Characterization of an NLRP1 Inflammasome from Zebrafish Reveals a Unique Sequential Activation Mechanism Underlying Inflammatory Caspases in Ancient Vertebrates

Abstract

NLRP1 inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, the existence of this inflammasome in nonmammalian species remains poorly understood. In this study, we report the molecular and functional identification of an NLRP1 homolog, <i>Danio rerio</i> NLRP1 (<i>Dr</i>NLRP1) from a zebrafish (<i>D. rerio</i>) model. This <i>Dr</i>NLRP1 possesses similar structural architecture to mammalian NLRP1s. It can trigger the formation of a classical inflammasome for the activation of zebrafish inflammatory caspases (<i>D. rerio</i> Caspase [<i>Dr</i>Caspase]-A and <i>Dr</i>Caspase-B) and maturation of <i>D. rerio</i> IL-1β in a <i>D. rerio</i> ASC (<i>Dr</i>ASC)-dependent manner. In this process, <i>Dr</i>NLRP1 promotes the aggregation of <i>Dr</i>ASC into a filament with <i>Dr</i>ASC^CARD core and <i>Dr</i>ASC^PYD cluster. The assembly of <i>Dr</i>NLRP1 inflammasome depends on the CARD-CARD homotypic interaction between <i>Dr</i>NLRP1 and <i>Dr</i>ASC^CARD core, and PYD-PYD interaction between <i>Dr</i>Caspase-A/B and <i>Dr</i>ASC^PYD cluster. The FIIND domain in <i>Dr</i>NLRP1 is necessary for inflammasome assembly. To understand the mechanism of how the two <i>Dr</i>Caspases are coordinated in <i>Dr</i>NLRP1 inflammasome, we propose a two-step sequential activation model. In this model, the recruitment and activation of <i>Dr</i>Caspase-A/B in the inflammasome is shown in an alternate manner, with a preference for <i>Dr</i>Caspase-A followed by a subsequent selection for <i>Dr</i>Caspase-B. By using morpholino oligonucleotide-based knockdown assays, the <i>Dr</i>NLRP1 inflammasome was verified to play important functional roles in antibacterial innate immunity in vivo. These observations demonstrate that the NLRP1 inflammasome originated as early as in teleost fish. This finding not only gives insights into the evolutionary history of inflammasomes but also provides a favorable animal model for the study of NLRP1 inflammasome-mediated immunology and diseases.