Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases.

The non-canonical inflammasome pathway was described which engages caspase-11 to mediate pyroptosis and the subsequent release of IL-1α, IL-1β and IL-18 in TLR4-independent way. Cathepsin B is capable of activating caspase-11 under cell-free conditions which may regulate non-canonical NLRP3 inflammasome pathway. In this study, we aimed to further investigate cathepsin B as potential activators of proinflammatory caspases which may be released upon proinflammatory stimuli and regulate non-canonical NLRP3 inflammasome pathway by modulating the activity of caspase-11. Pharmacological and gene-silencing approaches were used to evaluate the impact of cathepsin B on regulating non-canonical NLRP3 inflammasome pathway in wild-type and TLR4-/- Kupffer cells. A sepsis model was also created to investigate the effect of cathepsin B on survival. Meanwhile, cathepsin B activity and the expression level of caspase-4 were detected in human peripheral blood mononuclear cells (PBMC) which were separated from patients suffered from SIRS or sepsis and healthy volunteers. LPS stimulation caused cathepsin B activity and caspase-11 expression increase in TLR4-/- mice. Cathepsin B activity inhibition reduced the activation of caspase-11 and inflammasome and benefited survival in TLR4-/- mice. Upregulation of cathepsin B activity and caspase-4 activation was found in PBMC of patients with SIRS or sepsis. Our results suggest a critical role for cathepsin B as activators of proinflammatory caspases-11 and the regulatory effect in LPS-induced caspases-11-dependent necrosis.

The NLRP11 protein is only expressed in primates and participates in the activation of the canonical NLRP3 and non-canonical NLRP3 inflammasome activation after infection with gram-negative bacteria. Here, we generated a series of defined NLRP11 deletion mutants to further analyze the role of NLRP11 in NLRP3 inflammasome activation. Like the complete NLRP11 deletion mutant (NLRP11-/-), the NLRP11 mutant lacking the NAIP, C2TA, HET-E, and TP1 (NACHT) and leucine-rich repeat (LRR) domains (NLRP11∆N_LRR) showed reduced activation of the canonical NLRP3 inflammasome, whereas a pyrin domain mutant (NLRP11∆PYD) had no effect on NLRP3 activation. The NLRP11-/- and NLRP11∆N_LRR mutants, but not the NLRP11∆PYD mutant, also displayed reduced activation of caspase-4 during infection with the intracytosolic, gram-negative pathogen Shigella flexneri. We found that the human-adapted, acid-fast pathogen Mycobacterium tuberculosis and the opportunistic pathogen Mycobacterium kansasii both activate the non-canonical NLRP11 inflammasome in a caspase-4/caspase-5-dependent pathway. In conclusion, we show that NLRP11 functions in the non-canonical caspase-4/caspase-5 inflammasome activation pathway and the canonical NLRP3 inflammasome pathway and that NLRP11 is required for full recognition of mycobacteria by each of these pathways. Our work extends the spectrum of bacterial pathogen recognition by the non-canonical NLRP11-caspase4/caspase-5 pathway beyond gram-negative bacteria.IMPORTANCEThe activation of inflammasome complexes plays a crucial role in intracellular pathogen detection. NLRP11 and caspase-4 are essential for recognizing lipopolysaccharide (LPS), a molecule found in gram-negative bacteria such as the human pathogens Shigella spp., which activate both canonical NLRP3 and non-canonical inflammasome pathways. Through a series of deletion mutants, we demonstrate that the NACHT and LRR domains of NLRP11, but not its pyrin domain, are critical for detection of S. flexneri. Notably, our research reveals that the acid-fast bacterium M. tuberculosis is also detected by NLRP11 and caspase-4, despite not producing LPS. These findings significantly expand the range of pathogens recognized by NLRP11 and caspase-4 to now include acid-fast bacteria that do not contain LPS and underscore the versatility of these innate immune components in pathogen detection.

The non-canonical inflammasome, comprising caspase-4 in humans, initiates pyroptosis upon sensing cytosolic lipopolysaccharide (LPS) from Gram-negative bacteria. Caspase-4 activation also depends on several guanylate-binding proteins (GBPs), which associate with the surface of cytosolic bacteria. Here, we investigated how caspase-4 accesses its cognate ligand, the hydrophobic lipid A moiety of LPS, and the role of GBPs in this process. GBP1 was essential for caspase-4 activation during infection. Mechanistically, GBP1 deformed the LPS-containing outer membrane of cytosolic bacteria, acting as a GTP-dependent mechanoenzyme. In vitro, GBP1 fragmented LPS micelles and promoted caspase-4/LPS complex formation, thereby enhancing LPS-induced caspase-4 activation. Fragmented LPS micelles presented additional micelle tips that served as binding and activation sites for caspase-4, indicating that caspase-4 engages LPS membranes with defined geometry rather than individual LPS molecules. Thus, GBP-mediated deformation of the LPS-rich outer bacterial membrane generates regions of positive curvature that expose lipid A, enabling caspase-4 binding, oligomerization, and activation.

The gamma-interferon (IFNγ)-inducible guanylate-binding proteins (GBPs) promote host defense against gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate sensing of the gram-negative bacterial outer membrane component lipopolysaccharide (LPS) by the noncanonical caspase-4 inflammasome. There are seven human GBP paralogs, and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS. The GBP1 microcapsule recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates "free" LPS through protein polymerization. We demonstrate that supplementation of either recombinant polymerized GBP1 or GBP2 to an invitro reaction is sufficient to enhance LPS-induced caspase-4 activation. This provides a revised mechanistic framework for noncanonical inflammasome activation where GBP1 or GBP2 assembles cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to gram-negative bacterial infections.

Lipopolysaccharide (LPS) from gram-negative bacteria is a classical pathogen-associated molecular pattern and a strong inducer of immune responses. While the detection of LPS on the cell surface and in the endosome by Toll-like receptor 4 (TLR4) has been studied for some time, it has only recently been discovered that LPS can also be sensed in the cytosol of cells by a noncanonical inflammasome pathway, resulting in the activation of the cysteine protease caspase-11. Intriguingly, activation of this pathway requires the production of interferons (IFNs) and the induction of a class of IFN-induced GTPases called guanylate-binding proteins (GBPs), which have previously been linked to cell-autonomous killing of intracellular microbes. In this study, we review the recent advances in our understanding of cytosolic LPS sensing and the function of mammalian GBPs.

OPINION article Front. Microbiol., 21 March 2011Sec. Cellular and Infection Microbiology - closed section https://doi.org/10.3389/fmicb.2011.00057

Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1β, IL-18, and danger signals, as well as the induction of pyroptotic cell death. Inflammasome assembly and activation occurs in response to sensing of infectious, sterile and self-derived molecular patterns by cytosolic pattern recognition receptors, including the Nod-like receptor NLRP3. While these responses are essential for host defense, excessive and uncontrolled NLRP3 inflammasome responses cause and contribute to a wide spectrum of inflammatory diseases, including gout. A key step in NLRP3 inflammasome assembly is the sequentially nucleated polymerization of Pyrin domain (PYD)- and caspase recruitment domain (CARD)-containing inflammasome components. NLRP3 triggers polymerization of the adaptor protein ASC through PYD-PYD interactions, but ASC polymerization then proceeds in a self-perpetuating manner and represents a point of no return, which culminates in the activation of caspase-1 by induced proximity. In humans, small PYD-only proteins (POPs) lacking an effector domain regulate this key process through competitive binding, but limited information exists on their physiological role during health and disease. Here we demonstrate that POP1 expression in macrophages is sufficient to dampen MSU crystal-mediated inflammatory responses in animal models of gout. Whether MSU crystals are administered into a subcutaneous airpouch or into the ankle joint, the presence of POP1 significantly reduces neutrophil infiltration. Also, airpouch exudates have much reduced IL-1β and ASC, which are typical pro-inflammatory indicators that can also be detected in synovial fluids of gout patients. Exogenous expression of POP1 in mouse and human macrophages also blocks MSU crystal-induced NLRP3 inflammasome assembly, resulting in reduced IL-1β and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1β secretion. We further determined that the mechanism for the POP1-mediated inhibition of NLRP3 inflammasome activation is through its interference with the crucial NLRP3 and ASC interaction within the inflammasome complex. Strikingly, administration of an engineered cell permeable version of POP1 was able to ameliorate MSU crystal-mediated inflammation in vivo, as measured by neutrophil infiltration. Overall, we demonstrate that POP1 may play a crucial role in regulating inflammatory responses in gout.

Gossypol induces pyroptosis in mouse macrophages via a non-canonical inflammasome pathway

Bacteria-released components can modulate host innate immune response in the absence of direct host cell–bacteria interaction. In particular, bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide. However, further precise understanding of innate immune-modulation by bacterial OMVs remains elusive. Here, we present evidence that flagellated bacteria-released OMVs can trigger NLRC4 canonical inflammasome activation via flagellin delivery to the cytoplasm of host cells. Salmonella typhimurium-derived OMVs caused a robust NLRC4-mediated caspase-1 activation and interleukin-1β secretion in macrophages in an endocytosis-dependent, but guanylate-binding protein-independent manner. Notably, OMV-associated flagellin is crucial for Salmonella OMV-induced inflammasome response. Flagellated Pseudomonas aeruginosa-released OMVs consistently promoted robust NLRC4 inflammasome activation, while non-flagellated Escherichia coli-released OMVs induced NLRC4-independent non-canonical inflammasome activation leading to NLRP3-mediated interleukin-1β secretion. Flagellin-deficient Salmonella OMVs caused a weak interleukin-1β production in a NLRP3-dependent manner. These findings indicate that Salmonella OMV triggers NLRC4 inflammasome activation via OMV-associated flagellin in addition to a mild induction of non-canonical inflammasome signaling via OMV-bound lipopolysaccharide. Intriguingly, flagellated Salmonella-derived OMVs induced more rapid inflammasome response than flagellin-deficient Salmonella OMV and non-flagellated Escherichia coli-derived OMVs. Supporting these in vitro results, Nlrc4-deficient mice showed significantly reduced interleukin-1β production after intraperitoneal challenge with Salmonella-released OMVs. Taken together, our results here propose that NLRC4 inflammasome machinery is a rapid sensor of bacterial OMV-bound flagellin as a host defense mechanism against bacterial pathogen infection.

Interferon (IFN)-inducible guanylate binding proteins (GBPs) mediate cell-autonomous host resistance to bacterial pathogens and promote inflammasome activation. The prevailing model postulates that these two GBP-controlled activities are directly linked through GBP-dependent vacuolar lysis. It was proposed that the rupture of pathogen-containing vacuoles (PVs) by GBPs destroyed the microbial refuge and simultaneously contaminated the host cell cytosol with microbial activators of inflammasomes. Here, we demonstrate that GBP-mediated host resistance and GBP-mediated inflammatory responses can be uncoupled. We show that PVs formed by the rodent pathogen Chlamydia muridarum, so-called inclusions, remain free of GBPs and that C. muridarum is impervious to GBP-mediated restrictions on bacterial growth. Although GBPs neither bind to C. muridarum inclusions nor restrict C. muridarum growth, we find that GBPs promote inflammasome activation in C. muridarum-infected macrophages. We demonstrate that C. muridarum infections induce GBP-dependent pyroptosis through both caspase-11-dependent noncanonical and caspase-1-dependent canonical inflammasomes. Among canonical inflammasomes, we find that C. muridarum and the human pathogen Chlamydia trachomatis activate not only NLRP3 but also AIM2. Our data show that GBPs support fast-kinetics processing and secretion of interleukin-1β (IL-1β) and IL-18 by the NLRP3 inflammasome but are dispensable for the secretion of the same cytokines at later times postinfection. Because IFN-γ fails to induce IL-1β transcription, GBP-dependent fast-kinetics inflammasome activation can drive the preferential processing of constitutively expressed IL-18 in IFN-γ-primed macrophages in the absence of prior Toll-like receptor stimulation. Together, our results reveal that GBPs control the kinetics of inflammasome activation and thereby shape macrophage responses to Chlamydia infections.

Nonalcoholic fatty liver disease (NAFLD) is becoming increasingly common in countries with a Western style high fat diet. The caspase-1-dependent inflammasome pathway contributes to the pathogenesis of NAFLD and vascular inflammation. Current understandings are that caspase-1 and caspase-4 (caspase-11 in mice) work synergistically in the canonical inflammasome pathway. However, recent research indicates that caspase-4 can be activated independently via the non-canonical inflammasome pathway. While the pathogenesis of the canonical inflammasome pathway in NAFLD has been studied, the non-canonical inflammasome pathway is not well understood in this severe liver disease. Our data mining and experimental analysis revealed the following findings: 1) there is no overlap of the top ten upregulated pathways between the canonical and non-canonical inflammasome pathways; 2) there is no overlap of the top ten downregulated pathways between the canonical and non-canonical Inflammasome pathways; 3) the top pathways specifically mediating upregulation of non-canonical pathways included two cell biology pathways apoptosis signaling and death receptor signaling; 4) High fat feeding promotes the recruitment of F4/80 + / CCR2 + / CX3CR1 + hepatic macrophages in mouse livers; and 5) High fat feeding promotes the expression of caspase-11 in hepatic macrophages while activated GSDMD is predominately expressed in low density hepatic macrophages. Our results have demonstrated that canonical and non-canonical inflammasome pathways are differentially regulated in NAFLD and that three retinoid x receptor alpha signaling pathways may specifically inhibit the upregulation of non-canonical inflammasome pathways but not the canonical pathways. These findings have provided novel insights on differential regulatory mechanisms of canonical and non-canonical inflammasomes in the liver and their potential roles in sensing hyperlipidemic DAMPs and initiating the pathogenesis of NAFLD. Word count: 391. Funding source: NIH-NHLBI

Septic shock is the leading cause of morbidity and mortality in intensive care units worldwide. Gram-negative bacteria constitute one of the most common causes of sepsis that results in high fatality by initiating an excessive and uncontrolled host inflammatory response. At the epicenter of this response is the innate immune detection of lipopolysaccharides (LPS). In addition to TLR4 recognition of LPS, recent studies revealed a new LPS sensing mechanism in the cytosol. Inflammatory caspases, such as caspase-11, detect LPS to execute pyroptosis, an inflammatory form of cell death, and caspase-1 activation. A prerequisite for the activation of noncanonical inflammasome is the transcriptional induction of caspase-11, which is mediated by TLR4-TRIF, type I interferon, and complement signaling. Additionally, guanylate binding proteins (GBPs), interferon regulatory factor 1, and IRGB10 orchestrate the release of LPS from the vacuolar bacterial pathogens into the cytosol. While the host factors involved in LPS activation of caspase-11 are fairly characterized, the role of bacterial factors in this process is not clear. Our new findings from this study delineate the bacterial components necessary for the optimal engagement of the cytosolic LPS sensing pathway and eliciting noncanonical inflammasome responses.

Activating inflammatory caspases and releasing pro-inflammatory mediators are two essential functions of inflammasomes which are triggered in response to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). The canonical inflammasome pathway involves the activation of inflammasome and its downstream pathway via the adaptor ASC protein, which causes caspase 1 activation and, eventually, the cleavage of pro-IL-1b and pro-IL-18. The non-canonical inflammasome pathway is induced upon detecting cytosolic lipopolysaccharide (LPS) by NLRP3 inflammasome in Gram-negative bacteria. The activation of NLRP3 triggers the cleavage of murine caspase 11 (human caspase 4 or caspase 5), which results in the formation of pores (via gasdermin) to cause pyroptosis. Ehrlichia is an obligately intracellular bacterium which is responsible for causing human monocytic ehrlichiosis (HME), a potentially lethal disease similar to toxic shock syndrome and septic shock syndrome. Several studies have indicated that canonical and non-canonical inflammasome activation is a crucial pathogenic mechanism that induces dysregulated inflammation and host cellular death in the pathophysiology of HME. Mechanistically, the activation of canonical and non-canonical inflammasome pathways affected by virulent Ehrlichia infection is due to a block in autophagy. This review aims to explore the significance of non-canonical inflammasomes in ehrlichiosis, and how the pathways involving caspases (with the exception of caspase 1) contribute to the pathophysiology of severe and fatal ehrlichiosis. Improving our understanding of the non-canonical inflammatory pathway that cause cell death and inflammation in ehrlichiosis will help the advancement of innovative therapeutic, preventative, and diagnostic approaches to the treatment of ehrlichiosis.

Inflammasomes are intracellular protein complexes, members of the innate immune system, and their activation and regulation play an essential role in maintaining homeostatic conditions against exogenous and endogenous stimuli. Inflammasomes occur as cytosolic proteins and assemble into a complex during the recognition of pathogen-associated or danger-associated molecular patterns by pattern-recognition receptors in host cells. The formation of the inflammasome complex elicits signaling molecules of proinflammatory cytokines such as interleukin-1β and interleukin 18 via activation of caspase-1 in the canonical inflammasome pathway whereas caspase-11 in the case of a mouse and caspase-4 and caspase-5 in the case of humans in the non-canonical inflammasome pathway, resulting in pyroptotic or inflammatory cell death which ultimately leads to neuroinflammation and neurodegenerative diseases. Inflammasome activation, particularly in microglial cells and macrophages, has been linked to aging as well as age-related neurodegenerative diseases. The accumulation of abnormal/ misfolded proteins acts as a ligand for inflammasome activation in neurodegenerative diseases. Although recent studies have revealed the inflammasomes' functionality in both in vitro and in vivo models, many inflammasome signaling cascade activations during biological aging, neuroinflammation, and neurodegeneration are still ambiguous. In this review, we comprehensively unveil the cellular and molecular mechanisms of inflammasome activation during neuronal aging and age-related neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, prion disease, and amyotrophic lateral sclerosis.

Maltol (3-hydroxy-2-methyl-4-pyrone) is used widely as a food and cosmetic supplement, and it has antioxidant and anti-inflammatory activities. Inflammasome causes the maturation and secretion of interleukin (IL)-1β and -18 through the activation of caspase-1 (Casp1), which contributes to various inflammatory diseases. This study examined the effects of maltol on the inflammasome activation in macrophages and mice. Lipopolysaccharide (LPS)-primed macrophages were treated with a trigger of NLRP3, NLRC4, AIM2, or non-canonical (NC) inflammasomes in the presence of maltol. The secretion of IL-1β and IL-18 and the cleavage of Casp1 were analyzed as indices of inflammasome activation. Mice were injected with LPS and an NLRP3 trigger with or without maltol, and the peritoneal IL-1β secretions were observed. The effects of maltol on reactive oxygen species (ROS) production and Casp1 activity were analyzed to determine the mechanism. Maltol inhibited the activation of NLRP3 and NC inflammasomes, but it did not alter the other inflammasomes. Maltol also attenuated IL-1β secretion resulting from the inflammasome activation in mice. The anti-inflammatory mechanism of maltol was revealed by the inhibition of ROS production and Casp1 activity. Maltol is suggested to be promising as a anti-inflammasome molecule.