The combination of CRP isoforms with oxLDL decreases TNF-α and IL-6 release by U937-derived macrophages.
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ABSTRACT:
SUBMITTER: Krayem I
PROVIDER: S-EPMC5543421 | biostudies-other | 2017 Sep
REPOSITORIES: biostudies-other
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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.
| S-EPMC6036124 | biostudies-literature
Project description:oxLDL peptide vaccine and its antibody adoptive transferring have shown a significantly preventive or therapeutic effect in atherosclerotic animal model. The molecular mechanism behind this is obscure. Here, we report that oxLDL induces MCP-1 release in monocytes/macrophages through their TLR-4 (Toll-like receptor 4) and ERK MAPK pathway and is calcium/potassium channel-dependent. Using blocking antibodies against CD36, TLR-4, SR-AI and LOX-1, only TLR-4 antibody was found to have an inhibitory effect and ERK MAPK-specific inhibitor (PD98059) was found to have a dramatic inhibitory effect compared to inhibitors of other MAPK group members (p38 and JNK MAPKs) on oxLDL-induced MCP-1 release. The release of cytokines and chemokines needs influx of extracellular calcium and imbalance of efflux of potassium. Nifedipine, a voltage-dependent calcium channel (VDCC) inhibitor, and glyburide, an ATP-regulated potassium channel (K+ATP ) inhibitor, inhibit oxLDL-induced MCP-1 release. Potassium efflux and influx counterbalance maintains the negative potential of macrophages to open calcium channels, and our results suggest that oxLDL actually induces the closing of potassium influx channel - inward rectifier channel (Kir ) and ensuing the opening of calcium channel. ERK MAPK inhibitor PD98059 inhibits oxLDL-induced Ca2+ /Kir channel alterations. The interfering of oxLDL-induced MCP-1 release by its monoclonal antibody is through its Fc?RIIB (CD32). Using blocking antibodies against Fc?RI (CD64), Fc?RIIB (CD32) and Fc?RIII (CD16), only CD32 blocking antibody was found to reverse the inhibitory effect of oxLDL antibody on oxLDL-induced MCP-1 release. Interestingly, oxLDL antibody specifically inhibits oxLDL-induced ERK MAPK activation and ensuing Ca2+ /Kir channel alterations, and MCP-1 release. Thus, we found a molecular mechanism of oxLDL antibody on inhibition of oxLDL-induced ERK MAPK pathway and consequent MCP-1 release.
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Project description:Reprogramming of metabolic pathways in monocytes and macrophages can induce a proatherosclerotic inflammatory memory called trained innate immunity. Here, we have analyzed the role of the Liver X receptor (LXR), a crucial regulator of metabolism and inflammation, in oxidized low-density lipoprotein (oxLDL)-induced trained innate immunity. Human monocytes were incubated with LXR agonists, antagonists, and oxLDL for 24 h. After five days of resting time, cells were restimulated with the TLR-2 agonist Pam3cys. OxLDL priming induced the expression of LXRα but not LXRβ. Pharmacologic LXR activation was enhanced, while LXR inhibition prevented the oxLDL-induced inflammatory response. Furthermore, LXR inhibition blocked the metabolic changes necessary for epigenetic reprogramming associated with trained immunity. In fact, enrichment of activating histone marks at the IL-6 and TNFα promotor was reduced following LXR inhibition. Based on the differential expression of the LXR isoforms, we inhibited LXRα and LXRβ genes using siRNA in THP1 cells. As expected, siRNA-mediated knock-down of LXRα blocked the oxLDL-induced inflammatory response, while knock-down of LXRβ had no effect. We demonstrate a specific and novel role of the LXRα isoform in the regulation of oxLDL-induced trained immunity. Our data reveal important aspects of LXR signaling in innate immunity with relevance to atherosclerosis formation.
| S-EPMC9181299 | biostudies-literature
Project description:BackgroundExtravasation of macrophages and formation of lipid-laden foam cells are key events in the development and progression of atherosclerosis. The degradation of atherogenic lipoproteins subsequently leads to alterations in cellular lipid metabolism that influence inflammatory signaling. Especially sphingolipids and ceramides are known to be involved in these processes. We therefore analyzed monocyte derived macrophages during differentiation and after loading with enzymatically (eLDL) and oxidatively (oxLDL) modified low-density lipoproteins (LDL).MethodsPrimary human monocytes were isolated from healthy, normolipidemic blood donors using leukapheresis and counterflow elutriation. On the fourth day of MCSF-induced differentiation eLDL (40 ?g/ml) or oxLDL (80 ?g/ml) were added for 48h. Lipid species were analyzed by quantitative tandem mass spectrometry. Taqman qPCR was performed to investigate transcriptional changes in enzymes involved in sphingolipid metabolism. Furthermore, membrane lipids were studied using flow cytometry and confocal microscopy.ResultsMCSF dependent phagocytic differentiation of blood monocytes had only minor effects on the sphingolipid composition. Levels of total sphingomyelin and total ceramide remained unchanged, while lactosylceramides, cholesterylesters and free cholesterol decreased. At the species level most ceramide species showed a reduction upon phagocytic differentiation. Loading with eLDL preferentially increased cellular cholesterol while loading with oxLDL increased cellular ceramide content. Activation of the salvage pathway with a higher mRNA expression of acid and neutral sphingomyelinase, neutral sphingomyelinase activation associated factor and glucosylceramidase as well as increased surface expression of SMPD1 were identified as potentially underlying mechanisms. Moreover, flow-cytometric analysis revealed a higher cell-surface-expression of ceramide, lactosylceramide (CDw17), globotriaosylceramide (CD77), dodecasaccharide-ceramide (CD65s) and GM1 ganglioside upon oxLDL loading. ApoE in contrast to apoA-I preferentially bound to the ceramide enriched surfaces of oxLDL loaded cells. Confocal microscopy showed a co-localization of acid sphingomyelinase with ceramide rich membrane microdomains.ConclusioneLDL leads to the formation of lipid droplets and preferentially induces cholesterol/sphingomyelin rich membrane microdomains while oxLDL promotes the development of cholesterol/ceramide rich microdomains via activation of the salvage pathway.
| S-EPMC5117723 | biostudies-literature
Project description:ObjectivesBroad-spectrum antibiotics disrupt the intestinal microbiota. The microbiota is essential for physiological processes, such as the development of the gut immune system. Recent murine data suggest that the intestinal microbiota also modulates systemic innate immune responses; however, evidence in humans is lacking.MethodsTwelve healthy young men were given oral broad-spectrum antibiotics (ciprofloxacin 500 mg bid, vancomycin 500 mg tid and metronidazole 500 mg tid) for 7 days. At baseline, 1 day, and 6 weeks after antibiotics, blood and feces were sampled. Whole blood and isolated mononuclear cells were stimulated with selected Toll-like receptor agonists and heat-killed bacteria. Microbiota diversity and composition was determined using bacterial 16S rDNA sequencing.ResultsOne day after the antibiotic course, microbial diversity was significantly lower compared with baseline. After antibiotic therapy, systemic mononuclear cells produced lower levels of tumor necrosis factor (TNF)-α after ex vivo stimulation with lipopolysaccharide (LPS). This diminished capacity to produce TNF-α was restored 6 weeks after cessation of antibiotic therapy. In whole blood, a reduced capacity to release interleukin (IL)-1β and IL-6 was observed after LPS stimulation. Antibiotic treatment did not impact on differential leukocyte counts, phagocytosis, and cell surface markers of neutrophils and monocytes.ConclusionsIn this proof-of-principle study of healthy subjects, microbiota disruption by broad-spectrum antibiotics is reversibly associated with decreased systemic cellular responsiveness towards LPS. The implications of these findings in a clinical setting remain to be determined.
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Project description:Macrophages contribute to the pathogenesis of rheumatoid arthritis (RA). They can display different states of activation or "polarization," notably the so-called inflammatory "M1" and the various alternative "M2" polarizations, characterized by distinct functions. Data regarding the effects of RA anti-cytokine biological disease-modifying anti-rheumatic drugs (bDMARDs) on macrophage polarization are scarce. We aimed to assess in vitro modulation of macrophage polarization by bDMARDs targeting pro-inflammatory cytokines in RA. We generated monocyte derived macrophages using blood samples from 20 RA patients with active RA and 30 healthy controls. We evaluated in vitro the impact on M1 inflammatory macrophages of: etanercept (ETA), adalimumab (ADA), certolizumab (CZP), tocilizumab (TCZ), and rituximab (RTX). We assessed the impact on macrophage polarization using flow cytometry and RTqPCR to study the expression of surface markers and perform functional studies of cytokine production, phagocytosis, and negative feedback control of inflammation. Among evaluated bDMARDs, anti-TNF agents modulated the polarization of inflammatory macrophages by decreasing inflammatory surface markers (CD40, CD80) and favoring alternative markers (CD16, CD163, MerTK). Anti-TNF agents also induced alternative functions in macrophages activated in inflammatory condition with (i) the inhibition of inflammatory cytokines (TNF, IL-6, IL-12), (ii) an increase in phagocytosis. These findings were mechanistically related to an increase in early IL-10 production, responsible for higher negative feedback control of inflammation involving SOCS3 and Gas6. This IL-10 effect was STAT3-dependent. Anti-TNF agents not only inhibit in vitro inflammatory functions of macrophages, but also favor resolution of inflammation through polarization toward alternative features specifically involving the IL-10/STAT3 axis.
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Project description:The alarm cytokine interleukin-1β (IL-1β) is a potent activator of the inflammatory cascade following pathogen recognition. IL-1β production typically requires two signals: first, priming by recognition of pathogen-associated molecular patterns leads to the production of immature pro-IL-1β; subsequently, inflammasome activation by a secondary signal allows cleavage and maturation of IL-1β from its pro-form. However, despite the important role of IL-1β in controlling local and systemic inflammation, its overall regulation is still not fully understood. Here we demonstrate that peritoneal tissue-resident macrophages use an active inhibitory pathway, to suppress IL-1β processing, which can otherwise occur in the absence of a second signal. Programming by the transcription factor Gata6 controls the expression of prostacyclin synthase, which is required for prostacyclin production after lipopolysaccharide stimulation and optimal induction of IL-10. In the absence of secondary signal, IL-10 potently inhibits IL-1β processing, providing a previously unrecognized control of IL-1β in tissue-resident macrophages.
| S-EPMC7360975 | biostudies-literature
Project description: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.
| S-EPMC3491674 | biostudies-literature
Project description:BackgroundPyroptosis is a catabolic process relevant to periodontal disorders for which interleukin-1β (IL-1β) inflammation is central to the pathophysiology of the disease. Despite platelet-rich fibrin (PRF) anti-inflammatory properties and its application to support periodontal regeneration, the capacity of PRF to modulate pyroptosis, specifically the production and release of IL-1β, remains unknown. The question arises whether PRF could regulate IL-1β release from macrophages in vitro.MethodsTo answer this question, RAW 264.7 macrophages and primary macrophages obtained from murine bone marrow were primed with PRF before being challenged by lipopolysaccharide (LPS). Cells were then analysed for the pyroptosis signalling components by gene expression analyses and IL-1β secretion at the protein level. The release of mitochondrial reactive oxygen species (ROS) was also detected.ResultsPRF lowered the LPS-induced expression of IL-1β and NLRP3 inflammasome, caspase-11 and IL-18 in primary macrophages, and IL-1β and caspase-11 in RAW 264.7 cells. Additionally, PRF diminished the secretion of IL-1β at the protein level in LPS-induced RAW 264.7 cells. This was shown through immunoassays performed with the supernatant and further confirmed by analysing the lysates of permeabilised cells. Furthermore, PRF reduced the ROS release provoked by LPS in RAW 264.7 cells. Finally, to enhance IL-1β release from the LPS-primed macrophages, we introduced a second signal with adenosine triphosphate (ATP). In this setting, PRF significantly reduced IL-1β release in RAW 264.7 cells and a trend to diminish IL-1β release in primary macrophages.ConclusionThese findings suggest that PRF can reduce IL-1β release and, at least in part, inhibit pyroptosis-related factors in LPS-challenged macrophages.
| S-EPMC9368224 | biostudies-literature
Project description:ObjectiveWe aimed to evaluate the effects of combining the Simplified-Acute-Physiology-Score (SAPS) 2 or the SAPS 3 with Interleukin-6 (IL-6) or Procalcitonin (PCT) or C-Reactive Protein (CRP) concentrations for predicting in-hospital mortality.Material and methodsThis retrospective study was conducted in an interdisciplinary 22-bed intensive care unit (ICU) at a German university hospital. Within an 18-month period, SAPS 2 and SAPS 3 were calculated for 514 critically ill patients that were admitted to the internal medicine department. To evaluate discrimination performance, the area under the receiver operating characteristic curves (AUROCs) and the 95% confidence intervals (95% CIs) were calculated for each score, exclusively or in combination with IL-6 or PCT or CRP. DeLong test was used to compare different AUROCs.ResultsThe SAPS 2 exhibited a better discrimination performance than SAPS 3 with AUROCs of 0.81 (95% CI, 0.76-0.86) and 0.72 (95% CI, 0.66-0.78), respectively. Overall, combination of the SAPS 2 with IL-6 showed the best discrimination performance (AUROC 0.82; 95% CI, 0.77-0.87), albeit not significantly different from SAPS2. IL-6 performed better than PCT and CRP with AUROCs of 0.75 (95% CI, 0.69-0.81), 0.72 (95% CI, 0.66-0.77) and 0.65 (95% CI, 0.59-0.72), respectively. Performance of the SAPS 3 improved significantly when combined with IL-6 (AUROC 0.76; 95% CI, 0.69-0.81) or PCT (AUROC 0.73; 95% CI, 0.67-0.78).ConclusionsOur analysis provided evidence that the risk stratification performance of the SAPS 3 and, to a lesser degree, also of the SAPS 2 can increase when combined with IL-6. A more accurate detection of aberrant or dysregulated systemic immunological responses (by IL-6) may explain the higher performance achieved by SAPS 3 + IL-6 vs. SAPS 3. Thus, implementation of IL-6 in critical care scores can improve prediction outcomes, especially in patients experiencing acute inflammatory conditions; however, statistical results may vary across hospital types and/or patient populations with different case mix.
| S-EPMC7470390 | biostudies-literature