The biochemical pathways of apoptotic, necroptotic, pyroptotic, and ferroptotic cell death.
Molecular cell
Apoptosis, the first regulated form of cell death discovered in mammalian cells, is executed by caspase-3/7, which are dormant in living cells but become activated by upstream caspase-8 or caspase-9 in responding to extracellular cytokines or intracellular stress signals, respectively. The same cell death-inducing cytokines also cause necroptosis when caspase-8 is inhibited, resulting in the activation of receptor-interacting protein kinase 3 (RIPK3), which phosphorylates pseudokinase MLKL to trigger its oligomerization and membrane-disrupting activity. Caspase-1/4/5/11, known as inflammatory caspases, instead induce pyroptosis by cleaving gasdermin D, whose caspase-cleaved N terminus forms pores on the plasma membrane. The membrane protein NINJ1 amplifies the extent of membrane rupture initiated by gasdermin D. Additionally, disturbance of peroxidation of polyunsaturated fatty acid tails of membrane phospholipids triggers ferroptosis, an iron-dependent and caspases-independent necrotic death. This review will discuss how these regulated cell death pathways act individually and interconnectively in particular cell types to carry out specific physiological and pathological functions.
10.1016/j.molcel.2023.11.040
Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.
The Journal of biological chemistry
Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.
10.1016/j.jbc.2023.104792
Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis.
Newton Kim,Wickliffe Katherine E,Dugger Debra L,Maltzman Allie,Roose-Girma Merone,Dohse Monika,Kőműves László,Webster Joshua D,Dixit Vishva M
Nature
The aspartate-specific cysteine protease caspase-8 suppresses necroptotic cell death mediated by RIPK3 and MLKL. Indeed, mice that lack caspase-8 die in a RIPK3- and MLKL-dependent manner during embryogenesis. In humans, caspase-8 deficiency is associated with immunodeficiency or very early onset inflammatory bowel disease. The substrates that are cleaved by caspase-8 to prevent necroptosis in vivo have not been defined. Here we show that knock-in mice that express catalytically inactive caspase-8(C362A) die as embryos owing to MLKL-dependent necroptosis, similar to caspase-8-deficient mice. Thus, caspase-8 must cleave itself, other proteins or both to inhibit necroptosis. Mice that express caspase-8(D212A/D218A/D225A/D387A), which cannot cleave itself, were viable, as were mice that express c-FLIP or CYLD proteins that had been mutated to prevent cleavage by caspase-8. By contrast, mice that express RIPK1(D325A), in which the caspase-8 cleavage site Asp325 had been mutated, died mid-gestation. Embryonic lethality was prevented by inactivation of RIPK1, loss of TNFR1, or loss of both MLKL and the caspase-8 adaptor FADD, but not by loss of MLKL alone. Thus, RIPK1(D325A) appears to trigger cell death mediated by TNF, the kinase activity of RIPK1 and FADD-caspase-8. Accordingly, dying endothelial cells that contain cleaved caspase-3 were abnormally abundant in yolk sacs of Ripk1 embryos. Heterozygous Ripk1 cells and mice were viable, but were also more susceptible to TNF-induced cell death than were wild-type cells or mice. Our data show that Asp325 of RIPK1 is essential for limiting aberrant cell death in response to TNF, consistent with the idea that cleavage of RIPK1 by caspase-8 is a mechanism for dismantling death-inducing complexes.
10.1038/s41586-019-1548-x
FADD and Caspase-8 Regulate Gut Homeostasis and Inflammation by Controlling MLKL- and GSDMD-Mediated Death of Intestinal Epithelial Cells.
Schwarzer Robin,Jiao Huipeng,Wachsmuth Laurens,Tresch Achim,Pasparakis Manolis
Immunity
Pathways controlling intestinal epithelial cell (IEC) death regulate gut immune homeostasis and contribute to the pathogenesis of inflammatory bowel diseases. Here, we show that caspase-8 and its adapter FADD act in IECs to regulate intestinal inflammation downstream of Z-DNA binding protein 1 (ZBP1)- and tumor necrosis factor receptor-1 (TNFR1)-mediated receptor interacting protein kinase 1 (RIPK1) and RIPK3 signaling. Mice with IEC-specific FADD or caspase-8 deficiency developed colitis dependent on mixed lineage kinase-like (MLKL)-mediated epithelial cell necroptosis. However, MLKL deficiency fully prevented ileitis caused by epithelial caspase-8 ablation, but only partially ameliorated ileitis in mice lacking FADD in IECs. Our genetic studies revealed that caspase-8 and gasdermin-D (GSDMD) were both required for the development of MLKL-independent ileitis in mice with epithelial FADD deficiency. Therefore, FADD prevents intestinal inflammation downstream of ZBP1 and TNFR1 by inhibiting both MLKL-induced necroptosis and caspase-8-GSDMD-dependent pyroptosis-like death of epithelial cells.
10.1016/j.immuni.2020.04.002
RIPK1 and RIPK3: critical regulators of inflammation and cell death.
Newton Kim
Trends in cell biology
RIPK1 and RIPK3 (receptor-interacting serine/threonine protein kinases 1/3) interact by virtue of their RIP homotypic interaction motifs to mediate a form of cell death called necroptosis, although mice lacking these kinases have very different phenotypes. RIPK1-deficient mice die soon after birth, whereas RIPK3-deficient mice are healthy. Necroptosis involves cell rupture and is triggered by tumor necrosis factor (TNF), Toll-like receptors (TLRs), or the T cell receptor (TCR) when pro-apoptotic caspase-8 is inhibited. Various mouse models of disease are ameliorated by RIPK3 deficiency, suggesting that necroptosis contributes to pathology. Genetic rescue experiments now reveal why RIPK3-deficient are viable but RIPK1-deficient mice are not. These and other experiments indicate unexpected complexity in the regulation of both apoptosis and necroptosis by RIPK1 and RIPK3.
10.1016/j.tcb.2015.01.001
Sestrin2 maintains hepatic immune homeostasis and redox balance partially via inhibiting RIPK3-mediated necroptosis in metabolic dysfunction-associated steatohepatitis.
Molecular metabolism
BACKGROUND & AIMS:Necroptosis, a novel type of programmed cell death, is intricately associated with inflammatory response. Currently, most studies focus on the activation of necroptosis, while the mechanisms underlying the negative regulation of necroptosis remain poorly understood. METHODS:The effects of sestrin2 (SESN2) overexpression or knockdown on the regulation of necroptosis were assessed in the TNFα/Smac-mimetic/Z-VAD-FMK (T/S/Z)-induced necroptosis model and palmitic acid (PA)-induced lipotoxicity model. Western-blot, co-Immunoprecipitation, Glutathione S-transferase pull-down, and confocal assays were employed to explore the regulatory mechanisms including protein-protein interactions and post-translational modification. Furthermore, we used GSK'872, a specific inhibitor of receptor-interacting serine/threonine-protein kinase (RIPK) 3, to evaluate the relationship between SESN2-related alterations and RIPK3-mediated necroptosis in T/S/Z-induced necroptosis model, PA-induced lipotoxicity model, and high-fat high-cholesterol diet (HFHCD)-induced non-alcoholic steatohepatitis model. RESULTS:Our findings revealed that SESN2 was upregulated under conditions that induce necroptosis and functioned as a negative regulator of necroptosis. High levels of SESN2 could equipped hepatocytes with the ability to defend against necroptotic inflammation and oxidative stress. Mechanistically, SESN2 interacted with RIPK3 and tuned down necroptosis by inhibiting the phosphorylation of RIPK3, promoting the ubiquitination of RIPK3, and preventing the formation of the RIPK1/RIPK3 necrosome. The depletion of SESN2 resulted in excessive necroptosis, accompanied by increased fat accumulation, inflammation, and oxidative stress in the experimental steatohepatitis model. Blocking necroptosis by GSK'872 reduced the liberation of pro-inflammatory cytokines and reactive oxygen species generation, but not hepatocyte fat deposition, in both PA-treated SESN2 knockout cells and HFHCD-fed SESN2 knockout mice, suggesting that the activation of RIPK3-mediated necroptosis may partially account for the hyperinflammation and excessive oxidative stress induced by SESN2 deficiency. CONCLUSION:Our results suggested that SESN2 inhibited RIPK3-mediated necroptosis; this regulation is an important for the immune homeostasis and the redox balance in the liver.
10.1016/j.molmet.2023.101865
Ezetimibe ameliorates cisplatin-induced nephrotoxicity: A novel therapeutic approach via modulating AMPK/Nrf2/TXNIP signaling.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Cisplatin (Cis) is among the most powerful antineoplastic medications, nevertheless, its serious side effects; particularly nephrotoxicity designates a major concern. Previous studies reported that ezetimibe (Eze), a well-known antihyperlipidemic drug, exerts additional trivial pharmacological effects. In this work, we displayed Eze as an intriguing protective candidate in a cisplatin-induced nephrotoxicity rat model through AMPK activation. Eze (10 mg/kg, p.o.) was administered for two weeks and Cis (10 mg/kg, i.p.) was administered on the 10th day to induce nephrotoxicity in male Wistar rats. Treatment with Eze greatly augmented the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and the antioxidant regulator; nuclear factor erythroid 2-related factor 2 (Nrf2), thus, mitigating oxidative injury through induction of the antioxidant enzymes, such as heme oxygenase-1 (HO-1) and glutathione reductase (GR). As well, Eze relieved inflammation by reducing protein expression of thioredoxin-interacting protein (TXNIP) and nucleotide-binding domain-like receptor protein 3 (NLRP3), which led to a decrease in the release of caspase-1, in addition to, the inflammatory markers IL-18 and IL-1 β. Besides, Eze ameliorated apoptosis in the renal cells through inhibiting the phosphorylated Apoptosis signal-regulating kinase-1(p-ASK1), caspase-3 and reducing Bax/Bcl2ratio. Correspondingly, histopathological examination corroborated the previous biochemical findings. Collectively, Eze exerts significant renal protection against Cis-induced nephrotoxicity via antioxidant, anti-inflammatory and anti-apoptotic pathways that are probably mediated, at least partly, via activating AMPK/Nrf2/HO-1 pathway and conquering both TXNIP/NLRP3 inflammasome and TXNIP/ASK1 signaling pathways. To confirm the protective effect of Eze via AMPK-activation, an AMPK-inhibitor, dorsomorphin (Dors), when co-administered with Eze abolished its protective effect.
10.1096/fj.202302019R
Necroptosis, pyroptosis and apoptosis: an intricate game of cell death.
Cellular & molecular immunology
Cell death is a fundamental physiological process in all living organisms. Its roles extend from embryonic development, organ maintenance, and aging to the coordination of immune responses and autoimmunity. In recent years, our understanding of the mechanisms orchestrating cellular death and its consequences on immunity and homeostasis has increased substantially. Different modalities of what has become known as 'programmed cell death' have been described, and some key players in these processes have been identified. We have learned more about the intricacies that fine tune the activity of common players and ultimately shape the different types of cell death. These studies have highlighted the complex mechanisms tipping the balance between different cell fates. Here, we summarize the latest discoveries in the three most well understood modalities of cell death, namely, apoptosis, necroptosis, and pyroptosis, highlighting common and unique pathways and their effect on the surrounding cells and the organism as a whole.
10.1038/s41423-020-00630-3
Polystyrene microplastics lead to pyroptosis and apoptosis of ovarian granulosa cells via NLRP3/Caspase-1 signaling pathway in rats.
Hou Junyu,Lei Zhimin,Cui Linlu,Hou Yun,Yang Long,An Ru,Wang Qimeng,Li Shengda,Zhang Hongqin,Zhang Lianshuang
Ecotoxicology and environmental safety
Microplastics (MPs) considered as a new persistent environmental pollutant could enter into the circulatory system and result in decrease of sperm quantity and quality in mice. However, the effects of Polystyrene MPs (PS MPs) on the ovary and its mechanism in rats remained unclear. In this present study, thirty-two healthy female Wistar rats were exposed to different concentrations of 0.5 µm PS MPs dispersed in deionized water for 90 days. Using hematoxylin-eosin (HE) staining, the number of growing follicles was decreased compared to the control group. In addition, the activity of glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) were decreased while the expression level of malondialdehyde (MDA) was increased in ovary tissue. Confirmed by immunohistochemistry, the integrated optical density of NLRP3 and Cleaved-Caspase-1 had been elevated by 13.9 and 14 in granulosa cells in the 1.5 mg/kg/d group. Furthermore, compared to the control group, the level of AMH had been decreased by 23.3 pg/ml while IL-1β and IL-18 had been increased by 32 and 18.5 pg/ml in the 1.5 mg/kg/d group using the enzyme-linked immune sorbent assay (ELISA). Besides, the apoptosis of granulosa cells was elevated measured by terminal deoxyribonucleotide transferase-mediated nick end labeling (TUNEL) staining and flow cytometry. Moreover, western blot assays showed that the expressions of NLRP3/Caspase-1 signaling pathway related factors and Cleaved-Caspase-3 were increased. These results demonstrated that PS MPs could induce pyroptosis and apoptosis of ovarian granulosa cells via the NLRP3/Caspase-1 signaling pathway maybe triggered by oxidative stress. The present study suggested that exposure to microplastics had adverse effects on ovary and could be a potential risk factor for female infertility, which provided new insights into the toxicity of MPs on female reproduction.
10.1016/j.ecoenv.2021.112012
Green tea polyphenols alleviate TBBPA-induced gastric inflammation and apoptosis by modulating the ROS-PERK/IRE-1/ATF6 pathway in mouse models.
Food & function
Green tea polyphenols (GTP), an important phytochemical in the daily human diet, bind to various cellular receptors and exert anti-inflammatory and antioxidant benefits. The environmental contaminant tetrabromobisphenol A (TBBPA) enters the digestive system through multiple pathways, resulting in oxidative stress (OS), gastroenteritis, and mucosal injury. The aim of this study was to explore the molecular mechanisms of TBBPA-induced gastritis in mice treated with GTP and in an model. The results showed that exposure to TBBPA increased reactive oxygen species (ROS) levels, activated oxidative stress (OS) induced endoplasmic reticulum stress (ERS), and the expression of endoplasmic reticulum stress-related factors (, GRP78, PERK, IRE-1, ATF-6, .) increased. The inflammatory pathway NF-κB was activated, and the pro-inflammatory factors TNF-α, IL-1β, and IL-6 increased, while triggering a cascade reaction mediated by caspase-3. However, the addition of GTP could inhibit OS, restore the balance of endoplasmic reticulum homeostasis, and improve the inflammatory infiltration and apoptosis of gastric mucosal epithelial cells. Therefore, GTP alleviated ERS, reduced inflammation and apoptosis, and restored the gastric mucosal barrier by alleviating TBBPA-induced OS in mouse gastric tissues and GES-1 cells. This provides basic information for exploring the antioxidant mechanism of GTP and further investigating the toxic effects of TBBPA on mouse gastric mucosa.
10.1039/d4fo03012e
MMP-2 inhibition attenuates ER stress-mediated cell death during myocardial ischemia-reperfusion injury by preserving IRE1α.
Journal of molecular and cellular cardiology
Endoplasmic reticulum (ER) stress is one of the major events accompanying myocardial ischemia-reperfusion (IR) injury, as hypoxia and oxidative stress disrupt protein folding in the ER. As a result, the unfolded protein response (UPR) is activated through different sensors including inositol-requiring enzyme 1α (IRE1α) and protein kinase R-like ER kinase (PERK). Failure of the UPR to reduce ER stress induces cellular dysfunction. Matrix metalloproteinase-2 (MMP-2) is a ubiquitous protease that is activated intracellularly in response to oxidative stress and partially localizes near the ER. However, its role in ER homeostasis is unknown. We hypothesized that MMP-2 is involved in the regulation of the UPR and ER stress-mediated apoptosis during IR injury. Isolated mouse hearts subjected to IR injury showed impaired recovery of post-ischemic contractile function compared to aerobically perfused controls. Ventricular extracts from IR hearts had higher levels of glucose-regulated protein-78 and protein disulfide isomerase and lower levels of IRE1α and PERK compared to aerobic controls. MMP-2 inhibitors, ARP-100 or ONO-4817, given 10 min before ischemia, improved cardiac post-ischemic recovery and preserved IRE1α level in hearts subjected to 30 min ischemia/40 min reperfusion. IR also increased the levels of CHOP and mitochondrial Bax and caspase-3 and -9 activities, indicating induction of apoptosis, all of which were attenuated by MMP-2 inhibitors, regardless of the reperfusion time. Immunoprecipitation showed an association between MMP-2 and IRE1α in aerobic and IR hearts. During myocardial IR injury MMP-2 may impair the UPR and induce apoptosis by proteolysis of IRE1α. Inhibition of MMP-2 activity protects against cardiac contractile dysfunction in part by preserving IRE1α and preventing the progression to myocardial cell death.
10.1016/j.yjmcc.2024.11.013
DDIT3/CHOP promotes LPS/ATP-induced pyroptosis in osteoblasts via mitophagy inhibition.
Biochimica et biophysica acta. Molecular cell research
Inflammatory environments can trigger endoplasmic reticulum (ER) stress and lead to pyroptosis in various tissues and cells, including liver, brain, and immune cells. As a key factor of ER stress, DNA damage-inducible transcript 3 (DDIT3)/CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is upregulated in osteoblasts during inflammatory stimulation. DDIT3/CHOP may therefore regulate osteoblast pyroptosis in inflammatory conditions. During this investigation, we found that lipopolysaccharides (LPS)/adenosine 5'-triphosphate (ATP) stimulation in vitro induced osteoblasts to undergo pyroptosis, and the expression of DDIT3/CHOP was increased during this process. The overexpression of DDIT3/CHOP further promoted osteoblast pyroptosis as evidenced by the increased expression of the inflammasome NLR family pyrin domain containing 3 (NLRP3) and ratios of caspase-1 p20/caspase-1 and cleaved gasdermin D (GSDMD)/GSDMD. To explore the specific mechanism of this effect, we found through fluorescence imaging and Western blot analysis that LPS/ATP stimulation promoted PTEN-induced kinase 1 (PINK1)/E3 ubiquitin-protein ligase parkin (Parkin)-mediated mitophagy in osteoblasts, and this alteration was suppressed by the DDIT3/CHOP overexpression, resulting in increased ratio of pyroptosis compared with the control groups. The impact of DDIT3/CHOP on pyroptosis in osteoblasts was reversed by the application of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a specific mitophagy agonist. Therefore, our data demonstrated that DDIT3/CHOP promotes osteoblast pyroptosis by inhibiting PINK1/Parkin-mediated mitophagy in an inflammatory environment.
10.1016/j.bbamcr.2024.119712
The caspase-3/GSDME signal pathway as a switch between apoptosis and pyroptosis in cancer.
Jiang Mingxia,Qi Ling,Li Lisha,Li Yanjing
Cell death discovery
Apoptosis has long been recognized as a mechanism that kills the cancer cells by cytotoxic drugs. In recent years, studies have proved that pyroptosis can also shrink tumors and inhibit cells proliferation. Both apoptosis and pyroptosis are caspase-dependent programmed cell death pathways. Cysteinyl aspartate specific proteinase-3 (Caspase-3) is a common key protein in the apoptosis and pyroptosis pathways, and when activated, the expression level of tumor suppressor gene Gasdermin E (GSDME) determines the mechanism of tumor cell death. When GSDME is highly expressed, the active caspase-3 cuts it and releases the N-terminal domain to punch holes in the cell membrane, resulting in cell swelling, rupture, and death. When the expression of GSDME is low, it will lead to the classical mechanism of tumor cell death, which is apoptosis. More interestingly, researchers have found that GSDME can also be located upstream of caspase-3, connecting extrinsic, and intrinsic apoptotic pathways. Then, promoting caspase-3 activation, and forming a self-amplifying feed-forward loop. GSDME-mediated pyroptosis is correlated with the side effects of chemotherapy and anti-tumor immunity. This article mainly reviews the caspase-3/GSDME signal pathway as a switch between apoptosis and pyroptosis in cancer, to provide new strategies and targets for cancer treatment.
10.1038/s41420-020-00349-0
Caspase-11-Mediated Hepatocytic Pyroptosis Promotes the Progression of Nonalcoholic Steatohepatitis.
Cellular and molecular gastroenterology and hepatology
BACKGROUND:Nonalcoholic steatohepatitis (NASH) is an inflammatory disease with severe outcomes. Hepatocyte death, including apoptosis, necrosis, and pyroptosis, has been implicated in pathophysiology of NASH. Pyroptosis is mediated by inflammasome activation pathways including caspase-1-mediated canonical signaling pathway and caspase-11-mediated noncanonical signaling pathway. Until now, the precise role of caspase-11 in NASH remains unknown. In the present study, the potential roles of caspase-11 in NASH were explored. METHODS:We established methionine- and choline-deficient diet (MCD)-induced NASH mice model using wild-type caspase-11-deficient mice. The expression of caspase-11, liver injury, fibrosis, inflammation, and activation of gasdermin D and interleukin-1β were evaluated. RESULTS:Upregulated caspase-11 was detected in liver of mice with NASH. MCD-treated caspase-11-deficient mice had significantly decreased liver injury, fibrosis, and inflammation. The activation of gasdermin D and interleukin-1β was inhibited in caspase-11-deficient mice after MCD treatment. Overexpression of caspase-11 promoted steatohepatitis. CONCLUSIONS:Caspase-11-mediated hepatocytic pyroptosis promotes the progression of NASH.
10.1016/j.jcmgh.2021.04.009
Indole-3-propionic acid inhibits gut dysbiosis and endotoxin leakage to attenuate steatohepatitis in rats.
Experimental & molecular medicine
Microbial metabolites have emerged as critical components that mediate the metabolic effects of the gut microbiota. Here, we show that indole-3-propionic acid (IPA), a tryptophan metabolite produced by gut bacteria, is a potent anti-non-alcoholic steatohepatitis (NASH) microbial metabolite. Here, we demonstrate that administration of IPA modulates the microbiota composition in the gut and inhibits microbial dysbiosis in rats fed a high-fat diet. IPA induces the expression of tight junction proteins, such as ZO-1 and Occludin, and maintains intestinal epithelium homeostasis, leading to a reduction in plasma endotoxin levels. Interestingly, IPA inhibits NF-κB signaling and reduces the levels of proinflammatory cytokines, such as TNFα, IL-1β, and IL-6, in response to endotoxin in macrophages to repress hepatic inflammation and liver injury. Moreover, IPA is sufficient to inhibit the expression of fibrogenic and collagen genes and attenuate diet-induced NASH phenotypes. The beneficial effects of IPA on the liver are likely mediated through inhibiting the production of endotoxin in the gut. These findings suggest a protective role of IPA in the control of metabolism and uncover the gut microbiome and liver cross-talk in regulating the intestinal microenvironment and liver pathology via a novel dietary nutrient metabolite. IPA may provide a new therapeutic strategy for treating NASH.
10.1038/s12276-019-0304-5
Endotoxin tolerance ameliorates lipopolysaccharide/D-galactosamine-induced acute liver failure by negative regulation of the NF-κB/NLRP3 and activation of Nrf2/HO-1 via Sitr1.
International immunopharmacology
Acute liver failure (ALF) is a potentially fatal disorder characterized by extensive hepatocyte necrosis and rapid decline in liver function. Numerous factors, including oxidative stress, cell death, and inflammatory responses, are associated with its pathogenesis. Endotoxin tolerance (ET) refers to the phenomenon in which the body or cells exhibit low or no response to high-dose lipopolysaccharide (LPS) stimulation after pre-stimulation with low-dose LPS. However, the specific mechanism through which ET regulates LPS/D-galactosamine (D-GalN)-induced ALF remains unclear. An ALF mouse model was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (10 mg/kg). A low dose of LPS (0.1 mg/kg/d) was continuously administered to mice for 5 d before modeling to assess the protective effect of ET. The data from this study showed that ET alleviated the inflammatory response in mice with LPS/D-GalN-induced ALF. ET inhibited LPS-induced oxidative damage and pyroptosis in macrophages in vitro. RNA sequencing analysis showed that the NF-κB/NLRP3 pathway was linked to the anti-inflammatory and antioxidative effects of ET. Furthermore, using western blot, RT-qPCR, and immunofluorescence, we verified that ET inhibited the NF-κB/NLRP3 pathway and triggered the Nrf2/HO-1 signaling pathway to attenuate oxidative stress and cell pyroptosis. Sirt1 knockdown reversed this protective effect. In summary, our research elucidates that ET prevents ALF advancement by upregulating Sirt1 levels, triggering the Nrf2/HO-1 signaling axis, and suppressing the NF-κB/NLRP3 signaling cascade to inhibit oxidative stress and cell pyroptosis. Our results provide a mechanistic explanation for the protective effect of ET against ALF.
10.1016/j.intimp.2024.111994
Sodium Butyrate Ameliorates Oxidative Stress-Induced Intestinal Epithelium Barrier Injury and Mitochondrial Damage through AMPK-Mitophagy Pathway.
Li Xin,Wang Chunchun,Zhu Jiang,Lin Qian,Yu Minjie,Wen Jiashu,Feng Jie,Hu Caihong
Oxidative medicine and cellular longevity
Sodium butyrate has gained increasing attention for its vast beneficial effects. However, whether sodium butyrate could alleviate oxidative stress-induced intestinal dysfunction and mitochondrial damage of piglets and its underlying mechanism remains unclear. The present study used a hydrogen peroxide- (HO-) induced oxidative stress model to study whether sodium butyrate could alleviate oxidative stress, intestinal epithelium injury, and mitochondrial dysfunction of porcine intestinal epithelial cells (IPEC-J2) in AMPK-mitophagy-dependent pathway. The results indicated that sodium butyrate alleviated the HO-induced oxidative stress, decreased the level of reactive oxygen species (ROS), increased mitochondrial membrane potential (MMP), mitochondrial DNA (mtDNA), and mRNA expression of genes related to mitochondrial function, and inhibited the release of mitochondrial cytochrome c (Cyt c). Sodium butyrate reduced the protein expression of recombinant NLR family, pyrin domain-containing protein 3 (NLRP3) and fluorescein isothiocyanate dextran 4 kDa (FD4) permeability and increased transepithelial resistance (TER) and the protein expression of tight junction. Sodium butyrate increased the expression of light-chain-associated protein B (LC3B) and Beclin-1, reduced the expression of P62, and enhanced mitophagy. However, the use of AMPK inhibitor or mitophagy inhibitor weakened the protective effect of sodium butyrate on mitochondrial function and intestinal epithelium barrier function and suppressed the induction effect of sodium butyrate on mitophagy. In addition, we also found that after interference with AMPK, the protective effect of sodium butyrate on IPEC-J2 cells treated with HO was suppressed, indicating that AMPK is necessary for sodium butyrate to exert its protective effect. In summary, these results revealed that sodium butyrate induced mitophagy by activating AMPK, thereby alleviating oxidative stress, intestinal epithelium barrier injury, and mitochondrial dysfunction induced by HO.
10.1155/2022/3745135
Scutellarin ameliorates pulmonary fibrosis through inhibiting NF-κB/NLRP3-mediated epithelial-mesenchymal transition and inflammation.
Peng Ling,Wen Li,Shi Qing-Feng,Gao Feng,Huang Bin,Meng Jie,Hu Cheng-Ping,Wang Chang-Ming
Cell death & disease
Idiopathic pulmonary fibrosis (IPF) is featured with inflammation and extensive lung remodeling caused by overloaded deposition of extracellular matrix. Scutellarin is the major effective ingredient of breviscapine and its anti-inflammation efficacy has been reported before. Nevertheless, the impact of scutellarin on IPF and the downstream molecular mechanism remain unclear. In this study, scutellarin suppressed BLM-induced inflammation via NF-κB/NLRP3 pathway both in vivo and in vitro. BLM significantly elevated p-p65/p65 ratio, IκBα degradation, and levels of NLRP3, caspase-1, caspase-11, ASC, GSDMD, IL-1β, and IL-18, while scutellarin reversed the above alterations except for that of caspase-11. Scutellarin inhibited BLM-induced epithelial-mesenchymal transition (EMT) process in vivo and in vitro. The expression levels of EMT-related markers, including fibronectin, vimentin, N-cadherin, matrix metalloproteinase 2 (MMP-2) and MMP-9, were increased in BLM group, and suppressed by scutellarin. The expression level of E-cadherin showed the opposite changes. However, overexpression of NLRP3 eliminated the anti-inflammation and anti-EMT functions of scutellarin in vitro. In conclusion, scutellarin suppressed inflammation and EMT in BLM-induced pulmonary fibrosis through NF-κB/NLRP3 signaling.
10.1038/s41419-020-03178-2
The health benefits of dietary short-chain fatty acids in metabolic diseases.
Critical reviews in food science and nutrition
Short-chain fatty acids (SCFAs) are a subset of fatty acids that play crucial roles in maintaining normal physiology and developing metabolic diseases, such as obesity, diabetes, cardiovascular disease, and liver disease. Even though dairy products and vegetable oils are the direct dietary sources of SCFAs, their quantities are highly restricted. SCFAs are produced indirectly through microbial fermentation of fibers. The biological roles of SCFAs in human health and metabolic diseases are mainly due to their receptors, GPR41 and GPR43, FFAR2 and FFAR3. Additionally, it has been demonstrated that SCFAs modulate DNMTs and HDAC activities, inhibit NF-κB-STAT signaling, and regulate G(i/o)βγ-PLC-PKC-PTEN signaling and PPARγ-UCP2-AMPK autophagic signaling, thus mitigating metabolic diseases. Recent studies have uncovered that SCFAs play crucial roles in epigenetic modifications of DNAs, RNAs, and post-translational modifications of proteins, which are critical regulators of metabolic health and diseases. At the same time, dietary recommendations for the purpose of SCFAs have been proposed. The objective of the review is to summarize the most recent research on the role of dietary SCFAs in metabolic diseases, especially the signal transduction of SCFAs in metabolic diseases and their functional efficacy in different backgrounds and models of metabolic diseases, at the same time, to provide dietary and nutritional recommendations for using SCFAs as food ingredients to prevent metabolic diseases.
10.1080/10408398.2023.2297811
Free Fatty Acid Receptors (FFARs) in Adipose: Physiological Role and Therapeutic Outlook.
Cells
Fatty acids (FFAs) are important biological molecules that serve as a major energy source and are key components of biological membranes. In addition, FFAs play important roles in metabolic regulation and contribute to the development and progression of metabolic disorders like diabetes. Recent studies have shown that FFAs can act as important ligands of G-protein-coupled receptors (GPCRs) on the surface of cells and impact key physiological processes. Free fatty acid-activated receptors include FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), and FFAR4 (GPR120). FFAR2 and FFAR3 are activated by short-chain fatty acids like acetate, propionate, and butyrate, whereas FFAR1 and FFAR4 are activated by medium- and long-chain fatty acids like palmitate, oleate, linoleate, and others. FFARs have attracted considerable attention over the last few years and have become attractive pharmacological targets in the treatment of type 2 diabetes and metabolic syndrome. Several lines of evidence point to their importance in the regulation of whole-body metabolic homeostasis including adipose metabolism. Here, we summarize our current understanding of the physiological functions of FFAR isoforms in adipose biology and explore the prospect of FFAR-based therapies to treat patients with obesity and Type 2 diabetes.
10.3390/cells11040750
Free Fatty Acid Receptors in Health and Disease.
Kimura Ikuo,Ichimura Atsuhiko,Ohue-Kitano Ryuji,Igarashi Miki
Physiological reviews
Fatty acids are metabolized and synthesized as energy substrates during biological responses. Long- and medium-chain fatty acids derived mainly from dietary triglycerides, and short-chain fatty acids (SCFAs) produced by gut microbial fermentation of the otherwise indigestible dietary fiber, constitute the major sources of free fatty acids (FFAs) in the metabolic network. Recently, increasing evidence indicates that FFAs serve not only as energy sources but also as natural ligands for a group of orphan G protein-coupled receptors (GPCRs) termed free fatty acid receptors (FFARs), essentially intertwining metabolism and immunity in multiple ways, such as via inflammation regulation and secretion of peptide hormones. To date, several FFARs that are activated by the FFAs of various chain lengths have been identified and characterized. In particular, FFAR1 (GPR40) and FFAR4 (GPR120) are activated by long-chain saturated and unsaturated fatty acids, while FFAR3 (GPR41) and FFAR2 (GPR43) are activated by SCFAs, mainly acetate, butyrate, and propionate. In this review, we discuss the recent reports on the key physiological functions of the FFAR-mediated signaling transduction pathways in the regulation of metabolism and immune responses. We also attempt to reveal future research opportunities for developing therapeutics for metabolic and immune disorders.
10.1152/physrev.00041.2018
Short-chain fatty acids: linking diet, the microbiome and immunity.
Nature reviews. Immunology
The short-chain fatty acids (SCFAs) butyrate, propionate and acetate are microbial metabolites and their availability in the gut and other organs is determined by environmental factors, such as diet and use of antibiotics, that shape the diversity and metabolism of the microbiota. SCFAs regulate epithelial barrier function as well as mucosal and systemic immunity via evolutionary conserved processes that involve G protein-coupled receptor signalling or histone deacetylase activity. Indicatively, the anti-inflammatory role of butyrate is mediated through direct effects on the differentiation of intestinal epithelial cells, phagocytes, B cells and plasma cells, and regulatory and effector T cells. Intestinally derived SCFAs also directly and indirectly affect immunity at extra-intestinal sites, such as the liver, the lungs, the reproductive tract and the brain, and have been implicated in a range of disorders, including infections, intestinal inflammation, autoimmunity, food allergies, asthma and responses to cancer therapies. An ecological understanding of microbial communities and their interrelated metabolic states, as well as the engineering of butyrogenic bacteria may support SCFA-focused interventions for the prevention and treatment of immune-mediated diseases.
10.1038/s41577-024-01014-8
Bisphenol A exposure induces gut microbiota dysbiosis and consequent activation of gut-liver axis leading to hepatic steatosis in CD-1 mice.
Feng Dan,Zhang Hongmin,Jiang Xin,Zou Jun,Li Qingrong,Mai Haiyan,Su Dongfang,Ling Wenhua,Feng Xiang
Environmental pollution (Barking, Essex : 1987)
Interactions between the intestine and the liver, the so-called 'gut-liver axis', play a crucial role in the onset of hepatic steatosis and non-alcoholic fatty liver disease. However, not much is known about the impact of environmental pollutants on the gut-liver axis and consequent hepatic steatosis. Bisphenol A (BPA), a widely used plasticiser, is an important environmental contaminant that affects gut microbiota. We hypothesised that BPA induces hepatic steatosis by promoting gut microbiota dysbiosis and activating the gut-liver axis. In this study, male CD-1 mice were fed with diet containing BPA (50 μg/kg body weight/day) for 24 weeks. Dietary exposure to BPA increased lipid contents and fat accumulation in the liver. Analysis of 16 S rRNA gene sequencing revealed that the diversity of gut microbiota reduced and the composition of gut microbiota was altered in the BPA-fed mice. Further, the abundance of Proteobacteria, a marker of dysbacteria, increased, whereas the abundance of Akkermansia, a gut microbe associated with increased gut barrier function and reduced inflammation, markedly decreased. Expression levels of intestinal tight junction proteins (zona occludens-1 and occludin) also decreased drastically, leading to increased intestinal permeability and elevated levels of endotoxins. Furthermore, BPA up-regulated the expression of Toll-like receptor 4 (TLR4) and phosphorylation of nuclear factor-kappa B (NF-κB) in the liver and increased the production of inflammatory cytokines, including interleukin-1β, interleukin-18, tumour necrosis factor-α, and interleukin-6. Take together, our work indicated that dietary intake of BPA induced hepatic steatosis, and this was closely related to dysbiosis of gut microbiota, elevated endotoxin levels, and increased liver inflammation through the TLR4/NF-κB pathway.
10.1016/j.envpol.2020.114880
Combined bacterial translocation and cholestasis aggravates liver injury by activation pyroptosis in obstructive jaundice.
Heliyon
This study explores the mechanism by which obstructive jaundice (OJ) induces liver damage through pyroptosis. We induced OJ in rats via bile duct ligation and assessed liver damage using serum biochemical markers and histological analysis of liver tissue. Pyroptosis was investigated through immunofluorescence, ELISA, Western blot, and quantitative RT-PCR techniques. Additionally, we examined intestinal function and fecal microbiota alterations in the rats using 16S rDNA sequencing. In vitro experiments involved co-culturing Kupffer cells and hepatocytes, which were then exposed to bile and lipopolysaccharide (LPS). Our findings indicated that OJ modified the gut microbiota, increasing LPS levels, which, in conjunction with bile, initiated a cycle of inflammation, fibrosis, and cell death in the liver. Mechanistically, OJ elevated necrotic markers such as ATP, which in turn activated pyroptotic pathways. Increased levels of pyroptosis-related molecules, including NLRP3, caspase-1, gasdermin D, and IL-18, were confirmed. In our co-cultured cell model, bile exposure resulted in cell death and ATP release, leading to the activation of the NLRP3 inflammasome and its downstream effectors, caspase-1 and IL-18. The combination of bile and LPS significantly intensified pyroptotic responses. This study is the first to demonstrate that LPS and bile synergistically exacerbate liver injury by promoting necrosis and pyroptosis, unveiling a novel mechanism of OJ-associated hepatic damage and suggesting avenues for potential preventive or therapeutic interventions.
10.1016/j.heliyon.2024.e35793
Extract Alleviates NASH in Mice: Exploration of Inflammation and Gut Microbiota.
Nutrients
NASH (non-alcoholic steatohepatitis) is a severe liver disease characterized by hepatic chronic inflammation that can be associated with the gut microbiota. In this study, we explored the therapeutic effect of extract (GPE), a Chinese herbal extract, on methionine- and choline-deficient (MCD) diet-induced NASH mice. Based on the peak area, the top ten compounds in GPE were hydroxylinolenic acid, rutin, hydroxylinoleic acid, vanillic acid, methyl vanillate, quercetin, pheophorbide A, protocatechuic acid, aurantiamide acetate, and iso-rhamnetin. We found that four weeks of GPE treatment alleviated hepatic confluent zone inflammation, hepatocyte lipid accumulation, and lipid peroxidation in the mouse model. According to the 16S rRNA gene V3-V4 region sequencing of the colonic contents, the gut microbiota structure of the mice was significantly changed after GPE supplementation. Especially, GPE enriched the abundance of potentially beneficial bacteria such as and decreased the abundance of opportunistic pathogens such as . Moreover, RNA sequencing revealed that the GPE group showed an anti-inflammatory liver characterized by the repression of the NF-kappa B signaling pathway compared with the MCD group. Ingenuity Pathway Analysis (IPA) also showed that GPE downregulated the pathogen-induced cytokine storm pathway, which was associated with inflammation. A high dose of GPE (HGPE) significantly downregulated the expression levels of the tumor necrosis factor-α (TNF-α), myeloid differentiation factor 88 (Myd88), cluster of differentiation 14 (CD14), and Toll-like receptor 4 (TLR4) genes, as verified by real-time quantitative PCR (RT-qPCR). Our results suggested that the therapeutic potential of GPE for NASH mice may be related to improvements in the intestinal microenvironment and a reduction in liver inflammation.
10.3390/nu16111782
Acupuncture improved hepatic steatosis in HFD-induced NAFLD rats by regulating intestinal microbiota.
Frontiers in microbiology
Background:Intestinal dysbiosis has been increasingly implicated in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Acupuncture has been shown to have beneficial effects on NAFLD, but the mechanism is not yet clear. This study explores the potential beneficial effects of acupuncture on intestinal microbiota in NAFLD. Methods:An NAFLD model in Sprague Dawley rats was established using a high-fat diet (HFD) for 10 weeks. NAFLD rats were randomly divided into control, model, and acupuncture groups. Following acupuncture treatment over 6 weeks, automated biochemical analysis was used to measure serum lipid metabolism parameters, including levels of alanine transferase, aspartate transferase, alkaline phosphatase, total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. The level of serum inflammatory factors interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha (TNF-α) were measured by enzyme-linked immunosorbent assay. The characteristics of steatosis were evaluated using quantitative computed tomography, hematoxylin and eosin staining, and Oil Red O staining in the liver, while the intestinal microbiota was determined using 16S rRNA gene sequencing. Results:Acupuncture decreased the systemic inflammatory response, ameliorated dyslipidemia, and improved liver function indexes in NAFLD model rats. Tomography and staining indicated that acupuncture reduced steatosis and infiltration of inflammatory cells in the liver. 16S rRNA analysis showed that acupuncture reduced the Firmicutes to Bacteroidetes (F/B) ratio, increased the abundance of microbiota, including Bacteroidales_S24-7_group, Prevotellaceae, Bacteroidaceae, Blautia, norank_f_Bacteroidales_S24-7_group, Bacteroides, and Prevotella_9, and decreased the abundance of Ruminococcaceae_UCG-014. Correlation analysis suggested a close correlation between lipid metabolism, inflammation factors, hepatic steatosis, and the changed intestinal microbiota. Conclusion:Acupuncture can significantly improve lipid metabolism and the systemic inflammatory response in HFD-induced NAFLD rats, potentially by regulating intestinal microbiota composition.
10.3389/fmicb.2023.1131092
FEATURES OF INTESTINAL MICROBIOTA IN PATIENTS WITH NONALCOHOLIC FATTY LIVER DISEASE: EFFECTS ON MARKERS OF INFLAMMATION AND HEPATIC STEATOSIS.
Wiadomosci lekarskie (Warsaw, Poland : 1960)
OBJECTIVE:The aim: To study the state of the intestinal microbiota (ІМ) in patients with Nonalcoholic fatty liver disease (NAFLD) and to determine changes in its composition at the level of basic phylotypes. PATIENTS AND METHODS:Materials and methods: The study included 114 patients with NAFLD with metabolic disorders and 64 patients of control group. Determination of the composition of the ІМ at the level of major phylotypes was performed by identifying total bacterial DNA and DNA of Bacteroidetes, Firmicutes and Actinobacteria by quantitative polymerase chain reaction (PCR) in real time (qRT-PCR) using universal primers for the 16S rRNA gene and taxon-specific primers of production (Thermo Fisher Scientific). RESULTS:Results: It was defined the weak correlation between the content of Firmicutes and proinflammatory markers (C-reactive protein (CRP) and Tumor necrosis factor (TNF) alpha) (p <0.05) and inverse correlation of CRP with the content of Bacteroidetes (p <0.001). Also have been observed significant changes in the main intestinal phyla in the direction of increasing the content of Firmicutes in patients with NAFLD with a high degree of steatosis and elevated levels of proinflammatory cytokines (p <0.05). CONCLUSION:Conclusions: IM imbalance leads to excessive synthesis of pro-inflammatory cytokines, promotes the activation of cellular mechanisms, which increases the flow of fatty acids into hepatocytes and increases the degree of hepatic steatosis.
Gut-liver axis: Pathophysiological concepts and clinical implications.
Cell metabolism
Bidirectional crosstalk along the gut-liver axis controls gastrointestinal health and disease and exploits environmental and host mediators. Nutrients, microbial antigens, metabolites, and bile acids regulate metabolism and immune responses in the gut and liver, which reciprocally shape microbial community structure and function. Perturbation of such host-microbe interactions is observed in a variety of experimental liver diseases and is facilitated by an impaired intestinal barrier, which is fueling hepatic inflammation and disease progression. Clinical evidence describes perturbation of the gut-liver crosstalk in non-alcoholic fatty liver disease, alcoholic liver disease, and primary sclerosing cholangitis. In liver cirrhosis, a common sequela of these diseases, the intestinal microbiota and microbial pathogen-associated molecular patterns constitute liver inflammation and clinical complications, such as hepatic encephalopathy. Understanding the intricate metabolic interplay between the gut and liver in health and disease opens an avenue for targeted therapies in the future, which is probed in controlled clinical trials.
10.1016/j.cmet.2022.09.017
Host extracellular vesicles confer cytosolic access to systemic LPS licensing non-canonical inflammasome sensing and pyroptosis.
Nature cell biology
Intracellular surveillance for systemic microbial components during homeostasis and infections governs host physiology and immunity. However, a long-standing question is how circulating microbial ligands become accessible to intracellular receptors. Here we show a role for host-derived extracellular vesicles (EVs) in this process; human and murine plasma-derived and cell culture-derived EVs have an intrinsic capacity to bind bacterial lipopolysaccharide (LPS). Remarkably, circulating host EVs capture blood-borne LPS in vivo, and the LPS-laden EVs confer cytosolic access for LPS, triggering non-canonical inflammasome activation of gasdermin D and pyroptosis. Mechanistically, the interaction between the lipid bilayer of EVs and the lipid A of LPS underlies EV capture of LPS, and the intracellular transfer of LPS by EVs is mediated by CD14. Overall, this study demonstrates that EVs capture and escort systemic LPS to the cytosol licensing inflammasome responses, uncovering EVs as a previously unrecognized link between systemic microbial ligands and intracellular surveillance.
10.1038/s41556-023-01269-8
Gasdermin E-mediated target cell pyroptosis by CAR T cells triggers cytokine release syndrome.
Liu Yuying,Fang Yiliang,Chen Xinfeng,Wang Zhenfeng,Liang Xiaoyu,Zhang Tianzhen,Liu Mengyu,Zhou Nannan,Lv Jiadi,Tang Ke,Xie Jing,Gao Yunfeng,Cheng Feiran,Zhou Yabo,Zhang Zhen,Hu Yu,Zhang Xiaohui,Gao Quanli,Zhang Yi,Huang Bo
Science immunology
Cytokine release syndrome (CRS) counteracts the effectiveness of chimeric antigen receptor (CAR) T cell therapy in cancer patients, but the mechanism underlying CRS remains unclear. Here, we show that tumor cell pyroptosis triggers CRS during CAR T cell therapy. We find that CAR T cells rapidly activate caspase 3 in target cells through release of granzyme B. The latter cleaves gasdermin E (GSDME), a pore-forming protein highly expressed in B leukemic and other target cells, which results in extensive pyroptosis. Consequently, pyroptosis-released factors activate caspase 1 for GSDMD cleavage in macrophages, which results in the release of cytokines and subsequent CRS. Knocking out GSDME, depleting macrophages, or inhibiting caspase 1 eliminates CRS occurrence in mouse models. In patients, GSDME and lactate dehydrogenase levels are correlated with the severity of CRS. Notably, we find that the quantity of perforin/granzyme B used by CAR T cells rather than existing CD8 T cells is critical for CAR T cells to induce target cell pyroptosis.
10.1126/sciimmunol.aax7969
Protective CD8 T-cell response against Hantaan virus infection induced by immunization with designed linear multi-epitope peptides in HLA-A2.1/K transgenic mice.
Ma Ying,Tang Kang,Zhang Yusi,Zhang Chunmei,Cheng Linfeng,Zhang Fanglin,Zhuang Ran,Jin Boquan,Zhang Yun
Virology journal
BACKGROUND:An effective vaccine that prevents disease caused by hantaviruses is a global public health priority, but up to now, no vaccine has been approved for worldwide use. Therefore, novel vaccines with high prophylaxis efficacy are urgently needed. METHODS:Herein, we designed and synthesized Hantaan virus (HTNV) linear multi-epitope peptide consisting of HLA-A*02-restricted HTNV cytotoxic T cell (CTL) epitope and pan HLA-DR-binding epitope (PADRE), and evaluated the immunogenicity, as well as effectiveness, of multi-epitope peptides in HLA-A2.1/K transgenic mice with interferon (IFN)-γ enzyme-linked immunospot assay, cytotoxic mediator detection, proliferation assay and HTNV-challenge test. RESULTS:The results showed that a much higher frequency of specific IFN-γ-secreting CTLs, high levels of granzyme B production, and a strong proliferation capacity of specific CTLs were observed in splenocytes of mice immunized with multi-epitope peptide than in those of a single CTL epitope. Moreover, pre-immunization of multi-epitope peptide could reduce the levels of HTNV RNA loads in the liver, spleen and kidneys of mice, indicating that specific CTL responses induced by multi-epitope peptide could reduce HTNV RNA loads in vivo. CONCLUSIONS:This study may provide an important foundation for the development of novel peptide vaccines for HTNV prophylaxis.
10.1186/s12985-020-01421-y
Multimeric immunotherapeutic complexes activating natural killer cells towards HIV-1 cure.
Journal of translational medicine
BACKGROUND:Combination antiretroviral therapy (cART) has dramatically extended the life expectancy of people living with HIV-1 and improved their quality of life. There is nevertheless no cure for HIV-1 infection since HIV-1 persists in viral reservoirs of latently infected CD4 T cells. cART does not eradicate HIV-1 reservoirs or restore cytotoxic natural killer (NK) cells which are dramatically reduced by HIV-1 infection, and express the checkpoint inhibitors NKG2A or KIR2DL upregulated after HIV-1 infection. Cytotoxic NK cells expressing the homing receptor CXCR5 were recently described as key subsets controlling viral replication. METHODS:We designed and evaluated the potency of "Natural killer activating Multimeric immunotherapeutic compleXes", called as NaMiX, combining multimers of the IL-15/IL-15Rα complex with an anti-NKG2A or an anti-KIR single-chain fragment variable (scFv) to kill HIV-1 infected CD4 T cells. The oligomerization domain of the C4 binding protein was used to associate the IL-15/IL-15Rα complex to the scFv of each checkpoint inhibitor as well as to multimerize each entity into a heptamer (α form) or a dimer (β form). Each α or β form was compared in different in vitro models using one-way ANOVA and post-hoc Tukey's tests before evaluation in humanized NSG tg-huIL-15 mice having functional NK cells. RESULTS:All NaMiX significantly enhanced the cytolytic activity of NK and CD8 T cells against Raji tumour cells and HIV-1 ACH-2 cells by increasing degranulation, release of granzyme B, perforin and IFN-γ. Targeting NKG2A had a stronger effect than targeting KIR2DL due to higher expression of NKG2A on NK cells. In viral inhibition assays, NaMiX initially increased viral replication of CD4 T cells which was subsequently inhibited by cytotoxic NK cells. Importantly, anti-NKG2A NaMiX enhanced activation, cytotoxicity, IFN-γ production and CXCR5 expression of NK cells from HIV-1 positive individuals. In humanized NSG tg-huIL-15 mice, we confirmed enhanced activation, degranulation, cytotoxicity of NK cells, and killing of HIV-1 infected cells from mice injected with the anti-NKG2A.α NaMiX, as compared to control mice, as well as decreased total HIV-1 DNA in the lung. CONCLUSIONS:NK cell-mediated killing of HIV-1 infected cells by NaMiX represents a promising approach to support HIV-1 cure strategies.
10.1186/s12967-023-04669-4
Hepatocytes infected with hepatitis C virus change immunological features in the liver microenvironment.
Clinical and molecular hepatology
Hepatitis C virus (HCV) infection is remarkably efficient in establishing viral persistence, leading to the development of liver cirrhosis and hepatocellular carcinoma (HCC). Direct-acting antiviral agents (DAAs) are promising HCV therapies to clear the virus. However, recent reports indicate potential increased risk of HCC development among HCV patients with cirrhosis following DAA therapy. CD8+ T-cells participate in controlling HCV infection. However, in chronic hepatitis C patients, severe CD4+ and CD8+ T-cell dysfunctions have been observed. This suggests that HCV may employ mechanisms to counteract or suppress the host T-cell responses. The primary site of viral replication is within hepatocytes where infection can trigger the expression of costimulatory molecules and the secretion of immunoregulatory cytokines. Numerous studies indicate that HCV infection in hepatocytes impairs antiviral host immunity by modulating the expression of immunoregulatory molecules. Hepatocytes expressing whole HCV proteins upregulate the ligands of programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and transforming growth factor β (TGF-β) synthesis compared to those in hepatocytes in the absence of the HCV genome. Importantly, HCV-infected hepatocytes are capable of inducing regulatory CD4+ T-cells, releasing exosomes displaying TGF-β on exosome surfaces, and generating follicular regulatory T-cells. Recent studies report that the expression profile of exosome microRNAs provides biomarkers of HCV infection and HCV-related chronic liver diseases. A better understanding of the immunoregulatory mechanisms and identification of biomarkers associated with HCV infection will provide insight into designing vaccine against HCV to bypass HCV-induced immune dysregulation and prevent development of HCV-associated chronic liver diseases.
10.3350/cmh.2022.0032
HLA-A2 1 restricted peptides from the HBx antigen induce specific CTL responses in vitro and in vivo.
Hwang Yu Kyeong,Kim Nam Kyung,Park Jung Min,Lee Ki young,Han Won Kyo,Kim Hyung Il,Cheong Hong Seok
Vaccine
The HBx-derived, HLA-A2.1 restricted peptides, XEP-3, XEP-4, and XEP-6, induced activation of specific CTLs from patients with HBV in vitro. XEP-6 peptide induced the strongest response among the three peptides in CTLs from the blood samples of patients that were HBsAg positive. It was not clear whether the stage of disease (chronic infection, cirrhosis or hepatoma) was related to the responsiveness of the CTLs to each peptide. We vaccinated HLA-A2/K(b) transgenic mice with these peptides encapsulated in pH-sensitive liposomes at various concentrations and tested their ability to protect against challenge with rVV-HBx. Mice immunized with encapsulated peptides were protected against viral challenge whereas those immunized with empty liposomes were not. In general, 5 micro g of each peptide per head inoculation was sufficient to give protection after 2 weeks. After 3 weeks, this protective effect was increased. This effect of time was more important on the level of protection than the initial dose of the peptide. To explain the protective effect, IFN-gamma secreting CD8(+) cells isolated from mice 3 weeks after immunization were analyzed ex vivo. There was little dose dependency of peptide on IFN-gamma secretion except for XEP-3. The variations in the results may reflect the chemical properties of the peptides, such as solubility and binding affinity. In conclusion, epitope peptides derived from HBx can induce specific CTL activation and lead to cellular immunity in vitro and in vivo by inducing the peptide-specific CD8(+) CTLs. Thus, pH-sensitive liposomes increase the immune response following immunization with a peptide vaccine. This could be used for the treatment of HBV-related disease.
Effect of HBx on inflammation and mitochondrial oxidative stress in mouse hepatocytes.
Ling Li-Rong,Zheng Dan-Hua,Zhang Zhi-Yang,Xie Wen-Hui,Huang Yue-Hong,Chen Zhi-Xin,Wang Xiao-Zhong,Li Dan
Oncology letters
Hepatitis B virus × protein (HBx) serves an important role in the pathogenesis of the hepatitis B virus infection. Previous studies have reported that the interaction between HBx and hepatocyte mitochondria is an important factor leading to liver cell injury and apoptosis, ultimately inducing the formation of liver cancer. In the present study, a mouse model expressing HBx was constructed using hydrodynamic transfection based on the interaction between HBx and cytochrome oxidase (COX) subunit III. The specific mechanism of HBx-induced oxidative stress in mouse hepatocytes and the subsequent effect on mitochondrial function and inflammatory injury was assessed. The results demonstrated that HBx reduced the activity of COX and the expression of superoxide dismutase and upregulated the expression of malondialdehyde, NF-κB and phospho-AKT, thus increasing oxidative stress. In addition, HBx induced an increase in interleukin (IL)-6, IL-1β and IL-18 expression levels, which created an inflammatory microenvironment in the liver, further promoting hepatocyte inflammatory injury. Therefore, it was proposed that HBx may affect hepatocyte mitochondrial respiration by reducing the activity of cytochrome oxidase, leading to mitochondrial dysfunction and inducing hepatocyte inflammation and injury.
10.3892/ol.2020.11404
Hepatitis B Virus X Protein Induces Reactive Oxygen Species Generation via Activation of p53 in Human Hepatoma Cells.
Biomolecules
Hepatitis B virus (HBV), particularly through the HBx protein, induces oxidative stress during liver infections. This study reveals that HBx increases reactive oxygen species (ROS) via two distinct mechanisms. The first mechanism is p53-independent, likely involving mitochondrial dysfunction, as demonstrated by elevated ROS levels in p53-deficient Hep3B cells and p53-knocked-down HepG2 cells after HBx expression or HBV infection. The increase in ROS persisted even when p53 transcriptional activity was inhibited by pifithrin-α (PFT-α), a p53 inhibitor. The second mechanism is p53-dependent, wherein HBx activates p53, which then amplifies ROS production through a feedback loop involving ROS and p53. The ability of HBx to elevate ROS levels was higher in HepG2 than in Hep3B cells. Knocking down p53 in HepG2 cells lowered ROS levels, while ectopic p53 expression in Hep3B cells raised ROS. HBx-activated p53 downregulated catalase and upregulated manganese-dependent superoxide dismutase, contributing to ROS amplification. The transcriptional activity of p53 was crucial for these effects, as cells with a p53 R175H mutation or those treated with PFT-α generated less ROS. Additionally, HBx variants with Ser-101 increased p53 and ROS levels, whereas variants with Pro-101 did not. These dual mechanisms of HBx-induced ROS generation are likely significant in the pathogenesis of HBV and may contribute to liver diseases, including hepatocellular carcinoma.
10.3390/biom14101201
Dibutyl phthalate induces liver fibrosis via p38MAPK/NF-κB/NLRP3-mediated pyroptosis.
The Science of the total environment
Dibutyl phthalate (DBP) is one of the most employed plasticizers pervading the environment. DBP is a newly identified global organic pollutant that can activate NLRP3 inflammasomes and induce inflammatory liver injury. However, its hepatotoxicity remains poorly understood. The objective of this investigation was to investigate the probable pathways underlying DBP-induced liver injury. First, C57BL/6N mice were orally administered DBP at 10 and 50 mg/kg B.W. doses for 28 days. The observed results indicated a significant increase in liver collagen deposition and upregulated protein expression of fibrosis markers in mice. In addition, the p38MAPK/NF-κB signaling pathway and pyroptosis-related protein expression were upregulated. To establish a correlation between these changes, we conducted a conditioned medium co-culture of human hepatocellular carcinoma (HepG2) and human hepatic stellate (LX-2) cells. We performed inhibitor interventions to validate the mechanism of DBP-induced liver fibrosis in vitro. After treatment with p38MAPK (SB203580), NF-κB (PDTC), and NLRP3 (MCC950) inhibitors, the activation of LX-2 cells, the p38MAPK/NF-κB signaling pathway and pyroptosis due to DBP were alleviated. Therefore, DBP exposure leads to NLRP3-mediated pyroptosis of hepatocytes via the p38MAPK/NF-κB signaling pathway, activating LX-2 cells and causing liver fibrosis. Our findings offer a conceptual framework to understand the pathological underpinnings of DBP-induced liver injury while proposing novel ideas to prevent and treat DBP hepatotoxicity. Thus, targeting p38MAPK, NF-κB, and NLRP3 may prevent DBP-induced liver fibrosis.
10.1016/j.scitotenv.2023.165500
Naringenin attenuates non-alcoholic fatty liver disease by down-regulating the NLRP3/NF-κB pathway in mice.
British journal of pharmacology
BACKGROUND AND PURPOSE:Naringenin, a flavonoid compound with strong anti-inflammatory activity, attenuated non-alcoholic fatty liver disease (NAFLD) induced by a methionine-choline deficient (MCD) diet in mice. However, the mechanisms underlying this suppression of inflammation and NAFLD remain unknown. EXPERIMENTAL APPROACH:WT and NLRP3 mice were fed with MCD diet for 7 days to induce NAFLD and were given naringenin by gavage at the same time. in vitro experiments used HepG2 cells, primary hepatocytes, and Kupffer cells (KCs) stimulated by LPS or LPS plus oleic acid (OA). KEY RESULTS:Treating WT mice with naringenin (100 mg·kg ·day ) attenuated hepatic lipid accumulation and inflammation in the livers of mice given the MCD diet. NLRP3 mice showed less hepatic lipid accumulation than WT mice, but naringenin did not ameliorate hepatic lipid accumulation further in NLRP3 mice. Treating the HepG2 cells with naringenin or NLRP3 inhibitor MCC950 reduced lipid accumulation. Naringenin inhibited activation of the NLRP3/NF-κB pathway stimulated by OA together with LPS. In KCs isolated from WT mice, naringenin inhibited NLRP3 expression. Naringenin also inhibited lipid deposition, NLRP3 and IL-1β expression in WT hepatocytes but was not effective in NLRP3 hepatocytes. After re-expressing NLRP3 in NLRP3 hepatocytes by adenovirus, the anti-lipid deposition effect of naringenin was restored. CONCLUSION AND IMPLICATIONS:Naringenin prevented NAFLD via down-regulating the NLRP3/NF-κB signalling pathway both in KCs and in hepatocytes, thus attenuating inflammation in the mice livers.
10.1111/bph.14938
LncRNA NKILA inhibits HBV replication by repressing NF-κB signalling activation.
Virologica Sinica
Hepatitis B virus (HBV) infection results in liver cirrhosis and hepatocellular carcinoma (HCC). HBx/nuclear factor (NF)-κB pathway plays a role in HBV replication. However, whether NF-κB-interacting long noncoding RNA (NKILA), a suppressor of NF-κB activation, regulates HBV replication remains largely unknown. In this study, gain-and-loss experiments showed that NKILA inhibited HBV replication by inhibiting NF-κB activity. In turn, HBV infection down-regulated NKILA expression. In addition, expression levels of NKILA were lower in the peripheral blood-derived monocytes (PBMCs) of HBV-positive patients than in healthy individuals, which were correlated with HBV viral loads. And a negative correlation between NKILA expression level and HBV viral loads was observed in blood serum from HBV-positive patients. Lower levels of endogenous NKILA were also observed in HepG2 cells expressing a 1.3-fold HBV genome, HBV-infected HepG2-NTCP cells, stable HBV-producing HepG2.2.15 and HepAD38 cells, compared to those HBV-negative cells. Furthermore, HBx was required for NKILA-mediated inhibition on HBV replication. NKILA decreased HBx-induced NF-κB activation by interrupting the interaction between HBx and p65, whereas NKILA mutants lack of essential domains for NF-ĸB inhibition, lost the ability to inhibit HBV replication. Together, our data demonstrate that NKILA may serve as a suppressor of HBV replication via NF-ĸB signalling.
10.1016/j.virs.2023.10.002
Changing global epidemiology of liver cancer from 2010 to 2019: NASH is the fastest growing cause of liver cancer.
Cell metabolism
Liver cancer epidemiology is changing due to increasing alcohol consumption, rising prevalence of obesity, and advances in hepatitis B virus (HBV) and hepatitis C virus (HCV) treatment. However, the impact of these changes on global liver cancer burden remains unclear. We estimated global and regional temporal trends in the burden of liver cancer and the contributions of various liver disease etiologies using the methodology framework of the Global Burden of Disease study. Between 2010 and 2019, there was a 25% increase in liver cancer deaths. Age-standardized death rates (ASDRs) increased only in the Americas and remained stable or fell in all other regions. Between 2010 and 2019, non-alcoholic steatohepatitis (NASH) and alcohol had the fastest growing ASDRs, while HCV and HBV declined. Urgent measures are required at a global level to tackle underlying metabolic risk factors and slow the growing burden of NASH-associated liver cancer, especially in the Americas.
10.1016/j.cmet.2022.05.003
NAFLD and HBV interplay - related mechanisms underlying liver disease progression.
Frontiers in immunology
Non-alcoholic fatty liver disease (NAFLD) and Hepatitis B virus infection (HBV) constitute common chronic liver diseases with worldwide distribution. NAFLD burden is expected to grow in the coming decade, especially in western countries, considering the increased incidence of diabetes and obesity. Despite the organized HBV vaccinations and use of anti-viral therapies globally, HBV infection remains endemic and challenging public health issue. As both NAFLD and HBV have been associated with the development of progressive fibrosis, cirrhosis and hepatocellular carcinoma (HCC), the co-occurrence of both diseases has gained great research and clinical interest. The causative relationship between NAFLD and HBV infection has not been elucidated so far. Dysregulated fatty acid metabolism and lipotoxicity in NAFLD disease seems to initiate activation of signaling pathways that enhance pro-inflammatory responses and disrupt hepatocyte cell homeostasis, promoting progression of NAFLD disease to NASH, fibrosis and HCC and can affect HBV replication and immune encountering of HBV virus, which may further have impact on liver disease progression. Chronic HBV infection is suggested to have an influence on metabolic changes, which could lead to NAFLD development and the HBV-induced inflammatory responses and molecular pathways may constitute an aggravating factor in hepatic steatosis development. The observed altered immune homeostasis in both HBV infection and NAFLD could be associated with progression to HCC development. Elucidation of the possible mechanisms beyond HBV chronic infection and NAFLD diseases, which could lead to advanced liver disease or increase the risk for severe complications, in the case of HBV-NAFLD co-existence is of high clinical significance in the context of designing effective therapeutic targets.
10.3389/fimmu.2022.965548
Chronic hepatitis B with concurrent metabolic dysfunction-associated fatty liver disease: Challenges and perspectives.
Clinical and molecular hepatology
The prevalence of metabolic dysfunction-associated fatty liver disease (MAFLD) has increased among the general population and chronic hepatitis B (CHB) patients worldwide. Although fatty liver disease is a well-known risk factor for adverse liver outcomes like cirrhosis and hepatocellular carcinoma, its interactions with the hepatitis B virus (HBV) and clinical impacts seem complex. The presence of hepatic steatosis may suppress HBV viral activity, potentially leading to attenuated liver injury. In contrast, the associated co-morbidities like diabetes mellitus or obesity may increase the risk of developing adverse liver outcomes. These findings implicate that components of MAFLD may have diverse effects on the clinical manifestations of CHB. To this end, a clinical strategy is proposed for managing patients with concurrent CHB and MAFLD. This review article discusses the updated evidence regarding disease prevalence, interactions between steatosis and HBV, clinical impacts, and management strategies, aiming at optimizing holistic health care in the CHB population.
10.3350/cmh.2022.0422
Hepatitis B virus infection combined with nonalcoholic fatty liver disease: Interaction and prognosis.
Heliyon
Hepatitis B virus (HBV) infection is still one kind of the infectious diseases that seriously threaten human health. Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. HBV infection complicated with NAFLD is increasingly common. This review mainly describes the interaction between HBV infection and NAFLD, the interaction between steatosis and antiviral drugs, and the prognosis of HBV infection complicated with NAFLD. Most studies suggest that HBV infection may reduce the incidence of NAFLD. NAFLD can promote the spontaneous clearance of hepatitis B surface antigen (HBsAg), but whether it affects antiviral efficacy has been reported inconsistently. HBV infection combined with NAFLD can promote the progression of liver fibrosis, especially in patients with severe steatosis. The outcome of HBV infection combined with NAFLD predisposing to the progression of HCC remains controversial.
10.1016/j.heliyon.2023.e13113
Current perspectives of viral hepatitis.
World journal of gastroenterology
Viral hepatitis represents a major danger to public health, and is a globally leading cause of death. The five liver-specific viruses: Hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus, each have their own unique epidemiology, structural biology, transmission, endemic patterns, risk of liver complications, and response to antiviral therapies. There remain few options for treatment, in spite of the increasing prevalence of viral-hepatitis-caused liver disease. Furthermore, chronic viral hepatitis is a leading worldwide cause of both liver-related morbidity and mortality, even though effective treatments are available that could reduce or prevent most patients' complications. In 2016, the World Health Organization released its plan to eliminate viral hepatitis as a public health threat by the year 2030, along with a discussion of current gaps and prospects for both regional and global eradication of viral hepatitis. Today, treatment is sufficiently able to prevent the disease from reaching advanced phases. However, future therapies must be extremely safe, and should ideally limit the period of treatment necessary. A better understanding of pathogenesis will prove beneficial in the development of potential treatment strategies targeting infections by viral hepatitis. This review aims to summarize the current state of knowledge on each type of viral hepatitis, together with major innovations.
10.3748/wjg.v30.i18.2402
Dynamics and functions of lipid droplets.
Nature reviews. Molecular cell biology
Lipid droplets are storage organelles at the centre of lipid and energy homeostasis. They have a unique architecture consisting of a hydrophobic core of neutral lipids, which is enclosed by a phospholipid monolayer that is decorated by a specific set of proteins. Originating from the endoplasmic reticulum, lipid droplets can associate with most other cellular organelles through membrane contact sites. It is becoming apparent that these contacts between lipid droplets and other organelles are highly dynamic and coupled to the cycles of lipid droplet expansion and shrinkage. Importantly, lipid droplet biogenesis and degradation, as well as their interactions with other organelles, are tightly coupled to cellular metabolism and are critical to buffer the levels of toxic lipid species. Thus, lipid droplets facilitate the coordination and communication between different organelles and act as vital hubs of cellular metabolism.
10.1038/s41580-018-0085-z
Mechanism of inflammasomes in cancer and targeted therapies.
Frontiers in oncology
Inflammasomes, composed of the nucleotide-binding oligomerization domain(NOD)-like receptors (NLRs), are immune-functional protein multimers that are closely linked to the host defense mechanism. When NLRs sense pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), they assemble into inflammasomes. Inflammasomes can activate various inflammatory signaling pathways, including nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, and produce a large number of proinflammatory cytokines, which are closely associated with multiple cancers. They can also accelerate the occurrence and development of cancer by providing suitable tumor microenvironments, promoting tumor cell proliferation, and inhibiting tumor cell apoptosis. Therefore, the exploitation of novel targeted drugs against various inflammasomes and proinflammatory cytokines is a new idea for the treatment of cancer. In recent years, more than 50 natural extracts and synthetic small molecule targeted drugs have been reported to be in the research stage or have been applied to the clinic. Herein, we will overview the mechanisms of inflammasomes in common cancers and discuss the therapeutic prospects of natural extracts and synthetic targeted agents.
10.3389/fonc.2023.1133013
Natural compound Alternol actives multiple endoplasmic reticulum stress-responding pathways contributing to cell death.
Frontiers in pharmacology
Alternol is a small molecular compound isolated from the fermentation of a mutant fungus obtained from Taxus brevifolia bark. Our previous studies showed that Alternol treatment induced reactive oxygen species (ROS)-dependent immunogenic cell death. This study conducted a comprehensive investigation to explore the mechanisms involved in Alternol-induced immunogenic cell death. Prostate cancer PC-3, C4-2, and 22RV1 were used in this study. Alternol interaction with heat shock proteins (HSP) was determined using CETSA assay. Alternol-regulated ER stress proteins were assessed with Western blot assay. Extracellular adenosine triphosphate (ATP) was measured using ATPlite Luminescence Assay System. Our results showed that Alternol interacted with multiple cellular chaperone proteins and increased their expression levels, including endoplasmic reticulum (ER) chaperone hypoxia up-regulated 1 (HYOU1) and heat shock protein 90 alpha family class B member 1 (HSP90AB1), as well as cytosolic chaperone heat shock protein family A member 8 (HSPA8). These data represented a potential cause of unfolded protein response (UPR) after Alternol treatment. Further investigation revealed that Alternol treatment triggered ROS-dependent (ER) stress responses via R-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α). The double-stranded RNA-dependent protein kinase (PKR) but not activating transcription factor 6 (ATF6) cascades, leading to ATF-3/ATF-4 activation, C/EBP-homologous protein (CHOP) overexpression, and X-box binding protein XBP1 splicing induction. In addition, inhibition of these ER stress responses cascades blunted Alternol-induced extracellular adenosine triphosphate (ATP) release, one of the classical hallmarks of immunogenic cell death. Taken together, our data demonstrate that Alternol treatment triggered multiple ER stress cascades, leading to immunogenic cell death.
10.3389/fphar.2024.1397116
Tanshinone IIA alleviates NLRP3 inflammasome-mediated pyroptosis in Mycobacterium tuberculosis-(H37Ra-) infected macrophages by inhibiting endoplasmic reticulum stress.
Li Yinhong,Fu Yan,Sun Jinxia,Shen Jingjing,Liu Fanglin,Ning Bangzuo,Lu Zhenhui,Wei Luyao,Jiang Xin
Journal of ethnopharmacology
ETHNOPHARMACOLOGICAL RELEVANCE:Tanshinone IIA (Tan), extracted from Salvia miltiorrhiza Bunge, is a perennial herbal plant widely used as a folk remedy in Asian countries. Several studies have proved that Tanshinone IIA possesses many biological activities, such as anti-inflammatory, free-radical scavenging abilities, antioxidant properties, liver protection, and anti-cancer properties. AIM OF THE STUDY:The objective of the present study was to examine the anti-inflammatory effects of Tan. MATERIALS AND METHODS:The in vitro infection model of Mycobacterium tuberculosis-infected macrophages with the H37Ra strain was established. Murine macrophage Raw 264.7 and human monocyte THP-1 were used for the experiments. Cell viability was determined by the MTT assay. Western blot and lactate dehydrogenase (LDH) activity assays were used to detect the effects of Tan on cell pyroptosis and the level of NLRP3 inflammasome activation. Western blot, Co-immunoprecipitation and Immunofluorescence assays were used to observe the effect of Tan on the expression level of TXNIP. Immunofluorescence assays were applied to explore the effect of Tan on mtROS. Western blot and agarose gel electrophoresis were adopted to observe the effect of Tan on endoplasmic reticulum stress. The siRNA technique was applied to knockdown the expression levels of PERK/peIF2α, IRE1α and ATF6, and Western blot assay was employed to explore the NLRP3 inflammasome activation and possible molecular regulation mechanism of Tan. RESULTS:This study demonstrated that Tan decreased Mtb-induced cell pyroptosis by measuring GSDMD-N and LDH release provoked by NLRP3 inflammasome activation. Additionally, Tan inhibited endoplasmic reticulum stress (ERS), mitochondrial damage, and TXNIP protein expression, all of which acted as upstream signals of NLRP3 inflammasome activation in Mtb-infected macrophages. Significantly, NLRP3 inflammasome activation was suppressed by knocking down ERS pathway proteins, which further clarified that Tan partly targeted ERS to exert anti-inflammatory and immunoregulatory actions. CONCLUSION:This research confirms Tan's anti-inflammatory and immunoregulatory mechanisms in Mtb-infected macrophages by downregulating NLRP3 inflammasome activation-mediated pyroptosis provoked by ERS. Tan may function as an adjuvant drug to treat TB by adjusting host immune responses.
10.1016/j.jep.2021.114595
Endoplasmic reticulum stress related factor IRE1α regulates TXNIP/NLRP3-mediated pyroptosis in diabetic nephropathy.
Ke Ruiqiong,Wang Yan,Hong Shihua,Xiao Lixia
Experimental cell research
The nod-like receptor protein-3 (NLRP3)-mediated pyroptosis is involved in kidney diseases. Thioredoxin interacting protein (TXNIP) directly interacts with NLRP3. This study aimed to probe the mechanism of TXNIP and NLRP3 pathway in diabetic nephropathy (DN). Marker detection and histological staining indicated that in DN rats, the renal function was destroyed, and the TXNIP/NLRP3 axis was activated to induce inflammatory generation and pyroptosis. The protein levels of TXNIP, NLRP3 inflammatory components and endoplasmic reticulum stress (ERS)-related factors (ATF4, CHOP and IRE1α) were measured. DN rats were injected with LV-TXNIP-shRNA or IRE1α RNase specific inhibitor (STF-083010) to examine ERS- and pyroptosis-related proteins, and renal injury. Silencing TXNIP inhibited the NLRP3 axis and reduced renal damage in DN rats. ERS was activated in DN rats, and miR-200a expression was degraded by IRE1α. miR-200a bound to TXNIP. NRK-52E cells were induced by high glucose (HG) to simulate DN in vitro. The damage and pyroptosis of NRK-52E cells were analyzed. After inhibiting IRE1α, miR-200a expression increased and TXNIP expression decreased. miR-200a inhibition in HG-induced NRK-52E cells partially reversed the reduced pyroptosis by STF-083010. Overall, IRE1α upregulates miR-200a degradation in DN rats, and stimulates the TXINP/NLRP3 pathway-mediated pyroptosis and renal damage.
10.1016/j.yexcr.2020.112293
FXR Inhibits Endoplasmic Reticulum Stress-Induced NLRP3 Inflammasome in Hepatocytes and Ameliorates Liver Injury.
Han Chang Yeob,Rho Hyun Soo,Kim Ayoung,Kim Tae Hyun,Jang Kiseok,Jun Dae Won,Kim Jong Won,Kim Bumseok,Kim Sang Geon
Cell reports
Endoplasmic reticulum (ER) stress is associated with liver injury and fibrosis, and yet the hepatic factors that regulate ER stress-mediated inflammasome activation remain unknown. Here, we report that farnesoid X receptor (FXR) activation inhibits ER stress-induced NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome in hepatocytes. In patients with hepatitis B virus (HBV)-associated hepatic failure or non-alcoholic fatty liver disease, and in mice with liver injury, FXR levels in the liver inversely correlated with the extent of NLRP3 inflammasome activation. Fxr deficiency in mice augmented the ability of ER stress to induce NLRP3 and thioredoxin-interacting protein (TXNIP), whereas FXR ligand activation prevented it, ameliorating liver injury. FXR attenuates CCAAT-enhancer-binding protein homologous protein (CHOP)-dependent NLRP3 overexpression by inhibiting ER stress-mediated protein kinase RNA-like endoplasmic reticulum kinase (PERK) activation. Our findings implicate miR-186 and its target, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), in mediating the inhibition of ER stress by FXR. This study provides the insights on how FXR regulation of ER stress ameliorates hepatocyte death and liver injury and on the molecular basis of NLRP3 inflammasome activation.
10.1016/j.celrep.2018.07.068
Diabetes induces hepatocyte pyroptosis by promoting oxidative stress-mediated NLRP3 inflammasome activation during liver ischaemia and reperfusion injury.
Annals of translational medicine
BACKGROUND:Although diabetes mellitus has been reported to aggravate liver ischaemia and reperfusion (IR) injury, the basic mechanism remains largely unknown. The object of the present study was to determine the role of oxidative stress and hepatocellular pyroptosis in liver IR injury in diabetic mice. METHODS:Db/db and C57BL/6 mice at 8 weeks of age were subjected to liver IR injury. Liver injury and hepatocyte cell death were analyzed. A NOD-like receptor family pyrin domain-containing 3 protein (NLRP3) inflammasome antagonist (CY09) and a reactive oxygen species (ROS) antagonist (N-Acetyl-L-cysteine, NAC) were used to determine the role of ROS-mediated hepatocellular pyroptosis in diabetic mice post-IR. RESULTS:Aggravated liver IR injury was found in db/db mice compared to C57BL/6 control mice, as demonstrated by increased serum alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) levels, liver architecture damage and Suzuki scores. Interestingly, IR induces the pyroptosis of hepatocytes in db/db mice, as evidenced by enhanced NLRP3 inflammasome activation, increased numbers of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive hepatocytes and increased gene expression of interleukin-1β (IL-1β) and IL-18 in livers post-IR. The inhibitory effect of CY09, an NLRP3 antagonist, efficiently abrogated the exacerbation effects of diabetes on liver IR injury in db/db mice. Furthermore, increased ROS expression was detected in db/db mice compared to control mice after IR. ROS scavenging by NAC pretreatment markedly inhibited hepatocellular NLRP3 inflammasome activation and pyroptosis in the db/db mice post-IR, indicating that ROS play an essential role in mediating hepatocyte pyroptosis in the setting of diabetes mellitus. CONCLUSIONS:Our results demonstrate that diabetes induces hepatocyte pyroptosis by promoting oxidative stress-mediated NLRP3 inflammasome activation during liver IR injury. Strategies targeting ROS and NLRP3 inflammasome activation would be beneficial for preventing liver IR injury in diabetic patients.
10.21037/atm-20-1839
Activating Transcription Factor 6 Mediates Inflammatory Signals in Intestinal Epithelial Cells Upon Endoplasmic Reticulum Stress.
Stengel Stephanie T,Fazio Antonella,Lipinski Simone,Jahn Martin T,Aden Konrad,Ito Go,Wottawa Felix,Kuiper Jan W P,Coleman Olivia I,Tran Florian,Bordoni Dora,Bernardes Joana P,Jentzsch Marlene,Luzius Anne,Bierwirth Sandra,Messner Berith,Henning Anna,Welz Lina,Kakavand Nassim,Falk-Paulsen Maren,Imm Simon,Hinrichsen Finn,Zilbauer Matthias,Schreiber Stefan,Kaser Arthur,Blumberg Richard,Haller Dirk,Rosenstiel Philip
Gastroenterology
BACKGROUND & AIMS:Excess and unresolved endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) promotes intestinal inflammation. Activating transcription factor 6 (ATF6) is one of the signaling mediators of ER stress. We studied the pathways that regulate ATF6 and its role for inflammation in IECs. METHODS:We performed an RNA interference screen, using 23,349 unique small interfering RNAs targeting 7783 genes and a luciferase reporter controlled by an ATF6-dependent ERSE (ER stress-response element) promoter, to identify proteins that activate or inhibit the ATF6 signaling pathway in HEK293 cells. To validate the screening results, intestinal epithelial cell lines (Caco-2 cells) were transfected with small interfering RNAs or with a plasmid overexpressing a constitutively active form of ATF6. Caco-2 cells with a CRISPR-mediated disruption of autophagy related 16 like 1 gene (ATG16L1) were used to study the effect of ATF6 on ER stress in autophagy-deficient cells. We also studied intestinal organoids derived from mice that overexpress constitutively active ATF6, from mice with deletion of the autophagy related 16 like 1 or X-Box binding protein 1 gene in IECs (Atg16l1 or Xbp1, which both develop spontaneous ileitis), from patients with Crohn's disease (CD) and healthy individuals (controls). Cells and organoids were incubated with tunicamycin to induce ER stress and/or chemical inhibitors of newly identified activator proteins of ATF6 signaling, and analyzed by real-time polymerase chain reaction and immunoblots. Atg16l1 and control (Atg16l1) mice were given intraperitoneal injections of tunicamycin and were treated with chemical inhibitors of ATF6 activating proteins. RESULTS:We identified and validated 15 suppressors and 7 activators of the ATF6 signaling pathway; activators included the regulatory subunit of casein kinase 2 (CSNK2B) and acyl-CoA synthetase long chain family member 1 (ACSL1). Knockdown or chemical inhibition of CSNK2B and ACSL1 in Caco-2 cells reduced activity of the ATF6-dependent ERSE reporter gene, diminished transcription of the ATF6 target genes HSP90B1 and HSPA5 and reduced NF-κB reporter gene activation on tunicamycin stimulation. Atg16l1 and or Xbp1 organoids showed increased expression of ATF6 and its target genes. Inhibitors of ACSL1 or CSNK2B prevented activation of ATF6 and reduced CXCL1 and tumor necrosis factor (TNF) expression in these organoids on induction of ER stress with tunicamycin. Injection of mice with inhibitors of ACSL1 or CSNK2B significantly reduced tunicamycin-mediated intestinal inflammation and IEC death and expression of CXCL1 and TNF in Atg16l1 mice. Purified ileal IECs from patients with CD had higher levels of ATF6, CSNK2B, and HSPA5 messenger RNAs than controls; early-passage organoids from patients with active CD show increased levels of activated ATF6 protein, incubation of these organoids with inhibitors of ACSL1 or CSNK2B reduced transcription of ATF6 target genes, including TNF. CONCLUSIONS:Ileal IECs from patients with CD have higher levels of activated ATF6, which is regulated by CSNK2B and HSPA5. ATF6 increases expression of TNF and other inflammatory cytokines in response to ER stress in these cells and in organoids from Atg16l1 and Xbp1 mice. Strategies to inhibit the ATF6 signaling pathway might be developed for treatment of inflammatory bowel diseases.
10.1053/j.gastro.2020.06.088
Molecular mechanism of ATF6 in unfolded protein response and its role in disease.
Heliyon
Activating transcription factor 6 (ATF6), an important signaling molecule in unfolded protein response (UPR), plays a role in the pathogenesis of several diseases, including diseases such as congenital retinal disease, liver fibrosis and ankylosing spondylitis. After endoplasmic reticulum stress (ERS), ATF6 is activated after separation from binding immunoglobulin protein (GRP78/BiP) in the endoplasmic reticulum (ER) and transported to the Golgi apparatus to be hydrolyzed by site 1 and site 2 proteases into ATF6 fragments, which localize to the nucleus and regulate the transcription and expression of ERS-related genes. In these diseases, ERS leads to the activation of UPR, which ultimately lead to the occurrence and development of diseases by regulating the physiological state of cells through the ATF6 signaling pathway. Here, we discuss the evidence for the pathogenic importance of ATF6 signaling in different diseases and discuss preclinical results.
10.1016/j.heliyon.2024.e25937
Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease.
Lebeaupin Cynthia,Vallée Deborah,Hazari Younis,Hetz Claudio,Chevet Eric,Bailly-Maitre Béatrice
Journal of hepatology
The global epidemic of obesity has been accompanied by a rising burden of non-alcoholic fatty liver disease (NAFLD), with manifestations ranging from simple steatosis to non-alcoholic steatohepatitis, potentially developing into hepatocellular carcinoma. Although much attention has focused on NAFLD, its pathogenesis remains largely obscure. The hallmark of NAFLD is the hepatic accumulation of lipids, which subsequently leads to cellular stress and hepatic injury, eventually resulting in chronic liver disease. Abnormal lipid accumulation often coincides with insulin resistance in steatotic livers and is associated with perturbed endoplasmic reticulum (ER) proteostasis in hepatocytes. In response to chronic ER stress, an adaptive signalling pathway known as the unfolded protein response is triggered to restore ER proteostasis. However, the unfolded protein response can cause inflammation, inflammasome activation and, in the case of non-resolvable ER stress, the death of hepatocytes. Experimental data suggest that the unfolded protein response influences hepatic tumour development, aggressiveness and response to treatment, offering novel therapeutic avenues. Herein, we provide an overview of the evidence linking ER stress to NAFLD and discuss possible points of intervention.
10.1016/j.jhep.2018.06.008
MiR-149 attenuates endoplasmic reticulum stress-induced inflammation and apoptosis in nonalcoholic fatty liver disease by negatively targeting ATF6 pathway.
Chen Zhiyuan,Liu Yaling,Yang Li,Liu Peng,Zhang Yu,Wang Xiangyang
Immunology letters
This study aimed to research the effect of miR-149 on endoplasmic reticulum stress (ERS)-induced inflammation and apoptosis in non-alcoholic fatty liver disease (NAFLD). The mouse model with NAFLD was established by feeding with a high-fat diet, and the model establishment was subsequently confirmed by H&E staining and oil red O staining. MiR-149 agomir was injected into NAFLD mice to observe changes in liver tissues. After cell transfection, qRT-PCR and Western blot were performed to measure the expressions of lipid metabolism-related proteins (SCD-1, PPARα, and ABCA1), miR-149 and ATF6. Luciferase reporter gene assay was applied to verify the relationship between miR-149 and ATF6. Inflammatory factors (TNF-α, IL-1β, IL-6 and NF-κB) and apoptotic-related factors (caspase-12 and CHOP) were measured by ELISA and flow cytometry. qRT-PCR and Western blot were applied to detect expressions of ATF6 signaling pathway-related proteins (GRP94 and Akt). NAFLD progression was attenuated in mice injected with miR-149 agomir. The expression of miR-149 was reduced in liver tissues of NAFLD mice, while the expression of ATF6 was increased. Transfection of miR-149 can result in a decrease of ATF6 expression. ATF6 was a target gene of miR-149. MiR-149 could down-regulate the expressions of inflammatory factors and apoptotic-related factors. MiR-149 could down-regulate expressions of ATF6 signaling pathway-related proteins. MiR-149 alleviates ERS-induced inflammation and apoptosis by down-regulating the ATF6 signaling pathway, thus inhibiting the progression of NAFLD.
10.1016/j.imlet.2020.03.003
TSG-6 inhibits hypertrophic scar fibroblast proliferation by regulating IRE1α/TRAF2/NF-κB signalling.
International wound journal
TNF-stimulated gene (TSG-6) was reported to suppress hypertrophic scar (HS) formation in a rabbit ear model, and the overexpression of TSG-6 in human HS fibroblasts (HSFs) was found to induce their apoptotic death. The molecular basis for these findings, however, remains to be clarified. HSFs were subjected to TSG-6 treatment. Treatment with TSG-6 significantly suppressed HSF proliferation and induced them to undergo apoptosis. Moreover, TSG-6 exposure led to reductions in collagen I, collagen III, and α-SMA mRNA and protein levels, with a corresponding drop in proliferating cell nuclear antigen (PCNA) expression indicative of impaired proliferative activity. Endoplasmic reticulum (ER) stress was also suppressed in these HSFs as demonstrated by decreases in Bip and p-IRE1α expression, downstream inositol requiring enzyme 1 alpha (IRE1α) -Tumor necrosis factor receptor associated factor 2 (TRAF2) pathway signalling was inhibited and treated cells failed to induce NF-κB, TNF-α, IL-1β, and IL-6 expression. Overall, ER stress was found to trigger inflammatory activity in HSFs via the IRE1α-TRAF2 axis, as confirmed with the specific inhibitor of IRE1α STF083010. Additionally, the effects of TSG-6 on apoptosis, collagen I, collagen III, α-SMA, and PCNA of HSFs were reversed by the IRE1α activator thapsigargin (TG). These data suggest that TSG-6 administration can effectively suppress the proliferation of HSFs in part via the inhibition of IRE1α-mediated ER stress-induced inflammation (IRE1α/TRAF2/NF-κB signalling).
10.1111/iwj.13950
Astragaloside IV Alleviates Podocyte Injury in Diabetic Nephropathy through Regulating IRE-1α/NF-κ B/NLRP3 Pathway.
Chinese journal of integrative medicine
OBJECTIVE:To investigate the effects of astragaloside IV (AS-IV) on podocyte injury of diabetic nephropathy (DN) and reveal its potential mechanism. METHODS:In in vitro experiment, podocytes were divided into 4 groups, normal, high glucose (HG), inositol-requiring enzyme 1 (IRE-1) α activator (HG+thapsigargin 1 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups. Additionally, podocytes were divided into 4 groups, including normal, HG, AS-IV (HG+AS-IV 20 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups, respectively. After 24 h treatment, the morphology of podocytes and endoplasmic reticulum (ER) was observed by electron microscopy. The expressions of glucose-regulated protein 78 (GRP78) and IRE-1α were detected by cellular immunofluorescence. In in vivo experiment, DN rat model was established via a consecutive 3-day intraperitoneal streptozotocin (STZ) injections. A total of 40 rats were assigned into the normal, DN, AS-IV [AS-IV 40 mg/(kg·d)], and IRE-1α inhibitor [STF-083010, 10 mg/(kg·d)] groups (n=10), respectively. The general condition, 24-h urine volume, random blood glucose, urinary protein excretion rate (UAER), urea nitrogen (BUN), and serum creatinine (SCr) levels of rats were measured after 8 weeks of intervention. Pathological changes in the renal tissue were observed by hematoxylin and eosin (HE) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expressions of GRP78, IRE-1α, nuclear factor kappa Bp65 (NF-κBp65), interleukin (IL)-1β, NLR family pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), and nephrin at the mRNA and protein levels in vivo and in vitro, respectively. RESULTS:Cytoplasmic vacuolation and ER swelling were observed in the HG and IRE-1α activator groups. Podocyte morphology and ER expansion were improved in AS-IV and IRE-1α inhibitor groups compared with HG group. Cellular immunofluorescence showed that compared with the normal group, the fluorescence intensity of GRP78 and IRE-1α in the HG and IRE-1α activator groups were significantly increased whereas decreased in AS-IV and IRE-1α inhibitor groups (P<0.05). Compared with the normal group, the mRNA and protein expressions of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N in the HG group was increased (P<0.05). Compared with HG group, the expression of above indices was decreased in the AS-IV and IRE-1α inhibitor groups, and the expression in the IRE-1α activator group was increased (P<0.05). The expression of nephrin was decreased in the HG group, and increased in AS-IV and IRE-1α inhibitor groups (P<0.05). The in vivo experiment results revealed that compared to the normal group, the levels of blood glucose, triglyceride, total cholesterol, BUN, blood creatinine and urinary protein in the DN group were higher (P<0.05). Compared with DN group, the above indices in AS-IV and IRE-1α inhibitor groups were decreased (P<0.05). HE staining revealed glomerular hypertrophy, mesangial widening and mesangial cell proliferation in the renal tissue of the DN group. Compared with the DN group, the above pathological changes in renal tissue of AS-IV and IRE-1α inhibitor groups were alleviated. Quantitative RT-PCR and Western blot results of GRP78, IRE-1α, NF-κ Bp65, IL-1β, NLRP3, caspase-1 and GSDMD-N were consistent with immunofluorescence analysis. CONCLUSION:AS-IV could reduce ERS and inflammation, improve podocyte pyroptosis, thus exerting a podocyte-protective effect in DN, through regulating IRE-1α/NF-κ B/NLRP3 signaling pathway.
10.1007/s11655-024-3568-0
Modulation of endoplasmic reticulum stress via sulforaphane-mediated AMPK upregulation against nonalcoholic fatty liver disease in rats.
Cell stress & chaperones
Nonalcoholic fatty liver disease (NAFLD) is a major health concern. Endoplasmic reticulum (ER) stress, inflammation, and metabolic dysfunctions may be targeted to prevent the progress of nonalcoholic fatty liver disease. Sulforaphane (SFN), a sulfur-containing compound that is abundant in broccoli florets, seeds, and sprouts, has been reported to have beneficial effects on attenuating metabolic diseases. In light of this, the present study was designed to elucidate the mechanisms by which SFN ameliorated ER stress, inflammation, lipid metabolism, and insulin resistance - induced by a high-fat diet and ionizing radiation (IR) in rats. In our study, the rats were randomly divided into five groups: control, HFD, HFD + SFN, HFD + IR, and HFD + IR + SFN groups. After the last administration of SFN, liver and blood samples were taken. As a result, the lipid profile, liver enzymes, glucose, insulin, IL-1β, adipokines (leptin and resistin), and PI3K/AKT protein levels, as well as the mRNA gene expression of ER stress markers (IRE-1, sXBP-1, PERK, ATF4, and CHOP), fatty acid synthase (FAS), peroxisome proliferator-activated receptor-α (PPAR-α). Interestingly, SFN treatment modulated the levels of proinflammatory cytokine including IL-1β, metabolic indices (lipid profile, glucose, insulin, and adipokines), and ER stress markers in HFD and HFD + IR groups. SFN also increases the expression of PPAR-α and AMPK genes in the livers of HFD and HFD + IR groups. Meanwhile, the gene expression of FAS and CHOP was significantly attenuated in the SFN-treated groups. Our results clearly show that SFN inhibits liver toxicity induced by HFD and IR by ameliorating the ER stress events in the liver tissue through the upregulation of AMPK and PPAR-α accompanied by downregulation of FAS and CHOP gene expression.
10.1007/s12192-022-01286-w
Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming.
Nature chemical biology
Activation of the IRE1/XBP1s signaling arm of the unfolded protein response (UPR) is a promising strategy to correct defects in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. However, no pharmacologic activators of this pathway identified to date are suitable for ER proteostasis remodeling through selective activation of IRE1/XBP1s signaling. Here, we use high-throughput screening to identify non-toxic compounds that induce ER proteostasis remodeling through IRE1/XBP1s activation. We employ transcriptional profiling to stringently confirm that our prioritized compounds selectively activate IRE1/XBP1s signaling without activating other cellular stress-responsive signaling pathways. Furthermore, we demonstrate that our compounds improve ER proteostasis of destabilized variants of amyloid precursor protein (APP) through an IRE1-dependent mechanism and reduce APP-associated mitochondrial toxicity in cellular models. These results establish highly selective IRE1/XBP1s activating compounds that can be widely employed to define the functional importance of IRE1/XBP1s activity for ER proteostasis regulation in the context of health and disease.
10.1038/s41589-020-0584-z
An XBP1s-PIM-2 positive feedback loop controls IL-15-mediated survival of natural killer cells.
Science immunology
Spliced X-box-binding protein 1 (XBP1s) is an essential transcription factor downstream of interleukin-15 (IL-15) and AKT signaling, which controls cell survival and effector functions of human natural killer (NK) cells. However, the precise mechanisms, especially the downstream targets of XBP1s, remain unknown. In this study, by using XBP1 conditional knockout mice, we found that XBP1s is critical for IL-15-mediated NK cell survival but not proliferation in vitro and in vivo. Mechanistically, XBP1s regulates homeostatic NK cell survival by targeting PIM-2, a critical anti-apoptotic gene, which in turn stabilizes XBP1s protein by phosphorylating it at Thr. In addition, XBP1s enhances the effector functions and antitumor immunity of NK cells by recruiting T-bet to the promoter region of . Collectively, our findings identify a previously unknown mechanism by which IL-15-XBP1s signaling regulates the survival and effector functions of NK cells.
10.1126/sciimmunol.abn7993
Mammalian IRE1α dynamically and functionally coalesces with stress granules.
Nature cell biology
Upon endoplasmic reticulum (ER) stress, activation of the ER-resident transmembrane protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1) initiates a key branch of the unfolded protein response (UPR) through unconventional splicing generation of the transcription factor X-box-binding protein 1 (XBP1s). Activated IRE1 can form large clusters/foci, whose exact dynamic architectures and functional properties remain largely elusive. Here we report that, in mammalian cells, formation of IRE1α clusters is an ER membrane-bound phase separation event that is coupled to the assembly of stress granules (SGs). In response to different stressors, IRE1α clusters are dynamically tethered to SGs at the ER. The cytosolic linker portion of IRE1α possesses intrinsically disordered regions and is essential for its condensation with SGs. Furthermore, disruption of SG assembly abolishes IRE1α clustering and compromises XBP1 mRNA splicing, and such IRE1α-SG coalescence engenders enrichment of the biochemical components of the pro-survival IRE1α-XBP1 pathway during ER stress. Our findings unravel a phase transition mechanism for the spatiotemporal assembly of IRE1α-SG condensates to establish a more efficient IRE1α machinery, thus enabling higher stress-handling capacity.
10.1038/s41556-024-01418-7
Pharmacological Targeting of IRE1 in Cancer.
Raymundo Diana Pelizzari,Doultsinos Dimitrios,Guillory Xavier,Carlesso Antonio,Eriksson Leif A,Chevet Eric
Trends in cancer
IRE1α (inositol requiring enzyme 1 alpha) is one of the main transducers of the unfolded protein response (UPR). IRE1α plays instrumental protumoral roles in several cancers, and high IRE1α activity has been associated with poorer prognoses. In this context, IRE1α has been identified as a potentially relevant therapeutic target. Pharmacological inhibition of IRE1α activity can be achieved by targeting either the kinase domain or the RNase domain. Herein, the recent advances in IRE1α pharmacological targeting is summarized. We describe the identification and optimization of IRE1α inhibitors as well as their mode of action and limitations as anticancer drugs. The potential pitfalls and challenges that could be faced in the clinic, and the opportunities that IRE1α modulating strategies may present are discussed.
10.1016/j.trecan.2020.07.006
The metabolic ER stress sensor IRE1α suppresses alternative activation of macrophages and impairs energy expenditure in obesity.
Shan Bo,Wang Xiaoxia,Wu Ying,Xu Chi,Xia Zhixiong,Dai Jianli,Shao Mengle,Zhao Feng,He Shengqi,Yang Liu,Zhang Mingliang,Nan Fajun,Li Jia,Liu Jianmiao,Liu Jianfeng,Jia Weiping,Qiu Yifu,Song Baoliang,Han Jing-Dong J,Rui Liangyou,Duan Sheng-Zhong,Liu Yong
Nature immunology
Obesity is associated with metabolic inflammation and endoplasmic reticulum (ER) stress, both of which promote metabolic disease progression. Adipose tissue macrophages (ATMs) are key players orchestrating metabolic inflammation, and ER stress enhances macrophage activation. However, whether ER stress pathways underlie ATM regulation of energy homeostasis remains unclear. Here, we identified inositol-requiring enzyme 1α (IRE1α) as a critical switch governing M1-M2 macrophage polarization and energy balance. Myeloid-specific IRE1α abrogation in Ern1; Lyz2-Cre mice largely reversed high-fat diet (HFD)-induced M1-M2 imbalance in white adipose tissue (WAT) and blocked HFD-induced obesity, insulin resistance, hyperlipidemia and hepatic steatosis. Brown adipose tissue (BAT) activity, WAT browning and energy expenditure were significantly higher in Ern1; Lyz2-Cre mice. Furthermore, IRE1α ablation augmented M2 polarization of macrophages in a cell-autonomous manner. Thus, IRE1α senses protein unfolding and metabolic and immunological states, and consequently guides ATM polarization. The macrophage IRE1α pathway drives obesity and metabolic syndrome through impairing BAT activity and WAT browning.
10.1038/ni.3709
Inhibition of H2O2-induced cell death through FOXO1 modulation by EUK-172 in SK-N-MC cells.
Gheysarzadeh Ali,Yazdanparast Razieh
European journal of pharmacology
It has been suggested that excess accumulation of reactive oxygen species, termed oxidative stress, may lead to neuronal death resulting in neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. In oxidative stress-induced cell death numerous transcription factors are thought to be involved. One of them is Forkhead box protein O1 (FOXO1) that governs many genes involved in oxidative stress resistance, DNA repair, cell cycle arrest, proliferation and apoptosis. Apparently, FOXO1 activity is tightly linked to post translational modifications including phosphorylation and acetylation, which are modulated by many factors such as oxidative stress. Reactive oxygen species, as the major players in oxidative stress, guide FOXO1 nuclear localization at least by simultaneous c-Jun N-terminal kinase (JNK) activation and Akt/PKB activity suppression. Here, we showed that a synthetic salen-manganese derivative (EUK-172) with strong catalase activity reduced oxidative stress evident through marked reduction in intracellular reactive oxygen species, protein carbonylation and lipid peroxidation. In addition, our results indicated that EUK-172 not only reduced the FOXO1 protein content, but also it inhibited FOXO1 nuclear translocation in H(2)O(2)-exposed SK-N-MC cells. These events attenuated caspase-3 activity and bax/Bcl-2 ratio leading to higher viability of the H(2)O(2)-treated SK-N-MC cells.
10.1016/j.ejphar.2012.09.036
Myeloid FoxO1 depletion attenuates hepatic inflammation and prevents nonalcoholic steatohepatitis.
The Journal of clinical investigation
Hepatic inflammation is culpable for the evolution of asymptomatic steatosis to nonalcoholic steatohepatitis (NASH). Hepatic inflammation results from abnormal macrophage activation. We found that FoxO1 links overnutrition to hepatic inflammation by regulating macrophage polarization and activation. FoxO1 was upregulated in hepatic macrophages, correlating with hepatic inflammation, steatosis, and fibrosis in mice and patients with NASH. Myeloid cell conditional FoxO1 knockout skewed macrophage polarization from proinflammatory M1 to the antiinflammatory M2 phenotype, accompanied by a reduction in macrophage infiltration in liver. These effects mitigated overnutrition-induced hepatic inflammation and insulin resistance, contributing to improved hepatic metabolism and increased energy expenditure in myeloid cell FoxO1-knockout mice on a high-fat diet. When fed a NASH-inducing diet, myeloid cell FoxO1-knockout mice were protected from developing NASH, culminating in a reduction in hepatic inflammation, steatosis, and fibrosis. Mechanistically, FoxO1 counteracts Stat6 to skew macrophage polarization from M2 toward the M1 signature to perpetuate hepatic inflammation in NASH. FoxO1 appears to be a pivotal mediator of macrophage activation in response to overnutrition and a therapeutic target for ameliorating hepatic inflammation to stem the disease progression from benign steatosis to NASH.
10.1172/JCI154333
Trimethylamine N-Oxide Binds and Activates PERK to Promote Metabolic Dysfunction.
Chen Sifan,Henderson Ayana,Petriello Michael C,Romano Kymberleigh A,Gearing Mary,Miao Ji,Schell Mareike,Sandoval-Espinola Walter J,Tao Jiahui,Sha Bingdong,Graham Mark,Crooke Rosanne,Kleinridders Andre,Balskus Emily P,Rey Federico E,Morris Andrew J,Biddinger Sudha B
Cell metabolism
The gut-microbe-derived metabolite trimethylamine N-oxide (TMAO) is increased by insulin resistance and associated with several sequelae of metabolic syndrome in humans, including cardiovascular, renal, and neurodegenerative disease. The mechanism by which TMAO promotes disease is unclear. We now reveal the endoplasmic reticulum stress kinase PERK (EIF2AK3) as a receptor for TMAO: TMAO binds to PERK at physiologically relevant concentrations; selectively activates the PERK branch of the unfolded protein response; and induces the transcription factor FoxO1, a key driver of metabolic disease, in a PERK-dependent manner. Furthermore, interventions to reduce TMAO, either by manipulation of the gut microbiota or by inhibition of the TMAO synthesizing enzyme, flavin-containing monooxygenase 3, can reduce PERK activation and FoxO1 levels in the liver. Taken together, these data suggest TMAO and PERK may be central to the pathogenesis of the metabolic syndrome.
10.1016/j.cmet.2019.08.021
TARGETING S100A9-TLR2 AXIS CONTROLS MACROPHAGE NLRP3 INFLAMMASOME ACTIVATION IN FATTY LIVER ISCHEMIA REPERFUSION INJURY.
Shock (Augusta, Ga.)
ABSTRACT:Liver ischemia reperfusion (IR) injury significantly impacts clinical outcomes by increasing the risk of hepatic dysfunction after liver surgery. Fatty livers are more susceptible to IR stress. Recent studies have demonstrated that S100A9 plays a crucial role in both IR injury and the progression of liver steatosis. Nevertheless, the precise mechanisms underlying these effects remain unclear. In our study, transcriptome analysis of fatty livers subjected to IR insult in mice identified S100A9 as an important mediator. Employing loss-of-function approaches, we investigated the immune regulatory function of S100A9 and its downstream signaling in fatty liver IR injury. As expected, S100A9 emerged as one of the most significantly upregulated genes during the reperfusion stage in fatty livers. Genetic knockdown of S100A9 markedly ameliorated liver pathological damage, evidenced by reduced macrophage/neutrophil infiltration as well as the decreased expression of proinflammatory factors. Transcriptome/functional studies revealed that S100A9 triggered liver inflammatory response via regulating toll-like receptor 2 (TLR2)/activating transcription factor 4 (ATF4) signaling. Additionally, TLR2 expression was notably increased in macrophages from ischemic fatty livers. In vitro , recombinant S100A9-stimulated macrophages exhibited the elevated production of proinflammatory factors and TLR2/ATF4 pathway activation. Intriguingly, S100A9 facilitated ATF4 nuclear translocation and enhanced NEK7/NLRP3 inflammasome activation in macrophages. In conclusion, our study identified S100A9 as a key regulator responsible for macrophage NLRP3 inflammasome activation and subsequent inflammatory injury in fatty liver IR process. Targeting TLR2/ATF4 signaling may offer a novel therapeutic strategy for mitigating S100A9-mediated liver injury.
10.1097/SHK.0000000000002470
Endoplasmic reticulum stress in liver disease.
Journal of hepatology
The unfolded protein response (UPR) is activated upon the accumulation of misfolded proteins in the endoplasmic reticulum (ER) that are sensed by the binding immunoglobulin protein (BiP)/glucose-regulated protein 78 (GRP78). The accumulation of unfolded proteins sequesters BiP so it dissociates from three ER-transmembrane transducers leading to their activation. These transducers are inositol requiring (IRE) 1α, PKR-like ER kinase (PERK), and activating transcription factor (ATF) 6α. PERK phosphorylates eukaryotic initiation factor 2 alpha (eIF2α) resulting in global mRNA translation attenuation, and concurrently selectively increases the translation of several mRNAs, including the transcription factor ATF4, and its downstream target CHOP. IRE1α has kinase and endoribonuclease (RNase) activities. IRE1α autophosphorylation activates the RNase activity to splice XBP1 mRNA, to produce the active transcription factor sXBP1. IRE1α activation also recruits and activates the stress kinase JNK. ATF6α transits to the Golgi compartment where it is cleaved by intramembrane proteolysis to generate a soluble active transcription factor. These UPR pathways act in concert to increase ER content, expand the ER protein folding capacity, degrade misfolded proteins, and reduce the load of new proteins entering the ER. All of these are geared toward adaptation to resolve the protein folding defect. Faced with persistent ER stress, adaptation starts to fail and apoptosis occurs, possibly mediated through calcium perturbations, reactive oxygen species, and the proapoptotic transcription factor CHOP. The UPR is activated in several liver diseases; including obesity associated fatty liver disease, viral hepatitis, and alcohol-induced liver injury, all of which are associated with steatosis, raising the possibility that ER stress-dependent alteration in lipid homeostasis is the mechanism that underlies the steatosis. Hepatocyte apoptosis is a pathogenic event in several liver diseases, and may be linked to unresolved ER stress. If this is true, restoration of ER homeostasis prior to ER stress-induced cell death may provide a therapeutic rationale in these diseases. Herein we discuss each branch of the UPR and how they may impact hepatocyte function in different pathologic states.
10.1016/j.jhep.2010.11.005
Endoplasmic reticulum stress in liver diseases.
Hepatology (Baltimore, Md.)
The endoplasmic reticulum (ER) is an intracellular organelle that fosters the correct folding of linear polypeptides and proteins, a process tightly governed by the ER-resident enzymes and chaperones. Failure to shape the proper 3-dimensional architecture of proteins culminates in the accumulation of misfolded or unfolded proteins within the ER, disturbs ER homeostasis, and leads to canonically defined ER stress. Recent studies have elucidated that cellular perturbations, such as lipotoxicity, can also lead to ER stress. In response to ER stress, the unfolded protein response (UPR) is activated to reestablish ER homeostasis ("adaptive UPR"), or, conversely, to provoke cell death when ER stress is overwhelmed and sustained ("maladaptive UPR"). It is well documented that ER stress contributes to the onset and progression of multiple hepatic pathologies including NAFLD, alcohol-associated liver disease, viral hepatitis, liver ischemia, drug toxicity, and liver cancers. Here, we review key studies dealing with the emerging role of ER stress and the UPR in the pathophysiology of liver diseases from cellular, murine, and human models. Specifically, we will summarize current available knowledge on pharmacological and non-pharmacological interventions that may be used to target maladaptive UPR for the treatment of nonmalignant liver diseases.
10.1002/hep.32562
Dietary resveratrol supplementation alleviates cold exposure-induced pyroptosis and inflammation in broiler heart by modulating oxidative stress and endoplasmic reticulum stress.
Poultry science
To explore the potential protective effect of resveratrol (RES) on cold-exposed broilers, 360 21-day-old broilers were equally divided into 5 groups with 6 replicates. A control (CON) group was reared at the normal feeding temperature and received a basal diet, and 4 cold exposure (8 ± 1°C for 10 h/d from d 29 to 42) groups were fed the basal diet with 0 (CE), 250 (CE + RES250), 500 (CE + RES500), and 750 (CE + RES750) mg/kg RES from d 22 to 42. Broilers were slaughtered on d 42 and heart tissues were collected to measure the relevant indexes. The results showed that heart tissues of all CE-broilers had inflammatory cell infiltrations, and dietary RES supplementation reduced this phenomenon. Compared to CON group, the concentrations of MDA and HO were increased and activities of SOD and CAT were decreased in all CE-broilers (P < 0.05). mRNA expression of genes related to endoplasmic reticulum (ER) stress (GRP78, IRE1, PERK, EIF-2α, ATF4, ATF6, and CHOP), pyroptosis (NLRP3, ASC, Caspase1, GSDME, IL-18, and IL-1β), and proinflammation (TNF-α, IFN-γ, IL-2, and IL-6) was upregulated and that of ant-inflammatory cytokines (IL-4 and IL-10) was downregulated in CE and all CE + RES groups compared to CON group (P < 0.05). Compared to CE group, the activities of SOD and CAT and mRNA expression of anti-inflammatory genes were increased (P < 0.05), and concentrations of MDA and HO and mRNA expression of ER stress, pyroptosis and proinflammatory genes were reduced (P < 0.05) in 3 CE + RES groups. Additionally, protein levels of PERK, ATF4, CHOP, NLRP3, Caspase1, GSDMD, IL-18, IL-1β, TNF-α, and IL-10 were similar in their mRNA expression. Overall, cold exposure caused oxidative stress and ER stress, and induced pyroptosis and inflammatory response, resulting in heart injury in broilers, and dietary RES addition reduced heart damage by enhancing antioxidant defense function. This study indicates that RES can be a feed additive to alleviate cold exposure-induced heart injury in broilers, and a 500 mg RES/kg diet is the optimal supplemental level.
10.1016/j.psj.2024.104203
A new perspective on NAFLD: Focusing on lipid droplets.
Journal of hepatology
Lipid droplets (LDs) are complex and metabolically active organelles. They are composed of a neutral lipid core surrounded by a monolayer of phospholipids and proteins. LD accumulation in hepatocytes is the distinctive characteristic of non-alcoholic fatty liver disease (NAFLD), which is a chronic, heterogeneous liver condition that can progress to liver fibrosis and hepatocellular carcinoma. Though recent research has improved our understanding of the mechanisms linking LD accumulation to NAFLD progression, numerous aspects of LD biology are either poorly understood or unknown. In this review, we provide a description of several key mechanisms that contribute to LD accumulation in hepatocytes, favouring NAFLD progression. First, we highlight the importance of LD architecture and describe how the dysregulation of LD biogenesis leads to endoplasmic reticulum stress and inflammation. This is followed by an analysis of the causal nexus that exists between LD proteome composition and LD degradation. Finally, we describe how the increase in size of LDs causes activation of hepatic stellate cells, leading to liver fibrosis and hepatocellular carcinoma. We conclude that acquiring a more sophisticated understanding of LD biology will provide crucial insights into the heterogeneity of NAFLD and assist in the development of therapeutic approaches for this liver disease.
10.1016/j.jhep.2021.11.009
Endoplasmic reticulum stress: molecular mechanism and therapeutic targets.
Signal transduction and targeted therapy
The endoplasmic reticulum (ER) functions as a quality-control organelle for protein homeostasis, or "proteostasis". The protein quality control systems involve ER-associated degradation, protein chaperons, and autophagy. ER stress is activated when proteostasis is broken with an accumulation of misfolded and unfolded proteins in the ER. ER stress activates an adaptive unfolded protein response to restore proteostasis by initiating protein kinase R-like ER kinase, activating transcription factor 6, and inositol requiring enzyme 1. ER stress is multifaceted, and acts on aspects at the epigenetic level, including transcription and protein processing. Accumulated data indicates its key role in protein homeostasis and other diverse functions involved in various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, achromatopsia, cataracts, ocular tumors, ocular surface diseases, and myopia. This review summarizes the molecular mechanisms underlying the aforementioned ocular diseases from an ER stress perspective. Drugs (chemicals, neurotrophic factors, and nanoparticles), gene therapy, and stem cell therapy are used to treat ocular diseases by alleviating ER stress. We delineate the advancement of therapy targeting ER stress to provide new treatment strategies for ocular diseases.
10.1038/s41392-023-01570-w
Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.
Nature reviews. Molecular cell biology
'Reactive oxygen species' (ROS) is an umbrella term for an array of derivatives of molecular oxygen that occur as a normal attribute of aerobic life. Elevated formation of the different ROS leads to molecular damage, denoted as 'oxidative distress'. Here we focus on ROS at physiological levels and their central role in redox signalling via different post-translational modifications, denoted as 'oxidative eustress'. Two species, hydrogen peroxide (HO) and the superoxide anion radical (O), are key redox signalling agents generated under the control of growth factors and cytokines by more than 40 enzymes, prominently including NADPH oxidases and the mitochondrial electron transport chain. At the low physiological levels in the nanomolar range, HO is the major agent signalling through specific protein targets, which engage in metabolic regulation and stress responses to support cellular adaptation to a changing environment and stress. In addition, several other reactive species are involved in redox signalling, for instance nitric oxide, hydrogen sulfide and oxidized lipids. Recent methodological advances permit the assessment of molecular interactions of specific ROS molecules with specific targets in redox signalling pathways. Accordingly, major advances have occurred in understanding the role of these oxidants in physiology and disease, including the nervous, cardiovascular and immune systems, skeletal muscle and metabolic regulation as well as ageing and cancer. In the past, unspecific elimination of ROS by use of low molecular mass antioxidant compounds was not successful in counteracting disease initiation and progression in clinical trials. However, controlling specific ROS-mediated signalling pathways by selective targeting offers a perspective for a future of more refined redox medicine. This includes enzymatic defence systems such as those controlled by the stress-response transcription factors NRF2 and nuclear factor-κB, the role of trace elements such as selenium, the use of redox drugs and the modulation of environmental factors collectively known as the exposome (for example, nutrition, lifestyle and irradiation).
10.1038/s41580-020-0230-3
Targeting oxidative stress in disease: promise and limitations of antioxidant therapy.
Nature reviews. Drug discovery
Oxidative stress is a component of many diseases, including atherosclerosis, chronic obstructive pulmonary disease, Alzheimer disease and cancer. Although numerous small molecules evaluated as antioxidants have exhibited therapeutic potential in preclinical studies, clinical trial results have been disappointing. A greater understanding of the mechanisms through which antioxidants act and where and when they are effective may provide a rational approach that leads to greater pharmacological success. Here, we review the relationships between oxidative stress, redox signalling and disease, the mechanisms through which oxidative stress can contribute to pathology, how antioxidant defences work, what limits their effectiveness and how antioxidant defences can be increased through physiological signalling, dietary components and potential pharmaceutical intervention.
10.1038/s41573-021-00233-1
Aristolochic acid I induces proximal tubule injury through ROS/HMGB1/mt DNA mediated activation of TLRs.
Journal of cellular and molecular medicine
Aristolochic acids (AAs) are extracted from certain plants as folk remedies for centuries until their nephrotoxicity and carcinogenicity were recognized. Aristolochic acid I (AAI) is one of the main pathogenic compounds, and it has nephrotoxic, carcinogenic and mutagenic effects. Previous studies have shown that AAI acts mainly on proximal renal tubular epithelial cells; however, the mechanisms of AAI-induced proximal tubule cell damage are still not fully characterized. We exposed human kidney proximal tubule cells (PTCs; HK2 cell line) to AAI in vitro at different time/dose conditions and assessed cell proliferation, reactive oxygen species (ROS) generation, nitric oxide (NO) production, m-RNA/ protein expressions and mitochondrial dysfunction. AAI exposure decreased proliferation and increased apoptosis, ROS generation / NO production in PTCs significantly at 24 h. Gene/ protein expression studies demonstrated activation of innate immunity (TLRs 2, 3, 4 and 9, HMGB1), inflammatory (IL6, TNFA, IL1B, IL18, TGFB and NLRP3) and kidney injury (LCN2) markers. AAI also induced epithelial-mesenchymal transition (EMT) and mitochondrial dysfunction in HK2 cells. TLR9 knock-down and ROS inhibition were able to ameliorate the toxic effect of AAI. In conclusion, AAI treatment caused injury to PTCs through ROS-HMGB1/mitochondrial DNA (mt DNA)-mediated activation of TLRs and inflammatory response.
10.1111/jcmm.17451
Protective effect of N-(E)-p-coumaroyltyrosine on LPS-induced acute inflammatory injury and signaling pathway analysis.
Fish & shellfish immunology
N-trans-p-coumaroyltyrosine (N-(E)-p-coumaroyltyrosine, NPCT), extracted and purified from Abri Mollis Herba, is an amino acid amide. The defense mechanism of NPCT against inflammatory response is still unknown. In this study, lipopolysaccharide (LPS)-induced zebrafish acute inflammatory injury model was established to observe the inhibitory effect of NPCT on the aggregation of inflammatory cells in the yolk sac of zebrafish, as well as the inhibitory effect of NPCT on inflammatory and gas signaling factors. Results show that NPCT could inhibit inflammatory cell infiltration in zebrafish yolk sac, the migration and aggregation of macrophages and neutrophils to the site of inflammation, and the release of Nitric Oxide (NO) and Reactive Oxygen Species (ROS) in zebrafish, indicating that NPCT could substantially significantly prevent the development of LPS-induced acute systemic inflammation. In addition, the analysis results of RNA-seq showed that in the model group versus the administered group, the differentially expressed genes were mainly enriched to inflammatory signaling pathways, such as the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway, which were down-regulated in the administered group. The TLR4, MyD88, IRAK4, NF-κB, IκB, NLRP3, Caspase-1, ASC, IL-1β, and IL-6 genes were significantly different in the transcripts, and the overall trend of the qPCR results was consistent with the transcriptome sequencing results. Therefore, NPCT had a significant inhibitory effect on LPS-induced acute inflammatory injury in zebrafish, and its anti-inflammatory mechanism may be through the regulation of key genes on the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway, thereby affecting the release of relevant inflammatory cytokines.
10.1016/j.fsi.2023.109242
The PYRIN domain-only protein POP3 inhibits ALR inflammasomes and regulates responses to infection with DNA viruses.
Nature immunology
The innate immune system responds to infection and tissue damage by activating cytosolic sensory complexes called 'inflammasomes'. Cytosolic DNA is sensed by AIM2-like receptors (ALRs) during bacterial and viral infections and in autoimmune diseases. Subsequently, recruitment of the inflammasome adaptor ASC links ALRs to the activation of caspase-1. A controlled immune response is crucial for maintaining homeostasis, but the regulation of ALR inflammasomes is poorly understood. Here we identified the PYRIN domain (PYD)-only protein POP3, which competes with ASC for recruitment to ALRs, as an inhibitor of DNA virus-induced activation of ALR inflammasomes in vivo. Data obtained with a mouse model with macrophage-specific POP3 expression emphasize the importance of the regulation of ALR inflammasomes in monocytes and macrophages.
10.1038/ni.2829
The Trinity of cGAS, TLR9, and ALRs Guardians of the Cellular Galaxy Against Host-Derived Self-DNA.
Kumar Vijay
Frontiers in immunology
The immune system has evolved to protect the host from the pathogens and allergens surrounding their environment. The immune system develops in such a way to recognize self and non-self and develops self-tolerance against self-proteins, nucleic acids, and other larger molecules. However, the broken immunological self-tolerance leads to the development of autoimmune or autoinflammatory diseases. Pattern-recognition receptors (PRRs) are expressed by immunological cells on their cell membrane and in the cytosol. Different Toll-like receptors (TLRs), Nod-like receptors (NLRs) and absent in melanoma-2 (AIM-2)-like receptors (ALRs) forming inflammasomes in the cytosol, RIG (retinoic acid-inducible gene)-1-like receptors (RLRs), and C-type lectin receptors (CLRs) are some of the PRRs. The DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) is another PRR present in the cytosol and the nucleus. The present review describes the role of ALRs (AIM2), TLR9, and cGAS in recognizing the host cell DNA as a potent damage/danger-associated molecular pattern (DAMP), which moves out to the cytosol from its housing organelles (nucleus and mitochondria). The introduction opens with the concept that the immune system has evolved to recognize pathogens, the idea of ,and its failure due to the emergence of autoimmune diseases (ADs), and the discovery of PRRs revolutionizing immunology. The second section describes the cGAS-STING signaling pathway mediated cytosolic self-DNA recognition, its evolution, characteristics of self-DNAs activating it, and its role in different inflammatory conditions. The third section describes the role of TLR9 in recognizing self-DNA in the endolysosomes during infections depending on the self-DNA characteristics and various inflammatory diseases. The fourth section discusses about AIM2 (an ALR), which also binds cytosolic self-DNA (with 80-300 base pairs or bp) that inhibits cGAS-STING-dependent type 1 IFN generation but induces inflammation and pyroptosis during different inflammatory conditions. Hence, this trinity of PRRs has evolved to recognize self-DNA as a potential DAMP and comes into action to guard the cellular galaxy. However, their dysregulation proves dangerous to the host and leads to several inflammatory conditions, including sterile-inflammatory conditions autoinflammatory and ADs.
10.3389/fimmu.2020.624597
8-oxoguanine and 8-oxodeoxyguanosine Biomarkers of Oxidative DNA Damage: A Review on HPLC-ECD Determination.
Molecules (Basel, Switzerland)
Reactive oxygen species (ROS) are continuously produced in living cells due to metabolic and biochemical reactions and due to exposure to physical, chemical and biological agents. Excessive ROS cause oxidative stress and lead to oxidative DNA damage. Within ROS-mediated DNA lesions, 8-oxoguanine (8-oxoG) and its nucleotide 8-oxo-2'-deoxyguanosine (8-oxodG)-the guanine and deoxyguanosine oxidation products, respectively, are regarded as the most significant biomarkers for oxidative DNA damage. The quantification of 8-oxoG and 8-oxodG in urine, blood, tissue and saliva is essential, being employed to determine the overall effects of oxidative stress and to assess the risk, diagnose, and evaluate the treatment of autoimmune, inflammatory, neurodegenerative and cardiovascular diseases, diabetes, cancer and other age-related diseases. High-performance liquid chromatography with electrochemical detection (HPLC-ECD) is largely employed for 8-oxoG and 8-oxodG determination in biological samples due to its high selectivity and sensitivity, down to the femtomolar range. This review seeks to provide an exhaustive analysis of the most recent reports on the HPLC-ECD determination of 8-oxoG and 8-oxodG in cellular DNA and body fluids, which is relevant for health research.
10.3390/molecules27051620
Mitochondrial signal transduction.
Cell metabolism
The analogy of mitochondria as powerhouses has expired. Mitochondria are living, dynamic, maternally inherited, energy-transforming, biosynthetic, and signaling organelles that actively transduce biological information. We argue that mitochondria are the processor of the cell, and together with the nucleus and other organelles they constitute the mitochondrial information processing system (MIPS). In a three-step process, mitochondria (1) sense and respond to both endogenous and environmental inputs through morphological and functional remodeling; (2) integrate information through dynamic, network-based physical interactions and diffusion mechanisms; and (3) produce output signals that tune the functions of other organelles and systemically regulate physiology. This input-to-output transformation allows mitochondria to transduce metabolic, biochemical, neuroendocrine, and other local or systemic signals that enhance organismal adaptation. An explicit focus on mitochondrial signal transduction emphasizes the role of communication in mitochondrial biology. This framework also opens new avenues to understand how mitochondria mediate inter-organ processes underlying human health.
10.1016/j.cmet.2022.10.008
Mic19 depletion impairs endoplasmic reticulum-mitochondrial contacts and mitochondrial lipid metabolism and triggers liver disease.
Nature communications
Endoplasmic reticulum (ER)-mitochondria contacts are critical for the regulation of lipid transport, synthesis, and metabolism. However, the molecular mechanism and physiological function of endoplasmic reticulum-mitochondrial contacts remain unclear. Here, we show that Mic19, a key subunit of MICOS (mitochondrial contact site and cristae organizing system) complex, regulates ER-mitochondria contacts by the EMC2-SLC25A46-Mic19 axis. Mic19 liver specific knockout (LKO) leads to the reduction of ER-mitochondrial contacts, mitochondrial lipid metabolism disorder, disorganization of mitochondrial cristae and mitochondrial unfolded protein stress response in mouse hepatocytes, impairing liver mitochondrial fatty acid β-oxidation and lipid metabolism, which may spontaneously trigger nonalcoholic steatohepatitis (NASH) and liver fibrosis in mice. Whereas, the re-expression of Mic19 in Mic19 LKO hepatocytes blocks the development of liver disease in mice. In addition, Mic19 overexpression suppresses MCD-induced fatty liver disease. Thus, our findings uncover the EMC2-SLC25A46-Mic19 axis as a pathway regulating ER-mitochondria contacts, and reveal that impairment of ER-mitochondria contacts may be a mechanism associated with the development of NASH and liver fibrosis.
10.1038/s41467-023-44057-6
Mitochondrial alterations in fatty liver diseases.
Journal of hepatology
Fatty liver diseases can result from common metabolic diseases, as well as from xenobiotic exposure and excessive alcohol use, all of which have been shown to exert toxic effects on hepatic mitochondrial functionality and dynamics. Invasive or complex methodology limits large-scale investigations of mitochondria in human livers. Nevertheless, abnormal mitochondrial function, such as impaired fatty acid oxidation and oxidative phosphorylation, drives oxidative stress and has been identified as an important feature of human steatohepatitis. On the other hand, hepatic mitochondria can be flexible and adapt to the ambient metabolic condition to prevent triglyceride and lipotoxin accumulation in obesity. Experience from studies on xenobiotics has provided important insights into the regulation of hepatic mitochondria. Increasing awareness of the joint presence of metabolic disease-related (lipotoxic) and alcohol-related liver diseases further highlights the need to better understand their mutual interaction and potentiation in disease progression. Recent clinical studies have assessed the effects of diets or bariatric surgery on hepatic mitochondria, which are also evolving as an interesting therapeutic target in non-alcoholic fatty liver disease. This review summarises the current knowledge on hepatic mitochondria with a focus on fatty liver diseases linked to obesity, type 2 diabetes and xenobiotics.
10.1016/j.jhep.2022.09.020
Txnip mediates glucocorticoid-activated NLRP3 inflammatory signaling in mouse microglia.
Bharti Veni,Tan Hua,Zhou Hong,Wang Jun-Feng
Neurochemistry international
Many studies indicate that chronic stress and excessive stress hormone can cause an inflammatory response. Thioredoxin-interacting protein (Txnip) as an endogenous thioredoxin inhibitor suppresses thioredoxin-produced antioxidant effects. Txnip was also found to interact with nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), which activates NLRP3 inflammasome and promotes inflammatory processes. Recently our laboratory found that chronic stress can increase Txnip protein levels in mouse brain, indicating that Txnip may mediate chronic stress-induced inflammation. Microglia play an important role in neuroinflammation. The purpose of this study is to investigate the effect of chronic stress hormone treatment on Txnip and NLRP3 inflammasome signaling in cultured microglia cells. Our result showed that chronic treatment with stress hormone corticosterone increased Txnip protein levels and Txnip-NLRP3 binding in N9 mouse microglia, in primary cultured mouse microglia and in mouse brain. Our result also showed that chronic corticosterone treatment increased procaspase-1 cleavage, caspase-1 activity and interleukin-1β release in N9 microglia. Using CRISPR/Cas9 method we found that knocking out Txnip inhibited corticosterone-increased caspase-1 activity and interleukin-1β release. Our results suggest that chronic corticosterone treatment upregulates Txnip and increases Txnip-NLRP3 binding, which activates NLRP3 inflammasome, resulting in activation of caspase-1 and in further releasing of interleukin-1β. It is therefore likely that Txnip-activated NLRP3 inflammasome contributes to corticosterone-caused neuroinflammation.
10.1016/j.neuint.2019.104564
Novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death.
JHEP reports : innovation in hepatology
Background & Aims:The stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress. Methods:A mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIP) and TXNIP-proficient (TXNIP) mice. Results:The TXNIP mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIP controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIP inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-β (IFN-β) expression. Interestingly, TXNIP augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2',5' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis. Conclusions:Macrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases. Lay summary:Liver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.
10.1016/j.jhepr.2022.100532
Maresin 1 protects against lipopolysaccharide/d-galactosamine-induced acute liver injury by inhibiting macrophage pyroptosis and inflammatory response.
Yang Wenchang,Tao Kaixiong,Zhang Peng,Chen Xin,Sun Xiong,Li Ruidong
Biochemical pharmacology
BACKGROUND:Acute liver injury (ALI) caused by sepsis is a fearful disease with high mortality and poor prognosis. This study aimed to explore the roles and mechanism of Maresin 1 (MaR1) in lipopolysaccharide/d-galactosamine (LPS/D-GalN)-induced ALI. METHODS:We established an ALI mouse model induced by LPS/D-GalN. Each group was treated with or without LPS/D-GalN or MaR1. For the vitro experiments, RAW264.7, NCTC1469 cells, and bone marrow-derived macrophages (BMDMs) were stimulated with LPS. The effects of MaR1 on the reactive oxygen species (ROS), pyroptosis and inflammatory response in macrophages were investigated. RESULTS:MaR1 significantly inhibited an excessive inflammatory response and proinflammatory markers during LPS/D-GalN-induced ALI. MaR1 markedly decreased the levels of ROS, tumor necrosis factor-α, and interleukin-1β (IL-1β) in macrophages, and limited hepatocyte apoptosis in vitro. Upon exploring the mechanisms underlying the protective role of MaR1, we found MaR1 markedly upregulated the nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), and considerably reduced the phosphorylation of p38, ERK, and nuclear factor-kappa B (NF-κB)-p65. Knocking down Nrf2 decreased the effect of MaR1. Furthermore, we observed that MaR1 reduced inflammatory injury by inhibiting M1 macrophages and promoting M2 macrophage polarization. Finally, we observed that MaR1 could inhibit the production of gasdermin D N-terminus (GSDMD-N) in vivo. In vitro, MaR1 could significantly suppressed the expression of NLR family pyrin domain containing 3 (NLRP3) inflammasome, GSDMD-N, and IL-1β caused by LPS and nigericin stimulation in BMDMs. CONCLUSION:MaR1 could ameliorate inflammation during LPS/D-GalN induced ALI by suppressing mitogen-activated protein kinase /NF-κB signaling and NLRP3 inflammasome-induced pyroptosis, activating macrophage M1/M2 polarization and Nrf2/HO-1 signaling. This provides new evidence for the potential of developing MaR1 for ALI treatment.
10.1016/j.bcp.2021.114863
Apigenin Alleviated High-Fat-Diet-Induced Hepatic Pyroptosis by Mitophagy-ROS-CTSB-NLRP3 Pathway in Mice and AML12 Cells.
Journal of agricultural and food chemistry
Apigenin is considered the most-known natural flavonoid and is abundant in a wide variety of fruits and vegetables. A high fat diet (HFD) can induce liver injury and hepatocyte death in multiple ways. Pyroptosis is an innovative type of programmed cell death. Moreover, excessive pyroptosis of hepatocytes leads to liver injury. We used HFD to induce liver cell pyroptosis in C57BL/6J mice in this work. After gavage of apigenin, apigenin can significantly reduce the level of lactate dehydrogenase (LDH) in liver tissue ignited by HFD and reduce the levels of NLRP3 (NOD-like receptor family pyrin domain containing 3), the N-terminal domain of GSDMD (GSDMD-N), cleaved-caspase 1, cathepsin B (CTSB), interleukin-1β (IL-1β) and interleukin-18 (IL-18) protein expression and the colocalization of NLRP3 and CTSB and increase the level of lysosomal associated membrane protein-1 (LAMP-1) protein expression, thus alleviating cell pyroptosis. In a further in vitro mechanism study, we find that palmitic acid (PA) can induce pyroptosis in AML12 cells. After adding apigenin, apigenin can clear the damaged mitochondria through mitophagy and reduce the generation of intracellular reactive oxygen species (ROS), thus alleviating CTSB release caused by lysosomal membrane permeabilization (LMP), reducing the LDH release caused by PA and reducing the levels of NLRP3, GSDMD-N, cleaved-caspase 1, CTSB, IL-1β, and IL-18 protein expression. By adding the mitophagy inhibitor cyclosporin A (CsA), LC3-siRNA, the CTSB inhibitor CA-074 methyl ester (CA-074 Me), and the NLRP3 inhibitor MCC950, the aforementioned results were further confirmed. Therefore, our results show that HFD-fed and PA can damage mitochondria, promote the production of intracellular ROS, enhance the lysosomal membrane permeabilization (LMP), and cause the leakage of CTSB, thus activating the NLRP3 inflammatory body and inducing pyroptosis in C57BL/6J mice and AML12 cells, while apigenin alleviates this phenomenon through the mitophagy-ROS-CTSB-NLRP3 pathway.
10.1021/acs.jafc.2c07581
Irisin alleviates LPS-induced liver injury and inflammation through inhibition of NLRP3 inflammasome and NF-κB signaling.
Li Qian,Tan Ying,Chen Sainan,Xiao Xiaochan,Zhang Mingming,Wu Qi,Dong Maolong
Journal of receptor and signal transduction research
Lipopolysaccharide (LPS) provokes severe inflammation and cell death in sepsis, with liver being the major affected organ. Up-to-date, neither the mechanism of action nor target treatment is readily available for LPS-induced liver injury. This study examined the effect of irisin, an endogenous hormonal peptide, on LPS-induced liver injury using animal and cell models, and the mechanism involved with a special focus on pyroptosis. Irisin is known to regulate glucose metabolism, inflammation, and immune response, while our earlier work denoted the anti-inflammatory and anti-apoptotic properties for irisin. Inflammatory factors and AST/ALT were also detected. Pyroptosis, apoptosis, and reactive oxygen species (ROS) were evaluated using PI staining, TUNEL staining, DCFH-DA fluorescence, and western blot, respectively. Our results indicated that irisin attenuated LPS-induced liver injury and release of inflammatory cytokines. Increased activity of NLRP3 inflammasome was discovered in LPS-challenged Raw264.7 cells, along with elevated levels of inflammation and apoptosis, the effects of which were mediated by activation of ROS and nuclear factor κB (NF-κB) signaling. These changes were reversed following irisin treatment. Our study demonstrated that irisin countered LPS-mediated liver injury inhibiting apoptosis, NLRP3 inflammasome activation and NF-κB signaling. These findings revealed the role of irisin as a promising new anti-pyroptosis/apoptosis agent to reconcile the onset and progression of septic liver injury.
10.1080/10799893.2020.1808675
Palmitic acid activates NLRP3 inflammasome through NF-κB and AMPK-mitophagy-ROS pathways to induce IL-1β production in large yellow croaker (Larimichthys crocea).
Biochimica et biophysica acta. Molecular and cell biology of lipids
Studies on marine fish showed that vegetable oils substituted for excessive fish oil increased interleukin-1β (IL-1β) production. However, whether the nucleotide-binding oligomerization domain, leucine-rich repeat-containing family, pyrin domain-containing-3 (NLRP3) inflammasome has a substantial role in fatty acid-induced IL-1β production in fish remains unclear. The associated specific mechanism is also unknown. In this study, nlrp3, caspase-1 and apoptosis-associated speck-like protein containing a CARD (asc) were successfully cloned, and NLRP3 inflammasome consisted of NLRP3, caspase-1 and ASC in large yellow croaker. Primary hepatocytes of fish incubated with palmitic acid (PA) exhibited the highest expression of pro-inflammatory genes (il-1β and tnfα) and NLRP3 inflammasome related genes (nlrp3, caspase-1 and asc), caspase-1 activity and IL-1β production among different treatments. Furthermore, PA-induced NLRP3 inflammasome activation was confirmed to require two signals: the first signal was that PA promoted the NF-κB (P65) protein into the nucleus, and NF-κB increased NLRP3 promoter activity and nlrp3 transcription. The second signal was that PA inhibited AMPK phosphorylation and decreased mitophagy by inhibiting the expression of PINK and parkin proteins, thereby damaging the mitochondria that could not be effectively cleared. Mitochondrial damage generated excessive amounts of reactive oxygen species, which activated the NLRP3 inflammasome and then induced caspase-1 activity and IL-1β production. Therefore, excessive dietary PA activated NLRP3 inflammasome through NF-κB and AMPK-mitophagy-ROS pathways to induce IL-1β production, thereby leading to inflammation in fish.
10.1016/j.bbalip.2023.159428
CCN1/Integrin αβ Instigates Free Fatty Acid-Induced Hepatocyte Lipid Accumulation and Pyroptosis through NLRP3 Inflammasome Activation.
Nutrients
Hyperlipidemia with high blood levels of free fatty acids (FFA) is the leading cause of non-alcoholic steatohepatitis. CCN1 is a secreted matricellular protein that drives various cellular functions, including proliferation, migration, and differentiation. However, its role in mediating FFA-induced pro-inflammatory cell death and its underlying molecular mechanisms have not been characterized. In this study, we demonstrated that CCN1 was upregulated in the livers of obese mice. The increase in FFA-induced CCN1 was evaluated in vitro by treating hepatocytes with a combination of oleic acid and palmitic acid (2:1). Gene silencing using specific small interfering RNAs (siRNA) revealed that CCN1 participated in FFA-induced intracellular lipid accumulation, caspase-1 activation, and hepatocyte pyroptosis. Next, we identified integrin αβ as a potential receptor of CCN1. Co-immunoprecipitation demonstrated that the binding between CCN1 and integrin αβ increased in hepatocytes upon FFA stimulation in the livers of obese mice. Similarly, the protein levels of integrin α and β were increased in vitro and in vivo. Experiments with specific siRNAs confirmed that integrin αβ played a part in FFA-induced intracellular lipid accumulation, NLRP3 inflammasome activation, and pyroptosis in hepatocytes. In conclusion, these results provide novel evidence that the CCN1/integrin αβ is a novel mediator that drives hepatic lipotoxicity via NLRP3-dependent pyroptosis.
10.3390/nu14183871
Irisin Ameliorated Skeletal Muscle Atrophy by Inhibiting Fatty Acid Oxidation and Pyroptosis Induced by Palmitic Acid in Chronic Kidney Disease.
Kidney & blood pressure research
INTRODUCTION:Protein-energy waste (PEW) is a common complication in patients with chronic kidney disease (CKD), among which skeletal muscle atrophy is one of the most important clinical features of PEW. Pyroptosis is a type of proinflammatory, programmed cell death associated with skeletal muscle disease. Irisin, as a novel myokine, has attracted extensive attention for its protective role in the complications associated with CKD, but its role in muscle atrophy in CKD is unclear. METHODS:Palmitic acid (PA)-induced muscular atrophy was evaluated by a reduction in C2C12 myotube diameter. Muscle atrophy model was established in male C57BL/6J mice treated with 0.2% adenine for 4 weeks and then fed a 45% high-fat diet. Blood urea nitrogen and creatinine levels, body and muscle weight, and muscle histology were assessed. The expression of carnitine palmitoyltransferase 1A (CPT1A) and pyroptosis-related protein was analysed by Western blots or immunohistochemistry. The release of IL-1β was detected by enzyme-linked immunosorbent assay. RESULTS:In this study, we showed that PA-induced muscular atrophy manifested as a reduction in C2C12 myotube diameter. During this process, PA can also induce pyroptosis, as shown by the upregulation of NLRP3, cleaved caspase-1 and GSDMD-N expression and the increased IL-1β release and PI-positive cell rate. Inhibition of caspase-1 or NLRP3 attenuated PA-induced pyroptosis and myotube atrophy in C2C12 cells. Importantly, irisin treatment significantly ameliorated PA-induced skeletal muscle pyroptosis and atrophy. In terms of mechanism, PA upregulated CPT1A, a key enzyme of fatty acid oxidation (FAO), and irisin attenuated this effect, which was consistent with etomoxir (CPT1A inhibitor) treatment. Moreover, irisin improved skeletal muscle atrophy and pyroptosis in adenine-induced mice by regulating FAO. CONCLUSION:Our study firstly verifies that pyroptosis is a novel mechanism of skeletal muscle atrophy in CKD. Irisin ameliorates skeletal muscle atrophy by inhibiting FAO and pyroptosis in CKD, and irisin may be developed as a potential therapeutic agent for the treatment of muscle wasting in CKD patients.
10.1159/000533926
CD36, a signaling receptor and fatty acid transporter that regulates immune cell metabolism and fate.
The Journal of experimental medicine
CD36 is a type 2 cell surface scavenger receptor widely expressed in many immune and non-immune cells. It functions as both a signaling receptor responding to DAMPs and PAMPs, as well as a long chain free fatty acid transporter. Recent studies have indicated that CD36 can integrate cell signaling and metabolic pathways through its dual functions and thereby influence immune cell differentiation and activation, and ultimately help determine cell fate. Its expression along with its dual functions in both innate and adaptive immune cells contribute to pathogenesis of common diseases, including atherosclerosis and tumor progression, which makes CD36 and its downstream effectors potential therapeutic targets. This review comprehensively examines the dual functions of CD36 in a variety of immune cells, especially macrophages and T cells. We also briefly discuss CD36 function in non-immune cells, such as adipocytes and platelets, which impact the immune system via intercellular communication. Finally, outstanding questions in this field are provided for potential directions of future studies.
10.1084/jem.20211314
Deficiency of WTAP in hepatocytes induces lipoatrophy and non-alcoholic steatohepatitis (NASH).
Nature communications
Ectopic lipid accumulation and inflammation are the essential signs of NASH. However, the molecular mechanisms of ectopic lipid accumulation and inflammation during NASH progression are not fully understood. Here we reported that hepatic Wilms' tumor 1-associating protein (WTAP) is a key integrative regulator of ectopic lipid accumulation and inflammation during NASH progression. Hepatic deletion of Wtap leads to NASH due to the increased lipolysis in white adipose tissue, enhanced hepatic free fatty acids uptake and induced inflammation, all of which are mediated by IGFBP1, CD36 and cytochemokines such as CCL2, respectively. WTAP binds to specific DNA motifs which are enriched in the promoters and suppresses gene expression (e.g., Igfbp1, Cd36 and Ccl2) with the involvement of HDAC1. In NASH, WTAP is tranlocated from nucleus to cytosol, which is related to CDK9-mediated phosphorylation. These data uncover a mechanism by which hepatic WTAP regulates ectopic lipid accumulation and inflammation during NASH progression.
10.1038/s41467-022-32163-w
FALCON systematically interrogates free fatty acid biology and identifies a novel mediator of lipotoxicity.
Cell metabolism
Cellular exposure to free fatty acids (FFAs) is implicated in the pathogenesis of obesity-associated diseases. However, there are no scalable approaches to comprehensively assess the diverse FFAs circulating in human plasma. Furthermore, assessing how FFA-mediated processes interact with genetic risk for disease remains elusive. Here, we report the design and implementation of fatty acid library for comprehensive ontologies (FALCON), an unbiased, scalable, and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids associated with decreased membrane fluidity. Furthermore, we prioritized genes that reflect the combined effects of harmful FFA exposure and genetic risk for type 2 diabetes (T2D). We found that c-MAF-inducing protein (CMIP) protects cells from FFA exposure by modulating Akt signaling. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism.
10.1016/j.cmet.2023.03.018
HILPDA promotes NASH-driven HCC development by restraining intracellular fatty acid flux in hypoxia.
Journal of hepatology
BACKGROUND & AIMS:The prevalence of non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is rising rapidly, yet its underlying mechanisms remain unclear. Herein, we aim to determine the role of hypoxia-inducible lipid droplet associated protein (HILPDA)/hypoxia-inducible gene 2 (HIG2), a selective inhibitor of intracellular lipolysis, in NASH-driven HCC. METHODS:The clinical significance of HILPDA was assessed in human NASH-driven HCC specimens by immunohistochemistry and transcriptomics analyses. The oncogenic effect of HILPDA was assessed in human HCC cells and in 3D epithelial spheroids upon exposure to free fatty acids and either normoxia or hypoxia. Lipidomics profiling of wild-type and HILPDA knockout HCC cells was assessed via shotgun and targeted approaches. Wild-type (Hilpda) and hepatocyte-specific Hilpda knockout (Hilpda) mice were fed a Western diet and high sugar in drinking water while receiving carbon tetrachloride to induce NASH-driven HCC. RESULTS:In patients with NASH-driven HCC, upregulated HILPDA expression is strongly associated with poor survival. In oxygen-deprived and lipid-loaded culture conditions, HILPDA promotes viability of human hepatoma cells and growth of 3D epithelial spheroids. Lack of HILPDA triggered flux of polyunsaturated fatty acids to membrane phospholipids and of saturated fatty acids to ceramide synthesis, exacerbating lipid peroxidation and apoptosis in hypoxia. The apoptosis induced by HILPDA deficiency was reversed by pharmacological inhibition of ceramide synthesis. In our experimental mouse model of NASH-driven HCC, Hilpda exhibited reduced hepatic steatosis and tumorigenesis but increased oxidative stress in the liver. Single-cell analysis supports a dual role of hepatic HILPDA in protecting HCC cells and facilitating the establishment of a pro-tumorigenic immune microenvironment in NASH. CONCLUSIONS:Hepatic HILPDA is a pivotal oncometabolic factor in the NASH liver microenvironment and represents a potential novel therapeutic target. IMPACT AND IMPLICATIONS:Non-alcoholic steatohepatitis (NASH, chronic metabolic liver disease caused by buildup of fat, inflammation and damage in the liver) is emerging as the leading risk factor and the fastest growing cause of hepatocellular carcinoma (HCC), the most common form of liver cancer. While curative therapeutic options exist for HCC, it frequently presents at a late stage when such options are no longer effective and only systemic therapies are available. However, systemic therapies are still associated with poor efficacy and some side effects. In addition, no approved drugs are available for NASH. Therefore, understanding the underlying metabolic alterations occurring during NASH-driven HCC is key to identifying new cancer treatments that target the unique metabolic needs of cancer cells.
10.1016/j.jhep.2023.03.041
Mechanisms and disease consequences of nonalcoholic fatty liver disease.
Cell
Nonalcoholic fatty liver disease (NAFLD) is the leading chronic liver disease worldwide. Its more advanced subtype, nonalcoholic steatohepatitis (NASH), connotes progressive liver injury that can lead to cirrhosis and hepatocellular carcinoma. Here we provide an in-depth discussion of the underlying pathogenetic mechanisms that lead to progressive liver injury, including the metabolic origins of NAFLD, the effect of NAFLD on hepatic glucose and lipid metabolism, bile acid toxicity, macrophage dysfunction, and hepatic stellate cell activation, and consider the role of genetic, epigenetic, and environmental factors that promote fibrosis progression and risk of hepatocellular carcinoma in NASH.
10.1016/j.cell.2021.04.015
Acetyl-CoA carboxylase inhibition disrupts metabolic reprogramming during hepatic stellate cell activation.
Bates Jamie,Vijayakumar Archana,Ghoshal Sarani,Marchand Bruno,Yi Saili,Kornyeyev Dmytro,Zagorska Anna,Hollenback David,Walker Katie,Liu Kathy,Pendem Swetha,Newstrom David,Brockett Robert,Mikaelian Igor,Kusam Saritha,Ramirez Ricardo,Lopez David,Li Li,Fuchs Bryan C,Breckenridge David G
Journal of hepatology
BACKGROUND & AIMS:Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step of de novo lipogenesis and regulates fatty acid β-oxidation in hepatocytes. ACC inhibition reduces hepatic fat content and markers of liver injury in patients with NASH; however, the effect of ACC inhibition on liver fibrosis has not been reported. METHODS:A direct role for ACC in fibrosis was evaluated by measuring de novo lipogenesis, procollagen production, gene expression, glycolysis, and mitochondrial respiration in hepatic stellate cells (HSCs) in the absence or presence of small molecule inhibitors of ACC. ACC inhibitors were evaluated in rodent models of liver fibrosis induced by diet or the hepatotoxin, diethylnitrosamine. Fibrosis and hepatic steatosis were evaluated by histological and biochemical assessments. RESULTS:Inhibition of ACC reduced the activation of TGF-β-stimulated HSCs, as measured by both α-SMA expression and collagen production. ACC inhibition prevented a metabolic switch necessary for induction of glycolysis and oxidative phosphorylation during HSC activation. While the molecular mechanism by which inhibition of de novo lipogenesis blocks glycolysis and oxidative phosphorylation is unknown, we definitively show that HSCs require de novo lipogenesis for activation. Consistent with this direct antifibrotic mechanism in HSCs, ACC inhibition reduced liver fibrosis in a rat choline-deficient, high-fat diet model and in response to chronic diethylnitrosamine-induced liver injury (in the absence of hepatic lipid accumulation). CONCLUSIONS:In addition to reducing lipid accumulation in hepatocytes, ACC inhibition also directly impairs the profibrogenic activity of HSCs. Thus, small molecule inhibitors of ACC may lessen fibrosis by reducing lipotoxicity in hepatocytes and by preventing HSC activation, providing a mechanistic rationale for the treatment of patients with advanced liver fibrosis due to NASH. LAY SUMMARY:Hepatic fibrosis is the most important predictor of liver-related outcomes in patients with non-alcoholic steatohepatitis (NASH). Small molecule inhibitors of acetyl-CoA carboxylase (ACC) reduce hepatic fat content and markers of liver injury in patients with NASH. Herein, we report that inhibition of ACC and de novo lipogenesis also directly suppress the activation of hepatic stellate cells - the primary cell responsible for generating fibrotic scar in the liver - and thus fibrosis. These data provide further evidence for the use of ACC inhibitors to treat patients with NASH and advanced fibrosis.
10.1016/j.jhep.2020.04.037
Immune mechanisms linking metabolic injury to inflammation and fibrosis in fatty liver disease - novel insights into cellular communication circuits.
Journal of hepatology
Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease and is emerging as the leading cause of cirrhosis, liver transplantation and hepatocellular carcinoma (HCC). NAFLD is a metabolic disease that is considered the hepatic manifestation of the metabolic syndrome; however, during the evolution of NAFLD from steatosis to non-alcoholic steatohepatitis (NASH), to more advanced stages of NASH with liver fibrosis, the immune system plays an integral role. Triggers for inflammation are rooted in hepatic (lipid overload, lipotoxicity, oxidative stress) and extrahepatic (gut-liver axis, adipose tissue, skeletal muscle) systems, resulting in unique immune-mediated pathomechanisms in NAFLD. In recent years, the implementation of single-cell RNA-sequencing and high dimensional multi-omics (proteogenomics, lipidomics) and spatial transcriptomics have tremendously advanced our understanding of the complex heterogeneity of various liver immune cell subsets in health and disease. In NAFLD, several emerging inflammatory mechanisms have been uncovered, including profound macrophage heterogeneity, auto-aggressive T cells, the role of unconventional T cells and platelet-immune cell interactions, potentially yielding novel therapeutics. In this review, we will highlight the recent discoveries related to inflammation in NAFLD, discuss the role of immune cell subsets during the different stages of the disease (including disease regression) and integrate the multiple systems driving inflammation. We propose a refined concept by which the immune system contributes to all stages of NAFLD and discuss open scientific questions arising from this paradigm shift that need to be unravelled in the coming years. Finally, we discuss novel therapeutic approaches to target the multiple triggers of inflammation, including combination therapy via nuclear receptors (FXR agonists, PPAR agonists).
10.1016/j.jhep.2022.06.012
Lipotoxicity-induced STING1 activation stimulates MTORC1 and restricts hepatic lipophagy.
Autophagy
Lipid accumulation often leads to lipotoxic injuries to hepatocytes, which can cause nonalcoholic steatohepatitis. The association of inflammation with lipid accumulation in liver tissue has been studied for decades; however, key mechanisms have been identified only recently. In particular, it is still unknown how hepatic inflammation regulates lipid metabolism in hepatocytes. Herein, we found that PA treatment or direct stimulation of STING1 promoted, whereas STING1 deficiency impaired, MTORC1 activation, suggesting that STING1 is involved in PA-induced MTORC1 activation. Mechanistic studies revealed that STING1 interacted with several components of the MTORC1 complex and played an important role in the complex formation of MTORC1 under PA treatment. The involvement of STING1 in MTORC1 activation was dependent on SQSTM1, a key regulator of the MTORC1 pathway. In SQSTM1-deficient cells, the interaction of STING1 with the components of MTORC1 was weak. Furthermore, the impaired activity of MTORC1 via rapamycin treatment or STING1 deficiency decreased the numbers of LDs in cells. PA treatment inhibited lipophagy, which was not observed in STING1-deficient cells or rapamycin-treated cells. Restoration of MTORC1 activity via treatment with amino acids blocked lipophagy and LDs degradation. Finally, increased MTORC1 activation concomitant with STING1 activation was observed in liver tissues of nonalcoholic fatty liver disease patients, which provided clinical evidence for the involvement of STING1 in MTORC1 activation. In summary, we identified a novel regulatory loop of STING1-MTORC1 and explain how hepatic inflammation regulates lipid accumulation. Our findings may facilitate the development of new strategies for clinical treatment of hepatic steatosis.: AA: amino acid; ACTB: actin beta; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; DEPTOR: DEP domain containing MTOR interacting protein; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FFAs: free fatty acids; GFP: green fluorescent protein; HFD: high-fat diet; HT-DNA: herring testis DNA; IL1B: interleukin 1 beta; LAMP1: lysosomal associated membrane protein 1; LDs: lipid droplets; MAP1LC3: microtubule associated protein 1 light chain 3; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryonic fibroblasts; MLST8: MTOR associated protein, LST8 homolog; MT-ND1: mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 1; mtDNA: mitochondrial DNA; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NAFL: nonalcoholic fatty liver; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; NPCs: non-parenchymal cells; PA: palmitic acid; PLIN2: perilipin 2; RD: regular diet; RELA: RELA proto-oncogene, NF-kB subunit; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; RPTOR: regulatory associated protein of MTOR complex 1; RRAGA: Ras related GTP binding A; RRAGC: Ras related GTP binding C; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TGs: triglycerides; TREX1: three prime repair exonuclease 1.
10.1080/15548627.2021.1961072
Caspases in Cell Death, Inflammation, and Disease.
Van Opdenbosch Nina,Lamkanfi Mohamed
Immunity
Caspases are an evolutionary conserved family of cysteine proteases that are centrally involved in cell death and inflammation responses. A wealth of foundational insight into the molecular mechanisms that control caspase activation has emerged in recent years. Important advancements include the identification of additional inflammasome platforms and pathways that regulate activation of inflammatory caspases; the discovery of gasdermin D as the effector of pyroptosis and interleukin (IL)-1 and IL-18 secretion; and the existence of substantial crosstalk between inflammatory and apoptotic initiator caspases. A better understanding of the mechanisms regulating caspase activation has supported initial efforts to modulate dysfunctional cell death and inflammation pathways in a suite of communicable, inflammatory, malignant, metabolic, and neurodegenerative diseases. Here, we review current understanding of caspase biology with a prime focus on the inflammatory caspases and outline important topics for future experimentation.
10.1016/j.immuni.2019.05.020
NLRP3 and pyroptosis blockers for treating inflammatory diseases.
Trends in pharmacological sciences
The nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) inflammasome has emerged as a key mediator of pathological inflammation in many diseases and is an exciting drug target. Here, we review the molecular basis of NLRP3 inhibition by drug-like small molecules under development as novel therapeutics. We also summarize recent strategies to block pyroptosis as a novel approach to suppress chronic inflammation. Major recent developments in this area include the elucidation of mechanisms of action (MoAs) by which small molecules block NLRP3 inflammasome assembly and gasdermin D (GSDMD)-induced pyroptosis. We also discuss the status of clinical trials using agents that block specific components of the NLRP3 pathway, including their potential clinical applications for the treatment of many diseases.
10.1016/j.tips.2022.04.003
Pyroptosis-Induced Inflammation and Tissue Damage.
Journal of molecular biology
Programmed cell deaths are pathways involving cells playing an active role in their own destruction. Depending on the signaling system of the process, programmed cell death can be divided into two categories, pro-inflammatory and non-inflammatory. Pyroptosis is a pro-inflammatory form of programmed cell death. Upon cell death, a plethora of cytokines are released and trigger a cascade of responses from the neighboring cells. The pyroptosis process is a double-edged sword, could be both beneficial and detrimental in various inflammatory disorders and disease conditions. A physiological outcome of these responses is tissue damage, and sometimes death of the host. In this review, we focus on the inflammatory response triggered by pyroptosis, and resulting tissue damage in selected organs.
10.1016/j.jmb.2021.167301
Caspases in Cell Death, Inflammation, and Pyroptosis.
Kesavardhana Sannula,Malireddi R K Subbarao,Kanneganti Thirumala-Devi
Annual review of immunology
Caspases are a family of conserved cysteine proteases that play key roles in programmed cell death and inflammation. In multicellular organisms, caspases are activated via macromolecular signaling complexes that bring inactive procaspases together and promote their proximity-induced autoactivation and proteolytic processing. Activation of caspases ultimately results in programmed execution of cell death, and the nature of this cell death is determined by the specific caspases involved. Pioneering new research has unraveled distinct roles and cross talk of caspases in the regulation of programmed cell death, inflammation, and innate immune responses. In-depth understanding of these mechanisms is essential to foster the development of precise therapeutic targets to treat autoinflammatory disorders, infectious diseases, and cancer. This review focuses on mechanisms governing caspase activation and programmed cell death with special emphasis on the recent progress in caspase cross talk and caspase-driven gasdermin D-induced pyroptosis.
10.1146/annurev-immunol-073119-095439
Natural products in drug discovery and development: Synthesis and medicinal perspective of leonurine.
Frontiers in chemistry
Natural products, those molecules derived from nature, have been used by humans for thousands of years to treat ailments and diseases. More recently, these compounds have inspired chemists to use natural products as structural templates in the development of new drug molecules. One such compound is leonurine, a molecule isolated and characterized in the tissues of . This molecule has received attention from scientists in recent years due to its potent anti-oxidant, anti-apoptotic, and anti-inflammatory properties. More recently researchers have shown leonurine to be useful in the treatment of cardiovascular and nervous system diseases. Like other natural products such as paclitaxel and artemisinin, the historical development of leonurine as a therapeutic is very interesting. Therefore, this review provided an overview of natural product discovery, through to the development of a potential new drug. Content will summarize known plant sources, the pathway used in the synthesis of leonurine, and descriptions of leonurine's pharmacological properties in mammalian systems.
10.3389/fchem.2022.1036329
Application of Mathematical Modeling and Computational Tools in the Modern Drug Design and Development Process.
Molecules (Basel, Switzerland)
The conventional drug discovery approach is an expensive and time-consuming process, but its limitations have been overcome with the help of mathematical modeling and computational drug design approaches. Previously, finding a small molecular candidate as a drug against a disease was very costly and required a long time to screen a compound against a specific target. The development of novel targets and small molecular candidates against different diseases including emerging and reemerging diseases remains a major concern and necessitates the development of novel therapeutic targets as well as drug candidates as early as possible. In this regard, computational and mathematical modeling approaches for drug development are advantageous due to their fastest predictive ability and cost-effectiveness features. Computer-aided drug design (CADD) techniques utilize different computer programs as well as mathematics formulas to comprehend the interaction of a target and drugs. Traditional methods to determine small-molecule candidates as a drug have several limitations, but CADD utilizes novel methods that require little time and accurately predict a compound against a specific disease with minimal cost. Therefore, this review aims to provide a brief insight into the mathematical modeling and computational approaches for identifying a novel target and small molecular candidates for curing a specific disease. The comprehensive review mainly focuses on biological target prediction, structure-based and ligand-based drug design methods, molecular docking, virtual screening, pharmacophore modeling, quantitative structure-activity relationship (QSAR) models, molecular dynamics simulation, and MM-GBSA/MM-PBSA approaches along with valuable database resources and tools for identifying novel targets and therapeutics against a disease. This review will help researchers in a way that may open the road for the development of effective drugs and preventative measures against a disease in the future as early as possible.
10.3390/molecules27134169
High throughput virtual screening (HTVS) of peptide library: Technological advancement in ligand discovery.
European journal of medicinal chemistry
High-throughput virtual screening (HTVS) is a leading biopharmaceutical technology that employs computational algorithms to uncover biologically active compounds from large-scale collections of chemical compound libraries. In addition, this method often leverages the precedence of screening focused libraries for assessing their binding affinities and improving physicochemical properties. Usually, developing a drug sometimes takes ages, and lessons are learnt from FDA-approved drugs. This screening strategy saves resources and time compared to laboratory testing in certain stages of drug discovery. Yet in-silico investigations remain challenging in some cases of drug discovery. For the last few decades, peptide-based drug discoveries have received remarkable momentum for several advantages over small molecules. Therefore, developing a high-fidelity HTVS platform for chemically versatile peptide libraries is highly desired. This review summarises the modern and frequently appreciated HTVS strategies for peptide libraries from 2011 to 2021. In addition, we focus on the software used for preparing peptide libraries, their screening techniques and shortcomings. An index of various HTVS methods reported here should assist researchers in identifying tools that could be beneficial for their peptide library screening projects.
10.1016/j.ejmech.2022.114766
An integrated view of anti-inflammatory and antifibrotic targets for the treatment of NASH.
Journal of hepatology
Successful development of treatments for non-alcoholic fatty liver disease and its progressive form, non-alcoholic steatohepatitis (NASH), has been challenging. Because NASH and fibrosis lead to progression towards cirrhosis and clinical outcomes, approaches have either sought to attenuate metabolic dysregulation and cell injury, or directly target the inflammation and fibrosis that ensue. Targets for reducing the activation of inflammatory cascades include nuclear receptor agonists (e.g. resmetirom, lanifibranor, obeticholic acid), modulators of lipotoxicity (e.g. aramchol, acetyl-CoA carboxylase inhibitors) or modification of genetic variants (e.g. PNPLA3 gene silencing). Extrahepatic inflammatory signals from the circulation, adipose tissue or gut are targets of hormonal agonists (semaglutide, tirzepatide, FGF19/FGF21 analogues), microbiota or lifestyle interventions. Stress signals and hepatocyte death activate immune responses, engaging innate (macrophages, innate lymphocyte populations) and adaptive (auto-aggressive T cells) mechanisms. Therapies have also been developed to blunt immune cell activation, recruitment (chemokine receptor inhibitors), and responses (e.g. galectin-3 inhibitors, anti-platelet drugs). The disease-driving pathways of NASH converge to elicit fibrosis, which is reversible. The activation of hepatic stellate cells into matrix-producing myofibroblasts can be inhibited by antagonising soluble factors (e.g. integrins, cytokines), cellular crosstalk (e.g. with macrophages), and agonising nuclear receptor signalling. In advanced fibrosis, cell therapy with restorative macrophages or reprogrammed (CAR) T cells may accelerate repair through hepatic stellate cell deactivation or killing, or by enhancing matrix degradation. Heterogeneity of disease - either due to genetics or divergent disease drivers - is an obstacle to defining effective drugs for all patients with NASH that will be overcome incrementally.
10.1016/j.jhep.2023.03.038
Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease.
Cellular and molecular life sciences : CMLS
Non-alcoholic fatty liver disease (NAFLD) is currently the world's most common liver disease, estimated to affect up to one-fourth of the population. Hallmarked by hepatic steatosis, NAFLD is associated with a multitude of detrimental effects and increased mortality. This narrative review investigates the molecular mechanisms of hepatic steatosis in NAFLD, focusing on the four major pathways contributing to lipid homeostasis in the liver. Hepatic steatosis is a consequence of lipid acquisition exceeding lipid disposal, i.e., the uptake of fatty acids and de novo lipogenesis surpassing fatty acid oxidation and export. In NAFLD, hepatic uptake and de novo lipogenesis are increased, while a compensatory enhancement of fatty acid oxidation is insufficient in normalizing lipid levels and may even promote cellular damage and disease progression by inducing oxidative stress, especially with compromised mitochondrial function and increased oxidation in peroxisomes and cytochromes. While lipid export initially increases, it plateaus and may even decrease with disease progression, sustaining the accumulation of lipids. Fueled by lipo-apoptosis, hepatic steatosis leads to systemic metabolic disarray that adversely affects multiple organs, placing abnormal lipid metabolism associated with NAFLD in close relation to many of the current life-style-related diseases.
10.1007/s00018-018-2860-6
TLR8 agonist selgantolimod regulates Kupffer cell differentiation status and impairs HBV entry into hepatocytes via an IL-6-dependent mechanism.
Gut
OBJECTIVE:Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment. DESIGN:We identified -expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN's indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN's effect were validated using transcriptomic data from HBV-infected patients. RESULTS:Hepatic expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, ) and downregulated genes associated with the KC identity (eg, ). Treatment of hepatocytes with SLGN-CM downregulated and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition. CONCLUSION:Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection.
10.1136/gutjnl-2023-331396
S100A6 Activates Kupffer Cells via the p-P38 and p-JNK Pathways to Induce Inflammation, Mononuclear/macrophage Infiltration Sterile Liver Injury in Mice.
Inflammation
Noninfectious liver injury, including the effects of chemical material, drugs and diet, is a major cause of liver diseases worldwide. In chemical and drugs-induced liver injury, innate inflammatory responses are mediated by extracellular danger signals. The S100 protein can act as danger signals, which can promote the migration and chemotaxis of immune cells, promote the release of various inflammatory cytokines, and regulate the body's inflammatory and immune responses. However, the role of S100A6 in inflammatory response in chemical and drugs-induced sterile liver injury remains unclear. We constructed the model of sterile liver injury induced by carbon tetrachloride (CCl)/Paracetamol (APAP) and performed RNA sequencing (RNA-seq) on the liver tissues after injury (days 2 and 5). We analyzed inflammatory protein secretion in the liver tissue supernatant by enzyme-linked immunosorbent assay (ELISA), determined the inflammation response by bioinformatic analysis during sterile liver injury, and assessed mononuclear/macrophage infiltration by immunohistochemistry and flow cytometry. Immunohistochemistry was used to analyze the location of S100A6. We conducted inflammatory factor expression analysis and molecular mechanistic studies in Kupffer cells (KCs) induced by S100A6 using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), ELISA, and western blot in vitro experiments. We performed chemokine CCL2 expression analysis and molecular mechanism studies using the same method. We used a Transwell assay to show the infiltration of mononuclear/macrophage. We here observed that aggravated inflammatory response was shown in CCl and APAP-administrated mice, as evidenced by enhanced production of inflammatory cytokines (TNF-α, IL-1β), and elevated mononuclear/macrophage infiltration and activation of immunity. The expression of S100A6 was significantly increased on day 2 after sterile liver injury, which is primarily produced by injured liver cells. Mechanistic studies established that S100A6 activates Kupffer cells (KCs) via the p-P38, p-JNK and P65 pathways to induce inflammation in vitro. Furthermore, TNF-α can stimulate liver cells via the p-P38 and p-JNK pathways to produce CCL2 and promote the infiltration of mononuclear/macrophage. In summary, we showed that S100A6 plays an important role in regulating inflammation, thus influencing sterile liver injury. Our findings provide novel evidence that S100A6 can as a danger signal that contributes to pro-inflammatory activation through p-P38 and p-JNK pathways in CCl and APAP-induced sterile liver injury in mice. In addition, the inflammatory factor TNF-α induces a large amount of CCL2 production in normal liver cells surrounding the injured area through a paracrine action, which is chemotactic for blood mononuclear/macrophage infiltration.
10.1007/s10753-022-01750-w
Methods on improvements of the poor oral bioavailability of ginsenosides: Pre-processing, structural modification, drug combination, and micro- or nano- delivery system.
Journal of ginseng research
Panax ginseng Meyer is a traditional Chinese medicine that is widely used as tonic in Asia. The main pharmacologically active components of ginseng are the dammarane-type ginsenosides, which have been shown to have anti-cancer, anti-inflammatory, immunoregulatory, neuroprotective, and metabolic regulatory activities. Moreover, some of ginsenosides (eg, Rh2 and Rg3) have been developed into nutraceuticals. However, the utilization of ginsenosides in clinic is restrictive due to poor permeability in cells and low bioavailability in human body. Obviously, the dammarane skeleton and glycosyls of ginsenosides are responsible for these limitations. Therefore, improving the oral bioavailability of ginsenosides has become a pressing issue. Here, based on the structures of ginsenosides, we summarized the understanding of the factors affecting the oral bioavailability of ginsenosides, introduced the methods to enhance the oral bioavailability and proposed the future perspectives on improving the oral bioavailability of ginsenosides.
10.1016/j.jgr.2023.07.005
The Influence of Nanoparticle Properties on Oral Bioavailability of Drugs.
Wang Yuanyuan,Pi Chao,Feng Xianhu,Hou Yi,Zhao Ling,Wei Yumeng
International journal of nanomedicine
Oral administration has been the most common therapeutic regimen in various diseases because of its high safety, convenience, lower costs, and high compliance of patients. However, susceptible in hostile gastrointestinal (GI) environment, many drugs show poor permeability across GI tract mucus and intestinal epithelium with poor oral absorption and limited therapeutic efficacy. In recent years, nanoparticulate drug delivery systems (NDDS) have become a hot research spot because of their unique advantages including protecting drug from premature degrading and interacting with the physiological environment, increasing intracellular penetration, and enhancing drug absorption. However, a slight change in physicochemistry of nanoparticles can significantly impact their interaction with biological pathways and alter the oral bioavailability of drugs. Hence, this review focuses on the factors affecting oral bioavailability from two aspects. On the one hand, the factors are the biochemical and physiological barriers in oral drugs delivery. On the other hand, the factors are the nanoparticle properties including size, surface properties, and shape of nanoparticles.
10.2147/IJN.S257269
Curcumin-piperine co-supplementation and human health: A comprehensive review of preclinical and clinical studies.
Phytotherapy research : PTR
Curcumin is extracted from the rhizomes Curcuma longa L. It is known for its anti-inflammatory and anti-oxidant activities. Despite its safety and potential for use against various diseases, curcumin's utility is restricted due to its low oral bioavailability. Co-administration of curcumin along with piperine could potentially improve the bioavailability of curcumin. The present review aimed to provide an overview of the efficacy and safety of curcumin-piperine co-supplementation in human health. The findings of this comprehensive review show the beneficial effects of curcumin-piperine in improving glycemic indices, lipid profile and antioxidant status in diabetes, improving the inflammatory status caused by obesity and metabolic syndrome, reducing oxidative stress and depression in chronic stress and neurological disorders, also improving chronic respiratory diseases, asthma and COVID-19. Further high-quality clinical trial studies are needed to firmly establish the clinical efficacy of the curcumin-piperine supplement.
10.1002/ptr.7737
Influence of intermittent fasting on autophagy in the liver.
Bioscience trends
Studies have found that intermittent fasting (IF) can prevent diabetes, cancer, heart disease, and neuropathy, while in humans it has helped to alleviate metabolic syndrome, asthma, rheumatoid arthritis, Alzheimer's disease, and many other disorders. IF involves a series of coordinated metabolic and hormonal changes to maintain the organism's metabolic balance and cellular homeostasis. More importantly, IF can activate hepatic autophagy, which is important for maintaining cellular homeostasis and energy balance, quality control, cell and tissue remodeling, and defense against extracellular damage and pathogens. IF affects hepatic autophagy through multiple interacting pathways and molecular mechanisms, including adenosine monophosphate (AMP)-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), silent mating-type information regulatory 2 homolog-1 (SIRT1), peroxisomal proliferator-activated receptor alpha (PPARα) and farnesoid X receptor (FXR), as well as signaling pathways and molecular mechanisms such as glucagon and fibroblast growth factor 21 (FGF21). These pathways can stimulate the pro-inflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), play a cytoprotective role, downregulate the expression of aging-related molecules, and prevent the development of steatosis-associated liver tumors. By influencing the metabolism of energy and oxygen radicals as well as cellular stress response systems, IF protects hepatocytes from genetic and environmental factors. By activating hepatic autophagy, IF has a potential role in treating a variety of liver diseases, including non-alcoholic fatty liver disease, drug-induced liver injury, viral hepatitis, hepatic fibrosis, and hepatocellular carcinoma. A better understanding of the effects of IF on liver autophagy may lead to new approaches for the prevention and treatment of liver disease.
10.5582/bst.2023.01207
Ginsenosides from as Key Modulators of NF-κB Signaling Are Powerful Anti-Inflammatory and Anticancer Agents.
International journal of molecular sciences
Nuclear factor kappa B (NF-κB) signaling pathways progress inflammation and immune cell differentiation in the host immune response; however, the uncontrollable stimulation of NF-κB signaling is responsible for several inflammatory illnesses regardless of whether the conditions are acute or chronic. Innate immune cells, such as macrophages, microglia, and Kupffer cells, secrete pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, via the activation of NF-κB subunits, which may lead to the damage of normal cells, including neurons, cardiomyocytes, hepatocytes, and alveolar cells. This results in the occurrence of neurodegenerative disorders, cardiac infarction, or liver injury, which may eventually lead to systemic inflammation or cancer. Recently, ginsenosides from , a historical herbal plant used in East Asia, have been used as possible options for curing inflammatory diseases. All of the ginsenosides tested target different steps of the NF-κB signaling pathway, ameliorating the symptoms of severe illnesses. Moreover, ginsenosides inhibit the NF-κB-mediated activation of cancer metastasis and immune resistance, significantly attenuating the expression of MMPs, Snail, Slug, TWIST1, and PD-L1. This review introduces current studies on the therapeutic efficacy of ginsenosides in alleviating NF-κB responses and emphasizes the critical role of ginsenosides in severe inflammatory diseases as well as cancers.
10.3390/ijms24076119
Kupffer cell-derived TNF-α promotes hepatocytes to produce CXCL1 and mobilize neutrophils in response to necrotic cells.
Su Li,Li Na,Tang Hua,Lou Ziyang,Chong Xiaodan,Zhang Chenxi,Su Jiacan,Dong Xin
Cell death & disease
The damage-associated molecular pattern molecules (DAMPs) released by necrotic cells can trigger inflammatory response, which will facilitate the clearance of these dead cells. Neutrophil mobilization is a very important step for the dead cell clearance, however the detailed mechanisms for DAMPs induce neutrophil mobilization remains largely elusive. In this study, by using a necrotic cell-induced neutrophil mobilization mice model, we found that both neutrophil number and percentage rapidly (as early as 30 min) increased with necrotic cells but not live cell treatment. CXCL1 was rapidly increased in the serum and was responsible for the neutrophil mobilization when treated with necrotic cells. We further demonstrated that the hepatocytes in the liver were the main source of CXCL1 production in response to necrotic cells challenge. However, the hepatocytes did not express CXCL1 when incubating with necrotic cells alone. When Kupffer cells were ablated, the increased CXCL1 levels as well as neutrophil mobilization were abolished with necrotic cells challenge. Moreover, we clarified Kupffer cells-derived TNF-α activates the NF-κB pathway in hepatocytes and promote hepatocytes to express CXCL1. In summary, we showed that the liver is the main source for necrotic cell-induced CXCL1 production and neutrophil mobilization. Kupffer cells in the liver sense DAMPs and release TNF-α to activate the NF-κB pathway in hepatocytes. The interaction between Kupffer cells and hepatocytes is critical for CXCL1 production.
10.1038/s41419-018-0377-4
Innate immunity to intracellular LPS.
Nature immunology
Monitoring of the cytosolic compartment by the innate immune system for pathogen-encoded products or pathogen activities often enables the activation of a subset of caspases. In most cases, the cytosolic surveillance pathways are coupled to activation of caspase-1 via canonical inflammasome complexes. A related set of caspases, caspase-11 in rodents and caspase-4 and caspase-5 in humans, monitors the cytosol for bacterial lipopolysaccharide (LPS). Direct activation of caspase-11, caspase-4 and caspase-5 by intracellular LPS elicits the lytic cell death called 'pyroptosis', which occurs in multiple cell types. The pyroptosis is executed by the pore-forming protein GSDMD, which is activated by cleavage mediated by caspase-11, caspase-4 or caspase-5. In monocytes, formation of GSDMD pores can induce activation of the NLRP3 inflammasome for maturation of the cytokines IL-1β and IL-18. Caspase-11-mediated pyroptosis in response to cytosolic LPS is critical for antibacterial defense and septic shock. Here we review the emerging literature on the sensing of cytosolic LPS and its regulation and pathophysiological functions.
10.1038/s41590-019-0368-3
Chinese Herbal Medicine Suyin Detoxification Granule Inhibits Pyroptosis and Epithelial-Mesenchymal Transition by Downregulating MAVS/NLRP3 to Alleviate Renal Injury.
Journal of inflammation research
PURPOSE:Proteinuria is an independent risk factor of chronic kidney disease (CKD). Albumin-induced tubulointerstitial inflammation and epithelial-mesenchymal transition (EMT) via the activation of NLRP3 inflammasome is a potential therapeutic target for CKD. Suyin Detoxification Granule (SDG) improves proteinuria and postpones renal failure. However, the underlying mechanism is still unknown. METHODS:Firstly, the rat model of renal failure was established using intragastric administration of adenine. Renal function, proteinuria, inflammatory indicators in serum, and renal pathology were assessed, and renal immunohistochemical staining of NLRP3 inflammasomes was performed after intervention with low and high concentrations of SDG. Secondly, the model of renal tubular epithelial HK-2 cells was established using albumin in vitro, and the cell viability, EMT phenotype, and the expression of proteins in the NLRP3 inflammasome signaling pathway were measured after the freeze-dried powder of Suyin Detoxification Prescription (SDP) and CY-09, which is a selective and direct NLRP3 inhibitor, were co-incubated with albumin. ATP, SOD, mitochondrial membrane potential, and ROS were further measured in vitro, and changes in the mitochondrial function after SDP intervention were observed. The mitochondrial antiviral signaling protein (MAVS) was knocked down using siRNA, and the interaction between MAVS and NLRP3 was verified using Western blotting, polymerase chain reaction (PCR), and immunofluorescence. RESULTS:SDG improved renal function and proteinuria, alleviated renal fibrosis, and reduced serum inflammation and the expression of the components of the NLRP3 inflammasome in the kidney. In vitro, SDP and CY-09 enhanced cell viability after injury with albumin and inhibited pyroptosis induced by the NLRP3 inflammatory signaling pathway and expression of proteins involved in EMT. It was further found that SDP alleviated the mitochondrial dysfunction caused by albumin. The knockdown of MAVS reduced the expression of NLRP3 pathway proteins and their mRNA levels and also weakened the co-localization of NLRP3, thus, reducing cell pyroptosis. CONCLUSION:SDP protected renal tubular epithelial cells from cell pyroptosis and EMT by regulating the albumin-induced mitochondrial dysfunction/ MAVS/ NLRP3-ASC-caspase-1 inflammasome signaling pathway.
10.2147/JIR.S341598
Extended cleavage specificities of human granzymes A and K, two closely related enzymes with conserved but still poorly defined functions in T and NK cell-mediated immunity.
Frontiers in immunology
Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTL) and natural killer cells (NK). Granzyme A is the most abundant of the different granzymes (gzms) expressed by these two cell types. Gzms A and K are found in all jawed vertebrates and are the most well conserved of all hematopoietic serine proteases. Their potential functions have been studied extensively for many years, however, without clear conclusions. Gzm A was for many years thought to serve as a key component in the defense against viral infection by the induction of apoptosis in virus-infected cells, similar to gzm B. However, later studies have questioned this role and instead indicated that gzm A may act as a potent inducer of inflammatory cytokines and chemokines. Gzms A and K form clearly separate branches in a phylogenetic tree indicating separate functions. Transcriptional analyses presented here demonstrate the presence of gzm A and K transcripts in both CD4 and CD8 T cells. To enable screening for their primary biological targets we have made a detailed analysis of their extended cleavage specificities. Phage display analysis of the cleavage specificity of the recombinant enzymes showed that both gzms A and K are strict tryptases with high selectivity for Arg over Lys in the P1 position. The major differences in the specificities of these two enzymes are located N-terminally of the cleavage site, where gzm A prefers small amino acids such as Gly in the P3 position and shows a relatively relaxed selectivity in the P2 position. In contrast, gzm K prefers large amino acids such as Phe, Tyr, and Trp in both the P2 and P3 positions and does not tolerate negatively charged residues in the P2 position. This major distinction in extended specificities is likely reflected also in preferred targets of these two enzymes. This information can now be utilized for high-precision screening of primary targets for gzms A and K in search of their highly conserved but still poorly defined functions in vertebrate immunity.
10.3389/fimmu.2023.1211295
T Cells in Fibrosis and Fibrotic Diseases.
Zhang Mengjuan,Zhang Song
Frontiers in immunology
Fibrosis is the extensive deposition of fibrous connective tissue, and it is characterized by the accumulation of collagen and other extracellular matrix (ECM) components. Fibrosis is essential for wound healing and tissue repair in response to a variety of triggers, which include infection, inflammation, autoimmune disorder, degenerative disease, tumor, and injury. Fibrotic remodeling in various diseases, such as liver cirrhosis, pulmonary fibrosis, renal interstitial fibrosis, myocardial infarction, systemic sclerosis (SSc), and graft-versus-host disease (GVHD), can impair organ function, causing high morbidity and mortality. Both innate and adaptive immunity are involved in fibrogenesis. Although the roles of macrophages in fibrogenesis have been studied for many years, the underlying mechanisms concerning the manner in which T cells regulate fibrosis are not completely understood. The T cell receptor (TCR) engages the antigen and shapes the repertoire of antigen-specific T cells. Based on the divergent expression of surface molecules and cell functions, T cells are subdivided into natural killer T (NKT) cells, γδ T cells, CD8 cytotoxic T lymphocytes (CTL), regulatory T (Treg) cells, T follicular regulatory (Tfr) cells, and T helper cells, including Th1, Th2, Th9, Th17, Th22, and T follicular helper (Tfh) cells. In this review, we summarize the pro-fibrotic or anti-fibrotic roles and distinct mechanisms of different T cell subsets. On reviewing the literature, we conclude that the T cell regulations are commonly disease-specific and tissue-specific. Finally, we provide perspectives on microbiota, viral infection, and metabolism, and discuss the current advancements of technologies for identifying novel targets and developing immunotherapies for intervention in fibrosis and fibrotic diseases.
10.3389/fimmu.2020.01142
Bruton's Tyrosine Kinase Inhibitor Attenuates Warm Hepatic Ischemia/Reperfusion Injury via Modulation of the NLR Family Pyrin Domain Containing 3 Inflammasome.
Song Shao-Hua,Liu Fang,Zhao Yuan-Yu,Sun Ke-Yan,Guo Meng,Li Pei-Lei,Liu Hao,Ding Guo-Shan,Fu Zhi-Ren
Transplantation proceedings
The NLR family pyrin domain containing 3 (NLRP3) inflammasome is a widely studied inflammasome that plays a critical role in inflammatory responses. Many triggers, including microbial pathogens (ie, bacteria and viruses) and other signals (ie, reactive oxygen species, adenosine triphosphate, urate, silicon, and asbestos), can stimulate the NLRP3 inflammasome. Liver ischemia/reperfusion (I/R) injury is a common pathologic process during liver surgery and shock and can induce severe liver damage. Although its pathogenesis is still unclear, oxidative stress and overproduction of the inflammatory response are likely to contribute to I/R injury. The NLRP3 inflammasome is activated during the I/R process, resulting in further recruitment and activation of caspase-1. Activated caspase-1 cleaves the pro-forms of interleukin-1β and interleukin-18 and results in their maturation, triggering a proinflammatory cytokine cascade and causing liver damage. Bruton's tyrosine kinase is a critical molecule involved in diverse cellular pathways, such as proliferation, apoptosis, inflammation, and angiogenesis. Intrahepatic Bruton's tyrosine kinase is mainly expressed on Kupffer cells and sinusoidal endothelial cells, and the inflammasome is activated in Kupffer cells. Our study found that inhibition of Bruton's tyrosine kinase effectively attenuated liver I/R injury by suppressing activation of the NLRP3 inflammasome in Kupffer cells.
10.1016/j.transproceed.2019.10.024
Antrodia camphorata polysaccharide improves inflammatory response in liver injury via the ROS/TLR4/NF-κB signal.
Journal of cellular and molecular medicine
Antrodia Camphorata Polysaccharide (ACP) refers to a kind of polysaccharide extracted from the natural porous fungus Antrodia camphorata. This study investigated the mechanism of action of ACP in protecting the liver. The results showed that ACP suppressed the LPS-induced KC cell activation, reduced the expression of inflammatory factors, increased the SOD level and suppressed ROS expression. In addition, N-acetylcysteine (NAC) was adopted for pre-treatment to suppress ROS. The results indicated that NAC synergistically exerted its effect with ACP, suggesting that ACP played its role through suppressing ROS. Further detection revealed that ACP activated the Nrf2 signal. It was discovered in the mouse model that, ACP effectively improved liver injury in mice, decreased ALT and AST levels, and suppressed the expression of inflammatory factors. This study suggests that ACP can exert its effect against oxidative stress via the Nrf2-ARE signalling, which further improves the production of ROS and the activation of TLR4-NF-κB signalling, and protects the liver against liver injury.
10.1111/jcmm.17283
Trilobatin rescues fulminant hepatic failure by targeting COX2: Involvement of ROS/TLR4/NLRP3 signaling.
Phytomedicine : international journal of phytotherapy and phytopharmacology
BACKGROUND:Fulminant hepatic failure (FHF) lacks efficient therapies notwithstanding increased comprehending of the inflammatory response and oxidative stress play crucial roles in the pathogenesis of this type of hepatic damage. Trilobatin (TLB), a naturally occurring food additive, is endowed with anti-inflammation and antioxidant properties. PURPOSE:In current study, we evaluated the effect of TLB on FHF with a mouse model with d-galactosamine/lipopolysaccharide (GalN/LPS)-induced FHF and LPS-stimulated Kupffer cells (KCs) injury. METHODS:Mice were randomly divided into seven groups: control group, TLB 40 mg/kg + control group, GalN/LPS group, TLB 10 mg/kg + GalN/LPS group, TLB 20 mg/kg + GalN/LPS group, TLB 40 mg/kg + GalN/LPS group, bifendate 150 mg/kg + GalN/LPS group. The mice were administered intragastrically TLB (10, 20 and 40 mg/kg) for 7 days (twice a day) prior to injection of GalN (700 mg/kg)/LPS (100 µg/kg). The KCs were pretreated with TLB (2.5, 5, 10 μM) for 2 h or its analogue (10 μM) or COX2 inhibitor (10 μM), and thereafter challenged by LPS (1 μg/ml) for 24 h. RESULTS:TLB effectively rescued GalN/LPS-induced FHF. Furthermore, TLB inhibited TLR 4/NLRP3/pyroptosis pathway, and caspase 3-dependent apoptosis pathway, along with reducing excessive cellular and mitochondrial ROS generation and enhancing mitochondrial biogenesis. Intriguingly, TLB directly bound to COX2 as reflected by transcriptomics, molecular docking technique and surface plasmon resonance assay. Furthermore, TLB failed to attenuate LPS-induced inflammation and oxidative stress in KCs in the absence of COX2. CONCLUSION:Our findings discover a novel pharmacological effect of TLB: protecting against FHF-induced pyroptosis and apoptosis through mediating ROS/TLR4/NLRP3 signaling pathway and reducing inflammation and oxidative stress. TLB may be a promising agent with outstanding safety profile to treat FHF.
10.1016/j.phymed.2023.155059
Sclareol ameliorates liver injury by inhibiting nuclear factor-kappa B/NOD-like receptor protein 3-mediated inflammation and lipid metabolism disorder in diabetic mice.
International journal of immunopathology and pharmacology
: Sclareol (SCL) is a natural diterpene with anti-inflammation and antioxidant properties. This study aimed to assess the hepatoprotective effects of SCL in diabetic mice. : SCL (10 mg/kg) was administered intragastrically to C57BL/6 mice with streptozotocin-induced diabetes daily for 5 weeks to evaluate its beneficial effects in liver injury. Body and liver weight and blood glucose levels were measured. Liver histopathology, fibrosis, and lipid accumulation were evaluated using hematoxylin and eosin, Masson's trichrome, and Oil Red O staining, respectively. Serum hepatic enzyme and lipid levels were measured using an automatic biochemical analyzer. Hepatocellular apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Oxidative stress markers and reactive oxygen species (ROS) were measured using appropriate assay kits. The effects of sclareol on inflammation and lipid metabolism was evaluated by enzyme-linked immunosorbent assay (ELISA), immunohistochemical analysis, and Western blot assays. : SCL significantly decreased serum liver enzymes and lipids levels, and alleviated adipogenesis and fibrosis. Moreover, the protein levels of acetyl-CoA carboxylase and sterol response element-binding protein 1 were downregulated, whereas the expression of carnitine palmitoyl transferase 1 was upregulated. SCL increased the antioxidant activity, and decreased ROS levels. SCL alleviated hepatic mitochondrial damage. Furthermore, SCL inhibited Kupffer cell infiltration and reduced serum inflammatory cytokine levels. SCL significantly downregulated the protein expression of nuclear factor-kappa B (NF-κB) P65, NOD-like receptor protein 3 (NLRP3), caspase 1, and interleukin-1β. Our findings suggest that SCL improves diabetes-induced liver injury by alleviating the NF-κB/NLRP3-mediated inflammation and lipid metabolism disorder.
10.1177/03946320231223644
Ginsenoside Rb1 Reduces D-GalN/LPS-induced Acute Liver Injury by Regulating TLR4/NF-κB Signaling and NLRP3 Inflammasome.
Journal of clinical and translational hepatology
Background and Aims:The effect of ginsenoside Rb1 on D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced acute liver injury (ALI) is unknown. The aim of this study was to evaluate the effect of ginsenoside Rb1 on ALI and its underlying mechanisms. Methods:Mice were pretreated with ginsenoside Rb1 by intraperitoneal injection for 3 days before D-GalN/LPS treatment, to induce ALI. The survival rate was monitored every hour for 24 h, and serum biochemical parameters, hepatic index and histopathological analysis were evaluated to measure the degree of liver injury. ELISA was used to detect oxidative stress and inflammatory cytokines in hepatic tissue and serum. Immunohistochemistry staining, RT-PCR and western blotting were performed to evaluate the expression of toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB), and NLR family, pyrin domain-containing 3 protein (NLRP3) in liver tissue and Kupffer cells (KCs). Results:Ginsenoside Rb1 improved survival with D-GalN/LPS-induced ALI by up to 80%, significantly ameliorated the increased alanine and aspartate transaminase, restored the hepatic pathological changes and reduced the levels of oxidative stress and inflammatory cytokines altered by D-GalN/LPS. Compared to the control group, the KCs were increased in the D-GalN/LPS groups but did not increase significantly with Rb1 pretreatment. D-GalN/LPS could upregulate while Rb1 pretreatment could downregulate the expression of interleukin (IL)-1β, IL-18, NLRP3, apoptosis associated speck-like protein containing CARD (ASC) and caspase-1 in isolated KCs. Furthermore, ginsenoside Rb1 inhibited activation of the TLR4/NF-κB signaling pathway and NLRP3 inflammasome induced by D-GalN/LPS administration. Conclusions:Ginsenoside Rb1 protects mice against D-GalN/LPS-induced ALI by attenuating oxidative stress and the inflammatory response through the TLR4/NF-κB signaling pathway and NLRP3 inflammasome activation.
10.14218/JCTH.2021.00072
Allicin alleviated acrylamide-induced NLRP3 inflammasome activation via oxidative stress and endoplasmic reticulum stress in Kupffer cells and SD rats liver.
Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association
Acrylamide (AA) in heat-processed food leads to widespread concerns due to its hepatotoxicity. Allicin, a plant-derived antioxidant, possesses a significant protective effect on AA-induced hepatotoxicity, but the mechanism is still unclear. Herein, we investigated the mechanism in Kupffer cells and SD rats liver. Molecular docking, molecular dynamics simulation and LigPlus software speculated that allicin inhibited the activity of CYP2E1 expression by binding to its amino acid residues Phe116, Phe207, Leu210, Phe298, Ala299, Thr303, Val364 and Phe478 through hydrophobic interactions. Allicin decreased the reactive oxygen species (ROS) release and CYP2E1 protein expression and then alleviated the appearance of OS. Meanwhile, allicin significantly reduced ERS characteristic proteins GRP78, CHOP and UPR branch IRE1α pathway key proteins p-IRE, p-ASK, TRAF2 and XBP-1s expression. Simultaneously, allicin ameliorated OS and ERS activation, which inhibited the activation of the MAPK and NF-κB pathways, and down-regulated JNK, ERK, p38, p65 and IκBα phosphorylation. Allicin pre-treatment inhibited AA-induced inflammation as evidenced by reducing NLRP3 inflammasome activation, decreasing Cleaved-Caspase-1 expression as well as IL-1β, IL-18, IL-6 and TNF-α secretion. Taken together, our data provide new insights into possible signaling pathways involved in allicin attenuating AA-induced hepatotoxicity in vivo and in vitro.
10.1016/j.fct.2020.111937
Silencing lncRNA Lfar1 alleviates the classical activation and pyoptosis of macrophage in hepatic fibrosis.
Zhang Kun,Shi Zhemin,Zhang Mengxia,Dong Xueyi,Zheng Lina,Li Guantong,Han Xiaohui,Yao Zhi,Han Tao,Hong Wei
Cell death & disease
Hepatic fibrosis is a common pathological consequence of a sustained wound healing response to continuous liver injury, characterized by increased production and accumulation of extracellular matrix. If unresolved, the fibrotic process results in organ failure, and eventually death after the development of cirrhosis. It has been suggested that macrophages play central role in the progression of hepatic fibrosis, which is related to inflammation and pyroptosis, a novel programmed and proinflammatory cell death. However, it remains far less clear if, or how, lncRNAs regulates the activation and pyroptosis of macrophage in hepatic fibrosis. In the present study, we demonstrated that the liver-enriched lncRNA Lfar1, which has been reported to promote hepatic fibrosis through inducing hepatic stellate cells activation and hepatocytes apoptosis, was dysregulated during proinflammatory M1 activation and pyroptosis of macrophage. Our study revealed that silencing lnc-Lfar1 by a lentivirus-shRNA alleviated CCl- and BDL-induced proinflammatory M1 macrophage activation and NLRP3 inflammasome-mediated pyroptosis. Furthermore, the in vitro experiments demonstrated that lnc-Lfar1 knockdown significantly suppressed LPS- and IFN-γ-induced proinflammatory activation of macrophages, and inhibited LPS/ATP- and LPS/Nigericin-induced NLRP3 inflammasome-mediated pyroptosis. Mechanistically, lnc-Lfar1 regulated LPS- and IFN-γ-induced proinflammatory activation of macrophages through the NF-ĸB pathway. All these data supported our conclusion that lnc-Lfar1 plays a vital role in controlling the activation and pyroptosis of macrophage, thus providing a possible therapeutic target against inflammation-related disorders including hepatic fibrosis.
10.1038/s41419-020-2323-5
Adipokines, Hepatokines and Myokines: Focus on Their Role and Molecular Mechanisms in Adipose Tissue Inflammation.
Frontiers in endocrinology
Chronic low-grade inflammation in adipose tissue (AT) is a hallmark of obesity and contributes to various metabolic disorders, such as type 2 diabetes and cardiovascular diseases. Inflammation in ATs is characterized by macrophage infiltration and the activation of inflammatory pathways mediated by NF-κB, JNK, and NLRP3 inflammasomes. Adipokines, hepatokines and myokines - proteins secreted from AT, the liver and skeletal muscle play regulatory roles in AT inflammation endocrine, paracrine, and autocrine pathways. For example, obesity is associated with elevated levels of pro-inflammatory adipokines (e.g., leptin, resistin, chemerin, progranulin, RBP4, WISP1, FABP4, PAI-1, Follistatin-like1, MCP-1, SPARC, SPARCL1, and SAA) and reduced levels of anti-inflammatory adipokines such as adiponectin, omentin, ZAG, SFRP5, CTRP3, vaspin, and IL-10. Moreover, some hepatokines (Fetuin A, DPP4, FGF21, GDF15, and MANF) and myokines (irisin, IL-6, and DEL-1) also play pro- or anti-inflammatory roles in AT inflammation. This review aims to provide an updated understanding of these organokines and their role in AT inflammation and related metabolic abnormalities. It serves to highlight the molecular mechanisms underlying the effects of these organokines and their clinical significance. Insights into the roles and mechanisms of these organokines could provide novel and potential therapeutic targets for obesity-induced inflammation.
10.3389/fendo.2022.873699
Eritoran Attenuates Hepatic Inflammation and Fibrosis in Mice with Chronic Liver Injury.
Hsieh Yun-Cheng,Lee Kuei-Chuan,Wu Pei-Shan,Huo Teh-Ia,Huang Yi-Hsiang,Hou Ming-Chih,Lin Han-Chieh
Cells
Toll-like receptor 4 (TLR4) signaling plays a key role in liver inflammation and fibrosis. The therapeutic effects of eritoran, a TLR4 antagonist, in mice with chronic liver injury remained unclear. C57BL/6 mice were fed a fast-food diet (FFD) or treated with carbon tetrachloride (CCl) to induce chronic liver injury. Eritoran (10 mg/kg) or a vehicle was randomly intraperitoneally administered to the FFD-fed mice and the CCl-injured mice. Primary mouse liver cells were cultured with lipopolysaccharide (LPS) or eritoran. In both FFD and CCl mouse models, eritoran significantly reduced serum ALT levels and decreased hepatic inflammatory cell infiltration without altering hepatic steatosis. Additionally, eritoran attenuated liver fibrosis by decreasing hepatic stellate cells (HSCs) activation and the abundance of α-smooth muscle actin and transforming growth factor-β1. Hepatic TLR4 downstream signaling including MyD88 expression, NF-κB p65 nuclear translocation, p38 and JNK phosphorylation were successfully inhibited by eritoran. In the in vitro study, LPS-induced nuclear translocation of NF-κB in primary HSCs and Kupffer cells was significantly suppressed by eritoran. In conclusion, eritoran attenuated hepatic inflammation and fibrosis by inhibition of the TLR4 signaling pathway in mice with chronic liver injury. Eritoran may serve as a potential drug for chronic liver disease.
10.3390/cells10061562
Deletion of TLR4 attenuates lipopolysaccharide-induced acute liver injury by inhibiting inflammation and apoptosis.
Acta pharmacologica Sinica
Septic acute liver injury is one of the leading causes of fatalities in patients with sepsis. Toll-like receptor 4 (TLR4) plays a vital role in response to lipopolysaccharide (LPS) challenge, but the mechanisms underlying TLR4 function in septic injury remains unclear. In this study, we investigated the role of TLR4 in LPS-induced acute liver injury (ALI) in mice with a focus on inflammation and apoptosis. Wild-type (WT) and TLR4-knockout (TLR4) mice were challenged with LPS (4 mg/kg) for 6 h. TLR4 signaling cascade markers (TLR4, MyD88, and NF-κB), inflammatory markers (TNFα, IL-1β, and IL-6), and apoptotic markers (Bax, Bcl-2, and caspase 3) were evaluated. We showed that LPS challenge markedly increased the levels of serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and other liver pathological changes in WT mice. In addition, LPS challenge elevated the levels of liver carbonyl proteins and serum inflammatory cytokines, upregulated the expression of TLR4, MyD88, and phosphorylated NF-κB in liver tissues. Moreover, LPS challenge significantly increased hepatocyte apoptosis, caspase 3 activity, and Bax level while suppressing Bcl-2 expression in liver tissues. These pathological changes were greatly attenuated in TLR4 mice. Similar pathological responses were provoked in primary hepatic Kupffer cells isolated from WT and TLR4 mice following LPS (1 μg/mL, 6 h) challenge. In summary, these results demonstrate that silencing of TLR4 attenuates LPS-induced liver injury through inhibition of inflammation and apoptosis via TLR4/MyD88/NF-κB signaling pathway. TLR4 deletion confers hepatoprotection against ALI induced by LPS, possibly by repressing macrophage inflammation and apoptosis.
10.1038/s41401-020-00597-x
Chronic Inflammation in Non-Alcoholic Steatohepatitis: Molecular Mechanisms and Therapeutic Strategies.
Luci Carmelo,Bourinet Manon,Leclère Pierre S,Anty Rodolphe,Gual Philippe
Frontiers in endocrinology
Non-Alcoholic Steatohepatitis (NASH) is the progressive form of Non-Alcoholic Fatty Liver Disease (NAFLD), the main cause of chronic liver complications. The development of NASH is the consequence of aberrant activation of hepatic conventional immune, parenchymal, and endothelial cells in response to inflammatory mediators from the liver, adipose tissue, and gut. Hepatocytes, Kupffer cells and liver sinusoidal endothelial cells contribute to the significant accumulation of bone-marrow derived-macrophages and neutrophils in the liver, a hallmark of NASH. The aberrant activation of these immune cells elicits harmful inflammation and liver injury, leading to NASH progression. In this review, we highlight the processes triggering the recruitment and/or activation of hepatic innate immune cells, with a focus on macrophages, neutrophils, and innate lymphoid cells as well as the contribution of hepatocytes and endothelial cells in driving liver inflammation/fibrosis. On-going studies and preliminary results from global and specific therapeutic strategies to manage this NASH-related inflammation will also be discussed.
10.3389/fendo.2020.597648
Role of immune responses in the development of NAFLD-associated liver cancer and prospects for therapeutic modulation.
Journal of hepatology
The liver is the central metabolic organ of the body, regulating energy and lipid metabolism, while also having potent immunological functions. Overwhelming the metabolic capacity of the liver via obesity and a sedentary lifestyle leads to hepatic lipid accumulation, chronic necro-inflammation, enhanced mitochondrial/endoplasmic reticulum stress and development of non-alcoholic fatty liver disease (NAFLD), and its more severe form non-alcoholic steatohepatitis (NASH). Based on an improved understanding of pathophysiological mechanisms, specifically targeting metabolic pathways to prevent or slow down the progression of NAFLD to liver cancer will become possible. Genetic/environmental factors are also known to contribute to the development of NASH and progression to liver cancer. The complex pathophysiology of NAFLD-NASH is reflected by environmental factors, particularly the gut microbiome and its metabolic products. NAFLD-associated HCC most often occurs in the context of a chronically inflamed and cirrhotic liver. Recognition of environmental alarmins or metabolites derived from the gut microbiota and the metabolically injured liver create a strong inflammatory milieu supported by innate and adaptive immunity. Several recent studies indicate that chronic steatosis induces auto-aggressive CD8+CXCR6+PD1+ T cells that eliminate parenchymal and non-parenchymal cells in an antigen-independent manner. This promotes chronic liver damage and a pro-tumorigenic environment. CD8+CXCR6+PD1+ T cells possess an exhausted, hyperactivated, resident phenotype; they trigger the NASH to HCC transition and might be responsible for weaker responses to immune checkpoint inhibitors - in particular atezolizumab/bevacizumab. Here, we provide an overview of NASH-related inflammation/pathogenesis, focusing on new discoveries on the role of T cells. This review discusses preventive measures to halt disease progression to liver cancer and therapeutic strategies to manage patients with NASH-HCC.
10.1016/j.jhep.2023.02.033
Natural Killer Cells: From Innate to Adaptive Features.
Mujal Adriana M,Delconte Rebecca B,Sun Joseph C
Annual review of immunology
Natural killer (NK) cells are innate lymphocytes that provide critical host defense against pathogens and cancer. Originally heralded for their early and rapid effector activity, NK cells have been recognized over the last decade for their ability to undergo adaptive immune processes, including antigen-driven clonal expansion and generation of long-lived memory. This review presents an overview of how NK cells lithely partake in both innate and adaptive responses and how this versatility is manifest in human NK cell-mediated immunity.
10.1146/annurev-immunol-101819-074948
Regulation of cell polarity by interactions of Msb3 and Msb4 with Cdc42 and polarisome components.
Molecular and cellular biology
In Saccharomyces cerevisiae, polarized growth depends on interactions between the actin cytoskeleton and the secretory machinery. Here we show that the Rab GTPase-activating proteins (GAPs) Msb3 and Msb4 interact directly with Spa2, a scaffold protein of the "polarisome" that also interacts with the formin Bni1. Spa2 is required for the polarized localization of Msb3 and Msb4 at the bud tip. We also show that Msb3 and Msb4 bind specifically to Cdc42-GDP and Rho1-GDP in vitro and that Msb3 and Rho GDP dissociation inhibitor act independently but oppositely on Cdc42. Finally, we show that Msb3 and Msb4 are involved in Bni1-nucleated actin assembly in vivo. These results suggest that Msb3 and Msb4 regulate polarized growth by multiple mechanisms, directly regulating exocytosis through their GAP activity toward Sec4 and potentially coordinating the functions of Cdc42, Rho1, and Bni1 in the polarisome through their binding to these GTPases. A functional equivalent of the polarisome probably exists in other fungi and mammals.
10.1128/MCB.25.19.8567-8580.2005
Structure and mechanism of bactericidal mammalian perforin-2, an ancient agent of innate immunity.
Science advances
Perforin-2 (MPEG1) is thought to enable the killing of invading microbes engulfed by macrophages and other phagocytes, forming pores in their membranes. Loss of perforin-2 renders individual phagocytes and whole organisms significantly more susceptible to bacterial pathogens. Here, we reveal the mechanism of perforin-2 activation and activity using atomic structures of pre-pore and pore assemblies, high-speed atomic force microscopy, and functional assays. Perforin-2 forms a pre-pore assembly in which its pore-forming domain points in the opposite direction to its membrane-targeting domain. Acidification then triggers pore formation, via a 180° conformational change. This novel and unexpected mechanism prevents premature bactericidal attack and may have played a key role in the evolution of all perforin family proteins.
10.1126/sciadv.aax8286
The pore conformation of lymphocyte perforin.
Science advances
Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with proapoptotic granzymes, to bind to a target cell membrane where it oligomerizes and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here, we present a 4-Å resolution cryo-electron microscopy structure of the perforin pore, revealing previously unidentified inter- and intramolecular interactions stabilizing the assembly. During pore formation, the helix-turn-helix motif moves away from the bend in the central β sheet to form an intermolecular contact. Cryo-electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerization and membrane insertion, and explain how several pathogenic mutations affect function.
10.1126/sciadv.abk3147
Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells.
Zhou Zhiwei,He Huabin,Wang Kun,Shi Xuyan,Wang Yupeng,Su Ya,Wang Yao,Li Da,Liu Wang,Zhang Yongliang,Shen Lianjun,Han Weidong,Shen Lin,Ding Jingjin,Shao Feng
Science (New York, N.Y.)
Cytotoxic lymphocyte-mediated immunity relies on granzymes. Granzymes are thought to kill target cells by inducing apoptosis, although the underlying mechanisms are not fully understood. Here, we report that natural killer cells and cytotoxic T lymphocytes kill gasdermin B (GSDMB)-positive cells through pyroptosis, a form of proinflammatory cell death executed by the gasdermin family of pore-forming proteins. Killing results from the cleavage of GSDMB by lymphocyte-derived granzyme A (GZMA), which unleashes its pore-forming activity. Interferon-γ (IFN-γ) up-regulates GSDMB expression and promotes pyroptosis. is highly expressed in certain tissues, particularly digestive tract epithelia, including derived tumors. Introducing GZMA-cleavable GSDMB into mouse cancer cells promotes tumor clearance in mice. This study establishes gasdermin-mediated pyroptosis as a cytotoxic lymphocyte-killing mechanism, which may enhance antitumor immunity.
10.1126/science.aaz7548
Activity of caspase-8 determines plasticity between cell death pathways.
Newton Kim,Wickliffe Katherine E,Maltzman Allie,Dugger Debra L,Reja Rohit,Zhang Yue,Roose-Girma Merone,Modrusan Zora,Sagolla Meredith S,Webster Joshua D,Dixit Vishva M
Nature
Caspase-8 is a protease with both pro-death and pro-survival functions: it mediates apoptosis induced by death receptors such as TNFR1, and suppresses necroptosis mediated by the kinase RIPK3 and the pseudokinase MLKL. Mice that lack caspase-8 display MLKL-dependent embryonic lethality, as do mice that express catalytically inactive CASP8(C362A). Casp8Mlkl mice die during the perinatal period, whereas Casp8Mlkl mice are viable, which indicates that inactive caspase-8 also has a pro-death scaffolding function. Here we show that mutant CASP8(C362A) induces the formation of ASC (also known as PYCARD) specks, and caspase-1-dependent cleavage of GSDMD and caspases 3 and 7 in MLKL-deficient mouse intestines around embryonic day 18. Caspase-1 and its adaptor ASC contributed to the perinatal lethal phenotype because a number of Casp8MlklCasp1 and Casp8MlklAsc mice survived beyond weaning. Transfection studies suggest that inactive caspase-8 adopts a distinct conformation to active caspase-8, enabling its prodomain to engage ASC. Upregulation of the lipopolysaccharide sensor caspase-11 in the intestines of both Casp8Mlkl and Casp8MlklCasp1 mice also contributed to lethality because Casp8MlklCasp1Casp11 (Casp11 is also known as Casp4) neonates survived more often than Casp8MlklCasp1 neonates. Finally, Casp8Ripk3Casp1Casp11 mice survived longer than Casp8MlklCasp1Casp11 mice, indicating that a necroptosis-independent function of RIPK3 also contributes to lethality. Thus, unanticipated plasticity in death pathways is revealed when caspase-8-dependent apoptosis and MLKL-dependent necroptosis are inhibited.
10.1038/s41586-019-1752-8
Caspase-8 inactivation drives autophagy-dependent inflammasome activation in myeloid cells.
Science advances
Caspase-8 activity controls the switch from cell death to pyroptosis when apoptosis and necroptosis are blocked, yet how caspase-8 inactivation induces inflammasome assembly remains unclear. We show that caspase-8 inhibition via IETD treatment in Toll-like receptor (TLR)-primed myeloid cells promoted interleukin-1β (IL-1β) and IL-18 production through inflammasome activation. Caspase-8, caspase-1/11, and functional GSDMD, but not NLRP3 or RIPK1 activity, proved essential for IETD-triggered inflammasome activation. Autophagy became prominent in IETD-treated macrophages, and inhibiting it attenuated IETD-induced cell death and IL-1β/IL-18 production. In contrast, inhibiting GSDMD or autophagy did not prevent IETD-induced septic shock in mice, implying distinct death processes in other cell types. Cathepsin-B contributes to IETD-mediated inflammasome activation, as its inhibition or down-regulation limited IETD-elicited IL-1β production. Therefore, the autophagy and cathepsin-B axis represents one of the pathways leading to atypical inflammasome activation when apoptosis and necroptosis are suppressed and capase-8 is inhibited in myeloid cells.
10.1126/sciadv.abn9912
Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis.
Fritsch Melanie,Günther Saskia D,Schwarzer Robin,Albert Marie-Christine,Schorn Fabian,Werthenbach J Paul,Schiffmann Lars M,Stair Neil,Stocks Hannah,Seeger Jens M,Lamkanfi Mohamed,Krönke Martin,Pasparakis Manolis,Kashkar Hamid
Nature
Caspase-8 is the initiator caspase of extrinsic apoptosis and inhibits necroptosis mediated by RIPK3 and MLKL. Accordingly, caspase-8 deficiency in mice causes embryonic lethality, which can be rescued by deletion of either Ripk3 or Mlkl. Here we show that the expression of enzymatically inactive CASP8(C362S) causes embryonic lethality in mice by inducing necroptosis and pyroptosis. Similar to Casp8 mice, Casp8 mouse embryos died after endothelial cell necroptosis leading to cardiovascular defects. MLKL deficiency rescued the cardiovascular phenotype but unexpectedly caused perinatal lethality in Casp8 mice, indicating that CASP8(C362S) causes necroptosis-independent death at later stages of embryonic development. Specific loss of the catalytic activity of caspase-8 in intestinal epithelial cells induced intestinal inflammation similar to intestinal epithelial cell-specific Casp8 knockout mice. Inhibition of necroptosis by additional deletion of Mlkl severely aggravated intestinal inflammation and caused premature lethality in Mlkl knockout mice with specific loss of caspase-8 catalytic activity in intestinal epithelial cells. Expression of CASP8(C362S) triggered the formation of ASC specks, activation of caspase-1 and secretion of IL-1β. Both embryonic lethality and premature death were completely rescued in Casp8MlklAsc or Casp8MlklCasp1 mice, indicating that the activation of the inflammasome promotes CASP8(C362S)-mediated tissue pathology when necroptosis is blocked. Therefore, caspase-8 represents the molecular switch that controls apoptosis, necroptosis and pyroptosis, and prevents tissue damage during embryonic development and adulthood.
10.1038/s41586-019-1770-6
Pyroptosis and Apoptosis Pathways Engage in Bidirectional Crosstalk in Monocytes and Macrophages.
Taabazuing Cornelius Y,Okondo Marian C,Bachovchin Daniel A
Cell chemical biology
Pyroptosis is a lytic form of programmed cell death mediated by the inflammatory caspase-1, -4, and -5. We recently discovered that small-molecule inhibitors of the serine peptidases DPP8 and DPP9 (DPP8/9) induce pro-caspase-1-dependent pyroptosis in monocytes and macrophages. Notably, DPP8/9 inhibitors, unlike microbial agents, absolutely require caspase-1 to induce cell death. Therefore, DPP8/9 inhibitors are useful probes to study caspase-1 in cells. Here, we show that, in the absence of the pyroptosis-mediating substrate gasdermin D (GSDMD), caspase-1 activates caspase-3 and -7 and induces apoptosis, demonstrating that GSDMD is the only caspase-1 substrate that induces pyroptosis. Conversely, we found that, during apoptosis, caspase-3/-7 specifically block pyroptosis by cleaving GSDMD at a distinct site from the inflammatory caspases that inactivates the protein. Overall, this work reveals bidirectional crosstalk between apoptosis and pyroptosis in monocytes and macrophages, further illuminating the complex interplay between cell death pathways in the innate immune system.
10.1016/j.chembiol.2017.03.009
Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7.
Science advances
Apoptosis is a complex multi-step process driven by caspase-dependent proteolytic cleavage cascades. Dysregulation of apoptosis promotes tumorigenesis and limits the efficacy of chemotherapy. To assess the complex interactions among caspases during apoptosis, we disrupted caspase-8, -9, -3, -7, or -6 and combinations thereof, using CRISPR-based genome editing in living human leukemia cells. While loss of apical initiator caspase-8 or -9 partially blocked extrinsic or intrinsic apoptosis, respectively, only combined loss of caspase-3 and -7 fully inhibited both apoptotic pathways, with no discernible effect of caspase-6 deficiency alone or in combination. Caspase-3/7 double knockout cells exhibited almost complete inhibition of caspase-8 or -9 activation. Furthermore, deletion of caspase-3 and -7 decreased mitochondrial depolarization and cytochrome c release upon apoptosis activation. Thus, activation of effector caspase-3 or -7 sets off explosive feedback amplification of upstream apoptotic events, which is a key feature of apoptotic signaling essential for efficient apoptotic cell death.
10.1126/sciadv.aau9433
Cytochrome c speeds up caspase cascade activation by blocking 14-3-3ε-dependent Apaf-1 inhibition.
Elena-Real Carlos A,Díaz-Quintana Antonio,González-Arzola Katiuska,Velázquez-Campoy Adrián,Orzáez Mar,López-Rivas Abelardo,Gil-Caballero Sergio,De la Rosa Miguel Á,Díaz-Moreno Irene
Cell death & disease
Apoptosis is a highly regulated form of programmed cell death, essential to the development and homeostasis of multicellular organisms. Cytochrome c is a central figure in the activation of the apoptotic intrinsic pathway, thereby activating the caspase cascade through its interaction with Apaf-1. Our recent studies have revealed 14-3-3ε (a direct inhibitor of Apaf-1) as a cytosolic cytochrome c target. Here we explore the cytochrome c / 14-3-3ε interaction and show the ability of cytochrome c to block 14-3-3ε-mediated Apaf-1 inhibition, thereby unveiling a novel function for cytochrome c as an indirect activator of caspase-9/3. We have used calorimetry, NMR spectroscopy, site mutagenesis and computational calculations to provide an insight into the structural features of the cytochrome c / 14-3-3ε complex. Overall, these findings suggest an additional cytochrome c-mediated mechanism to modulate apoptosome formation, shedding light onto the rigorous apoptotic regulation network.
10.1038/s41419-018-0408-1
Caspase-9 activation results in downstream caspase-8 activation and bid cleavage in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease.
The Journal of neuroscience : the official journal of the Society for Neuroscience
Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity are both associated with dopaminergic neuron death in the substantia nigra (SN). Apoptosis has been implicated in this cell loss; however, whether or not it is a major component of disease pathology remains controversial. Caspases are a major class of proteases involved in the apoptotic process. To evaluate the role of caspases in PD, we analyzed caspase activation in MPTP-treated mice, in cultured dopaminergic cells, and in postmortem PD brain tissue. MPTP was found to elicit not only the activation of the effector caspase-3 but also the initiators caspase-8 and caspase-9, mitochondrial cytochrome c release, and Bid cleavage in the SN of wild-type mice. These changes were attenuated in transgenic mice neuronally expressing the general caspase inhibitor protein baculoviral p35. These mice also displayed increased resistance to the cytotoxic effects of the drug. MPTP-associated toxicity in culture was found temporally to involve cytochrome c release, activation of caspase-9, caspase-3, and caspase-8, and Bid cleavage. Caspase-9 inhibition prevented the activation of both caspase-3 and caspase-8 and also inhibited Bid cleavage, but not cytochrome c release. Activated caspase-8 and caspase-9 were immunologically detectable within MPP(+)-treated mesencephalic dopaminergic neurons, dopaminergic nigral neurons from MPTP-treated mice, and autopsied Parkinsonian tissue from late-onset sporadic cases of the disease. These data demonstrate that MPTP-mediated activation of caspase-9 via cytochrome c release results in the activation of caspase-8 and Bid cleavage, which we speculate may be involved in the amplification of caspase-mediated dopaminergic cell death. These data suggest that caspase inhibitors constitute a plausible therapeutic for PD.
No longer married to inflammasome signaling: the diverse interacting pathways leading to pyroptotic cell death.
The Biochemical journal
For over 15 years the lytic cell death termed pyroptosis was defined by its dependency on the inflammatory caspase, caspase-1, which, upon pathogen sensing, is activated by innate immune cytoplasmic protein complexes known as inflammasomes. However, this definition of pyroptosis changed when the pore-forming protein gasdermin D (GSDMD) was identified as the caspase-1 (and caspase-11) substrate required to mediate pyroptotic cell death. Consequently, pyroptosis has been redefined as a gasdermin-dependent cell death. Studies now show that, upon liberation of the N-terminal domain, five gasdermin family members, GSDMA, GSDMB, GSDMC, GSDMD and GSDME can all form plasma membrane pores to induce pyroptosis. Here, we review recent research into the diverse stimuli and cell death signaling pathways involved in the activation of gasdermins; death and toll-like receptor triggered caspase-8 activation of GSDMD or GSMDC, apoptotic caspase-3 activation of GSDME, perforin-granzyme A activation of GSDMB, and bacterial protease activation of GSDMA. We highlight findings that have begun to unravel the physiological situations and disease states that result from gasdermin signaling downstream of inflammasome activation, death receptor and mitochondrial apoptosis, and necroptosis. This new era in cell death research therefore holds significant promise in identifying how distinct, yet often networked, pyroptotic cell death pathways might be manipulated for therapeutic benefit to treat a range of malignant conditions associated with inflammation, infection and cancer.
10.1042/BCJ20210711
Caspase cleavage of RIPK3 after Asp is dispensable for mouse embryogenesis.
Cell death and differentiation
The proteolytic activity of caspase-8 suppresses lethal RIPK1-, RIPK3- and MLKL-dependent necroptosis during mouse embryogenesis. Caspase-8 is reported to cleave RIPK3 in addition to the RIPK3-interacting kinase RIPK1, but whether cleavage of RIPK3 is crucial for necroptosis suppression is unclear. Here we show that caspase-8-driven cleavage of endogenous mouse RIPK3 after Asp is dependent on downstream caspase-3. Consistent with RIPK3 cleavage being a consequence of apoptosis rather than a critical brake on necroptosis, Ripk3 knock-in mice lacking the Asp cleavage site are viable and develop normally. Moreover, in contrast to mice lacking caspase-8 in their intestinal epithelial cells, Ripk3 mice do not exhibit increased sensitivity to high dose tumor necrosis factor (TNF). Ripk3 macrophages died at the same rate as wild-type (WT) macrophages in response to TNF plus cycloheximide, TNF plus emricasan, or infection with murine cytomegalovirus (MCMV) lacking M36 and M45 to inhibit caspase-8 and RIPK3 activation, respectively. We conclude that caspase cleavage of RIPK3 is dispensable for mouse development, and that cleavage of caspase-8 substrates, including RIPK1, is sufficient to prevent necroptosis.
10.1038/s41418-023-01255-5
Death receptor-induced signaling pathways are differentially regulated by gamma interferon upstream of caspase 8 processing.
Siegmund Daniela,Wicovsky Andreas,Schmitz Ingo,Schulze-Osthoff Klaus,Kreuz Sebastian,Leverkus Martin,Dittrich-Breiholz Oliver,Kracht Michael,Wajant Harald
Molecular and cellular biology
FasL and gamma interferon (IFN-gamma) are produced by activated T cells and NK cells and synergistically induce apoptosis. Although both cytokines can also elicit proinflammatory responses, a possible cross talk of these ligands with respect to nonapoptotic signaling has been poorly addressed. Here, we show that IFN-gamma sensitizes KB cells for apoptosis induction by facilitating death-inducing signaling complex (DISC)-mediated caspase 8 processing. Moreover, after protection against death receptor-induced apoptosis by caspase inhibition or Bcl2 overexpression, IFN-gamma also sensitized for Fas- and TRAIL death receptor-mediated NF-kappaB activation leading to synergistic upregulation of a variety of proinflammatory genes. In contrast, Fas-mediated activation of JNK, p38, and p42/44 occurred essentially independent from IFN-gamma sensitization, indicating that the apoptosis- and NF-kappaB-related FasL-IFN-gamma cross talk was not due to a simple global enhancement of Fas signaling. Overexpression of FLIP(L) and FLIP(S) inhibited Fas- as well as TRAIL-mediated NF-kappaB activation and apoptosis induction in IFN-gamma-primed cells suggesting that both responses are coregulated at the level of the DISC.
10.1128/MCB.25.15.6363-6379.2005
The metabolite α-KG induces GSDMC-dependent pyroptosis through death receptor 6-activated caspase-8.
Cell research
Pyroptosis is a form of regulated cell death mediated by gasdermin family members, among which the function of GSDMC has not been clearly described. Herein, we demonstrate that the metabolite α-ketoglutarate (α-KG) induces pyroptosis through caspase-8-mediated cleavage of GSDMC. Treatment with DM-αKG, a cell-permeable derivative of α-KG, elevates ROS levels, which leads to oxidation of the plasma membrane-localized death receptor DR6. Oxidation of DR6 triggers its endocytosis, and then recruits both pro-caspase-8 and GSDMC to a DR6 receptosome through protein-protein interactions. The DR6 receptosome herein provides a platform for the cleavage of GSDMC by active caspase-8, thereby leading to pyroptosis. Moreover, this α-KG-induced pyroptosis could inhibit tumor growth and metastasis in mouse models. Interestingly, the efficiency of α-KG in inducing pyroptosis relies on an acidic environment in which α-KG is reduced by MDH1 and converted to L-2HG that further boosts ROS levels. Treatment with lactic acid, the end product of glycolysis, builds an improved acidic environment to facilitate more production of L-2HG, which makes the originally pyroptosis-resistant cancer cells more susceptible to α-KG-induced pyroptosis. This study not only illustrates a pyroptotic pathway linked with metabolites but also identifies an unreported principal axis extending from ROS-initiated DR6 endocytosis to caspase-8-mediated cleavage of GSDMC for potential clinical application in tumor therapy.
10.1038/s41422-021-00506-9
Role of pyroptosis in inflammation and cancer.
Cellular & molecular immunology
Pyroptosis is a form of programmed cell death mediated by gasdermin and is a product of continuous cell expansion until the cytomembrane ruptures, resulting in the release of cellular contents that can activate strong inflammatory and immune responses. Pyroptosis, an innate immune response, can be triggered by the activation of inflammasomes by various influencing factors. Activation of these inflammasomes can induce the maturation of caspase-1 or caspase-4/5/11, both of which cleave gasdermin D to release its N-terminal domain, which can bind membrane lipids and perforate the cell membrane. Here, we review the latest advancements in research on the mechanisms of pyroptosis, newly discovered influencing factors, antitumoral properties, and applications in various diseases. Moreover, this review also provides updates on potential targeted therapies for inflammation and cancers, methods for clinical prevention, and finally challenges and future directions in the field.
10.1038/s41423-022-00905-x
Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival.
Gurcel Laure,Abrami Laurence,Girardin Stephen,Tschopp Jurg,van der Goot F Gisou
Cell
Many pathogenic organisms produce pore-forming toxins as virulence factors. Target cells however mount a response to such membrane damage. Here we show that toxin-induced membrane permeabilization leads to a decrease in cytoplasmic potassium, which promotes the formation of a multiprotein oligomeric innate immune complex, called the inflammasome, and the activation of caspase-1. Further, we find that when rendered proteolytic in this context caspase-1 induces the activation of the central regulators of membrane biogenesis, the Sterol Regulatory Element Binding Proteins (SREBPs), which in turn promote cell survival upon toxin challenge possibly by facilitating membrane repair. This study highlights that, in addition to its well-established role in triggering inflammation via the processing of the precursor forms of interleukins, caspase-1 has a broader role, in particular linking the intracellular ion composition to lipid metabolic pathways, membrane biogenesis, and survival.
10.1016/j.cell.2006.07.033
The NLRP3 inflammasome: activation and regulation.
Trends in biochemical sciences
The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome is a cytoplasmic supramolecular complex that is activated in response to cellular perturbations triggered by infection and sterile injury. Assembly of the NLRP3 inflammasome leads to activation of caspase-1, which induces the maturation and release of interleukin-1β (IL-1β) and IL-18, as well as cleavage of gasdermin D (GSDMD), which promotes a lytic form of cell death. Production of IL-1β via NLRP3 can contribute to the pathogenesis of inflammatory disease, whereas aberrant IL-1β secretion through inherited NLRP3 mutations causes autoinflammatory disorders. In this review, we discuss recent developments in the structure of the NLRP3 inflammasome, and the cellular processes and signaling events controlling its assembly and activation.
10.1016/j.tibs.2022.10.002
Gasdermin D pore structure reveals preferential release of mature interleukin-1.
Nature
As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1β and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-1. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1β and IL-18 over that of their precursors. Consistent with these findings, living-but not pyroptotic-macrophages preferentially release mature IL-1β upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.
10.1038/s41586-021-03478-3
NLRP3 inflammasome activation and cell death.
Cellular & molecular immunology
The NLRP3 inflammasome is a cytosolic multiprotein complex composed of the innate immune receptor protein NLRP3, adapter protein ASC, and inflammatory protease caspase-1 that responds to microbial infection, endogenous danger signals, and environmental stimuli. The assembled NLRP3 inflammasome can activate the protease caspase-1 to induce gasdermin D-dependent pyroptosis and facilitate the release of IL-1β and IL-18, which contribute to innate immune defense and homeostatic maintenance. However, aberrant activation of the NLRP3 inflammasome is associated with the pathogenesis of various inflammatory diseases, such as diabetes, cancer, and Alzheimer's disease. Recent studies have revealed that NLRP3 inflammasome activation contributes to not only pyroptosis but also other types of cell death, including apoptosis, necroptosis, and ferroptosis. In addition, various effectors of cell death have been reported to regulate NLRP3 inflammasome activation, suggesting that cell death is closely related to NLRP3 inflammasome activation. In this review, we summarize the inextricable link between NLRP3 inflammasome activation and cell death and discuss potential therapeutics that target cell death effectors in NLRP3 inflammasome-associated diseases.
10.1038/s41423-021-00740-6
Parthenolide targets NLRP3 to treat inflammasome-related diseases.
International immunopharmacology
Natural products have attracted extensive attention from researchers in medical fields due to their abundant biological activities. Parthenolide (PTL) is a sesquiterpene lactone originally purified from herb Feverfew (Tanacetum parthenium), recent studies have showed its potential activities of anti-cancer and anti-inflammatory. Acting as the most studied inflammasome, NLRP3 inflammasome played an important role in human diseases including type-2 diabetes (T2D), Alzheimer's disease (AD) and cryopyrin-associated periodic syndromes (CAPS). In this article, we show that PTL specially inhibits the activation of NLRP3 inflammation by block the upstream signal and prevent the assembly of NLRP3 inflammasome complex. Furthermore, we showed the treatment of PTL significantly attenuates the symptoms of lipopolysaccharide (LPS)-induced systemic inflammation and dextran sulfate sodium (DSS)-induced colitis in mice models. Thus, our results demonstrate that PTL alleviates inflammation by targeting NLRP3 inflammasome, which indicate that PTL acting as a promising natural product for the treatment of NLRP3 inflammasome-related diseases.
10.1016/j.intimp.2023.110229
Propofol directly induces caspase-1-dependent macrophage pyroptosis through the NLRP3-ASC inflammasome.
Sun Lingbin,Ma Wei,Gao Wenli,Xing Yanmei,Chen Lixin,Xia Zhengyuan,Zhang Zhongjun,Dai Zhongliang
Cell death & disease
Propofol infusion syndrome (PRIS) is an uncommon life-threatening complication observed most often in patients receiving high-dose propofol. High-dose propofol treatment with a prolonged duration can damage the immune system. However, the associated molecular mechanisms remain unclear. An increasing number of clinical and experimental observations have demonstrated that tissue-resident macrophages play a critical role in immune regulation during anaesthesia and procedural sedation. Since the inflammatory response is essential for mediating propofol-induced cell death and proinflammatory reactions, we hypothesised that propofol overdose induces macrophage pyroptosis through inflammasomes. Using primary cultured bone marrow-derived macrophages, murine macrophage cell lines (RAW264.7, RAW-asc and J774) and a mouse model, we investigated the role of NLRP3 inflammasome activation and secondary pyroptosis in propofol-induced cell death. We found that high-dose propofol strongly cleaved caspase-1 but not caspase-11 and biosynthesis of downstream interleukin (IL)-1β and IL-18. Inhibition of caspase-1 activity blocks IL-1β production. Moreover, NLRP3 deletion moderately suppressed cleaved caspase-1 as well as the proportion of pyroptosis, while levels of AIM2 were increased, triggering a compensatory pathway to pyroptosis in NLRP3 macrophages. Here, we show that propofol-induced mitochondrial reactive oxygen species (ROS) can trigger NLRP3 inflammasome activation. Furthermore, apoptosis-associated speck-like protein (ASC) was found to mediate NLRP3 and AIM2 signalling and contribute to propofol-induced macrophage pyroptosis. In addition, our work shows that propofol-induced apoptotic initiator caspase (caspase-9) subsequently cleaved effector caspases (caspase-3 and 7), indicating that both apoptotic and pyroptotic cellular death pathways are activated after propofol exposure. Our studies suggest, for the first time, that propofol-induced pyroptosis might be restricted to macrophage through an NLRP3/ASC/caspase-1 pathway, which provides potential targets for limiting adverse reactions during propofol application. These findings demonstrate that propofol overdose can trigger cell death through caspase-1 activation and offer new insights into the use of anaesthetic drugs.
10.1038/s41419-019-1761-4
Digital signaling network drives the assembly of the AIM2-ASC inflammasome.
Matyszewski Mariusz,Morrone Seamus R,Sohn Jungsan
Proceedings of the National Academy of Sciences of the United States of America
The AIM2-ASC inflammasome is a filamentous signaling platform essential for mounting host defense against cytoplasmic dsDNA arising not only from invading pathogens but also from damaged organelles. Currently, the design principles of its underlying signaling network remain poorly understood at the molecular level. We show here that longer dsDNA is more effective in inducing AIM2 assembly, its self-propagation, and downstream ASC polymerization. This observation is related to the increased probability of forming the base of AIM2 filaments, and indicates that the assembly discerns small dsDNA as noise at each signaling step. Filaments assembled by receptor AIM2, downstream ASC, and their joint complex all persist regardless of dsDNA, consequently generating sustained signal amplification and hysteresis. Furthermore, multiple positive feedback loops reinforce the assembly, as AIM2 and ASC filaments accelerate the assembly of nascent AIM2 with or without dsDNA. Together with a quantitative model of the assembly, our results indicate that an ultrasensitive digital circuit drives the assembly of the AIM2-ASC inflammasome.
10.1073/pnas.1712860115
AIM2 Inflammasome's First Decade of Discovery: Focus on Oral Diseases.
Wang Lufei,Sun Lu,Byrd Kevin M,Ko Ching-Chang,Zhao Zhenxing,Fang Jie
Frontiers in immunology
A common feature of many acute and chronic oral diseases is microbial-induced inflammation. Innate immune responses are the first line of defense against pathogenic microorganisms and are initiated by pattern recognition receptors (PRRs) that specifically recognize pathogen-associated molecular patterns and danger-associated molecular patterns. The activation of certain PRRs can lead to the assembly of macromolecular oligomers termed , which are responsible for pro-inflammatory cytokine maturation and secretion and thus activate host inflammatory responses. About 10 years ago, the absent in melanoma 2 (AIM2) was independently discovered by four research groups, and among the "canonical" inflammasomes [including AIM2, NLR family pyrin domain (NLRP)1, NLRP3, NLR family apoptosis inhibitory protein (NAIP)/NLR family, caspase activation and recruitment domain (CARD) containing (NLRC)4, and pyrin], AIM2 so far is the only one that simultaneously acts as a cytosolic DNA sensor due to its DNA-binding ability. Undoubtedly, such a double-faceted role gives AIM2 greater mission and more potential in the mediation of innate immune responses. Therefore, AIM2 has garnered much attention from the broad scientific community during its first 10 years of discovery (2009-2019). How the AIM2 inflammasome is related to oral diseases has aroused debate over the past few years and is under active investigation. AIM2 inflammasome may potentially be a key link between oral diseases and innate immunity. In this review, we highlight the current knowledge of the AIM2 inflammasome and its critical role in the pathogenesis of various oral diseases, which might offer future possibilities for disease prevention and targeted therapy utilizing this continued understanding.
10.3389/fimmu.2020.01487
Structural Mechanisms of NLRP3 Inflammasome Assembly and Activation.
Annual review of immunology
As an important sensor in the innate immune system, NLRP3 detects exogenous pathogenic invasions and endogenous cellular damage and responds by forming the NLRP3 inflammasome, a supramolecular complex that activates caspase-1. The three major components of the NLRP3 inflammasome are NLRP3, which captures the danger signals and recruits downstream molecules; caspase-1, which elicits maturation of the cytokines IL-1β and IL-18 and processing of gasdermin D to mediate cytokine release and pyroptosis; and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), which functions as a bridge connecting NLRP3 and caspase-1. In this article, we review the structural information that has been obtained on the NLRP3 inflammasome and its components or subcomplexes, with special focus on the inactive NLRP3 cage, the active NLRP3-NEK7 (NIMA-related kinase 7)-ASC inflammasome disk, and the PYD-PYD and CARD-CARD homotypic filamentous scaffolds of the inflammasome. We further implicate structure-derived mechanisms for the assembly and activation of the NLRP3 inflammasome.
10.1146/annurev-immunol-081022-021207
The NLRP3 inflammasome: molecular activation and regulation to therapeutics.
Nature reviews. Immunology
NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) is an intracellular sensor that detects a broad range of microbial motifs, endogenous danger signals and environmental irritants, resulting in the formation and activation of the NLRP3 inflammasome. Assembly of the NLRP3 inflammasome leads to caspase 1-dependent release of the pro-inflammatory cytokines IL-1β and IL-18, as well as to gasdermin D-mediated pyroptotic cell death. Recent studies have revealed new regulators of the NLRP3 inflammasome, including new interacting or regulatory proteins, metabolic pathways and a regulatory mitochondrial hub. In this Review, we present the molecular, cell biological and biochemical bases of NLRP3 activation and regulation and describe how this mechanistic understanding is leading to potential therapeutics that target the NLRP3 inflammasome.
10.1038/s41577-019-0165-0
Decoding Toll-like receptors: Recent insights and perspectives in innate immunity.
Immunity
Toll-like receptors (TLRs) are an evolutionarily conserved family in the innate immune system and are the first line of host defense against microbial pathogens by recognizing pathogen-associated molecular patterns (PAMPs). TLRs, categorized into cell surface and endosomal subfamilies, recognize diverse PAMPs, and structural elucidation of TLRs and PAMP complexes has revealed their intricate mechanisms. TLRs activate common and specific signaling pathways to shape immune responses. Recent studies have shown the importance of post-transcriptional regulation in TLR-mediated inflammatory responses. Despite their protective functions, aberrant responses of TLRs contribute to inflammatory and autoimmune disorders. Understanding the delicate balance between TLR activation and regulatory mechanisms is crucial for deciphering their dual role in immune defense and disease pathogenesis. This review provides an overview of recent insights into the history of TLR discovery, elucidation of TLR ligands and signaling pathways, and their relevance to various diseases.
10.1016/j.immuni.2024.03.004
Cryo-EM structures of the ATP release channel pannexin 1.
Deng Zengqin,He Zhihui,Maksaev Grigory,Bitter Ryan M,Rau Michael,Fitzpatrick James A J,Yuan Peng
Nature structural & molecular biology
The plasma membrane adenosine triphosphate (ATP) release channel pannexin 1 (PANX1) has been implicated in many physiological and pathophysiological processes associated with purinergic signaling, including cancer progression, apoptotic cell clearance, inflammation, blood pressure regulation, oocyte development, epilepsy and neuropathic pain. Here we present near-atomic-resolution structures of human and frog PANX1 determined by cryo-electron microscopy that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the size of permeable substrates. The amino and carboxyl termini, not resolved in the density map, appeared to be structurally dynamic and might contribute to narrowing of the pore during channel gating. In combination with functional characterization, this work elucidates the previously unknown architecture of pannexin channels and establishes a foundation for understanding their unique channel properties.
10.1038/s41594-020-0401-0
ATP transporters in the joints.
Purinergic signalling
Extracellular adenosine triphosphate (ATP) plays a central role in a wide variety of joint diseases. ATP is generated intracellularly, and the concentration of the extracellular ATP pool is determined by the regulation of its transport out of the cell. A variety of ATP transporters have been described, with connexins and pannexins the most commonly cited. Both form intercellular channels, known as gap junctions, that facilitate the transport of various small molecules between cells and mediate cell-cell communication. Connexins and pannexins also form pores, or hemichannels, that are permeable to certain molecules, including ATP. All joint tissues express one or more connexins and pannexins, and their expression is altered in some pathological conditions, such as osteoarthritis (OA) and rheumatoid arthritis (RA), indicating that they may be involved in the onset and progression of these pathologies. The aging of the global population, along with increases in the prevalence of obesity and metabolic dysfunction, is associated with a rising frequency of joint diseases along with the increased costs and burden of related illness. The modulation of connexins and pannexins represents an attractive therapeutic target in joint disease, but their complex regulation, their combination of gap-junction-dependent and -independent functions, and their interplay between gap junction and hemichannel formation are not yet fully elucidated. In this review, we try to shed light on the regulation of these proteins and their roles in ATP transport to the extracellular space in the context of joint disease, and specifically OA and RA.
10.1007/s11302-021-09810-w
A conserved family of immune effectors cleaves cellular ATP upon viral infection.
Cell
During viral infection, cells can deploy immune strategies that deprive viruses of molecules essential for their replication. Here, we report a family of immune effectors in bacteria that, upon phage infection, degrade cellular adenosine triphosphate (ATP) and deoxyadenosine triphosphate (dATP) by cleaving the N-glycosidic bond between the adenine and sugar moieties. These ATP nucleosidase effectors are widely distributed within multiple bacterial defense systems, including cyclic oligonucleotide-based antiviral signaling systems (CBASS), prokaryotic argonautes, and nucleotide-binding leucine-rich repeat (NLR)-like proteins, and we show that ATP and dATP degradation during infection halts phage propagation. By analyzing homologs of the immune ATP nucleosidase domain, we discover and characterize Detocs, a family of bacterial defense systems with a two-component phosphotransfer-signaling architecture. The immune ATP nucleosidase domain is also encoded within diverse eukaryotic proteins with immune-like architectures, and we show biochemically that eukaryotic homologs preserve the ATP nucleosidase activity. Our findings suggest that ATP and dATP degradation is a cell-autonomous innate immune strategy conserved across the tree of life.
10.1016/j.cell.2023.07.020
Extrinsic and intrinsic apoptosis activate pannexin-1 to drive NLRP3 inflammasome assembly.
Chen Kaiwen W,Demarco Benjamin,Heilig Rosalie,Shkarina Kateryna,Boettcher Andreas,Farady Christopher J,Pelczar Pawel,Broz Petr
The EMBO journal
Pyroptosis is a form of lytic inflammatory cell death driven by inflammatory caspase-1, caspase-4, caspase-5 and caspase-11. These caspases cleave and activate the pore-forming protein gasdermin D (GSDMD) to induce membrane damage. By contrast, apoptosis is driven by apoptotic caspase-8 or caspase-9 and has traditionally been classified as an immunologically silent form of cell death. Emerging evidence suggests that therapeutics designed for cancer chemotherapy or inflammatory disorders such as SMAC mimetics, TAK1 inhibitors and BH3 mimetics promote caspase-8 or caspase-9-dependent inflammatory cell death and NLRP3 inflammasome activation. However, the mechanism by which caspase-8 or caspase-9 triggers cell lysis and NLRP3 activation is still undefined. Here, we demonstrate that during extrinsic apoptosis, caspase-1 and caspase-8 cleave GSDMD to promote lytic cell death. By engineering a novel D88A knock-in mouse, we further demonstrate that this proinflammatory function of caspase-8 is counteracted by caspase-3-dependent cleavage and inactivation of GSDMD at aspartate 88, and is essential to suppress GSDMD-dependent cell lysis during caspase-8-dependent apoptosis. Lastly, we provide evidence that channel-forming glycoprotein pannexin-1, but not GSDMD or GSDME promotes NLRP3 inflammasome activation during caspase-8 or caspase-9-dependent apoptosis.
10.15252/embj.2019101638
Gasdermins and pannexin-1 mediate pathways of chemotherapy-induced cell lysis in hematopoietic malignancies.
Science signaling
Pyroptosis is a mechanism of programmed, necrotic cell death mediated by gasdermins, a family of pore-forming proteins. Caspase-1 activates gasdermin D (GSDMD) under inflammatory conditions, whereas caspase-3 activates GSDME under apoptotic conditions, such as those induced by chemotherapy. These pathways are thought to be separate. However, we found that they are part of an integrated network of gatekeepers that enables pyroptotic cell death. We observed that GSDMD was the primary pyroptotic mediator in cultured blood cells in response to doxorubicin and etoposide, two common chemotherapies for hematopoietic malignancies. Upon treatment, the channel protein pannexin-1 (PANX1), which is stimulated by the initiation of apoptosis, increased membrane permeability to induce K efflux-driven activation of the NLRP3 inflammasome and GSDMD. However, either PANX1 or GSDME could also be the primary mediator of chemotherapy-induced pyroptosis when present at higher amounts. The most abundant pore-forming protein in acute myeloid leukemias from patients predicted the cell death pathway in response to chemotherapy. This interconnected network, a multistep switch that converts apoptosis to pyroptosis, could be clinically titratated to modulate cell death with regard to antitumor immunity or tumor lysis syndrome in patients.
10.1126/scisignal.abl6781
Caspase-8-driven apoptotic and pyroptotic crosstalk causes cell death and IL-1β release in X-linked inhibitor of apoptosis (XIAP) deficiency.
The EMBO journal
Genetic lesions in X-linked inhibitor of apoptosis (XIAP) pre-dispose humans to cell death-associated inflammatory diseases, although the underlying mechanisms remain unclear. Here, we report that two patients with XIAP deficiency-associated inflammatory bowel disease display increased inflammatory IL-1β maturation as well as cell death-associated caspase-8 and Gasdermin D (GSDMD) processing in diseased tissue, which is reduced upon patient treatment. Loss of XIAP leads to caspase-8-driven cell death and bioactive IL-1β release that is only abrogated by combined deletion of the apoptotic and pyroptotic cell death machinery. Namely, extrinsic apoptotic caspase-8 promotes pyroptotic GSDMD processing that kills macrophages lacking both inflammasome and apoptosis signalling components (caspase-1, -3, -7, -11 and BID), while caspase-8 can still cause cell death in the absence of both GSDMD and GSDME when caspase-3 and caspase-7 are present. Neither caspase-3 and caspase-7-mediated activation of the pannexin-1 channel, or GSDMD loss, prevented NLRP3 inflammasome assembly and consequent caspase-1 and IL-1β maturation downstream of XIAP inhibition and caspase-8 activation, even though the pannexin-1 channel was required for NLRP3 triggering upon mitochondrial apoptosis. These findings uncouple the mechanisms of cell death and NLRP3 activation resulting from extrinsic and intrinsic apoptosis signalling, reveal how XIAP loss can co-opt dual cell death programs, and uncover strategies for targeting the cell death and inflammatory pathways that result from XIAP deficiency.
10.15252/embj.2021110468
PANX1-mediated ATP release confers FAM3A's suppression effects on hepatic gluconeogenesis and lipogenesis.
Military Medical Research
BACKGROUND:Extracellular adenosine triphosphate (ATP) is an important signal molecule. In previous studies, intensive research had revealed the crucial roles of family with sequence similarity 3 member A (FAM3A) in controlling hepatic glucolipid metabolism, islet β cell function, adipocyte differentiation, blood pressure, and other biological and pathophysiological processes. Although mitochondrial protein FAM3A plays crucial roles in the regulation of glucolipid metabolism via stimulating ATP release to activate P2 receptor pathways, its mechanism in promoting ATP release in hepatocytes remains unrevealed. METHODS:db/db, high-fat diet (HFD)-fed, and global pannexin 1 (PANX1) knockout mice, as well as liver sections of individuals, were used in this study. Adenoviruses and adeno-associated viruses were utilized for in vivo gene overexpression or inhibition. To evaluate the metabolic status in mice, oral glucose tolerance test (OGTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT), and magnetic resonance imaging (MRI) were conducted. Protein-protein interactions were determined by coimmunoprecipitation with mass spectrometry (MS) assays. RESULTS:In livers of individuals and mice with steatosis, the expression of ATP-permeable channel PANX1 was increased (P < 0.01). Hepatic PANX1 overexpression ameliorated the dysregulated glucolipid metabolism in obese mice. Mice with hepatic PANX1 knockdown or global PANX1 knockout exhibited disturbed glucolipid metabolism. Restoration of hepatic PANX1 rescued the metabolic disorders of PANX1-deficient mice (P < 0.05). Mechanistically, ATP release is mediated by the PANX1-activated protein kinase B-forkhead box protein O1 (Akt-FOXO1) pathway to inhibit gluconeogenesis via P2Y receptors in hepatocytes. PANX1-mediated ATP release also activated calmodulin (CaM) (P < 0.01), which interacted with c-Jun N-terminal kinase (JNK) to inhibit its activity, thereby deactivating the transcription factor activator protein-1 (AP1) and repressing fatty acid synthase (FAS) expression and lipid synthesis (P < 0.05). FAM3A stimulated the expression of PANX1 via heat shock factor 1 (HSF1) in hepatocytes (P < 0.05). Notably, FAM3A overexpression failed to promote ATP release, inhibit the expression of gluconeogenic and lipogenic genes, and suppress gluconeogenesis and lipid deposition in PANX1-deficient hepatocytes and livers. CONCLUSIONS:PANX1-mediated release of ATP plays a crucial role in maintaining hepatic glucolipid homeostasis, and it confers FAM3A's suppressive effects on hepatic gluconeogenesis and lipogenesis.
10.1186/s40779-024-00543-6
Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells.
Csak Timea,Ganz Michal,Pespisa Justin,Kodys Karen,Dolganiuc Angela,Szabo Gyongyi
Hepatology (Baltimore, Md.)
UNLABELLED:The pathogenesis of nonalcoholic steatohepatitis (NASH) and inflammasome activation involves sequential hits. The inflammasome, which cleaves pro-interleukin-1β (pro-IL-1β) into secreted IL-1β, is induced by endogenous and exogenous danger signals. Lipopolysaccharide (LPS), a toll-like receptor 4 ligand, plays a role in NASH and also activates the inflammasome. In this study, we hypothesized that the inflammasome is activated in NASH by multiple hits involving endogenous and exogenous danger signals. Using mouse models of methionine choline-deficient (MCD) diet-induced NASH and high-fat diet-induced NASH, we found up-regulation of the inflammasome [including NACHT, LRR, and PYD domains-containing protein 3 (NALP3; cryopyrin), apoptosis-associated speck-like CARD-domain containing protein, pannexin-1, and pro-caspase-1] at the messenger RNA (mRNA) level increased caspase-1 activity, and mature IL-1β protein levels in mice with steatohepatitis in comparison with control livers. There was no inflammasome activation in mice with only steatosis. The MCD diet sensitized mice to LPS-induced increases in NALP3, pannexin-1, IL-1β mRNA, and mature IL-1β protein levels in the liver. We demonstrate for the first time that inflammasome activation occurs in isolated hepatocytes in steatohepatitis. Our novel data show that the saturated fatty acid (FA) palmitic acid (PA) activates the inflammasome and induces sensitization to LPS-induced IL-1β release in hepatocytes. Furthermore, PA triggers the release of danger signals from hepatocytes in a caspase-dependent manner. These hepatocyte-derived danger signals, in turn, activate inflammasome, IL-1β, and tumor necrosis factor α release in liver mononuclear cells. CONCLUSION:Our novel findings indicate that saturated FAs represent an endogenous danger in the form of a first hit, up-regulate the inflammasome in NASH, and induce sensitization to a second hit with LPS for IL-β release in hepatocytes. Furthermore, hepatocytes exposed to saturated FAs release danger signals that trigger inflammasome activation in immune cells. Thus, hepatocytes play a key role in orchestrating tissue responses to danger signals in NASH.
10.1002/hep.24341
Pannexin1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated free fatty acids in liver cells.
Xiao Feng,Waldrop Shar L,Khimji Al-karim,Kilic Gordan
American journal of physiology. Cell physiology
Hepatocyte lipoapoptosis induced by saturated free fatty acids (FFA) contributes to hepatic inflammation in lipotoxic liver injury, and the cellular mechanisms involved have not been defined. Recent studies have shown that apoptosis in nonhepatic cells stimulates ATP release via activation of pannexin1 (panx1), and extracellular ATP functions as a proinflammatory signal for recruitment and activation of the inflammatory cells. However, it is not known whether lipoapoptosis stimulates ATP release in liver cells. We found that lipoapoptosis induced by saturated FFA stimulated ATP release in liver cells that increased extracellular ATP concentration by more than fivefold above the values observed in healthy cells. This sustained pathophysiological ATP release was not dependent on caspase-3/7 activation. Inhibition of c-Jun NH(2)-terminal kinase (JNK), a key mediator of lipoapoptosis, with SP600125 blocked pathophysiological ATP release in a dose-dependent manner. RT-PCR analysis indicated that panx1 is expressed in hepatocytes and multiple liver cell lines. Notably, inhibition of panx1 expression with short hairpin (sh)RNA inhibited in part pathophysiological ATP release. Moreover, lipoapoptosis stimulated uptake of a membrane impermeable dye YoPro-1 (indicative of panx1 activation), which was inhibited by panx1 shRNA, probenecid, and mefloquine. These results suggest that panx1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated FFA. Thus panx1 may play an important role in hepatic inflammation by mediating an increase in extracellular ATP concentration in lipotoxic liver injury.
10.1152/ajpcell.00175.2012
Structures of human pannexin 1 reveal ion pathways and mechanism of gating.
Nature
Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation, apoptotic cell clearance and human oocyte development. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.
10.1038/s41586-020-2357-y
Cryo-EM structures of pannexin 1 and 3 reveal differences among pannexin isoforms.
Nature communications
Pannexins are single-membrane large-pore channels that release ions and ATP upon activation. Three isoforms of pannexins 1, 2, and 3, perform diverse cellular roles and differ in their pore lining residues. In this study, we report the cryo-EM structure of pannexin 3 at 3.9 Å and analyze its structural differences with pannexin isoforms 1 and 2. The pannexin 3 vestibule has two distinct chambers and a wider pore radius in comparison to pannexins 1 and 2. We further report two cryo-EM structures of pannexin 1, with pore substitutions W74R/R75D that mimic the pore lining residues of pannexin 2 and a germline mutant of pannexin 1, R217H at resolutions of 3.2 Å and 3.9 Å, respectively. Substitution of cationic residues in the vestibule of pannexin 1 results in reduced ATP interaction propensities to the channel. The germline mutant R217H in transmembrane helix 3 (TM3), leads to a partially constricted pore, reduced ATP interaction and weakened voltage sensitivity. The study compares the three pannexin isoform structures, the effects of substitutions of pore and vestibule-lining residues and allosteric effects of a pathological substitution on channel structure and function thereby enhancing our understanding of this vital group of ATP-release channels.
10.1038/s41467-024-47142-6
Emerging Insights into Noncanonical Inflammasome Recognition of Microbes.
Russo Ashley J,Behl Bharat,Banerjee Ishita,Rathinam Vijay A K
Journal of molecular biology
Inflammasomes are cytosolic multi-molecular complexes that sense intracellular microbial danger signals and metabolic perturbations. Inflammasome activation leads to the activation of caspase-1 and the release of pro-inflammatory cytokines IL-1β and IL-18 accompanied by cell death. An inflammasome-based surveillance machinery for Gram-negative bacterial infections has been recently discovered. This noncanonical inflammasome relies on sensing the cytosolic presence of lipopolysaccharide of Gram-negative bacteria via inflammatory caspases such as caspase-4, -5, and -11. This review discusses the recent findings related to the mechanism of activation of the noncanonical inflammasome and its biological functions.
10.1016/j.jmb.2017.10.003
Extended subsite profiling of the pyroptosis effector protein gasdermin D reveals a region recognized by inflammatory caspase-11.
Bibo-Verdugo Betsaida,Snipas Scott J,Kolt Sonia,Poreba Marcin,Salvesen Guy S
The Journal of biological chemistry
Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1β (pro-IL18/pro-IL1β). Here, cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1'-P4', the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1'-P4' region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1β cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1'-P4' region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1'-P4'-based inhibitors could be exploited to specifically target inflammatory caspases.
10.1074/jbc.RA120.014259
Pyroptotic and non-pyroptotic effector functions of caspase-11.
Immunological reviews
Innate immune cells, epithelial cells, and many other cell types are capable of detecting infection or tissue injury, thus mounting regulated immune response. Inflammasomes are highly sophisticated and effective orchestrators of innate immunity. These oligomerized multiprotein complexes are at the center of various innate immune pathways, including modulation of the cytoskeleton, production and maturation of cytokines, and control of bacterial growth and cell death. Inflammasome assembly often results in caspase-1 activation, which is an inflammatory caspase that is involved in pyroptotic cell death and release of inflammatory cytokines in response to pathogen patterns and endogenous danger stimuli. However, the nature of stimuli and inflammasome components are diverse. Caspase-1 activation mediated release of mature IL-1β and IL-18 in response to canonical stimuli initiated by NOD-like receptor (NLR), and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). On the other hand, caspase-11 delineates a non-canonical inflammasome that promotes pyroptotic cell death and non-pyroptotic functions in response to non-canonical stimuli. Caspase-11 in mice and its homologues in humans (caspase-4/5) belong to caspase-1 family of cysteine proteases, and play a role in inflammation. Knockout mice provided new genetic tools to study inflammatory caspases and revealed the role of caspase-11 in mediating septic shock in response to lethal doses of lipopolysaccharide (LPS). Recognition of LPS mediates caspase-11 activation, which promotes a myriad of downstream effects that include pyroptotic and non-pyroptotic effector functions. Therefore, the physiological functions of caspase-11 are much broader than its previously established roles in apoptosis and cytokine maturation. Inflammation induced by exogenous or endogenous agents can be detrimental and, if excessive, can result in organ and tissue damage. Consequently, the existence of sophisticated mechanisms that tightly regulate the specificity and sensitivity of inflammasome pathways provides a fine-tuning balance between adequate immune response and minimal tissue damage. In this review, we summarize effector functions of caspase-11.
10.1111/imr.12910
Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.
Shi Jianjin,Zhao Yue,Wang Kun,Shi Xuyan,Wang Yue,Huang Huanwei,Zhuang Yinghua,Cai Tao,Wang Fengchao,Shao Feng
Nature
Inflammatory caspases (caspase-1, -4, -5 and -11) are critical for innate defences. Caspase-1 is activated by ligands of various canonical inflammasomes, and caspase-4, -5 and -11 directly recognize bacterial lipopolysaccharide, both of which trigger pyroptosis. Despite the crucial role in immunity and endotoxic shock, the mechanism for pyroptosis induction by inflammatory caspases is unknown. Here we identify gasdermin D (Gsdmd) by genome-wide clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease screens of caspase-11- and caspase-1-mediated pyroptosis in mouse bone marrow macrophages. GSDMD-deficient cells resisted the induction of pyroptosis by cytosolic lipopolysaccharide and known canonical inflammasome ligands. Interleukin-1β release was also diminished in Gsdmd(-/-) cells, despite intact processing by caspase-1. Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in GSDMD, which was required and sufficient for pyroptosis. The cleavage released the intramolecular inhibition on the gasdermin-N domain that showed intrinsic pyroptosis-inducing activity. Other gasdermin family members were not cleaved by inflammatory caspases but shared the autoinhibition; gain-of-function mutations in Gsdma3 that cause alopecia and skin defects disrupted the autoinhibition, allowing its gasdermin-N domain to trigger pyroptosis. These findings offer insight into inflammasome-mediated immunity/diseases and also change our understanding of pyroptosis and programmed necrosis.
10.1038/nature15514
Recognition and maturation of IL-18 by caspase-4 noncanonical inflammasome.
Nature
The canonical (caspase-1) and noncanonical (comprising caspases 4, 5 and 11; hereafter, caspase-4/5/11) inflammasomes both cleave gasdermin D (GSDMD) to induce pyroptosis. Whereas caspase-1 processes IL-1β and IL-18 for maturation, no cytokine target has been firmly established for lipopolysaccharide-activated caspase-4/5/11. Here we show that activated human caspase-4, but not mouse caspase-11, directly and efficiently processes IL-18 in vitro and during bacterial infections. Caspase-4 cleaves the same tetrapeptide site in pro-IL-18 as caspase-1. The crystal structure of the caspase-4-pro-IL-18 complex reveals a two-site (binary) substrate-recognition mechanism; the catalytic pocket engages the tetrapeptide, and a unique exosite that critically recognizes GSDMD similarly binds to a specific structure formed jointly by the propeptide and post-cleavage-site sequences in pro-IL-18. This binary recognition is also used by caspase-5 as well as caspase-1 to process pro-IL-18. In caspase-11, a structural deviation around the exosite underlies its inability to target pro-IL-18, which is restored by rationally designed mutations. The structure of pro-IL-18 features autoinhibitory interactions between the propeptide and the post-cleavage-site region, preventing recognition by the IL-18Rα receptor. Cleavage by caspase-1, -4 or -5 induces substantial conformational changes of IL-18 to generate two critical receptor-binding sites. Our study establishes IL-18 as a target of lipopolysaccharide-activated caspase-4/5. The finding is paradigm shifting in the understanding of noncanonical-inflammasome-mediated defences and also the function of IL-18 in immunity and disease.
10.1038/s41586-023-06742-w
Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice.
Xu Bing,Jiang Mingzuo,Chu Yi,Wang Weijie,Chen Di,Li Xiaowei,Zhang Zhao,Zhang Di,Fan Daiming,Nie Yongzhan,Shao Feng,Wu Kaichun,Liang Jie
Journal of hepatology
BACKGROUND & AIMS:Gasdermin D (GSDMD)-executed programmed necrosis is involved in inflammation and controls interleukin (IL)-1β release. However, the role of GSDMD in non-alcoholic steatohepatitis (NASH) remains unclear. We investigated the role of GSDMD in the pathogenesis of steatohepatitis. METHODS:Human liver tissues from patients with non-alcoholic fatty liver disease (NAFLD) and control individuals were obtained to evaluate GSDMD expression. Gsdmd knockout (Gsdmd) mice, obese db/db mice and their wild-type (WT) littermates were fed with methionine-choline deficient (MCD) or control diet to induce steatohepatitis. The Gsdmd and WT mice were also used in a high-fat diet (HFD)-induced NAFLD model. In addition, Alb-Cre mice were administered an adeno-associated virus (AAV) vector that expressed the gasdermin-N domain (AAV9-FLEX-GSDMD-N) and were fed with either MCD or control diet for 10 days. RESULTS:GSDMD and its pyroptosis-inducing fragment GSDMD-N were upregulated in liver tissues of human NAFLD/NASH. Importantly, hepatic GSDMD-N protein levels were significantly higher in human NASH and correlated with the NAFLD activity score and fibrosis. GSDMD-N remained a potential biomarker for the diagnosis of NASH. MCD-fed Gsdmd mice exhibit decreased severity of steatosis and inflammation compared with WT littermates. GSDMD was associated with the secretion of pro-inflammatory cytokines (IL-1β, TNF-α, and MCP-1 [CCL2]) and persistent activation of the NF-ĸB signaling pathway. Gsdmd mice showed lower steatosis, mainly because of reduced expression of the lipogenic gene Srebp1c (Srebf1) and upregulated expression of lipolytic genes, including Pparα, Aco [Klk15], Lcad [Acadl], Cyp4a10 and Cyp4a14. Alb-Cre mice administered with AAV9-FLEX-GSDMD-N showed significantly aggravated steatohepatitis when fed with MCD diet. CONCLUSION:As an executor of pyroptosis, GSDMD plays a key role in the pathogenesis of steatohepatitis, by controlling cytokine secretion, NF-ĸB activation, and lipogenesis. LAY SUMMARY:Non-alcoholic fatty liver disease has become one of the most feared chronic liver diseases, because it is the most rapidly growing indication for adult liver transplantation and a major cause of hepatocellular carcinoma. However, the mechanisms involved in the transformation of simple steatosis to steatohepatitis remain unclear. Herein, we show that gasdermin D driven pyroptosis is prominent in patients with non-alcoholic steatohepatitis (NASH), and gasdermin-N domain remains a potential biomarker for the diagnosis of NASH. Gasdermin D plays a key role in the pathogenesis of NASH by regulating lipogenesis, the inflammatory response, and the NF-ĸB signaling pathway, revealing potential treatment targets for NASH in humans.
10.1016/j.jhep.2017.11.040
Orally administered saccharide-sequestering nanocomplex to manage carbohydrate metabolism disorders.
Science advances
Excessive carbohydrate intake is linked to the growing prevalence of diabetes, nonalcoholic fatty liver disease (NAFLD), and obesity. α-Glucosidases inhibitor, the only Food and Drug Administration-approved drug for limiting the absorption of polysaccharides and disaccharides, is ineffective for monosaccharides. Here, we develop a boronic acid-containing polymer nanocomplex (Nano-Poly-BA), absorbing all saccharides into nanocomplex with the diol/boronic acid molar ratio far above 1, to prevent saccharides' absorption in the gut. The orally administered Nano-Poly-BA is nonabsorbable and nontoxic. When tested against four kinds of carbohydrates and three real-world foods (coke, blueberry jam, and porridge), Nano-Poly-BA shows remarkable after-meal blood glucose reductions in wild-type, type 1, and type 2 diabetic mouse models. In a NAFLD mouse model induced by fructose, Nano-Poly-BA shows substantial reduction of hepatic lipogenesis. In short, the orally administered saccharide-sequestering polymer nanocomplex may help prediabetic, diabetic, overweight, and even healthy people to manage sugar intake.
10.1126/sciadv.abf7311
The protective effect of quercetin on macrophage pyroptosis via TLR2/Myd88/NF-κB and ROS/AMPK pathway.
Luo Xing,Bao Xiaoyi,Weng Xiuzhu,Bai Xiaoxuan,Feng Yi,Huang Jianxin,Liu Shaoyu,Jia Haibo,Yu Bo
Life sciences
AIMS:Pyroptosis is a pro-inflammatory form of programmed cell death, which plays a vital role in the development of inflammatory diseases. As a natural flavonoid, quercetin has been shown to possess anti-inflammatory activity, but its effects on macrophage pyroptosis is still unclear. Therefore, this study aims to investigate the effects of quercetin on macrophage pyroptosis and the underlying mechanism. MATERIAL AND METHODS:LPS/ATP treatment was used to induce THP-1 macrophage pyroptosis. Cell counting kit-8 (CCK-8) assay was used to evaluate cell viability. Scanning electron microscope (SEM) was used to detect cell morphology. Hoechst/propidium iodide (PI) staining and lactate dehydrogenase (LDH) assay were performed to evaluate the cell membrane integrity. The expression of key components and effectors of nod-like receptors3 (NLRP3) inflammasome were examined by real-time PCR and western blot. Immunofluorescence staining was used to detect reactive oxygen species (ROS) level and P65 nuclear translocation. KEY FINDINGS:Our results showed that quercetin prevented THP-1 macrophage pyroptosis by reducing the expression of NLRP3 and cleaved-caspase1, as well as IL-1β and N-GSDMD in a concentration dependent manner. Quercetin suppressed NLRP3 inflammasome activation by inhibiting ROS overproduction. Moreover, quercetin inhibited the phosphorylation of P65 and its translocation from cytoplasm into nuclear. In addition, we found that quercetin suppressed the increase of TLR2/Myd88 and p-AMPK induced by LPS/ATP, while both TLR2 and AMPK agonist weakened the inhibitory effect of quercetin on the activity of NLRP3 inflammasome and alleviated the protective effect on macrophages pyroptosis. SIGNIFICANCE:Quercetin possesses a protective effect on macrophages pyroptosis via TLR2/Myd88/NF-κB and ROS/AMPK pathway.
10.1016/j.lfs.2021.120064
cGAS-STING, inflammasomes and pyroptosis: an overview of crosstalk mechanism of activation and regulation.
Cell communication and signaling : CCS
BACKGROUND:Intracellular DNA-sensing pathway cGAS-STING, inflammasomes and pyroptosis act as critical natural immune signaling axes for microbial infection, chronic inflammation, cancer progression and organ degeneration, but the mechanism and regulation of the crosstalk network remain unclear. Cellular stress disrupts mitochondrial homeostasis, facilitates the opening of mitochondrial permeability transition pore and the leakage of mitochondrial DNA to cell membrane, triggers inflammatory responses by activating cGAS-STING signaling, and subsequently induces inflammasomes activation and the onset of pyroptosis. Meanwhile, the inflammasome-associated protein caspase-1, Gasdermin D, the CARD domain of ASC and the potassium channel are involved in regulating cGAS-STING pathway. Importantly, this crosstalk network has a cascade amplification effect that exacerbates the immuno-inflammatory response, worsening the pathological process of inflammatory and autoimmune diseases. Given the importance of this crosstalk network of cGAS-STING, inflammasomes and pyroptosis in the regulation of innate immunity, it is emerging as a new avenue to explore the mechanisms of multiple disease pathogenesis. Therefore, efforts to define strategies to selectively modulate cGAS-STING, inflammasomes and pyroptosis in different disease settings have been or are ongoing. In this review, we will describe how this mechanistic understanding is driving possible therapeutics targeting this crosstalk network, focusing on the interacting or regulatory proteins, pathways, and a regulatory mitochondrial hub between cGAS-STING, inflammasomes, and pyroptosis. SHORT CONCLUSION:This review aims to provide insight into the critical roles and regulatory mechanisms of the crosstalk network of cGAS-STING, inflammasomes and pyroptosis, and to highlight some promising directions for future research and intervention.
10.1186/s12964-023-01466-w
Channelling inflammation: gasdermins in physiology and disease.
Nature reviews. Drug discovery
Gasdermins were recently identified as the mediators of pyroptosis - inflammatory cell death triggered by cytosolic sensing of invasive infection and danger signals. Upon activation, gasdermins form cell membrane pores, which release pro-inflammatory cytokines and alarmins and damage the integrity of the cell membrane. Roles for gasdermins in autoimmune and inflammatory diseases, infectious diseases, deafness and cancer are emerging, revealing potential novel therapeutic avenues. Here, we review current knowledge of the family of gasdermins, focusing on their mechanisms of action and roles in normal physiology and disease. Efforts to develop drugs to modulate gasdermin activity to reduce inflammation or activate more potent immune responses are highlighted.
10.1038/s41573-021-00154-z
Pyroptosis-induced inflammation and tissue damage.
Seminars in immunology
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.
10.1016/j.smim.2023.101781
The gasdermins, a protein family executing cell death and inflammation.
Broz Petr,Pelegrín Pablo,Shao Feng
Nature reviews. Immunology
The gasdermins are a family of recently identified pore-forming effector proteins that cause membrane permeabilization and pyroptosis, a lytic pro-inflammatory type of cell death. Gasdermins contain a cytotoxic N-terminal domain and a C-terminal repressor domain connected by a flexible linker. Proteolytic cleavage between these two domains releases the intramolecular inhibition on the cytotoxic domain, allowing it to insert into cell membranes and form large oligomeric pores, which disrupts ion homeostasis and induces cell death. Gasdermin-induced pyroptosis plays a prominent role in many hereditary diseases and (auto)inflammatory disorders as well as in cancer. In this Review, we discuss recent developments in gasdermin research with a focus on mechanisms that control gasdermin activation, pore formation and functional consequences of gasdermin-induced membrane permeabilization.
10.1038/s41577-019-0228-2
The p-STAT3/ANXA2 axis promotes caspase-1-mediated hepatocyte pyroptosis in non-alcoholic steatohepatitis.
Journal of translational medicine
BACKGROUND:To explore the roles of Annexin A2 (ANXA2) on hepatocyte pyroptosis and hepatic fibrosis in nonalcoholic steatohepatitis (NASH) and underlying molecular mechanism. METHODS:Bioinformatics analyses were performed on transcriptome data of liver tissues from mice and patients with liver fibrosis for screening the hepatocyte pyroptosis-related differential genes. The in vivo NASH mouse model and in vitro NASH cellular model were established. The expression levels of Anxa2/ANXA2 were quantified. Then, the upstream transcription factor of Anxa2 was screened by ChIP-Seq and experimentally verified. The effects of the p-STAT3/ANXA2 axis on Caspase-1 mediated pyroptosis and fibrosis were explored by in vivo and in vitro experiments. RESULTS:Bioinformatics analyses suggested that the expression of Anxa2/ANXA2 was significantly up-regulated in liver tissues of both NASH mice and patients scoring with high pyroptotic activity. Experimental data showed that the ANXA2 expression was positively associated with the development of hepatocyte pyroptosis and fibrosis. As a transcription factor of ANXA2, p-STAT3 can bind to the promoter of Anxa2 and promote its transcription. The inhibition of p-STAT3 can significantly suppress hepatocyte pyroptosis and fibrosis, which was significantly reversed after the over-expression of Anxa2. Caspase-1 was verified as the player of the p-STAT3/ANXA2 axis to promote pyroptosis and fibrosis. By specifically inhibiting Caspase-1, the promotion effect of the p-STAT3/ANXA2 axis on pyroptosis and fibrosis can be significantly weakened. CONCLUSION:The p-STAT3 promoted Anxa2 expression at the transcription level, thus activating the Caspase-1 mediated hepatocyte pyroptosis and fibrosis in NASH.
10.1186/s12967-022-03692-1
Caspase-11 promotes high-fat diet-induced NAFLD by increasing glycolysis, OXPHOS, and pyroptosis in macrophages.
Frontiers in immunology
Introduction:Non-alcoholic fatty liver disease (NAFLD) has a global prevalence of 25% of the population and is a leading cause of cirrhosis and hepatocellular carcinoma. NAFLD ranges from simple steatosis (non-alcoholic fatty liver) to non-alcoholic steatohepatitis (NASH). Hepatic macrophages, specifically Kupffer cells (KCs) and monocyte-derived macrophages, act as key players in the progression of NAFLD. Caspases are a family of endoproteases that provide critical connections to cell regulatory networks that sense disease risk factors, control inflammation, and mediate inflammatory cell death (pyroptosis). Caspase-11 can cleave gasdermin D (GSDMD) to induce pyroptosis and specifically defends against bacterial pathogens that invade the cytosol. However, it's still unknown whether high fat diet (HFD)-facilitated gut microbiota-generated cytoplasmic lipopolysaccharides (LPS) activate caspase-11 and promote NAFLD. Methods:To examine this hypothesis, we performed liver pathological analysis, RNA-seq, FACS, Western blots, Seahorse mitochondrial stress analyses of macrophages and bone marrow transplantation on HFD-induced NAFLD in WT and Casp11-/- mice. Results and Discussion:Our results showed that 1) HFD increases body wight, liver wight, plasma cholesterol levels, liver fat deposition, and NAFLD activity score (NAS score) in wild-type (WT) mice; 2) HFD increases the expression of caspase-11, GSDMD, interleukin-1β, and guanylate-binding proteins in WT mice; 3) Caspase-11 deficiency decreases fat liver deposition and NAS score; 4) Caspase-11 deficiency decreases bone marrow monocyte-derived macrophage (MDM) pyroptosis (inflammatory cell death) and inflammatory monocyte (IM) surface GSDMD expression; 5) Caspase-11 deficiency re-programs liver transcriptomes and reduces HFD-induced NAFLD; 6) Caspase-11 deficiency decreases extracellular acidification rates (glycolysis) and oxidative phosphorylation (OXPHOS) in inflammatory fatty acid palmitic acid-stimulated macrophages, indicating that caspase-11 significantly contributes to maintain dual fuel bioenergetics-glycolysis and OXPHOS for promoting pyroptosis in macrophages. These results provide novel insights on the roles of the caspase-11-GSDMD pathway in promoting hepatic macrophage inflammation and pyroptosis and novel targets for future therapeutic interventions involving the transition of NAFLD to NASH, hyperlipidemia, type II diabetes, metabolic syndrome, metabolically healthy obesity, atherosclerotic cardiovascular diseases, autoimmune diseases, liver transplantation, and hepatic cancers.
10.3389/fimmu.2023.1113883
Gasdermin D-mediated pyroptosis: mechanisms, diseases, and inhibitors.
Frontiers in immunology
Gasdermin D (GSDMD)-mediated pyroptosis and downstream inflammation are important self-protection mechanisms against stimuli and infections. Hosts can defend against intracellular bacterial infections by inducing cell pyroptosis, which triggers the clearance of pathogens. However, pyroptosis is a double-edged sword. Numerous studies have revealed the relationship between abnormal GSDMD activation and various inflammatory diseases, including sepsis, coronavirus disease 2019 (COVID-19), neurodegenerative diseases, nonalcoholic steatohepatitis (NASH), inflammatory bowel disease (IBD), and malignant tumors. GSDMD, a key pyroptosis-executing protein, is linked to inflammatory signal transduction, activation of various inflammasomes, and the release of downstream inflammatory cytokines. Thus, inhibiting GSDMD activation is considered an effective strategy for treating related inflammatory diseases. The study of the mechanism of GSDMD activation, the formation of GSDMD membrane pores, and the regulatory strategy of GSDMD-mediated pyroptosis is currently a hot topic. Moreover, studies of the structure of caspase-GSDMD complexes and more in-depth molecular mechanisms provide multiple strategies for the development of GSDMD inhibitors. This review will mainly discuss the structures of GSDMD and GSDMD pores, activation pathways, GSDMD-mediated diseases, and the development of GSDMD inhibitors.
10.3389/fimmu.2023.1178662
Role of the Inflammasome in Liver Disease.
Annual review of pathology
The involvement of inflammasomes in the proinflammatory response observed in chronic liver diseases, such as alcohol-associated liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD), is widely recognized. Although there are different types of inflammasomes, most studies to date have given attention to NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3) in the pathogenesis of ALD, NAFLD/nonalcoholic steatohepatitis, and fibrosis. Canonical inflammasomes are intracellular multiprotein complexes that are assembled after the sensing of danger signals and activate caspase-1, which matures interleukin (IL)-1β, IL-18, and IL-37 and also induces a form of cell death called pyroptosis. Noncanonical inflammasomes activate caspase-11 to induce pyroptosis. We discuss the different types of inflammasomes involved in liver diseases with a focus on () signals and mechanisms of inflammasome activation, () the role of different types of inflammasomes and their products in the pathogenesis of liver diseases, and () potential therapeutic strategies targeting components of the inflammasomes or cytokines produced upon inflammasome activation.
10.1146/annurev-pathmechdis-032521-102529
Sphingomyelin synthase 1 mediates hepatocyte pyroptosis to trigger non-alcoholic steatohepatitis.
Koh Eun Hee,Yoon Ji Eun,Ko Myoung Seok,Leem Jaechan,Yun Ji-Young,Hong Chung Hwan,Cho Yun Kyung,Lee Seung Eun,Jang Jung Eun,Baek Ji Yeon,Yoo Hyun Ju,Kim Su Jung,Sung Chang Ohk,Lim Joon Seo,Jeong Won-Il,Back Sung Hoon,Baek In-Jeoung,Torres Sandra,Solsona-Vilarrasa Estel,Conde de la Rosa Laura,Garcia-Ruiz Carmen,Feldstein Ariel E,Fernandez-Checa Jose C,Lee Ki-Up
Gut
OBJECTIVE:Lipotoxic hepatocyte injury is a primary event in non-alcoholic steatohepatitis (NASH), but the mechanisms of lipotoxicity are not fully defined. Sphingolipids and free cholesterol (FC) mediate hepatocyte injury, but their link in NASH has not been explored. We examined the role of free cholesterol and sphingomyelin synthases (SMSs) that generate sphingomyelin (SM) and diacylglycerol (DAG) in hepatocyte pyroptosis, a specific form of programmed cell death associated with inflammasome activation, and NASH. DESIGN:Wild-type C57BL/6J mice were fed a high fat and high cholesterol diet (HFHCD) to induce NASH. Hepatic SMS1 and SMS2 expressions were examined in various mouse models including HFHCD-fed mice and patients with NASH. Pyroptosis was estimated by the generation of the gasdermin-D N-terminal fragment. NASH susceptibility and pyroptosis were examined following knockdown of SMS1, protein kinase Cδ (PKCδ), or the NLR family CARD domain-containing protein 4 (NLRC4). RESULTS:HFHCD increased the hepatic levels of SM and DAG while decreasing the level of phosphatidylcholine. Hepatic expression of but not was higher in mouse models and patients with NASH. FC in hepatocytes induced expression, and knockdown prevented HFHCD-induced NASH. DAG produced by SMS1 activated PKCδ and NLRC4 inflammasome to induce hepatocyte pyroptosis. Depletion of prevented hepatocyte pyroptosis and the development of NASH. Conditioned media from pyroptotic hepatocytes activated the NOD-like receptor family pyrin domain containing 3 inflammasome (NLRP3) in Kupffer cells, but knockout mice were not protected against HFHCD-induced hepatocyte pyroptosis. CONCLUSION:SMS1 mediates hepatocyte pyroptosis through a novel DAG-PKCδ-NLRC4 axis and holds promise as a therapeutic target for NASH.
10.1136/gutjnl-2020-322509
Hepatocyte pyroptosis and release of inflammasome particles induce stellate cell activation and liver fibrosis.
Journal of hepatology
BACKGROUND & AIMS:Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS:We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3CreA mice, and Gsdmd mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS:We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3CreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1β secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS:These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY:Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.
10.1016/j.jhep.2020.07.041
Non-invasive diagnosis and monitoring of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.
The lancet. Gastroenterology & hepatology
Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent form of chronic liver disease that poses challenges in diagnosis and risk stratification. Non-alcoholic steatohepatitis (NASH), the more progressive form of NAFLD, is particularly challenging to diagnose in the absence of histology. Liver biopsy is infrequently performed due to its invasive nature, potential for sampling error, and lack of inter-rater reliability. Non-invasive tests that can accurately identify patients with at-risk NASH (ie, individuals with biopsy-proven NASH with NAFLD activity score [NAS] ≥4 and fibrosis stage ≥2) are key tools to identify candidates for pharmacologic therapy in registrational trials for the treatment of NASH-related fibrosis. With emerging pharmacotherapy, non-invasive tests are required to track treatment response. Lastly, there is an unmet need for non-invasive tests to assess risk for clinical outcomes including progression to cirrhosis, hepatic decompensation, liver-related mortality, and overall mortality. In this Review we examine advances in non-invasive tests to diagnose and monitor NAFLD and NASH.
10.1016/S2468-1253(23)00066-3
Cell death.
Cell
Cell death supports morphogenesis during development and homeostasis after birth by removing damaged or obsolete cells. It also curtails the spread of pathogens by eliminating infected cells. Cell death can be induced by the genetically programmed suicide mechanisms of apoptosis, necroptosis, and pyroptosis, or it can be a consequence of dysregulated metabolism, as in ferroptosis. Here, we review the signaling mechanisms underlying each cell-death pathway, discuss how impaired or excessive activation of the distinct cell-death processes can promote disease, and highlight existing and potential therapies for redressing imbalances in cell death in cancer and other diseases.
10.1016/j.cell.2023.11.044
Challenges and opportunities in NASH drug development.
Nature medicine
Nonalcoholic fatty liver disease (NAFLD) and its more severe form, nonalcoholic steatohepatitis (NASH), represent a growing worldwide epidemic and a high unmet medical need, as no licensed drugs have been approved thus far. Currently, histopathological assessment of liver biopsies is mandatory as a primary endpoint for conditional drug approval. This requirement represents one of the main challenges in the field, as there is substantial variability in this invasive histopathological assessment, which leads to dramatically high screen-failure rates in clinical trials. Over the past decades, several non-invasive tests have been developed to correlate with liver histology and, eventually, outcomes to assess disease severity and longitudinal changes non-invasively. However, further data are needed to ensure their endorsement by regulatory authorities as alternatives to histological endpoints in phase 3 trials. This Review describes the challenges of drug development in NAFLD-NASH trials and potential mitigating strategies to move the field forward.
10.1038/s41591-023-02242-6
Circadian regulation of liver function: from molecular mechanisms to disease pathophysiology.
Nature reviews. Gastroenterology & hepatology
A wide variety of liver functions are regulated daily by the liver circadian clock and via systemic circadian control by other organs and cells within the gastrointestinal tract as well as the microbiome and immune cells. Disruption of the circadian system, as occurs during jetlag, shift work or an unhealthy lifestyle, is implicated in several liver-related pathologies, ranging from metabolic diseases such as obesity, type 2 diabetes mellitus and nonalcoholic fatty liver disease to liver malignancies such as hepatocellular carcinoma. In this Review, we cover the molecular, cellular and organismal aspects of various liver pathologies from a circadian viewpoint, and in particular how circadian dysregulation has a role in the development and progression of these diseases. Finally, we discuss therapeutic and lifestyle interventions that carry health benefits through support of a functional circadian clock that acts in synchrony with the environment.
10.1038/s41575-023-00792-1
Clinical course of non-alcoholic fatty liver disease and the implications for clinical trial design.
Journal of hepatology
BACKGROUND & AIMS:The predicted risk and timeline to progression to liver-related outcomes in the population with NAFLD are not well-characterized. We aimed to examine the risk and time to progression to cirrhosis, hepatic decompensation and death in a contemporary population over a long follow-up period, to obtain information to guide endpoint selection and sample size calculations for clinical trials on NAFLD-related cirrhosis. METHODS:This is a retrospective study of prospectively collected data in a medical record linkage system, including all adults diagnosed with NAFLD between 1996-2016 by clinical, biochemical and radiological criteria in Olmsted County, Minnesota and followed until 2019. Liver-related outcomes and death were ascertained and validated by individual medical record review. Time and risk of progression from NAFLD to cirrhosis to decompensation and death were assessed using multistate modeling. RESULTS:A total of 5,123 individuals with NAFLD (median age 52 years, 53% women) were followed for a median of 6.4 (range 1-23) years. The risk of progression was as follows: from NAFLD to cirrhosis: 3% in 15 years; compensated cirrhosis to first decompensation: 33% in 4 years (8%/year); first decompensation to ≥2 decompensations: 48% in 2 years. Albumin, bilirubin, non-bleeding esophageal varices and diabetes were independent predictors of decompensation. Among the 575 deaths, 6% were liver related. Therapeutic trials in compensated cirrhosis would require enrolment of a minimum of 2,886 individuals followed for >2 years to detect at least a 15% relative decrease in liver-related endpoints. CONCLUSION:In this population-based cohort with 23 years of longitudinal follow-up, NAFLD was slowly progressive, with liver-related outcomes affecting only a small proportion of people. Large sample sizes and long follow-up are required to detect reductions in liver-related endpoints in clinical trials. LAY SUMMARY:For patients with compensated non-alcoholic steatohepatitis-related cirrhosis, the time spent in this state and the risk of progression to decompensation are not well-known in the population. We examined the clinical course of a large population-based cohort over 23 years of follow-up. We identified that adults with compensated cirrhosis spend a mean time of 4 years in this state and have a 10% per year risk of progression to decompensation or death. The risk of further progression is 3-fold higher in adults with cirrhosis and one decompensating event. These results are reflective of placebo arm risks in drug clinical trials and are essential in the estimation of adequate sample sizes.
10.1016/j.jhep.2022.07.004
Global prevalence of non-alcoholic fatty liver disease in type 2 diabetes mellitus: an updated systematic review and meta-analysis.
Gut
INTRODUCTION:Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, with type 2 diabetes mellitus (T2DM) as a major predictor. Insulin resistance and chronic inflammation are key pathways in the pathogenesis of T2DM leading to NAFLD and vice versa, with the synergistic effect of NAFLD and T2DM increasing morbidity and mortality risks. This meta-analysis aims to quantify the prevalence of NAFLD and the prevalence of clinically significant and advanced fibrosis in people with T2DM. METHODS:MEDLINE and Embase databases were searched from inception until 13 February 2023. The primary outcomes were the prevalence of NAFLD, non-alcoholic steatohepatitis (NASH) and fibrosis in people with T2DM. A generalised linear mixed model with Clopper-Pearson intervals was used for the analysis of proportions with sensitivity analysis conducted to explore heterogeneity between studies. RESULTS:156 studies met the inclusion criteria, and a pooled analysis of 1 832 125 patients determined that the prevalence rates of NAFLD and NASH in T2DM were 65.04% (95% CI 61.79% to 68.15%, I=99.90%) and 31.55% (95% CI 17.12% to 50.70%, I=97.70%), respectively. 35.54% (95% CI 19.56% to 55.56%, I=100.00%) of individuals with T2DM with NAFLD had clinically significant fibrosis (F2-F4), while 14.95% (95% CI 11.03% to 19.95%, I=99.00%) had advanced fibrosis (F3-F4). CONCLUSION:This study determined a high prevalence of NAFLD, NASH and fibrosis in people with T2DM. Increased efforts are required to prevent T2DM to combat the rising burden of NAFLD. PROSPERO REGISTRATION NUMBER:CRD42022360251.
10.1136/gutjnl-2023-330110
Global prevalence of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in the overweight and obese population: a systematic review and meta-analysis.
The lancet. Gastroenterology & hepatology
BACKGROUND:The global burden of non-alcoholic fatty liver disease (NAFLD) parallels the increase in obesity rates across the world. Although overweight and obesity status are thought to be an effective indicator for NAFLD screening, the exact prevalence of NAFLD in this population remains unknown. We aimed to report the prevalence of NAFLD, non-alcoholic fatty liver (NAFL), and non-alcoholic steatohepatitis (NASH) in the overweight and obese population. METHODS:In this systematic review and meta-analysis, we searched Medline and Embase from database inception until March 6, 2022, using search terms including but not limited to "non-alcoholic fatty liver disease", "overweight", "obesity", and "prevalence". Cross-sectional and longitudinal observational studies published after Jan 1, 2000, written in or translated into English were eligible for inclusion; paediatric studies were excluded. Articles were included if the number of NAFLD, NAFL, or NASH events in an overweight and obese population could be extracted. Summary data were extracted from published reports. The primary outcomes were the prevalence of NAFLD, NAFL, and NASH in an overweight and obese population and the prevalence of fibrosis in individuals who were overweight or obese and who had NAFLD. A meta-analysis of proportions was done with the generalised linear mixed model. This study is registered with PROSPERO (CRD42022344526). FINDINGS:The search identified 7389 articles. 151 studies met the inclusion criteria and were included in the meta-analysis. In the pooled analysis comprising 101 028 individuals, the prevalence of NAFLD in the overweight population was 69·99% (95% CI 65·40-74·21 I=99·10%), the prevalence of NAFL was 42·49% (32·55-53·08, I=96·40%), and the prevalence of NASH was 33·50% (28·38-39·04, I=95·60%). Similar prevalence estimates were reported in the obese population for NAFLD (75·27% [95% CI 70·90-79·18]; I=98·50%), NAFL (43·05% [32·78-53·97]; I=96·30%) and NASH (33·67% [28·45-39·31]; I=95·60%). The prevalence of NAFLD in the overweight population was the highest in the region of the Americas (75·34% [95% CI: 67·31-81·93]; I=99·00%). Clinically significant fibrosis (stages F2-4) was present in 20·27% (95% CI 11·32-33·62; I= 93·00%) of overweight individuals with NAFLD and in 21·60% (11·47-36·92; I=95·00%) of obese patients with NAFLD while 6·65% (4·35-10·01; I=58·00%) of overweight individuals with NAFLD and 6·85% (3·85-11·90; I=90·00%) of obese individuals with NAFLD had advanced fibrosis (stages F3-4). INTERPRETATION:This study summarises the estimated global prevalence of NAFLD, NAFL, and NASH in overweight and obese individuals; these findings are important for improving the understanding of the global NAFLD burden and supporting disease management in the at-risk overweight and obese population. FUNDING:None.
10.1016/S2468-1253(22)00317-X
Changing epidemiology, global trends and implications for outcomes of NAFLD.
Journal of hepatology
Non-alcoholic fatty liver disease (NAFLD) has rapidly become the most common liver disease globally and is currently estimated to affect 38% of the global population. Only a minority of patients with NAFLD will progress to cirrhosis or hepatocellular carcinoma, but from this vast population the total number of patients who are at risk of such severe outcomes is increasing. Worryingly, individuals are increasingly being affected by NAFLD at an earlier age, meaning there is more time for them to develop severe complications. With considerable changes in dietary composition and urbanisation, alongside the growth in obesity and type 2 diabetes in the global population, in particular in developing countries, the global proportion of persons affected by NAFLD is projected to increase further. Yet, there are large geographical discrepancies in the prevalence rates of NAFLD and its inflammatory component non-alcoholic steatohepatitis (NASH). Such differences are partly related to differing socio-economic milieus, but also to genetic predisposition. In this narrative review, we discuss recent changes in the epidemiology of NAFLD and NASH from regional and global perspectives, as well as in special populations. We also discuss the potential consequences of these changes on hepatic and extrahepatic events.
10.1016/j.jhep.2023.04.036
Nonalcoholic Fatty Liver Disease 2020: The State of the Disease.
Cotter Thomas G,Rinella Mary
Gastroenterology
Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease, with a worldwide prevalence of 25%. In the United States, NAFLD and its subtype, nonalcoholic steatohepatitis, affect 30% and 5% of the population, respectively. Considering the ongoing obesity epidemic beginning in childhood, the rise in diabetes, and other factors, the prevalence of NAFLD along with the proportion of those with advanced liver disease is projected to continue to increase. This will have an important impact on public health reflected in health care costs, including impact on the need for liver transplantation, for which nonalcoholic steatohepatitis is already close to becoming the most common indication. NAFLD patients with evidence of nonalcoholic steatohepatitis and advanced fibrosis are at markedly increased risk of adverse outcomes, including overall mortality, and liver-specific morbidity and mortality, respectively. Identification of this cohort of NAFLD patients is paramount, given the associated poorer outcomes, in order to target resources to those who need it most. Various noninvasive tools have been developed in this regard. This review provides an update on the epidemiology, clinical and prognostic features, and diagnostic approach to patients with NAFLD.
10.1053/j.gastro.2020.01.052
Baicalin attenuates hepatic injury in non-alcoholic steatohepatitis cell model by suppressing inflammasome-dependent GSDMD-mediated cell pyroptosis.
Shi Huilian,Zhang Yanliang,Xing Jing,Liu Lina,Qiao Fei,Li Jun,Chen Yuanyuan
International immunopharmacology
Baicalin (BA), a flavone glycoside, is the constituent of Scutellaria baicalensis, a Chinese herbal medicine used to treat non-alcoholic steatohepatitis (NASH). However, the mechanism of BA on NASH is still not clear. Here, the improving effect of BA on hepatocyte through inhibition of pyroprosis was investigated in vitro. With a cell model of NASH exposing HepG2 cells in free fatty acids (FFA), we revealed that BA could improve hepatocyte from FFA-induced morphological damage and death. And then through transcriptomes screening, a significant down-regulation of NLR pyrin domain containing 3 (Nlrp3), gasdermin D (Gsdmd), andinterleukin-1 beta (IL-1β) expression were found after BA treatment. Further analysis confirmed that BA could decrease the levels of NLRP3 and GSDMD, as well as the release of IL-1β and IL-18, resulting in the reduction of pyroptosis. Moreover, the improving effect of BA could be attenuated by Gsdmd knockdown. In conclusion, BA can reduce pyroptosis of hepatocyte by blocking NLRP3-GSDMD signaling in vitro.
10.1016/j.intimp.2020.106195
Role of pyroptosis in liver diseases.
Al Mamun Abdullah,Wu Yanqing,Jia Chang,Munir Fahad,Sathy Kasfia Jahan,Sarker Tamanna,Monalisa Ilma,Zhou Kailiang,Xiao Jian
International immunopharmacology
Pyroptosis is known as a novel form of pro-inflammatory cell death program, which is exceptional from other types of cell death programs. Particularly, pyroptosis is characterized by Gasdermin family-mediated pore formation and subsequently cellular lysis, also release of several pro-inflammatory intracellular cytokines. In terms of mechanism, there are two signaling pathways involved in pyroptosis, including caspase-1, and caspase-4/5/11 mediated pathways. However, pyroptosis plays important roles in immune defense mechanisms. Recent studies have demonstrated that pyroptosis plays significant roles in the development of liver diseases. In our review, we have focused on the role of pyroptosis based on the molecular and pathophysiological mechanisms in the development of liver diseases. We have also highlighted targeting of pyroptosis for the therapeutic implications in liver diseases in the near future.
10.1016/j.intimp.2020.106489
Vitamin D ameliorates high-fat-diet-induced hepatic injury via inhibiting pyroptosis and alters gut microbiota in rats.
Archives of biochemistry and biophysics
Accumulating evidence suggests that vitamin D (VD) has a therapeutic effect on non-alcoholic fatty liver disease (NAFLD). Pyroptosis and gut microbiota have been recognized as critical factors of the progression of NAFLD. However, the effect of VD on the pyroptosis and gut microbiota in NAFLD remains inconclusive. Herein, rats were fed high fat diet (HFD) for 12 weeks and concurrently treated with 5 μg/kg 1,25(OH)D twice a week. BRL-3A cells were stimulated with 0.4 mmol/L palmitic acid (PA) and 1 μg/ml lipopolysaccharide (LPS) for 16 h and treated with 10 mol/L 1,25(OH)D. Effect of VD on the hepatic injury, lipid accumulation, activation of NLRP3 inflammasome and pyroptosis was determined in vivo and in vitro. Next, gasdermin D N-terminal (GSDMD-N) fragment was overexpressed in BRL-3A cells to investigate the role of pyroptosis in the therapeutic effect of VD on NAFLD. In addition, gut microbiota in NAFLD rats was also analyzed. Results showed that VD attenuated HFD-induced hepatic injury in vivo and PA-LPS-induced impairment of cell viability in vitro, and inhibited lipid accumulation, activation of NLRP3 inflammasome and pyroptosis in vivo and in vitro. GSDMD-N fragment overexpression suppressed the protective effect of VD on PA-LPS-induced activation of NLRP3 inflammasome, impairment of cell viability and lipid accumulation, indicating that VD might attenuate NAFLD through inhibiting pyroptosis. Additionally, VD also restored HFD-induced gut microbiota dysbiosis by increasing the relative abundance of Lactobacillus and reducing that of Acetatifactor, Oscillibacter and Flavonifractor. This study provides a novel mechanism underlying VD therapy against NAFLD.
10.1016/j.abb.2021.108894