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Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages
Published on: May 21, 2018
Transient Receptor Potential Melastatin 2 Negatively Regulates LPS-ATP-Induced Caspase-1-Dependent Pyroptosis of Bone
Haihong Wang1, Xinyi Zhou2, Hui Li3
1Department of Anesthesiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou 310016, China.
Background:
Pyroptosis, a new form of cell death, which has special morphological characteristics, depends on caspase-1 activation and occupies an important role in inflammatory immune diseases and ischemia-reperfusion injury. ROS is a common activator of NLR/caspase-1. Transient receptor potential melastatin 2 (TRPM2), a selective cation channel, is involved in inflammatory regulation. This study was designed to explore the role of TRPM2 in activating caspase-1 and caspase-1-dependent pyroptosis of mouse BMDMs.
Methods:
BMDMs isolated from WT and TRPM2-/- mice were treated with LPS and ATP, along with ROS inhibitor (NAC and DPI), caspase-1 inhibitor (Z-YVAD), or not. The activation of caspase-1 was measured by western blot. EtBr and EthD-2 staining were used to assess the incidence of pyroptosis.
Results:
Compared with WT, the activated caspase-1-P10 was higher and the percentage of EtBr positive cells was also increased in TRPM2-/- group, which were both inhibited by Z-YVAD, NAC, or DPI. ASC oligomerization was increased in TRPM2-/- group.
Conclusion:
Deletion of TRPM2 can enhance the activation of caspase-1 and pyroptosis, which may be via modulating ROS production, suggesting that TRPM2 plays a critical role in immune adjustment.
Insights
Transient receptor potential melastatin 2 (TRPM2) deletion enhances caspase-1 activation and pyroptosis in mouse macrophages. This suggests TRPM2 plays a key role in regulating immune responses, potentially by modulating reactive oxygen species (ROS) production.
Area of Science:
- Immunology
- Cell Biology
- Molecular Medicine
Background:
- Pyroptosis is a caspase-1 dependent inflammatory cell death pathway implicated in immune diseases and injury.
- Reactive oxygen species (ROS) commonly activate the NLR/caspase-1 inflammasome.
- Transient receptor potential melastatin 2 (TRPM2) channels are involved in inflammatory regulation.
Purpose of the Study:
- To investigate the role of TRPM2 in activating caspase-1.
- To determine TRPM2's involvement in caspase-1-dependent pyroptosis in mouse bone marrow-derived macrophages (BMDMs).
Main Methods:
- BMDMs from wild-type (WT) and TRPM2 knockout (TRPM2-/-) mice were utilized.
- Cells were treated with LPS, ATP, ROS inhibitors (NAC, DPI), or a caspase-1 inhibitor (Z-YVAD).
- Caspase-1 activation was assessed via western blot, and pyroptosis incidence was measured using EtBr and EthD-2 staining.
Main Results:
- TRPM2-/- BMDMs exhibited increased caspase-1 activation (caspase-1-P10) and pyroptosis compared to WT.
- These effects were significantly inhibited by Z-YVAD, NAC, and DPI.
- ASC oligomerization was also elevated in TRPM2-/- BMDMs.
Conclusions:
- TRPM2 deletion enhances caspase-1 activation and pyroptosis.
- This enhancement may occur through modulation of ROS production.
- TRPM2 plays a critical role in immune system regulation.
