Inflammasomes are multiprotein complexes of the innate immune system that orchestrate development of inflammation by activating the secretion of proinflammatory cytokines, IL-1β and IL-18. The LPS of Gram-negative bacteria have been shown to activate a novel, noncanonical inflammasome by directly binding in the cytosol to human caspase-4 and mouse caspase-11. Activation of noncanonical inflammasome exerts two major effects: it activates the NLRP3–caspase-1–mediated processing and secretion of IL-1β and IL-18 and induces the inflammatory cell death, pyroptosis, via gasdermin D. This previously unexpected cytosolic LPS sensing of the innate immune system provides critical hints for host response to Gram-negative bacterial infections and development of different inflammatory diseases. However, many of its molecular regulatory mechanisms are yet to be discovered. In this review, we provide comprehensive analysis of current understanding of intracellular LPS detection and pyroptosis via noncanonical inflammasome and discuss the recently proposed mechanisms of its function and regulation.

The discovery of the cytoplasmic inflammasome protein complex in 2002 was a breakthrough in our understanding of how the immune system triggers inflammation. The inflammasomes are protein complexes of the innate immune system that induce inflammation in response to microbial infection and endogenous danger signals. Inflammasomes activate inflammatory caspases, cysteine-dependent aspartate-directed proteases, which mediate proteolytic processing and secretion of the proinflammatory cytokines IL-1β and IL-18 and induce a lytic type of cell death, pyroptosis.

The inflammasomes include both canonical and noncanonical inflammasomes. Canonical NLRP3 inflammasome is activated by various microbial pathogen-associated molecular patterns (PAMPs) or cell-derived damage-associated molecular patterns (DAMPs) (1). The structural diversity of its activators suggests that NLRP3 inflammasome does not directly recognize particular molecular structures. It is likely that PAMPs and DAMPs activate NLRP3 inflammasome by inducing changes in cellular homeostasis that trigger its activation. The upstream events that activate NLRP3 inflammasome include potassium efflux, calcium signaling, lysosomal disruption, mitochondrial reactive oxygen species production, and the release of oxidized mitochondrial DNA (reviewed in Ref. 2). Canonical NLRP3 inflammasome activation results in the recruitment of procaspase-1 through adapter molecule apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) to the inflammasome complex. Subsequently, caspase-1 is activated, and it proteolytically processes proinflammatory cytokines IL-1β and IL-18, resulting in their secretion from innate immune cells like macrophages and dendritic cells (1).

A noncanonical caspase-11 inflammasome was described in 2011 in mice. The group of V. Dixit (3) discovered that the originally used caspase-1 knockout mouse strain contained a caspase-11 protein product lacking most of the small catalytic subunit and therefore encoding a nonfunctional caspase-11. The authors microinjected a caspase-11 bacterial artificial chromosome transgene into the Casp1/11−/− double-knockout embryo to generate mice lacking only caspase-1 (3). Comparative studies involving Casp1−/− and Casp1/11−/− knockout mice showed that caspase-1 is essential for the secretion of IL-1β and IL-18, whereas caspase-11 activates pyroptosis, an inflammatory form of cell death, in response to infections with Gram-negative bacteria (3). Subsequent studies have shown that caspase-4 and caspase-5, the orthologs of mouse caspase-11, function as a noncanonical inflammasome in human cells (46). In this article, we review the current knowledge on noncanonical caspase-4/5/11 inflammasomes with focus on their role in host defense and inflammation.

Bacterial LPS, a major immunogenic constituent of the outer leaflet of the cell wall of Gram-negative bacteria, is the essential PAMP responsible for development of inflammation during Gram-negative bacterial infections. LPS is recognized in extracellular space by TLR4, a pattern-recognition receptor of the innate immunity that activates signaling cascades that initialize and orchestrate antibacterial inflammatory response (7). Upon activation, the TLR4R dimerizes and recruits myeloid differentiation factor 88 (MyD88) adapter, which initiates signaling cascade inducing transcription of proinflammatory cytokines IL-1β, IL-6 and TNF by NF-κB (reviewed in Ref. 8). This MyD88-dependent signaling also serves as a priming step for canonical inflammasome activation by inducing NLRP3 gene expression (9). During LPS recognition, the TLR4R is also endocytosed, and in the endosomal compartment, it signals through TIR domain–containing adaptor-inducing IFN-β (TRIF), inducing type I IFN production (10).

In 2011, Kayagaki et al. (3) demonstrated for the first time that caspase-11 is necessary for proteolytic processing and secretion of IL-1β in mouse macrophages infected with Gram-negative bacteria but dispensable when the same cells were infected with Gram-positive bacteria or treated with nonbacterial inflammasome activators. Importantly, caspase-11 rather than caspase-1 was required for the pyroptotic cell death to occur, whereas NLRP3 and ASC were dispensable. This discovery implied that caspase-11 confers specific sensitivity to a ligand characteristic for Gram-negative bacteria (3). This novel caspase-11 function was later confirmed in further studies involving other Gram-negative bacteria (11). Subsequently, it was demonstrated that guanylate-binding protein (GBP)–mediated bacterial escape from the vacuoles was essential for caspase-11 activation (12, 13), indicating that caspase-11–activating factor must be an integral component of the internalized bacteria. Hagar et al. (14) and Shi et al. (15) showed that this activating factor is hexa- and penta-acylated, but not tetra-acylated LPS. LPS and its lipid A component directly interact with the caspase activation and recruitment domain (CARD) of the caspase-11 in the cytosol of infected cells (15). This interaction was shown to be the essential trigger of endotoxic shock, independently of TLR4 (14), by inducing autoproteolysis of caspase-11 (16, 17).

Caspase-4 and caspase-5 are human orthologs of mouse caspase-11 (18, 19). Many of the conclusions regarding their immunological functions have been based on research on murine caspase-11. Despite the similarities between caspase-11 and caspase-5, most publications consider caspase-4 to be the functional homolog of caspase-11. Caspase-4 has been shown to be directly involved in LPS sensing. The role of caspase-5 in LPS detection has remained more unclear. Casson et al. (5) showed that caspase-4 mediates both IL-1β secretion and pyroptosis in human macrophages infected with intracellular Gram-negative bacteria expressing type III and type IV secretion systems, which export LPS from vacuolar compartment to cytosol. Interestingly, caspase-5 seemed not to play any detectable role in recognition of these pathogens, suggesting distinct functions for caspase-4 and caspase-5 in human macrophages. In addition, caspase-5, but not caspase-4, is LPS and IFN inducible (20), also suggesting differential function for these caspases. Like caspase-11, the human caspase-4 directly interacts with LPS and purified lipid A through the CARD domains (15). In vitro study demonstrated that caspase-4 interacts with LPS micelles via CARD domain and disaggregates them to promote its autoactivation (21). However, unlike caspase-11, human caspase-4 binds with high affinity to tetra-acylated LPS (22), demonstrating broader specificity of caspase-4 compared with its mouse counterpart.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC) belongs to naturally occurring phospholipids that have a broad range of biological activities including modulation of inflammatory response. OxPAPC is a DAMP released by dying cells, reaching high concentrations in damaged tissues (23). OxPAPC has both proinflammatory and anti-inflammatory effects. It has been shown to function as a TLR4 agonist and antagonist (24, 25). Zanoni et al. (26) showed that oxPAPC activates IL-1β processing and secretion of biologically active cytokine through caspase-11–dependent pathway in mouse dendritic cells without inducing pyroptosis. In contrast to this work, Chu et al. (27) demonstrated that oxPAPC inhibits the noncanonical inflammasome in macrophages: oxPAPC directly bound caspase-4 and caspase-11 competing with LPS binding and consequently inhibited LPS-induced pyroptosis and IL-1β release. These studies suggest that inflammatory effects of oxPAPC are cell specific. In vivo study with septic shock mouse models revealed that oxPAPC has antagonistic activity during sepsis (27). This report supports the role of oxPAPC as an anti-inflammatory regulator and therefore provides a basis for novel therapies targeting noncanonical inflammasomes during Gram-negative bacterial sepsis.

In search for the effector of inflammatory cell death, three independent groups identified gasdermin D as the executioner of pyroptosis (18, 28, 29). Gasdermin D turned out to be a direct substrate of caspase-11 and critical for cell death and IL-1β release, but not its maturation (18). Interestingly, caspase-4, caspase-5, and caspase-11 are capable of processing gasdermin D independently of the NLRP3 and its adapter protein ASC (29). However, in an in vitro assay, caspase-1 was able to efficiently process both proinflammatory cytokines and gasdermin D, whereas caspase-11 proteolytic activity was limited to gasdermin D only (30). Upon processing, gasdermin D is cleaved at Asp275, and its N-terminal fragment forms 13–35-nM-diameter pores in the cellular membrane, causing cell lysis (31, 32). Overexpression of N-terminal gasdermin D fragment is sufficient to induce pyroptosis (28). Recently, it was shown that site-specific autoprocessing of caspase-4 or caspase-11 generates a p10 product of these caspases, which is required for the cleavage of gasdermin D and induction of pyroptosis (33). It is tempting to speculate that differential processing of the caspase-4/11 may have a role in switching these caspases from NLRP3 inflammasome-activating to pyroptosis-inducing form.

In contrast to gasdermin D processing ability, noncanonical caspases-4/5/11 are less efficient in direct processing of proinflammatory cytokines IL-1β and IL-18 compared with caspase-1. It has been shown that these caspases require NLRP3 inflammasome for processing and secretion of IL-1β and IL-18 (3). However, caspase-4–induced activation of NLRP3 is dependent on potassium efflux in contrast to caspase-4–induced pyroptotic cell death in human and mouse macrophages (6, 34). Other signaling events that precede caspase-4–induced NLRP3 activation have remained largely uncharacterized. Innate immune cells can secrete proteins through the conventional endoplasmic reticulum–Golgi secretory pathway as well as through unconventional, extracellular vesicle-mediated pathway. Our previous studies have shown that several NLRP3 activators induce robust vesicle-mediated protein secretion in human macrophages (3538). Recently, we have shown that the noncanonical caspase-4 inflammasome also activates vesicle-mediated protein secretion in macrophages (39). It is highly likely that this caspase-4–induced protein secretion is dependent on NLRP3 inflammasome. These results also highlight that vesicle-mediated protein secretion is an essential part of innate immune response following canonical and noncanonical inflammasome activation, possibly serving as an auxiliary mechanism of the cytokine delivery (40).

Under stressful conditions such as intestinal dysbiosis, Gram-negative bacteria secrete outer membrane vesicles (OMVs) that contain LPS. Vanaja et al. (41) first demonstrated that OMVs secreted by Gram-negative bacteria are taken up by murine macrophages through clathrin-mediated endocytosis, and the LPS they carry reaches the cytosol by escaping from early endosomal compartment. This OMVs-mediated LPS delivery triggered robust caspase-11–dependent pyroptosis and inflammation both in vitro and in vivo (41). Wacker et al. (42) proposed that upon delivery to cytosol, the LPS-rich OMV surface recruits multiple caspase-4 molecules, bringing them in close proximity and facilitating their autoactivation. This mechanism may also explain robust activation of caspase-11/caspase-4 inflammasome in infections by intracellular bacteria. Similarly to intracellular bacteria vacuole escape, the process of immune recognition of endocytosed OMVs requires the action of a family of small IFN-induced GTPases, GBP, in particular GBP2 (13, 43). In an attempt of elucidating the mechanism of GBP involvement in OMVs sensing, Santos et al. (44) investigated the signaling involved in response of mouse bone marrow–derived macrophages to purified Escherichia coli–derived OMVs. They found that GBP1, GBP2, and GBP5 were rapidly but transiently recruited to the internalized OMVs, binding to LPS. Moreover, GBP recruitment to OMVs increased the affinity of internalized OMVs to caspase-11. Interestingly, recent study suggested that internalization of OMVs for caspase-11 signaling also requires TLR4-TRIF signal (45). Therefore, at initial stage of interaction with host cells, bacterial OMVs are likely recognized by TLR4 in a similar manner as extracellular bacteria but being small enough to be internalized, may subsequently reach the cytosol to exert noncanonical inflammasome activation. This initial extracellular recognition of OMVs seems to be critical for pyroptosis to occur (45). Importantly, a recent report pointed to the possible distinct roles of caspase-4 and caspase-5 in recognition of OMVs in human cells—Bitto et al. (46) found that in human monocytes, caspase-4 is critical for recognition of Pseudomonas aeruginosa–LPS, whereas caspase-5 is rather involved in recognition of P. aeruginosa–OMVs. This finding supports the concept that human caspase-4 and caspase-5 have distinct roles in human innate immunity, and it is possible that these caspases require distinct molecular environment surrounding LPS-rich surfaces or complexing of LPS with additional factors for recognition.

Of note, another study investigating the mechanism of injury caused by OMVs from enterohemorrhagic E. coli suggested that non-LPS components of OMVs might also contribute to host cell killing. Bielaszewska et al. (47) found that Shiga toxin 2a, cytolethal distending toxin V, hemolysin, and flagellin are trafficked inside the cell in OMVs, where they exert cell cycle arrest and caspase-9–dependent apoptosis. Although this observation was true in nonleukocytic cells, it is conceivable that similar mechanisms can also be involved in macrophage and dendritic cell responses to OMVs. It remains to be determined if caspase-11 or its human orthologs are involved in OMVs-induced apoptosis.

Endocytosis of bacterial OMVs is not the only route for extracellular LPS to reach cytosol and activate caspase-4 and/or caspase-11. Deng et al. (48) demonstrated that high-mobility group box 1 (HMGB1) protein binds and delivers extracellular LPS to the cytosol of mouse macrophages to activate caspase-11. HMGB1 directly binds LPS extracellularly, and HMGB1–LPS complex is internalized to lysosomes by the receptor for advanced glycation end products (RAGE). Leakage of lysosomal LPS to cytosol resulted in caspase-1 activation. In mouse model of sepsis, it was shown that HMGB1 is required for caspase-11–dependent pyroptosis and lethality (48). It was also shown that in vivo LPS stimulation of liver hepatocytes promotes HMGB1 production in a TLR4-dependent manner (48). These findings highlight the role of HMGB1, TLR4, and noncanonical inflammasome in lethal Gram-negative bacterial sepsis.

The cyclic GMP-AMP synthase (cGAS)/stimulator of IFN genes (STING) pathway is a component of the innate immune system that functions to detect the presence of cytosolic DNA and/or DNA damage (49). The cGAS/STING pathway has an important role in tumorigenesis, as well as in recognition of DNA viruses and some intracellular bacteria. Upon binding to DNA, cGAS activates STING, resulting in production of type I IFNs (49). Two recent reports have shown that there is cross-talk between cGAS/STING and caspase-4/11 pathways. Kerur et al. (50) demonstrated that cGAS drives noncanonical inflammasome activation in age-related macular degeneration, which is characterized by retinal pigmented epithelial cell death. The cGAS-driven IFN signaling was shown to function as a conduit for mitochondrial damage-induced caspase-4 activation in human cells and caspase-11 in mice (50). This finding expands the innate immune-sensing repertoire of cGAS and caspase-4 to noninfectious human disease. Zhang et al. (51) in turn showed that STING, which is also known as transmembrane protein 173 (TMEM173), drives lethal coagulation in sepsis. They demonstrated that the STING/TMEM173-dependent increase in cytosolic calcium drives caspase-11–induced gasdermin D cleavage and activation. Gasdermin D activation triggered the release of F3, the key initiator of blood coagulation. Genetic or pharmacological inhibition of the STING/TMEM173, gasdermin D, or F3 protein blocked systemic coagulation and improved animal survival in mice models of sepsis (51). In conclusion, this study shows that STING and gasdermin D cross-talk is involved in blood clotting during lethal bacterial infections.

The noncanonical inflammasome-mediated responses must be tightly controlled because of detrimental role of prolonged inflammasome activity, resulting in pyroptosis. It is now becoming clear that caspase-4/5/11 activity is a subject of a number of direct and indirect regulatory mechanisms (Fig. 1).

FIGURE 1.

Noncanonical caspase-4/5/11 inflammasome activation. Activation of IFNR and TLR4/MD-2 by IFN and LPS, respectively, provides signal 1 transcriptional activation of the expression of inflammasome components and proinflammatory cytokines. LPS is taken up by the cells in the form of a complex with HMGB1 in receptor-mediated endocytosis or during the endocytosis of bacteria or their OMVs. Subsequently, LPS can reach the cytosol of host cells by escaping the endosomal pathway, facilitated by the action of GBPs. Liberated LPS in the cytosol is recognized by caspase-4/5/11 directly through binding to its CARD domain. Upon LPS recognition, caspase-4/5/11 multimerizes and undergoes autoactivation in the process of noncanonical inflammasome activation. In a similar manner, oxPAPC also may directly interact with caspase-4/5/11 in the cytosol. Active caspase-4/5/11 complexes process the gasdermin D, whose N-terminal fragment forms pores in cellular membrane–triggering pyroptosis and activate the NLRP3 inflammasome through unknown pathway (red arrow). NLRP3 inflammasome activity is perpetuated by signal 2 transmembrane calcium/potassium fluxes, which are activated through gasdermin D pores in the cell membrane. Activity of inflammasome is also associated with increased secretion of extracellular vesicles and modulation of their cargo.

FIGURE 1.

Noncanonical caspase-4/5/11 inflammasome activation. Activation of IFNR and TLR4/MD-2 by IFN and LPS, respectively, provides signal 1 transcriptional activation of the expression of inflammasome components and proinflammatory cytokines. LPS is taken up by the cells in the form of a complex with HMGB1 in receptor-mediated endocytosis or during the endocytosis of bacteria or their OMVs. Subsequently, LPS can reach the cytosol of host cells by escaping the endosomal pathway, facilitated by the action of GBPs. Liberated LPS in the cytosol is recognized by caspase-4/5/11 directly through binding to its CARD domain. Upon LPS recognition, caspase-4/5/11 multimerizes and undergoes autoactivation in the process of noncanonical inflammasome activation. In a similar manner, oxPAPC also may directly interact with caspase-4/5/11 in the cytosol. Active caspase-4/5/11 complexes process the gasdermin D, whose N-terminal fragment forms pores in cellular membrane–triggering pyroptosis and activate the NLRP3 inflammasome through unknown pathway (red arrow). NLRP3 inflammasome activity is perpetuated by signal 2 transmembrane calcium/potassium fluxes, which are activated through gasdermin D pores in the cell membrane. Activity of inflammasome is also associated with increased secretion of extracellular vesicles and modulation of their cargo.

Close modal

Transmembrane calcium and potassium ion fluxes are known regulators of NLRP3 inflammasome as primary probes of membrane integrity. Potassium efflux has been shown to be a prerequisite for caspase-11 activation (52). Interestingly, transient receptor potential channel 1 (TRPC1) protein, a member of a family of nonselective ion channels, has been identified as a direct substrate for caspase-11 (53). Caspase-11, but not caspase-1, degrades TRPC1 upon activation, leading to increased secretion of IL-1β, but not affecting cell death (53). Similarly, bone marrow–derived macrophages from Trpc1 knockout mice developed stronger inflammatory response than wild-type cells upon infection with Gram-negative bacteria (53). The currently available data suggest that potassium/calcium fluxes exert important roles in regulation of the noncanonical inflammasome activation. It seems plausible that formation of gasdermin D pores during pyroptosis dramatically changes transmembrane cationic equilibria, further amplifying the inflammatory responses (40, 54). However, precise mechanisms affected by transient and local modulations of cation concentrations, in particular whether they directly modulate caspase-4 activity, remain to be characterized in detail. Specifically, whether transmembrane cationic mobilization acts downstream of gasdermin D pore formation, or is activated earlier in the signaling cascade for activation of caspase-11, as suggested by Case et al. (52), remains an open question.

Significant efforts of current research are focused on finding direct molecular regulators of noncanonical inflammasome signaling. Among these, GBP family of small molecular GTPases, highly upregulated by IFN, emerges as a group of critical regulators of caspase-4/11–mediated inflammation. GBPs have initially been shown to mediate disassembly of bacteria-containing vacuoles and internalized OMVs to expose LPS for noncanonical inflammasome activation (12, 44); however, they also seem to play proinflammatory role after the LPS-carrying bacteria reach the cytosol by facilitating activation of caspase-4/11 (13, 55). Moreover, IFN-induced GBP expression was shown to amplify caspase-11 activation in intracellular bacteria recognition, although it was not essential for pyroptosis to occur (56). Hence, it is likely that the function of GBPs in regulation of caspase-4/11 is rather associated with the NLRP3 inflammasome activation axis and not with pyroptosis.

In the search for previously unidentified caspase-4 regulators, Benaoudia et al. (57) used a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screen in human monocytic U937 cells. They identified IFN regulatory factor (IRF) 2 to be essential for the pyroptosis upon cytosolic LPS sensing. IRF2 was required for caspase-4 function in human monocytic cells, and it directly bound to caspase-4 promoter to transcription of caspase-4 gene. Induction of another member of the IRF family, IRF1, with IFN-γ was able to restore the pyroptosis in IRF2 knockout cells. Pyroptosis and IL-1β release were completely abolished in IRF1/IRF2 double-knockout cells upon infection with Gram-negative bacteria (57). These results show a cooperative role between transcription factors of the IRF family in regulation of caspase-4 gene expression. Concurrently, Kayagaki et al. (58) showed that IRF2 directly regulates gasdermin D expression, but not that of caspase-11, in mouse macrophages. Similarly to findings described above, IRF1 was able to compensate for IRF2 deficiency to induce transcriptional upregulation of gasdermin D gene expression (58). Together, these recent findings underline a critical role of IFN signaling in noncanonical inflammasome response. The reports also show that there are species-dependent differences in regulation of noncanonical inflammasome activation.

It was recently shown that other members of the gasdermin family also serve as potential regulators of noncanonical inflammasome function. Gasdermin B, a substrate of both inflammatory and apoptotic caspases, was shown to interact with caspase-4 CARD domain, promoting its activation in a process proposed as “noncanonical pyroptosis” (59). Interestingly, gasdermin B was highly expressed in the leukocytes of septic shock patients, which was associated with increased release of the gasdermin D (59). Gasdermin E, which has been associated with caspase-3–mediated apoptotic signaling, has also been shown to compensate for pyroptosis in the absence of canonical inflammasome pathway (60). Recently, the propyroptotic role of gasdermin E has also been identified. Zhang et al. (61) found that granzyme B produced by NK cells cleaves tumor-expressed gasdermin E, resulting in tumor cell pyroptosis, without engaging proinflammatory caspases. This mechanism may augment antitumor immunity. Despite these recent findings, little is known about functional redundancy in the gasdermin family and their regulatory roles in the function of noncanonical inflammasome.

There are several studies showing that the activity of NLRP3 is regulated by posttranslational mechanisms like deubiquitination and phosphorylation (reviewed in Ref. 2). Latest discoveries indicate that noncanonical caspase-11 is also controlled by posttranslational mechanisms. Polyubiquitination of proteins is a well-described mechanism of targeting unwanted proteins to proteasome for degradation. In one study, the E3 ubiquitin ligase Nedd4 has been shown to target caspase-11 for degradation to limit the extent of pyroptosis (62). Although it is yet unknown what signals regulate the polyubiquitination of noncanonical caspases, it certainly indicates that noncanonical inflammasome activity is also under control of conserved protein degradation mechanisms. Whether protein phosphorylation signaling cascades regulate caspase-4/5/11 activity is currently unknown. A study by Chen et al. (63) recently identified a novel mechanism limiting the caspase-11–dependent pyroptosis through l-adrenaline–induced production of cAMP, indicating that modulation of intracellular cAMP levels could be a promising target for future therapies of septic shock.

Inflammasomes are critical to both local and systemic inflammation and form the major signaling hub that regulates inflammation. They have a key role in antimicrobial defense, and their role in development and pathology of atherosclerosis, diabetes, neurodegenerative, autoimmune diseases, and endotoxemia-induced sepsis is evident. It can be expected that treatments specifically targeting inflammasomes will have a major effect on human health in the coming 10–15 y. Discovery of noncanonical inflammasome and roles of caspase-4 and caspase-11 in pyroptosis have a profound impact on our understanding of Gram-negative bacterial infections and etiology of sepsis and possibly other inflammatory diseases. Pyroptosis may play an important role in the pathogenesis of atherosclerosis. Caspase-11 is expressed in endothelial cells (64), and direct activation of these cells by LPS could represent a novel mechanism triggering cardiovascular inflammation. Hyperactivation of caspase-4 pathway, as is the case in sepsis, is associated with multiorgan failure and may result in the death of the host if intensive care is not implemented. Treatment of sepsis and many other inflammatory diseases may be significantly improved when components and regulators of noncanonical inflammasome are targeted in therapy. These could include specific targeting of intracellular pathways regulating the delivery of LPS to caspase-4 and possible interference with gasdermin D–mediated cell membrane permeabilization. Studies throughout recent years provided knowledge on intermediate regulators of LPS trafficking from outside the cell to the cytosol. Inhibition of HMBG1–RAGE interaction could have beneficial effect in limiting the amount of cytosolic LPS, in consequence possibly constraining the pyroptotic effect. Similarly, targeting GBP could inhibit the extent of noncanonical inflammasome activation. Currently, ongoing research in the area provides novel findings about regulation of noncanonical inflammatory responses and will reveal novel drug targets and therapeutic opportunities in management of Gram-negative bacterial sepsis and metabolic diseases with underlying inflammatory background.

This work was supported by National Science Centre, Poland Grant 2018/28/C/NZ6/00069 (to W.C.) and The Academy of Finland Decision 322638.

Abbreviations used in this article:

ASC

apoptosis-associated speck-like protein containing a caspase activation and recruitment domain

CARD

caspase activation and recruitment domain

cGAS

cyclic GMP-AMP synthase

DAMP

damage-associated molecular pattern

GBP

guanylate-binding protein

HMGB1

high-mobility group box 1

IRF

IFN regulatory factor

OMV

outer membrane vesicle

oxPAPC

oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine

PAMP

pathogen-associated molecular pattern

STING

stimulator of IFN genes

TMEM173

transmembrane protein 173

TRPC1

transient receptor potential channel 1.

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The authors have no financial conflicts of interest.