A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells

Abstract

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.

Priming Is Dispensable for NLRP3 Inflammasome Activation in Human Monocytes In Vitro

“…While pro-IL-1b is not expressed basally, pro-IL-18 is constitutively expressed in different cell types including macrophages, monocytes, dendritic cells, astrocytes and microglia (16). There are several reports that have described caspase-1 activation and IL-18 release in the absence of a priming step and just in response to the second or activating signal, especially in human cells (17)(18)(19)(20)(21). Despite this evidence, whether this process is dependent on the NLRP3 inflammasome has not been explored.…”

<p>In vitro expression of NLRP inflammasome-induced active Caspase-1 expression in normal human epidermal keratinocytes (NHEK) by various exogenous threats and subsequent inhibition by naturally derived ingredient blends</p>

“…35 In 2017, O'Brien et al, reported on a new bioluminescence assay that directly detects the release of ACasp-1 from NLRP inflammasomes. 36 Activation of the NLRP inflammasomes can occur via various agonists such as UV energy or chemical initiators. 37 The assay has been used to examine Caspase-1 expression in skin fibroblasts and keratinocytes.…”

Zoledronic acid regulates the synthesis and secretion of IL-1β through Histone methylation in macrophages

“…Cleavage of GSDMD relieves an intramolecular autoinhibition and enables translocation of the N-terminal fragment of GSDMD to the inner leaflet of the plasma membrane to form pores. Thus, IL-1β and IL-18 are released extracellularly to stimulate inflammatory responses 8,9 . Pyroptosis has been suggested to contribute to innate immunity against microbial infection 10 , it is also involved in the pathogenesis of endotoxin shock, acquired immune deficiency syndrome and other diseases 11 .…”