Mode of actions of nanomaterial-induced respiratory toxicity

Inhalation of particles can induce lung inflammation, leading to both acute and chronic health issues. The development of reliable safety tests for inhaled nanomaterials is hindered by a limited understanding of cell-specific responses. To address this, we conducted a longitudinal analysis of cellular perturbations in mice exposed to carbon black (CNP), double-walled (DWCNT), and multi-walled carbon nanotubes (MWCNT) via pulmonary delivery using single-cell RNA sequencing. At selected doses, all NMs induced similar levels of neutrophil influx into the airspace at 12h, with continued elevation observed until d6 for DWCNT and until d28 for MWCNT. Each NM exhibited unique patterns of protein release into the airspace. CNP triggered GM-CSF and CXCL1 release, primarily from alveolar epithelial cells, while DWCNT induced CCL2 and CCL3 release involving interstitial macrophages and monocytes. MWCNT elicited the most extensive cytokine response, including Th2 cytokines CCL11 and CCL19, crucial for fibrosis development. BAL analysis showed a significant increase in total macrophage numbers at d6 post-exposure to CNTs, coinciding with the loss of specific alveolar macrophage populations, particularly notable for MWCNT. Intravital microscopy confirmed MWCNT-induced depletion of alveolar macrophages. Histological analysis revealed cell death in various lung cell types, scRNA seq indicated necroptosis and cuproptosis in macrophages and ferroptosis in epithelial cells, supported by immunohistology for ACSL4. The insights gained from this study will significantly enhance the precision of future in vitro testing methods, providing valuable information on the mode of actions of nanomaterials and their impact on respiratory health.