Cultures Of Human Bronchial Epithelial Cells Research Articles

Liquid application dosing alters the physiology of air-liquid interface (ALI) primary human bronchial epithelial cell/lung fibroblast co-cultures and in vitro testing relevant endpoints.

Differentiated primary human bronchial epithelial cell (dpHBEC) cultures grown under air-liquid interface (ALI) conditions exhibit key features of the human respiratory tract and are thus critical for respiratory research as well as efficacy and toxicity testing of inhaled substances (e.g., consumer products, industrial chemicals, and pharmaceuticals). Many inhalable substances (e.g., particles, aerosols, hydrophobic substances, reactive substances) have physiochemical properties that challenge their evaluation under ALI conditions in vitro. Evaluation of the effects of these methodologically challenging chemicals (MCCs) in vitro is typically conducted by "liquid application," involving the direct application of a solution containing the test substance to the apical, air-exposed surface of dpHBEC-ALI cultures. We report that the application of liquid to the apical surface of a dpHBEC-ALI co-culture model results in significant reprogramming of the dpHBEC transcriptome and biological pathway activity, alternative regulation of cellular signaling pathways, increased secretion of pro-inflammatory cytokines and growth factors, and decreased epithelial barrier integrity. Given the prevalence of liquid application in the delivery of test substances to ALI systems, understanding its effects provides critical infrastructure for the use of in vitro systems in respiratory research as well as in the safety and efficacy testing of inhalable substances.

Isolation of Membrane Bound Mucins from Human Bronchial Epithelial Cells.

Membrane-bound mucins constitute a large portion of the periciliary layer of lung epithelial surfaces, and thus play an important role in many aspects of innate defense. The biophysical and biochemical properties of the membrane-bound mucins have important implications for mucociliary clearance, viral penetration, and potential therapeutics delivered to the airway surface. Hence, isolating them and determining these properties is important in understanding airways disease and ultimately in developing treatments. Here, we describe a method using isopycnic centrifugation to enrich and isolate shed membrane-bound mucins from the washings of human bronchial epithelial cell cultures.

Variability in Donor Lung Culture and Relative Humidity Impact the Stability of 2009 Pandemic H1N1 Influenza Virus on Nonporous Surfaces.

Respiratory viruses can be transmitted by multiple modes, including contaminated surfaces, commonly referred to as fomites. Efficient fomite transmission requires that a virus remain infectious on a given surface material over a wide range of environmental conditions, including different relative humidities. Prior work examining the stability of influenza viruses on surfaces has relied upon virus grown in media or eggs, which does not mimic the composition of virus-containing droplets expelled from the human respiratory tract. In this study, we examined the stability of the 2009 pandemic H1N1 (H1N1pdm09) virus on a variety of nonporous surface materials at four different humidities. Importantly, we used virus grown in primary human bronchial epithelial cell (HBE) cultures from different donors to recapitulate the physiological microenvironment of expelled viruses. We observed rapid inactivation of H1N1pdm09 on copper under all experimental conditions. In contrast to copper, viruses were stable on polystyrene plastic, stainless steel, aluminum, and glass, at multiple relative humidities, but greater decay on acrylonitrile butadiene styrene (ABS) plastic was observed at short time points. However, the half-lives of viruses at 23% relative humidity were similar among noncopper surfaces and ranged from 4.5 to 5.9 h. Assessment of H1N1pdm09 longevity on nonporous surfaces revealed that virus persistence was governed more by differences among HBE culture donors than by surface material. Our findings highlight the potential role of an individual's respiratory fluid on viral persistence and could help explain heterogeneity in transmission dynamics. IMPORTANCE Seasonal epidemics and sporadic pandemics of influenza cause a large public health burden. Although influenza viruses disseminate through the environment in respiratory secretions expelled from infected individuals, they can also be transmitted by contaminated surfaces where virus-laden expulsions can be deposited. Understanding virus stability on surfaces within the indoor environment is critical to assessing influenza transmission risk. We found that influenza virus stability is affected by the host respiratory secretion in which the virus is expelled, the surface material on which the droplet lands, and the ambient relative humidity of the environment. Influenza viruses can remain infectious on many common surfaces for prolonged periods, with half-lives of 4.5 to 5.9 h. These data imply that influenza viruses are persistent in indoor environments in biologically relevant matrices. Decontamination and engineering controls should be used to mitigate influenza virus transmission.

Evaluation of the pulmonary toxicity of PSNPs using a Transwell-based normal human bronchial epithelial cell culture system

To reduce the nanoplastics (NPs) toxicity assessment error, we established a Transwell-based bronchial epithelial cell exposure system to assess the pulmonary toxicity of polystyrene NPs (PSNPs). Transwell exposure system was more sensitive than submerged culture for toxicity detection of PSNPs. PSNPs adhered to the BEAS-2B cell surface, were ingested by the cell, and accumulated in the cytoplasm. PSNPs induced oxidative stress and inhibited cell growth through apoptosis and autophagy. A noncytotoxic dose of PSNPs (1 ng/cm2) increased the expression levels of inflammatory factors (ROCK-1, NF-κB, NLRP3, ICAM-1, etc) in BEAS-2B cells, whereas a cytotoxic dose (1000 ng/cm2) induced apoptosis and autophagy, which might inhibit the activation of ROCK-1 and contribute to reducing inflammation. In addition, the noncytotoxic dose increased the expression levels of zonula occludens-2 (ZO-2) and α1-antitrypsin (α-AT) proteins in BEAS-2B cells. Therefore, in response to PSNP exposure, a compensatory increase in the activities of inflammatory factors, ZO-2, and α-AT may be triggered at low doses as a mechanism to preserve the survival of BEAS-2B cells. In contrast, exposure to a high dose of PSNPs elicits a noncompensatory response in BEAS-2B cells. Overall, these findings suggest that PSNPs may be harmful to human pulmonary health even at an ultralow concentration.

An in vitro evaluation of e-vapor products: The contributions of chemical adulteration, concentration, and device power.

Homemade e-liquids and power-adjustable vaping devices may carry higher risks than commercial formulations and fixed-power devices. This study used human macrophage-like and bronchial epithelial (NHBE) cell cultures to investigate toxicity of homemade e-liquids containing propylene glycol and vegetable glycerin (PG/VG), nicotine, vitamin E acetate (VEA), medium-chain fatty acids (MCFAs), phytol, and cannabidiol (CBD). SmallAir™ organotypic epithelial cultures were exposed to aerosols generated at different power settings (10-50 W). Carbonyl levels were measured, and endpoints reflecting epithelial function (ciliary beating frequency [CBF]), integrity (transepithelial electrical resistance [TEER]), and structure (histology) were investigated. Treatment with nicotine or VEA alone or with PG/VG did not impact cell viability. CBD, phytol, and lauric acid caused cytotoxicity in both culture systems and increased lipid-laden macrophages. Exposure of SmallAir™ organotypic cultures to CBD-containing aerosols resulted in tissue injury and loss of CBF and TEER, while PG/VG alone or with nicotine or VEA did not. Aerosols generated with higher power settings had higher carbonyl concentrations. In conclusion, the presence and concentration of certain chemicals and device power may induce cytotoxicity in vitro. These results raise concerns that power-adjustable devices may generate toxic compounds and suggest that toxicity assessments should be conducted for both e-liquid formulations and their aerosols.

Oxidative Stress Effects of Multiple Pollutants in an Indoor Environment on Human Bronchial Epithelial Cells

Benzene, toluene, and xylene (denoted as BTX) are normally used in coatings, sealants, curing agents and other home decoration products, which can cause harm to human health. However, traditional studies mostly focus on the toxicity evaluation of a single pollution source, and little attention has been paid to the toxicity reports of multiple pollutants in a complex system. To evaluate the impact of indoor BTX on human health at the cellular level, the oxidative stress effect of BTX on human bronchial epithelial cells was assessed, including cell cytotoxicity, intracellular ROS, cell mitochondrial membrane potential, cell apoptosis, and CYP2E1 expression. The concentrations of BTX introduced into the human bronchial epithelial cell culture medium were determined based on both the tested distribution in 143 newly decorated rooms and the limited concentrations in the indoor air quality (denoted as IAQ) standards. Our study showed that the concentration in line with the standard limit may still pose a serious risk to health. The cellular biology effect studies of BTX showed that BTX, even at concentrations lower than the national standard limit, can still induce observable oxidative stress effects which warrant attention.

Optimization of Primary Human Bronchial Epithelial 3D Cell Culture with Donor-Matched Fibroblasts and Comparison of Two Different Culture Media.

In vitro airway models are increasingly important for pathomechanistic analyses of respiratory diseases. Existing models are limited in their validity by their incomplete cellular complexity. We therefore aimed to generate a more complex and meaningful three-dimensional (3D) airway model. Primary human bronchial epithelial cells (hbEC) were propagated in airway epithelial cell growth (AECG) or PneumaCult ExPlus medium. Generating 3D models, hbEC were airlifted and cultured on a collagen matrix with donor-matched bronchial fibroblasts for 21 days comparing two media (AECG or PneumaCult ALI (PC ALI)). 3D models were characterized by histology and immunofluorescence staining. The epithelial barrier function was quantified by transepithelial electrical resistance (TEER) measurements. The presence and function of ciliated epithelium were determined by Western blot and microscopy with high-speed camera. In 2D cultures, an increased number of cytokeratin 14-positive hbEC was present with AECG medium. In 3D models, AECG medium accounted for high proliferation, resulting in hypertrophic epithelium and fluctuating TEER values. Models cultured with PC ALI medium developed a functional ciliated epithelium with a stable epithelial barrier. Here, we established a 3D model with high in vivo-in vitro correlation, which has the potential to close the translational gap for investigations of the human respiratory epithelium in pharmacological, infectiological, and inflammatory research.

Abstract A010: Chemopreventive effects of carvedilol and nebivolol in benzo(a)pyrene-induced transformation of non-tumorous human bronchial epithelial cell

Abstract Lung cancer is among the most common cancers worldwide. The primary risk factor for the development of lung cancer is cigarette smoking and there are a large amount of studies has confirmed the association between smoking and lung cancer. Chemopreventive drug would be beneficial among patients who are in high risk due to chronic exposure to carcinogens. In the previous study of chemopreventive activity of carvedilol, it significantly delayed and reduced skin squamous cell carcinoma development. The aim of the present study was to explore the role of carvedilol and nebivolol in benzo(a)pyrene induced transformation of the non-tumorous human bronchial epithelial cells. To evaluate the effects of carvedilol and nebivolol on chemopreventive activity against BPDE-induced lung carcinogenesis, anchorage-independent growth assay in soft agar was used to determine the degree of malignant transformation of BEAS-2b cells. Before the investigation of carvedilol and nebivolol, the appropriate drug concentration aiming minimum cytotoxicity on normal BEAS-2b cells were determined by MTT assay. The MTT assay was used to measure cellular metabolic activity as an indicator of cytotoxicity. Carvedilol at 10 μM or lower and Nebivolol at 1 μM or lower did not cause significant cytotoxicity. In non-tumorigenic human bronchial epithelial cell culture BEAS-2B vitro assays, carvedilol and nebivolol exert a protective effect against BPDE-indued transformation at non-toxic concentrations in a dose dependent manner. As shown in the figure below, Nebivolol (1 μM and 0.5 μM) and Carvedilol (1 μM and 0.5 μM) both significantly attenuated colony formation compared with BPDE control. However, the anti-transformation mechanism is unknown, further studies of carvedilol and nebivolol are needed to investigate their chemoprevention activity. Citation Format: Carol Lin. Chemopreventive effects of carvedilol and nebivolol in benzo(a)pyrene-induced transformation of non-tumorous human bronchial epithelial cell [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A010.

Abstract A017: Lung cancer chemoprevention with the β-blocker carvedilol

Abstract Chronic exposure to carcinogens that are present in tobacco smoke is a causative factor for lung carcinogenesis. Although smoking cessation successfully reduced the prevalence of cigarette use in the US, lung cancer risk for current and former smokers remains high, for whom no effective chemopreventive agent currently exists. In the present study, carvedilol, an FDA-approved β-blocker was examined on lung carcinogenesis induced by the tobacco carcinogen benzo(a)pyrene [B(a)P] in vitro and in vivo. In non-tumorigenic human bronchial epithelial cell culture BEAS-2B, carvedilol inhibited benzo(a)pyrene diol epoxide (BPDE)-induced malignant transformation at non-toxic concentrations in a dose-dependent manner. Although β-adrenergic receptors (β-ARs) are expressed in BEAS-2B cells, the anticancer activity of carvedilol is independent of β-blockade since the non-β-blocking R-carvedilol enantiomer also blocked transformation while the β-blockers atenolol (β1-AR selective blocker), ICI-118,551 (β2-AR selective blocker), and propranolol (nonselective blocker) had no effect. Carvedilol’s anti-transformation activity is possibly mediated by aryl hydrocarbon receptor signaling because carvedilol, R- and S-carvedilol all inhibited B(a)P-activated xenobiotic responsive element (XRE) promoter and CYP1A1 mRNA expression. In a B(a)P-induced acute lung toxicity model in CD-1 mice, pretreatment with carvedilol, R- or S-carvedilol (20 mg/kg/day) for 7 days significantly attenuated increased plasma levels of lactate dehydrogenase and malondialdehyde, inflammatory cell infiltration, histopathologic changes, and overexpression of COX-2 in the lung. In a B(a)P-induced lung carcinogenesis model in A/J mice, carvedilol at 3.2 and 20 mg/kg significantly attenuated tumor multiplicity and burden, to a similar degree as the EGFR inhibitor gefitinib. Our study reveals a previously unexplored role for the FDA approved cardiovascular drug carvedilol in the prevention of tobacco carcinogen-associated lung cancer. Citation Format: Ayaz Shahid, Mengbing Chen, Carol Lin, Bradley T. Andresen, Cyrus Parsa, Robert Orlando, Ying Huang. Lung cancer chemoprevention with the β-blocker carvedilol [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A017.

Human Bronchial Epithelial Cell Culture Models for Cigarette Smoke and Vaping Studies.

Despite the continuing public health efforts to stop or reduce smoking, cigarette smoke use remains popular in the youth and adult population. A recent surge in the use of electronic cigarette and vaping products has created another major health challenge in public health. There is an urgent need to use physiologically relevant models to study the health effect of smoking or vaping in human subjects. Airway diseases such as bronchitis (Landman et al., CMAJ 191:E1321-E1331, 2019; Goniewicz, et al. Harm Reduct J 17:91, 2020; Xie et al., JAMA Netw Open 3:e2020816, 2020) have been described in people who smoke, vape, or both. Here, we will describe methods to collect, expand, and culture human airway epithelial cells from endobronchial brushings and expose these cells cultured at the air-liquid interface to cigarette smoke or electronic cigarette vapor.

Assessing the Toxicological Relevance of Nanomaterial Agglomerates and Aggregates Using Realistic Exposure In Vitro.

Low dose repeated exposures are considered more relevant/realistic in assessing the health risks of nanomaterials (NM), as human exposure such as in workplace occurs in low doses and in a repeated manner. Thus, in a three-week study, we assessed the biological effects (cell viability, cell proliferation, oxidative stress, pro-inflammatory response, and DNA damage) of titanium-di-oxide nanoparticle (TiO2 NP) agglomerates and synthetic amorphous silica (SAS) aggregates of different sizes in human bronchial epithelial (HBE), colon epithelial (Caco2), and human monocytic (THP-1) cell lines repeatedly exposed to a non-cytotoxic dose (0.76 µg/cm2). We noticed that neither of the two TiO2 NPs nor their agglomeration states induced any effects (compared to control) in any of the cell lines tested while SAS aggregates induced some significant effects only in HBE cell cultures. In a second set of experiments, HBE cell cultures were exposed repeatedly to different SAS suspensions for two weeks (first and second exposure cycle) and allowed to recover (without SAS exposure, recovery period) for a week. We observed that SAS aggregates of larger sizes (size ~2.5 µm) significantly affected the cell proliferation, IL-6, IL-8, and total glutathione at the end of both exposure cycle while their nanosized counterparts (size less than 100 nm) induced more pronounced effects only at the end of the first exposure cycle. As noticed in our previous short-term (24 h) exposure study, large aggregates of SAS did appear to be similarly potent as nano sized aggregates. This study also suggests that aggregates of SAS of size greater than 100 nm are toxicologically relevant and should be considered in risk assessment.

Can Resveratrol-Inhaled Formulations Be Considered Potential Adjunct Treatments for COVID-19?

The pandemic caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has led to an extraordinary threat to the global healthcare system. This infection disease, named COVID-19, is characterized by a wide clinical spectrum, ranging from asymptomatic or mild upper respiratory tract illness to severe viral pneumonia with fulminant cytokine storm, which leads to respiratory failure. To improve patient outcomes, both the inhibition of viral replication and of the unwarranted excessive inflammatory response are crucial. Since no specific antiviral drug has been proven effective for the treatment of patients and the only upcoming promising agents are monoclonal antibodies, inexpensive, safe, and widely available treatments are urgently needed. A potential anti-inflammatory molecule to be evaluated, which possesses antiviral activities in several experimental models, is the polyphenol resveratrol. This compound has been shown to inhibit SARS-CoV-2 replication in human primary bronchial epithelial cell cultures and to downregulate several pathogenetic mechanisms involved in COVID-19 severity. The use of resveratrol in clinical practice is limited by the low bioavailability following oral administration, due to the pharmacokinetic and metabolic characteristics of the molecule. Therefore, topical administration through inhaled formulations could allow us to achieve sufficiently high concentrations of the compound in the airways, the entry route of SARS-CoV-2.

Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells

Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air–liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5ʹ-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO) sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells, plus submersion, and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids, this region exhibited basal eRNA production, RNA polymerase II enrichment, and looping to the TSS, plausibly underlying constitutive KLF9 expression. Post glucocorticoid treatment, interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity, but negligibly affected KLF9 expression. Overall, a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell lines can translate to physiologically relevant systems.

1265. AT-527, an Oral Purine Nucleotide Prodrug Exhibiting Potent In Vitro Antiviral Activity Against Human Coronaviruses, Including SARS-CoV-2

BackgroundCoronaviruses (CoVs) are the causative pathogens of several human diseases, including seasonal respiratory infections (HCoV-229E and HCoV-OC43), Middle East respiratory syndrome (MERS-CoV), severe acute respiratory syndrome (SARS-CoV-1) and the novel CoV recently identified as the virus responsible for the current COVID-19 pandemic, SARS-CoV-2. AT-527 is currently in Phase 2 clinical trials and has demonstrated potent activity and a well-tolerated safety profile in HCV-infected subjects. Here we report the in vitro activity of AT-511, the free base form of AT-527, against SARS-CoV-2 and other CoVs.MethodsBHK-21, Huh-7, RD and differentiated normal human bronchial epithelial (dNHBE) cell cultures were exposed to virus and serial dilutions of test compounds. Independent assessments of antiviral activity were obtained by determining effective concentrations of test compounds required to 1) prevent half-maximal (EC50) virus-induced cytopathic effect (CPE) using MTT or neutral red staining and 2) produce virus yield reductions (VYR) by 90% (EC90) using standard endpoint dilution CCID50 assays in Vero 76 cells. Half maximal cytotoxicity of test compounds was determined by dye (MTT or neutral red) staining in the absence of added virus or by microscopic inspection (dNHBE cells only).ResultsTable 1 presents the in vitro activities of AT-511 against several coronaviruses. Also included in these assays are the antiviral activities of potential COVID-19 oral treatments, including chloroquine, hydroxychloroquine and N4-hydroxycytidine.Table 1. In Vitro Activity of AT-511 Against Various Human Coronaviruses ConclusionThe data demonstrate the potent in vitro activity of AT-511 against several CoVs, with individual EC90 values ranging from 0.34 to 1.2 µM against HCoV-229E, HCoV-OC43, SARS-CoV-1 and SARS-CoV-2 and less activity against MERS-CoV (average EC90 = 36 µM). The potent in vitro antiviral activity of AT-511 against SARS-CoV-2 (EC90 = 0.55 µM), associated with the AT-527 safety profile in treated HCV patients, support the ongoing clinical evaluation of the safety and efficacy of AT-527 in COVID-19 patients.DisclosuresSteven S. Good, MS, Atea Pharmaceuticals, Inc. (Employee) Adel Moussa, PhD, Atea Pharmaceuticals, Inc. (Employee) Xiao-Jian Zhou, PhD, Atea Pharmaceuticals, Inc. (Employee) Keith Pietropaolo, B.A., Atea Pharmaceuticals, Inc. (Employee) Jean-Pierre Sommadossi, PhD, Atea Pharmaceuticals, Inc. (Board Member)

Retinoic acid improves baseline barrier function and attenuates TNF-α-induced barrier leak in human bronchial epithelial cell culture model, 16HBE 14o.

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14C-mannitol and 80% of the leak to 14C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward “band-shift” in occludin Western immunoblots. The presence of RA partially reversed TNF-α’s effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a “cytokine storm” may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.

Flow-Cytometric Analysis and Purification of Airway Epithelial-Cell Subsets.

The human airway epithelium is essential in homeostasis, and epithelial dysfunction contributes to chronic airway disease. Development of flow-cytometric methods to characterize subsets of airway epithelial cells will enable further dissection of airway epithelial biology. Leveraging single-cell RNA-sequencing data in combination with known cell type-specific markers, we developed panels of antibodies to characterize and isolate the major airway epithelial subsets (basal, ciliated, and secretory cells) from human bronchial epithelial-cell cultures. We also identified molecularly distinct subpopulations of secretory cells and demonstrated cell subset-specific expression of low-abundance transcripts and microRNAs that are challenging to analyze with current single-cell RNA-sequencing methods. These new tools will be valuable for analyzing and separating airway epithelial subsets and interrogating airway epithelial biology.

RNA oxidation in chromatin modification and DNA-damage response following exposure to formaldehyde

Formaldehyde is an environmental and occupational chemical carcinogen implicated in the damage of proteins and nucleic acids. However, whether formaldehyde provokes modifications of RNAs such as 8-oxo-7,8-dihydroguanine (8-oxoG) and the role that these modifications play on conferring long-term adverse health effects remains unexplored. Here, we profile 8-oxoG modifications using RNA-immunoprecipitation and RNA sequencing (8-oxoG RIP-seq) to identify 343 RNA transcripts heavily enriched in oxidations in human bronchial epithelial BEAS-2B cell cultures exposed to 1 ppm formaldehyde for 2 h. RNA oxidation altered expression of many transcripts involved in chromatin modification and p53-mediated DNA-damage responses, two pathways that play key roles in sustaining genome integrity and typically deregulated in tumorigenesis. Given that these observations were identified in normal cells exhibiting minimal cell stress and death phenotypes (for example, lack of nuclear shrinkage, F-actin alterations or increased LDH activity); we hypothesize that oxidative modification of specific RNA transcripts following formaldehyde exposure denotes an early process occurring in carcinogenesis analogous to the oxidative events surfacing at early stages of neurodegenerative diseases. As such, we provide initial investigations of RNA oxidation as a potentially novel mechanism underlying formaldehyde-induced tumorigenesis.

Host-Pathogen Responses to Pandemic Influenza H1N1pdm09 in a Human Respiratory Airway Model.

The respiratory Influenza A Viruses (IAVs) and emerging zoonotic viruses such as Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pose a significant threat to human health. To accelerate our understanding of the host–pathogen response to respiratory viruses, the use of more complex in vitro systems such as normal human bronchial epithelial (NHBE) cell culture models has gained prominence as an alternative to animal models. NHBE cells were differentiated under air-liquid interface (ALI) conditions to form an in vitro pseudostratified epithelium. The responses of well-differentiated (wd) NHBE cells were examined following infection with the 2009 pandemic Influenza A/H1N1pdm09 strain or following challenge with the dsRNA mimic, poly(I:C). At 30 h postinfection with H1N1pdm09, the integrity of the airway epithelium was severely impaired and apical junction complex damage was exhibited by the disassembly of zona occludens-1 (ZO-1) from the cell cytoskeleton. wdNHBE cells produced an innate immune response to IAV-infection with increased transcription of pro- and anti-inflammatory cytokines and chemokines and the antiviral viperin but reduced expression of the mucin-encoding MUC5B, which may impair mucociliary clearance. Poly(I:C) produced similar responses to IAV, with the exception of MUC5B expression which was more than 3-fold higher than for control cells. This study demonstrates that wdNHBE cells are an appropriate ex-vivo model system to investigate the pathogenesis of respiratory viruses.

Selection of reference genes for quantitative PCR: identifying reference genes for airway epithelial cells exposed to Pseudomonas aeruginosa.

Most quantitative PCR (qPCR) experiments report differential expression relative to the expression of one or more reference genes. Therefore, when experimental conditions alter reference gene expression, qPCR results may be compromised. Little is known about the magnitude of this problem in practice. We found that reference gene responses are common and hard to predict and that their stability should be demonstrated in each experiment. Our reanalysis of 15 airway epithelia microarray data sets retrieved from the National Center for Biotechnology Information (NCBI) identified no common reference gene that was reliable in all 15 studies. Reanalysis of published RNA sequencing (RNA-seq) data in which human bronchial epithelial cells (HBEC) were exposed to Pseudomonas aeruginosa revealed that minor experimental details, including bacterial strain, may alter reference gene responses. Direct measurement of 32 TaqMan reference genes in primary cultures of HBEC exposed to P. aeruginosa (strain PA14) demonstrated that choosing an unstable reference gene could make it impossible to observe statistically significant changes in IL8 gene expression. We found that reference gene instability is a general phenomenon and not limited to studies of airway epithelial cells. In a diverse compendium of 986 human microarray experiments retrieved from the NCBI, reference genes were differentially expressed in 42% of studies. Experimentally induced changes in reference gene expression ranged from 21% to 212%. These results highlight the importance of identifying adequate reference genes for each experimental system and documenting their response to treatment in each experiment. This will enhance experimental rigor and reproducibility in qPCR studies.

Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.