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Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages
Published on: May 21, 2018
OLT1177 (Dapansutrile) inhibits Gasdermin D-dependent IL-1β Release and Pyroptotic Cell Death in Bone Marrow-derived
Karl A Pankratz1, Masoom Raza1, Jarren Ypil1
1University of Colorado Anschutz Medical Campus, Aurora, CO 80045.
Abstract:
Gasdermins are a family of pore-forming proteins that regulate the release of pro-inflammatory cytokine, interleukin-1β (IL-1β) from infected or PAMP-stimulated cells. During infection or injury, IL-1β is released by both human and mouse macrophages. IL-1β release from mouse macrophages is associated with cell death, often termed "pyroptosis". Mouse macrophages undergoing pyroptosis assemble an exit channel termed gasdermin D (GSDMD). Both the processing of IL-1β and the formation of the exit channel are caspase-1 dependent. Here, in bacterial endotoxin, lipopolysaccharide (LPS), treated mouse bone marrow-derived macrophages (BMDMs), we studied the pharmacologic inhibition of the intracellular nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome by OLT1177. BMDMs stimulated with LPS plus the potassium efflux inducer nigericin triggered the formation of the NLRP3 inflammasome. Treatment of these BMDMs with OLT1177 suppressed cell death by 42% and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)-speck formation by approximately 60%. In addition, OLT1177 dose-dependently inhibited IL-1β, CCL3, and myeloperoxidase (MPO) secretion and the pore-forming (GSDMD) from LPS-primed BMDMs, suggesting the existence of a vicious cycle controlled by IL-1β release. Overall, our study demonstrates that OLT1177 prevents IL-1β release from BMDMs by inhibiting caspase-1 and the conversion of (GSDMD) into its active N-terminal fragment (GSDMD-N). This study thus supports the concept that orally administered OLT1177 can be used to prevent local as well as systemic inflammation in humans.
Insights
The drug OLT1177 inhibits the NLRP3 inflammasome, preventing gasdermin D (GSDMD) activation and pro-inflammatory cytokine release. This demonstrates OLT1177
Area of Science:
- Immunology and Molecular Biology
- Inflammasome signaling pathways
- Cell death mechanisms
Background:
- Gasdermins are pore-forming proteins crucial for releasing pro-inflammatory cytokines like IL-1β.
- IL-1β release from macrophages is linked to pyroptosis, a form of programmed cell death.
- The NLRP3 inflammasome and caspase-1 activation are key regulators of IL-1β processing and pyroptosis.
Purpose of the Study:
- To investigate the pharmacologic inhibition of the NLRP3 inflammasome using OLT1177 in macrophages.
- To determine OLT1177's effect on IL-1β release, pyroptosis, and gasdermin D (GSDMD) processing.
Main Methods:
- Mouse bone marrow-derived macrophages (BMDMs) were stimulated with lipopolysaccharide (LPS) and nigericin.
- The NLRP3 inflammasome activation was induced, and its inhibition by OLT1177 was assessed.
- Measurements included cell death, ASC-speck formation, cytokine secretion (IL-1β, CCL3), and GSDMD cleavage.
Main Results:
- OLT1177 treatment suppressed LPS-induced cell death by 42% and ASC-speck formation by approximately 60%.
- OLT1177 dose-dependently inhibited IL-1β, CCL3, and myeloperoxidase (MPO) secretion.
- OLT1177 inhibited the conversion of GSDMD into its active N-terminal fragment (GSDMD-N), thereby blocking IL-1β release.
Conclusions:
- OLT1177 effectively prevents IL-1β release by inhibiting caspase-1 and GSDMD activation.
- The findings suggest a self-amplifying inflammatory loop involving IL-1β release that OLT1177 can disrupt.
- Orally administered OLT1177 holds potential for treating local and systemic inflammation in humans.
