Caspase-1, -4, -5 and -11 activate gasdermin D (GSDMD) pores, causing pyroptotic cell death and the release of the interleukins IL-1β and IL-18 (ref. 1). Blocking this pathway holds therapeutic promise for the treatment of inflammatory disorders, but cell-permeable caspase inhibitors have not proved successful in clinical trials2. Here we describe covalent caspase inhibitors that selectively block pyroptosis and IL-1β secretion despite being excluded from healthy cells. These inhibitors did not prevent caspase-driven apoptosis, implying that GSDMD pores facilitated their uptake. Membrane-impermeable dyes entered the cells rescued from pyroptosis, consistent with transient membrane permeabilization by GSDMD pores. Caspase inhibition prevented rather than delayed cell death, consistent with membrane repair mechanisms neutralizing the initial GSDMD pores. Inhibiting caspase-1 and caspase-11 suppressed IL-1β and IL-18 production in a mouse model of endotoxic shock, underscoring the therapeutic potential of exploiting GSDMD pores for targeted caspase inhibition in inflammatory diseases.

NINJ1, a cell surface protein, mediates plasma membrane rupture (PMR) in cells undergoing pyroptosis or other form of cell death. Catastrophic PMR releases proinflammatory cytoplasmic molecules termed damage-associated molecular patterns (DAMPs). Some mice with Ninj1-deficient alleles (Ninj1−/−) die perinatally due to developmental hydrocephalus with variable frequency. While Ninj1−/− mice are invaluable for studying cell lysis, their utility especially in large-scale in vivo disease models has been severely bottlenecked by this sporadic hydrocephalus. Using novel knock-in mouse models, we show under physiological conditions that the conserved, N-terminal amphipathic α1 domain of NINJ1 is strictly required for PMR, yet completely dispensable for embryonic development. Ninj1K45Q/K45Q, harboring a point mutation in the α1 domain, resembles Ninj1 deficiency by attenuating PMR in cultured bone marrow-derived macrophages (BMDMs) and liver injury in vivo. In contrast, Ninj2−/− alleles or Ninj1W29A/W29A alleles that encode a point mutation in a reported adhesion segment of the N-terminus region, exhibit unaltered PMR in BMDMs. Crucially, unlike Ninj1−/− animals, Ninj1K45Q/K45Q mice and Ninj1W29A/W29A mice exhibit normal Mendelian survival and are free from developmental hydrocephalus. Our genetic findings decouple the lytic function of NINJ1 from its homeostatic role in development, establishing Ninj1K45Q/K45Q mice as a powerful, hydrocephalus-free resource to accelerate in vivo and in vitro investigations of PMR and inflammatory diseases.

Caspase-1, -4, -5, and -11 activate Gasdermin D (GSDMD) to form pores in the plasma membrane. In addition to releasing interleukin (IL)-1β and IL-18, GSDMD pores cause a lytic, proinflammatory form of cell death called pyroptosis. Blocking this pathway holds therapeutic promise for the treatment of inflammatory disorders, but clinical trials of cell permeable caspase inhibitors have been unsuccessful. Here, we describe covalent inhibitors of proinflammatory caspases that are impermeable to healthy cells but effectively block caspase-1 driven pyroptosis and IL-1β secretion. Their failure to inhibit apoptosis implies inhibitor entry through GSDMD pores. Propidium iodide entered rescued cells, confirming transient membrane permeabilization via caspase-1 and GSDMD. Caspase-1 inhibition prevented rather than delayed cell death, likely due to the activation of membrane repair mechanisms neutralizing the initial GSDMD pores. Notably, inhibiting caspase-1 and -11 suppressed IL-1β and IL-18 production in a mouse model of endotoxic shock. These findings underscore the therapeutic potential of exploiting GSDMD pores for the delivery of caspase inhibitors, offering a novel strategy for treating inflammatory diseases. ### Competing Interest Statement K.M.G., M.E.T., I.S., B.L., R.S.J., E.S.L., P.K., W.P.L, J.Z., N.K., K.N., and V.M.D. were employees of Genentech.

Lytic cell death culminates in plasma membrane rupture, which releases large intracellular molecules to augment the inflammatory response. Plasma membrane rupture is mediated by the effector membrane protein ninjurin-1 (NINJ1)(1), which polymerizes and ruptures the membrane via its hydrophilic face(1-4). How NINJ1 is restrained under steady-state conditions to ensure cell survival remains unknown. Here we describe the molecular underpinnings of NINJ1 inhibition. Using cryogenic electron microscopy, we determined the structure of inactive-state mouse NINJ1 bound to the newly developed nanobody Nb538. Inactive NINJ1 forms a face-to-face homodimer by adopting a three-helix conformation with unkinked transmembrane helix 1 (TM1), in contrast to the four-helix TM1-kinked active conformation(2-4). Accordingly, endogenous NINJ1 from primary macrophages is a dimer under steady-state conditions. Inactive dimers sequester the membrane rupture-inducing hydrophilic face of NINJ1 and occlude the binding site for kinked TM1 from neighbouring activated NINJ1 molecules. Mutagenesis studies in cells show that destabilization of inactive face-to-face dimers leads to NINJ1-mediated cell death, whereas stabilization of face-to-face dimers inhibits NINJ1 activity. Moreover, destabilizing mutations prompt spontaneous TM1 kink formation, a hallmark of NINJ1 activation. Collectively, our data demonstrate that dimeric NINJ1 is autoinhibited in trans to prevent unprovoked plasma membrane rupture and cell death.

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ1 1 . PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus d -galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia–reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia–reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response(1-3). The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein(4-8), which contains two transmembrane regions, has 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 were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and 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 wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.

Significance Inflammasomes, cytosolic multicomponent complexes, sense patterns of pathogenesis or metabolic changes and initiate a cellular response via the activation of the inflammatory caspases (iCasps). A more comprehensive substrate analysis for iCasps will greatly improve our understanding of this pathway and provide potential blood-based biomarkers for inflammasome activation in disease. Antibodies typically exhibit high affinity and specificity for a single modification site, in direct contrast to a protease that exhibits a more degenerate recognition profile. Here, we generate antibodies that defy this convention and exhibit a degenerate recognition motif similar to the iCasps and apply these new tools to elucidate hundreds of putative iCasp substrates. Our findings reveal insights into inflammasome function.

The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1β and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants.

Pyroptosis requires the induction of gasdermin D expression by the transcription factor IRF2.

[This corrects the article DOI: 10.1371/journal.pbio.3000354.].

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.

Intracellular LPS sensing by caspase-4/5/11 triggers proteolytic activation of pore-forming gasdermin D (GSDMD), leading to pyroptotic cell death in Gram-negative bacteria-infected cells. Involvement of caspase-4/5/11 and GSDMD in inflammatory responses, such as lethal sepsis, makes them highly desirable drug targets. Using knock-in (KI) mouse strains, we herein provide genetic evidence to show that caspase-11 auto-cleavage at the inter-subunit linker is essential for optimal catalytic activity and subsequent proteolytic cleavage of GSDMD. Macrophages from caspase-11–processing dead KI mice (Casp11Prc D285A/D285A) exhibit defective caspase-11 auto-processing and phenocopy Casp11−/− and caspase-11 enzymatically dead KI (Casp11Enz C254A/C254A) macrophages in attenuating responses to cytoplasmic LPS or Gram-negative bacteria infection. GsdmdD276A/D276A KI macrophages also fail to cleave GSDMD and are hypo-responsive to inflammasome stimuli, confirming that the GSDMD Asp276 residue is a nonredundant and indispensable site for proteolytic activation of GSDMD. Our data highlight the role of caspase-11 self-cleavage as a critical regulatory step for GSDMD processing and response against Gram-negative bacteria.

As a front line of defense against pathogenic microbes, our body employs a primitive, yet highly sophisticated and potent innate immune response pathway collectively referred to as the inflammasome. Innate immune cells, epithelial cells, and many other cell types are capable of detecting infection or tissue injury and mounting a coordinated molecular defense. For example, Gram-negative bacteria are specifically detected via a surveillance mechanism that involves activation of extracellular receptors such as Toll-like receptors (TLRs) followed by intracellular recognition and activation of pathways such as caspase-11 (caspase-4/5 in humans). Importantly, lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is a strong trigger of these pathways. Extracellular LPS primarily stimulates TLR4, which can serve as a priming signal for expression of inflammasome components. Intracellular LPS can then trigger caspase-11-dependent inflammasome activation in the cytoplasm. Here, we briefly review the burgeoning caspase-11-dependent non-canonical inflammasome field, focusing mainly on the innate sensing of LPS.

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.