Myxoma virus lacking the pyrin-like protein M013 is sensed in human myeloid cells by both NLRP3 and multiple Toll-like receptors, which independently activate the inflammasome and NF-κB innate response pathways

The myxoma virus (MYXV)-encoded pyrin domain-containing protein M013 coregulates inflammatory responses mediated by both the inflammasome and the NF-κB pathways. Infection of human THP-1 monocytic cells with a MYXV construct deleted for the M013 gene (vMyxM013-KO), but not the parental MYXV, activates both the inflammasome and NF-κB pathways and induces a spectrum of proinflammatory cytokines and chemokines, like interleukin-1β (IL-1β), tumor necrosis factor (TNF), IL-6, and monocyte chemoattractant protein 1. Here, we report that vMyxM013-KO virus-mediated activation of inflammasomes and secretion of IL-1β are dependent on the adaptor protein ASC, caspase-1, and NLRP3 receptor. However, vMyxM013-KO virus-mediated activation of NF-κB signaling, which induces TNF secretion, was independent of ASC, caspase-1, and either the NLRP3 or AIM2 inflammasome receptors. We also report that early synthesis of pro-IL-1β in response to vMyxM013-KO infection is dependent upon the components of the inflammasome complex. Activation of the NLRP3 inflammasome and secretion of IL-1β was also dependent on the release of cathepsin B and production of reactive oxygen species (ROS). By using small interfering RNA screening, we further demonstrated that, among the RIG-I-like receptors (RLRs) and Toll-like receptors (TLRs), only TLR2, TLR6, TLR7, and TLR9 contribute to the NF-κB-dependent secretion of TNF and the inflammasome-dependent secretion of IL-1β in response to vMyxM013-KO virus infection. Additionally, we demonstrate that early triggering of the mitogen-activated protein kinase pathway by vMyxM013-KO virus infection of THP-1 cells plays a critical common upstream role in the coordinate induction of both NF-κB and inflammasome pathways. We conclude that an additional cellular sensor(s)/receptor(s) in addition to the known RLRs/TLRs plays a role in the M013 knockout virus-induced activation of NF-κB pathway signaling, but the activation of inflammasomes entirely depends on sensing by the NLRP3 receptor in response to vMyxM013-KO infection of human myeloid cells.     

Pyrin levels in human monocytes and monocyte-derived macrophages regulate IL-1beta processing and release

Macrophages and their precursors, monocytes, are key cells involved in the innate immune response. Although both monocytes and macrophages produce caspase-1, the key enzyme responsible for pro-IL-1beta processing; macrophages are limited in their ability to activate the enzyme and release functional IL-1beta. In this context, because mutations in the pyrin gene (MEFV) cause the inflammatory disorder familial Mediterranean fever, pyrin is believed to regulate IL-1beta processing. To determine whether variations in pyrin expression explain the difference between monocytes and macrophages in IL-1beta processing and release, pyrin was studied in human monocytes and monocyte-derived macrophages. Although monocytes express pyrin mRNA and protein, which is readily inducible by endotoxin, monocyte-derived macrophages express significantly less pyrin mRNA and protein. Pyrin levels directly correlated with IL-1beta processing in monocytes and macrophages; therefore, we asked whether pyrin might promote IL-1beta processing and release. HEK293 cells were transfected with pyrin, caspase-1, apoptotic speck protein with a caspase recruitment domain, and IL-1beta. Pyrin induced IL-1beta processing and release in a dose-dependent manner. Conversely, pyrin small interference RNA suppressed pro-IL-1beta processing in both THP-1 cells and fresh human monocytes. In summary, both pyrin expression and IL-1beta processing and release are diminished upon the maturation of monocytes to macrophages. When pyrin is ectopically expressed or silenced, IL-1beta processing and release parallels the level of pyrin. In conclusion, in the context of endotoxin-induced activation of mononuclear phagocytes, pyrin augments IL-1beta processing and release.     

Serum amyloid A activates the NLRP3 inflammasome via P2X7 receptor and a cathepsin B-sensitive pathway

Serum amyloid A (SAA) is an acute-phase protein, the serum levels of which can increase up to 1000-fold during inflammation. SAA has a pathogenic role in amyloid A-type amyloidosis, and increased serum levels of SAA correlate with the risk for cardiovascular diseases. IL-1β is a key proinflammatory cytokine, and its secretion is strictly controlled by the inflammasomes. We studied the role of SAA in the regulation of IL-1β production and activation of the inflammasome cascade in human and mouse macrophages, as well as in THP-1 cells. SAA could provide a signal for the induction of pro-IL-1β expression and for inflammasome activation, resulting in secretion of mature IL-1β. Blocking TLR2 and TLR4 attenuated SAA-induced expression of IL1B, whereas inhibition of caspase-1 and the ATP receptor P2X(7) abrogated the release of mature IL-1β. NLRP3 inflammasome consists of the NLRP3 receptor and the adaptor protein apoptosis-associated speck-like protein containing CARD (a caspase-recruitment domain) (ASC). SAA-mediated IL-1β secretion was markedly reduced in ASC(-/-) macrophages, and silencing NLRP3 decreased IL-1β secretion, confirming NLRP3 as the SAA-responsive inflammasome. Inflammasome activation was dependent on cathepsin B activity, but it was not associated with lysosomal destabilization. SAA also induced secretion of cathepsin B and ASC. In conclusion, SAA can induce the expression of pro-IL-1β and activation of the NLRP3 inflammasome via P2X(7) receptor and a cathepsin B-sensitive pathway. Thus, during systemic inflammation, SAA may promote the production of IL-1β in tissues. Furthermore, the SAA-induced secretion of active cathepsin B may lead to extracellular processing of SAA and, thus, potentially to the development of amyloid A amyloidosis.     

Burkholderia cenocepacia Type VI Secretion System Mediates Escape of Type II Secreted Proteins into the Cytoplasm of Infected Macrophages

Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs) resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.     

Varicella-zoster virus infection triggers formation of an interleukin-1β (IL-1β)-processing inflammasome complex

Innate cellular immunity is the immediate host response against pathogens, and activation of innate immunity also modulates the induction of adaptive immunity. The nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular receptors that recognize conserved patterns associated with intracellular pathogens, but information about their role in the host defense against DNA viruses is limited. Here we report that varicella-zoster virus (VZV), an alphaherpesvirus that is the causative agent of varicella and herpes zoster, induces formation of the NLRP3 inflammasome and the associated processing of the proinflammatory cytokine IL-1β by activated caspase-1 in infected cells. NLRP3 inflammasome formation was induced in VZV-infected human THP-1 cells, which are a transformed monocyte cell line, primary lung fibroblasts, and melanoma cells. Absent in melanoma gene-2 (AIM2) is an interferon-inducible protein that can form an alternative inflammasome complex with caspase-1 in virus-infected cells. Experiments in VZV-infected melanoma cells showed that NLRP3 protein recruits the adaptor protein ASC and caspase-1 to form an NLRP3 inflammasome complex independent of AIM2 protein and in the absence of free radical reactive oxygen species release. NLRP3 was also expressed extensively in infected skin xenografts in the severe combined immunodeficiency mouse model of VZV pathogenesis in vivo. We conclude that NLRP3 inflammasome formation is an innate cellular response to infection with this common pathogenic human herpesvirus.     

The rate of interleukin-1beta secretion in different myeloid cells varies with the extent of redox response to Toll-like receptor triggering

Human myeloid cells activate the NLRP3 inflammasome and secrete interleukin (IL)-1β in response to various Toll-like receptor (TLR) ligands, but the rate of secretion is much higher in primary human monocytes than in cultured macrophages or THP-1 cells. The different myeloid cells also display different redox status under resting conditions and redox response to TLR activation. Resting monocytes display a balanced redox state, with low production of reactive oxygen species (ROS) and antioxidants. TLR engagement induces an effective redox response with increased ROS generation followed by a sustained antioxidant response, parallelled by efficient IL-1β secretion. Drugs blocking ROS production or the antioxidant response prevent the secretion of mature IL-1β but not the biosynthesis of pro-IL-1β, indicating that redox remodeling is responsible for IL-1β processing and release. Unlike monocytes, THP-1 cells and cultured macrophages have up-regulated antioxidant systems that buffer the oxidative hit provided by TLR triggering and suppress the consequent redox response. This aborted redox remodeling is paralleled by low efficiency IL-1β processing and secretion. High doses (5 mM) of H(2)O(2) overcome the high antioxidant capacity of THP-1 cells, restore an efficient redox response, and increase the rate of IL-1β secretion. Together these data indicate that a tightly controlled redox homeostasis in resting cells is a prerequisite for a robust redox response to TLR ligands, in turn necessary for the efficient inflammasome activation. Inflammasome activation by bacterial DNA is not modulated by redox responses, suggesting that redox-dependent regulation of IL-1β secretion is restricted to some inflammasomes including NLRP3 but excluding AIM-2.     

Measles virus V protein inhibits NLRP3 inflammasome-mediated interleukin-1β secretion

Inflammasomes are cytosolic protein complexes that stimulate the activation of caspase-1, which in turn induces the secretion of the inflammatory cytokines Interleukin-1β (IL-1β) and IL-18. Recent studies have indicated that the inflammasome known as the NOD-like-receptor-family, pyrin domain-containing 3 (NLRP3) inflammasome recognizes several RNA viruses, including the influenza and encephalomyocarditis viruses, whereas the retinoic acid-inducible gene I (RIG-I) inflammasome may detect vesicular stomatitis virus. We demonstrate that measles virus (MV) infection induces caspase-1-dependent IL-1β secretion in the human macrophage-like cell line THP-1. Gene knockdown experiments indicated that IL-1β secretion in MV-infected THP-1 cells was mediated by the NLRP3 inflammasome but not the RIG-I inflammasome. MV produces the nonstructural V protein, which has been shown to antagonize host innate immune responses. The recombinant MV lacking the V protein induced more IL-1β than the parental virus. THP-1 cells stably expressing the V protein suppressed NLRP3 inflammasome-mediated IL-1β secretion. Furthermore, coimmunoprecipitation assays revealed that the V protein interacts with NLRP3 through its carboxyl-terminal domain. NLRP3 was located in cytoplasmic granular structures in THP-1 cells stably expressing the V protein, but upon inflammasome activation, NLRP3 was redistributed to the perinuclear region, where it colocalized with the V protein. These results indicate that the V protein of MV suppresses NLRP3 inflammasome-mediated IL-1β secretion by directly or indirectly interacting with NLRP3.     

TLR2/MyD88/NF-κB pathway, reactive oxygen species, potassium efflux activates NLRP3/ASC inflammasome during respiratory syncytial virus infection

Human respiratory syncytial virus (RSV) constitute highly pathogenic virus that cause severe respiratory diseases in newborn, children, elderly and immuno-compromised individuals. Airway inflammation is a critical regulator of disease outcome in RSV infected hosts. Although "controlled" inflammation is required for virus clearance, aberrant and exaggerated inflammation during RSV infection results in development of inflammatory diseases like pneumonia and bronchiolitis. Interleukin-1β (IL-1β) plays an important role in inflammation by orchestrating the pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 to yield mature IL-1β that is secreted extracellularly. Activation of caspase-1 is mediated by a multi-protein complex known as the inflammasome. Although RSV infection results in IL-1β release, the mechanism is unknown. Here in, we have characterized the mechanism of IL-1β secretion following RSV infection. Our study revealed that NLRP3/ASC inflammasome activation is crucial for IL-1β production during RSV infection. Further studies illustrated that prior to inflammasome formation; the "first signal" constitutes activation of toll-like receptor-2 (TLR2)/MyD88/NF-κB pathway. TLR2/MyD88/NF-κB signaling is required for pro-IL-1β and NLRP3 gene expression during RSV infection. Following expression of these genes, two "second signals" are essential for triggering inflammasome activation. Intracellular reactive oxygen species (ROS) and potassium (K(+)) efflux due to stimulation of ATP-sensitive ion channel promote inflammasome activation following RSV infection. Thus, our studies have underscored the requirement of TLR2/MyD88/NF-κB pathway (first signal) and ROS/potassium efflux (second signal) for NLRP3/ASC inflammasome formation, leading to caspase-1 activation and subsequent IL-1β release during RSV infection.     

Critical roles of ASC inflammasomes in caspase-1 activation and host innate resistance to Streptococcus pneumoniae infection

Streptococcus pneumoniae is a Gram-positive, extracellular bacterium that is responsible for significant mortality and morbidity worldwide. Pneumolysin (PLY), a cytolysin produced by all clinical isolates of the pneumococcus, is one of the most important virulence factors of this pathogen. We have previously reported that PLY is an essential factor for activation of caspase-1 and consequent secretion of IL-1β and IL-18 in macrophages infected with S. pneumoniae. However, the host molecular factors involved in caspase-1 activation are still unclear. To further elucidate the mechanism of caspase-1 activation in macrophages infected with S. pneumoniae, we examined the involvement of inflammasomes in inducing this cellular response. Our study revealed that apoptosis-associated specklike protein containing a caspase recruitment domain (ASC), an adaptor protein for inflammasome receptors such as nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2), is essentially required for the induction of caspase-1 activation by S. pneumoniae. Caspase-1 activation was partially impaired in NLRP3(-/-) macrophages, whereas knockdown and knockout of AIM2 resulted in a clear decrease in caspase-1 activation in response to S. pneumoniae. These results suggest that ASC inflammasomes, including AIM2 and NLRP3, are critical for caspase-1 activation induced by S. pneumoniae. Furthermore, ASC(-/-) mice were more susceptible than wild-type mice to S. pneumoniae, with impaired secretion of IL-1β and IL-18 into the bronchoalveolar lavage after intranasal infection, suggesting that ASC inflammasomes contribute to the protection of host from infection with PLY-producing S. pneumoniae.     

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.     

The pyrin inflammasome in host–microbe interactions

A) RhoA regulation of pyrin. B) Inactivation of RhoA and control of pyrin inflammasome activation by bacterial effectors and toxins.A) In steady-state conditions, RhoA is constantly being turned on and off by GEFs and GAPs that exchange GDP for GTP or accelerate conversion of GTP to GDP, respectively. RhoA regulates the pyrin inflammasome through interactions with transducers of RhoA signaling, PRKs, that phosphorylate pyrin creating a 14-3-3 binding site. Phosphorylated and 14-3-3 bound pyrin is locked in an off state where its conformation prevents interaction with inflammasome components. B) (1) Bacteria deliver toxins or effecter proteins to host cells using T3SS, T6SS, secretion via ECVs or other general secretion mechanisms. There are several classes of RhoA modifying toxins including, those that covalently modify RhoA, those that proteolytically cleave RhoA and GAPs. (2) Toxins and effectors inactivate RhoA stopping PRK signaling to pyrin. (3) The lack of negative signal by PRKs and possible action of a phosphatase leads to the dephosphorylation of pyrin on its regulatory serine residues and loss of 14-3-3 binding. (4) Another class of toxins and effectors are those that inhibit pyrin inflammasome formation. YopM, an inhibitor of pyrin inflammasome formation represented in blue, recruits kinases RSK and PRK to pyrin to keep it phosphorylated. (5) When pyrin is dephosphorylated it then binds to the ASC adapter protein which binds and dimerizes pro-caspase-1 forming the inflammasome complex. Additionally, evidence suggests that microtubule polymerization is required for inflammasome formation. Upon dimerization, pro-caspase-1 undergoes autoproteolytic cleavage fully activating the enzyme. Mature caspase-1 then cleaves pro-IL-1β, pro-IL-18 and GSDMD to their mature forms. (6) Gasdermin-D then oligomerizes and forms pores in the plasma membrane which allows secretion of IL-1β and IL-18. If enough pores in the plasma membrane form the cell will undergo cell death via lysis known as pyroptosis.Abbreviations: GDP: guanosine diphosphate; GTP: guanosine triphosphate; PRK: protein kinase C-related kinase; T3SS: type 3 secretion system; T6SS: type 6 secretion system; ECVs: extracellular vesicles; GAP: GTPase activating proteins; ASC: apoptosis-associated speck-like protein; IL: interleukin; GSDMD: gasdermin-D; PSP: protein serine/threonine phosphatase.

1. Download : Download high-res image (106KB)
2. Download : Download full-size image

     

Vascular endothelial cells senescence is associated with NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation via reactive oxygen species (ROS)/thioredoxin-interacting protein (TXNIP) pathway

Endothelial dysfunction caused by endothelial cells senescence and chronic inflammation is tightly linked to the development of cardiovascular diseases. NLRP3 (NOD-like receptor family pyrin domain-containing3) inflammasome plays a central role in inflammatory response that is associated with diverse inflammatory diseases. This study explores the effects and possible mechanisms of NLRP3 inflammasome in endothelial cells senescence. Results show an increment of pro-inflammatory cytokine interleukin (IL) −1β secretion and caspase-1 activation during the senescence of endothelial cells induced by bleomycin. Moreover, secreted IL-1β promoted endothelial cells senescence through up-regulation of p53/p21 protein expression. NLRP3 inflammasome was found to mediate IL-1β secretion through the production of ROS (reactive oxygen species) during the senescence of endothelial cells. Furthermore, the association of TXNIP (thioredoxin-interacting protein) with NLRP3 induced by ROS promoted NLRP3 inflammasome activation in senescent endothelial cells. In addition, the expressions of NLRP3 inflammasome related genes, ASC (apoptosis associated speck-like protein containing a CARD), TXNIP, cleaved caspase-1 and IL-1β, were also increased in vitro and in vivo studies. These findings indicate that endothelial senescence could be mediated through ROS and NLRP3 inflammasome signaling pathways, suggesting a potential target for the prevention of endothelial senescence-related cardiovascular diseases.     

The NLRP3 inflammasome contributes to brain injury in pneumococcal meningitis and is activated through ATP-dependent lysosomal cathepsin B release

Streptococcus pneumoniae meningitis causes brain damage through inflammation-related pathways whose identity and mechanisms of action are yet unclear. We previously identified caspase-1, which activates precursor IL-1 type cytokines, as a central mediator of inflammation in pneumococcal meningitis. In this study, we demonstrate that lack of the inflammasome components ASC or NLRP3 that are centrally involved in caspase-1 activation decreases scores of clinical and histological disease severity as well as brain inflammation in murine pneumococcal meningitis. Using specific inhibitors (anakinra and rIL-18-binding protein), we further show that ASC- and NLRP3-dependent pathologic alterations are solely related to secretion of both IL-1β and IL-18. Moreover, using differentiated human THP-1 cells, we demonstrate that the pneumococcal pore-forming toxin pneumolysin is a key inducer of IL-1β expression and inflammasome activation upon pneumococcal challenge. The latter depends on the release of ATP, lysosomal destabilization (but not disruption), and cathepsin B activation. The in vivo importance of this pathway is supported by our observation that the lack of pneumolysin and cathepsin B inhibition is associated with a better clinical course and less brain inflammation in murine pneumococcal meningitis. Collectively, our study indicates a central role of the NLRP3 inflammasome in the pathology of pneumococcal meningitis. Thus, interference with inflammasome activation might be a promising target for adjunctive therapy of this disease.     

Activation of an NLRP3 inflammasome restricts Mycobacterium kansasii infection

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1β secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1β derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii.     

Cytosolic,Autocrine Alpha-1 Proteinase Inhibitor (A1PI) Inhibits Caspase-1 and Blocks IL-1β Dependent Cytokine Release in Monocytes

Rationale

Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli.

Methods

IL-1 β expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro.

Results

IL-1 β expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1β and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1β and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner.

Conclusions

Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1β secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.     

Enteropathogenic Escherichia coli Uses NleA to Inhibit NLRP3 Inflammasome Activation

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are related strains capable of inducing severe gastrointestinal disease. For optimal infection, these pathogens actively modulate cellular functions through the deployment of effector proteins in a type three secretion system (T3SS)-dependent manner. In response to enteric pathogen invasion, the Nod-like receptor pyrin domain containing (NLRP) inflammasome has been increasingly recognized as an important cytoplasmic sensor against microbial infection by activating caspase-1 and releasing IL-1β. EPEC and EHEC are known to elicit inflammasome activation in macrophages and epithelial cells; however, whether the pathogens actively counteract such innate immune responses is unknown. Using a series of compound effector-gene deletion strains of EPEC, we screened and identified NleA, which could subdue host IL-1β secretion. It was found that the reduction is not because of blocked NF-κB activity; instead, the reduction results from inhibited caspase-1 activation by NleA. Immunostaining of human macrophage-like cells following infection revealed limited formation of inflammasome foci with constituents of total caspase-1, ASC and NLRP3 in the presence of NleA. Pulldown of PMA-induced differentiated THP-1 lysate with purified MBP-NleA reveals that NLRP3 is a target of NleA. The interaction was verified by an immunoprecipitation assay and direct interaction assay in which purified MBP-NleA and GST-NLRP3 were used. We further showed that the effector interacts with regions of NLRP3 containing the PYD and LRR domains. Additionally, NleA was found to associate with non-ubiquitinated and ubiquitinated NLRP3 and to interrupt de-ubiquitination of NLRP3, which is a required process for inflammasome activation. Cumulatively, our findings provide the first example of EPEC-mediated suppression of inflammasome activity in which NieA plays a novel role in controlling the host immune response through targeting of NLRP3.