Neutrophil-Enriched Biomarkers and Long-Term Prognosis in Acute Coronary Syndrome: a Systematic Review and Meta-analysis.

Activated neutrophils release a range of inflammatory products that represent potential biomarkers, and there is interest in the prognostic value of these in acute coronary syndrome (ACS) patients. We conducted a systematic review to examine neutrophil-enriched biomarkers and the occurrence of major adverse cardiovascular events (MACE) in patients with ACS. We identified twenty-seven studies including 17,831 patients with ACS. The most studied biomarkers were neutrophil gelatinase-associated lipocalin (NGAL) and myeloperoxidase (MPO). Meta-analyses showed that elevated NGAL was associated with higher MACE rates (unadjusted risk ratio (RR) 1.52, 95% CI 1.12-2.06, p = 0.006) as were elevated MPO levels (unadjusted RR 1.61, 95% CI 1.22-2.13, p = 0.01). There was limited data suggesting that increased levels of calprotectin, proteinase-3 and double-stranded DNA were also associated with MACE. These results suggest that higher levels of neutrophil-enriched biomarkers may be predictive of MACE in patients with ACS, although higher-quality studies are needed to confirm these observations.

Ensuring Full Spectrum Flow Cytometry Data Quality for High-Dimensional Data Analysis.

Full spectrum flow cytometry (FSFC) allows for the analysis of more than 40 parameters at the single-cell level. Compared to the practice of manual gating, high-dimensional data analysis can be used to fully explore single-cell datasets and reduce analysis time. As panel size and complexity increases so too does the detail and time required to prepare and validate the quality of the resulting data for use in downstream high-dimensional data analyses. To ensure data analysis algorithms can be used efficiently and to avoid artifacts, some important steps should be considered. These include data cleaning (such as eliminating variable signal change over time, removing cell doublets, and antibody aggregates), proper unmixing of full spectrum data, ensuring correct scale transformation, and correcting for batch effects. We have developed a methodical step-by-step protocol to prepare full spectrum high-dimensional data for use with high-dimensional data analyses, with a focus on visualizing the impact of each step of data preparation using dimensionality reduction algorithms. Application of our workflow will aid FSFC users in their efforts to apply quality control methods to their datasets for use in high-dimensional analysis, and help them to obtain valid and reproducible results. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Data cleaning Basic Protocol 2: Validating the quality of unmixing Basic Protocol 3: Data scaling Basic Protocol 4: Batch-to-batch normalization.

Increased levels of low density neutrophils (LDNs) in myocardial infarction.

BACKGROUND: Recent evidence suggests that neutrophils are highly plastic cells that can display heterogeneous phenotypes. Low-density neutrophils (LDNs) have been described in many inflammatory conditions, and are thought to represent an immature, hyperactivated subtype of neutrophils. Neutrophils are significantly involved in the inflammatory response to myocardial infarction (MI), although we do not know the extent to which LDNs exist, or function, in MI. This study sought to determine the frequency and phenotype of LDNs in MI patients, compared to healthy subjects (HS). METHODS: LDNs and normal-density neutrophils (NDNs) were isolated from the peripheral blood of MI subjects (n = 12) and HSs (n = 12) using density gradient centrifugation. LDNs and NDNs were analysed by flow cytometry to identify neutrophils (CD66b+CD15+CD14-CD3-CD19- cells) and examine neutrophil activation (CD11b, CD66b and CD15) and maturity (CD33 and CD16). RESULTS: We identified LDNs within the peripheral blood mononuclear cell (PBMC) fraction of blood, and this population is significantly enriched in MI patients (1.04 ± 0.75% of PBMCs), compared to HS (0.29 ± 0.24%, p = .003). Across both cohorts, LDNs express significantly higher levels of CD66b and CD15, indicating a heightened state of activation compared to NDNs. In this study, LDNs were described as CD33highCD16low, compared to CD33lowCD16high NDNs, indicating the immaturity of this neutrophil subtype. CONCLUSIONS: An increase in the frequency of hyperactivated, immature LDNs is an immunological feature of MI. We highlight a potential pathological role of LDNs in MI, which underscores the need to expand our current understanding of this subtype in MI and other cardiovascular diseases (CVDs).

A combined biomarker approach for characterising extracellular matrix profiles in acute myocardial infarction.

Extracellular matrix (ECM) biomarkers are useful for measuring underlying molecular activity associated with cardiac repair following acute myocardial infarction (AMI). The aim of this study was to conduct exploratory factor analysis (EFA) to examine the interrelationships between ECM biomarkers, and cluster analysis to identify if distinct ECM profiles could distinguish patient risk in AMI. Ten ECM biomarkers were measured from plasma in 140 AMI patients: MMP-2, -3, -8, -9, periostin, procollagen I N-Terminal propeptide, osteopontin, TGF-ß1, TIMP-1 and -4. EFA grouped eight ECM biomarkers into a two-factor solution, which comprised three biomarkers in Factor 1 and five biomarkers in Factor 2. Notably, ECM biomarkers were not separated based on biological function. Cluster analysis grouped AMI patients into three distinct clusters. Cluster One (n = 54) had increased levels of MMP-8, MMP-9, and TGF-B1. Cluster Two (n = 43) had elevated levels of MMP-2, MMP-3, osteopontin, periostin and TIMP-1, and increased high-sensitivity troponin T and GRACE scores. Cluster Three (n = 43) had decreased levels of ECM biomarkers. Circulating ECM biomarkers demonstrated collinearity and entwined biological functions based on EFA analysis. Using cluster analysis, patients with similar clinical presentations could be separated into distinct ECM profiles that were associated with differential patient risk. Clinical significance remains to be determined.

Linking Neutrophil Extracellular Traps and Platelet Activation: A Composite Biomarker Score for Predicting Outcomes after Acute Myocardial Infarction.

BACKGROUND: Activation of both platelets and neutrophils can contribute to the risk of major adverse cardiovascular events (MACE) following acute myocardial infarction (AMI). Neutrophil extracellular traps (NETs) are an important product of the platelet-neutrophil axis and exaggerate vascular damage in cardiovascular disease. Additionally, activated platelets can drive NETosis and are directly linked to thromboembolic risk. Investigating the combined effect of biomarkers for NETosis and platelet activation represents a novel approach to risk prediction post-AMI. Here, we examined the utility of a composite biomarker score, inclusive of both pathways, for predicting MACE post-AMI. METHODS AND RESULTS: In a case-control design, 100 case patients who experienced MACE within 1 year of index admission were matched in a 1:2 ratio with control patients. Serum levels of myeloperoxidase-DNA, neutrophil elastase-DNA, and citrullinated histone H3 were assayed by enzyme-linked immunosorbent assay (ELISA) as markers of NET burden. To measure platelet activation, soluble P-selectin was assayed by ELISA in parallel. Platelet and neutrophil counts were also recorded. Composite biomarker scores, inclusive of biomarkers for NETosis and platelet activation, were assessed using multivariate regression modeling. These composite biomarker scores were independent predictors of 1-year MACE. The strongest association with MACE was observed using a composite of platelet count, soluble P-selectin, and all NET markers (odds ratio: 1.94; 1.16-3.25). CONCLUSION: Here, we demonstrate the importance of combining biomarkers of NETosis and platelet activation for risk prediction in patients with AMI. Combining biomarkers from closely linked, but distinct, biological pathways was more effective than utilizing either type of biomarker alone.

An IL-6-IL-8 score derived from principal component analysis is predictive of adverse outcome in acute myocardial infarction.

INTRODUCTION: Many studies have shown that elevated biomarkers of inflammation following acute myocardial infarction (AMI) are associated with major adverse cardiovascular events (MACE). However, the optimal way of measuring the complex inflammatory response following AMI has not been determined. In this study we explore the use of principal component analysis (PCA) utilising multiple inflammatory cytokines to generate a combined cytokine score that may be predictive of MACE post-AMI. METHODS: Thirteen inflammatory cytokines were measured in plasma of 317 AMI patients, drawn 48-72 h following symptom onset. Patients were followed-up for one year to determine the incidence of MACE. PCA was used to generate a combined score using six cytokines that were detectable in the majority of patients (IL-1ß, -6, -8, and -10; MCP-1; and RANTES), and using a subset of cytokines that were associated with MACE on univariate analysis. Multivariate models using baseline characteristics, elevated individual cytokines and PCA-derived scores determined independent predictors of MACE. RESULTS: IL-6 and IL-8 were significantly associated with MACE on univariate analysis and were combined using PCA into an IL-6-IL-8 score. The combined cytokine score and IL-6-IL-8 PCA-derived score were both significantly associated with MACE on univariate analysis. In multivariate models IL-6-IL-8 scores (OR = 2.77, p = 0.007) and IL-6 levels (OR = 2.18, p = 0.035) were found to be independent predictors of MACE. CONCLUSION: An IL-6-IL-8 score derived from PCA was found to independently predict MACE at one year and was a stronger predictor than any individual cytokine, which suggests this may be an appropriate strategy to quantify inflammation post-AMI. Further investigation is required to determine the optimal set of cytokines to measure in this context.

Immunoglobulin G levels predicts risk of recurrent adverse cardiovascular events in myocardial infarction patients.

Background: Patients with myocardial infarction (MI) are at an increased risk of experiencing recurrent major adverse cardiovascular events (MACE) but predicting MACE has remained challenging. Immunoglobulins are implicated in cardiovascular disease, although the predictive value of total immunoglobulin G (IgG) has not yet been evaluated in a secondary prevention setting. This study examined whether total IgG is predictive of MACE in an MI population, and how total IgG compared to the predictive value of C-reactive protein (CRP), an acute inflammatory marker. Methods: We conducted a case-control study with 40 MI subjects (cases) who experienced MACE within 1 year of their index admission. Cases were matched for age, sex, diabetes and presentation with 77 controls who did not have MACE. Pre-discharge plasma samples were analysed for total IgG and CRP. Results: We observed higher levels of total plasma IgG in MI subjects with MACE (24.9 (16.2-43.7) mg/mL) compared to controls (18.4 (9.1-37.3) mg/mL; p < 0.05). Higher levels of IgG were associated with increased risk of MACE in our MI population. MI subjects within quartiles 3 and 4 of total IgG had 6 times and 4 times, respectively, the rate of MACE compared to subjects in quartile 1. There was no difference in CRP levels between cases and controls (1.1 (0.5-3.0) vs. 1.9 (0.6-6.1) mg/mL, p = 0.10), and no relationship was observed between CRP and MACE. Conclusion: Pre-discharge IgG level was a better marker for predicting MACE post-MI than CRP, which had no predictive value in this study.

Platelets modulate multiple markers of neutrophil function in response to in vitro Toll-like receptor stimulation.

INTRODUCTION: In addition to their role in facilitating leukocyte-mediated inflammation, platelets can dampen leukocyte pro-inflammatory responses in some contexts. Consequently, platelets are increasingly appreciated as regulators of inflammation. Together, platelets and neutrophils play a role in inflammation through Toll-like receptor (TLR) expression, although we do not fully understand how platelets shape neutrophil responses to TLR stimulation. Here, we aimed to determine the extent to which platelets can modulate neutrophil function in response to in vitro stimulation with TLR4, TLR2/1, and TLR2/6 agonists. METHODS: Neutrophils from 10 healthy individuals were cultured alone or with autologous platelets. Neutrophils ± platelets were left unstimulated or were stimulated with 1 or 100 ng/mL lipopolysaccharide (LPS; a TLR4 agonist), Pam3CSK4 (a TLR2/1 agonist) and fibroblast-stimulating lipopeptide (FSL)-1 (a TLR2/6 agonist). Neutrophil activation and phagocytic activity were assessed by flow cytometry, and elastase and interleukin-8 secretion were assessed by ELISA. RESULTS: The addition of platelets attenuated neutrophil CD66b and CD11b expression in response to various doses of Pam3CSK4 and FSL-1. Furthermore, platelet co-culture was associated with higher CD62L expression (indicating reduced CD62L shedding) in response to these TLR agonists. Platelets also reduced elastase secretion in unstimulated cultures and in response to low-dose TLR stimulation. Conversely, platelet co-culture increased neutrophil phagocytosis in unstimulated cultures and in response to low-dose Pam3CSK4 and FSL-1. Platelets also increased IL-8 secretion in response to low-dose LPS. CONCLUSION: Platelets are complex immunomodulators that can attenuate some, and simultaneously augment other, neutrophil functions. This modulation can occur both in the absence and presence of TLR stimulation.

The effects of aspirin and ticagrelor on Toll-like receptor (TLR)-mediated platelet activation: results of a randomized, cross-over trial.

Platelet activation underlies the pathology of an acute myocardial infarction (AMI), and dual antiplatelet therapy (DAPT) is administered post-AMI to limit this activation. Platelets express Toll-like receptors (TLRs) 1, 2, and 4 and become potently activated in response to TLR2/1 and TLR4 stimulation. However, it is unknown whether antiplatelet agents can protect against platelet activation via these TLR pathways. This study aimed to determine the extent to which TLR-mediated platelet activation can be inhibited by currently used antiplatelet agents. Ten healthy subjects were enrolled into a single-blinded randomized cross-over trial. Subjects received either aspirin monotherapy or DAPT (aspirin in combination with ticagrelor) for 1 week, were washed out, and crossed over to the other drug regimen. Platelet activation was assessed in response to Pam3CSK4 (a TLR2/1 agonist) and lipopolysaccharide (LPS; a TLR4 agonist) at baseline and after each antiplatelet drug regimen. Platelet-surface expression of CD62p and PAC1 by flow cytometry was measured as markers of platelet activation. At baseline, expression of CD62p and PAC1 increased significantly in response to high-dose LPS and in a dose-dependent manner in response to Pam3CSK4. Aspirin monotherapy did not inhibit platelet activation in response to any TLR agonist tested. DAPT with aspirin and ticagrelor only modestly inhibited expression of both activation markers in response to high doses of Pam3CSK4 and LPS. However, incubation with these TLR agonists led to substantial platelet activation despite treatment with these anti-platelet agents. Platelet-TLR2/1 and platelet-TLR4 represent intact on-treatment platelet activation pathways, which may contribute to on-going platelet activation post-AMI.

Platelets regulate leucocyte responses to Toll-like receptor stimulation.

OBJECTIVES: Platelets are important regulators of vascular thrombosis and inflammation and are known to express Toll-like receptors (TLRs). Through TLRs, platelets mediate a number of responses by interacting with leucocytes. Here, we report the extent to which platelets modulate in vitro peripheral blood mononuclear cells (PBMCs) and granulocyte responses to TLR4, TLR2/1 and TLR2/6 stimulation in healthy subjects. METHODS: Peripheral blood mononuclear cells and granulocytes from 10 healthy volunteers were cultured alone or cocultured with platelets. Cultures were left unstimulated or stimulated with 1 or 100 ng mL-1 of either LPS (TLR4 agonist), Pam3CSK4 (TLR2/1 agonist) or fibroblast-stimulating lipopeptide (FSL)-1 (TLR2/6 agonist). Neutrophil activation (CD66b expression), monocyte activation (HLA-DR), granulocyte elastase production and PBMC cytokine and chemokine production were examined. RESULTS: Platelet coculture decreased neutrophil CD66b expression in response to LPS, Pam3CSK4 and FSL-1, and modestly decreased monocyte HLA-DR expression in response to low-dose LPS. Platelets reduced granulocyte elastase secretion in response to low doses of all TLR agonists tested. In response to LPS, platelet coculture reduced IL-6, tumor necrosis factor (TNF)-α and MIP-1ß production, and increased IL-10 production by PBMCs. In response to FSL-1, platelets increased IL-6, IL-10 and MIP-1ß production, but reduced TNF-α production. Platelet coculture did not alter PBMC cytokine/chemokine production in response to Pam3CSK4. CONCLUSION: This study challenges the notion that platelets act solely in a pro-inflammatory manner. Rather, platelets are complex immunomodulators that regulate leucocyte responses to TLR stimulation in a TLR agonist-specific manner. Platelets may modulate leucocyte responses to dampen inflammation, and this platelet effect may play an important role in reducing inflammation-mediated host damage.

Platelet Toll-like receptor (TLR) expression and TLR-mediated platelet activation in acute myocardial infarction.

Both platelets and Toll-like receptors (TLRs) contribute to acute myocardial infarction (AMI). Platelet activation can occur post-AMI and despite treatment with anti-platelet therapy. TLRs may represent an alternative platelet activation pathway, although the role of platelet-TLRs in AMI is poorly characterized. The aim of this study was to examine platelet-TLR expression and TLR-mediated platelet activation in healthy and AMI subjects. Here, we report that platelets from AMI patients exhibit upregulation of some, but not other, TLRs. When examined by western blotting, platelet-TLR1 and TLR4 were significantly upregulated in AMI subjects compared to healthy subjects (both p<0.05). Platelet-TLR2 was slightly, but non-significantly, upregulated in AMI patients and platelet-TLR6 expression did not change across cohorts. Platelets from both healthy and AMI subjects exhibited distinct activation patterns in response to various TLR agonists (0.1-100µg/mL), as determined by flow cytometry. Healthy and AMI platelets became dose-dependently and directly activated in response to Pam3CSK4, a TLR2/1 agonist, but were directly potently activated only in response to the highest dose (100µg) of lipopolysaccharide (LPS), a TLR4 agonist. Platelet activation in response to both of these agonists was similar across cohorts, despite treatment with anti-platelet therapy in the AMI cohort. At all doses used in this study, platelets were unable to become directly activated by FSL-1, a TLR2/6 agonist. We conclude that the platelet-TLR2/1 activation pathway is functional post-AMI and despite treatment with anti-platelet therapy. The platelet-TLR4 pathway appears to be less likely, and the platelet-TLR2/6 pathways unlikely, to contribute to post-AMI platelet activation.

Toll-like receptor 9 expression and activation in acute coronary syndrome patients on dual anti-platelet therapy.

INTRODUCTION: The Toll-like receptor 9 (TLR9) pathway can activate platelets but its role in acute coronary syndromes (ACS) is unknown. This study examined TLR9 expression and platelet activation in response to ODN2006, a TLR9 agonist, in healthy subjects and in ACS subjects treated with dual anti-platelet therapy (DAPT). MATERIALS AND METHODS: TLR9 expression was examined in both resting and thrombin receptor activator peptide (TRAP)-activated platelets (1 and 10µM) from healthy and ACS subjects by flow cytometry. In both cohorts, ODN2006-mediated platelet activation (5µM) was examined in whole blood (WB) and platelet-rich plasma (PRP) using cell-surface CD62p and CD63 expression by flow cytometry. RESULTS: Baseline TLR9 expression was significantly greater in ACS subjects compared to healthy subjects (p<0.01). Following TRAP activation, TLR9 expression increased dose-dependently in healthy subjects. However, no difference in TLR9 expression was seen in ACS platelets following TRAP activation. ODN2006 treatment resulted in significant increases in cell-surface expression of CD62p and CD63 in both WB (all p<0.001) and PRP (all p<0.001) in comparison to unstimulated platelets in healthy subjects. Despite DAPT, ODN2006 treatment produced significant increases in both activation markers in the ACS cohort across WB and PRP (all p<0.0001). Elevated baseline expression of TLR9 in ACS platelets may indicate increased sensitivity to TLR9 agonists and contribute to increased platelet activation in these patients. Furthermore, ODN2006 stimulation can activate platelets in ACS subjects despite treatment with DAPT. CONCLUSION: This study demonstrates TLR9 expression and activation to be of potential therapeutic importance in ASC patients.