The bronchial epithelium is continuously exposed to a multitude of noxious challenges in inhaled air. Cellular contact with most damaging agents is reduced by the action of the mucociliary apparatus ...and by formation of a physical barrier that controls passage of ions and macromolecules. In conjunction with these defensive barrier functions, immunomodulatory cross-talk between the bronchial epithelium and tissue-resident immune cells controls the tissue microenvironment and barrier homeostasis. This is achieved by expression of an array of sensors that detect a wide variety of viral, bacterial, and nonmicrobial (toxins and irritants) agents, resulting in production of many different soluble and cell-surface molecules that signal to cells of the immune system. The ability of the bronchial epithelium to control the balance of inhibitory and activating signals is essential for orchestrating appropriate inflammatory and immune responses and for temporally modulating these responses to limit tissue injury and control the resolution of inflammation during tissue repair. In asthmatic patients abnormalities in many aspects of epithelial barrier function have been identified. We postulate that such abnormalities play a causal role in immune dysregulation in the airways by translating gene-environment interactions that underpin disease pathogenesis and exacerbation.
Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or ...stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 'alveolar' cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham's F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.
The bronchial epithelium is constantly exposed to a wide range of environmental materials present in inhaled air, including noxious gases and anthropogenic and natural particulates, such as gas and ...particles from car emissions, tobacco smoke, pollens, animal dander, and pathogens. As a fully differentiated, pseudostratified mucociliary epithelium, the bronchial epithelium protects the internal milieu of the lung from these agents by forming a physical barrier involving adhesive complexes and a chemical barrier involving secretion of mucus, which traps inhaled particles that can be cleared by the mucociliary escalator. It is a testament to the effectiveness of these two barriers that most environmental challenges are largely overcome without the need to develop an inflammatory response. However, as the initial cell of contact with the environment, the bronchial epithelium also plays a pivotal role in immune surveillance and appropriate activation of immune effector cells and antigen presenting cells in the presence of pathogens or other danger signals. Thus, the bronchial epithelium plays a central role in controlling tissue homeostasis and innate immunity. This review will discuss these barrier properties and how dysregulation of these homeostatic mechanisms can contribute to disease pathologies such as asthma.
Angiotensin-converting enzyme 2 (ACE2) is the main entry point in airway epithelial cells for SARS-CoV-2. ACE2 binding to the SARS-CoV-2 protein spike triggers viral fusion with the cell plasma ...membrane, resulting in viral RNA genome delivery into the host. Despite ACE2's critical role in SARS-CoV-2 infection, full understanding of ACE2 expression, including in response to viral infection, remains unclear. ACE2 was thought to encode five transcripts and one protein of 805 amino acids. In the present study, we identify a novel short isoform of ACE2 expressed in the airway epithelium, the main site of SARS-CoV-2 infection. Short ACE2 is substantially upregulated in response to interferon stimulation and rhinovirus infection, but not SARS-CoV-2 infection. This short isoform lacks SARS-CoV-2 spike high-affinity binding sites and, altogether, our data are consistent with a model where short ACE2 is unlikely to directly contribute to host susceptibility to SARS-CoV-2 infection.
Asthma is a common chronic disease characterized by variable respiratory distress with underlying airway inflammation and airflow obstruction. The incidence of asthma has risen inexorably over the ...past 50 years, suggesting that environmental factors are important in its etiology. All inhaled environmental stimuli interact with the lung at the respiratory epithelium, and it is a testament to the effectiveness of the airway innate defenses that the majority of inhaled substances are cleared without the need to elicit an inflammatory response. However, once this barrier is breached, effective communication with immune and inflammatory cells is required to protect the internal milieu of the lung. In asthma, the respiratory epithelium is known to be structurally and functionally abnormal. Structurally, the epithelium shows evidence of damage and has more mucus-producing cells than normal airways. Functionally, the airway epithelial barrier can be more permeable and more sensitive to oxidants and show a deficient innate immune response to respiratory virus infection compared with that in normal individuals. The potential of a susceptible epithelium and the underlying mesenchyme to create a microenvironment that enables deviation of immune and inflammatory responses to external stimuli may be crucial in the development and progression of asthma. In this review, we consider three important groups of environmental stimuli on the epithelium in asthma: oxidants, such as environmental pollution and acetaminophen; viruses, including rhinovirus; and agents that cause barrier disruption, such as house dust mite allergens. The pathology associated with each stimulus is considered, and potential future treatments arising from research on their effects are presented.
Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have ...previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy.
Defective epithelial barrier function in asthma Xiao, Chang, PhD; Puddicombe, Sarah M., PhD; Field, Sarah, PhD ...
Journal of allergy and clinical immunology,
09/2011, Volume:
128, Issue:
3
Journal Article
Peer reviewed
Background Asthma is a complex disease involving gene and environment interactions. Although atopy is a strong predisposing risk factor for asthma, local tissue susceptibilities are required for ...disease expression. The bronchial epithelium forms the interface with the external environment and is pivotally involved in controlling tissue homeostasis through provision of a physical barrier controlled by tight junction (TJ) complexes. Objectives To explain the link between environment exposures and airway vulnerability, we hypothesized that epithelial TJs are abnormal in asthma, leading to increased susceptibility to environmental agents. Methods Localization of TJs in bronchial biopsies and differentiated epithelial cultures was assessed by electron microscopy or immunostaining. Baseline permeability and the effect of cigarette smoke and growth factor were assessed by measurement of transepithelial electrical resistance and passage of fluorescently labeled dextrans. Results By using immunostaining, we found that bronchial biopsies from asthmatic subjects displayed patchy disruption of TJs. In differentiated bronchial epithelial cultures, TJ formation and transepithelial electrical resistance were significantly lower ( P < .05) in cultures from asthmatic donors (n = 43) than from normal controls (n = 40) and inversely correlated with macromolecular permeability. Cultures from asthmatic donors were also more sensitive to disruption by cigarette smoke extract. Epidermal growth factor enhanced basal TJ formation in cultures from asthmatic subjects ( P < .01) and protected against cigarette smoke–induced barrier disruption ( P < .01). Conclusions Our results show that the bronchial epithelial barrier in asthma is compromised. This defect may facilitate the passage of allergens and other agents into the airway tissue, leading to immune activation and may thus contribute to the end organ expression of asthma.
Background
Effective treatment for severe asthma is a significant unmet need. While eosinophilic inflammation caused by type 2 cytokines is responsive to corticosteroid and biologic therapies, many ...severe asthmatics exhibit corticosteroid‐unresponsive mixed granulocytic inflammation.
Objective
Here, we tested the hypothesis that the pro‐allergic cytokine, IL‐13, can drive both corticosteroid‐sensitive and corticosteroid‐resistant responses.
Results
By integration of in vivo and in vitro models of IL‐13–driven inflammation, we identify a role for the epidermal growth factor receptor (EGFR/ERBB1) as a mediator of corticosteroid‐unresponsive inflammation and bronchial hyperresponsiveness driven by IL‐13. Topological data analysis using human epithelial transcriptomic data from the U‐BIOPRED cohort identified severe asthma groups with features consistent with the presence of IL‐13 and EGFR/ERBB activation, with involvement of distinct EGFR ligands. Our data suggest that IL–13 may play a dual role in severe asthma: on the one hand driving pathologic corticosteroid‐refractory mixed granulocytic inflammation, but on the other hand underpinning beneficial epithelial repair responses, which may confound responses in clinical trials.
Conclusion and Clinical Relevance
Detailed dissection of those molecular pathways that are downstream of IL‐13 and utilize the ERBB receptor and ligand family to drive corticosteroid‐refractory inflammation should enhance the development of new treatments that target this sub‐phenotype(s) of severe asthma, where there is an unmet need.
MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in ...co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-β, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-β, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-β and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other microRNAs when studying disease.
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