Pyroptosis is a form of regulated cell death that depends on the formation of plasma membrane pores by members of the gasdermin (GSDM) protein family, often due to inflammatory caspase activation (Galluzzi et al., 2018). In the 1990s, pyroptosis was initially described as a caspase-1-dependent and bacteria-induced necrotic cell death; present in macrophages infected with Salmonella typhimurium (Zychlinsky et al., 1992; Monack et al., 1996). The term “pyroptosis” was coined by Cookson and Brennan in 2001 (from the Greek “pyro”, meaning fire or fever, and “ptosis”, denoting a falling) (Cookson and Brennan, 2001). Pyroptotic cell death is an important contributor to physiologic and pathologic processes dependent on the activation of the pore-forming gasdermin protein family. Once initiated, signaling cascades trigger the inflammasome-dependent and -independent formation of cell membrane pores, resulting in rupturing of the cell membrane, releasing its content such as inflammatory molecules and danger signals that promote inflammation and cellular infiltration. Besides its physiologic role in regulating cell death, pyroptosis has been associated with the pathogenesis of a variety of diseases, including metabolic dysfunction-associated steatohepatitis (MASH), formerly known as nonalcoholic steatohepatitis (NASH), inflammatory bowel disease (IBD), atherosclerosis, diabetes, and cancer (Rao et al., 2022). The regulation of cell death is a central part of physiologic cell development and homeostasis. The highly orchestrated homeostasis is severely impaired in dysregulated inflammation and chronic exposure to pathogenic or danger signals. Two different pathways regulate the mechanisms of pyroptosis, namely the canonical and the non-canonical pathway. The classical or canonical pathway includes the inflammasome activation and caspase-1-dependent cleavage of gasdermin D (GSDMD), whereas the non-canonical pathway initiates inflammasome-independent GSDMD cleavage via caspase-4/5/11. Cells undergoing pyroptosis can be observed exhibiting distinct morphological features such as chromatin condensation with an intact nucleus, mild cell swelling, membrane blebbing, the formation of pyroptotic bodies, and plasma membrane permeabilization, which is caused by the pore formation of cleaved gasdermins (Jorgensen and Miao, 2015; Yu et al., 2021). The pores lead to uncontrolled ion flux and the secretion of damage-associated molecular patterns (DAMPs) and proinflammatory cytokines intrinsic to pyroptotic cell death (IL-1β and IL-18). Recent advancements improved the understanding of the interactions between different upstream and downstream effectors of the inflammasome, which is at the center of the canonical pathway. Of high interest is the role of the pore-forming gasdermin family. Gasdermin activation however is not solely governed by canonical and non-canonical pyroptotic pathways, given the increasing evidence that multiple (cell death) pathways influence the activation or inhibition of the gasdermin family. Moreover, it is not completely understood when GSDMD activation only leads to the release of proinflammatory cytokines and DAMPs or irreversibly results in cell death. In this review, we will summarize new data on the interplay of pyroptosis and pathways regulating gasdermin activation, discuss their roles in MASH, and potentials as therapeutic targets.

Gasdermins are expressed in various cell and tissue types and were initially identified in the murine gastrointestinal tract and epidermis, hence, their “gasdermin” nomenclature (Tamura et al., 2007). The six known members of the GSDM family (Box 2) are GSDMA, GSDMB, GSDMC, GSDMD, GSDME (also known as DNFA5), and PJVK (DFNB59). All gasdermins (except PJVK, which has a truncated C-terminal domain) share a similar two-domain architecture in which the C-terminal domain functions as an inhibitory domain to restrict the N-terminal pore-forming function. Both domains are connected through a linker region unique to each gasdermin protein. Beyond the first observations of GSDMD mediating caspase-1 and 11-dependent pyroptosis in immune cells, subsequent studies revealed many similar and unique features that characterize gasdermins, including their cellular source, activation pathways, and biological functions (He et al., 2015; Kayagaki et al., 2015; Shi et al., 2015).

The caspase cleavage site residues in the linker region vary among gasdermin family members, and not all gasdermins are cleaved by caspases (Figure 1C). Even though the role of GSDMD as the executioner of pyroptosis signaling is well described, GSDMD deficiency does not completely suppress pyroptosis, pointing out the various overlapping and redundant pathways (Kayagaki et al., 2015; Galluzzi et al., 2018). Upon closer examination of the available evidence regarding gasdermin regulation, it can be concluded that (1) the repertoire of pathways leading to gasdermin activation expands beyond the caspase family, (2) gasdermins function differently in different cell types based on the stimuli, (3) pyroptosis and caspase-pathways might be fully dependent on one another to regulate pore formation via different gasdermins.

Apoptotic cell death is executed by caspase-8 (extrinsic apoptosis) or caspase-9 (intrinsic apoptosis). Both initiator caspases, in turn, activate the executioner caspases-3 and -7 (46). Recent evidence showed that gasdermin activation is not always caspase-1-dependent but is also regulated by known apoptosis-regulating caspases. Caspase-3, for instance, was identified to cleave GSDME in the linker region to liberate GSDME^NT, which activates pyroptosis in a similar mechanism to that of GSDMD^NT. In addition, caspase-3 was also shown to cleave GSDMD; however, cleavage occurs within the N-terminal region rather than in the linker region, leading to the inhibition of GSDMD by generating an inactive NT fragment (Rogers et al., 2017; Wang et al., 2017; Shojaie et al., 2020). In virus-infected macrophages, caspase-3-mediated cleavage of GSDME was observed to induce pyroptosis and cell rupture after cells already have entered apoptosis, which has been termed “secondary necrosis” (or post-apoptosis) (Rogers et al., 2017). In contrast, Wang et al. (2017) postulated that the caspase-3-GSDME axis can directly induce pyroptotic cell death in chemotherapy-treated cells lacking apoptotic features. Inhibition of caspase-1 as the main regulator of GSDMD activation seems logical to prevent pyroptotic cell death. However, it is likely that even when caspase-1 is inhibited, cells remain doomed by activation of compensatory signaling pathways. In the absence of caspase-1, cells can still undergo lytic cell death. In murine caspase-1-deficient BMDMs treated with flagellin, a bacterial PAMP and activator of the NLRC4 inflammasome, cell death was induced via NLRC4/caspase-8, resulting in apoptosis, plasma cell membrane damage, and subsequent secondary necrosis (Lee et al., 2018b). This cell death (of an unknown program) was independent of the GSDME-caspase-3 axis. The gasdermin activation via caspase-8 seems to be cell-specific: In response to the Yersinia bacteria infection, caspase-8 directly cleaves GSDMD, whereas GSDME is dispensable for macrophage cell lysis downstream of the ripoptosome (Chen et al., 2019). The authors proposed that—under the control of the ripoptosome (RIPK1 and RIPK3)-caspase-8-axis, which can control necroptosis and apoptosis—NLRP3 inflammasome activation was induced by potassium efflux through the pannexin-1 channel rather than GSDMD (Chen et al., 2019), which is in contrast to a previous study that showed that ion efflux and NLRP3 activation were caspase-8-dependent (Orning et al., 2018). However, pore formation via pannexin-1 would explain why GSDMD deficiency does not completely abolish IL-1β secretion. Apoptosis is negatively regulated by X-linked inhibitor of apoptosis (XIAP). Loss of XIAP increased cell death and inflammatory responses in both the colonic mucosa and peripheral blood mononuclear cells (PBMCs) of XIAP-deficient IBD patients reflecting heightened caspase-8, GSDMD, and IL-1β activation (Hughes et al., 2023). It appears that the current studies have only scratched the surface of our comprehension of the intricate mechanisms that regulate programmed cell death, which is essential for a controlled immune response and the prevention of adverse effects caused by an uncontrolled inflammatory response. Gasdermins, which are expressed in various cell types, have diverse functions depending on the cell type and pathological condition. Briefly, GSDMD activation in macrophages induces the release of proinflammatory cytokines and the release of GSDMD^NT fragments that can bind to bacterial cardiolipin leading to the rupture of bacterial membranes and efficient host defense. In neutrophils, the canonical, caspase-1-mediated activation of GSDMD induces pyroptosis but is dispensable for NETosis, a neutrophil-specific cell death characterized by the production of neutrophil extracellular traps (NETs) (Chauhan et al., 2022). Conversely, caspase-1-independent cleavage of GSDMD by either neutrophil-specific serine protease, neutrophil elastase (ELANE), or non-canonical caspases of the inflammasome was shown to be involved in NETosis by possibly forming membrane pores and allowing extrusion of DNA fragments that can form NETs (Chen et al., 2018; Kambara et al., 2018; Sollberger et al., 2018; Burgener et al., 2019). Of note, in an E.coli-induced peritonitis model GSDMD deficiency led to improved host defense by delaying neutrophil death and increased levels of proinflammatory cytokines in the peritoneal cavity, showing an anti-inflammatory effect of ELANE-dependent GSDMD activation in neutrophils (Kambara et al., 2018). Thus, GSDMD can play a pleiotropic and context-dependent role, exerting both pro- and anti-inflammatory effects. Further insights into the gasdermin pathway regulation in neutrophils were gained by the study of Burgener et al. (2019) The authors showed that CathepsinG activates both executors of apoptosis and pyroptosis, namely caspase-7 and GSDMD.

The incidence of metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease, is increasing worldwide (Younossi et al., 2018). The disease is complicated by its potential progression as a result of liver inflammation and fibrosis, termed metabolic dysfunction-associated steatohepatitis (MASH, formerly NASH), which increases the risk for the development of cirrhosis, liver failure, or the development of primary liver cancer. Approximately 10% of MASLD patients will progress to MASH, and 20%–25% of these patients will develop fibrosis that will progress to cirrhosis (Younossi et al., 2018). MASH is diagnosed via liver biopsy and is pathologically characterized by lobular inflammation, accumulation of intracellular lipids (steatosis), and hepatocyte ballooning, with or without fibrosis. However, the molecular and inflammatory mechanisms causing the progression to MASH are not yet fully understood. Hepatocyte cell death as a result of lipotoxicity is one of the key drivers that causes the progression of MASLD to MASH. The persistent release of DAMPs by stressed and injured hepatocytes is recognized by immune cells and can lead to chronic inflammation. However, cell death in the setting of MASLD/MASH is not restricted to hepatocytes but also occurs in various subsets of immune cells and other non parenchymal liver cells which can influence disease progression (Luedde et al., 2014). For a detailed description of different types of cell death in MASLD/MASH we refer the reader to extensive reviews on the topic as we will focus on pyroptosis and gasdermins in MASH (Schuster et al., 2018; Schwabe et al., 2020). Here, we will describe the role of NLRP3 and GSDMD in liver inflammation and MASH and discuss cell type-specific effects of pyroptosis in murine and human MASH.

In the liver, the first demonstration of pyroptotic cell death as a novel form of cell death contributing to tissue injury came from a study using Nlrp3 knockin mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3), resulting in a hyperactive NLRP3 (Wree et al. 2014a). NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation in these mice. In the setting of diet-induced liver inflammation and fibrosis, levels of inflammasome components, including NLRP3, pro-IL-18, pro-IL-1β, ASC, and caspase-1 were found to be elevated in both MASH patients and experimental MASH animal models (Wree et al., 2014b; Gaul et al., 2021). These results indicated that NLRP3 is also a driver platform of inflammation in MASLD and facilitates progression to MASH or worsens MASH. However, studies using models of isolated steatosis initially suggested that at these early stages of the disease, global inhibition of inflammasomes may be detrimental to the liver. Indeed, Henao-Mejia et al. (2012) found that in models of isolated hepatic steatosis, including a short-term (4 weeks) methionine and choline-deficient diet (MCD) diet model, various global inflammasome-related knockouts (Asc^−/− , Caspase-1^−/− , Il-18^−/− , Nlrp6^−/− , Nlrp3^−/− , Nlrc4^−/− ) had increased levels of ALT and AST as well as increased MASLD activity scores, with similar results in the leptin-receptor deficient animals (db/db) MASLD mouse model which was additionally co-housed with Asc^−/− mice resulting in comparable levels of liver injury. Using murine models associated with more advanced diseases resembling human MASH and fibrosis, Wree et al. (2014b) demonstrated that the global Nlrp3 knockout mice were protected from MASH-associated hepatomegaly, liver injury, and infiltration of activated macrophages. Further, hepatic fibrosis was attenuated but steatosis development seemed to be unaffected. In a second mouse model, a short-term choline-deficient L-amino-defined (CDAA) diet was fed for 4 weeks to induce steatosis in WT mice and tamoxifen-inducible Nlrp3 knockin mice with the alanine 350 to valine (A350V) mutation. WT animals showed isolated hepatic steatosis while inducible Nlrp3 knockin mice showed severe liver inflammation, with increased infiltration of activated macrophages and early signs of liver fibrosis (Wree et al., 2014b).

Since GSDMD is identified as the executor of pyroptosis by forming pores in the cell membrane, it has been intriguing to consider its crucial, proinflammatory role in the development of MASH. As suspected, protein levels of GSDMD and its pyroptosis-inducing fragment GSDMD^NT were increased in liver tissues of human MASLD/MASH compared to healthy controls (Xu et al., 2018). The GSDMD^NT levels correlated with both the MASLD activity score and liver fibrosis. In the same study, GSDMD-deficient mice were either fed MCD to induce MASH or high-fat diet (HFD) to induce MASLD. In both models, GSDMD deficiency safeguarded the mice from steatohepatitis and fibrosis. Further, it was demonstrated that GSDMD triggered the expression of proinflammatory cytokines (such as IL-1β, TNF-α, and MCP-1), resulting in the activation of the NF-κB signaling pathway and the subsequent recruitment of macrophages in MASH, implying that the pyroptosis-GSDMD axis plays a crucial role in the development of MASH. Hepatocytes isolated from C57BL/6J mice fed a high-fat, high-cholesterol diet (HFHCD) for 12 weeks showed increased levels of GSDMD^NT, suggesting that pyroptosis in hepatocytes reflects diet-induced liver injury (Koh et al., 2021). In addition, the knockdown of the NLRC4 inflammasome prevented MASH and significantly decreased GSDMD^NT levels. Interestingly, isolated hepatocytes from HFHCD-fed Nlrp3 knockout mice in the same study had increased levels of GSDMD^NT fragments and increased cell death, whereas in vivo experiments indicated that caspase-1 and Nlrp3 knockout mice were protected from HFHCD-induced hepatic inflammation and fibrosis, which is in line with previous results (Dixon et al., 2012; Mridha et al., 2017). The authors concluded that NLRP3 does not directly mediate hepatocyte-specific GSDMD-dependent pyroptosis, and the beneficial effects of NLRP3 inhibition are rather the results of its effects dampening inflammation triggered by infiltrating pro-inflammatory macrophages. In a diet model where mice were either fed a high-fat diet or additionally received the carcinogen 9,10-dimethyl-1,2-benzanthracene (DMBA) for hepatocellular carcinoma development, cleaved-caspase-1, caspase-11, and cleaved-GSDMD were upregulated in freshly isolated hepatic stellate cells (HSCs) (Yamagishi et al., 2022). A transient knockdown of GSDMD in cultured murine HSCs reduced the release of IL-1β, whereas the stimulation with lipoteichoic acid (TLR2 ligand) facilitated the expression and maturation of caspase-11, leading to the creation of N-terminal fragments and pore formation as well as increased expression of IL-1β and IL-33. The diet-induced MASH mouse models are summarized in Table 1. The pleiotropic effects of the inflammasome-gasdermin signaling axis in different stages of the MASLD-MASH sequence need further elucidation in future studies. All studies discussed above have in common that the transgenic mice were full-body knock outs and not cell type- or liver-specific. Given the cell-dependent effects of pyroptosis, it is of utmost importance to look at the cell type-specific role of NLRP3 in liver inflammation and MASH to understand such mechanisms better.

Apart from different disease stages, recent studies have allowed for a better understanding of pyroptosis and inflammasome activation in MASLD/MASH by focusing on the cell-specific effects of pyroptosis. Liver inflammation and fibrogenesis in MASH are complex and dynamic processes, necessitating intricate interplays amongst diverse cell populations, cytokines, and signaling pathways. The liver consists of hepatocytes as well as nonparenchymal cells including HSCs, liver sinusoidal endothelial cells (LSECs), Kupffer cells, and other immune cells. Recent evidence has shed light on the distinct roles played by these various cell types in MASH pathogenesis, unveiling specific subsets within immune cells that can exert varying effects on the disease. The detailed mechanisms of cellular interactions governing inflammation and fibrosis in steatotic liver disease have been extensively reviewed in other sources (Peiseler et al., 2022). Here we will focus on the cell type-specific-effect of pyroptosis in liver inflammation and MASH (Figure 2).

To investigate the cell-specific impact of NLRP3, Gaul et al. used a hepatocyte-specific Nlrp3 ^L351P/+Alb-Cre knockin mouse. It was shown that hepatocytes may undergo NLRP3-mediated pyroptosis and thereby amplify inflammasome-driven hepatic fibrogenesis (Gaul et al., 2021). The study also added to the knowledge on pyroptosis-related intercellular communication by uncovering that HSCs engulf extracellular NLRP3 inflammasome particles, increasing IL-1β secretion and α-smooth muscle actin expression in these cells. HSCs are known to be responsible for extracellular collagen deposition and play a central role in liver fibrogenesis. The role of pyroptosis in HSCs was further elucidated by an overexpression model of NLRP3. In the study by Inzaugarat et al. (2019) HSC-specific Nlrp3 ^L351P/+ knockin mice were generated and histological examination revealed increased collagen deposition in the liver as well as increased markers of fibrosis compared to WT littermates, indicating that pyroptosis can stimulate the transdifferentiation of quiescent HSCs to collagen-producing myofibroblasts. Markers for activation of macrophages and neutrophils were similar between Nlrp3 ^L351P/+Lrat-Cre knockin mice and WT. Neutrophils are also an abundant cell type in MASH livers and play a dual role in the pathobiology of MASH. They can trigger liver injury and fibrosis associated with diet-induced MASH in mice, presumably by releasing toxic molecules including proteases, oxidants, cytokines and NETs but depletion in the resolution phase of MASH leads to defective repair and remodeling processes (Kim et al., 2021). Their role as resolving effector cells in liver fibrosis that induce pro-inflammatory macrophages into a restorative phenotype and thereby reversing inflammation once the injury trigger ceases is potentially exerted via miR-223, a critical negative regulator of NLRP3 (Calvente et al., 2019). However, the role of NLRP3 activation in neutrophils during liver inflammation and fibrosis is not completely understood and available data is scarce. To shed light on these mechanisms, our group has developed mouse models with neutrophil-specific mutant NLRP3 (Kaufmann et al., 2022a). Mutant mice developed severe liver inflammation and lethal autoinflammation that was comparable to mice with a systemic expression of mutant NLRP3. NLRP3 activation in neutrophils leads to a pro-inflammatory cytokine and chemokine profile in the liver, infiltration by neutrophils and macrophages, and an increase in cell death. Furthermore, mutant mice develop liver fibrosis associated with increased expression of pro-fibrogenic genes. Details of the different pyroptosis-inducing models can be found in Table 2. Even though these studies gave broad insight into the cell-specific role of pyroptosis in liver fibrosis and inflammation, comprehensive data for the cell-specific impact of NLRP3 on MASLD/MASH is still scarce. To that end, Kaufmann et al. (2022b) compared mice that either had a hepatocyte-, HSC- or myeloid cell-specific deletion of Nlrp3 and were fed a MASH-inducing diet (CDAA-HFD) or a Western diet. Nlrp3 deletion in myeloid-derived cells led to reduced liver fibrosis and reduced markers for HSC activation (Col1a, Col3a). In contrast, the deletion of Nlrp3 in HSCs or hepatocytes was not protective. In co-culture experiments, NLRP3 was activated in WT and NLRP3 KO THP-1 cells (human monocytes), by stimulation with LPS and nigericin and then co-cultured with human LX-2 cells (HSCs). The HSCs were activated by the inflammasome-stimulated monocytes, and this effect was significantly reduced where NLRP3 was downregulated in monocytes. The findings highlight the pyroptosis-related crosstalk between monocytes and HSC contributing to the transdifferentiation of HSCs into active myofibroblasts.

Great efforts have been made to develop compounds and drugs to inhibit key drivers of the pyroptosis pathways. The inflammasomes (mainly NLRP3) and the gasdermins are at the focus of drug development for various inflammatory diseases, including MASH. Considering the crucial role of GSDMD in inflammasome-mediated cell death and cytokine secretion, the blocking of gasdermin pores is currently being explored as a new therapeutic target for anti-inflammatory interventions. Regarding the GSDM family, Liu et al. (2019) analyzed mutations interfering with the lipid binding and oligomerization of gasdermins and thereby impeding cell lysis. These regions are responsible for lipid binding, oligomerization, and pore formation, making them appealing targets for developing therapeutic molecules through structure-based design. However, as for MASH, the development of a therapy has been challenging and no drugs have been approved for the treatment of the disease. Drug approval for MASH treatment requires a substantial histological improvement, a resolution of MASH and/or an improvement in fibrosis stage proven by liver biopsy. Seeing as fibrosis is an important predictor of morbidity and mortality outcomes and since pyroptosis contributes to hepatic inflammation and fibrosis, targeting pyroptosis (amongst other pro-inflammatory and profibrotic pathways) can be a therapeutic option. It should be mentioned that a variety of drugs directed against metabolic, proinflammatory and pro-fibrotic pathways associated with MASH are under investigation and are described in detail elsewhere (Tacke et al., 2023). In the following sections, we will highlight the current status and recent advances in the development of antipyroptotic therapeutics.

New insights have enhanced our understanding of the mechanisms governing cell death, particularly shedding more light on the interplay between different types of cell death. In the context of MASLD and MASH, it is crucial to apply our knowledge of these pathways to better target the key cell types involved in MASH development. This can be accomplished by utilizing rodent models that are cell and liver specific. It is clear that we are only at the initial stages of comprehending how different cell types and their subsets contribute to aggravating or resolving MASH.

Upcoming studies will provide much needed insights into unanswered questions, including the role of the entire gasdermin family or negative regulators of pyroptosis in MASH. Although completely specific gasdermin inhibitors are still undergoing preclinical testing, drugs targeting multiple pathways, including pyroptosis, are already being examined in clinical studies. This underscores the significance of further exploring the diverse regulatory pathways of gasdermin-dependent cell death to identify new therapeutic targets for treating inflammatory diseases. Future studies will need to investigate the interactions between different cell death pathways and the cell-specific effects of inhibiting cell death effector proteins. The improved understanding of these intricate and complex processes will fuel advancements in drug development for targeting inflammatory disorders and MASH.