A secretomics analysis reveals major differences in the macrophage responses towards different types of carbon nanotubes

Our plain-English summary of A secretomics analysis reveals major differences in the macrophage responses towards different types of carbon nanotubes, by J. Palomäki, J. Sund, M. Vippola, et al (Nanotoxicology, 2015).

The study

• Authors: J. Palomäki, J. Sund, M. Vippola, P. Kinaret, D. Greco, K. Savolainen, A. Puustinen, H. Alenius
• Journal: Nanotoxicology
• Published: 2015;9(6):719-28
• Cited by: 25 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

Certain types of carbon nanotubes (CNT) can evoke inflammation, fibrosis and mesothelioma in vivo, raising concerns about their potential health effects. It has been recently postulated that NLRP3 inflammasome activation is important in the CNT-induced toxicity. However, more comprehensive studies of the protein secretion induced by CNT can provide new information about their possible pathogenic mechanisms. Here, we studied protein secretion from human macrophages with a proteomic approach in an unbiased way. Human monocyte-derived macrophages (MDM) were exposed to tangled or rigid, long multi-walled CNT (MWCNT) or crocidolite asbestos for 6 h. The growth media was concentrated and secreted proteins were analyzed using 2D-DIGE and DeCyder software. Subsequently, significantly up- or down-regulated protein spots were in-gel digested and identified with an LC-MS/MS approach. Bioinformatics analysis was performed to reveal the different patterns of protein secretion induced by these materials. The results show that both long rigid MWCNT and asbestos elicited ample and highly similar protein secretion. In contrast, exposure to long tangled MWCNT induced weaker protein secretion with a more distinct profile. Secretion of lysosomal proteins followed the exposure to all materials, suggesting lysosomal damage. However, only long rigid MWCNT was associated with apoptosis. This analysis suggests that the CNT toxicity in human MDM is mediated via vigorous secretion of inflammation-related proteins and apoptosis. This study provides new insights into the mechanisms of toxicity of high aspect ratio nanomaterials and indicates that not all types of CNT are as hazardous as asbestos fibers.

In plain English

This was a laboratory study of human immune cells, not of people. The authors grew macrophages from the blood of healthy donors in Finland. They exposed the cells for 6 hours to one of three materials: long rigid multi-walled carbon nanotubes, long tangled multi-walled carbon nanotubes, or crocidolite asbestos, which served as a positive control. They then measured the proteins the cells released. Rigid nanotubes changed the most protein spots (37), compared with 12 for tangled nanotubes and 5 for asbestos. The patterns after rigid nanotubes and asbestos resembled each other. The pattern after tangled nanotubes grouped with that of unexposed cells. Proteins linked to lysosomes, the cell compartments that break down material, were released after all three materials. After 18 hours, almost 30% of cells exposed to rigid nanotubes had died. Rigid nanotubes were the only material that caused apoptosis, a programmed form of cell death, in about 10% of cells. The authors concluded that rigid nanotubes most likely harm macrophages through apoptosis and pyroptosis, and that not all carbon nanotubes behave alike.

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: Laboratory (in vitro) experiment: proteomic analysis of proteins released by cultured human macrophages
• Who was studied: Macrophages grown from blood monocytes of healthy blood donors supplied by the Finnish Red Cross Blood Transfusion Service, Helsinki, Finland. Each sample pooled cells from three donors, and four independent repeat experiments were performed. The dates of the experiments are not given.
• Exposure: The materials were long rigid multi-walled carbon nanotubes (MWCNT-7), long tangled multi-walled carbon nanotubes, and crocidolite asbestos (UICC standard sample) as a positive control. The main dose is printed as 100 mg/ml, with an exposure time of 6 hours. Validation tests used doses of 3, 10, 30, 100 and 300 mg/ml as printed. Cell death was also measured at 18 hours.
• Main results:
  • Number of protein spots whose release differed significantly from unexposed cells: 37 for rigid nanotubes, 12 for tangled nanotubes, and 5 for asbestos. Altogether 29 different proteins were identified.
  • In clustering, the protein release patterns of rigid nanotube-exposed and asbestos-exposed cells were similar. The pattern of tangled nanotube-exposed cells grouped with that of untreated cells.
  • Four proteins were shared by the rigid nanotube and asbestos groups, and five by the rigid and tangled nanotube groups. No protein was common to all three groups, and asbestos and tangled nanotubes shared none.
  • The lysosomal pathway was enriched after exposure to all three materials.
  • At the 100 mg/ml dose (as printed), rigid nanotube-exposed cells released twice as much of the protein PPIA as cells exposed to tangled nanotubes or asbestos. They also released five times more active cathepsin D.
  • In the LDH cell-death test, 5 to 10% of cells died after 6 hours with rigid nanotubes and with asbestos. Almost 30% were dead after 18 hours with rigid nanotubes.
  • Only rigid nanotubes caused apoptosis: about 10% of cells showed signs of apoptosis after 18 hours.
  • Both rigid nanotubes and asbestos triggered release of IL-1 beta. Only rigid nanotubes, at 100 and 300 mg/ml (as printed), caused release of the cleaved caspase-1 p20 subunit.
• The authors' conclusion: The authors concluded that rigid multi-walled carbon nanotubes most likely harm macrophages through both apoptosis and pyroptosis pathways. They also concluded that not all carbon nanotubes are similar: in their view rigid ones cause severe toxicity, while tangled ones do not.
• Limitations the authors state:
  • The analysis misses proteins released very early after exposure.
  • Apoptosis caused by asbestos might not be observable within 6 hours.
  • A less strict statistical test was used for the pathway analysis, because the DAVID tool cannot give reliable results from small sets.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Crocidolite.

Medical terms in this study, explained in our Lexicon: Macrophage, Inflammation, Fibrosis, Mesothelioma, Crocidolite, Apoptosis, Aspect Ratio (Length-to-diameter ratio).

Related studies

• The mechanism of pleural inflammation by long carbon nanotubes: interaction of long fibres with macrophages stimulates them to amplify pro-inflammatory responses in mesothelial cells — F.A. Murphy, A. Schinwald, et al. (2012)
• Multi-walled carbon nanotubes injure the plasma membrane of macrophages — S. Hirano, S. Kanno, et al. (2008)
• Inhalation and Oropharyngeal Aspiration Exposure to Rod-Like Carbon Nanotubes Induce Similar Airway Inflammation and Biological Responses in Mouse Lungs — P. Kinaret, M. Ilves, et al. (2017)
• A Single Aspiration of Rod-like Carbon Nanotubes Induces Asbestos-like Pulmonary Inflammation Mediated in Part by the IL-1 Receptor — E.M. Rydman, M. Ilves, et al. (2015)
• Long, needle-like carbon nanotubes and asbestos activate the NLRP3 inflammasome through a similar mechanism — J. Palomäki, E. Välimäki, et al. (2011)
• Structural defects play a major role in the acute lung toxicity of multiwall carbon nanotubes: physicochemical aspects — I. Fenoglio, G. Greco, et al. (2008)

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