Toward a ToxAtlas of Carbon-Based Nanomaterials: Single-Cell RNA Sequencing Reveals Initiating Cell Circuits in Pulmonary Inflammation

Understanding how nanomaterial properties drive acute lung inflammation is critical for the development of safer materials, but for low solubility carbon-based nanomaterials (CBNs) the initiation of the inflammatory response is still poorly understood. Leveraging single-cell RNA sequencing of mouse lungs, 12 h after intratracheally instillation with different CBN spherical carbon nanoparticles (CNP), tangled double-walled (DWCNT), and rigid multiwalled carbon nanotubes (MWCNT) and lipopolysaccharide (LPS) as positive control, we identified 41 cell states and delineated material-specific molecular initiation events at single-cell resolution. CBN doses were chosen to cause equal levels of moderate inflammation, assessed by airspace neutrophilia, and exposure-triggered cellular activation was tested for <i>in vitro</i> reproducibility. To advance future development of cell-based assays, we developed a webtool, ToxAtlas, mapping CBN-specific gene responses of interest. Despite chemical similarity, CBN elicited distinct inflammatory cytokine and cell responses via different modes of action. CNP triggered neutrophilia through alveolar epithelial activation and <i>Cxcl1</i> and <i>Csf2</i> expression but without apparent cell damage or macrophage activation. In contrast, CNT induced epithelial and macrophage damage, with alarmin release (IL-1α, IL-33) dominating the MWCNT response. DWCNT caused alveolar epithelial injury, and pro-inflammatory macrophage and fibroblast-derived monocyte attractant (<i>Ccl2</i>, <i>Ccl7</i>) activation. Our initiating cell circuits identify epithelial as well as early fibroblast activation, especially from alveolar type 2 cell-adjacent lipofibroblasts, as central to orchestrating the initiation of CBN-induced inflammation. These findings support the role of mesenchymal cells in early pulmonary defense, eventually priming chronic inflammation, a known cause of MWCNT exposure.