Nasal Airway Epithelial Cells Research Articles

The triple combination of Remdesivir (GS-441524), Molnupiravir and Ribavirin is highly efficient in inhibiting coronavirus replication in human nasal airway epithelial cell cultures and in a hamster infection model

The use of fixed dose-combinations of antivirals with different mechanisms of action has proven key in the successful treatment of infections with HIV and HCV. For the treatment of infections with SARS-CoV-2 and possible future epi-/pandemic coronaviruses, it will be important to explore the efficacy of combinations of different drugs, in particular to avoid resistance development, such as in patients with immunodeficiencies. This work explores the effect of a combination of 3 broad-spectrum antiviral nucleosides on the replication of coronaviruses. To that end, we made use of primary human airway epithelial cell (HAEC) cultures grown at the air-liquid interface that were infected with the beta coronavirus OC43. We found that the triple combination of GS-441524 (the parent nucleoside of remdesivir), molnupiravir and ribavirin resulted in a more pronounced antiviral efficacy than what could be expected from a purely additive antiviral effect. The potency of this triple combination was next tested in SARS-CoV-2 infected hamsters in a prophylactic setup. To that end, for each of the drugs, intentionally suboptimal or even ineffective doses were selected. Yet, in the lungs of all hamsters that received triple prophylactic therapy (but not in those that received the respective double combinations) no infectious virus was detectable. Our findings indicate that co-administration of approved drugs for the treatment of coronavirus infections should be further explored but also against other families of viruses with epidemic and pandemic potential for which no effective antiviral treatment is available.

Transcriptomic analysis of primary nasal epithelial cells reveals altered interferon signalling in preterm birth survivors at one year of age.

Introduction: Many survivors of preterm birth (<37weeks gestation) have lifelong respiratory deficits, the drivers of which remain unknown. Influencers of pathophysiological outcomes are often detectable at the gene level and pinpointing these differences can help guide targeted research and interventions. This study provides the first transcriptomic analysis of primary nasal airway epithelial cells in survivors of preterm birth at approximately 1year of age. Methods: Nasal airway epithelial brushings were collected, and primary cell cultures established from term (>37 weeks gestation) and very preterm participants (≤32 weeks gestation). Ex vivo RNA was collected from brushings with sufficient cell numbers and in vitro RNA was extracted from cultured cells, with bulk RNA sequencing performed on both the sample types. Differential gene expression was assessed using the limma-trend pipeline and pathway enrichment identified using Reactome and GO analysis. To corroborate gene expression data, cytokine concentrations were measured in cell culture supernatant. Results: Transcriptomic analysis to compare term and preterm cells revealed 2,321 genes differentially expressed in ex vivo samples and 865 genes differentially expressed in cultured basal cell samples. Over one third of differentially expressed genes were related to host immunity, with interferon signalling pathways dominating the pathway enrichment analysis and IRF1 identified as a hub gene. Corroboration of disrupted interferon release showed that concentrations of IFN-α2 were below measurable limits in term samples but elevated in preterm samples [19.4 (76.7) pg/ml/µg protein, p = 0.03]. IFN-γ production was significantly higher in preterm samples [3.3 (1.5) vs. 9.4 (17.7) pg/ml/µg protein; p = 0.01] as was IFN-β [7.8 (2.5) vs. 13.6 (19.5) pg/ml/µg protein, p = 0.01]. Conclusion: Host immunity may be compromised in the preterm nasal airway epithelium in early life. Altered immune responses may lead to cycles of repeated infections, causing persistent inflammation and tissue damage which can have significant impacts on long-term respiratory function.

Single cell profiling to determine influence of wheeze and early-life viral infection on developmental programming of airway epithelium.

Nasal airway epithelial cells from children with wheeze are characterized by altered development and increased susceptibility to RSV infection.

In vitro and in vivo effects of Pelargonium sidoides DC. root extract EPs® 7630 and selected constituents against SARS-CoV-2 B.1, Delta AY.4/AY.117 and Omicron BA.2.

The occurrence of immune-evasive SARS-CoV-2 strains emphasizes the importance to search for broad-acting antiviral compounds. Our previous in vitro study showed that Pelargonium sidoides DC. root extract EPs® 7630 has combined antiviral and immunomodulatory properties in SARS-CoV-2-infected human lung cells. Here we assessed in vivo effects of EPs® 7630 in SARS-CoV-2-infected hamsters, and investigated properties of EPs® 7630 and its functionally relevant constituents in context of phenotypically distinct SARS-CoV-2 variants. We show that EPs® 7630 reduced viral load early in the course of infection and displayed significant immunomodulatory properties positively modulating disease progression in hamsters. In addition, we find that EPs® 7630 differentially inhibits SARS-CoV-2 variants in nasal and bronchial human airway epithelial cells. Antiviral effects were more pronounced against Omicron BA.2 compared to B.1 and Delta, the latter two preferring TMPRSS2-mediated fusion with the plasma membrane for cell entry instead of receptor-mediated low pH-dependent endocytosis. By using SARS-CoV-2 Spike VSV-based pseudo particles (VSVpp), we confirm higher EPs® 7630 activity against Omicron Spike-VSVpp, which seems independent of the serine protease TMPRSS2, suggesting that EPs® 7630 targets endosomal entry. We identify at least two molecular constituents of EPs® 7630, i.e., (-)-epigallocatechin and taxifolin with antiviral effects on SARS-CoV-2 replication and cell entry. In summary, our study shows that EPs® 7630 ameliorates disease outcome in SARS-CoV-2-infected hamsters and has enhanced activity against Omicron, apparently by limiting late endosomal SARS-CoV-2 entry.

In-vitro and in-vivo anti-allergic effects of magnolol on allergic rhinitis via inhibition of ORAI1 and ANO1 channels

Ethnopharmacological relevanceFlos Magnoliae (the dried flower buds of Magnolia biondii Pamp, FM) is a known herbal traditional medicine used for the symptomatic relief of nasal congestion and rhinorrhea caused by rhinitis and sinusitis. Magnolol, a neolignan from the magnolia family, is a secondary metabolite known to have anti-allergic and anti-inflammatory effects. However, the underlying mechanisms and therapeutic effect of magnolol in the treatment of allergic rhinitis (AR) remain elusive. Aims of the studyAnoctamin 1 (ANO1), a calcium-activated anion channel, mediates mucus and electrolyte secretion in nasal airway epithelial cells, whereas calcium release-activated calcium channel protein 1 (ORAI1) participates in the activation of T-lymphocytes and mast cells. The aim of our study is to understand the mechanisms of action of magnolol against AR, i.e., whether it acts through the modulation of ANO1 and ORAI1 channels that are expressed in nasal epithelial cells and T-lymphocytes, respectively. Materials and methodsWhole-cell patch clamp was used to record the activity of ORAI1 and ANO1 ion channels in ORAI1 or ANO1 overexpressed HEK293T cells, while the Ussing chamber apparatus was used to measure electrolyte transport via the epithelium, in Calu-3 cells cultured in an air-liquid interface. Additionally, calcium imaging of Jurkat T-lymphocytes was used to assess changes in the intracellular calcium concentration. Magnolol toxicity was assessed using the CCK-8 assay, and its effect on T-lymphocyte proliferation was measured by labeling human primary T-lymphocytes with carboxyfluorescein succinimidyl ester. Finally, OVA-induced Balb/c mice were employed to evaluate the effect of magnolol on nasal symptoms, as well as cytokine and eosinophil infiltration in AR. ResultsMagnolol inhibits ORAI1 and ANO1 channels in a concentration-dependent manner. Magnolol (30 μM) inhibits anti-CD3 induced cellular proliferation and production of IL-2 via ORAI1 channels in T-lymphocytes. Further, ATP-induced electrolyte transport mediated by ANO1 channels is significantly inhibited by magnolol in IL-4 sensitized Calu-3 cells. Notably, 300 μM magnolol significantly attenuates cytokine and eosinophil infiltration, thus alleviating AR symptoms in mice OVA-induced AR. ConclusionMagnolol may be a promising therapeutic agent for the treatment and prevention of AR.

Cellular and systemic energy metabolic dysregulation in asthma development—a hypothesis-generating approach

The roles of systemic and airway-specific epithelial energy metabolism in altering the developmental programming of airway epithelial cells (AECs) in early life are poorly understood. Our aim was to assess carbohydrate metabolism in developing AECs among children with and without wheeze and test the association of infant plasma energy biomarkers with subsequent recurrent wheeze and asthma outcomes. We measured cellular carbohydrate metabolism in live nasal AECs collected at age 2 years from 15 male subjects with and without a history of wheeze and performed a principal component analysis to visually assess clustering of data on AEC metabolism of glycolitic metabolites and simple sugars. Among 237 children with available year 1 plasma samples, we tested the associations of year 1 plasma energy biomarkers and recurrent wheeze and asthma by using generalized estimating equations and logistic regression. Children with a history of wheeze had lower utilization of glucose in their nasal AECs than did children with no wheeze. Systemically, a higher plasma glucose concentration at year 1 (within the normal range) was associated with decreased odds ofasthma at age 5 years (adjusted odds ratio= 0.56; 95% CI=0.35-0.90). Insulin concentration, glucose-to-insulin ratio, C-peptide concentration, and leptin concentration at year 1 were associated with recurrent wheeze from age 2 years to age 5 years. These results suggest that there is significant energy metabolism dysregulation in early life, which likely affects AEC development. These pertubations of epithelial cell metabolism in infancy may have lasting effects on lung development that could render the airway more susceptible to allergic sensitization.

Metabolic Reprogramming of Nasal Airway Epithelial Cells Following Infant Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is a seasonal mucosal pathogen that infects the ciliated respiratory epithelium and results in the most severe morbidity in the first six months of life. RSV is a common cause of acute respiratory infection during infancy and is an important early-life risk factor strongly associated with asthma development. While this association has been repeatedly demonstrated, limited progress has been made on the mechanistic understanding in humans of the contribution of infant RSV infection to airway epithelial dysfunction. An active infection of epithelial cells with RSV in vitro results in heightened central metabolism and overall hypermetabolic state; however, little is known about whether natural infection with RSV in vivo results in lasting metabolic reprogramming of the airway epithelium in infancy. To address this gap, we performed functional metabolomics, 13C glucose metabolic flux analysis, and RNA-seq gene expression analysis of nasal airway epithelial cells (NAECs) sampled from infants between 2–3 years of age, with RSV infection or not during the first year of life. We found that RSV infection in infancy was associated with lasting epithelial metabolic reprogramming, which was characterized by (1) significant increase in glucose uptake and differential utilization of glucose by epithelium; (2) altered preferences for metabolism of several carbon and energy sources; and (3) significant sexual dimorphism in metabolic parameters, with RSV-induced metabolic changes most pronounced in male epithelium. In summary, our study supports the proposed phenomenon of metabolic reprogramming of epithelial cells associated with RSV infection in infancy and opens exciting new venues for pursuing mechanisms of RSV-induced epithelial barrier dysfunction in early life.

Transcriptome Analysis Identifies Doublesex and Mab-3 Related Transcription Factor (DMRT3) in Nasal Polyp Epithelial Cells of Patients Suffering from Non-Steroidal Anti-Inflammatory Drug-Exacerbated Respiratory Disease (AERD).

Background: Aspirin-exacerbated respiratory disease (AERD) is a syndrome characterised by chronic rhinosinusitis, nasal polyps, asthma and aspirin intolerance. An imbalance of eicosanoid metabolism with anover-production of cysteinyl leukotrienes (CysLTs) has been associated with AERD. However, the precise mechanisms underlying AERD are unknown. Objective: To establish the transcriptome of the nasal polyp airway epithelial cells derived from AERD patients to discover gene expression patterns in this disease. Methods: Nasal airway epithelial cells were isolated from 12 AERD polyps and 8 AERD non-polyp nasal mucosa samples as controls from the same subjects. Utilising the Illumina HiSeq 2500 platform, RNA samples were sequenced. Potential gene candidate DMRT3 was selected from the differentially-expressed genes for validation. Results: Comparative transcriptome profiling of nasal epithelial cells was accomplished in AERD. A total of 20 genes had twofold mean regulation expression differences or greater. In addition, 8 genes were upregulated, including doublesex and mab-3 related transcription factor 3 (DMRT3), and 12 genes were downregulated. Differentially regulated genes comprised roles in inflammation, defence and immunity. Metabolic process and embryonic development pathways were significantly enriched. Enzyme-linked immune sorbent assay (ELISA) results of DMRT3 in AERD patients were significantly upregulated compared to controls (p = 0.03). Immunohistochemistry (IHC) of AERD nasal polyps localised DMRT3 and was predominantly released in the airway epithelia. Conclusion: Findings suggest that DMRT3 could be potentially involved in nasal polyp development in AERD patients. Furthermore, several genes are downregulated, hinting at the dedifferentiation phenomenon in AERD polyps. However, further studies are imperative to confirm the exact mechanism of polyp formation in AERD patients.

CXCL10 production induced by high levels of IKKε in nasal airway epithelial cells in the setting of chronic inflammation

The airway is the major entry route of pathogens due to continuous gas exchange with the environment. In particular, the nasal epithelial layer is the common site of airborne mucotropic virus infections. The inflammatory response to such infections must be tightly controlled due to its non-specific nature. Unrestrained inflammation breaks down the physiological mucosal defense system and leads to secondary bacterial or fungal infections. Chronic rhinosinusitis (CRS) is a prevalent inflammatory disease that compromises quality of life. In spite of its importance in the initiation of inflammation, the role of interferon signaling in nasal airway epithelial cells is largely unknown. We analyzed the expression of interferon signaling genes using clinical lavage specimens and nasal airway epithelial cells collected from CRS patients and controls. Basal expression of IFNAs, IKBKE, STAT1, and some CXC chemokines was elevated in samples from CRS patients. In subsequent in vitro studies, we found IKKε to be the key molecule and augmented CXCL10 secretion. Based on our findings and review of the literature, we hypothesized that high levels of IKKε might induce intractable inflammation via CXCL10. We tested the hypothesis in an animal model and found not only that IKKε induced severe eosinophilic inflammation with CXCL10 over-production, but also that inhibition of IKKε resolved the inflammation.

Influence of gestational and developmental age on human airway epithelial innate immune responses to Respiratory Syncytial Virus (RSV) in early life

Background RSV is a major respiratory pathogen in infants, with preterm and young infants being at increased risk of severe disease. Airway epithelial cell (AEC) innate immune responses represent an important first line of defence. Development of these responses in early life is poorly understood. Methods Well differentiated cultures were generated using nasal AECs harvested from term and preterm infants at birth and from the same infants at one-year old. Cultures were infected with RSV and resulting cytopathology and innate immune responses studied. Results RSV growth kinetics were similar between preterm and term newborn cohorts at birth and in the one-year-old repeat cohort. Secretion of interferon lambda-1 (IFN-l1), CXCL10 (IP-10), CCL5 (RANTES) and CXCL8 (IL-8) were similar between RSV-infected preterm and term newborn AECs. Following RSV infection, significantly higher secretion of IFN-l1 (P=0.0034), CXCL10 (P&lt;0.05) and CCL5 (P&lt;0.05) was noted in one-year-derived AECs compared to newborn AECs. We also identified differential expression of a novel endogenously expressed anti-RSV protein, pleiotrophin (PTN), which interacts with nucleolin, a cofactor for RSV entry. PTN expression/secretion was significantly decreased in preterm versus term AECs (P=0.014) and in newborn compared to one-year WD-PNECs (P=0.0008). Conclusion These exciting findings represent the first description of age- and prematurity-related differences in AEC innate immune responses, demonstrating greater RSV-induced expression of pro-inflammatory chemokines with increasing age and developmental differences in expression of the novel anti-RSV protein PTN. These findings may, in part, explain the increased susceptibility of preterm and very young infants to severe RSV disease.

Endoplasmic Reticulum Stress Induces MUC5AC and MUC5B Expression in Human Nasal Airway Epithelial Cells.

ObjectivesEndoplasmic reticulum (ER) stress is known to be associated with inflammatory airway diseases, and three major transmembrane receptors: double-stranded RNA-activated protein kinase-like ER kinase, inositol requiring enzyme 1, and activating transcription factor 6 (ATF6) play important roles in ER stress-related proinflammatory signaling. However, the effects of ER stress and these three major signaling pathways on the regulation of the production of airway mucins in human nasal airway epithelial cells have not been elucidated.MethodsIn primary human nasal epithelial cells, the effect of tunicamycin (an ER stress inducer) and 4-phenylbutyric acid (4-PBA, ER stress inhibitor) on the expression of MUC5AC and MUC5B was investigated by reverse transcriptasepolymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and immunoblot analysis. Small interfering RNA (siRNA) transfection was used to identify the mechanisms involved.ResultsTunicamycin increased the expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules, including spliced X-box binding protein 1 (XBP-1), transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP), and ATF6. In addition, 4-PBA attenuated the tunicamycin-induced expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules. Furthermore, siRNA knockdowns of XBP-1, CHOP, and ATF6 blocked the tunicamycin-induced mRNA expressions and glycoprotein productions of MUC5AC and MUC5B.Conclusion. These results demonstrate that ER stress plays an important role in the regulation of MUC5AC and MUC5B via the activations of XBP-1, CHOP, and ATF6 in human nasal airway epithelial cells.

Regional Differences in Airway Epithelial Cells Reveal Tradeoff between Defense against Oxidative Stress and Defense against Rhinovirus

SUMMARYRhinovirus is a leading cause of acute respiratory infections and asthma attacks, but infections are also frequently cleared from the nasal mucosa without causing symptoms. We sought to better understand host defense against rhinovirus by investigating antiviral defense in primary human nasal and bronchial airway epithelial cells cultured ex vivo. Surprisingly, upon rhinovirus infection or RIG-I stimulation, nasal-derived epithelial cells exhibited much more robust antiviral responses than bronchial-derived cells. Conversely, RIG-I stimulation triggered more robust activation of the NRF2-dependent oxidative stress response in bronchial cells compared to nasal cells. NRF2 activation dampened epithelial antiviral responses, whereas NRF2 knockdown enhanced antiviral responses and was protective during rhinovirus infection. These findings demonstrate a tradeoff in epithelial defense against distinct types of airway damage, namely, viral versus oxidative, and reveal differential calibration of defense responses in cells derived from different airway microenvironments.

Comparison of paired human nasal and bronchial airway epithelial cell responses to rhinovirus infection and IL-13 treatment

BackgroundBecause of its advantage as a minimally invasive procedure, nasal brushings have been increasingly used and proposed as a valuable approach to study lower airway diseases in lieu of bronchial epithelial cells. However, there is limited or conflicting evidence pertaining to whether nasal samples can be surrogates to bronchial samples. The goal of the present study is to test whether nasal epithelial cells have similar antiviral and inflammatory responses to IL-13 treatment and rhinovirus infection, a condition mimicking virally induced asthma exacerbation. Nasal and bronchial airway epithelial cells taken from the same patient were cultured under submerged and air–liquid interface (ALI) culture in the absence or presence of rhinovirus and IL-13 treatment. Inflammatory cytokines IP-10 and eotaxin-3, antiviral gene Mx1 and viral levels were measured.ResultsIn the absence of IL-13 treatment, nasal and bronchial cells showed a similar IP-10 response in both ALI and submerged cultures. Under the ALI culture, short term (e.g., 3 days) IL-13 treatment had a minimal effect on viral and Mx1 levels in both cell types. However, prolonged (e.g., 14 days) IL-13 treatments in both cell types decreased viral load and Mx1 expression. Under the submerged culture, IL-13 treatment in both cell types has minimal effects on viral load, IP-10 and Mx1. IL-13-induced eotaxin-3 production was similar in both types of cells under either submerged or ALI culture, which was not affected by viral infection.ConclusionsOur data suggest that nasal epithelial cells could serve as a surrogate to bronchial epithelial cells in future studies aimed at defining the role of type 2 cytokine IL-13 in regulating pro-inflammatory and antiviral responses.

Pro-inflammatory mediator responses from neonatal airway epithelial cells and early childhood wheeze.

Airway epithelial cell (AEC) function differs between children with and without asthma. Here, we associated neonatal AEC function with asthma symptoms at 4 years of age. Nasal AEC were collected from neonates within 48 h of birth. Cells were cultured and stimulated with tumor necrosis factor alpha/interleukin-1 beta (TNFα/IL-1β), lipopolysaccharide (LPS), or house dust mite (HDM). Absolute concentrations of pro-inflammatory mediators in the culture supernatant were quantified and expressed as median [interquartile range] in pg/mg protein. A parent-completed respiratory questionnaire was returned when the child was 4 years old. AEC were successfully cultured in 139 neonates, of whom 120 were contacted at 4 years and 91 (76%) questionnaires were returned. Sixteen children had wheezed ever and 11 had recent wheeze. At birth, when compared to those with no recent wheeze, supernatants from cultured neonatal AEC from the children with recent wheeze had reduced median IL-8 (CXCL8) release after treatment with culture medium alone (P = 0.049), with TNFα/IL-1β (P < 0.001) and LPS (P = 0.004). Additionally, and when compared to those with no recent wheeze, 4 year olds with recent wheeze had reduced neonatal AEC release of IL-6 (P = 0.013), GMCSF (P = 0.012), and ICAM-1 (P = 0.017) after treatment with TNFα/IL-1β and reduced release of ICAM-1 (P = 0.038) and RANTES (P = 0.042) after treatment with HDM. Abnormalities in AEC function are present at birth before the onset of childhood wheeze. The relationship between reduced AEC function at birth and wheeze at 4 years was not exclusive, suggesting that post-natal factors are required for the AEC abnormality to translate into symptoms.

Nasal airway epithelial cell IL-6 and FKBP51 gene expression and steroid sensitivity in asthmatic children.

BackgroundMany asthmatic patients exhibit uncontrolled asthma despite high-dose inhaled corticosteroids (ICS). Airway epithelial cells (AEC) have distinct activation profiles that can influence ICS response.ObjectivesA pilot study to identify gene expression markers of AEC dysfunction and markers of corticosteroid sensitivity in asthmatic and non-asthmatic control children, for comparison with published reports in adults.MethodsAEC were obtained by nasal brushings and primary submerged cultures, and incubated in control conditions or in the presence of 10 ng/ml TNFalpha, 10-8M dexamethasone, or both. RT-PCR-based expression of FKBP51 (a steroid hormone receptor signalling regulator), NF-kB, IL-6, LIF (an IL-6 family neurotrophic cytokine), serpinB2 (which inhibits plasminogen activation and promotes fibrin deposition) and porin (a marker of mitochondrial mass) were determined.Results6 patients without asthma (median age 11yr; min-max: 7–13), 8 with controlled asthma (11yr, 7–13; median daily fluticasone dose = 100 μg), and 4 with uncontrolled asthma (12yr, 7–14; 1000 μg fluticasone daily) were included. Baseline expression of LIF mRNA was significantly increased in uncontrolled vs controlled asthmatic children. TNFalpha significantly increased LIF expression in uncontrolled asthma. A similar trend was observed regarding IL-6. Dexamethasone significantly upregulated FKBP51 expression in all groups but the response was blunted in asthmatic children. No significant upregulation was identified regarding NF-kB, serpinB2 and porin.ConclusionLIF and FKBP51 expression in epithelial cells were the most interesting markers of AEC dysfunction/response to corticosteroid treatment.

Airway Progenitor Clone Formation Is Enhanced by Y-27632-Dependent Changes in the Transcriptome.

The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 10(10) cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.

Using Drugs to Probe the Variability of Trans-Epithelial Airway Resistance.

BackgroundPrecision medicine aims to combat the variability of the therapeutic response to a given medicine by delivering the right medicine to the right patient. However, the application of precision medicine is predicated on a prior quantitation of the variance of the reference range of normality. Airway pathophysiology provides a good example due to a very variable first line of defence against airborne assault. Humans differ in their susceptibility to inhaled pollutants and pathogens in part due to the magnitude of trans-epithelial resistance that determines the degree of epithelial penetration to the submucosal space. This initial ‘set-point’ may drive a sentinel event in airway disease pathogenesis. Epithelia differentiated in vitro from airway biopsies are commonly used to model trans-epithelial resistance but the ‘reference range of normality’ remains problematic. We investigated the range of electrophysiological characteristics of human airway epithelia grown at air-liquid interface in vitro from healthy volunteers focusing on the inter- and intra-subject variability both at baseline and after sequential exposure to drugs modulating ion transport.Methodology/Principal FindingsBrushed nasal airway epithelial cells were differentiated at air-liquid interface generating 137 pseudostratified ciliated epithelia from 18 donors. A positively-skewed baseline range exists for trans-epithelial resistance (Min/Max: 309/2963 Ω·cm2), trans-epithelial voltage (-62.3/-1.8 mV) and calculated equivalent current (-125.0/-3.2 μA/cm2; all non-normal, P<0.001). A minority of healthy humans manifest a dramatic amiloride sensitivity to voltage and trans-epithelial resistance that is further discriminated by prior modulation of cAMP-stimulated chloride transport.Conclusions/SignificanceHealthy epithelia show log-order differences in their ion transport characteristics, likely reflective of their initial set-points of basal trans-epithelial resistance and sodium transport. Our data may guide the choice of the background set point in subjects with airway diseases and frame the reference range for the future delivery of precision airway medicine.

In Vitro Modeling of RSV Infection and Cytopathogenesis in Well-Differentiated Human Primary Airway Epithelial Cells (WD-PAECs).

The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis.

Ten-eleven translocation 1 (TET1) methylation is associated with childhood asthma and traffic-related air pollution

Asthma is a complex disorder influenced by genetics and the environment. Recent findings have linked abnormal DNA methylation in T cells with asthma; however, the potential dysregulation of methylation in airway epithelial cells is unknown. Studies of mouse models of asthma have observed greater levels of 5-hydroxymethylcytosine (5-hmC) and ten-eleven translocation 1 (TET1) expression in lungs. TET proteins are known to catalyze methylation through modification of 5-methylcytosine to 5-hmC. We sought to examine the association of TET1 methylation with asthma and traffic-related air pollution (TRAP). TET1 methylation levels from DNA derived from nasal airway epithelial cells collected from 12 African American children with physician-diagnosed asthma and their nonasthmatic siblings were measured by using Illumina 450K arrays. Regions of interest were verified by means of locus-specific pyrosequencing in 35 sibling pairs and replicated in an independent population (n = 186). Exposure to TRAP in participants' early life and at current home addresses was estimated by using a land-use regression model. Methylation studies in saliva, PBMCs, and human bronchial epithelial cells were done to support our findings. Loss of methylation at a single CpG site in the TET1 promoter (cg23602092) and increased global 5-hmC levels were significantly associated with asthma. In contrast, TRAP exposure at participants' current homes significantly increased methylation at the same site. Patterns were consistent across tissue sample types. 5-Aza-2'-deoxycytidine and diesel exhaust particle exposure in human bronchial epithelial cells was associated with altered TET1 methylation and expression and global 5-hmC levels. Our findings suggest a possible role of TET1 methylation in asthmatic patients and response to TRAP.

Primary airway epithelial cell culture and asthma in children-lessons learnt and yet to come.

Until recently the airway epithelial cell (AEC) was considered a simple barrier that prevented entry of inhaled matter into the lung parenchyma. The AEC is now recognized as having an important role in the inflammatory response of the respiratory system to inhaled exposures, and abnormalities of these responses are thought to be important to asthma pathogenesis. This review first explores how the challenges of studying nasal and bronchial AECs in children have been addressed and then summarizes the results of studies of primary AEC function in children with and without asthma. There is good evidence that nasal AECs may be a suitable surrogate for the study of certain aspects of bronchial AEC function, although bronchial AECs remain the gold standard for asthma research. There are consistent differences between children with and without asthma for nasal and bronchial AEC mediator release following exposure to a range of pro-inflammatory stimulants including interleukins (IL)-1β, IL-4, and IL-13. However, there are inconsistencies between studies, e.g., release of IL-6, an important pro-inflammatory cytokine, is not increased in children with asthma relative to controls in all studies. Future work should expand current understanding of the "upstream" signalling pathways in AEC, study AEC from children before the onset of asthma symptoms and in vitro models should be developed that replicate the in vivo status more completely, e.g., co-culture with dendritic cells. AECs are difficult to obtain from children and collaboration between centers is expected to yield meaningful advances in asthma understanding and ultimately help deliver novel therapies.