CRP Induces NETosis in Heart Failure Patients with or without Diabetes
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CRP Induces NETosis in Heart Failure Patients with or without Diabetes Branka Vulesevic, Simon S. Lavoie, Paul-Eduard Neagoe, Elizabeth Dumas, Agnès Räkel, Michel White and Martin G. Sirois Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 ImmunoHorizons 2019, 3 (8) 378-388 doi: https://doi.org/10.4049/immunohorizons.1900026 http://www.immunohorizons.org/content/3/8/378 This information is current as of April 18, 2021. References This article cites 57 articles, 17 of which you can access for free at: http://www.immunohorizons.org/content/3/8/378.full#ref-list-1 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://www.immunohorizons.org/alerts ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732.
RESEARCH ARTICLE Innate Immunity CRP Induces NETosis in Heart Failure Patients with or without Diabetes Branka Vulesevic,*,† Simon S. Lavoie,*,† Paul-Eduard Neagoe,* Elizabeth Dumas,*,† Agnès Räkel,‡,§ Michel White,*,‡ and Martin G. Sirois*,† *Centre de Recherche, Institut de Cardiologie de Montréal, Faculté de Médecine, Université de Montréal, Montreal, Quebec H1T 1C8, Canada; † Département de Pharmacologie et Physiologie, Faculté de Médecine, Université de Montréal, Montreal, Quebec H3C 3J7, Canada; ‡Département Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 de Médecine, Faculté de Médecine, Université de Montréal, Montreal, Quebec 3T 1J4, Canada; and §Centre de Recherche du Centre Hospitalier Universitaire de Montréal, Faculté de Médecine, Université de Montréal, Montreal, Quebec H2X 0A9, Canada ABSTRACT 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. ImmunoHorizons, 2019, 3: 378–388. INTRODUCTION as during exercise. In healthy men and women, the left ventricular ejection fraction (LVEF) ranges from 52 to 74%. Heart failure (HF) is deﬁned as a chronic and progressive Phenotypically, HF can be present with a reduced LVEF condition in which the heart is unable to meet the requirements (HFrEF) #40%, whereas patients with symptoms and signs of of metabolizing tissues in a situation of increased demand, such HF with LVEF $50% are classiﬁed as HF with a preserved LVEF Received for publication March 27, 2019. Accepted for publication July 23, 2019. Address correspondence and reprint requests to: Prof. Martin G. Sirois, Montreal Heart Institute, 5000 Belanger Street, Montreal, QC H1T 1C8, Canada. E-mail address: email@example.com ORCIDs: 0000-0002-3660-5410 (B.V.); 0000-0001-5594-121X (S.S.L.); 0000-0002-7945-1343 (M.W.). This work was supported by grants from the Canadian Institutes of Health Research (MOP-97943 to M.G.S.), Fonds de Recherche du Québec - Santé (FRQS) - Research Network on Cardiometabolic Health, Diabetes and Obesity (to M.W., A.R., and M.G.S.), and Fondation de l’Institut de Cardiologie de Montréal (FICM) (to M.G.S.). B.V. and E.D. were recipients of a fellowship and doctoral studentship, respectively, from FRQS, and S.S.L. was a recipient of an FICM studentship. M.W. is the recipient of the Carolyn and Richard Renaud Endowed Research Chair in Heart Failure of the Montreal Heart Institute. Abbreviations used in this article: CRP, C-reactive protein; HC, healthy control; HF, heart failure; HFpEF, HF with a preserved LVEF; HFrEF, HF with a reduced LVEF; hsCRP, high-sensitivity CRP; LDL, low-density lipoprotein; LVEF, left ventricular ejection fraction; MHI, Montreal Heart Institute; MPO, myeloperoxidase; NET, neutrophil extracellular trap; NYHA, New York Heart Association; pEF, preserved ejection fraction; rEF, reduced ejection fraction; rhCRP, recombinant human CRP; T2DM, type 2 diabetes. This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license. Copyright © 2019 The Authors 378 https://doi.org/10.4049/immunohorizons.1900026 ImmunoHorizons is published by The American Association of Immunologists, Inc.
ImmunoHorizons CRP INDUCES NETosis IN HEART FAILURE PATIENTS 379 (HFpEF). More recently, the European Society of Cardiology has MATERIALS AND METHODS proposed new guidelines for the inclusion of a third type of HF with LVEF between 41 and 49%, named HF with midrange Population ejection fraction (1, 2). Chronic, low-grade inﬂammation is one This was a prospective nonrandomized, noninterventional study of the major factors impacting the development and progres- that included HF patients with reduced ejection fraction (rEF) or sion of HF (3). It also contributes to an increase in a broad range preserved ejection fraction (pEF) with or without T2DM and of inﬂammatory cytokines and biomarkers in both HFrEF and patients diagnosed with T2DM but without any heart pathologic HFpEF (4). Moreover, coexistence of HF with other proin- condition. Nineteen patients with HF and 26 patients with HF plus ﬂammatory conditions, such as type 2 diabetes (T2DM) has been T2DM were recruited at the Montreal Heart Institute (MHI). associated with an increase of adverse outcomes and mortality Twenty-one T2DM patients with no symptoms or signs of HF (5), both in acute and chronic HFrEF and HFpEF (6). Despite were recruited from the Clinique d’Endocrinologie de Montréal. numerous diﬀerences in the pathophysiology and the clinical The blood collection from all patients (66) and healthy controls features of these two forms of HF, with or without T2DM, (HC; n = 25) was performed at the MHI. This study was approved signiﬁcant relationships between selected inﬂammatory markers by the Scientiﬁc Research Committee and the Ethics Committee of and adverse cardiovascular outcomes have been reported (7). the MHI (ethics No. ICM #01-406 and No. ICM #12-1374) and Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 One of these inﬂammatory markers and predictors of future conform to the principles outlined in the Declaration of Helsinki. cardiovascular events, C-reactive protein (CRP), produced in liver, Donors were informed about the procedures and signed a written- has been used as a common indicator of both acute infection and informed consent before participating in the study. subclinical inﬂammation (3). CRP is increased in patients with chronic stable HF (8), but the mechanisms connecting CRP and Selection criteria of healthy volunteers and patients the severity of HF remain unknown. Lately, multiple studies are HC volunteers recruited in this study were enrolled assuming they reporting a correlation between CRP and neutrophil counts (9) were not having any signiﬁcant medical conditions and were not and an association between increased neutrophil counts and on any anti-inﬂammatory medication for at least 14 d before blood higher prevalence of coronary disease (10), suggesting possible collection. All patients with T2DM alone had an HbA1c ,10% and connections between CRP, neutrophils, and progression of undiagnosed for HF conditions. These T2DM patients were cardiovascular disorders. Furthermore, mounting evidence is controlled by any available hypoglycemic medications and, as per reporting novel mechanisms by which neutrophils can promote guidelines, were treated with preventive hypertension medication. proinﬂammatory activities through the generation and release The characterization and recruitment of HF patients was in of neutrophil extracellular traps (NETs), termed NETosis (11). concordance with the guidelines set in the Treatment of Preserved NETosis occurs primarily through a cell death process, fol- Cardiac Function Heart Failure with an Aldosterone Antagonist lowing a nuclear envelope disassembly and nuclear chromatin clinical trial (24), which was prior to the setting of the new decondensation into the cytoplasm of intact cells, mixing with European Society for Cardiology guidelines (1). Therefore, HF cytoplasmic and granule components (12). Under physiological patients recruited from the MHI Heart Failure Clinic were conditions, this process takes 3–8 hours after neutrophil classiﬁed as HFrEF if their LVEF was #40% or HFpEF if their activation (12). However, it has been reported that neutrophils LVEF was $45% (24–26), as documented by contrast ventricu- from patients suﬀering from acute or low-inﬂammatory lography, magnetic resonance imaging, radionuclide ventriculog- pathologic conditions are primed to release NETs; thus, a rapid raphy, or quantitative echocardiography within the previous 12 mo release of NETs can be observed within minutes under various if no cardiac event occurred since the measurement of their LVEF conditions (13–18). NETs are composed of dsDNA decorated (24). In addition to the previous inclusion criteria for the HF with cytosolic and granule-derived proinﬂammatory cytokines patients, the HF plus T2DM patients had also an HbA1c ,10% and and enzymes (19); their composition depends on the state of were controlled by any available hypoglycemic medications and, neutrophil activation and possible conditions that aﬀect them as per guidelines, were treated with preventive hypertension (e.g., systemic lupus erythematosus, rheumatoid arthritis, cystic medication. HF patients had a New York Heart Association ﬁbrosis, and conditions associated with metabolic disorders) (NYHA) functional class II or III and, unless contraindicated, (17). One of the unchangeable and obligatory protein compo- were treated with an angiotensin-converting enzyme inhibitor nents of NETs is myeloperoxidase (MPO) (20). In vasculature, or with angiotensin II receptor blockers and stable doses of besides their original role as a bacterial traps, NETs can contribute b-blockers for at least 30 d. Patients with severe chronic to thrombi formation and vulnerable plaque destabilization (21). pulmonary disease, chronic active inﬂammatory disease, severe Previous studies reported a correlation between CRP and NETs renal failure (creatinine .250 mmol/l), liver dysfunction (trans- production under hemodialysis (22), rheumatoid arthritis, or sepsis aminases $3-fold upper normal values), and ongoing cancer conditions (17, 23). However, there are as yet no studies reporting malignancy were not eligible for the study. HF patients with or the relationship between CRP and NETs release in HF patients. without T2DM, T2DM patients, and HC having ongoing and/or Thus, our objectives were to characterize the changes in NETs in recent infection (within 2 wk prior to the study, as this would patients with HF with or without T2DM and to assess whether aﬀect neutrophil counts) or had CRP values higher than 15 mg/l there is a link between CRP level and NETs in these patients. (suggesting potential acute unrelated inﬂammatory state) were https://doi.org/10.4049/immunohorizons.1900026
380 CRP INDUCES NETosis IN HEART FAILURE PATIENTS ImmunoHorizons excluded from this study. Other exclusion factors were as follows: NETs and biomarkers quantiﬁcation recent myocardial infarction, recent stroke, HF functional NYHA As NETs are deﬁned as chromatin bound to MPO, for their class IV, unstable clinical condition, and recent open-heart surgery. detection in serum, an ELISA method that detects exclusively MPO-bound DNA complexes in serum samples was used, and Study protocol: serum and neutrophil collection results were analyzed by comparison of OD values between Venous blood samples (20 ml) were obtained from HC, T2DM groups, as previously described (28). The biomarkers IL-6 patients, and HF patients with or without T2DM in Vacutainer and MPO were quantiﬁed by ELISA kits (Bio-Techne, Minneapolis, serum separation tubes. Upon centrifugation (1000 3 g for 15 min) MN). High-sensitivity CRP (hsCRP) in serum samples was at least 3 ml of serum was collected, aliquoted, and frozen at quantiﬁed by nephelometry at the biochemistry laboratory 280°C. Another 25 ml sample of venous blood was mixed with at the MHI. Anticoagulant Citrate Dextrose Solution USP Formula A (MP Biomedicals, Solon, OH). Neutrophils were isolated and resus- NETs quantiﬁcation by confocal microscopy pended in RPMI medium supplemented with 25 mM HEPES and Neutrophils (1 3 106 cells per ml) in RPMI medium were 1% penicillin/streptomycin, as described previously (27). Contam- incubated at 37°C, 5% CO2 for 15 or 60 min with diﬀerent agonists: ination with PBMCs was ,0.1% as determined by morphological PBS and Tris (control buﬀer solutions), homopentamer re- analysis and ﬂow cytometry (data not shown), and viability Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 combinant human CRP (rhCRP; 1, 5, and 10 mg/l) (R&D Systems, was .98% (trypan blue dye exclusion) (27). Pure neutrophil Minneapolis, MN), IL-8 (25 nM, positive control) (PeproTech, population was used for all in vitro studies. Rocky Hill, NJ), blood serum collected from volunteers (HC, TABLE I. Baseline patient characteristics Control Group (n = 25) T2DM Group (n = 21) HF Group (n = 19) HF with T2DM Group (n = 26) p Value Age (y) 58.4 6 1.6 62.0 6 1.0 66.4 6 1.1 67.8 6 1.2 p # 0.03 Males n (%) 18 (72) 19 (90.5) 12 (63.2) 19 (73.1) NS NYHA classification n (%) Class II - - 15 (78.9) 19 (73.1) Class III - - 4 (21.1) 7 (26.9) LVEF n (%) - - 30.4 6 3 36.8 6 2.5 rEF n (%) - - 15 (78.9) 19 (73.1) pEF n (%) - - 4 (21.1) 7 (26.9) Ischemic HF n (%) - - 9 (47.4) 15 (57.7) Nonischemic HF n (%) 10 (52.6) 11 (42.3) Cardiomyopathy n (%) - - 3 (15.8) 1 (3.9) Valvular n (%) - - 3 (15.8) 3 (11.6) Others n (%) - - 4 (21.1) 7 (26.9) Medical conditions n (%) Hypertension - 8 (38.1) 15 (78.9) 10 (38.5) p = 0.006 Dyslipidemia - 9 (43.0) 15 (78.9) 19 (73.1) Stroke - 0 (0) 5 (26.4) 9 (34.6) Chronic kidney disease - 3 (14.3) 5 (26.4) 10 (38.5) Treatment n (%) ACEi - 8 (38.1) 3 (15.8) 9 (34.6) ARBs - 5 (23.8) 12 (63.2) 13 (50) b-blockers - 4 (19.1) 18 (94.7) 26 (100) Diuretic agent - 2 (9.5) 16 (84.2) 26 (100) Statin 6 (24) 17 (81) 16 (84.2) 21 (80.8) Anticoagulant 1 (4) 0 (0) 8 (42.1) 15 (57.7) Sulfonylureas - 5 (23.8) - 7 (26.9) DPP-4 inhibitor - 9 (42.9) 8 (42.1) 12 (46.2) a-Glucosidase inhibitors - 1 (4.8) 4 (21.1) 8 (30.8) Agonists GLP-1 - 3 (14.3) - - SGLT2 inhibitor - 5 (23.8) - - Metformin - 18 (85.7) - 5 (19.2) Insulin - 7 (33.3) - 4 (15.4) Creatinine (mmol/l) 78.5 79.4 126.4 125.8 p , 0.001 Glucose (mmol/l) 5.25 8.60 6.64 9.40 p , 0.001 Cholesterol (mmol/l) 4.97 3.52 3.63 3.10 NS Triglyceride (mmol/l) 1.79 1.57 1.56 1.93 NS LDL (mmol/l) 3.10 1.75 201 1.54 NS Values are mean 6 SE or percentage. ACEi, angiotensin-converting enzyme; ARBs, angiotensin receptor blocker; DPP-4 inhibitor, dipeptidyl peptidase-4 inhibitor; GLP-1, glucagon-like peptide 1; SGLT2, sodium/glucose cotransporter 2. https://doi.org/10.4049/immunohorizons.1900026
ImmunoHorizons CRP INDUCES NETosis IN HEART FAILURE PATIENTS 381 CRP-depleted serum and NETs induction Serum CRP depletion was performed by using agarose-coated beads with immobilized phosphorylcholine with speciﬁc aﬃnity for CRP (Life Technologies) (29). As negative control, serum aliquots were treated with noncoated agarose beads. As per protocol, after 30 min of incubation, the beads were centrifuged and hsCRP content in the depleted serum was analyzed by nephelom- etry at the biochemistry laboratory at the MHI. Incubation with phosphorylcholine-coated beads produced serum depleted of CRP (values ,0.16 mg/l), whereas agarose beads had no eﬀect on CRP values. Statistical analysis Any diﬀerences in variables between HC, T2DM patients, and the HF with or without T2DM population were evaluated using a Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 one-way ANOVA followed by a Dunnett post hoc test. The rela- tions between variables were assessed by using Pearson regression analyses. Diﬀerences between groups for in vitro studies were compared using a one-way ANOVA or a paired Student t test (for neutrophil response to CRP and CRP-depleted serum). Statistical signiﬁcance was set at p , 0.05. All analyses were performed using SPSS for Windows. RESULTS Patient characteristics Ninety-one volunteers were enrolled in this study. A total of 45 patients with HF NYHA functional class II to III were studied; 34 (75.6%) of them were having rEF with or without T2DM. Baseline FIGURE 1. NETs release by neutrophils. demographics and clinical characteristics are summarized in Neutrophils from patients with T2DM and HF with T2DM are precondi- Table I. The group of patients with HF with or without T2DM tioned to produce NETs as compared with neutrophils from HC. Rep- was signiﬁcantly older than the HC group. However, we resentative pictures of NETs release at 60 min from the following groups: performed a Pearson correlation analysis that showed no (a) HC, (b) T2DM, (c) HF and (d) HF with T2DM neutrophils. Neutrophils correlation between NETs concentration in serum and aging are labeled with wheat germ agglutinin (conjugated with Alexa 647; red) (p = 0.196) in this population. and NETs are labeled with SYTOX Green (green). Neutrophils collected from all three patient groups showed increased tendency for NETs re- lease at 15 and 60 min. Data shown as mean 6 SEM (n = 6–7 for each In vitro release of NETs from unstimulated neutrophils group, including HC). *p # 0.04 (e). We performed a time-dependent study to assess the optimal time needed for the release of NETs from patients with T2DM, HF, and HF with T2DM as compared with HC. In that study, we were able T2DM patients, HF patients, and HF patients with T2DM) and to test whether low-grade inﬂammation present in patients with CRP-depleted serums from HF patients and HF patients with T2DM and/or HF prompts neutrophils to undergo NETosis. In T2DM. Green-ﬂuorescent nuclear and chromosome counter- our study, we observed by confocal microscopy that neutrophils stain that is nonpermeable to live cells (SYTOX Green, 1 mM; from patients with T2DM, HF, and HF with T2DM were already Life Technologies, Burlington, ON, Canada) was then added to capable of promoting a signiﬁcant release of NETs as compared detect dsDNA NETs released by neutrophils. Images were with HC within 15–60 min postisolation (representative images obtained by confocal microscopy (LSM 710; Carl Zeiss, Toronto, in Fig. 1a–d). Neutrophils isolated from all three patient groups ON, Canada) and set to acquire a mosaic of pictures (5 3 5 (T2DM, HF, and HF with T2DM) under basal condition increased images) (Zen 2; Carl Zeiss) (magniﬁcation, 2003). To calculate NETs synthesis within 15 min by 5.5-, 6.2-, and 7.4-fold, respectively, the percentage of total area of image covered by NETs, an as compared with HC (Fig. 1e for HF and HF with T2DM, p # 0.04). algorithm in Image-Pro Plus 7 (Media Cybernetics, Rockville, After 60 min, NETosis was increased by 2.2-, 3.6-, and 4.1-fold in MD) was used, and a threshold to exclude low-ﬂuorescence patients with T2DM, HF, and HF with T2DM, respectively (Fig. 1e background was applied (27). for HF and HF with T2DM, p # 0.02). https://doi.org/10.4049/immunohorizons.1900026
382 CRP INDUCES NETosis IN HEART FAILURE PATIENTS ImmunoHorizons Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 FIGURE 2. Measures of inflammatory markers in the serum. Serum from all three patient groups was analyzed by ELISA for the presence of NETs, MPO, and IL-6 and by nephelometry for hsCRP. (a) NETs from HF patients were increased compared with HC, *p = 0.015. (b) The concentration of MPO in the HF and HF with T2DM serum was increased compared with HC, *p # 0.012, whereas (c) IL-6 and (d) CRP concentrations in all three groups were increased when compared with HC, *p # 0.025; (n = 25 for HC, n = 21 for T2DM, n = 19 for HF, and n = 26 for HF with T2DM for NETs and CRP; n = 14–18 in each group for MPO and n = 8–11 in each group for IL-6). NETs and biomarkers of inﬂammation in serum prompted us to diﬀerently regroup the collected data on NETs in The levels of NETs in serum from HF patients were signiﬁcantly serum. By grouping the patients and HC based on their serum CRP higher than in HC (2.12-fold, p = 0.011), whereas patients with levels into three diﬀerent categories (e.g., ,1, 1–3, and .3 mg/l), T2DM and HF with T2DM had 1.61- and 1.58-fold increase we found a new perspective of the CRP–NETs correlation. The compared with HC (Fig. 2a, p = 0.16). Our data showed a signiﬁcant group with CRP levels between 1 and 3 mg/l had a signiﬁcant increase of MPO levels (both chromatin bound and free) in both increase in NETs content (Fig. 4, Table II, p = 0.011). The increase HF patients and patients with HF with T2DM groups (Fig. 2b, in IL-6 was signiﬁcant (p = 0.034), whereas the ST2 increase was p # 0.011) but not in T2DM patients, compared with HC (p = 0.14). noticeable but NS (Table II, p = 0.39), and there was no signiﬁcant All three patient groups had a signiﬁcant increase of IL-6, whereas rise in total MPO (Table II). The group with a CRP concentration IL-6 (except for one healthy volunteer) was below the minimum .3 mg/l, besides having an increase in NETs serum level (Fig. 4, detectable concentration in the HC group (Fig. 2c, p # 0.026). CRP p = 0.015), had an increase in all analyzed inﬂammatory markers levels of all three patient groups were also signiﬁcantly increased (Table II, IL-6 and MPO, p , 0.001 and p = 0.034, respectively) (Fig. 2d, p # 0.039 compared with HC). more comorbidities (e.g., previous occurrence of stroke, renal disease, pulmonary hypertension, and previous history of gout), Correlations between NETs and inﬂammatory biomarkers higher fasting glucose and low-density lipoprotein (LDL), The presence of NETs in serum positively correlated with CRP and higher levels of ST2, a protein biomarker of cardiac stress (Fig. 3a, p = 0.033) and MPO levels (Fig. 3b, p = 0.003). IL-6 (Fig. 3c, (Table II). p = 0.234) and NETs had no correlation, but IL-6 was positively correlated with CRP (Fig. 3d, p = 0.039). Serum-mediated NETs release Neutrophils from HC were exposed to PBS (basal control) and four Elevated CRP concentration increases NETs content in serum types of serum (HC, T2DM, HF and HF with T2DM) for 60 min Examining the results of NETs content in the serum and the and observed by confocal microscopy (representative images Fig. correlation between CRP and NETs in all three patient groups 5a–e). Serum from HF and HF with T2DM patients signiﬁcantly https://doi.org/10.4049/immunohorizons.1900026
ImmunoHorizons CRP INDUCES NETosis IN HEART FAILURE PATIENTS 383 Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 FIGURE 3. Correlation between proinflammatory cytokines and NETs in serum. A significant positive correlation (Pearson regression) was found between NETs and CRP (a), NETs and MPO (b), CRP and IL-6 (d), and no correlation was found between NETs and IL-6 (c). augmented NETosis by 3.68- and 4.58-fold, respectively (Fig. 5f, NETosis was increased by 2.5-fold, and a treatment with 10 mg/l p # 0.03). The serums of HC and T2DM patients increased (but signiﬁcantly increased NETosis up to 3.5-fold (Fig. 6b, p # 0.025). nonsigniﬁcantly) the release of NETs by 1.95- (p = 0.33) and 2.88- In addition, we observed that rhCRP (10 mg/l) was as potent as fold (p = 0.06), respectively (Fig. 5f ). IL-8 (25 nM), a known inducer of NETosis (30) (Fig. 6b). Together, our results are summarized in an illustration showing how HF and CRP in serum stimulates NETs release diabetes are leading to NETs formation (Fig. 7). Using the serum from HF and HF with T2DM patients with CRP values of 4 6 1 mg/l, we tested if the CRP found in the serum is a possible inducer of NETosis. The same serum sample but CRP DISCUSSION depleted (,0.16 mg/l minimal detectable value) was used at the same time and on the same HC neutrophils to induce NETosis In the current study we observed that patients with HF and/or (Fig. 6a). Serum containing more than 4 mg/l of CRP signiﬁcantly T2DM who have higher CRP levels in their serum also have higher increased NETosis, (Fig. 6a, p # 0.04), whereas CRP-depleted NETs concentration, and their neutrophils are primed to serum had no eﬀect. synthesize NETs in vitro even in absence of stimulation. We also observed that a treatment with the serums from these patients is CRP stimulates NETs release capable of promoting NETosis in HC neutrophils, and this latter To demonstrate that CRP has a direct capacity to induce NETs eﬀect was lost when the serums were depleted from CRP. Finally, release, neutrophils from HC were treated for 60 min with rhCRP we also experimentally conﬁrmed the in vitro capacity of rhCRP to (1, 5, and 10 mg/l), which is comparable to the range of CRP promote NETosis. Our study provides, to our knowledge, the ﬁrst concentrations observed in donors’ blood (HC and patients). evidence that CRP is a direct inducer of NETosis and that elevated rhCRP induced NETosis in a concentration-dependent manner. At serum concentration of CRP participates in NETs formation. the lowest rhCRP concentration (1 mg/l), as observed in healthy It has been demonstrated that neutrophils are not just individuals, NETosis was increased by 1.5-fold, whereas at 5 mg/l, ﬁrst responders to acute infections but also active contributors https://doi.org/10.4049/immunohorizons.1900026
384 CRP INDUCES NETosis IN HEART FAILURE PATIENTS ImmunoHorizons T2DM cohort in two distinct groups (,3 antidiabetes drugs or $3 antidiabetes drugs), they did observe a signiﬁcant increase of circulating NETs in the serum of patients taking $3 antidiabetes drugs. In another study, it has also been reported that circulating NETs concentration is signiﬁcantly increased in newly diagnosed/ uncontrolled diabetic patients that returned to nonsigniﬁcant increase within 12 mo posttreatment on metformin (39). In our study, we observed a trend but nonsigniﬁcant increase of circulat- ing NETs in a cohort of well-controlled T2DM patients. Together, these data suggest that, depending on the glycemic status, medica- tion, and treatment duration since T2DM diagnosis (37–39), the increase of circulating NETs can ﬂuctuate from signiﬁcant to nonsigniﬁcant. To our knowledge, our study is the ﬁrst one to report a signiﬁcant increase of NETs levels in the serum of HF patients. As Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 FIGURE 4. Relationship between the levels of NETs and CRP in serum. T2DM is a common comorbidity factor in HF patients with a The concentration of NETs in serum is increased in patients with CRP signiﬁcant negative impact of prognosis (40), as well as higher levels .1 mg/l as compared with low CRP levels (,1 mg/l) (n = 25 in mortality rates among patients with HF with T2DM compared the CRP ,1 mg/l, n = 35 in the CRP = 1–3 mg/l, and n = 31 in the with HF alone (41), we assessed the comorbidity eﬀect of T2DM on CRP .3 mg/l groups, respectively). *p # 0.015. the release of NETs in HF patients. Although, we did not observe an increase of NETs levels in the serum of HF with T2DM patients to low-grade chronic inﬂammation (31), which can be explained, as compared with HF alone, we did observe an increasing trend on in part, by their capacity to release NETs (32). Despite growing the release of NETs from the isolated neutrophils of HF with evidences that various pathological conditions prime neutro- T2DM patients as compared with HF alone. CRP is one of the early phils for NETosis (17), the prognostic value of NETs release in markers of inﬂammation, and it is used to predict the likelihood serum is still debatable (33, 34). However, NETs can be considered of developing cardiovascular events (42) and coronary disease as a risk factor of future cardiovascular events because of their progression (43). Patients at risk for future vascular events present role in atherosclerosis, inﬂammation, and thrombosis in small stable elevations of CRP over time, probably because of sustained blood vessels (17, 22, 35, 36). vascular inﬂammation (44). Based on the study from Pearson et al. Previous ﬁndings reported a circulating increase of NETs in (45), it has been recommended by the Centers of Disease Control T2DM patients (37) and that local stimuli aﬀect spontaneous and Prevention/American Heart Association, to categorize pa- NETosis in isolated neutrophils from diabetic patients (18). In tients as low- (,1 mg/l CRP), mid- (1–3 mg/l CRP), or high-risk contrast, Miyoshi et al. (38) reported a nonsigniﬁcant increase in (.3 mg/l CRP) for cardiovascular events, and an inﬂammatory circulating NETs from well-controlled T2DM patients when status was set for CRP values $3 mg/l (45, 46). In our study, in each compared with HC. In their study, when the authors separated the of the three patient groups (T2DM, HF, and HF with T2DM), TABLE II. Characteristics of populations based on the CRP level CRP , 1 mg/l (n = 25) CRP = 1–3 mg/l (n = 35) CRP . 3 mg/l (n = 31) p Value NETs (OD) 0.079 6 0.010 0.160 6 0.022 0.158 6 0.026 p# 0.015 CRP (mg/l) 0.65 6 0.06 1.94 6 0.10 7.18 6 0.61 p# 0.016 MPO (ng/ml) 292.1 6 33.0 311.0 6 32 411.8 6 48.3 p= 0.034 IL-6 (pg/ml) 0 1.74 6 0.51 4.77 6 0.57 p= 0.0001 ST2 (ng/ml) 22.0 6 1.8 31.1 6 4.8 58.0 6 15.6 p= 0.045 Creatinine (mmol/l) 90.0 6 3 100.4 6 6.6 126.1 6 10 p= 0.002 Fasting glucose (mmol/l) 6.34 6 0.53 7.12 6 0.43 7.77 6 0.47 p= 0.036 Total cholesterol (mmol/l) 4.49 6 0.28 3.85 6 0.17 3.26 6 0.19 p= 0.001 LDL (mmol/l) 2.66 6 0.27 2.11 6 0.16 1.65 6 0.14 p= 0.001 Triglycerides (mmol/l) 1.80 6 0.15 1.81 6 0.17 1.58 6 0.15 Statins (%) 11 (44) 16 (45.7) 21 (67.7) Comorbidity n (%) 4 (16) 16 (45.7) 16 (51.6) p = 0.005 Age (y) 60.2 6 1.6 65.6 6 2 65.6 6 2 Sex: male n (%) 21 (84) 24 (68.6) 23 (74.2) HC n (%) 15 (60) 9 (25.7) 1 (3.2) T2DM n (%) 3 (12) 12 (34.3) 6 (19.4) HF n (%) 3 (12) 8 (22.9) 8 (25.8) HF with T2DM n (%) 4 (16) 6 (17.1) 16 (51.6) Values are mean 6 SE or percentage. ST2, cardiac damage biomarker. https://doi.org/10.4049/immunohorizons.1900026
ImmunoHorizons CRP INDUCES NETosis IN HEART FAILURE PATIENTS 385 of other clinical variables, but MPO has been shown as an inﬂuential factor in the progression of cardiovascular disease among these patients (48). We selected T2DM patients with stable Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 FIGURE 5. NETs release by neutrophils in presence of serum. Neutrophils were incubated for 60 min with serum from all three pa- tient groups (T2DM, HF, HF with T2DM) and from HC (a–e). A significant increase of NETs release was observed with the serum from HF and HF with T2DM (f). Data shown as mean 6 SEM (n = 7). *p # 0.03 as compared with PBS. 50% of them had CRP serum levels higher than 3 mg/l, even in absence of any acute inﬂammatory condition. In all four groups (T2DM, HF, HF with T2DM, and HC), we observed a correlation between the levels of NETs and CRP concentration. Therefore, we used CRP risk classiﬁcation (low, mid and high) to assess its eﬀect on NETs synthesis. The two groups with CRP levels in the mid- FIGURE 6. CRP is an essential serum element for NETosis induction. and high-risk range had a signiﬁcant increase of NETs in serum. Neutrophils were treated for 60 min with controls (PBS, PBS plus CRP- Surprisingly, the group with CRP .3 mg/l had lower cholesterol, depletion beads, and PBS plus agarose beads) with serums from patients (CRP LDL, and triglyceride levels compared with the other two groups. 4 mg/l), matched CRP-depleted serums (CRP ,0.16 mg/l), and matched This can be explained by lipid-lowering treatment routinely serums (CRP 4 mg/l) with control agarose beads (a). The serums from prescribed to T2DM and cardiovascular disease patients. Despite patients (CRP 4 mg/l) and matched serums (CRP 4 mg/l) with control these treatments, there was an increase in MPO and IL-6 serum beads significantly increased NETosis, *p # 0.04 compared with PBS, whereas concentrations in the group with CRP .3 mg/l as well as NETs. matched CRP-depleted serums (CRP ,0.16 mg/l) were unable to increase The correlation between MPO and NETs concentrations was NETs release (a). In another set of experiments, neutrophils were incubated expected, as both increase upon neutrophil activation (17). MPO with control vehicles (PBS and Tris buffer) for 60 min, IL-8 (25 nM; positive serum levels are characterized by pro-oxidative and proinﬂam- control), and rhCRP (1, 5, 10 mg/l). At the highest concentration, rhCRP in- matory properties and correlate with CRP levels and WBC count duced a significant increase in NETs release (b), *p # 0.025 compared with (47). T2DM is associated with a mild increase of MPO independent PBS. Data shown as mean 6 SEM (n = 6–7 for each group, including HC). https://doi.org/10.4049/immunohorizons.1900026
386 CRP INDUCES NETosis IN HEART FAILURE PATIENTS ImmunoHorizons Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 FIGURE 7. Proposed schematic illustration on the role of CRP in the induction of NETosis and its implication in HF and diabetes. HF and/or T2DM are marked by low-grade inflammation that increases concentration of inflammatory proteins in serum including IL-6 and CRP. IL-6 will increase hepatic CRP release in the bloodstream, which, in turn, activates neutrophils to induce NETosis, leading to further aggravated vasculature injury and associated cardiovascular events. and controlled glycaemia and without diagnosed heart condi- directly involved in NETosis, which is itself more accepted as a tions, which reﬂected in a nonsigniﬁcant MPO and NETs cause of cardiovascular complications. Recently, Martinod et al. increase in the serum. Similarly, the basal activation of healthy (52) proposed a role for NETs in age-related cardiac ﬁbrosis in neutrophils tested in vitro was not exacerbated by the serum of mice, but such a study has not yet been conducted in humans. T2DM patients. Furthermore, “netting” neutrophils may play important roles in All three groups of patients in our study had signiﬁcant the promotion of atherosclerosis, vasculitis of diﬀerent aetiologies, increases in IL-6 levels. Besides being the primary cytokine and other vascular disorders (17, 22). promoting hepatic CRP production, IL-6 can lead to cardiomyo- To further validate whether the CRP contained in the serum cytes hypertrophy, myocardial dysfunction, and muscle wasting plays a role in NETs release, we treated neutrophils with CRP- (49). Increased IL-6 concentrations have been previously shown depleted serums, showing that they lost their capacity to induce in the circulation of HF and T2DM patients (50). Although, in our NETosis. We also showed that rhCRP induces NETosis in a study, it did not correlate directly with NETs content in serum, the concentration-dependent manner. In addition, to our knowledge, indirect relationship can be assumed through induction of CRP our novel ﬁnding that CRP can promote NETosis, other studies produced in the liver. have reported the capacity of CRP to induce neutrophil phagocy- As it was previously described, elevated levels of ST2 tosis, motility, and binding to endothelium (53, 54). Together these (biomarker of cardiac stress) at baseline and follow-up were data reinforce the notion that CRP is not simply a predictive shown to be associated with an increased risk of adverse clinical biomarker of inﬂammation but also a proinﬂammatory agonist events (39, 40). In our study, a random selection of serum collected acting likely through its binding capacity onto FcgR expressed from each group of volunteers was tested for ST2, and the average on neutrophils (54–57). Forthcoming studies will be needed to value in the group with CRP concentration .3 mg/l was above the better delineate the cellular mechanisms involved in CRP-mediated diagnostic cut-oﬀ value for chronic HF (.35 ng/ml) (39). NETosis. In addition to its predictive role in determining cardiovascular In summary, our study proposes, to our knowledge, a novel risk, there is evidence that CRP might serve as an active participant mechanism by which CRP may increase the risk of cardiovascu- in atherogenesis, as it is detected in human atherosclerotic plaques lar events in these high-risk patients through NETs induction. (51). Our study proposes an additional mechanism by which CRP is In this study, we report that neutrophils can respond to chronic https://doi.org/10.4049/immunohorizons.1900026
ImmunoHorizons CRP INDUCES NETosis IN HEART FAILURE PATIENTS 387 inﬂammatory cytokines and can have a damaging eﬀect on the 8. Danesh, J., J. G. Wheeler, G. M. Hirschﬁeld, S. Eda, G. Eiriksdottir, overall inﬂammatory state (Fig. 7). Further studies that will A. Rumley, G. D. Lowe, M. B. Pepys, and V. Gudnason. 2004. C-reactive protein and other circulating markers of inﬂammation in the examine the relationship of anti-inﬂammatory therapies aiming prediction of coronary heart disease. N. Engl. J. Med. 350: 1387–1397. to reduce CRP levels and changes in NETosis are needed. 9. Shah, A. D., S. Denaxas, O. Nicholas, A. D. Hingorani, and H. Hemingway. 2017. Neutrophil counts and initial presentation of 12 cardiovascular Study limitations diseases: a CALIBER cohort study. [Published erratum appears in 2017 This is a small observational study with the primary goal to assess J. Am. Coll. Cardiol. 69: 3125–3126.] J. Am. Coll. Cardiol. 69: 1160–1169. the eﬀects of chronic inﬂammation in patients with HF and/or 10. Tracchi, I., G. Ghigliotti, M. Mura, S. Garibaldi, P. Spallarossa, C. Barisione, V. Boasi, M. Brunelli, L. Corsiglia, A. Barsotti, and T2DM on neutrophil activation. No discrimination was made C. Brunelli. 2009. Increased neutrophil lifespan in patients with between HFrEF and HFpEF. Despite their diﬀerent phenotypes, congestive heart failure. Eur. J. Heart Fail. 11: 378–385. the increase of inﬂammatory biomarkers has been reported in both 11. Kaplan, M. J., and M. Radic. 2012. Neutrophil extracellular traps: conditions. Nevertheless, additional studies are needed to deﬁne double-edged swords of innate immunity. J. Immunol. 189: 2689– the role of CRP and NETs in both forms of HF (4). As detailed 2695. earlier, although there is signiﬁcant diﬀerence between the age 12. Fuchs, T. A., U. Abed, C. Goosmann, R. Hurwitz, I. Schulze, V. Wahn, Y. Weinrauch, V. Brinkmann, and A. Zychlinsky. 2007. Novel cell of HC and patients with HF with T2DM, this did not aﬀect the death program leads to neutrophil extracellular traps. J. Cell Biol. 176: Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 correlation between NETs and CRP or other inﬂammatory 231–241. markers. 13. Yipp, B. G., B. Petri, D. Salina, C. N. Jenne, B. N. Scott, L. D. Zbytnuik, K. Pittman, M. Asaduzzaman, K. Wu, H. C. Meijndert, et al. 2012. Infection-induced NETosis is a dynamic process involving neutrophil DISCLOSURES multitasking in vivo. Nat. Med. 18: 1386–1393. 14. Yu, X., J. Tan, and S. L. Diamond. 2018. Hemodynamic force triggers The authors have no ﬁnancial conﬂicts of interest. rapid NETosis within sterile thrombotic occlusions. J. Thromb. Haemost. 16: 316–329. 15. Carestia, A., T. Kaufman, L. Rivadeneyra, V. I. Landoni, R. G. Pozner, ACKNOWLEDGMENTS S. Negrotto, L. P. D’Atri, R. M. Gómez, and M. Schattner. 2016. Me- diators and molecular pathways involved in the regulation of neu- We are thankful to the volunteers for kindly providing blood samples and trophil extracellular trap formation mediated by activated platelets. to Louis Villeneuve for confocal microscopy technical support. J. Leukoc. Biol. 99: 153–162. 16. Maugeri, N., L. Campana, M. Gavina, C. Covino, M. De Metrio, C. Panciroli, L. Maiuri, A. Maseri, A. D’Angelo, M. E. Bianchi, et al. REFERENCES 2014. Activated platelets present high mobility group box 1 to neu- trophils, inducing autophagy and promoting the extrusion of neu- trophil extracellular traps. J. Thromb. Haemost. 12: 2074–2088. 1. Ponikowski, P., A. A. Voors, S. D. Anker, H. Bueno, J. G. F. Cleland, 17. Mitsios, A., A. Arampatzioglou, S. Arelaki, I. Mitroulis, and K. Ritis. A. J. S. Coats, V. Falk, J. R. González-Juanatey, V. P. Harjola, 2017. NETopathies? unraveling the dark side of old diseases through E. A. Jankowska, et al; ESC Scientiﬁc Document Group. 2016. 2016 neutrophils. Front. Immunol. 7: 678. ESC guidelines for the diagnosis and treatment of acute and chronic heart failure: the task force for the diagnosis and treatment of acute 18. Wong, S. L., M. Demers, K. Martinod, M. Gallant, Y. Wang, and chronic heart failure of the European Society of Cardiology (ESC) A. B. Goldﬁne, C. R. Kahn, and D. D. Wagner. 2015. Diabetes primes developed with the special contribution of the Heart Failure Associ- neutrophils to undergo NETosis, which impairs wound healing. Nat. ation (HFA) of the ESC. [Published erratum appears in 2018 Eur. Med. 21: 815–819. Heart J. 39: 860.] Eur. Heart J. 37: 2129–2200. 19. Brinkmann, V., U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, 2. Dunlay, S. M., V. L. Roger, and M. M. Redﬁeld. 2017. Epidemiology of D. S. Weiss, Y. Weinrauch, and A. Zychlinsky. 2004. Neutrophil ex- heart failure with preserved ejection fraction. Nat. Rev. Cardiol. 14: tracellular traps kill bacteria. Science 303: 1532–1535. 591–602. 20. Metzler, K. D., T. A. Fuchs, W. M. Nauseef, D. Reumaux, J. Roesler, 3. Danesh, J., P. Whincup, M. Walker, L. Lennon, A. Thomson, I. Schulze, V. Wahn, V. Papayannopoulos, and A. Zychlinsky. 2011. P. Appleby, J. R. Gallimore, and M. B. Pepys. 2000. Low grade Myeloperoxidase is required for neutrophil extracellular trap for- inﬂammation and coronary heart disease: prospective study and mation: implications for innate immunity. Blood 117: 953–959. updated meta-analyses. BMJ 321: 199–204. 21. Rao, A. N., N. M. Kazzaz, and J. S. Knight. 2015. Do neutrophil ex- 4. Bozkurt, B., D. L. Mann, and A. Deswal. 2010. Biomarkers of in- tracellular traps contribute to the heightened risk of thrombosis in ﬂammation in heart failure. Heart Fail. Rev. 15: 331–341. inﬂammatory diseases? World J. Cardiol. 7: 829–842. 5. Benjamin, E. J., S. S. Virani, C. W. Callaway, A. M. Chamberlain, 22. Döring, Y., O. Soehnlein, and C. Weber. 2017. Neutrophil extracellular A. R. Chang, S. Cheng, S. E. Chiuve, M. Cushman, F. N. Delling, traps in atherosclerosis and atherothrombosis. Circ. Res. 120: 736–743. R. Deo, et al; American Heart Association Council on Epidemiology and 23. Xu, P. C., S. Lin, X. W. Yang, D. M. Gu, T. K. Yan, L. Wei, and Prevention Statistics Committee and Stroke Statistics Subcommittee. B. L. Wang. 2015. C-reactive protein enhances activation of coagula- 2018. Heart disease and stroke statistics-2018 update: a report from the tion system and inﬂammatory response through dissociating into American heart association. [Published erratum appears in 2018 Circu- monomeric form in antineutrophil cytoplasmic antibody-associated lation 137: e493.] Circulation 137: e67–e492. vasculitis. BMC Immunol. 16: 10. 6. Bahtiyar, G., D. Gutterman, and H. Lebovitz. 2016. Heart failure: a major 24. Pitt, B., M. A. Pfeﬀer, S. F. Assmann, R. Boineau, I. S. Anand, cardiovascular complication of diabetes mellitus. Curr. Diab. Rep. 16: 116. B. Claggett, N. Clausell, A. S. Desai, R. Diaz, J. L. Fleg, et al; TOPCAT 7. Dick, S. A., and S. Epelman. 2016. Chronic heart failure and inﬂam- Investigators. 2014. Spironolactone for heart failure with preserved mation: what do we really know? Circ. Res. 119: 159–176. ejection fraction. N. Engl. J. Med. 370: 1383–1392. https://doi.org/10.4049/immunohorizons.1900026
388 CRP INDUCES NETosis IN HEART FAILURE PATIENTS ImmunoHorizons 25. Grodin, J. L., S. Philips, W. Mullens, P. Nijst, P. Martens, J. C. Fang, and diabetes: JACC state-of-the-art review. J. Am. Coll. Cardiol. 73: M. H. Drazner, W. H. W. Tang, and A. Pandey. 2019. Prognostic im- 602–611. plications of plasma volume status estimates in heart failure with 41. Bajpai, A., and D. G. Tilley. 2018. The role of leukocytes in diabetic preserved ejection fraction: insights from TOPCAT. Eur. J. Heart Fail. cardiomyopathy. Front. Physiol. 9: 1547. 21: 634–642. 42. Pfützner, A., and T. Forst. 2006. High-sensitivity C-reactive protein as 26. Selvaraj, S., B. Claggett, S. J. Shah, I. Anand, J. L. Rouleau, E. O’Meara, cardiovascular risk marker in patients with diabetes mellitus. Diabetes A. S. Desai, E. F. Lewis, B. Pitt, N. K. Sweitzer, et al. 2018. Prognostic Technol. Ther. 8: 28–36. value of albuminuria and inﬂuence of spironolactone in heart failure 43. Fonseca, F. A., and M. C. Izar. 2016. High-sensitivity C-reactive with preserved ejection fraction. Circ. Heart Fail. 11: e005288. protein and cardiovascular disease across countries and ethnicities. 27. Lavoie, S. S., E. Dumas, B. Vulesevic, P.-E. Neagoe, M. White, and Clinics (São Paulo) 71: 235–242. M. G. Sirois. 2018. Synthesis of human neutrophil extracellular traps 44. Ridker, P. M. 2009. C-reactive protein: eighty years from discovery to contributes to angiopoietin-mediated in vitro proinﬂammatory and emergence as a major risk marker for cardiovascular disease. Clin. proangiogenic activities. J. Immunol. 200: 3801–3813. Chem. 55: 209–215. 28. Kessenbrock, K., M. Krumbholz, U. Schönermarck, W. Back, 45. Pearson, T. A., G. A. Mensah, R. W. Alexander, J. L. Anderson, W. L. Gross, Z. Werb, H. J. Gröne, V. Brinkmann, and D. E. Jenne. R. O. Cannon III, M. Criqui, Y. Y. Fadl, S. P. Fortmann, Y. Hong, 2009. Netting neutrophils in autoimmune small-vessel vasculitis. Nat. G. L. Myers, et al; American Heart Association. 2003. Markers of Med. 15: 623–625. inﬂammation and cardiovascular disease: application to clinical and 29. Kapur, R., K. M. Heitink-Pollé, L. Porcelijn, A. E. Bentlage, public health practice: a statement for healthcare professionals from Downloaded from http://www.immunohorizons.org/ by guest on April 18, 2021 M. C. Bruin, R. Visser, D. Roos, R. B. Schasfoort, M. de Haas, C. E. van the Centers for Disease Control and Prevention and the American der Schoot, and G. Vidarsson. 2015. C-reactive protein enhances IgG- Heart Association. Circulation 107: 499–511. mediated phagocyte responses and thrombocytopenia. Blood 125: 46. Sabatine, M. S., D. A. Morrow, K. A. Jablonski, M. M. Rice, 1793–1802. J. W. Warnica, M. J. Domanski, J. Hsia, B. J. Gersh, N. Rifai, 30. Hazeldine, J., P. Harris, I. L. Chapple, M. Grant, H. Greenwood, P. M. Ridker, et al; PEACE Investigators. 2007. Prognostic signiﬁ- A. Livesey, E. Sapey, and J. M. Lord. 2014. Impaired neutrophil ex- cance of the Centers for Disease Control/American Heart Association tracellular trap formation: a novel defect in the innate immune system high-sensitivity C-reactive protein cut points for cardiovascular and of aged individuals. Aging Cell 13: 690–698. other outcomes in patients with stable coronary artery disease. Cir- 31. Caielli, S., J. Banchereau, and V. Pascual. 2012. Neutrophils come of culation 115: 1528–1536. age in chronic inﬂammation. Curr. Opin. Immunol. 24: 671–677. 47. Loria, V., I. Dato, F. Graziani, and L. M. Biasucci. 2008. Myeloper- oxidase: a new biomarker of inﬂammation in ischemic heart disease 32. Pérez-Sánchez, C., P. Ruiz-Limón, M. A. Aguirre, Y. Jiménez-Gómez, and acute coronary syndromes. Mediators Inﬂamm. 2008: 135625. I. Arias-de la Rosa, M. C. Ábalos-Aguilera, A. Rodriguez-Ariza, 48. Wiersma, J. J., M. C. Meuwese, J. N. van Miert, A. Kastelein, M. C. Castro-Villegas, R. Ortega-Castro, P. Segui, et al. 2017. Di- J. G. Tijssen, J. J. Piek, and M. D. Trip. 2008. Diabetes mellitus type 2 agnostic potential of NETosis-derived products for disease activity, is associated with higher levels of myeloperoxidase. Med. Sci. Monit. atherosclerosis and therapeutic eﬀectiveness in Rheumatoid arthritis 14: CR406–CR410. patients. J. Autoimmun. 82: 31–40. 49. Kamimura, D., K. Ishihara, and T. Hirano. 2003. IL-6 signal trans- 33. Wang, H., L. L. Sha, T. T. Ma, L. X. Zhang, M. Chen, and M. H. Zhao. duction and its physiological roles: the signal orchestration model. 2016. Circulating level of neutrophil extracellular traps is not a useful Rev. Physiol. Biochem. Pharmacol. 149: 1–38. biomarker for assessing disease activity in antineutrophil cytoplasmic 50. Esteve, E., A. Castro, A. López-Bermejo, J. Vendrell, W. Ricart, and antibody-associated vasculitis. PLoS One 11: e0148197. J. M. Fernández-Real. 2007. Serum interleukin-6 correlates with 34. Arai, Y., K. Yamashita, K. Mizugishi, T. Watanabe, S. Sakamoto, endothelial dysfunction in healthy men independently of insulin T. Kitano, T. Kondo, H. Kawabata, N. Kadowaki, and A. Takaori-Kondo. sensitivity. Diabetes Care 30: 939–945. 2013. Serum neutrophil extracellular trap levels predict thrombotic 51. Paﬀen, E., and M. P. DeMaat. 2006. C-reactive protein in athero- microangiopathy after allogeneic stem cell transplantation. Biol. Blood sclerosis: a causal factor? Cardiovasc. Res. 71: 30–39. Marrow Transplant. 19: 1683–1689. 52. Martinod, K., T. Witsch, L. Erpenbeck, A. Savchenko, H. Hayashi, 35. Mozzini, C., U. Garbin, A. M. Fratta Pasini, and L. Cominacini. 2017. D. Cherpokova, M. Gallant, M. Mauler, S. M. Cifuni, and D. D. Wagner. An exploratory look at NETosis in atherosclerosis. Intern. Emerg. 2017. Peptidylarginine deiminase 4 promotes age-related organ ﬁbrosis. Med. 12: 13–22. J. Exp. Med. 214: 439–458. 36. Jorch, S. K., and P. Kubes. 2017. An emerging role for neutrophil 53. Schlayer, H. J., U. Karck, U. Ganter, R. Hermann, and K. Decker. 1987. extracellular traps in noninfectious disease. Nat. Med. 23: 279–287. Enhancement of neutrophil adherence to isolated rat liver sinusoidal 37. Menegazzo, L., S. Ciciliot, N. Poncina, M. Mazzucato, M. Persano, endothelial cells by supernatants of lipopolysaccharide-activated B. Bonora, M. Albiero, S. Vigili de Kreutzenberg, A. Avogaro, and monocytes. Role of tumor necrosis factor. J. Hepatol. 5: 311–321. G. P. Fadini. 2015. NETosis is induced by high glucose and associated 54. Ling, M. R., I. L. Chapple, A. J. Creese, and J. B. Matthews. 2014. with type 2 diabetes. Acta Diabetol. 52: 497–503. Eﬀects of C-reactive protein on the neutrophil respiratory burst in 38. Miyoshi, A., M. Yamada, H. Shida, D. Nakazawa, Y. Kusunoki, vitro. Innate Immun. 20: 339–349. A. Nakamura, H. Miyoshi, U. Tomaru, T. Atsumi, and A. Ishizu. 2016. 55. Bodman-Smith, K. B., A. J. Melendez, I. Campbell, P. T. Harrison, Circulating neutrophil extracellular trap levels in well-controlled type J. M. Allen, and J. G. Raynes. 2002. C-reactive protein-mediated 2 diabetes and pathway involved in their formation induced by high- phagocytosis and phospholipase D signalling through the high-aﬃnity dose glucose. Pathobiology 83: 243–251. receptor for immunoglobulin G (FcgammaRI). Immunology 107: 39. Carestia, A., G. Frechtel, G. Cerrone, M. A. Linari, C. D. Gonzalez, 252–260. P. Casais, and M. Schattner. 2016. NETosis before and after hyper- 56. Stein, M. P., J. C. Edberg, R. P. Kimberly, E. K. Mangan, D. Bharadwaj, glycemic control in type 2 diabetes mellitus patients. PLoS One 11: C. Mold, and T. W. Du Clos. 2000. C-reactive protein binding to e0168647. FcgammaRIIa on human monocytes and neutrophils is allele-speciﬁc. 40. McHugh, K., A. D. DeVore, J. Wu, R. A. Matsouaka, G. C. Fonarow, J. Clin. Invest. 105: 369–376. P. A. Heidenreich, C. W. Yancy, J. B. Green, N. Altman, and 57. Sproston, N. R., and J. J. Ashworth. 2018. Role of C-reactive protein at A. F. Hernandez. 2019. Heart failure with preserved ejection fraction sites of inﬂammation and infection. Front. Immunol. 9: 754. https://doi.org/10.4049/immunohorizons.1900026
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