U937 macrophages-like cells, palmitate and P. gingivalis
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ABSTRACT:
ORGANISM(S): Homo sapiens (human)
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PROVIDER: S-BSST910 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Metabolic diseases and low-grade chronic inflammation are interconnected: obese persons are at higher risk of developing periodontitis. However, the molecular mechanisms involved in the development and progression of periodontitis in an obesogenic microenvironment in response to periodontopathogens are still lacking. This study aims to investigate the combined effects of palmitate and Porphyromonas gingivalis on the secretion of pro-inflammatory cytokines and on transcriptional landscape modifications in macrophage-like cells. U937 macrophage-like cells were treated with palmitate and stimulated with P. gingivalis for 24h. Cytokines IL-1β, TNF-α and IL-6 were measured by ELISA in the culture medium and cell extracted RNA was submitted to a microarray analysis followed by Gene Ontology analyses. P. gingivalis, in presence of palmitate, potentiated IL-1β and TNF-α secretion in comparison to palmitate alone. Gene Ontology analyses also revealed that the combination palmitate-P. gingivalis potentiated the number of gene molecular functions implicated in the regulation of immune and inflammatory pathways compared to macrophages treated with palmitate alone. Our results provide the first comprehensive mapping of gene interconnections between palmitate and P. gingivalis during inflammatory responses in macrophage-like cells. These data highlight the importance of considering systemic conditions, specifically obesogenic microenvironment, in the management of periodontal disease in obese patients.
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Project description:The U937 cell culture is a pro-monocytic, human histiocytic lymphoma cell line. These monocytes can differentiate into either macrophages or dendritic cells (antigen-presenting cells) depending on the initiators. The U937 cells activated in the presence of phorbol 12-myristate 13-acetate (PMA) change their morphology into macrophage-like cells creating pseudopodia and adhering generously. Macrophages are known to produce reactive oxygen species (ROS) mostly during phagocytosis of foreign particles, an important non-specific immune response. Recently, we have focused on the role of hydroxyl radical (HO∙) and provide evidence on its importance for differentiation in U937 cells. Based on electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM), formation of HO∙ was confirmed within the cells undergoing differentiation and/or apoptosis during the PMA treatment. This study aims to increase our knowledge of ROS metabolism in model cell lines used in human research.
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Project description:BackgroundMacrophage-derived factors contribute to whole-body insulin resistance, partly by impinging on metabolically active tissues. As proof of principle for this interaction, conditioned medium from macrophages treated with palmitate (CM-PA) reduces insulin action and glucose uptake in muscle cells. However, the mechanism whereby CM-PA confers this negative response onto muscle cells remains unknown.Methodology/principal findingsL6-GLUT4myc myoblasts were exposed for 24 h to palmitate-free conditioned medium from RAW 264.7 macrophages pre-treated with 0.5 mM palmitate for 6 h. This palmitate-free CM-PA, containing selective cytokines and chemokines, inhibited myoblast insulin-stimulated insulin receptor substrate 1 (IRS1) tyrosine phosphorylation, AS160 phosphorylation, GLUT4 translocation and glucose uptake. These effects were accompanied by a rise in c-Jun N-terminal kinase (JNK) activation, degradation of Inhibitor of ?B? (I?B?), and elevated expression of proinflammatory cytokines in myoblasts. Notably, CM-PA caused IRS1 phosphorylation on Ser1101, and phosphorylation of novel PKC? and ?. Co-incubation of myoblasts with CM-PA and the novel and conventional PKC inhibitor Gö6983 (but not with the conventional PKC inhibitor Gö6976) prevented PKC? and ? activation, JNK phosphorylation, restored I?B? mass and reduced proinflammatory cytokine production. Gö6983 also restored insulin signalling and glucose uptake in myoblasts. Moreover, co-silencing both novel PKC ? and ? isoforms in myoblasts by RNA interference, but not their individual silencing, prevented the inflammatory response and restored insulin sensitivity to CM-PA-treated myoblasts.Conclusions/clinical significanceThe results suggest that the block in muscle insulin action caused by CM-PA is mediated by novel PKC? and PKC?. This study re-establishes the participation of macrophages as a relay in the action of fatty acids on muscle cells, and further identifies PKC? and PKC? as key elements in the inflammatory and insulin resistance responses of muscle cells to macrophage products. Furthermore, it portrays these PKC isoforms as potential targets for the treatment of fatty acid-induced, inflammation-linked insulin resistance.
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Project description:PurposeInterleukin-37 (IL-37) is an anti-inflammatory cytokine that inhibits a broad spectrum of inflammatory responses in various human cells, including neutrophils, macrophages, and endothelial cells. The aim of this study was to identify the role of IL-37 in toll-like receptor 9 (TLR9) signaling in human macrophages.Materials and methodsHuman macrophage U937 cells treated with CpG-oligonucleotides (CpG-ODN), recombinant IL-37, or dexamethasone were used in an in vitro study. IL-37 small interfering RNA (siRNA) and TLR9 siRNA were used to silence endogenous IL-37 and TLR9, respectively. Expression levels of phosphorylated nuclear factor-κB (NF-κB), IκBα, IL-37, IL-1β, tumor necrosis factor-α (TNF-α), and IL-6 protein were assessed by real-time quantitative polymerase chain reaction and Western blotting. CpG-ODN-mediated IL-37 expression stimulated by dexamethasone was detected using immunofluorescent analysis.ResultsU937 cells treated with CpG-ODN induced activation of the NF-κB pathway and increased the expression of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6, but reduced that of IL-37. Recombinant IL-37 attenuated phosphorylation of NF-κB and IκBα and the expression of IL-1β, TNF-α, and IL-6 stimulated by CpG-ODN. Human macrophages transfected with IL-37 siRNA augmented the expression of IL-1β, TNF-α, and IL-6 mRNA and protein in cells treated with CpG-ODN. Dexamethasone markedly inhibited expression of pro-inflammatory cytokines in U937 cells, whereas IL-37 expression was increased with the addition of dexamethasone. Inflammatory responses elicited by CpG-ODN were dependent on an MyD88-TRAF6 pathway. IL-37 inhibited CpG-ODN-induced ubiquitination of TRAF6 in U937 macrophages.ConclusionIL-37 inhibits CpG-ODN-mediated inflammatory responses through regulation of a TRAF6-NF-κB pathway in human macrophages.
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Project description:Inflammation is regulated by many endogenous factors including estrogen, a steroid hormone that declines with increasing age, leading to excessive inflammation in the elderly. C-reactive protein (CRP) is an acute phase inflammatory protein that exists in two forms, native CRP (nCRP) and monomeric CRP (mCRP), which mediate distinct biological activities. It is unclear how each CRP isoform mediates nitric oxide (NO), a signaling molecule generated by NO synthase (NOS). This study investigated whether CRP isoforms have distinct effects on NO production by unstimulated and lipopolysaccharide (LPS)-activated monocytes/macrophages and whether estrogen mediates CRP-induced NO production in an in vitro model of aging. NO and inducible NOS (iNOS) were measured (n = 12) by the Griess assay and an enzyme-linked immunosorbent assay, respectively following incubation (24 h) of human-derived U937 monocytes/macrophages with CRP isoforms [(nCRP) = 500 and 1,000 µg/ml; (mCRP) = 100 and 250 µg/ml] in the absence or presence of 17 beta-estradiol (1 × 10-7, 1 × 10-8, and 1 × 10-9 M). The response to each CRP isoform and estrogen was dependent on the differentiation and activation status of cells. Monocytes with or without prior LPS-activation significantly increased (P < 0.01) NO/iNOS production when treated with mCRP. The mCRP isoform had no effect (P > 0.05) on NO/iNOS production by unactivated or LPS-activated macrophages, whereas nCRP significantly (P < 0.05) reduced NO/iNOS production by macrophages, with or without prior LPS-activation. The nCRP isoform had opposing actions on monocytes, significantly (P < 0.01) increasing and reducing NO/iNOS by unactivated and LPS-activated monocytes, respectively. Estrogen significantly (P < 0.01) reversed nCRP-mediated NO inhibition by unactivated macrophages but decreased CRP-induced NO by unactivated monocytes treated with nCRP or mCRP and LPS-activated monocytes treated with mCRP. NO was differentially mediated by CRP isoforms in a cell-type/state-specific manner, with production corresponding to concomitant changes in iNOS levels. Collectively, the findings indicate nCRP and estrogen predominantly reduce NO production, whereas mCRP increases NO production. This supports growing evidence that mCRP exacerbates inflammation while nCRP and estrogen dampen the overall inflammatory response. Therapeutic strategies that restore estrogen levels to those found in youth and promote the stability of nCRP or/and prevent the formation of mCRP may reduce NO production in age-related inflammatory conditions.
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