AIM2-Inhibitory Nanobodies Reveal Differential Activation of AIM2 and NLRP3 Inflammasomes by Poxvirus DNA Genomes in Primary Human Cells

Abstract

Poxviruses are large double-stranded (ds)DNA viruses that replicate in the cytoplasm. While variola virus (VARV) caused millions of deaths before its eradication in 1980, monkeypox virus (MPXV) has been declared a public health emergency of international concern with over 100,000 reported cases. Inflammasomes are large macromolecular complexes that coordinate the activation of caspase-1, maturation of pro-inflammatory cytokines, and pyroptotic cell death. Poxviruses and cytosolic dsDNA have been reported to activate the AIM2 inflammasome in mice and the NLRP3 inflammasome in human monocytes. However, the inflammasome responses to poxvirus infections have not been adequately addressed in human primary cells. <br /> To study the inflammasome response to poxviruses, we used the model poxvirus vaccinia virus (VACV) and MPXV. We employed the inflammasome reporter caspase-1-CARD-EGFP encoded by VACV to quantify inflammasome activation in primary human macrophages, CD14 monocytes, and normal human epidermal keratinocytes (NHEKs). VACV infection triggered robust inflammasome activation in IFN-γ treated primary macrophages and keratinocytes, AIM2 activation in IFN-γ treated THP-1 macrophages, and NLRP3 activation in primary CD14 monocytes. To investigate the role of AIM2 in primary cells, we generated and characterized nanobodies targeting the AIM2 pyrin domain (PYD) and identified three AIM2-inhibitory nanobodies. We solved the crystal structure of the inhibitory nanobodies in complex with AIM2 PYD and revealed that they inhibit AIM2 activation by preventing AIM2 PYD polymerization. Exploiting nanobodies with different binding epitopes, we developed bivalent nanobodies that completely block AIM2 inflammasome activation. Using VACV as both a vector and a trigger, we engineered recombinant VACV encoding AIM2-inhibitory bivalent nanobodies to express nanobodies in primary cells. We revealed that VACV infection activates the AIM2 inflammasome in primary human macrophages and keratinocytes, asserting AIM2 as a relevant sensor in human myeloid cells and primary keratinocytes. Additionally, we demonstrated that incoming VACV genomes are sufficient to trigger AIM2 inflammasome activation, while the VACV B2 poxin selectively restricts NLRP3 inflammasome activation in primary monocytes. <br /> In conclusion, this study provides the first evidence of cell-type-specific inflammasome responses to poxvirus infection in human primary cells, uncovering potential inflammasome regulatory mechanisms that remain unknown. Additionally, we have developed the first AIM2-inhibitory nanobodies, which serve as valuable molecular tools to dissect AIM2 functions and hold potential for therapeutic application in AIM2-associated diseases.