NLRP3 is expressed in innate immune cells, mainly macrophages, and NLRP3 is one of the most widely studied inflammasome-forming NLRs. The NLRP3 inflammasome is activated by an increasing number of stimulators, including viruses, fungi, bacteria, pore-forming toxins, reactive oxygen species (ROS), extracellular ATP, potassium efflux and calcium influx, mitochondrial dysfunction, nigericin, urate crystals, amyloid-B, and various environmental insults such as asbestos and silica. Direct binding of a ligand to NLRP3, however, remains unclear since all stimuli are structurally and chemically very different (23). Instead, it is believed that NLRP3 can sense a common cellular event that is induced by the wide variety of stimuli, such as potassium efflux or production of ROS, however the underlying mechanism remains to be determined. The NLRP3 inflammasome is important for maintaining a balanced in the gastrointestinal tract since Nlrp3^-/- mice displayed a unique intestinal microbiota and are more susceptible to experimental colitis (24–26). Nlrp3^-/- mice were also found to have reduced expression of AMPs, which could potentially influence the microbiota composition (25).

NLRP3 is also involved in the non-canonical inflammasome which is activated by the binding of cytosolic lipopolysaccharides (LPS) of Gram-negative bacteria to the murine inflammatory caspase-11 (27, 28). In humans, the orthologs of murine caspase-11 are caspase-4 and -5 (29). Cytosolic LPS induces self-cleavage and oligomerization of caspase-11 that subsequently processes gasdermin D to induce pore formation in the plasma membrane. The pore formation leads to potassium efflux, which in turn activates the NLRP3inflammasome resulting in the processing of pro-IL-1β and pro-IL-18 and secretion of IL-1β and IL-18 (30, 31). Caspase-11 is widely expressed in a variety of cells of both the hematopoietic- and non-hematopoietic lineage, including epithelial cells and macrophages (32). It was suggested that since caspase-11 directly binds to LPS it only contributes to defense against gram-negative bacteria, however, a recent study reported that caspase-11 plays a crucial role in generating immune responses against Listeria monocytogenes and Staphylococcus aureus infection (see paragraph 2.3. The NLRP6 inflammasome) (33).

The NLRC4 inflammasome is activated in response to flagellin and the needle and inner rod protein of Type III and Type IV secretion systems (T3SS, T4SS) of Gram-negative bacteria, such as Salmonella Typhimurium, Shigella flexneri, Legionella pneumophila and Pseudomonas aeruginosa. These ligands do not directly bind to NLRC4, but instead, NLRC4 requires the NLR family apoptosis inhibitory proteins (NAIPs) to sense and bind to the bacterial ligands. Murine NAIP1 binds the needle protein, NAIP2 the inner rod protein and NAIP5/6 binds flagellin (34–36). Humans have two isoforms, NAIP and the extended NAIP*. NAIP* binds to flagellin, whereas NAIP binds the needle and rod proteins (37–39). Direct binding of flagellin to NAIP5 changes the conformation of NAIP5 allowing recruitment and binding of NLRC4, which promotes further recruitment and activation of the next incoming NLRC4 protein, resulting in a wheel-shaped structure consisting of a single flagellin-NAIP5 and NLRC4 protomers (40–44). Upon completion of the formation of the wheel-shaped structure, the CARDs of NLRC4 interact and recruit ASC molecules to initiate ASC polymerization into long filaments, known as the ASC specks. The CARD of ASC is exposed on the outside as serves as a docking station for pro-caspase-1 (45). The NLRC4 inflammasome can also function without ASC, since macrophages deficient of ASC display similar levels of pyroptosis in response to NLRC4 ligands (46, 47). The gram-positive bacteria Staphylococcus aureus and Listeria monocytogenes have also been demonstrated to activate the NLRC4 inflammasome. The specific ligand and the exact underlying mechanism how S. aureus (non-flagellated, no T3SS or T4SS) can activate the NLRC4 inflammasome is currently unknown (48). L. monocytogenes flagellin activates the NLRC4 inflammasome (49, 50).

NLPR6 is a lesser studied NLR and is predominantly expressed in the intestine. Similar to the NLRP3 and NLRC4 inflammasome, NLRP6 assembles into a multiprotein complex consisting of ASC and caspase-1, promoting cleavage and secretion of inflammatory cytokines and cleavage of gasdermin D to initiate pyroptosis (51). One of the first reports on NLRP6 demonstrated that it is important in maintaining homeostasis of the microbiome (52). Changes in the composition of the microbiome results in an imbalance in the microbial community that is associated with a wide range of human diseases, such as inflammatory bowel disease, cancer, obesity, and diabetes. Dysbiosis can be established in multiple different ways, for example during infections, caused by mutation in host factors or changes to the diet. Additionally, the host immune system has also been demonstrated to be important in maintaining homeostasis of the microbiome. NLRP6 activation was one of the first host factors of the innate immune system suggested to regulate the microbiome composition (52). It was demonstrated that Asc^-/- , Caspase-1^-/- , Nlrp6^-/- and Il18^-/- mice, but not Il1b^-/- or Il1R^-/- mice, developed severe colitis after dextran sodium sulphate (DSS) treatment and that co-housed wild type mice were also more susceptible to DSS treatment, indicating that the microbiota is responsible for the exacerbation of DSS-induced colitis (51, 52). These findings, however, were later challenged as it was demonstrated that Nlrp6^-/- and Asc^-/- mice and littermate control mice did not reveal any differences in the microbiome composition, nor were they more susceptible to DSS treatment (53). They argued that the differences in the microbiome could be associated with the facilities where the mice are housed. In the report by Levy and co-workers, it was demonstrated that the microbiota-modulated metabolite taurine can activate the NLRP6 inflammasome resulting in pro-IL-18 processing and IL-18 secretion. IL-18 mediates production of antimicrobial peptides (AMPs) and a balanced AMP landscape is required for the maintenance of intestinal homeostasis (51). In contrast, the levels of the metabolites histamine and spermine were increased in Asc^-/- mice, and both histamine and spermine suppressed IL-18 production in colon explants. These data indicate that microbiota-modulated metabolites can either positively or negatively regulate the NLRP6 inflammasome and their absence/presence in the gastrointestinal tract can have major effects on gut health and disease (51). NLRP6 expression in intestinal epithelial cells protects against colorectal cancer and colitis via a mechanism dependent on caspase-1 mediated IL-18 production (52–55). NLRP6 has also been demonstrated to be expressed in goblet cells where it regulates mucus production (56). Interestingly, Nlrp6^-/- mice are more resistant to infection with Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli, demonstrating NLRP6 can act as a negative regulator in innate immune signaling and host defense (57). Similarly, NLRP6 serves as a negative regulator during infection with Staphylococcus aureus (58). In a later report it was demonstrated that in response to Listeria monocytogenes and Staphylococcus aureus infection, NLRP6 directly binds to lipoteichoic acid (LTA) in macrophages leading to the recruitment of both caspase-1 and caspase-11 resulting in IL-18 secretion (33). In addition to its role in the host defense against bacterial species, NLRP6 can also bind to RNA from enteric viruses, and it can sense microbiota-associated metabolites (51, 59). In response to viral RNA, however, the NLRP6 does not form an inflammasome with ASC and caspase-1, but instead binds the RNA helicase DHX15 and interacts with mitochondrial antiviral signaling protein (MAVS) to induce a type I interferon (T1IFN) response (59). Although NLRP6 can form the classical inflammasome, many aspects of NLRP6 are still poorly understood; NLRP6 plays a major role in maintenance of microbiota homeostasis, can bind distinct ligands, is anti-bacterial and anti-viral, but is also a negative regulator of innate immune signaling. Future studies will have to determine the underlying mechanisms of NLRP6 activation and immune signaling during infections.

Absent in melanoma 2 (AIM2) is a member of the HIN200 family and is a cytosolic sensor of double-stranded DNA (dsDNA). It is the first non-NLR receptor identified that can form an inflammasome with ASC and caspase-1 (60–63). Upon binding of bacterial, viral and mitochondrial dsDNA to the HIN200 domain of AIM2, multiple sensors cluster together resulting in binding of ASC via PYD-PYD interaction. The CARD domain of ASC subsequently recruits procaspase-1 to the complex allowing for autocleavage of caspase-1, processing of pro-IL-1β, pro-IL18 and gasdermin D, resulting in cytokine secretion and pyroptosis. Gram-positive bacteria, including but not limited to Staphylococcus aureus and Listeria monocytogenes release dsDNA in the cytosol after escaping the phagolysosome that activates the AIM2 inflammasome (64, 65).

L. monocytogenes is a Gram-positive bacterium responsible for listeriosis acquired by contaminated food. Murine or human macrophages infected with L. monocytogenes demonstrated activation of multiple inflammasomes, including AIM2-ASC, NLRP3-caspase-1, NAIP5-NLRC4, NLRP6-caspase-11, caspase-1, caspase-11, and NLRP1B (69, 70). In addition, cytosolic listeriolysin O (LLO) and lipoteichoic acid (LTA) have also been implicated in inflammasome activation ( Table 1 ).

After endocytosis, L. monocytogenes promotes lysosomal disruption, leading to the release of lysosomal contents, translocation of Listeria to the cytoplasm and inflammasome activation. NLRP3 is activated when L. monocytogenes escapes the vacuole in conditions with limited cytosolic replication and lysosomal damage, and NLRC4 is activated by the bacterial flagellum. However, AIM2 recognition may predominate in conditions with high cytosolic replication. AIM2-ASC-caspase-1 is the inflammasome sensor of L. monocytogenes DNA and its expression requires type I IFN signaling (106). AIM2 connects DNA directly, resulting in atypical inflammasome with the activation of caspase-1, IL-1β and IL-18 and consequent induction of pyroptosis (71, 107).

Gao and co-workers (72) showed that infection by L. monocytogenes induced the activation of the NLRP3 inflammasome through the Mst1/2-ALK pathways; as well as the participation of the interaction between Nek7 and NLRP3 via JNK that promoted the intensification of the host defense against Listeria infection by increasing the maturation and release of the pro-inflammatory cytokine IL-1β. Furthermore, pore-forming toxin and LLO played a critical role in the process. Recently, an increase in transcriptional levels of inflammatory factors was demonstrated, where levels of proteins associated with NLRP3, NF-κB and ERK were increased after Listeria infection and consequent inflammation of the central nervous system (73). Therefore, blocking inflammasome activation could decrease inflammation and improve patient survival and prognosis as a potential therapeutic intervention target for L. monocytogenes infection (108).

Interestingly, mouse macrophages infected with L. monocytogenes activated NLRP6 and caspase-11. NLRP6, activated by Gram-positive bacteria or lipoteichoic acid (LTA) in the host cytosol, has a function to limit inflammation and to regulate systemic infection by pathogens (109, 110). Activation of NLRP6 in macrophages infected by L. monocytogenes or LTA induced caspase-11 processing which promoted IL-1β/IL-18 maturation, caspase-1 activation and regulation of IL-18 secretion, revealing a novel signaling pathway by which the NLRP6 inflammasome was activated by cytosolic LTA, recruiting pro-inflammatory caspases in systemic infection by Gram-positive bacterial pathogens (33).

NLRP1B is activated under conditions that lead to a decrease in cytosolic ATP (76), suggesting that energy reduction may allow NLRP1B to detect intracellular pathogens, including Gram-positive bacteria. The pore-forming toxin and LLO were required for the rupture of the vacuolar membrane, reduction of ATP levels and energy stress through disruption of mitochondrial function by L. monocytogenes. In addition, Listeria invasion into the cytosol may contribute to ATP depletion. Listeria-infected fibroblasts expressed NLRP1B, pro-caspase-1 and IL-1β, suggesting that NLRP1B detected a signal generated by metabolic stress (74). Another study showed that LLO was recruited and oligomerized leading to activation of Lyn and Syk kinases in lipid membrane rafts. Upregulation of ASC phosphorylation was verified in a secondary step, showing that ASC phosphorylation can utilize another signaling pathway in addition to LLO-mediated Lyn-Syk kinases. These data demonstrated that pathogens could activate different inflammasomes in order to control immune responses and benefit the spread of the pathogen in the host (75).

During inflammasome activation, the increased influx of macrophages may be detrimental to the defense against L. monocytogenes in vivo. The Mint3-mediated pathway, which regulates ATP production in macrophages, has contributed to the regulation of severe listeriosis in mice, suggesting a potential therapeutic target (111). In this way, the regulation of gasdermin D by caspase-1 was enhanced in Mint3–/– mice infected with Listeria, accompanied by a strong induction of pyroptosis and bacterial shedding (112). Other molecules have also been described as activating inflammasomes; among them the P2X7 receptor, a potent activator of NLRP3 inflammasome and the release of the pro-inflammatory cytokines IL-1β and IL-18 (113). However, very little is known about P2X5, belong to the P2X family, in the immune system. Recently, P2X5 has been shown to play an important role in protective immune responses associated with inflammasome activity, including in vivo infection by L. monocytogenes (114).

The protein kinase, p38-regulated/activated protein kinase (PRAK), contributes to the regulation of cell proliferation, stress responses and cell migration. Furthermore, PRAK dysfunction impaired the antibacterial activity of neutrophils and the formation of neutrophil extracellular traps (NETs) (115). Assays with PRAK-deficient mice showed high mortality when compared to wild-type mice after infection by L. monocytogenes. In addition, Listeria-induced autophagic activities were reduced in the absence of PRAK. In this way, PRAK potentiated bactericidal activity with increased ROS production, inflammasome activation and autophagy induction, providing a new perspective for innate immunity against listeriosis (116).

Staphylococcus aureus is responsible for human infections such as pneumonia, endocarditis and sepsis with high morbidity/mortality and consequent increase in hospital costs (117). S. aureus can manipulate cell death by apoptosis, necrosis or pyroptosis to colonize and establish infection in different hosts (118). The inflammasome plays a crucial role in rising an effective immune response against S. aureus infection, controlling bacterial load and the magnitude of the adaptive immune response (7). S. aureus pore-forming toxins (α-toxin, LukAB and PVL) induced NLRP3-ASC inflammasome activation in human and murine phagocytes in vitro. In addition, methicillin-resistant S. aureus strain activated NLRP3-ASC inflammasomes through K+ efflux, caspase-1 activation, and release of IL-1β and IL-18 cytokines with induction of pyroptosis (77, 78). Peptidoglycan resistant to lysozyme strongly inhibited IL-1β production in response to infection in vitro and in vivo, suggesting that the S. aureus subverts IL-1β secretion by modifying its peptidoglycan from cell wall (119). Thus, new therapeutic approaches targeting peptidoglycan degradation in host immune cells could control bacterial infections.

ASC assembly and AIM2 activation by intracellular S. aureus after suppression of perforin-2 were described for the first time by Pastar and co-workers (79). In this work, S. aureus-infected diabetic foot ulcers triggered the AIM2 inflammasome activation resulting in activation of gasdermin D, and increased levels of IL-1β in tissue samples from chronic ulcers in diabetic patients, contributing to prolonged inflammation. Additionally, phagocytosis and bacterial degradation were related to the activation of NLRP3 inflammasomes and IL-1β secretion in response to both live S. aureus and S. aureus peptidoglycan. Interestingly, IL-1β production was mediated by the NLRP3 activation, and the secretion of IL-6, KC and CCL2 was AIM2-dependent (120). Together, the NLRP3/AIM2 inflammasomes played a critical role in S. aureus infection and could be a therapeutic target to control unwanted inflammation.

The involvement of extracellular vesicles serving as a secretory pathway for the transport of protected bacterial cargo (virulence factors, surface glycoproteins, proteases, etc) to host cells have been reported in several Gram-positive bacteria. Activation of NLRP3 by S. aureus extracellular vesicles have been shown to be dependent on the load of pore-forming toxins and K+ efflux. The critical role of pore-forming toxins was associated with S. aureus extracellular vesicles in triggering inflammasome activation, modulating of innate host immune response during S. aureus infection. In this way, extracellular vesicles serve as an important secretion system that allows the transport of molecules to host cells during infections to modulate cellular functions (121). Similarly, after endocytosis of S. aureus by bovine mammary epithelial cells, the bacteria escaped from the endosomal vesicles, causing inflammation and cell death through the K+ efflux and NLRP3 activation, and consequently pyroptosis of MAC-T cells (80).

Staphylococcal enterotoxin A promotes intestinal barrier dysfunction, triggering an inflammatory response with activation of the NLRP3 inflammasome and the NF-κB/MAPK signaling pathways. Selective inhibitors of NF-κB/MAPK pathways in THP-1 cells attenuated the expression of inflammasome-associated proteins that were upregulated by staphylococcal enterotoxin A. Thus, the development of compounds acting on enterotoxin A may be a promising strategy for the prevention and treatment of diseases caused by the toxin (81). Moreover, the triggering of receptors expressed in myeloid cells 2 (TREM2) is considered a protective factor for the host against bacterial infection; however, their role is unclear. TREM2 or its adapter molecule DAP12 activates β-catenin, the molecule responsible for maintaining cell viability and preventing cell death, in myeloid cells stimulated by M-CSF. Study showed that TREM2 inhibited macrophage pyroptosis induced by S. aureus and Streptococcus pneumoniae, providing a new host defense mechanism against pathogenic microorganisms (122–124). In addition, TREM2 promoted the stabilization and phosphorylation of β-catenin in the nucleus and cytoplasm and down-regulation of NLRP3 inflammasome activation. TREM2/β-catenin also inhibited ASC oligomerization and ASC-NLRP3 association with suppressed pyroptosis in macrophages infected by pyogenic bacteria (80).

Potential role of staphylokinase in the virulence of community associated S. aureus remains unknown. Furthermore, a significant role of staphylokinase in the acute pneumonia model involved the activation of innate immune signals, especially pathways related to the NLRP3 inflammasome. The investigations revealed that staphylokinase contributed to NLRP3 inflammasome activation, increasing K+ efflux, ROS production and NF-κB signaling, exacerbating pulmonary infection. These data improve our understanding of the mechanism of action of staphylokinase in bacterial pathogenesis (80).

Recently, researchers showed that Staphylococcus epidermidis strains induced caspase-1-independent IL-1β release and NLRP3 inflammasome activation in keratinocytes, providing important information during the S. epidermidis-keratinocyte interaction to activate the innate host defense (82).

Cutibacterium acnes (formerly called Propionibacterium acnes) plays a significant role in the development of acne, triggering immunologic defense that are vital for the pathogenesis and clinical appearance of acne vulgaris (148). In addition, C. acnes can also induce intervertebral disc degeneration (149). C. acnes produces proteases that participate in the inflammatory response of acne causing degradation of the extracellular matrix and proteolytic detachment of follicular keratinocytes. Activation of the NLRP3 inflammasome was dependent on metalloproteinase expression by C. acnes and ROS generation in sebocytes (97). C. acnes also induced ROS production, NLRP3 inflammasome activation and IL-8 release in HaCaT keratinocytes. Furthermore, the authors described the anti-inflammatory potential of the medicinal herb Paris polyphylla in reducing the inflammatory responses, proliferation and migration of keratinocytes caused by C. acnes, suggesting a new therapeutic for the treatment of acne (150). In addition, celastrol isolated from the Celastraceae family also exhibited anti-inflammatory activities by suppressing NLRP3 inflammasome activation promoted by C. acnes through inhibition of K63 deubiquitination of NLRP3, and providing evidence for its application in disease therapy (151).

Previously published data demonstrated increased activation of the NLRP3 inflammasome, caspase-1, caspase-5, IL-1β, IL-18 and gasdermin D after infection of C. acnes-infected nucleus pulposus tissue. In this study, it was also shown that the addition of the inflammasome inhibitor NLRP3 MCC950 and the thioredoxin binding protein (TXNIP) reduced the secretions of IL-1β and IL-18, implying an inflammatory response activated by C. acnes via the TXNIP-NLRP3 pathway. In this way, MCC950 can alleviate the inflammatory lesion and pyroptosis of C. acnes-infected cells, slowing the progression of disc degeneration, providing a new direction for the treatment of intervertebral disc degeneration (98).

The NLRP3 inflammasome plays a key role in acne lesions, raising interest in the suppression of this mechanism in the treatment of acne. Activation of NF-κB induced an increase in the expression of inflammatory factors during NLRP3 inflammasome activation. Thus, the suppression of the NF-κB pathway and the activation of the ERK1/2 MAPK signal transduction pathways caused inhibition of caspase-1 and NLRP3 expression, which could be considered therapeutic targets in the treatment of acne (99).

Corynebacterium pseudotuberculosis, a facultative intracellular microorganism, belongs to the Corynebacterium-Mycobacterium-Nocardia-Rhodococcus group responsible for caseous lymphadenitis in goats and sheep. C. pseudotuberculosis is reported worldwide as the cause of significant economic losses affecting meat, wool and milk production, being difficult to detect in subclinically infected animals, to control and eradicate (152). C. pseudotuberculosis infections cause pyogranulomas and abscesses in organs and lymph nodes, with an increase in inflammatory cytokines. IL-1β is an important cytokine during the inflammatory phase in response to pathogens. Zhou and colleagues first described the activation of the NLRP3 inflammasome in IL-1β secretion in macrophages after infection by C. pseudotuberculosis. Activation of NLRP3 was independent of AIM2, but dependent on NF-κB, p38MAPK and partially dependent on TLR4 pathways. These results contribute to the knowledge of the mechanism of IL-1β secretion and of the host’s pro-inflammatory immune response in macrophages infected with C. pseudotuberculosis (100).

Bacillus anthracis, spore-forming Gram-positive bacteria, efficiently kills infected hosts through the systemic action of secreted edema toxin (EdTx) and lethal toxin (LeTx) (153). LeTx targets polymorphonuclear and mononuclear cells during the early stages of infection, paralyzing the host’s innate immune defenses and allowing bacteria to spread and cause systemic infection (154). Furthermore, direct or direct activation of caspase-1 by the NLRP1B inflammasome in response to LeTx was related to macrophage killing (101). Recent publication has shown the involvement of TNFR1, TNFR2 receptors, and the MAPKK/p38/MK2 signaling pathway in LeTx-intoxicated macrophages during NLRP3 inflammasome activation dependent on RIPK1 activity (155).

In contrast to activation of the NLRP3 sensor, little is known about the requirements for NLRP1B activation. Cell death was induced by LeTx toxin and mediated by the NLRP1B inflammasome and proteasome activity. However, inhibition of components such as caspase-1, K⁺ channel, cathepsin B and heat shock can block this activity. In this way, proteasome inhibition could delay the time of death caused by the LeTx toxin (102). When IL-1β and IL-18 are present, the LeTx toxin activates NLRP1B in toxin-responsive rodent macrophages, while resistant macrophages only suffer the consequences of cleavage of LeTx’s mitogen-activated protein kinase and others subtracts. Importantly, LeTx was not able to regulate or activate IL-1β release or induce the formation of murine neutrophil NETs. LeTx also did not induce neutrophil pyroptosis, despite cleaving cytosolic mitogen-activated protein kinase substrates, suggesting a complex mechanism of interaction between different cell types and mediators in vivo that differs from in vitro or ex vivo models (156). Therefore, it is important to know the inflammasome activation in a multicellular context of inflammatory response to a bacterial toxin.

Clostridioides difficile is the leading cause of hospital-acquired infection, resulting in pseudomembranous colitis, toxic megacolon, and even death. The diagnosis of high inflammatory level is a predictor of severe disease with high morbidity and mortality. Toxins A and B produced by C. difficile can stimulate a potent pro-inflammatory response with activation of IL-1β, IL-6, IL-8 and TNFα with inflammasome activation. C. difficile toxins A/B also synergize with MyD88 in the production of IL-23. Furthermore, inhibition of Rho protein GTPase activity by toxins A/B induced the cleavage of pro-IL-1β and IL-8, resulting in the recruitment of innate immune cells (157).

The pyrine inflammasome does not directly recognize pathogens or host-derived molecules, but responds to disturbances in cytoplasmic homeostasis caused by infections leading to inactivation of RhoA GTPase. Activation of these signaling pathways increases IL-1β production and promotes IL-1 receptor expression with uncontrolled activation of the pyrin inflammasome in neutrophils (158). Furthermore, pyrin inflammasome activation can be affected by RIPK3, which modulates the rapamycin (mTOR) pathway, regulating the expression of the gene encoding pyrin and pyrin inflammasome activation (103).

Interactions between bacterial virulence factors and cell receptors are fundamental as possible targets for treating bacterial infectious diseases. Surface layer proteins of C. difficile (SLPs) bind to the lipid rafts, induced caspase-1, and IL-1β production in dose-dependent manners (159). The production of IL-1β induced by C. difficile in macrophages was dependent on caspase-1, MyD88 and TLR2, considered critical in the production of pro-IL-1β. Additionally, the toxigenic C. difficile in the cytosol was necessary for inflammasome activation through the ATP-P2X7 pathway with consequent loss of membrane integrity, release of intracellular content and caspase-1-dependent cell death. In addition, SLPs were released from pyroptotic cells. Consequently, MyD88 and TLR2 were essential components in pro-IL-1β cleavage and inflammasome activation via ATP-P2X7, responsible for inflammation-mediated bacterial clearance during C. difficile infection (104). Together, these results contribute to the identification and understanding of the essential factors of infection caused by C. difficile, allowing new therapeutic strategies to prevent the disease.

Recently, evidence has demonstrated the involvement of mixed-lineage protein kinase (MLKL) in mediating the immune response against severe gas gangrene and enterocolitis caused by Clostridium perfringens. MLKL plays an important role in innate immunity during the process of necroptosis. Regarding C. perfringens, MLKL promoted bacterial eradication, increased host survival and resolution of inflammation, demonstrating that MLKL-NLRP3 inflammasome confers protection to invading pathogens (160).

Another species, Clostridium septicum, a pathogen that causes sepsis and gas gangrene, was able to activate the inflammasome complex in mice and humans. C. septicum secretes a α-toxin responsible for binding to glycosylphosphatidylinositol proteins anchored in the plasma membrane of host cells, forming pores that allow the efflux of Mg²⁺ and K⁺ ions. The efflux of these cytosolic ions triggers NLRP3 inflammasome activation, caspase-1 and gasdermin D activation, IL-1β and IL-18 secretion. The innate detection of the α-toxin is essential for the recognition of C. septicum infection, whose therapeutic blockade may be able to prevent sepsis and death caused by microbial toxins (105).