Immunomodulation of the NLRP3 Inflammasome through Structure-Based Activator Design and Functional Regulation via Lysosomal Rupture

[Image: see text] The NLRP3 inflammasome plays a role in the inflammatory response to vaccines, in antimicrobial host defense, and in autoimmune diseases. However, its mechanism of action remains incompletely understood. NLRP3 has been shown to be activated by diverse stimuli including microbial toxins, ATP, particulate matter, etc. that activate multiple cellular processes. There have been two major challenges in translating inflammasome activators into controlled adjuvants. Both stem from their chemical and structural diversity. First, it is difficult to identify a minimum requirement for inflammasome activation. Second, no current activator can be tuned to generate a desired degree of activation. Thus, in order to design such immunomodulatory biomaterials, we developed a new tunable lysosomal rupture probe that leads to significant differences in inflammasome activation owing to structural changes as small as a single amino acid. Using these probes, we conduct experiments that suggest that rupturing lysosomes is a critical, initial step necessary to activate an inflammasome and that it precedes other pathways of activation. We demonstrate that each molecule differentially activates the inflammasome based solely on their degree of lysosomal rupture. We have employed this understanding of chemical control in structure-based design of immunomodulatory NLRP3 agonists on a semipredictive basis. This information may guide therapeutic interventions to prevent or mitigate lysosomal rupture and will also provide a predictive framework for dosable activation of the NLRP3 inflammasome for potential applications in vaccines and immunotherapies.