195 research outputs found
Nod2 Downregulates TLR2/1 Mediated IL1Ξ² Gene Expression in Mouse Peritoneal Macrophages
Nod2 is a cytosolic pattern recognition receptor. It has been implicated in many inflammatory conditions. Its signaling has been suggested to modulate TLR responses in a variety of ways, yet little is known about the mechanistic details of the process. We show in this study that Nod2 knockdown mouse peritoneal macrophages secrete more IL1Ξ² than normal macrophages when stimulated with peptidoglycan (PGN). Muramyl dipeptide (MDP, a Nod2 ligand) + PGN co-stimulated macrophages have lower expression of IL1Ξ² than PGN (TLR2/1 ligand) stimulated macrophages. MDP co-stimulation have similar effects on Pam3CSK4 (synthetic TLR2/1 ligand) mediated IL1Ξ² expression suggesting that MDP mediated down regulating effects are receptor dependent and ligand independent. MDP mediated down regulation was specific for TLR2/1 signaling as MDP does not affect LPS (TLR4 ligand) or zymosan A (TLR2/6 ligand) mediated IL1Ξ² expression. Mechanistically, MDP exerts its down regulating effects by lowering PGN/Pam3CSK4 mediated nuclear cRel levels. Lower nuclear cRel level were observed to be because of enhanced transporting back rather than reduced nuclear translocation of cRel in MDP + PGN stimulated macrophages. These results demonstrate that Nod2 and TLR2/1 signaling pathways are independent and do not interact at the level of MAPK or NF-ΞΊB activation
CIN85 drives B cell responses by linking BCR signals to the canonical NF-ΞΊB pathway
CIN85 transduces B cell receptor signals to IKK-Ξ², and its expression in B cells is essential for T cellβindependent type II antibody responses in mice
Computational modelling of NF-ΞΊB activation by IL-1RI and its co-receptor TILRR, predicts a role for Cytoskeletal Sequestration of IΞΊBΞ± in inflammatory signalling.
The transcription factor NF-ΞΊB (nuclear factor kappa B) is activated by Toll-like receptors and controlled by mechanotransduction and changes in the cytoskeleton. In this study we combine 3-D predictive protein modelling and in vitro experiments with in silico simulations to determine the role of the cytoskeleton in regulation of NF-ΞΊB. Simulations used a comprehensive agent-based model of the NF-ΞΊB pathway, which includes the type 1 IL-1 receptor (IL-1R1) complex and signalling intermediates, as well as cytoskeletal components. Agent based modelling relies on in silico reproductions of systems through the interactions of its components, and provides a reliable tool in investigations of biological processes, which require spatial considerations and involve complex formation and translocation of regulatory components. We show that our model faithfully reproduces the multiple steps comprising the NF-ΞΊB pathway, and provides a framework from which we can explore novel aspects of the system. The analysis, using 3-D predictive protein modelling and in vitro assays, demonstrated that the NF-ΞΊB inhibitor, IΞΊBΞ± is sequestered to the actin/spectrin complex within the cytoskeleton of the resting cell, and released during IL-1 stimulation, through a process controlled by the IL-1RI co-receptor TILRR (Toll-like and IL-1 receptor regulator). In silico simulations using the agent-based model predict that the cytoskeletal pool of IΞΊBΞ± is released to adjust signal amplification in relation to input levels. The results suggest that the process provides a mechanism for signal calibration and enables efficient, activation-sensitive regulation of NF-ΞΊB and inflammatory responses
NOD2 controls the nature of the inflammatory response and subsequent fate of Mycobacterium tuberculosis and M. bovis BCG in human macrophages
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92022/1/j.1462-5822.2010.01544.x.pd
Differential effects of selenium and knock-down of glutathione peroxidases on TNFΞ± and flagellin inflammatory responses in gut epithelial cells
Selenium (Se) is essential for human health. Despite evidence that Se intake affects inflammatory responses, the mechanisms by which Se and the selenoproteins modulate inflammatory signalling, especially in the gut, are not yet defined. The aim of this work was to assess effects of altered Se supply and knock-down of individual selenoproteins on NF-ΞΊB activation in gut epithelial cells. Caco-2 cells were stably transfected with gene constructs expressing luciferase linked either to three upstream NF-ΞΊB response elements and a TATA box or only a TATA box. TNFΞ± and flagellin activated NF-ΞΊB-dependent luciferase activity and increased IL-8 expression. Se depletion decreased expression of glutathione peroxidase1 (GPX1) and selenoproteins H and W and increased TNFΞ±-stimulated luciferase activity, endogenous IL-8 expression and reactive oxygen species (ROS) production. These effects were not mimicked by independent knock-down of either GPX1, selenoprotein H or W; indeed, GPX1 knock-down lowered TNFΞ±-induced NF-ΞΊB activation and did not affect ROS levels. GPX4 knock-down decreased NF-ΞΊB activation by flagellin but not by TNFΞ±. We hypothesise that Se depletion alters the pattern of expression of multiple selenoproteins that in turn increases ROS and modulates NF-ΞΊB activation in epithelial cells, but that the effect of GPX1 knock-down is ROS-independent
NIK Stabilization in Osteoclasts Results in Osteoporosis and Enhanced Inflammatory Osteolysis
Maintenance of healthy bone requires the balanced activities of osteoclasts (OCs), which resorb bone, and osteoblasts, which build bone. Disproportionate action of OCs is responsible for the bone loss associated with postmenopausal osteoporosis and rheumatoid arthritis. NF-ΞΊB inducing kinase (NIK) controls activation of the alternative NF-ΞΊB pathway, a critical pathway for OC differentiation. Under basal conditions, TRAF3-mediated NIK degradation prevents downstream signaling, and disruption of the NIK:TRAF3 interaction stabilizes NIK leading to constitutive activation of the alternative NF-ΞΊB pathway.Using transgenic mice with OC-lineage expression of NIK lacking its TRAF3 binding domain (NT3), we now find that alternative NF-ΞΊB activation enhances not only OC differentiation but also OC function. Activating NT3 with either lysozyme M Cre or cathepsinK Cre causes high turnover osteoporosis with increased activity of OCs and osteoblasts. In vitro, NT3-expressing precursors form OCs more quickly and at lower doses of RANKL. When cultured on bone, they exhibit larger actin rings and increased resorptive activity. OC-specific NT3 transgenic mice also have an exaggerated osteolytic response to the serum transfer model of arthritis.Constitutive activation of NIK drives enhanced osteoclastogenesis and bone resorption, both in basal conditions and in response to inflammatory stimuli
Characterization of NF-ΞΊB reporter U937 cells and their application for the detection of inflammatory immune-complexes
Our study tested the hypothesis that immunoglobulins differ in their ability to activate the nuclear factor-ΞΊB pathway mediated cellular responses. These responses are modulated by several properties of the immune complex, including the ratio of antibody isotypes binding to antigen. Immunoassays allow the measurement of antigen specific antibodies belonging to distinct immunoglobulin classes and subclasses but not the net biological effect of the combination of these antibodies. We set out to develop a biosensor that is suitable for the detection and characterization of antigen specific serum antibodies. We genetically modified the monocytoid U937 cell line carrying Fc receptors with a plasmid encoding NF-ΞΊB promoter-driven GFP. This clone, U937-NF-ΞΊB, was characterized with respect to FcR expression and response to solid-phase immunoglobulins. Human IgG3, IgG4 and IgG1 induced GFP production in a time- and dose-dependent manner, in this order of efficacy, while IgG2 triggered no activation at the concentrations tested. IgA elicited no response alone but showed significant synergism with IgG3 and IgG4. We confirmed the importance of activation via FcΞ³RI by direct stimulation with monoclonal antibody and by competition assays. We used citrullinated peptides and serum from rheumatoid arthritis patients to generate immune complexes and to study the activation of U937-NF-ΞΊB, observing again a synergistic effect between IgG and IgA. Our results show that immunoglobulins have distinct pro-inflammatory potential, and that U937-NF-ΞΊB is suitable for the estimation of biological effects of immune-complexes, offering insight into monocyte activation and pathogenesis of antibody mediated diseases
Polymorphisms in NFkB, PXR, LXR and risk of colorectal cancer in a prospective study of Danes
<p>Abstract</p> <p>Background</p> <p>Transcription factors and nuclear receptors constitute a link between exposure to heterocyclic amines and polycyclic aromatic hydrocarbons from meat and tobacco smoke and colorectal cancer (CRC) risk. The aim of this study was to investigate if polymorphisms in nuclear factor kappa-B, pregnane X receptor, and liver X receptor were associated with risk of CRC, and to investigate possible interactions with lifestyle factors such as smoking, meat consumption, and NSAID use.</p> <p>Methods</p> <p>The polymorphisms nuclear factor kappa-B (<it>NFkB, NFKB1) </it>-94 insertion/deletion ATTG (rs28362491), pregnane X receptor (<it>PXR, NR1I2) </it>A-24381C (rs1523127), C8055T (rs2276707), A7635G (rs6785049), liver X receptor (<it>LXR-Ξ², NR1H3) </it>C-rs1405655T, T-rs2695121C were assessed together with lifestyle factors in a nested case-cohort study of 378 CRC cases and 756 random participants from the Danish prospective Diet, Cancer and Health study of 57,053 persons.</p> <p>Results</p> <p>Carriers of <it>NFkB </it>-94deletion were at 1.45-fold higher risk of CRC than homozygous carriers of the insertion allele (incidence rate ratio (IRR) = 1.45, 95% confidence interval (95% CI): 1.10-1.92). There was interaction between this polymorphism and intake of red and processed meat in relation to CRC risk. Carriers of <it>NFkB </it>-94deletion were at 3% increased risk pr 25 gram meat per day (95% CI: 0.98-1.09) whereas homozygous carriers of the insertion were not at increased risk (p for interaction = 0.03). <it>PXR </it>and <it>LXR </it>polymorphisms were not associated with CRC risk. There was no interaction between use of nonsteroid antiinflammatory drugs (NSAID) or smoking status and <it>NFkB</it>, <it>PXR </it>or <it>LXR </it>polymorphisms.</p> <p>Conclusions</p> <p>A polymorphism in <it>NFkB </it>was associated with CRC risk and there was interaction between this polymorphism and meat intake in relation to CRC risk. This study suggests a role for NFkB in CRC aetiology.</p
GEF-H1 Mediated Control of NOD1 Dependent NF-ΞΊB Activation by Shigella Effectors
Shigella flexneri has evolved the ability to modify host cell function with intracellular active effectors to overcome the intestinal barrier. The detection of these microbial effectors and the initiation of innate immune responses are critical for rapid mucosal defense activation. The guanine nucleotide exchange factor H1 (GEF-H1) mediates RhoA activation required for cell invasion by the enteroinvasive pathogen Shigella flexneri. Surprisingly, GEF-H1 is requisite for NF-ΞΊB activation in response to Shigella infection. GEF-H1 interacts with NOD1 and is required for RIP2 dependent NF-ΞΊB activation by H-Ala-D-Ξ³Glu-DAP (Ξ³TriDAP). GEF-H1 is essential for NF-ΞΊB activation by the Shigella effectors IpgB2 and OspB, which were found to signal in a NOD1 and RhoA Kinase (ROCK) dependent manner. Our results demonstrate that GEF-H1 is a critical component of cellular defenses forming an intracellular sensing system with NOD1 for the detection of microbial effectors during cell invasion by pathogens
Rac1 Regulates the NLRP3 Inflammasome Which Mediates IL-1beta Production in Chlamydophila pneumoniae Infected Human Mononuclear Cells
Chlamydophila pneumoniae causes acute respiratory tract infections and has been associated with development of asthma and atherosclerosis. The production of IL-1Ξ², a key mediator of acute and chronic inflammation, is regulated on a transcriptional level and additionally on a posttranslational level by inflammasomes. In the present study we show that C. pneumoniae-infected human mononuclear cells produce IL-1Ξ² protein depending on an inflammasome consisting of NLRP3, the adapter protein ASC and caspase-1. We further found that the small GTPase Rac1 is activated in C. pneumoniae-infected cells. Importantly, studies with specific inhibitors as well as siRNA show that Rac1 regulates inflammasome activation in C. pneumoniae-infected cells. In conclusion, C. pneumoniae infection of mononuclear cells stimulates IL-1Ξ² production dependent on a NLRP3 inflammasome-mediated processing of proIL-1Ξ² which is controlled by Rac1
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