Gasdermin-induced necrotic cell death called pyroptosis has been defined as a new type of cell death (1). Initially, pyroptosis was regarded as caspase-1–dependent necrosis for a long time to describe proinflammatory programmed cell death (2). As time passed and research advanced, especially with the discovery of inflammasome and the inflammasome pathway, pyroptosis was redefined as inflammasome-related cell death (3, 4). Furthermore, as the gasdermin protein family was identified to play a significant role in pyroptosis, particularly gasdermin D (GSDMD) serves as the key executor, the definition of pyroptosis was further integrated (5–8). In response to certain inflammatory signals, a diversity of inflammatory caspases like caspase-1, 4, 5 (in humans) and caspase-11 (in mice) are activated (6). Subsequently, with the disruption of plasma membrane integrity, tremendous inflammatory factors, such as Interleukin-1β (IL-1β), Interleukin-18 (IL-18), and tumor necrosis factor α (TNFα) were released (3, 9). Thus, pyroptosis is a unique cell death that has special mechanism and pathways.

There is no denying that GSDMD is an indispensable molecule in pyroptosis. GSDMD (also called GSDMDC1, DFNA5L, or FKSG10) was initially found in the homologues of GSDMA (10). Saeki et al. showed that GSDMD was widely expressed in different tissues and immune cells, including intestinal epithelial cells (IECs) and different subsets of leukocytes (11, 12). In terms of structure, GSDMD consists of an N-terminal domain (NTD, containing 242 amino acids) and a C-terminal domain (CTD, containing a 43–amino acid linker and 199 amino acids) (13). Notably, the GSDMD-NTD can induce pyroptosis by pore formation, whereas the expression of GSDMD-CTD is able to protect cells from pyroptosis (5). In normal status, GSDMD-NTD is in a special state called intramolecular autoinhibition by binding with GSDMD-CTD (5, 7). However, as long as the inflammatory pathway is activated and inflammatory caspases cleave the GSDMD to disrupt the autoinhibition (5, 6, 14, 15), the oligomerization of the GSDMD-NTD will occur (5, 6, 14, 16–18). Subsequently, numerous pyroptotic pores are formed, which are composed of GSDMD-NTD oligomers (5, 6, 19). Consequently, the GSDMD-NTD punch the cell membrane to form pores that can trigger pyroptosis and IL-1β release.

GSDMD also functions as the shared common effector of multiple inflammasomes that actively involved in a body of inflammatory and autoimmune diseases. Because of the critical function of GSDMD in inflammasome-related pyroptosis, GSDMD represents an ideal and novel target for therapeutic intervention. Early in 2006, Fink et al. demonstrated that pore formation of pyroptosis leads to release of caspase-1–activated cytokines from Salmonella-infected macrophages, suggesting pyroptosis as a novel pathway of inflammatory programmed cell death, whereas GSDMD-induced inflammatory pyroptosis was uncovered (20). Excessive or inappropriate pyroptosis can be highly pathological and lead to the development of numerous diseases (21). Conceivably, GSDMD acts as the crucial executor of pyroptosis and plays a significant role in various diseases, including autoimmune diseases, infectious inflammatory diseases, and other pyroptosis-associated diseases (22). GSDMD also has been confirmed as a key regulator and a therapeutic target in several diseases, like multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE) (23, 24), cryopyrin-associated periodic syndromes (CAPS) (25–27), and Gram-negative bacteria–induced sepsis (6, 28, 29). Notably, the function and effect of GSDMD on other pyroptosis-associated diseases require closer study, such as Familial Mediterranean fever (FMF) (30–32), gout (33), Alzheimer’s disease (AD) (34), and Ischemic brain injury (35–37). Thus, carrying out the research of prohibiting pyroptosis via GSDMD signaling inhibitor has profound implications.

Up to date, the strategies of inhibiting GSDMD directly to repress pyroptosis in diseases have been evidenced. The mechanistic strategies include preventing the cleavage of GSDMD, as well as the oligomerization of GSDMD-NT fragments to reduce the formation of pyroptotic pores. In recent studies, several small-molecule inhibitors have emerged, like necrosulfonamide (NSA) (19), LDC7559 (38), magnesium (Mg²⁺) (39), disulfiram (40), and succination of GSDMD (41). These inhibitors can impede the cleavage of GSDMD or block the oligomerization of GSDMD-NT to mitigate pyroptosis.

In this review, we summarized the current advances of GSDMD, including GSDMD protein expression, stabilization, modification, activation, pore formation, and repair during pyroptosis. In addition to the lytic and non-lytic function of GSDMD, we also described some recent findings, including GSDMD complex in polyubiquitination and non-pyroptosis release of IL-1β, ADP-riboxanation, NINJ1 in pore forming, GSDMD binding protein TRIM21, GSDMD succination, and Regulator-Rag-mTOR-ROS regulation of GSDMD. Finally, we summarized that GSDMD is a key regulator and a therapeutic target in several diseases.

At the transcription level, two functioned binding sites of NF-κB were reported in the GSDMD promoter, including 250- to 100-bp and 720- to 250-bp upstream of the initiation site (42). Furthermore, attenuation of nuclear factor κB (NF-κB)–related GSDMD upregulation in adipocytes by melatonin, leading to the inhibition of inflammasome-induced and GSDMD-mediated pyroptosis (42). Consistently, the IL-1β release was also reduced in adipocyte after nuclear factor κB (NF-κB) inhibition. In addition, the hypermethylation of the promoter region mediated by DNMT (DNA methyltransferases) results in the reduced expression of GSDMD in NK92 cells, and enhancement of DNMT-mediated methylation-silencing of GSDMD by dimethyl fumarate (DMF) or monomethyl fumarate (MMF) was found to inhibit pyroptosis and IL-1β release (43), suggesting hypermethylation of GSDMD promoter region may serve as a critical checkpoint to transcriptionally modify the production of GSDMD in pyroptosis.

Moreover, recent studies have demonstrated that interferon regulatory factor 2 (IRF2) is crucial to enhance the transcription of GSDMD for the execution of pyroptosis (44). IRF2 is one of the transcription factors of interferon regulatory factor family, which can bind to the specific site in GSDMD promoter leading to promotion of GSDMD transcription and expression (44). GSDMD expression was profoundly scaled down in IRF2-deficient endothelial cells and macrophages. Disruption of IRF2-binding site abolished the pyroptosis mediated by both the canonical and non-canonical inflammasomes significantly (44). Whereas, IRF1 was reported to regulate GSDMD gene expression in EA.hy926 cells in the absence of IRF2. It has been shown that the expression of GSDMD in IRF2-deficient cells could be rescued by IFNs induced IRF1 (44, 45). Interestingly, these regulation of GSDMD through IFNs and NF-κB was occurred in certain specific cells at the transcription level, due to the low stability of GSDMD in such cells. In conclusion, the regulation of GSDMD at the transcription level provided us a unique thought on pyroptosis suppression ( Figure 1 ).

GSDMD-mediated pyroptosis has been be classified as lytic cell death featured by membrane punching, cell swelling, and pore forming ( Figure 2 ). In the pathway of canonical inflammasome-mediated GSDMD activation, certain pattern recognition receptors (PRRs), like Pyrin, AIM2, NAIP-NLRC4, NLRP3, and NLRP1, were activated after recognizing PAMPs and DAMPs and stimulation by second signaling agonist, which lead to the assemble of the canonical inflammasome (3, 7, 46). The formation of inflammsome requires the interaction between caspase recruitment domain PYD/CARD of different PRRs, the apoptosis-associated speck-like protein with a caspase recruitment domain (ASC) and pro–caspase-1 (7, 47). Pro–caspase-1 was auto-processed in the inflammasome to generate the activated caspase-1, which can cleave the proinflmmatory cytokines, such as pro–IL-1β and pro–IL-18, into bioactive forms (3, 48). Meanwhile, the autoinhibition state of GSDMD is also disrupted by activated caspase-1 to form the GSDMD-NTD (7). Subsequently, the GSDMD-NT migrates and interacts with the cell membrane by lipid binding site, followed by GSDMD-NT oligomerization, resulting in the pore forming, cell pyroptosis, and cytokine release. Moreover, the pores also lead to severe leakage of liposomes as well as dissolution of membranes including cellular membranes and organelle membranes (1, 5, 6, 49).

The GSDMD-mediated IL-1β release is generally considered to be taken place during pyroptosis. However, a few studies have reported that IL-1β can also be released dependent on a unique non-lytic function of GSDMD (50–52). The mechanism of non-lytic release of IL-1β has been determined in detail. IL-1β was first delivered to the extracellular space via aggregating in membrane vesicles like exosomes or secretory autophagy (53–56). It is noteworthy to mention that the Bulek et al. reported that the full-length GSDMD played an important role in the release of IL-1β within small extracellular vesicles (sEVs) from non-myeloid cells, such as IECs (53). Full-length GSDMD, along with a spectrum of novel GSDMD-interacting proteins, formed a new complex chaperoned by the Hsp90 and CDC37, NEDD4 (an E3 ligase), and caspase-8 in lipopolysaccharide (LPS)–induced young adult mice colonic (YAMC) epithelial cells. NEDD4 is known as an E3 ligase but plays a pivotal role in catalyzing polyubiquitination of pro–IL-1β. Along with the activation and assembly of caspase-8 inflammasome, NEDD4 catalyzes the polyubiquitination of pro–IL-1β and subsequently promotes the complex loading into secretory vesicles for IL-1β secretion (53), which provides a novel insight for us to understand the non-lytic function of GSDMD and inflammatory IL-1β secretion ( Figure 2 ).

To cope with the infection of exogenous pathogens and certain endogenous damages, the immune system can protect the host by innate immunity and adaptive immunity. The innate immune system recognizes a set of pathogen-specific model molecules, called pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) (57, 58). These PAMPS and DAMPs include some structural components of bacteria, such as LPS, peptidoglycan (PGN), or nucleic acid molecules of viruses (57). Furthermore, the host possesses a battery of receptors to recognize these PAMPs, termed as PRRs (59). PRRs recognize a series of PAMPS during host defense and the occurrence of inflammation, which leads to the formation of inflammasome (4, 33, 60–64). The inflammasome is capable of activating a body of inflammatory caspases, containing caspase-1/4/5 in human and caspase-1/11 in mice (64, 65) ( Figure 3 ). In the canonical inflammasome pathway, different inflammasomes are activated upon the stimulation of unique pathogens or non-pathogens (46). A host of studies have revealed that NLRP3 can recognize ATP or nigericin (9), NLRC4 can identify flagellin in the Salmonella enterica serovar Typhimuriumor (66, 67), AIM2 can identify the dsDNA ligand poly(dA:dT) (61, 68, 69), and Clostridium difficile toxin B can be recognized by Pyrin (30, 70). As reported, all of the above inflammasome could serve as the upstream of GSDMD and resulted in the cleavage of GSDMD by Casp to execute the pyroptosis. Notably, the activation of GSDMD could also feed-back control the activation of inflammasome. For example, potassium can be released from the GSDMD pores, causing the activation of NLRP3 inflammasome (7, 71), providing a new connection between canonical inflammasome and GSDMD activation ( Figure 3A ).

Unlike the canonical inflammasome pathway, the non-canonical inflammasome pathway leads to the activation of caspase-4/5 in humans and caspase-11 in mice (4, 72) ( Figure 3B ). LPS from Gram-negative bacteria can bind with these caspases and induce the activation and oligomerization of GSDMD without the involvement of inflammasome (63, 73–76). Likewise, the activated caspase-11 (caspase-4 and caspase-5 in humans) directly cleaves GSDMD and releases GSDMD-NTD, leading to the formation of membrane pores and pyroptosis (6, 15, 76). In addition to Casp-11, Mandal et al. showed that the collaboration between caspase-11 and caspase-8 supports the occurrence of endotoxic shock (77), supporting a critical role of Casp-8 in GSDMD-mediated pyroptosis. In analogy to canonical inflammasome pathway, activation of non-canonical inflammasome casp-11 pathway also results in the cleavage of pro–IL-1β/18 into mature IL-1β/18, which are subsequently secreted via GSDMD pores ( Figure 3B ).

Recently, some other pathways of GSDMD-activating pyroptosis have been recognized beyond the canonical and non-canonical inflammasome pathway ( Figure 3C ). It has been demonstrated that Yersinia infection-induced inhibition of the transforming growth factor β–activated kinase (TAK1) or pharmacological inhibitor of TAK1 by 5Z-7-oxozeaenol (5z7) contributes to the cleavage of GSDMD without the participation of caspase-1/11 (78, 79). Mechanistically, GSDMD cleavage is directly driven by active caspase-8 in vivo and in vitro upon TAK1 inhibition (80). In detail, TAK1 strictly restrains the phosphorylation of receptor-interacting protein 1 (RIPK1) and the NLRP3 inflammasome (81, 82). In contrast, inhibition of TAK1 by TNF stimulation or Toll-like receptor 3/4 (TLR3 and TLR4) activation results in the release of RIPK1 (7, 78). The RIPK1 recruits Fas-associated via death domain (FADD) and pro–caspase-8 to assemble a complex called complex II b or Ripoptosome to activate the caspase-8 (7, 78, 79), which allow the activated caspase-8 subsequently to elicit the cleavage of GSDMD to induce pyroptosis (64). Meanwhile, the IL-1β is cleaved after NLRP3 inflammasome activation and secreted through the GSDMD membrane pores (83) ( Figure 3C ).

Except for the inflammatory caspases-related GSDMD activation, the neutrophil elastase (ELANE) in aging neutrophils has been recognized as an important molecule to cleavage GSDMD (84). In contrast to macrophage, where GSDMD-NT fragment was produced by the cleavage of a cascade of inflammatory caspases, the ELANE-dependent GSDMD activation and cleavage in neutrophils were caspase independent. Of note, ELANE-mediated GSDMD cleavage was suggested as the upstream of the caspase cleavage (85). The production of GSDMD cleavage mediated by ELANE in neutrophil is termed as ELANE-derived NT fragment (GSDMD-eNT), and it is capable of forming membrane pores to induce pyroptosis as caspase induced GSDMD-NT (85). In addition, the formation of neutrophil extracellular traps (NETs), a unique kind of neutrophil cell death that releases chromatin structures to the extracellular space, is reported to be correlated with GSDMD (38, 86). Particularly, ELANE-mediated cleavage of GSDMD promotes the formation of NETs in response to extracellular stimuli that trigger the activation of non-canonical inflammasome (64, 86). In addition, GSDMD-dependent death renders neutrophils produce antimicrobial NETs against cytosolic bacteria for defense (38, 86). All of these results suggest a distinct role of ELANE in GSDMD activation ( Figure 3C ).

In addition to the certain PRRs, like Pyrin, AIM2, NAIP-NLRC4, NLRP3, and NLRP1, the caspase recruitment domain-containing protein 8 (CARD8) also functions as a pathogen PRR to mediate the inflammatory process (87). CARD8 not only is associated with inflammasome-mediated pyroptosis in macrophages (88–90) but also recognizes all clinically isolated human immunodeficiency virus (HIV) subtypes to promote the pyroptosis in CD4⁺ T cells (87). In cells infected with HIV, the bioactive C-terminal CARD:caspase recruitment domain fragment of CARD8 is released for the assembly of inflammasomes, leading to GSDMD cleavage and GSDMD-mediated pyroptosis (87). As a critical mechanism of host defense, human immune system can recognize cells infected with HIV by CARD8 and then induces the activation of GSDMD-related pyroptosis to eliminate the latent virus and protects our body against AIDS (87) ( Figure 3 ).

On account of GSDMD that plays pivotal role in the occurrence of pyroptosis, the modification of GSDMD is growing evidenced. Humphries et al. discovered that GSDMD and GSDME can be modified with 2-(succinyl) on specific cysteine ​​sites by fumaric acid produced during metabolism (41). This succination modification of GSDMD inhibit the cleavage of full-length GSDMD as well as the oligomerization of GSDMD-NT to form membrane pores and prohibit the development of pyroptosis (41). GSDMD succination suppresses its interaction with caspases, which limits GSDMD processing, oligomerization, and the capacity to induce cell death, suggesting that GSDMD succination is an attractive strategy to restrain GSDMD activation ( Figure 4 ). As early studies reported, 2-(succinyl)-cysteine ​​modification is an irreversible modification of DMF covalently to cysteine. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and kelch like ECH associated protein 1 (KEAP1) has been confirmed can be modified by this (91, 92). Although it was demonstrated that Cys191 in human GSDMD (Cys192 in mouse) has the capacity of meditating GSDMD oligomerization (17), it can be modified with 2-succinyl in higher abundance (41). Of note, as a metabolic intermediate in the tricarboxylic acid cycle, fumaric acid demonstrated unique impacts on the modification of GSDMD and pyroptosis.

The cell pyroptosis is featured by the presence of plasma membrane rupture (PMR), which is correlated with the production of damage-associated molecular patterns (DAMPs) and inflammatory response (93). As mentioned above, the process of pyroptosis requires the oligomerization of GSDMD-NT and the formation of the membrane pores, leading to the release IL-1β and LDH, the standard markers of the inflammation and PMR respectively. Ruan et al. found that LPS exposure promotes the release of a lectin called Galectin-1 via GSDMD-driven membrane pores and triggers inflammatory response (94). As reported, the size of GSDMD pores is about 21.5 nm inner diameter (95), which is much larger than IL-1β (about 4.5 nm diameter) and some large DAMPs. Although the importance of GSDMD in pore forming is known, the underlying mechanism of PMR is still to be determined. In 2021, Dixit et al. demonstrated that NINJ1 mediated PMR in lytic cell death (96). NINJ1, widely expresses in central nervous system (CNS), is served as an adhesion molecule related to inflammation and tumor inhibition (97). The absence of NINJ1 in macrophage results in impaired PMR and release of HMGB1 (a known DAMP) and LDH, supporting a dispensable role of NINJ1 in formation of membrane pores (96). In unstimulated BMDMs, NINJ1 exists in the form of dimer or trimer. However, upon the death stimuli, NINJ1 uses an evolutionarily conserved extracellular domain for self-oligomerization to induce PMR characterized by cell shrinkage and bleb formation (96). Interestingly, bleb formation is not prevented in NINJ1 deficiency BMDMs; however, a persistent ballooned morphology is developed to inhibit PMR (96). Although GSDMD is capable of inducing the formation of membrane pores, NINJ1 acts as a mediator of PMR on the other hand, supporting that cell death–related PMR is an active but not passive event ( Figure 4 ).

Although the process of GSDMD cleavage, oligomerization, and pore forming has been well recognized, the mechanism of modification and regulating of GSDMD is still elusive. In 2021, Li et al. reported that Shigella flexneri infection can evade the occurrence of GSDMD-mediated cell pyroptosis (98). Interestingly, Shigella flexneri mediated a new type of post-translational modification through the secretion of the effector protein OspC3 to inhibit caspase-4/11. OspC3 interacts with caspase-4/11 to block its self-cleavage and activation induced by LPS. This process was dependent on a novel type of post-translational modification called arginine ADP-riboxanation. OspC3-modified caspase-4/11 lose the ability to bind and cleave GSDMD, leading to the escape of Shigella flexneri from the recognition and elimination during immune defense (98) ( Figure 4 ). In addition, GSDMD can also be regulated by binding with other proteins, such as TRIM21. Gao et al. identified TRIM21 as a new regulator of GSDMD-mediated pyroptosis (99). TRIM21 directly interacts with GSDMD through the PRY-SPRY domain in vivo and in vitro to maintain the stable expression of GSDMD in cell (99). The discovery of this regulatory factor also provides a new target for fine-tuning and treatment of inflammation-related diseases. In addition, neutrophil serine proteases (NSPs), including cathepsin G (CATG), participated in GSDMD-associated immune response through a variety of ways (100, 101). Weinrauch et al. demonstrated that NSP is related to neutrophil death; however, the underlying mechanism has not yet been fully elucidated (102). In 2019, Burgener et al. found that while Cathepsin G cleaved GSDMD at Leu274 residue to generate a p30 fragment in neutrophil, the intracellular protease inhibitors Serpinb1a and Serpinb6a have the ability to prevent cathepsin G-dependent neutrophil death (103). In other words, Serpinb1a and Serpinb6a indirectly affect GSDMD-related neutrophil pyroptosis by inhibiting cathepsin G, supporting the important regulating role of these molecules in GSDMD activation in neutrophil ( Figure 4 ).

It has been reported that pathogenic Yersinia was able to inhibit TAK1 to initiate Casp-8–triggered GSDMD cleavage and pore forming in macrophages; however, the regulator of GSDMD activation is not clear. The Evavold et al. established an experimental system to screen the factors of GSDMD-mediated membrane pore formation in the process of pyroptosis (104). Ragulator-Rag was discovered through genetic screening and identified as the key in the process of the formation of membrane pores. In 2021, two back-to-back studies reported Rag-Ragulator complex that functions as a novel key factor to regulate GSDMD-mediated pyroptosis (104, 105). Of note, this complex promotes the GSDMD activation independent of the canonical and non-canonical inflammasomes signaling. Specifically, the activity of mTOR (downstream of Ragulator-Rag) is indeed necessary for GSDMD-mediated pore formation. Notably, mTORC1, but not mTORC2, is participated in pyroptosis (104). While knockout of RagA or RagC does not affect the localization of GSDMD on the membrane, it does significantly impact the oligomerization of GSDMD (104). Furthermore, Zheng et al. explored the mechanism of Rag-Ragulator complex in FADD-RIPK1-Caspase-8–mediated pyroptosis (105). They found loss of Rag-Ragulator significantly inhibited a series of biological processes including the complexIIformation, RIPK1 phosphorylation, and caspase-8 activation, confirming that Rag-Ragulator complex (including RagA, RagC, and Lamtor1-5) actively participates in the regulation of Yersinia infection-induced GSDMD-mediated pyroptosis (105) ( Figure 4 ).

Although activation of GSDMD results in the pore forming of cell membrane, pyroptosis does not necessarily to be the outcome of plasma membrane damage, because the repair programs like shedding as exosomes or the endocytosis of damaged membranes can be triggered by the influx of Ca²⁺ (106–109). Rühl et al. revealed that the endosomal sorting complexes required for transport (ESCRT) complex could be recruited to the damaged cell membrane during pyroptosis and subsequently initiates the repair process (52). ESCRT-III controls the integrity of plasma membrane and formed “bubbles” in plasma membrane disruption portions. With the shedding of “bubbles” from intact cells, the pores are repaired completely to preserve the common function of plasma membrane (110). In contrast, the inhibition of the ESCRT-III complex will remarkably aggravate the pyroptic cell death and IL-1β release in both human and mouse cells, whereas the cleavage of caspase-1 and GSDMD are grossly unaffected (52). In conclusion, ESCRT acts as a negative regulatory element of the downstream of GSDMD during pyroptosis and is able to repair the damaged plasma membrane ( Figure 3 ).

For infectious diseases, LPS produced by Gram-negative bacteria can be delivered into the cytosol with outer membrane vesicles (OMVs) and trigger caspase-11–dependent pyroptosis (29, 143, 144). Consequently, several studies demonstrated that GSDMD is of great importance in LPS-induced sepsis (6, 28). Consistently, GSDMD deficiency protected mice from the lethality in LPS-induced sepsis (6) ( Table 2 ).

In addition to sepsis caused by bacteria, certain viral infectious diseases are also tightly correlated with pyroptosis, such as HIV (160, 161). CD4⁺T cell intrinsic NLRP3–Caspase-1 mediates pyroptosis and drives the T cell depletion after HIV-1 infection, whereas the role of GSDMD in this process remains to be eliminated (161). Similar to HIV, both DNA virus and RNA virus infection, like influenza A virus and zika, lead to the formation of inflammasomes and the production of various inflammatory cytokines, including IL-1β (145, 146). However, the activation of canonical and non-canonical inflammasome block cGAS-dependent signaling and impacts the host defense during DNA virus infection (145). Enterovirus 71 (EV71), a virus that is capable to trigger hand-foot-and-mouth disease (HFMD), can elicit cleavage of GSDMD independent of caspase-1 (147). All of these studies might provide a crucial role of GSDMD prevention and treatment of viral infection diseases. In addition to bacteria and virus infection, the role of GSDMD has also been proposed during fungal infection. Indeed, caspase-1/11–induced poroptosis was initiated immediately after fungal infection, especially in immunocompromised patients (162). IL-1β has been proven as a crucial factor for the immune response to fungal infections (163). Several studies have shown that NLRP3-deficient mice are more susceptible to Candida albicans infection (150). Karki et al. revealed that Aspergillus fumigatus induce the assembly of AIM2 and NLRP3 inflammasome (151). Although the role of GSDMD in fungal infectious diseases has not been defined exactly, as the downstream of AIM2 and NLRP3 inflammasome, GSDMD is believed to play critical role in these diseases ( Table 2 ).

In certain kidney diseases of the urinary system, the renal cells are capable of triggering acute tubular necrosis, autoimmunity, ischemia-reperfusion injury (IRI), and necrotizing glomerulonephritis (152). Renal cell dysfunction often drives inflammasome-associated caspases to cleavage and release IL-1β or IL-18 via GSDMD-mediated plasma membrane pores (152, 153). Similar to renal cell, the neuron in brain could also be induced to pyroptosis. It has been demonstrated that β-amyloid was capable of inducing neuronal pyroptosis in AD (34, 154). Aβ_1-42 exposure leads to the upregulation of GSDMD-NT (p30), which was cleaved by the NLRP3–caspase-1 inflammasome in mice cortical neurons (MCNs) (34). The pyroptosis of neuron also takes place in stroke, one of the Top2 causes of death and disability worldwide (164). In middle cerebral artery occlusion/reperfusion (MCAO/R) stroke model, the elevation of GSDMD, IL-1β, and IL-18 was observed (37). It is demonstrated that not only GSDMD (37) but also NLRC4 and NLRP3 have been identified to promote microglia or neuronal pyroptotic cell death in ischemic stroke (35, 36). Consistently, disruption of NLRC4 inflammasome reduced the production of IL-1β and IL-18 and attenuated the disease severity of cerebral ischemia injury (35). These results suggested that NLRC4-GSDMD signaling plays an important role in promoting the disease progression ( Table 2 ). Whereas over-activated pryoptosis of the tissue cells results in the impairment of the normal function of our body, the dysregulation of pyroptosis is actively engaged the development and progression of various cancers, including cervical cancer, lung cancer, and gastrointestinal tumors (155–157). For example, the activation of NLRP6 and NLRP6-dependent pyroptosis and IL-18 secretion has been characterized to improve the antitumor immunity by maintaining a healthy gut microbiota (157). Elsewhere, the inhibition of IL-1β by canakinumab significantly reduces the incidence and mortality of lung cancer as observed in a clinical trial, suggesting that pyroptosis may play distinct role in different cancers (156). Despite the importance of pyroptosis has been recognized in cancers, the unique mechanism of GSDMD in tumor growth, invasiveness, and metastasis still needs future exploration ( Table 2 ).

Because GSDMD was identified as the direct executor of pyroptosis, completely understanding the regulatory mechanisms of GSDMD, such as GSDMD protein expression, stabilization, modification, activation, pore formation, and repair during pyroptosis, is critical in the field of biological science and clinic. Although the GSDMD cleavage by classic inflammatory caspases like caspase-1/11/8 is well known, the neutrophil elastase (ELANE) can also cleave the full-length of GSDMD, suggesting that the mechanism of GSDMD cleavage may be cell specific. In addition, the knowledge on the outcome of cells with GSDMD activation has also been advanced. The GSDMD-NT could migrate to the cell membrane and oligomerization to form the GSDMD-NT complex, rendering the release of cytosolic contents and resulting in lytic pyroptosis. In contrast, the non-lytic function of GSDMD has been summarized, providing a unique insight of GSDMD on cell function and disease. Currently, almost all of the GSDMD-related literatures found IL-1β was released via the cell membrane pores in macrophage; however, the secretion of IL-1β in gut epithelial cell is dependent on the full-length GSDMD in colitis. Whether the later mechanism still take place in other cells is still to be eliminated.

Because GSDMD serves as the common co-effector of inflammasome, blockade of GSDMD by prohibition against the cleavage or oligomerization of GSDMD via certain small molecule has been proven to work in various diseases. So far, the currently available inhibitors of GSDMD (such as NAS, DMF, and Disulfiram) also impact the upstream signaling of GSDMD, such as NF-κB, casp-1 cleavage by modified reactive cysteines covalently. In-depth recognition of mechanisms of regulating pyroptosis and executive function of GSDMD will not only expand our current knowledge but also enable to develop novel and more specific inhibitors. Furthermore, despite of the fact that GSDMD is involved in numerous diseases, the inhibitors targeting GSDMD have not been successfully developed in clinic until now. Some clinical drugs, such as DMF and disulfiram, are well-tolerance and has the potential for clinical trials by conventional drug in new use. It is critical to further discover the more specific GSDMD inhibitors that may directly block GSDMD-NT activity to repress the pore formation. To this end, our group found that a small molecular C202-2729 specifically interacts with GSDMD and blocks its lipid-binding site without impacting on the activation of GSDMD upstream proteins. Although more selective GSDMD inhibitors are still required, the identification and optimization inhibitors will undoubtedly promote the clinical translation usage.

In regard to pore formation, emerging work presents a new mechanism about membrane repair by recruiting the endosomal sorting complexes required for transport (ESCRT) that can negatively regulate pyroptosis (52, 110). Some regulating mechanisms of GSDMD are also uncovered, such as GSDMD complex in polyubiquitination and non-lytic function of GSDMD in IL-1β release, ADP-riboxanation, NINJ1 in pore forming, GSDMD binding protein TRIM21, GSDMD succination, and Regulator-Rag-mTOR-ROS regulation of GSDMD. All of the above mechanism will be favorable for drug discovery and development. For example, as inspired by fumarate produced metabolites, promotion of the succination of GSDMD by DMF to repress GSDMD activation come to light (41). Other endogenous metabolite compounds like heparin are capable of blocking the transportation of LPS into the cytoplasm to prevent caspase-11–related septic lethality and pyroptosis (165). Endogenous metabolite compounds are also becoming attractive candidates to inhibit the GSDMD-dependent pyroptosis. In conclusion, although the regulation mechanism of GSDMD has achieved great advances, the unique pattern of GSDMD activation in distinct cell type or disease is still required further exploration. Despite multiple GSDMD inhibitors have been identified, more selective and tolerance inhibitors are still required for clinical translation. GSDMD is becoming a very attractive checkpoint in multiple diseases, and it is opening new window for therapeutic intervention against a body of GSDMD-involved diseases.

C-JZ and YX conceived the study. C-JZ, ZL, SJ, and MJ wrote the manuscript. All authors contributed to the article and approved the submitted version.

This study was supported by the National Natural Science Fund for Excellent Young Scholars (82022019 to C-JZ), Nanjing special fund for Medical Science and Technology Development Projects for Distinguished Young Scholars (JQX19005 to C-JZ), and National Natural Science Foundation of China (82101414 to MJ, 81701235 to C-JZ, and 81991514 to C-JZ).

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.

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