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Diabetic patients face an increased risk of cataract development. This study investigates the molecular mechanism by which oxidative stress activates the NLRP3/caspase-1/gasdermin-D (GSDMD) pathway, leading to inducing pyroptosis in human lens epithelial cells (HLECs) exposed to high glucose (HG) environments. In vitro, HLECs were cultured in HG medium and treated with N-acetylcysteine (NAC), MCC950 (an NLRP3 inflammasome inhibitor), and VX-765 (a caspase-1 inhibitor). Cell viability was assessed using CCK-8. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), lactate dehydrogenase (LDH) levels and activities of superoxide dismutase (SOD) and catalase (CAT) were measured using biochemical kits. Active interleukin (IL)-18 and IL-1β levels in cell culture supernatants were quantified using ELISA kits. Western blot analysis determined levels of GSDMD, GSDMD-N, NLRP3, apoptosis-associated speck-like protein containing CARD (ASC) and cleaved caspase-1. HG induced oxidative stress in HLECs, as indicated by elevated cellular ROS and MDA levels, decreased GSH level, and reduced SOD and CAT activities. HG triggered HLEC pyroptosis, characterized by increased LDH release, levels of cell pyroptosis-related proteins GSDMD-N/GSDMD ratio, and active IL-1β and IL-18 in cell culture supernatants. Inhibition of oxidative stress by NAC partially reversed HG-induced pyroptosis in HLECs. HG induced oxidative stress, activating the NLRP3 inflammasome activation and cleaving caspase-1 cleavage in HLECs. MCC950-suppressed NLRP3 inflammasome activation or VX765-inhibited caspase-1 cleavage partially reversed HG-induced HLEC pyroptosis. Oxidative stress in HLECs under HG conditions induces pyroptosis via the NLRP3/caspase-1/GSDMD pathway. [ABSTRACT FROM AUTHOR]

Aim: Gasdermin-D (GSDMD) and pannexin-1 (PANX1), which are proinflammatory proteins, may be involved in the pathogenesis of complications in pregnant women with obesity. We aimed to investigate the expression of GSDMD and PANX1 biochemically in samples of maternal omental and placental tissue obtained from pregnant women with or without obesity, and their correlation with maternal, obstetric, and fetal/neonatal variables. Methods: The prospective observational study included 79 pregnant women who underwent elective cesarean sections with a pregnancy between 37 and 41 weeks from December 2021 to April 2022. They were divided into three study groups according to their body mass index (BMI): normal weight (with a BMI 30) (n=26). Omental and placental GSDMD and PANX1 levels were measured by enzyme-linked immunosorbent assay between the groups. Results: In the homogenate of omental tissue, the median levels of GSDMD and PANX1 protein in the overweight and obese groups were significantly lower than those in the normal weight groups (p=0.0154 and p=0.0184, respectively). In the placental tissue samples, the median levels of GSDMD and PANX1 proteins in the normal weight, overweight, and obese groups were similar. Conclusion: GSDMD and PANX1 expressions were shown for the first time in the omentum and placenta together; which has the potential to be used as a predictive and diagnostic test panel after further studies in obese pregnant women.

Atopic dermatitis (AD) is an inflammatory skin disease with intense pruritus, and chronic skin colonization by Staphylococcus aureus. To understand the inflammatory status in AD, we investigated the inflammasome complex, that activates ASC (Apoptosis-associated speck-like protein containing a CARD), caspase-1 and GSDMD (gasdermin-D), and production of IL-1β and IL-18. We aimed to evaluate the expression of the inflammasome pathway in the skin of adults with AD. Thirty patients with moderate to severe AD and 20 healthy controls were enrolled in the study. We performed the analysis of the inflammasome components NLRP1, NLRP3, AIM-2, IL-1β, IL-18, Caspase-1, ASC, GSDMD, and CD68 expression (macrophage marker) by immunohistochemistry and immunofluorescence. The main findings included increased expression of NLRP3, NLRP1 and AIM-2 at dermal level of severe AD; augmented IL-18 and IL-1β expression at epidermis of moderate and severe patients, and in the dermis of severe AD; augmented expression of ASC, caspase-1 and GSDMD in both epidermis and dermis of moderate and severe AD. We detected positive correlation between caspase-1, GSDMD and IL-1β (epidermis) and caspase-1 (dermis) and AD severity; NLRP3, AIM-2 and IL-1β, and NLRP3 with IL-18 in the epidermis; ASC, GSDMD and IL-1β, and NLRP3, AIM-2, caspase-1, and IL-18 in the dermis. We also evidenced the presence of CD68+ macrophages secreting GSDMD, ASC and IL-1β in moderate and severe AD. Cutaneous macrophages, early detected in moderate AD, have its role in the disease inflammatory mechanisms. Our study indicates a canonical activation pathway of inflammasomes, reinforced by the chronic status of inflammation in AD. The analysis of the inflammasome complex evidenced an imbalance in its regulation, with increased expression of the evaluated components, which is remarkably in severe AD, emphasizing its relevance as potential disease biomarkers and targets for immunomodulatory interventions. [ABSTRACT FROM AUTHOR]

Aim: Gasdermin-D (GSDMD) and pannexin-1 (PANX1), which are proinflammatory proteins, may be involved in the pathogenesis of complications in pregnant women with obesity. We aimed to investigate the expression of GSDMD and PANX1 biochemically in samples of maternal omental and placental tissue obtained from pregnant women with or without obesity, and their correlation with maternal, obstetric, and fetal/neonatal variables. Methods: The prospective observational study included 79 pregnant women who underwent elective cesarean sections with a pregnancy between 37 and 41 weeks from December 2021 to April 2022. They were divided into three study groups according to their body mass index (BMI): normal weight (with a BMI <25) (n=25), overweight (BMI between 25 and 30) (n=28), and obese (with a BMI >30) (n=26). Omental and placental GSDMD and PANX1 levels were measured by enzyme-linked immunosorbent assay between the groups. Results: In the homogenate of omental tissue, the median levels of GSDMD and PANX1 protein in the overweight and obese groups were significantly lower than those in the normal weight groups (p=0.0154 and p=0.0184, respectively). In the placental tissue samples, the median levels of GSDMD and PANX1 proteins in the normal weight, overweight, and obese groups were similar. Conclusion: GSDMD and PANX1 expressions were shown for the first time in the omentum and placenta together; which has the potential to be used as a predictive and diagnostic test panel after further studies in obese pregnant women. [ABSTRACT FROM AUTHOR]

We analyzed actin cytoskeleton alterations during NET extrusion by neutrophil-like dHL-60 cells and human neutrophils in the absence of DNase1 containing serum to avoid chromatin degradation and microfilament disassembly. NET-formation by dHL-60 cells and neutrophils was induced by Ionomycin or phorbol-12-myristat-13-acetate (PMA). Subsequent staining with anti-actin and TRITC-phalloidin showed depolymerization of the cortical F-actin at spatially confined areas, the NET extrusion sites, effected by transient activation of the monooxygenase MICAL-1 supported by the G-actin binding proteins cofilin, profilin, thymosin ß4 and probably the F-actin fragmenting activity of gelsolin and/or its fragments, which also decorated the formed NETs. MICAL-1 itself appeared to be proteolyzed by neutrophil elastase possibly to confine its activity to the NET-extrusion area. The F-actin oxidization activity of MICAL-1 is inhibited by Levosimendan leading to reduced NET-formation. Anti-gasdermin-D immunohistochemistry showed a cytoplasmic distribution in non-stimulated cells. After stimulation the NET-extrusion pore displayed reduced anti-gasdermin-D staining but accumulated underneath the plasma membrane of the remaining cell body. A similar distribution was observed for myosin that concentrated together with cortical F-actin along the periphery of the remaining cell body suggesting force production by acto-myosin interactions supporting NET expulsion as indicated by the inhibitory action of the myosin ATPase inhibitor blebbistatin. Isolated human neutrophils displayed differences in their content of certain cytoskeletal proteins. After stimulation neutrophils with high gelsolin content preferentially formed “cloud”-like NETs, whereas those with low or no gelsolin formed long “filamentous” NETs.

Fangqing Zhu,1 Sheng Li,2 Qiuping Gu,1 Ningsheng Xie,1 Yinxia Wu3 1Department of Gastroenterology, Ganzhou People’s Hospital, Ganzhou, Jiangxi, 341000, People’s Republic of China; 2Department of Gastroenterology, Yuebei People’s Hospital, Shantou University Medical College, Shaoguan, Guangdong, 512026, People’s Republic of China; 3Department of Rehabilitation, Ganzhou People’s Hospital, Ganzhou, Jiangxi, 341000, People’s Republic of ChinaCorrespondence: Fangqing Zhu; Yinxia Wu, Email wanliqingkong0707@163.com; xiaflora0608@163.comBackground: Pyroptosis is a form of proinfammatory gasdermin-mediated programmed cell death. Abnormal infammation in the intestine is a critical risk factor for Ulcerative colitis (UC). However, at present, it is not clear whether pyroptosis of colonic fibroblasts is involved in the pathogenesis and progression of UC.Methods: In this study, key genes associated with UC were identified by bioinformatics analysis. Datasets were downloaded from the Gene Expression Omnibus (GEO) database (GSE193677). The differentially expressed genes were analyzed, and the hub genes were screened by weighted gene co-expression network analysis (WGCNA) and differentially expressed genes. We also downloaded the dataset from GEO for single-cell RNA sequencing (GSE231993). The expression of key genes was verified by immunohistochemistry, immunofluorescence and Western blot, and the specific pathways of key genes inducing pyroptosis in cell lines were explored.Results: The results of bioinformatics analysis showed that the expression of APOL1 and CXCL1 in UC tissues was significantly higher than that in normal tissues. The results of single-cell analysis showed that the two genes were co-localized to fibroblasts. These results were consistent with the results of immunohistochemistry and immunofluorescence colocalization in human intestinal mucosa specimens. Furthermore, APOL1 overexpression induced NLRP3-caspase1-GSDMD-mediated pyroptosis of fibroblasts, which was confirmed by Western blot.Conclusion: APOL1 induces pyroptosis of fibroblasts mediated by NLRP3-Caspase1-GSDMD signaling pathway and promote the release of chemokines CXCL1. Fibroblasts may play a crucial role in the pathogenesis and progression of UC.Keywords: apolipoprotein L1, pyroptosis, fibroblasts, gasdermin-D, Ulcerative colitis

Gasdermin-D (GSDMD) belongs to the Gasdermin family (GSDM), which are pore-forming effector proteins that facilitate inflammatory cell death, also known as pyroptosis. This type of programmed cell death is dependent on inflammatory caspase activation, which cleaves gasdermin-D (GSDMD) to form membrane pores and initiates the release of pro-inflammatory cytokines. Pyroptosis plays an important role in achieving immune regulation and homeostasis within various organ systems. The role of GSDMD in pyroptosis has been extensively studied in recent years. In this review, we summarize the role of GSDMD in cellular and organ injury mediated by pyroptosis. We will also provide an outlook on GSDMD therapeutic targets in various organ systems. [ABSTRACT FROM AUTHOR]

ABSTRACTGasdermin-D (GSDMD) belongs to the Gasdermin family (GSDM), which are pore-forming effector proteins that facilitate inflammatory cell death, also known as pyroptosis. This type of programmed cell death is dependent on inflammatory caspase activation, which cleaves gasdermin-D (GSDMD) to form membrane pores and initiates the release of pro-inflammatory cytokines. Pyroptosis plays an important role in achieving immune regulation and homeostasis within various organ systems. The role of GSDMD in pyroptosis has been extensively studied in recent years. In this review, we summarize the role of GSDMD in cellular and organ injury mediated by pyroptosis. We will also provide an outlook on GSDMD therapeutic targets in various organ systems.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it mostly arises as a consequence of persistent chronic inflammation. Recently, NLRP3 inflammasome has caught the attention of many research groups due to its involvement in different types of cancer. However, its direct role in HCC remains elusive. Our study aimed to evaluate the role of NLRP3 inflammasome and pyroptosis in HCC and to clarify the potential mechanism by which 17β-estradiol (E2) can be used as a protective factor against HCC. NLRP3, caspase-1 (CASP1) as well as gasdermin-D (GSDMD) mRNA expression levels were assessed in human HCC tissues and adjacent non-cancerous liver tissues. Also, HepG2 HCC cells were cultured and treated with E2, followed by detection of the mRNA levels of these three genes. Our results revealed that NLRP3, CASP1, and GSDMD mRNA expressions were significantly lower in HCC tissues than in controls, and this under-expression was closely correlated with advanced HCC stages and grades. In contrast, HepG2 HCC cells displayed significantly higher expression levels of NLRP3 inflammasome components and GSDMD in the two E2-treated groups compared to the untreated group. Also, NLRP3, CASP1, and GSDMD mRNA expression levels were positively correlated with each other. This study confirmed that lack of NLRP3 inflammasome is involved in HCC progression and 17β-estradiol-induced activation of NLRP3 inflammasome may be effective in HCC treatment as it inhibited tumor cell growth and proliferation by triggering CASP1-dependent pyroptosis in HCC cells. [ABSTRACT FROM AUTHOR]

Inflammasome activation is associated with a myriad of inflammatory diseases. However, existing methods provides a limited understanding of spatiotemporal kinetics of inflammasome activation, with restricted scope for early detection of associated treatment efficacy. This limitation offers an opportunity for the development of biocompatible in‐vivo inflammasome monitoring tools with translational prospects. To achieve this, they report developing a pair of lipid‐based nanoparticle systems, a reporter nanoparticle consisting of a caspase‐1 activatable probe alone, and a theranostic nanoparticle combining the probe with an inflammasome‐inhibiting drug. This biocompatible platform enhances the probe's residence time in circulation by preventing its opsonization and allowing its sustained release over time. Their results demonstrate the specificity of reporter nanoparticles towards caspase‐1 activity and provides early‐on monitoring of inflammasome activation both in‐vitro as well as in‐vivo. Additionally, the delivery of disulfiram, an inflammasome‐inhibiting drug, along with reporter probe using theranostic nanoparticles enables real‐time tracking of treatment efficacy in the gouty‐arthritis inflammatory model. In summary, they report an unparalleled pair of the inflammasome‐associated reporter and theranostic platforms suited not only for diagnostic applications but can also detect inflammasome‐targeted treatment efficiency in real‐time. These findings establish two novel, sensitive nanotools for non‐invasive evaluation of inflammasome‐targeted immunotherapy. [ABSTRACT FROM AUTHOR]

Introduction: Gingivitis is inflammation that often attacks the gum tissue. Bacteria are the most important cause. Pyroptosis is a type of programmed cell death caused infection trough expressed pro-inflammatory cytokines. Objective of this study is to know that pyroptosis was occurs in the gingivitis due to bacterial triggers by expressing Gasdermin-D and inflammasome-NLRP-3 proteins. Material and Methods: The experimental is analytic research with the case and control groups design. Event gingivitis was triggered by Aggregatibacter actinomycetemcomitans. After secrified a biopsy was performed on the distal portion of teeth and fixed with 10% buffered formalin. Hematoxylin eosin are stained to make sure which is healthy or gingivitis. Immunohistochemical stain was done using anti Gasdermin-D and inflammasome-NLRP-3 monoclonal antibodies. The interval data from 0 to 4 is analyzed to determine the differences in expression of Gasdermin D and inflammasome-NLRP-3 proteins between healthy pericorona and pericoronitis tissue. Results: The median of expression Gasdermin-D reflected in healthy gum and gingivitis are 1.75 : 3.125, while for Inflammasome-NLPR-3 are 2 : 3.625. Found differences between healthy gum and gingivitis both in Gasdermin D (p=0.001) and inflammasoeme-NLRP-3 (p=0,009). Conclusion: Pyroptosis was occurs in the gingivitis due to bacterial triggers by expressing Gasdermin-D and inflammasome-NLRP-3 proteins. [ABSTRACT FROM AUTHOR]

Abstract Inflammasome activation is associated with a myriad of inflammatory diseases. However, existing methods provides a limited understanding of spatiotemporal kinetics of inflammasome activation, with restricted scope for early detection of associated treatment efficacy. This limitation offers an opportunity for the development of biocompatible in‐vivo inflammasome monitoring tools with translational prospects. To achieve this, they report developing a pair of lipid‐based nanoparticle systems, a reporter nanoparticle consisting of a caspase‐1 activatable probe alone, and a theranostic nanoparticle combining the probe with an inflammasome‐inhibiting drug. This biocompatible platform enhances the probe's residence time in circulation by preventing its opsonization and allowing its sustained release over time. Their results demonstrate the specificity of reporter nanoparticles towards caspase‐1 activity and provides early‐on monitoring of inflammasome activation both in‐vitro as well as in‐vivo. Additionally, the delivery of disulfiram, an inflammasome‐inhibiting drug, along with reporter probe using theranostic nanoparticles enables real‐time tracking of treatment efficacy in the gouty‐arthritis inflammatory model. In summary, they report an unparalleled pair of the inflammasome‐associated reporter and theranostic platforms suited not only for diagnostic applications but can also detect inflammasome‐targeted treatment efficiency in real‐time. These findings establish two novel, sensitive nanotools for non‐invasive evaluation of inflammasome‐targeted immunotherapy.

Background: Pyroptosis, gasdermin-mediated programmed cell death, is readily induced in macrophages by activation of the canonical inflammasome (caspase-1) or by intracellular lipopolysaccharide (LPS)-mediated non-canonical inflammasome (caspase-11) activation. However, whether pyroptosis is induced similarly in hepatocytes is still largely controversial but highly relevant to liver pathologies such as alcoholic/nonalcoholic liver disease, drug-induced liver injury, ischemia-reperfusion and liver transplant injury, or organ damage secondary to sepsis. Methods and Results: In this study we found that hepatocytes activate and cleave gasdermin-D (GSDMD) at low levels after treatment with LPS. Overexpression of caspase-1 or caspase-11 p10/p20 activated domains was able to induce typical GSDMD-dependent pyroptosis in hepatocytes both in vitro and in vivo. However, morphologic features of pyroptosis in macrophages (eg, pyroptotic bodies, cell flattening, loss of cell structure) did not occur in pyroptotic hepatocytes, with cell structure remaining relatively intact despite the cell membrane being breached. Our results suggest that hepatocytes activate pyroptosis pathways and cleave GSDMD, but this does not result in cell rupture and confer the same pyroptotic morphologic changes as previously reported in macrophages. This is true even with caspase-1 or caspase-11 artificial overexpression way above levels seen endogenously even after priming or in pathologic conditions. Conclusions: Our novel findings characterize hepatocyte morphology in pyroptosis and suggest alternative use for canonical/non-canonical inflammasome activation/signaling and subsequent GSDMD cleavage because there is no rapid cell death as in macrophages. Improved understanding and recognition of the role of these pathways in hepatocytes may result in novel therapeutics for a range of liver diseases.

Pyroptosis is an inflammatory programed cell death process that plays a crucial role in cancer therapeutic, while Gasdermin-D is a critical effector protein for pyroptosis execution. This review discusses the intricate interactions between Gasdermin-D and some non-coding RNAs (lncRNA, miRNA, siRNA) and their potential application in the regulation of pyroptosis as an anticancer therapy. Correspondingly, these ncRNAs significantly implicate in Gasdermin-D expression and function regarding the pyroptosis pathway. Functioning as competing endogenous RNAs (ceRNAs), these ncRNAs might regulate Gasdermin-D at the molecular level, underlying fatal cell death caused by cancer and tumor propagation. Therefore, these interactions appeal to therapeutics, offering new avenues for cancer treatment. It address this research gap by discussing the possible roles of ncRNAs as mediators of gasdermin-D regulation. It suggest therapeutic strategies based on the current research findings to ensure the interchange between the ideal pyroptosis and cancer cell death. [Display omitted] [ABSTRACT FROM AUTHOR]

• The AIM2 inflammasome signaling pathway was upregulated in human ascending thoracic aortic dissection and was associated with smooth muscle cell pyroptosis. • Aim2 deficiency in mice prevents aortic dissection formation in both the thoracic and abdominal aorta. • Transfecting SMCs with AIM2 siRNA and GSDMD siRNA reduced dsDNA-induced SMC death. The absent in melanoma 2 (AIM2) inflammasome induces pyroptosis, tissue inflammation, and extracellular matrix destruction. We tested the hypothesis that the AIM2 inflammasome contributes to aortic aneurysm and dissection (AAD) development by promoting pyroptosis in smooth muscle cells (SMCs). We examined AIM2 expression in aortic tissues from patients with ascending thoracic aortic aneurysm (ATAA) and aortic dissection (ATAD) and from organ donor controls. AIM2's role in AAD development was evaluated in AIM2-deficient mice in a sporadic AAD model induced by challenging mice with a high-fat diet and angiotensin II infusion. The direct effects of dsDNA on SMC death in vitro were studied. Western blot analyses showed that AIM2 was increased in ATAD compared to ATAA and control tissue. Immunofluorescence demonstrated increased AIM2 in SMCs and macrophages in the aortic media and adventitia of dissected tissue. Increased AIM2 abundance was associated with increased cleavage of caspase-1 and cleavage of gasdermin-D, indicating activation of pyroptosis. In a mouse model of sporadic AAD induced by high-fat diet and angiotensin II infusion, AIM2-deficient mice showed significant reduction in aortic dissection, but not aneurysm formation in all aortic segments, versus wild-type mice. Finally, treating cultured human aortic SMCs with double-stranded DNA induced AIM2 expression, caspase-1 cleavage, and gasdermin-D cleavage; these effects were reduced by silencing AIM2 and caspase-1 genes, suggesting involvement of the AIM2 inflammasome in cytosolic DNA-induced activation of SMC pyroptosis. Activation of the AIM2 inflammasome cascade contributes to aortic degeneration and dissection, in part, by activating pyroptosis. [ABSTRACT FROM AUTHOR]

We analyzed actin cytoskeleton alterations during NET extrusion by neutrophil-like dHL-60 cells and human neutrophils in the absence of DNase1 containing serum to avoid chromatin degradation and microfilament disassembly. NET-formation by dHL-60 cells and neutrophils was induced by Ionomycin or phorbol-12-myristat-13-acetate (PMA). Subsequent staining with anti-actin and TRITC-phalloidin showed depolymerization of the cortical F-actin at spatially confined areas, the NET extrusion sites, effected by transient activation of the monooxygenase MICAL-1 supported by the G-actin binding proteins cofilin, profilin, thymosin ß4 and probably the F-actin fragmenting activity of gelsolin and/or its fragments, which also decorated the formed NETs. MICAL-1 itself appeared to be proteolyzed by neutrophil elastase possibly to confine its activity to the NET-extrusion area. The F-actin oxidization activity of MICAL-1 is inhibited by Levosimendan leading to reduced NET-formation. Anti-gasdermin-D immunohistochemistry showed a cytoplasmic distribution in non-stimulated cells. After stimulation the NET-extrusion pore displayed reduced anti-gasdermin-D staining but accumulated underneath the plasma membrane of the remaining cell body. A similar distribution was observed for myosin that concentrated together with cortical F-actin along the periphery of the remaining cell body suggesting force production by acto-myosin interactions supporting NET expulsion as indicated by the inhibitory action of the myosin ATPase inhibitor blebbistatin. Isolated human neutrophils displayed differences in their content of certain cytoskeletal proteins. After stimulation neutrophils with high gelsolin content preferentially formed "cloud"-like NETs, whereas those with low or no gelsolin formed long "filamentous" NETs. • Actin cytoskeleton alterations during NET extrusion by neutrophil-like dHL-60 cells and human neutrophils were analyzed in the absence of DNase1 containing serum to avoid chromatin degradation and microfilament disassembly. F-actin fragmenting activity by the monooxygenase MICAL-1 supported by the G-actin binding proteins gelsolin cofilin, profilin, and thymosin ß4 was identified to be essential for NET-extrusion. [ABSTRACT FROM AUTHOR]

The dynamic balance between cell proliferation, differentiation, and death regulates the homeostasis and pathological processes in multicellular organisms. Cell death was considered to be mainly in two types, apoptosis (programmed cell death) and necrosis. However, in recent years, several different ways of cell death are specified, one of which is pyroptosis. The newly discovered pyroptosis consists of a process mediated by the gasdermin family with an inflammatory and immune system response. The effects of pyroptosis on cancer are complex and may vary with the genetic structure. Pyroptosis inhibits tumor formation and development. On the other hand, it supports the microenvironment that is appropriate for tumor formation. Moreover, manipulations of pyroptosis in tumor cells may provide an alternative for cancer treatment. The present review is aimed to provide an overview of the current knowledge of signal transduction systems in pyroptosis, classical (canonical), non-classical pathways, the Gasdermin protein family, the pore-forming mechanism of this family, its relationship with cancers, and the manipulations of pyroptosis in treatment. [ABSTRACT FROM AUTHOR]

Gasdermin D (GSDMD) has been proven to be a key protein in the activation of pyroptosis. Pyroptosis of renal tubular epithelial cells contributes to the progression of tubular injury in kidney diseases. However, it remains elusive whether and how GSDMD is involved in the regulation of diabetic kidney disease (DKD). In this study, we found that tubular injury is accompanied by the up-regulation of Toll-like receptor 4 (TLR4) and GSDMD in patients with diabetic kidney disease. In addition, we discovered that the expressions of cleaved Caspase-1, active N-terminal fragments of GSDMD (GSDMD-NT), IL-18, and the secretion of IL-1β also increased in the kidneys of db/db mice. These changes were partially ameliorated following intraperitoneal injection of TAK-242, an inhibitor of TLR4. Similar results were observed in human tubular cells (HK-2) subjected to high-glucose (HG) conditions and treated with TAK-242 or parthenolide (inhibitor of NF-κB) by Western blot, Enzyme-linked immunosorbent assay (ELISA), and flow cytometry. These results indicated that TLR4/NF-κB signaling could induce GSDMD-mediated pyroptosis in tubular cells in DKD.

Objectives: To observe the effects on tyrosine hydroxylase (TH), α-synaptic nucleoprotein (α-syn), sirtuin 3 (Sirt3), NOD-like receptor 3 (NLRP3) and gasdermin-D (GSDMD) in the substantia nigra of midbrain after electroacupuncture (EA) at "Fengfu"(GV16), "Taichong" (LR3) and "Zusanli" (ST36) in rats of Parkinson's disease (PD), so as to explore the mechanism of EA in treatment of PD., Methods: SD rats were randomly divided into control, model and EA groups, with 10 rats in each group. The PD model was established by injecting rotenone into the neck and back, lasting 28 days. In the EA group, EA was applied to GV16, LR3 and ST36, 30 min each time, once daily, consecutively for 28 days. The open-field test was adopted to detect the total distance of autonomic movement of rats, and the pole climbing test was used to detect the body coordination ability of rats. In the substania nigra of midbrain, the positive expression of TH was determined using immunohistochemistry, the mRNA expression levels of α - syn, Sirt3, NLRP3 and GSDMD were detected by quantitative real-time fluorescence PCR, and the protein expression levels of NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and cysteinyl aspartate specific proteinase (Caspase)-1 were detected by Western blot., Results: Compared with the control group, the total distance of autonomous movement was decreased ( P <0.01) in the model group, and the score of pole climbing experiment was increased ( P <0.01)；in the midbrain substantia nigra the positive expression of TH was decreased ( P <0.01)；the mRNA expression level of Sirt3 was decreased ( P <0.01), and those of α-syn, NLRP3 and GSDMD were increased ( P <0.01)；while the protein expression levels of NLRP3, ASC and Caspase-1 were increased ( P <0.01). When compared with the model group, the total distance of autonomous movement in open field experiment was increased ( P <0.01) in the EA group and the score of pole climbing experiment was lower ( P <0.05)；in the midbrain substantia nigra the positive expression of TH was increased ( P <0.01)；the mRNA expression level of Sirt3 in the midbrain substantia nigra was increased ( P <0.01), and those of α-syn, NLRP3 and GSDMD were reduced ( P <0.01)；while the protein expression levels of NLRP3, ASC and Caspase-1 decreased ( P <0.01, P <0.05)., Conclusions: EA at "GV16" "LR3" and "ST36" can repair the neuronal injury, clear the abnormal accumulation of α-syn in the substania nigra of midbrain, and ameliorate mitochondrial damage in PD rats, which may be obtained by regulating Sirt3/NLRP3/GSDMD signaling pathway, so as to delay the occurrence and development of Parkinson's disease.

Disrupted mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation are often associated with macrophage pyroptosis. It remains unclear how these forms of mitochondrial dysfunction relate to inflammasome activation and gasdermin-D (Gsdmd) cleavage, two central steps of the pyroptotic process. Here, we also found MMP collapse and ROS generation induced by Nlrp3 inflammasome activation as previous studies reported. The elimination of ROS alleviated the cleavage of Gsdmd, suggesting that Gsdmd cleavage occurs downstream of ROS release. Consistent with this result, hydrogen peroxide treatment augmented the cleavage of Gsdmd by caspase-1. Indeed, four amino acid residues of Gsdmd were oxidized under oxidative stress in macrophages. The efficiency of Gsdmd cleavage by inflammatory caspase-1 was dramatically reduced when oxidative modification was blocked by mutation of these amino acid residues. These results demonstrate that Gsdmd oxidation serves as a de novo mechanism by which mitochondrial ROS promote Nlrp3 inflammasome-dependent pyroptotic cell death. [ABSTRACT FROM AUTHOR]

Gasdermin D (GSDMD) has been proven to be a key protein in the activation of pyroptosis. Pyroptosis of renal tubular epithelial cells contributes to the progression of tubular injury in kidney diseases. However, it remains elusive whether and how GSDMD is involved in the regulation of diabetic kidney disease (DKD). In this study, we found that tubular injury is accompanied by the up-regulation of Toll-like receptor 4 (TLR4) and GSDMD in patients with diabetic kidney disease. In addition, we discovered that the expressions of cleaved Caspase-1, active N-terminal fragments of GSDMD (GSDMD-NT), IL-18, and the secretion of IL-1β also increased in the kidneys of db/db mice. These changes were partially ameliorated following intraperitoneal injection of TAK-242, an inhibitor of TLR4. Similar results were observed in human tubular cells (HK-2) subjected to high-glucose (HG) conditions and treated with TAK-242 or parthenolide (inhibitor of NF-κB) by Western blot, Enzyme-linked immunosorbent assay (ELISA), and flow cytometry. These results indicated that TLR4/NF-κB signaling could induce GSDMD-mediated pyroptosis in tubular cells in DKD. [ABSTRACT FROM AUTHOR]

The immune system is armed with a broad range of receptors to detect and initiate the elimination of bacterial pathogens. Inflammasomes are molecular platforms that sense a diverse range of microbial insults to develop appropriate host response. In that context, noncanonical inflammasome arose as a sensor for Gram‐negative bacteria‐derived LPS leading to the control of infections. This review describes the role of caspase‐11/gasdermin‐D‐dependent immune response against Gram‐negative bacteria and presents an overview of guanylate‐binding proteins (GBPs) at the interface of noncanonical inflammasome activation. Indeed, caspase‐11 acts as a receptor for LPS and this interaction elicits caspase‐11 autoproteolysis that is required for its optimal catalytic activity. Gasdermin‐D is cleaved by activated caspase‐11 generating an N‐terminal domain that is inserted into the plasmatic membrane to form pores that induce pyroptosis, a cell death program involved in intracellular bacteria elimination. This mechanism also promotes IL‐1β release and potassium efflux that connects caspase‐11 to NLRP3 activation. Furthermore, GBPs display many features to allow LPS recognition by caspase‐11, initiating the noncanonical inflammasome response prompting the immune system to control bacterial infections. In this review, we discuss the recent findings and nuances related to this mechanism and its biological functions. [ABSTRACT FROM AUTHOR]

It has been shown that cyclic stretch could induce inflammatory response such as pyroptosis and the release of IL-1β in human periodontal ligament cells, through activating inflammasome and related caspases. Though gasdermin-d (GSDMD) has been reported to be present in some inflammatory diseases and function as a crucial executioner of pyroptosis, the role of GSDMD in the stretch-induced inflammatory response in human periodontal ligament cells (HPDLCs) has not been well clarified. In this study, it was found that GSDMD was activated by cyclic stretch, and its activation affected the pyroptotic rate in HPDLCs, leading to the maturation and secretion of IL-1β and IL-18 ultimately. In addition, GSDMD was found to be regulated by caspase-1 directly. Nevertheless, the exact relationship between inflammasomes and GSDMD in the stretch-induced inflammatory response still needs to be further elucidated. [ABSTRACT FROM AUTHOR]

Wound healing is a complex process that can leave pathological scars, especially in case of infections from opportunistic microorganisms. In this context, herbal medicines open up great possibilities for investigation. One of the species of interest native to Brazil is Garcinia brasiliensis ("bacupari"). Traditionally known for treating wounds and ulcers, G. brasiliensis presents anti-inflammatory, antioxidant and antimicrobials properties. But, its wound healing profile in experimental models, in order to validate its efficacy, is still litle studied. Thus, the objective of this work was to evaluate, in an infected cutanous wound model, the potential of formulations incorporated with G. brasiliensis leaves extracts. Crude extract (CE), Ethyl Acetate Fraction (EAF) and Hexanic Fraction (HF) were submitted to phytochemical assays, high performance thin layer chromatography (HTPLC) and cytotoxicity studies. CE and EAF were also tested for microbicidal properties and incorporated in cream and gel formulations at 10% concentration. After stability testing, the gel formulations with CE or EAF at 10% were selected and applied to skin wounds infected or not with Staphylococcus aureus in Wistar rats. The healing potenttial of the extracts was verified by the expression of the protein Annexin A1 (AnxA1), related to the processes of inflammation and antifibrotic function, the cells immunostaining for Gasdermin-D (GSDM-D), a marker of pyroptotic cell death, and the dosage of interleukin-10 (IL-10) and monocyte chemotactic protein (MCP)-1 inflammatory mediators. Phytochemical studies indicated the presence of compounds of pharmacological interest, including Catechin, Quercetin and Berberine in addition to low cytotoxicity of CE and EAF at 10%. After the 6-day topical treatments, CE and EAF gel formulations demonstrated to control the pruritus formation process. The treatments decreased AnxA1 expression and the amount of cells immunostained for GSDM-D, and increased the expression of MCP-1 in infected wounds. Together, the results show important anti-inflammatory profile and skin healing potential of CE and EAF from G. brasiliensis leaves, even in infected lesions, with therapeutic perspectives. [Display omitted] [ABSTRACT FROM AUTHOR]

Aflatoxin B1 (AFB1) disrupts the blood-brain barrier by poisoning the vascular endothelial cells and astrocytes that maintain it. It is important to examine whether aflatoxin B1 or its metabolite, aflatoxin M1 (AFM1), affect microglia, which as the "immune cells" in the brain may amplify their damaging effects. Here we evaluated the toxicity of AFB1 and AFM1 against primary microglia and found that both aflatoxins decreased the viability of primary microglia and increased the leakage of lactate dehydrogenase, gamma-H2AX expression, nuclear lysis, necrosis and apoptosis in a dose-dependent manner. The potential contribution of microglia to the toxic effects of aflatoxins was assessed in transwell co-culture experiments involving microglia, neurons, astrocytes, oligodendrocytes or neural stem/precursor cells. And we found that the toxic effects of both aflatoxins on various types of nervous system cells were greater in the presence of microglia than in their absence. We also found that both aflatoxins induced gasdermin D-mediated microglial pyroptosis and inflammatory cytokine expression by activating the NLRP3 inflammasome. Blockade of gasdermin D activity in AFB1- or AFM1-treated primary microglia using dimethyl fumarate (DMF) reduced the release of IL-1β, IL-18 and nitric oxide, as well as the neurotoxicity of microglia-conditioned medium to neurons, astrocytes, oligodendrocytes and neural stem/precursor cells. These data suggested that the toxicity of AFB1 and AFM1 on various cells of the central nervous system is due, remarkably, the gasdermin D-mediated microglial pyroptosis exacerbates their neurotoxicity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Inflammasomes are multimeric complexes that can sense pathogens and danger signals in the environment. Upon detection of stimuli, caspase-1 is recruited to the inflammasome complex that cleaves and activates pro-inflammatory cytokines, thus initiating a cascade of inflammatory events. While inflammasomes form a crucial component of the host response to pathogens and danger molecules, their unchecked activation can result in the development of autoimmune diseases, metabolic disorders, and pathological outcomes. This chapter describes some assays to detect the measurable outcomes of inflammasome formation and activation. The protocol describes the methods to study the inflammasome pathway using an in vitro assay in primary macrophages. It can be applied to studies investigating the pathway mechanisms and potential therapeutics in the form of inhibitors or activators., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Pyroptosis, gasdermin-mediated programmed cell death, is readily induced in macrophages by activation of the canonical inflammasome (caspase-1) or by intracellular lipopolysaccharide (LPS)-mediated non-canonical inflammasome (caspase-11) activation. However, whether pyroptosis is induced similarly in hepatocytes is still largely controversial but highly relevant to liver pathologies such as alcoholic/nonalcoholic liver disease, drug-induced liver injury, ischemia-reperfusion and liver transplant injury, or organ damage secondary to sepsis., Methods and Results: In this study we found that hepatocytes activate and cleave gasdermin-D (GSDMD) at low levels after treatment with LPS. Overexpression of caspase-1 or caspase-11 p10/p20 activated domains was able to induce typical GSDMD-dependent pyroptosis in hepatocytes both in vitro and in vivo. However, morphologic features of pyroptosis in macrophages (eg, pyroptotic bodies, cell flattening, loss of cell structure) did not occur in pyroptotic hepatocytes, with cell structure remaining relatively intact despite the cell membrane being breached. Our results suggest that hepatocytes activate pyroptosis pathways and cleave GSDMD, but this does not result in cell rupture and confer the same pyroptotic morphologic changes as previously reported in macrophages. This is true even with caspase-1 or caspase-11 artificial overexpression way above levels seen endogenously even after priming or in pathologic conditions., Conclusions: Our novel findings characterize hepatocyte morphology in pyroptosis and suggest alternative use for canonical/non-canonical inflammasome activation/signaling and subsequent GSDMD cleavage because there is no rapid cell death as in macrophages. Improved understanding and recognition of the role of these pathways in hepatocytes may result in novel therapeutics for a range of liver diseases., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Usnic acid (UA) is one of the well-known lichen metabolites that induces liver injury. It is mainly extracted from Usnea longissima and U. diffracta in China or from other lichens in other countries. U. longissima has been used as traditional Chinese medicine for treatment of cough, pain, indigestion, wound healing and infection. More than 20 incidences with hepatitis and liver failure have been reported by the US Food and Drug Administration since 2000. UA is an uncoupler of oxidative phosphorylation causing glutathione and ATP depletion. Previous histological studies observed extensive cell and organelle swellings accompanied with hydrotropic vacuolization of hepatocytes. This study was to investigate the mechanism of UA-induced liver toxicity in normal human L02 liver cells and ICR mice using various techniques, such as immunoblotting and siRNA transfection. Assays were performed to evaluate the oxidative stress and levels of GSH, MDA and SOD. Double flouresencence staining was used for the detection of apoptotic cell death. The protein expressions, such as glutathione S transferase, glutathione reductase, glutathione peroxidase 4, catalase, c-Jun N-terminal protein kinase, caspases, gastamin-D and porimin were detected by Western blotting. Comparisons between transfected and non-transfected cells were applied for the elucidation of the role of porimin in UA-induced hepatotoxicity. Histopathological examination of mice liver tissue, serum total bilirubin and hepatic enzymes of alanine aminotransferase and aspatate aminotransferase were also studied. The protein expressions of glutathione reductase, glutathione S transferase and glutathione peroxidase-4 were increased significantly in normal human L02 liver cells. Catalase expression was diminished in dose-dependent manner. Moreover, (+)-UA did not induce the activation of caspase-3, caspase-1 or gasdermin-D. No evidence showed the occurrence of pyroptosis. However, the porimin expressions were increased significantly. In addition, (+)-UA caused no cytotoxicity in the porimin silencing L02 cells. In conclusion, (+)-UA induces oncotic L02 cell death via increasing protein porimin and the formation of irreversible membrane pores. This may be the potential research area for future investigation in different aspects especially bioactivity and toxicology. Image 1 [ABSTRACT FROM AUTHOR]

The key to further improving outcomes in sepsis lies in understanding and abrogating the dysfunctional immune response that leads to organ failure. Activation of gasdermin-D, a pore-forming protein within the inflammasome cascade, has recently been recognized as the critical step in pyroptosis and organ dysfunction. In this study, we sought to investigate the presence of gasdermin-D in critically ill subjects., Design Setting and Patients: Prospective pilot study comparing microparticulate active gasdermin-D levels in critically ill patients admitted to the medical ICU at The Ohio State University Medical Center to healthy donors and clinical outcomes., Interventions: None., Measurements and Main Results: Plasma was collected from subjects upon consent and microparticles were isolated by ultracentrifugation. Proteins of interest were identified by immunoblot analysis of microparticle lysates. Quantification was accomplished by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD). Investigators were then unblinded and compared microparticulate active gasdermin-D levels to physician adjudicated clinical diagnoses and outcomes. No appreciable levels of active gasdermin-D were observed in microparticles from healthy volunteers and nonseptic critically ill patients. However, elevated levels of gasdermin-D were noted in microparticles from the septic cohort of critically ill patients. Furthermore, a significant positive correlation by linear regression was noted when microparticulate active gasdermin-D levels were compared with microparticulate levels of CD63, an exosomal marker, CD14, a monocyte marker, and CD69, a marker of monocyte activation ( R 2 = 0.37, p = 0.0011, R 2 = 0.85, p < 0.0001, and R 2 = 0.43, p = 0.0003, respectively)., Conclusions: This is the first study to demonstrate circulating active gasdermin-D in septic patients in the intensive care setting. Our findings also suggest that active gasdermin-D in septic patients is encapsulated in exosomes derived from activated monocytes. Further characterization in the clinical setting is warranted., Competing Interests: Supported, in part, by grants from National Institutes of Health (NIH) (RO1HL24325 to Dr. Sarkar and RO1GM108928 to Drs. Wewers and Sarkar). The remaining authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2019 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

As an essential part of programmed cell death, pyroptosis is an inflammatory response that is elicited upon infection by intracellular pathogens. Metabolic diseases, atherosclerosis and vital organ damage occur if pyroptosis is over-activated. Macrophages are the main cells that induce pyroptosis with the help of intracellular pattern-recognition receptors stimulated by danger signals and pathogenic microorganisms in the cytosol of host cells. Activated inflammatory caspases induce pyroptosis and produce pro-inflammatory cytokines, such as interleukin-1β and interleukin-18. Inflammatory programmed cell death is classified as canonical or non-canonical based on inflammatory caspases, which includes caspase-1 (in human and mouse) and caspase-11 (in mouse) or caspase-4 and -5 (in humans). Activated inflammatory caspases cleave the pore-forming effector protein, gasdermin-D, inducing osmotic pressure deregulation of internal fluids and subsequently rupturing the cell membranes. Inflammatory caspases could be attractive therapeutic targets for inflammatory bowel disease (IBD) in which pyroptosis may play an important role. This article reviews the current understanding of the mechanism of pyroptosis, focusing on the regulation of inflammatory caspases and therapeutic strategies for IBD.

The cells of the gastrointestinal (GI) epithelium are the first to contact the microbiota and food components. As a direct consequence of this, these cells are the first line of defence and key players in priming the immune response. One of the first responses against GI insults is the formation of the inflammasome, a multiprotein complex assembled in response to environmental threats. The formation of the inflammasome regulates caspase-1 by cleaving it into its active form. Once activated, caspase-1 can cleave interleukin-1β (IL-1β), which promotes adaptive and humoral immunity. Some strains, like Lactobacillus johnsonii N6.2, are able to modulate the biosynthesis of important host metabolites mediating inflammation. Of these metabolites are the pro-inflammatory kynurenines. L. johnsonii N6.2 is able to downregulate kynurenines biosynthesis via a redox active mechanism negatively affecting indoleamine 2,3-dioxygenase activity. In this study, we evaluated the effects of L. johnsonii N6.2 combined with the natural antioxidant and anti-inflammatory molecule rosmarinic acid (RA). Inflammasome assembly and the kynurenine pathway were evaluated in GI samples of BioBreeding diabetes-prone (BB-DP) rats. In this work, BB-DP rats were fed daily with RA, L. johnsonii N6.2; or both combined. The transcriptional rate and proteins levels of inflammasome and kynurenine pathway components in ileum tissue were evaluated. Elevated levels of pro-caspase-1 were observed in rats fed with L. johnsonii, while RA had no effect on pro-caspase-1 expression. Western blot assays demonstrated that L. johnsonii fed rats showed lower levels of mature caspase-1, when compared to the other treatments. Furthermore, IL-1β maturation followed a similar pattern across the treatments. Differences were also observed between treatments in expression levels of key enzymes in the kynurenine pathway. These findings support the role of L. johnsonii in modulating the assembly of the inflammasome as well as some steps of the pro-inflammatory kynurenine pathway.

Gasdermin-D (also known as GSDMD), the newly identified executioner of pyroptotic cell death, is cleaved by activated caspase-1 downstream of canonical inflammasome activation or caspase-4, 5, and 11 upon their ligation and activation by cytosolic LPS. Upon a single cleavage between the two domains in Gasdermin-D, the N-terminal domain binds to membrane lipids and lyses cells by forming pores of an inner diameter of 10-14 nm within the membrane. The inter-domain cleavage of Gasdermin-D is a reliable marker for the activation of inflammatory caspases and cell pyroptosis. Here, we describe the methods for examining Gasdermin-D cleavage by activated inflammatory caspases in vitro and upon inflammasome activation in vivo.