Human non-canonical inflammasomes activate CASP3 to limit intracellular Salmonella replication in macrophages.

Inflammasomes are signaling platforms that activate inflammatory caspases to initiate innate immune responses. Canonical inflammasomes sense diverse threats and activate CASP1, which cleaves the pro-inflammatory cytokines IL-1β and IL-18 and the pore-forming protein gasdermin D (GSDMD) to induce pyroptosis. In contrast, the non-canonical inflammasome senses bacterial lipopolysaccharide (LPS) through CASP4 and CASP5 to induce pyroptosis. While CASP1 substrates are well defined, those of CASP4 and CASP5 remain less understood. Here, we show that intracellular LPS and the gram-negative bacterial pathogen Salmonella activate CASP4/5 in macrophages to directly cleave and activate CASP3 and CASP7. Activated CASP3 subsequently cleaves gasdermin E (GSDME). Surprisingly, CASP3, but not GSDME, was required for restricting intracellular Salmonella replication, suggesting a protective role for apoptotic signaling. We further find that most GSDMD cleavage during non-canonical signaling is mediated by CASP1. Consistent with this, loss of GSDMD, but not GSDME, reduced LDH release, establishing GSDMD as the primary driver of pyroptosis during LPS transfection. In contrast, during Salmonella infection, cell lysis occurred independently of both GSDMD and GSDME, suggesting the involvement of alternative lytic mechanisms. Finally, we demonstrate that CASP4/5 activation of CASP3/7 and GSDME occurs in human primary macrophages, defining CASP4/5 as dual apoptotic initiator and inflammatory caspases in innate immunity.