CD5L as a promising biological therapeutic for treating sepsis.

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.

Dectin-1/SYK Activation Induces Antimicrobial Peptide and Negative Regulator of NF-κB Signaling in Human Oral Epithelial Cells.

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 ß-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 ß-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 ß-glucan-rich zymosan did not alter the mRNA expression of chemokines nor of interleukin (IL)6, IL8, IL1ß, IL17A, and IL17F. Zymosan induced the expression of antimicrobial peptides ß-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 ß-glucan, which activates SYK and induces the expression of antimicrobial peptides and negative regulators of NF-κB, potentially maintaining oral homeostasis.

RNA-seq revealed the anti-pyroptotic effect of suramin by suppressing NLRP3/caspase-1/GSDMD pathway in LPS-induced MH-S alveolar macrophages.

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.

Simvastatin induces pyroptosis via ROS/caspase-1/GSDMD pathway in colon cancer.

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-1ß, 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.

ERRα promotes glycolytic metabolism and targets the NLRP3/caspase-1/GSDMD pathway to regulate pyroptosis in endometrial cancer.

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.

METTL14-upregulated miR-6858 triggers cell apoptosis in keratinocytes of oral lichen planus through decreasing GSDMC.

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 N6-adenosine methylation (m6A) levels in the epithelial layer of OLP. m6A 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 m6A modification regulates miR-6858 production to decrease GSDMC expression and to trigger keratinocyte apoptosis in the context of OLP.

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

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritus, erythema, and skin barrier dysfunction. Gasdermin D (GSDMD) is the key executioner of an inflammatory cell death mechanism known as pyroptosis. However, the role of GSDMD in the pathogenesis of AD remains unclear. Through the analysis of publicly available Gene Expression Omnibus (GEO) datasets, we observed an upregulation of Gsdmd mRNA in the skin tissue of AD patients. Moreover, we delved into the impact of GSDMD deletion and inhibition on AD-like skin lesions using a mouse model induced by the topical application of oxazolone (Oxa). We found that mice lacking GSDMD exhibited relieved AD signs and symptoms in terms of reduced skin thickness, scarring and scratching behavior compared to wild-type mice after induction of AD-like skin lesions. This was associated with decreased infiltration of inflammatory cells, reduced epidermal thickness, and decreased serum levels of IgE and IL-4. Western blot analysis further revealed decreased GSDMD cleavage in the skin of GSDMD knockout mice, and reduced expression of IL-1ß 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.

Polydatin alleviates mycoplasma pneumoniae-induced injury via inhibition of Caspase-1/GSDMD-dependent pyroptosis.

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-1ß (IL-1ß) 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.

LncRNA Gm44206 Promotes Microglial Pyroptosis Through NLRP3/Caspase-1/GSDMD Axis and Aggravate Cerebral Ischemia-Reperfusion Injury.

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)-1ß, 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.

HPV18 E6 inhibits α-ketoglutarate-induced pyroptosis of esophageal squamous cell carcinoma cells via the P53/MDH1/ROS/GSDMC pathway.

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.

Saikosaponin-D induces the pyroptosis of lung cancer by increasing ROS and activating the NF-κB/NLRP3/caspase-1/GSDMD pathway.

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-1ß 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.

Jinyinqingre Oral Liquid alleviates LPS-induced acute lung injury by inhibiting the NF-κB/NLRP3/GSDMD pathway.

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.

Tou Nong powder obstructs ulcerative colitis through the regulation of NF-κB/NLRP3/Caspase-1/GSDMD inflammasome pyroptotic pathway.

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-1ß and IL-18.

Perforin-2 is a pore-forming effector of endocytic escape in cross-presenting dendritic cells.

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.

Channeling antigens to CD8+ T cells.

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

Pyroptosis-induced inflammation and tissue damage.

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.

The Pyroptotic and Nonpyroptotic Roles of Gasdermins in Modulating Cancer Progression and Their Perspectives on Cancer Therapeutics.

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-1ß 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.

PD-L1 promotes GSDMD-mediated NET release by maintaining the transcriptional activity of Stat3 in sepsis-associated encephalopathy.

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.

Microglial and Neuronal Cell Pyroptosis Induced by Oxygen-Glucose Deprivation/Reoxygenation Aggravates Cell Injury via Activation of the Caspase-1/GSDMD Signaling Pathway.

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.

LDC7559 inhibits microglial activation and GSDMD-dependent pyroptosis after subarachnoid hemorrhage.

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.