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

Our plain-English summary of Proteomic Profiling of Alveolar Macrophages Identifies Loss of Lysosomal Content as an Indicator of Nanofiber-Induced Frustrated Phagocytosis, by T. Stobernack, A. Vennemann, C. Ribalta, et al (Small, 2026).

The study

• Authors: T. Stobernack, A. Vennemann, C. Ribalta, J. Schendel, O. Gräb, R. Ledwith, M. Pink, A. Haase, M. Wiemann, V.I. Dumit
• Journal: Small
• Published: 2026;22(11):e10530
• Cited by: 2 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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.

© 2026 The Author(s). Small published by Wiley‐VCH GmbH.

In plain English

This was a laboratory study in cultured cells; it did not test asbestos. The authors exposed rat lung immune cells (the NR8383 alveolar macrophage cell line) to silicon carbide nanofibers, to Mitsui-7 multi-walled carbon nanotubes, and to Printex-90, a carbon material made of spherical particles. In 16-hour screening tests at 22.5 to 180 µg/mL, all three materials damaged cells in a dose-dependent way. Only the nanofibers caused a dose-dependent rise in the inflammatory signal TNF-α. At 22.5 µg/mL, a concentration that caused no detectable cell damage in the screening tests, the authors measured proteins after 18 hours. More proteins changed with the nanofibers than with Printex-90. Inside the cells, 78 proteins changed with Printex-90, 201 with silicon carbide and 286 with Mitsui-7. With the nanofibers, most proteins from inside lysosomes (the cell's digestive compartments) fell inside the cells and rose in the surrounding fluid. Lysosomal membrane proteins were not found in the fluid. The authors concluded this was most likely an early event of frustrated phagocytosis, which happens when a cell cannot fully engulf a long fiber. They proposed 17 marker proteins for testing nanofibers without animal experiments.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: In vitro laboratory experiment on a rat alveolar macrophage cell line: toxicity screening assays plus proteomic analysis (mass spectrometry) of cell lysates and culture supernatants
• Who was studied: NR8383 rat alveolar macrophage cell line (from ATCC), grown in culture; proteomic experiments used three biological replicates per condition, each measured three times. No humans or animals were studied.
• Exposure: Silicon carbide (SiC) nanofibers (mean diameter 190 nm, mean length 11.7 µm), Mitsui-7 multi-walled carbon nanotubes (mean diameter 49 ± 13.4 nm, length 3.86 ± 1.94 µm), and Printex-90 spherical carbon particles (14 nm). Screening doses were 22.5, 45, 90 and 180 µg/mL for 16 hours, without serum. Proteomics used 22.5 µg/mL for 18 hours. No asbestos was tested.
• Main results:
  • All three materials caused dose-dependent release of LDH, a marker of cell damage. Membrane damage became significant at 45 to 90 µg/mL.
  • Release of the lysosomal enzyme glucuronidase was more pronounced with SiC and Mitsui-7 nanofibers than with Printex-90.
  • TNF-α, an inflammation signal, rose with dose after SiC and Mitsui-7 nanofiber treatment but not after Printex-90 treatment.
  • The screening assay identified 22.5 µg/mL of any of the materials as a non-cytotoxic concentration, which was then used for proteomic profiling.
  • In cell lysates at 22.5 µg/mL, the number of significantly altered proteins was 78 with Printex-90, 201 with SiC nanofibers and 286 with Mitsui-7.
  • In culture supernatants, the number of significantly altered proteins was 61 with Printex-90, 133 with SiC nanofibers and 313 with Mitsui-7.
  • 124 proteins in lysates and 89 in supernatants were altered by both nanofibers but not by Printex-90. Pathway analysis of these proteins pointed to the lysosome pathway in both sample types.
  • With SiC nanofibers and Mitsui-7, most lysosomal proteins were reduced in whole-cell lysates and increased in culture supernatants.
  • No lysosomal membrane proteins were detected in the culture supernatants.
  • Of the 25 altered luminal lysosomal proteins shown for cell lysates, 20 were hydrolases. These included six cathepsins (Ctsa, Ctsb, Ctsc, Ctsd, Ctsh, Ctsz).
  • Of 58 proteins in the authors' earlier preliminary fingerprint, 41 were detected in cell lysates. The 17 most consistently and significantly altered were selected as a refined marker set.
• The authors' conclusion: The authors concluded that they provided the first direct evidence that putatively pathogenic nanofibers cause alveolar macrophages to release proteins from inside lysosomes. They considered this most likely an early event of frustrated phagocytosis. They proposed the protein signature as a readout for animal-free testing methods.
• Limitations the authors state:
  • The proteomic method cannot detect protein activation states, such as activated NALP3.
  • Two proteins, Gm2a and CD68, did not fit the authors' hypothesis. Further experiments are needed to understand these changes.
  • NALP3 activation may not be specific to fibers. It may instead be part of a broader, general cellular stress response.
  • The proposed mechanism may not be limited to high-aspect-ratio materials.

Topics

Medical terms in this study, explained in our Lexicon: Alveolar Macrophage, Frustrated Phagocytosis, Fibrosis, Lung Cancer, Mesothelioma, Macrophage, Biomarker.

Related studies

• Fluorescence imaging of reactive oxygen metabolites generated in single macrophage cells (NR8383) upon phagocytosis of natural zeolite (erionite) fibers — J.F. Long, P.K. Dutta, et al. (1997)
• Use of back-scatter electron signals to visualise cell/nanowires interactions in vitro and in vivo; frustrated phagocytosis of long fibres in macrophages and compartmentalisation in mesothelial cells in vivo — A. Schinwald, K. Donaldson (2012)
• Effects of chrysotile on a lysosomal enzyme preparation and on the hydrolytic enzyme activity of cultured alveolar macrophages — R. Desai, R.J. Richards (1983)
• Iron and reactive oxygen species in the asbestos-induced tumor necrosis factor-alpha response from alveolar macrophages — P.P. Simeonova, M.I. Luster (1995)
• Alveolar macrophage cytokine and growth factor production in a rat model of crocidolite-induced pulmonary inflammation and fibrosis — K.E. Driscoll, J.K. Maurer, et al. (1995)
• Distinctive profile of alveolar macrophage-derived cytokine release induced by fibrogenic and nonfibrogenic mineral dusts — I. Lemaire, S. Ouellet (1996)

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