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Pore-forming toxins (PFTs) are released by one cell to directly inflict damage on another cell. Hosts use PFTs, including members of the membrane attack complex/perforin protein family, to fight infections and cancer, while bacteria and parasites deploy PFTs to promote infection. Apicomplexan parasites secrete perforin-like proteins as PFTs to egress from infected cells and traverse tissue barriers. Other protozoa, along with helminth parasites, utilize saposin-like PFTs prospectively for nutrient acquisition during infection. This review discusses seminal and more recent advances in understanding how parasite PFTs promote infection and describes how they are regulated and fulfill their roles without causing parasite self-harm. Although exciting progress has been made in defining mechanisms of pore formation by PFTs, many open questions remain to be addressed to gain additional key insights into these remarkable determinants of parasitic infections.

The molecular mechanisms regulating CD8+ cytotoxic T lymphocytes (CTL) are not fully understood. Here, we show that the peroxisome proliferator-activated receptor δ (PPARδ) suppresses CTL cytotoxicity by inhibiting RelA DNA binding. Treatment of ApcMin/+ mice with the PPARδ agonist GW501516 reduced the activation of normal and tumor-associated intestinal CD8+ T cells and increased intestinal adenoma burden. PPARδ knockout or knockdown in CTLs increased their cytotoxicity against colorectal cancer cells, whereas overexpression of PPARδ or agonist treatment decreased it. Correspondingly, perforin, granzyme B, and IFNγ protein and mRNA levels were higher in PPARδ knockout or knockdown CTLs and lower in PPARδ overexpressing or agonist-treated CTLs. Mechanistically, we found that PPARδ binds to RelA, interfering with RelA-p50 heterodimer formation in the nucleus, thereby inhibiting its DNA binding in CTLs. Thus, PPARδ is a critical regulator of CTL effector function. Significance: Here, we provide the first direct evidence that PPARδ plays a critical role in suppressing the immune response against tumors by downregulating RelA DNA-binding activity. This results in decreased expression of perforin, granzyme B, and IFNγ. Thus, PPARδ may serve as a valuable target for developing future cancer immunotherapies., (©2024 The Authors; Published by the American Association for Cancer Research.)

Pyroptosis, a typical inflammatory cell death mode, has been increasingly demonstrated to have therapeutic value in inflammatory diseases such as sepsis. However, the mechanisms and therapeutic targets of sepsis remain elusive. Here, we reported that REGγ inhibition promoted pyroptosis by regulating members of the gasdermin family in macrophages. Mechanistically, REGγ directly degraded Bim, a factor of the Bcl-2 family that can inhibit the cleavage of GSDMD/E, ultimately preventing the occurrence of pyroptosis. Furthermore, cecal ligation and puncture (CLP)-induced sepsis model mice showed downregulation of REGγ at both the RNA and protein levels. Gasdermin-mediated pyroptosis was augmented in REGγ-knockout mice, and these mice exhibited more severe sepsis-related tissue injury. More importantly, we found that REGγ expression was downregulated in clinical sepsis samples, such as those from patients with Pseudomonas aeruginosa (PA) infection. Finally, PA-infected mice showed decreased REGγ levels in the lung. In summary, our study reveals that the REGγ-Bim-GSDMD/E pathway is a novel regulatory mechanism of pyroptosis in sepsis-related tissue injury., (© 2024. The Author(s).)

Intrinsically disordered protein regions (IDPRs) are pivotal in regulation of transcription and facilitation of signal transduction. Because of their multiple conformational states of structure, characterizing the highly flexible structures of IDPRs becomes challenging. Herein, we employed the wild-type (WT) aerolysin nanopore as a real-time biosensor for identification and monitoring of long peptides containing IDPRs. This sensor successfully identified three intrinsically disordered peptides, with the lengths up to 43 amino acids, by distinguishing the unique signatures of blockade current and duration time. The analysis of the binding constant revealed that interactions between the nanopore and peptides are critical for peptide translocation, which suggests that mechanisms beyond mere volume exclusion. Furthermore, we were able to compare the conformational stabilities of various IDPRs by examining the detailed current traces of blockade events. Our approach can detect the conformational changes of IDPR in a confined nanopore space. These insights broaden the understanding of peptide structural changes. The nanopore biosensor showed the potential to study the conformations change of IDPRs, IDPRs transmembrane interactions, and protein drug discovery., (© 2024 Wiley-VCH GmbH.)

The gasdermins are a family of pore-forming proteins that have recently emerged as executors of pyroptosis, a lytic form of cell death that is induced by the innate immune system to eradicate infected or malignant cells. Mammalian gasdermins comprise a cytotoxic N-terminal domain, a flexible linker and a C-terminal repressor domain. Proteolytic cleavage in the linker releases the cytotoxic domain, thereby allowing it to form β-barrel membrane pores. Formation of gasdermin pores in the plasma membrane eventually leads to a loss of the electrochemical gradient, cell death and membrane rupture. Here we review recent work that has expanded our understanding of gasdermin biology and function in mammals by revealing their activation mechanism, their regulation and their roles in autoimmunity, host defence and cancer. We further highlight fungal and bacterial gasdermin pore formation pointing to a conserved mechanism of cell death induction., (© 2024. Springer Nature Limited.)

Pore-forming toxins (PFTs) are proteins that form lesions in biological membranes. Better understanding of the structure and function of these proteins will be beneficial in a number of biotechnological applications, including the development of new pest control methods in agriculture. When searching for new pore formers, existing sequence homology-based methods fail to discover truly novel proteins with low sequence identity to known proteins. Search methodologies based on protein structures would help us move beyond this limitation. As the number of known structures for PFTs is very limited, it's quite challenging to identify new proteins having similar structures using computational approaches like deep learning. In this article, we therefore propose a sample-efficient graphical model, where a protein structure graph is first constructed according to consensus secondary structures. A semi-Markov conditional random fields model is then developed to perform protein sequence segmentation. We demonstrate that our method is able to distinguish structurally similar proteins even in the absence of sequence similarity (pairwise sequence identity < 0.4)-a feat not achievable by traditional approaches like HMMs. To extract proteins of interest from a genome-wide protein database for further study, we also develop an efficient framework for UniRef50 with 43 million proteins., (© 2024 Wiley Periodicals LLC.)

Genome editing has the potential to improve the unsatisfactory therapeutic effect of antitumor immunotherapy. However, the cell plasma membrane prevents the entry of almost all free genome-manipulation agents. Therefore, a system can be spatiotemporally controlled and can instantly open the cellular membrane to allow the entry of genome-editing agents into target cells is needed. Here, inspired by the ability of T cells to deliver cytotoxins to cancer cells by perforation, an ultrasound (US)-controlled perforation system (UPS) is established to enhance the delivery of free genome-manipulating agents. The UPS can perforate the tumor cell membrane while maintaining cell viability via a controllable lipid peroxidation reaction. In vitro, transmembrane-incapable plasmids can enter cells and perform genome editing with the assistance of UPS, achieving an efficiency of up to 90%. In vivo, the UPS is biodegradable, nonimmunogenic, and tumor-targeting, enabling the puncturing of tumor cells under US. With the application of UPS-assisted genome editing, gasdermin-E expression in 4T1 tumor-bearing mice is successfully restored, which leads to pyroptosis-mediated antitumor immunotherapy via low-dose X-ray irradiation. This study provides new insights for designing a sonoporation system for genome editing. Moreover, the results demonstrate that restoring gasdermin expression by genome editing significantly improves the efficacy of radioimmunotherapy., (© 2024 Wiley‐VCH GmbH.)

Animal venoms are a complex mixture of highly specialized toxic molecules. Among them, pore-forming proteins (PFPs) or toxins (PFTs) are one of the major disease-causing toxic elements. The ability of the PFPs in defense and toxicity through pore formation on the host cell surface makes them unique among the toxin proteins. These features made them attractive for academic and research purposes for years in the areas of microbiology as well as structural biology. All the PFPs share a common mechanism of action for the attack of host cells and pore formation in which the selected pore-forming motifs of the host cell membrane-bound protein molecules drive to the lipid bilayer of the cell membrane and eventually produces water-filled pores. But surprisingly their sequence similarity is very poor. Their existence can be seen both in a soluble state and also in transmembrane complexes in the cell membrane. PFPs are prevalent toxic factors that are predominately produced by all kingdoms of life such as virulence bacteria, nematodes, fungi, protozoan parasites, frogs, plants, and also from higher organisms. Nowadays, multiple approaches to applications of PFPs have been conducted by researchers both in basic as well as applied biological research. Although PFPs are very devastating for human health nowadays researchers have been successful in making these toxic proteins into therapeutics through the preparation of immunotoxins. We have discussed the structural, and functional mechanism of action, evolutionary significance through dendrogram, domain organization, and practical applications for various approaches. This review aims to emphasize the PFTs to summarize toxic proteins together for basic knowledge as well as to highlight the current challenges, and literature gap along with the perspective of promising biotechnological applications for their future research., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Sepsis results from systemic, dysregulated inflammatory responses to infection, culminating in multiple organ failure. Here, we demonstrate the utility of CD5L for treating experimental sepsis caused by cecal ligation and puncture (CLP). We show that CD5L's important features include its ability to enhance neutrophil recruitment and activation by increasing circulating levels of CXCL1, and to promote neutrophil phagocytosis. CD5L-deficient mice exhibit impaired neutrophil recruitment and compromised bacterial control, rendering them susceptible to attenuated CLP. CD5L -/- peritoneal cells from mice subjected to medium-grade CLP exhibit a heightened pro-inflammatory transcriptional profile, reflecting a loss of control of the immune response to the infection. Intravenous administration of recombinant CD5L (rCD5L) in immunocompetent C57BL/6 wild-type (WT) mice significantly ameliorates measures of disease in the setting of high-grade CLP-induced sepsis. Furthermore, rCD5L lowers endotoxin and damage-associated molecular pattern (DAMP) levels, and protects WT mice from LPS-induced endotoxic shock. These findings warrant the investigation of rCD5L as a possible treatment for sepsis in humans., (© 2024. The Author(s).)

A specialized population of mast cells residing within epithelial layers, currently known as intraepithelial mast cells (IEMCs), was originally observed over a century ago, yet their physiological functions have remained enigmatic. In this study, we unveil an unexpected and crucial role of IEMCs in driving gasdermin C-mediated type 2 immunity. During helminth infection, αEβ7 integrin-positive IEMCs engaged in extensive intercellular crosstalk with neighboring intestinal epithelial cells (IECs). Through the action of IEMC-derived proteases, gasdermin C proteins intrinsic to the epithelial cells underwent cleavage, leading to the release of a critical type 2 cytokine, interleukin-33 (IL-33). Notably, mast cell deficiency abolished the gasdermin C-mediated immune cascade initiated by epithelium. These findings shed light on the functions of IEMCs, uncover a previously unrecognized phase of type 2 immunity involving mast cell-epithelial cell crosstalk, and advance our understanding of the cellular mechanisms underlying gasdermin C activation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The pathogenicity of many bacteria, including Bacillus cereus and Staphylococcus aureus , depends on pore-forming toxins (PFTs), which cause the lysis of host cells by forming pores in the membranes of eukaryotic cells. Bioinformatic analysis revealed a region homologous to the Lys171-Gly250 sequence in hemolysin II (HlyII) from B. cereus in over 600 PFTs, which we designated as a "homologous peptide". Three β-barrel PFTs were used for a detailed comparative analysis. Two of them-HlyII and cytotoxin K2 (CytK2)-are synthesized in Bacillus cereus sensu lato ; the third, S. aureus α-toxin (Hla), is the most investigated representative of the family. Protein modeling showed certain amino acids of the homologous peptide to be located on the surface of the monomeric forms of these β-barrel PFTs. We obtained monoclonal antibodies against both a cloned homologous peptide and a 14-membered synthetic peptide, DSFNTFYGNQLFMK, as part of the homologous peptide. The HlyII, CytK2, and Hla regions recognized by the obtained antibodies, as well as an antibody capable of suppressing the hemolytic activity of CytK2, were identified in the course of this work. Antibodies capable of recognizing PFTs of various origins can be useful tools for both identification and suppression of the cytolytic activity of PFTs.

Background/aim: Oral epithelial cells serve as the primary defense against microbial exposure in the oral cavity, including the fungus Candida albicans. Dectin-1 is crucial for recognition of β-glucan in fungi. However, expression and function of Dectin-1 in oral epithelial cells remain unclear., Materials and Methods: We assessed Dectin-1 expression in Ca9-22 (gingiva), HSC-2 (mouth), HSC-3 (tongue), and HSC-4 (tongue) human oral epithelial cells using flow cytometry and real-time polymerase chain reaction. Cell treated with β-glucan-rich zymosan were evaluated using real-time polymerase chain reaction. Phosphorylation of spleen-associated tyrosine kinase (SYK) was analyzed by western blotting., Results: Dectin-1 was expressed in all four cell types, with high expression in Ca9-22 and HSC-2. In Ca9-22 cells, exposure to β-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1β, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides β-defensin-1 and LL-37, but not S100 calcium-binding protein A8 (S100A8) and S100A9. Furthermore, the expression of cylindromatosis (CYLD), a negative regulator of nuclear factor kappa B (NF-κB) signaling, was induced. In HSC-2 cells, zymosan induced the expression of IL17A. The expression of tumor necrosis factor alpha-induced protein 3 (TNFAIP3), a negative regulator of NF-κB signaling, was also induced. Expression of other cytokines and antimicrobial peptides remained unchanged. Zymosan induced phosphorylation of SYK in Ca9-22 cells, as well as NF-κB., Conclusion: Oral epithelial cells express Dectin-1 and recognize β-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), acting as one common sepsis-associated organ injury, induces uncontrolled and self-amplifies pulmonary inflammation. Given the lack of clinically effective approaches, the mortality rate of it still remains high. Suramin(SUR), as an antiparasitic drug initially, was found to ameliorate sepsis associated ALI in our previous work. However, the underlying mechanism of its protective effects has not been clarified. Pyroptosis, categorized as an inflammatory form of programmed cell death, could aggravate lung inflammatory responses via inducing alveolar macrophages (AM) pyroptosis., Methods: MH-S AM cell line was stimulated with or without lipopolysaccharide (LPS) or suramin, and the differential expression genes (DEGs) were excavated using RNA sequencing (RNA-seq). To identify the regulatory roles of these genes, pyroptosis-related genes (PRGs), GO/KEGG and GSEA analysis were conducted. We also performed WB, qRTPCR and ELISA to validate the RNA-seq results and further expound the protective effect of suramin., Results: 624 DEGs were identified between control (CON) and lipopolysaccharide (LPS) groups, and enrichment analysis of these genes revealed significantly enriched pathways that related to immune system and signal transduction. Meanwhile, 500 DEGs were identified in LPS/SUR+LPS group. In addition to the pathways mentioned above, IL-17 pathway and C-type lectin receptor signaling pathway were also enriched. All 6 pathways were connected with pyroptosis. Concurrently, the "DESeq2" R package was used to identify differentially expressed PRGs. Nod1, Nod2, interleukin (IL)-1b, IL-6, tumor necrosis factor (TNF), NLRP3 were upregulated under LPS stimulation. Then, in SUR+LPS group, Nod2, IL-6, IL-1b, NLRP3 were downregulated. The validation results of WB, qRT-PCR, and ELISA showed: the protein and mRNA expression levels of NLRP3, caspase-1, GSDMD and the concentrations of IL-1b, IL-18 were decreased when treated with suramin and LPS., Conclusion: Suramin could inhibit NLRP3/caspase-1/GSDMD canonical pyroptosis pathway in LPS-induced MH-S alveolar macrophages., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Tou Nong Powder (TNP), a classical Chinese medicinal formula originated from the Chinese Ming Dynasty, has been applied to treat skin ulcers in patients with deficient constitutions. According to theory of traditional Chinese medicine, colonic ulcers share similar pathological conditions with skin ulcers, and consequently, TNP has been applied to ulcerative colitis (UC) safely and effectively., Aim of Study: To investigate whether TNP obstructs 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced enteric inflammation through regulation of NLRP3 inflammasome and attenuating enteric pyroptosis., Materials and Methods: Network pharmacology and UPLC-Q-TOF/MS were operated to identify compounds and pharmacological potential targets. The therapeutic effects of TNP were assessed on TNBS induced colitis via general symptoms (disease activity index, colonic weight and length) and histopathological observation. The NF-κB/NLRP3/Caspase-1/GSDMD signaling pathway regulation was investigated by Western blot and real time reverse transcription polymerase chain reaction (RT-qPCR)., Results: TNP ameliorates the disease activity index, reverses the increase of colonic weight increase, alleviates colonic shortening and colonic histopathological injury. A decrease in tumor necrosis factor α (TNF-α), diamine oxidase (DAO), intercellular adhesion molecule-1 (ICAM-1), and endo-toxin (ET) were investigated in peripheral circulation. Moreover, TNP significantly obstructed the NF-κB/NLRP3/Caspase-1/GSDMD signaling pathway., Conclusion: TNP displays a promising therapeutic effect on UC via suppressing NF-κB/NLRP3/Caspase-1/GSDMD signaling pathway and reducing the expression of IL-1β and IL-18., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: The outcome of patients with colon cancer is still unsatisfied nowadays. Simvastatin is a type of statins with anti-cancer activity, but its effect on colon cancer cells remains unclear. The present study is intended to determine the underlying mechanism of simvastatin in treatment of colon cancer., Methods: The viability and pyroptosis rate of cells treated and untreated with simvastatin were analysed by CCK-8 and flow cytometry assays, respectively. We used DCFH-DA and flow cytometry to detect reactive oxygen species (ROS) production. Levels of pyroptosis markers were detected by western blotting analysis or immunofluorescence staining. Besides, the anticancer properties of simvastatin on colon cancer were further demonstrated using a cell line based xenograft tumor model., Results: Simvastatin treatment in HCT116 and SW620 induced pyroptosis and suppressed cell proliferation, with changes in the expression level of NLPR3, ASC, cleaved-caspase-1, mature IL-1β, IL-18 and GSDMD-N. Moreover, inhibition of caspase-1 and ROS attenuated the effects of simvastatin on cancer cell viability. In addition, it was identified that simvastatin has an anti-tumor effect by down-regulating ROS production and inducing downstream caspase-1 dependent pyroptosis in the subcutaneous transplantation tumors of HCT116 cells in BALB/c nude mice., Conclusions: Our in vitro and in vivo results indicated that simvastatin induced pyroptosis through ROS/caspase-1/GSDMD pathway, thereby serving as a potential agent for colon cancer treatment. Video Abstract., (© 2023. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Tumor cells can resist chemotherapy-induced pyroptosis through glycolytic reprogramming. Estrogen-related receptor alpha (ERRα) is a central regulator of cellular energy metabolism associated with poor cancer prognosis. Herein, we refine the oncogenic role of ERRα in the pyroptosis pathway and glycolytic metabolism., Methods: The interaction between ERRα and HIF-1α was verified using co-immunoprecipitation. The transcriptional binding sites of ERRα and NLRP3 were confirmed using dual-luciferase reporter assay and cleavage under targets and tagmentation (CUT&Tag). Flow cytometry, transmission electron microscopy, scanning electron microscopy, cell mito stress test, and extracellular acidification rate analysis were performed to investigate the effects of ERRα on the pyroptosis pathway and glycolytic metabolism. The results of these experiments were further confirmed in endometrial cancer (EC)-derived organoids and nude mice. In addition, the expression of ERRα-related pyroptosis genes was analyzed using The Cancer Genome Atlas and Gene Expression Omnibus database., Results: Triggered by a hypoxic microenvironment, highly expressed ERRα could bind to the promoter of NLRP3 and inhibit caspase-1/GSDMD signaling, which reduced inflammasome activation and increased pyroptosis resistance, thereby resulting in the resistance of cancer cells to cisplatin. Moreover, ERRα activated glycolytic rate-limiting enzyme to bridge glycolytic metabolism and pyroptosis in EC. This phenomenon was further confirmed in EC-derived organoids and nude mice. CUT & Tag sequencing and The Cancer Genome Atlas database analysis showed that ERRα participated in glycolysis and programmed cell death, which resulted in EC progression., Conclusions: ERRα inhibits pyroptosis in an NLRP3-dependent manner and induces glycolytic metabolism, resulting in cisplatin resistance in EC cells., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

Oral lichen planus (OLP), a chronic inflammatory disorder, is characterized by the massive cell apoptosis in the keratinocytes of oral mucosa. However, the mechanism responsible for triggering oral keratinocyte apoptosis is not fully explained. Here, we identify that Gasdermin C (GSDMC) downregulation contributes to apoptosis in human oral keratinocytes. Mechanistically, we describe that activated nuclear factor kappa B (NF-κB) pathway induces overexpression of methyltransferase-like 14 (METTL14), which increases N 6 -adenosine methylation (m 6 A) levels in the epithelial layer of OLP. m 6 A modification is capable of regulating primary miR-6858 processing and alternative splicing, leading to miR-6858 increases. miR-6858 can bind and promote GSDMC mRNA degradation. Forced expression of GSDMC is able to rescue cell apoptosis in human oral keratinocyte models resembling OLP. Collectively, our data unveil that m 6 A modification regulates miR-6858 production to decrease GSDMC expression and to trigger keratinocyte apoptosis in the context of OLP., (© 2023. The Author(s).)

Atopic dermatitis is featured with impaired skin barrier. The stratum corneum and the intercellular tight junctions constitute the permeability barrier, which is essential to protect water loss in the host and prevent pathogen entry. The epidermal barrier is constantly renewed by differentiating keratinocytes through cornification, during which autophagy contributes to elimination of organelles and nucleus. The human GSDMA and its mouse homologs Gsdma1-3 are expressed in the suprabasal epidermis. Although a pyroptotic role of GSDMA/Gsdma1 in host defense against Streptococcus pyogenes has been reported, the physiological function of Gsdma1/a2/a3 in epidermal homeostasis remains elusive. Here, through repeated epidermal barrier disruption, we found that tight junction formation and stratum corneum maturation were defective in the Gsdma1/a3-deficient epidermis. Using comparative gene profiling analysis, mitochondrial respiration measurement, and in vivo tracing of mitophagy, our data indicate that Gsdma1/a3 activation leads to mitochondrial dysfunction and subsequently facilitates mitochondrial turnover and epidermal cornification. In calcipotriol (MC903)-induced atopic dermatitis-like animal model, we showed that Gsdma1/a3-deficiency selectively enhanced the T helper type 2 response. Remarkably, the GSDMA expression is reduced in the epidermis of patients with atopic dermatitis compared with that of normal individuals. Gsdma1/a3-deficiency might be involved in atopic dermatitis pathogenesis, likely through GSDMA-mediated epidermal differentiation and cornification., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mycoplasma pneumoniae (MP) is one of the main pathogens causing community acquired pneumonia (CAP) in children and adults. Previous pharmacological and clinical studies have shown that Polydatin (PD) exerts anti-inflammatory action by conferring protective benefit in MP pneumonia. However, the mechanism underlying the of PD on MP infection remains unclear. It was found that PD alleviated MP-induced injury by inhibiting caspase-1/gasdermin D (GSDMD)-mediated epithelial pyroptosis. The results demonstrated that PD inhibited the transformation of GSDMD to N-terminal gasdermin-N (GSDMD-N) by decreasing caspase-1 activation, as well as suppressed the formation and secretion of interleukin-1β (IL-1β) and interleukin-18 (IL-18), reversed Na, K-ATPase reduction, and suppressed LDH release both in vitro and vivo. Taken together, epithelial pyroptosis in BEAS-2B cells and lung injury in mice were prevented by PD. In conclusion, PD suppressed pulmonary injury triggered by MP infection, by inhibiting the caspase-1/GSDMD-mediated epithelial pyroptosis signaling pathway. Thus, PD may be regarded as a potential therapy for MP-induced inflammation., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Pyroptosis is a programmed necrotic cell death executed by gasdermins, a family of pore-forming proteins. The cleavage of gasdermins by specific proteases enables their pore-forming activity. The activation of the prototype member of the gasdermin family, gasdermin D (GSDMD), is linked to innate immune monitoring by inflammasomes. Additional gasdermins such as GSDMA, GSDMB, GSDMC, and GSDME are activated by inflammasome-independent mechanisms. Pyroptosis is emerging as a key host defense strategy against pathogens. However, excessive pyroptosis causes cytokine storm and detrimental inflammation leading to tissue damage and organ dysfunction. Consequently, dysregulated pyroptotic responses contribute to the pathogenesis of various diseases, including sepsis, atherosclerosis, acute respiratory distress syndrome, and neurodegenerative disorders. This review will discuss the inflammatory consequences of pyroptosis and the mechanisms of pyroptosis-induced tissue damage and disease pathogenesis., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Inhibition of the inflammatory response triggered by microglial pyroptosis inflammatory activation may be one of the effective ways to alleviate cerebral ischemia-reperfusion injury, the specific mechanism of which remains unclear. In this study, BV-2 microglia with or without oxygen-glucose deprivation/reoxygenation (OGD/R) or long noncoding RNA (lncRNA) Gm44206 knockdown were used as cell models to conduct an in vitro study. Detection of lactate dehydrogenase release and pyroptosis-related protein levels was performed using a corresponding kit and western blotting, respectively. Proliferation of microglia was evaluated by CCK8 assay. Enzyme-linked immunosorbent assay was applied for measuring levels of proinflammatory cytokines. This study verified the involvement of microglial pyroptosis as well as upregulation of NLRP3, Caspase-1, GSDMD, and Apoptosis-associated Speck-like protein containing a C-terminal caspase-recruitment domain (ASC) in cerebral ischemia-reperfusion injury. Moreover, knockdown of lncRNA Gm44206 could alleviate OGD/R-induced microglial pyroptosis and cell proliferation inhibition through the NLRP3/Caspase-1/GSDMD pathway, thus decreasing the release of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-18, and tumor necrosis factor-alpha. In conclusion, this study established a correlation between microglial pyroptosis and cerebral ischemia-reperfusion injury and identified lncRNA Gm44206 as a potential regulator of NLRP3/Caspase-1/GSDMD axis-mediated microglial pyroptosis, which could be considered a promising therapeutic target.

Pyroptosis is a new type of programmed cell death, which induces a strong pro-inflammatory reaction. However, the mechanism of pyroptosis after brain ischemia/reperfusion (I/R) and the interaction between different neural cell types are still unclear. This study comprehensively explored the mechanisms and interactions of microglial and neuronal pyroptosisin the simulated I/R environment in vitro. The BV2 (as microglial) and HT22(as neuronal) cells were treated by oxygen-glucose deprivation/reoxygenation (OGD/R). Both BV2 and HT22 cells underwent pyroptosis after OGD/R, and the pyroptosis occurred at earlier time point in HT22than that of BV2. Caspase-11 and Gasdermin E expression in BV2 and HT22 cells did not change significantly after OGD/R. Inhibition of caspase-1 or GSDMD activity, or down-regulation of GSDMD expression, alleviated pyroptosis in both BV2 and HT22 cells after OGD/R. Transwell studies further showed that OGD/R-treated HT22 or BV2 cells aggravated pyroptosis of adjacent non-OGD/R-treated cells, which could be relieved by inhibition of caspase-1 or GSDMD. These results suggested that OGD/R induces pyroptosis of microglia and neuronal cells and aggravates cell injury via activation of caspase-1/GSDMD signaling pathway. Our findings indicated that caspase-1 and GSDMD may be therapeutic targets after cerebral I/R., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Saikosaponin-D (SSD), an active ingredient in Bupleurum chinense, exerts anticancer effects in various cancers by inhibiting cancer proliferation and inducing apoptosis. However, whether SSD can induce other forms of cell death is unknown. The current study aims to demonstrate that SSD can induce pyroptosis in non-small-cell lung cancer. In this study, HCC827 and A549 non-small-cell lung cancer cells were treated with different concentrations of SSD for 1.5 h. HE and TUNEL staining were used to verify cell damage caused by SSD. Immunofluorescence and western blotting were performed to verify the effect of SSD on the NF-κB/NLRP3/caspase-1/gasdermin D (GSDMD) pathway. Changes in inflammatory factors were detected by ELISAs. Finally, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) was introduced to verify that SSD induces pyroptosis through the ROS/NF-κB pathway. The results of the HE and TUNEL staining showed that SSD resulted in balloon-like swelling of NSCLC cells accompanied by increased DNA damage. Immunofluorescence and western blot assays confirmed that SSD treatment activated the NLRP3/caspase-1/GSDMD pathway, stimulated an increase in ROS levels and activated NF-κB in lung cancer cells. The ROS scavenger N-acetylcysteine significantly attenuated SSD-induced NF-κB/NLRP3/caspase-1/GSDMD pathway activation and inhibited the release of the inflammatory cytokines IL-1β and IL-18. In conclusion, SSD induced lung cancer cell pyroptosis by inducing ROS accumulation and activating the NF-κB/NLRP3/caspase-1/GSDMD pathway. These experiments lay the foundation for the application of SSD in the treatment of non-small-cell lung cancer and regulation of the lung cancer immune microenvironment., (© 2023 Wiley Periodicals LLC.)

Oncogene E6 plays a critical role in the development and progression of esophageal cancer caused by human papillomavirus (HPV) infection. Alpha-ketoglutarate (AKG) is a key metabolite in the tricarboxylic acid cycle and has been widely used as a dietary and anti-ageing supplement. In this study, we found that treating esophageal squamous carcinoma cells with a high dose of AKG can induce cell pyroptosis. Furthermore, our research confirms that HPV18 E6 inhibits AKG-induced pyroptosis of esophageal squamous carcinoma cells by lowering P53 expression. P53 downregulates malate dehydrogenase 1 (MDH1) expression; however, MDH1 downregulates L-2-hydroxyglutarate (L-2HG) expression, which inhibits a rise in reactive oxygen species (ROS) levels-as L-2HG is responsible for excessive ROS. This study reveals the actuating mechanism behind cell pyroptosis of esophageal squamous carcinoma cells induced by high concentrations of AKG, and we posit the molecular pathway via which the HPV E6 oncoprotein inhibits cell pyroptosis., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of endocytic compartments in DCs, whereby internalized proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell type-specific effectors of endocytic escape. We devised an assay suitable for genetic screening and identified a pore-forming protein, perforin-2 ( Mpeg1 ), as a dedicated effector exclusive to cross-presenting cells. Perforin-2 was recruited to antigen-containing compartments, where it underwent maturation, releasing its pore-forming domain. Mpeg1 -/- mice failed to efficiently prime CD8 + T cells to cell-associated antigens, revealing an important role for perforin-2 in cytosolic entry of antigens during cross-presentation.

Perforin-2 facilitates antigen translocation to the cytosol in cross-presenting dendritic cells.

Background: Neuronal loss is a vital pathological feature of temporal lobe epilepsy (TLE). However, the exact mechanism of neuronal loss in TLE is not fully understood. Pyroptosis, a novel form of programmed cell death (PCD), has been considered a contributor to the pathogenesis of TLE. However, recent studies have implicated extensive molecular crosstalk among pyroptosis, apoptosis, and necroptosis in various diseases, and they can be transformed to each other according to different contexts. This study aimed to investigate whether gasdermin D (GSDMD)-mediated pyroptosis is involved in the pathogenesis of TLE and whether crosstalk exists in the process of the modulation of pyroptosis., Methods: The TLE model was established by intra-amygdala injection of kainic acid. The Racine score and local field potential (LFP) recordings were used to assess seizure severity. Western blotting and immunofluorescence were applied to detect the levels and cellular localization of GSDMD. The neuronal loss and type of neuronal death in the bilateral hippocampus were assessed by Nissl staining and flow cytometry analysis. The underlying crosstalk among pyroptosis, apoptosis, and necroptosis was explored by western blot and verified by VX765., Results: GSDMD was significantly upregulated and mainly expressed within the neurons of the hippocampus in the TLE model. Inhibition of pyroptosis by GSDMD knockdown triggered caspase-3-mediated apoptosis, leading to excess neuronal loss and deterioration of epileptic behaviors. Blocking caspase-1 markedly inhibited caspase-3-mediated apoptosis and improved epileptic behaviors under GSDMD knockdown., Conclusions: Our results demonstrate that GSDMD-mediated pyroptosis is involved in the pathogenesis of TLE. However, inhibition of GSDMD triggers caspase-1-mediated crosstalk between pyroptosis and apoptosis, which exacerbates neuronal loss and seizure susceptibility. Therefore, the complex crosstalk among different forms of PCD should be considered when a potential molecular target in the single PCD pathway is modulated. On the other hand, along with further studies of molecular crosstalk among the PCD pathways, taking advantage of crosstalk to attenuate neuronal loss may provide new insight for the clinical therapy of TLE., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Acute lung injury (ALI) is a prevalent and severe clinical condition characterized by inflammatory damage to the lung endothelial and epithelial barriers, resulting in high incidence and mortality rates. Currently, there is a lack of safe and effective drugs for the treatment of ALI. In a previous clinical study, we observed that Jinyinqingre oral liquid (JYQR), a Traditional Chinese Medicine formulation prepared by the Taihe Hospital, Affiliated Hospital of Hubei University of Medicine, exhibited notable efficacy in treating inflammation-related hepatitis and cholecystitis in clinical settings. However, the potential role of JYQR in ALI/acute respiratory distress syndrome (ARDS) and its anti-inflammatory mechanism remains unexplored. Thus, the present study aimed to investigate the therapeutic effects and underlying molecular mechanisms of JYQR in ALI using a mouse model of lipopolysaccharide (LPS)-induced ALI and an in vitro RAW264.7 cell model. JYQR yielded substantial improvements in LPS-induced histological alterations in lung tissues. Additionally, JYQR administration led to a noteworthy reduction in total protein levels within the BALF, a decrease in MPAP, and attenuation of pleural thickness. These findings collectively highlight the remarkable efficacy of JYQR in mitigating the deleterious effects of LPS-induced ALI. Mechanistic investigations revealed that JYQR pretreatment significantly inhibited NF-κB activation and downregulated the expressions of the downstream proteins, namely NLRP3 and GSDMD, as well as proinflammatory cytokine levels in mice and RAW2647 cells. Consequently, JYQR alleviated LPS-induced ALI by inhibiting the NF-κB/NLRP3/GSDMD pathway. JYQR exerts a protective effect against LPS-induced ALI in mice, and its mechanism of action involves the downregulation of the NF-κB/NLRP3/GSDMD inflammatory pathway., (Copyright © 2023 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Gasdermins (GSDMs) are a protein family encoded by six paralogous genes in humans, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME (also known as DFNA5), and DFNB59 (also known as pejvakin). Structurally, members of the GSDM family possess a C-terminus (an autoinhibitory domain) and a positively charged N-terminus (a pore-forming domain) linked with divergent peptide linkers. Recently, GSDMs have been identified as key executors of pyroptosis (an immunogenic programmed cell death) due to their pore-forming activities on the plasma membrane when proteolytically cleaved by caspases or serine proteases. Accumulating studies suggest that chemoresistance is attributed to dysregulation of apoptotic machinery and that inducing pyroptosis to bypass aberrant apoptosis can potently resensitize apoptosis-resistant cancer to chemotherapeutics. Pyroptosis is initiated by pore formation and culminates with plasma membrane rupture; these processes enable the release of proinflammatory cytokines (e.g., IL-1β and IL-18) and damage-associated molecular patterns, which further modulate antitumor immunity within the tumor microenvironment. Although pyroptosis is considered a promising strategy to boost antitumor effects, it is also reported to cause unwanted tissue damage (e.g., gut damage and nephrotoxicity). Intriguingly, mounting evidence has uncovered nonpyroptotic roles of GSDMs in tumorigenesis, such as proliferation, invasion, metastasis, and drug resistance. Thus, this provides a rationale for GSDMs as potential therapeutic targets. Taken together, we shed unbiased light on the pyroptosis-dependent roles of GSDMs in cancer progression and highlighted how GSDMs modulate tumorigenesis in a pyroptosis-independent manner. It is evident that targeting GSDMs seems profound in cancer management; however, several problems require further investigation to target GSDMs from bench to bedside, which is elucidated in the discussion section., (© 2023. L. Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland.)

Gasdermin (GSDM)-mediated pyroptosis is functionally involved in multiple diseases, but Gasdermin-B (GSDMB) exhibit cell death-dependent and independent activities in several pathologies including cancer. When the GSDMB pore-forming N-terminal domain is released by Granzyme-A cleavage, it provokes cancer cell death, but uncleaved GSDMB promotes multiple pro-tumoral effects (invasion, metastasis, and drug resistance). To uncover the mechanisms of GSDMB pyroptosis, here we determined the GSDMB regions essential for cell death and described for the first time a differential role of the four translated GSDMB isoforms (GSDMB1-4, that differ in the alternative usage of exons 6-7) in this process. Accordingly, we here prove that exon 6 translation is essential for GSDMB mediated pyroptosis, and therefore, GSDMB isoforms lacking this exon (GSDMB1-2) cannot provoke cancer cell death. Consistently, in breast carcinomas the expression of GSDMB2, and not exon 6-containing variants (GSDMB3-4), associates with unfavourable clinical-pathological parameters. Mechanistically, we show that GSDMB N-terminal constructs containing exon-6 provoke cell membrane lysis and a concomitant mitochondrial damage. Moreover, we have identified specific residues within exon 6 and other regions of the N-terminal domain that are important for GSDMB-triggered cell death as well as for mitochondrial impairment. Additionally, we demonstrated that GSDMB cleavage by specific proteases (Granzyme-A, Neutrophil Elastase and caspases) have different effects on pyroptosis regulation. Thus, immunocyte-derived Granzyme-A can cleave all GSDMB isoforms, but in only those containing exon 6, this processing results in pyroptosis induction. By contrast, the cleavage of GSDMB isoforms by Neutrophil Elastase or caspases produces short N-terminal fragments with no cytotoxic activity, thus suggesting that these proteases act as inhibitory mechanisms of pyroptosis. Summarizing, our results have important implications for understanding the complex roles of GSDMB isoforms in cancer or other pathologies and for the future design of GSDMB-targeted therapies., (© 2023. The Author(s).)

Purpose: Pyroptosis is a newly discovered form of programmed pro-inflammatory cell death. The main signaling pathways include the classical scorch death pathway that depends on NLRP3 inflammatory vesicles and other activation caspase-1 and the non-classical scorch death pathway that depends on caspase-4 /5/11. The substrate of all inflammatory caspases is GSDMD; a large number of studies have confirmed that pyroptosis is associated with certain infectious diseases, atherosclerotic diseases, metabolic diseases, and aseptic inflammatory diseases of important organs. In recent years, pyroptosis has been studied partially in the ocular field. So, this article reviews the recent literature intending to help readers understand the main mechanisms of cellular scorch death and the progress of GSDMD-mediated cellular scorch death in retinal vascular inflammatory diseases., Method: A detailed review of the literature related to pyroptosis and inflammatory diseases of the retinal vasculature is presented. The following 6 electronic databases were searched: CNKI, Wanfang, VIP, PubMed, The Cochrane Library, and Embase Databases, and the search period was from the database to May 2022. The main search keywords include "Pyroptosis," " GSDMD," "Retinal Vascular Inflammatory Disease," "Diabetic retinopathy," "Retinal vasculitis." The discovery of pyroptosis, the main molecular mechanisms, key proteins, and their pathogenesis and therapeutic prospects in retinal vasculitis diseases are extensively studied and summarized., Result: The mechanisms of gasdermin D-mediated pyroptosis are elaborated and analyzed, with particular emphasis on their key role and potential in the pathogenesis and treatment of inflammatory retinal vascular lesions., Conclusion: Gasdermin D-mediated pyroptosis is a well-studied form of programmed pro-inflammatory cell death, which has a bidirectional regulatory effect on a variety of immune and inflammatory diseases. The literature reveals that pyroptosis is closely related to the pathogenesis of retinal vascular inflammatory diseases, and it may be an important therapeutic target for diabetic retinopathy and other retinal vasculitis eye diseases in the future., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Gasdermins (GSDMs) are pore-forming proteins that play critical roles in host defence through pyroptosis 1,2 . Among GSDMs, GSDMB is unique owing to its distinct lipid-binding profile and a lack of consensus on its pyroptotic potential 3-7 . Recently, GSDMB was shown to exhibit direct bactericidal activity through its pore-forming activity 4 . Shigella, an intracellular, human-adapted enteropathogen, evades this GSDMB-mediated host defence by secreting IpaH7.8, a virulence effector that triggers ubiquitination-dependent proteasomal degradation of GSDMB 4 . Here, we report the cryogenic electron microscopy structures of human GSDMB in complex with Shigella IpaH7.8 and the GSDMB pore. The structure of the GSDMB-IpaH7.8 complex identifies a motif of three negatively charged residues in GSDMB as the structural determinant recognized by IpaH7.8. Human, but not mouse, GSDMD contains this conserved motif, explaining the species specificity of IpaH7.8. The GSDMB pore structure shows the alternative splicing-regulated interdomain linker in GSDMB as a regulator of GSDMB pore formation. GSDMB isoforms with a canonical interdomain linker exhibit normal pyroptotic activity whereas other isoforms exhibit attenuated or no pyroptotic activity. Overall, this work sheds light on the molecular mechanisms of Shigella IpaH7.8 recognition and targeting of GSDMs and shows a structural determinant in GSDMB critical for its pyroptotic activity., (© 2023. The Author(s).)

Cytotoxic lymphocyte-derived granzyme A (GZMA) cleaves GSDMB, a gasdermin-family pore-forming protein 1,2 , to trigger target cell pyroptosis 3 . GSDMB and the charter gasdermin family member GSDMD 4,5 have been inconsistently reported to be degraded by the Shigella flexneri ubiquitin-ligase virulence factor IpaH7.8 (refs. 6,7 ). Whether and how IpaH7.8 targets both gasdermins is undefined, and the pyroptosis function of GSDMB has even been questioned recently 6,8 . Here we report the crystal structure of the IpaH7.8-GSDMB complex, which shows how IpaH7.8 recognizes the GSDMB pore-forming domain. We clarify that IpaH7.8 targets human (but not mouse) GSDMD through a similar mechanism. The structure of full-length GSDMB suggests stronger autoinhibition than in other gasdermins 9,10 . GSDMB has multiple splicing isoforms that are equally targeted by IpaH7.8 but exhibit contrasting pyroptotic activities. Presence of exon 6 in the isoforms dictates the pore-forming, pyroptotic activity in GSDMB. We determine the cryo-electron microscopy structure of the 27-fold-symmetric GSDMB pore and depict conformational changes that drive pore formation. The structure uncovers an essential role for exon-6-derived elements in pore assembly, explaining pyroptosis deficiency in the non-canonical splicing isoform used in recent studies 6,8 . Different cancer cell lines have markedly different isoform compositions, correlating with the onset and extent of pyroptosis following GZMA stimulation. Our study illustrates fine regulation of GSDMB pore-forming activity by pathogenic bacteria and mRNA splicing and defines the underlying structural mechanisms., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Mounting evidence indicates that inhibition of microglial activation and neuronal pyroptosis plays important roles in brain function recovery after subarachnoid hemorrhage (SAH). LDC7559 is a newly discovered gasdermin D (GSDMD) inhibitor. Previous studies have demonstrated that LDC7559 could inhibit microglial proliferation and pyroptosis. However, the beneficial effects of LDC7559 on SAH remain obscure. Based on this background, we investigated the potential role and the mechanism of LDC7559 on SAH-induced brain damage both in vivo and in vitro . The findings revealed that microglial activation and neuronal pyroptosis were evidently increased after SAH, which could be markedly suppressed by LDC7559 both in vivo and in vitro. Meanwhile, LDC7559 treatment reduced neuronal apoptosis and improved behavior function. Mechanistically, LDC7559 decreased the levels of GSDMD and cleaved GSDMD after SAH. In contrast, nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation by nigericin increased GSDMD-mediated pyroptosis and abated the beneficial effects of LDC7559 on SAH-induced brain damage. However, LDC7559 treatment did not significantly affect the expression of NLRP3 after SAH. Taken together, LDC7559 might suppress neuronal pyroptosis and microglial activation after SAH by inhibiting GSDMD, thereby promoting brain functional recovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cai, Wu, Lai, Yao, Zeng, Fang, Lin, Chen, Xu and Chen.)

Sepsis-associated encephalopathy (SAE), as shown as acute and long-term cognitive impairment, is associated with increased mortality of sepsis. The causative factors of SAE are diverse and the underlying pathological mechanisms of SAE remain to be fully elucidated. Multiple studies have demonstrated a crucial role of microglia in the development of SAE, but the role of neutrophils and neutrophil extracellular traps (NETs) in SAE is still unclear. Here, we firstly show that in murine sepsis model, neutrophils and NETs promote blood-brain barrier (BBB) disruption, neuronal apoptosis and microglia activation in hippocampus and induce hippocampus-dependent memory impairment. Anti-Gr-1 antibody or DNase I treatment attenuates these sepsis-induced changes. Then, we find that genetic deletion of neutrophil GSDMD or PD-L1 reduces NET release and improves SAE in murine sepsis model. Finally, in human septic neutrophils, p-Y705-Stat3 binds to PD-L1, promotes PD-L1 nuclear translocation and enhances transcription of the gasdermin D (GSDMD) gene. In summary, our findings firstly identify a novel function of PD-L1 in maintaining transcriptional activity of p-Y705-Stat3 to promote GSDMD-dependent NET release in septic neutrophils, which plays a critical role in the development of SAE., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Gasdermin (GSDM) B is a member of the GSDM family, which is a protein that may be involved in the cell pyroptosis process and is associated with inflammatory diseases., Objective: To explore the correlation between GSDMB and psoriasis vulgaris., Methods: Skin lesions from 33 patients with psoriasis vulgaris and 69 normal controls were collected. ELISA and Western blot were adopted to detect proteins. The HaCaT cell line was transfected with 3 sets of interfering sequence siRNA, and the mRNA and protein levels before and after the transfection were measured by qPCR and Western blot respectively, so as to establish a cell model with low GSDMB gene expression; the MTT method was used to detect cells viability, flow cytometry to detect cell apoptosis., Results: The level of GSDMB protein in the skin lesions of patients with psoriasis vulgaris was lower than that in normal skin tissues (P < 0.05). The mRNA and protein expression levels of the target gene in the siRNA-GSDMB-3 group were lower than those in the control group (P < 0.05). The proliferation of HaCaT cells was decreased by MTT method and flow cytometry, and the apoptosis rate was increased (P < 0.05)., Conclusion: The expression level of GSDMB in psoriasis vulgaris lesion tissue is lower than that of normal skin tissue. The down-regulation of GSDMB expression can inhibit cell proliferation and promote cell apoptosis. GSDMB may play a role in the pathogenesis of psoriasis by affecting the differentiation of keratinocytes and the function of T cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ji et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are among the most common morbidities affecting extremely premature infants who receive oxygen therapy. Many clinical studies indicate that BPD is associated with advanced ROP. However, the mechanistic link between hyperoxia, BPD, and ROP remains to be explored. Gasdermin D (GSDMD) is a key executor of inflammasome-induced pyroptosis and inflammation. Inhibition of GSDMD has been shown to attenuate hyperoxia-induced BPD and brain injury in neonatal mice. The objective of this study was to further define the mechanistic roles of GSDMD in the pathogenesis of hyperoxia-induced BPD and ROP in mouse models. Here we show that global GSDMD knockout (GSDMD-KO) protects against hyperoxia-induced BPD by reducing macrophage infiltration, improving alveolarization and vascular development, and decreasing cell death. In addition, GSDMD deficiency prevented hyperoxia-induced ROP by reducing vasoobliteration and neovascularization, improving thinning of multiple retinal tissue layers, and decreasing microglial activation. RNA sequencing analyses of lungs and retinas showed that similar genes, including those from inflammatory, cell death, tissue remodeling, and tissue and vascular developmental signaling pathways, were induced by hyperoxia and impacted by GSDMD-KO in both models. These data highlight the importance of GSDMD in the pathogenesis of BPD and ROP and suggest that targeting GSDMD may be beneficial in preventing and treating BPD and ROP in premature infants., (© 2023. The Author(s).)

The gasdermins (GSDM), a family of pore-forming proteins, consist of gasdermin A (GSDMA), gasdermin B (GSDMB), gasdermin C (GSDMC), gasdermin D (GSDMD), gasdermin E (GSDME) and DFNB59 (Pejvakin (PJVK)) in humans. These proteins play an important role in pyroptosis. Among them, GSDMD is the most extensively studied protein and is identified as the executioner of pyroptosis. Other family members have also been implicated in certain cancers. As a unique form of programmed cell death, pyroptosis is closely related to tumor progression, and the inflammasome, an innate immune mechanism that induces inflammation and pyroptosis. In this review, we explore the current developments of pyroptosis, the inflammasome, and especially we review the gasdermin family members and their role in inducing pyroptosis and the possible therapeutic values in antitumor effects.

Background: Pyroptosis of endothelial cells is a new cause of endothelial dysfunction in multiple diseases. Ceramide acts as a potential bioactive mediator of inflammation and increases vascular endothelial permeability in many diseases, whether it can aggravate vascular endothelial injury by inducing cell pyroptosis remains unknown. This study was established to explore the effects of C8-ceramide (C8-Cer) on human umbilical vein vascular endothelial cells (HUVECs) and its possible underlying mechanism., Methods: HUVECs were exposed to various concentrations of C8-Cer for 12 h, 24 h, 48 h. The cell survival rate was measured using the cell counting kit-8 assay. Western blotting and Real-time polymerase chain reaction (RT-PCR) were used to detect the pyroptosis-releated protein and mRNA expressions, respectively. Caspase-1 activity assay was used to detect caspase-1 activity. Hoechst 33342/propidium iodide double staining and flow cytometry were adopted to measure positive staining of cells. Lactate dehydrogenase release assay and enzyme-linked immunosorbent assay were adopted to measure leakage of cellular contents. FITC method was used to detect the permeability of endothelial cells. ROS fluorescence intensity were detected by flow cytometry., Results: The viability of HUVECs decreased gradually with the increase in ceramide concentration and time. Ceramide upregulated the expression of thioredoxin interacting protein (TXNIP), NLRP3, GSDMD, GSDMD-NT, caspase-1 and Casp1 p20 at the protein and mRNA level in a dose-dependent manner. It also enhanced the PI uptake in HUVECs and upregulated caspase-1 activity. Moreover, it promoted the release of lactate dehydrogenase, interleukin-1β, and interleukin-18. Meanwhile, we found that ceramide led to increased vascular permeability. The inhibitor of NLRP3 inflammasome assembly, MCC950, was able to disrupt the aforementioned positive loop, thus alleviating vascular endothelial cell damage. Interestingly, inhibition of TXNIP either chemically using verapamil or genetically using small interfering RNA (siRNA) can effectively inhibit ceramide-induced pyroptosis and improved cell permeability. In addition, ceramide stimulated reactive oxygen species (ROS) generation. The pretreatment of antioxidant N-acetylcysteine (NAC), ROS scavenger, blocked the expression of pyroptosis markers induced by C8-cer in HUVECs., Conclusion: The current study demonstrated that C8-Cer could aggravate vascular endothelial cell damage and increased cell permeability by inducing cell pyroptosis. The results documented that the ROS-dependent TXNIP/NLRP3/GSDMD signalling pathway plays an essential role in the ceramide-induced pyroptosis in HUVECs., (© 2022. The Author(s).)

Colorectal cancer (CRC) remains the third cause of cancer-related mortality in Western countries, metastases are the main cause of death. CRC treatment remains limited by systemic toxicity and chemotherapy resistance. Therefore, nanoparticle-mediated delivery of cytotoxic agents selectively to cancer cells represents an efficient strategy to increase the therapeutic index and overcome drug resistance. We have developed the T22-PE24-H6 therapeutic protein-only nanoparticle that incorporates the exotoxin A from Pseudomonas aeruginosa to selectively target CRC cells because of its multivalent ligand display that triggers a high selectivity interaction with the CXCR4 receptor overexpressed on the surface of CRC stem cells. We here observed a CXCR4-dependent cytotoxic effect for T22-PE24-H6, which was not mediated by apoptosis, but instead capable of inducing a time-dependent and sequential activation of pyroptotic markers in CRC cells in vitro . Next, we demonstrated that repeated doses of T22-PE24-H6 inhibit tumor growth in a subcutaneous CXCR4 + CRC model, also through pyroptotic activation. Most importantly, this nanoparticle also blocked the development of lymphatic and hematogenous metastases, in a highly aggressive CXCR4 + SW1417 orthotopic CRC model, in the absence of systemic toxicity. This targeted drug delivery approach supports for the first time the clinical relevance of inducing GSDMD-dependent pyroptosis, a cell death mechanism alternative to apoptosis, in CRC models, leading to the selective elimination of CXCR4 + cancer stem cells, which are associated with resistance, metastases and anti-apoptotic upregulation.

Acute lung injury (ALI) is considered as a severe respiratory disease with aggravated inflammatory responses. Krüppel-like factor 9 ( KLF9 ), a member of KLF family, has been reported to be involved in inflammatory disorders. However, the effect of KLF9 in ALI has not been elucidated. Here the present study was to clarify the role of KLF9 and its mechanism in ALI. The ALI in vitro model was established with lipopolysaccharide (LPS)-treated RAW264.7 cells. Mice were injected with LPS to conduct an ALI in vivo model. The expression of KLF9 and gasdermin D ( GSDMD ) was examined using quantitative reverse transcription-PCR, haematoxylin-eosin/immunohistochemistry staining and western blot assays. Enzyme-linked immunosorbent assay was employed to detect the levels of inflammatory cytokines. JASPAR database was used to predict the recognition motif of KLF9 , and the relationship between KLF9 and GSDMD was determined by luciferase reporter assay and chromatin immunoprecipitation analysis. The number of neutrophils in bronchoalveolar lavage fluid as well as the wet/dry weight ratio was caculated. The results showed that The expression of KLF9 in lung was significantly increased in LPS-stimulated mice. Moreover, KLF9 knockout relieved the lung injury in ALI mice. GSDMD is one of targets genes of the transcription factor KLF9 . KLF9 knockout induced a decreased expression of GSDMD in LPS-treated mice. Furthermore, in RAW264.7 cells after LPS administration, KLF9 knockdown reduced the levels of inflammatory factors and suppressed the expression of GSDMD . In summarise, these findings exhibited that KLF9 knockout could mitigate the lung injury and inflammatory responses in ALI mice by directly regulating GSDMD .

Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1β secretion and neutrophil cell death remain unclear. Here, we show that neutrophil-targeted expression of the disease-associated gain-of-function Nlrp3 A350V mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1β in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1β are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91 Phox -deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively., (© 2022 The Authors.)

Leukemia is a fatal hematopoietic disorder with a poor prognosis. Drug resistance is inevitable after the long‑term use of chemotherapeutic agents. Liproxstatin‑1, commonly known as a ferroptosis inhibitor, has never been reported to have anticancer effects. In the present study, the antileukemic role of liproxstatin‑1 in K562 leukemia cells was investigated. Liproxstatin‑1 inhibited K562 cell proliferation in a dose‑ and time‑dependent manner. RNA sequencing revealed several pathways that were affected by liproxstatin‑1, such as the G1/S transition of the mitotic cell cycle and extrinsic or intrinsic apoptotic signaling pathways. The results of flow cytometry indicated that liproxstatin‑1 arrests the cell cycle at the G1 phase, and even at the G2/M phase. p21 WAF1/CIP1 , a cyclin‑dependent kinase inhibitor, was upregulated. It was also determined that liproxstatin‑1 induced BAX and TNF‑α expression, which was accompanied by cleavage of caspase‑3 and PARP. The caspase‑3‑specific inhibitor z‑DEVD‑FMK rescued some of the apoptotic cells. Interestingly, K562 cells were characterized by swelling and plasma membrane rupture when treated with a high concentration of liproxstatin‑1, which was inconsistent with the typical apoptotic appearance. Thus, it was hypothesized that apoptosis‑mediated pyroptosis occurs during liproxstatin‑1‑induced cell death. The expression of the hallmark of pyroptosis, the cleaved N‑terminal GSDME, increased. Additionally, it was observed that endoplasmic reticulum stress and autophagy were involved in liproxstatin‑1‑induced cell death. Collectively, liproxstatin‑1 induced cell cycle arrest, apoptosis, and caspase‑3/GSDME‑dependent secondary pyroptosis in K562 leukemia cells, which provides new hope for the treatment of leukemia.

Perforin is a granular effector pore-forming protein formed in NK cells and Cytotoxic T lymphocytes. These cytotoxic proteins are part of the first-line immune defense in the human body. They ensure apoptosis of pathogen-infected cells or tumor cells in the human body. Activation of receptors on NK cell or T cell triggers secondary proteins in these cells. Further, it leads to Ca 2+ dependent perforin egress towards the target cell, ensued by PI3K signaling pathway. Perforin undergoes oligomerization over the target cell membrane and forms transmembrane pores with the membrane-spanning domain-MACPF domain. Granzymes, proapoptotic serine proteases are released through these pores and initiate the target cell apoptotic pathway leading to the cell death. Although perforin is a savior for humans from tumor and viral infections, uncontrolled expression of the perforins leads to the autoimmune conditions, including Familial Hemophagocytic lymphohistiocytosis, insulin-dependent diabetes, and cerebral myocarditis. The present review is the concerted effort to highlight the mechanistic pathways concerning perforin secretion, NK cell and T cell-mediated cytotoxicity towards virus-infected and transformed cells. This is followed by the discussion on synthetic derivatives tested so far to inhibit the perforin in pre and clinical arena for certain unusual conditions., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Raj Kumar reports financial support and administrative support were provided by Central University of Punjab. Raj Kumar reports a relationship with Central University of Punjab that includes: employment. Raj Kumar has patent No with royalties paid to Pending. Authors do not have any conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Toll-like receptor 4 (TLR4) mediated pathway plays a pivotal role in promoting tubulointerstitial inflammation and contributes to the progression in type 2 diabetic kidney disease (T2DKD). As the first identified key pyroptosis executor, gasdermin D (GSDMD) is activated by caspases and might be the key protein to switch apoptosis to pyroptosis. It remains unclear that role of TLR4 on canonical pyroptosis pathway, and whether GSDMD is involved in switching from apoptosis to pyroptosis in the TLR4-related tubular injury in T2DKD., Methods: Immunohistochemistry staining was used to detect the expression of pyroptosis-related proteins in renal tissues of T2DKD patients. T2DKD models was induced in TLR4 knockout (TLR4 -/- ) mice through a high-fat diet combined with streptozotocin. Pyroptosis (caspase-1, GSDMD, interleukin 18(IL-18), interleukin 1β(IL-1β)) and apoptosis levels (caspase-3, Bax and Bcl-2) were detected by Western blot. HK-2 cells were cultured under high-glucose (HG) conditions as an in vitro model and then challenged with a TLR4-specific antagonist (TAK-242). GSDMD small interfering RNA (siRNA) and overexpression plasmid were transfected into HK-2 cells to down- or up-regulate GSDMD. The pyroptosis and apoptosis rates were determined by flow cytometry., Results: The expression levels of caspase-1, GSDMD, IL-18 and IL-1β were increased in renal biopsy tissues of T2DKD patients and GSDMD expression was positively correlated with tubular injury. Silencing GSDMD attenuated HG-induced IL-18, IL-1β, FN and α-SMA, and reduced pyroptotic cells rate in HK-2 cells. Up-regulation of GSDMD inhibited HG-induced expression of Bax and cleaved caspase-3 and reduced apoptosis rate. TLR4 knockout alleviated tubular injury and interstitial macrophages infiltration, improved impaired renal dysfunction, and decreased the expressions of active N-terminal of GSDMD(GSDMD-N), cleaved caspase-1(cl-caspase-1) and cleaved caspase-3(cl-caspase-3) in T2DKD mice. TLR4 inhibition reduced HG-induced pyroptosis and apoptosis level in HK-2 cells, while GSDMD up-regulation increased pyroptosis rate and decreased apoptosis rate., Conclusions: TLR4 could exacerbate tubular injury and fibrosis via GSDMD-mediated canonical pyroptosis pathway in T2DKD. Activation of GSDMD could inhibit apoptosis and activate pyroptosis, which may involve the potential switch mechanism between TLR4-mediated pyroptosis and apoptosis in T2DKD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cytotoxic T lymphocytes (CTLs) lyse target cells by delivering lytic granules that contain the pore former perforin to the cytotoxic immunological synapse. Here, we establish that opposing cytoskeletal forces drive lytic granule polarization and simultaneously shape T cell synapse topography to enhance target perforation. At the cell rear, actomyosin contractility drives the anterograde movement of lytic granules toward the nucleus. At the synapse, dynein-derived forces induce negatively curved membrane pockets to which granules are transported around the nucleus. These highly concave degranulation pockets are located directly opposite positively curved bulges on the target cell membrane. We identify a curvature bias in the action of perforin, which preferentially perforates positively curved tumor cell membrane. Together, these findings demonstrate murine and human T cell-mediated cytotoxicity to be a highly tuned mechano-biochemical system, in which the forces that polarize lytic granules locally bend the synaptic membrane to favor the unidirectional perforation of the target cell., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Gasdermin D (GSDMD) plays an essential role in the pathway of pyroptosis. However, whether GSDMD participates in myocardial ischaemia/reperfusion injury (MI/RI) remains poorly understood., Methods: Serum levels of GSDMD and IL-18 in ST-segment elevation myocardial infarction (STEMI) patients were measured by ELISA. The expression of GSDMD and GSDMD N-terminal (GSDMD-NT) in vivo and in vitro was assessed by western blot and immunofluorescence staining. GSDMD -/- mice and wild type (WT) mice were induced MI/RI, followed by cardiac ultrasound and histological analysis., Results: Clinically, patients suffering from STEMI after percutaneous coronary intervention (PCI) exhibited higher levels of GSDMD and IL-18 than that in the controls. In vitro , the cleavage of GSDMD was significantly upregulated in macrophages exposed to hypoxia/reoxygenation or H 2 O 2 . In vivo , the levels of GSDMD and GSDMD-NT increased notably after MI/RI, especially in macrophages infiltrating in the infarct area. Moreover, compared with WT mice, GSDMD -/- mice showed reduced infarct size (25.45 ± 3.07% versus 36.47 ± 3.72%), improved left ventricular ejection fraction (37.71 ± 1.81% versus 29.44 ± 2.28%) and left ventricular fractional shortening (18.01 ± 0.97% versus 13.62 ± 1.15%) as well as attenuated pathological damage after I/R injury, along with reduced levels of proinflammatory cytokines and decreased infiltration of neutrophils., Conclusions: Our study revealed that GSDMD deficiency significantly alleviated the inflammatory response by regulating pyroptosis, reduced the infarct size and preserved cardiac function after MI/RI, thus providing a potential strategy for the treatment of myocardial reperfusion injury., Competing Interests: Author ZW was employed by TAU Cambridge Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ye, Zhang, Zhang, Luan, Xu, Wu, Ju and Hu.)

Sepsis is a systemic inflammatory response syndrome with high mortality. Acute liver injury is an independent predictor for poor prognosis in septic patients. Polygonatum sibiricum polysaccharides (PSP) have been reported to possess anti-inflammatory and hepatoprotective activities. To evaluate the effects of PSP on septic liver injury and demonstrate the potential molecular mechanisms, the septic acute liver injury (SALI) model was established in BALB/c mice via intraperitoneal injection of lipopolysaccharide (LPS). We found that PSP treatment could remarkably reduce the 48 h mortality rate of septic mice; alleviate liver histopathologic damage; lower the activity of neutrophil infiltration marker MPO in liver tissue; and decrease the levels of liver function indexes AST, ALT, ALP, and TBIL, inflammatory cytokines TNFα and IL-6, and pyroptosis-related inflammatory cytokines IL-18 and IL-1β in serum. TUNEL staining and detecting GSDMD-NT protein expression level in liver tissue revealed that PSP could restrain excessive pyroptosis. In addition, PSP treatment reversed the upregulations of mRNA expression levels of the NLRP3/GSDMD signals in the liver. Our results indicated the potential protective role of PSP against SALI by inhibiting pyroptosis via NLRP3/GSDMD signals.

Background: Reports in recent years have shown that pancreatic β-cell pyroptosis represents a critical mechanism involved with the progressive failure of pancreatic function. Previous research from our laboratory has indicated that artemether can increase the number of cells in pancreatic islets of db/db mice. In this study, we further examined whether artesunate (ART) protects pancreatic β-cells from the damage of streptozotocin (STZ) by inhibiting pyroptosis., Materials and Methods: In vitro, MIN6 cells exposed to 1 mM STZ were treated with ART (0.8 or 1.6 μM). The effects of ART on STZ-treated cells were evaluated through CCK-8 assay, flow cytometry and western blot, and further compared the effects of ART with the NLRP3 inhibitor, Mcc950 upon pyroptosis pathway proteins using western blot. In vivo, Male C57 mice were administered with a single intraperitoneal injection of STZ, and those with confirmed diabetes mellitus were given ART (0.5 or 1.0 mg/ml in drinking water) for 18 days. The effects of ART on STZ-induced diabetes were assessed by the observation of the general situation, glucose tolerance test, hematoxylin-eosin (HE) staining and immunohistochemistry., Results: In MIN6 cells treated with STZ, we found that ART increased cell viability, decreased the number of late apoptotic cells (including pyroptosis cells) and inhibited the expression of proteins associated with the pyroptosis pathway. In STZ-induced animal model, the administration of ART reduced blood glucose levels, improved the consumption status within this diabetic mouse model and inhibited the expression of proteins include in the pyroptosis pathway in mice pancreats., Conclusions: Inhibition of pyroptosis may be a critical mechanism through which artesunate exerts protective effects upon pancreatic β cells., (Copyright © 2022 Elsevier Inc. All rights reserved.)