Establishing toxicologically relevant primary alveolar macrophage models to enable a realistic assessment of nanomaterials safety

Abstract

Alveolar macrophages (AMs) are key sentinels of the distal lung, maintaining surfactant homeostasis and clearing inhaled particles through continuous surveillance and phagocytosis. Mechanistic studies of particle-cell interactions, important to assess the respiratory safety or toxicity of particles, however, are limited by the lack of suitable in vitro models that preserve the core features of resident AMs. Here, we benchmarked six murine AM models, including short-term cultured primary AMs (AMs 3 h, AMs 24 h), primary AMs maintained with GM-CSF, TGF-{beta}, and the PPAR-{gamma} agonist rosiglitazone (AMs GTR), bone marrow-derived AM-like cells (BM-AMs), the immortalized AM cell line MH-S, and the non-transformed fetal liver-derived AM-like cell line MPI, against in vivo AMs based on surface phenotype, transcriptomic identity, and their responses to three carbon-based nanomaterials (CBNs). Both cell lines showed limited AM identity, characterized by low CD11c and SiglecF but high CD11b surface expression. In contrast, AMs GTR and BM-AMs more closely preserved resident AM-like surface marker profiles. Transcriptomic features, although differing for alterations in metabolism-related pathways, particularly oxidative phosphorylation, revealed most in vivo-like for the primary AM models. Functional, toxicological assessment using soot-like carbon nanoparticles (CNP), double-walled carbon nanotubes (DWCNT) and multi-walled carbon nanotubes (MWCNT) demonstrated strong model-dependent responses. Consistent with MWCNT-induced reduction in AM numbers observed in vivo, AMs GTR and BM-AMs exhibited the strongest in vitro responses, including cytotoxicity and inflammatory cytokine release. MH-S cells in contrast stood out for their cytotoxic sensitivity supporting their use for high throughput cytotoxicity tests. Overall, these findings identify AMs GTR and BM-AMs as the closest models for studying AMs biology and AM-intrinsic responses to poorly soluble nanoparticles like CBNs, and with regard to availability of higher cell numbers, BM-AMs might provide a scalable platform for mechanistic and higher-throughput nanotoxicology studies.