Apoptosis was the first type of programmed cell death to be described, initially based on morphological features that distinguished it from necrosis, an uncontrolled, accidental form of cell death observed upon extreme physicochemical insults (9). In this regard, apoptosis is characterized by chromatin condensation, nuclear fragmentation, cell shrinkage with formation of cellular membrane blebs, and, finally, cellular disintegration into fragments known as apoptotic bodies (10). Importantly, during apoptosis, the plasma membrane integrity is preserved, avoiding the release of intracellular contents to the extracellular milieu. This feature contributes to the concept that apoptosis is an (relatively) inflammatory-silent form of cell death. Indeed, recognition and elimination of apoptotic cells during physiological circumstances, such as tissue/organ sculpture during development and tissue homeostasis, occurs without the cardinal signs of inflammation. In addition, it is well established that recognition of apoptotic cells by macrophages, in particular, results in the production of anti-inflammatory molecules, such as TGF-β and PGE₂ (11). On the other hand, it is also known that apoptotic cells release a series of so-called “find-me” signals, such as extracellular ATP and lysophosphatidylcholine (LPC), capable of recruiting phagocytes to the site of apoptotic corpses, characterizing, therefore, at least one aspect of an inflammatory reaction (12, 13). Besides, more recently, it was shown that apoptosis initiated via the FAS/CD95 death receptor is associated with the release of chemokines and other immunologically active proteins that coordinates the migration of phagocytes and proper removal of apoptotic cells (14). Taken together, it is reasonable to say that although not completely “silent,” apoptosis is a form of cell death that does not trigger an overt inflammatory response.

From the molecular point of view, much of our knowledge about the regulation of apoptosis came from works with the nematode Caenorhabditis elegans. In a series of elegant studies, Bob Horvitz and colleagues identified four crucial genes (Ced-3, Ced-4, Ced-9, and Egl-1) responsible for the control of developmental cell death in C. elegans (15), which granted him the Nobel Prize in Physiology or Medicine in 2002, together with John Sulston and Sidney Brenner, “for their discoveries concerning genetic regulation of organ development and programmed cell death.” Soon after Horvitz discoveries, it became clear that cell death in C. elegans and apoptosis in mammals shared a very similar, phylogenetically conserved mechanism. Apoptosis is executed by certain members of a family of cysteine aspartate-specific proteases called caspases (16–18). Importantly, not all caspases induces apoptosis. Caspases-1, -4, -5, -11, -12, -13, and-14 are inflammatory caspases not related to the initiation or execution of the apoptotic program. Caspases are produced as an inactive pro-form (zymogen) that can be activated either through proteolytic processing by upstream caspases (in the case of caspases-3, -6, and-7) or via dimerization in the context of multimolecular platforms, such as the apoptosome (caspase-9), the DISC (death-inducing signaling complex) (caspases-8 and-10), the PIDDosome (caspase-2), and the inflammasome (caspase-1 and-11) (16). Executioner or effector caspases, such as caspase-3, -6, and-7 (and CED-3 in C. elegans), are responsible for the induction of the morphological as well as the biochemical features associated with apoptosis, including oligonucleosomal DNA fragmentation and externalization of phosphatidylserine (PS) residues from the inner to the outer leaflet of the plasma membrane (19). Interestingly, in mammals, although the inhibition of effector caspases prevents apoptosis, it does not preclude cell death, which proceeds with different morphological and biochemical characteristics (20). Because of this, it has been proposed that apoptosis in mammals may not be actually a cell death mechanism, but perhaps a termination step of a cell-death program aimed to properly dispose damaged or unwanted cells without initiating inflammatory responses (18).

There are two signaling pathways of apoptosis (Figure 1). The intrinsic pathway deals with signals derived from intracellular stress, such as DNA damage, oxidative stress, dysregulation of Ca²⁺ homeostasis, interference with the cytoskeleton structure, endoplasmic reticulum stress, etc. Its first layer of regulation comprises the differential expression/activation of BCL-2 family members, responsible for controlling the mitochondria outer membrane permeabilization (MOMP) (21). When the pro-apoptotic stress is too strong for a given cell, MOMP allows the selective release of certain mitochondrial proteins, such as SMAC (second mitochondria-derived activator of caspases)/Diablo (direct IAP binding protein with low pI), HtrA2 (high temperature requirement protein A2)/Omi, and cytochrome c to the cytosol. Cytochrome c associates with APAF-1 (apoptosis-activating factor-1), the mammalian CED-4 homolog, and pro-caspase-9, thereby assembling the apoptosome and enabling caspase-9 to activate the downstream effector caspases. SMAC/Diablo and HtrA2/Omi facilitate apoptosis by preventing the inhibitory action of the inhibitors of apoptosis proteins (IAPs) on the effector caspases. The extrinsic pathway, in comparison, is initiated by the interaction of trimeric, extracellular ligands (TNF-α, CD95L, and TRAIL) to their cognate receptors (TNFR1, CD95 and TRAILRI, or TRAILRII, respectively) present on the plasma membrane (10, 22, 23). The stimulation of these so-called death receptors (DRs) leads to the recruitment of adaptor molecules, such as TRADD (Tumor necrosis factor receptor type 1-associated death domain protein) and/or FADD (Fas-associated protein with death domain), and the pro-caspase-8, giving rise to the conventional DISC. Next, caspase-8 directly activates the effector caspases or amplifies the cell death signal by engaging BID (BH3 interacting-domain death agonist), a pro-apoptotic member of the BCL-2 (B-cell lymphoma 2) family, leading to MOMP, cytochrome c release and assembly of the apoptosome (Figure 1). It is important to mention that the activation of caspase-8 in the context of DISC can be regulated by c-FLIP (cellular FLICE-like inhibitory protein), a catalytically-dead caspase-8 homolog (24).

In some instances, apoptosis can also be triggered by TLR stimulation, as a defense mechanism against infection. TLR2 was the first PRR to be associated with induction of apoptosis, by virtue of its ability to recruit FADD via MyD88 (Myeloid differentiation primary response 88), and the consequent activation of caspase-8 (25). Likewise, bacterial lipoproteins were reported to trigger apoptosis through this TLR2 pathway (26, 27) and Mycobacterium tuberculosis was also shown to induce TLR-2/caspase-8-dependent apoptosis in macrophages (28). Interestingly, TLR3-induced apoptosis is mediated via TRIF (TIR-domain-containing adapter-inducing interferon-β), which interacts with RIPK1 (Receptor Interacting Serine/Threonine Kinase 1) through its RHIM (RIP homotypic interaction motif) domain (please refer to necroptosis section for further information on these protein-protein interactions). FADD is then recruited, and activates caspase-8 leading to apoptosis (25, 29). In human keratinocytes, poly I:C-induced apoptosis required the stimulation of TLR3 and its adaptor TRIF, thus inducing caspase-8 activation (30); the same molecules were shown to induce apoptosis in human breast cancer cells (31). Not surprisingly, TLR4 can induce apoptosis either via MyD88 or TRIF, and depending on the cell type or conditions engage the extrinsic or intrinsic pathways. For instance, Yersinia was shown to induce TLR4-mediated apoptosis of macrophages through TRIF (32, 33). TRIF-mediated apoptosis seems to be executed through the extrinsic pathway, with no evidence of the involvement of the mitochondrial pathway (34). Interestingly, UV irradiation was shown to induce apoptosis in murine macrophages through TLR4 and MyD88 (35). Despite these observations and a number of other examples that we have not presented here, it is important to emphasize that PRR-induced apoptosis is a relatively minor event compared to all other triggers of apoptosis and that PRR activation leads preferentially to other forms of regulated cell death, as we will discuss below.

Evidence of a molecularly controlled necrotic cell death was first provided by studies showing that Tumor Necrosis Factor Receptor 1 (TNFR1) and CD95 ligation were capable of inducing necrosis, particularly when caspase activity was inhibited (36, 37). This idea was further supported by a study that demonstrated that the cowpox virus could induce necrosis in porcine kidney cells when it harbored the caspase inhibitor CrmA (cytokine response modifier A) (38). This cell death mode was named “Necroptosis,” as it reflects the existence of a molecular pathway (like apoptosis) but with a necrotic phenotype.

The first molecule to be identified in the necroptotic pathway was RIPK1 as its kinase activity inhibitor, necrostatin-1 (Nec-1), was shown to suppress cell death triggered by caspase inhibition during TNFR1/Fas stimulation (39). RIPK1 has been previously involved in apoptotic and survival pathways, functioning as a scaffold protein to the assembly of the respective signaling platforms (40). Contrastingly, the RIPK1 kinase activity is indispensible for death receptor-triggered necroptosis, as its auto-phosphorylation induces a conformational change that allows RIPK1 to recruit, via their respective RHIM domains, the next member of this pathway, namely RIPK3 (41–43). Once recruited, RIPK3 gets activated by auto-phosphorylation and forms an amyloid-like structure, which promotes the recruitment and activation of Mixed Lineage Kinase Domain-Like (MLKL) (42, 44–47). RIPK3-phosphorylated MLKL oligomerizes and translocates to the plasma membrane, where it interacts with phosphatidylinositides and induces plasma membrane disruption [(48–51); Figure 2). Distinct effector mechanisms were raised to account for the MLKL-driven permeabilization of the plasma membrane, either directly by pore or cation channel formation, or indirectly, by activation of TRPM or other ion channels (48–52). It is still unclear, however, which of these mechanisms are physiologically relevant. Nonetheless, in all cases, MLKL induces a loss of osmolality control, which causes cell swelling and membrane rupture. Recently, ESCRT-III machinery was suggested to counter these effects by shedding out the MLKL-damaged plasma membrane regions (53).

Necroptosis can be initiated by a variety of signals. The first to be described and most thoroughly studied was TNFR1 ligation (36). Upon its ligation, TNFR1 typically assembles a multimolecular complex (Complex I) composed by TRADD, RIPK1, TRAF2, TRAF5, cIAP1, cIAP2, and LUBAC (linear ubiquitin chain assembly complex), which is involved in NF-κB activation, pro-inflammatory cytokines synthesis and cell survival (54). Sustained TNFRI ligation leads to CYLD-mediated deubiquitination of this complex, which disassembles, allowing the formation of a secondary complex (Complex II) in the cytosol, constituted by TRADD, FADD, RIPK1, caspase-8, and occasionally c-FLIP (54). As pointed out above, when c-FLIP levels are low, caspase-8 forms active homodimers and triggers downstream events that culminate in apoptosis. However, in the absence of FADD, c-FLIP or a functional caspase-8, TNFRI signaling results in the recruitment of TRADD and RIPK1, forming a platform called complex IIb or necrosome, wherein RIPK3 and MLKL are activated to execute necroptosis (55). Although slightly differing on how RIPK1 is brought to the complex, this molecule has also a central role in Fas and TRAILR-induced necroptosis, as RIPK1 is, in all these cases, mandatory to recruit RIPK3 via their RHIM homotypic domain interactions (54).

Necroptosis can also be triggered by PRRs, such as TLR3 and TLR4, intracellular sensing proteins, such as DAI, RIG-I and MDA-5 as well as interferon signaling [(56–59); Figure 2). Intriguingly, however, RIPK1 is dispensable for or even inhibitory of the necrosome formation during TLR3-, TLR4-, DAI-, and interferon-mediated necroptosis (57, 60). In these cases, RIPK3 is directly recruited to the signaling platforms, and the presence of RIPK1 slows down or halts the RIPK3-mediated activation of MLKL (60, 61). The ability of RIPK1 to recruit FADD, and consequently, caspase-8 and FLIP accounts, at least in part, for its inhibitory property. Therefore, from the molecular point of view, necroptosis ought to be defined as a RIPK3-dependent form of cell death.

Many other stimuli have been described as capable to induce necroptosis, ranging from UV irradiation, chemotherapeutic drugs (such as cisplatin, etoposide, and staurosporine), natural compounds (such as shikonin and its analogs), to DNA damage, hypoxia, ischemia/reperfusion and oxidative stress (62). The signaling pathways that lead to necroptosis in each of these cases are still to be fully elucidated. Further studies are required to evaluate whether they are dependent on RIPK1 and also whether they directly signal to a RIPK3-activating platform or indirectly, via up regulation of a classic necroptotic inducer, such as TNF or FasL. For example, UV irradiation was reported to induce necroptosis via TNF upregulation but also via spontaneous aggregation of RIPK1 and RIPK3, independently of any death receptor ligation (29, 63). Particularly puzzling is the fact that shikonin, a naphthoquinone compound obtained from a plant extract, can induce necroptosis even in the absence of FADD/caspase-8/FLIP inhibition, which is thought to be mandatory for this type of cell death (64). Thus, either this compound can itself somehow block their activity, or it shall be instrumental to decipher alternative ways in which MLKL is activated and necroptosis is executed.

Nonetheless, despite the different mechanisms that initiate necroptosis, in all cases cells undergo rapid MLKL-mediated plasma membrane permeabilization with consequent release of intracellular contents, including many DAMPs, such as lysosomal proteases, DNA, mtDNA, ATP, and HMGB1 [(55); Table 1). Therefore, similarly to pyroptosis (see below), necroptosis is considered a pro-inflammatory form of cell death. Even so, it is still to be determined whether the pro-inflammatory properties of necroptotic cells are the result of the intracellular content leakage or, rather, they can actively produce and/or modify specific DAMPs. Evidence for the latter comes from ESCRT-III-deficient cells that undergo necroptosis much faster, which limits the amount of inflammatory cytokines and chemokines produced and hinders antigen cross-presentation (53). Moreover, both RIPK3 and MLKL have been associated with inflammasome and NF-κB activation, supporting the notion that the pro-inflammatory potential of necroptotic cells goes beyond the passive release of their intracellular content (110–112).

Necroptotic cells not only induce a potent inflammatory response but they are also highly immunogenic, which may be instrumental against infection and during anti-tumoral responses. For example, mice injected with necroptotic cells present a higher CD8⁺ T cell cross-priming and increased tumor immunity when compared with animals injected with apoptotic cells (113, 114). Likewise, RIPK3 deficiency in mice inhibits immune cell infiltration and attenuates organ injury during sepsis (115). Therefore, given that necroptosis is highly immunogenic, disruption in the necroptotic pathway would be expected in some pathophysiological conditions. Indeed, it was reported that most of the in vitro transformed cells as well as human tumor samples have low or no expression of RIPK3 (116), and a cohort of chronic lymphocytic leukemia patients present down regulation of CYLD (117). Furthermore, patients with lower expression of RIPK3 or MLKL have worse prognosis for breast cancer or ovarian cancer, respectively (116, 118), suggesting that resistance to necroptosis is positively selected during tumor growth and/or development. This may be associated with an increased ability to evade immune attack, either by prolonging the lifespan of the transformed cells, by decreasing the availability of DAMPs, or by avoiding the activation of antigen-presenting cells during the immune responses. Therefore, induction of necroptosis in tumors may change its immunogenicity and promote a better immune response against it. This is particularly exciting, as we are currently witnessing novel and promising approaches in tumor treatment that are based on stimulation of the immune system. On the other hand, it is possible that the inflammation generated by necroptosis may promote tumor development by stimulating angiogenesis and metastasis (119). Therefore, thorough investigation of the benefits and pitfalls of inducing inflammatory cell death for each cancer type will be required in order to determine whether inducing necroptosis is indeed a good option in the specific cancer treatment.

Besides its impact on tumorigenesis and tumor progression, deficient necroptotic signaling can be detrimental during viral infection. Mice lacking RIPK3 are highly sensitive to vaccinia virus due to widespread infection (120). Likewise, RIPK3-deficient mice are more susceptible to Influenza A virus (IAV) than the wild-type animals (121). Remarkably, seasonal IAV, but not the 1918 and 2009 pandemic IAV strains, induces RIPK3-mediated immunogenic death of dendritic cells (122). The pandemic strains' ability to suppress necroptosis was mapped to the hemagglutinin (HA) genomic segment (122), indicating that either the pandemic strains' HA do not induce necroptosis or it may directly interfere with the necroptotic signaling pathway.

Keeping with the notion that suppressing necroptosis is advantageous to the infectious agent, there is accumulating evidence that viruses can encode molecules that are able to directly interfere with the necroptotic signaling. vIRA, a molecule expressed by MCMV that contains a RHIM-like domain blocks RIPK3 recruitment to RIPK1 and to DAI (57). MCMV expressing vIRA mutated in its RHIM domain produces an attenuated viremia in wild-type mice, which is reverted in RIPK3-deficient animals (57). Likewise, HSV-1 and HSV-2 express ICP-6 and ICP-10, respectively, which are able to suppress necroptosis in human cells through a similar RHIM-dependent mechanism (123, 124). Curiously, in mice, ICP-6 was shown to promote necroptosis through direct aggregation with RIPK3, restricting virus propagation (124, 125). A different mode of action was reported for the IE1-regulated gene product expressed by HCMV, which suppresses necroptosis downstream of RIPK3 activation and MLKL recruitment (126).

Bacteria can also induce necroptosis, at least in vitro. It is less clear, though, whether necroptosis plays a central role in bacterial infections in vivo. Loss of RIPK3 in combination with deletion or inhibition of caspase-8 or FADD renders mice susceptible to a number of pathogens, including Yersinia and Citrobacter (127, 128). However, the relative contribution of necroptosis and caspase-8-mediated apoptosis in these models were not yet tested, as caspase-8- or FADD–deficient animals are not viable (129–131).

Necroptosis, though, may not always be protective against infection. Macrophage death by necroptosis correlates with increased susceptibility to Salmonella infection (132). Also, HIV-specific CD8⁺ T cell response, which is a key indicator of infection control, is impaired due to increased necroptosis levels in this cell population (133). Taken together, necroptosis seems to be detrimental when it eliminates the population that is central for the immune control of the infection. In the other cases, necroptosis limits infection, mostly likely by destroying the pathogen's replicative niche through a cell death mode that generates a pro-inflammatory and immunogenic environment. However, it is important to note that, as mentioned above, RIPK3 and MLKL were shown to participate of additional signaling platforms, including inflammasome activation, NF-κB signaling and even apoptosis induction (134). Therefore, in the light of these novel RIPK3 and MLKL roles, it is essential to reevaluate the relative contribution of necroptosis to the phenotypes observed. A good illustration comes from the fact that while RIPK3-deficient mice are more susceptible to IAV, MLKL-deficient animals are not, indicating that necroptosis is not the sole RIPK3-mediated mechanism important in IAV control (121). In fact, it was shown that IAV also triggers RIPK3-mediated apoptosis, via recruitment of RIPK1, FADD and caspase-8. This was further supported by the fact that MLKL-caspase-8 double deficient mice present similar levels of susceptibility to IAV infection observed with the RIPK3-deficient animals (121). Another example is that RIPK3-deficient mice are less susceptible to Staphylococcus aureus lung damage and present reduced bacterial loads and inflammation, while MLKL-deficient animals present an opposite outcome, suggesting that these molecules have independent, non-necroptotic roles (135).

Pyroptosis is a necrotic form of regulated cell death distinct from necroptosis, mainly due to the requirement of inflammatory caspase-1 and/or caspase-11 (murine caspase-11 corresponds to caspases-4 and -5 in humans) [(136); Figure 3). It is the result of pore formation in the plasma membrane that increases osmotic pressure ensuing in osmotic lysis and, consequently, the release of the intracellular content, including pro-inflammatory cytokines and DAMPs (137). Although distinct from the typical oligonucleosomal fragmentation observed during apoptosis, DNA fragmentation is also a hallmark of pyroptosis, which seems to occur independently of the caspase-activated DNase (CAD) (138).

Pyroptosis is a form of cell death initiated in response to the engagement of certain members of the PRRs, which are capable of assembling complex structures called inflammasomes. These platforms are composed by a sensor protein, either from the NLR or the pyrin and HIN domain-containing protein (PYHIN) families of cytosolic PRRs, in addition to the adaptor molecule apoptosis-associated speck-like protein containing a caspase activating and recruitment domain (ASC) and pro-caspase-1. There are five major types of the so-called canonical inflammasomes—NLR pyrin domain-containing 3 (NLRP3), NLRP1, neuronal apoptosis inhibitory protein (NAIP)/NLR CARD-containing 4 (NLRC4), absent in melanoma 2 (AIM2), and PYRIN inflammasomes (139). Activation of one of these cytosolic sensors in response to PAMPs, DAMPs or cytosolic disturbances such as ionic imbalance leads to the recruitment and activation of caspase-1 either directly or through the ASC adaptor molecule [(140); Figure 3). Besides the induction of pyroptosis, caspase-1 also leads to the processing and release of the pro-inflammatory cytokines IL-1β and IL-18 (139).

In contrast to the canonical inflammasomes, which require multicomplex structures, the non-canonical inflammasome seems to be composed solely by pro-caspase-11, which plays the role of the sensor as well as the executor (141, 142). During intracellular gram-negative bacteria infections, Lipid A, a component of LPS, can directly bind to the CARD domain of pro-caspase-11 (143), which gets activated and induces pyroptosis. Interestingly, the non-canonical caspase-11 inflammasome acts independently of LPS recognition by TLR4 and does not directly induce IL1-β and IL-18 maturation [(141, 142); Figure 3). In monocytes, however, non-canonical inflammasome stimulation may result in minor production of IL-1β and IL-18 through the bystander induction of NLRP3 activation (144). Interestingly, LPS-induced lethal shock is driven by the activation of the non-canonical inflammasome. Since IL-1β and IL-18 release are not a major outcome of caspase-11 activation, the exacerbated inflammatory response observed in sepsis seems to be mainly driven by pyroptosis, probably due to the efflux of DAMPs, such as High Mobility Group Box 1 (HMGB1) and IL-1α [Table 1; (145)].

Such non-canonical inflammasome-mediated responses have drawn the attention of different research groups that became interested in unraveling the relevant as well as the pathogenic caspase-11 downstream targets. In 2015, two concurrent studies reported that Gasdermin D (GSDMD), a member of the GSDM family, was the effector component of the non-canonical inflammasome pathway (146, 147), which was later confirmed by a third study (148). Kayagaki and colleagues performed N-ethyl-N-nitrosourea mutagenesis screening for mutations that compromised LPS-induced IL-1β release and pyroptosis (146) while Shi and colleagues employed the clustered regularly interspaced palindromic repeat (CRISPR)/Cas9 genome-editing screens in TLR4 deficient mouse bone marrow-derived macrophages for guide RNAs that protected from LPS-induced cell death (147). Both studies hit GSDMD as a substrate for caspase-11 and the effector of pyroptosis. GSDMD is composed by a C-terminal and a N-terminal domain linked by a long loop. Caspase-11 cleaves an aspartate residue within the linking loop, releasing the N-terminal fragment from the inhibitory C-terminus (146, 147). The N-terminal domain, also called Pore-Forming Domain (PFD) (149) oligomerizes and associates with lipids in the inner plasma membrane to form 10–33 nm pores leading to cell swelling and eventually to cell lysis (150–153). Importantly, it was also demonstrated that caspase-1 cleaves GSDMD at the same site as caspase-11, establishing that GSDMD is also required for the canonical inflammasome-driven pyroptosis (147).

Until the discovery of GSDMD as the pyroptosis executioner, the physiological function of GSDM proteins was largely unknown. However, recent studies described that the PFD is highly conserved among several members the GSMD family. Indeed, expression of PFD from GSDMA, GSDMA3, GSDMB, GSDMC, GSDME, or GSDMA3 in HEK293 was able to induce pore formation and a cell death phenotype similar to pyroptosis (147, 151). Moreover, GSDMA3 cleavage by caspase-3 in HEK293 and macrophages results in a secondary necrotic cell death after apoptosis (154). This necrotic cell death might contribute to hearing loss in GSDMA3 spontaneous mutations that are associated with deafness (155). Thus, given the cytotoxic activity of different GSDM PFD, some authors have proposed a redefinition of pyroptosis as a GSDM-mediated cell death (146). However, it is controversial how other GSDM members are activated and whether these proteins participate in cell death pathways. Also, GSDMD seems not to be required for pyroptosis during prolonged inflammasome activation in response to the classical agonists, ATP, and flagellin (146). Moreover, in the absence of caspase-1 protease activity, caspase-8 accounts for GSDMD-independent cell death in response to inflammasome agonists (156–158). Since some of these processes share features of pyroptosis, it is hard to define pyroptosis solely as being a process of cell death regulated by inflammatory caspases or mediated by GSDM proteins, since we can find exceptions to the rules that govern both concepts.

From the biological point-of-view, cell death by pyroptosis results in a fast removal of infected cell leading to the elimination of the replication niche. Conversely to the previous idea of liberation of bacteria to the extracellular milieu by pyroptotic cells (159), the current knowledge predicts that, instead, the damaged bacteria remain trapped within the pyroptotic corpses. This structure is called pore-induced trap (PIT) and it prevents bacterial dissemination (160, 161). Despite that PIT does not directly kill intracellular bacteria, pyroptosis renders them more susceptible to H₂0₂, to the antimicrobial peptide polymyxin B and to the antibiotic ciprofloxacin (157). As a consequence, the recovered bacteria from PIT are less capable to infect neighbor cells.

The inflammatory milieu created by the release of the intracellular content from pyroptotic cells recruits circulating phagocytes to the infectious site. Subsequently, neutrophils efferocyte the PIT and kill the pathogen by a mechanism dependent on reactive oxygen species (ROS) (161). Extracellular bacteria can also be controlled by the action of antimicrobial peptides (160, 161) and potentially by the GSDMD N-terminal domain released during cell lysis due to its affinity to cardiolipin and phosphatidylserine expressed in some bacterial cell membranes, such as Escherichia coli and Listeria monocytogenes (152, 162). Interestingly, canonical and non-canonical inflammasomes are required for intestinal epithelial cells (IECs) responses to infections (163, 164). The activation of NLRC4 inflammasomes in IECs results in a lytic cell death prior to a non-conventional process of cell expulsion that contributes to control bacterial replication. Although caspase-1 and Gasdermin-D were required for IEC pyroptosis, both molecules were dispensable for cell expulsion, demonstrating that coordinated inflammasome responses in IECs are important to prevent bacterial translocation to deeper tissues (163, 164).

Interestingly, neutrophils seems to be more resistant to pyroptosis than macrophages in response to Salmonella and are able to maintain a sustained IL-1β production and secretion, which could be important to control the infection (165). However, Kambara et al. (166) recently described that a specific neutrophil elastase (ELANE) is able to cleave GSDMD independently of caspases activity, promoting a lytic cell death in these cells. Interestingly, these authors demonstrated that GSDMD-dependent neutrophil death impairs the control of extracellular bacteria E. coli, thus suggesting that GSDMD could exert an anti-inflammatory role depending on the infection context.

In addition to its role in the elimination of replicative niche, the pyroptosis machinery is involved in IL-1β and IL-18 release. As these cytokines lack the signal peptide, their release is considered to occur by non-conventional pathways (167). Among the different pathways that have been proposed to explain their secretion, mechanisms involving cell death are particularly subject to intense debate in the literature. Growing evidences suggest that IL-1β can be released by viable monocytes (168), dendritic cells (DCs) (169), and macrophages (170). GSDMD pore is large enough to allow IL-1β release concomitant with the influx of cationic ions (148). Notably, in viable cells, GSDMD seems to be required for IL-1β translocation to the extracellular space in response to stimuli that hyperactivate phagocytes, such as oxidized phospholipids (oxPAPC) in DCs or LPS in human monocytes (170–172). Nonetheless, it is difficult to establish whether the cells were actually viable, since cell death can precede cell lysis, thus suggesting that pyroptosis and cell lysis can be uncoupled events (173). Moreover, the assessment of cell death by the detection of lactate dehydrogenase release (LDH), used in several studies as the only viability assay, might be insufficient to discriminate viable cells from dying cells since both viable and unviable cells can release LDH to the cell culture (170, 173, 174).

Although many studies have demonstrated the requirement of canonical and non-canonical inflammasomes to host defense against pathogens, the precise contribution of pyroptosis and other inflammasome-related mechanisms are poorly understood and arose mainly from in vitro assays or bacterial infection models in mice deficient for molecules that compose these platforms (159, 175). In L. monocytogenes, S. typhimurium, and B. thailandensis in vivo infections, the lack of caspase-1/11 was more deleterious to the host than IL-1β/IL-18 deficiency (176–179), while in mice infected with F. novicida, the treatment with recombinant IL-1β/IL-18 only partially recovered the resistance to infection, suggesting that cytokine secretion was not sufficient to protect the host (180). The susceptibility of GSDMD-deficient mice seems to correlate with that demonstrated by caspase-1/11-deficient mice, although the deletion of GSDMD also culminated in reduced IL-1β/IL-18 release (146–148). Even though (a) IL-1β/IL-18 secretion might occur independently of pyroptosis and (b) caspase-1 or caspase-11 deletion is more severe than IL-1β/IL-18 ablation in some bacterial infections, usually cytokine release and cell death are overlapping events necessary for optimal host defense (159). In any case, the susceptibility of GSDMD and other GSDM deficient mouse strains to infectious agents and its comparison to mice lacking caspase-1, caspase-11, IL-1β, and/or IL-18 remains to be established, especially in non-bacterial infection contexts. For example, despite clear evidences of the involvement of inflammatory caspases in the host control of some fungal infections such as Candida albicans, Aspergillus fumigatus, and Paracoccidioides brasiliensis (172), the requirement of GSDMD to cell death and the consequences to the host resistance against these infections is still to be elucidated.

Notwithstanding, the highly pro inflammatory outcome of pyroptosis as well as the cell loss can be prejudicial to the host during the response to pathogens. In HIV patients, the quiescent CD4 T cells depletion seems to be mainly mediated by pyroptosis (181, 182). During HIV abortive infection, the engagement of the interferon-gamma-inducible-protein 16 (IFI16) in response to cytosolic viral DNA leads to inflammasome assembly and caspase-1 mediated CD4 T cells pyroptosis in lymphoid tissues (181, 182). Interestingly, co-cultivation of lymphoid-derived cells sensitizes blood-derived CD4 T cells to HIV-induced pyroptosis (183). Moreover, pyroptotic peripheral blood CD14⁺CD16⁻ monocytes from HIV-infected patients release ASC specks, a hallmark of inflammasome activation. Therefore, besides the depletion of CD4 T cells, pyroptosis of CD4 T cells and monocytes contributes to the chronic inflammation that characterizes the disease (184).

The identification of the non-canonical inflammasome and the discovery of GSDMD as the executioner of pyroptosis have expanded our understanding of the mechanisms driving this type of cell death. However, further studies are necessary to elucidate the precise role of inflammatory and non-inflammatory caspases and the participation of members from GSDM family and/or other effector proteases in the molecular regulation of pyroptosis. In addition, the understanding of its role during infection or inflammatory processes in vivo will contribute to better understand the biological relevance of this regulated cell death induced in response to the PRRs activation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer FD and handling Editor declared their shared affiliation at the time of review.