Abstract 1319: Monitoring inflammasome activation with a bioluminescent caspase-1 assay

Inflammatory responses and immune modulation play important and complex roles in cancer development and therapy, but methods to monitor caspase-1 activity associated with inflammasome activation have been limited. Inflammasomes are protein complexes induced by diverse inflammatory stimuli. Caspase-1, an essential component of the inflammasome, is activated in response to these stimuli, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. Western blots and ELISA are the primary, but indirect, methods for monitoring caspase-1 activity currently in use. To simplify and provide a more direct means of detecting cell-based caspase-1 activity, we developed a sensitive, homogeneous, plate-based assay that eliminates the need for significant sample processing. The assay employs a single-step, bioluminescent format combining a caspase-1 substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized reagent subsequently added to treated cells in an assay well. The coupled-enzyme system quickly reaches a steady-state between caspase cleavage of the substrate and luciferase conversion of the aminoluciferin, with light generated proportional to the amount of caspase-1 activity present in the sample. In addition to substrate selection, assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor, MG132, in the reagent and by the subsequent use of a caspase-1 inhibitor, Ac-YVAD-CHO, to confirm activity. This approach enables clear determination of caspase-1 activity even in the context of apoptotic cells. Studies with Casp1 −/− cells further demonstrate the effectiveness of this assay system to specifically detect cell-based, caspase-1 activity. Using this novel assay system, caspase-1 activation has been quantitatively determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, monosodium urate crystals, R-848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages from mice, as well as in human primary monocytes. Of note, caspase-1 activity can be monitored either directly in cells or released into the culture medium following cell treatment with various inflammatory stimuli. Monitoring released caspase-1 activity in supernatants is fast, sensitive, and nondestructive, thereby enabling subsequent multiplexing of the biological sample with other assays to monitor additional cell parameters, such as IL-1β release or cell death. Therefore, this assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators. Citation Format: Martha O9Brien, Danielle Moehring, Raul Munoz-Planillo, Gabriel Nunez, Justin Callaway, Jenny Ting, Mike Scurria, Tim Ugo, Laurent Bernad, James Cali, Dan Lazar. Monitoring inflammasome activation with a bioluminescent caspase-1 assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1319. doi:10.1158/1538-7445.AM2015-1319