Molecular and cellular mechanisms of the pro-fibrotic effects of carbon nanotubes

Carbon nanotubes (CNT) are molecular-scale tubes of graphene sheets rolled into cylinders with peculiar characteristics that make them highly attractive for numerous industrial applications. Thus, investigating the health hazards of CNT is of great importance in view of the increased potential for human exposure within occupational, environmental, and consumer environments. Several studies have already shown that CNT can induce inflammatory, fibrotic or carcinogenic reactions in the lung of experimental animals. These toxic responses appear, however, determined by the physico-chemical characteristics of CNT. This work focuses on lung fibrosis, a process involving the proliferation and activation of fibroblasts. The main objectives were (1) to understand the mechanisms mediating the fibrogenic activity of CNT by investigating their direct and indirect (via macrophages and epithelial cells) effects on fibroblasts and (2) to develop valid in vitro models to predict the fibrogenic activity of CNT. An additional objective was to assess and to identify physico-chemical properties of CNT that determine their fibrogenic activity. As CNT have been shown to interact with several toxicological assays, we first designed an adapted protocol of the WST-1 cell viability assay to avoid or take into account interferences of nanomaterials. Our data demonstrate that the direct in vitro activity of CNT on fibroblast proliferation strongly reflects their fibrogenic activity in vivo, supporting a predictive value of this in vitro endpoint. Kinase receptors, ERK 1/2 signaling and endocytosis were identified as mechanisms involved in fibroblast proliferation induced by fibrogenic CNT. In addition, we showed that CNT indirectly stimulate fibroblast differentiation, via epithelial cells and macrophages, highlighting the contribution of these cells in the development of fibrosis. We found that the release of IL-6 from epithelial cells is another useful biomarker, in addition to the proliferative activity of CNT on fibroblasts, to predict the toxic potential of CNT. The results also confirm that the length and diameter of CNT constitute important physico-chemical determinants of their capacity to induce lung fibrosis. Finally, we have organized, analyzed and integrated all the current knowledge concerning the mechanisms of action of CNT relevant for their pro-fibrotic activity in a tentative adverse outcome pathway (AOP). This provides a global picture of the complex network of events contributing to the fibrogenic activity of CNT, and allows identifying predictive in vitro endpoints useful for the toxicological assessment of new and emerging CNT.