Gasdermin D Cleavage Research Articles

A Novel Bioluminescent Biosensor Quantifying Intramolecular Interaction and Levels of Pyroptosis Effector GSDMD.

Gasdermin D (GSDMD) is a key executor of pyroptosis, a form of inflammation-induced programmed cell death. Recently, GSDMD has been shown to play important roles in the development of various inflammatory-related human diseases including heart failure and cancer, suggesting that it is a promising therapeutic target for these diseases. While extensive studies on GSDMD's role in pyroptosis have been reported, it is challenging to study its function due to the lack of enzymatic activity of GSDMD. In this study, we used the NanoBiT technology to develop a novel GSDMD bioluminescent biosensor (GSDMD-BS) that detects the amount of non-cleaved GSDMD. This sensor allows us to quantify GSDMD's intramolecular interactions, the amounts of uncleaved GSDMD after caspase-1 cleavage, and expression levels in living cells. In vitro experiments using purified GSDMD-BS also confirmed the sensor's accuracy in reporting GSDMD levels and cleavage. Moreover, the potential for in vivo application was demonstrated in a xenograft mouse model. In conclusion, we have developed a GSDMD biosensor that is a valuable tool for real-time monitoring of GSDMD dynamics and pyroptosis. This biosensor will significantly expedite pyroptosis research and can be used for high-throughput screening for drugs targeting GSDMD for the therapy of many inflammation-related diseases.

Angelica sinensis Polysaccharide Suppresses Pyroptosis in Myocardial Ischemia/Reperfusion Injury via the FN1/NF-κB/NLRP3 Pathway

Background Pyroptosis, a form of inflammatory programmed cell death, has recently emerged as a pivotal factor in the pathogenesis of myocardial ischemia/reperfusion (MI/R) injury. Despite its significance, effective therapeutic strategies targeting MI/R-induced pyroptosis remain elusive in current clinical practice. Previous studies have demonstrated the promising anti-inflammatory effects of Angelica sinensis polysaccharide (ASP) in the context of certain inflammatory disorders. Objectives We aimed to investigate the effects of ASP on pyroptosis in MI/R injury and elucidate the potential molecular mechanisms by combining transcriptomic analysis with complementary in vivo and in vitro experiments. Materials and methods H9c2 cells were used to establish a hypoxia/reoxygenation (H/R) model, and MI/R injury was induced in rats by ligating and releasing the left anterior coronary artery. Myocardial tissue samples were harvested for transcriptomic sequencing and bioinformatic analyses. Cardioprotective effects were evaluated through electrocardiography, echocardiography, and histological examination. Enzyme-linked immunosorbent assay (ELISA) was employed to quantify levels of inflammatory mediators. Biochemical assays were conducted to assess myocardial injury biomarkers and Caspase-1 activity. Western blotting was performed to analyze the protein expression levels of Fibronectin 1 (FN1), Nuclear factor kappa B (NF-κB) p65, phosphorylated NF-κB p65 (p-NF-κB p65), Nod-like receptor protein 3 (NLRP3), and Gasdermin-D (GSDMD). Results Pretreatment with ASP conferred potent cardioprotective effects in a rat model of MI/R injury, as evidenced by significant attenuation of infarct size, myocardial enzyme levels, and ST-segment elevation on electrocardiography, alongside notable improvements in cardiac function. Transcriptomic profiling unveiled that differentially expressed genes modulated by ASP treatment were predominantly implicated in the pyroptosis-elicited inflammatory response. Concordantly, both in vivo and in vitro experiments substantiated that ASP treatment effectively attenuated MI/R-induced pyroptosis, as manifested by diminished levels of pyroptosis-related indicators, encompassing the proportion of TUNEL-positive cells, Caspase-1 activation, GSDMD cleavage, and the liberation of pro-inflammatory cytokines. Further mechanistic investigations revealed ASP inhibition of the FN1/NF-κB/NLRP3 signaling pathway. Conclusions Our investigation, leveraging system-level transcriptomic profiling, demonstrates that ASP confers cardioprotective effects against MI/R injury through the suppression of cardiomyocyte pyroptosis, culminating from downregulation of the FN1/NF-κB/NLRP3 pathway.

Investigation of the role of NLRP3 inflammasome activation in new-generation BCR-ABL1 tyrosine kinase inhibitors-induced hepatotoxicity

New generation BCR-ABL1 TKIs raised attention regarding their adverse effects, including hepatotoxicity. Indeed, bosutinib and nilotinib were associated with severe hepatotoxicity compared with imatinib. Moreover, ponatinib has a boxed warning due to its potential to cause inflammatory liver damage, even death. However, the underlying mechanisms remain unclear. This study aimed to investigate the role of NLRP3 inflammasome activation in the underlying mechanism of ponatinib and bosutinib-induced hepatotoxicity. Furthermore, we determined the initiating event of this adverse outcome pathway by measuring the levels of reactive oxygen species as well as mitochondrial membrane potential in AML12 cells. The results demonstrated that ponatinib or bosutinib markedly inhibited cell viability and caused cytosolic membrane damage in cells. Moreover, drugs (IC50) dramatically induced oxidative stress and mitochondrial membrane potential disruption, which led to upregulation in the expression levels of NLRP3 inflammasome-related genes and proteins, activation of NLRP3 inflammasomes, cleavage of gasdermin-D and caspase-1, secretion of IL-1β, and cytosolic membrane damage. Furthermore, MCC950, a well-known specific inhibitor of NLRP3 inflammasome, and antioxidant N-acetyl-l-cysteine reversed the effects of drugs on the NLRP3 signaling pathway and cytosolic membranes. In summary, NLRP3 inflammasome activation is involved in new-generation BCR-ABL1 TKIs-triggered hepatotoxicity. Mitochondrial damage and reactive oxygen species accumulation were significant upstream signaling events in this signaling pathway.

Septins promote macrophage pyroptosis by regulating gasdermin D cleavage and ninjurin-1-mediated plasma membrane rupture

The septin cytoskeleton is primarily known for roles in cell division and host defense against bacterial infection. Despite recent insights, the full breadth of roles for septins in host defense is poorly understood. In macrophages, Shigella induces pyroptosis, a pro-inflammatory form of cell death dependent upon gasdermin D (GSDMD) pores at the plasma membrane and cell surface protein ninjurin-1 (NINJ1) for membrane rupture. Here, we discover that septins promote macrophage pyroptosis induced by lipopolysaccharide (LPS)/nigericin and Shigella infection, but do not affect cytokine expression or release. We observe that septin filaments assemble at the plasma membrane, and cleavage of GSDMD is impaired in septin-depleted cells. We found that septins regulate mitochondrial dynamics and the expression of NINJ1. Using a Shigella-zebrafish infection model, we show that septin-mediated pyroptosis is an invivo mechanism of infection control. The discovery of septins as a mediator of pyroptosis may inspire innovative anti-bacterial and anti-inflammatory treatments.

Bavachinin, a main compound of Psoraleae Fructus, facilitates GSDMD-mediated pyroptosis and causes hepatotoxicity in mice

Psoraleae Fructus (PF, Psoralea corylifolia L.), a traditional medicine with a long history of application, is widely used clinically for the treatment of various diseases. However, the reports of PF-related adverse reactions, such as hepatotoxicity, phototoxic dermatitis, and allergy, are increasing year by year, with liver injury being the mostly common. Our previous studies have demonstrated that PF and its preparations can cause liver injury in lipopolysaccharide (LPS)-mediated susceptibility mouse model, but the mechanism of PF-related liver injury is unclear. In this study, we showed that PF and bavachinin, a major component of PF, can directly induce the expression of caspase-1 and interleukin-1β (IL-1β), indicating that PF and bavachinin can directly triggered the activation of inflammasome. Furthermore, pretreatment with NLR family pyrin domain-containing 3 (NLRP3), NLR family CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome inhibitors, containing MCC950, ODN TTAGGG (ODN) and carnosol, all significantly reversed bavachinin-induced inflammasome activation. Mechanistically, bavachinin dose-dependently promote Gasdermin D (GSDMD) post-shear activation and then induce mitochondrial reactive oxygen species (mtROS) production and this effect is markedly inhibited by pretreatment with N-Acetylcysteine amide (NAC). In addition, combination treatment of LPS and bavachinin significantly induced liver injury in mice, but not LPS or bavachinin alone, and transcriptome analysis further validated these results. Thus, PF and bavachinin can induce the activation of inflammasome by promoting GSDMD cleavage and cause hepatotoxicity in mice. Therefore, PF, bavachinin, and PF-related preparations should be avoided in patients with inflammasome activation-associated diseases.

Human Coronavirus 229E Infection Inactivates Pyroptosis Executioner Gasdermin D but Ultimately Leads to Lytic Cell Death Partly Mediated by Gasdermin E.

Human coronavirus 229E (HCoV-229E) is associated with upper respiratory tract infections and generally causes mild respiratory symptoms. HCoV-229E infection can cause cell death, but the molecular pathways that lead to virus-induced cell death as well as the interplay between viral proteins and cellular cell death effectors remain poorly characterized for HCoV-229E. Studying how HCoV-229E and other common cold coronaviruses interact with and affect cell death pathways may help to understand its pathogenesis and compare it to that of highly pathogenic coronaviruses. Here, we report that the main protease (Mpro) of HCoV-229E can cleave gasdermin D (GSDMD) at two different sites (Q29 and Q193) within its active N-terminal domain to generate fragments that are now unable to cause pyroptosis, a form of lytic cell death normally executed by this protein. Despite GSDMD cleavage by HCoV-229E Mpro, we show that HCoV-229E infection still leads to lytic cell death. We demonstrate that during virus infection caspase-3 cleaves and activates gasdermin E (GSDME), another key executioner of pyroptosis. Accordingly, GSDME knockout cells show a significant decrease in lytic cell death upon virus infection. Finally, we show that HCoV-229E infection leads to increased lytic cell death levels in cells expressing a GSDMD mutant uncleavable by Mpro (GSDMD Q29A+Q193A). We conclude that GSDMD is inactivated by Mpro during HCoV-229E infection, preventing GSDMD-mediated cell death, and point to the caspase-3/GSDME axis as an important player in the execution of virus-induced cell death. In the context of similar reported findings for highly pathogenic coronaviruses, our results suggest that these mechanisms do not contribute to differences in pathogenicity among coronaviruses. Nonetheless, understanding the interactions of common cold-associated coronaviruses and their proteins with the programmed cell death machineries may lead to new clues for coronavirus control strategies.

Contribution of Gasdermin D Pore Formation to NLRP3 Inflammasome Product Secretion and Pyroptosis in Podocytes during Obesity-Induced Glomerulopathy

NLRP3 inflammasome activation in podocytes has been reported to play an important role in the development of glomerular inflammation and sclerosis during obesity. Although, the molecular mechanism mediating the secretion of NLRP3 inflammasome products to induce local inflammatory response remains poorly understood. Given that gasdermin D (GSDMD) has been found to form oligomeric pores on plasma membrane for release of inflammasome product and execution of pyroptosis, the present study tested whether GSDMD pore contributes to NLRP3 inflammasome product secretion and pyroptosis in podocytes and thereby initiates glomerular inflammation and sclerosis in obesity-induced glomerulopathy (ORG). By Western blot analysis, we demonstrated that palmitic acid (PA) as an obesity-related danger factor dose-dependently stimulated the reduction of total GSDMD and elevation of cleaved GSDMD (GSDMD-NT) in podocytes. In vivo, glomerular decrease in total GSDMD and increase in GSDMD-NT were remarkable in wild type (WT/WT) mice on high-fat diet (HFD) compared to control mice. Urinary excretion of NLRP3 inflammasome products was also significantly increased by HFD treatment. Moreover, we found that pre-treatment with amitriptyline or MCC950 blocked NLRP3 inflammasome activation as well as GSDMD pore formation on plasma membrane of podocytes, while disulfiram prevented GSDMD pore formation without affecting NLRP3 inflammasome activation. Since acid sphingomyelinase (ASM)-ceramide signaling pathway plays a vital role in the development of ORG, we examined whether ASM activity determines GSDMD pore formation in podocytes. By atom force microscopy, a 3D topography map of a single podocyte was generated, and pore-like structures were observed in the membrane of PA-treated WT/WT podocytes. Podocytes lacking Smpd1 (gene code of ASM) gene did not show pore formation, but Smpd1 gene overexpression amplified PA-induced pore formation in podocytes. Moreover, it was found that HFD-induced GSDMD cleavage and pyroptosis in glomeruli were blocked by Smpd1 gene deletion in Smpd1−/− mice. On the contrary, podocyte-specific Smpd1 gene overexpression amplified GSDMD-NT production and pyroptosis in glomeruli. Correspondingly, HFD-induced podocyte foot process effacement and glomerular sclerosis in mice were inhibited by Smpd1 gene knockout but exaggerated by podocyte-specific Smpd1 gene overexpression. Taken together, our findings suggest that ASM-ceramide signaling pathway may control GSDMD pore formation in podocytes to determine glomerular inflammation and sclerosis in ORG. This study was supported by NIH grants DK054927 and DK120491. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ROS-dependent S-palmitoylation activates cleaved and intact gasdermin D.

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation and was previously shown to form large transmembrane pores after cleavage by inflammatory caspases to generate the GSDMD N-terminal domain (GSDMD-NT)1-10. Here we report that GSDMD Cys191 is S-palmitoylated and thatpalmitoylation is required for pore formation. S-palmitoylation, which does not affect GSDMD cleavage, is augmented by mitochondria-generated reactive oxygen species (ROS). Cleavage-deficient GSDMD(D275A) is also palmitoylated after inflammasome stimulation or treatment with ROS activators and causes pyroptosis, although less efficiently than palmitoylated GSDMD-NT. Palmitoylated, but not unpalmitoylated, full-length GSDMD induces liposome leakage and forms a pore similar in structure to GSDMD-NT pores shown by cryogenic electron microscopy. ZDHHC5 and ZDHHC9 are the major palmitoyltransferases that mediate GSDMD palmitoylation, and their expression is upregulated by inflammasome activation and ROS. The other human gasdermins are also palmitoylated at their N termini. These data challenge the concept that cleavage is the only trigger for GSDMD activation. They suggest that reversible palmitoylation is a checkpoint for pore formation by both GSDMD-NT and intact GSDMD that functions as a general switch for the activation of this pore-forming family.

Protective Effect of Silibinin on Lipopolysaccharide-Induced Endotoxemia by Inhibiting Caspase-11-Dependent Cell Pyroptosis.

To explore the protective effect and the underlying mechanism of silibinin (SIB), one of the active compounds from Silybum marianum (L.) Gaertn in endotoxemia. Mouse peritoneal macrophage were isolated via intraperitoneally injection of BALB/c mice with thioglycolate medium. Cell viability was assessed using the cell counting kit-8, while cytotoxicity was determined through lactate dehydrogenase cytotoxicity assay. The protein expressions of interleukin (IL)-1 α, IL-1 β, and IL-18 were determined by enzyme-linked immunosorbent assay. Intracellular lipopolysaccharide (LPS) levels were measured by employing both the limulus amoebocyte lysate assay and flow cytometry. Additionally, proximity ligation assay was employed for the LPS and caspase-11 interaction. Mice were divided into 4 groups: the control, LPS, high-dose-SIB (100 mg/kg), and low-dose-SIB (100 mg/kg) groups (n=8). Zebrafish were divided into 4 groups: the control, LPS, high-dose-SIB (200 εmol/L), and low-dose-SIB (100 εmol/L) groups (n=30 for survival experiment and n=10 for gene expression analysis). The expression of caspase-11, gasdermin D (GSDMD), and N-GSDMD was determined by Western blot and the expressions of caspy2, gsdmeb, and IL-1 β were detected using quantitative real-time PCR. Histopathological observation was performed through hematoxylineosin staining, and protein levels in bronchoalveolar lavage fluid were quantified using the bicinchoninicacid protein assay. SIB noticeably decreased caspase-11 and GSDMD-mediated pyroptosis and suppressed the secretion of IL-1 α, IL-1 β, and IL-18 induced by LPS (P<0.05). Moreover, SIB inhibited the translocation of LPS into the cytoplasm and the binding of caspase-11 and intracellular LPS (P<0.05). SIB also attenuated the expression of caspase-11 and N-terminal fragments of GSDMD, inhibited the relative cytokines, prolonged the survival time, and up-regulated the survival rate in the endotoxemia models (P<0.05). SIB can inhibit pyroptosis in the LPS-mediated endotoxemia model, at least in part, by inhibiting the caspase-11-mediated cleavage of GSDMD. Additionally, SIB inhibits the interaction of LPS and caspase-11 and inhibits the LPS-mediated up-regulation of caspase-11 expression, which relieves caspase-11-dependent cell pyroptosis and consequently attenuates LPS-mediated lethality.

A palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation in pyroptosis.

Gasdermin D (GSDMD) is the executor of pyroptosis, which is important for host defence against pathogen infection. Following activation, caspase-mediated cleavage of GSDMD releases an amino-terminal fragment (GSDMD-NT), which oligomerizes and forms pores in the plasma membrane, leading to cell death and release of proinflammatory cytokines. The spatial and temporal regulation of this process in cells remains unclear. Here we identify GSDMD as a substrate for reversible S-palmitoylation on C192 during pyroptosis. The palmitoyl acyltransferase DHHC7 palmitoylates GSDMD to direct its cleavage by caspases. Subsequently, palmitoylation of GSDMD-NT promotes its translocation to the plasma membrane, where APT2 depalmitoylates GSDMD-NT to unmask the C192 residue and promote GSDMD-NT oligomerization. Perturbation of either palmitoylation or depalmitoylation suppresses pyroptosis, leading to increased survival of mice with lipopolysaccharide-induced lethal septic shock and increased sensitivity to bacterial infection. Our findings reveal a model through which a palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation during pyroptosis.

Mitochondrial reactive oxygen species initiate gasdermin D-mediated pyroptosis and contribute to paraquat-induced nephrotoxicity

Paraquat (PQ)-induced acute kidney injury (AKI) progresses rapidly and is associated with high mortality rates; however, no specific antidote for PQ has been identified. Poor understanding of toxicological mechanisms underlying PQ has hindered the development of suitable treatments to combat PQ exposure. Gasdermin D (GSDMD), a key executor of pyroptosis, has recently been shown to enhance nephrotoxicity in drug-induced AKI. To explore the role of pyroptosis in PQ-induced AKI, the plasma membrane damage of the cells was detected by LDH release assay. Western blot was performed to detect the cleavage of GSDMD. RNA sequencing analysis was performed to explore the mechanism of PQ induced nephrotoxicity. Herein, we demonstrated that PQ could induce pyroptosis in HK-2 cells and nephridial tissues. Mechanistically, PQ initiated GSDMD cleavage, and GSDMD knockout attenuated PQ-induced nephrotoxicity in vivo. Further analysis revealed that the accumulation of mitochondrial reactive oxygen species (ROS) induced p38 activation, contributing to PQ-induced pyroptosis. Furthermore, mitoquinone, a mitochondria-targeted antioxidant, reduced mitochondrial ROS levels and inhibited pyroptosis. Collectively, these findings provide insights into the role of GSDMD-dependent pyroptosis as a novel mechanism of PQ-induced AKI.

Discovery of Potent, Orally Bioavailable, Tricyclic NLRP3 Inhibitors.

NLRP3 is a molecular sensor recognizing a wide range of danger signals. Its activation leads to the assembly of an inflammasome that allows for activation of caspase-1 and subsequent maturation of IL-1β and IL-18, as well as cleavage of Gasdermin-d and pyroptotic cell death. The NLRP3 inflammasome has been implicated in a plethora of diseases including gout, type 2 diabetes, atherosclerosis, Alzheimer's disease, and cancer. In this publication, we describe the discovery of a novel, tricyclic, NLRP3-binding scaffold by high-throughput screening. The hit (1) could be optimized into an advanced compound NP3-562 demonstrating excellent potency in human whole blood and full inhibition of IL-1β release in a mouse acute peritonitis model at 30 mg/kg po dose. An X-ray structure of NP3-562 bound to the NLRP3 NACHT domain revealed a unique binding mode as compared to the known sulfonylurea-based inhibitors. In addition, NP3-562 shows also a good overall development profile.

Quercetin-induced pyroptosis in colon cancer through NEK7-mediated NLRP3 inflammasome-GSDMD signaling pathway activation.

Pyroptosis, a gasdermin-mediated lytic cell death, is a new hotspot topic in cancer research, and induction of tumor pyroptosis has emerged as a new target in cancer management. Quercetin (Que), a natural substance, demonstrates promising anticancer action. However, further information is required to fully comprehend the function and mechanism of Que in pyroptosis in colon cancer. This study revealed the underlying mechanism of Que-induced pyroptosis in colon cancer in vitro and in vivo. Que inhibited colon cancer cell growth through gasdermin D (GSDMD)-mediated pyroptosis. Depletion of GSDMD, rather than gasdermin E (GSDME), reversed the cytotoxic effects of Que on colon cancer cells. Que treatment upregulated NIMA-related kinase 7 (NEK7) protein expression, thus facilitating the assembly of the NLRP3 inflammasome and cleavage of GSDMD. NEK7 silencing resulted in colon cancer cell growth in vitro and in vivo. Mechanistically, NEK7 depression restrained the activation of the NLRP3 inflammasome-GSDMD pathway, thus attenuating pyroptosis triggered by Que in colon cancer cells. Furthermore, lower NEK7 and NLRP3 expression levels indicated colon cancer progression. Our results unveiled a novel pattern of anti-colon cancer activity of Que, and activation of NEK7-mediated pyroptosis is potentially a promising therapeutic target for colon cancer, which provides novel experimental proof for the clinical application of Que.

Phagocytic cell death leads to enhanced release of pro-inflammatory S100A12 in familial Mediterranean fever

BackgroundFamilial Mediterranean fever (FMF) is a prototypical autoinflammatory syndrome associated with phagocytic cell activation. Pyrin mutations are the genetic basis of this disease, and its expression has been shown in monocytes, granulocytes, dendritic cells, and synovial fibroblasts. Pyrin functions as a cytosolic pattern recognition receptor and forms a distinct pyrin inflammasome. The phagocyte-specific protein S100A12 is predominantly expressed in granulocytes and belongs to the group of damage associated molecular patterns (DAMP). S100A12 can be detected at massively elevated levels in the serum of FMF patients, even in clinically inactive disease. Whether this is crucial for FMF pathogenesis is as yet unknown, and we therefore investigated the mechanisms of S100A12 release from granulocytes of FMF patients presenting clinically inactive.ResultsWe demonstrate that FMF neutrophils from patients in clinical inactive disease possess an intrinsic activity leading to cell death even in exogenously unstimulated neutrophils. Cell death resembles NETosis and is dependent on ROS and pore forming protein gasdermin D (GSDMD), as inhibitors for both are capable of completely block cell death and S100A12 release. When pyrin-activator TcdA (Clostridium difficile toxin A) is used to stimulate, neutrophilic cell death and S100A12 release are significantly enhanced in neutrophils from FMF patients compared to neutrophils from HC.ConclusionsWe are able to demonstrate that activation threshold of neutrophils from inactive FMF patients is decreased, most likely by pre-activated pyrin. FMF neutrophils present with intrinsically higher ROS production, when cultured ex vivo. This higher baseline ROS activity leads to increased GSDMD cleavage and subsequent release of, e.g., S100A12, and to increased cell death with features of NETosis and pyroptosis. We show for the first time that cell death pathways in neutrophils of inactive FMF patients are easily triggered and lead to ROS- and GSDMD-dependent activation mechanisms and possibly pathology. This could be therapeutically addressed by blocking ROS or GSDMD cleavage to decrease inflammatory outbreaks when becoming highly active.

O-GlcNAc modification of GSDMD attenuates LPS-induced endothelial cells pyroptosis

ObjectiveIncreased O-linked β-N-acetylglucosamine (O-GlcNAc) stimulation has been reported to protect against sepsis associated mortality and cardiovascular derangement. Previous studies, including our own research, have indicated that gasdermin-D(GSDMD)-mediated endothelial cells pyroptosis contributes to sepsis-associated endothelial injury. This study explored the functions and mechanisms of O-GlcNAc modification on lipopolysaccharide (LPS)-induced pyroptosis and its effects on the function of GSDMD.MethodsA LPS-induced septic mouse model administrated with O-GlcNAcase (OGA) inhibitor thiamet-G (TMG) was used to assess the effects of O-GlcNAcylation on sepsis-associated vascular dysfunction and pyroptosis. We conducted experiments on human umbilical vein endothelial cells (HUVECs) by challenging them with LPS and TMG to investigate the impact of O-GlcNAcylation on endothelial cell pyroptosis and implications of GSDMD. Additionally, we identified potential O-GlcNAcylation sites in GSDMD by utilizing four public O-GlcNAcylation site prediction database, and these sites were ultimately established through gene mutation.ResultsSeptic mice with increased O-GlcNAc stimulation exhibited reduced endothelial injury, GSDMD cleavage (a marker of pyroptosis). O-GlcNAc modification of GSDMD mitigates LPS-induced pyroptosis in endothelial cells by preventing its interaction with caspase-11 (a human homologous of caspases-4/5). We also identified GSDMD Serine 338 (S338) as a novel site of O-GlcNAc modification, leading to decreased association with caspases-4 in HEK293T cells.ConclusionsOur findings identified a novel post-translational modification of GSDMD and elucidated the O-GlcNAcylation of GSDMD inhibits LPS-induced endothelial injury, suggesting that O-GlcNAc modification-based treatments could serve as potential interventions for sepsis-associated vascular endothelial injury.

Gasdermin D promotes hyperinflammation and immunopathology during severe influenza A virus infection

Excessive inflammation and tissue damage during severe influenza A virus (IAV) infection can lead to the development of fatal pulmonary disease. Pyroptosis is a lytic and pro-inflammatory form of cell death executed by the pore-forming protein gasdermin D (GSDMD). In this study, we investigated a potential role for GSDMD in promoting the development of severe IAV disease. IAV infection resulted in cleavage of GSDMD in vivo and in vitro in lung epithelial cells. Mice genetically deficient in GSDMD (Gsdmd−/−) developed less severe IAV disease than wildtype mice and displayed improved survival outcomes. GSDMD deficiency significantly reduced neutrophil infiltration into the airways as well as the levels of pro-inflammatory cytokines TNF, IL-6, MCP-1, and IL-1α and neutrophil-attracting chemokines CXCL1 and CXCL2. In contrast, IL-1β and IL-18 responses were not largely impacted by GSDMD deficiency. In addition, Gsdmd−/− mice displayed significantly improved influenza disease resistance with reduced viral burden and less severe pulmonary pathology, including decreased epithelial damage and cell death. These findings indicate a major role for GSDMD in promoting damaging inflammation and the development of severe IAV disease.

Inhibition of gasdermin D (GSDMD) as a promising therapeutic approach for atopic dermatitis

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritus, erythema, and skin barrier dysfunction. Gasdermin D (GSDMD) is the key executioner of an inflammatory cell death mechanism known as pyroptosis. However, the role of GSDMD in the pathogenesis of AD remains unclear. Through the analysis of publicly available Gene Expression Omnibus (GEO) datasets, we observed an upregulation of Gsdmd mRNA in the skin tissue of AD patients. Moreover, we delved into the impact of GSDMD deletion and inhibition on AD-like skin lesions using a mouse model induced by the topical application of oxazolone (Oxa). We found that mice lacking GSDMD exhibited relieved AD signs and symptoms in terms of reduced skin thickness, scarring and scratching behavior compared to wild-type mice after induction of AD-like skin lesions. This was associated with decreased infiltration of inflammatory cells, reduced epidermal thickness, and decreased serum levels of IgE and IL-4. Western blot analysis further revealed decreased GSDMD cleavage in the skin of GSDMD knockout mice, and reduced expression of IL-1β and IL-18. Inhibition of GSDMD using the pharmacological agent disulfiram or the herbal compound matrine significantly attenuated the symptoms of AD-like skin lesions in wild-type mice, GSDMD cleavage and pro-inflammatory cytokines were reduced as well. Our results suggest that GSDMD-mediated pyroptosis plays a critical role in the development of AD-like skin lesions, and targeting GSDMD may be a promising therapeutic strategy for AD.

Gasdermin D-mediated pyroptosis is regulated by AMPK-mediated phosphorylation in tumor cells

Gasdermin D (GSDMD) is a critical mediator of pyroptosis, which consists of a N-terminal pore-forming domain and a C-terminal autoinhibitory domain. Its cytolytic activity is sequestered by the intramolecular autoinhibitory mechanism. Upon caspase-1/11 mediated cleavage of GSDMD, the N-terminal pore-forming domain (GD-NT) is released to mediate pyroptosis. However, it remains unclear how GD-NT is regulated once it is generated. In the current study, we developed a TetOn system in which GD-NT was selectively induced in tumor cells to explore how the cytolytic activity of GD-NT is regulated. We found that the cytolytic activity of GD-NT was negatively regulated by the AMP-activated protein kinase (AMPK) and AMPK activation rendered tumor cells resistant to GD-NT-mediated pyroptosis. Mechanistically, AMPK phosphorylated GD-NT at the serine 46 (pS46-GD), which altered GD-NT oligomerization and subsequently eliminated its pore-forming ability. In our in vivo tumor model, AMPK-mediated phosphorylation abolished GD-NT-induced anti-tumor activity and resulted in an aggressive tumor growth. Thus, our data demonstrate the critical role of AMPK in negatively regulating the cytolytic activity of GD-NT. Our data also highlight an unexpected link between GSDMD-mediated pyroptosis and the AMPK signaling pathway in certain tumor cells.

Murine Eosinophils Release IL-1β via a Non-Lytic Mechanism Dependent on Gasdermin D (GSDMD) Expression but Independent of GSDMD Proteolytic Processing

Abstract Physiological roles for gasdermin D (GSDMD) in pyroptosis and IL-1β release during inflammasome signaling have been well-characterized in macrophages. This involves GSDMD cleavage by caspase-1 to generate N-GSDMD products that assemble as macropores in the plasma membrane (PM) to mediate direct IL-1β efflux and eventual pyroptosis. Granulocytes, including neutrophils and eosinophils, are also important sources of IL-1β in multiple innate immune responses. In neutrophils, we have previously reported that GSDMD expression and proteolytic processing to N-GSDMD is required for IL-1β release but is independent from accumulation of PM N-GSDMD pores and pyroptosis due to alternative trafficking of N-GSDMD to azurophilic granules and autophagosomes; this atypical GSDMD-dependent IL-1β release is mediated in part by secretory autophagy. We have now tested whether this alternative GSDMD-dependent mechanism for non-lytic IL-1β release is also operative in eosinophils using primary murine bone marrow-derived eosinophils (BMDE) from wildtype (C57Bl/6) or Gsdmd−/−mice and stimulated with activators of NLRP3 inflammasome signaling. NLRP3 inflammasome-activated BMDE rapidly process and release IL-1β via a mechanism dependent on GSDMD expression but independent of GSDMD proteolytic processing, N-GSDMD pore accumulation in the PM, and pyroptosis. This requirement for GSDMD in IL-1β release from BMDE is operative during the initial phases (~60 min) of NLRP3 inflammasome activation but is then complemented by a yet-be-defined GSDMD-independent pathway with prolonged stimulation. These findings indicate a role for GSDMD as a chaperone rather than pore-forming protein and distinct from mechanisms defined in neutrophils. Supported by grants from (NIH P01-AI141350, R01-EY014362).

Reconstitution of human pyroptotic cell death in Saccharomyces cerevisiae

Pyroptosis is a lytic form of programmed cell death induced by the activation of gasdermins. The precise mechanism of gasdermin activation by upstream proteases remains incompletely understood. Here, we reconstituted human pyroptotic cell death in yeast by inducible expression of caspases and gasdermins. Functional interactions were reflected by the detection of cleaved gasdermin-D (GSDMD) and gasdermin-E (GSDME), plasma membrane permeabilization, and reduced growth and proliferative potential. Following overexpression of human caspases-1, -4, -5, and -8, GSDMD was cleaved. Similarly, active caspase-3 induced proteolytic cleavage of co-expressed GSDME. Caspase-mediated cleavage of GSDMD or GSDME liberated the ~ 30 kDa cytotoxic N-terminal fragments of these proteins, permeabilized the plasma membrane and compromised yeast growth and proliferation potential. Interestingly, the observation of yeast lethality mediated by co-expression of caspases-1 or -2 with GSDME signified functional cooperation between these proteins in yeast. The small molecule pan-caspase inhibitor Q-VD-OPh reduced caspase-mediated yeast toxicity, allowing us to expand the utility of this yeast model to investigate the activation of gasdermins by caspases that would otherwise be highly lethal to yeast. These yeast biological models provide handy platforms to study pyroptotic cell death and to screen for and characterize potential necroptotic inhibitors.