Continuous mucociliary transport by primary human airway epithelial cells in vitro.
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ABSTRACT:
SUBMITTER: Sears PR
PROVIDER: S-EPMC4504973 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The coronavirus disease (COVID-19) pandemic, caused by SARS-CoV-2 coronavirus, is devastatingly impacting human health. A prominent component of COVID-19 is the infection and destruction of the ciliated respiratory cells, which perpetuates dissemination and disrupts protective mucociliary transport (MCT) function, an innate defense of the respiratory tract. Thus, drugs that augment MCT could improve barrier function of the airway epithelium, reduce viral replication and, ultimately, COVID-19 outcomes. We tested five agents known to increase MCT through distinct mechanisms for activity against SARS-CoV-2 infection using a model of human respiratory epithelial cells terminally differentiated in an air/liquid interphase. Three of the five mucoactive compounds tested showed significant inhibitory activity against SARS-CoV-2 replication. An archetype mucoactive agent, ARINA-1, blocked viral replication and therefore epithelial cell injury, thus, it was further studied using biochemical, genetic and biophysical methods to ascertain mechanism of action via improvement of MCT. ARINA-1 antiviral activity was dependent on enhancing the MCT cellular response, since terminal differentiation, intact ciliary expression and motion was required for ARINA-1-mediated anti-SARS-CoV2 protection. Ultimately, we showed that improvement of cilia movement was caused by ARINA-1-mediated regulation of the redox state of the intracellular environment, which benefited MCT. Our study indicates that Intact MCT reduces SARS-CoV-2 infection, and its pharmacologic activation may be effective as an anti-COVID-19 treatment.
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Project description:The lung is continuously exposed to particles, toxicants, and microbial pathogens that are cleared by a complex mechanical, innate, and acquired immune system. Mucociliary clearance, mediated by the actions of diverse conducting airway and submucosal gland epithelial cells, plays a critical role in a multilayered defense system by secreting fluids, electrolytes, antimicrobial and antiinflammatory proteins, and mucus onto airway surfaces. The mucociliary escalator removes particles and pathogens by the mechanical actions of cilia and cough. Abnormalities in mucociliary clearance, whether related to impaired fluid secretion, ciliary dysfunction, lack of cough, or the disruption of epithelial cells lining the respiratory tract, contribute to the pathogenesis of common chronic pulmonary disorders. Although mucus and other airway epithelial secretions play a critical role in protecting the lung during acute injury, impaired mucus clearance after chronic mucus hyperproduction causes airway obstruction and infection, which contribute to morbidity in common pulmonary disorders, including chronic obstructive pulmonary disease, asthma, idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis, and primary ciliary dyskinesia. In this summary, the molecular and cellular mechanisms mediating airway mucociliary clearance, as well as the role of goblet cell metaplasia and mucus hyperproduction, in the pathogenesis of chronic respiratory diseases are considered.
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Project description:Secreted mucus is a frontline defense against respiratory infection, enabling the capture and swift removal of infectious or irritating agents from the lungs. Airway mucus is composed of two mucins: mucin 5B (MUC5B) and 5AC (MUC5AC). Together, they form a hydrogel that can be actively transported by cilia along the airway surface. In chronic respiratory diseases, abnormal expression of these mucins is directly implicated in dysfunctional mucus clearance. Yet, the role of each mucin in supporting normal mucus transport remains unclear. Here, we generate human airway epithelial tissue cultures deficient in either MUC5B or MUC5AC to understand their individual contributions to mucus transport. We find that MUC5B and MUC5AC deficiency results in impaired and discoordinated mucociliary transport, respectively, demonstrating the importance of each mucin to airway clearance.
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Project description:In vitro differentiation of airway epithelium is of interest for respiratory tissue engineering and studying airway diseases. Both applications benefit from the use of primary cells to maintain a mucociliated phenotype and thus physiological functionality. Complex differentiation procedures often lack standardization and reproducibility. To alleviate these shortfalls, we compared differentiation behavior of human nasal epithelial cells in four differentiation media. Cells were differentiated at the air-liquid interface (ALI) on collagen-coated inserts. Mucociliary differentiation status after five weeks was analyzed by electron microscopy, histology and immunohistochemistry. The amount of ciliation was estimated and growth factor concentrations were evaluated using ELISA. We found that retinoic-acid-supplemented mixture of DMEM and Airway Epithelial Cell Growth Medium gave most promising results to obtain ciliated and mucus producing nasal epithelium in vitro. We discovered the balance between retinoic acid (RA), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor ? (FGF-?) to be relevant for differentiation. We could show that low VEGF, EGF and FGF-? concentrations in medium correspond to absent ciliation in specific donors. Therefore, our results may in future facilitate donor selection and non-invasive monitoring of ALI cultures and by this contribute to improved standardization of epithelial in vitro culture.
| S-EPMC7181704 | biostudies-literature
Project description:Differentiated air-liquid interface models are the current standard to assess the mucociliary phenotype using clinically-derived samples in a controlled environment. However, obtaining basal progenitor airway epithelial cells (AEC) from the lungs is invasive and resource-intensive. Hence, we applied a tissue engineering approach to generate organotypic sinonasal AEC (nAEC) epithelia to determine whether they are predictive of bronchial AEC (bAEC) models. Basal progenitor AEC were isolated from healthy participants using a cytological brushing method and differentiated into epithelia on transwells until the mucociliary phenotype was observed. Tissue architecture was assessed using H&E and alcian blue/Verhoeff-Van Gieson staining, immunofluorescence (for cilia via acetylated α-tubulin labelling) and scanning electron microscopy. Differentiation and the formation of tight-junctions were monitored over the culture period (day 1-32) by quantifying trans-epithelial electrical resistance. End point (day 32) tight junction protein expression was assessed using Western blot analysis of ZO-1, Occludin-1 and Claudin-1. Reverse transcription qPCR-array was used to assess immunomodulatory and autophagy-specific transcript profiles. All outcome measures were assessed using R-statistical software. Mucociliary architecture was comparable for nAEC and bAEC-derived cultures, e.g. cell density P = 0.55, epithelial height P = 0.88 and cilia abundance P = 0.41. Trans-epithelial electrical resistance measures were distinct from day 1-14, converged over days 16-32, and were statistically similar over the entire culture period (global P < 0.001). This agreed with end-point (day 32) measures of tight junction protein abundance which were non-significant for each analyte (P > 0.05). Transcript analysis for inflammatory markers demonstrated significant variation between nAEC and bAEC epithelial cultures, and favoured increased abundance in the nAEC model (e.g. TGFβ and IL-1β; P < 0.05). Conversely, the abundance of autophagy-related transcripts were comparable and the range of outcome measures for either model exhibited a considerably more confined uncertainty distribution than those observed for the inflammatory markers. Organotypic air-liquid interface models of nAEC are predictive of outcomes related to barrier function, mucociliary architecture and autophagy gene activity in corresponding bAEC models. However, inflammatory markers exhibited wide variation which may be explained by the sentinel immunological surveillance role of the sinonasal epithelium.
| S-EPMC9648462 | biostudies-literature
Project description:BackgroundAirway tuft cells, formerly called brush cells have long been described only morphologically in human airways. More recent RNAseq studies described a chemosensory cell population, which includes tuft cells, by a distinct gene transcription signature. Yet, until which level in the tracheobronchial tree in native human airway epithelium tuft cells occur and if they function as regulators of innate immunity, e.g., by regulating mucociliary clearance, remained largely elusive.MethodsWe performed immunohistochemistry, RT-PCR and immunoblotting analyses for various tuft cell markers to confirm the presence of this cell type in human tracheal samples. Immunohistochemistry was conducted to study the distribution of tuft cells along the intrapulmonary airways in humans. We assessed the influence of bitter substances and the taste transduction pathway on mucociliary clearance in mouse and human tracheal samples by measuring particle transport speed.ResultsTuft cells identified by the expression of their well-established marker POU class 2 homeobox 3 (POU2F3) were present from the trachea to the bronchioles. We identified choline acetyltransferase in POU2F3 expressing cells as well as the transient receptor potential melastatin 5 (TRPM5) channel in a small population of tracheal epithelial cells with morphological appearance of tuft cells. Application of bitter substances, such as denatonium, led to an increase in mucociliary clearance in human tracheal preparations. This was dependent on activation of the TRPM5 channel and involved cholinergic and nitric oxide signalling, indicating a functional role for human tuft cells in the regulation of mucociliary clearance.ConclusionsWe were able to detect tuft cells in the tracheobronchial tree down to the level of the bronchioles. Moreover, taste transduction and cholinergic signalling occur in the same cells and regulate mucociliary clearance. Thus, tuft cells are potentially involved in the regulation of innate immunity in human airways.
| S-EPMC10625704 | biostudies-literature
Project description:The development of pathologic mucus, which is not readily cleared from the airways, is an important contributor to the morbidity and mortality associated with asthma. It is not clear how the major airway mucins MUC5AC and MUC5B are organized within the mucus gel or how this gel contributes to airway obstruction in asthma. Here, we demonstrated that mucus plugs from individuals with fatal asthma are heterogeneous gels with distinct MUC5AC- and MUC5B-containing domains. Stimulation of cultured human bronchial epithelial cells with IL-13, a key mediator in asthma, induced the formation of heterogeneous mucus gels and dramatically impaired mucociliary transport. Impaired transport was not associated with defects in ciliary function but instead was related to tethering of MUC5AC-containing mucus gel domains to mucus-producing cells in the epithelium. Replacement of tethered mucus with untethered mucus restored mucociliary transport. Together, our results indicate that tethering of MUC5AC-containing domains to the epithelium causes mucostasis and likely represents a major cause of mucus plugging in asthma.
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Project description:Mast cells (MCs) are found in increased numbers at airway mucosal surfaces in asthmatic patients. Because human airway epithelial cells (HAECs) actively participate in airway inflammatory responses and are in direct contact with MCs in the mucosa, we hypothesized that HAEC-MC interactions may contribute to the differentiation and survival of MCs in the airway mucosa. Here, we show that HAECs express mRNA and protein for soluble and membrane-bound stem cell factor, releasing soluble stem cell factor into the cell culture supernatant at a concentration of 5.9 +/- 0.1 ng per 10(6) HAEC. HAECs were able to support MC survival in coculture in the absence of any exogenous cytokines for at least 4 d. Before the initiation of coculture, MCs were uniformly tryptase and chymase (MC(TC)) double positive, but by 2 d of coculture the majority of MCs expressed tryptase (MC(T)) alone. MCs supported in coculture generated low amounts of cysteinyl-leukotrienes (cys-LT) after FcepsilonRI-dependent activation (0.2 +/- 0.1 ng of cys-LT per 10(6) cells) and required priming with IL-4 and IL-3 during coculture to achieve a quantity of cys-LT generation within the range expected for human lung mucosal MC (26.5 +/- 16 ng of cys-LT per 10(6) cells). In these culture conditions, HAECs were able to direct mucosal MC protease phenotype, but T cell-derived Th2 cytokines were required for the expression of a functional airway MC eicosanoid phenotype. Thus, distinct cell types may direct unique aspects of reactive mucosal MC phenotype in the airways.
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Project description: Not available
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Project description:BACKGROUND: An abnormally high incidence of lung disease has been observed in the residents of Libby, Montana, which has been attributed to occupational and environmental exposure to fibrous amphiboles originating from a nearby contaminated vermiculite mine. The composition of Libby amphibole (LA) is complex and minimal toxicity data are available. In this study, we conduct a comparative particle toxicity analysis of LA compared with standard reference asbestiform amphibole samples. METHODS: Primary human airway epithelial cells (HAEC) were exposed to two different LA samples as well as standard amphibole reference samples. Analysis of the samples included a complete particle size distribution analysis, calculation of surface area by electron microscopy and by gas adsorption and quantification of surface-conjugated iron and hydroxyl radical production by the fibers. Interleukin-8 mRNA levels were quantified by qRT-PCR to measure relative pro-inflammatory response induced in HAEC in response to amphibole fiber exposure. The relative contribution of key physicochemical determinants on the observed pro-inflammatory response were also evaluated. RESULTS: The RTI amosite reference sample contained the longest fibers and demonstrated the greatest potency at increasing IL-8 transcript levels when evaluated on an equal mass basis. The two LA samples and the UICC amosite reference sample consisted of similar particle numbers per milligram as well as similar particle size distributions and induced comparable levels of IL-8 mRNA. A strong correlation was observed between the elongated particle (aspect ratio ?3:1) dose metrics of length and external surface area. Expression of the IL-8 data with respect to either of these metrics eliminated the differential response between the RTI amosite sample and the other samples that was observed when HAEC were exposed on an equal mass basis. CONCLUSIONS: On an equal mass basis, LA is as potent as the UICC amosite reference sample at inducing a pro-inflammatory response in HAEC but is less potent than the RTI amosite sample. The results of this study show that the particle length and particle surface area are highly correlated metrics that contribute significantly to the toxicological potential of these amphibole samples with respect to the inflammogenic response induced in airway epithelial cells.
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