Multilayered alveolar 3D in vitro models: a new approach to study the molecular hallmarks of asbestos toxicity/pathogenicity

The prolonged exposure to mineral fibres represents a serious occupational and environmental hazard, since it has been associated with the insurgence of various diseases (e.g., pneumoconiosis, pulmonary fibrosis, malignant mesothelioma and lung cancer). In fact, inhaled mineral fibres may reach the lower parts of the respiratory tract, ultimately embedding in the alveolar tissue, where the activation of the local immune system —mediated by resident macrophages— leads to the instauration of a persistent inflammatory state. In this regard, the development of physiologically relevant 3D in vitro models of the human alveolar tissue is crucial to overcome the massive use of poorly relatable and ethically concerning animal models when evaluating the potential damaging effects of inhaled substances. To this aim, we propose a multilayered 3D model of the human alveolar tissue which reproduces the thinnest part of the alveoli by stratification of human alveolar and endothelial cells separated by an electrospun PCL-Gel membrane mimicking the alveolar basement membrane. Moreover, the addition of THP-1-derived human macrophages to this 3D model ensures an accurate in vitro reproduction of the inflammatory process initiated by hazardous mineral fibres in the alveolar tissue. This novel 3D in vitro system was employed to obtain a more in-depth understanding of the molecular mechanisms underlying the toxicity/pathogenicity of different asbestos fibres (i.e., chrysotile and crocidolite). To this aim, several well-known hallmarks of tissue damage and inflammation were investigated up to 7 days, while changes in the structural architecture of the 3D multilayer were monitored by confocal microscopy. Thus, the gene expression levels of pro-inflammatory cytokines and oxidative stress regulators were evaluated by qPCR; then, several markers of asbestos-induced malignancy (i.e., γ-H2AX histone phosphorylation, 8-oxoguanine DNA glycosylase levels and miR-126 expression) were analysed.