TNF/TNF-R1 pathway is involved in doxorubicin-induced acute sterile inflammation

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Citation: Cell Death and Disease (2013) 4,e961; doi:10.1038/cddis.2013.496

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TNF/TNF-R1 pathway is involved in doxorubicin-induced acute sterile inammation

A Kaczmarek1,2, O Krysko3, L Heyndrickx1,2, T Lve Aaes1,2, T Delvaeye1,2,4, C Bachert3, L Leybaert4, P Vandenabeele*,1,2,5

and DV Krysko*,1,2,5

Cell Death and Disease (2013) 4, e961; doi:http://dx.doi.org/10.1038/cddis.2013.496

Web End =10.1038/cddis.2013.496 ; published online 12 December 2013 Subject Category: Immunity

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 inammation. 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 inammation in peritoneum, which is associated with apoptosis.5 Doxorubicin-killed cells could be also a source of damage-associated molecular patterns (DAMPs) leading to inammation and tumor necrosis factor (TNF) production, which would amplify the inammatory 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 inammation.6 This study aimed to examine whether TNF contributes to the doxorubicin-induced acute sterile inammation.

Intraperitoneal injection of doxorubicin provoked an acute inammatory response accompanied by the inux 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 uid collected 16 h after doxorubicin injection contained increased levels of TNF as compared to vehicle-injected group (0.090.14 versus2.110.84 pg/ml, Po0.0001).

We next tested the involvement of TNF-R1 and TNF-R2 in

the sterile inammation 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 inammation was 3.4-fold lower in TNF-R1-decient mice than in controls (Figure 1a), but in TNF-R2-decient mice, there was no difference as compared

to wild-type mice (Figure 1b). These results show that TNF-R1 was involved in mediating the inammatory 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.

0 Veh Dox Veh Dox

1.6106

1.6106

**

ns

n=5

1.4106

1.4106

Number of Neutrophils

1.2106

1.2106

1.0106

1.0106

8.0105

Number of Neutrophils

8.0105

6.0105

6.0105

4.0105

4.0105

2.0105

2.0105

n=8

n=8

n=6

n=5

n=5

n=7

n=9

0 Veh Dox Veh Dox

WT

TNFR1-/- TNFR2-/-

WT

1.6106

**

1.4106

Number of Neutrophils

1.2106

1.0106

8.0105

6.0105

4.0105

2.0105

n=5 n=6

n=7

n=7

0 Veh Veh+E Dox Dox+E

Figure 1 Role of TNF-R1 and TNF-R2 and the effect of inhibiting soluble TNF on doxorubicin-induced acute inammatory response. The number of neutrophils in peritoneal lavage was decreased in TNF-R1 / (a) but not in TNF-R2 / (b).Treatment of wild-type mice with Etanercept signicantly reduced the recruitment of neutrophils 16 h after intraperitoneal injection of doxorubicin (Dox E) (c). Negative

controls were injected with vehicle alone or with vehicle plus Enanercept (Veh E).

Means and S.E.M. are for pooled data, and n is the number of mice in each group. **Po0.002. MannWhitney test was used to evaluate differences between groups (GraphPad Prism-5). The number of monocytes/macrophages and neutrophils in the peritoneal exudate cells was determined as described previously.5

1Inammation Research Center, VIB, Ghent, Belgium; 2Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium; 3Upper Airway Research Laboratory, Department of Oto-Rhino-Laryngology, Ghent University Hospital, Ghent, Belgium and 4Department of Basic Medical Sciences, Physiology Group, Ghent University, Ghent, Belgium*Corresponding authors: P Vandenabeele or DV Krysko, Inammation Research Center, VIB-Ghent University, Technologiepark 927 B-9052, Ghent (Zwijnaarde), Belgium. Tel: [phone] (PV) or [phone] (DVK); Fax: [phone]; E-mail: mailto:[email protected]

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5These authors contributed equally to the work.

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2

Further, we investigated whether acute inammation 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 signicantly reduced the recruitment of neutrophils (Figure 1c). This further conrms the importance of TNF in doxorubicin-induced acute inammation.

In conclusion, we report that intraperitoneal injection of doxorubicin in mice leads to increase in TNF levels in the lavage uid. 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 inammation. We demonstrated that the acute inammatory response to doxorubicin was reduced in TNF-R1- but not TNF-R2-decient 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 inammatory response to doxorubicin.

Conict of InterestThe authors declare no conict of interest.

Acknowledgements. We thank Dr. A Bredan for editing the manuscript. This work was supported by project grants from the Fund for Scientic Research Flanders (FWO-Vlaanderen, G.0728.10, G060713N, G0A5413N to DVK; 3G067512 to DVK and OK) and by an individual research grant from FWOVlaanderen (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. Vandenabeeles 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.

1. Wang S et al. J Biol Chem 2004; 279: 2553525543.2. Bournazou I et al. J Clin Invest 2009; 119: 2032.3. Apetoh L et al. Trends Mol Med 2008; 14: 141151.4. Krysko DV et al. Nat Rev Cancer 2012; 12: 860875.5. Krysko DV et al. Cell Death Differ 2011; 18: 13161325.6. Balkwill F. Nat Rev Cancer 2009; 9: 361371.7. Fantuzzi F et al. Expert Opin Ther Targets 2008; 12: 10851096.8. Aluise CD et al. Free Radic Biol Med 2011; 50: 16301638.9. Niiya M et al. Cancer Chemother Pharmacol 2003; 52: 391398.10. Nozaki N et al. Circulation 2004; 110: 28692874.

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