Strain- and host species-specific inflammasome activation,IL-1β release,and cell death in macrophages infected with uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1β (IL-1β) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1β secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1β secretion pathway in human macrophages. This has important implications for understanding UTI in humans.     

An NLRP7-containing inflammasome mediates recognition of microbial lipopeptides in human macrophages

Cytosolic pathogen- and damage-associated molecular patterns are sensed by pattern recognition receptors, including members of the nucleotide-binding domain and leucine-rich repeat-containing gene family (NLR), which cause inflammasome assembly and caspase-1 activation to promote maturation and release of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and induction of pyroptosis. However, the contribution of most of the NLRs to innate immunity, host defense, and inflammasome activation and their specific agonists are still unknown. Here we describe identification and characterization of an NLRP7 inflammasome in human macrophages, which is induced in response to microbial acylated lipopeptides. Activation of NLRP7 promoted ASC-dependent caspase-1 activation, IL-1β and IL-18 maturation, and restriction of intracellular bacterial replication, but not caspase-1-independent secretion of the proinflammatory cytokines IL-6 and tumor necrosis factor-α. Our study therefore increases our currently limited understanding of NLR activation, inflammasome assembly, and maturation of IL-1β and IL-18 in human macrophages.     

阿托伐他汀通过下调NLRP1炎性体表达抑制IL-1β和IL-18的释放

 目的:探讨核苷酸结合寡聚化结构域样受体蛋白1(NLRP1)炎性体在阿托伐他汀抑制THP-1巨噬细胞白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)分泌中的作用。方法:用10 μg/L脂多糖诱导THP-1巨噬细胞分泌IL-1β和IL-18,以不同浓度阿托伐他汀(1、10和20 μmol/L)孵育细胞24 h,或以10 μmol/L阿托伐他汀处理细胞不同时间(12、24和48 h),或转染NLRP1 siRNA以沉默细胞内NLRP1的表达。采用RT-PCR检测细胞内NLRP1炎性体mRNA的表达,Western blot检测细胞内NLRP1炎性体蛋白的表达,ELISA检测细胞上清液中IL-1β和IL-18的含量。结果:阿托伐他汀可抑制THP-1巨噬细胞NLRP1炎性体mRNA和蛋白的表达,且这种效应呈浓度和时间依赖性。转染NLRP1 siRNA后,THP-1巨噬细胞NLRP1的蛋白表达明显下降,且阿托伐他汀对IL-1β和IL-18分泌的抑制作用明显增强。结论:阿托伐他汀通过抑制NLRP1炎性体表达减少巨噬细胞IL-1β和IL-18的释放,发挥抗炎作用,进而延缓动脉粥样硬化进展。     

Sphingosine regulates the NLRP3-inflammasome and IL-1β release from macrophages

Interleukin-1β (IL-1β) is a pro-inflammatory cytokine that regulates inflammatory responses to injury and infection. IL-1β secretion requires the protease caspase-1, which is activated following recruitment to inflammasomes. Endogenous danger-associated molecular patterns (DAMPs) released from necrotic cells activate caspase-1 through an NLRP3-inflammasome. Here, we show that the endogenous lipid metabolite sphingosine (Sph) acts as a DAMP by inducing the NLRP3-inflammasome-dependent secretion of IL-1β from macrophages. This process was dependent upon serine/threonine protein phosphatases since the PP1/PP2A inhibitors okadaic acid and calyculin A inhibited Sph-induced IL-1β release. IL-1β release induced by other well-characterized NLRP3-inflammasome activators, such as ATP and uric acid crystals, in addition to NLRC4 and AIM2 inflammasome activators was also blocked by these inhibitors. Thus, we propose Sph as a new DAMP, and that a serine/threonine phosphatase (PP1/PP2A)-dependent signal is central to the endogenous host mechanism through which diverse stimuli regulate inflammasome activation.     

The murine neutrophil NLRP3 inflammasome is activated by soluble but not particulate or crystalline agonists

Neutrophils express pattern recognition receptors (PRRs) and regulate immune responses via PRR‐dependent cytokine production. An emerging theme is that neutrophil PRRs often exhibit cell type‐specific adaptations in their signalling pathways. This prompted us to examine inflammasome signalling by the PRR NLRP3 in murine neutrophils, in comparison to well‐established NLRP3 signalling pathways in macrophages. Here, we demonstrate that while murine neutrophils can indeed signal via the NLRP3 inflammasome, neutrophil NLRP3 selectively responds to soluble agonists but not to the particulate/crystalline agonists that trigger NLRP3 activation in macrophages via phagolysosomal rupture. In keeping with this, alum did not trigger IL‐1β production from human PMN, and the lysosomotropic peptide Leu‐Leu‐OMe stimulated only weak NLRP3‐dependent IL‐1β production from murine neutrophils, suggesting that lysosomal rupture is not a strong stimulus for NLRP3 activation in neutrophils. We validated our in vitro findings for poor neutrophil NLRP3 responses to particles in vivo, where we demonstrated that neutrophils do not significantly contribute to alum‐induced IL‐1β production in mice. In all, our studies highlight that myeloid cell identity and the nature of the danger signal can strongly influence signalling by a single PRR, thus shaping the nature of the resultant immune response.     

ADP/P2Y1 aggravates inflammatory bowel disease through ERK5-mediated NLRP3 inflammasome activation

Inflammasomes are essential for inflammation and pathogen elimination in response to microbial infection and endogenous danger signals. However, the mechanism of inflammasome activation by endogenous danger signals mediated posttranslational modification and the connection between inflammasomes and inflammatory diseases remains elusive. In this study, we found that ADP was highly released from injured colonic tissue as a danger signal during inflammatory bowel disease. Consequently, extracellular ADP activated the NLRP3 inflammasome through P2Y₁ receptor-mediated calcium signaling, which led to the maturation and secretion of IL-1β and further aggravation of experimental colitis. Genetic ablation or pharmacological blockade of the P2Y₁ receptor significantly ameliorated DSS-induced colitis and endotoxic shock through reducing NLRP3 inflammasome activation. Moreover, ERK5-mediated tyrosine phosphorylation of ASC was essential for activation of the NLRP3 inflammasome. Thus, our study provides a novel theoretical basis for posttranslational modification of ASC in NLRP3 inflammasome activation and revealed that ADP/P2Y₁ is a potential drug target for inflammatory bowel disease.     

The protein kinase PKR is critical for LPS‐induced iNOS production but dispensable for inflammasome activation in macrophages

Inflammasomes are multi‐protein platforms that drive the activation of caspase‐1 leading to the processing and secretion of biologically active IL‐1β and IL‐18. Different inflammasomes including NOD‐like receptor (NLR) family pyrin domain‐containing 3 (NLRP3), NLR caspase‐recruitment domain‐containing 4 (NLRC4) and absent in melanoma 2 (AIM2) are activated and assembled in response to distinct microbial or endogenous stimuli. However, the mechanisms by which upstream stimuli trigger inflammasome activation remain poorly understood. Double‐stranded RNA‐activated protein kinase (PKR), a protein kinase activated by viral infection, has been recently shown to be required for the activation of the inflammasomes. Using macrophages from two different mouse strains deficient in PKR, we found that PKR is important for the induction of the inducible nitric oxide synthase (iNOS). However, PKR was dispensable for caspase‐1 activation, processing of pro‐IL‐1β/IL‐18 and secretion of IL‐1β induced by stimuli that trigger the activation of NLRP3, NLRC4 and AIM2. These results indicate that PKR is not required for inflammasome activation in macrophages.     

Sensing damage by the NLRP3 inflammasome

The NLRP3 inflammasome is activated in response to a variety of signals that are indicative of damage to the host including tissue damage, metabolic stress, and infection. Upon activation, the NLRP3 inflammasome serves as a platform for activation of the cysteine protease caspase-1, which leads to the processing and secretion of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Dysregulated NLRP3 inflammasome activation is associated with both heritable and acquired inflammatory diseases. Here, we review new insights into the mechanism of NLRP3 inflammasome activation and its role in disease pathogenesis.     

Cirtical role for Salmonella effector SopB in regulating inflammasome activation

ObjectiveSalmonella is known to evolve many mechanisms to avoid or delay inflammasome activation which remain largely unknown. In this study, we investigated whether the SopB protein critical to bacteria virulence capacity was an effector that involved in the regulation of inflammasome activation.MethodsBMDMs from NLRC4-, NLRP3-, caspase-1/-11-, IFI16- and AIM2-deficient mice were pretreated with LPS, and subsequently stimulated with a series of SopB-related strains of Salmonella, inflammasome induced cell death, IL-1β secretion, cleaved caspase-1 production and ASC speckle formation were detected.ResultsWe found that SopB could inhibit host IL-1β secretion, caspase-1 activation and inflammasome induced cell death using a series of SopB-related strains of Salmonella; however the reduction of IL-1β secretion was not dependent on sensor that contain PYD domain, such as NLRP3, AIM2 or IFI16, but dependent on NLRC4. Notably, SopB specifically prevented ASC oligomerization and the enzymatic activity of SopB was responsible for the inflammasome inhibition. Furthermore, inhibition of Akt signaling induced enhanced inflammasome activation.ConclusionsThese results revealed a novel role in inhibition of NLRC4 inflammasome for Salmonella effector SopB.     

Mechanisms of NLRP3 inflammasome activation and its role in NSAID-induced enteropathy

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce cytokines, including tumor necrosis factor-α and interleukins (ILs), in the small intestine via a Toll-like receptor 4 (TLR4)-dependent pathway, leading to intestinal ulceration. Activation of the inflammasome promotes pro-caspase-1 cleavage, leading to pro-IL-1β maturation. We examined the role of NLRP3 inflammasome in NSAID-induced enteropathy. Small intestinal damage developed 3 h after indomethacin administration, accompanied by increases in IL-1β and NLRP3 mRNA expression and mature caspase-1 and IL-1β levels. In vivo blocking of IL-1β using neutralizing antibodies attenuated indomethacin-induced damage, whereas exogenous IL-1β aggravated it. NLRP3^−/− and caspase-1^−/− mice exhibited resistance to the damage with reduction of mature IL-1β production. This resistance was abolished by exogenous IL-1β. TLR4 deficiency prevented intestinal damage and inhibited upregulation of NLRP3 and IL-1β mRNAs and maturation of pro-caspase-1 and pro-IL-1β, whereas TLR4 activation by its agonists exerted opposite effects. Apyrase, an adenosine triphosphate (ATP) scavenger, or Brilliant Blue G, a purinergic P2X₇ receptor antagonist, inhibited the damage as well as caspase-1 activation and IL-1β processing, despite there being sufficient amounts of pro-IL-1β and NLRP3. These results suggest that NLRP3 inflammasome-derived IL-1β plays a crucial role in NSAID-induced enteropathy and that both TLR4- and P2X₇-dependent pathways are required for NLRP3 inflammasome activation.     

Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis

We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1β (IL-1β). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The antiapoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1β production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1β secretion could be competitively inhibited by the oxidized nucleoside 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.     

Histones trigger sterile inflammation by activating the NLRP3 inflammasome

Sterile cell death mediated inflammation is linked to several pathological disorders and involves danger recognition of intracellular molecules released by necrotic cells that activate different groups of innate pattern recognition receptors. Toll‐like receptors directly interact with their extrinsic or intrinsic agonists and induce multiple proinflammatory mediators. In contrast, the NLRP3 inflammasome is rather thought to represent a downstream element integrating various indirect stimuli into proteolytic cleavage of interleukin (IL)–1β and IL‐18. Here, we report that histones released from necrotic cells induce IL‐1β secretion in an NLRP3–ASC‐caspase‐1‐dependent manner. Genetic deletion of NLRP3 in mice significantly attenuated histone‐induced IL‐1β production and neutrophil recruitment. Furthermore, necrotic cells induced neutrophil recruitment, which was significantly reduced by histone‐neutralizing antibodies or depleting extracellular histones via enzymatic degradation. These results identify cytosolic uptake of necrotic cell‐derived histones as a triggering mechanism of sterile inflammation, which involves NLRP3 inflammasome activation and IL‐1β secretion via oxidative stress.     

Brucella abortus nitric oxide metabolite regulates inflammasome activation and IL‐1β secretion in murine macrophages

NLRP3 inflammasome is a protein complex crucial to caspase‐1 activation and IL‐1β and IL‐18 maturation. This receptor participates in innate immune responses to different pathogens, including the bacteria of genus Brucella. Our group recently demonstrated that Brucella abortus‐induced IL‐1β secretion involves NLRP3 inflammasome and it is partially dependent on mitochondrial ROS production. However, other factors could be involved, such as P2X7‐dependent potassium efflux, membrane destabilization, and cathepsin release. Moreover, there is increasing evidence that nitric oxide acts as a modulator of NLRP3 inflammasome. The aim of this study was to unravel the mechanism of NLRP3 inflammasome activation induced by B. abortus, as well as the involvement of bacterial nitric oxide (NO) as a modulator of this inflammasome pathway. We demonstrated that NO produced by B. abortus can be used by the bacteria to modulate IL‐1β secretion in infected murine macrophages. Additionally, our results suggest that B. abortus‐induced IL‐1β secretion depends on a P2X7‐independent potassium efflux, lysosomal acidification, cathepsin release, mechanisms clearly associated to NLRP3 inflammasome. In summary, our results help to elucidate the molecular mechanisms of NLRP3 activation and regulation during an intracellular bacterial infection.     

NLRP3 inflammasome activation regulated by NF-κB and DAPK contributed to paraquat-induced acute kidney injury

Paraquat can result in dysfunction of multiple organs after ingestion in human. However, the mechanisms of nucleotide-binding domain and leucine-rich repeat containing protein 3 (NLRP3) inflammasome activation in acute kidney injury have not been clearly demonstrated. The aim of this study was to determine the effect of NLRP3 inflammasome activation and its regulation by nuclear factor-kappa B (NF-κB) and death-associated protein kinase (DAPK). Male Wistar rats were treated with intraperitoneal injection of paraquat at 20 mg/kg, and NF-κB inhibitor BAY 11-7082 was pretreated at 10 mg/kg 1 h before paraquat exposure. Additionally, rat renal tubular epithelial cells (NRK-52E) were transfected with small interfering RNA (siRNA) against DAPK to evaluate its role in NLRP3 inflammasome activation. DAPK and NLRP3 inflammasome were evaluated by immunohistochemistry staining or Western blot; the pro-inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and interleukin-18 (IL-18) were measured via ELISA. The results showed that NF-κB, DAPK, and NLRP3 inflammasome were activated in paraquat (PQ)-treated rat kidney; the secretion of pro-inflammatory cytokines was significantly increased. These toxic effects were attenuated by NF-κB inhibitor. Besides, the activation of NLRP3 inflammasome and secretion of IL-1β and IL-18 in paraquat-treated rat renal tubular epithelial cells were inhibited by siRNA against DAPK. In conclusion, NLRP3 inflammasome activation regulated by NF-κB and DAPK played an important role in paraquat-induced acute kidney injury.     

The NLRP3 inflammasome: a therapeutic target for inflammation-associated cancers

ABSTRACT

Introduction: Inflammasomes are large multimeric intracellular complexes that are capable of maturation and secretion of pro-inflammatory cytokines, IL-1β and IL-18, in response to danger signal molecules. As a member of the inflammasome family, the NLRP3 inflammasome has recently been under intense investigation revealing its possible role in several human diseases especially cancers.

Areas covered: In this review, we will discuss the biology and mechanism of NLRP3 inflammasome activation, its role in specific types of tumors and the novel therapeutic modalities targeting this complex.

Expert opinion: The NLRP3 inflammasome and its components including the adapter apoptosis-associated speck-like (ASC) protein and caspase-1 impose different and sometimes contrasting effects in tumorigenesis depending on various contexts. Considering the novel role of this complex in the initiation and progression of neoplasia, the NLRP3 inflammasome and its pathways provide desirable therapeutic targets for prevention, treatment, and prognosis of certain types of cancer. To date, several agents have been introduced for this purpose, some of which have shown promising results in the clinic.     

Negative regulation of NLRP3 inflammasome by SIRT1 in vascular endothelial cells

NLRP3 inflammasome not only functions as a critical effector in innate immunity, but also triggers the production of proinflammatory cytokines involved in inflammation-associated diseases. Sirtuin 1 (SIRT1) plays an important role in the regulation of cellular inflammation. However, whether the activation of NLRP3 inflammasome is regulated by SIRT1 remains unknown. In this study, we investigated the regulatory effect of SIRT1 on NLRP3 inflammasome and the underlying mechanisms. We found that lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced the activation of NLRP3 inflammasome in human umbilical vein endothelial cells (HUVECs). Activation of SIRT1 inhibited NLRP3 inflammasome activation and subsequent caspase-1 cleavage as well as interleukin (IL)-1β secretion, whereas SIRT1 knockdown obviously enhanced the activation of NLRP3 inflammasome in HUVECs. Importantly, gene silencing of SIRT1 abrogated the inhibitory effect of SIRT1 activator on NLRP3 inflammasome formation and IL-1β production in HUVECs stimulated with LPS plus ATP. Further study indicated that cluster of differentiation 40 (CD40) may be involved in the regulation of NLRP3 inflammasome by SIRT1. In vivo studies indicated that implantation of the periarterial carotid collar increased the arterial expression levels of CD40 and CD40 Ligand (CD40L), but inhibited arterial SIRT1 expression in the rabbits. Moreover, treatment with SIRT1 activator decreased CD40 and CD40L levels in collared arteries. Meanwhile, serum IL-1β level, the marker of inflammasome activation, was also inhibited by SIRT1 activation. Taken together, these findings revealed a novel regulatory mechanism of NLRP3 inflammasome by SIRT1, which may be related to suppression of CD40.     

Caspase‐11 activates a canonical NLRP3 inflammasome by promoting K+ efflux

Recognition of microbe‐associated molecular patterns or endogenous danger signals by a subset of cytosolic PRRs results in the assembly of multiprotein signaling complexes, the so‐called inflammasomes. Canonical inflammasomes are assembled by NOD‐like receptor (NLR) or PYHIN family members and activate caspase‐1, which promotes the induction of pyroptosis and the release of mature interleukin‐1β/‐18. Recently, a noncanonical inflammasome pathway was discovered that results in caspase‐11 activation in response to bacterial lipopolysaccharide (LPS) in the cytosol. Interestingly, caspase‐11 induces pyroptosis by itself, but requires NLRP3, the inflammasome adapter ASC, and caspase‐1 to promote cytokine secretion. Here, we have studied the mechanism by which caspase‐11 controls IL‐1β secretion. Investigating NLRP3/ASC complex formation, we find that caspase‐11 functions upstream of a canonical NLRP3 inflammasome. The activation of NLRP3 by caspase‐11 during LPS transfection is a cell‐intrinsic process and is independent of the release of danger signals. Furthermore, we show that active caspase‐11 leads to a drop of intracellular potassium levels, which is necessary to activate NLRP3. Our study, therefore, sheds new light on the mechanism of noncanonical inflammasome signaling.     

The NLRP3/ASC/Caspase-1 axis regulates IL-1β processing in neutrophils

Neutrophils play a pivotal role in the defense against bacterial, viral and fungal infections and are important mediators in the acute inflammatory response. At the same time, neutrophils are also in volved in sterile inflammatory responses that are triggered by endogenous ligands. A series of immediate effector functions and the expression of proinflammatory genes enable neutrophils to initiate the immune response against the injurious agent. Among these, interleukin-1β (IL-1β) plays a key role in the orchestration of the inflammatory response. Induction of IL-1β expression leads to production of cytosolic pro-IL-1β, which requires further processing by a proteolytic cleavage event. Caspase-1 was initially identified as the main IL-1β-converting enzyme, and the upstream events leading to caspase-1 activation were identified as so-called inflammasome complexes. Up to now, the inflammasome system has mainly been studied in macrophages, whereas the inflammasome was thought to play a redundant or no role in the cell intrinsic processing of pro-IL-1β in neutrophils. Here, we identify the expression of the components of the NLRP3 inflammasome complex in neutrophils and show that the NLRP3 inflammasome pathway is indeed operational in neutrophils. Our findings establish the NLRP3 inflammasome as a key step in the secretion of matured IL-1β by neutrophils.     

Secreted aspartic proteases of Candida albicans activate the NLRP3 inflammasome

In a recent report, we demonstrated that distinct members of the secreted aspartic protease (Sap) family of Candida albicans are able to induce secretion of proinflammatory cytokines by human monocytes, independently of their proteolytic activity and specific pH optima. In particular, C. albicans Sap2 and Sap6 potently induced IL‐1β, TNF‐α, and IL‐6 production. Here, we demonstrate that Sap2 and Sap6 proteins trigger IL‐1β and IL‐18 production through inflammasome activation. This occurs via NLRP3 and caspase‐1 activation, which cleaves pro‐IL‐1β into secreted bioactive IL‐1β, a cytokine that was induced by Saps in monocytes, in monocyte‐derived macrophages and in dendritic cells. Downregulation of NLRP3 by RNA interference strongly reduced the secretion of bioactive IL‐1β. Inflammasome activation required Sap internalization via a clathrin‐dependent mechanism, intracellular induction of K⁺ efflux, and ROS production. Inflammasome activation of monocytes induced by Sap2 and Sap6 differed from that induced by LPS‐ATP in several aspects. Our data reveal novel immunoregulatory mechanisms of C. albicans and suggest that Saps contribute to the pathogenesis of candidiasis by fostering rather than evading host immunity.