Airway Mucosa Research Articles

Identification of neutrophils and eosinophils in upper airway mucosa with immunofluorescence multiplex image cytometry.

Characterization of inflammation in chronic rhinosinusitis with (CRSwNP) and without nasal polyps (CRSsNP) is an ongoing research process. To overcome limitations of current cytologic techniques, we investigated whether immunofluorescence multiplex image cytometry could quantify intact neutrophils, eosinophils, and other immune cells in solid upper airway mucosa. We used a four-channel immunofluorescence-microscopy technique for the simultaneous detection of the leukocyte marker CD45, the neutrophil marker myeloperoxidase, two eosinophil markers, i.e., major basic protein and eosinophil peroxidase, and DAPI (4',6-diamidin-2-phenylindole), in formalin-fixed paraffin-embedded upper airway tissue samples of patients with CRSwNP and CRSsNP, as well as of patients free of CRS with inferior turbinate hypertrophy (controls). Image acquisition and analysis were performed with TissueFAXS and StrataQuest (TissueGnostics, Vienna, Austria), respectively. Positive and negative immunostaining were differentiated with a specific fluorescence signal/background signal ratio. Isotype controls were used as negative controls. In six controls, nine patients with CRSsNP, and 11 patients with CRSwNP, the median area scanned and median cell count per patient were 14.2mm2 and 34,356, respectively. In CRSwNP, the number of eosinophils was three times higher (23%) than that of neutrophils (7%). Three times more immune cells were encountered in CRSwNP (33%) compared to CRSsNP (11%). In controls, inflammation was balanced between the epithelial layer and lamina propria, in contrast to CRS (three times more pronounced inflammation in the lamina propria). The quantification of intact neutrophils, eosinophils, and other immune cells in solid tissue with undisrupted architecture seems feasible with immunofluorescence multiplex image cytometry.

Role of TRPV1 and TRPA1 in TSLP production in nasal epithelial cells

BackgroundTRP protein is sensitive to external temperature changes, but its pathogenic mechanism in the upper airway mucosa is still unclear. ObjectiveTo investigate the mechanism of TRPV1and TRPA1 in regulating the secretion of inflammatory factors in nasal epithelial cells. MethodsThe expression of TRPV1 and TRPA1 in nasal mucosal epithelial cells was investigated using immunofluorescence assays. Epithelial cells were stimulated with TRPV1 and TRPA1 agonists and antagonists, and changes in Ca2+ release and inflammatory factor secretion in epithelial cells were detected. TSLP secretion stimulated with the calcium chelating agent EGTA was evaluated. The transcription factor NFAT was observed by immunofluorescence staining. ResultsTRPV1 and TRPA1 expression was detected in nasal epithelial cells, and Ca2+ influx was increased after stimulation with agonists. After the activation of TRPV1 and TRPA1, the gene expression of TSLP, IL-25, and IL-33 and the protein expression levels of TSLP and IL-33 were increased, and only TSLP could be inhibited by antagonists and siRNAs. After administration of EGTA, the secretion of TSLP was inhibited significantly, and the expression of the transcription factor NFAT in the nucleus was observed after activation of the TRPV1 and TRPA1 proteins in epithelial cells. ConclusionActivation of TRPV1 and TRPA1 on nasal epithelial cells stimulates the generation of TSLP through the Ca2+/NFAT pathway. It also induces upregulation of IL-25 and IL-33 gene expression levels and increased levels of IL-33 protein, leading to the development of airway inflammation.

Cryptococcus neoformans rapidly invades the murine brain by sequential breaching of airway and endothelial tissues barriers, followed by engulfment by microglia.

Cryptococcus neoformans causes lethal meningitis and accounts for approximately 10%-15% of AIDS-associated deaths worldwide. There are major gaps in our understanding of how this fungus invades the mammalian brain. To investigate the dynamics of C. neoformans tissue invasion, we mapped fungal localization and host cell interactions in infected brain, lung, and upper airways using mouse models of systemic and airway infection. To enable this, we developed an in situ imaging pipeline capable of measuring large volumes of tissue while preserving anatomical and cellular information by combining thick tissue sections, tissue clarification, and confocal imaging. We confirm high fungal burden in mouse upper airway after nasal inoculation. Yeast in turbinates were frequently titan cells, with faster kinetics than reported in mouse lungs. Importantly, we observed one instance of fungal cells enmeshed in lamina propria of the upper airways, suggesting penetration of airway mucosa as a possible route of tissue invasion and dissemination to the bloodstream. We extend previous literature positing bloodstream dissemination of C. neoformans, by finding viable fungi in the bloodstream of mice a few days after intranasal infection. As early as 24 h post systemic infection, the majority of C. neoformans cells traversed the blood-brain barrier, and were engulfed or in close proximity to microglia. Our work presents a new method for investigating microbial invasion, establishes that C. neoformans can breach multiple tissue barriers within the first days of infection, and demonstrates microglia as the first cells responding to C. neoformans invasion of the brain.IMPORTANCECryptococcal meningitis causes 10%-15% of AIDS-associated deaths globally. Still, brain-specific immunity to cryptococci is a conundrum. By employing innovative imaging, this study reveals what occurs during the first days of infection in brain and in airways. We found that titan cells predominate in upper airways and that cryptococci breach the upper airway mucosa, which implies that, at least in mice, the upper airways are a site for fungal dissemination. This would signify that mucosal immunity of the upper airway needs to be better understood. Importantly, we also show that microglia, the brain-resident macrophages, are the first responders to infection, and microglia clusters are formed surrounding cryptococci. This study opens the field to detailed molecular investigations on airway immune response, how fungus traverses the blood-brain barrier, how microglia respond to infection, and ultimately how microglia monitor the blood-brain barrier to preserve brain function.

Similarity Network Analysis of the Adaptive Immune Response in the Proximal Airway.

Recent immunologic study of the adaptive immune repertoire in the subglottic airway demonstrated high-frequency T cell clones that do not overlap between individuals. However, the anatomic distribution and antigenic target of the T cell repertoire in the proximal airway mucosa remain unresolved. Single-cell RNA sequencing of matched scar and unaffected mucosa from idiopathic subglottic stenosis patients (iSGS, n = 32) was performed and compared with airway mucosa from healthy controls (n = 10). T cell receptor (TCR) sequences were interrogated via similarity network analysis to explore antigenic targets using the published algorithm: Grouping of Lymphocyte Interactions by Paratope Hotspots (GLIPH2). The mucosal T cell repertoire in healthy control airways consisted of highly expressed T cell clones conserved across anatomic subsites (trachea, bronchi, bronchioles, and lung). In iSGS, high-frequency clones were equally represented in both scar and adjacent non-scar tissue. Significant differences in repertoire structure between iSGS scar and unaffected mucosa was observed, driven by unique low-frequency clones. GLIPH2 results suggest low-frequency clones share targets between multiple iSGS patients. Healthy airway mucosa has a highly conserved T cell repertoire across multiple anatomic subsites. Similarly, iSGS patients have highly expressed T cell clones present in both scar and unaffected mucosa. iSGS airway scar possesses an abundance of less highly expanded clones with predicted antigen targets shared between patients. Interrogation of these shared motifs suggests abundant adaptive immunity to viral targets in iSGS airway scar. These results provide insight into disease pathogenesis and illuminate new treatment strategies in iSGS. Level NA Laryngoscope, 2024.

A Clinical Study on Video Bronchoscopy-guided Coblation for Benign Central Airway Stenosis.

Background: Benign central airway stenosis poses a significant challenge to respiratory and thoracic surgeons due to the high recurrence rate associated with current treatment methods, causing severe breathing difficulties and potentially life-threatening complications. This article aims to investigate the therapeutic efficacy and prospects of using coblation in the management of benign central airway stenosis in adults. Moreover, the pathogenesis of benign central airway stenosis was deeply explored to provide better guidance for future clinical treatments. Materials and Methods: This retrospective study examined patients with benign central airway stenosis who were treated at The Second Hospital of Hebei Medical University from 2017 to 2020. In addition, a comparative analysis of whole-genome sequencing was conducted between the aforementioned patient group and healthy populations to investigate the underlying etiology of this stenotic condition. Results: The present study encompassed 32 patients who underwent 43 treatments in total between 2017 and 2020. All patients exhibited alleviation of airway stenosis and an improvement in clinical symptoms following surgery, without any significant surgical or postoperative complications. Whole-genome analysis revealed significant changes in gene expression in the airway mucosa of patients with benign airway stenosis in comparison to healthy populations. A total of 91 differentially expressed genes were identified, among which 44 upregulated genes displayed characteristics of promoting inflammatory responses. Conclusion: Coblation demonstrates promise as an efficacious treatment modality for adults suffering from benign central airway stenosis, and its widespread application in clinical settings is anticipated. The direct pathogenesis of benign central airway stenosis involves airway lumen narrowing and obstruction resulting from excessive inflammation and proliferative granulation.

IL-5 antagonism reverses priming and activation of eosinophils in severe eosinophilic asthma

Eosinophils are key effector cells mediating airway inflammation and exacerbation in patients with severe eosinophilic asthma. They are present in increased number and activation state in the airway mucosa and lumen. Interleukin-5 (IL-5) is the key eosinophil growth factor that is thought to play a role in eosinophil priming and activation. However, the mechanism of these effects is still not fully understood. The anti-IL-5 antibody mepolizumab reduces eosinophil counts in the airway modestly but has a large beneficial effect on the frequency of exacerbations of severe eosinophilic asthma, suggesting that reduction in eosinophil priming and activation is of central mechanistic importance. In this study, we used the therapeutic effect of mepolizumab and single cell RNAseq to investigate the mechanism of eosinophil priming and activation by IL-5. We demonstrated that IL-5 is a dominant driver of eosinophil priming and plays multifaceted roles in eosinophil function. It enhances eosinophil responses to other stimulators of migration, survival and activation by activating PI3K, MAPK and p38 signal pathways. It also enhances the pro-fibrotic roles of eosinophils in airway remodeling via TGF-β pathway. These findings provide mechanistic understanding of eosinophil priming in severe eosinophilic asthma and the therapeutic effect of anti-IL-5 approaches in the disease.

Genomic attributes of airway commensal bacteria and mucosa

Microbial communities at the airway mucosal barrier are conserved and highly ordered, in likelihood reflecting co-evolution with human host factors. Freed of selection to digest nutrients, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the initial results of systematic culture and whole-genome sequencing of the thoracic airway bacteria, identifying 52 novel species amongst 126 organisms that constitute 75% of commensals typically present in heathy individuals. Clinically relevant genes encode antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. Using whole-genome content we identify dysbiotic features that may influence asthma and chronic obstructive pulmonary disease. We match isolate gene content to transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways to sustain host interactions with microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosa in lung diseases of global significance.

Impact of social distancing from the COVID-19 pandemic on the immuno-inflammatory response of older adults

BackgroundOlder adults, as the population considered at increased risk for severe COVID-19, were the most impacted by social isolation. Thus, this study aimed to assess the salivary immune/inflammatory response of older adults before and during the COVID-19 pandemic.MethodsA cohort of 11 older adults (mean age 66.8 ± 6.1) was followed at three different time points: before (S1) and after 6 (S2) and 20 months (S3) of the beginning of the COVID-19 pandemic in Brazil. Unstimulated saliva samples were obtained to assess the levels of antibodies (secretory IgA, IgG and IgM) by ELISA and cytokines (IL-2, IL-5, IL-6, IL-8 and IL-10, TSLP, IFN-γ, TNF-α) by multiplex analysis. Significant differences were evaluated using the Kruskal–Wallis test with Dunn's post-test.ResultsNone volunteer presented periodontal disease or caries. All volunteers received at least two doses of the COVID-19 vaccines after S2 and before S3. A tendency to increase salivary levels of SIgA and IgM at S2 and of IgG at S3 were observed compared to the values found at S1 and S2. Significantly decreased levels of IL-2 and IL-5 were found at S2 and S3 (p < 0.001) time points. Lower levels of IFN-γ were found at S2 as compared to the values observed at S1 (p < 0.01). A significant decrease in the IFN-γ/IL-10 ratio was found at S2 (p < 0.01). When assessing the Th1/Th2 ratios, a significant decrease was found in the IFN-γ/TSLP ratio at S2 (p < 0.001) and S3 (p < 0.001) when compared to the values at S1. In addition, a significant increase was observed in the TNF-α/IL-5 ratio at S2 (p < 0.001) and S3 (p < 0.001) in comparison to the values at S1. In a similar way, an increase in the TNF-α/IL-6 ratio (Fig. 5E) was observed at S3 (p < 0.001) when compared to the values at S1.ConclusionsOverall, this study provides valuable insights into the impact of COVID-19-induced social isolation on immune/inflammatory responses in the upper airway mucosa, particularly those present in oral cavity, of older adults. It demonstrates that a controlled shift in Th1 and Th2 immune responses, both during infection and post-vaccination, can create favorable conditions to combat viral infections without exacerbating the immune response or worsening the pathology.

Eosinophil granulocytes in chronic inflammatory respiratory diseases and CRSwNP: Function, immunological basis, and clinical significance.

Eosinophils play an important regulatory and immunomodulatory role in airway mucosa and have antiparasitic and antiviral properties as well as pro-inflammatory effects that may also cause persistence of inflammation with tissue remodeling. The number of eosinophils and the detection of specific mediators in biological samples from, e.g., blood, nasal secretions, and bronchial fluid can serve as biomarkers that reflect the underlying pathophysiology of certain diseases, predict treatment success, and detect therapy effects. A literature search was conducted to determine the immunologic basis, mode of action, clinical significance, and available evidence for therapeutic approaches using eosinophil-targeted monoclonal antibodies by searching Medline, Pubmed, and the national and international trial database (ClinicalTrials.gov) and guideline registries as well as the Cochrane Library. Human studies published on the topic in the period up to and including 10/2023 were considered. Based on the international literature and previous experience, the results are summarized, and recommendations are given. The important role of eosinophils in immunological processes in the airway mucosa is comprehensively analyzed and can serve as a basis for current and future treatment approaches.

EGPA: Eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome) as a special presentation of chronic rhinosinusitis with nasal polyps (CRSwNP).

Eosinophilic granulomatosis with polyangiitis (EGPA) was formerly known as Churg-Strauss syndrome. The condition is characterized by disseminated necrotizing vasculitis with extravascular granulomas associated with hypereosinophilia. The vasculitides affect small vessels and are associated with antineutrophil cytoplasmic antibodies (ANCAs) detectable in the blood. Distinguishing between type 2-mediated chronic airway inflammation such as chronic rhinosinusitis with nasal polyps (CRSwNP) without vasculitis can be clinically challenging and should be considered. Immunological background, diagnosis, and therapy of EGPA were identified through literature searches in Medline, PubMed, as well as national and international studies (ClinicalTrials.gov) and the Cochrane Library. Human studies published up to and including 10/2023 on the topic were considered. In cases of deteriorating general health with previously known eosinophilic inflammation of the upper and lower airways, EGPA and its interdisciplinary investigation should be considered. Various types of eosinophilic inflammation and syndromes must be considered differentially. Characterization of mucosal airway inflammation through biomarker determination is meaningful and occasionally makes the difference for targeted therapy.

Nasal polyps show decreased mucociliary transport despite vigorous ciliary beating

ObjectiveMucociliary transport function in the airway mucosa is essential for maintaining a clean mucosal surface. This function is impaired in upper and lower airway diseases. Nasal polyps are a noticeable pathological feature that develop in some of the patients with chronic rhinosinusitis. Like ordinary nasal mucosae, nasal polyps have a ciliated pseudostratified epithelium with vigorous ciliary beating. We measured ex vivo Mucociliary Transport Velocity (MCTV) and Ciliary Beat Frequency (CBF) and explored the expressions of Planar Cell Polarity (PCP) proteins in nasal polyps in comparison with turbinate mucosae. MethodsInferior turbinates and nasal polyps were surgically collected from patients with chronic rhinosinusitis. Ex vivo MCTV and CBF were measured using a high-speed digital imaging system. Expressions of PCP proteins were explored by fluorescence immunohistochemistry and quantitative RT-PCR. ResultsThe MCTV of nasal polyps was significantly lower than that of the turbinates (7.43 ± 2.01 vs. 14.56 ± 2.09 μm/s; p = 0.0361), whereas CBF did not differ between the two tissues. The MCTV vector was pointed to the posteroinferior direction in all turbinates with an average inclination angle of 41.0 degrees. Immunohistochemical expressions of Dishevelled-1, Dishevelled-3, Frizzled3, Frizzled6, Prickle2 and Vangl2 were lower in the nasal polyps than in the turbinates. Confocal laser scanning microscopy showed that Frizzled3 was localized along the cell junction on the apical surface. The expression levels of mRNAs for Dishevelled-1, Dishevelled-3 and Frizzled3 in the nasal polyps were also decreased in comparison with the turbinates. ConclusionThese results indicate that muco ciliary transport in nasal polyps is impaired although vigorous ciliary beating is maintained, and that the impairment may be caused by a decrease in Dishevelled/Frizzled proteins and resultant PCP disarrangement. Level of evidenceLevel 3.

MYCOPLASMATIC (ENZOOTIC) PNEUMONIA OF PIGS AS A HEALTH PROBLEM IN FATTENING UNITS

Mycopasmatic or enzootic pneumonia is the most common disease of the respiratory system under in the intensive pig production. It is clinically manifested by coughing, a chronic inflammatory process in the lungs, high morbidity and a relatively low percentage of deaths. The infection can be transmitted horizontally and vertically. Mycoplasma hyopneumoniae invades the epithelial cells of the trachea, bronchi, bronchioles and alveoli and disrupts the function of the ciliary body. Evagination of epithelial cells occurs, so that the cleansing of the airway mucosa by the mucociliary apparatus is inhibited. As a result, bacterial complications (Pasteurella, Bordetella, Klebsiella, Actinobacillus, Hemophilus) are common.M.hyopneminia can play imortant role in PRDC. The development of Mycoplasma hyopneumoniae is favoured by large congregations of pigs in small spaces, inadequate environmental conditions (microclimate), parasitic infections and inadequate nutrition. Mycoplasma hyopneumonia can be a significant health problem on the fattening farm, exacerbated by the influence of non-specific factors as well as the spread of other bacterial pathogens.

Transient Anti-Interferon Auto Antibodies in the Airway Are Associated with Recovery in Mild COVID-19

Pre-existing blood anti-interferon autoantibodies have been associated with susceptibility to severe COVID-19. However, nothing is known about the autoantibody response in the airway mucosa of mild to severe COVID-19 patients. We profiled longitudinal samples collected from the airway and matching blood of more than 100 donors over &amp;gt;18 months, spanning from mild COVID-19 to hospitalized patients in the ICU. We found that transiently induced anti-type I interferon autoantibodies in the nasal mucosa are a common feature and are associated with recovery from mild and moderate COVID-19. To quantify the breadth of antibody response in blood and the airway site of infection, we developed flowBEAT, a novel flow cytometry-based assay capable of simultaneously measuring all 8 human antibody isotypes against 22 SARS-CoV-2 structural and non-structural antigens, including autoantigens. We identified the induction of nasal anti-type I interferon autoantibodies within the first two weeks of infection, waning with disease recovery. Notably, there was an inverse association between nasal and blood anti-interferon responses, while nasal anti-interferon was linked to recovery in cases of mild/moderate disease. In contrast, near-ubiquitous anti-interferon in the endotracheal aspirates of critical cases correlated with heightened airway hyper-inflammation. In both blood and mucosa, anti-interferon positively correlated with a broad anti-viral antibody response, including non-structural proteins. While nasal anti-interferon was universally transient, we identified a group of donors sustaining long-term blood anti-interferon autoantibodies who may be at risk for a more severe subsequent viral infection. Within our longitudinal cohort, we assessed 500 infected-recovered and vaccinated samples and showed that nasal anti-SARS-CoV-2 antibodies are sustained over 18 months by repeated vaccination and boosting. We identified isotype signatures associated with disease severity and an autoantibody signature predictive of vaccine non-response. Thus, our findings emphasize the importance of continued vaccination/boosting to sustain nasal immunity and help prevent ongoing community spread. More importantly, our studies reveal predictive blood markers of protective nasal immunity and highlight a protective role for nasal anti-interferon autoantibodies in the immunopathology of COVID-19.

Pressure-controlled ventilation-volume guaranteed mode improves bronchial mucus transport velocity in patients during laparoscopic surgery for gynecological oncology: a randomized controlled study

BackgroundMechanical ventilation during general anesthesia may impair airway mucosal function. This study aimed to investigate the effect of pressure-controlled ventilation-volume guaranteed (PCV-VG) on bronchial mucus transport velocity (BTV) in patients during laparoscopic surgery for gynecological oncology compared with volume controlled ventilation (VCV).Methods66 patients undergoing elective a laparoscopic surgery for gynecological oncology. The patients were randomized into two group receiving either PCV-VG or VCV. a drop of methylene blue was placed on the surface of the airway mucosa under the bronchoscopeand, then the distance the dye movement was measured after 2, 4, and 6 min. Outcomes were assessed at T0 (5 min after endotracheal intubation and before initiation of pneumoperitoneum), T1 and T2 (1 and 2 h after stabilization of pneumoperitoneum respectively). BTV at T0, T1 and T2 was the primary outcome. Secondary outcomes included heart rate (HR), mean arterial pressure (MAP), body temperature, end-tidal CO2 pressure (PETCO2), tidal volume(VT), peak inspiratory pressure (PIP), mean inspiratory pressure (Pmean), respiratory rate (RR), and dynamic compliance (CDyn) at T0, T1, and T2.Results64 patients were included in the analysis. The median [interquartile range] BTV was significantly lower in VCV group at T1 and T2 that at T0 (P < 0.05). Furthermore, BTV was slightly reduced in PCV-VG compared with VCV. BTV in PCV-VG was significantly decreased at T2 compared with BTV at T0 (P < 0.05) and slightly decreased at T1 compared with T0(P > 0.05). Compared with the PCV-VG group, BTV in VCV group significantly decreased at T2 (P < 0.05). No participants experienced respiratory complications.ConclusionsPCV-VG is more suitable for patients undergoing laparoscopic surgery for gynecological oncology than VCV since it can protect mucociliary clearance function.Trial registrationThis trial is registered on https://www.chictr.org.cn/ in Chinese Clinical Trial Registry (ChiCTR.2200064564: Date of registration 11/10/2022).

A robust mRNA signature obtained via recursive ensemble feature selection predicts the responsiveness of omalizumab in moderate-to-severe asthma.

Not being well controlled by therapy with inhaled corticosteroids and long-acting β2 agonist bronchodilators is a major concern for severe-asthma patients. The current treatment option for these patients is the use of biologicals such as anti-IgE treatment, omalizumab, as an add-on therapy. Despite the accepted use of omalizumab, patients do not always benefit from it. Therefore, there is a need to identify reliable biomarkers as predictors of omalizumab response. Two novel computational algorithms, machine-learning based Recursive Ensemble Feature Selection (REFS) and rule-based algorithm Logic Explainable Networks (LEN), were used on open accessible mRNA expression data from moderate-to-severe asthma patients to identify genes as predictors of omalizumab response. With REFS, the number of features was reduced from 28,402 genes to 5 genes while obtaining a cross-validated accuracy of 0.975. The 5 responsiveness predictive genes encode the following proteins: Coiled-coil domain- containing protein 113 (CCDC113), Solute Carrier Family 26 Member 8 (SLC26A), Protein Phosphatase 1 Regulatory Subunit 3D (PPP1R3D), C-Type lectin Domain Family 4 member C (CLEC4C) and LOC100131780 (not annotated). The LEN algorithm found 4 identical genes with REFS: CCDC113, SLC26A8 PPP1R3D and LOC100131780. Literature research showed that the 4 identified responsiveness predicting genes are associated with mucosal immunity, cell metabolism, and airway remodeling. Both computational methods show 4 identical genes as predictors of omalizumab response in moderate-to-severe asthma patients. The obtained high accuracy indicates that our approach has potential in clinical settings. Future studies in relevant cohort data should validate our computational approach.

Computational fluid dynamics modeling of aerosol particle transport through lung airway mucosa

Delivery of aerosols to the lung can treat various lung diseases. However, the conducting airways are coated by a protective mucus layer with complex properties that make this form of delivery difficult. Mucus is a non-Newtonian fluid and is cleared from the lungs over time by ciliated cells. Further, its gel-like structure hinders the diffusion of particles through it. Any aerosolized treatment of lung diseases must penetrate the mucosal barrier. Using computational fluid dynamics, a model of the airway mucus and periciliary layer was constructed to simulate the transport of impacted aerosol particles. The model predicts the dosage fraction of particles of a certain size that penetrate the mucus and reach the underlying tissue, as well as the distance downstream of the dosage site where tissue concentration is maximized. Reactions that may occur in the mucus are also considered, with simulated data for the interaction of a model virus and an antibody.

Nanoparticle-Mediated Strategies for Enhanced Drug Penetration and Retention in the Airway Mucosa.

Airway mucus is a complex viscoelastic gel composed mainly of water, glycoproteins, lipids, enzymes, minerals, etc. Among them, glycoproteins are the main factors determining mucus's gel-like rheology. Airway mucus forms a protective barrier by secreting mucin, which represents a barrier for absorption, especially for more lipophilic drugs. It rapidly removes drugs from the airway through the physiological mucus clearance mechanism so drugs cannot remain in the lungs or reach the airway epithelial tissue for a long time. Significant progress has been made in enhancing drug lung deposition recently, but strategies are still needed to help drugs break through the lung mucosal barrier. Based on the physiopathological mechanisms of airway mucus, this paper reviews and summarizes strategies to enhance drug penetration and retention in the airway mucosa mediated by nano-delivery systems, including mucosal permeation systems, mucosal adhesion systems, and enzyme-modified delivery systems. On this basis, the potential and challenges of nano-delivery systems for improving airway mucus clearance are revealed. New ideas and approaches are provided for designing novel nano-delivery systems that effectively improve drug retention and penetration in the airway mucus layer.

Autologous Airway Basal Cell Transplantation Alleviates Airway Epithelium Defect in Recurrent Benign Tracheal Stenosis.

Airway epithelium defects are a hallmark of recurrent benign tracheal stenosis (RBTS). Reconstructing an intact airway epithelium is of great importance in airway homeostasis and epithelial wound healing and has great potential for treating tracheal stenosis. An experimental study was conducted in canines to explore the therapeutic effect of autologous basal cell transplantation in restoring airway homeostasis. First, airway mucosae from human patients with recurrent tracheal stenosis were analyzed by single-cell RNA sequencing. Canines were then randomly divided into tracheal stenosis, Stent, Stent + Cells, and Stent + Cells + Biogel groups. Autologous airway basal cells of canines in the Stent + Cells and Stent + Cells + Biogel groups were transplanted onto the stenotic airway after modeling. A biogel was coated on the airway prior to basal cell transplantation in the Stent + Cells + Biogel group. After bronchoscopic treatments, canines were followed up for 16 weeks. Single-cell RNA sequencing demonstrated packed airway basal cells and an absence of normal airway epithelial cells in patients with RBTS. Autologous airway basal cell transplantation, together with biogel coating, was successfully performed in the canine model. Follow-up observation indicated that survival time in the Stent + Cells + Biogel group was significantly prolonged, with a higher (100%) survival rate compared with the other groups. In terms of pathological and bronchoscopic findings, canines that received autologous basal cell transplantation showed a reduction in granulation hyperplasia as well as airway re-epithelialization with functionally mature epithelial cells. Autologous airway basal cell transplantation might serve as a novel regenerative therapy for airway re-epithelialization and inhibit recurrent granulation hyperplasia in benign tracheal stenosis.

Characterizing the T Cell Repertoire in the Proximal Airway in Health and Disease.

Recent translational scientific efforts in subglottic stenosis (SGS) support a disease model where epithelial alterations facilitate microbiome displacement, dysregulated immune activation, and localized fibrosis. Given the observed immune cell infiltrate in SGS, we sought to test the hypothesis that SGS cases possessed a low diversity (highly clonal) adaptive immune response when compared with healthy controls. Single cell RNA sequencing (scRNA-seq) of subglottic mucosal scar in iSGS (n = 24), iLTS (n = 8), and healthy controls (n = 7) was performed. T cell receptor (TCR) sequences were extracted, analyzed, and used to construct repertoire structure, compare diversity, interrogate overlap, and define antigenic targets using the Immunarch bioinformatics pipeline. The proximal airway mucosa in health and disease are equally diverse via Hill framework quantitation (iSGS vs. iLTS vs. Control, p > 0.05). Repertoires do not significantly overlap between individuals (Morisita <0.02). Among iSGS patients, clonality of the TCR repertoire is driven by CD8+ T cells, and iSGS patients possess numerous TCRs targeting viral and intercellular pathogens. High frequency clonotypes do not map to known targets in public datasets. SGS cases do not possess a lower diversity adaptive immune infiltrate when compared with healthy controls. Interestingly, the TCR repertoire in both health and disease contains a restricted number of high frequency clonotypes that do not significantly overlap between individuals. The target of the high frequency clonotypes in health and disease remain unresolved. NA Laryngoscope, 134:1757-1764, 2024.

Single-cell RNA sequencing of bronchoscopy specimens: development of a rapid minimal handling protocol.

Single-cell RNA sequencing (scRNA-seq) is an important tool for understanding disease pathophysiology, including airway diseases. Currently, the majority of scRNA-seq studies in airway diseases have used invasive methods (airway biopsy, surgical resection), which carry inherent risks and thus present a major limitation to scRNA-seq investigation of airway pathobiology. Bronchial brushing, where the airway mucosa is sampled using a cytological brush, is a viable, less invasive method of obtaining airway cells for scRNA-seq. Here we describe the development of a rapid and minimal handling protocol for preparing single-cell suspensions from bronchial brush specimens for scRNA-seq. Our optimized protocol maximizes cell recovery and cell quality and facilitates large-scale profiling of the airway transcriptome at single-cell resolution.