Poxvirus dsDNA genomes differentially activate AIM2 or NLRP3 inflammasomes in human primary cells

The innate immune system is known for its ability to recognize cytosolic DNA as evidence of infection, but detailed studies of this process have been mostly limited to mice and cell lines. To investigate inflammasome responses in human primary cells, we used engineered viruses encoding the inflammasome reporter caspase-1^CARD-EGFP. We show that released genomes of vaccinia virus and monkeypox virus trigger robust inflammasome assembly in human primary cells. To determine the involved inflammasome sensors, we generated nanobodies against AIM2. Three of them inhibit AIM2 inflammasome assembly by blocking the polymerization of the AIM2 Pyrin domain, most potently as bivalent nanobodies. Utilizing an engineered vaccinia virus expressing bivalent AIM2 nanobodies, we demonstrate that inflammasomes in primary human macrophages and keratinocytes are nucleated by AIM2, while CD14⁺ monocytes assemble NLRP3 inflammasomes. This finding resolves the discrepancy between the previously reported activation of AIM2 inflammasomes in mice and NLRP3 inflammasomes in humans, and provides the first evidence for cell-type-specific regulation of DNA-triggered inflammasome activation. The newly developed AIM2-specific nanobodies offer a precise tool to dissect and potentially target AIM2 inflammasome assembly in other disease contexts.