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 Transplantation

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 1

Ferroptosis regulates cell death and promotes neutrophil recruitment in injured hearts.

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Ferroptosis regulates cell death and promotes neutrophil recruitment in ...
Intravital 2-photon imaging of neutrophil (green) behavior in (A) control WT cardiac grafts, (B) after treatment of recipient mice with Nec-1, (C) in RIPK3-deficient donor hearts, and (D) after administration of Fer-1 to heart recipients. Vessels were labeled red after injection of quantum dots. n = 4 per experimental group. Scale bars: 30 μm (A–D). (E) Intravascular rolling velocities of neutrophils, (F) density of neutrophils, and (G) percentage of extravasated neutrophils in the experimental conditions displayed in A–D. Data in E–G represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA followed by post hoc Tukey’s multiple comparisons test. (H) Neutrophil recruitment per minute to coronary veins in control cardiac grafts and after treatment of recipient mice with Fer-1. (I) Cardiomyocyte death determined by ethidium bromide in cardiac grafts after treatment of recipient mice with vehicle or Fer-1. n = 4 per experimental group. Original magnification, ×200. (J) Flow cytometric assessment of death (DAPI+) of fibroblasts and endothelial cells in B6 cardiac grafts after transplantation into vehicle- or Fer-1–treated syngeneic recipient mice. n = 6 per experimental group. (K) LC-MS/MS assessment of pro-ferroptotic PE molecular species. MS spectrum of PE obtained from WT mice. Inset: MS spectrum in the range of m/z from 798.49 to 798.58. The content of PE(38:4)+OO molecular species (HOO-AA-PE, ferroptotic cell death signal) in cardiac grafts after treatment of recipient mice with vehicle or Fer-1. Data in H–K represent the mean ± SEM. *P < 0.05, by 2-sided Mann-Whitney U test.