89 Lung Single Cell Transcriptomics to Guide the Development of AOP Anchored Cell-Based Assays in Response to Nanoparticle Exposure

Abstract

Abstract Inhaled nanoparticles (NP) can cause acute and chronic inflammation. Here we seek to identify NP-specific cellular perturbation pathways and the underlying key cell types to inform adverse outcome pathway (AOP) anchored in vitro studies, by single cell transcriptomics. We exposed mice intratracheally to carbon black (CNP), tangled double-walled (DWCNT) and rigid, multi-walled carbon nanotubes (MWCNT). All lungs underwent single-cell RNA sequencing (scRNA-seq), histology and BAL analysis. At the chosen doses all NPs caused comparable airspace neutrophilia at 12h, which increased until d6 for the CNTs but remained elevated until d28 only for MWCNT, indicating the key event (KE) acute and chronic inflammation. Comparing scRNA-seq and BAL cytokine levels at 12h, differentiated the fate of inflammation NP specifically. In agreement with airspace neutrophilia, CNP uniquely triggered GM-CSF and CXCL1 release, with Csf2 and Cxcl1 mRNAs being expressed mainly in alveolar epithelial cells. DWCNT caused CCL2, -3 and -4 release involving interstitial macrophages and monocytes, associated with acute high lung monocyte and at d6 high BAL macrophage numbers. MWCNT in contrast caused ample BAL cytokine increases involving different cell types and specifically caused a Th2 cytokine response (CCL11, IL10), a crucial KE of fibrosis AOPs. MWCNT triggered cell communication analysis uncovered NP-specific networks including epithelial cells, fibroblasts, macrophages, dendritic cells and endothelial cells. Our study uncovers early NP-specific cell perturbations and identified first specific cellular response pattern to guide AOP predictive cell-based testing strategies. Further analysis of the cellular dynamics shall enhance our understanding of NP specific cell circuits and related pathologies.