Antagonistic nanobodies reveal mechanism of GSDMD pore formation and unexpected therapeutic potential

Abstract

Activation of various inflammasomes converges on the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases, followed by oligomerization of the N-terminal domain (GSDMDNT) and the assembly of pores penetrating target membranes. Yet, it remained unclear what triggers the conformational changes that allow membrane insertion, as methods to study pore formation in living cells were limited. We raised nanobodies specific for human GSDMD and found two nanobodies that prevent pyroptosis and IL-1β release when expressed in the cytosol of human macrophages. Nanobody binding to GSDMDNTblocked its oligomerization, while inflammasome assembly and GSDMD processing itself were not affected. The nanobody-stabilized monomers of GSDMDNTpartitioned into the plasma membrane, suggesting that pore formation is initiated by insertion of monomers, followed by oligomerization in the target membrane. When GSDMD pore formation was inhibited, cells still underwent caspase-1-dependent apoptosis, likely due to the substantially augmented caspase-1 activity. This hints at a novel layer of regulation of caspase-1 activity by GSDMD pores. Moreover, we revealed the unexpected therapeutic potential of antagonistic GSDMD nanobodies, as recombinant nanobodies added to the medium prevented cell death by pyroptosis, likely by entering through GSDMD pores and curtailing the assembly of additional pores. GSDMD nanobodies may thus be suitable to treat the ever-growing list of diseases caused by activation of the (non-) canonical inflammasomes.

Pyroptosis inhibiting nanobodies block Gasdermin D pore formation

“…Our SPR-based binding experiments were performed in the presence of 5 mM DTT, indicating that the nanobodies retain their binding ability even under reducing conditions and should not be affected by the reducing milieu of the cytoplasm. This observation is consistent with the inhibition of GSDMD pore formation in THP-1 cells 32 .…”

“…By sterically inhibiting oligomerization instead of targeting reactive cysteine residues in GSDMD, our nanobodies provide a new mechanism of pyroptosis inhibition. Comprehensive analysis of cytosolically expressed VHHGSDMD-1 and VHHGSDMD-2 in the human macrophage-like cell line THP-1 as well as in primary human macrophages reveals complete inhibition of pyroptosis and IL-1β secretion 32 . GSDMD is an intracellular protein and the delivery of antibodies and nanobodies to the cytoplasm has long been limited due to their inability to cross the plasma membrane 38,39 .…”

“…GSDMD is an intracellular protein and the delivery of antibodies and nanobodies to the cytoplasm has long been limited due to their inability to cross the plasma membrane 38,39 . Transient GSDMD pore formation after inflammasome activation seems to be able to allow access of inhibitory GSDMD nanobodies to the cytosol and prevents cell death by pyroptosis 32 . In addition, recent studies showed that nanobodies can be delivered to the cytoplasm of cells using cell penetrating peptide fusions, and nanobody mRNA could be delivered using gene therapy approaches [39][40][41][42][43] .…”

“…Together with the accompanying study 32 , we here describe the identification of inhibitory and non-inhibitory nanobodies targeting human GSDMD that can be used in a variety of ways from studying GSDMD biology and pyroptosis to compound screening approaches or the application as anti-inflammasomal inhibitors.…”

“…GSDMD targeting nanobodies were raised by immunization of an alpaca with full-length recombinant human GSDMD protein. Identification by phage display and initial characterization of binding analyzed by enzyme-linked immunosorbent assay (ELISA) and LUMIER assays are described by Schiffelers et al 32 . The potential binders differed by at least 7.6% in their amino acid sequence and showed great variety in the lengths and composition of their complementarity determining region 3 (CDR3) (Fig.…”