Development of a specific, active human IL-18 assay for assessing inflammasome activation

Abstract

Abstract Interleukin-18 (IL-18) was first identified for its ability to induce interferon-γ expression. IL-18 is a proinflammatory cytokine and its release along with IL-1β is a primary output of inflammasome activation. We have developed convenient tools for monitoring inflammasome activation, including assays for caspase-1 activity and IL-1β release. While both IL-1β and IL-18 are expressed as inactive zymogens requiring processing to active forms, they differ in expression and regulation. IL-1β requires upregulation for expression and is primarily restricted to myeloid cells, whereas IL-18 is constitutively expressed more broadly in epithelial cells, as well as myeloid cells. IL-18 is also uniquely regulated by an IL-18 binding protein (IL-18bp). The IL-18bp has a very high affinity for active IL-18 and effectively blocks receptor activation. IL-18-stimulated interferon-γ release stimulates IL-18bp release in a negative feedback loop. The Lumit™ technology is a Nanoluc®-based platform that enables direct, no wash assays for analyte detection using standard luminometers. For Lumit IL-18 assays, we either NanoBiT®-labeled two IL-18 antibodies or NanoBiT®-labeled the IL-18bp as well as one IL-18 antibody. These resulted in, respectively, one assay that measures both IL-18 and proIL-18 and another assay specific for active IL-18. The specific assay was used to measure active IL-18 release from canonical inflammasome-activated THP-1 cells and PBMCs, as well as non-canonical inflammasome-activated lung A549 epithelial cells. Enabling direct quantitation of unbound, active IL-18 is essential for elucidating IL-18’s role in inflammation and disease. A Lumit™ IL-18 assay adds to the toolbox for monitoring inflammasome activation.