A novel reporter to visualize and quantify endogenous inflammasomes and caspase-1 recruitment

Abstract Inflammasomes are signaling complexes that coordinate inflammation by inducing rapid cytokine secretion and that protect against invading pathogens by initiating death of infected cells. Yet, current methods do not allow us to specifically monitor inflammasome activation in real-time at endogenous protein levels. To overcome this shortcoming, we developed and characterized a novel fluorescent reporter that visualizes inflammasome assembly and reports on the recruitment of the effector protein caspase-1 without impairing downstream signaling. The reporter permits the analysis of inflammasome assembly over time as well as precise quantification of cells with assembled inflammasomes. We have successfully applied the reporter in lentivirus-transduced human and murine cell lines and primary cells but have also applied recombinant viruses that encode the reporter in their genome to detect inflammasome responses in primary cells. Mouse intestinal enteroids expressing the reporter allowed us to visualize how Salmonella infection triggers NAIP/NLRC4 inflammasome assembly, cell death, and expulsion in complex tissues. Lastly, we apply the new tool to gain mechanistic insights and prove that caspase-1 activation on inflammasomes relies on the assembly of filaments of caspase-1CARD, which can be terminated by the CARD-only protein CARD17 to shut down cytokine secretion. We anticipate broad application of the reporter in fundamental and applied research, as it permits the quantitative assessment of inflammasome assembly at both high temporal and spatial resolution or in high throughput. Competing Interest Statement F.I. Schmidt is cofounder and consultant of Odyssey Therapeutics. The other authors declare no competing interests.