Longitudinal improvements in clinical and functional outcomes following initiation of elexacaftor/tezacaftor/ivacaftor in patients with cystic fibrosis.

Background: Use of elexacaftor/tezacaftor/ivacaftor (ETI) for treatment of cystic fibrosis (CF) has resulted in unprecedented clinical improvements necessitating development of outcome measures for monitoring disease course. Intranasal micro-optical coherence tomography (µOCT) has previously helped detect and characterize mucociliary abnormalities in patients with CF. This study was done to determine if µOCT can define the effects of ETI on nasal mucociliary clearance and monitor changes conferred to understand mechanistic effects of CFTR modulators beyond CFTR activation. Methods: 26 subjects, with at least 1 F508del mutation were recruited and followed at baseline (visit 1), +1 month (visit 2) and +6 months (visit 4) following initiation of ETI therapy. Clinical outcomes were computed at visits 1, 2 and 4. Intranasal µOCT imaging and functional metrics analysis including mucociliary transport rate (MCT) estimation were done at visits 1 and 2. Results: Percent predicted forced expiratory volume in 1 s (ppFEV1) showed a significant increase of +10.9 % at visit 2, which sustained at visit 4 (+10.6 %). Sweat chloride levels significantly decreased by -36.6 mmol/L and -41.3 mmol/L at visits 2 and 4, respectively. µOCT analysis revealed significant improvement in MCT rate (2.8 ± 1.5, visit 1 vs 4.0 ± 1.5 mm/min, visit 2; P = 0.048). Conclusions: Treatment with ETI resulted in significant and sustained clinical improvements over 6 months. Functional improvements in MCT rate were evident within a month after initiation of ETI therapy indicating that µOCT imaging is sensitive to the treatment effect of HEMT and suggests improved mucociliary transport as a probable mechanism of action underlying the clinical benefits.

Transgenic ferret models define pulmonary ionocyte diversity and function.

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.

Mucociliary transport deficiency and disease progression in Syrian hamsters with SARS-CoV-2 infection.

Substantial clinical evidence supports the notion that ciliary function in the airways is important in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, the extent or nature of impairment of mucociliary transport (MCT) in in vivo models remains unknown. We hypothesize that SARS-CoV-2 infection results in MCT deficiency in the airways of golden Syrian hamsters that precedes pathological injury in lung parenchyma. Micro-optical coherence tomography was used to quantitate functional changes in the MCT apparatus. Both genomic and subgenomic viral RNA pathological and physiological changes were monitored in parallel. We show that SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 days postinfection (dpi) in hamsters, principally due to 79% diminished airway coverage of motile cilia. Correlating quantitation of physiological, virological, and pathological changes reveals steadily descending infection from the upper airways to lower airways to lung parenchyma within 7 dpi. Our results indicate that functional deficits of the MCT apparatus are a key aspect of COVID-19 pathogenesis, may extend viral retention, and could pose a risk factor for secondary infection. Clinically, monitoring abnormal ciliated cell function may indicate disease progression. Therapies directed toward the MCT apparatus deserve further investigation.

STAT3 mutation-associated airway epithelial defects in Job syndrome.

BACKGROUND: Job syndrome is a disease of autosomal dominant hyper-IgE syndrome (AD-HIES). Patients harboring STAT3 mutation are particularly prone to airway remodeling and airway infections. OBJECTIVES: Airway epithelial cells play a central role as the first line of defense against pathogenic infection and express high levels of STAT3. This study thus interrogates how AD-HIES STAT3 mutations impact the physiological functions of airway epithelial cells. METHODS: This study created human airway basal cells expressing 4 common AD-HIES STAT3 mutants (R382W, V463del, V637M, and Y657S). In addition, primary airway epithelial cells were isolated from a patient with Job syndrome who was harboring a STAT3-S560del mutation and from mice harboring a STAT3-V463del mutation. Cell proliferation, differentiation, barrier function, bacterial elimination, and innate immune responses to pathogenic infection were quantitatively analyzed. RESULTS: STAT3 mutations reduce STAT3 protein phosphorylation, nuclear translocation, transcription activity, and protein stability in airway basal cells. As a consequence, STAT3-mutated airway basal cells give rise to airway epithelial cells with abnormal cellular composition and loss of coordinated mucociliary clearance. Notably, AD-HIES STAT3 airway epithelial cells are defective in bacterial killing and fail to initiate vigorous proinflammatory responses and neutrophil transepithelial migration in response to an experimental model of Pseudomonas aeruginosa infection. CONCLUSIONS: AD-HIES STAT3 mutations confer numerous abnormalities to airway epithelial cells in cell differentiation and host innate immunity, emphasizing their involvement in the pathogenesis of lung complications in Job syndrome. Therefore, therapies must address the epithelial defects as well as the previously noted immune cell defects to alleviate chronic infections in patients with Job syndrome.

COVID-19 Causes Ciliary Dysfunction as Demonstrated by Human Intranasal Micro-Optical Coherence Tomography Imaging.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2), causative agent of coronavirus disease 2019 (COVID-19), binds via ACE2 receptors, highly expressed in ciliated cells of the nasal epithelium. Micro-optical coherence tomography (µOCT) is a minimally invasive intranasal imaging technique that can determine cellular and functional dynamics of respiratory epithelia at 1-µm resolution, enabling real time visualization and quantification of epithelial anatomy, ciliary motion, and mucus transport. We hypothesized that respiratory epithelial cell dysfunction in COVID-19 will manifest as reduced ciliated cell function and mucociliary abnormalities, features readily visualized by µOCT. Symptomatic outpatients with SARS-CoV-2 aged ≥ 18 years were recruited within 14 days of symptom onset. Data was interpreted for subjects with COVID-19 (n=13) in comparison to healthy controls (n=8). Significant reduction in functional cilia, diminished ciliary beat frequency, and abnormal ciliary activity were evident. Other abnormalities included denuded epithelium, presence of mucus rafts, and increased inflammatory cells. Our results indicate that subjects with mild but symptomatic COVID-19 exhibit functional abnormalities of the respiratory mucosa underscoring the importance of mucociliary health in viral illness and disease transmission. Ciliary imaging enables investigation of early pathogenic mechanisms of COVID-19 and may be useful for evaluating disease progression and therapeutic response.

Mucociliary Transport Deficiency and Disease Progression in Syrian Hamsters with SARS-CoV-2 Infection.

Substantial clinical evidence supports the notion that ciliary function in the airways plays an important role in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, consequent impaired mucociliary transport (MCT) remains unknown for the intact MCT apparatus from an in vivo model of disease. Using golden Syrian hamsters, a common animal model that recapitulates human COVID-19, we quantitatively followed the time course of physiological, virological, and pathological changes upon SARS-CoV-2 infection, as well as the deficiency of the MCT apparatus using micro-optical coherence tomography, a novel method to visualize and simultaneously quantitate multiple aspects of the functional microanatomy of intact airways. Corresponding to progressive weight loss up to 7 days post-infection (dpi), viral detection and histopathological analysis in both the trachea and lung revealed steadily descending infection from the upper airways, as the main target of viral invasion, to lower airways and parenchymal lung, which are likely injured through indirect mechanisms. SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 dpi, largely due to diminished motile ciliation coverage, but not airway surface liquid depth, periciliary liquid depth, or cilia beat frequency of residual motile cilia. Further analysis indicated that the fewer motile cilia combined with abnormal ciliary motion of residual cilia contributed to the delayed MCT. The time course of physiological, virological, and pathological progression suggest that functional deficits of the MCT apparatus predispose to COVID-19 pathogenesis by extending viral retention and may be a risk factor for secondary infection. As a consequence, therapies directed towards the MCT apparatus deserve further investigation as a treatment modality.

Evaluation of a novel CFTR potentiator in COPD ferrets with acquired CFTR dysfunction.

RATIONALE: The majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been tested in vivo, given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure. OBJECTIVES: To evaluate CFTR dysfunction induced by smoking and test its pharmacological reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis. METHODS: Ferrets were exposed for 6 months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1 month. Electrophysiological measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle-tracking microrheology, microcomputed tomography imaging, histopathological analysis and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes. MEASUREMENTS AND MAIN RESULTS: Following cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced bronchial wall thickening and airway epithelial hypertrophy. CONCLUSIONS: The pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathological features of chronic bronchitis in a COPD ferret model.

Neutrophil dysfunction in cystic fibrosis.

BACKGROUND: Excessive neutrophil inflammation is the hallmark of cystic fibrosis (CF) airway disease. Novel technologies for characterizing neutrophil dysfunction may provide insight into the nature of these abnormalities, revealing a greater mechanistic understanding and new avenues for CF therapies that target these mechanisms. METHODS: Blood was collected from individuals with CF in the outpatient clinic, CF individuals hospitalized for a pulmonary exacerbation, and non-CF controls. Using microfluidic assays and advanced imaging technologies, we characterized 1) spontaneous neutrophil migration using microfluidic motility mazes, 2) neutrophil migration to and phagocytosis of Staphylococcal aureus particles in a microfluidic arena, 3) neutrophil swarming on Candida albicans clusters, and 4) Pseudomonas aeruginosa-induced neutrophil transepithelial migration using micro-optical coherence technology (µOCT). RESULTS: Participants included 44 individuals: 16 Outpatient CF, 13 Hospitalized CF, and 15 Non-CF individuals. While no differences were seen with spontaneous migration, CF neutrophils migrated towards S. aureus particles more quickly than non-CF neutrophils (p < 0.05). CF neutrophils, especially Hospitalized CF neutrophils, generated significantly larger aggregates around S. aureus particles over time. Hospitalized CF neutrophils were more likely to have dysfunctional swarming (p < 0.01) and less efficient clearing of C. albicans (p < 0.0001). When comparing trans-epithelial migration towards Pseudomonas aeruginosa epithelial infection, Outpatient CF neutrophils displayed an increase in the magnitude of transmigration and adherence to the epithelium (p < 0.05). CONCLUSIONS: Advanced technologies for characterizing CF neutrophil function reveal significantly altered migratory responses, cell-to-cell clustering, and microbe containment. Future investigations will probe mechanistic basis for abnormal responses in CF to identify potential avenues for novel anti-inflammatory therapeutics.

Stromal Nerve Imaging and Tracking Using Micro-Optical Coherence Tomography.

Purpose: To image, track and map the nerve fiber distribution in excised rabbit corneas over the entire stromal thickness using micro-optical coherence tomography (µOCT) to develop a screening tool for early peripheral neuropathy. Methods: Excised rabbit corneas were consecutively imaged by a custom-designed µOCT prototype and a commercial laser scanning fluorescence confocal microscope. The µOCT images with a field of view of approximately 1 × 1 mm were recorded with axial and transverse resolutions of approximately 1 µm and approximately 4 µm, respectively. In the volumetric µOCT image data, network maps of hyper-reflective, branched structures traversing different stromal compartments were segmented using semiautomatic image processing algorithms. Furthermore, the same corneas received ßIII-tubulin antibody immunostaining before digital confocal microscopy, and a comparison between µOCT image data and immunohistochemistry analysis was performed to validate the nerval origin of the tracked network structures. Results: Semiautomatic tracing of the nerves with a high range of different thicknesses was possible through the whole corneal volumes, creating a skeleton of the traced nerves. There was a good conformity between the hyper-reflective structures in the µOCT data and the stained nerval structures in the immunohistochemistry data. Conclusions: This article demonstrates nerval imaging and tracking as well as a spatial correlation between µOCT and a fluorescence corneal nerve standard for larger nerves throughout the full thickness of the cornea ex vivo. Translational Relevance: Owing to its advantageous properties, µOCT may become useful as a noncontact method for assessing nerval structures in humans to screen for early peripheral neuropathy.

Imaging intracellular motion with dynamic micro-optical coherence tomography.

This paper describes a new technology that uses 1-µm-resolution optical coherence tomography (µOCT) to obtain cross-sectional images of intracellular dynamics with dramatically enhanced image contrast. This so-called dynamic µOCT (d-µOCT) is accomplished by acquiring a time series of µOCT images and conducting power frequency analysis of the temporal fluctuations that arise from intracellular motion on a pixel-per-pixel basis. Here, we demonstrate d-µOCT imaging of freshly excised human esophageal and cervical biopsy samples. Depth-resolved d-µOCT images of intact tissue show that intracellular dynamics provides a new contrast mechanism for µOCT that highlights subcellular morphology and activity in epithelial surface maturation patterns.

A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance.

Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells1,2. Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies3. Previous studies have identified mutations that affect ciliary structure and nucleation in PCD4, but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance.

Author Correction: A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Aspergillus fumigatus Cell Wall Promotes Apical Airway Epithelial Recruitment of Human Neutrophils.

Aspergillus fumigatus is a ubiquitous fungal pathogen capable of causing multiple pulmonary diseases, including invasive aspergillosis, chronic necrotizing aspergillosis, fungal colonization, and allergic bronchopulmonary aspergillosis. Intact mucociliary barrier function and early airway neutrophil responses are critical for clearing fungal conidia from the host airways prior to establishing disease. Following inhalation, Aspergillus conidia deposit in the small airways, where they are likely to make their initial host encounter with epithelial cells. Challenges in airway infection models have limited the ability to explore early steps in the interactions between A. fumigatus and the human airway epithelium. Here, we use inverted air-liquid interface cultures to demonstrate that the human airway epithelium responds to apical stimulation by A. fumigatus to promote the transepithelial migration of neutrophils from the basolateral membrane surface to the apical airway surface. Promoting epithelial transmigration with Aspergillus required prolonged exposure with live resting conidia. Swollen conidia did not expedite epithelial transmigration. Using A. fumigatus strains containing deletions of genes for cell wall components, we identified that deletion of the hydrophobic rodlet layer or dihydroxynaphthalene-melanin in the conidial cell wall amplified the epithelial transmigration of neutrophils, using primary human airway epithelium. Ultimately, we show that an as-yet-unidentified nonsecreted cell wall protein is required to promote the early epithelial transmigration of human neutrophils into the airspace in response to A. fumigatus Together, these data provide critical insight into the initial epithelial host response to Aspergillus.

Intranasal micro-optical coherence tomography imaging for cystic fibrosis studies.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Although impairment of mucociliary clearance contributes to severe morbidity and mortality in people with CF, a clear understanding of the pathophysiology is lacking. This is, in part, due to the absence of clinical imaging techniques capable of capturing CFTR-dependent functional metrics at the cellular level. Here, we report the clinical translation of a 1-µm resolution micro-optical coherence tomography (µOCT) technology to quantitatively characterize the functional microanatomy of human upper airways. Using a minimally invasive intranasal imaging approach, we performed a clinical study on age- and sex-matched CF and control groups. We observed delayed mucociliary transport rate at the cellular level, depletion of periciliary liquid layer, and prevalent loss of ciliation in subjects with CF. Distinctive morphological differences in mucus and various forms of epithelial injury were also revealed by µOCT imaging and had prominent effects on the mucociliary transport apparatus. Elevated mucus reflectance intensity in CF, a proxy for viscosity in situ, had a dominant effect. These results demonstrate the utility of µOCT to determine epithelial function and monitor disease status of CF airways on a per-patient basis, with applicability for other diseases of mucus clearance.

A glycopolymer improves vascoelasticity and mucociliary transport of abnormal cystic fibrosis mucus.

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.

The airways of the lung are the primary sites of disease in asthma and cystic fibrosis. Here we study the cellular composition and hierarchy of the mouse tracheal epithelium by single-cell RNA-sequencing (scRNA-seq) and in vivo lineage tracing. We identify a rare cell type, the Foxi1+ pulmonary ionocyte; functional variations in club cells based on their location; a distinct cell type in high turnover squamous epithelial structures that we term 'hillocks'; and disease-relevant subsets of tuft and goblet cells. We developed 'pulse-seq', combining scRNA-seq and lineage tracing, to show that tuft, neuroendocrine and ionocyte cells are continually and directly replenished by basal progenitor cells. Ionocytes are the major source of transcripts of the cystic fibrosis transmembrane conductance regulator in both mouse (Cftr) and human (CFTR). Knockout of Foxi1 in mouse ionocytes causes loss of Cftr expression and disrupts airway fluid and mucus physiology, phenotypes that are characteristic of cystic fibrosis. By associating cell-type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease.

Flexible, high-resolution micro-optical coherence tomography endobronchial probe toward in vivo imaging of cilia.

We report the design and fabrication of a flexible, longitudinally scanning high-resolution micro-optical coherence tomography (µOCT) endobronchial probe, optimized for micro-anatomical imaging in airways. The 2.4 mm diameter and flexibility of the probe allows it to be inserted into the instrument channel of a standard bronchoscope, enabling real-time video guidance of probe placement. To generate a depth-of-focus enhancing annular beam, we utilized a new fabrication method, whereby a hollow glass ferrule was angle-polished and gold-coated to produce an elongated annular reflector. We present validation data that verifies the preservation of linear scanning, despite the use of flexible materials. When utilized on excised, cultured mouse trachea, the probe acquired images of comparable quality to those obtained by a benchtop µOCT system.

Micro-optical coherence tomography of the mammalian cochlea.

The mammalian cochlea has historically resisted attempts at high-resolution, non-invasive imaging due to its small size, complex three-dimensional structure, and embedded location within the temporal bone. As a result, little is known about the relationship between an individual's cochlear pathology and hearing function, and otologists must rely on physiological testing and imaging methods that offer limited resolution to obtain information about the inner ear prior to performing surgery. Micro-optical coherence tomography (µOCT) is a non-invasive, low-coherence interferometric imaging technique capable of resolving cellular-level anatomic structures. To determine whether µOCT is capable of resolving mammalian intracochlear anatomy, fixed guinea pig inner ears were imaged as whole temporal bones with cochlea in situ. Anatomical structures such as the tunnel of Corti, space of Nuel, modiolus, scalae, and cell groupings were visualized, in addition to individual cell types such as neuronal fibers, hair cells, and supporting cells. Visualization of these structures, via volumetrically-reconstructed image stacks and endoscopic perspective videos, represents an improvement over previous efforts using conventional OCT. These are the first µOCT images of mammalian cochlear anatomy, and they demonstrate µOCT's potential utility as an imaging tool in otology research.