| The innate immune system is many organisms first line of defense against pathogenic insult or tissue damage. This defense strategy is intent on restoring homeostasis upon perturbation. Upon activation of the innate immune system in humans, an oligomeric protein complex termed the "Inflammasome" forms in myeloid cells. The canonical output of activation of any subset of inflammasome is Caspase-1-mediated secretion of pro-inflammatory cytokines IL1beta and IL18. Chronic or uncontrolled inflammasome activation is at the core of myriad economically burdening diseases. In many of these diseases, endogenous factors chronically engage the innate immune system. To study these diseases in in vivo, over time as they progress, in a transgenic organism will segue into the development of next generation therapeutics. In this study we sought to utilize a modified Firefly Luciferase (pGLO) construct to measure Caspase-1, and thus, inflammasome activity. pGLO has been engineered to be catalytically activated in response to targeted cleavage by a protease. We report that insertion of a short peptide sequence targeted by Caspase-1 into the linker region of a circularly permuted Firefly Luciferase (pGLO) has allowed us to quantitatively assess the level of inflammasome activation in live cells. A successful in vitro experiment demonstrating sensitive and specific quantification of Caspase-1 activity should lead to an in vivo mouse model for the study of inflammatory disease. |
