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Updated: Apr 5, 2026
Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1β in Human Monocyte-derived Dendritic Cells
Published on: May 22, 2014
Reconstituted AIM2 inflammasome in cell-free system
Naoe Kaneko1, Yuki Ito1, Tomoyuki Iwasaki1
1Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan.
Abstract:
Absent in melanoma 2 (AIM2) is an intracellular pattern-recognition receptor, which is a member of the PYHIN protein family, consisting of a PYD domain and an IFN-inducible nuclear localization (HIN) domain. AIM2 is reported to oligomerize with adaptor protein ASC upon sensing bacterial and viral cytosolic DNA in order to form the AIM2 inflammasome, which activates caspase-1 leading to IL-1β secretion. Dysregulation of AIM2 inflammasome is supposed to result in autoinflammatory and autoimmune diseases. Thus, the development of new targeted drugs against AIM2 inflammasome would be important for the treatment of these diseases. However, since AIM2 inflammasome is an intracellular receptor, enforced internalization of both ligands and candidate molecules is necessary for the screening of AIM2-inflammasome-targeted molecules. We developed a reconstituted AIM2 inflammasome in a cell-free system with amplified luminescent proximity homogeneous assay (Alpha). Strong Alpha signal was detected upon incubation with poly-deoxyadenylic-deoxythymidylic acid, poly(dA:dT), whereas no Alpha signal was detected upon incubation with muramyl dipeptide, one of the NLR ligands of Nod2 ligand. The interaction between AIM2 and ASC was disrupted by an anti-human ASC monoclonal antibody, CRID3, a class of diarylsulfonylurea-containing compounds, and glycyrrhizin, a substance found in liquorice root. Thus, the reconstituted AIM2 inflammasome in a cell-free system is useful for screening AIM2-inflammasome-targeted therapeutic molecules.
Insights
A new cell-free system using Alpha assay effectively screens for AIM2 inflammasome inhibitors. This method aids in developing targeted drugs for autoinflammatory and autoimmune diseases by assessing AIM2 inflammasome activity.
Area of Science:
- Immunology
- Molecular Biology
Background:
- Absent in melanoma 2 (AIM2) is an intracellular pattern-recognition receptor crucial for innate immunity.
- AIM2 forms inflammasomes upon sensing cytosolic DNA, activating caspase-1 and IL-1β secretion.
- Dysregulation of AIM2 inflammasome is linked to autoinflammatory and autoimmune diseases.
Purpose of the Study:
- To develop a cell-free system for screening AIM2 inflammasome-targeted therapeutic molecules.
- To overcome challenges of intracellular targeting for drug development.
Main Methods:
- Development of a reconstituted AIM2 inflammasome in a cell-free system.
- Utilized amplified luminescent proximity homogeneous assay (Alpha) for signal detection.
- Tested poly(dA:dT) as a ligand and evaluated inhibitors like CRID3 and glycyrrhizin.
Main Results:
- The cell-free system demonstrated a strong Alpha signal with the DNA ligand poly(dA:dT).
- No signal was detected with a Nod2 ligand, confirming specificity.
- The interaction between AIM2 and ASC was successfully inhibited by known compounds.
Conclusions:
- The reconstituted cell-free AIM2 inflammasome system is a viable tool for high-throughput screening.
- This platform facilitates the discovery of novel therapeutic agents targeting AIM2 inflammasome-mediated diseases.
