Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes

Abstract

Expansion in production and commercial use of nanomaterials increases the potential human exposure during the lifecycle of these materials (production, use, and disposal). Inhalation is a primary route of exposure to nanomaterials; therefore it is critical to assess their potential respiratory hazard. Herein, we developed a three-dimensional alveolar model (EpiAlveolar) consisting of human primary alveolar epithelial cells, fibroblasts, and endothelial cells, with or without macrophages for predicting long-term responses to aerosols. Following thorough characterization of the model, proinflammatory and profibrotic responses based on the adverse outcome pathway concept for lung fibrosis were assessed upon repeated subchronic exposures (up to 21 days) to two types of multiwalled carbon nanotubes (MWCNTs) and silica quartz particles. We simulate occupational exposure doses for the MWCNTs (1−30 μg/cm 2 ) using an air−liquid interface exposure device (VITROCELL Cloud) with repeated exposures over 3 weeks. Specific key events leading to lung fibrosis, such as barrier integrity and release of proinflammatory and profibrotic markers, show the responsiveness of the model. Nanocyl induced, in general, a less pronounced reaction than Mitsui-7, and the cultures with human monocyte-derived macrophages (MDMs) showed the proinflammatory response at later time points than those without MDMs. In conclusion, we present a robust alveolar model to predict inflammatory and fibrotic responses upon exposure to MWCNTs.

An In Vitro Lung System to Assess the Proinflammatory Hazard of Carbon Nanotube Aerosols

“…Direct exposures of fibroblasts to CNTs stimulated progressive cell proliferation or collagen production, the known fibrotic markers [25,26]. Submerged exposure of fibroblasts, epithelial cells, and macrophages used in the presented study was previously used as a first screening tool, and it was shown that exposure to CNTs can induce proinflammatory and profibrotic responses [27,28] An increased proinflammatory response upon repeated exposure to CNT aerosols was also observed in a 3D lung co-culture model (in the presence of immune cells) consisting of cell-lines [29] or primary cells including fibroblasts [30].…”

“…The underlying drive behind the development of an inflammatory response is to clear the foreign material from the tissue and to eventually initiate the repairing pathway, which is facilitated by growth factors, such as the TGF-β. TGF-β was used as a positive fibrotic control because of its involvement in the development of fibrosis in different organs, disturbances of the homeostatic microenvironment, promotion of cell activation, migration, invasion, and excessive extracellular matrix production [30,46]. No statistically significant increase for any of the cytokine release measurements was observed at any exposure time-points upon exposures to any of the tested materials.…”

“…It is crucial to gain insight into the pulmonary hazard of prolonged (subchronic) CNT exposure at repeated doses that can realistically mimic the in vivo conditions. We recently showed that a lower respiratory tract 3D co-culture model with primary human cells, i.e., EpiAlveolar ™ , could be a promising tool to predict the development of pulmonary fibrosis in response to fibrotic substances such as CNTs [30]. In addition to a proinflammatory and profibrotic response as assessed by cytokine measurements, a disruption in barrier integrity (determined by transepithelial electrical resistance (TEER) measurements) and an increase in alveolar tissue thickening were observed in response to the chemical positive control, transforming growth factor β (TGF-β) [30].…”

“…We recently showed that a lower respiratory tract 3D co-culture model with primary human cells, i.e., EpiAlveolar ™ , could be a promising tool to predict the development of pulmonary fibrosis in response to fibrotic substances such as CNTs [30]. In addition to a proinflammatory and profibrotic response as assessed by cytokine measurements, a disruption in barrier integrity (determined by transepithelial electrical resistance (TEER) measurements) and an increase in alveolar tissue thickening were observed in response to the chemical positive control, transforming growth factor β (TGF-β) [30]. Within the light of this conclusion, the current study was designed to assess the suitability of a 3D lung co-culture model consisting of cell lines (i.e., epithelial cells, macrophages, and fibroblasts) to acute (24 h) and prolonged (96 h) exposures of realistic CNT exposure doses.…”

An Alternative Perspective towards Reducing the Risk of Engineered Nanomaterials to Human Health

“…[63] In theory, by enabling analysis of each KE (e.g., pro-inflammatory mediator release, cell proliferation, pro-fibrotic mediator release, collagen production), it is possible to use an in vitro model to determine if it predicts the KE(s), and thus the AO. Recently, a study by Barosova and colleagues [64] used AOP173 to assess the pro-inflammatory and pro-fibrotic impacts of different multi-walled CNTs upon an advanced in vitro lung cell system. These approaches are starting to lay the foundation for the use of (advanced) in vitro models to predict the hazardous outcome of human exposure to specific xenobiotics, such as ENMs.…”