Airway Surface Liquid Layer Research Articles

Specialized proresolving mediator resolvin E1 corrects the altered cystic fibrosis nasal epithelium cilia beating dynamics

In cystic fibrosis (CF), impaired mucociliary clearance leads to chronic infection and inflammation. However, cilia beating features in a CF altered environment, consisting of dehydrated airway surface liquid layer and abnormal mucus, have not been fully characterized. Furthermore, acute inflammation is normally followed by an active resolution phase requiring specialized proresolving lipid mediators (SPMs) and allowing return to homeostasis. However, altered SPMs biosynthesis has been reported in CF. Here, we explored cilia beating dynamics in CF airways primary cultures and its response to the SPMs, resolvin E1 (RvE1) and lipoxin B4 (LXB4). Human nasal epithelial cells (hNECs) from CF and non-CF donors were grown at air-liquid interface. The ciliary beat frequency, synchronization, orientation, and density were analyzed from high-speed video microscopy using a multiscale Differential Dynamic Microscopy algorithm and an in-house developed method. Mucins and ASL layer height were studied by qRT-PCR and confocal microscopy. Principal component analysis showed that CF and non-CF hNEC had distinct cilia beating phenotypes, which was mostly explained by differences in cilia beat organization rather than frequency. Exposure to RvE1 (10 nM) and to LXB4 (10 nM) restored a non-CF-like cilia beating phenotype. Furthermore, RvE1 increased the airway surface liquid (ASL) layer height and reduced the mucin MUC5AC thickness. The calcium-activated chloride channel, TMEM16A, was involved in the RvE1 effect on cilia beating, hydration, and mucus. Altogether, our results provide evidence for defective cilia beating in CF airway epithelium and a role of RvE1 and LXB4 to restore the main epithelial functions involved in the mucociliary clearance.

Bicarbonate Effects on Antibacterial Immunity and Mucus Glycobiology in the Cystic Fibrosis Lung: A Review With Selected Experimental Observations.

The primary defect in cystic fibrosis (CF) is abnormal chloride and bicarbonate transport in the cystic fibrosis transmembrane conductance regulator (CFTR) epithelial ion channel. The apical surface of the respiratory tract is lined by an airway surface liquid layer (ASL) composed of mucin comprising mainly MUC5A and MUC5B glycoproteins. ASL homeostasis depends on sodium bicarbonate secretion into the airways and secretion deficits alter mucus properties leading to airway obstruction, inflammation, and infections. Downstream effects of abnormal ion transport in the lungs include altered intrinsic immune defenses. We observed that neutrophils killed Pseudomonas aeruginosa more efficiently when it had been exposed to sodium bicarbonate, and formation of neutrophil extracellular traps (NETs) by neutrophils was augmented in the presence of increasing bicarbonate concentrations. Physiological levels of bicarbonate sensitized P. aeruginosa to the antimicrobial peptide cathelicidin LL-37, which is present in both lung ASL and in NETs. Sodium bicarbonate has various uses in clinical medicine and in the care of CF patients, and could be further explored as a therapeutic adjunct against Pseudomonas infections.

Effects of continuous and discrete boundary conditions on the movement of upper-convected maxwell and Newtonian mucus layers in coughing and sneezing.

Mucociliary clearance is an important phenomenon inside the respiratory system as a first defensive mechanism against pathogens. Therefore, any assumption considered for the mucociliary clearance and affects its functionality must be validated. The present research deals with the effects of boundary conditions on the movement of upper-convected Maxwell and high viscosity Newtonian mucus layers, numerically. Furthermore, the validity of replacing the viscoelastic mucus layer with a high viscosity Newtonian layer is evaluated. The airway surface liquid layer is considered a two-layer model including non-Newtonian mucus and Newtonian periciliary layers. Four cyclic boundary conditions are imposed at the mucus-periciliary interface as the cilia movement to obtain variations of mucociliary clearance. The upper boundary of the mucus layer is also exposed to different shear stress levels including free slip, cough, and sneeze conditions. By investigation of velocity variations inside mucus and periciliary layers, it is concluded the differences between viscoelastic and Newtonian mucus are not negligible. The maximum velocity differences between the two fluids are more than 52% and 215% during cough and sneeze, respectively. The results show there is a high order of dependency between the relaxation time and the imposed boundary conditions at the mucus-periciliary interface that leads to the invalidation of replacing two fluids with each other. Moreover, the results show substituting the viscoelastic mucus with a high viscosity Newtonian one depends on the mucus-periciliary interface boundary condition. If an independent time-varying boundary condition is used, the substitution leads to an error less than 7% under different shear stress levels. However, time-varying boundary condition shows 38% and 88% differences between high viscosity Newtonian and viscoelastic mucus layers. Furthermore, neglecting the recovery stroke leads to a velocity underestimation up to 50% by substituting viscoelastic mucus with a high viscosity Newtonian one. Therefore, replacing the viscoelastic mucus with a high viscosity Newtonian one is not acceptable for numerical simulations.

Effect of Alpha-1 Antitrypsin on CFTR Levels in Primary Human Airway Epithelial Cells Grown at the Air-Liquid-Interface.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is influenced by the fundamental cellular processes like epithelial differentiation/polarization, regeneration and epithelial–mesenchymal transition. Defects in CFTR protein levels and/or function lead to decreased airway surface liquid layer facilitating microbial colonization and inflammation. The SERPINA1 gene, encoding alpha1-antitrypsin (AAT) protein, is one of the genes implicated in CF, however it remains unknown whether AAT has any influence on CFTR levels. In this study we assessed CFTR protein levels in primary human lung epithelial cells grown at the air-liquid-interface (ALI) alone or pre-incubated with AAT by Western blots and immunohistochemistry. Histological analysis of ALI inserts revealed CFTR- and AAT-positive cells but no AAT-CFTR co-localization. When 0.5 mg/mL of AAT was added to apical or basolateral compartments of pro-inflammatory activated ALI cultures, CFTR levels increased relative to activated ALIs. This finding suggests that AAT is CFTR-modulating protein, albeit its effects may depend on the concentration and the route of administration. Human lung epithelial ALI cultures provide a useful tool for studies in detail how AAT or other pharmaceuticals affect the levels and activity of CFTR.

Transport and deposition of hygroscopic particles in asthmatic subjects with and without airway narrowing

This study numerically investigates the effect of hygroscopicity on transport and deposition of particles in severe asthmatic lungs with distinct airway structures. The study human subjects were selected from two imaging-based severe asthmatic clusters with one characterized by non-constricted airways and the other by constricted airways in the lower left lobe (LLL). We compared the deposition fractions of sodium chloride (NaCl) particles with a range of aerodynamic diameters (1–8 μm) in cluster archetypes under conditions with and without hygroscopic growth. The temperature and water vapor distributions in the airways were simulated with an airway wall boundary condition that accounts for variable temperature and water vapor evaporation at the interface between the lumen and the airway surface liquid layer. On average, the deposition fraction increased by about 6% due to hygroscopic particle growth in the cluster subjects with constricted airways, while it increased by only about 0.5% in those with non-constricted airways. The effect of particle growth was most significant for particles with an initial diameter of 2μm in the cluster subjects with constricted airways. The effect diminished with increasing particle size, especially for particles with an initial diameter larger than 4 μm. This suggests the necessity to differentiate asthmatic subjects by cluster in engineering the aerosol size for tailored treatment. Specifically, the treatment of severe asthmatic subjects who have constricted airways with inhalation aerosols may need submicron-sized hygroscopic particles to compensate for particle growth, if one targets for delivering to the peripheral region. These results could potentially inform the choice of particle size for inhalational drug delivery in a cluster-specific manner.

Effect of continuous positive airway pressure treatment on permeability, inflammation and mucus production of human epithelial cells.

Continuous positive airway pressure (CPAP) therapy is the gold standard treatment for obstructive sleep apnoea, which affects millions of people worldwide. However, this therapy normally results in symptoms such as dryness, sneezing, rhinorrhoea, post-nasal drip, nasal congestion and epistaxis in the upper airways.Using bronchial epithelial (Calu-3) and nasal epithelial (RPMI 2650) cells in an in vitro respiratory model, this study, for the first time, investigates the effect of CPAP positive pressure on the human respiratory epithelial mechanisms that regulate upper airways lubrication characteristics. To understand how the epithelium and mucus are affected by this therapy, several parameters were determined before and after positive pressure application.This work demonstrates that the positive pressure not only compresses the cells, but also reduces their permeability and mucus secretion rate, thus drying the airway surface liquid layer and altering the mucus/water ratio. It is also observed that the respiratory epithelia is equally inflamed without or with external humidification during CPAP application.These findings clearly identify the causes of the side-effects reported by patients under CPAP therapy.

Ablation of IL-33 Suppresses Th2 Responses but Is Accompanied by Sustained Mucus Obstruction in the Scnn1b Transgenic Mouse Model.

Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. Th2 immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of Th2 immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., Scnn1b-Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33KO) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33HET) Tg+ mice. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major Th2 cytokine. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had significantly reduced levels of Th2-associated gene signatures (Slc26a4, Clca1, Retnla, and Chi3l4), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33HET/Tg+ mice, although the IL-33KO/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33KO/Tg+ mice compared with IL-33HET/Tg+ mice. Taken together, our data indicate that although IL-33 modulates Th2 inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33.

Thickness of the airway surface liquid layer in the lung is affected in cystic fibrosis by compromised synergistic regulation of the ENaC ion channel.

The lung epithelium is lined with a layer of airway surface liquid (ASL) that is crucial for healthy lung function. ASL thickness is controlled by two ion channels: epithelium sodium channel (ENaC) and cystic fibrosis (CF) transmembrane conductance regulator (CFTR). Here, we present a minimal mathematical model of ENaC, CFTR and ASL regulation that sheds light on the control of ENaC by the short palate lung and nasal epithelial clone 1 (SPLUNC1) protein and by phosphatidylinositol 4,5-biphosphate (PI(4,5)P2). The model, despite its simplicity, yields a good fit to experimental observations and is an effective tool for exploring the interplay between ENaC, CFTR and ASL. Steady-state data and dynamic information constrain the model's parameters without ambiguities. Testing the hypothesis that PI(4,5)P2 protects ENaC from ubiquitination suggests that this protection does not improve the model results and that the control of the ENaC opening probability by PI(4,5)P2 is sufficient to explain all available data. The model analysis further demonstrates that ASL at the steady state is sensitive to small changes in PI(4,5)P2 abundance, particularly in CF conditions, which suggests that manipulation of phosphoinositide metabolism may promote therapeutic benefits for CF patients.

Concurrent absorption and secretion of airway surface liquids and bicarbonate secretion in human bronchioles.

Although small airways account for the largest fraction of the total conducting airway surfaces, the epithelial fluid and electrolyte transport in small, native airway epithelia has not been well characterized. Investigations have been limited, no doubt, by the complex tissue architecture as well as by its inaccessibility, small dimensions, and lack of applicable assays, especially in human tissues. To better understand how the critically thin layer of airway surface liquid (ASL) is maintained, we applied a "capillary"-Ussing chamber (area ≈1 mm2) to measure ion transport properties of bronchioles with diameters of ~2 mm isolated from resected specimens of excised human lungs. We found that the small human airway, constitutively and concurrently, secretes and absorbs fluid as observed in porcine small airways (50). We found that the human bronchiolar epithelium is also highly anion selective and constitutively secretes bicarbonate ( ), which can be enhanced pharmacologically by cAMP as well as Ca2+-mediated agonists. Concurrent secretion and absorption of surface liquid along with secretion help explain how the delicate volume of the fluid lining the human small airway is physiologically buffered and maintained in a steady state that avoids desiccating or flooding the small airway with ASL.

Construction of a Suite of Computable Biological Network Models Focused on Mucociliary Clearance in the Respiratory Tract.

Mucociliary clearance (MCC), considered as a collaboration of mucus secreted from goblet cells, the airway surface liquid layer, and the beating of cilia of ciliated cells, is the airways’ defense system against airborne contaminants. Because the process is well described at the molecular level, we gathered the available information into a suite of comprehensive causal biological network (CBN) models. The suite consists of three independent models that represent (1) cilium assembly, (2) ciliary beating, and (3) goblet cell hyperplasia/metaplasia and that were built in the Biological Expression Language, which is both human-readable and computable. The network analysis of highly connected nodes and pathways demonstrated that the relevant biology was captured in the MCC models. We also show the scoring of transcriptomic data onto these network models and demonstrate that the models capture the perturbation in each dataset accurately. This work is a continuation of our approach to use computational biological network models and mathematical algorithms that allow for the interpretation of high-throughput molecular datasets in the context of known biology. The MCC network model suite can be a valuable tool in personalized medicine to further understand heterogeneity and individual drug responses in complex respiratory diseases.

A Continuum Model of Mucosa with Glycan‐Ion Pairing

AbstractAdvances in the study of glycosoaminoglycan biohydrogels, label‐free electrokinetic analysis of soft‐diffuse layers in contact with saline solutions, and elucidation of ion‐specific behavior in many biochemical systems offer the opportunity to marry these principal features in a new mathematical model of the mucosal glycocalyx. The model is based on the electroquasistatic subset of Maxwell's equations in the form of the steady‐state continuum Poisson–Boltzmann equation for electrostatics with explicit incorporation of pairwise binding of ions to fixed charged‐groups in the hydrogel. The pairwise association is modeled using reversible bimolecular reactions via stoichiometric dissociation constants that represent the rule of matching water affinities—the observation that similar hydration structures of the pair results in less dissociation. Applications of the model to specific gels and salts, including a heparin star polyethylene glycol (starPEG) biohydrogel and the airway surface liquid layer in cystic fibrosis, are presented to postulate some quantitative consequences of glycocalyx ion partitioning.

Effect of airway surface liquid on the forces on the pharyngeal wall: Experimental fluid-structure interaction study.

Obstructive sleep apnoea syndrome (OSAS) is a breathing disorder with a multifactorial etiology. The respiratory epithelium is lined with a thin layer of airway surface liquid preventing interactions between the airflow and epithelium. The effect of the liquid lining in OSAS pathogenesis remains poorly understood despite clinical research. Previous studies have shown that the physical properties of the airway surface liquid or altered stimulation of the airway mechanoreceptors could alleviate or intensify OSAS; however, these studies do not provide a clear physical interpretation. To study the forces transmitted from the airflow to the liquid-lined compliant wall and to discuss the effects of the airway surface liquid properties on the stimulation of the mechanoreceptors, a novel and simplified experimental system mimicking the upper airway fundamental characteristics (i.e., liquid-lined compliant wall and complex unsteady airflow features) was constructed. The fluctuating force on the compliant wall was reduced through a damping mechanism when the liquid film thickness and/or the viscosity were increased. Conversely, the liquid film damping was reduced when the surface tension decreased. Based on the experimental data, empirical correlations were developed to predict the damping potential of the liquid film. In the future, this will enable us to extend the existing computational fluid-structure interaction simulations of airflow in the human upper airway by incorporating the airway surface liquid effect without adopting two-phase flow interface tracking methods. Furthermore, the experimental system developed in this study could be used to investigate the fundamental principles of the complex once/twice-coupled physical phenomena.

Cilia and Mucociliary Clearance.

Mucociliary clearance (MCC) is the primary innate defense mechanism of the lung. The functional components are the protective mucous layer, the airway surface liquid layer, and the cilia on the surface of ciliated cells. The cilia are specialized organelles that beat in metachronal waves to propel pathogens and inhaled particles trapped in the mucous layer out of the airways. In health this clearance mechanism is effective, but in patients with primary cilia dyskinesia (PCD) the cilia are abnormal, resulting in deficient MCC and chronic lung disease. This demonstrates the critical importance of the cilia for human health. In this review, we summarize the current knowledge of the components of the MCC apparatus, focusing on the role of cilia in MCC.

Thick airway surface liquid volume and weak mucin expression in pendrin-deficient human airway epithelia.

Pendrin is an anion exchanger whose mutations are known to cause hearing loss. However, recent data support the linkage between pendrin expression and airway diseases, such as asthma. To evaluate the role of pendrin in the regulation of the airway surface liquid (ASL) volume and mucin expression, we investigated the function and expression of pendrin and ion channels and anion exchangers. Human nasal epithelial cells were cultured from 16 deaf patients carrying pendrin mutations (DFNB4) and 17 controls. The cells were treated with IL-13 to induce mucus hypersecretion. Airway surface liquid thickness was measured and real-time polymerase chain reaction was performed targeting various transporters and MUC5AC. Anion exchanger activity was measured using a pH-sensitive fluorescent probe. Periodic acid-Schiff staining was performed on the cultured cells and inferior turbinate tissues. The ASL layer of the nasal epithelia from DFNB4 subjects was thicker than the controls, and the difference became more prominent following IL-13 stimulation. There was no difference in anion exchange activity after IL-13 treatment in the cells from DFNB4 patients, while it increased in the controls. Goblet cell metaplasia induced by IL-13 treatment seen in the controls was not observed in the DFNB4 cells. Furthermore, the periodic acid-Schiff staining-positive area was lesser in the inferior turbinate tissues from DFNB4 patients that those from controls. Pendrin plays a critical role in ASL volume regulation and mucin expression as pendrin-deficient airway epithelial cells are refractory to stimulation with IL-13. Specific blockers targeting pendrin in the airways may therefore have therapeutic potential in the treatment of allergic airway diseases.

Physiological impact of abnormal lipoxin A₄ production on cystic fibrosis airway epithelium and therapeutic potential.

Lipoxin A4 has been described as a major signal for the resolution of inflammation and is abnormally produced in the lungs of patients with cystic fibrosis (CF). In CF, the loss of chloride transport caused by the mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel gene results in dehydration, mucus plugging, and reduction of the airway surface liquid layer (ASL) height which favour chronic lung infection and neutrophil based inflammation leading to progressive lung destruction and early death of people with CF. This review highlights the unique ability of LXA4 to restore airway surface hydration, to stimulate airway epithelial repair, and to antagonise the proinflammatory program of the CF airway, circumventing some of the most difficult aspects of CF pathophysiology. The report points out novel aspects of the cellular mechanism involved in the physiological response to LXA4, including release of ATP from airway epithelial cell via pannexin channel and subsequent activation of and P2Y11 purinoreceptor. Therefore, inadequate endogenous LXA4 biosynthesis reported in CF exacerbates the ion transport abnormality and defective mucociliary clearance, in addition to impairing the resolution of inflammation, thus amplifying the vicious circle of airway dehydration, chronic infection, and inflammation.

Pseudomonas aeruginosa triggers CFTR-mediated airway surface liquid secretion in swine trachea.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations in the gene encoding for the anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Several organs are affected in CF, but most of the morbidity and mortality comes from lung disease. Recent data show that the initial consequence of CFTR mutation is the failure to eradicate bacteria before the development of inflammation and airway remodeling. Bacterial clearance depends on a layer of airway surface liquid (ASL) consisting of both a mucus layer that traps, kills, and inactivates bacteria and a periciliary liquid layer that keeps the mucus at an optimum distance from the underlying epithelia, to maximize ciliary motility and clearance of bacteria. The airways in CF patients and animal models of CF demonstrate abnormal ASL secretion and reduced antimicrobial properties. Thus, it has been proposed that abnormal ASL secretion in response to bacteria may facilitate the development of the infection and inflammation that characterize CF airway disease. Whether the inhalation of bacteria triggers ASL secretion, and the role of CFTR, have never been tested, however. We developed a synchrotron-based imaging technique to visualize the ASL layer and measure the effect of bacteria on ASL secretion. We show that the introduction of Pseudomonas aeruginosa and other bacteria into the lumen of intact isolated swine tracheas triggers CFTR-dependent ASL secretion by the submucosal glands. This response requires expression of the bacterial protein flagellin. In patients with CF, the inhalation of bacteria would fail to trigger ASL secretion, leading to infection and inflammation.

Cystic Fibrosis Transmembrane Conductance Regulator and Pseudomonas

Cystic Fibrosis Transmembrane Conductance Regulator and <i>Pseudomonas</i>

Activation of P2RY11 and ATP Release by Lipoxin A4 Restores the Airway Surface Liquid Layer and Epithelial Repair in Cystic Fibrosis

In cystic fibrosis (CF), the airway surface liquid (ASL) height is reduced as a result of impaired ion transport, which favors bacterial colonization and inflammation of the airway and leads to progressive lung destruction. Lipoxin (LX)A4, which promotes resolution of inflammation, is inadequately produced in the airways of patients with CF. We previously demonstrated that LXA4 stimulates an ASL height increase and epithelial repair. Here we report the molecular mechanisms involved in these processes. We found that LXA4 (1 nM) induced an apical ATP release from non-CF (NuLi-1) and CF (CuFi-1) airway epithelial cell lines and CF primary cultures. The ATP release induced by LXA4 was completely inhibited by antagonists of the ALX/FPR2 receptor and Pannexin-1 channels. LXA4 induced an increase in intracellular cAMP and calcium, which were abolished by the selective inhibition of the P2RY11 purinoreceptor. Pannexin-1 and ATP hydrolysis inhibition and P2RY11 purinoreceptor knockdown all abolished the increase of ASL height induced by LXA4. Inhibition of the A2b adenosine receptor did not affect the ASL height increase induced by LXA4, whereas the PKA inhibitor partially inhibited this response. The stimulation of NuLi-1 and CuFi-1 cell proliferation, migration, and wound repair by LXA4 was inhibited by the antagonists of Pannexin-1 channel and P2RY11 purinoreceptor. Taken together, our results provide evidence for a novel role of LXA4 in stimulating apical ATP secretion via Pannexin-1 channels and P2RY11 purinoreceptors activation leading to an ASL height increase and epithelial repair.

Bioengineered Lysozyme Reduces Bacterial Burden and Inflammation in a Murine Model of Mucoid Pseudomonas aeruginosa Lung Infection

The spread of drug-resistant bacterial pathogens is a growing global concern and has prompted an effort to explore potential adjuvant and alternative therapies derived from nature's repertoire of bactericidal proteins and peptides. In humans, the airway surface liquid layer is a rich source of antibiotics, and lysozyme represents one of the most abundant and effective antimicrobial components of airway secretions. Human lysozyme is active against both Gram-positive and Gram-negative bacteria, acting through several mechanisms, including catalytic degradation of cell wall peptidoglycan and subsequent bacterial lysis. In the infected lung, however, lysozyme's dense cationic character can result in sequestration and inhibition by polyanions associated with airway inflammation. As a result, the efficacy of the native enzyme may be compromised in the infected and inflamed lung. To address this limitation, we previously constructed a charge-engineered variant of human lysozyme that was less prone to electrostatic-mediated inhibition in vitro. Here, we employ a murine model to show that this engineered enzyme is superior to wild-type human lysozyme as a treatment for mucoid Pseudomonas aeruginosa lung infections. The engineered enzyme effectively decreases the bacterial burden and reduces markers of inflammation and lung injury. Importantly, we found no evidence of acute toxicity or allergic hypersensitivity upon repeated administration of the engineered biotherapeutic. Thus, the charge-engineered lysozyme represents an interesting therapeutic candidate for P. aeruginosa lung infections.

Chapter Four - Cellular and Molecular Biology of Airway Mucins

Airway mucus constitutes a thin layer of airway surface liquid with component macromolecules that covers the luminal surface of the respiratory tract. The major function of mucus is to protect the lungs through mucociliary clearance of inhaled foreign particles and noxious chemicals. Mucus is comprised of water, ions, mucin glycoproteins, and a variety of other macromolecules, some of which possess anti-microbial, anti-protease, and anti-oxidant activities. Mucins comprise the major protein component of mucus and exist as secreted and cell-associated glycoproteins. Secreted, gel-forming mucins are mainly responsible for the viscoelastic property of mucus, which is crucial for effective mucociliary clearance. Cell-associated mucins shield the epithelial surface from pathogens through their extracellular domains and regulate intracellular signaling through their cytoplasmic regions. However, neither the exact structures of mucin glycoproteins, nor the manner through which their expression is regulated, are completely understood. This chapter reviews what is currently known about the cellular and molecular properties of airway mucins.