Pyroptosis: A promising therapeutic target for noninfectious diseases

Abstract Pyroptosis, which is characterized by gasdermin family protein‐mediated pore formation, cellular lysis and the release of pro‐inflammatory cytokines, is a form of programmed cell death associated with intracellular pathogens‐induced infection. However, emerging evidence indicates that pyroptosis also contributes to sterile inflammation. In this review, we will first illustrate the biological process of pyroptosis. Then, we will focus on the pathogenic effects of pyroptosis on multiple noninfectious disorders. At last, we will characterize several specific pyroptotic inhibitors targeting the pyroptotic signalling pathway. These data demonstrate that pyroptosis plays a prominent role in sterile diseases, thereby providing a promising approach to the treatment of noninfective inflammatory disorders.

Inflammasomes are multi-protein complexes that assemble in the cytosol after sensing pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). 14 Inflammasomes can be divided into canonical inflammasomes and noncanonical inflammasomes according to their different compo- an adaptor apoptosis-associated speck-like (ASC) and caspases-1, whereas noncanonical inflammasomes are just assembled by human caspase-4/5 (mouse orthologs caspase-11). 15,16 The canonical and noncanonical inflammasomes can be activated by a repertoire of infectious and sterile stimuli. For an instant, NLRP1 inflammasome responds to Bacillus anthracis. NLRC4 inflammasome senses Salmonella. NLRP3 responds to ROS and K + . AIM2 inflammasome detects DNA virus. Pyrin inflammasome discerns toxins. In addition, noncanonical inflammasome senses lipopolysaccharide (LPS) of Gram-negative Bacilli. 17 Of interest, pyroptosis is first identified to be triggered by caspase-1 downstream of canonical inflammasomes (Figure 1). In 2015, He et al and Shi et al 18,19 indicated that caspase-1, which is activated downstream of NLRP3, pyrin and AIM2 inflammasomes activation, can induce pyroptotic cell death by cleaving gasdermin D (GSDMD) into a 31 kDa pore-forming N-terminal GSDMD NT fragment in marrow-derived macrophages (BMDMs). Similarly, Linder et al 20 showed that caspase-1, which is activated downstream of CARD8 inflammasome upon dipeptidyl-peptidase (DPP) inhibition, induces pyroptosis in human resting T cells. Mechanistically, the GSDMD NT fragment, which is cleaved by active caspase-1, binds to and then inserts into the lipid bilayer of the plasma membrane. 21,22 Using high-resolution atomic force microscopy (AFM) and cryoelectron microscopy (cryo-EM), Mulvihill et al and Xia et al 23,24 further showed that the β1-β2 loop of GSDMD NT prepore is critical for its insertion into the lipid bilayer. They found that the hydrophobic tips of β1-β2 loop serve as an anchor for insertion whilst the surrounding basic residues interact with the acidic lipids. Once inserted, GSDMD NT fragment oligomerizes and forms ring-like membranespanning pores through regulator-rag-mTORC1-mitochondrial reactive oxygen species (ROS) pathway. 25 Interestingly, Xia et al 24 proposed that GSDMD NT prepore, which enriches with negative potentials, preferentially releases positively charged mature IL-1β but not negatively charged pro-IL-1β through an electrostaticsdependent way.

| Pro-apoptotic caspase-induced pyroptosis
Apart from pro-inflammatory caspases, several studies have demonstrated that pyroptosis can be induced via pro-apoptotic caspases independent of the canonical or noncanonical inflammasomes Mechanistically, TNFα/TAK1 inhibitor, which can promote TNF complex IIb formation, induces the autoprocessing of caspase-8 into active p18 fragment and forms dimerization, finally triggering GSDMD cleavage and GDSMD NT -mediated pyroptosis. 31 Interestingly, caspase-8 can also shear gasdermin C (GSDMC) into GDSMC NT fragments and subsequently switch apoptosis to pyroptosis in breast cancer cells with TNFα treatment. 32 Consistently, Zhang et al 33 recently showed that the caspase-8, which is recruited to ROS-oxidized and internalized death receptor 6 (DR6) after application with dimethylα-ketoglutarate (DMα-KG), can cleave GSDMC at Asp240 and subsequently trigger GSDMC-mediated pyroptotic cell death in many human gastric cancer cells (SGC-7901 and BGC-823), human colon cancer cells (HCT116) and human hepatoma cells (Huh7).

| Granzyme-triggered pyroptosis
In 2020, two independent studies illustrated for the first time that pyroptosis can be triggered through granzyme, which is independent of inflammatory or pro-apoptotic caspases ( Figure 3). Zhang et al 36 found that serine protease Granzyme B (GzmB) from cytotoxic lymphocytes can induce GSDME cleavage directly in target cells. Similarly, Zhou and colleagues gave another evidence that Granzyme A (GzmA), which is also released from cytotoxic lymphocytes, can cleave gasdermins B (GSDMB) directly and eventually give rise to GSDMB-mediated pyroptosis in target cells. 37 These studies rewrite the conclusion that pyroptosis can only be activated by caspases, which expands our understanding of the activation modes of pyroptosis.

| PYROP TOS IS IN NONINFEC TI OUS DISE A SE S
In normal physiology, pyroptosis plays a critical role in anti-microbial innate immune defences. 38,39 However, excessive cell deaths and inflammatory responses caused by pyroptosis may also have deleterious effects on various sterile, noninfectious disorders ( Figure 4).

| Pyroptosis in sterile inflammatory diseases
The sterile inflammatory response, which is in the absence of infection, is required for organ development, tissue repair and host defence. However, dysregulated sterile inflammation may lead to many inflammatory diseases, including lung inflammation, type 2 diabetes and liver sterile diseases. Given the critical effects of pyroptosis on driving inflammation, it has been hypothesized that pyroptosis may function as a potential contributor in several sterile inflammatory diseases.
Previously, a series of genome-wide association studies (GWAS) indicated that GSDMA and GSDMB polymorphisms are associated with asthma. [40][41][42] However, the mechanism whereby GSDMA and GSDMB promote the onset of asthma is largely unknown. Recently, Panganiban et al 43 proved that GSDMB might contribute to asthma through GSDMB-dependent pyroptosis in airway epithelial cells.
Additionally, a splicing variant (rs11078928) of GSDMB can reduce asthma risk as this variant can abolish GSDMB-mediated pyroptosis by deleting 13 amino acids in the N-terminus of GSDMB.
Furthermore, inflammasomes (such as NLRP3) and caspases (such as caspase-1/11) have been reported to participate in asthma.
Toluene diisocyanate (TDI) was found to exacerbate asthmatic airway inflammation by inducing NLRP3 inflammasome activation.
Mechanistically, active NLRP3 inflammasome in epithelial cells activate caspase-1 to cleave GSDMD, finally increasing the IL-1β release and aggravating the airway inflammation in asthma. 44 Moreover, Zaslona et al 45 identified that caspase-11-driven pyroptosis in macrophages is a critical regulator of allergic airway inflammation. Taken together, these studies advance our knowledge of the contributory role of pyroptosis in asthma.
Type 2 diabetes (T2D) is a chronic disease characterized by hyperglycemia and relative insulin deficiency due to the progressive loss of insulin secretion. Islet inflammation has proved to be a major pathological cause of insulin secretion deficiency. Recently, Chang et al 46 identified that NLRP3 inflammasome-induced pyroptosis contributes to islet inflammation in type 2 diabetes mellitus patients and rats. Mechanistically, NEK7 (NIMA-related kinase 7), which can be suppressed by miR-23a-3p, is highly expressed in type 2 diabetes mellitus patients and rats. Upregulated NEK7 triggers NLRP3 inflammasome activation and then promotes caspase-1-GSDMD-mediated F I G U R E 3 Granzyme-induced pyroptosis. GSDMB-or GSDME-regulated pyroptosis can be induced through granzymes, which are released from killer cytotoxic lymphocytes (including NK cell/ CD8 + T cell) and chimeric antigen receptor T cell (CAR-T cell). CAR-T cell, chimeric antigen receptor T cell; GzmA, granzyme A; GzmB, granzyme B; NK cells, natural killer cells

F I G U R E 4 Pyroptosis in noninfectious
diseases. Pyroptosis contributes to multiple noninfectious disorders including neuronal diseases, sterile inflammatory diseases, cancer, atherosclerosis, acute injury, adverse pregnancy complications and autoimmune diseases via lytic cell death, cytokines releases, dysfunction of organelles and auto-antigens release. Meanwhile, pyroptosis can also inhibit the development of some specific tumours via pyroptotic cell death and antitumour immunity. Redline and text mean promotion. Greenline and text indicate suppression. AT1-AA, angiotensin II type 1 receptor autoantibody pyroptotic cell death and IL-1β releases in BMDM, eventually resulting in islet inflammation and T2D onset. Moreover, pyroptosis also exerts contributory effects on multiple diabetic complications, including diabetic cardiomyopathy and diabetic retinopathy.
Pyroptosis also contributes to sterile inflammatory liver diseases, including alcoholic hepatitis (AH), nonalcoholic fatty liver disease these studies provide experimental evidence to understand how the pyroptotic signalling pathway enhances sterile inflammation in various liver diseases, which provide potential therapeutic targets for sterile inflammatory liver diseases.

| Pyroptosis in autoimmune diseases
Autoimmune diseases are characterized by the production of autoreactive antibodies that react with immune effector cells or host tissues. Accumulating evidence suggests that pyroptosis is involved in the pathogenesis of autoimmune diseases.
Systemic lupus erythematosus (SLE), which is an autoimmune disease with multi-system damage, is characterized by the presence of autoreactive antibodies, immune complex formation and deposition in the, joints, kidneys and serosal membranes. Accumulated evidence indicates that pyroptosis is crucial for SLE. Pyroptosis in monocytes and macrophages, which is activated by canonical inflammasomes downstream of interaction with dsDNA/dsDNA antibody or U1 small riboprotein (U1-snRNP)/anti-U1-snRNP antibody, can potentiate the inflammatory responses in SLE patients by releasing IL-1β, IL-18 and HMGB1. 54,55 Interestingly, recent evidence illustrated that the intact nuclei, which is released from pyroptotic monocytes or macrophages, might serve as a newly identified autoantigen for SLE. 56 Consequently, targeting pyroptosis might be a good way to treat SLE.

| Pyroptosis in neuronal diseases
Accumulating evidence suggests that pyroptosis might participate in the pathology of neuronal diseases through multiple pathways. First, pyroptosis can induce perforation in the plasma membrane of neurons, microglia and astrocytes, which leads to pyroptotic cell death directly. Second, pyroptosis potentiates neuroinflammation via proinflammatory cytokines release. Third, pyroptosis might cause organelle dysfunctions by forming pores in their membrane.
Ischemic stroke, the second leading cause of death in the world, is originally from blocks or plugs in a blood vessel in the brain by that downregulated low-density lipoprotein receptor (LDLR) promotes NLRP3-mediated neuronal pyroptosis, ultimately leading to neuronal injury in ischemia. Additionally, GSDMD-mediated pyroptosis in microglia, astrocytes and infiltrating macrophages downstream of canonical/non-canonical inflammasomes activation facilitate the passage of intracellular inflammatory factors, ultimately promoting ischemic brain injury. [65][66][67] Furthermore, pyroptosis in neurons might also induce mitochondria dysfunctions, finally resulting in increasing ROS levels and aggravating ischemic injuries. 63 Combined, these studies suggest that pyroptosis might be a promising therapeutic target for ischemic stroke.  Additionally, the pyroptotic pathway, which is activated by Aβ and hyperphosphorylated tau, is also implicated in AD. Mechanistically, inflammasomes, including NLRP1, AIM2 and NLRP3 inflammasome, can be activated by Aβ or hyperphosphorylated tau, ultimately leading to GSDMD-dependent neuronal pyroptosis in vitro and in vivo. [73][74][75] These studies extend our understanding of the pathogenesis of AD, which points to the modulation of pyroptosis as a novel therapeutic strategy for AD.

| Pyroptosis in cancer
The role of pyroptosis in cancer is much more complex, which is influenced by many factors, including tissues source and genetic backgrounds ( Figure 5). On the one hand, pyroptotic proteins might function as oncogenes in multiple tumours. For example, GSDMB may act as an oncogene to promote tumorigenesis in the liver, gastric tissues, uterine, cervical and breast cancers. 76  On the other hand, several lines of evidence implicated that pyroptotic proteins may serve as tumour suppressors. Sasaki et al showed that GSDMA, which is downstream of transforming growth factorβ (TGFβ), is highly expressed in the gastric epithelial cell lines but appears silenced in gastric cancer cell lines. 81,82 Similarly, GSDME, which is also highly expressed in normal tissue, is downregulated by promoter DNA methylation in colorectal cancer and breast cancer. 83,84 Since GSDMA and GSDME exert critical tumour-suppressive effects on tumorigenesis, upregulation of GSDMA/GSDME and induction of GSDMA/GSDME-related pyroptosis might be a promising therapeutic target for the treatment of CD8 + T lymphocytes, can directly cleave GSDME and consequently enhance the anti-tumour immunity by activating GSDME-mediated pyroptosis in breast cancer cells and melanoma. Analogously, Zhou et al 37 showed that GzmA, which is also derived from cytotoxic T cells and NK cells, can enhance tumour clearance via directly triggering GSDMB-mediated cancer cell pyroptosis. Additionally, CAR T cellreleased GzmB triggers GSDME-mediated pyroptosis in target tumour cells. 89 Interestingly, Wang and colleagues further illustrated that pyroptosis can also augment antitumour immunity by sensitizing 4T1 tumours to anti-PD1 therapy in a bioorthogonal system. 90 Collectively, these results indicate that pyroptosis and pyroptotic proteins can exert tumour suppressive effects via induction of pyroptotic cancer cell death and enhancement of anti-tumour immunity.
Taken together, these studies provide a comprehensive view of pyroptosis in cancer. The specific role and mechanism of pyroptosis in tumorigenesis warrant further investigations. Pathologically, AIM2 inflammasome, which is upregulated by oxLDL, mediates GSDMD-dependent VSMCs pyroptosis through ASC/ caspase1 pathway. 105 Additionally, NLRP3 inflammasome can be activated by oxLDL, finally resulting in VSMCs pyroptosis and progressing the pathological condition of AS. 106

| Pyroptosis in acute injury
An acute injury is an injury that usually results from a specific impact or trauma in the brain, lung or kidney. Inflammation and pyroptotic cell death, which are triggered via cytoplasmic inflammasome complexes, are regarded as key contributors to acute injuries. Traumatic brain injury (TBI) is sudden traumatic damage in the brain with oedema, axonal shearing, neuronal death and vascular damage. The post-TBI primary insult typically leads to secondary damage, including neuroinflammation, 108 neuronal cell death 109 and mitochondrial dysfunction. 110 Growing research has revealed that neuroinflammation and neuronal pyroptotic cell death mediated by active caspase-1 downstream of NLRP1/NLRP3/AIM2 inflammasome activation is pivotal mechanisms of brain injury responses in TBI. [111][112][113] Additionally, pyroptosis of infiltrating CD11 + leukocytes and activated microglia contributes to the pathophysiology of secondary injury after severe TBI. 114 Furthermore, canonical inflammasome-induced pyroptosis in brain microvascular endothelial cells (BMVECs) results in blood-brain barrier (BBB) leakage and brain oedema, ultimately aggravating damages after TBI. 115 Collectively, these results advance our understanding of pyroptosis in TBI.
Acute lung injury (ALI), which is characterized by acute severe hypoxia, is lung inflammation and VECs damage arising from a wide variety of both pulmonary and generalized acute diseases.
Pathological studies indicate that alveolar macrophage activation which is another critical executor of pyroptosis, is also involved in cisplatin-or I/R-induced AKI. Mechanistically, active caspase-3, which is activated after I/R-or cisplatin-treatment, cleaves GSDME and consequently contributes to I/R-or cisplatin-induced AKI by triggering GSDME-mediated pyroptosis in TECs. Together, all of these new findings confirmed that pyroptosis of TECs plays a critical role in AKI, indicating the potential for developing novel treatment against AKI by targeting the pyroptotic signalling pathway.

| Pyroptosis in adverse pregnancy complications
Pregnancy complications, such as preeclampsia (PE), gestational diabetes and preterm birth, are health problems that occur during preg-

| Pyroptosis as therapeutic targets
Given that pyroptosis takes a prominent role in these noninfective diseases, the development of small molecular inhibitors targeting pyroptotic proteins and signalling pathway is a promising therapeutic strategy ( Table 1). familial Mediterranean fever (FMF) mouse model. 158

| CON CLUS I ON S AND FUTURE PER S PEC TIVE S
Pyroptosis, a kind of inflammatory cell programmed death mediated by gasdermins protein, is an important part of innate immunity. Moreover, pyroptosis also exerts a vital role in noninfective inflammatory disorders. Our cognition of pyroptosis has gone through several stages. From the beginning, researchers just focused on the essential role of canonical or noncanonical inflammasomes (such as NLRP1, NLRP3, NLRC4 and AIM2 inflammasome) and pro-inflammatory caspases (caspases-1/4/5/11) in pyroptosis.
Subsequently, researchers discovered that pro-apoptosis caspases (caspases-3/6/8) also participate in the process of pyroptosis. Until recently, several researches illustrated that granzymes (GzmA/ GzmB) can initiate pyroptosis without caspases participation. These studies refresh our understanding of pyroptosis. Further studies will be continued to explore the precise activation modes of pyroptosis in the future.
Growing evidence indicates that pytoptosis is implicated in multiple noninfective diseases, such as sterile inflammatory diseases, autoimmune diseases, neuronal diseases, atherosclerosis, acute injuries and various cancers, thereby providing a new entry point for the treatment of these disorders. However, some inhibitors, such as caspase antagonists, NSA and disulfiram, might lead to unexpected side effects due to the lack of sufficient specificity.
Further researches are needed to improve the specificity of pyroptotic inhibitors.
Although pytoptosis exerts pathogenic effects on noninfective diseases, it also has beneficial effects on tumour suppression in some contexts. For instance, Wang and his colleagues demonstrated that a small amount of pyroptotic tumour cell death (less than 15%) is sufficient to clear the entire tumour graft. 90 Additionally, several studies demonstrated that GSDMB or GSDME-mediated pyroptosis, which is triggered by granzymes released from cytotoxic lymphocytes, can potently suppress tumour growth. 36,37 These studies suggest that enhancing pyroptosis does open novel therapeutic avenues for cancer clearance via increasing pyroptotic cell death and anti-tumour immunity. However, pyroptosis might be a double-edged sword.
Extensive pyroptosis can cause severe tissues damages. Shen et al 160 indicated that GSDME-mediated pyroptosis in renal TECs is responsible for cisplatin-or doxorubicin-induced nephrotoxicity. Moreover, GSDME-dependent pyroptosis and subsequent IL-1β/IL-6 releases in macrophages contribute to cytokine release syndrome (CRS) during CAR T cell therapy. 89 Thus, specific activation of a pyroptotic sig- flexneri. 161 This study suggests that post-translational modification, such as ubiquitination, might serve as an important way to enhance or antagonize pyroptosis. It will be very attractive to figure out the potential regulatory effects of other post-translational modifications on pyroptosis in the future.
In conclusion, pyroptosis, which is an important kind of inflammatory program cell death, plays a key role in noninfective inflammatory disorders. Future studies are needed to further demonstrate its definite role in human diseases, providing a unique therapeutic opportunity for the treatment of multiple sterile inflammatory disorders.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflicting interests.

AUTH O R CO NTR I B UTI O N
T.L. and L.T. wrote the manuscript; G.Z., B.L. and L.T. edited the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.