Inflammatory Caspases: Toward a Unified Model for Caspase Activation by Inflammasomes.

Inflammasomes are inflammatory signaling complexes that provide molecular platforms to activate the protease function of inflammatory caspases. Caspases-1, -4, -5, and -11 are inflammatory caspases activated by inflammasomes to drive lytic cell death and inflammatory mediator production, thereby activating host-protective and pathological immune responses. Here, we comprehensively review the mechanisms that govern the activity of inflammatory caspases. We discuss inflammatory caspase activation and deactivation mechanisms, alongside the physiological importance of caspase activity kinetics. We also examine mechanisms of caspase substrate selection and how inflammasome and cell identities influence caspase activity and resultant inflammatory and pyroptotic cellular programs. Understanding how inflammatory caspases are regulated may offer new strategies for treating infection and inflammasome-driven disease.

A 360° view of the inflammasome: Mechanisms of activation, cell death, and diseases.

Inflammasomes are critical sentinels of the innate immune system that respond to threats to the host through recognition of distinct molecules, known as pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), or disruptions of cellular homeostasis, referred to as homeostasis-altering molecular processes (HAMPs) or effector-triggered immunity (ETI). Several distinct proteins nucleate inflammasomes, including NLRP1, CARD8, NLRP3, NLRP6, NLRC4/NAIP, AIM2, pyrin, and caspases-4/-5/-11. This diverse array of sensors strengthens the inflammasome response through redundancy and plasticity. Here, we present an overview of these pathways, outlining the mechanisms of inflammasome formation, subcellular regulation, and pyroptosis, and discuss the wide-reaching effects of inflammasomes in human disease.

Structural Mechanism for GSDMD Targeting by Autoprocessed Caspases in Pyroptosis.

The pyroptosis execution protein GSDMD is cleaved by inflammasome-activated caspase-1 and LPS-activated caspase-11/4/5. The cleavage unmasks the pore-forming domain from GSDMD-C-terminal domain. How the caspases recognize GSDMD and its connection with caspase activation are unknown. Here, we show site-specific caspase-4/11 autoprocessing, generating a p10 product, is required and sufficient for cleaving GSDMD and inducing pyroptosis. The p10-form autoprocessed caspase-4/11 binds the GSDMD-C domain with a high affinity. Structural comparison of autoprocessed and unprocessed capase-11 identifies a ß sheet induced by the autoprocessing. In caspase-4/11-GSDMD-C complex crystal structures, the ß sheet organizes a hydrophobic GSDMD-binding interface that is only possible for p10-form caspase-4/11. The binding promotes dimerization-mediated caspase activation, rendering a cleavage independently of the cleavage-site tetrapeptide sequence. Crystal structure of caspase-1-GSDMD-C complex shows a similar GSDMD-recognition mode. Our study reveals an unprecedented substrate-targeting mechanism for caspases. The hydrophobic interface suggests an additional space for developing inhibitors specific for pyroptotic caspases.

Adrenergic Signaling in Muscularis Macrophages Limits Infection-Induced Neuronal Loss.

Enteric-associated neurons (EANs) are closely associated with immune cells and continuously monitor and modulate homeostatic intestinal functions, including motility and nutrient sensing. Bidirectional interactions between neuronal and immune cells are altered during disease processes such as neurodegeneration or irritable bowel syndrome. We investigated the effects of infection-induced inflammation on intrinsic EANs (iEANs) and the role of intestinal muscularis macrophages (MMs) in this context. Using murine models of enteric infections, we observed long-term gastrointestinal symptoms, including reduced motility and loss of excitatory iEANs, which was mediated by a Nlrp6- and Casp11-dependent mechanism, depended on infection history, and could be reversed by manipulation of the microbiota. MMs responded to luminal infection by upregulating a neuroprotective program via ß2-adrenergic receptor (ß2-AR) signaling and mediated neuronal protection through an arginase 1-polyamine axis. Our results identify a mechanism of neuronal death post-infection and point to a role for tissue-resident MMs in limiting neuronal damage.

The NLRP6 Inflammasome Recognizes Lipoteichoic Acid and Regulates Gram-Positive Pathogen Infection.

The activator and composition of the NLRP6 inflammasome remain poorly understood. We find that lipoteichoic acid (LTA), a molecule produced by Gram-positive bacteria, binds and activates NLRP6. In response to cytosolic LTA or infection with Listeria monocytogenes, NLRP6 recruited caspase-11 and caspase-1 via the adaptor ASC. NLRP6 activation by LTA induced processing of caspase-11, which promoted caspase-1 activation and interleukin-1ß (IL-1ß)/IL-18 maturation in macrophages. Nlrp6-/- and Casp11-/- mice were less susceptible to L. monocytogenes infection, which was associated with reduced pathogen loads and impaired IL-18 production. Administration of IL-18 to Nlrp6-/- or Casp11-/- mice restored the susceptibility of mutant mice to L. monocytogenes infection. These results reveal a previously unrecognized innate immunity pathway triggered by cytosolic LTA that is sensed by NLRP6 and exacerbates systemic Gram-positive pathogen infection via the production of IL-18.

The orphan receptor Nur77 binds cytoplasmic LPS to activate the non-canonical NLRP3 inflammasome.

Intracellular sensing of lipopolysaccharide (LPS) by murine caspase-11 or human caspase-4 initiates a protease cascade, termed the non-canonical inflammasome, that results in gasdermin D (GSDMD) processing and subsequent NLRP3 inflammasome activation. In an effort aimed at identifying additional sensors for intracellular LPS by biochemical screening, we identified the nuclear orphan receptor Nur77 as an LPS-binding protein in macrophage lysates. Nr4a1-/- macrophages exhibited impaired activation of the NLRP3 inflammasome, but not caspase-11, in response to LPS. Biochemical mapping revealed that Nur77 bound LPS directly through a domain in its C terminus. Yeast two-hybrid assays identified NLRP3 as a binding partner for Nur77. The association between Nur77 and NLRP3 required the presence of LPS and dsDNA. The source of dsDNA was the mitochondria, requiring the formation of gasdermin-D pores. In vivo, Nur77 deficiency ameliorated host response to endotoxins. Thus, Nur77 functions as an intracellular LPS sensor, binding mitochondrial DNA and LPS to activate the non-canonical NLRP3 inflammasome.

Heparin prevents caspase-11-dependent septic lethality independent of anticoagulant properties.

Heparin, a mammalian polysaccharide, is a widely used anticoagulant medicine to treat thrombotic disorders. It is also known to improve outcomes in sepsis, a leading cause of mortality resulted from infection-induced immune dysfunction. Whereas it is relatively clear how heparin exerts its anticoagulant effect, the immunomodulatory mechanisms enabled by heparin remain enigmatic. Here, we show that heparin prevented caspase-11-dependent immune responses and lethality in sepsis independent of its anticoagulant properties. Heparin or a chemically modified form of heparin without anticoagulant function inhibited the alarmin HMGB1-lipopolysaccharide (LPS) interaction and prevented the macrophage glycocalyx degradation by heparanase. These events blocked the cytosolic delivery of LPS in macrophages and the activation of caspase-11, a cytosolic LPS receptor that mediates lethality in sepsis. Survival was higher in septic patients treated with heparin than those without heparin treatment. The identification of this previously unrecognized heparin function establishes a link between innate immune responses and coagulation.

Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.

Bacterial Endotoxin Activates the Coagulation Cascade through Gasdermin D-Dependent Phosphatidylserine Exposure.

Excessive activation of the coagulation system leads to life-threatening disseminated intravascular coagulation (DIC). Here, we examined the mechanisms underlying the activation of coagulation by lipopolysaccharide (LPS), the major cell-wall component of Gram-negative bacteria. We found that caspase-11, a cytosolic LPS receptor, activated the coagulation cascade. Caspase-11 enhanced the activation of tissue factor (TF), an initiator of coagulation, through triggering the formation of gasdermin D (GSDMD) pores and subsequent phosphatidylserine exposure, in a manner independent of cell death. GSDMD pores mediated calcium influx, which induced phosphatidylserine exposure through transmembrane protein 16F, a calcium-dependent phospholipid scramblase. Deletion of Casp11, ablation of Gsdmd, or neutralization of phosphatidylserine or TF prevented LPS-induced DIC. In septic patients, plasma concentrations of interleukin (IL)-1α and IL-1ß, biomarkers of GSDMD activation, correlated with phosphatidylserine exposure in peripheral leukocytes and DIC scores. Our findings mechanistically link immune recognition of LPS to coagulation, with implications for the treatment of DIC.

Bacterial pathogen manipulation of host membrane trafficking.

Pathogens use a vast number of strategies to alter host membrane dynamics. Targeting the host membrane machinery is important for the survival and pathogenesis of several extracellular, vacuolar, and cytosolic bacteria. Membrane manipulation promotes bacterial replication while suppressing host responses, allowing the bacterium to thrive in a hostile environment. This review provides a comprehensive summary of various strategies used by both extracellular and intracellular bacteria to hijack host membrane trafficking machinery. We start with mechanisms used by bacteria to alter the plasma membrane, delve into the hijacking of various vesicle trafficking pathways, and conclude by summarizing bacterial adaptation to host immune responses. Understanding bacterial manipulation of host membrane trafficking provides insights into bacterial pathogenesis and uncovers the molecular mechanisms behind various processes within a eukaryotic cell.

Molecular mechanisms of emerging inflammasome complexes and their activation and signaling in inflammation and pyroptosis.

Inflammasomes are multi-protein complexes that assemble within the cytoplasm of mammalian cells in response to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), driving the secretion of the pro-inflammatory cytokines IL-1ß and IL-18, and pyroptosis. The best-characterized inflammasome complexes are the NLRP3, NAIP-NLRC4, NLRP1, AIM2, and Pyrin canonical caspase-1-containing inflammasomes, and the caspase-11 non-canonical inflammasome. Newer inflammasome sensor proteins have been identified, including NLRP6, NLRP7, NLRP9, NLRP10, NLRP11, NLRP12, CARD8, and MxA. These inflammasome sensors can sense PAMPs from bacteria, viruses and protozoa, or DAMPs in the form of mitochondrial damage, ROS, stress and heme. The mechanisms of action, physiological relevance, consequences in human diseases, and avenues for therapeutic intervention for these novel inflammasomes are beginning to be realized. Here, we discuss these emerging inflammasome complexes and their putative activation mechanisms, molecular and signaling pathways, and physiological roles in health and disease.

The Endotoxin Delivery Protein HMGB1 Mediates Caspase-11-Dependent Lethality in Sepsis.

Caspase-11, a cytosolic endotoxin (lipopolysaccharide: LPS) receptor, mediates pyroptosis, a lytic form of cell death. Caspase-11-dependent pyroptosis mediates lethality in endotoxemia, but it is unclear how LPS is delivered into the cytosol for the activation of caspase-11. Here we discovered that hepatocyte-released high mobility group box 1 (HMGB1) was required for caspase-11-dependent pyroptosis and lethality in endotoxemia and bacterial sepsis. Mechanistically, hepatocyte-released HMGB1 bound LPS and targeted its internalization into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycation end-products (RAGE). Subsequently, HMGB1 permeabilized the phospholipid bilayer in the acidic environment of lysosomes. This resulted in LPS leakage into the cytosol and caspase-11 activation. Depletion of hepatocyte HMGB1, inhibition of hepatocyte HMGB1 release, neutralizing extracellular HMGB1, or RAGE deficiency prevented caspase-11-dependent pyroptosis and death in endotoxemia and bacterial sepsis. These findings indicate that HMGB1 interacts with LPS to mediate caspase-11-dependent pyroptosis in lethal sepsis.

Caspase-11/GSDMD contributes to the progression of hyperuricemic nephropathy by promoting NETs formation.

Hyperuricemia is an independent risk factor for chronic kidney disease (CKD) and promotes renal fibrosis, but the underlying mechanism remains largely unknown. Unresolved inflammation is strongly associated with renal fibrosis and is a well-known significant contributor to the progression of CKD, including hyperuricemia nephropathy. In the current study, we elucidated the impact of Caspase-11/Gasdermin D (GSDMD)-dependent neutrophil extracellular traps (NETs) on progressive hyperuricemic nephropathy. We found that the Caspase-11/GSDMD signaling were markedly activated in the kidneys of hyperuricemic nephropathy. Deletion of Gsdmd or Caspase-11 protects against the progression of hyperuricemic nephropathy by reducing kidney inflammation, proinflammatory and profibrogenic factors expression, NETs generation, α-smooth muscle actin expression, and fibrosis. Furthermore, specific deletion of Gsdmd or Caspase-11 in hematopoietic cells showed a protective effect on renal fibrosis in hyperuricemic nephropathy. Additionally, in vitro studies unveiled the capability of uric acid in inducing Caspase-11/GSDMD-dependent NETs formation, consequently enhancing α-smooth muscle actin production in macrophages. In summary, this study demonstrated the contributory role of Caspase-11/GSDMD in the progression of hyperuricemic nephropathy by promoting NETs formation, which may shed new light on the therapeutic approach to treating and reversing hyperuricemic nephropathy.

Role of caspase-11 non-canonical inflammasomes in retinal ischemia/reperfusion injury.

BACKGROUND: Retinal ischemia/reperfusion (IR) injury is a common pathological process in many ophthalmic diseases. Interleukin-1ß (IL-1ß) is an important inflammatory factor involved in the pathology of retinal IR injury, but the mechanism by which IL-1ß is regulated in such injury remains unclear. Caspase-11 non-canonical inflammasomes can regulate the synthesis and secretion of IL-1ß, but its role in retinal IR injury has not been elucidated. This study aimed to evaluate the role of caspase-11 non-canonical inflammasomes in retinal IR injury. METHODS: Retinal IR injury was induced in C57BL/6J mice by increasing the intraocular pressure to 110 mmHg for 60 min. The post-injury changes in retinal morphology and function and in IL-1ß expression were compared between caspase-11 gene knockout (caspase-11-/-) mice and wild-type (WT) mice. Morphological and functional changes were evaluated using hematoxylin-eosin staining and retinal whole mount staining and using electroretinography (ERG), respectively. IL-1ß expression in the retina was measured using enzyme-linked immunosorbent assay (ELISA). The levels of caspase-11-related protein were measured using western blot analysis. The location of caspase-11 in the retina was determined via immunofluorescence staining. Mouse type I astrocytes C8-D1A cells were used to validate the effects of caspase-11 simulation via hypoxia in vitro. Small-interfering RNA targeting caspase-11 was constructed. Cell viability was evaluated using the MTT assay. IL-1ß expression in supernatant and cell lysate was measured using ELISA. The levels of caspase-11-related protein were measured using western blot analysis. RESULTS: Retinal ganglion cell death and retinal edema were more ameliorated, and the ERG b-wave amplitude was better after retinal IR injury in caspase-11-/- mice than in WT mice. Further, caspase-11-/- mice showed lower protein expressions of IL-1ß, cleaved caspase-1, and gasdermin D (GSDMD) in the retina after retinal IR injury. Caspase-11 protein was expressed in retinal glial cells, and caspase-11 knockdown played a protective role against hypoxia in C8-D1A cells. The expression levels of IL-1ß, cleaved caspase-1, and GSDMD were inhibited after hypoxia in the si-caspase-11 constructed cells. CONCLUSIONS: Retinal IR injury activates caspase-11 non-canonical inflammasomes in glial cells of the retina. This results in increased protein levels of GSDMD and IL-1ß and leads to damage in the inner layer of the retina.

Fusobacterium nucleatum infection activates the noncanonical inflammasome and exacerbates inflammatory response in DSS-induced colitis.

Caspase activation results in pyroptosis, an inflammatory cell death that contributes to several inflammatory diseases by releasing inflammatory cytokines and cellular contents. Fusobacterium nucleatum is a periodontal pathogen frequently detected in human cancer and inflammatory bowel diseases. Studies have reported that F. nucleatum infection leads to NLRP3 activation and pyroptosis, but the precise activation process and disease association remain poorly understood. This study demonstrated that F. nucleatum infection exacerbates acute colitis in mice and activates pyroptosis through caspase-11-mediated gasdermin D cleavage in macrophages. Furthermore, F. nucleatum infection in colitis mice induces the enhancement of IL-1⍺ secretion from the colon, affecting weight loss and severe disease activities. Neutralization of IL-1⍺ protects F. nucleatum infected mice from severe colitis. Therefore, F. nucleatum infection facilitates inflammation in acute colitis with IL-1⍺ from colon tissue by activating noncanonical inflammasome through gasdermin D cleavage.

Dectin-1 aggravates neutrophil inflammation through caspase-11/4-mediated macrophage pyroptosis in asthma.

BACKGROUND: The pattern recognition receptor Dectin-1 was initially discovered to play a pivotal role in mediating pulmonary antifungal immunity and promoting neutrophil-driven inflammation. Recent studies have revealed that Dectin-1 is overexpressed in asthma, but the specific mechanism remains elusive. Additionally, Dectin-1 has been implicated in promoting pyroptosis, a hallmark of severe asthma airway inflammation. Nevertheless, the involvement of the non-classical pyroptosis signal caspase-11/4 and its upstream regulatory mechanisms in asthma has not been completely explored. METHODS: House dust mite (HDM)-induced mice was treated with Dectin-1 agonist Curdlan, Dectin-1 inhibitor Laminarin, and caspase-11 inhibitor wedelolactone separately. Subsequently, inflammatory cells in bronchoalveolar lavage fluid (BALF) were analyzed. Western blotting was performed to measure the protein expression of caspase-11 and gasdermin D (GSDMD). Cell pyroptosis and the expression of chemokine were detected in vitro. The correlation between Dectin-1 expression, pyroptosis factors and neutrophils in the induced sputum of asthma patients was analyzed. RESULTS: Curdlan appeared to exacerbate neutrophil airway inflammation in asthmatic mice, whereas wedelolactone effectively alleviated airway inflammation aggravated by Curdlan. Moreover, Curdlan enhanced the release of caspase-11 activation fragments and N-terminal fragments of gasdermin D (GSDMD-N) stimulated by HDM both in vivo or in vitro. In mouse alveolar macrophages (MH-S cells), Curdlan/HDM stimulation resulted in vacuolar degeneration and elevated lactate dehydrogenase (LDH) release. In addition, there was an upregulation of neutrophil chemokines CXCL1, CXCL3, CXCL5 and their receptor CXCR2, which was suppressed by wedelolactone. In asthma patients, a positive correlation was observed between the expression of Dectin-1 on macrophages and caspase-4 (the human homology of caspase-11), and the proportion of neutrophils in induced sputum. CONCLUSION: Dectin-1 activation in asthma induced caspase-11/4 mediated macrophage pyroptosis, which subsequently stimulated the secretion of chemokines, leading to the exacerbation of airway neutrophil inflammation.

Plexin C1 influences immune response to intracellular LPS and survival in murine sepsis.

BACKGROUND: Intracellular sensing of lipopolysaccharide (LPS) is essential for the immune response against gram-negative bacteria and results in activation of caspase-11 and pyroptotic cell death with fatal consequences in sepsis. We found the neuronal guidance receptor plexin C1 (PLXNC1) influences the intracellular response to LPS. METHODS: We employed a murine model of sepsis via cecal ligation and binding (CLP), using PLXNC1-/- mice and littermate controls, and additionally transfected murine bone-marrow-derived macrophages (BMDMs) from both genotypes with LPS to achieve activation of the noncanonical inflammasome ex vivo. Additionally, we transfected the PLXNC1 ligand SL4c-d in vivo and ex vivo to examine its effect on intracellular LPS response. RESULTS: We found the neuronal guidance receptor PLXNC1 dampens the intracellular response to LPS by interacting with adenylate cyclase 4 (ADCY4) and protein kinase A activity, which in turn diminishes caspase-11 expression. The absence of PLXNC1 results in excessive inflammation marked by increased cytokine release, increased secondary organ injury and reduced sepsis survival in a murine sepsis model induced by CLP. Notably, administration of SL4c-d-peptide ligand of PLXNC1-reduces the inflammatory response during CLP-induced sepsis and improves survival. CONCLUSIONS: These results elucidate a previously unknown mechanism for PLXNC1 suppressing excessive noncanonical inflammasome activity and offer a new potential target for treatment of sepsis with its detrimental effects.

Pyroptosis: Caspase-11 Unlocks the Gates of Death.

How inflammatory caspases trigger pyroptotic cell death is mostly unexplained. In this issue of Immunity, Núñez and colleagues report that caspase-11 cleaves the transmembrane channel pannexin-1, causing an efflux of cellular ATP that promotes a P2X7 receptor-dependent pyroptosis.

Sulconazole induces pyroptosis promoted by interferon-γ in monocyte/macrophage lineage cells.

Imidazole derivatives are commonly used as antifungal agents. Here, we aimed to investigate the functions of imidazole derivatives on macrophage lineage cells. We assessed the expression levels of inflammatory cytokines in mouse monocyte/macrophage lineage (RAW264.7) cells. All six imidazole derivatives examined, namely ketoconazole, sulconazole, isoconazole, luliconazole, clotrimazole, and bifonazole, reduced the expression levels of inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor-α, after induction by lipopolysaccharide (LPS) in RAW264.7 cells. These imidazole derivatives also induced cell death in RAW264.7 cells, regardless of the presence or absence of LPS. Since the cell death was characteristic in morphology, we investigated the mode of the cell death. An imidazole derivative, sulconazole, induced gasdermin D degradation together with caspase-11 activation, namely, pyroptosis in RAW264.7 cells and peritoneal macrophages. Furthermore, priming with interferon-γ promoted sulconazole-induced pyroptosis in RAW264.7 cells and macrophages and reduced the secretion of the inflammatory cytokine, IL-1ß, from sulconazole-treated macrophages. Our results suggest that imidazole derivatives suppress inflammation by inducing macrophage pyroptosis, highlighting their modulatory potential for inflammatory diseases.

The role of NLRP6 in the development and progression of neurological diseases.

The nervous system possesses the remarkable ability to undergo changes in order to store information; however, it is also susceptible to damage caused by invading pathogens or neurodegenerative processes. As a member of nucleotide-binding oligomerization domain-like receptor (NLR) family, the NLRP6 inflammasome serves as a cytoplasmic innate immune sensor responsible for detecting microbe-associated molecular patterns. Upon activation, NLRP6 can recruit the adapter protein apoptosis-associated speck-like protein (ASC) and the inflammatory factors caspase-1 or caspase-11. Consequently, inflammasomes are formed, facilitating the maturation and secretion of pro-inflammatory cytokines such as inflammatory factors-18 (IL-18) and inflammatory factors-1ß (IL-1ß). Precise regulation of NLRP6 is crucial for maintaining tissue homeostasis, as dysregulated inflammasome activation can contribute to the development of various diseases. Furthermore, NLRP6 may also play a role in the regulation of extraintestinal diseases. In cells of the brain, such as astrocytes and neurons, NLRP6 inflammasome are also present. Here, the assembly and subsequent activation of caspase-1 mediated by NLRP6 contribute to disease progression. This review aims to discuss the structure and function of NLRP6, explain clearly the mechanisms that induce and activate NLRP6, and explore its role within the central and peripheral nervous system.