Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

Gram-negative bacteria including Escherichia coli, Citrobacter rodentium, Salmonella typhimurium, and Shigella flexneri are sensed in an ill-defined manner by an intracellular inflammasome complex that activates caspase-11. We show that macrophages loaded with synthetic lipid A, E. coli lipopolysaccharide (LPS), or S. typhimurium LPS activate caspase-11 independently of the LPS receptor Toll-like receptor 4 (TLR4). Consistent with lipid A triggering the noncanonical inflammasome, LPS containing a divergent lipid A structure antagonized caspase-11 activation in response to E. coli LPS or Gram-negative bacteria. Moreover, LPS-mutant E. coli failed to activate caspase-11. Tlr4(-/-) mice primed with TLR3 agonist polyinosinic:polycytidylic acid [poly(I:C)] to induce pro-caspase-11 expression were as susceptible as wild-type mice were to sepsis induced by E. coli LPS. These data unveil a TLR4-independent mechanism for innate immune recognition of LPS.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules termed damage-associated molecular patterns (DAMPs) that propagate the inflammatory response. The underlying mechanism for PMR, however, is unknown. Here we show that the ill-characterized nerve injury-induced protein 1 (NINJ1) -a cell surface protein with two transmembrane regions -plays an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1 -/macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and failed to release numerous intracellular proteins including High Mobility Group Box 1 (HMGB1, a known DAMP) and Lactate Dehydrogenase (LDH, a standard measure of PMR). Ninj1 -/macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1 -/mice were more susceptible than wildtype mice to Citrobacter rodentium, suggesting a role for PMR in anti-bacterial host defense.Mechanistically, NINJ1 utilized an evolutionarily conserved extracellular α-helical domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held dogma that cell death-related PMR is a passive event.Pyroptosis is a potent inflammatory mode of lytic cell death triggered by diverse infectious and sterile insults 1-3 . It is driven by the pore-forming fragment of gasdermin D (GSDMD) 4-7 and releases two exemplar proteins: interleukin-1β (IL-1β), a pro-inflammatory cytokine, and LDH, a standard marker of PMR and lytic cell death. An early landmark study 8 predicted two sequential steps for pyroptosis: (1) initial formation of a small plasma membrane pore causing IL-1β release and non-selective ionic fluxes, and (2) subsequent PMR attributable to oncotic cell swelling. PMR releases LDH (140 kDa) and large DAMPs. While the predicted size of gasdermin pores (~18 nm inner diameter 9 ) is large enough to release IL-1β (17 kDa, ~4.5 nm diameter), the underlying mechanism for subsequent PMR has been considered a passive osmotic lysis event. An unbiased forward genetic screen identifies NINJ1To identify essential mediators of PMR, we performed a forward genetic screen using bone marrow-derived macrophages (BMDMs) from N-ethyl-N-nitrosourea (ENU)-mutagenized mice.

The Ras/mitogen-activated protein kinase (MAPK) pathway plays a critical role in transducing mitogenic signals from receptor tyrosine kinases. Loss-of-function mutations in one feedback regulator of Ras/MAPK signaling, SPRED1 (Sprouty-related protein with an EVH1 domain), cause Legius syndrome, an autosomal dominant human disorder that resembles Neurofibromatosis-1 (NF1). Spred1 functions as a negative regulator of the Ras/MAPK pathway; however, the underlying molecular mechanism is poorly understood. Here we show that neurofibromin, the NF1 gene product, is a Spred1-interacting protein that is necessary for Spred1's inhibitory function. We show that Spred1 binding induces the plasma membrane localization of NF1, which subsequently down-regulates Ras-GTP levels. This novel mechanism for the regulation of neurofibromin provides a molecular bridge for understanding the overlapping pathophysiology of NF1 and Legius syndrome.

The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4. Furthermore, caspase-1 catalytically dead knock-in (Casp1 C284A KI) BMDMs genetically segregate pyroptosis and apoptosis, and confirm that caspase-1 does not functionally compete with ASC for NLRC4 interactions. We show that NLRC4/caspase-8-mediated apoptotic cells eventually undergo plasma cell membrane damage in vitro, suggesting that this pathway can lead to secondary necrosis. Unexpectedly, we found that DFNA5/GSDME, a member of the pore-forming gasdermin family, is dispensable for the secondary necrosis that follows NLRC4-mediated apoptosis in macrophages. Together, our data confirm the existence of an alternative caspase-8 activation pathway diverging from the NLRC4 inflammasome in primary macrophages.