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Updated: Jun 4, 2025
Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages
Published on: May 21, 2018
Time-of-day control of mitochondria regulates NLRP3 inflammasome activation in macrophages
James R O'Siorain1, Shannon L Cox1, Cloé Payet1
1Curtis Clock Laboratory, School of Pharmacy and Biomolecular Sciences (PBS), Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
Abstract:
Macrophages are innate immune cells that orchestrate the process of inflammation, which varies across time of day. This ensures appropriate biological timing of the immune response with the external environment. The NLRP3 inflammasome mediates IL-1-family cytokine release via pyroptosis. Mitochondria play a multifaceted role regulating NLRP3 inflammasome activity. Mitochondria exhibit distinct metabolic changes across time of day, which are influenced by clock genes. However, whether the macrophage clock regulates the NLRP3 inflammasome via mitochondrial control remains unclear. We find heightened mitochondrial membrane potential (Δψm) and enhanced NLRP3 inflammasome activation from peritoneal exudate cells (PECs) isolated at circadian time (CT) 12 compared to CT 0. In vitro time-of-day synchronization of bone-marrow derived macrophages (BMDMs) induced time-dependent differences in NLRP3 inflammasome activation. Myeloid-specific Bmal1-deletion enhanced NLRP3 inflammasome activity in PECs at CT0 and in unsynchronized BMDMs compared to controls. Pharmacologically disrupting Δψm in synchronized cells reduced NLRP3 inflammasome activation to comparable levels, and the same occurred with Bmal1-deletion. These results further demonstrate circadian clock timing of the NLRP3 inflammasome, which is dependent on mitochondrial function and driven through the circadian gene Bmal1.
Insights
The macrophage circadian clock regulates the NLRP3 inflammasome, a key inflammation pathway. This timing depends on mitochondrial function and the Bmal1 gene, impacting immune responses.
Area of Science:
- Immunology
- Chronobiology
- Cellular Biology
Background:
- Macrophages are innate immune cells controlling inflammation, with daily variations linked to environmental cues.
- The NLRP3 inflammasome triggers IL-1 cytokine release and pyroptosis, crucial inflammatory processes.
- Mitochondria influence NLRP3 inflammasome activity, exhibiting daily metabolic shifts regulated by clock genes.
Purpose of the Study:
- To investigate if the macrophage circadian clock controls NLRP3 inflammasome activity through mitochondrial regulation.
- To elucidate the role of the Bmal1 gene in this circadian control mechanism.
Main Methods:
- Comparing NLRP3 inflammasome activation and mitochondrial membrane potential (Δψm) in peritoneal exudate cells (PECs) at different circadian times (CT0 vs. CT12).
- Synchronizing bone-marrow derived macrophages (BMDMs) in vitro to assess time-dependent NLRP3 inflammasome activation.
- Analyzing the impact of myeloid-specific Bmal1 deletion on NLRP3 inflammasome activity.
- Investigating the effect of pharmacologically disrupting Δψm on inflammasome activation.
Main Results:
- Heightened mitochondrial membrane potential (Δψm) and NLRP3 inflammasome activation were observed at CT12 compared to CT0.
- In vitro synchronized BMDMs showed time-dependent differences in NLRP3 inflammasome activation.
- Myeloid-specific Bmal1 deletion increased NLRP3 inflammasome activity.
- Disrupting Δψm or deleting Bmal1 reduced NLRP3 inflammasome activation.
Conclusions:
- The circadian clock in macrophages regulates NLRP3 inflammasome activation.
- Mitochondrial function, specifically Δψm, is critical for this circadian control.
- The circadian gene Bmal1 drives this clock-dependent regulation of the NLRP3 inflammasome.
