Recessively Inherited Deficiency of Secreted WFDC2 (HE4) Causes Nasal Polyposis and Bronchiectasis.

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 bi-allelic pathogenic variants in 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. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

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.

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.

The role of SPAG1 in the assembly of axonemal dyneins in human airway epithelia.

Mutations in SPAG1, a dynein axonemal assembly factor (DNAAF) that facilitates the assembly of dynein arms in the cytoplasm before their transport into the cilium, result in primary ciliary dyskinesia (PCD), a genetically heterogenous disorder characterized by chronic oto-sino-pulmonary disease, infertility and laterality defects. To further elucidate the role of SPAG1 in dynein assembly, we examined its expression, interactions and ciliary defects in control and PCD human airway epithelia. Immunoprecipitations showed that SPAG1 interacts with multiple DNAAFs, dynein chains and canonical components of the R2TP complex. Protein levels of dynein heavy chains (DHCs) and interactions between DHCs and dynein intermediate chains (DICs) were reduced in SPAG1 mutants. We also identified a previously uncharacterized 60 kDa SPAG1 isoform, through examination of PCD subjects with an atypical ultrastructural defect for SPAG1 variants, that can partially compensate for the absence of full-length SPAG1 to assemble a reduced number of outer dynein arms. In summary, our data show that SPAG1 is necessary for axonemal dynein arm assembly by scaffolding R2TP-like complexes composed of several DNAAFs that facilitate the folding and/or binding of the DHCs to the DIC complex.

Expression of a Truncated Form of ODAD1 Associated with an Unusually Mild Primary Ciliary Dyskinesia Phenotype.

Primary ciliary dyskinesia (PCD) is a rare lung disease caused by mutations that impair the function of motile cilia, resulting in chronic upper and lower respiratory disease, reduced fertility, and a high prevalence of situs abnormalities. The disease is genetically and phenotypically heterogeneous, with causative mutations in > 50 genes identified, and clinical phenotypes ranging from mild to severe. Absence of ODAD1 (CCDC114), a component of the outer dynein arm docking complex, results in a failure to assemble outer dynein arms (ODAs), mostly immotile cilia, and a typical PCD phenotype. We identified a female (now 34 years old) with an unusually mild clinical phenotype who has a homozygous non-canonical splice mutation (c.1502+5G>A) in ODAD1. To investigate the mechanism for the unusual phenotype, we performed molecular and functional studies of cultured nasal epithelial cells. We demonstrate that this splice mutation results in the expression of a truncated protein that is attached to the axoneme, indicating that the mutant protein retains partial function. This allows for the assembly of some ODAs and a significant level of ciliary activity that may result in the atypically mild clinical phenotype. The results also suggest that partial restoration of ciliary function by therapeutic agents could lead to significant improvement of disease symptoms.

Structural insights into the cause of human RSPH4A primary ciliary dyskinesia.

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Component 4A (RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we present the native three-dimensional structure of RSPH4A-/- human respiratory cilia using samples collected noninvasively from a PCD patient. Using cryo-electron tomography (cryo-ET) and subtomogram averaging, we compared the structures of control and RSPH4A-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A-/- cilia. However, RSPH4A-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination.

Induction of ciliary orientation by matrix patterning and characterization of mucociliary transport.

Impaired mucociliary clearance (MCC) is a key feature of many airway diseases, including asthma, bronchiectasis, chronic obstructive pulmonary disease, cystic fibrosis, and primary ciliary dyskinesia. To improve MCC and develop new treatments for these diseases requires a thorough understanding of how mucus concentration, mucus composition, and ciliary activity affect MCC, and how different therapeutics impact this process. Although differentiated cultures of human airway epithelial cells are useful for investigations of MCC, the extent of ciliary coordination in these cultures varies, and the mechanisms controlling ciliary orientation are not completely understood. By introducing a pattern of ridges and grooves into the underlying collagen substrate, we demonstrate for the first time, to our knowledge, that changes in the extracellular matrix can induce ciliary alignment. Remarkably, 90% of human airway epithelial cultures achieved continuous directional mucociliary transport (MCT) when grown on the patterned substrate. These cultures maintain transport for months, allowing carefully controlled investigations of MCC over a wide range of normal and pathological conditions. To characterize the system, we measured the transport of bovine submaxillary gland mucin (BSM) under several conditions. Transport of 5% BSM was significantly reduced compared with that of 2% BSM, and treatment of 5% BSM with the reducing agent tris(2-carboxyethyl)phosphine (TCEP) reduced viscosity and increased the rate of MCT by approximately twofold. Addition of a small amount of high-molecular-weight DNA increased mucus viscosity and reduced MCT by ∼75%, demonstrating that the composition of mucus, as well as the concentration, can have significant effects on MCT. Our results demonstrate that a simple patterning of the collagen substrate results in highly coordinated ciliated cultures that develop directional MCT, and can be used to investigate the mechanisms controlling the regulation of ciliary orientation. Furthermore, the results demonstrate that this method provides an improved system for studying the effects of mucus composition and therapeutic agents on MCC.

Motile cilia genetics and cell biology: big results from little mice.

Our understanding of motile cilia and their role in disease has increased tremendously over the last two decades, with critical information and insight coming from the analysis of mouse models. Motile cilia form on specific epithelial cell types and typically beat in a coordinated, whip-like manner to facilitate the flow and clearance of fluids along the cell surface. Defects in formation and function of motile cilia result in primary ciliary dyskinesia (PCD), a genetically heterogeneous disorder with a well-characterized phenotype but no effective treatment. A number of model systems, ranging from unicellular eukaryotes to mammals, have provided information about the genetics, biochemistry, and structure of motile cilia. However, with remarkable resources available for genetic manipulation and developmental, pathological, and physiological analysis of phenotype, the mouse has risen to the forefront of understanding mammalian motile cilia and modeling PCD. This is evidenced by a large number of relevant mouse lines and an extensive body of genetic and phenotypic data. More recently, application of innovative cell biological techniques to these models has enabled substantial advancement in elucidating the molecular and cellular mechanisms underlying the biogenesis and function of mammalian motile cilia. In this article, we will review genetic and cell biological studies of motile cilia in mouse models and their contributions to our understanding of motile cilia and PCD pathogenesis.

Secretory Cells Dominate Airway CFTR Expression and Function in Human Airway Superficial Epithelia.

Rationale: Identification of the specific cell types expressing CFTR (cystic fibrosis [CF] transmembrane conductance regulator) is required for precision medicine therapies for CF. However, a full characterization of CFTR expression in normal human airway epithelia is missing. Objectives: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single-cell RNA (scRNA) sequencing (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. scRNA in situ hybridization (ISH) and single-cell qRT-PCR were performed for validation. In vitro culture systems correlated CFTR function with cell types. Lentiviruses were used for cell type-specific transduction of wild-type CFTR in CF cells. Measurements and Main Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and, particularly, small airway superficial epithelia, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, whereas the expression in ciliated cells was infrequent and low. scRNA ISH and single-cell qRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to those of normal control subjects. CFTR mediated Cl- secretion in cultures tracked secretory cell, but not ionocyte, densities. Furthermore, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells and not with ionocytes. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not in ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.

Mutation of CFAP57, a protein required for the asymmetric targeting of a subset of inner dynein arms in Chlamydomonas, causes primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, reduced fertility, and randomization of the left/right body axis. It is caused by defects of motile cilia and sperm flagella. We screened a cohort of affected individuals that lack an obvious axonemal defect for pathogenic variants using whole exome capture, next generation sequencing, and bioinformatic analysis assuming an autosomal recessive trait. We identified one subject with an apparently homozygous nonsense variant [(c.1762C>T), p.(Arg588*)] in the uncharacterized CFAP57 gene. Interestingly, the variant results in the skipping of exon 11 (58 amino acids), which may be due to disruption of an exonic splicing enhancer. In normal human nasal epithelial cells, CFAP57 localizes throughout the ciliary axoneme. Nasal cells from the PCD patient express a shorter, mutant version of CFAP57 and the protein is not incorporated into the axoneme. The missing 58 amino acids include portions of WD repeats that may be important for loading onto the intraflagellar transport (IFT) complexes for transport or docking onto the axoneme. A reduced beat frequency and an alteration in ciliary waveform was observed. Knockdown of CFAP57 in human tracheobronchial epithelial cells (hTECs) recapitulates these findings. Phylogenetic analysis showed that CFAP57 is highly conserved in organisms that assemble motile cilia. CFAP57 is allelic with the BOP2/IDA8/FAP57 gene identified previously in Chlamydomonas reinhardtii. Two independent, insertional fap57 Chlamydomonas mutant strains show reduced swimming velocity and altered waveforms. Tandem mass tag (TMT) mass spectroscopy shows that FAP57 is missing, and the "g" inner dyneins (DHC7 and DHC3) and the "d" inner dynein (DHC2) are reduced, but the FAP57 paralog FBB7 is increased. Together, our data identify a homozygous variant in CFAP57 that causes PCD that is likely due to a defect in the inner dynein arm assembly process.

Identification of genetic variants in CFAP221 as a cause of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare disorder that affects the biogenesis or function of motile cilia resulting in chronic airway disease. PCD is genetically and phenotypically heterogeneous, with causative mutations identified in over 40 genes; however, the genetic basis of many cases is unknown. Using whole-exome sequencing, we identified three affected siblings with clinical symptoms of PCD but normal ciliary structure, carrying compound heterozygous loss-of-function variants in CFAP221. Computational analysis suggests that these variants are the most damaging alleles shared by all three siblings. Nasal epithelial cells from one of the subjects demonstrated slightly reduced beat frequency (16.5 Hz vs 17.7 Hz, p = 0.16); however, waveform analysis revealed that the CFAP221 defective cilia beat in an aberrant circular pattern. These results show that genetic variants in CFAP221 cause PCD and that CFAP221 should be considered a candidate gene in cases where PCD is suspected but cilia structure and beat frequency appear normal.

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.

Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance.

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD.

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.

Quantitative Proteomic Analysis of Human Airway Cilia Identifies Previously Uncharacterized Proteins of High Abundance.

Cilia are essential to many diverse cellular processes. Although many major axonemal components have been identified and studied, how they interact to form a functional axoneme is not completely understood. To further our understanding of the protein composition of human airway cilia, we performed a semiquantitative analysis of ciliary axonemes using label-free LC/MSE, which identified over 400 proteins with high confidence. Tubulins were the most abundant proteins identified, with evidence of 20 different isoforms obtained. Twelve different isoforms of axonemal dynein heavy chain were also identified. Absolute quantification of the nontubulin components demonstrated a greater than 75-fold range of protein abundance (RSPH9;1850 fmol vs CCDC103;24 fmol), adding another level of complexity to axonemal structure. Of the identified proteins, ∼70% are known axonemal proteins. In addition, many previously uncharacterized proteins were identified. Unexpectedly, several of these, including ERICH3, C1orf87, and CCDC181, were present at high relative abundance in the cilia. RT-PCR analysis and immunoblotting confirmed cilia-specific expression for eight uncharacterized proteins, and fluorescence microscopy demonstrated unique axonemal localizations. These studies have provided the first quantitative analysis of the ciliary proteome and have identified and characterized several previously unknown proteins as major constituents of human airway cilia.

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.

High-speed and high-sensitivity parallel spectral-domain optical coherence tomography using a supercontinuum light source.

The three most important metrics in optical coherence tomography (OCT) are resolution, speed, and sensitivity. Because there is a complex interplay between these metrics, no previous work has obtained the best performance in all three metrics simultaneously. We demonstrate that a high-power supercontinuum source, in combination with parallel spectral-domain OCT, achieves an unparalleled combination of resolution, speed, and sensitivity. This system captures cross-sectional images spanning 4 mm×0.5 mm at 1,024,000 lines/s with 2×14 µm resolution (axial×transverse) at a sensitivity of 113 dB. Imaging using the proposed system is demonstrated on highly differentiated human bronchial epithelial cells to capture and spatially localize ciliary dynamics.

Diffusion-sensitive optical coherence tomography for real-time monitoring of mucus thinning treatments.

Mucus hydration (wt%) has become an increasingly useful metric in real-time assessment of respiratory health in diseases like cystic fibrosis and COPD, with higher wt% indicative of diseased states. However, available in vivo rheological techniques are lacking. Gold nanorods (GNRs) are attractive biological probes whose diffusion through tissue is sensitive to the correlation length of comprising biopolymers. Through employment of dynamic light scattering theory on OCT signals from GNRs, we find that weakly-constrained GNR diffusion predictably decreases with increasing wt% (more disease-like) mucus. Previously, we determined this method is robust against mucus transport on human bronchial epithelial (hBE) air-liquid interface cultures (R2=0.976). Here we introduce diffusion-sensitive OCT (DS-OCT), where we collect M-mode image ensembles, from which we derive depth- and temporally-resolved GNR diffusion rates. DS-OCT allows for real-time monitoring of changing GNR diffusion as a result of topically applied mucus-thinning agents, enabling monitoring of the dynamics of mucus hydration never before seen. Cultured human airway epithelial cells (Calu-3) with a layer of endogenous mucus were doped with topically deposited GNRs (80×22nm), and subsequently treated with hypertonic saline (HS) or isotonic saline (IS). DS-OCT provided imaging of the mucus thinning response up to a depth of 600µm with 4.65µm resolution, over a total of 8 minutes in increments of ≥3 seconds. For both IS and HS conditions, DS-OCT captured changes in the pattern of mucus hydration over time. DS-OCT opens a new window into understanding mechanisms of mucus thinning during treatment, enabling real-time efficacy feedback needed to optimize and tailor treatments for individual patients.