Granzyme A amplifies human neutrophil pyroptosis

Abstract Neutrophil granule serine proteases contribute to antimicrobial defence and regulate inflammatory cell death programs. Granzyme A (GrA) expression in neutrophils is controversial and its role in neutrophil cell death remains unknown. Here, we show that pharmacological inhibition of GrA with the selective covalent inhibitor iSK15 delays and attenuates pyroptotic death induced by nigericin and granule permeabilization, while leaving spontaneous apoptosis intact. Pan-caspase inhibition was less efficient than iSK15 at inhibiting pyroptosis, suggesting that GrA amplifies pyroptotic execution through a caspase-independent mechanism downstream of inflammasome activation. GrA directly cleaved gasdermin D (GSDMD) and GSDME, but did not generate stable canonical N-terminal pore-forming fragments, indicating that GrA enhances pyroptosis via gasdermin-independent pathways. During pyroptosis, active GrA remained predominantly intracellular, relocating to the cytosol and was released from cells only at late stages, most likely as a consequence of terminal cell lysis. GrA inhibition also reduced reactive oxygen species generation. GrA-deficient mouse neutrophils did not differ from wild-type neutrophils upon nigericin stimulation, consistent with species-specific differences in GrA structure and substrate specificity. In contrast, the role of GrA was confirmed in human iPSC-derived neutrophil-like cells. Together, our findings identify GrA as a non-canonical regulator of neutrophil pyroptosis and support selective GrA inhibition as a potentially tractable strategy for modulating neutrophil-driven inflammatory responses.