In vitro models of the respiratory barrier: a case study on zinc oxide nanoparticles

Background: In the last decade, nanoparticles (NPs) have brought a huge revolution in the many fields, especially in toxicology studies. Their small size (diameter lower than 100 nm) consent them to have unique chemical and physical properties, which, besides their positive impact in industrial use, may induce adverse health effects too. The production and the emission of zinc oxide nanoparticles (nZnO) is highly increased during the last years, for their industrial, cosmetic, anti-microbial and biomedical applications. Several in vitro and in vivo studies have shown that nZnO are toxic to different mammalian cells inducing inflammation, oxidative stress and systemic effects. However, many questions about the mechanisms of nZnO induced toxicity are still unanswered. Inhalation is the major way of entry and NPs, for their small dimension, can evade the clearance of the respiratory tree and reach the alveoli. NPs can exert their effects through two routes: 1) they may translocate across the air-blood barrier (ABB) and directly enter the endothelial cells; 2) they may induce the epithelial cells to release inflammatory mediators, which subsequently promote the release of endothelial dysfunction markers from the endothelium. According to the REACH regulation (No 1907/2006/EC) and the 3Rs (Refinement, Reduction, Replacement) principle, in vitro alternative strategies are needed to test new chemical compounds, such as NPs. Thus, the choice of an appropriate in vitro model becomes crucial for the evaluation of NPs-induced effects. Co-cultures on Transwell inserts represent a useful tool for the study of NPs effects on different cells. With this model, systemic effects are evaluable, as well as cells exposure to the air-liquid interface (ALI), a more suitable exposure scenario. The aim of this work was to evaluate the effects of nZnO, especially potential effects on inflammation and endothelial dysfunction, using different new in vitro models that mimic the respiratory barrier. Methods and results: Cytotoxicity and biological effects (inflammatory and cellular stress response) on monocultures of human epithelial alveolar cells (NCI-H441) have been tested. A co-culture, composed of epithelial lung cells and human endothelial microvascular pulmonary cells (HPMEC-ST1.6R), and a tri-culture adding monocytes, have been set-up. The results obtained have shown that nZnO (20 µg/ml) have a cytotoxic effect on monocultures, while at the lower dose (10 µg/ml) they do not affect the ABB integrity. However, nZnO induce inflammatory response and the release of soluble adhesion molecules from the endothelial cells in the co-culture system. These data showed for the first time that nZnO are able to induce endothelial inflammation and dysfunction in a 3D-in vitro model of the ABB in which the cells are not directly exposed to nZnO, but the release of inflammatory mediators is regulated by the overlaying epithelium that receives NPs. Furthermore, we observed that the addition of immune cells in the tri-culture modulated the cellular response to nZnO. Finally, a third different system was set-up in order to compare nZnO effects in submerged and ALI conditions. A co-culture of alveolar macrophages and epithelial cells have been set-up and exposed to nZnO suspension or aerosol. This experimental approach has shown that different ways of NPs administration influence the cellular response, demonstrating that cells are more sensitive to nZnO when they are exposed to the ALI system. Conclusions: This research evidenced that the crosstalk between different cells of the respiratory epithelium is crucial when NPs toxicity has to be evaluated. Different in vitro ABB are useful tools to understand the mechanisms of NPs-induced effects, especially their inflammatory potential and their effects on the cardiovascular system. Moreover, NPs administration too has a pivotal role in the biological outcomes occurring at NPs exposure.