Normal Human Airway Epithelium Research Articles

Secretory Cells Dominate Airway CFTR Expression and Function in Human Airway Superficial Epithelia.

Rationale: Identification of the specific cell types expressing CFTR (cystic fibrosis [CF] transmembrane conductance regulator) is required for precision medicine therapies for CF. However, a full characterization of CFTR expression in normal human airway epithelia is missing. Objectives: To identify the cell types that contribute to CFTR expression and function within the proximal-distal axis of the normal human lung. Methods: Single-cell RNA (scRNA) sequencing (scRNA-seq) was performed on freshly isolated human large and small airway epithelial cells. scRNA in situ hybridization (ISH) and single-cell qRT-PCR were performed for validation. In vitro culture systems correlated CFTR function with cell types. Lentiviruses were used for cell type-specific transduction of wild-type CFTR in CF cells. Measurements and Main Results: scRNA-seq identified secretory cells as dominating CFTR expression in normal human large and, particularly, small airway superficial epithelia, followed by basal cells. Ionocytes expressed the highest CFTR levels but were rare, whereas the expression in ciliated cells was infrequent and low. scRNA ISH and single-cell qRT-PCR confirmed the scRNA-seq findings. CF lungs exhibited distributions of CFTR and ionocytes similar to those of normal control subjects. CFTR mediated Cl- secretion in cultures tracked secretory cell, but not ionocyte, densities. Furthermore, the nucleotide-purinergic regulatory system that controls CFTR-mediated hydration was associated with secretory cells and not with ionocytes. Lentiviral transduction of wild-type CFTR produced CFTR-mediated Cl- secretion in CF airway secretory cells but not in ciliated cells. Conclusions: Secretory cells dominate CFTR expression and function in human airway superficial epithelia. CFTR therapies may need to restore CFTR function to multiple cell types, with a focus on secretory cells.

Isolation of Human Coronaviruses OC43, HKU1, NL63, and 229E in Yamagata, Japan, Using Primary Human Airway Epithelium Cells Cultured by Employing an Air-Liquid Interface Culture.

Isolation of seasonal coronaviruses, which include human coronavirus (HCoV) OC43, HCoV-HKU1, and HCoV-NL63, from primary cultures is difficult because it requires experienced handling, an exception being HCoV-229E, which can be isolated using cell lines such as RD-18S and HeLa-ACE2-TMPRSS2. We aimed to isolate seasonal CoVs in Yamagata, Japan to obtain infective virions useful for further research and to accelerate fundamental studies on HCoVs and SARS-CoV-2. Using modified air-liquid interface (ALI) culture of the normal human airway epithelium from earlier studies, we isolated 29 HCoVs (80.6%: 16, 6, 6, and 1 isolates of HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E, respectively) from 36 cryopreserved nasopharyngeal specimens. In ALI cultures of HCoV-OC43 and HCoV-NL63, the harvested medium contained more than 1 × 104 genome copies/µL at every tested time point during the more than 100 days of culture. Four isolates of HCoV-NL63 were further subcultured and successfully propagated in an LLC-MK2 cell line. Our results suggest that ALI culture is useful for isolating seasonal CoVs and sustainably obtaining HCoV-OC43 and HCoV-NL63 virions. Furthermore, the LLC-MK2 cell line in combination with ALI cultures can be used for the large-scale culturing of HCoV-NL63. Further investigations are necessary to develop methods for culturing difficult-to-culture seasonal CoVs in cell lines.

Novel instrument to generate representative e-cigarette vapors for physicochemical particle characterization and in-vitro toxicity

The use of “electronic cigarettes” (e-cigs) has exponentially increased during the last decade. This drastic growth has been observed in smokers and never smokers, women and teenagers, with indicators for adverse effects related to nicotine, but also to e-liquid and vapor components. The aerosol characteristics depend on the e-cig features. Therefore, the introduction of many different e-cig types on the market leads to concerns regarding their potential health effects. Our study aims to develop an instrument to generate representative vapors from e-cigarette devices, characterize the physicochemical properties of the aerosols and elucidate the adverse effects of inhaled vapors on normal human airway epithelia. E-cig vapors were produced using a specially developed aerosol generation instrument mimicking user puffing behavior. Aerosol particle size distribution and concentration were characterized with a Scanning Mobility Particle Sizer. Air-liquid interface cultures of differentiated human bronchial epithelial cells were exposed to 1 puff topography, i.e. to 21 puffs of 3 s with 22-second puff intervals using the Nano-Aerosol Chamber for In-Vitro Toxicity, which simulates particle deposition in the respiratory tract. Cell morphology was evaluated by light microcopy and induction of cell death by release of lactate dehydrogenase at 4 and 24 h after aerosol exposure. Particle size in e-cig generated aerosols ranged from 20 to 430 nm and concentrations exceeded 1 × 106 particles/cm3. Deposited particle doses were 0.9–3 μg/cm2, depending on e-device type and number of puffs. A single exposure to e-cig vapors caused changes in epithelial cell shape but no massive cell death. We successfully developed an aerosol generation system to produce and deposit realistic vapors from e-cigs. The results obtained indicate that a single exposure to e-cig aerosols affects epithelial morphology and slightly increases cell death. Our interdisciplinary approach combining aerosol technology and biology allows identifying adverse effects of e-cigs to the respiratory tract in vitro.

POU2AF1 Functions in the Human Airway Epithelium To Regulate Expression of Host Defense Genes.

In the process of seeking novel lung host defense regulators by analyzing genome-wide RNA sequence data from normal human airway epithelium, we detected expression of POU domain class 2-associating factor 1 (POU2AF1), a known transcription cofactor previously thought to be expressed only in lymphocytes. Lymphocyte contamination of human airway epithelial samples obtained by bronchoscopy and brushing was excluded by immunohistochemistry staining, the observation of upregulation of POU2AF1 in purified airway basal stem/progenitor cells undergoing differentiation, and analysis of differentiating single basal cell clones. Lentivirus-mediated upregulation of POU2AF1 in airway basal cells induced upregulation of host defense genes, including MX1, IFIT3, IFITM, and known POU2AF1 downstream genes HLA-DRA, ID2, ID3, IL6, and BCL6. Interestingly, expression of these genes paralleled changes of POU2AF1 expression during airway epithelium differentiation in vitro, suggesting POU2AF1 helps to maintain a host defense tone even in pathogen-free condition. Cigarette smoke, a known risk factor for airway infection, suppressed POU2AF1 expression both in vivo in humans and in vitro in human airway epithelial cultures, accompanied by deregulation of POU2AF1 downstream genes. Finally, enhancing POU2AF1 expression in human airway epithelium attenuated the suppression of host defense genes by smoking. Together, these findings suggest a novel function of POU2AF1 as a potential regulator of host defense genes in the human airway epithelium.

Early events in the pathogenesis of chronic obstructive pulmonary disease. Smoking-induced reprogramming of airway epithelial basal progenitor cells.

The airway epithelium is the primary site of the earliest pathologic changes induced by smoking, contributing to the development of chronic obstructive pulmonary disease (COPD). The normal human airway epithelium is composed of several major cell types, including differentiated ciliated and secretory cells, intermediate undifferentiated cells, and basal cells (BC). BC contain the stem/progenitor cell population responsible for maintenance of the normally differentiated airway epithelium. Although inflammatory and immune processes play a significant role in the pathogenesis of COPD, the earliest lesions include hyperplasia of the BC population, suggesting that the disease may start with this cell type. Apart from BC hyperplasia, smoking induces a number of COPD-relevant airway epithelial remodeling phenotypes that are likely initiated in the BC population, including mucous cell hyperplasia, squamous cell metaplasia, epithelial-mesenchymal transition, altered ciliated and nonmucous secretory cell differentiation, and suppression of junctional barrier integrity. Significant progress has been recently made in understanding the biology of human airway BC, including gene expression features, stem/progenitor, and other functions, including interaction with other airway cell types. Accumulating evidence suggests that human airway BC function as both sensors and cellular sources of various cytokines and growth factors relevant to smoking-associated airway injury, as well as the origin of various molecular and histological phenotypes relevant to the pathogenesis of COPD. In the context of these considerations, we suggest that early BC-specific smoking-induced molecular changes are critical to the pathogenesis of COPD, and these represent a candidate target for novel therapeutic approaches to prevent COPD progression in susceptible individuals.

The efficacy of Echinacea in a 3‐D tissue model of human airway epithelium

We evaluated the antirhinovirus efficacy of a standardized preparation of Echinacea purpurea (Echinaforce) in a 3-dimensional organotypic model of normal human airway epithelium (EpiAirway tissue). Individual replicate tissue samples, maintained as inserts in culture for 3 days or 3 weeks, were infected with rhinovirus type 1A (RV1A), Echinacea alone, a combination of the two, or medium only. None of the treatments affected the histological appearance or integrity of the tissues, all of which maintained a high level of cell viability and preservation of cilia. RV infection resulted in increased mucopolysaccharide inclusions in the goblet cells, but this feature was reversed by Echinacea treatment. This result was confirmed by measurements of mucin secretion, which was stimulated by RV but reversed by Echinacea, suggesting that mucus production during colds could be ameliorated by Echinacea. We did not find evidence of virus replication, although the RV-infected tissues secreted substantial amounts of the pro-inflammatory cytokines IL-6 and IL-8 (CXCL8), and this response was reversed by Echinacea treatment. These results confirmed previous findings derived from studies of bronchial and lung epithelial cell lines, namely, that RV infection results in a substantial inflammatory response in the absence of virus replication.

Effects of prostagladin E2 on gel-forming mucin secretion in normal human nasal epithelial cells

Conclusion. The findings of this study indicate that prostaglandin E2 (PGE2) induces MUC5AC gene expression and mucin secretion via the EP4 receptor in cultured normal human nasal epithelial cells. Objectives. Recently, PGE2 was found to induce MUC5AC production via an agonist of EP2/EP4, but not EP1/EP3, in normal human airway epithelium. However, the receptor that mediates MUC5AC has not been determined. This study aimed to investigate the MUC5AC mucin gene and mucin secretion by PGE2 and its receptors in cultured normal human nasal epithelial cells. Methods. After treatment with PGE2 and/or PGE2 antagonist, gel-forming mucin mRNA expression was determined by reverse transcription-polymerase chain reaction. Total mucin and MUC5AC mucin levels were measured using an immuno-dot blotting assay. Results. PGE2 increased only MUC5AC gene expression and MUC5AC mucin, but not expression of other gel-forming mucin genes. An EP2 receptor antagonist (AH 6809) did not suppress the PGE2-induced MUC5AC gene expression or MUC5AC mucin. However, an EP4 receptor antagonist (AH23848) significantly suppressed the level of PGE2-induced MUC5AC gene expression and MUC5AC mucin.

Cystic Fibrosis Airway Epithelial Ca2+i Signaling: THE MECHANISM FOR THE LARGER AGONIST-MEDIATED Ca2+i SIGNALS IN HUMAN CYSTIC FIBROSIS AIRWAY EPITHELIA

In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca(2+)(i)) mobilization. We have shown that Ca(2+)(i) signals resulting from apical purinoceptor (P2Y(2)-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y(2)-R-dependent Ca(2+)(i) signals in CF human airway epithelia. We show that the increased Ca(2+)(i) mobilization in CF was not specific to P2Y(2)-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y(2)-R or a more efficient coupling between P2Y(2)-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y(2)-R activation-promoted Ca(2+)(i) signals in CF epithelia resulted from an increased density and Ca(2+) storage capacity of apically confined endoplasmic reticulum (ER) Ca(2+) stores. To address whether the ER up-regulation resulted from ER retention of misfolded DeltaF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of DeltaF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, DeltaF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y(2)-R activation-mobilized Ca(2+)(i), which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether luminal infection/inflammation triggers an up-regulation of the apically confined ER Ca(2+) stores, normal airway epithelia were chronically exposed to supernatant from mucopurulent material from CF airways. Supernatant treatment expanded the apically confined ER, resulting in larger apical P2Y(2)-R activation-dependent Ca(2+)(i) responses, which reproduced the increased Ca(2+)(i) signals observed in CF epithelia. In conclusion, the mechanism for the larger Ca(2+)(i) signals elicited by apical P2Y(2)-R activation in CF airway epithelia is an expansion of the apical ER Ca(2+) stores triggered by chronic luminal airway infection/inflammation. Greater ER-derived Ca(2+)(i) signals may provide a compensatory mechanism to restore, at least acutely, mucus clearance in CF airways.

Swelling-activated Ca2+ Entry via TRPV4 Channel Is Defective in Cystic Fibrosis Airway Epithelia

The vertebrate transient receptor potential cationic channel TRPV4 has been proposed as an osmo- and mechanosensor channel. Studies using knock-out animal models have further emphasized the relevance of the TRPV4 channel in the maintenance of the internal osmotic equilibrium and mechanosensation. However, at the cellular level, there is still one important question to answer: does the TRPV4 channel generate the Ca(2+) signal in those cells undergoing a Ca(2+)-dependent regulatory volume decrease (RVD) response? RVD in human airway epithelia requires the generation of a Ca(2+) signal to activate Ca(2+)-dependent K(+) channels. The RVD response is lost in airway epithelia affected with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator channel. We have previously shown that the defective RVD in CF epithelia is linked to the lack of swelling-dependent activation of Ca(2+)-dependent K(+) channels. In the present study, we show the expression of TRPV4 in normal human airway epithelia, where it functions as the Ca(2+) entry pathway that triggers the RVD response after hypotonic stress, as demonstrated by TRPV4 antisense experiments. However, cell swelling failed to trigger Ca(2+) entry via TRPV4 channels in CF airway epithelia, although the channel's response to a specific synthetic activator, 4 alpha-phorbol 12,13-didecanoate, was maintained. Furthermore, RVD was recovered in CF airway epithelia treated with 4 alpha-phorbol 12,13-didecanoate. Together, these results suggest that defective RVD in CF airway epithelia might be caused by the absence of a TRPV4-mediated Ca(2+) signal and the subsequent activation of Ca(2+)-dependent K(+) channels.

Number and proliferation of clara cells in normal human airway epithelium.

Experimental pathologic studies suggest that Clara cells are one of the types of airway stem cells but the proliferation of Clara cells in human lungs has not yet been examined. The purpose of this study was to assess in conducting airways of normal human lungs: (1) the number of Clara cells; and (2) the contribution of Clara cells to the proliferation compartment. Samples of histologic normal tissue were taken from seven lungs obtained by autopsy. A triple sequential (immuno)histochemical staining was performed, using MIB-1 as a proliferation marker and anti-CC10 for the identification of Clara cells; subsequently, a PAS stain was carried out as a marker for goblet cells, as these cells were reported to be CC10-immunoreactive in an unknown proportion. Clara cells were virtually absent in the proximal airway epithelium. The number of Clara cells in the terminal bronchioles was 11 +/- 3% (mean +/- SD) and in respiratory bronchioles 22 +/- 5%. The overall proliferation compartment of the conducting airway epithelium was 0.83 +/- 0.47%; the contribution of Clara cells was 9%. In the terminal bronchioles 15% of proliferating airway epithelial cells were Clara cells, and in the respiratory bronchioles this number increased to 44%. The contribution of Clara cells to the proliferation compartment of normal human tracheobronchial epithelium is substantial, demonstrating a role of the Clara cell in the maintenance of the normal epithelium of the distal conducting airways in humans.

Number and proliferation of basal and parabasal cells in normal human airway epithelium.

Two roles have been suggested for basal cells on the basis of studies performed with laboratory animals: (1) anchoring of the tracheobronchial epithelium; and (2) being the epithelial stem cell. Parabasal cells located just above the basal cells have also been shown to contribute to cell renewal. However, a systematic study of the composition and proliferation of basal and parabasal cells in normal human lungs is lacking. The aims of this study were to determine in normal human conducting-airway epithelium: (1) the number of basal and parabasal cells; and (2) the contribution of basal and parabasal cells to the proliferation fraction. Samples of histologically normal tissue, free of pulmonary disease, were taken from seven lungs obtained by autopsy. Immunohistochemical staining was performed with the primary antibody MIB-1 as a proliferation marker and the antikeratin antibody 34betaE12 as a marker for basal and parabasal cells. In the largest conducting airways (diameter >= 4 mm), the percentages of basal and parabasal cells were 31% and 7%, respectively; the contribution to the proliferation compartment was 51% for basal and 33% for parabasal cells. In the smallest airways (diameter < 0.5 mm), 6% of epithelial cells were basal cells, with a 30% contribution to the proliferation compartment, whereas parabasal cells were absent. The high fraction of basal and parabasal cells contributing to the proliferation compartment of normal human conducting-airway epithelium supports the theory that cells at or near the basement membrane are likely to be progenitor cells.

Number and proliferation of neuroendocrine cells in normal human airway epithelium.

Pulmonary neuroendocrine cells (PNECs) are ubiquitous in human adult airway epithelium, and are implicated in pulmonary disease as PNEC hyperplasia has been described in a great number of different pulmonary lesions. The purpose of this study was to determine the baseline quantity and proliferative fraction of adult PNECs in the conducting airway epithelium of normal lungs. Autopsy was performed within 6 h of death on 250 subjects. In 9 of 250 cases without pulmonary disease, seven sequential samples were taken from trachea down to respiratory bronchioles, fixed in formalin, and embedded in paraffin. After microwave pretreatment in citrate buffer, sequential immunohistochemical staining was carried out, using MIB-1 (antibody directed against recombinant parts of the Ki-67 antigen) as marker for the proliferation fraction and chromogranin-A (CgA) for the identification of PNECs. Quantification of all epithelial cells of the conducting airways, MIB-1, and CgA immunoreactive cells, and length of basement membrane was interactively performed using image analysis. The results show that microwave treatment significantly enhances the sensitivity of CgA without loss of specificity. In total, 326,500 epithelial cells and 105 cm of basement membrane (BM) length were counted (3,101 cells/cm BM). The proliferation fraction of all epithelial cells was 0.76 +/- 0.53% (mean +/- SD). No mitosis was seen. The mean quantity of PNECs was 0.41 +/- 0.17% of all epithelial cells (12.5 cells/cm BM). Many dendritic PNEC processes parallel to the basement membrane could be appreciated between adjacent epithelial cells. No difference in the number of PNECs was found between large and small conducting airways. PNECs were not observed in the alveoli. Only 13 PNECs were immunoreactive for MIB-1; the proliferative fraction of PNECs was 1 to 2%. In histologically normal adult human conducting airway epithelium, the average overall proliferation fraction is 0.76%. The proliferation fraction of PNECs is equal to the overall proliferative fraction. The fraction of PNECs is 0.41%, using anti-CgA antiserum after microwave pretreatment of slides, 12 times more than previously reported. Many dendritic neuroendocrine cell processes were seen between non-neuroendocrine epithelial cells adjacent to the basement membrane. The widely held belief that neuroendocrine cells are of little importance in the adult lung is contradicted by the relative high density of PNECs and its cytoplasmic processers.

Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo.

Nitric oxide (NO) is an important mediator of inflammatory responses in the lung and a key regulator of bronchomotor tone. An airway NO synthase (NOS; EC 1.14.13.39) has been proposed as a source of endogenous NO in the lung but has not been clearly defined. Through molecular cloning, we conclusively demonstrate that NO synthesis in normal human airways is due to the continuous expression of the inducible NOS (iNOS) isoform in airway epithelial cells. Although iNOS mRNA expression is abundant in airway epithelial cells, expression is not detected in other pulmonary cell types, indicating that airway epithelial cells are unique in the continuous pattern of iNOS expression in the lung. In situ analysis reveals all airway epithelial cell types express iNOS. However, removal of epithelial cells from the in vivo airway environment leads to rapid loss of iNOS expression, which suggests expression is dependent upon conditions and/or factors present in the airway. Quantitation of NOS activity in epithelial cell lysates indicates nanomolar levels of NO synthesis occur in vivo. Remarkably, the high-level iNOS expression is constant in airway epithelium of normal individuals over time. However, expression is strikingly decreased by inhaled corticosteroids and beta-adrenergic agonists, medications commonly used in treatment of inflammatory airway diseases. Based upon these findings, we propose that respiratory epithelial cells are key inflammatory cells in the airway, functioning in host defense and potentially playing a role in airway inflammation.