelium

Dampening neutrophil-driven inflammation in the airways remains a challenge in treating cystic fibrosis (CF) lung disease. Myeloperoxidase (MPO) is a neutrophilic enzyme that produces reactive oxygen species and is highly concentrated in CF sputum samples. Greater MPO concentrations have been previously correlated with increased mucus plugging in bronchiectasis, suggesting that the enzyme could impair mucociliary transport. MPO reacts competitively with either thiocyanate (SCN-) or chloride (Cl-) in the airways to catalyze the production of hypothiocyanous acid (HOSCN) or hypochlorous acid (HOCl), respectively. HOCl has proved in prior studies to be extremely cytotoxic, while HOSCN can drastically reduce cytotoxicity. The concentration of SCN- in the airways is largely dependent on transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which is dysfunctional in individuals with CF and causes low SCN- concentrations. CFTR modulator therapies likely raise the concentration of SCN- and enhance the production of HOSCN in the airways. We found that MPO inhibits mucociliary transport in vitro in regardless of SCN- concentrations primarily due to increasing the macromolecular components and effective viscosity of airway surface liquid. The impairment of mucus clearance by MPO was similar to neutrophil elastase (NE), another neutrophilic granular enzyme that damages the host tissues and induces the secretion of mucin proteins by the airway epithelium. Overall, these findings identify MPO as a therapeutic target to resolve deficits in airway clearance function in CF and other related muco-obstructive lung diseases.

cell biology↗

bioRxiv2025

Female sex hormones enhance gonococcal colonization at the endocervix by modifying cervical mucus

Neisseria gonorrhoeae is a human-exclusive pathogen that causes gonorrhea. Gonococci (GC) initiate female infections by colonizing the cervix, which can remain asymptomatic, cause cervicitis, or ascend to the upper female reproductive tract (FRT), leading to severe tissue damage. The FRT undergoes sex hormone-mediated changes during the menstrual cycle, which have long been implicated in the vulnerability to GC infection. One of the major changes is the increase and decrease in the production of the gel-forming mucin MUC5B by the endocervix in response to the level of estradiol (E2). This study examined the impact of sex hormones on GC infection of the human cervix, utilizing a human cervical tissue explant model. Tissue explants were treated without and with E2 alone or in combination with progesterone (E2+P4) to mimic various menstrual cycle phases. Treatment of E2 or E2+P4 enhanced GC colonization at the endocervix exclusively, but did not affect epithelial transmigration. While both treatments increased the number of GC microcolonies, E2+P4 increased GC colony size on the endocervical epithelium. These increases were independent of GC host receptors, carcinoembryonic antigen-related cell adhesion molecules. GC effectively diffused through cervical mucus to interact with the cervical epithelium under all hormone conditions and through mucin hydrogels with different MUC5B and MUC5AC compositions. Mucus gels collected from cervical explants and animal mucin mixtures enhanced GC aggregation in vitro. GC diffusion through mucin-hydrogels and aggregation in the presence of cervical mucus or animal mucins decreased as the MUC5B concentration increased. Our results suggest that female sex hormones promote GC colonization at the human endocervix by modulating the cervical mucus production, regulating womens susceptibility to GC infection, and further reveal the ability of GC to evade the mucus defense barrier for infection. Author SummaryNeisseria gonorrhoeae is a bacterial pathogen that primarily infects the human genital and female reproductive tracts, causing gonorrhea. While this bacterium can infect both men and women, the infection can lead to severe and permanent damage to womens reproductive systems. Currently, the relationship of gonococcal infection with the menstrual cycle is unknown. Here, we utilize a human cervical tissue explant model that mimics gonococcal infection in women to examine the impact of female sex hormones that drive the menstrual cycle on gonococcal infection. We found that estrogen alone or in combination with progesterone enhanced gonococcal colonization, increasing both the number and size of bacterial microcolonies on the cervical luminal surface, through regulating mucus production. Gonococci effectively penetrate through mucus layers to reach cervical epithelial cells and also prefer to aggregate with each other in the presence of mucus. Our results reveal that hormone-regulated mucus production changes the vulnerability of women to gonococcal infection, and that gonococci convert the mucus defense barrier into a colonization facilitator.

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

Synthetic mucus barrier arrays as a nanoparticle formulation screening platform

A mucus gel layer lines the luminal surface of tissues throughout the body to protect them from infectious agents and particulates. As a result, nanoparticle drug delivery systems delivered to these sites may become trapped in mucus and subsequently cleared before they can reach target cells. As such, optimizing the properties of nanoparticle delivery vehicles, such as their surface chemistry and size, is essential to improving their penetration through the mucus barrier. In previous work, we developed a mucin-based hydrogel that has viscoelastic properties like that of native mucus which can be further tailored to mimic specific mucosal tissues and disease states. Using this biomimetic hydrogel system, a 3D-printed array containing synthetic mucus barriers was created that is compatible with a 96-well plate enabling its use as a high-throughput screening platform for nanoparticle drug delivery applications. To validate this system, we evaluated several established design parameters to determine their impact on nanoparticle penetration through synthetic mucus barriers. Consistent with the literature, we found nanoparticles of smaller size and coated with a protective PEG layer more efficiently penetrated through synthetic mucus barriers. In addition, we evaluated a mucolytic (tris (2-carboxyethyl) phosphine, TCEP) for use as a permeation enhancer for mucosal drug delivery. In comparison to N-acetyl cysteine (NAC), we found TCEP significantly improved nanoparticle penetration through a disease-like synthetic mucus barrier. Overall, our results establish a new high-throughput screening approach using synthetic mucus barrier arrays to identify promising nanoparticle formulation strategies for drug delivery to mucosal tissues.

bioengineering↗

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↗

bioRxiv2023

Engineering in vitro models of cystic fibrosis lung disease using neutrophil extracellular trap inspired biomaterials

Cystic fibrosis (CF) is a muco-obstructive lung disease where inflammatory responses due to chronic infection result in the accumulation of neutrophil extracellular traps (NETs) in the airways. NETs are web-like complexes comprised mainly of decondensed chromatin that function to capture and kill bacteria. Prior studies have established excess release of NETs in CF airways increases viscoelasticity of mucus secretions and reduces mucociliary clearance. Despite the pivotal role of NETs in CF disease pathogenesis, current in vitro models of this disease do not account for their contribution. Motivated by this, we developed a new approach to study the pathobiological effects of NETs in CF by combining synthetic NET-like biomaterials, composed of DNA and histones, with an in vitro human airway epithelial cell culture model. To determine the impact of synthetic NETs on airway clearance function, we incorporated synthetic NETs into mucin hydrogels and cell culture derived airway mucus to assess their rheological and transport properties. We found that the addition of synthetic NETs significantly increases mucin hydrogel and native mucus viscoelasticity. As a result, mucociliary transport in vitro was significantly reduced with the addition of mucus containing synthetic NETs. Given the prevalence of bacterial infection in the CF lung, we also evaluated the growth of Pseudomonas aeruginosa in mucus with or without synthetic NETs. We found mucus containing synthetic NETs promoted microcolony growth and prolonged bacterial survival. Together, this work establishes a new biomaterial enabled approach to study innate immunity mediated airway dysfunction in CF.

bioengineering↗