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Showing papers on "Respiratory epithelium published in 2019"


Journal ArticleDOI
TL;DR: This work shows that the CXCR6/CXCL16 axis governs the partitioning of TRM cells to different compartments of the lung and maintains the airway TRM cell pool.
Abstract: Resident memory T cells (TRM cells) are an important first-line defense against respiratory pathogens, but the unique contributions of lung TRM cell populations to protective immunity and the factors that govern their localization to different compartments of the lung are not well understood. Here, we show that airway and interstitial TRM cells have distinct effector functions and that CXCR6 controls the partitioning of TRM cells within the lung by recruiting CD8 TRM cells to the airways. The absence of CXCR6 significantly decreases airway CD8 TRM cells due to altered trafficking of CXCR6-/- cells within the lung, and not decreased survival in the airways. CXCL16, the ligand for CXCR6, is localized primarily at the respiratory epithelium, and mice lacking CXCL16 also had decreased CD8 TRM cells in the airways. Finally, blocking CXCL16 inhibited the steady-state maintenance of airway TRM cells. Thus, the CXCR6/CXCL16 signaling axis controls the localization of TRM cells to different compartments of the lung and maintains airway TRM cells.

186 citations


Journal ArticleDOI
TL;DR: Using single-cell transcriptomics and lineage inference, trajectories from basal to luminal cells are unraveled, providing novel markers for specific populations and offers new insights into the molecular mechanisms occurring during mucociliary epithelium regeneration.
Abstract: The upper airway epithelium, which is mainly composed of multiciliated, goblet, club and basal cells, ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases, defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference, we have unraveled trajectories from basal to luminal cells, providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells, which we have entitled deuterosomal cells, is defined by specific markers, such as DEUP1, FOXN4, YPEL1, HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells, thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process, such as keratins or components of the Notch, Wnt or BMP/TGFβ pathways, can be identified. Confirmation of our results on fresh human and pig airway samples, and on mouse tracheal cells, extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation.

171 citations


Book ChapterDOI
TL;DR: Asthma is a genetically and phenotypically complex disease that has a major impact on global health, and epithelial-derived cytokines are important in the recruitment and activation of immune cells in the airway.
Abstract: Asthma is a genetically and phenotypically complex disease that has a major impact on global health. Signs and symptoms of asthma are caused by the obstruction of airflow through the airways. The epithelium that lines the airways plays a major role in maintaining airway patency and in host defense. The epithelium initiates responses to inhaled or aspirated substances, including allergens, viruses, and bacteria, and epithelial-derived cytokines are important in the recruitment and activation of immune cells in the airway. Changes in the structure and function of the airway epithelium are a prominent feature of asthma. Approximately half of individuals with asthma have evidence of active type 2 immune responses in the airway. In these individuals, epithelial cytokines promote type 2 responses, and responses to type 2 cytokines result in increased epithelial mucus production and other effects that cause airway obstruction. Recent work also implicates other epithelial responses, including interleukin-17, interferon and ER stress responses, that may contribute to asthma pathogenesis and provide new targets for therapy.

121 citations


Journal ArticleDOI
TL;DR: It is reported that inactivation of Fgfr2b in BESCs impairs their contribution to both alveolar epithelial regeneration and honeycomb cysts after bleomycin injury, which enhances fibrosis resolution by favoring the more desirable outcome of alve solar epithelium regeneration over the development of pathologic honeycomb Cysts.
Abstract: Summary Idiopathic pulmonary fibrosis is a common form of interstitial lung disease resulting in alveolar remodeling and progressive loss of pulmonary function because of chronic alveolar injury and failure to regenerate the respiratory epithelium. Histologically, fibrotic lesions and honeycomb structures expressing atypical proximal airway epithelial markers replace alveolar structures, the latter normally lined by alveolar type 1 (AT1) and AT2 cells. Bronchial epithelial stem cells (BESCs) can give rise to AT2 and AT1 cells or honeycomb cysts following bleomycin-mediated lung injury. However, little is known about what controls this binary decision or whether this decision can be reversed. Here we report that inactivation of Fgfr2b in BESCs impairs their contribution to both alveolar epithelial regeneration and honeycomb cysts after bleomycin injury. By contrast overexpression of Fgf10 in BESCs enhances fibrosis resolution by favoring the more desirable outcome of alveolar epithelial regeneration over the development of pathologic honeycomb cysts.

83 citations


Journal ArticleDOI
TL;DR: It is reported that the flavoring chemicals induce transcriptomic changes and perturb cilia function in the airway epithelium and likely contribute to the adverse effects of e-cig in the lung.
Abstract: The widespread use of electronic cigarettes (e-cigarettes or e-cig) is a growing public health concern. Diacetyl and its chemical cousin 2,3-pentanedione are commonly used to add flavors to e-cig; however, little is known about how the flavoring chemicals may impair lung function. Here we report that the flavoring chemicals induce transcriptomic changes and perturb cilia function in the airway epithelium. Using RNA-Seq, we identified a total of 163 and 568 differentially expressed genes in primary normal human bronchial epithelial (NHBE) cells that were exposed to diacetyl and 2,3-pentanedione, respectively. DAVID pathway analysis revealed an enrichment of cellular pathways involved in cytoskeletal and cilia processes among the set of common genes (142 genes) perturbed by both diacetyl and 2,3-pentanedione. Consistent with this, qRT-PCR confirmed that the expression of multiple genes involved in cilia biogenesis was significantly downregulated by diacetyl and 2,3-pentanedione in NHBE cells. Furthermore, immunofluorescence staining showed that the number of ciliated cells was significantly decreased by the flavoring chemicals. Our study indicates that the two widely used e-cig flavoring chemicals impair the cilia function in airway epithelium and likely contribute to the adverse effects of e-cig in the lung.

82 citations


Journal ArticleDOI
TL;DR: It is suggested that the transfer of exosomal cargo between airway epithelial cells significantly alters the qualitative and quantitative profiles of airway secretions, including mucin hypersecretion, and the miRNA cargo ofExosomes in target cells.
Abstract: Airway epithelium structure/function can be altered by local inflammatory/immune signals, and this process is called epithelial remodeling. The mechanism by which this innate response is regulated,...

74 citations


Journal ArticleDOI
TL;DR: Different anti-influenza virus schemes of innate immune cells are addressed and how these cells fine-tune the balance between immunoprotection and immunopathology during IAV infection is addressed.
Abstract: Influenza virus infection is a serious threat to humans and animals, with the potential to cause severe pneumonia and death. Annual vaccination strategies are a mainstay to prevent complications related to influenza. However, protection from the emerging subtypes of influenza A viruses (IAV) even in vaccinated individuals is challenging. Innate immune cells are the first cells to respond to IAV infection in the respiratory tract. Virus replication-induced production of cytokines from airway epithelium recruits innate immune cells to the site of infection. These leukocytes, namely, neutrophils, monocytes, macrophages, dendritic cells, eosinophils, natural killer cells, innate lymphoid cells, and γδ T cells, become activated in response to IAV, to contain the virus and protect the airway epithelium while triggering the adaptive arm of the immune system. This review addresses different anti-influenza virus schemes of innate immune cells and how these cells fine-tune the balance between immunoprotection and immunopathology during IAV infection. Detailed understanding on how these innate responders execute anti-influenza activity will help to identify novel therapeutic targets to halt IAV replication and associated immunopathology.

67 citations


Journal ArticleDOI
TL;DR: The role of cytokines transforming growth factor β, interleukin (IL-) 10, IL-27, and IL-35 are discussed, highlighting the critical importance of cellular source and immunological context for the effects of these cytokines in vivo.

66 citations


Journal ArticleDOI
TL;DR: Genetic variants in genes important in epithelial cell function and phenotype contribute to a diversity of responses toAir pollution in the population at the individual and group levels and suggest a need for personalized approaches to attenuate the respiratory mucosal immune responses to air pollution.
Abstract: Every day, we breathe in more than 10,000 L of air that contains a variety of air pollutants that can pose negative consequences to lung health. The respiratory mucosa formed by the airway epithelium is the first point of contact for air pollution in the lung, functioning as a mechanical and immunologic barrier. Under normal circumstances, airway epithelial cells connected by tight junctions secrete mucus, airway surface lining fluid, host defense peptides, and antioxidants and express innate immune pattern recognition receptors to respond to inhaled foreign substances and pathogens. Under conditions of air pollution exposure, the defenses of the airway epithelium are compromised by reductions in barrier function, impaired host defense to pathogens, and exaggerated inflammatory responses. Central to the mechanical and immunologic changes induced by air pollution are activation of redox-sensitive pathways and a role for antioxidants in normalizing these negative effects. Genetic variants in genes important in epithelial cell function and phenotype contribute to a diversity of responses to air pollution in the population at the individual and group levels and suggest a need for personalized approaches to attenuate the respiratory mucosal immune responses to air pollution.

63 citations


Journal ArticleDOI
TL;DR: It is found that the number of viruses infecting a cell influences the magnitude of virus antagonism of the host innate antiviral response, and Infections performed at high MOIs resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOIs.
Abstract: Early interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The study of global cellular innate immune responses often masks multiple aspects of the mechanisms by which populations of cells work as organized and heterogeneous systems to defeat virus infection, and how the virus counteracts these systems. In this study, we experimentally dissected the dynamics of IAV and human epithelial respiratory cell interaction during early infection at the single-cell level. We found that the number of viruses infecting a cell (multiplicity of infection [MOI]) influences the magnitude of virus antagonism of the host innate antiviral response. Infections performed at high MOIs resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOIs. In addition, single-cell patterns of expression of interferons (IFN) and IFN-stimulated genes (ISGs) provided important insights into the contributions of the infected and bystander cells to the innate immune responses during infection. Specifically, the expression of multiple ISGs was lower in infected than in bystander cells. In contrast with other IFNs, IFN lambda 1 (IFNL1) showed a widespread pattern of expression, suggesting a different cell-to-cell propagation mechanism more reliant on paracrine signaling. Finally, we measured the dynamics of the antiviral response in primary human epithelial cells, which highlighted the importance of early innate immune responses at inhibiting virus spread. IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics of seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for defense against the virus, and therefore, it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells functions as a complex system to orchestrate the response to virus infection and how the virus counteracts this system.

59 citations


Journal ArticleDOI
TL;DR: The artificial trachea created via a tissue engineering method using 3D bio-printing was effective in the regeneration of respiratory epithelium, but not in cartilage regeneration.
Abstract: Various treatment methods for tracheal defects have been attempted, such as artificial implants, allografts, autogenous grafts, and tissue engineering; however, no perfect method has been established. We attempted to create an effective artificial trachea via a tissue engineering method using 3D bio-printing. A multi-layered scaffold was fabricated using a 3D printer. Polycaprolactone (PCL) and hydrogel were used with nasal epithelial and auricular cartilage cells in the printing process. An artificial trachea was transplanted into 15 rabbits and a PCL scaffold without the addition of cells was transplanted into 6 rabbits (controls). All animals were followed up with radiography, CT, and endoscopy at 3, 6, and 12 months. In the control group, 3 out of 6 rabbits died from respiratory symptoms. Surviving rabbits in control group had narrowed tracheas due to the formation of granulation tissue and absence of epithelium regeneration. In the experimental group, 13 of 15 animals survived, and the histologic examination confirmed the regeneration of epithelial cells. Neonatal cartilage was also confirmed at 6 and 12 months. Our artificial trachea was effective in the regeneration of respiratory epithelium, but not in cartilage regeneration. Additional studies are needed to promote cartilage regeneration and improve implant stability.

Journal ArticleDOI
TL;DR: Evidence is provided for a crucial role of TMEM16A in fusion of mucus-filled granules with the apical plasma membrane and cellular exocytosis, and taken together, TMEM 16A supports fluid secretion by ciliated airway epithelial cells, but also maintains excessive mucus secretion during inflammatory airway disease.
Abstract: The inflammatory airway disease cystic fibrosis (CF) is characterized by airway obstruction due to mucus hypersecretion, airway plugging, and bronchoconstriction. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is dysfunctional in CF, leading to defects in epithelial transport. Although CF pathogenesis is still disputed, activation of alternative Cl- channels is assumed to improve lung function in CF. Two suitable non-CFTR Cl- channels are present in the airway epithelium, the Ca2+ activated channel TMEM16A and SLC26A9. Activation of these channels is thought to be feasible to improve hydration of the airway mucus and to increase mucociliary clearance. Interestingly, both channels are upregulated during inflammatory lung disease. They are assumed to support fluid secretion, necessary to hydrate excess mucus and to maintain mucus clearance. During inflammation, however, TMEM16A is upregulated particularly in mucus producing cells, with only little expression in ciliated cells. Recently it was shown that knockout of TMEM16A in ciliated cells strongly compromises Cl- conductance and attenuated mucus secretion, but does not lead to a CF-like lung disease and airway plugging. Along this line, activation of TMEM16A by denufosol, a stable purinergic ligand, failed to demonstrate any benefit to CF patients in earlier studies. It rather induced adverse effects such as cough. A number of studies suggest that TMEM16A is essential for mucus secretion and possibly also for mucus production. Evidence is now provided for a crucial role of TMEM16A in fusion of mucus-filled granules with the apical plasma membrane and cellular exocytosis. This is probably due to local Ca2+ signals facilitated by TMEM16A. Taken together, TMEM16A supports fluid secretion by ciliated airway epithelial cells, but also maintains excessive mucus secretion during inflammatory airway disease. Because TMEM16A also supports airway smooth muscle contraction, inhibition rather than activation of TMEM16A might be the appropriate treatment for CF lung disease, asthma and COPD. As a number of FDA-approved and well-tolerated drugs have been shown to inhibit TMEM16A, evaluation in clinical trials appears timely.

Journal ArticleDOI
TL;DR: Ex vivo modeling of primary airway epithelial cells in organotypic co-culture with mast cells has shown that epithelial-derived IL-33 uniquely induced type-2 cytokines in mast cells, which regulated the expression of epithelial IL33 in a feedforward loop.
Abstract: Asthma is a heterogeneous syndrome that has been subdivided into physiologic phenotypes and molecular endotypes. The most specific phenotypic manifestation of asthma is indirect airway hyperresponsiveness (AHR), and a prominent molecular endotype is the presence of type 2 inflammation. The underlying basis for type 2 inflammation and its relationship to AHR are incompletely understood. We assessed the expression of type 2 cytokines in the airways of subjects with and without asthma who were extensively characterized for AHR. Using quantitative morphometry of the airway wall, we identified a shift in mast cells from the submucosa to the airway epithelium specifically associated with both type 2 inflammation and indirect AHR. Using ex vivo modeling of primary airway epithelial cells in organotypic coculture with mast cells, we show that epithelial-derived IL-33 uniquely induced type 2 cytokines in mast cells, which regulated the expression of epithelial IL33 in a feed-forward loop. This feed-forward loop was accentuated in epithelial cells derived from subjects with asthma. These results demonstrate that type 2 inflammation and indirect AHR in asthma are related to a shift in mast cell infiltration to the airway epithelium, and that mast cells cooperate with epithelial cells through IL-33 signaling to regulate type 2 inflammation.

Journal ArticleDOI
TL;DR: The current study showed that the NLRP3 inflammasome signaling axis could functionally mediate hRV-induced inflammation, pyroptosis, and mucus production in airway epithelium, which might be an essential mechanism associated with hRv-induced airway remodeling.
Abstract: Background The airway epithelium maintains mucosal homeostasis and effectively responds to pathogens. The roles of the epithelial NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in human rhinovirus (hRV) infection and its effects mediating epithelial functional changes remain poorly understood. Objective We investigated the mechanisms and cellular functions mediated by the epithelial NLRP3 inflammasome on hRV infection. Methods Using models of primary human nasal epithelial progenitor cells and differentiated human nasal epithelial cells (hNECs) infected by hRV, we functionally examined key factors for NLRP3 inflammasome activation, cell death, and mucus production. Furthermore, NLRP3 and IL-1β in human epithelium from nasal mucosal inflammation induced by hRV were evaluated. Results The inflammasome-mediated IL-1β secretion and pyroptosis in human nasal epithelial progenitor cells and hNECs on hRV infection were dependent on the DDX33/DDX58–NLRP3–caspase-1–GSDMD axis. In differentiated hNECs hRV could also promote major airway epithelial mucin (MUC5AC) production through this axis. Our results further confirmed that the NLRP3 inflammasome signaling pathway was responsible for suppressing hRV replication in airway epithelium. Finally, hRV infection in chronically inflamed nasal mucosa was associated with epithelial mucus hyperproduction, whereas NLRP3 and IL-1β expression levels were significantly increased in hRV-infected epithelium with goblet cell hyperplasia compared with normal epithelium without viral infection. Conclusion The current study showed that the NLRP3 inflammasome signaling axis could functionally mediate hRV-induced inflammation, pyroptosis, and mucus production in airway epithelium, which might be an essential mechanism associated with hRV-induced airway remodeling.

Journal ArticleDOI
TL;DR: Genetic deletion and genotype-specific studies in primary AECs indicate CDHR3 is critical to HRV-C infection of ciliated cells and the rs6967330 SNP confers risk of severe childhood asthma exacerbations.
Abstract: Background Research in transformed immortalized cell lines indicates the cadherin-related family member 3 (CDHR3) protein serves as a receptor for human rhinovirus (HRV)–C. Similar experiments indicate that the CDHR3 coding variant rs6967330 increases CDHR3 protein surface expression. Objective We sought to determine whether CDHR3 is necessary for HRV-C infection of primary airway epithelial cells (AECs) and to identify molecular mechanisms by which CDHR3 variants confer risk for asthma exacerbations. Methods CDHR3 function and influence on HRV-C infection were investigated by using single-cell transcriptomics, CRISPR-Cas9 gene knockout, and genotype-specific donor experiments performed in primary AECs. Nasal airway epithelium cis–expression quantitative trait locus (eQTL) analysis of CDHR3 was performed, followed by association testing for asthma hospitalization in minority children. Results CDHR3 lung expression is exclusive to ciliated AECs and associated with basal bodies during and after motile ciliogenesis. Knockout of CDHR3 in human AECs did not prevent ciliated cell differentiation but was associated with a decrease in transepithelial resistance and an 80% decrease in HRV-C infection of the mucociliary epithelium. AECs from subjects homozygous for the risk-associated rs6967330 single nucleotide polymorphism (SNP) exhibited greater HRV-C infection compared with cells homozygous for the nonrisk allele. AEC cis-eQTL analysis indicated that rs6967330 and other SNPs are eQTLs for CDHR3. Only the eQTL block containing the rs6967330 SNP showed a significant association with childhood asthma hospitalization. Conclusions Genetic deletion and genotype-specific studies in primary AECs indicate CDHR3 is critical to HRV-C infection of ciliated cells. The rs6967330 SNP confers risk of severe childhood asthma exacerbations, likely through increasing HRV-C infection levels and protein surface localization.

Journal ArticleDOI
TL;DR: The role of AQPs in the lung with regard to fluid homeostasis across the respiratory epithelium is discussed, where AQPs provide a transcellular route for water transport across epithelia.
Abstract: The lung is the interface between air and blood where the exchange of oxygen and carbon dioxide occurs. The surface liquid that is directly exposed to the gaseous compartment covers both conducting airways and respiratory zone and forms the air-liquid interface. The barrier that separates this lining fluid of the airways and alveoli from the extracellular compartment is the pulmonary epithelium. The volume of the lining fluid must be kept in a range that guarantees an appropriate gas exchange and other functions, such as mucociliary clearance. It is generally accepted that this is maintained by balancing resorptive and secretory fluid transport across the pulmonary epithelium. Whereas osmosis is considered as the exclusive principle of fluid transport in the airways, filtration may contribute to alveolar fluid accumulation under pathologic conditions. Aquaporins (AQP) facilitate water flux across cell membranes, and as such, they provide a transcellular route for water transport across epithelia. However, their contribution to near-isosmolar fluid conditions in the lung still remains elusive. Herein, we discuss the role of AQPs in the lung with regard to fluid homeostasis across the respiratory epithelium.

Journal ArticleDOI
TL;DR: It is suggested that autophagy and glucose metabolism are essential for the maintenance of airway epithelium at steady state and during allergic inflammation.
Abstract: Efficient repair of injured epithelium by airway progenitor cells could prevent acute inflammation from progressing into chronic phase in lung. Here, we used small molecules, genetic loss-of-function, organoid cultures, and in vivo lung-injury models to show that autophagy is essential for maintaining the pool of airway stem-like vClub cells by promoting their proliferation during ovalbumin-induced acute inflammation. Mechanistically, impaired autophagy disrupted glucose uptake in vClub progenitor cells, and either reduced accessibility to glucose or partial inhibition of glycolysis promoted the proliferative capacity of vClub progenitor cells and their daughter Club cells. However, glucose deprivation or glycolysis blockade abrogated the proliferative capacity of airway vClub cells and Club cells but promoted ciliated and goblet cell differentiation. Deficiency of glucose transporter-1 suppressed the proliferative capacity of airway progenitor cells after ovalbumin challenge. These findings suggested that autophagy and glucose metabolism are essential for the maintenance of airway epithelium at steady state and during allergic inflammation.

Journal ArticleDOI
TL;DR: The COPD airway epithelium displays altered differentiation for ciliated cells, which recapitulates in vitro, at least in part through TGF-β1, which is hypothesized to cause bronchial epithelial cell lineage specification to be dysregulated.
Abstract: In COPD, epithelial changes are prominent features in the airways, such as goblet cell hyperplasia and squamous metaplasia. In contrast, it remains unclear whether ciliated cells are reduced and which pathways dysregulate epithelial differentiation. We hypothesized that bronchial epithelial cell lineage specification is dysregulated in COPD because of an aberrant reprogramming through transforming growth factor (TGF)-β1. Surgical lung tissue from 81 COPD and 61 control (smokers and non-smokers) patients was assessed for bronchial epithelial cell phenotyping by immunohistochemistry, both in situ and in vitro in reconstituted air-liquid interface (ALI) cultures. The role of TGF-β1 was studied in vitro. COPD epithelium in large airways, when compared to controls, showed decreased β-tubulin IV + ciliated cells (4.4%, 2.5–8.8% versus 8.5%, 6.3–11.8% of surface staining, median and IQR, p = 0.0009) and increased MUC5AC + goblet cells (34.8%, 24.4–41.9% versus 10.3%, 5.1–17.6%, p < 0.0001). Both features were recapitulated in the ALI-cultured epithelium from COPD patients. Exogenous TGF-β1 reduced mucociliary differentiation while neutralizing TGF-β1 during ALI increased both specialized cell types. The COPD airway epithelium displays altered differentiation for ciliated cells, which recapitulates in vitro, at least in part through TGF-β1.

Journal ArticleDOI
TL;DR: This review focuses on specialized innate-like lymphocytes present in the lung that act as key sensors of lung insults and direct the pulmonary immune response and the contribution of other ILCs and unconventional T cells during chronic lung inflammation is poorly described.
Abstract: The lungs are continuously subjected to environmental insults making them susceptible to infection and injury. They are protected by the respiratory epithelium, which not only serves as a physical barrier but also a reactive one that can release cytokines, chemokines, and other defense proteins in response to danger signals, and can undergo conversion to protective mucus-producing goblet cells. The lungs are also guarded by a complex network of highly specialized immune cells and their mediators to support tissue homeostasis and resolve integrity deviation. This review focuses on specialized innate-like lymphocytes present in the lung that act as key sensors of lung insults and direct the pulmonary immune response. Included amongst these tissue-resident lymphocytes are innate lymphoid cells (ILCs), which are classified into five distinct subsets (natural killer, ILC1, ILC2, ILC3, lymphoid tissue-inducer cells), and unconventional T cells including natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ-T cells. While ILCs and unconventional T cells together comprise only a small proportion of the total immune cells in the lung, they have been found to promote lung homeostasis and are emerging as contributors to a variety of chronic lung diseases including pulmonary fibrosis, allergic airway inflammation, and chronic obstructive pulmonary disease (COPD). A particularly intriguing trait of ILCs that has recently emerged is their plasticity and ability to alter their gene expression profiles and adapt their function in response to environmental cues. The malleable nature of these cells may aid in rapid responses to pathogen but may also have downstream pathological consequences. The role of ILC2s in Th2 allergic airway responses is becoming apparent but the contribution of other ILCs and unconventional T cells during chronic lung inflammation is poorly described. This review presents an overview of our current understanding of the involvement of ILCs and unconventional T cells in chronic pulmonary diseases.

Journal ArticleDOI
TL;DR: A broad review of the cytokines and chemokines secreted from human airway epithelial cell models during respiratory syncytial virus (RSV) infection based on a comprehensive literature review suggests chemo-attraction of peripheral immune cells as a key function of the epithelium.
Abstract: The airway epithelium is the primary target of respiratory syncytial virus infection. It is an important component of the antiviral immune response. It contributes to the recruitment and activation of innate immune cells from the periphery through the secretion of cytokines and chemokines. This paper provides a broad review of the cytokines and chemokines secreted from human airway epithelial cell models during respiratory syncytial virus (RSV) infection based on a comprehensive literature review. Epithelium-derived chemokines constitute most inflammatory mediators secreted from the epithelium during RSV infection. This suggests chemo-attraction of peripheral immune cells, such as monocytes, neutrophils, eosinophils, and natural killer cells as a key function of the epithelium. The reports of epithelium-derived cytokines are limited. Recent research has started to identify novel cytokines, the functions of which remain largely unknown in the wider context of the RSV immune response. It is argued that the correct choice of in vitro models used for investigations of epithelial immune functions during RSV infection could facilitate greater progress in this field.

Journal ArticleDOI
TL;DR: It is found that branching morphogenesis in the embryonic chicken lung requires extracellular matrix (ECM) remodeling driven by reciprocal interactions between the epithelium and mesenchyme, and novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branch morphogenesis are revealed.
Abstract: Reciprocal epithelial-mesenchymal signaling is essential for morphogenesis, including branching of the lung. In the mouse, mesenchymal cells differentiate into airway smooth muscle that wraps around epithelial branches, but this contractile tissue is absent from the early avian lung. Here, we have found that branching morphogenesis in the embryonic chicken lung requires extracellular matrix (ECM) remodeling driven by reciprocal interactions between the epithelium and mesenchyme. Before branching, the basement membrane wraps the airway epithelium as a spatially uniform sheath. After branch initiation, however, the basement membrane thins at branch tips; this remodeling requires mesenchymal expression of matrix metalloproteinase 2, which is necessary for branch extension but for not branch initiation. As branches extend, tenascin C (TNC) accumulates in the mesenchyme several cell diameters away from the epithelium. Despite its pattern of accumulation, TNC is expressed exclusively by epithelial cells. Branch extension coincides with deformation of adjacent mesenchymal cells, which correlates with an increase in mesenchymal fluidity at branch tips that may transport TNC away from the epithelium. These data reveal novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branching morphogenesis.

Journal ArticleDOI
TL;DR: Repeated exposure to E171 or TiO2-NPs, in vitro, induce moderate inflammation and mucus secretion in intestinal cells.
Abstract: Environmental contamination with TiO2 and the use of TiO2 as a food additive (E171) or in cosmetics result in human exposure to TiO2 via inhalation, ingestion, and through skin contact. When inhaled, most TiO2 particles are cleared via the mucociliary escalator and are then swallowed. Together with the ingestion of E171, this process results in a significant exposure of the human gastro-intestinal tract to TiO2. One of the functions of the intestine is to protect the body from external aggression, via the so-called intestinal barrier function. The aim of this study was to determine whether, and through which mechanisms, TiO2 affects this function. Caco-2 and HT29-MTX cells were co-cultured to reconstitute an in vitro mucus-secreting intestinal epithelium. This epithelium was exposed to TiO2-NPs, either pure anatase or mixed anatase/rutile, or to E171. Two exposure scenarii were used: acute exposure for 6 h or 48 h after cell differentiation (21 days post-seeding), or repeated exposure during the course of cell differentiation, i.e., twice a week for 21 days post-seeding. Epithelial cells repeatedly exposed to TiO2 developed an inflammatory profile, together with increased mucus secretion. Epithelial integrity was unaltered, but the content of ATP-binding cassette (ABC) family xenobiotic efflux pumps was modified. Taken together, these data show that TiO2 moderately but significantly dysregulates several features that contribute to the protective function of the intestine.

Journal ArticleDOI
TL;DR: The cultivation conditions and structural features of the biodegradable non‐woven material are investigated in order to obtain a well‐differentiated human airway epithelium.
Abstract: Objectives The conversion of tissue engineering into a routine clinical tool cannot be achieved without a deep understanding of the interaction between cells and scaffolds during the process of tissue formation in an artificial environment. Here, we have investigated the cultivation conditions and structural features of the biodegradable non-woven material in order to obtain a well-differentiated human airway epithelium. Materials and methods The bilayered scaffold was fabricated by electrospinning technology. The efficiency of the scaffold has been evaluated using MTT cell proliferation assay, histology, immunofluorescence and electron microscopy. Results With the use of a copolymer of chitosan-gelatin-poly-l-lactide, a bilayered non-woven scaffold was generated and characterized. The optimal structural parameters of both layers for cell proliferation and differentiation were determined. The basal airway epithelial cells differentiated into ciliary and goblet cells and formed pseudostratified epithelial layer on the surface of the scaffold. In addition, keratinocytes formed a skin equivalent when seeded on the same scaffold. A comparative analysis of growth and differentiation for both types of epithelium was performed. Conclusions The structural parameters of nanofibres should be selected experimentally depending on polymer composition. The major challenges on the way to obtain the well-differentiated equivalent of respiratory epithelium on non-woven scaffold include the following: the balance between scaffold permeability and thickness, proper combination of synthetic and natural components, and culture conditions sufficient for co-culturing of airway epithelial cells and fibroblasts. For generation of skin equivalent, the lack of diffusion is not so critical as for pseudostratified airway epithelium.

Journal ArticleDOI
Tianyu Zhou1, Yan Hu1, Yunxia Wang1, Chao Sun1, Yijue Zhong1, Jiping Liao1, Guangfa Wang1 
TL;DR: PM2.5 aggravated apoptosis in cigarette-inflamed bronchial epithelial cells and the responses could be suppressed by Z-VAD-FMK, a pan-caspase inhibitor, and the results gave a new idea about the mechanism of PM2.

Journal ArticleDOI
TL;DR: In this article, the authors characterized the cellular source of GABA in the lungs of nonhuman primates and humans and assessed GABA secretion and signaling in primate disease models, and found that pulmonary neuroendocrine cells (PNECs) were the major source of GA in the primate lungs.
Abstract: Mucus overproduction is a major contributor to morbidity and mortality in asthma. Mucus overproduction is induced by orchestrated actions of multiple factors that include inflammatory cytokines and γ-aminobutyric acid (GABA). GABA is produced only by pulmonary neuroendocrine cells (PNECs) in the mouse lung. Recent studies in a neonatal mouse model of allergic inflammation have shown that PNECs play an essential role in mucus overproduction by GABA hypersecretion. Whether PNECs mediate dysregulated GABA signaling for mucus overproduction in asthma is unknown. In this study, we characterized the cellular source of GABA in the lungs of nonhuman primates and humans and assessed GABA secretion and signaling in primate disease models. We found that like in mice, PNECs were the major source of GABA in primate lungs. In addition, an infant nonhuman primate model of asthma exhibited an increase in GABA secretion. Furthermore, subjects with asthma had elevated levels of expression of a subset of GABA type α (GABAα) and type β (GABAβ) receptors in airway epithelium compared with those of healthy control subjects. Last, employing a normal human bronchial epithelial cell model of preinduced mucus overproduction, we showed pharmaceutical blockade of GABAα and GABAβ receptor signaling reversed the effect of IL-13 on MUC5AC gene expression and goblet cell proliferation. Together, our data demonstrate an evolutionarily conserved intraepithelial GABA signaling that, in concert with IL-13, plays an essential role in mucus overproduction. Our findings may offer new strategies to ameliorate mucus overproduction in patients with asthma by targeting PNEC secretion and GABA signaling.

Journal ArticleDOI
TL;DR: The role of MMP-2 and -9 in CSE-induced EMT is highlighted and its molecular cascade through EGFR/AKT/ERK/β-catenin axis is curated, which could be restored by MMP/ -9 inhibitor and fisetin.

Journal ArticleDOI
TL;DR: In this article, Eact is shown to be an activator of the Ca2+ -permeable TRPV4 channel in recombinant Fischer rat thyroid (FRT) cells.
Abstract: Key points Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca2+ elevation by TRPV4 stimulation leads to CFTR channel activation. Abstract TMEM16A is a Ca2+ -activated Cl- channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4 stimulation induced activation of the CFTR Cl- channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl- secretory processes.

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TL;DR: The established primary models display an appropriate model for nasal mucosa with secreted MUC5AC, beating cilia and a functional epithelial barrier, which is suitable for long-term evaluation of sustained release dosage forms, and translational mucosal models are needed.
Abstract: Background: The epithelial layer of the nasal mucosa is the first barrier for drug permeation during intranasal drug delivery. With increasing interest for intranasal pathways, adequate in vitro models are required. Here, porcine olfactory (OEPC) and respiratory (REPC) primary cells were characterised against the nasal tumour cell line RPMI 2650. Methods: Culture conditions for primary cells from porcine nasal mucosa were optimized and the cells characterised via light microscope, RT-PCR and immunofluorescence. Epithelial barrier function was analysed via transepithelial electrical resistance (TEER), and FITC-dextran was used as model substance for transepithelial permeation. Beating cilia necessary for mucociliary clearance were studied by immunoreactivity against acetylated tubulin. Results: OEPC and REPC barrier models differ in TEER, transepithelial permeation and MUC5AC levels. In contrast, RPMI 2650 displayed lower levels of MUC5AC, cilia markers and TEER, and higher FITC-dextran flux rates. Conclusion: To screen pharmaceutical formulations for intranasal delivery in vitro, translational mucosal models are needed. Here, a novel and comprehensive characterisation of OEPC and REPC against RPMI 2650 is presented. The established primary models display an appropriate model for nasal mucosa with secreted MUC5AC, beating cilia and a functional epithelial barrier, which is suitable for long-term evaluation of sustained release dosage forms.

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TL;DR: The applicability of ALI models for the investigation of the early embryonic microenvironment and for its implications in assisted reproductive technologies are discussed.
Abstract: The air-liquid interface (ALI) approach is primarily used to mimic respiratory tract epithelia in vitro. It is also known to support excellent differentiation of 3D multilayered skin models. To establish an ALI culture, epithelial cells are seeded into compartmentalized culture systems on porous filter supports or gel substrata. After an initial propagation period, the culture medium is removed from the apical side of the epithelium, exposing the cells to the surrounding air. Therefore, nutritive supply to the cells is warranted only by the basolateral cell pole. Under these conditions, the epithelial cells differentiate and regain full baso-apical polarity. Some types of epithelia even generate in vivo-like apical fluid or mucus. Interestingly, the ALI culture approach has also been shown to support morphological and functional differentiation of epithelial cells that are not normally exposed to ambient air in vivo. This review aims at giving a brief overview on the characteristics of ALI cultures in general and ALI models of female reproductive tract epithelia in particular. We discuss the applicability of ALI models for the investigation of the early embryonic microenvironment and for its implications in assisted reproductive technologies.

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TL;DR: It is demonstrated that HCA attenuates the inflammatory response and reparative potential of primary human small airway epithelial and capillary endothelium and induces mitochondrial dysfunction and mesenchymal stem cells promote lung epithelial wound repair via the transfer of functional mitochondria.
Abstract: Acute respiratory distress syndrome (ARDS) is a devastating disorder characterized by diffuse inflammation and edema formation. The main management strategy, low tidal volume ventilation, can be associated with the development of hypercapnic acidosis (HCA). Mesenchymal stem cells (MSCs) are a promising therapeutic candidate currently in early-phase clinical trials. The effects of HCA on the alveolar epithelium and capillary endothelium are not well established. The therapeutic efficacy of MSCs has never been reported in HCA. In the present study, we evaluated the effects of HCA on inflammatory response and reparative potential of the primary human small airway epithelial and lung microvasculature endothelial cells as well as on the capacity of bone marrow-derived MSCs to promote wound healing in vitro. We demonstrate that HCA attenuates the inflammatory response and reparative potential of primary human small airway epithelium and capillary endothelium and induces mitochondrial dysfunction. It was found that MSCs promote lung epithelial wound repair via the transfer of functional mitochondria; however, this proreparative effect of MSCs was lost in the setting of HCA. Therefore, HCA may adversely impact recovery from ARDS at the cellular level, whereas MSCs may not be therapeutically beneficial in patients with ARDS who develop HCA.-Fergie, N., Todd, N., McClements, L., McAuley, D., O'Kane, C., Krasnodembskaya, A. Hypercapnic acidosis induces mitochondrial dysfunction and impairs the ability of mesenchymal stem cells to promote distal lung epithelial repair.