Viral lung infections are a major cause of morbidity and mortality worldwide. Despite significant advances in vaccines and antivirals, there remains a tremendous need for broadly applicable treatments that can be utilized across viral infections. Before infecting epithelial cells, viruses interact with the epithelial glycocalyx, which contains high-molecular weight hyaluronan (HMWHA), a glycosaminoglycan that has beneficial effects in lung injury. In this study, we sought to determine the role of HMWHA in viral pneumonia. We infected mice with influenza or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and treated them with prophylactic or therapeutic doses of HMWHA or saline control. We performed in vitro experiments of infection with viruses of respiratory and nonrespiratory human and animal cells and evaluated the effect of HMWHA on infection. We analyzed existing databases for expression of hyaluronan and the transcription factor E2F1. Finally, we performed a clinical trial with HMWHA in patients with severe coronavirus disease (COVID-19). Exogenously applied HMWHA improved survival in SARS-CoV-2 and influenza infection in mice by ameliorating inflammation through the inhibition of E2F1. In a clinical study, inhaled HMWHA improved outcomes in patients with severe COVID-19. Furthermore, airway epithelia naturally express HMWHA, which is induced during viral infection and prevents infection through the macromolecular crowding of viruses. Our data provide a mechanistic justification for the use of HMWHA as a broadly effective prophylactic and therapeutic agent in viral airway infection.

BACKGROUND:Eosinophilic esophagitis is associated with epithelial barrier dysfunction. Epidemiologic studies suggest environmental factors promote disease pathogenesis. The common household detergent sodium dodecyl sulfate (SDS) induces epithelial barrier dysfunction and eosinophilic inflammation in mice. We hypothesized that acute SDS exposure would compromise the esophageal mucosal barrier in humans. METHODS:Healthy adults brushed with 2 g of an SDS-containing toothpaste. Esophageal impedance was measured continuously pre-/post-toothbrushing to assess the effect of toothpaste on epithelial barrier function. SDS was measured in saliva using a methylene blue anionic substances assay. Participants completed 1-h esophageal string tests (ESTs) pre-/post-toothbrushing and protein isolates from EST eluates from the proximal and distal esophagus were analyzed by tandem mass tag mass spectrometry. RESULTS:Ten of twelve subjects completed the clinical study. SDS in the initial expectorate measured immediately after toothbrushing varied markedly (median: 321.40 μg/mL; range: 169.90-1243.00 μg/mL) and was detectable in saliva in 80% of subjects (median: 9.55 μg/mL; range: 0-123.40 μg/mL) at 60 min post toothbrushing. Esophageal mucosal impedance decreased within 30 min of toothbrushing (p < 0.01). Toothbrushing increased salivary viscosity and proteins associated with mucosal defense in EST eluates. The tight junction pathway was downregulated while the salivary secretion and complement and coagulation cascades pathways were upregulated in the proximal esophagus. CONCLUSION:In healthy individuals, SDS remained detectable in saliva for a prolonged period after toothbrushing at concentrations previously shown to disrupt esophageal epithelial barrier function in vitro. Toothbrushing with an SDS-containing toothpaste was associated with reduced esophageal mucosal impedance, altered salivary properties, and activated mucosal immune responses.

BACKGROUND:Intentional destruction of ultrasound microbubble (MB) contrast agents by induced cavitation is a well-established technique in contrast imaging applications and may translate to pulmonary imaging. However, the safety of exposing human bronchial epithelial (hBE) cells to an inhalable contrast agent with intentional MB destruction is not known. METHODS:We conducted an in vitro evaluation by exposing hBE cell cultures to a mucus-targeting, cationic MB contrast agent. A total of 12 hBE cell cultures, from 3 healthy donors (4 cultures each), were exposed to 4 experimental conditions: (i) No-contrast imaging with no MBs, using cadence contrast pulse sequencing followed by a MB destruction pulse exposure (mechanical index [MI] ramped from 0.14 to 1.90); (ii) contrast imaging using MBs with contrast pulse sequencing at low MI (MI maintained at 0.14); (iii) contrast imaging with intentional MB destruction by MB destruction pulse exposure; and (iv) Triton-X (positive control, inducing cellular death). Cell culture viability was evaluated pre- and 24 hours post-exposure by quantifying percent ciliated area and ciliary beat frequency. RESULTS:The contrast signal was visible for all cultures administered MBs, with signal loss only apparent after the MB destruction pulse. Culture viability (percent ciliated area) was comparable at pre- and post-exposure, for all imaging conditions (coefficient of variation range, 3%-8%), with no decline in ciliary beat frequency. No ciliated area persisted after exposure to Triton-X. CONCLUSIONS:Intentional MB destruction induced by a high MI does not induce cellular death to the respiratory epithelium. These findings support further development of an inhaled MB contrast agent for pulmonary imaging applications.

The design and implementation of biomedical devices for both diagnostic and direct medical applications have revolutionized patient care, paving the way for improved patient outcomes. Understanding the characteristics of materials used in the design of new devices is essential for their advancement. In this review, our goal is to assist biomedical researchers in appreciating the importance of these properties and the role of selecting the proper measurement. We discuss how the nanoscopic molecular composition, arrangement, and interactions generate the properties of liquids, solids, viscoelastic materials, and colloids and discuss the measurement techniques that can be used to assess these properties from the nanoscale to the macroscale. We explore the linear and nonlinear mechanical responses of materials, elucidate their behaviors under varying conditions, and discuss corresponding measurement techniques. Finally, we highlight the importance of tailoring measurements to the underlying biological processes and applications being investigated.

Rationale: Hyper-IgE syndrome (STAT3-HIES), also known as Job's syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES is characterized by chronic pulmonary infection and inflammation, suggesting impaired innate host defense. Objectives: To identify airway epithelial host defense defects caused by STAT3 mutations that, together with immune dysfunction, contribute to recurrent pulmonary infections in STAT3-HIES. Methods: STAT3-HIES sputum was analyzed for biochemical and biophysical properties. STAT3-HIES excised lungs were harvested for histology, and bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific R382W mutation, expressed via lentivirus, and STAT3 knockout (CRISPR/Cas9) were studied in normal human bronchial epithelial cells under basal or inflammatory (IL1β)-stimulated conditions. Effects of STAT3 deficiency on transcriptomics, epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Measurements and Main Results: STAT3-HIES sputum showed increased mucus concentration and viscoelasticity. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1β expression. STAT3 mutations reduced CFTR mRNA and protein amounts, impaired CFTR-dependent fluid and mucin secretion, suppressed antimicrobial peptide, cytokine, and chemokine expression, and acidified airway surface liquid at baseline and after IL1β exposure. Notably, mutant STAT3 suppressed IL1R1 expression. Furthermore, STAT3 mutations impaired multiciliogenesis by blocking commitment to ciliated cell lineages through inhibition of HES6, leading to defective mucociliary transport. Administration of a γ-secretase inhibitor restored HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. Conclusions: STAT3 dysfunction leads to multicomponent defects in airway epithelial innate defense, which, in conjunction with immune deficiency, contributes to chronic pulmonary infection in STAT3-HIES.

Tracheal and distal airway imaging enhance the evaluation of mucociliary clearance (MCC) and respiratory health. Herein, we characterize in vivo pulmonary imaging performance of a microbubble (MB) contrast agent optimized for muco-adhesion. A three-way crossover trial (12 mice, 3 imaging timepoints each) was conducted to evaluate tracheal ultrasound image enhancement following oropharyngeal instillation of standard MBs, our optimized MB formulation (TAP-cationic MBs), and lipid solution control. The feasibility of delivering our TAP-cationic MBs as an aerosol to the distal airways was also evaluated using a porcine model. Contrast imaging procedures were well-tolerated by both animal models. In mice, tracheal delineation was comparably enhanced with TAP-cationic MBs (contrast-to-noise ratio [CNR]: 42.26 dB) and standard MBs (CNR: 45.09 dB). Both exceeded lipid solution control (CNR: 11.9 dB, p < .05). In the porcine model, nebulized administration of TAP-cationic MBs yielded MB accumulation in the distal airways visible on transcutaneous ultrasound. Modifying the standard MB formulation to optimize muco-adhesion does not diminish image enhancement when administered oropharyngeally as a liquid solution, and when administered as an aerosol, TAP-cationic MBs deposit, and can be visualized in the distal lung airways. These findings support further development of MB contrast agents for pulmonary applications.

Impaired mucociliary transport is a distinguishing sign of cystic fibrosis, but current methods of evaluation are invasive or expose young patients to ionizing radiation. Contrast-enhanced ultrasound imaging may provide a feasible alternative. We formulated a cationic microbubble ultrasound contrast agent, to optimize adhesion to the respiratory mucus layer when inhaled. Potential toxicity was evaluated in human bronchial epithelial cell (hBEC) cultures following a 24-hour exposure, compared to positive and negative control conditions. In vivo tolerability and pulmonary image enhancement feasibility were evaluated in mice, comparing oropharyngeal administration of contrast agent to saline control. When induced to flow across mucus plated on microscope slides, cationic microbubbles demonstrated greater affinity for target samples than standard microbubbles. Cationic microbubbles elicited no proinflammatory or cytotoxic response in hBECs, nor were any cross-links to the cilia observed. Unlike standard microbubbles, cationic microbubbles mixed into the mucus layer, without epithelial absorption, and were observed to move with the mucus layer by the action of mucociliary transport. When administered to mice, cationic microbubbles enhanced sonographic visualization of the trachea, and were well-tolerated with no adverse effects. This developmental work supports the safety and feasibility of a mucus-targeting contrast agent that may be useful for pulmonary ultrasound applications.

Rationale: Bronchiectasis is a pathological dilatation of the bronchi in the respiratory airways associated with environmental or genetic causes (e.g., cystic fibrosis, primary ciliary dyskinesia, and primary immunodeficiency disorders), but most cases remain idiopathic. Objectives: To identify novel genetic defects in unsolved cases of bronchiectasis presenting with severe rhinosinusitis, nasal polyposis, and pulmonary Pseudomonas aeruginosa infection. Methods: DNA was analyzed by next-generation or targeted Sanger sequencing. RNA was analyzed by quantitative PCR and single-cell RNA sequencing. Patient-derived cells, cell cultures, and secretions (mucus, saliva, seminal fluid) were analyzed by Western blotting and immunofluorescence microscopy, and mucociliary activity was measured. Blood serum was analyzed by electrochemiluminescence immunoassay. Protein structure and proteomic analyses were used to assess the impact of a disease-causing founder variant. Measurements and Main Results: We identified biallelic pathogenic variants in WAP four-disulfide core domain 2 (WFDC2) in 11 individuals from 10 unrelated families originating from the United States, Europe, Asia, and Africa. Expression of WFDC2 was detected predominantly in secretory cells of control airway epithelium and also in submucosal glands. We demonstrate that WFDC2 is below the limit of detection in blood serum and hardly detectable in samples of saliva, seminal fluid, and airway surface liquid from WFDC2-deficient individuals. Computer simulations and deglycosylation assays indicate that the disease-causing founder variant p.Cys49Arg structurally hampers glycosylation and, thus, secretion of mature WFDC2. Conclusions: WFDC2 dysfunction defines a novel molecular etiology of bronchiectasis characterized by the deficiency of a secreted component of the airways. A commercially available blood test combined with genetic testing allows its diagnosis.

In a healthy colon, the stratified mucus layer serves as a crucial innate immune barrier to protect the epithelium from microbes. Mucins are complex glycoproteins that serve as a nutrient source for resident microflora and can be exploited by pathogens. We aimed to understand how the intestinal pathogen, Clostridioides difficile, independently uses or manipulates mucus to its benefit, without contributions from members of the microbiota. Using a 2-D primary human intestinal epithelial cell model to generate physiologic mucus, we assessed C. difficile-mucus interactions through growth assays, RNA-Seq, biophysical characterization of mucus, and contextualized metabolic modeling. We found that host-derived mucus promotes C. difficile growth both in vitro and in an infection model. RNA-Seq revealed significant upregulation of genes related to central metabolism in response to mucus, including genes involved in sugar uptake, the Wood-Ljungdahl pathway, and the glycine cleavage system. In addition, we identified differential expression of genes related to sensing and transcriptional control. Analysis of mutants with deletions in highly upregulated genes reflected the complexity of C. difficile-mucus interactions, with potential interplay between sensing and growth. Mucus also stimulated biofilm formation in vitro, which may in turn alter the viscoelastic properties of mucus. Context-specific metabolic modeling confirmed differential metabolism and the predicted importance of enzymes related to serine and glycine catabolism with mucus. Subsequent growth experiments supported these findings, indicating mucus is an important source of serine. Our results better define responses of C. difficile to human gastrointestinal mucus and highlight flexibility in metabolism that may influence pathogenesis. IMPORTANCE Clostridioides difficile results in upward of 250,000 infections and 12,000 deaths annually in the United States. Community-acquired infections continue to rise, and recurrent disease is common, emphasizing a vital need to understand C. difficile pathogenesis. C. difficile undoubtedly interacts with colonic mucus, but the extent to which the pathogen can independently respond to and take advantage of this niche has not been explored extensively. Moreover, the metabolic complexity of C. difficile remains poorly understood but likely impacts its capacity to grow and persist in the host. Here, we demonstrate that C. difficile uses native colonic mucus for growth, indicating C. difficile possesses mechanisms to exploit the mucosal niche. Furthermore, mucus induces metabolic shifts and biofilm formation in C. difficile, which has potential ramifications for intestinal colonization. Overall, our work is crucial to better understand the dynamics of C. difficile-mucus interactions in the context of the human gut.

Significance: Assessing the nanostructure of polymer solutions and biofluids is broadly useful for understanding drug delivery and disease progression and for monitoring therapy. Aim: Our objective is to quantify bronchial mucus solids concentration (wt. %) during hypertonic saline (HTS) treatment in vitro via nanostructurally constrained diffusion of gold nanorods (GNRs) monitored by polarization-sensitive optical coherence tomography (PS-OCT). Approach: Using PS-OCT, we quantified GNR translational (DT) and rotational (DR) diffusion coefficients within polyethylene oxide solutions (0 to 3 wt. %) and human bronchial epithelial cell (hBEC) mucus (0 to 6.4 wt. %). Interpolation of DT and DR data is used to develop an assay to quantify mucus concentration. The assay is demonstrated on the mucus layer of an air-liquid interface hBEC culture during HTS treatment. Results: In polymer solutions and mucus, DT and DR monotonically decrease with increasing concentration. DR is more sensitive than DT to changes above 1.5 wt. % of mucus and exhibits less intrasample variability. Mucus on HTS-treated hBEC cultures exhibits dynamic mixing from cilia. A region of hard-packed mucus is revealed by DR measurements. Conclusions: The extended dynamic range afforded by simultaneous measurement of DT and DR of GNRs using PS-OCT enables resolving concentration of the bronchial mucus layer over a range from healthy to disease in depth and time during HTS treatment in vitro. (c) The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa, which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic tolerance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro. We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa. Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. IMPORTANCE:Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro, is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.

Rationale:Hyper IgE syndrome (STAT3-HIES), also known as Job's syndrome, is a rare immunodeficiency disease typically caused by dominant-negative STAT3 mutations. STAT3-HIES syndrome is characterized by chronic pulmonary infection and inflammation, suggesting impairment of pulmonary innate host defense. Objectives:To identify airway epithelial host defense defects consequent to STAT3 mutations that, in addition to reported mutant STAT3 immunologic abnormalities, produce pulmonary infection. Methods:STAT3-HIES sputum was evaluated for biochemical/biophysical properties. STAT3-HIES excised lungs were harvested for histology; bronchial brush samples were collected for RNA sequencing and in vitro culture. A STAT3-HIES-specific mutation (R382W), expressed by lentiviruses, and a STAT3 knockout, generated by CRISPR/Cas9, were maintained in normal human bronchial epithelia under basal or inflammatory (IL1β) conditions. Effects of STAT3 deficiency on transcriptomics, and epithelial ion channel, secretory, antimicrobial, and ciliary functions were assessed. Measurements and Main Results:Mucus concentrations and viscoelasticity were increased in STAT3-HIES sputum. STAT3-HIES excised lungs exhibited mucus obstruction and elevated IL1β expression. STAT3 deficiency impaired CFTR-dependent fluid and mucin secretion, inhibited expression of antimicrobial peptides, cytokines, and chemokines, and acidified airway surface liquid at baseline and post-IL1β exposure in vitro. Notably, mutant STAT3 suppressed IL1R1 expression. STAT3 mutations also inhibited ciliogenesis in vivo and impaired mucociliary transport in vitro, a process mediated via HES6 suppression. Administration of a γ-secretase inhibitor increased HES6 expression and improved ciliogenesis in STAT3 R382W mutant cells. Conclusions:STAT3 dysfunction leads to multi-component defects in airway epithelial innate defense, which, in conjunction with STAT3-HIES immune deficiency, contributes to chronic pulmonary infection.

Human respiratory mucus (HRM) is extremely soft, compelling passive microrheology for linear viscoelastic characterization. We focus this study on the use of passive microrheology to characterize HRM heterogeneity, a phenomenon in normal HRM that becomes extreme during cystic fibrosis (CF) disease. Specifically, a fraction of the mucin polymers comprising HRM phase-separate into insoluble structures, called flakes, dispersed in mucin-depleted solution. We first reconstitute HRM samples to the MUC5B:MUC5AC mucin ratios consistent with normal and CF clinical samples, which we show recapitulate progressive flake formation and heterogeneity. We then employ passive particle tracking with 200 nm and 1 mu m diameter beads in each reconstituted sample. To robustly analyze the tracking data, we introduce statistical denoising methods for low signal-to-noise tracking data within flakes, tested and verified using model-generated synthetic data. These statistical methods provide a fractional Brownian motion classifier of all successfully denoised, tracked beads in flakes and the dilute solution. From the ensemble of classifier data, per bead diameter and mucus sample, we then employ clustering methods to learn and infer multiple levels of heterogeneity: (i) tracked bead data within vs. outside flakes and (ii) within-flake data buried within or distinguishable from the experimental noise floor. Simulated data consistent with experimental data (within and outside flakes) are used to explore form(s) of the generalized Stokes-Einstein relation (GSER) that recover the dynamic moduli of homogeneous and heterogeneous truth sets of purely flakelike, dilute solution, and mixture samples. The appropriate form of GSER is applied to experimental data to show (i) flakes are heterogeneous with gel and sol domains; (ii) dilute solutions are heterogeneous with only sol domains; and (iii) flake and dilute solution properties vary with probe diameter.

AbstractPeople with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grownPseudomonas aeruginosabiofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models.Author SummaryOne of the major challenges associated with chronic respiratory infections in cystic fibrosis and chronic obstructive pulmonary disease is difficult to treat biofilms that form in hyperconcentrated mucus. Biofilms are mechanically robust due to an exterior polymeric matrix that protects from the immune response and antibiotics. Antibiotics like tobramycin alone have little impact on the biofilm matrix, but disruption of viscous mucus and the biofilm architecture has previously been shown to improve antibiotic efficacy. Combination treatments must be used to break up mucus and biofilms and simultaneously kill bacteria. The most promising combination in this study includes the surfactant NP40, the biopolymer hyaluronic acid, and the antibiotic tobramycin which together decreased biofilm viscosity, reduced bacterial load, and increased mucociliary transport rates. The results from this study may be translated to an infected animal study for eventual clinical trials.