NLRP1-Dependent Pyroptosis Leads to Acute Lung Injury and Morbidity in Mice

Acute inflammation in response to both exogenous and endogenous danger signals can lead to the assembly of cytoplasmic inflammasomes that stimulate the activation of caspase-1. Subsequently, caspase-1 facilitates the maturation and release of cytokines and also, under some circumstances, the induction of cell death by pyroptosis. Using a mouse line lacking expression of NLRP1, we show that assembly of this inflammasome in cells is triggered by a toxin from anthrax and that it initiates caspase-1 activation and release of IL-1β. Furthermore, NLRP1 inflammasome activation also leads to cell death, which escalates over 3 d following exposure to the toxin and culminates in acute lung injury and death of the mice. We show that these events are not dependent on production of IL-1β by the inflammasome but are dependent on caspase-1 expression. In contrast, muramyl dipeptide-mediated inflammasome formation is not dependent on NLRP1 but NLRP3. Taken together, our findings show that assembly of the NLRP1 inflammasome is sufficient to initiate pyroptosis, which subsequently leads to a self-amplifying cascade of cell injury within the lung from which the lung cannot recover, eventually resulting in catastrophic consequences for the organism.     

Induction of inflammasome-dependent pyroptosis by carbon black nanoparticles

Inhalation of nanoparticles has been implicated in respiratory morbidity and mortality. In particular, carbon black nanoparticles are found in many different environmental exposures. Macrophages take up inhaled nanoparticles and respond via release of inflammatory mediators and in some cases cell death. Based on new data, we propose that exposure of macrophages (both a macrophage cell line and primary human alveolar macrophages) to carbon black nanoparticles induces pyroptosis, an inflammasome-dependent form of cell death. Exposure of macrophages to carbon black nanoparticles resulted in inflammasome activation as defined by cleavage of caspase 1 to its active form and downstream IL-1β release. The cell death that occurred with carbon black nanoparticle exposure was identified as pyroptosis by the protective effect of a caspase 1 inhibitor and a pyroptosis inhibitor. These data demonstrate that carbon black nanoparticle exposure activates caspase 1, increases IL-1β release after LPS priming, and induces the proinflammatory cell death, pyroptosis. The identification of pyroptosis as a cellular response to carbon nanoparticle exposure is novel and relates to environmental and health impacts of carbon-based particulates.     

Antimicrobial Cathelicidin Peptide LL-37 Inhibits the LPS/ATP-Induced Pyroptosis of Macrophages by Dual Mechanism

Pyroptosis is a caspase-1 dependent cell death, associated with proinflammatory cytokine production, and is considered to play a crucial role in sepsis. Pyroptosis is induced by the two distinct stimuli, microbial PAMPs (pathogen associated molecular patterns) and endogenous DAMPs (damage associated molecular patterns). Importantly, cathelicidin-related AMPs (antimicrobial peptides) have a role in innate immune defense. Notably, human cathelicidin LL-37 exhibits the protective effect on the septic animal models. Thus, in this study, to elucidate the mechanism for the protective action of LL-37 on sepsis, we utilized LPS (lipopolysaccharide) and ATP (adenosine triphosphate) as a PAMP and a DAMP, respectively, and examined the effect of LL-37 on the LPS/ATP-induced pyroptosis of macrophage-like J774 cells. The data indicated that the stimulation of J774 cells with LPS and ATP induces the features of pyroptosis, including the expression of IL-1β mRNA and protein, activation of caspase-1, inflammasome formation and cell death. Moreover, LL-37 inhibits the LPS/ATP-induced IL-1β expression, caspase-1 activation, inflammasome formation, as well as cell death. Notably, LL-37 suppressed the LPS binding to target cells and ATP-induced/P2X₇-mediated caspase-1 activation. Together these observations suggest that LL-37 potently inhibits the LPS/ATP-induced pyroptosis by both neutralizing the action of LPS and inhibiting the response of P2X₇ to ATP. Thus, the present finding may provide a novel insight into the modulation of sepsis utilizing LL-37 with a dual action on the LPS binding and P2X₇ activation.     

Cytolethal distending toxin‐induced release of interleukin‐1β by human macrophages is dependent upon activation of glycogen synthase kinase 3β, spleen tyrosine kinase (Syk) and the noncanonical inflammasome

Cytolethal distending toxins (Cdt) are a family of toxins produced by several human pathogens which infect mucocutaneous tissue and induce inflammatory disease. We have previously demonstrated that the Aggregatibacter actinomycetemcomitans Cdt induces a pro‐inflammatory response from human macrophages which involves activation of the NLRP3 inflammasome. We now demonstrate that in addition to activating caspase‐1 (canonical inflammasome), Cdt treatment leads to caspase‐4 activation and involvement of the noncanonical inflammasome. Cdt‐treated cells exhibit pyroptosis characterised by cleavage of gasdermin‐D (GSDMD), release of HMGB1 at 24 hr and LDH at 48 hr. Inhibition of either the canonical (caspase‐1) or noncanonical (caspase‐4) inflammasome blocks both Cdt‐induced release of IL‐1β and induction of pyroptosis. Analysis of upstream events indicates that Cdt induces Syk phosphorylation (activation); furthermore, blockade of Syk expression and inhibition of pSyk activity inhibit both Cdt‐induced cytokine release and pyroptosis. Finally, we demonstrate that increases in pSyk are dependent upon Cdt‐induced activation of GSK3β. These studies advance our understanding of Cdt function and provide new insight into the virulence potential of Cdt in mediating the pathogenesis of disease caused by Cdt‐producing organisms such as A. actinomycetemcomitans.     

Serum amyloid A and inflammasome activation: A link to breast cancer progression?

Breast cancer is the most frequently diagnosed cancer in women globally. Although there have been many significant advances made in the diagnosis and treatment of breast cancer, numerous unresolved challenges remain, which include prevention, early diagnosis, metastasis and recurrence. The role of inflammation in cancer development is well established and is believed to be one of the leading hallmarks of cancer progression. Recently, the role of the inflammasome, a cytosolic multiprotein complex, has received attention in different cancers. By contributing to the activation of inflammatory cytokines the inflammasome intensifies the inflammatory cascade. The inflammasome can be activated through several pathways, which include the binding of pattern associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) to toll-like receptors (TLRs). Serum amyloid A (SAA), a non-specific acute-phase protein, can function as an endogenous DAMP by binding to pattern recognition receptors like TLRs on both breast cancer cells and cancer associated fibroblasts (CAFs). SAA can thus stimulate the production of IL-1β, thereby creating a favourable inflammatory environment to support tumour growth. The aim of this review is to highlight the possible role of SAA as an endogenous DAMP in the tumour microenvironment (TME) thereby promoting breast cancer growth through the activation of the NLRP3 inflammasome.     

Anthrax Lethal Factor Cleavage of Nlrp1 Is Required for Activation of the Inflammasome

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1β and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.     

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.     

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion

Objective and backgroundActivation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice.MethodsPlasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury.ResultsIn patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects.ConclusionHMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.     

Adenovirus membrane penetration activates the NLRP3 inflammasome

Adenovirus type 5 (Ad5) infection of macrophages results in rapid secretion of interleukin-1β (IL-1β) and is dependent on the inflammasome components NLRP3 and ASC and the catalytic activity of caspase-1. Using lentivirus-expressed short hairpin RNA (shRNA) and competitive inhibitors, we show that Ad-induced IL-1β release is dependent upon Toll-like receptor 9 (TLR9) sensing of the Ad5 double-stranded DNA (dsDNA) genome in human cell lines and primary monocyte-derived macrophages but not in mouse macrophages. Additionally, a temperature-sensitive mutant of Ad5 unable to penetrate endosomal membranes, ts1, is unable to induce IL-1β release in TLR2-primed THP-1 cells, suggesting that penetration of endosomal membranes is required for IL-1β release. Disruption of lysosomal membranes and the release of cathepsin B into the cytoplasm are required for Ad-induced NLRP3 activation. Ad5 cell entry also induces reactive oxygen species (ROS) production, and inhibitors of ROS prevent Ad-induced IL-1β release. Ad5 activation of NLRP3 also induces necrotic cell death, resulting in the release of the proinflammatory molecule HMGB1. This work further defines the mechanisms of virally induced inflammasome activation.     

Regulation of HMGB1 release by inflammasomes

High mobility group box 1 (HMGB1) is an evolutionarily conserved non-histone chromatin-binding protein. During infection or injury, activated immune cells and damaged cells release HMGB1 into the extracellular space, where HMGB1 functions as a proinflammatory mediator and contributes importantly to the pathogenesis of infl ammatory diseases. Recent studies reveal that inflammasomes, intracellular protein complexes, critically regulate HMGB1 release from activated immune cells in response to a variety of exogenous and endogenous danger signals. Double stranded RNA dependent kinase (PKR), an intracellular danger-sensing molecule, physically interacts with inflammasome components and is important for inflammasome activation and HMGB1 release. Together, these studies not only unravel novel mechanisms of HMGB1 release during infl ammation, but also provide potential therapeutic targets to treat HMGB1-related infl ammatory diseases.     

TLR2 signaling contributes to rapid inflammasome activation during F. novicida infection

BACKGROUND: Early detection of microorganisms by the innate immune system is provided by surface-expressed and endosomal pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Detection of microbial components by TLRs initiates a signaling cascade leading to the expression of proinflammatory cytokines including IL-6 and IL-1β. Some intracellular bacteria subvert the TLR response by rapidly escaping the phagosome and entering the cytosol. However, these bacteria may be recognized by the inflammasome, a multi-protein complex comprised of a sensor protein, ASC and the cysteine protease caspase-1. Inflammasome activation leads to release of the proinflammatory cytokines IL-1β and IL-18 and death of the infected cell, an important host defense that eliminates the pathogen's replicative niche. While TLRs and inflammasomes are critical for controlling bacterial infections, it is unknown whether these distinct host pathways cooperate to activate defenses against intracellular bacteria. METHODOLOGY/SIGNIFICANT FINDINGS: Using the intracellular bacterium Francisella novicida as a model, we show that TLR2(-/-) macrophages exhibited delayed inflammasome activation compared to wild-type macrophages as measured by inflammasome assembly, caspase-1 activation, cell death and IL-18 release. TLR2 also contributed to inflammasome activation in response to infection by the cytosolic bacterium Listeria monocytogenes. Components of the TLR2 signaling pathway, MyD88 and NF-κB, were required for rapid inflammasome activation. Furthermore, TLR2(-/-) mice exhibited lower levels of cell death, caspase-1 activation, and IL-18 production than wild-type mice upon F. novicida infection. CONCLUSIONS/SIGNIFICANCE: These results show that TLR2 is required for rapid inflammasome activation in response to infection by cytosolic bacterial pathogens. In addition to further characterizing the role of TLR2 in host defense, these findings broaden our understanding of how the host integrates signals from spatiotemporally separated PRRs to coordinate an innate response against intracellular bacteria.     

Differential regulation of caspase-1 activation via NLRP3/NLRC4 inflammasomes mediated by aerolysin and type III secretion system during Aeromonas veronii infection

Aeromonas spp. are Gram-negative bacteria that cause serious infectious disease in humans. Such bacteria have been shown to induce apoptosis in infected macrophages, yet the host responses triggered by macrophage death are largely unknown. In this study, we demonstrate that the infection of mouse bone marrow-derived macrophages with Aeromonas veronii biotype sobria triggers activation of caspase-1 with the ensuing release of IL-1β and pyroptosis. Caspase-1 activation in response to A. veronii infection requires the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain and both the NLRP3 and NLRC4 inflammasomes. Furthermore, caspase-1 activation requires aerolysin and a functional type III secretion system in A. veronii. Aerolysin-inducing caspase-1 activation is mediated through the NLRP3 inflammasome, with aerolysin-mediated cell death being largely dependent on the NLRP3 inflammasome. In contrast, the type III secretion system activates both the NLRP3 and NLRC4 inflammasomes. Inflammasome-mediated caspase-1 activation is also involved in host defenses against systemic A. veronii infection in mice. Our results indicated that multiple factors from both the bacteria and the host play a role in eliciting caspase-1 activation during A. veronii infection.     

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.     

Ubiquitination is a major modulator for the activation of inflammasomes and pyroptosis

Inflammasomes are a central node of the innate immune defense system against the threat of homeostatic perturbance caused by pathogenic organisms or host-derived molecules. Inflammasomes are generally composed of multimeric protein complexes that assemble in the cytosol after sensing danger signals. Activated inflammasomes promote downstream proteolytic activation, which triggers the release of pro-inflammatory cytokines therefore inducing pyroptotic cell death. The inflammasome pathway is finely tuned by various mechanisms. Recent studies found that protein post-translational modifications such as ubiquitination also modulate inflammasome activation. Targeting the ubiquitination modification of the inflammasome pathway might be a promising strategy for related diseases. In this review, we extensively discuss the advances in inflammasome activation and pyroptosis modulated by ubiquitination which help in-depth understanding and controlling the inflammasome and pyroptosis in various diseases.     

Macrophage endocytosis of high-mobility group box 1 triggers pyroptosis

Macrophages can be activated and regulated by high-mobility group box 1 (HMGB1), a highly conserved nuclear protein. Inflammatory functions of HMGB1 are mediated by binding to cell surface receptors, including the receptor for advanced glycation end products (RAGE), Toll-like receptor (TLR)2, TLR4, and TLR9. Pyroptosis is a caspase-1-dependent programmed cell death, which features rapid plasma membrane rupture, DNA fragmentation, and release of proinflammatory intracellular contents. Pyroptosis can be triggered by various stimuli, however, the mechanism underlying pyroptosis remains unclear. In this study, we identify a novel pathway of HMGB1-induced macrophage pyroptosis. We demonstrate that HMGB1, acting through RAGE and dynamin-dependent signaling, initiates HMGB1endocytosis, which in turn induces cell pyroptosis. The endocytosis of HMGB1 triggers a cascade of molecular events, including cathepsin B release from ruptured lysosomes followed by pyroptosome formation and caspase-1 activation. We further confirm that HMGB1-induced macrophage pyroptosis also occurs in vivo during endotoxemia, suggesting a pathophysiological significance for this form of pyroptosis in the development of inflammation. These findings shed light on the regulatory role of ligand-receptor internalization in directing cell fate, which may have an important role in the progress of inflammation following infection and injury.Infection and injury, the most challenging factors to the survival of species throughout evolution, are still major causes of human death worldwide. Infection and severe trauma share many overlapping features in the mechanism of activation of the innate immune system through pathogen-associated molecular pattern molecules derived from microbes and damage-associated molecular pattern (DAMP) molecules released by damaged tissues.^{1, 2} High-mobility group box 1 (HMGB1), a highly conserved ubiquitous protein present in the nucleus and cytoplasm of nearly all cell types, is the prototypic DAMP molecule.³ During infection and sterile tissue injury, HMGB1 is released from cells and serves as a necessary and sufficient mediator of inflammation to induce a variety of cellular responses including cell chemotaxis and release of pro-inflammatory cytokines.^{4, 5} Inflammatory functions of HMGB1 are mediated by binding to the cell surface receptors, including the receptor for advanced glycation end products (RAGEs), Toll-like receptor (TLR)2, TLR4, and TLR9.^{6, 7}RAGE is a type I transmembrane protein and a member of the immunoglobulin superfamily expressed in many cell populations, including endothelial cells, vascular smooth muscle cells, neurons, neutrophils, and macrophages/monocytes.⁸ RAGE has been implicated as a receptor mediating the chemotaxis and cytokine activity of HMGB1 in macrophages and tumor cells.^{7, 9, 10} RAGE engagement by multiple ligands is linked to a range of signaling pathways including activation of NF-κB,^{11, 12} PI3K/Akt,¹³ MAPKs,^{14, 15} Jak/STAT,¹⁶ and Rho GTPases,¹⁷ although how RAGE transduces the signaling is not fully addressed.Pyroptosis is a caspase-1-dependent programmed cell death, which is morphologically and mechanistically distinct from other forms of cell death such as apoptosis and necrosis.¹⁸ Previous observations suggested that pyroptosis features rapid plasma membrane rupture and release of proinflammatory intracellular contents, contrasting with the characteristic of apoptosis, which packs cellular contents and induces non-inflammatory phagocytic uptake of the apoptotic bodies by macrophages.¹⁹ Pyroptosis can be triggered by various pathological stimuli, such as microbial infection, stroke, heart attack, or cancer;¹⁸ however, the mechanism that underlies pyroptosis formation in response to inflammatory mediators remains unclear. In this study, we identify a novel pathway of HMGB1-induced pyroptosis. We demonstrate that HMGB1 acting through RAGE on macrophages triggers dynamin-dependent endocytosis of HMGB1, which in turn induces cell pyroptosis. The endocytosis of HMGB1 initiates a cascade of molecular events, including cathepsin B (CatB) activation and release from ruptured lysosomes, followed by pyroptosome formation and caspase-1 activation. We further confirm that this in vitro observation of HMGB1-induced macrophage pyroptosis also occurs in vivo during endotoxemia, suggesting a pathophysiological significance for pyroptosis in the development of inflammation.     

Role of lysosome rupture in controlling Nlrp3 signaling and necrotic cell death

The Nod-like receptor, Nlrp3, has been linked to inflammatory diseases and adjuvant-mediated immune responses. A wide array of structurally diverse agents does not interact directly with Nlrp3, but is thought to activate the Nlrp3 inflammasome by inducing a common upstream signal, such as lysosome rupture. To test the connection between lysosome integrity and Nlrp3 signaling, we analyzed inflammasome activation following stimulation of murine macrophages with lysosome-destabilizing agents and pyroptosis inducers. Here we provide evidence that lysosomal rupture and the corresponding release of lysosomal hydrolases is an early event in macrophages exposed to the lysosome-destabilizing adjuvants LLOMe and alum. Lysosome rupture preceded cell death induction mediated by these agents and was associated with the degradation of low-molecular weight proteins, including the inflammasome component caspase-1. Proteolysis of caspase-1 was controlled by specific cathepsins, but was independent of autocatalytic processes and Nlrp3 signaling. Consistent with these findings, lysosome-disrupting agents triggered only minimal caspase-1 activation and failed to cause caspase-1-dependent cell death (pyroptosis), generally associated with Nlrp3 signaling. In contrast, lysosome rupture was a late event in macrophages exposed to prototypical pyroptosis inducers. These agents triggered extensive Nlrp3 signaling prior to lysosome rupture with only minimal impact on the cellular proteome. Taken together, our findings suggest that lysosome impairment triggers a cascade of events culminating in cell death but is not crucial for Nlrp3 signaling. The significant differences observed between lysosome-disrupting agents and pyroptosis inducers might explain the distinct immunologic responses associated with these compounds.     

Hemorrhagic shock primes for lung vascular endothelial cell pyroptosis: role in pulmonary inflammation following LPS

Hemorrhagic shock (HS) often renders patients more susceptible to lung injury by priming for an exaggerated response to a second infectious stimulus. Acute lung injury (ALI) is a major component of multiple organ dysfunction syndrome following HS and regularly serves as a major cause of patient mortality. The lung vascular endothelium is an active organ that has a central role in the development of ALI through synthesizing and releasing of a number of inflammatory mediators. Cell pyroptosis is a caspase-1-dependent regulated cell death, which features rapid plasma membrane rupture and release of proinflammatory intracellular contents. In this study, we demonstrated an important role of HS in priming for LPS-induced lung endothelial cell (EC) pyroptosis. We showed that LPS through TLR4 activates Nlrp3 (NACHT, LRR, and PYD domains containing protein 3) inflammasome in mouse lung vascular EC, and subsequently induces caspase-1 activation. However, HS induced release of high-mobility group box 1 (HMGB1), which acting through the receptor for advanced glycation end products initiates EC endocytosis of HMGB1, and subsequently triggers a cascade of molecular events, including cathepsin B release from ruptured lysosomes followed by pyroptosome formation and caspase-1 activation. These HS-induced events enhance LPS-induced EC pyroptosis. We further showed that lung vascular EC pyroptosis significantly exaggerates lung inflammation and injury. The present study explores a novel mechanism underlying HS-primed ALI and thus presents a potential therapeutic target for post-HS ALI.Hemorrhagic shock (HS) often renders patients more susceptible to a secondary stimulus (e.g., infection) resulting in the development of systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS) by activating and priming the inflammatory process. The underlying mechanism of how HS leads to SIRS and MODS has yet to be fully determined. Acute lung injury (ALI) is a major component of MODS and often serves as a direct cause of patient mortality.^{1, 2} The lung vascular endothelium is an active organ that critically contributes to the pathogenesis of ALI following trauma, sepsis, and shock by affecting pulmonary and systemic homeostasis, including secretion of cytokines, chemokines, and adhesion molecules.^{3, 4} There is a significant gap in our knowledge concerning the mechanisms of HS regulation of lung endothelial cell (EC) activation and death, and subsequent promotion of lung inflammation.Pyroptosis is a caspase-1-dependent form of regulated cell death that is stimulated by a range of microbial infections and non-infectious stimuli.^{5, 6} Morphologically, pyroptosis is characterized by plasma membrane rupture, which results in the release of intracellular contents,^{7, 8, 9, 10, 11} and cleavage of chromosomal DNA.^{7, 11, 12, 13, 14}The magnitude of caspase-1 activation is important for the fate of cells. Low level of activation of caspase-1 might be necessary for cell survival in response to external stimulations.¹⁵ However, over-activation of caspase-1 may serve as a premise for cell pyroptosis.^{5, 6} The platform for caspase-1 activation includes inflammasome and pyroptosome. The former comprises of NOD-like receptors (NLRs) or AIM2 receptor, caspase-1, and apoptosis-associated speck-like protein containing a CARD domain (ASC); and the latter is composed of oligomerized ASC dimers.¹⁶ We have previously reported that HMGB1 has a critical role in activation of inflammasome and pyroptosome in macrophages in a setting of HS.^{17, 18}In this study, we demonstrated an important role of HS in priming for LPS-induced lung EC pyroptosis. We showed that LPS through TLR4 activates Nlrp3 inflammasome in mouse lung vascular EC (MLVEC), and subsequently induces caspase-1 activation. However, HS induced release of high-mobility group box 1 (HMGB1), which acting through the receptor for advanced glycation end products (RAGE) initiates EC endocytosis of HMGB1, and subsequently triggers a cascade of molecular events, including cathepsin B (CatB) release from ruptured lysosomes followed by pyroptosome formation and caspase-1 activation. These HS-induced events enhance LPS-induced EC pyroptosis. We further showed that lung vascular EC pyroptosis significantly exaggerates lung inflammation and injury. The present study explores a novel mechanism underlying HS-primed ALI and thus presents a potential therapeutic target for ALI induced after HS.     

炎性小体激活与细胞焦亡的研究进展

细胞焦亡是一种依赖天冬氨酸特异性半胱氨酸蛋白酶1(cysteinyl aspartate specific proteinase 1,caspase-1)/caspase-11的程序性细胞死亡方式。炎性小体的激活在细胞焦亡过程中扮演重要角色。当病原体入侵时,核苷酸结合寡聚化结构域样受体(nucleotide-binding oligomerization domain-like receptor,NLR)和黑色素瘤缺乏因子2(absent in melanoma 2,AIM2)等胞内模式识别受体(pattern recognition receptor,PRR)与相应配体结合,导致炎性小体多蛋白复合物组装和caspase-1/caspase-11激活,进而诱导细胞焦亡发生。深入研究炎性小体激活和细胞焦亡的相关机制,对认识炎症性疾病的发生发展非常重要。本文就炎性小体激活与细胞焦亡的研究进展进行综述。     

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.     

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.