A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly

Abstract

Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro–caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASCCARD interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASCPYD filaments for the first time. These data revealed that cross-linking of ASCPYD filaments via ASCCARD mediates the assembly of ASC foci.