Towards the optimization of a NLRP3 inflammasome model system in J774A.1 murine macrophages and THP-1 human monocytes

Abstract

The NLRP3 inflammasome is a cytosolic, multimeric protein complex, that detects and responds to modulations in tissue homeostasis disruptions such as pathogen detection, tissue damage, and environmental stressors. Dysregulated NLRP3 inflammasome activity has been linked to various autoinflammatory and autoimmune diseases including diabetes and Alzheimer’s disease, rendering the inflammasome an attractive potential therapeutic target. However, NLRP3 activation mechanisms are complex and can be induced by a multitude of stimulatory conditions, leading to challenges with developing a reliable model system. In this study, we aimed to optimize appropriate conditions to detect NLRP3 inflammasome activation in both J774A.1 murine macrophages and THP-1 human monocytes. In J774A.1 cells, we detected upregulated expression of pro-IL-1β following LPS-treatment, indicative of NLRP3 inflammasome priming. Following nigericin stimulation, we observed diminished pro-IL-1β levels and no mature IL-1β. We were able to differentiate THP-1 monocytic cells into macrophage-like phenotypes using 500nM PMA treatment for 24 hours. Following nigericin treatment, ASC levels decreased in LPS-undifferentiated, and increased in PMA-differentiated THP-1 cells relative to their respective untreated controls. We observed nigericin-induced, duration-dependent increases in cell death for both THP-1 phenotypes using a cell viability assay. In conclusion, we confirmed prior findings regarding J774A.1 NLRP3 inflammasome signaling and initiated the optimization of NLRP3 inflammasome activation in THP-1 cells. Further enhancements are required to conclusively identify NLRP3 activation and optimize a reliable cell line model system.