The zebrafish NLRP3 inflammasome has functional roles in ASC-dependent interleukin-1β maturation and gasdermin E–mediated pyroptosis

The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog ( Dr NLRP3) in a zebrafish ( Danio rerio ) model. We found that Dr NLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase ( Dr Caspase-A/-B) activation and interleukin 1β ( Dr IL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which Dr NLRP3 organizes Dr ASC into a filament that recruits Dr Caspase-A/-B by homotypic pyrin domain (PYD)–PYD interactions. Dr Caspase-A/-B activation in the Dr NLRP3 inflammasome occurred in two steps, with Dr Caspase-A being activated first and Dr Caspase-B second. Dr NLRP3 also directly activated full-length Dr Caspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by Dr NLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down Dr NLRP3 in zebrafish embryos and generating a Dr ASC-knockout ( Dr ASC −/− ) fish clone, we characterized the function of the Dr NLRP3 inflammasome in anti-bacterial immunity in vivo . The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis.