Dear Editor,
Doxorubicin is an anthracycline antibiotic, which is used in treatment of cancer and it works by induction of apoptosis in cancerous cells.1 In vivo, apoptotic cells are rapidly cleared preferentially by monocytes and neutrophil migration is inhibited2 to limit tissue injury and inflammation. More recently, few anti-cancer drugs, including anthracyclines (doxorubicin) were shown to evoke apoptosis in cancerous cells, which is associated with immune system activation (i.e. immunogenic apoptosis).3, 4 In our previous study we reported that doxorubicin can induce acute inflammation in peritoneum, which is associated with apoptosis.5 Doxorubicin-killed cells could be also a source of damage-associated molecular patterns (DAMPs) leading to inflammation and tumor necrosis factor (TNF) production, which would amplify the inflammatory response triggered by DAMPs. TNF is a pleiotropic cytokine that binds to receptors TNF-R1 and TNF-R2 and, depending on cell type, triggers different signaling pathways, including cell death and inflammation.6 This study aimed to examine whether TNF contributes to the doxorubicin-induced acute sterile inflammation.
Intraperitoneal injection of doxorubicin provoked an acute inflammatory response accompanied by the influx of neutrophils.5 We observed an increased level of LDH, a marker of tissue damage and secondary necrotic cells, already after 6 h with further increase after 16 h post injection of doxorubicin (Supplementary Figure 1a). Lavage fluid collected 16 h after doxorubicin injection contained increased levels of TNF as compared to vehicle-injected group (0.09±0.14 versus 2.11±0.84 pg/ml, P<0.0001).
We next tested the involvement of TNF-R1 and TNF-R2 in the sterile inflammation in response to doxorubicin. Wild-type mice had abundant neutrophils in their abdominal cavities, but this response was markedly decreased in TNF-R1/2 double knockout mice (by 2.4-fold, Supplementary Figure 1b). The number of neutrophils attracted in response to doxorubicin-induced acute inflammation was 3.4-fold lower in TNF-R1-deficient mice than in controls (Figure 1a), but in TNF-R2-deficient mice, there was no difference as compared to wild-type mice (Figure 1b). These results show that TNF-R1 was involved in mediating the inflammatory response but TNF-R2 was not. Importantly, cell death in TNF-R1/2 double knockout mice was not affected in comparison to wild-type mice (data not shown) indicating that TNF does not contribute to the cell death process itself.
Further, we investigated whether acute inflammation can be reduced in wild-type mice by the administration of Etanercept, a recombinant dimeric soluble form of TNF-R2 that blocks the interaction of TNF with its cell surface receptors.7 Wild-type mice were injected intraperitoneally with Etanercept together with doxorubicin and 16 h later the recruited cells were phenotyped. Etanercept significantly reduced the recruitment of neutrophils (Figure 1c). This further confirms the importance of TNF in doxorubicin-induced acute inflammation.
In conclusion, we report that intraperitoneal injection of doxorubicin in mice leads to increase in TNF levels in the lavage fluid. It has been also reported that patients undergoing chemotherapy with doxorubicin had elevated levels of TNF in their plasma.8 Similarly, mouse in vitro and in vivo studies demonstrated up-regulation of TNF after doxorubicin administration both on mRNA and protein levels.9, 10 Previously, we found that the majority of cells that died apoptotically due to doxorubicin treatment were monocytes/macrophages with some minor neutrophils.5 Therefore, it is possible that either dying macrophages or attracted neutrophils might be a source of TNF, and that by binding to TNF-R1 it could amplify the inflammation. We demonstrated that the acute inflammatory response to doxorubicin was reduced in TNF-R1- but not TNF-R2-deficient mice. In addition, Etanercept decreases the attraction of neutrophils after doxorubicin administration. These studies show that TNF and the TNF-R1 signaling pathway are key elements in the acute sterile inflammatory response to doxorubicin.
References
Wang S et al J Biol Chem 2004; 279: 25535–25543.
Bournazou I et al J Clin Invest 2009; 119: 20–32.
Apetoh L et al Trends Mol Med 2008; 14: 141–151.
Krysko DV et al Nat Rev Cancer 2012; 12: 860–875.
Krysko DV et al Cell Death Differ 2011; 18: 1316–1325.
Balkwill F . Nat Rev Cancer 2009; 9: 361–371.
Fantuzzi F et al Expert Opin Ther Targets 2008; 12: 1085–1096.
Aluise CD et al Free Radic Biol Med 2011; 50: 1630–1638.
Niiya M et al Cancer Chemother Pharmacol 2003; 52: 391–398.
Nozaki N et al Circulation 2004; 110: 2869–2874.
Acknowledgements
We thank Dr. A Bredan for editing the manuscript. This work was supported by project grants from the Fund for Scientific Research Flanders (FWO-Vlaanderen, G.0728.10, G060713N, G0A5413N to DVK; 3G067512 to DVK and OK) and by an individual research grant from FWO-Vlaanderen (31507110 to DVK). DVK is a senior postdoctoral researcher paid by a fellowship from FWO-Vlaanderen. AK is a recipient of an Emmanuel van der Schueren scholarship from the Flemish League Against Cancer. Vandenabeele’s group is supported by VIB, Ghent University (GROUP-ID Consortium of the UGent MRP initiative), FWO-Vlaanderen (G.0875.11, G.0973.11, G.0A45.12N), Federal Research Program (IAP 7/32), PV holds a Methusalem grant (BOF09/01M00709) from the Flemish Government.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies this paper on Cell Death and Disease website
Supplementary information
Rights and permissions
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
About this article
Cite this article
Kaczmarek, A., Krysko, O., Heyndrickx, L. et al. TNF/TNF-R1 pathway is involved in doxorubicin-induced acute sterile inflammation. Cell Death Dis 4, e961 (2013). https://doi.org/10.1038/cddis.2013.496
Published:
Issue Date:
DOI: https://doi.org/10.1038/cddis.2013.496
This article is cited by
-
RGS7 balances acetylation/de-acetylation of p65 to control chemotherapy-dependent cardiac inflammation
Cellular and Molecular Life Sciences (2023)