Lower Airway Epithelium Research Articles

Nasal epithelial gene expression and total IgE in children and adolescents with asthma

Little is known about nasal epithelial gene expression and total IgE in youth. We aimed to identify genes whose nasal epithelial expression differs by total IgE in youth, and group them into modules that could be mapped to airway epithelial cell types. We conducted a transcriptome-wide association study of total IgE in 469 Puerto Ricans aged 9 to 20 years who participated in the Epigenetic Variation and Childhood Asthma in Puerto Ricans study, separately in all subjects and in those with asthma. We then attempted to replicate top findings for each analysis using data from 3 cohorts. Genes with a Benjamini-Hochberg-adjusted P value of less than .05 in the Epigenetic Variation and Childhood Asthma in Puerto Ricans study and a P value of less than .05 in the same direction of association in 1 or more replication cohort were considered differentially expressed genes (DEGs). DEGs for total IgE in subjects with asthma were further dissected into gene modules using coexpression analysis, and such modules were mapped to specific cell types in airway epithelia using public single-cell RNA-sequencing data. A higher number of DEGs for total IgE were identified in subjects with asthma (n= 1179 DEGs) than in all subjects (n= 631 DEGs). In subjects with asthma, DEGs were mapped to 11 gene modules. The top module for positive correlation with total IgE was mapped to myoepithelial and mucus secretory cells in lower airway epithelia and was regulated by IL-4, IL5, IL-13, and IL-33. Within this module, hub genes included CDH26, FETUB, NTRK2, CCBL1, CST1, and CST2. Furthermore, an enrichment analysis showed overrepresentation of genes in signaling pathways for synaptogenesis, IL-13, and ferroptosis, supporting interactions between interleukin- and acetylcholine-induced responses. Our findings for nasal epithelial gene expression support neuroimmune coregulation of total IgE in youth with asthma.

Human rhinovirus serotypes induces different immune responses

BackgroundDifferent species of human rhinovirus (HRV) can induce varied antiviral and inflammatory responses in human blood macrophages and lower airway epithelium. Although human nasal epithelial cells (HNECs) are a primary infection route of HRV, differences between major and minor groups of HRV in the upper airway epithelium have not been studied in detail. In this study, we investigated viral replications and immune responses of major and minor groups of HRV in the HNECs.MethodsViral replication, immune responses of IFN-β, IFN-λ, proinflammatory cytokines, and viral receptors, and mRNA expression of transcription factors of HRV16 (major group) and HRV1B (minor group) in the HNECs were assessed.ResultsCompared with HRV16, HRV1B replicated more actively without excessive cell death and produced higher IFN-β, IFN-λ1/3, CXCL10, IL-6, IL-8, and IL-18 levels. Furthermore, low-density lipoprotein receptor (LDLR), TLR3, MDA5, NF-κB, STAT1, and STAT2 mRNA levels increased in HRV1B-infected HNECs.ConclusionHRV1B induces a stronger antiviral and inflammatory response from cell entry to downstream signaling compared with HRV16.

Immunoproteasomes as a novel antiviral mechanism in rhinovirus-infected airways

Rhinovirus (RV) infection is involved in acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). RV primarily infects upper and lower airway epithelium. Immunoproteasomes (IP) are proteolytic machineries with multiple functions including the regulation of MHC class I antigen processing during viral infection. However, the role of IP in RV infection has not been explored. We sought to investigate the expression and function of IP during airway RV infection. Primary human tracheobronchial epithelial (HTBE) cells were cultured at air-liquid interface (ALI) and treated with RV16, RV1B, or interferon (IFN)-λ in the absence or presence of an IP inhibitor (ONX-0914). IP gene (i.e. LMP2) deficient mouse tracheal epithelial cells (mTECs) were cultured for the mechanistic studies. LMP2-deficient mouse model was used to define the in vivo role of IP in RV infection. IP subunits LMP2 and LMP7, antiviral genes MX1 and OAS1 and viral load were measured. Both RV16 and RV1B significantly increased the expression of LMP2 and LMP7 mRNA and proteins, and IFN-λ mRNA in HTBE cells. ONX-0914 down-regulated MX1 and OAS1, and increased RV16 load in HTBE cells. LMP2-deficient mTECs showed a significant increase in RV1B load compared with the wild-type (WT) cells. LMP2-deficient (compared with WT) mice increased viral load and neutrophils in bronchoalveolar lavage (BAL) fluid after 24 h of RV1B infection. Mechanistically, IFN-λ induction by RV infection contributed to LMP2 and LMP7 up-regulation in HTBE cells. Our data suggest that IP are induced during airway RV infection, which in turn may serve as an antiviral and anti-inflammatory mechanism.

Use of ferrets for electrophysiologic monitoring of ion transport.

Limited success achieved in translating basic science discoveries into clinical applications for chronic airway diseases is attributed to differences in respiratory anatomy and physiology, poor approximation of pathologic processes, and lack of correlative clinical endpoints between humans and laboratory animal models. Here, we discuss advantages of using ferrets (Mustela putorus furo) as a model for improved understanding of human airway physiology and demonstrate assays for quantifying airway epithelial ion transport in vivo and ex vivo, and establish air-liquid interface cultures of ferret airway epithelial cells as a complementary in vitro model for mechanistic studies. We present data here that establishes the feasibility of measuring these human disease endpoints in ferrets. Briefly, potential difference across the nasal and the lower airway epithelium in ferrets could be consistently assessed, were highly reproducible, and responsive to experimental interventions. Additionally, ferret airway epithelial cells were amenable to primary cell culture methods for in vitro experiments as was the use of ferret tracheal explants as an ex vivo system for assessing ion transport. The feasibility of conducting multiple assessments of disease outcomes supports the adoption of ferrets as a highly relevant model for research in obstructive airway diseases.

Preceding human metapneumovirus infection increases adherence of Streptococcus pneumoniae and severity of murine pneumococcal pneumonia.

Coinfection with respiratory virus and Streptococcus pneumoniae has been frequently reported in several epidemiologic studies. The aim of this study was to explore the effect of preceding human metapneumovirus (hMPV) inoculation on subsequent pneumococcal infection. Hep-2 and A549 cells were infected with hMPV then inoculated with S. pneumoniae. Bacterial adhesion was measured using colony forming unit and cytometric-fluorescence assays. In vivo bacterial adhesion was examined in hMPV-infected mice after inoculation of fluorescence-conjugated S. pneumoniae. Pulmonary inflammation (bacterial titers, cytokine levels, and histopathology) of hMPV-infected mice was investigated after inoculation with S. pneumoniae. In vitro results of bacterial infection with S. pneumoniae on A549 and Hep-2 monolayer cells showed that even though cellular adherence was variable among different serotypes, there was significantly enhanced bacterial adherence in A549 cells with preceding hMPV infection. In addition, in vivo study of hMPV-infected mice showed increased adhesion of S. pneumoniae on the bronchial epithelium with delayed bacterial clearance and exacerbated histopathology. Furthermore, mice with preceding hMPV infection showed repressed recruitment of airway neutrophils with decreased expression of neutrophil chemoattractants during pneumococcal infection. These results suggest that hMPV-infected airway cells, especially the lower airway epithelium, express increased adherence with S. pneumoniae. Furthermore, hMPV-infected mice showed impaired recruitment of airway neutrophils, possibly leading to delayed bacterial clearance and exacerbated pulmonary inflammation, after secondary infection with pneumococcal isolates.

The Impact of Allergic Rhinitis and Asthma on Human Nasal and Bronchial Epithelial Gene Expression

BackgroundThe link between upper and lower airways in patients with both asthma and allergic rhinitis is still poorly understood. As the biological complexity of these disorders can be captured by gene expression profiling we hypothesized that the clinical expression of rhinitis and/or asthma is related to differential gene expression between upper and lower airways epithelium.ObjectiveDefining gene expression profiles of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles.MethodsThis cross-sectional study included 18 subjects (6 allergic asthma and allergic rhinitis; 6 allergic rhinitis; 6 healthy controls). The estimated false discovery rate comparing 6 subjects per group was approximately 5%. RNA was extracted from isolated and cultured epithelial cells from bronchial brushings and nasal biopsies, and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and Bioconductor Limma package. For gene ontology GeneSpring GX12 was used.ResultsThe study was successfully completed by 17 subjects (6 allergic asthma and allergic rhinitis; 5 allergic rhinitis; 6 healthy controls). Using correction for multiple testing, 1988 genes were differentially expressed between healthy lower and upper airway epithelium, whereas in allergic rhinitis with or without asthma this was only 40 and 301 genes, respectively. Genes influenced by allergic rhinitis with or without asthma were linked to lung development, remodeling, regulation of peptidases and normal epithelial barrier functions.ConclusionsDifferences in epithelial gene expression between the upper and lower airway epithelium, as observed in healthy subjects, largely disappear in patients with allergic rhinitis with or without asthma, whilst new differences emerge. The present data identify several pathways and genes that might be potential targets for future drug development.

Effect and influence of mitogen-activated protein kinases pathway on airway inflammation and epithefium apoptosis in asthmatic guinea pigs

Objective To study the airway inflammation, epithefium apoptosis, p38 MAPK expression level in asthmatic guinea pigs and the effect of dexmethasone therapy. Methods Forty-eight guinea pigs were randomly divided into 4 groups: control group, asthma group, dexmethasone-treated group and MAPK pathway inhibitor SB203580 trea-ted group, 12 guinea pigs in each group.Hematoxylin and eosin staining, Massong Trichrome stain, Periodic Acid Schifgtaining were performed.Histological changes were detected under the optical or the electron microscope.Immunohistochemistry assays were used to determine the status of IL-5 and phosphorylated p38 MAPK in airway epitheliam.Inflammatory factor level was detected by enzyme linked immunosorbent assay(ELISA). Immunohistochemistry assays of cleaved caspase 3 was used to detect the level of epithefium apoptosis. Results Compared with the control group, guinea pigs in the asthmatic group showed significantly higher airway epithelium apoptosis(detected as the cleaved caspase-3 imumunoreactive cells), lung tissue phosphorylated p38 MAPK expression level, IL-5 level in BALF(P 0.05). Compared with SB203580 group, guinea pigs in the DEX group showed significantly lower airway epithelium apoptosis, lung tissue phosphorylated p38 MAPK expression level, and IL-5 level in BALF(P<0.01). Conclusions Airway inflammation is involved in epithefium apoptosis in the asthmatic guinea pigs.The interfering effect of apoptosis by DEX may be partly related to inhibition of lung tissue phosphorylated p38 MAPK expression level, and p38 MAPK may participate in the asthmatic inflammation and induce airway epithelium apoptosis. Key words: Bronchial asthma; Apoptosis; Inflammation; Mitogen-activated protein kinase; Dexmethasone; Interleukin

P24 Pharmacokinetics of peramivir in upper and lower airway epithelia and plasma

P24 Pharmacokinetics of peramivir in upper and lower airway epithelia and plasma

944 Preceding Human Metapneumovirus Infection Increases Adherence of Streptococcus Pneumoniae and Severity of Murine Pneumococcal Pneumonia

Co-infections with respiratory virus and Streptococcus pneumoniae have been frequently reported in epidemiologic studies. Here, we explore how inoculating A549 and Hep-2 cell monolayers with human metapneumovirus (hMPV) affected subsequent...

Regional expression of epithelial MDR1/P‐glycoprotein in chronic rhinosinusitis with and without nasal polyposis

P-glycoprotein (P-gp) is a 170-kDa transmembrane glycoprotein encoded by the MDR1 (ABCB1) gene and is constitutively expressed on lower airway epithelium. P-gp has been shown to function as an immunomodulator regulating efflux of T-helper 1/T-helper 2 (Th1/Th2) cytokines from its host cell; however, its association with sinonasal inflammation has not been described. The purpose of this study is to determine the pattern and degree of epithelial P-gp expression in chronic rhinosinusitis (CRS) with nasal polyposis (CRSwNP) or CRS without nasal polyposis (CRSsNP). Institutional Review Board (IRB)-approved study utilizing sinus, septal, and inferior turbinate mucosa in patients with no disease, CRS, and CRSwNP (n = 4 each). Quantitative fluorescent immunohistochemistry (Q-FIHC) was performed using an anti-P-gp antibody and a secondary fluorescein isothiocyanate (FITC)-conjugated Fc specific fragment. Protein expression was quantified by calculating the epithelial to nonspecific background intensity ratio (4 images/subsite). Scores less than 1 suggested negligible expression. Staining ratios between patient groups and subsites were compared using a 2-tailed Student t test. Among the sinus mucosa, P-gp expression in CRSwNP (1.570 ± 0.354) was significantly greater than both CRS (1.224 ± 0.248) and control (0.762 ± 0.128) (p < 0.001, p = 0.002; respectively). CRS scores were significantly greater than control (p = 0.002). Among the septal mucosa, there was no significant difference between CRSwNP (0.914 ± 0.264), CRS (1.126 ± 0.476), or control (0.966 ± 0.327). Among the inferior turbinate mucosa, there was no significant difference between CRSwNP (1.047 ± 0.157), CRS (1.099 ± 0.362), or control (0.824 ± 0.181). MDR1/P-gp is overexpressed in the epithelial layer of sinus mucosa in patients with both CRSwNP and CRS relative to other sinonasal subsites. Expression in healthy mucosa is negligible. Given its known immunomodulatory function this suggests that P-gp may play a role in the pathogenesis or maintenance of chronic sinonasal inflammation.

Raised interleukin-17 is immunolocalised to neutrophils in cystic fibrosis lung disease.

Interleukin (IL)-17 is pivotal in orchestrating the activity of neutrophils. Neutrophilic inflammation is the dominant pathology in cystic fibrosis (CF) lung disease. We investigated IL-17 protein expression in the lower airway in CF, its cellular immunolocalisation and the effects of IL-17 on CF primary bronchial epithelial cells. Immunohistochemistry was performed on explanted CF lungs and compared with the non-suppurative condition pulmonary hypertension (PH). Airway lavages and epithelial cultures were generated from explanted CF lungs. Immunoreactivity for IL-17 was significantly increased in the lower airway epithelium in CF (median 14.1%) compared with PH (2.95%, p=0.0001). The number of cells staining positive for IL-17 in the lower airway mucosa was also increased (64 cells·mm(-1) compared with 9 cells·mm(-1) basement membrane, p=0.0005) and included both neutrophils in addition to mononuclear cells. IL-17 was detectable in airway lavages from explanted CF lungs. Treatment of epithelial cultures with IL-17 increased production of IL-8, IL-6 and granulocyte macrophage colony-stimulating factor. In conclusion, immunoreactive IL-17 is raised in the lower airway of people with CF and localises to both neutrophils and mononuclear cells. IL-17 increases production of pro-neutrophilic mediators by CF epithelial cells, suggesting potential for a positive feedback element in airway inflammation.

How Viral Infections Cause Exacerbation of Airway Diseases

Exacerbations of asthma and COPD are major causes of morbidity, mortality, and health-care costs. Over the last decade, studies using new molecular diagnostic techniques have established that respiratory viruses are a major cause of exacerbations of both asthma and COPD. The most prevalent viruses detected during exacerbations are the rhinoviruses. Despite the burden of disease associated with exacerbations, little is known about the mechanisms of virus-induced exacerbations of airway diseases. Exacerbations are associated with increased airway inflammation in patients with both asthma and COPD, but many questions remain unanswered regarding the key inflammatory cells and mediators involved. Identifying the key inflammatory mediators involved in exacerbations holds the promise of developing diagnostic and prognostic markers of exacerbation. In addition, such studies can identify new therapeutic targets for the development of novel drugs for the prevention and treatment of exacerbations.

Similar Frequency of Rhinovirus‐Infectible Cells in Upper and Lower Airway Epithelium

Rhinovirus (RV) infections can alter lower airway physiology and inflammation, yet the characteristics of RV replication in lower airway cells are incompletely understood. An RV serotype 16 (RV16)-specific monoclonal antibody was identified. Immunohistochemistry and an infectious center assay were used to quantitate the infectivity of RV16 in primary bronchial and adenoidal epithelial cells. The proportion of infectible epithelial cells increased with the inoculum but did not exceed 10%. Analysis of bronchial tissue samples infected ex vivo demonstrated a small subset of RV-infected cells in the epithelial layer. These data confirm previous reports that RV infects only a small subset of epithelial cells in upper airway tissues and indicate that lower airway epithelial cells have a similar susceptibility to RV infection. In confirming that RV can infect cells in the lower airway, these results suggest that lower airway dysfunction occurs through this mechanism in susceptible persons.

Role of aquaporin water channels in airway fluid transport, humidification, and surface liquid hydration.

Several aquaporin-type water channels are expressed in mammalian airways and lung: AQP1 in microvascular endothelia, AQP3 in upper airway epithelia, AQP4 in upper and lower airway epithelia, and AQP5 in alveolar epithelia. Novel quantitative methods were developed to compare airway fluid transport-related functions in wild-type mice and knockout mice deficient in these aquaporins. Lower airway humidification, measured from the moisture content of expired air during mechanical ventilation with dry air through a tracheotomy, was 54-56% efficient in wild-type mice, and reduced by only 3-4% in AQP1/AQP5 or AQP3/AQP4 double knockout mice. Upper airway humidification, measured from the moisture gained by dry air passed through the upper airways in mice breathing through a tracheotomy, decreased from 91 to 50% with increasing ventilation from 20 to 220 ml/min, and reduced by 3-5% in AQP3/AQP4 knockout mice. The depth and salt concentration of the airway surface liquid in trachea was measured in vivo using fluorescent probes and confocal and ratio imaging microscopy. Airway surface liquid depth was 45 +/- 5 microm and [Na(+)] was 115 +/- 4 mM in wild-type mice, and not significantly different in AQP3/AQP4 knockout mice. Osmotic water permeability in upper airways, measured by an in vivo instillation/sample method, was reduced by approximately 40% by AQP3/AQP4 deletion. In doing these measurements, we discovered a novel amiloride-sensitive isosmolar fluid absorption process in upper airways (13% in 5 min) that was not affected by aquaporin deletion. These results establish the fluid transporting properties of mouse airways, and indicate that aquaporins play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption.

Na+/K+ATPase in Lower Airway Epithelium from Cystic Fibrosis and Non-Cystic-Fibrosis Lung

The basolateral Na+/K+ATPase plays a critical role in sodium reabsorption across airway epithelium. Nasal epithelium shows increased Na+/K+ATPase activity in cystic fibrosis (CF) but Na+/K+ATPase has not been characterized in human lung epithelium or compared in CF and normal lung. We measured3[H] ouabain binding and Na+/K+ATPase activity in human tracheal epithelium and compared Na/K ATPase activity in bronchial epithelium in CF and control subjects. In tracheal epithelium Na+/K+ATPase pumps were abundant and of high3[H] ouabain affinity (Kd 4.7 nM, Bmax38 pmol/mg) and Na+/K+ATPase activity was 55±8 nmol/mg protein/min. Bronchial epithelial Na+/K+ATPase activity was twofold higher in CF patients than in controls. The increased Na+/K+ATPase activity may contribute to the increased sodium reabsorption seen in cystic fibrosis.

NAD(P)H nitroblue tetrazolium reductase levels in apparently normoxic tissues: a histochemical study correlating enzyme activity with binding of radiolabelled misonidazole

Hack and Helmy's method for the histochemical identification of NAD(P)H nitroblue tetrazolium reductase activity was employed to pinpoint reductase activity in certain cells in the mouse. High activity was observed in the following: lower airway epithelium, liver (centrilobular zone), eyelid (meibomian and sebaceous glands), vulval gland and parotid gland (striated cells of intralobular ducts). All of these cells had previously been identified as sites of binding of the reactive metabolites formed from the enzymic reduction of misonidazole (MISO) (Cobb et al., 1989). It had previously been thought that MISO binding would only take place in significant amounts in hypoxic tissues (tumour and possibly liver) since in normoxic tissues oxygen should reverse the initial one electron enzymic reduction, thus preventing progressive reduction to reactive species. We suggest that the very high levels of reductase in the above listed, probably normoxic, tissues contribute significantly to the accumulation of bound reactive MISO metabolite(s).