Development of an in vitro human alveolar epithelial air-liquid interface model using a small molecule inhibitor cocktail.

BACKGROUND: The alveolar epithelium is exposed to numerous stimuli, such as chemicals, viruses, and bacteria that cause a variety of pulmonary diseases through inhalation. Alveolar epithelial cells (AECs) cultured in vitro are a valuable tool for studying the impacts of these stimuli and developing therapies for associated diseases. However, maintaining the proliferative capacity of AECs in vitro is challenging. In this study, we used a cocktail of three small molecule inhibitors to cultivate AECs: Y-27632, A-83-01, and CHIR99021 (YAC). These inhibitors reportedly maintain the proliferative capacity of several types of stem/progenitor cells. RESULTS: Primary human AECs cultured in medium containing YAC proliferated for more than 50 days (over nine passages) under submerged conditions. YAC-treated AECs were subsequently cultured at the air-liquid interface (ALI) to promote differentiation. YAC-treated AECs on ALI day 7 formed a monolayer of epithelial tissue with strong expression of the surfactant protein-encoding genes SFTPA1, SFTPB, SFTPC, and SFTPD, which are markers for type II AECs (AECIIs). Immunohistochemical analysis revealed that paraffin sections of YAC-treated AECs on ALI day 7 were mainly composed of cells expressing surfactant protein B and prosurfactant protein C. CONCLUSIONS: Our results indicate that YAC-containing medium could be useful for expansion of AECIIs, which are recognized as local stem/progenitor cells, in the alveoli.

Development of an In Vitro Sensitisation Test Using a Coculture System of Human Bronchial Epithelium and Immune Cells.

Chemical respiratory sensitisation is a serious health problem. However, to date, there are no validated test methods available for identifying respiratory sensitisers. The aim of this study was to develop an in vitro sensitisation test by modifying the human cell line activation test (h-CLAT) to detect respiratory sensitisers and distinguish them from skin sensitisers. THP-1 cells were exposed to the test chemicals (two skin sensitisers and six respiratory sensitisers), either as monocultures or as cocultures with air-liquid interface-cultured reconstructed human bronchial epithelium. The responses were analysed by measuring the expression levels of surface markers on THP-1 cells (CD86, CD54 and OX40L) and the concentrations of cytokines in the culture media (interleukin (IL)-8, IL-33 and thymic stromal lymphopoietin (TSLP)). The cocultures exhibited increased CD54 expression on THP-1 cells; moreover, in the cocultures but not in the monocultures, exposure to two uronium salts (i.e. respiratory sensitisers) increased CD54 expression on THP-1 cells to levels above the criteria for a positive h-CLAT result. Additionally, exposure to the respiratory sensitiser abietic acid, significantly increased IL-8 concentration in the culture medium, but only in the cocultures. Although further optimisation of the method is needed to distinguish respiratory from skin sensitisers by using these potential markers (OX40L, IL-33 and TSLP), the coculture of THP-1 cells with bronchial epithelial cells offers a potentially useful approach for the detection of respiratory sensitisers.

Development of a micronucleus test using the EpiAirway™ organotypic human airway model.

BACKGROUND: The use of organotypic human tissue models in genotoxicity has increased as an alternative to animal testing. Genotoxicity is generally examined using a battery of in vitro assays such as Ames and micronucleus (MN) tests that cover gene mutations and structural and numerical chromosome aberrations. At the 7th International Workshop on Genotoxicity Testing, working group members agreed that the skin models have reached an advanced stage of maturity, while further efforts in liver and airway models are needed [Pfuhler et al., Mutat. Res. 850-851 (2020) 503135]. Organotypic human airway model is composed of fully differentiated and functional respiratory epithelium. However, because cell proliferation in organotypic airway models is thought to be less active, assessing their MN-inducing potential is an issue, even in the cytokinesis-blocking approach using cytochalasin B (CB) [Wang et al., Environ. Mol. Mutagen. 62 (2021) 306-318]. Here, we developed a MN test using EpiAirway™ in which epidermal growth factor (EGF) was included as a stimulant of cell division. RESULTS: By incubating EpiAirway™ tissue with medium containing various concentrations of CB, we found that the percentage of binucleated cells (%BNCs) almost plateaued at 3 µg/mL CB for 72 h incubation. Additionally, we confirmed that EGF stimulation with CB incubation produced an additional increase in %BNCs with a peak at 5 ng/mL EGF. Transepithelial electrical resistance measurement and tissue histology revealed that CB incubation caused the reduced barrier integrity and cyst formation in EpiAirway™. Adenylate kinase assay confirmed that the cytotoxicity increased with each day of culture in the CB incubation period with EGF stimulation. These results indicated that chemical treatment should be conducted prior to CB incubation. Under these experimental conditions, it was confirmed that the frequency of micronucleated cells was dose-dependently increased by apical applications of two clastogens, mitomycin C and methyl methanesulfonate, and an aneugen, colchicine, at the subcytotoxic concentrations assessed in %BNCs. CONCLUSIONS: Well-studied genotoxicants demonstrated capability in an organotypic human airway model as a MN test system. For further utilization, investigations of aerosol exposure, repeating exposure protocol, and metabolic activation are required.

Donor-to-donor variability of a human three-dimensional bronchial epithelial model: A case study of cigarette smoke exposure.

Three-dimensional (3D) cultured primary cells are used to predict the toxicity of substances towards humans because these 3D cultures closely mimic the physiological architecture of tissues. Nonetheless, it is important to consider primary-cell-specific variability for endpoint selection and appropriate evaluation of toxicity because donor-dependent characteristics may be retained even in in vitro cell cultures. In this report, 3D differentiated bronchial epithelial cells from three donors were used to investigate donor-to-donor variability, with an aqueous extract of cigarette smoke (CS) used as the test substance. Ciliary function, cytokine secretion, and histopathology, which are affected by CS, were examined, and transcriptomic analysis was also performed. The results revealed that interleukin-8 secretion and oxidative stress-related gene expression were consistently altered for all donors; however, their amplitudes varied. Moreover, one of the donors showed unique responses to CS, suggesting that this donor was an outlier. This donor showed intrinsic differences in histology, cytokine secretion, and gene expression profile. Such donors may help evaluate potential toxicological concerns and aid our understanding of disease pathogenesis. Conversely, these donors may confound toxicological assessment and endpoint selection. Fit-for-purpose handling of inter-donor variability is warranted.

In vitro long-term repeated exposure and exposure switching of a novel tobacco vapor product in a human organotypic culture of bronchial epithelial cells.

Next-generation tobacco products and nicotine delivery systems such as heat-not-burn tobacco products and electronic cigarettes, the usage of which is expected to have a beneficial impact on public health, have gained popularity over the past decade. However, the risks associated with the long-term use of such products are still incompletely understood. Although the risks of these products should be clarified through epidemiological studies, such studies are normally performed based on each product category, not product-by-product. Therefore, investigation of the risk on a product-by-product basis is important to provide specific scientific evidence. In the current study, we performed the 40-day repeated exposure of in vitro human bronchial epithelial tissues to cigarette smoke (CS) or vapor from our proprietary novel tobacco vapor product (NTV). In addition, tissue samples exposed to CS were switched to NTV or CS exposure was stopped at 20 days to reflect a situation where smokers switched to NTV or ceased to smoke. All tissue samples were assessed in terms of toxicity, inflammation and transcriptomic alterations. Tissue samples switched to NTV and the cessation of exposure samples showed recovery from CS-induced damage although there was a time-course difference. Moreover, repeated exposure to NTV produced negligible effects on the tissue samples while CS produced cumulative effects. Our results suggest that the use of NTV, including switching to NTV from cigarette smoking, has fewer effects on bronchial epithelial tissues than continuing smoking.

Regulation of NRF2, AP-1 and NF-κB by cigarette smoke exposure in three-dimensional human bronchial epithelial cells.

Cigarette smoke (CS) is a complex mixture of chemicals and interacts with various physiological processes. We previously reported that nuclear factor erythroid 2-related factor 2 (NRF2) was the most sensitive transcription factor to aqueous CS extract (AqCSE) exposure in monolayer cultured human bronchial epithelial cell lines. Recently, in vitro three-dimensional (3D) culture models have been used to supplement pharmacological and toxicological assessments. Bronchial epithelium models in particular are useful for the evaluation of substances that directly contact the respiratory tract, such as CS. In the present study, we used 3D-cultured human bronchial epithelial cells (HBECs) to assess activation of transcription factors and relevant gene expression in response to AqCSE, primarily focusing on NRF2 and nuclear factor-kappa B (NF-κB) pathways. The 3D-cultured HBECs exposed to AqCSE showed expression of NRF2 and its nuclear translocation in addition to upregulation of genes related to oxidative stress. Our results suggest that the NRF2 pathway was the dominant pathway when 3D-cultured HBECs were exposed to AqCSE at a low dose, supporting our previous findings that NRF2 was the most sensitive transcription factor in response to AqCSE. Expression and nuclear translocation of NF-κB were not increased, although proinflammatory genes were upregulated. However, another inflammation-related transcription factor, activation protein 1, was induced by AqCSE. Gene classification analysis suggested that induction of the inflammatory response by AqCSE was dependent on NRF2 and activation protein 1 rather than NF-κB.

Effects of repeated cigarette smoke extract exposure over one month on human bronchial epithelial organotypic culture.

Cigarette smoke is a known risk factor for inflammatory diseases in the respiratory tract, and inflammatory exacerbation is considered pivotal to the pathogenesis of these diseases. Here, we performed two repeated exposure studies in which we exposed human bronchial epithelial tissues in an organotypic culture model to cigarette smoke extract (CSE); the first study was conducted over a four-day period to determine the suitable dose range for the extended exposure period, and the second was a one-month exposure study to elucidate the exposure-by-exposure effects in bronchial tissues. We focused on matrix metalloproteinase (MMP)-9 and -1/3 and the inflammatory cytokines interleukin (IL)-8 and growth factor related oncogene to evaluate the transition into an inflammatory state. Even at CSE doses with no or low toxicity for a single exposure, the repetition of exposure induced cumulative effects on both the inflammatory responses, specifically the IL-8 and MMPs levels, and tissue morphology. Interestingly, untreated controls initially had relatively high baseline levels of these secreted proteins; these levels gradually declined, after which they showed periodic level changes, suggesting an acclimation period may be needed for this system. These results demonstrate the usability of this system for the elucidation of sub-chronic effects in vitro.

Oxidative stress responses in human bronchial epithelial cells exposed to cigarette smoke and vapor from tobacco- and nicotine-containing products.

The use of novel tobacco- and nicotine-containing vapor products that do not combust tobacco leaves is on the rise worldwide. The emissions of these products typically contain lower numbers and levels of potentially harmful chemicals compared with conventional cigarette smoke. These vapor products may therefore elicit fewer adverse biological effects. We compared the effects of emissions from different types of such products, i.e., our proprietary novel tobacco vapor product (NTV), a commercially available heat-not-burn tobacco product (HnB), and e-cigarette (E-CIG), and a combustible cigarette in a human bronchial epithelial cell line. The aqueous extract (AqE) of the test product was prepared by bubbling the produced aerosol into medium. Cells were exposed to the AqEs of test products, and then glutathione oxidation, Nrf2 activation, and secretion of IL-8 and GM-CSF were examined. We found that all endpoints were similarly perturbed by exposure to each AqE, but the effective dose ranges were different between cigarette smoke and the tobacco- and nicotine-containing vapors. These results demonstrate that the employed assays detect differences between product exposures, and thus may be useful to understand the relative potential biological effects of tobacco- and nicotine-containing products.

Application of a direct aerosol exposure system for the assessment of biological effects of cigarette smoke and novel tobacco product vapor on human bronchial epithelial cultures.

Recent advancements in in vitro exposure systems and cell culture technology enable direct exposure to cigarette smoke (CS) of human organotypic bronchial epithelial cultures. MucilAir organotypic bronchial epithelial cultures were exposed, using a Vitrocell exposure system, to mainstream aerosols from the 3R4F cigarette or from a recently developed novel tobacco vapor product (NTV). The exposure aerosol dose was controlled by dilution flow and the number of products smoked; there were five exposure conditions for 3R4F smoke and three for NTV vapor. The amount of nicotine delivered to the tissues under each condition was analyzed and that of the total particulate matter (TPM) was estimated using nicotine data. The nicotine dose was similar for the two products at the highest dose, but the estimated TPM levels from the NTV were 3.7 times the levels from the 3R4F. Following 3R4F smoke exposure, a dose dependent increase was observed in cytotoxicity, cytokine secretion, and differential gene expression. However, no changes were detected in these endpoints following NTV vapor exposure, suggesting the biological effects of NTV vapor are lower than those of conventional combustible CS. Our study design, which includes collection of biological data and dosimetry data, is applicable to assessing novel tobacco products.

A 3D epithelial-mesenchymal co-culture model of human bronchial tissue recapitulates multiple features of airway tissue remodeling by TGF-ß1 treatment.

BACKGROUND: The collagen gel contraction assay measures gel size to assess the contraction of cells embedded in collagen gel matrices. Using the assay with lung fibroblasts is useful in studying the lung tissue remodeling process in wound healing and disease development. However, the involvement of bronchial epithelial cells in this process should also be investigated. METHODS: We applied a layer of mucociliary differentiated bronchial epithelial cells onto collagen gel matrices with lung fibroblasts. This co-culture model enables direct contact between epithelial and mesenchymal cells. We stimulated the culture with transforming growth factor (TGF) ß1 as an inducer of tissue remodeling for 21 days, and measured gel size, histological changes, and expression of factors related to extracellular matrix homeostasis. RESULTS: TGF-ß1 exerted a concentration-dependent effect on collagen gel contraction and on contractile myofibroblasts in the mesenchymal collagen layer. TGF-ß1 also induced expression of the mesenchymal marker vimentin in the basal layer of the epithelium, suggesting the induction of epithelial-mesenchymal transition. In addition, the expression of various genes encoding extracellular matrix proteins was upregulated. Fibrotic tenascin-C accumulated in the sub-epithelial region of the co-culture model. CONCLUSION: Our findings indicate that TGF-ß1 can affect both epithelial and mesenchymal cells, and induce gel contraction and structural changes. Our novel in vitro co-culture model will be a useful tool for investigating the roles of epithelial cells, fibroblasts, and their interactions in the airway remodeling process.