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Mol. Cells

Published online December 20, 2021

© The Korean Society for Molecular and Cellular Biology

Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages

Yuan-Shen Chen1 , Wei-Chu Chuang2 , Hsiu-Ni Kung3 , Ching-Yuan Cheng2 , Duen-Yi Huang2 , Ponarulselvam Sekar4 , and Wan-Wan Lin2,4,5,*

1Department of Neurosurgery, National Taiwan University Hospital Yunlin Branch, Douliu 64041, Taiwan, 2Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 10617, Taiwan, 3Graduate Institute of Anatomy and Cell Biology, National Taiwan University, Taipei 10617, Taiwan, 4Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 11031, Taiwan, 5Department of Pharmacology, National Defense Medical Center, Taipei 11490, Taiwan

Correspondence to : wwllaura1119@ntu.edu.tw

Received: July 23, 2021; Revised: September 24, 2021; Accepted: October 15, 2021

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.

Abstract

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

Keywords autophagy, interferon, JAK/STAT1, macrophage, necrosis, zVAD

Article

On-line First

Mol. Cells

Published online December 20, 2021

Copyright © The Korean Society for Molecular and Cellular Biology.

Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages

Yuan-Shen Chen1 , Wei-Chu Chuang2 , Hsiu-Ni Kung3 , Ching-Yuan Cheng2 , Duen-Yi Huang2 , Ponarulselvam Sekar4 , and Wan-Wan Lin2,4,5,*

1Department of Neurosurgery, National Taiwan University Hospital Yunlin Branch, Douliu 64041, Taiwan, 2Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 10617, Taiwan, 3Graduate Institute of Anatomy and Cell Biology, National Taiwan University, Taipei 10617, Taiwan, 4Graduate Institute of Medical Sciences, Taipei Medical University, Taipei 11031, Taiwan, 5Department of Pharmacology, National Defense Medical Center, Taipei 11490, Taiwan

Correspondence to:wwllaura1119@ntu.edu.tw

Received: July 23, 2021; Revised: September 24, 2021; Accepted: October 15, 2021

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.

Abstract

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNβ, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

Keywords: autophagy, interferon, JAK/STAT1, macrophage, necrosis, zVAD

Mol. Cells
Dec 31, 2021 Vol.44 No.12, pp. 861~919
COVER PICTURE
Structure of the fly peripheral neurons in the fly head. Flies have basic sensory organs including eyes for vision, antennae and maxillary palps for olfaction, and proboscis (magenta) for gustation which can be labelled with monoclonal antibody 22C10. The figure is a 3D reconstructed image with 30 slices of confocal sections with 3 μm interval. It shows that the proboscis is required for sensing attractive carboxylic acids such as glycolic acid, citric acid, and lactic acid (Shrestha and Lee, pp. 900-910).

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