RIPK1 activates distinct gasdermins in macrophages and neutrophils upon pathogen blockade of innate immune signaling

Injection of effector proteins to block host innate immune signaling is a common strategy used by many pathogenic organisms to establish an infection. For example, pathogenic Yersinia species inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signaling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1–caspase-8 death–inducing platform that confers antibacterial defense. While recent studies revealed that caspase-8 cleaves the pore-forming protein gasdermin D to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defense is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase–dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1β release, which is critical for anti-Yersinia defense. During in vivo infection, IL-1β neutralization increases bacterial burden in wild-type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signaling.

Gasdermins are a family of recently described pore-forming proteins and are emerging as key drivers of cell death and inflammation. Gasdermins comprise a cytotoxic N-terminal domain connected to an inhibitory carboxyl-terminal domain and are activated upon proteolytic cleavage (1, 2). This cleavage event releases the cytotoxic N-terminal fragment, which creates membrane pores and triggers a form of lytic cell death called pyroptosis (3–6). Gasdermin D (GSDMD) is arguably the best characterized family member to date and is activated upon proteolysis by caspase-1, 4, 5, 8, and 11 and serine proteases (7–14). Active GSDMD promotes host defense by eliminating the replicating niche of intracellular pathogens (15) and inducing the extrusion of antimicrobial neutrophil extracellular traps (NETs) (16). In addition, GSDMD pores act as a conduit for bioactive IL-1β release (17–19), a potent proinflammatory cytokine that similarly requires proteolytic cleavage by caspase-1 or -8 to gain biological activity (20). By contrast, gasdermin E (GSMDE [also known as DFNA5]) is activated by apoptotic caspase-3 and 7 and granzyme B, which drives tumor cell pyroptosis and anti-tumor immunity (21–23). The physiological function of GSDME in primary immune cells and its potential role in host defense remain unresolved and have not been reported.Pathogenic Yersinia are a group of Gram-negative extracellular bacteria that causes disease ranging from gastroenteritis (Yersinia pseudotuberculosis) to plague (Y. pestis). A major mechanism by which pathogenic Yersinia establish systemic infection is by injecting the effector protein YopJ, an acetyltransferase that blocks transforming growth factor beta-activated kinase 1 (TAK1), to inhibit host innate immune signaling and proinflammatory cytokine production (24). To counteract this, detection of YopJ activity by myeloid cells induces the assembly of a cytoplasmic death–inducing complex that comprises receptor-interacting serine/threonine protein kinase 1 (RIPK1), fas-associated protein with death domain, and caspase-8 (24–26). During in vivo infection, RIPK1/caspase-8–dependent cell death in myeloid cells restricts bacterial dissemination and replication at distal sites by inducing proinflammatory cytokine production from uninfected bystander cells (24). More recently, GSDMD was identified as a caspase-8 substrate during Yersinia infection that drives antimicrobial defense in vivo (11, 12, 27). However, whether RIPK1 activates additional substrates to restrict Yersinia infection is unclear and is a focus of this study. Here, we identify GSDME as a substrate activated downstream of RIPK1 that confers host resistance against Yersinia. Gsdme-deficient mice failed to control bacterial replication in the spleen and liver and consequently are more susceptible to Yersinia infection than wild-type (WT) animals. Mechanistically, our data reveal that RIPK1 promotes caspase-3–dependent GSDME activation and IL-1β release in neutrophils, but not macrophages. Neutralization of IL-1β impaired bacterial clearance in WT, but not Gsdme^−/−, animals, indicating that IL-1β is mainly secreted through GSDME pores during Yersinia challenge in vivo.