Executor Of Pyroptosis Research Articles

NLRP3 inflammasome-mediated microglial pyroptosis is critically involved in the development of post-cardiac arrest brain injury

BackgroundBrain injury is the leading cause of death and disability in survivors of cardiac arrest, where neuroinflammation is believed to play a pivotal role, but the underlying mechanism remains unclear. Pyroptosis is a pro-inflammatory form of programmed cell death that triggers inflammatory response upon infection or other stimuli. This study aims to understand the role of microglial pyroptosis in post-cardiac arrest brain injury.MethodsSprague-Dawley male rats underwent 10-min asphyxial cardiac arrest and cardiopulmonary resuscitation or sham-operation. Flow cytometry analysis, Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), co-immunoprecipitation, and immunofluorescence were used to evaluate activated microglia and CD11b-positive leukocytes after cardiac arrest and assess inflammasome activation and pyroptosis of specific cellular populations. To further explore the underlying mechanism, MCC950 or Ac-YVAD-cmk was administered to block nod-like receptor family protein 3 (NLRP3) or caspase-1, respectively.ResultsOur results showed that, in a rat model, successful resuscitation from cardiac arrest resulted in microglial pyroptosis and consequential inflammatory infiltration which was mediated by the activation of NLRP3 inflammasome. Targeting NLRP3 and caspase-1, the executor of pyroptosis, with selective inhibitors MCC950 and Ac-YVAD-cmk treatment significantly prevented microglial pyroptosis, reduced infiltration of leukocytes, improved neurologic outcome, and alleviated neuro-pathological damages after cardiac arrest in modeling rats.ConclusionsThis study demonstrates that microglial pyroptosis mediated by NLRP3 inflammasome is critically involved in the pathogenesis of post-cardiac arrest brain injury and provides a new therapeutic strategy.

Photoreceptors Degenerate Through Pyroptosis After Experimental Retinal Detachment.

PurposeGasdermin D (GSDMD) is crucial in neuronal pyroptosis. GSDMD-N and GSDMD-C are two subdomains of the protein GSDMD. GSDMD-N is an executor of pyroptosis, and GSDMD-C has an inhibitory effect on pyroptotic cell death. This study evaluated the role of GSDMD in photoreceptor cell pyroptosis caused by retinal detachment (RD).MethodsRD models were established in rats, and GSDMD cleavage was detected by western blotting. The morphology of photoreceptors was assessed by transmission electron microscopy. Some rats were given subretinal injections of recombinant adeno-associated virus 2/8 (rAAV2/8)–GSDMD-C before RD surgery. We documented the expression of caspase-1 and GSDMD-N in retinas by western blot. Levels of IL-1β, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) were detected by quantitative RT-PCR. The membrane integrity of photoreceptors was evaluated by TOTO-3 iodide staining. Retinal function was measured by electroretinography, and the thickness of the outer nuclear layer was also recorded. We measured the activation of glial fibrillary acidic protein (GFAP), F4/80, and ionized calcium binding adaptor molecule 1 (Iba-1) by immunofluorescence.ResultsThe cleavage of GSDMD peaked at 1 day after RD. The administration of rAAV2/8–GSDMD-C reduced the pyroptosis and subsequent apoptosis of photoreceptors and preserved the retinal function after RD. Expression of IL-1, TNF-α, and MCP-1 was decreased in the rAAV2/8–GSDMD-C group. In addition, the activation of GFAP, Iba-1, and F4/80 in retinas was alleviated by administering rAAV2/8–GSDMD-C after RD.ConclusionsGSDMD participates in the pyroptosis of photoreceptor after RD. Overexpression of GSDMD-C may block GSDMD cleavage and attenuate photoreceptor degeneration.

Gasdermin D Mediates the Maturation and Release of IL-1α Downstream of Inflammasomes

IL-1α serves as a pro-inflammatory cytokine. Although pro-IL-1α has cytokine activity, proteolytic maturation increases its potency and release from cells. IL-1α maturation occurs in a caspase-1-dependent manner following inflammasome activation. However, pro-IL-1α is not a substrate of caspase-1, and it remains unclear what mediates the maturation of this cytokine downstream of inflammasomes. Here, we show that gasdermin D (GSDMD), an executor of pyroptosis, is required for the rapid induction of IL-1α maturation by non-particulate inflammasome activators. Ablation of GSDMD abrogated the maturation of IL-1α, but not of IL-1β. Inflammasome-induced maturation of IL-1α relied on extracellular Ca2+ and calpains. Ca2+ influx and calpain activation were induced in a GSDMD-dependent manner. Glycine, which inhibits cell lysis, but not GSDMD pore formation, did not affect IL-1α maturation. These results suggest that during inflammasome activation, GSDMD processed by caspase-1 forms plasma membrane pores that mediate Ca2+ influx, resulting in calpain-dependent maturation of IL-1α.

Berberine Inhibits Nod-Like Receptor Family Pyrin Domain Containing 3 Inflammasome Activation and Pyroptosis in Nonalcoholic Steatohepatitis via the ROS/TXNIP Axis.

Berberine (BBR), an isoquinoline alkaloid originating from herbal plants, has been deemed beneficial for non-alcoholic fatty liver disease. Increasing evidence has demonstrated that Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and the subsequent pyroptosis contribute to the progression of non-alcoholic steatohepatitis (NASH). However, whether BBR impacts NLRP3 inflammasome activation and pyroptosis in NASH and the potential mechanism remains unclear. In the current study, we found that BBR significantly decreased lipid accumulation, ameliorated reactive oxygen species (ROS) and lipid peroxides, Tumor necrosis factor alpha (TNF-α) expression, and phosphorylation of Nuclear factor kappa B (NF-κB) p65 both in vivo and in vitro. In particular, BBR significantly inhibited NLRP3 expression, caspase-1 activity, and the pyroptosis executor, GSDMD-N, expression. In addition, BBR displayed similar inhibitory effects on NLRP3 inflammasome and pyroptosis with a decrease in ROS levels and TXNIP expression as N-acetyl-cysteine, a ROS scavenger, did. Whereas, the inhibitory effect of BBR on ROS, TXNIP expression, NLRP3 inflammasome activation and pyroptosis could be reversed by H2O2 in AML12 cells. This study demonstrates that BBR's inhibitory effect on NLRP3 inflammasome activation and pyroptosis may be mediated by ROS/TXNIP axis in vitro for the first time. Our findings suggest BBR is a potential candidate for the treatment of NASH.

The role of Caspase-1/GSDMD-mediated pyroptosis in Taxol-induced cell death and a Taxol-resistant phenotype in nasopharyngeal carcinoma regulated by autophagy.

Taxol has been widely used as a first-line chemotherapeutic agent for the treatment of advanced nasopharyngeal carcinoma (NPC). However, acquired drug resistance has caused great difficulties in clinical treatment. Pyroptosis is a newly discovered programmed cell death pathway, and Caspase-1 and gasdermin D (GSDMD) play key roles in driving canonical pyroptosis. Increasing evidence suggests that pyroptosis is associated with the development of cancer; however, the function and mechanism of pyroptosis in NPC remain obscure. In this study, we observed that Taxol treatment caused pyroptotic cell death, along with activation of Caspase-1 and maturation of IL-1β, as well as cleavage of GSDMD, which is the canonical pyroptosis executor. Furthermore, Taxol-induced pyroptotic cell death could be suppressed by Caspase-1 inhibitor (Z-YVAD-FMK) and GSDMD knockout. Moreover, NPC parental cells demonstrated higher levels of pyroptosis than Taxol-resistant cells, and pyroptosis mediated by Caspase-1/GSDMD suppression induced by a Caspase-1 inhibitor and GSDMD knockout could induce a Taxol-resistant phenotype in vitro and in vivo. By transfecting an siRNA targeting Beclin-1 into NPC Taxol-resistant cells, we discovered that autophagy could negatively regulate pyroptosis by inhibiting Caspase-1/GSDMD activation. Taken together, our results indicated that Caspase-1/GSDMD mediated Taxol-induced pyroptosis and a Taxol-resistant phenotype in NPC cell lines, which may be regulated by autophagy. Thus, we provide novel insight into the mechanisms of Taxol-induced cell death and a promising approach to improve the therapeutic outcomes of patients with advanced NPC.

The advances in pyroptosis initiated by inflammasome in inflammatory and immune diseases.

Pyroptosis is a programmed and inflammatory cell death initiated by inflammasome. During pyroptosis, cytosolic pattern recognition receptors, apoptosis-associated speck-like protein and pro-Caspase-1 form activated inflammasome together. Caspase-1 activated by inflammasome results in generating an N-terminal cleavage product of gasdermin D (GSDMD), which is a major executor of pyroptosis. As a consequence of pyroptosis, a large number of pro-inflammatory cytokines are released including IL-1β and IL-18. Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and absent in melanoma 2 (AIM2)-like receptors (ALRs) belong to cytosolic pattern recognition receptors and assemble inflammasomes by detecting host cell damage signals. Pyroptosis pathways are divided into canonical and non-canonical pathways according to the identification of damage signals by cytoplasmic protein sensors. Pyroptosis not only plays an important role in infection, but also plays a vital role in inflammatory immune diseases. This article reviews the advances research of pyroptosis initiated by inflammasome in inflammatory and immune diseases.

Molecular mechanism and immunological function of pyroptosis

Pyroptosis is a form of programmed cell death that is highly proinflammatory. Pyroptosis was originally observed in macrophages in response to bacterial infection or bacterial toxin stimulation. This cell death, then misclassified as a type of apoptosis specifically in macrophages, requires caspase-1 that also processes interleukin-1β mainly expressed in monocytic cells. Caspase-1 is activated by canonical inflammasomes, a major cytosolic innate immune surveillance mechanism that can detect various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Studies on innate immunity to cytosolic lipopolysaccharide (LPS) have established that murine caspase-11 and its human counterparts caspase-4/5 can also mediate pyroptosis upon directly binding to the lipid A moiety of LPS. Caspase-11/4/5 do not process interleukin and caspase-11/4/5-mediated pyroptosis is prevalent in non-monocytic cells. Very recent studies have identified gasdermin D (GSDMD) as the pyroptotic substrate for both caspase-1 and caspase-11/4/5. GSDMD represents a large gasdermin family that shares membrane pore-forming activity, rendering a new concept of pyroptosis as gasdermin-mediated programmed necrotic cell death. In this article, we review the concept evolvement of pyroptotic cell death from a historic perspective, covering the innate immune pathways upstream of caspase-1 and caspase-11/4/5. We also discuss the recent advances on pyroptosis, particularly mechanistic and functional understandings of GSDMD and the gasdermin family of pyroptosis executor proteins.

Molecular mechanisms of gasdermin D autoinhibition and recognition by inflammatory caspases

Abstract Pyroptosis is an inflammatory form of programmed cell death that plays important roles in immune protection against infections and in inflammatory disorders. Gasdermin D (GSDMD) is an executor of pyroptosis upon cleavage by caspases-1/4/5/11 following canonical and noncanonical inflammasome activation. GSDMD N-terminal domain assembles membrane pores to induce cytolysis, whereas its C-terminal domain inhibits cell death through intramolecular association with the N domain. The crystal structures of the human and murine GSDMD C-terminal domains differ from those of the full-length murine GSDMA3 and the human GSDMB C-terminal domain. Mutations of GSDMD C-domain residues predicted to locate at its interface with the N-domain enhanced pyroptosis, in agreement with the role of the GSDMD C-terminal domain as an autoinhibition domain. We further demonstrate that the full-length GSDMD and its cleavage site peptide, FLTD, can directly bind the catalytic domains of inflammatory caspases. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, as well as suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. The present study not only contributes to our understanding of the distinct mode of GSDMD autoinhibition and recognition by inflammatory caspases, but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.

Oleic acid ameliorates palmitic acid induced pyroptosis by attenuating endoplasmic reticulum stress in HepG2 cells

BackgroundIn the liver, chronic exposure to high FFAs in the context of hyperlipidemia or ectopic deposition of fat will result in nonalcoholic fatty liver disease (NAFLD) ultimately. In the present study, the mechanisms of saturated fatty acids induced cell death have been investigated. Particular attentions have been pay on caspase‐1 mediated pyroptosis and endoplasmic reticulum stress (ERS). In addition, the protective effects of a monounsaturated fatty acid, oleic acid (OA) against hepatic lipotoxicity was explored.MethodsHuman hepatoma cell line HepG2 was challenged by palmitic acid (PA) to induce lipotoxicity, and OA was supplemented concomitantly with PA to investigate its protective effects. Cell viability was determined by CCK8 assay and apoptosis was measured by flow cytometry. The expression of ER stress and pyroptosis related proteins was measured by Western blot or real‐time PCR. The morphology of pyroptosis was presented by PI staining and AO/EB Dyeingy. The expression level of gasdermin D (GSDMD), an executor of pyroptosis, was determined by Western blot and immunofluorescence assay.Results1) PA deteriorated cell viability of HepG2 cells in dose and time dependent manner. PA of 0.4 mM has remarkably decreased the cell viability (60%) compared to controls after 24 h incubation. Stimulation with OA of 0.2 mM was nontoxic to cell viability, but the combination of OA and PA robustly restored the cell viability to normal level. 2) Inconsistent with the results in cell viability, PA challenge for 24 h has not significantly increased cell apoptosis in HepG2. However, the expression of inflammasome and pyroptosis markers, including caspase‐1/p20, IL‐1 beta, IL‐18, NLRP3 and GSDMD was significantly up‐regulated, indicating that pyroptosis was activated obviously. In addition, the occurrence of pyroptosis was convinced by the increased AO/EB double positive cells as well as increased GSDMD expression after PA stimulation, suggesting a kind of gasdermin‐mediated programmed necrosis, which is a typical feature of pyroptosis. Interestingly, OA substantially suppressed the PA induced activation of pyroptosis, evidenced by significantly down‐regulated expression of above markers. 3) Meanwhile, the expression of ER stress related markers was found notably up‐regulated after 24 h PA exposure, including CHOP, phosphorylated PERK, and GRP78 on protein levels, and UPR genes, such as XBP1, CHOP, and GRP78 on mRNA levels. In consistence, OA attenuated PA triggered ER stress in HepG2 cells.ConclusionCompared to apoptosis, caspase‐1 mediated pyroptosis might be an essential form of cell death driven by excessive uptake of saturated fatty acid. Monounsaturated oleic acid was able to robustly protect HepG2 cells against lipotoxicity. The down‐regulation of GSDMD expression and consequently amelioration of pyroptosis may involve in the mechanisms of OA mediated protective action, and the OA mediated alleviation of PA triggered ER stress might be a possible upstream mechanism of its effects on pyroptosis.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gasdermin Family: a Promising Therapeutic Target for Stroke.

Stroke is one of the leading causes of death worldwide and therapies are limited. According to the mechanisms underlying stroke, direct cell demise, destruction of organelles, blood-brain barrier disruption, neuroinflammation are potential therapeutic targets. Gasdermin (GSDM) family is a series of recently discovered proteins, which acts as the executor of pyroptosis and protagonist of membrane pore formation. It can be cleaved by inflammatory caspases that are activated by multi-protein complexes, named as inflammasomes, and divided into two domains. The N-terminal domain interacts with lipid localized in the plasma membrane as well as organelle membrane and perforates them. After the formation of pores, the dysfunctional structures suffer lytic death and their contents are released. In this review, we discuss the biological characteristic of GSDM family and their possible roles in stroke-induced brain injury, and based on the review, we hypothesize the feasibility of using GSDMs as a potential target for stroke treatment.

Classical swine fever virus induces pyroptosis in the peripheral lymphoid organs of infected pigs

Classical swine fever virus (CSFV) causes a highly lethal disease in pigs, which is characterized by immunosuppression. Leukopenia is known to be a possible mechanism of immunosuppression during CSFV infection. As a new and specialized form of cell death, pyroptosis is the key response of the innate immune system to pathogens, and is widely involved in the occurrence and development of infectious diseases. However, the relationship between CSFV and pyroptosis has not been explored. In this study, we investigated the occurrence of pyroptosis in pigs following CSFV infection. According to qRT-PCR assay results, the prevalence of this virus in peripheral lymphoid organs (tonsils, lymph nodes, and spleen) was much higher than that in other organs. Severe bleeding, necrosis, and a significant reduction in lymphocytes were found in the peripheral lymphoid organs of CSFV-infected pigs based on histological examination. In-depth studies showed that an increased ratio of deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells were present in the peripheral lymphoid organs of the CSFV-infected group according to immunohistochemistry. Meanwhile, the p10 subunit and activity of caspase-1, which is a regulator of pyroptosis, the N-terminal domain of gasdermin D, which is an executor of pyroptosis, and the cleavage and secretion of IL-1b, which is a product of pyroptosis were increased in the peripheral lymphoid organs of the CSFV-infected group. Together, these results demonstrated that pyroptosis is involved in CSFV-induced cell death in vivo, which provides a new understanding of the mechanism associated with lymphocyte depletion and immunosuppression in pigs infected with this virus.

Structures of the Gasdermin D C-Terminal Domains Reveal Mechanisms of Autoinhibition

Pyroptosis is an inflammatory form of programmed cell death that plays important roles in immune protection against infections and in inflammatory disorders. Gasdermin D (GSDMD) is an executor of pyroptosis upon cleavage by caspases-1/4/5/11 following canonical and noncanonical inflammasome activation. GSDMD N-terminal domain assembles membrane pores to induce cytolysis, whereas its C-terminal domain inhibits cell death through intramolecular association with the N domain. The molecular mechanisms of autoinhibition for GSDMD are poorly characterized. Here we report the crystal structures of the human and murine GSDMD C-terminal domains, which differ from those of the full-length murine GSDMA3 and the human GSDMB C-terminal domain. Mutations of GSDMD C-domain residues predicted to locate at its interface with the N-domain enhanced pyroptosis. Our results suggest that GSDMDs may employ a distinct mode of intramolecular domain interaction and autoinhibition, which may be relevant to its unique role in pyroptosis downstream of inflammasome activation.

From the Editor’s desk....: April 2018

There is a clear need to identify molecular mechanisms of non-alcoholic steatohepatitis (NASH) to develop novel targeted therapies. Pyroptosis is a highly inflammatory form of programmed cell death. Gasdermin D (GSDMD)-executed programmed necrosis is involved in inflammation and controls interleukin (IL)-1β release. In this issue of the Journal, Xu et al. studied its role in human and experimental NASH. GSDMD and its pyroptosis-inducing fragment GSDMD-N were upregulated in liver tissues of human NASH and correlated with disease severity. Gsdmd−/− mice with experimental NASH exhibit decreased severity of steatosis and inflammation. GSDMD expression was associated with the secretion of pro-inflammatory cytokines and persistent activation of the NF-κB signaling pathway. Reduced steatosis in Gsdmd−/− mice was due to decreased expression of the lipogenic gene Srebp-1c and upregulated expression of lipolytic genes. This translational study reveals that GSDMD plays a key role as a pyroptosis executor in the pathogenesis of steatohepatitis by controlling cytokine secretion and lipogenesis.

Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice

Gasdermin D (GSDMD)-executed programmed necrosis is involved in inflammation and controls interleukin (IL)-1β release. However, the role of GSDMD in non-alcoholic steatohepatitis (NASH) remains unclear. We investigated the role of GSDMD in the pathogenesis of steatohepatitis. Human liver tissues from patients with non-alcoholic fatty liver disease (NAFLD) and control individuals were obtained to evaluate GSDMD expression. Gsdmd knockout (Gsdmd-/-) mice, obese db/db mice and their wild-type (WT) littermates were fed with methionine-choline deficient (MCD) or control diet to induce steatohepatitis. The Gsdmd-/- and WT mice were also used in a high-fat diet (HFD)-induced NAFLD model. In addition, Alb-Cre mice were administered an adeno-associated virus (AAV) vector that expressed the gasdermin-N domain (AAV9-FLEX-GSDMD-N) and were fed with either MCD or control diet for 10 days. GSDMD and its pyroptosis-inducing fragment GSDMD-N were upregulated in liver tissues of human NAFLD/NASH. Importantly, hepatic GSDMD-N protein levels were significantly higher in human NASH and correlated with the NAFLD activity score and fibrosis. GSDMD-N remained a potential biomarker for the diagnosis of NASH. MCD-fed Gsdmd-/- mice exhibit decreased severity of steatosis and inflammation compared with WT littermates. GSDMD was associated with the secretion of pro-inflammatory cytokines (IL-1β, TNF-α, and MCP-1 [CCL2]) and persistent activation of the NF-ĸB signaling pathway. Gsdmd-/- mice showed lower steatosis, mainly because of reduced expression of the lipogenic gene Srebp1c (Srebf1) and upregulated expression of lipolytic genes, including Pparα, Aco [Klk15], Lcad [Acadl], Cyp4a10 and Cyp4a14. Alb-Cre mice administered with AAV9-FLEX-GSDMD-N showed significantly aggravated steatohepatitis when fed with MCD diet. As an executor of pyroptosis, GSDMD plays a key role in the pathogenesis of steatohepatitis, by controlling cytokine secretion, NF-ĸB activation, and lipogenesis. Non-alcoholic fatty liver disease has become one of the most feared chronic liver diseases, because it is the most rapidly growing indication for adult liver transplantation and a major cause of hepatocellular carcinoma. However, the mechanisms involved in the transformation of simple steatosis to steatohepatitis remain unclear. Herein, we show that gasdermin D driven pyroptosis is prominent in patients with non-alcoholic steatohepatitis (NASH), and gasdermin-N domain remains a potential biomarker for the diagnosis of NASH. Gasdermin D plays a key role in the pathogenesis of NASH by regulating lipogenesis, the inflammatory response, and the NF-ĸB signaling pathway, revealing potential treatment targets for NASH in humans.

Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion.

Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1β (IL-1β) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1β in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1β secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd−/− cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd−/− cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.