Key Component Of Inflammasome Research Articles

Inflammasome Activation in an In Vitro Sepsis Model Recapitulates Increased Monocyte Distribution Width Seen in Patients With Sepsis.

OBJECTIVES:Increased monocyte distribution width (MDW) has recently been shown to be a reliable indicator of early sepsis detection. This study therefore sought to determine if inflammasome activation can be linked to monocyte size changes in sepsis.DESIGN:An in vitro sepsis model using bacterial endotoxin (lipopolysaccharide [LPS]) to study the effect of inflammasome activation on monocyte cell size distribution by microscopy and MDW measurements using a standard clinical hematology analyzer.SETTING:University research laboratory.SUBJECTS:Healthy adult volunteers and cultured human monocyte cells in wild-type state and after clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 knockout of key inflammasome components (apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, gasdermin-D).INTERVENTIONS:In vitro treatment of specimens with bacterial LPS.MEASUREMENTS AND MAIN RESULTS:Wild-type THP1 cells demonstrated a significant increase in cell area (207 μm2 [159–400 μm2] vs 160 μm2 [134–198 μm2]; p < 0.001) and distribution width (198 vs 55 μm2; p < 0.0001) by microscopy following treatment with LPS. Increased MDW correlated with inflammasome activation as demonstrated by release of interleukin (IL)-1β and with the presence of large distended pyroptotic cells by microscopy. All of these effects were blocked in the inflammasome knockout cells. Whole blood samples treated similarly also demonstrated IL-1β release and increased MDW (median 24.7 U [22.2–27.2 U] vs 16.3 U [15.1–17.6 U]; p = 0.008) as measured using the Beckman-Coulter Unicel DxH900 analyzer. When peripheral blood mononuclear cells were isolated prior to treatment with LPS, microscopy confirmed the presence of large pyroptotic cells correlating to IL-1β release in the human subject samples as well.CONCLUSIONS:The increased MDW seen in patients with sepsis can be reproduced in an in vitro sepsis model and blocked using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 technology to inactivate the inflammasome. These findings suggest that pyroptotic cellular swelling underlies changes in MDW in septic patients and connect MDW to early events in the inflammatory cascade of sepsis.

Inflammasome activation: from molecular mechanisms to autoinflammation.

Inflammasomes are assembled by innate immune sensors that cells employ to detect a range of danger signals and respond with pro‐inflammatory signalling. Inflammasomes activate inflammatory caspases, which trigger a cascade of molecular events with the potential to compromise cellular integrity and release the IL‐1β and IL‐18 pro‐inflammatory cytokines. Several molecular mechanisms, working in concert, ensure that inflammasome activation is tightly regulated; these include NLRP3 post‐translational modifications, ubiquitination and phosphorylation, as well as single‐domain proteins that competitively bind to key inflammasome components, such as the CARD‐only proteins (COPs) and PYD‐only proteins (POPs). These diverse regulatory systems ensure that a suitable level of inflammation is initiated to counteract any cellular insult, while simultaneously preserving tissue architecture. When inflammasomes are aberrantly activated can drive excessive production of pro‐inflammatory cytokines and cell death, leading to tissue damage. In several autoinflammatory conditions, inflammasomes are aberrantly activated with subsequent development of clinical features that reflect the degree of underlying tissue and organ damage. Several of the resulting disease complications may be successfully controlled by anti‐inflammatory drugs and/or specific cytokine inhibitors, in addition to more recently developed small‐molecule inhibitors. In this review, we will explore the molecular processes underlying the activation of several inflammasomes and highlight their role during health and disease. We also describe the detrimental effects of these inflammasome complexes, in some pathological conditions, and review current therapeutic approaches as well as future prospective treatments.

Glucagon-like peptide-1 receptor mediates the beneficial effect of liraglutide in an acute lung injury mouse model involving the thioredoxin-interacting protein.

Repurposing clinically used drugs is among the important strategies in drug discovery. Glucagon-like peptide-1 (GLP-1) and its diabetes-based drugs, such as liraglutide, possess a spectrum of extra-pancreatic functions, while GLP-1 receptor (GLP-1R) is most abundantly expressed in the lung. Recent studies have suggested that GLP-1-based drugs exert beneficial effects in chronic, as well as acute, lung injury rodent models. Here, we show that liraglutide pretreatment reduced LPS induced acute lung injury in mice. It significantly reduced lung injury score, wet/dry lung weight ratio, bronchoalveolar lavage fluid immune cell count and protein concentration, and cell apoptosis in the lung, and it was associated with reduced lung inflammatory cytokine and chemokine gene expression. Importantly, these effects were virtually absent in GLP-1R−/− mice. A well-known function of GLP-1 and GLP-based drugs in pancreatic β-cells is the attenuation of high-glucose stimulated expression of thioredoxin-interacting protein (TxNIP), a key component of inflammasome. LPS-challenged lungs showed elevated TxNIP mRNA and protein expression, which was attenuated by liraglutide treatment in a GLP-1R-dependent manner. Hence, our observations suggest that GLP-1R is essential in mediating beneficial effects of liraglutide in acute lung injury, with the inflammasome component TxNIP as a potential target.

Non-canonical Inflammasome-Mediated IL-1β Production by Primary Endometrial Epithelial and Stromal Fibroblast Cells Is NLRP3 and Caspase-4 Dependent.

Inflammation of the post-partum uterus is a normal physiological event, crucial for tissue involution and repair. However, in the bovine, some cows fail to resolve this inflammation, resulting in endometritis, which compromises fertility. Earlier work has identified upregulated expression of the potent inflammatory cytokine IL-1β early post-partum, in cows which subsequently develop endometritis. As a result, targeting IL-1β expression holds potential as a novel treatment for this disease, yet the regulatory mechanisms contributing to IL-1β expression in the bovine endometrium remain unknown. To investigate this, endometrial tissue samples were obtained 7 and 21 days post-partum (DPP) from cows that were diagnosed with endometritis at 21 DPP and cows that experienced a physiological level of inflammation throughout involution. IL-1β was measured by qPCR, ELISA, and immunohistochemistry. Seven DPP, endometrial IL-1β protein levels were significantly higher in animals that proceeded to develop endometritis at 21 DPP. IL-1β production could be detected in luminal and glandular epithelium, in underlying stromal fibroblasts as well as infiltrating immune cells. To investigate the mechanisms regulating IL-1β expression, primary endometrial epithelial cells, stromal fibroblasts and PBMCs were stimulated with LPS and the inflammasome activator nigericin. Stromal fibroblast cells were particularly potent producers of IL-1β. Basolateral LPS stimulation of polarized epithelial cells induced IL1B mRNA and a previously undescribed IL-1β protein isoform, with preferential protein secretion into the apical compartment. Key inflammasome components [nod-like receptor protein 3 (NLRP3), nima-related kinase-7 (NEK7), apoptosis speck like protein containing a CARD (ASC), and gasdermin-D] were expressed by endometrial cells following stimulation. Endometrial cell stimulation in the presence of NLRP3 receptor (MCC950) and pan-caspase (Z-VAD-FMK) inhibitors blocked IL-1β production, demonstrating its dependence on the NLRP3 inflammasome and on caspase activity. Furthermore, caspase-4 specific siRNA prevented IL-1β production, confirming that inflammasome activation in endometrial cells is caspase-4 but not caspase-1 dependent, as shown in other species. Identifying the tissue- and species-specificity of inflammasome assembly and activation has critical relevance for our understanding of inflammation and suggests new therapeutic targets to enhance the resolution of inflammatory pathologies including endometritis in cattle.

Key component of inflammasome, NLRC4, was identified in the lesional epidermis of psoriatic patients.

Inflammasomes are multimolecular complexes that control the inflammatory response. The function of inflammasomes in the pathogenesis of psoriasis is still unclear. To clarify the relationship between inflammasomes and the pathophysiology of psoriasis, and in particular, to identify molecules interacting with caspase-1, a crucial component of inflammasomes, scale extracts obtained from patients with psoriasis were immunoprecipitated with anti-caspase-1 antibody and analyzed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS). The expression of the inflammasome component was assessed by immunohistochemical analysis and an in vitro assay. We identified several candidates for caspase-1-interacting proteins from the psoriatic scale extracts by immunoprecipitation and LC-MS/MS. Nucleotide-binding oligomerization domain-containing protein-like receptor family CARD domain-containing protein 4 (NLRC4) was the only inflammasome component among the candidates; thus, the protein is considered to be a key factor of inflammasomes in psoriasis. No inflammasome component was found in the extracts of atopic dermatitis or normal skin by LC-MS/MS. Immunohistochemical analysis demonstrated upregulation of NLRC4 in the lesional epidermis of some psoriatic patients whereas weak expression of NLRC4 was detected in the normal and non-lesional epidermis. The mRNA expression of the NLRC4 gene increased in keratinocytes at confluency, 48 h after air exposure and after the addition of 1.5 mmol/L calcium chloride. Our findings suggest that NLRC4 may be involved in the exacerbation or modification of psoriatic lesions.

Lack of chemopreventive effects of P2X7R inhibitors against pancreatic cancer.

Pancreatic cancer (PC) is an almost uniformly lethal disease with inflammation playing an important role in its progression. Sustained stimulation of purinergic receptor P2X7 drives induction of NLRP inflammasome activation. To understand the role of P2X7 receptor and inflammasome, we performed transcriptomic analysis of p48Cre/+-LSL-KrasG12D/+ mice pancreatic tumors by next generation sequencing. Results showed that P2X7R's key inflammasome components, IL-1β and caspase-1 are highly expressed (p < 0.05) in pancreatic tumors. Hence, to target P2X7R, we tested effects of two P2X7R antagonists, A438079 and AZ10606120, on pancreatic intraepithelial neoplasms (PanINs) and their progression to PC in p48Cre/+-LSL-KrasG12D/+ mice. Following dose optimization studies, for chemoprevention efficacy, six-week-old p48Cre/+-LSL-KrasG12D/+ mice (24–36/group) were fed modified AIN-76A diets containing 0, 50 or 100 ppm A438079 and AZ10606120 for 38 weeks. Pancreata were collected, weighed, and evaluated for PanINs and PDAC. Control diet-fed male mice showed 50% PDAC incidence. Dietary A438079 and AZ10606120 showed 60% PDAC incidence. A marginal increase of PanIN 3 (carcinoma in-situ) was observed in drug-treated mice. Importantly, the carcinoma spread in untreated mice was 24% compared to 43–53% in treatment groups. Reduced survival rates were observed in mice exposed to P2X7R inhibitors. Both drugs showed a decrease in caspase-3, caspase-1, p21 and Cdc25c. Dietary A438079 showed modest inhibition of P2X7R, NLRP3, and IL-33, whereas AZ10606120 had no effects. In summary, targeting the P2X7R pathway by A438079 and AZ10606120 failed to show chemopreventive effects against PC and slightly enhanced PanIN progression to PDAC. Hence, caution is needed while treating high-risk individuals with P2X7R inhibitors.

Abstract 5255: Lack of chemopreventive effects of dietary P2×7R inhibitors against pancreatic cancer in p48Cre/+-LSL-KrasG12D/+ mice

Abstract Pancreatic cancer (PC) still remains a devastating disease that is almost uniformly lethal. Despite advances in the field of molecular genetics in human pancreatic cancers, targeted therapies has not yet translated to an improved overall survival for patients. Chronic inflammation is hallmark of many cancers, including PC. P2×7R is the most potent of the membrane receptors responsible for inflammasome activation and release of inflammatory cytokines. Sustained stimulation of P2×7R drives induction of NLRP inflammasome activation. To understand the role of P2×7 receptor and inflammasome in pancreatic tumor progression, we carried transcriptomic analysis of LSL-Kras pancreatic tumors by next generation sequencing. Results showed that the P2×7 receptor (∼20-fold); key inflammasome components, IL-1β (∼45-fold), caspase-1 (15-fold), IL-18 (∼35-fold), IL-33 (∼93 folds), TNF-α (∼13-fold) and COX-2 (∼41-fold) are highly expressed (p&amp;lt;0.05). Analysis of human PC cell lines, human and mouse PC tissues by western immunoblotting, real time (RT)-polymerase chain reaction (PCR), immunohistochemistry and/or immunofluorescence suggest that the P2×7R is a critical contributor to the progression of pancreatic tumor growth. Hence, to target P2×7R, we tested the effects of two P2×7R antagonists A438079 and AZ10606120 at two doses (50 and 100 ppm) on pancreatic intraepithelial neoplasms (PanINs) and their progression to PDAC in p48Cre/+-LSL-KrasG12D/+ mice. Six-week old KrasG12D/+ (24-36/group) mice were fed (AIN-76A) diets containing 0, 50 or 100 ppm of A438079 and AZ10606120 for 38 weeks. Pancreata were collected, weighed, and evaluated histopathologically for PanINs and PDAC. To understand molecular mechanisms, we analyzed levels of proliferation, inflammatory and cell cycle makers; PCNA, p21, P2×7R, NLRP, COX-2, IL-33, caspase-3, caspase-1, Cdc25c and p53 expressions by IHC, IHF, Western blotting, and/or RT-PCR methods. Results suggest that control diet fed mice showed 50% incidence of PDAC in male mice. Dietary A4338079 and AZ10606120 showed up to 60% incidence of PDAC. CT-PET imaging analysis showed enhanced tumor growth in treatment groups. Importantly, the carcinoma spread in untreated mice was 24% as compared to 43-53% in treatment groups. Also, marginal increase of PanIN 3 (carcinoma in-situ) was observed in drug treated mice. Both drugs showed a decrease in caspase-3, caspase-1, p21 and Cdc25c. Dietary A438079 showed modest inhibition of PCNA, P2×7R, NLRP, and IL-33 whereas AZ10606120 had no effects. In summary, targeting the P2×7R pathway by A438079 and AZ10606120 failed to show significant chemopreventive effects in suppression of PC and hence caution is needed while treating high risk individuals for PC with P2×7R inhibitors. Also, it is not clear whether dietary route of administration might be reason for lack of efficacy. {Supported by NCI-CN-N01- 25008-78}. Citation Format: Altaf Mohammed, Naveena B. Janakiram, Venkateshwar Madka, Gopal Pathuri, Qian Li, Rebekah Ritchie, Laura Biddick, Hannah Kutsche, Yuting Zhang, Anil Singh, Hariprasad Gali, Stan Lightfoot, Vernon E. Steele, Chen S. Suen, Chinthalapally V. Rao. Lack of chemopreventive effects of dietary P2×7R inhibitors against pancreatic cancer in p48Cre/+-LSL-KrasG12D/+ mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5255.

P38δ MAPK: A Novel Regulator of NLRP3 Inflammasome Activation With Increased Expression in Coronary Atherogenesis.

Activation of the inflammasome pathway in macrophages results in the secretion of 2 potent proinflammatory and proatherogenic cytokines, interleukin (IL)-1β, and IL-18. Atherosclerotic lesions are characterized by the presence of various endogenous activators of the NLR family pyrin domain containing 3 (NLRP3) inflammasome, including cholesterol crystals and extracellular ATP. The aim of this study was to comprehensively characterize the expression of inflammasome pathway components and regulators in human atherosclerotic lesions. Twenty human coronary artery RNA samples from 10 explanted hearts were analyzed using an inflammasome pathway-focused quantitative polymerase chain reaction array. Advanced atherosclerotic plaques, when compared with early-to-intermediate lesions from the same coronary trees, displayed significant upregulation of 12 target genes, including the key inflammasome components apoptosis-associated speck-like protein containing a CARD domain, caspase-1, and IL-18. Immunohistochemical stainings of the advanced plaques revealed macrophage foam cells positive for NLRP3 inflammasome components around the necrotic lipid cores. The polymerase chain reaction array target p38δ mitogen-activated protein kinase was upregulated in advanced plaques and strongly expressed by lesional macrophage foam cells. In cultured human monocyte-derived macrophages, the p38δ mitogen-activated protein kinase was activated by intracellular stress signals triggered during ATP- and cholesterol crystal-induced NLRP3 inflammasome activation and was required for NLRP3-mediated IL-1β secretion. Increased expression of the key inflammasome components in advanced coronary lesions implies enhanced activity of the inflammasome pathway in progression of coronary atherosclerosis. The p38δ mitogen-activated protein kinase was identified as a novel regulator of NLRP3 inflammasome activation in primary human macrophages, and thus, represents a potential target for modulation of atherosclerotic inflammation.

Regulation of NOD-like receptors and inflammasome activation in cerebral endothelial cells.

Cerebral endothelial cells (CECs) forming the blood-brain barrier are at the interface of the immune and the central nervous systems and thus may play an important role in the functional integration of the two systems. Here, we investigated how CECs recognize and respond to pathogen- and damage-associated molecular patterns to regulate the functions of the neurovascular unit. First we detected the expression of several NOD-like receptors (NLRs) - including NOD1, NOD2, NLRC4, NLRC5, NLRP1, NLRP3, NLRP5, NLRP9, NLRP10, NLRP12, NLRA, and NLRX - in human brain endothelial cells. Inflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, and IL-1β had stimulatory effects on the transcription of many of these receptors. Expression of key inflammasome components (NOD2, NLRP3, and caspase 1) along with caspase-cleaved interleukins IL-1β and IL-33 could be induced by priming with lipopolysaccharide and activation with muramyl dipeptide. In addition, combined treatment with lipopolysaccharide and muramyl dipeptide resulted in IL-1β secretion in a caspase- and ERK1/2 kinase-dependent manner. Our findings demonstrate that NLRs and inflammasomes can be activated in cerebral endothelial cells, which may confer a yet unexplored role to the blood-brain barrier in neuroimmune and neuroinflammatory processes.

Nucleoside diphosphate kinase and flagellin from Pseudomonas aeruginosa induce interleukin 1 expression via the Akt/NF-κB signaling pathways.

Inflammatory responses are a first line of host defense against a range of invading pathogens, consisting of the release of proinflammatory cytokines followed by attraction of polymorphonuclear neutrophils (PMNs) to the site of inflammation. Among the many virulence factors that contribute to the pathogenesis of infections, nucleoside diphosphate kinase (Ndk) mediates bacterially induced toxicity against eukaryotic cells. However, no study has examined how Ndk affects inflammatory responses. The present study examined the mechanisms by which Pseudomonas aeruginosa activates inflammatory responses upon infection of cells. The results showed that bacterial Ndk, with the aid of an additional bacterial factor, flagellin, induced expression of the proinflammatory cytokines interleukin-1α (IL-1α) and IL-1β. Cytokine induction appeared to be dependent on the kinase activity of Ndk and was mediated via the NF-κB signaling pathway. Notably, Ndk activated the Akt signaling pathway, which acts upstream of NF-κB, as well as caspase-1, which is a key component of inflammasome. Thus, this study demonstrated that P. aeruginosa, through the combined effects of Ndk and flagellin, upregulates the expression of proinflammatory cytokines via the Akt/NF-κB signaling pathways.