Proteomic Profiling of Alveolar Macrophages Identifies Loss of Lysosomal Content as an Indicator of Nanofiber‐Induced Frustrated Phagocytosis

ABSTRACT Toxicological research on inhalable fibers, such as asbestos, has identified material morphology (i.e., length and diameter) and bio‐persistence as drivers of adverse health effects (e.g., fibrosis, lung cancer, mesothelioma). Although nanofibers may meet these criteria, their small diameters may enable them to adopt different shapes, affecting their toxicity. While nanofiber pathogenicity is still assessed using animal models, the development of alternative in vitro methods relies on a mechanistic understanding of toxicity. Here, we address nanofiber‐induced protein changes in alveolar macrophages by analyzing whole cell lysates and supernatants of NR8383 cells exposed to silicon carbide nanofibers, Mitsui‐7 carbon nanotubes, and Printex‐90. While all materials elicited a similar dose‐dependent cytotoxicity, there was a nanofiber‐specific release of TNF‐α and glucuronidase. Proteomic profiling after treatment with low, non‐cytotoxic concentrations confirmed the inflammatory response and revealed a release of 20 lysosomal, luminal hydrolases, including six cathepsins, into the extracellular supernatant. In cell lysates, these hydrolases were decreased, while membrane‐associated lysosomal proteins remained unchanged, suggesting that macrophages engulfing long nanofibers release lysosomal content from open membrane pouches during frustrated phagocytosis. Additionally, 17 biomarkers of nanofiber‐induced toxicity were identified as potential targets for predictive, animal‐free screening. These early markers may be of value for assessing nanofiber toxicity.