The NAIP5/NLRC4 inflammasome is perhaps the best-studied inflammasome complex with regard to resistance to infections. Their involvement has been reported against infections such as S. typhimurium (31, 32), L. pneumophila (25), P. aeruginosa (33, 34), Y. pestis (35), S. flexneri (36), and A. veronii (37). NAIP/NLRC4-mediated responses are related to the restriction of bacterial growth due to the active caspase-1-mediated canonical and non-canonical effector mechanisms, highlighting the importance of this inflammasome as a host defense mechanism against a large number of bacterial infections. The best elucidated effector mechanisms involved in the control of infections mediated by caspase-1 are the secretion of inflammatory cytokines IL-1β and IL-18 and the induction of pyroptosis (38).

The term pyroptosis (from the Greek “pyro” meaning fire or fever, and “ptosis” to a fault) was coined in 2001 to describe a pro-inflammatory programed cell death during S. typhimurium infection (39). Morphological and biochemical changes displayed by S. typhimurium-infected dying cells were more closely related to those found in classic necrosis compared with those observed during apoptosis, including the following: (1) diffuse DNA fragmentation with no chromatin condensation; (2) early loss of membrane integrity observed by the simultaneous uptake of annexin V with an impermeable membrane dye; (3) lactate dehydrogenase (LDH) release, suggesting a loss of intracellular content; and (4) independence of any apoptotic caspase. Although cells dying by pyroptosis displayed features of necrosis with an inflammatory outcome, the authors found that this process was highly regulated by active caspase-1, as the addition of inhibitors of caspase-1 (z-YVAD-fmk) abolished S. typhimurium-induced cell death.

The induction of pyroptosis by pathogenic bacteria depends on an active secretion system that translocates bacterial proteins into the cell cytosol, such as the T3SS (SPI-1) of S. typhimurium and type IV (T4SS) of L. pneumophila (12, 13, 40–42). Mutant L. pneumophila (43) or P. aeruginosa (34, 44) lacking flagellin fail to activate caspase-1 and, therefore, are not able to induce pyroptosis and IL-1β secretion in infected macrophages. Accordingly, the transfection of purified flagellin from L. pneumophila and S. typhimurium directly into the cell cytosol is sufficient to trigger caspase-1-dependent pore formation, pyroptosis, and IL-1β secretion (45, 46). Importantly, infection with the non-flagellated bacteria S. flexneri also induces NLRC4-mediated pyroptosis, most likely in response to the inner rod component of T3SS (36).

Although the molecular mechanisms that regulate pyroptosis remain to be elucidated, the model of S. typhimurium infection has given us important knowledge about this form of cell death. The cell lysis observed during pyroptosis seems to result from a highly regulated process of pore formation in the plasma membrane (45, 46). Pores dissipate cellular ionic gradients but allow the retention of larger cytoplasmic constituents, leading to increased liquid osmotic pressure and water influx. These events are followed by cell swelling and subsequent osmotic lysis with the release of intracellular contents, which are potentially inflammatory (45, 46). Caspase-1-dependent DNA cleavage also occurs during pyroptosis (45, 47). However, the DNA cleavage observed during S. typhimurium-induced pyroptosis is independent of caspase-activated DNase (CAD) (45, 47), unlike what is observed during apoptosis, in which the proteolysis of inhibitor of CAD (ICAD) by apoptotic caspases mediates the release of CAD to the nucleus, where it cleaves DNA between nucleosomes. Therefore, pyroptotic cells do not display the typical pattern of oligonucleosomal fragmentation observed during apoptosis, a fact that can be used to distinguish between these two processes of cell death (48).

There is good evidence implicating pyroptosis as an important host defense mechanism mediated by NAIP/NLRC4 that clears intracellular pathogens in vitro. The death of infected macrophages by pyroptosis seems to correlate with a rapid loss of the replicative niche and high bacterial loads are recovered from macrophages deficient in components of inflammasomes or infected with mutant bacterial strains that fail to trigger their activation [reviewed by Bortoluci and Medzhitov (1) and Bergsbaken et al. (49)]. Moreover, a study conducted in vivo demonstrated that the NLRC4-dependent flagellin-mediated lysis of bacteria-containing macrophages not only results in the early loss of the intracellular replication niche but also creates an inflammatory milieu with the recruitment of effector cells to the infection site, which are involved in pathogen clearance (32). Although the possible targets of caspase-1 and caspase-11 mobilized during pyroptosis remain unidentified, the studies involving NAIP/NLRC4 hugely contribute to the idea that this inflammatory form of cell death is an important effector mechanism against infections.

IL-1 was the first identified cytokine and has been related to several inflammatory processes. IL-1 plays a role in virtually all cells and organs, ranging from fever and resistance to microorganisms to the activation of the hypothalamus–pituitary–adrenal axis (HPA) (50–56). IL-18 was first described in 1989 as a potent IFN-γ-inducing factor and an important component of polarized type-1 T helper cells (Th1) and type-1 macrophages (M1) responses, cells with a pro-inflammatory profile (57–59). Macrophages, monocytes, lymphocytes, keratinocytes, microglia, neutrophils, dendritic cells, and other cells are described as important sources of IL-1β and IL-18 (60–64). IL-18 and IL-1β have similar processing; they are both synthesized in an inactive form that requires processing by active caspase-1 to become biologically active (61, 65, 66). Although extensively studied, the mechanism responsible for IL-1β and IL-18 release has not been fully elucidated. These cytokines can be passively released during cell lysis; however, there is recent evidence supporting the existence of active mechanisms involved in the secretion of IL-1β and IL-18, such as caspase-1-induced membrane pores, vesicle shedding and lysosomal exocytosis (45, 49).

Although the precise effector mechanisms of IL-1β and IL-18 remain to be elucidated, these cytokines have been reported to be important mediators induced by NAIP/NLRC4 to host resistance to bacterial infections (67). In addition to the effects of IL-1β and IL-18 in the activation and recruitment of innate immune cells, these cytokines have important roles in the activation and differentiation of T lymphocytes (52). IL-1β and IL-18 have been shown to drive the establishment of T CD4⁺ adaptive responses in mice and in humans and are responsible for the differentiation of Th17 and Th1, respectively (68–70). However, little is known about the involvement of IL-1β and IL-18 in NAIP/NLRC4-induced adaptive immune responses. Kupz et al. demonstrated that IL-18, when produced by the activation of NLRC4 during infection by S. typhimurium, is required for the activation of non-cognate CD8⁺ T cells and the production of IFN-γ (71), supporting a role for this cytokine in the induction of cellular responses.

Additional evidence of the role of NAIP/NLRC4 in the activation of T cells came from an experimental vaccination with irradiated flagellin-expressing tumor cells. Authors demonstrated that the immunization of mice with flagellin-fused tumor cells induced tumor-specific CD4⁺ and CD8⁺ T cell responses and prevented parental tumor growth. Despite the well-known role of TLR5, the recognition of flagellin by the NAIP5/NLRC4 inflammasome was also required for the induction of a protective CD8⁺ T cell response and tumor suppression. Although the NAIP5/NLRC4 inflammasome-mediated IL-1β secretion in response to the injection of flagellin-modified tumor cells, it is unclear whether the involvement of this cytokine was necessary for the success of this immunotherapy. The role of IL-1β and IL-18 in tumorigenesis remains controversial. There is strong evidence supporting pro-tumorigenic properties of these cytokines via the induction of chronic inflammation. Although the induction of Tregs and Th17 could impair the immune response against tumor cells, it is reasonable to consider that the activation of Th1 and cytotoxic CD8 T cells by IL-1β and IL-18 may be beneficial to the host (72, 73).

In addition to the well-characterized functions of NAIP/NLRC4 inflammasomes described above, non-canonical effector mechanisms have emerged. Recent data describe a range of effector functions mediated by NAIP/NLRC4 inflammasomes that operate independently of IL-1β, IL-18 and pyroptosis. The NAIP5/NLRC4 inflammasome has been implicated in the activation of phospholipase A2 (cPLA2) with a consequent production of lipid mediators, such as prostaglandins and leukotrienes (74). Authors demonstrated that systemic cytosolic flagellin stimulation leads to an “eicosanoid storm” that initiates inflammation and the loss of vascular fluids, resulting in a very fast death in mice. Of note, these effects are mediated by NAIP5/NLRC4 and occur independently of IL-1β/IL-18 or pyroptosis.

Inflammasomes have also been implicated in the active secretion of endogenous molecules known as DAMPs, challenging the idea that these molecules are only passively released during the process of cell lysis (75). IL-1α is an alarmin, whose release has been recently linked to inflammasomes. Both IL-1β and IL-1α present some common features, such as belonging to the same family, synthesis in the cytoplasm and secretion by an unconventional pathway independent of the endoplasmic reticulum and Golgi complex (55); additionally, they are released simultaneously by various stimuli, and they act on the same receptor, IL-1R1, thus sharing some biological functions (52). However, despite these similarities, there are some important differences in the production, secretion, and function of these cytokines. Unprocessed forms of both IL-1α and IL-1β are thought to be produced in response to TLR ligands, but they have distinct activities. Unlike IL-1β, which needs to be processed by caspase-1 to become biologically active (65), the uncleaved form of IL-1α is able to engage IL-1R1 (60, 76), although it’s full activity seems to require cleavage by calpain (77). Although IL-1α is not a substrate for caspase-1, there are some reports that have demonstrated that macrophages from caspase-1-deficient mice release less IL-1α (27, 78–80), suggesting the involvement of inflammasomes.

The mechanism by which caspase-1 mediates IL-1α secretion is still a matter of debate. A recent study demonstrated that the requirement of inflammasomes for IL-1α secretion depends on the nature of agonists (81). Caspase-1 has been described as a shuttle that facilitates the secretion of leaderless proteins, such as IL-1α (80). However, it is not clear whether active caspase-1 is the shuttle itself or whether it activates another enginery that is dependent on its activity, e.g., IL-1β (82) or IL-1R2 (77), as has been proposed for the secretion of IL-1α in response to NLRP3 agonists. The involvement of NLRC4 inflammasomes in IL-1α secretion is poorly understood. In one previous study, infection by S. typhimurium resulted in NLRC4- and caspase-1-dependent secretion of IL-1α (81). Interestingly, in contrast with most of the NLRP3 agonists, the secretion of IL-1α in response to S. typhimurium was completely independent of ASC, indicating a differential requirement for this adaptor molecule in cytokine secretion in response to NLRC4 agonists, as IL-1β is entirely dependent on ASC (2). However, Barry et al. showed that IL-1α initiates the inflammatory response driven by L. pneumophila independent of caspase-1 and NLRC4 (83). We recently reported that the activation of macrophages with purified flagellin inserted into lipidic vesicles induced IL-1α secretion in a manner partially dependent on caspase-1 and cathepsin B (27). Therefore, the reasons for the discrepancies in the literature and the precise mechanisms involved in the cross talk between IL-1α and NLRC4/caspase-1 axis remain to be addressed.

Another factor whose secretion has been linked to inflammasomes is the “High Mobility group box-1” (HMGB-1). HMGB-1 is a nuclear protein involved in the regulation of nucleosome function and DNA transcription that functions as an inflammatory mediator when released to the extracellular milieu (84). Lamkanfi et al. reported a critical role for HMGB-1 secreted through the NLRP3/ASC/caspase-1 axis in LPS-induced endotoxic shock (85). Interestingly, macrophages infected with S. typhimurium released significant amounts of HMGB-1 in a NLRC4 and caspase-1-dependent manner but independently of ASC, which is similar to previous reports of IL-1α secretion (81). During pyroptosis induced by a variety of stimuli, including S. typhimurium infection, HMGB-1 did not undergo caspase-1-mediated processing before its secretion, but extracellular HMGB-1 was hyperacetylated at the nuclear localization sequences (NLSs) (86). Because this translational modification is essential for HMGB-1 translocation from the nucleus to the cytoplasm (87, 88), HMGB-1 release upon inflammasome activation seems to be a coordinated process. More recently, Nystrom et al. (89) reported that NLRC4-mediated pyroptosis is the prevalent factor in the regulation of HMGB-1 secretion, leading to the release of the chemotactic acetylated HMGB-1 isoform without requiring TLR-derived priming. Although the mechanisms by which inflammasome components can regulate DAMPs secretion still need to be better understood, DAMPs are already considered important therapeutic targets because of their role in host resistance against infection and their involvement in inflammatory disorders.

With respect to antibodies production NLRC4, NAIP5, and caspase-1 have been reported to have a redundant role with TLR5 in the induction of total IgG (90) or IgG1 (91) against flagellin or co-administered OVA and an additive effect to TLR5 in the induction of IgG2a (91). In the absence of MyD88, in which TLR5, IL-1β, IL-1α, and IL-18 signaling is compromised, the production of antibodies induced by flagellin was reduced but not abolished, and a large amount of antibodies was still produced (91). The same results were obtained with TLR5/caspase-1 double-knockout mice (91), supporting previous data that demonstrated that no significant difference was observed in specific anti-flagellin IgG titers in mice deficient for IL-18 (92) or IL-1R (93). These reports suggest that some yet-undiscovered mechanism that acts in addition to TLR5 and inflammasome-mediated cytokines could be involved in the adjuvant properties of flagellin, requiring new investigations into this agonist.

In addition to inflammatory mediators and cell death processes, some cellular effector mechanisms mediated by NLRC4 have emerged. Previous studies from our group described a requirement of NAIP5, NLRC4, and caspase-1 for the activation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) secretion in response to cytosolic flagellin (94). Interestingly, cytosolic flagellin-induced iNOS activation is preserved in the absence of MYD88, ruling out the participation of TLR5, IL-1β, and IL-18. Moreover, NO secretion through the NAIP5/NLRC4-caspase-1 axis in response to flagellin is involved in the control of L. pneumophila (94) and S. typhimurium (unpublished data from our group) by macrophages, pointing to this pathway as an additional effector mechanism mediated by NAIP5/NLRC4.

Autophagy is another effector mechanism used by NAIP5/NLRC4 to control L. pneumophila. In the presence of NAIP5, NLRC4 macrophages present a rapid turnover of LC3⁺ L. pneumophila-containing vesicles, preventing the establishment of secondary infections (95). This response is mediated by the detection of flagellin, and the inhibition of autophagy in macrophages infected with flagellin-sufficient L. pneumophila increased the rate of pyroptosis in these cells. These data confirm a previous study that demonstrated that NLRC4 plays a role in the regulation of autophagy by binding Beclin-1 in steady-state conditions (96). Because the initiation of autophagy seems to precede the induction of pyroptosis, autophagy can be considered a pathway through which macrophages raise the threshold of contaminants necessary to result in the loss of cell by inflammatory cell death. NAIP5/NLRC4 can also restrict flagellin-competent L. pneumophila replication by promoting the delivery of L. pneumophila-containing phagosomes (LCP) to lysosomes for degradation (43, 97). In the absence of NAIP5/NLRC4/caspase-1, LCP avoids fusion with lysosomes, which allows the pathogen to exponentially replicate inside macrophages. This effect is dependent on caspase-1-mediated caspase-7 processing and does not require IL-1β/IL-18 and the classical apoptosis pathway involving caspase-8 and -9 (98). These data corroborate a previous report that demonstrated a requirement of NLRC4, caspase-1, and ASC for caspase-7 processing during infection with flagellin-competent S. typhimurium (99). NLRC4 and ASC-dependent caspase-8 proteolysis was also reported during S. typhimurium infection (100). Interestingly, caspase-8 contributes to Salmonella-induced IL-1β production, but it is dispensable for inducing pyroptosis, whereas caspase-1 processes pro-IL-1β and coordinates pyroptosis. These data highlight the fact that inflammasomes are dynamic complexes that are able to recruit distinct members of the caspase family to induce diverse effector functions in response to Salmonella infection.

Similar to what has been demonstrated during apoptosis (101, 102) and necrosis (103), the cleavage of PARP1 (also called ARTD1) was also observed during pyroptosis induced by S. typhimurium (104). PARP1 processing in S. typhimurium-infected macrophages was abrogated in Nlrc4^−/− but not in Nlrp3^−/− cells, consistent with the role of the NAIP5/NLRC4 inflammasome in the induction of pyroptosis during S. typhimurium infection (12, 31, 105). PARP1 is a nuclear chromatin-associated multifunctional enzyme that catalyzes the polymerization of ADP-ribose units from donor NAD⁺ molecules (106, 107). Although it has been historically studied in the context of genotoxic stress signaling and consequent apoptosis, PARP1 has been related to chromatin structure regulation, transcription, and chromosomal organization (108, 109). Previous reports showed that inflammasomes are able to use PARP1 to induce the transcription of NF-κB-dependent target genes independently of any type of programed cell death (110). Upon LPS stimulation, caspase-7 is activated by caspase-1, which is translocated to the nucleus to induce PARP1 cleavage at the promoters of a subset of NF-κB-dependent target genes that are negatively regulated by PARP1. Mutating the PARP1 cleavage site D214 renders PARP1 uncleavable and inhibits PARP1 release from chromatin and, therefore, chromatin decondensation, thereby restraining the expression of cleavage-dependent NF-κB target genes, such as il-6, cfs2, and lif, but not ip-10 (110). Preliminary and unpublished data from our group suggest the involvement of caspase-1-dependent PARP1 cleavage in iNOS gene expression upon cytosolic flagellin stimulation, as iNOS expression is significantly reduced in macrophages that harbor non-cleavable PARP1 (D214N). This is important evidence of the involvement of inflammasomes in epigenetic regulation and gene expression, although many of these outputs require further evaluation.

An important process of lysosomal exocytosis occurs during pyroptosis. Bergsbaken and Cookson (111) demonstrated that caspase-1-mediated pore formation induced during S. typhimurium infection promotes an influx of extracellular calcium, which is critical for lysosomal exocytosis. The release of lysosomal proteases with known antimicrobial activity contributes to the control of extracellular bacteria. In addition to the effect of lysosomal contents in the extracellular compartment, recent data from our group demonstrated that cytosolic flagellin is also able to activate a lysosomal pathway that culminates in an inflammasome-independent inflammatory form of cell death. This inflammasome-independent cell death induced by cytosolic flagellin is regulated by cathepsins B and D and is temporally correlated with the restriction of S. typhimurium infection by macrophages (27). Together, these data indicate a cross talk between lysosomes and NAIP/NLRC4 inflammasomes that impact the control of bacterial infections and opens new avenues for the development of inflammasome-based therapeutic strategies for non-infectious pathologies such as tumors. In fact, lysosomes have been considered important targets for the development of anti-tumor drugs (112). Lysosomes from cancer cells appear to be less stable than normal cells, which has given rise to the development of therapies based on lysosomotropic detergents. In this sense, flagellin could be an alternative that in addition to the induction of lysosomal cell death, is able to mediate several effector mechanisms as described throughout this review (Figure 1).