Abstract The development of in vitro safety tests for inhaled nanomaterials is hindered by limited understanding of cell-specific responses, particularly epithelial activation and inflammation. We performed single-cell RNA sequencing (scRNAseq) analysis of lung cells from intratracheally exposed mice with carbon-based nanomaterials (CBN), ie carbon nanoparticles (CNP), double-walled (DWCNT), or multi-walled carbon nanotubes (MWCNT). Based on identified cellular circuits, we explore murine alveolar epithelial lung organoids cultured with supporting fibroblasts to reproduce signatures of epithelial injury and inflammatory activation. All CBN induced comparable neutrophilia at 12 h, sustained until day 6 for DWCNT and day 28 for MWCNT. CNT exposure caused epithelial and alveolar macrophage injury, reflected by alveolar barrier disruption and alarmin release (Il33, Il1α). Furthermore, the epithelial niche showed the transient accumulation of KRT8+ alveolar differentiation intermediates (ADI), known for their relevance in injury response and fibrotic remodelling. Interestingly, early fibroblast activation supported sustained inflammation and monocyte recruitment, leading to a later observed profibrotic environment and tissue remodelling (Voss & Han, 2025, ACS Nano). Lung organoids reproduced key epithelial signatures based on alarmin release, including significantly elevated Krt8 and Hopx gene expression for ADI, and inflammatory activation of type 2 epithelial cells (Lcn2, GM-Csf, Lamp3). In vitro, we observed substantial IL6 release with increased organoid forming capacity indicating regenerative activity upon alarmin release from damaged cells. Macrophage conditioned medium induced upregulation of Col1a2, Col3a1 and Ccl11 in lung organoids, demonstrating fibroblast activation and ongoing matrix remodelling. These findings support the integration of scRNAseq data into murine lung organoids as relevant new approach methodology for predicting CBN-induced epithelial toxicity in vitro.
Lei et al. (Thu,) studied this question.