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

Caspase-1 processes pro-IL-1 beta 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-1 beta 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-1 beta, TNF alpha, 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-1 beta and less suppression of TNF alpha, IL-6 and IL-8, while pan-caspase inhibitor, z-VAD-fmk, only weakly suppressed Il-1 beta 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-1 beta response. In contrast pretreatment with inhibitors prior to LPS stimulation is required to achieve marked decreases in IL-1 beta production. Thereby also demonstrating IL-1 beta release by cells in whole blood culture stimulated with LPS is a rapid response. Thus studying inflammasome/caspase-1/IL-1 beta axis requires appropriate selection and application of inhibitors. (C) 2014 Published by Elsevier B.V.