Chemokine CCL19 promotes type 2 T-cell differentiation and allergic airway inflammation.

BACKGROUND: Allergic asthma is driven largely by allergen-specific TH2 cells, which develop in regional lymph nodes on the interaction of naive CD4+ T cells with allergen-bearing dendritic cells that migrate from the lung. This migration event is dependent on CCR7 and its chemokine ligand, CCL21. However, is has been unclear whether the other CCR7 ligand, CCL19, has a role in allergic airway disease. OBJECTIVE: This study sought to define the role of CCL19 in TH2 differentiation and allergic airway disease. METHODS: Ccl19-deficient mice were studied in an animal model of allergic asthma. Dendritic cells or fibroblastic reticular cells from wild-type and Ccl19-deficient mice were cultured with naive CD4+ T cells, and cytokine production was measured by ELISA. Recombinant CCL19 was added to CD4+ T-cell cultures, and gene expression was assessed by RNA-sequencing and quantitative PCR. Transcription factor activation was assessed by flow cytometry. RESULTS: Lungs of Ccl19-deficient mice had less allergic airway inflammation, reduced airway hyperresponsiveness, and less IL-4 and IL-13 production compared with lungs of Ccl19-sufficient animals. Naive CD4+ T cells cocultured with Ccl19-deficient dendritic cells or fibroblastic reticular cells produced lower amounts of type 2 cytokines than did T cells cocultured with their wild-type counterparts. Recombinant CCL19 increased phosphorylation of STAT5 and induced expression of genes associated with TH2 cell and IL-2 signaling pathways. CONCLUSIONS: These results reveal a novel, TH2 cell-inducing function of CCL19 in allergic airway disease and suggest that strategies to block this pathway might help to reduce the incidence or severity of allergic asthma.

BPIFB1 loss alters airway mucus properties and diminishes mucociliary clearance.

Airway mucociliary clearance (MCC) is required for host defense and is often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the gene Bpifb1/Lplunc1 as a regulator of airway MUC5B protein levels using genetic approaches. Here, we show that BPIFB1 is required for effective MCC in vivo using Bpifb1 knockout (KO) mice. Reduced MCC in Bpifb1 KO mice occurred in the absence of defects in epithelial ion transport or reduced ciliary beat frequency. Loss of BPIFB1 in vivo and in vitro altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and the protein mesh of secreted mucus in human airway epithelia cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.NEW & NOTEWORTHY BPIFB1, also known as LPLUNC1, was found to regulate mucociliary clearance (MCC), a key aspect of host defense in the airway. Loss of this protein was also associated with altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC.

Mucolytic treatment of chronic rhinosinusitis in a murine model of primary ciliary dyskinesia.

Background: Genetic defects in motile cilia cause primary ciliary dyskinesia (PCD), a rare disease with no specific therapeutics. Individuals with PCD often have impaired fertility and laterality defects and universally suffer from upper and lower airway diseases. Chronic rhinosinusitis is a universal feature of PCD, and mucus accumulation and subsequent infections of the sinonasal cavity cause significant morbidity in individuals with PCD. Despite this, there are no approved treatments that specifically target mucus. Objective: The goals of this study were to determine whether computed tomography (CT) imaging could be used to quantify mucus accumulation and whether the use of a mucolytic agent to reduce disulfide cross-links present in mucins would improve the effectiveness of nasal lavage at removing mucus in a murine model of PCD. Methods: Adult mice with a deletion of the axonemal dynein Dnaic1 were imaged using CT scanning to characterize mucus accumulation. The animals were then treated by nasal lavage with saline, with/without the disulfide-reducing agent tris(2-carboxyethyl)phosphine. Post-treatment CT scans were used to quantify improvement in the sinonasal cavity. Results: Mucus accumulation in the nasal cavity was readily quantified by CT. Compared to sham-treated control animals, nasal lavage with/without a mucolytic agent resulted in a significant reduction of accumulated mucus (p < 0.01). Treatment with the mucolytic agent showed a greater reduction of accumulated mucus than treatment with saline alone. Conclusion: The results suggest that inclusion of a mucolytic agent may increase the effectiveness of nasal lavage at reducing mucus burden in PCD.

Chronic airway epithelial hypoxia exacerbates injury in muco-obstructive lung disease through mucus hyperconcentration.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O2, airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na+ and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of ß and γENaC (epithelial Na+ channel) subunit expression. The combination of increased Na+ absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA-in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

Animal models of cystic fibrosis in the era of highly effective modulator therapies.

Few human genetic diseases can rely on the availability of as many and as diverse animal models as cystic fibrosis (CF), a multiorgan syndrome caused by functional absence of cystic fibrosis transmembrane regulator (CFTR). The recent development of highly effective CFTR modulator drug therapies simultaneously highlighted the remarkable clinical improvement achievable with these treatments, the lack of therapeutic alternatives for non-responders, and the need to understand the kinetics of disease upon early life/chronic treatment. These advances have rekindled efforts to leverage animal models to address critical knowledge gaps in CF. This article provides a concise overview of the areas of interests for therapeutic intervention in the current CF landscape, focusing on the contributions of in vivo models to understand CF pathogenesis, identify therapeutic windows, and develop novel therapies for all CFTR mutations.

Regional Differences in Mucociliary Clearance in the Upper and Lower Airways.

As the nasal cavity is the portal of entry for inspired air in mammals, this region is exposed to the highest concentration of inhaled particulate matter and pathogens, which must be removed to keep the lower airways sterile. Thus, one might expect vigorous removal of these substances via mucociliary clearance (MCC) in this region. We have investigated the rate of MCC in the murine nasal cavity compared to the more distal airways (trachea). The rate of MCC in the nasal cavity (posterior nasopharynx, PNP) was ∼3-4× greater than on the tracheal wall. This appeared to be due to a more abundant population of ciliated cells in the nasal cavity (∼80%) compared to the more sparsely ciliated trachea (∼40%). Interestingly, the tracheal ventral wall exhibited a significantly lower rate of MCC than the tracheal posterior membrane. The trachealis muscle underlying the ciliated epithelium on the posterior membrane appeared to control the surface architecture and likely in part the rate of MCC in this tracheal region. In one of our mouse models (Bpifb1 KO) exhibiting a 3-fold increase in MUC5B protein in lavage fluid, MCC particle transport on the tracheal walls was severely compromised, yet normal MCC occurred on the tracheal posterior membrane. While a blanket of mucus covered the surface of both the PNP and trachea, this mucus appeared to be transported as a blanket by MCC only in the PNP. In contrast, particles appeared to be transported as discrete patches or streams of mucus in the trachea. In addition, particle transport in the PNP was fairly linear, in contrast transport of particles in the trachea often followed a more non-linear route. The thick, viscoelastic mucus blanket that covered the PNP, which exhibited ∼10-fold greater mass of mucus than did the blanket covering the surface of the trachea, could be transported over large areas completely devoid of cells (made by a breach in the epithelial layer). In contrast, particles could not be transported over even a small epithelial breach in the trachea. The thick mucus blanket in the PNP likely aids in particle transport over the non-ciliated olfactory cells in the nasal cavity and likely contributes to humidification and more efficient particle trapping in this upper airway region.

Inhaled gene therapy of preclinical muco-obstructive lung diseases by nanoparticles capable of breaching the airway mucus barrier.

INTRODUCTION: Inhaled gene therapy of muco-obstructive lung diseases requires a strategy to achieve therapeutically relevant gene transfer to airway epithelium covered by particularly dehydrated and condensed mucus gel layer. Here, we introduce a synthetic DNA-loaded mucus-penetrating particle (DNA-MPP) capable of providing safe, widespread and robust transgene expression in in vivo and in vitro models of muco-obstructive lung diseases. METHODS: We investigated the ability of DNA-MPP to mediate reporter and/or therapeutic transgene expression in lung airways of a transgenic mouse model of muco-obstructive lung diseases (ie, Scnn1b-Tg) and in air-liquid interface cultures of primary human bronchial epithelial cells harvested from an individual with cystic fibrosis. A plasmid designed to silence epithelial sodium channel (ENaC) hyperactivity, which causes airway surface dehydration and mucus stasis, was intratracheally administered via DNA-MPP to evaluate therapeutic effects in vivo with or without pretreatment with hypertonic saline, a clinically used mucus-rehydrating agent. RESULTS: DNA-MPP exhibited marked greater reporter transgene expression compared with a mucus-impermeable formulation in in vivo and in vitro models of muco-obstructive lung diseases. DNA-MPP carrying ENaC-silencing plasmids provided efficient downregulation of ENaC and reduction of mucus burden in the lungs of Scnn1b-Tg mice, and synergistic impacts on both gene transfer efficacy and therapeutic effects were achieved when DNA-MPP was adjuvanted with hypertonic saline. DISCUSSION: DNA-MPP constitutes one of the rare gene delivery systems providing therapeutically meaningful gene transfer efficacy in highly relevant in vivo and in vitro models of muco-obstructive lung diseases due to its unique ability to efficiently penetrate airway mucus.

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

Content and Performance of the MiniMUGA Genotyping Array: A New Tool To Improve Rigor and Reproducibility in Mouse Research.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

IL-1ß dominates the promucin secretory cytokine profile in cystic fibrosis.

Cystic fibrosis (CF) lung disease is characterized by early and persistent mucus accumulation and neutrophilic inflammation in the distal airways. Identification of the factors in CF mucopurulent secretions that perpetuate CF mucoinflammation may provide strategies for novel CF pharmacotherapies. We show that IL-1ß, with IL-1α, dominated the mucin prosecretory activities of supernatants of airway mucopurulent secretions (SAMS). Like SAMS, IL-1ß alone induced MUC5B and MUC5AC protein secretion and mucus hyperconcentration in CF human bronchial epithelial (HBE) cells. Mechanistically, IL-1ß induced the sterile α motif-pointed domain containing ETS transcription factor (SPDEF) and downstream endoplasmic reticulum to nucleus signaling 2 (ERN2) to upregulate mucin gene expression. Increased mRNA levels of IL1B, SPDEF, and ERN2 were associated with increased MUC5B and MUC5AC expression in the distal airways of excised CF lungs. Administration of an IL-1 receptor antagonist (IL-1Ra) blocked SAMS-induced expression of mucins and proinflammatory mediators in CF HBE cells. In conclusion, IL-1α and IL-1ß are upstream components of a signaling pathway, including IL-1R1 and downstream SPDEF and ERN2, that generate a positive feedback cycle capable of producing persistent mucus hyperconcentration and IL-1α and/or IL-1ß-mediated neutrophilic inflammation in the absence of infection in CF airways. Targeting this pathway therapeutically may ameliorate mucus obstruction and inflammation-induced structural damage in young CF children.

The Cystic Fibrosis-Like Airway Surface Layer Is not a Significant Barrier for Delivery of Eluforsen to Airway Epithelial Cells.

Background: Eluforsen (previously known as QR-010) is a 33-mer antisense oligonucleotide under development for oral inhalation in cystic fibrosis (CF) patients with the delta F508 mutation. Previous work has shown that eluforsen restores CF transmembrane conductance regulator (CFTR) function in vitro and in vivo. To be effective, eluforsen has first to reach its primary target, the lung epithelial cells. Therefore, it has to diffuse through the CF airway surface layer (ASL), which in CF is characterized by the presence of thick and viscous mucus, impaired mucociliary clearance, and persistent infections. The goal of this study was to assess delivery of eluforsen through CF-like ASL. Methods and Results: First, air-liquid interface studies with cultured primary airway epithelial cells revealed that eluforsen rapidly diffuses through CF-like mucus at clinically relevant doses when nebulized once or repeatedly, over a range of testing doses. Furthermore, eluforsen concentrations remained stable in CF patient sputum for at least 48 hours, and eluforsen remained intact in the presence of various inhaled CF medications for at least 24 hours. When testing biodistribution of eluforsen after orotracheal administration in vivo, no differences in lung, liver, trachea, and kidney eluforsen concentration were observed between mice with a CF-like lung phenotype (ENaC-overexpressing mice) and control wild-type (WT) littermates. Also, eluforsen was visualized in the airway epithelial cell layer of CF-like muco-obstructed mice and WT littermates. Finally, studies of eluforsen uptake and binding to bacteria prevalent in CF lungs, and diffusion through bacterial biofilms showed that eluforsen was stable and not absorbed by, or bound to bacteria. In addition, eluforsen was found to be able to penetrate Pseudomonas aeruginosa biofilms. Conclusions: The thickened and concentrated CF ASL does not constitute a significant barrier for delivery of eluforsen, and feasibility of oral inhalation of eluforsen is supported by these data.

XBP1S Regulates MUC5B in a Promoter Variant-Dependent Pathway in Idiopathic Pulmonary Fibrosis Airway Epithelia.

Rationale: The goal was to connect elements of idiopathic pulmonary fibrosis (IPF) pathogenesis, including chronic endoplasmic reticulum stress in respiratory epithelia associated with injury/inflammation and remodeling, distal airway mucus obstruction and honeycomb cyst formation with accumulation of MUC5B (mucin 5B), and associations between IPF risk and polymorphisms in the MUC5B promoter. Objectives: To test whether the endoplasmic reticulum (ER) stress sensor protein ERN2 (ER-to-nucleus signaling 2) and its downstream effector, the spliced form of XBP1S (X-box-binding protein 1), regulate MUC5B expression and differentially activate the MUC5B promoter variant in respiratory epithelia. Methods: Primary human airway epithelial (HAE) cells, transgenic mouse models, human IPF lung tissues, and cell lines expressing XBP1S and MUC5B promoters were used to explore relationships between the ERN2/XBP1S pathway and MUC5B. An inhibitor of the pathway, KIRA6, and XBP1 CRISPR-Cas9 were used in HAE cells to explore therapeutic potential. Measurements and Main Results: ERN2 regulated MUC5B and MUC5AC mRNAs. Downstream XBP1S selectively promoted MUC5B expression in vitro and in distal murine airway epithelia in vivo. XBP1S bound to the proximal region of the MUC5B promoter and differentially upregulated MUC5B expression in the context of the MUC5B promoter rs35705950 variant. High levels of ERN2 and XBP1S were associated with excessive MUC5B mRNAs in distal airways of human IPF lungs. Cytokine-induced MUC5B expression in HAE cells was inhibited by KIRA6 and XBP1 CRISPR-Cas9. Conclusions: A positive feedback bistable ERN2-XBP1S pathway regulates MUC5B-dominated mucus obstruction in IPF, providing an unfolded protein response-dependent mechanism linking the MUC5B promoter rs35705950 polymorphism with IPF pathogenesis. Inhibiting ERN2-dependent pathways/elements may provide a therapeutic option for IPF.

Mice with a Deletion of Rsph1 Exhibit a Low Level of Mucociliary Clearance and Develop a Primary Ciliary Dyskinesia Phenotype.

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disease caused by mutations in over 40 different genes. Individuals with PCD caused by mutations in RSPH1 (radial spoke head 1 homolog) have been reported to have a milder phenotype than other individuals with PCD, as evidenced by a lower incidence of neonatal respiratory distress, higher nasal nitric oxide concentrations, and better lung function. To better understand genotype-phenotype relationships in PCD, we have characterized a mutant mouse model with a deletion of Rsph1. Approximately 50% of cilia from Rsph1-/- cells appeared normal by transmission EM, whereas the remaining cilia revealed a range of defects, primarily transpositions or a missing central pair. Ciliary beat frequency in Rsph1-/- cells was significantly lower than in control cells (20.2 ± 0.8 vs. 25.0 ± 0.9 Hz), and the cilia exhibited an aberrant rotational waveform. Young Rsph1-/- animals demonstrated a low rate of mucociliary clearance in the nasopharynx that was reduced to zero by about 1 month of age. Rsph1-/- animals accumulated mucus in the nasal cavity but had a lower bacterial burden than animals with a deletion of dynein axonemal intermediate chain 1 (Dnaic1-/-). Thus, Rsph1-/- mice display a PCD phenotype similar to but less severe than that observed in Dnaic1-/- mice, similar to what has been observed in humans. The results suggest that some individuals with PCD may not have a complete loss of mucociliary clearance and further suggest that early diagnosis and intervention may be important to maintain this low amount of clearance.

An Improved Inhaled Mucolytic to Treat Airway Muco-obstructive Diseases.

RATIONALE: Airways obstruction with thick, adherent mucus is a pathophysiologic and clinical feature of muco-obstructive respiratory diseases, including chronic obstructive pulmonary disease, asthma, and cystic fibrosis (CF). Mucins, the dominant biopolymer in mucus, organize into complex polymeric networks via the formation of covalent disulfide bonds, which govern the viscoelastic properties of the mucus gel. For decades, inhaled N-acetylcysteine (NAC) has been used as a mucolytic to reduce mucin disulfide bonds with little, if any, therapeutic effects. Improvement of mucolytic therapy requires the identification of NAC deficiencies and the development of compounds that overcome them. OBJECTIVES: Elucidate the pharmacological limitations of NAC and test a novel mucin-reducing agent, P3001, in preclinical settings. METHODS: The study used biochemical (e.g., Western blotting, mass spectrometry) and biophysical assays (e.g., microrheology/macrorheology, spinnability, mucus velocity measurements) to test compound efficacy and toxicity in in vitro and in vivo models and patient sputa. MEASUREMENTS AND MAIN RESULTS: Dithiothreitol and P3001 were directly compared with NAC in vitro and both exhibited superior reducing activities. In vivo, P3001 significantly decreased lung mucus burden in ßENaC-overexpressing mice, whereas NAC did not (n = 6-24 mice per group). In NAC-treated CF subjects (n = 5), aerosolized NAC was rapidly cleared from the lungs and did not alter sputum biophysical properties. In contrast, P3001 acted faster and at lower concentrations than did NAC, and it was more effective than DNase in CF sputum ex vivo. CONCLUSIONS: These results suggest that reducing the viscoelasticity of airway mucus is an achievable therapeutic goal with P3001 class mucolytic agents.

Localization of Secretory Mucins MUC5AC and MUC5B in Normal/Healthy Human Airways.

RATIONALE: MUC5AC and MUC5B are the predominant gel-forming mucins in the mucus layer of human airways. Each mucin has distinct functions and site-specific expression. However, the regional distribution of expression and cell types that secrete each mucin in normal/healthy human airways are not fully understood. OBJECTIVES: To characterize the regional distribution of MUC5B and MUC5AC in normal/healthy human airways and assess which cell types produce these mucins, referenced to the club cell secretory protein (CCSP). METHODS: Multiple airway regions from 16 nonsmoker lungs without a history of lung disease were studied. MUC5AC, MUC5B, and CCSP expression/colocalization were assessed by RNA in situ hybridization and immunohistochemistry in five lungs with histologically healthy airways. Droplet digital PCR and cell cultures were performed for absolute quantification of MUC5AC/5B ratios and protein secretion, respectively. MEASUREMENTS AND MAIN RESULTS: Submucosal glands expressed MUC5B, but not MUC5AC. However, MUC5B was also extensively expressed in superficial epithelia throughout the airways except for the terminal bronchioles. Morphometric calculations revealed that the distal airway superficial epithelium was the predominant site for MUC5B expression, whereas MUC5AC expression was concentrated in proximal, cartilaginous airways. RNA in situ hybridization revealed MUC5AC and MUC5B were colocalized with CCSP-positive secretory cells in proximal superficial epithelia, whereas MUC5B and CCSP-copositive cells dominated distal regions. CONCLUSIONS: In normal/healthy human airways, MUC5B is the dominant secretory mucin in the superficial epithelium and glands, with distal airways being a major site of expression. MUC5B and MUC5AC expression is a property of CCSP-positive secretory cells in superficial airway epithelia.

Mucociliary Clearance in Mice Measured by Tracking Trans-tracheal Fluorescence of Nasally Aerosolized Beads.

Mucociliary clearance (MCC) is the first line of defense in clearing airways. In genetically engineered mice, each component of this system (ciliary beat, mucus, airway surface hydration) can be studied separately to determine its contribution to MCC. Because MCC is difficult to measure in mice, MCC measurements are often omitted from these studies. We report a simple method to measure MCC in mice involving nasal inhalation of aerosolized fluorescent beads and trans-tracheal bead tracking. This method has a number of advantages over existing methods: (1) a small volume of liquid is deposited thus minimally disturbing the airway surface; (2) bead behavior on airways can be visualized; (3) useful for adult or neonatal mice; (4) the equipment is relatively inexpensive and easily obtainable. The type of anesthetic had no significant effect on the rate of MCC, but overloading the airways with beads significantly decreased MCC. In addition, the rate of bead transport was not different in alive (3.11 mm/min) vs recently euthanized mice (3.10 mm/min). A 5-min aerosolization of beads in a solution containing UTP significantly increased the rate of MCC, demonstrating that our method would be of value in testing the role of various pharmacological agents on MCC.

An Adeno-Associated Viral Vector Capable of Penetrating the Mucus Barrier to Inhaled Gene Therapy.

Diffusion of the viral vectors evaluated in inhaled gene therapy clinical trials to date are largely hindered within airway mucus, which limits their access to, and transduction of, the underlying airway epithelium prior to clearance from the lung. Here, we discovered that adeno-associated virus (AAV) serotype 6 was able to rapidly diffuse through mucus collected from cystic fibrosis (CF) patients, unlike previously tested AAV serotypes. A point mutation of the AAV6 capsid suggests a potential mechanism by which AAV6 avoids adhesion to the mucus mesh. Significantly greater transgene expression was achieved with AAV6 compared to a mucoadhesive serotype, AAV1, in air-liquid interface cultures of human CF bronchial epithelium with naturally secreted mucus or induced mucus hypersecretion. In addition, AAV6 achieved superior distribution and overall level of transgene expression compared to AAV1 in the airways and whole lungs, respectively, of transgenic mice with airway mucus obstruction. Our findings motivate further evaluation and clinical development of AAV6 for inhaled gene therapy.

Effect of LysM+ macrophage depletion on lung pathology in mice with chronic bronchitis.

Macrophages (MΦ) are key sentinels of respiratory exposure to inhaled environmental stimuli. In normal "healthy" tissues, MΦ are believed to be a dormant cell type that, upon exposure to stress-causing stimuli, may get activated to exhibit pro- or anti-inflammatory roles. To test whether stress present in chronic bronchitic (CB) airways triggers MΦ to manifest protective or detrimental responses, the DTA+ (LysM-regulated Diphtheria Toxin A expressing) strain with partial MΦ-deficiency was crossed with the Scnn1b-Tg mouse model of CB and the progenies were studied at 4-5 weeks of age. Compared with DTA- littermates, the DTA+ mice had ~50% reduction in bronchoalveolar lavage (BAL) MΦ, and the recovered MΦ were immature, phenotypically distinct, and functionally defective. DTA+/Scnn1b-Tg mice exhibited a similar depletion of LysM+ MΦ offset by a significant increase in LysM- MΦ in the BAL. In DTA+/Scnn1b-Tg mice, lung disease was more severe than in DTA-/Scnn1b-Tg littermates, as indicated by an increased incidence of mucus plugging, mucous cells, airway inflammation, higher levels of cytokines/chemokines (KC, TNF-α, MIP-2, M-CSF, IL-5, and IL-17), and worsened alveolar airspace enlargement. DTA+/Scnn1b-Tg mice exhibited increased occurrence of lymphoid nodules, which was concomitant with elevated levels of immunoglobulins in BAL. Collectively, these data indicate that numerical deficiency of MΦ in stressed airspaces is responded via compensatory increase in the recruitment of immature MΦ and altered non-MΦ effector cell-centered responses, for example, mucus production and adaptive immune defense. Overall, these data identify dynamic roles of MΦ in moderating, rather than exacerbating, the severity of lung disease in a model of CB.