Transcriptional activation of inflammasome components by Libby amphibole and the role of iron

Our plain-English summary of Transcriptional activation of inflammasome components by Libby amphibole and the role of iron, by J.H. Shannahan, A.J. Ghio, M.C. Schladweiler, et al (Inhalation Toxicology, 2012).

The study

• Authors: J.H. Shannahan, A.J. Ghio, M.C. Schladweiler, J.H. Richards, D. Andrews, S.H. Gavett, U.P. Kodavanti
• Journal: Inhalation Toxicology
• Published: 2012;24(1):60-9
• Cited by: 12 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

Abstract

The induction of the NALP3 inflammasome complex is shown to be necessary for the development of fibrosis after asbestos exposure. Libby amphibole (LA) induces lung inflammation and fibrosis, while complexation of iron (Fe) on fibers inhibits inflammation. In this study we examined the ability of LA to induce the inflammasome cascade and the role of Fe in modulating inflammasome activity. Spontaneously hypertensive rats were exposed intratracheally to either saline (300 μl), deferoxamine (Def) (1 mg), FeCl(3) (21 μg), LA (0.5 mg), Fe-loaded LA (Fe + LA), or LA + Def. Activities of oxidative stress-sensitive enzymes, expression of inflammasome-specific genes, and cytokine proteins in bronchoalveolar lavage fluid were analyzed. Lung enzymes at 4 h and 24 h post-exposure were unchanged. LA increased lung expression of genes including interleukin-1β (IL-1β), cathepsin-B, ASC, NALP3, interleukin (IL)-6 and NFκB. LA+Fe significantly reduced IL-1β and NFκB with a trend of reduction in ASC, NALP3, cathepsin-B and IL-6 expression. Def treatment did not reverse the inhibitory effect of Fe on IL-1β and ASC but reversed IL-6 expression. CCL-7, CCL-12, CXCL-3 and COX-2 were induced by LA while LA+Fe tended to reduce these responses. Phosphorylation of ERK but not MEK was increased at 4 h after LA but not LA+Fe exposure. In conclusion, components of the NALP3 inflammasome are transcriptionally activated acutely during LA-induced inflammation. The key inflammatory regulators IL-1β and NFκB were inhibited in the presence of surface-complexed Fe possibly through decreased ERK signaling upstream of the NALP3 inflammasome. The inflammasome activation by LA may contribute to fibrosis, and Fe may reduce this response and alter compensatory mechanisms in individuals exposed to LA.

In plain English

This was an animal experiment in which male spontaneously hypertensive rats were given a single intratracheal instillation of saline, deferoxamine (Def), FeCl3, Libby amphibole (LA), iron-loaded LA (LA + Fe) or LA + Def, and examined 4 h or 24 h later. Using a rat array of 84 inflammasome-related genes, the authors found that LA increased lung mRNA for cathepsin-B, ASC, NALP3, IL-1β, NFκB and IL-6, while interferon-γ and IL-18 mRNA were unchanged. LA + Fe significantly reduced the IL-1β response and returned NFκB to control levels, with reduction trends for other inflammasome genes; Def removed the inhibition for only some genes. Lung mRNA for the macrophage cytokines CCL-7, CCL-12 and CXCL-3, and for COX-2, also rose after LA. Oxidant-sensitive lung enzyme activities did not change at 4 h or 24 h. Phosphorylated ERK, but not MEK, was increased after LA. In lavage fluid, CXCl-1 and IL-4 proteins were lower in LA and LA + Def rats. The authors concluded LA transcriptionally induces NALP3 inflammasome components and that surface iron reduces this, probably via reduced ERK activation.

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: controlled animal experiment (in vivo rat intratracheal instillation study with gene-expression, protein and enzyme endpoints)
• Who was studied: 96 male spontaneously hypertensive rats, 11-12 weeks old, studied at a US EPA laboratory; lungs examined 4 h or 1 day after instillation (n = 8 per group for lavage, n = 6 per group for RNA and signaling)
• Exposure: Single intratracheal instillation of Libby amphibole (0.5 mg, respirable PM2.5 fraction from the Rainy Creek Complex, described by the authors as a mixture of winchite, richterite (printed as 'richerite') and tremolite), LA loaded with iron, or LA plus deferoxamine, compared with saline, deferoxamine or FeCl3 alone
• Main results:
  • At 4 h, LA increased lung mRNA expression of cathepsin-B, ASC, NALP3, IL-1β, NFκB and IL-6 relative to saline controls; cathepsin-B, ASC and NALP3 were not increased above saline control in rats given LA + Fe or LA + Def
  • Iron-loading of LA significantly lowered the LA-induced IL-1β mRNA response and brought NFκB mRNA back to control levels
  • Pulmonary interferon-γ and IL-18 mRNA were not altered by LA, LA + Fe or LA + Def
  • Lung mRNA for the macrophage cytokines CCL-7, CCL-12 and CXCL-3 increased after LA, and less so after LA + Fe
  • Lung COX-2 mRNA increased after Def, LA, LA + Fe and LA + Def compared with controls
  • Oxidant-sensitive lung enzyme activities (NADPH quinone oxidoreductase, total SOD, CuZnSOD, MnSOD) showed no significant change after LA or LA + Fe at either time point
  • In cell-free lavage fluid, CXCl-1 fell from 1030 ± 213 pg/mg protein in saline rats to 436 ± 63 in LA rats and 250 ± 35 in LA + Def rats, and IL-4 from 2.58 ± 0.34 to 1.20 ± 0.12 in LA rats (significant versus saline); IFN-γ, IL-1β, IL-5 and TNF-α protein were not significantly altered by LA
  • The ratio of phosphorylated to total ERK rose after LA, LA + Fe and LA + Def, with less pERK in LA + Fe rats than LA rats, while the pMEK to total MEK ratio was unchanged
• The authors' conclusion: The authors concluded that LA transcriptionally induces components of the NALP3 inflammasome and specifically IL-1β in the lung, that surface-complexed iron reduces NFκB and IL-1β transcription probably by reducing ERK activation, and that inflammasome activation might be necessary for fibrosis and chronic disease after LA exposure
• Limitations the authors state:
  • The LA sample was a mixture of fiber types, so the authors state they cannot say which fiber type caused the inflammasome activation
  • Caspase-1 activity could not be measured reliably because heme in lung homogenate interfered with the assay
  • The authors suggest lavage cytokine proteins may have bound to fibers and been removed during centrifugation, limiting antibody-based protein quantification
  • The authors state the mechanism by which deferoxamine reversed iron's inhibition of inflammation without increasing transcription of some NALP3 components needs further study

Mines named here

Asbestos mines this study is about. Each mine's page says who ran it, when it worked, and what it produced.

• Libby vermiculite mine, Montana

All asbestos mines

Topics

Medical terms in this study, explained in our Lexicon: Libby amphiboles, Fibrosis, Asbestos Exposure, Inflammation, Oxidative Stress, Cytokine, Bronchoalveolar Lavage (BAL).

Related studies

• The role of iron in Libby amphibole-induced acute lung injury and inflammation — J.H. Shannahan, A.J. Ghio, et al. (2011)
• Differential activation of the inflammasome in THP-1 cells exposed to chrysotile asbestos and Libby "six-mix" amphiboles and subsequent activation of BEAS-2B cells — M. Li, M.E. Gunter, et al. (2012)
• Vascular and thrombogenic effects of pulmonary exposure to Libby amphibole — J.H. Shannahan, M.C. Schladweiler, et al. (2012)
• Pulmonary inflammatory and fibrotic responses in Fischer 344 rats after intratracheal instillation exposure to Libby amphibole — D.J. Padilla-Carlin, M.C. Schladweiler, et al. (2011)
• Sumas Mountain chrysotile induces greater lung fibrosis in Fischer344 rats than Libby amphibole, El Dorado tremolite, and Ontario ferroactinolite — J.M. Cyphert, A. Nyska, et al. (2012)
• Comparative long-term toxicity of Libby amphibole and amosite asbestos in rats after single or multiple intratracheal exposures — J.M. Cyphert, D.J. Carlin, et al. (2015)

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