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Neutrophil extracellular traps and monocyte subsets at the culprit lesion site of myocardial infarction patients.

ABSTRACT: Neutrophils release their chromatin into the extracellular space upon activation. These web-like structures are called neutrophil extracellular traps (NETs) and have potent prothrombotic and proinflammatory properties. In ST-elevation myocardial infarction (STEMI), NETs correlate with increased infarct size. The interplay of neutrophils and monocytes impacts cardiac remodeling. Monocyte subsets are classified as classical, intermediate and non-classical monocytes. In the present study, in vitro stimulation with NETs led to an increase of intermediate monocytes and reduced expression of CX3CR1 in all subsets. Intermediate monocytes have been associated with poor outcome, while non-classical CX3CR1-positive monocytes could have reparative function after STEMI. We characterized monocyte subsets and NET markers at the culprit lesion site of STEMI patients (n?=?91). NET surrogate markers were increased and correlated with larger infarct size and with fewer non-classical monocytes. Intermediate and especially non-classical monocytes were increased at the culprit site compared to the femoral site. Low CX3CR1 expression of monocytes correlated with high NET markers and increased infarct size. In this translational system, causality cannot be proven. However, our data suggest that NETs interfere with monocytic differentiation and receptor expression, presumably promoting a subset shift at the culprit lesion site. Reduced monocyte CX3CR1 expression may compromise myocardial salvage.

SUBMITTER: Mangold A

PROVIDER: S-EPMC6841683 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Neutrophil extracellular traps and monocyte subsets at the culprit lesion site of myocardial infarction patients.

Mangold Andreas A Hofbauer Thomas M TM Ondracek Anna S AS Artner Tyler T Scherz Thomas T Speidl Walter S WS Krychtiuk Konstantin A KA Sadushi-Kolici Roela R Jakowitsch Johannes J Lang Irene M IM

Scientific reports 20191108 1

Neutrophils release their chromatin into the extracellular space upon activation. These web-like structures are called neutrophil extracellular traps (NETs) and have potent prothrombotic and proinflammatory properties. In ST-elevation myocardial infarction (STEMI), NETs correlate with increased infarct size. The interplay of neutrophils and monocytes impacts cardiac remodeling. Monocyte subsets are classified as classical, intermediate and non-classical monocytes. In the present study, in vitro ...[more]

PMID: 31704966

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Project description:BackgroundLeukocyte-mediated inflammation is crucial in ST-segment elevation myocardial infarction (STEMI). We recently observed that neutrophil extracellular traps (NETs) are increased at the culprit site, promoting activation and differentiation of fibrocytes, cells with mesenchymal and leukocytic properties. Fibrocyte migration is mediated by monocyte chemoattractant protein (MCP)-1 and C-C chemokine receptor type 2 (CCR2). We investigated the interplay between NETs, fibrocyte function, and MCP-1 in STEMI.MethodsCulprit site and peripheral blood samples of STEMI patients were drawn during primary percutaneous coronary intervention. MCP-1 and the NET marker citrullinated histone H3 (citH3) were measured by ELISA while double-stranded DNA was stained with a fluorescent dye. The influence of MCP-1 on NET formation in vitro was assessed using isolated healthy donor neutrophils. Human coronary artery endothelial cells (hCAECs) were stimulated with isolated NETs, and MCP-1 gene expression was measured by ELISA and qPCR. CCR2 expression of culprit site and peripheral blood fibrocytes was characterized by flow cytometry. Healthy donor fibrocyte receptor expression and chemotaxis were investigated in response to stimulation with MCP-1 and NETs in vitro.ResultsNETs and concentrations of MCP-1 were increased at the culprit site of 50 consecutive STEMI patients. NET stimulation of hCAECs induced transcription of ICAM-1, IL-6, and MCP-1, and secretion of MCP-1. MCP-1 promoted NET formation of healthy donor neutrophils in vitro. An increasing MCP-1 gradient correlated with fibrocyte accumulation at the culprit site. Locally increased MCP-1 levels were negatively correlated with CCR2 expression on fibrocytes. MCP-1 and NETs induced CCR2 downregulation on fibrocytes in vitro. NETs did not function as a chemotactic stimulus for fibrocytes or monocytes and could block migration in response to MCP-1 for both cell populations.ConclusionNETs function as signaling scaffolds at the culprit site of STEMI. NETs assist MCP-1 and ICAM-1 release from culprit site coronary artery endothelial cells. MCP-1 facilitates further NETosis. Monocytes enter the culprit site along an MCP-1 gradient, to transdifferentiate into fibrocytes in the presence of NETs.

Project description:Complement activation and neutrophil extracellular traps (NETs) have both been suggested to drive atherosclerotic plaque progression. Although experimental studies suggest interplay between these two innate immunity components, the relevance in patients with coronary artery disease (CAD) is unclear. The aim of this study was to assess associations between complement activation and NETs in patients with stable CAD and examine the role of complement activation on clinical outcome. Blood samples from a cohort of patients with angiographically verified stable CAD (n = 1001) were analyzed by ELISA for the terminal complement complex (TCC) and by relative quantification for gene expression of the C5a receptor 1 (C5aR1) as markers of complement activation. As markers of NETs, dsDNA was analyzed by fluorescent nucleic acid stain and myeloperoxidase-DNA (MPO-DNA) by ELISA. Clinical outcome was defined as unstable angina, nonhemorrhagic stroke, acute myocardial infarction (MI), or death (n = 106, whereof 36 MI). Levels of TCC and C5aR1 were not significantly correlated to dsDNA (TCC: r = -0.045, p = 0.153; C5aR1: r = -0.060, p = 0.434) or MPO-DNA (TCC: r = 0.026, p = 0.414; C5aR1: r = 0.123, p = 0.107). When dividing TCC and C5aR1 levels into quartiles (Q), levels of MPO-DNA differed significantly across quartiles (TCC: p = 0.008, C5aR1: 0.049), while dsDNA did not (TCC: p = 0.181, C5aR1: p = 0.771). Patients with TCC levels in Q4 had significantly higher levels of MPO-DNA than Q1-3 (p = 0.019), and C5aR1 levels in Q3-4 had significantly higher levels of MPO-DNA than Q1-2 (p = 0.046). TCC levels did not differ between patients experiencing a clinical endpoint or not, but high levels were associated with increased risk of acute MI (OR. 1.97, 95% CI: 0.99-3.90, p = 0.053) during two-year follow up, also when adjusted for relevant covariates. In conclusion, TCC and C5aR1 were moderately associated with the NET marker MPO-DNA, and TCC levels were related to the risk of future MI in this cohort of patients with stable CAD.

Project description:Innate immune responses and rapid recruitment of leukocytes, which regulate inflammation and subsequent healing, play a key role in acute myocardial infarction (MI). Peptidylarginine deiminase 4 (PAD4) is critically involved in chromatin decondensation during the release of Neutrophil Extracellular Traps (NETs) by activated neutrophils. Alternatively, activated macrophages (M2) and accurate collagen deposition determine the repair of the infarcted heart. In this study, we investigated the impact of NETs on macrophage polarization and their role for acute cardiac inflammation and subsequent cardiac healing in a mouse model of acute MI. NETs were found to promote in vitro macrophage polarization toward a reparative phenotype. NETs suppressed pro-inflammatory macrophages (M1) under hypoxia and diminished IL-6 and TNF-? expression. Further on, NETs strongly supported M2b polarization and IL-10 expression. In cardiac fibroblasts, NETs increased TGF-ß expression under hypoxic culture conditions. PAD4-/- mice subjected to permanent ligation of the left anterior descending artery suffered from overwhelming inflammation in the acute phase of MI. Noteworthy, PAD4-/- neutrophils were unable to release NETs upon ex vivo stimulation with ionomycin or PMA, but produced significantly higher amounts of reactive oxygen species (ROS). Increased levels of circulating cell-free DNA, mitochondrial DNA and cardiac troponin were found in PAD4-/- mice in the acute phase of MI when compared to WT mice. Reduced cardiac expression of IL-6, IL-10, and M2 marker genes, as well as increased TNF-? expression, suggested a pro-inflammatory state. PAD4-/- mice displayed significantly increased cardiac MMP-2 expression under baseline conditions. At day 1, post-MI, PAD4-/- mice showed increased end-diastolic volume and increased thinning of the left ventricular wall. Interestingly, improved cardiac function, as demonstrated by significantly increased ejection fraction, was found at day 21. Altogether, our results indicate that NETs support macrophage polarization toward an M2 phenotype, thus displaying anti-inflammatory properties. PAD4 deficiency aggravates acute inflammation and increases tissue damage post-MI, partially due to the lack of NETs.

Project description:Severe coronavirus disease 2019 (COVID-19) is characterized by the intense formation of neutrophil extracellular traps (NETs), leading to the occlusion of microvessels, as shown in pulmonary samples. The occurrence of ST-elevated myocardial infarction (STEMI) is a serious cardiac manifestation of COVID-19; the intrinsic mechanism of coronary thrombosis appears to still be unknown. To determine the role of NETs in coronary thrombosis in patients with COVID-19. This was a consecutive series of patients with COVID-19 at an academic tertiary hospital in Madrid, Spain, who underwent primary coronary interventions for STEMI in which coronary aspirates were obtained in the catheterization laboratory using a thrombus aspiration device. Patients with COVID-19 who experienced a STEMI between March 23 and April 11, 2020, from whom coronary thrombus samples were aspirated during primary coronary intervention, were included in the analysis. These patients were compared with a series conducted from July 2015 to December 2015 of patients with STEMI. The presence and quantity of NETs in coronary aspirates from patients with STEMI and COVID-19. The method for the analysis of NETs in paraffin-embedded coronary thrombi was based on the use of confocal microscopy technology and image analysis for the colocalization of myeloperoxidase-DNA complexes and citrullinated histone H3. Immunohistochemical analysis of thrombi was also performed. Clinical and angiographic variables were prospectively collected. Five patients with COVID-19 were included (4 men [80%]; mean [SD] age, 62 [14] years); the comparison group included 50 patients (44 males [88%]; mean [SD] age, 58 [12] years). NETs were detected in the samples of all 5 patients with COVID-19, and the median density of NETs was 61% (95% CI, 43%-91%). In the historical series of patients with STEMI, NETs were found in 34 of 50 thrombi (68%), and the median NET density was 19% (95% CI, 13%-22%; P < .001). All thrombi from patients with COVID-19 were composed of fibrin and polymorphonuclear cells. None of them showed fragments of atherosclerotic plaque or iron deposits indicative of previous episodes of plaque rupture. In this small case series of patients with COVID-19 and myocardial infarction, NETs seem to play a major role in the pathogenesis of STEMI in COVID-19 disease. Our findings support the idea that targeting intravascular NETs might be a relevant goal of treatment and a feasible way to prevent coronary thrombosis in patients with severe COVID-19 disease.

Dataset Information

Neutrophil extracellular traps and monocyte subsets at the culprit lesion site of myocardial infarction patients.

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Neutrophil extracellular traps and monocyte subsets at the culprit lesion site of myocardial infarction patients.

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