Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin

Our plain-English summary of Ferroptosis-dependent extracellular vesicles from macrophage contribute to asbestos-induced mesothelial carcinogenesis through loading ferritin, by F. Ito, K. Kato, I. Yanatori, et al (Redox Biology, 2021).

The study

• Authors: F. Ito, K. Kato, I. Yanatori, T. Murohara, S. Toyokuni
• Journal: Redox Biology
• Published: 2021;47:102174
• Cited by: 77 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Asbestos-associated diseases remain a social burden worldwide. Our previous studies identified asbestos-induced iron-rich milieu for mesothelial cells with ceaseless macrophage ferroptosis. However, molecular mechanisms how this mutagenic milieu influences mesothelial cells have not been elucidated yet. Here, we propose a novel mechanism that extracellular vesicles (EVs) mediate asbestos-associated mutagenic factors to mesothelial cells. In a mice model of intraperitoneal crocidolite injection, mutagenic milieu highly expressed CD63, an exosomal marker. We then used a GFP-CD63 labeled THP-1 macrophage model exposed to crocidolite/iron, which generated EVs under ferroptotic process. We observed that MeT-5A mesothelial cells can receive and internalize these EVs. Furthermore, we comprehensively analyzed the ferroptosis-dependent EVs (FedEVs) for transported proteins and identified ferritin heavy/light chains as major components. Therefore, we inferred that FedEVs transport iron from ferroptotic macrophages to mesothelial cells. RNA sequencing revealed that the mesothelial cells receiving higher amounts of the FedEVs were mitotic, especially at the S and G2/M phases, by the use of Fucci mesothelial cells. Nuclear 8-hydroxy-2'-deoxyguanosine and γ-H2AX were significantly increased in the recipient mesothelial cells after exposure to FedEVs. Collectively, we here demonstrate a novel mechanism that FedEVs act as a key mutagenic mediator by transporting iron, which contribute to asbestos-induced mesothelial carcinogenesis.

Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.

In plain English

This laboratory study combined a mouse model with cell-culture experiments to ask how asbestos-exposed macrophages affect mesothelial cells. Male C57BL/6 N mice (n = 9) were injected into the peritoneal cavity with 3 mg crocidolite and examined six months later. The fibers were found inside CD68-positive macrophages, not in mesothelial cells, and CD63 (an extracellular-vesicle marker) was abundant in a granular pattern in mesothelial and myofibroblast cells near the granuloma. In culture, THP1 macrophages exposed to crocidolite (15 micrograms per square centimetre) with ferric ammonium citrate, and HT1080 cells exposed to RSL3, secreted more extracellular vesicles, which the authors called ferroptosis-dependent EVs (FedEVs). MeT-5A mesothelial cells internalized these vesicles within 6 to 9 hours and showed increased catalytic Fe(II). Proteomic analysis identified ferritin light and heavy chains as major vesicle proteins. RNA sequencing comparing mesothelial cells with high versus low FedEV uptake gave 245 differentially expressed genes (163 up, 82 down) linked to mitosis. Nuclear 8-OHdG and gamma-H2AX increased after FedEV exposure. The authors concluded FedEVs transport iron as a mutagenic mediator.

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: Experimental laboratory study combining an in vivo mouse intraperitoneal crocidolite model with in vitro cell-culture, proteomic and RNA-sequencing experiments
• Who was studied: Male C57BL/6 N mice, 6 weeks old, n = 9, examined 6 months after injection, plus human cell lines MeT-5A mesothelial, THP1 macrophage, HT1080 fibrosarcoma and HEK293T; Nagoya University, Japan
• Exposure: Crocidolite (UICC) asbestos: 3 mg intraperitoneally in mice. In vitro: 15 micrograms per square centimetre crocidolite with or without ferric ammonium citrate 100 micrograms per ml for up to 96 h. RSL3 10 microM for 24 h was used as a ferroptosis inducer.
• Main results:
  • In the mouse model six months after crocidolite injection, asbestos fibers were inside CD68-positive macrophages and not in myofibroblasts or mesothelial cells. CD63 was abundant in mesothelial and myofibroblast cells near the granuloma.
  • Extracellular vesicle numbers from THP1 macrophages were highest with crocidolite exposure under iron-rich conditions, with the size peak shifting to 250 nm and 350 nm
  • MeT-5A mesothelial cells completely internalized GFP-CD63-labelled FedEVs at 6 to 9 hours
  • Mesothelial cells taking up FedEVs showed significantly increased intracellular catalytic Fe(II) at 24 hours
  • Proteomic analysis of FedEVs from macrophages identified ferritin light and heavy chains. Only vesicles from iron-treated or iron-plus-crocidolite-treated cells contained high FtL and FtH.
  • RNA sequencing comparing mesothelial cells with high versus low FedEV uptake gave 245 differentially expressed genes (163 upregulated, 82 downregulated). Gene ontology analysis linked these genes to the mitotic cell cycle.
  • In Fucci-labelled mesothelial cells, BFP-CD63 from FedEVs increased in cells in S and G2/M phases
  • Nuclear 8-hydroxy-2'-deoxyguanosine and gamma-H2AX increased in mesothelial cells 24 hours after exposure to crocidolite-derived FedEVs from THP1 cells
  • The iron chelator desferal reduced catalytic Fe(II) in recipient mesothelial cells dose-dependently, but the level remained notably higher than the control level
• The authors' conclusion: The authors concluded that extracellular vesicles from ferroptotic macrophages, loaded with ferritin, deliver iron to mesothelial cells. In their view this is a remote mutagenic mechanism in asbestos-induced mesothelial carcinogenesis and a basis for prevention by iron reduction.
• Limitations the authors state:
  • The authors stated that the possible EV receptor findings (ITGB1, CD44, CD11a, ICAM1) need further investigation
  • The authors stated that further studies are warranted to confirm the current results
  • The authors noted that their view that an iron-rich stromal environment is critical for ferritin-rich vesicles was based on in vitro experiments

Topics

Medical terms in this study, explained in our Lexicon: Macrophage, Carcinogenesis, Ferritin, Mesothelial Cells, Crocidolite.

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