Applying caspase-1 inhibitors for inflammasome assays in human whole blood

Caspase-1 processes pro-IL-1β and pro-IL-18 into bioactive forms. Caspase-1 activity is regulated by a multiprotein complex known as an inflammasome. Multiple danger and damage associated signals drive inflammasome formation. Currently, evaluation of inflammasome activity is of particular interest as its role in chronic and acute inflammatory pathologies becomes evident. Specific inhibitors are therefore required to evaluate the contributions of the inflammasome and IL-1β to these disease processes. While several inhibitors are available for caspase-1 blocking experiments, in this study we show effects of two commonly used caspase inhibitors: z-VAD-fmk and ac-YVAD-cmk on secretion of pro-inflammatory cytokines: IL-1β, TNFα, IL-8 and IL-6 in whole blood stimulated with LPS. We demonstrate ac-YVAD-cmk is a specific caspase-1 inhibitor resulting in pronounced decreases in IL-1β and less suppression of TNFα, IL-6 and IL-8, while pan-caspase inhibitor, z-VAD-fmk, only weakly suppressed Il-1β while acting strongly on the other three cytokines. Furthermore, we demonstrated that simultaneous treatment of whole blood cultures with inhibitor and LPS fails to attenuate the IL-1β response. In contrast, pretreatment with inhibitors prior to LPS stimulation is required to achieve marked decreases in IL-1β production. Thereby also demonstrating IL-1β release by cells in whole blood culture stimulated with LPS is a rapid response. Thus studying inflammasome/caspase-1/IL-1β axis requires appropriate selection and application of inhibitors.