Low density neutrophils and neutrophil extracellular traps (NETs) are new inflammatory players in heart failure.

BACKGROUND: Heart failure with reduced (HFrEF) or preserved ejection fraction (HFpEF) is characterized by low-grade chronic inflammation. Circulating neutrophils regroup two subtypes termed high- and low-density neutrophils (HDNs and LDNs). LDNs represent less than 2% of total neutrophil under physiological conditions, but their count increase in multiple pathologies, releasing more inflammatory cytokines and neutrophil extracellular traps (NETs). The aims of this study were to assess the differential count and role of HDNs, LDNs and NETs-related activities in HF patients. METHODS: HDNs and LDNs were isolated from human blood by density gradient and purified by FACS and their counts obtained by flow cytometry. NETs formation (NETosis) was quantified by confocal microscopy. Circulating inflammatory and NETosis biomarkers were measured by ELISA. Neutrophil adhesion onto human extracellular matrix (hECM) was assessed by optical microscopy. RESULTS: A total of 140 individuals were enrolled, including 33 healthy volunteers (HV), 41 HFrEF (19 stable patients and 22 presenting acute decompensated HF; ADHF) and 66 HFpEF patients (36 stable patients and 30 presenting HF decompensation). HDNs and LDNs counts were significantly increased up to 39% and 2740% respectively in HF patients compared to HV. In HF patients, the correlations between LDNs counts and circulating inflammatory (CRP, IL-6 and -8), Troponin T, NT-proBNP and NETosis components were all significant. In vitro, LDNs expressed more H3Cit and NETs and were more pro-adhesive, with ADHFpEF patients presenting the highest pro-inflammatory profile. CONCLUSIONS: HFpEF patients present higher levels of circulating LDNs and NETs related activities, which are the highest in the context of acute HF decompensation.

Low-Density Neutrophils Contribute to Subclinical Inflammation in Patients with Type 2 Diabetes.

Type 2 diabetes (T2D) is characterized by low-grade inflammation. Low-density neutrophils (LDNs) represent normally less than 2% of total neutrophils but increase in multiple pathologies, releasing inflammatory cytokines and neutrophil extracellular traps (NETs). We assessed the count and role of high-density neutrophils (HDNs), LDNs, and NET-related activities in patients with T2D. HDNs and LDNs were purified by fluorescence-activated cell sorting (FACS) and counted by flow cytometry. Circulating inflammatory and NETs biomarkers were measured by ELISA (Enzyme Linked Immunosorbent Assay). NET formation was quantified by confocal microscopy. Neutrophil adhesion onto a human extracellular matrix (hECM) was assessed by optical microscopy. We recruited 22 healthy volunteers (HVs) and 18 patients with T2D. LDN counts in patients with diabetes were significantly higher (160%), along with circulating NETs biomarkers (citrullinated H3 histone (H3Cit), myeloperoxidase (MPO), and MPO-DNA (137%, 175%, and 69%, respectively) versus HV. Circulating interleukins (IL-6 and IL-8) and C-Reactive Protein (CRP) were significantly increased by 117%, 171%, and 79%, respectively, in patients compared to HVs. Isolated LDNs from patients expressed more H3Cit, MPO, and NETs, formed more NETs, and adhered more on hECM compared to LDNs from HVs. Patients with T2D present higher levels of circulating LDN- and NET-related biomarkers and associated pro-inflammatory activities.

Correlation between Neutrophil Extracellular Traps (NETs) Expression and Primary Graft Dysfunction Following Human Lung Transplantation.

Primary graft dysfunction (PGD) is characterized by alveolar epithelial and vascular endothelial damage and inflammation, lung edema and hypoxemia. Up to one-third of recipients develop the most severe form of PGD (Grade 3; PGD3). Animal studies suggest that neutrophils contribute to the inflammatory process through neutrophil extracellular traps (NETs) release (NETosis). NETs are composed of DNA filaments decorated with granular proteins contributing to vascular occlusion associated with PGD. The main objective was to correlate NETosis in PGD3 (n = 9) versus non-PGD3 (n = 27) recipients in an exploratory study. Clinical data and blood samples were collected from donors and recipients pre-, intra- and postoperatively (up to 72 h). Inflammatory inducers of NETs' release (IL-8, IL-6 and C-reactive protein [CRP]) and components (myeloperoxidase [MPO], MPO-DNA complexes and cell-free DNA [cfDNA]) were quantified by ELISA. When available, histology, immunohistochemistry and immunofluorescence techniques were performed on lung biopsies from donor grafts collected during the surgery to evaluate the presence of activated neutrophils and NETs. Lung biopsies from donor grafts collected during transplantation presented various degrees of vascular occlusion including neutrophils undergoing NETosis. Additionally, in recipients intra- and postoperatively, circulating inflammatory (IL-6, IL-8) and NETosis biomarkers (MPO-DNA, MPO, cfDNA) were up to 4-fold higher in PGD3 recipients compared to non-PGD3 (p = 0.041 to 0.001). In summary, perioperative elevation of NETosis biomarkers is associated with PGD3 following human lung transplantation and these biomarkers might serve to identify recipients at risk of PGD3 and initiate preventive therapies.

Neutrophils and Circulating Inflammatory Biomarkers in Diabetes Mellitus and Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is characterized by low-grade chronic inflammation, which could be exacerbated by type 2 diabetes mellitus (DM). We hypothesized that neutrophils in patients with DM and patients with HFpEF with/without DM contribute to low-grade inflammation through the release of pro-inflammatory cytokines. Venous blood was withdrawn from patients with DM (n = 22), HFpEF (n = 15), HFpEF with DM (n = 13), and healthy controls (CTL) (n = 21). Levels of circulating cytokines and in vitro cytokines released by isolated neutrophils were assessed by enzyme-linked immunosorbent assay. Compared with CTL, there was a significant decrease in circulating nitric oxide in patients with DM (p ≤0.001), HFpEF (p ≤0.05), and HFpEF with DM (p ≤0.001) up to 44%. Circulating soluble intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 levels increased (up to 2.5-fold and 1.9-fold, respectively; p ≤0.001) in patients with HFpEF and patients with HFpEF and DM, whereas soluble E-selectin only increased in patients with HFpEF and DM (1.4-fold, p ≤0.001). Circulating vascular endothelial growth factor levels were similar in CTL and patients with DM but were decreased in patients with HFpEF with/without DM (up to 94%; p ≤0.001). Circulating C-reactive protein, interleukin (IL)-8, IL-6, and IL-receptor antagonist increased in all patient groups with a maximum of 3.3-fold, 4.7-fold, 4.8-fold, and 1.6-fold, respectively, in patients with HFpEF and patients with DM. In vitro, lipopolysaccharide increased neutrophils IL-6 release from HFpEF with DM (3.7-fold; p ≤0.001), and IL-8 release from DM and HFpEF with DM versus CTL (2.8-fold and 10.1-fold, respectively; p ≤0.001). IL-1 receptor antagonist and vascular endothelial growth factor release from HFpEF neutrophils significantly decreased up to 87.0% and 92.2%, respectively, versus CTL. Neutrophils from patients with DM and HFpEF release more cytokines than CTL. This increase in pro-inflammatory status may explain the greater event rate in patients with HFpEF and DM.

Acute effect of passive heat exposure on markers of cardiometabolic function in adults with type 2 diabetes mellitus.

Heat therapy is a promising strategy to improve cardiometabolic health. This study evaluated the acute physiological responses to hot water immersion in adults with type 2 diabetes mellitus (T2DM). On separate days in randomized order, 13 adults with T2DM [8 males/5 females, 62 ± 12 yr, body mass index (BMI): 30.1 ± 4.6 kg/m2] were immersed in thermoneutral (34°C, 90 min) or hot (41°C, core temperature ≥38.5°C for 60 min) water. Insulin sensitivity was quantified via the minimal oral model during an oral glucose tolerance test (OGTT) performed 60 min after immersion. Brachial artery flow-mediated dilation (FMD) and reactive hyperemia were evaluated before and 40 min after immersion. Blood samples were drawn to quantify protein concentrations and mRNA levels of HSP70 and HSP90, and circulating concentrations of cytokines. Relative to thermoneutral water immersion, hot water immersion increased core temperature (+1.66°C [+1.47, +1.87], P < 0.01), heart rate (+34 beats/min [+24, +44], P < 0.01), antegrade shear rate (+96 s-1 [+57, +134], P < 0.01), and IL-6 (+1.38 pg/mL [+0.31, +2.45], P = 0.01). Hot water immersion did not exert an acute change in insulin sensitivity (-0.3 dL/kg/min/µU/mL [-0.9, +0.2], P = 0.18), FMD (-1.0% [-3.6, +1.6], P = 0.56), peak (+0.36 mL/min/mmHg [-0.71, +1.43], P = 0.64), and total (+0.11 mL/min/mmHg × min [-0.46, +0.68], P = 0.87) reactive hyperemia. There was also no change in eHSP70 (P = 0.64), iHSP70 (P = 0.06), eHSP90 (P = 0.80), iHSP90 (P = 0.51), IL1-RA (P = 0.11), GLP-1 (P = 0.59), and NF-κB (P = 0.56) after hot water immersion. The physiological responses elicited by hot water immersion do not acutely improve markers of cardiometabolic function in adults with T2DM.NEW & NOTEWORTHY Heat therapy has been shown to improve markers of cardiometabolic health in preclinical and clinical studies. However, the effects of heat therapy in individuals with type 2 diabetes mellitus (T2DM) remain understudied. We examined the acute effect of hot water immersion on glucose tolerance, flow-mediated dilation, reactive hyperemia, inflammatory markers, and heat shock proteins in adults with T2DM. Hot water immersion did not acutely improve the markers studied.

Neutrophils pro-inflammatory and anti-inflammatory cytokine release in patients with heart failure and reduced ejection fraction.

AIMS: Heart failure with reduced ejection fraction (HFrEF) is characterized by sub-clinical inflammation. Changes in selected biomarkers of inflammation concomitant with the release of pro-inflammatory and anti-inflammatory cytokines by neutrophils have not been investigated in patients with HFrEF. METHODS AND RESULTS: Fifty-two patients, aged 68.8 ± 1.7 years, with HFrEF and left ventricular ejection fraction 28.7 ± 1.0%, and 21 healthy controls (CTL) were recruited. Twenty-five HF patients had type 2 diabetes. Venous blood samples from HF and CTL were collected once. Neutrophil-derived pro-inflammatory and anti-inflammatory cytokine levels were assessed in plasma by ELISA. Plasma biomarkers assessed included: C-reactive protein (CRP), vascular endothelial growth factor (VEGF), interleukins (IL)-6, -8, -1 receptor antagonist (-1RA), nitric oxide (NO), soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule 1 (sVCAM-1) and E-Selectin (sE-Sel). Neutrophils were isolated and stimulated with various agonists to promote VEGF, IL-6, IL-8, and IL-1RA release. Compared with CTL, HFrEF patients showed a marked decrease in circulating VEGF [178.0 (interquartile range; IQR 99.6; 239.2) vs. 16.2 (IQR 9.3; 20.2) pg/mL, P ≤ 0.001] and NO [45.2 (IQR 42.1; 57.6) vs. 40.6 (IQR 30.4; 47.1) pg/mL, P = 0.0234]. All other circulating biomarkers were significantly elevated. Neutrophils isolated from patients with HFrEF exhibited a greater IL-8 release in response to LPS [1.2 ± 0.1 (CTL); 10.4 ± 1.6 ng/mL (HFrEF) and 12.4 ± 1.6 ng/mL (HFrEF and DM), P ≤ 0.001]. IL-6 release in response to LPS was not changed in HFrEF patients without diabetes, whereas it was significantly increased in patients with HFrEF and diabetes [46.7 ± 3.9 (CTL) vs. 165.8 ± 48.0 pg/mL (HFrEF), P = 0.1713 and vs. 397.7 ± 67.4 pg/mL (HFrEF and DM), P ≤ 0.001]. In contrast, the release of VEGF and IL-1RA was significantly reduced in HFrEF (VEGF; TNF-α: 38.6 ± 3.1 and LPS: 25.3 ± 2.6 pg/mL; IL1RA; TNF-α: 0.6 ± 0.04 and LPS: 0.3 ± 0.02 ng/mL) compared with CTL (VEGF; TNF-α: 60.0 ± 9.4 and LPS: 41.2 ± 5.9 pg/mL; IL1RA; TNF-α: 3.3 ± 0.2 and LPS: 2.3 ± 0.1 ng/mL). CONCLUSIONS: Patients with HFrEF exhibit a significant decrease in circulating VEGF. The release of VEGF and both pro-inflammatory and anti-inflammatory cytokines from the stimulated neutrophils is markedly altered in these patients. The clinical significance of these findings deserves further investigation.

Angiopoietin 1 release from human neutrophils is independent from neutrophil extracellular traps (NETs).

BACKGROUND: Neutrophils induce the synthesis and release of angiopoietin 1 (Ang1), a cytosolic growth factor involved in angiogenesis and capable of inducing several pro-inflammatory activities in neutrophils. Neutrophils also synthesize and release neutrophil extracellular traps (NETs), comprised from decondensed nuclear DNA filaments carrying proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), proteinase 3 (PR3) and calprotectin (S100A8/S100A9), which together, contribute to the innate immune response against pathogens (e.g., bacteria). NETs are involved in various pathological conditions through pro-inflammatory, pro-thrombotic and endothelial dysfunction effects and have recently been found in heart failure (HF) and type 2 diabetes (T2DM) patients. The aim of the present study was to investigate the role of NETs on the synthesis and release of Ang1 by the neutrophils in patients with T2DM and HF with preserved ejection fraction (HFpEF) (stable or acute decompensated; ADHFpEF) with or without T2DM. RESULTS: Our data show that at basal level (PBS) and upon treatment with LPS, levels of NETs are slightly increased in patients suffering from T2DM, HFpEF ± T2DM and ADHF without (w/o) T2DM, whereas this increase was significant in ADHFpEF + T2DM patients compared to healthy control (HC) volunteers and ADHFpEF w/o T2DM. We also observed that treatments with PMA or A23187 increase the synthesis of Ang1 (from 150 to 250%) in HC and this effect is amplified in T2DM and in all cohorts of HF patients. Ang1 is completely released (100%) by neutrophils of all groups and does not bind to NETs as opposed to calprotectin. CONCLUSIONS: Our study suggests that severely ill patients with HFpEF and diabetes synthesize and release a greater abundance of NETs while Ang1 exocytosis is independent of NETs synthesis.

Acute Vascular Benefits of Finnish Sauna Bathing in Patients With Stable Coronary Artery Disease.

BACKGROUND: Finnish sauna bathing habits are associated with a decreased risk of cardiovascular mortality. The physiologic adaptations mediating this association remain to be fully elucidated. This study tested the hypothesis that Finnish sauna bathing acutely improves peripheral flow-mediated dilation (FMD) in middle-aged and older adults with stable coronary artery disease (CAD). METHODS: Twenty-two adults (20 male, 2 female; 67 ± 10 years) with stable CAD underwent 2 periods of 10 minutes in a Finnish sauna (81.3 ± 2.7°C, 23 ± 3% humidity) separated by 10 minutes of thermoneutral rest. Before and 51 ± 8 minutes after sauna bathing, brachial artery FMD and postocclusive reactive hyperemia (PORH) were evaluated by means of Doppler ultrasound. RESULTS: Sauna bathing increased core temperature (mean +0.66°C [95% CI 0.54-0.77], P < 0.01) and heart rate (+27 beats/min [24-29], P < 0.01), and decreased systolic (-19 mm Hg [-31 to -6]; P < 0.01) and diastolic (-6 mm Hg [-11 to -1], P < 0.01) blood pressures. Brachial artery FMD was greater after sauna bathing (+1.21% [0.16-2.26], P = 0.04), whereas PORH was unchanged (peak: +0.51 mL/min/mm Hg [-0.13 to 1.15], P = 0.11; area under the curve: +0.21 mL/mm Hg [-0.12 to 0.54]; P = 0.19). CONCLUSIONS: A typical Finnish sauna bathing session acutely improves peripheral FMD in middle-aged and older adults with stable CAD.

Colchicine reduces lung injury in experimental acute respiratory distress syndrome.

The acute respiratory distress syndrome (ARDS) is characterized by intense dysregulated inflammation leading to acute lung injury (ALI) and respiratory failure. There are no effective pharmacologic therapies for ARDS. Colchicine is a low-cost, widely available drug, effective in the treatment of inflammatory conditions. We studied the effects of colchicine pre-treatment on oleic acid-induced ARDS in rats. Rats were treated with colchicine (1 mg/kg) or placebo for three days prior to intravenous oleic acid-induced ALI (150 mg/kg). Four hours later they were studied and compared to a sham group. Colchicine reduced the area of histological lung injury by 61%, reduced lung edema, and markedly improved oxygenation by increasing PaO2/FiO2 from 66 ± 13 mmHg (mean ± SEM) to 246 ± 45 mmHg compared to 380 ± 18 mmHg in sham animals. Colchicine also reduced PaCO2 and respiratory acidosis. Lung neutrophil recruitment, assessed by myeloperoxidase immunostaining, was greatly increased after injury from 1.16 ± 0.19% to 8.86 ± 0.66% and significantly reduced by colchicine to 5.95 ± 1.13%. Increased lung NETosis was also reduced by therapy. Circulating leukocytosis after ALI was not reduced by colchicine therapy, but neutrophils reactivity and CD4 and CD8 cell surface expression on lymphocyte populations were restored. Colchicine reduces ALI and respiratory failure in experimental ARDS in relation with reduced lung neutrophil recruitment and reduced circulating leukocyte activation. This study supports the clinical development of colchicine for the prevention of ARDS in conditions causing ALI.

Impact of Finnish sauna bathing on circulating markers of inflammation in healthy middle-aged and older adults: A crossover study.

OBJECTIVES: Finnish sauna bathing is associated with a reduced risk of adverse health outcomes. The acute physiological responses elicited by Finnish sauna bathing that could explain this association remain understudied. This study characterized the acute effect of Finnish sauna bathing on circulating markers of inflammation in healthy middle-aged and older adults. DESIGN: With the use of a crossover study design, 20 healthy middle-aged and older adults (9 men/11 women, 66 ±â€¯6 years old) performed 3 interventions in random order: 1) 1 x 10 min of Finnish sauna bathing (80 °C, 20 % humidity); 2) 2 x 10 min of Finnish sauna bathing; 3) a time-control period during which participants sat outside of the sauna for 10 min. MAIN OUTCOMES: Venous blood samples were obtained before (≤15 min) and after (∼65 min) each intervention to determine circulating concentrations of interleukin 6 (IL-6), interleukin 1 receptor antagonist (IL-1RA), and C-reactive protein (CRP). RESULTS: IL-6 increased in response to 2 x 10 min of sauna bathing (+0.92 pg/mL [+0.16, +1.68], P = 0.02), but not following the 1 x 10 min session (+0.17 pg/mL [-0.13, +0.47], P = 0.26). IL1-RA increased during the 1 x 10 min (+51.27 pg/mL [+20.89, +81.65], P < 0.01) and 2 x 10 min (+30.78 pg/mL [+3.44, +58.12], P = 0.03) sessions. CRP did not change in response to either sauna session (P = 0.34). CONCLUSION: These results demonstrate that typical Finnish sauna bathing sessions acutely increase IL-6 and IL1-RA in healthy middle-aged and older adults.

Increased Circulating Levels of Neutrophil Extracellular Traps During Cardiopulmonary Bypass.

BACKGROUND: The intensity of inflammatory response triggered by cardiopulmonary bypass (CPB) during cardiac surgery has been associated with adverse outcomes. Neutrophils might contribute to organ injury through the liberation of DNA histone-based structures named "neutrophil extracellular traps" (NETs). Our objective was to assess circulating NETs levels before and after cardiac surgery in low-risk and high-risk patients. METHODS: This prospective cohort study included 2 groups of patients undergoing elective cardiac surgery with the use of CPB. The first group consisted of low-risk patients (European System for Cardiac Operative Risk Evaluation II ≤ 1%), and the second group included high-risk patients (European System for Cardiac Operative Risk Evaluation II ≥ 5%). Blood samples were drawn pre-CPB and 3 hours post-CPB separation. Measurements of circulating NETs, interleukin-6, C-reactive protein, myeloperoxidase, citrullinated histone 3, and pentraxin-related protein 3 levels were performed at each time point. RESULTS: Twenty-four patients, 12 high-risk and 12 low-risk patients, were included. Circulating NETs measurements changed from a median of 0.054 before CPB to 0.084 at 3 hours post-CPB separation, with a median increase of 0.037 (P < 0.001) per patient. No difference was noted between the high-risk and low-risk groups. A linear relationship was found between the circulating NETs measurements 3 hours post-CPB and CPB duration (ß = 0.047; confidence interval, 0.012-0.081; P = 0.01 R2 = 0.27). A correlation was found between the change in NETs with citrullinated histone 3 and myeloperoxidase levels, but not between NETs and other inflammatory biomarkers. CONCLUSIONS: Circulating NETs measurements increases during cardiac surgery with postsurgical levels proportional to CPB duration. The clinical significance of NETs production during cardiac surgery should be further investigated.

CONTEXTE: L'intensité de la réponse inflammatoire déclenchée par la circulation extracorporelle en cours de chirurgie cardiaque a été associée à des résultats défavorables. Les neutrophiles pourraient contribuer à des lésions organiques par la libération de structures d'ADN lié à des histones appelées « pièges extracellulaires des neutrophiles ¼ (ou NETs, de l'anglais neutrophil extracellular traps). Nous nous sommes donné pour objectif d'évaluer les taux de ces pièges extracellulaires dans la circulation avant et après la chirurgie cardiaque chez des patients exposés à des risques faibles et à des risques élevés. MÉTHODOLOGIE: Cette étude prospective de cohortes a été menée chez deux groupes de patients subissant une chirurgie cardiaque non urgente au cours de laquelle nous avons eu recours à la circulation extracorporelle. Le premier groupe était composé de patients exposés à un faible risque (EuroSCORE II [European System for Cardiac Operative Risk Evaluation II] ≤ 1 %), et le second, de patients exposés à un risque élevé (EuroSCORE II ≥ 5 %). Des échantillons sanguins étaient prélevés avant la mise sous circulation extracorporelle, puis 3 heures après le sevrage de la circulation extracorporelle. Les taux de NETs circulants, d'interleukine-6, de protéine C réactive, de myéloperoxydase, d'histones 3 citrullinées et de protéines apparentées à la pentraxine 3 ont été mesurés à chaque point d'évaluation. RÉSULTATS: Les données obtenues chez vingt-quatre patients, 12 dans le groupe à risque élevé et 12 dans le groupe à faible risque, ont été incluses dans l'analyse. Le taux médian de NETs circulants est passé de 0,054 avant la circulation extracorporelle à 0,084 trois heures après le sevrage, avec une augmentation médiane de 0,037 (p < 0,001) par patient. Aucune différence n'a été notée entre les deux groupes. Une relation linéaire a été observée entre la mesure du taux de NETs circulants 3 heures après le sevrage et la durée de la circulation extracorporelle (ß = 0,047; intervalle de confiance : 0,012-0,081; p = 0,01; R2= 0,27). Une corrélation a été notée entre la variation du taux de NETs comportant des histones 3 citrullinés et du taux de myéloperoxydase, mais pas entre le taux de NETs et ceux des autres biomarqueurs de l'inflammation. CONCLUSIONS: Le taux de NETs circulants augmente pendant une chirurgie cardiaque, le taux après la chirurgie étant proportionnel à la durée de la circulation extracorporelle. L'importance clinique de la production de NETs pendant la chirurgie cardiaque doit faire l'objet d'études plus approfondies.

CRP Induces NETosis in Heart Failure Patients with or without Diabetes.

C-reactive protein (CRP) is recognized as a biomarker of chronic, low-grade inflammation associated with vascular disorders. Lately, the role of neutrophils and neutrophil extracellular traps (NETs) has been investigated as a potential source of chronic inflammation and cardiovascular complications. This study investigated NETs as a marker of inflammation in patients with symptomatic heart failure (HF) with or without type 2 diabetes (T2DM) and examined the correlation between NETs and CRP. We performed a noninterventional study including patients with HF with or without T2DM, T2DM, and a healthy control (HC) group. NETs and other inflammatory markers in serum were measured by ELISA. The release of NETs (NETosis) in vitro under various stimuli was measured by confocal microscopy. The levels of NETs in the serum of HF patients were significantly higher compared with HC (112%). Serum CRP concentrations were significantly increased in HF and HF plus T2DM patients compared with HC, and a positive correlation was observed between serum CRP and NETs levels. Neutrophils from HF and HF plus T2DM patients underwent in vitro NETs release faster than T2DM and HC without any stimuli. In vitro, serum collected from the HF and the HF plus T2DM group induced NETosis in healthy neutrophils significantly more when compared with HC and T2DM, which was prevented by depletion from CRP. We confirmed in vitro that CRP induces a concentration-dependent NETs synthesis. This study proposes a mechanism by which CRP increases the risk of future cardiovascular events and supports mounting evidences on the role of neutrophils in chronic low-grade inflammation associated with HF.

A comparison of the effects of selective and non-selective mineralocorticoid antagonism on glucose homeostasis of heart failure patients with glucose intolerance or type II diabetes: A randomized controlled double-blind trial.

Mineralocorticoid receptor antagonists (MRAs) decrease morbidity and mortality in patients with heart failure (HF). However, spironolactone, a non-selective MRA, has been shown to exert a harmful effect on glucose homeostasis. The objective of this multicenter, randomized, controlled, double-blind trial was to compare the effects of spironolactone to those of the selective MRA eplerenone on glucose homeostasis among 62 HF patients with glucose intolerance or type II diabetes. Trial registration number:NCT01586442.

Synthesis of Human Neutrophil Extracellular Traps Contributes to Angiopoietin-Mediated In Vitro Proinflammatory and Proangiogenic Activities.

Neutrophil extracellular traps (NETs) are composed of nuclear DNA in a web-like structure extruded from neutrophils in response to either bacterial infection or inflammation. We previously reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of both angiopoietins (Ang1 and Ang2) to induce proinflammatory activities, such as synthesis and release of platelet-activating factor, upregulation of ß2 integrin complex (CD11/CD18), and neutrophil chemotaxis. In contrast, only Ang1 but not Ang2 is capable of promoting translational and transcriptional activities in neutrophils. In this article, we addressed whether Ang1 and/or Ang2 could modulate the release of NETs and if they contribute to angiopoietin-mediated proinflammatory activities. We observed that Ang1 and Ang2, alone or combined (10 nM, 3 h), increase NET synthesis and release by ≈2.5-fold as compared with PBS-treated neutrophils. The release of NETs is Tie2 dependent and requires downstream intracellular participation of PI3K, p38, and p42/44 MAPK pathways; reactive oxygen species production; intracellular calcium store depletion; and protein arginine deiminase 4 activation. These isolated NETs induced neutrophil and endothelial cell activation, leading to neutrophil adhesion onto human extracellular matrix and HUVEC and in vitro formation of capillary-like tubes by endothelial cells. Our study reports the capacity of Ang1 and Ang2 to promote the release of NETs and that these NETs contribute to angiopoietin-mediated in vitro proinflammatory and proangiogenic activities.

New Insights into the Pro-Inflammatory Activities of Ang1 on Neutrophils: Induction of MIP-1ß Synthesis and Release.

We reported the expression of angiopoietin Tie2 receptor on human neutrophils and the capacity of angiopoietins (Ang1 and Ang2) to induce pro-inflammatory activities, such as platelet-activating factor synthesis, ß2-integrin activation and neutrophil migration. Recently, we observed differential effects between both angiopoietins, namely, the capacity of Ang1, but not Ang2, to promote rapid interleukin-8 synthesis and release, as well as neutrophil viability. Herein, we addressed whether Ang1 and/or Ang2 could modulate the synthesis and release of macrophage inflammatory protein-1ß (MIP-1ß) by neutrophils. Neutrophils were isolated from blood of healthy volunteers; intracellular and extracellular MIP-1ß protein concentrations were assessed by ELISA. After 24 hours, the basal intracellular and extracellular MIP-1ß protein concentrations were ≈500 and 100 pg/106 neutrophils, respectively. Treatment with Ang1 (10 nM) increased neutrophil intracellular and extracellular MIP-1ß concentrations by 310 and 388% respectively. Pretreatment with PI3K (LY294002), p38 MAPK (SB203580) and MEK (U0126) inhibitors completely inhibited Ang1-mediated increase of MIP-1ß intracellular and extracellular protein levels. Pretreatment with NF-κB complex inhibitors, namely Bay11-7085 and IKK inhibitor VII or with a transcription inhibitor (actinomycin D) and protein synthesis inhibitor (cycloheximide), did also abrogate Ang1-mediated increase of MIP-1ß intracellular and extracellular protein levels. We validated by RT-qPCR analyses the effect of Ang1 on the induction of MIP-1ß mRNA levels. Our study is the first one to report Ang1 capacity to induce MIP-1ß gene expression, protein synthesis and release from neutrophils, and that these effects are mediated by PI3K, p38 MAPK and MEK activation and downstream NF-κB activation.

Decreased circulating and neutrophil mediated VEGF-A165 release in stable long-term cardiac transplant recipients.

BACKGROUND: Vascular endothelial growth factor (VEGF) may play a role on the allograft remodelling following cardiac transplantation (CTx). We measured the circulating levels of VEGF-A165 concomitantly with the proinflammatory (Interleukin-8; IL-8), anti-inflammatory (IL-1 receptor antagonist; IL-1RA) and their release from neutrophils of CTx recipients. METHODS: Eighteen CTx recipients aged 49.6 ± 3.1 years, being transplanted for 145 ± 20 months were age-matched to 35 healthy control (HC) subjects. Concomitantly to plasma assessment, circulating neutrophils were isolated, purified and stimulated by vehicle (PBS), N-formyl-Met-Leu-Phe (fMLP, 10(-7) M), bacterial lipopolysaccharide (LPS, 1 µg/mL), or tumour necrosis factor alpha (TNF-α, 10 ng/mL). RESULTS: Compared with HC, CTx recipients exhibited a decrease (-80%) in plasmatic levels of VEGF-A165 (225 ± 42 (HC) vs 44 ± 10 pg/mL (CTx); (p < 0.001). There were no differences in the levels of IL-8 and IL-1RA. Under basal or stimulated conditions, neutrophils from CTx patients exhibited a marked decrease ranging from -30 to -88% on their capacity to release VEGF-A165, IL-8 and IL-1RA upon stimulation. CONCLUSIONS: Long-term CTx recipients exhibit a marked reduction in the circulating levels of VEGF-A165, as well as neutrophil-mediated release of VEGF-A165, IL-1RA and IL-8 compared to healthy volunteers. The mechanisms and physiological impacts of these findings deserve additional investigations.

Effect of everolimus on the immunomodulation of the human neutrophil inflammatory response and activation.

The primary cause of mortality at 5 years following a cardiac transplantation is the development of atherosclerosis, termed coronary allograft vasculopathy (CAV). This pathology is characterized by diffused intimal hyperplasia and emanates from coronary arterial injuries caused by immune inflammatory cells. Neutrophils play an important role in this inflammatory process; however, their potential participation in the pathogenesis of CAV is poorly understood. Despite their essential contribution to the prevention of graft rejection, immunosuppressive drugs could have detrimental effects owing to their pro-inflammatory activities. Thus, we investigated the impact of different immunosuppressive drugs on the inflammatory response of neutrophils isolated from the blood of healthy volunteers. Under basal conditions, mammalian target of rapamycin (mTOR) inhibitors (sirolimus and everolimus) had the most potent anti-inflammatory effect, decreasing both IL-8 release (≈-80%) and vascular endothelial growth factor (VEGF) release (≈-65%) and preserving the release of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RA). In TNF-α-treated neutrophils, pre-incubation with everolimus provided the most potent effect, simultaneously reducing the release of both VEGF and IL-8 while doubling the release of IL-1RA. This latter effect of everolimus was maintained even when administered in combination with other immunosuppressive drugs. Sirolimus and everolimus decreased the tumor necrosis factor (TNF)-α-induced adhesion of neutrophils to human endothelial cells and human extracellular matrix. This effect was largely dependent on the ability of these compounds to alter ß2-integrin/CD18 activation. Our results suggest a potential mechanism for the beneficial effect of everolimus in the prevention of CAV in heart transplant recipients.

Heart failure with anemia: novel findings on the roles of renal disease, interleukins, and specific left ventricular remodeling processes.

BACKGROUND: Anemia is a highly prevalent and strong independent prognostic marker in heart failure (HF), yet this association is not completely understood. Whether anemia is simply a marker of disease severity and concomitant chronic kidney disease or represents the activation of other detrimental pathways remains uncertain. We sought to determine which pathophysiological pathways are exacerbated in patients with HF, reduced ejection fraction (HFrEF) and anemia in comparison with those without anemia. METHODS AND RESULTS: In a prospective study involving 151 patients, selected biomarkers were analyzed, each representing proposed contributive mechanisms in the pathophysiology of anemia in HF. We compared clinical, echocardiographic, and circulating biomarkers profiles among patients with HFrEF and anemia (group 1), HFrEF without anemia (group 2), and chronic kidney disease with preserved EF, without established HF (chronic kidney disease control group 3). We demonstrate here that many processes other than those related to chronic kidney disease are involved in the anemia-HF relationship. These are linked to the pathophysiological mechanisms pertaining to left ventricular systolic dysfunction and remodeling, systemic inflammation and volume overload. We found that levels of interleukin-6 and interleukin-10, specific markers of cardiac remodeling (procollagen type III N-terminal peptide, matrix metalloproteinase-2, tissue inhibitor of matrix metalloproteinase 1, left atrial volume), myocardial stretch (NT-proBNP [N-terminal probrain natriuretic peptide]), and myocyte death (troponin T) are related to anemia in HFrEF. CONCLUSIONS: Anemia is strongly associated not only with markers of more advanced and active heart disease but also with the level of renal dysfunction in HFrEF. Increased myocardial remodeling, inflammation, and volume overload are the hallmarks of patients with anemia and HF. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT00834691.

Angiopoietin-1 but not angiopoietin-2 promotes neutrophil viability: Role of interleukin-8 and platelet-activating factor.

We previously reported the expression of angiopoietin receptor Tie2 on human neutrophils. Both angiopoietins (Ang1 and Ang2) induce platelet activating factor (PAF) synthesis from endothelial cells (ECs) and neutrophils. Both angiopoietins can also modulate EC viability and since PAF can promote pro-survival activity on neutrophils, we addressed whether Ang1 and/or Ang2 could modulate neutrophil viability. Neutrophils were isolated from venous blood of healthy volunteers and neutrophil viability was assessed by flow cytometry using apoptotic and necrotic markers (annexin-V and propidium iodide (P.I.), respectively). Basal neutrophil viability from 0 to 24 h post-isolation decreased from 98% to ≈45%. Treatment with anti-apoptotic mediators such as interleukin-8 (IL-8; 25 nM) and PAF (100 nM) increased neutrophil basal viability by 34 and 26% (raising it from 43 to 58 and 55%) respectively. Treatment with Ang1 (0.001-50 nM) increased neutrophil viability by up to 41%, while Ang2 had no significant effect. Combination of IL-8 (25 nM) or PAF (100 nM) with Ang1 (10 nM) further increased neutrophil viability by 56 and 60% respectively. We also observed that Ang1, but not Ang2 can promote IL-8 release and that a pretreatment of the neutrophils with blocking anti-IL-8 antibodies inhibited the anti-apoptotic effect of IL-8 and Ang1 by 92 and 81% respectively. Pretreatment with a selective PAF receptor antagonist (BN 52021), did abrogate PAF pro-survival activity, without affecting Ang1-induced neutrophil viability. Our data are the first ones to report Ang1 pro-survival activity on neutrophils, which is mainly driven through IL-8 release.

Angiopoietin-1 but not angiopoietin-2 induces IL-8 synthesis and release by human neutrophils.

We previously reported Tie2 receptor expression on human neutrophils, which promote chemotactic activities upon activation by both angiopoietins (Ang1 and Ang2). Moreover, we observed that neutrophil pretreatment with Ang1 or Ang2 enhances interleukin-8 (IL-8) chemotactic effect. Therefore, we assessed the capacity of Ang1 and/or Ang2 to modulate neutrophil IL-8 synthesis and release. Neutrophils isolated from healthy donors were stimulated in a time- (1-6 h) and concentration-(10(-10) -10(-8) M) dependent manner with both angiopoietins. IL-8 mRNA production was measured by RT-qPCR, whereas its protein synthesis and release from neutrophils was assessed by ELISA. Ang1 (10(-8) M) induced a significant and maximal increase of IL-8 mRNA (4.7-fold) within 1 h, and promoted maximal IL-8 protein synthesis (3.6-fold) and release (5.5-fold) within 2 h as compared to control PBS-treated neutrophils. Treatment with Ang2 alone did not modulate IL-8 synthesis or release, and its combination to Ang1 did not affect Ang1 activity. Neutrophil pretreatment with a protein synthesis inhibitor (CHX) increased IL-8 mRNA synthesis by 18-fold, and reduced Ang1-mediated IL-8 protein synthesis and release by 96% and 92%, respectively. Pretreatment with a transcription inhibitor (ActD) reduced IL-8 mRNA synthesis by 54% and IL-8 protein synthesis and release by 52% and 79%, respectively. Using specific kinase inhibitors, we observed that Ang1-driven IL-8 mRNA and protein synthesis is p42/44 MAPK-dependent and -independent from p38 MAPK and PI3K activity. Our study is the first to report the capacity of Ang1 (as opposed to Ang2) to promote neutrophil IL-8 synthesis and release through the activation of p42/44 MAPK pathway.