Nanobodies dismantle post‐pyroptotic ASC specks and counteract inflammation in vivo

Inflammasomes sense intracellular clues of infection, damage, or metabolic imbalances. Activated inflammasome sensors polymerize the adaptor ASC into micron‐sized “specks” to maximize caspase‐1 activation and the maturation of IL‐1 cytokines. Caspase‐1 also drives pyroptosis, a lytic cell death characterized by leakage of intracellular content to the extracellular space. ASC specks are released among cytosolic content, and accumulate in tissues of patients with chronic inflammation. However, if extracellular ASC specks contribute to disease, or are merely inert remnants of cell death remains unknown. Here, we show that camelid‐derived nanobodies against ASC (VHH ASC ) target and disassemble post‐pyroptotic inflammasomes, neutralizing their prionoid, and inflammatory functions. Notably, pyroptosis‐driven membrane perforation and exposure of ASC specks to the extracellular environment allowed VHH ASC to target inflammasomes while preserving pre‐pyroptotic IL‐1β release, essential to host defense. Systemically administrated mouse‐specific VHH ASC attenuated inflammation and clinical gout, and antigen‐induced arthritis disease. Hence, VHH ASC neutralized post‐pyroptotic inflammasomes revealing a previously unappreciated role for these complexes in disease. VHH ASC are the first biologicals that disassemble pre‐formed inflammasomes while preserving their functions in host defense. SYNOPSIS Inflammasome activation results in a lytic cell death named Pyroptosis. Following pyroptosis, inflammasome platforms called ASC specks are released into the extracellular space, where they spread inflammation through their pro‐inflammatory and prion‐like activities. However, if extracellular ASC specks have functions in the development of disease remains ill‐defined. The present study tackles this question using ASC‐targeting nanobodies. ASC‐specific nanobodies (VHHASC) block the pro‐inflammatory and prionoid activities of extracellular ASC specks. Pyroptosis and GSDMD membrane pores allow VHHASC to target intracellular ASC specks in pyroptotic cells, while maintaining IL‐1β responses. VHHASC treatment in vivo ameliorates inflammation in MSU‐ and antigen‐induced arthritis. VHHASC reveal a role for post‐pyroptotic inflammasomes in the establishment of arthritic inflammation, and represent a new alternative for clinical treatment of inflammasome‐dependent disease. Graphical Abstract Inflammasome activation results in a lytic cell death named Pyroptosis. Following pyroptosis, in