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.

Innate immunity to intracellular LPS.

Monitoring of the cytosolic compartment by the innate immune system for pathogen-encoded products or pathogen activities often enables the activation of a subset of caspases. In most cases, the cytosolic surveillance pathways are coupled to activation of caspase-1 via canonical inflammasome complexes. A related set of caspases, caspase-11 in rodents and caspase-4 and caspase-5 in humans, monitors the cytosol for bacterial lipopolysaccharide (LPS). Direct activation of caspase-11, caspase-4 and caspase-5 by intracellular LPS elicits the lytic cell death called 'pyroptosis', which occurs in multiple cell types. The pyroptosis is executed by the pore-forming protein GSDMD, which is activated by cleavage mediated by caspase-11, caspase-4 or caspase-5. In monocytes, formation of GSDMD pores can induce activation of the NLRP3 inflammasome for maturation of the cytokines IL-1ß and IL-18. Caspase-11-mediated pyroptosis in response to cytosolic LPS is critical for antibacterial defense and septic shock. Here we review the emerging literature on the sensing of cytosolic LPS and its regulation and pathophysiological functions.

SERPINB1-mediated checkpoint of inflammatory caspase activation.

Inflammatory caspases (caspase-1, caspase-4, caspase-5 and caspase-11 (caspase-1/-4/-5/-11)) mediate host defense against microbial infections, processing pro-inflammatory cytokines and triggering pyroptosis. However, precise checkpoints are required to prevent their unsolicited activation. Here we report that serpin family B member 1 (SERPINB1) limited the activity of those caspases by suppressing their caspase-recruitment domain (CARD) oligomerization and enzymatic activation. While the reactive center loop of SERPINB1 inhibits neutrophil serine proteases, its carboxy-terminal CARD-binding motif restrained the activation of pro-caspase-1/-4/-5/-11. Consequently, knockdown or deletion of SERPINB1 prompted spontaneous activation of caspase-1/-4/-5/-11, release of the cytokine IL-1ß and pyroptosis, inducing elevated inflammation after non-hygienic co-housing with pet-store mice and enhanced sensitivity to lipopolysaccharide- or Acinetobacter baumannii-induced endotoxemia. Our results reveal that SERPINB1 acts as a vital gatekeeper of inflammation by restraining neutrophil serine proteases and inflammatory caspases in a genetically and functionally separable manner.

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.

The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response.

Assembly of the NLRP3 inflammasome activates caspase-1 and mediates the processing and release of the leaderless cytokine IL-1ß and thereby serves a central role in the inflammatory response and in diverse human diseases. Here we found that upon activation of caspase-1, oligomeric NLRP3 inflammasome particles were released from macrophages. Recombinant oligomeric protein particles composed of the adaptor ASC or the p.D303N mutant form of NLRP3 associated with cryopyrin-associated periodic syndromes (CAPS) stimulated further activation of caspase-1 extracellularly, as well as intracellularly after phagocytosis by surrounding macrophages. We found oligomeric ASC particles in the serum of patients with active CAPS but not in that of patients with other inherited autoinflammatory diseases. Our findings support a model whereby the NLRP3 inflammasome, acting as an extracellular oligomeric complex, amplifies the inflammatory response.

Harnessing proteases for T regulatory cell immunotherapy.

The immune system is tightly regulated by a subset of T cells defined as regulatory T cells (Tregs). Tregs maintain immune homeostasis by restraining unwarranted immune cell activation and effector function. Here, we discuss an important but underappreciated role of proteases in controlling Treg function. Proteases regulate a number of vital processes that determine T cell immune responses and some of them such as furin, ADAM (through regulating LAG receptor), MALT, and asparaginyl endopeptidase are implicated in Treg immunobiology. Targeted protease inhibition, using either small molecule inhibitors or gene deficient mice has demonstrated their specificity in modulating Treg function in experimental murine models. These data further highlight the ability of proteases to specifically regulate Tregs but no other T effector lineages. Taken together, it is apparent that incorporating proteases as targets within Treg cell engineering protocols may enable generation of robust Treg cellular therapeutics. These engineered Tregs may possess enhanced regulatory function along with resistance to lineage deviation in inflammatory disease such as colitis and graft versus host disease. Within this review, we summarize research on the role of proteases in regulating Treg function and discuss the translational potential of harnessing Treg function by targeting protease driven regulatory pathways.

TLR11-independent inflammasome activation is critical for CD4+ T cell-derived IFN-γ production and host resistance to Toxoplasma gondii.

Innate recognition of invading intracellular pathogens is essential for regulating robust and rapid CD4+ T cell effector function, which is critical for host-mediated immunity. The intracellular apicomplexan parasite, Toxoplasma gondii, is capable of infecting almost any nucleated cell of warm-blooded animals, including humans, and establishing tissue cysts that persist throughout the lifetime of the host. Recognition of T. gondii by TLRs is essential for robust IL-12 and IFN-γ production, two major cytokines involved in host resistance to the parasite. In the murine model of infection, robust IL-12 and IFN-γ production have been largely attributed to T. gondii profilin recognition by the TLR11 and TLR12 heterodimer complex, resulting in Myd88-dependent IL-12 production. However, TLR11 or TLR12 deficiency failed to recapitulate the acute susceptibility to T. gondii infection seen in Myd88-/- mice. T. gondii triggers inflammasome activation in a caspase-1-dependent manner resulting in cytokine release; however, it remains undetermined if parasite-mediated inflammasome activation impacts IFN-γ production and host resistance to the parasite. Using mice which lack different inflammasome components, we observed that the inflammasome played a limited role in host resistance when TLR11 remained functional. Strikingly, in the absence of TLR11, caspase-1 and -11 played a significant role for robust CD4+ TH1-derived IFN-γ responses and host survival. Moreover, we demonstrated that in the absence of TLR11, production of the caspase-1-dependent cytokine IL-18 was sufficient and necessary for CD4+ T cell-derived IFN-γ responses. Mechanistically, we established that T. gondii-mediated activation of the inflammasome and IL-18 were critical to maintain robust CD4+ TH1 IFN-γ responses during parasite infection in the absence of TLR11.

Great balls of fire: activation and signalling of inflammatory caspases.

Innate immune responses are tightly regulated by various pathways to control infections and maintain homeostasis. One of these pathways, the inflammasome pathway, activates a family of cysteine proteases called inflammatory caspases. They orchestrate an immune response by cleaving specific cellular substrates. Canonical inflammasomes activate caspase-1, whereas non-canonical inflammasomes activate caspase-4 and -5 in humans and caspase-11 in mice. Caspases are highly specific enzymes that select their substrates through diverse mechanisms. During inflammation, caspase activity is responsible for the secretion of inflammatory cytokines and the execution of a form of lytic and inflammatory cell death called pyroptosis. This review aims to bring together our current knowledge of the biochemical processes behind inflammatory caspase activation, substrate specificity, and substrate signalling.

Regulation, Activation and Function of Caspase-11 during Health and Disease.

Caspase-11 is a pro-inflammatory enzyme that is stringently regulated during its expression and activation. As caspase-11 is not constitutively expressed in cells, it requires a priming step for its upregulation, which occurs following the stimulation of pathogen and cytokine receptors. Once expressed, caspase-11 activation is triggered by its interaction with lipopolysaccharide (LPS) from Gram-negative bacteria. Being an initiator caspase, activated caspase-11 functions primarily through its cleavage of key substrates. Gasdermin D (GSDMD) is the primary substrate of caspase-11, and the GSDMD cleavage fragment generated is responsible for the inflammatory form of cell death, pyroptosis, via its formation of pores in the plasma membrane. Thus, caspase-11 functions as an intracellular sensor for LPS and an immune effector. This review provides an overview of caspase-11-describing its structure and the transcriptional mechanisms that govern its expression, in addition to its activation, which is reported to be regulated by factors such as guanylate-binding proteins (GBPs), high mobility group box 1 (HMGB1) protein, and oxidized phospholipids. We also discuss the functional outcomes of caspase-11 activation, which include the non-canonical inflammasome, modulation of actin dynamics, and the initiation of blood coagulation, highlighting the importance of inflammatory caspase-11 during infection and disease.

Functional crosstalk between non-canonical caspase-11 and canonical NLRP3 inflammasomes during infection-mediated inflammation.

Inflammation is a part of the body's immune response for protection against pathogenic infections and other cellular damages; however, chronic inflammation is a major cause of various diseases. One key step in the inflammatory response is the activation of inflammasomes, intracellular protein complexes comprising pattern recognition receptors and other inflammatory molecules. The role of the NLRP3 inflammasome in inflammatory responses has been extensively investigated; however, the caspase-11 inflammasome has been recently identified and has been classified as a 'non-canonical' inflammasome, and emerging studies have highlighted its role in inflammatory responses. Because the ligands and the mechanisms for the activation of these two inflammasomes are different, studies to date have separately described their roles, although recent studies have reported the functional cooperation between these two inflammasomes during an inflammatory response. This review discusses the studies investigating the functional crosstalk between non-canonical caspase-11 and canonical NLRP3 inflammasomes in the context of inflammatory responses; moreover, it provides insight for the development of novel anti-inflammatory therapeutics to prevent and treat infectious and inflammatory diseases.

Guanylate-binding protein 5 licenses caspase-11 for Gasdermin-D mediated host resistance to Brucella abortus infection.

Innate immune response against Brucella abortus involves activation of Toll-like receptors (TLRs) and NOD-like receptors (NLRs). Among the NLRs involved in the recognition of B. abortus are NLRP3 and AIM2. Here, we demonstrate that B. abortus triggers non-canonical inflammasome activation dependent on caspase-11 and gasdermin-D (GSDMD). Additionally, we identify that Brucella-LPS is the ligand for caspase-11 activation. Interestingly, we determine that B. abortus is able to trigger pyroptosis leading to pore formation and cell death, and this process is dependent on caspase-11 and GSDMD but independently of caspase-1 protease activity and NLRP3. Mice lacking either caspase-11 or GSDMD were significantly more susceptible to infection with B. abortus than caspase-1 knockout or wild-type animals. Additionally, guanylate-binding proteins (GBPs) present in mouse chromosome 3 participate in the recognition of LPS by caspase-11 contributing to non-canonical inflammasome activation as observed by the response of Gbpchr3-/- BMDMs to bacterial stimulation. We further determined by siRNA knockdown that among the GBPs contained in mouse chromosome 3, GBP5 is the most important for Brucella LPS to be recognized by caspase-11 triggering IL-1ß secretion and LDH release. Additionally, we observed a reduction in neutrophil, dendritic cell and macrophage influx in spleens of Casp11-/- and Gsdmd-/- compared to wild-type mice, indicating that caspase-11 and GSDMD are implicated in the recruitment and activation of immune cells during Brucella infection. Finally, depletion of neutrophils renders wild-type mice more susceptible to Brucella infection. Taken together, these data suggest that caspase-11/GSDMD-dependent pyroptosis triggered by B. abortus is important to infection restriction in vivo and contributes to immune cell recruitment and activation.

Identification and functional characterization of three caspases in Takifugu obscurus in response to bacterial infection.

Caspases are evolutionarily conserved proteases, which are inextricably linked with the apoptosis and immune system in mammals. However, the expression pattern and function of some caspases remain largely unknown in pufferfish. In this study, three different pufferfish caspases (caspase-2 (Pfcasp-2), caspase-3 (Pfcasp-3), and caspase-8 (Pfcasp-8)) were characterized, and their expression patterns and functions were determined following Aeromonas hydrophila infection. The open reading frames of Pfcasp-2, -3, and -8 are 1,320, 846, and 1455 bp, respectively. Analyses of sequence alignment and phylogenetic tree showed that casp-2, -3, and -8 share 52%-65%, 33%-40%, 63%-78% overall sequence identities with those of other vertebrates, respectively. 3D structures of Pfcasp-2, -3, and -8 enjoy conservation in core area together, while each owns a distinctive profile. Comparisons of deduced amino acid sequences indicated that Pfcaspases possessed the caspase domain and conserved active sites like 'HG' and 'QACXG' (X for R or G). qRT-PCR results revealed that Pfcasp-2, -3, and -8 were expressed constitutively in a wide range of organs, especially in immune-related organs including whole blood and kidney. In vitro, the expressions of the three caspases (Pfcasp-2, 3, and -8) and immune-related genes (IgM and IL-8) were significantly up-regulated in kidney leukocytes after A. Hydrophila challenge and inhibitors treatment. The expressions of Pfcasp-2 and Pfcasp-3 were successfully inhibited in the kidney leukocytes by Ac-DEVD-CHO (an inhibitor to caspase-3), but the expression of Pfcasp-8 was not affected. Cellular localization analysis showed that the distribution of Pfcasp-2, -3, and -8 was in cytoplasm. Further, overexpression of Pfcasp-2, -3, or -8 was found to cause DNA damage and apoptosis, suggesting that three caspases may be related to apoptosis and mediate different apoptosis pathways in pufferfish. Moreover, the expressions of these caspases were also up-regulated in whole blood and kidney after A. hydrophila challenge, indicating their possible involvement in the immune response against A. hydrophia stimulation. Taken together, the results of this study suggest that the caspase-2,-3, and -8 may play an important role in the apoptosis and immune response in pufferfish.

An effector caspase Sp-caspase first identified in mud crab Scylla paramamosain exhibiting immune response and cell apoptosis.

Apoptosis plays a key role in the immune defense against pathogen infection, and caspase is one of the most important protease enzyme families, which could initiate and execute apoptosis. Among crustaceans, several caspase genes have been reported. However, caspase in mud crab Scylla paramamosain, have not been identified yet. Here, in the present study, we characterized a new caspase, named as Sp-caspase, from S. paramamosain. The full-length cDNA sequence of Sp-caspase contained 966 bp open reading frame, encoding 322 amino acids, and its molecular weight was 36 kDa. This gene has three conserved domains of the caspase family, a prodomain, a large subunit P20 and a small subunit P10. Phylogenetic analysis showed that Sp-caspase was clustered into an effector caspase group. Sp-caspase mainly distributed in midgut, hepatopancreas, hemocytes and female ovaries, and the transcript was significantly regulated in different tissues after being challenged with Vibrio parahaemolyticus, Vibrio alginolyticus or LPS. After infection with V. alginolyticus, the apoptosis rate of hemocytes notably increased, while the mRNA level of Sp-caspase and hydrolysis activity of caspase 3/7 significantly decreased. Furthermore, in vitro assays showed that the recombinant protein tSp-caspase (deletion of Sp-caspase prodomain) could efficiently recognize and cleave human caspase 3/7 substrate Ac-DEVD-pNA, functioning as an effector caspase. Meanwhile, heterologous expression of Sp-caspase in several cell lines (HEK293T cells, HeLa cells and HighFive cells) could specifically induce cell apoptosis. Taken together, these data demonstrated that Sp-caspase could perform apoptosis as an effector caspase. In addition, it might be a negative regulator of hemocytes apoptosis under pathogen infection, which would contribute to homeostasis and immune defense of hemocytes in S. paramamosain.

Inflammatory caspases are innate immune receptors for intracellular LPS.

The murine caspase-11 non-canonical inflammasome responds to various bacterial infections. Caspase-11 activation-induced pyroptosis, in response to cytoplasmic lipopolysaccharide (LPS), is critical for endotoxic shock in mice. The mechanism underlying cytosolic LPS sensing and the responsible pattern recognition receptor are unknown. Here we show that human monocytes, epithelial cells and keratinocytes undergo necrosis upon cytoplasmic delivery of LPS. LPS-induced cytotoxicity was mediated by human caspase-4 that could functionally complement murine caspase-11. Human caspase-4 and the mouse homologue caspase-11 (hereafter referred to as caspase-4/11) and also human caspase-5, directly bound to LPS and lipid A with high specificity and affinity. LPS associated with endogenous caspase-11 in pyroptotic cells. Insect-cell purified caspase-4/11 underwent oligomerization upon LPS binding, resulting in activation of the caspases. Underacylated lipid IVa and lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) could bind to caspase-4/11 but failed to induce their oligomerization and activation. LPS binding was mediated by the CARD domain of the caspase. Binding-deficient CARD-domain point mutants did not respond to LPS with oligomerization or activation and failed to induce pyroptosis upon LPS electroporation or bacterial infections. The function of caspase-4/5/11 represents a new mode of pattern recognition in immunity and also an unprecedented means of caspase activation.

A perspective on mammalian caspases as positive and negative regulators of inflammation.

Members of the caspase family of cysteine proteases coordinate the morphological and biochemical events that typify apoptosis. However, neutralization of caspase activity in mammals fails to block death in response to most proapoptotic stimuli. This is because many cell death triggers provoke mitochondrial dysfunction upstream of caspase activation as a consequence of BAX/BAK channel opening. Although genetic or pharmacological inactivation of caspases fails to block cell death in most instances, it does convert the phenotype from apoptosis to necrosis. This has important implications for how the immune system responds to such cells, as necrotic cells provoke inflammation whereas apoptotic cells typically do not. Here, we propose an alternative perspective on apoptosis-associated caspase function by suggesting that these proteases are activated, not to kill, but to extinguish the proinflammatory properties of dying cells. This perspective unifies the mammalian caspase family as either positive or negative regulators of inflammation.

Neutrophil pyroptosis: new perspectives on sepsis.

Pyroptosis is a caspase-1 or caspase-4/5/11-dependent programmed cell death associated with inflammation, which is initiated by inflammasomes or cytosolic LPS in innate immunity. Sepsis is a life-threatening organ dysfunction caused by an imbalance in the body's response to infection. It is a complex interaction between the pathogen and the host's immune system. Neutrophils play the role of a double-edged sword in sepsis, and a number of studies have previously shown that regulation of neutrophils is the most crucial part of sepsis treatment. Pyroptosis is one of the important forms for neutrophils to function, which is increasingly understood as a host active immune response. There is ample evidence that neutrophil pyroptosis may play an important role in sepsis. In recent years, a breakthrough in pyroptosis research has revealed the main mechanism of pyroptosis. However, the potential value of neutrophil pyroptosis in the treatment of sepsis did not draw enough attention. A literature review was performed on the main mechanism of pyroptosis in sepsis and the potential value of neutrophils pyroptosis in sepsis, which may be suitable targets for sepsis treatment in future.

Molecular characterization of three caspases from Bostrychus sinensis and their transcriptional responses to bacteria and viruses.

The caspase family proteins are aspartate-specific cysteine proteases that transmit extracellular signals to cells, ultimately cause apoptosis and therefore play a key role in cellular immunity. In this study, we cloned and characterized three caspases from Chinese black sleeper (Bostrychus sinensis), Bscasp-1, Bscasp-8 and Bscasp-9. Real-time PCR analysis showed that Bscasp-1, Bscasp-8 and Bscasp-9 were universally expressed in all tested tissues of B. sinensis. Expression analyses showed that after poly(I:C) stimulation and bacterial (Vibrio parahaemolyticus) infection, the three caspases were significantly upregulated. After poly(I:C) stimulation, the change of Bscasp-1 expression in the head kidney was the most obvious; peak expression was about 80.78-fold more than that of the control. In addition, the expression of Bscasp-8 and Bscasp-9 in the peripheral blood and liver was 167.99- and 17.98-fold higher than that in the control group, respectively. After V. parahaemolyticus infection, the expression peaks of Bscasp-1 and Bscasp-8 in the peripheral blood and spleen were 85.82-fold and 280.83-fold that of the control. However, the expression of Bscasp-9 in the peripheral blood was upregulated only 8.33-fold higher than that in the control group. These results indicate that Bscasp-1, Bscasp-8 and Bscasp-9 are likely involved in response to viral and bacterial infection.

Recent Advances in Lipopolysaccharide Recognition Systems.

Lipopolysaccharide (LPS), commonly known as endotoxin, is ubiquitous and the most-studied pathogen-associated molecular pattern. A component of Gram-negative bacteria, extracellular LPS is sensed by our immune system via the toll-like receptor (TLR)-4. Given that TLR4 is membrane bound, it recognizes LPS in the extracellular milieu or within endosomes. Whether additional sensors, if any, play a role in LPS recognition within the cytoplasm remained unknown until recently. The last decade has seen an unprecedented unfolding of TLR4-independent LPS sensing pathways. First, transient receptor potential (TRP) channels have been identified as non-TLR membrane-bound sensors of LPS and, second, caspase-4/5 (and caspase-11 in mice) have been established as the cytoplasmic sensors for LPS. Here in this review, we detail the brief history of LPS discovery, followed by the discovery of TLR4, TRP as the membrane-bound sensor, and our current understanding of caspase-4/5/11 as cytoplasmic sensors.

Emerging Proviral Roles of Caspases during Lytic Replication of Gammaherpesviruses.

Due to their roles in the regulation of programmed cell death and inflammation, the cellular caspase proteases are considered antiviral factors. However, recent studies have revealed examples of proviral functions for caspases. Here, we review a growing body of literature on the role of caspases in promoting the replication of human gammaherpesviruses. We propose that gammaherpesviruses have evolved ways to redirect these enzymes and to use their activation to support viral replication and immune evasion.

Leucocyte integrins, but neither caspases nor NLR inflammasome are associated with lipopolysaccharide recognition and response in barramundi (Lates calcarifer).

The inflammatory response of fish to LPS is subdued, attributed to absence of TLR4, a key pro-inflammatory receptor for LPS in mammals. Nevertheless, LPS is processed in fish in a T-independent manner and is a protective antigen in fish vaccines, yet pathways for processing LPS in fish remain to be elucidated. Here, we report that caspases and NOD-like receptor inflammasomes typically responsible for LPS recognition and processing in mammals lack critical domains or are absent in barramundi (Lates calcarifer). On the other hand, leucocyte integrins MAC-1 and LFA-1 were detected on the surface of neutrophil- and lymphocyte-like cells respectively in the barramundi spleen by immunocytochemistry, and leucocytes displaying MAC-1 or LFA-1 bound to Factor X and ESM-1 respectively. Exposure to MAC-1 and LFA-1 induced significant IL-1ß expression post-stimulation with LPS compared to unstimulated and isotype controls, but the differences observed in TNF-α expression were inconclusive. Our findings implicate MAC-1 and LFA-1 involvement in immune processing of LPS in barramundi and in antigen processing in fish.