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

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

Zinc depletion regulates the processing and secretion of IL-1β

Sterile inflammation contributes to many common and serious human diseases. The pro-inflammatory cytokine interleukin-1β (IL-1β) drives sterile inflammatory responses and is thus a very attractive therapeutic target. Activation of IL-1β in sterile diseases commonly requires an intracellular multi-protein complex called the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome. A number of disease-associated danger molecules are known to activate the NLRP3 inflammasome. We show here that depletion of zinc from macrophages, a paradigm for zinc deficiency, also activates the NLRP3 inflammasome and induces IL-1β secretion. Our data suggest that zinc depletion damages the integrity of lysosomes and that this event is important for NLRP3 activation. These data provide new mechanistic insight to how zinc deficiency contributes to inflammation and further unravel the mechanisms of NLRP3 inflammasome activation.     

Caspase-11 Activation in Response to Bacterial Secretion Systems that Access the Host Cytosol

Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1β. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1β. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1β, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1β release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1β, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1β release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense.     

Activation of the NLRP3 Inflammasome Complex is Not Required for Stress-Induced Death of Pancreatic Islets

Loss of pancreatic beta cells is a feature of type-2 diabetes. High glucose concentrations induce endoplasmic reticulum (ER) and oxidative stress-mediated apoptosis of islet cells in vitro. ER stress, oxidative stress and high glucose concentrations may also activate the NLRP3 inflammasome leading to interleukin (IL)-1β production and caspase-1 dependent pyroptosis. However, whether IL-1β or intrinsic NLRP3 inflammasome activation contributes to beta cell death is controversial. This possibility was examined in mouse islets. Exposure of islets lacking functional NLRP3 or caspase-1 to H₂O_2, rotenone or thapsigargin induced similar cell death as in wild-type islets. This suggests that oxidative or ER stress do not cause inflammasome-mediated cell death. Similarly, deficiency of NLRP3 inflammasome components did not provide any protection from glucose, ribose or gluco-lipotoxicity. Finally, genetic activation of NLRP3 specifically in beta cells did not increase IL-1β production or cell death, even in response to glucolipotoxicity. Overall, our results show that glucose-, ER stress- or oxidative stress-induced cell death in islet cells is not dependent on intrinsic activation of the NLRP3 inflammasome.     

Localization and Functionality of the Inflammasome in Neutrophils

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.     

Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of Trypanosoma cruzi Replication and Survival Is Dispensable in NLRP3?/? Macrophages

In this study, we have utilized wild-type (WT), ASC^−/−, and NLRP3^−/− macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1β production in mφs in comparison to LPS-treated controls. When WT and ASC^−/− macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3^−/− macrophages, despite an inability to elicit IL-1β activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3^−/− macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1β-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1β/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.     

Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL

Cellular FADD-like interleukin-1β–converting enzyme inhibitory proteins (c-FLIPs; isoforms c-FLIP long [c-FLIP_L], c-FLIP short [c-FLIP_S], and c-FLIP Raji [c-FLIP_R]) regulate caspase-8 activation and death receptor (DR)–induced apoptosis. In this study, using a combination of mathematical modeling, imaging, and quantitative Western blots, we present a new mathematical model describing caspase-8 activation in quantitative terms, which highlights the influence of c-FLIP proteins on this process directly at the CD95 death-inducing signaling complex. We quantitatively define how the stoichiometry of c-FLIP proteins determines sensitivity toward CD95-induced apoptosis. We show that c-FLIP_L has a proapoptotic role only upon moderate expression in combination with strong receptor stimulation or in the presence of high amounts of one of the short c-FLIP isoforms, c-FLIP_S or c-FLIP_R. Our findings resolve the present controversial discussion on the function of c-FLIP_L as a pro- or antiapoptotic protein in DR-mediated apoptosis and are important for understanding the regulation of CD95-induced apoptosis, where subtle differences in c-FLIP concentrations determine life or death of the cells.     

NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy in a Type 2 Diabetes Rat Model

Background

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is associated with metabolic disorder and cell death, which are important triggers in diabetic cardiomyopathy (DCM). We aimed to explore whether NLRP3 inflammasome activation contributes to DCM and the mechanism involved.

Methods

Type 2 diabetic rat model was induced by high fat diet and low dose streptozotocin. The characteristics of type 2 DCM were evaluated by metabolic tests, echocardiography and histopathology. Gene silencing therapy was used to investigate the role of NLRP3 in the pathogenesis of DCM. High glucose treated H9c2 cardiomyocytes were used to determine the mechanism by which NLRP3 modulated the DCM. The cell death in vitro was detected by TUNEL and EthD-III staining. TXNIP-siRNA and pharmacological inhibitors of ROS and NF-kB were used to explore the mechanism of NLRP3 inflammasome activation.

Results

Diabetic rats showed severe metabolic disorder, cardiac inflammation, cell death, disorganized ultrastructure, fibrosis and excessive activation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), pro-caspase-1, activated caspase-1 and mature interleukin-1β (IL-1β). Evidence for pyroptosis was found in vivo, and the caspase-1 dependent pyroptosis was found in vitro. Silencing of NLRP3 in vivo did not attenuate systemic metabolic disturbances. However, NLRP3 gene silencing therapy ameliorated cardiac inflammation, pyroptosis, fibrosis and cardiac function. Silencing of NLRP3 in H9c2 cardiomyocytes suppressed pyroptosis under high glucose. ROS inhibition markedly decreased nuclear factor-kB (NF-kB) phosphorylation, thioredoxin interacting/inhibiting protein (TXNIP), NLRP3 inflammasome, and mature IL-1β in high glucose treated H9c2 cells. Inhibition of NF-kB reduced the activation of NLRP3 inflammasome. TXNIP-siRNA decreased the activation of caspase-1 and IL-1β.

Conclusion

NLRP3 inflammasome contributed to the development of DCM. NF-κB and TXNIP mediated the ROS-induced caspase-1 and IL-1β activation, which are the effectors of NLRP3 inflammasome. NLRP3 gene silencing may exert a protective effect on DCM.     

Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke

Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen–glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1β and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1β and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.     

Malaria-Induced NLRP12/NLRP3-Dependent Caspase-1 Activation Mediates Inflammation and Hypersensitivity to Bacterial Superinfection

Cyclic paroxysm and high fever are hallmarks of malaria and are associated with high levels of pyrogenic cytokines, including IL-1β. In this report, we describe a signature for the expression of inflammasome-related genes and caspase-1 activation in malaria. Indeed, when we infected mice, Plasmodium infection was sufficient to promote MyD88-mediated caspase-1 activation, dependent on IFN-γ-priming and the expression of inflammasome components ASC, P2X7R, NLRP3 and/or NLRP12. Pro-IL-1β expression required a second stimulation with LPS and was also dependent on IFN-γ-priming and functional TNFR1. As a consequence of Plasmodium-induced caspase-1 activation, mice produced extremely high levels of IL-1β upon a second microbial stimulus, and became hypersensitive to septic shock. Therapeutic intervention with IL-1 receptor antagonist prevented bacterial-induced lethality in rodents. Similar to mice, we observed a significantly increased frequency of circulating CD14⁺CD16⁻Caspase-1⁺ and CD14^dimCD16⁺Caspase-1⁺ monocytes in peripheral blood mononuclear cells from febrile malaria patients. These cells readily produced large amounts of IL-1β after stimulation with LPS. Furthermore, we observed the presence of inflammasome complexes in monocytes from malaria patients containing either NLRP3 or NLRP12 pyroptosomes. We conclude that NLRP12/NLRP3-dependent activation of caspase-1 is likely to be a key event in mediating systemic production of IL-1β and hypersensitivity to secondary bacterial infection during malaria.     

Virulent Mycobacterium bovis Beijing Strain Activates the NLRP7 Inflammasome in THP-1 Macrophages

Mycobacterium bovis is the causative agent of tuberculosis in a wide range of mammals, including humans. Macrophages are the first line of host defense. They secrete proinflammatory cytokines, such as interleukin-1 beta (IL-1β), in response to mycobacterial infection, but the underlying mechanisms by which human macrophages are activated and release IL-1β following M. bovis infection are poorly understood. Here we show that the ‘nucleotide binding and oligomerization of domain-like receptor (NLR) family pyrin domain containing 7 protein’ (NLRP7) inflammasome is involved in IL-1β secretion and caspase-1 activation induced by M. bovis infection in THP-1 macrophages. NLRP7 inflammasome activation promotes the induction of pyroptosis as well as the expression of tumor necrosis factor alpha (TNF-α), Chemokine (C-C motif) ligand 3 (CCL3) and IL-1β mRNAs. Thus, the NLRP7 inflammasome contributes to IL-1β secretion and induction of pyroptosis in response to M. bovis infection in THP-1 macrophages.     

Nod-like Receptor Protein 3 (NLRP3) Inflammasome Activation and Podocyte Injury via Thioredoxin-Interacting Protein (TXNIP) during Hyperhomocysteinemia

NADPH oxidase-derived reactive oxygen species (ROS) have been reported to activate NLRP3 inflammasomes resulting in podocyte and glomerular injury during hyperhomocysteinemia (hHcys). However, the mechanism by which the inflammasome senses ROS is still unknown in podocytes upon hHcys stimulation. The current study explored whether thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of the antioxidant thioredoxin and ROS sensor, mediates hHcys-induced NLRP3 inflammasome activation and consequent glomerular injury. In cultured podocytes, size exclusion chromatography and confocal microscopy showed that inhibition of TXNIP by siRNA or verapamil prevented Hcys-induced TXNIP protein recruitment to form NLRP3 inflammasomes and abolished Hcys-induced increases in caspase-1 activity and IL-1β production. TXNIP inhibition protected podocytes from injury as shown by normal expression levels of podocyte markers, podocin and desmin. In vivo, adult C57BL/6J male mice were fed a folate-free diet for 4 weeks to induce hHcys, and TXNIP was inhibited by verapamil (1 mg/ml in drinking water) or by local microbubble-ultrasound TXNIP shRNA transfection. Evidenced by immunofluorescence and co-immunoprecipitation studies, glomerular inflammasome formation and TXNIP binding to NLRP3 were markedly increased in mice with hHcys but not in TXNIP shRNA-transfected mice or those receiving verapamil. Furthermore, TXNIP inhibition significantly reduced caspase-1 activity and IL-1β production in glomeruli of mice with hHcys. Correspondingly, TXNIP shRNA transfection and verapamil attenuated hHcys-induced proteinuria, albuminuria, glomerular damage, and podocyte injury. In conclusion, our results demonstrate that TXNIP binding to NLRP3 is a key signaling mechanism necessary for hHcys-induced NLRP3 inflammasome formation and activation and subsequent glomerular injury.     

The c-FLIPL Cleavage Product p43FLIP Promotes Activation of Extracellular Signal-regulated Kinase (ERK), Nuclear Factor κB (NF-κB), and Caspase-8 and T Cell Survival

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIP_L can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIP_L. This triggers cleavage of c-FLIP_L at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.     

Mycobacterium tuberculosis inhibits the NLRP3 inflammasome activation via its phosphokinase PknF

Mycobacterium tuberculosis (Mtb) has evolved to evade host innate immunity by interfering with macrophage functions. Interleukin-1β (IL-1β) is secreted by macrophages after the activation of the inflammasome complex and is crucial for host defense against Mtb infections. We have previously shown that Mtb is able to inhibit activation of the AIM2 inflammasome and subsequent pyroptosis. Here we show that Mtb is also able to inhibit host cell NLRP3 inflammasome activation and pyroptosis. We identified the serine/threonine kinase PknF as one protein of Mtb involved in the NLRP3 inflammasome inhibition, since the pknF deletion mutant of Mtb induces increased production of IL-1β in bone marrow-derived macrophages (BMDMs). The increased production of IL-1β was dependent on NLRP3, the adaptor protein ASC and the protease caspase-1, as revealed by studies performed in gene-deficient BMDMs. Additionally, infection of BMDMs with the pknF deletion mutant resulted in increased pyroptosis, while the IL-6 production remained unchanged compared to Mtb-infected cells, suggesting that the mutant did not affect the priming step of inflammasome activation. In contrast, the activation step was affected since potassium efflux, chloride efflux and the generation of reactive oxygen species played a significant role in inflammasome activation and subsequent pyroptosis mediated by the Mtb pknF mutant strain. In conclusion, we reveal here that the serine/threonine kinase PknF of Mtb plays an important role in innate immune evasion through inhibition of the NLRP3 inflammasome.