Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart transplantation
Wenjun Li, … , Kory J. Lavine, Daniel Kreisel
Wenjun Li, … , Kory J. Lavine, Daniel Kreisel
Published February 26, 2019
Citation Information: J Clin Invest. 2019;129(6):2293-2304. https://doi.org/10.1172/JCI126428.
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Research Article Inflammation

Ferroptotic cell death and TLR4/Trif signaling initiate neutrophil recruitment after heart transplantation

Abstract

Nonapoptotic forms of cell death can trigger sterile inflammation through the release of damage-associated molecular patterns, which are recognized by innate immune receptors. However, despite years of investigation, the mechanisms that initiate inflammatory responses after heart transplantation remain elusive. Here, we demonstrate that ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, decreases the levels of pro-ferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamine, reduces cardiomyocyte cell death, and blocks neutrophil recruitment following heart transplantation. Inhibition of necroptosis had no effect on neutrophil trafficking in cardiac grafts. We extend these observations to a model of coronary artery ligation–induced myocardial ischemia reperfusion injury (IRI), in which inhibition of ferroptosis resulted in reduced infarct size, improved left ventricular (LV) systolic function, and reduced LV remodeling. Using intravital imaging of cardiac transplants, we show that ferroptosis orchestrates neutrophil recruitment to injured myocardium by promoting adhesion of neutrophils to coronary vascular endothelial cells through a TLR4/Trif/type I IFN signaling pathway. Thus, we have discovered that inflammatory responses after cardiac transplantation are initiated through ferroptotic cell death and TLR4/Trif-dependent signaling in graft endothelial cells. These findings provide a platform for the development of therapeutic strategies for heart transplant recipients and patients who are vulnerable to IRI following restoration of coronary blood flow.

Authors

Wenjun Li, Guoshuai Feng, Jason M. Gauthier, Inessa Lokshina, Ryuji Higashikubo, Sarah Evans, Xinping Liu, Adil Hassan, Satona Tanaka, Markus Cicka, Hsi-Min Hsiao, Daniel Ruiz-Perez, Andrea Bredemeyer, Richard W. Gross, Douglas L. Mann, Yulia Y. Tyurina, Andrew E. Gelman, Valerian E. Kagan, Andreas Linkermann, Kory J. Lavine, Daniel Kreisel

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Figure 2

Ferroptosis regulates cardiomyocyte cell death and LV remodeling following myocardial infarction.

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Ferroptosis regulates cardiomyocyte cell death and LV remodeling followi...
(A) Measurement of total CK activity in control hearts and hearts subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion using Langendorff preparations. Mice were treated with either vehicle control or Fer-1 two hours before harvesting. n = 5 per group. (B) Serial measurement of CK activity following reperfusion in control hearts and vehicle- and Fer-1–treated hearts subjected to IRI. n = 5 per group. *P < 0.05 versus control; **P < 0.05 versus other groups, by 2-sided Mann-Whitney U test. (C) Arachidonic acid metabolites measured by LC-MS/MS in control hearts and vehicle- and Fer-1–treated hearts subjected to IRI. Data are displayed as box-and-whisker plots. Lines indicate the mean value. *P < 0.05 versus control; **P < 0.05 versus other groups; #P < 0.05 versus the vehicle IRI group. (D) TTC staining and measurement of infarct area in hearts obtained from vehicle- and Fer-1–treated mice 48 hours following 90 minutes of IRI. *P < 0.05 compared with vehicle control. Original magnification, ×10. (E) Ly6G immunostaining and quantification 48 hours following 90 minutes of IRI in vehicle- and Fer-1–treated hearts. Blue indicates DAPI staining. Original magnification, ×200. *P < 0.05 versus vehicle control. (F) Echocardiography of vehicle- and Fer-1–treated mice 4 weeks following 90 minutes of IRI. Yellow dashed line indicates the akinetic area. (G–I) Quantification of LV ejection fraction, LV diastolic and systolic volumes, and akinetic area in vehicle- and Fer-1–treated mice 4 weeks after IRI. The akinetic area is expressed as a percentage of the LV area. *P < 0.05 versus vehicle control, by Mann-Whitney U test. (J) Picrosirius red staining (red) and infarct size quantification 4 weeks following 90 minutes of IRI in vehicle- and Fer-1–treated hearts. Lines represent the mean. *P < 0.05, by 2-sided Mann-Whitney U test.