Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1 mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. 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.
Caspase-1, an essential component of the inflammasome, is activated in response to inflammatory stimuli, resulting in 1) the processing and release of IL-1b and IL-18, and 2) pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized reagent added directly to cultured cells. The assay can also measure caspase-1 activity released into the medium. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor, MG-132, in the reagent and by use of a caspase-1 inhibitor, Ac-YVAD-CHO, to confirm activity. Using this assay system, caspase-1 activity has been monitored in THP-1 cells, J774A.1 mouse macrophages, mouse bone marrow-derived macrophages (BMDMs), and human primary monocytes treated with known inflammasome inducers. Caspase-1 activity was not detected in BMDMs derived from Casp 1−/−mice. Measuring caspase-1 activity released into medium enables multiplexing of the sample to monitor additional cell parameters. Multiplexing to monitor caspase-1 activity, cell death, and IL-1b release revealed that unprimed, undifferentiated THP-1 cells stimulated with nigericin or a-hemolysin activate caspase-1 and undergo pyroptosis, but do not release any IL-1b. This suggests that K+ efflux alone can engage the inflammasome and activate caspase-1 in THP-1 cells, providing an interesting system to assess pyroptosis in the absence of IL-1b. This convenient caspase-1 assay will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators.
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 O'Brien, Danielle Moehring, Raúl Muñoz-Planillo, Gabriel Núñez, 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
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