We recently showed that interleukin (IL)-6 inhibition by tocilizumab improves myocardial salvage in ST-elevation myocardial infarction (STEMI). However, the mechanisms for this effect are not clear.
...In this exploratory sub-study of the ASSAIL-MI trial, we examined leukocyte differential counts and their relation to myocardial salvage and peak troponin T (TnT) in STEMI patients randomised to tocilizumab (n = 101) or placebo (n = 98). We performed RNA-sequencing on whole blood (n = 40) and T cells (n = 20). B and T cell subpopulations were examined by flow cytometry (n = 69).
(i) STEMI patients had higher neutrophil counts at hospitalisation compared with stable angina patients. (ii) After percutaneous coronary intervention there was a gradual decline in neutrophils, which was significantly more pronounced in the tocilizumab group. (iii) The decrease in neutrophils in the tocilizumab group was associated with improved myocardial salvage and lower peak TnT. (iv) RNA-sequencing suggested that neutrophil function was also attenuated by tocilizumab. (v) B and T cell sub-populations changed only minimally after STEMI with minor effects of tocilizumab, supported as well by RNA-sequencing analyses of T cells. (vi) However, a low CD8+ count was associated with improved myocardial salvage in patients admitted to the hospital > 3 h after symptom onset.
Tocilizumab induced a rapid reduction in neutrophils and seemed to attenuate neutrophil function in STEMI patients potentially related to the beneficial effects of tocilizumab on myocardial salvage.
South-Eastern Norway Regional Health Authority (Nos. 2019067, 2017084), the Central Norway Regional Health Authority and Norwegian Research Council (No. 283867).
Background Interleukin 6 concentration is associated with myocardial injury, heart failure, and mortality after myocardial infarction. In the Norwegian tocilizumab non-ST-segment-elevation myocardial ...infarction trial, the first randomized trial of interleukin 6 blockade in myocardial infarction, concentration of both C-reactive protein and troponin T were reduced in the active treatment arm. In this follow-up study, an aptamer-based proteomic approach was employed to discover additional plasma proteins modulated by tocilizumab treatment to gain novel insights into the effects of this therapeutic approach. Methods and Results Plasma from percutaneous coronary intervention-treated patients, 24 in the active intervention and 24 in the placebo-control arm, drawn 48 hours postrandomization were randomly selected for analysis with the SOMAscan assay. Employing slow off-rate aptamers, the relative abundance of 1074 circulating proteins was measured. Proteins identified as being significantly different between groups were subsequently measured by enzyme immunoassay in the whole trial cohort (117 patients) at all time points (days 1-3 7 time points and 3 and 6 months). Five proteins identified by the SOMAscan assay, and subsequently confirmed by enzyme immunoassay, were significantly altered by tocilizumab administration. The acute-phase proteins lipopolysaccharide-binding protein, hepcidin, and insulin-like growth factor-binding protein 4 were all reduced during the hospitalization phase, as was the monocyte chemoattractant C-C motif chemokine ligand 23. Proteinase 3, released primarily from neutrophils, was significantly elevated. Conclusions Employing the SOMAscan aptamer-based proteomics platform, 5 proteins were newly identified that are modulated by interleukin 6 antagonism and may mediate the therapeutic effects of tocilizumab in non-ST-segment-elevation myocardial infarction.
Elevated interleukin-6 (IL-6) and complement activation are associated with detrimental effects of inflammation in coronary artery disease (CAD). The complement anaphylatoxins C5a and C3a interact ...with their receptors; the highly inflammatory C5aR1, and the C5aR2 and C3aR. We evaluated the effect of the IL-6 receptor (IL-6R)-antagonist tocilizumab on the expression of the anaphylatoxin receptors in whole blood from non-ST-elevation myocardial infarction (NSTEMI) patients. Separately, anaphylatoxin receptor expression in peripheral blood mononuclear cells (PBMC) from patients with different entities of CAD was investigated.
NSTEMI patients were randomized to one dose of tocilizumab (
= 28) or placebo (
= 32) and observed for 6 months. Whole blood samples drawn at inclusion, at day 2, 3 and after 6 months were used for mRNA isolation. Plasma was prepared for analysis of complement activation measured as sC5b-9 by ELISA. Furthermore, patients with different CAD entities comprising stable angina pectoris (SAP,
= 22), non-ST-elevation acute coronary syndrome (NSTE-ACS,
= 21) and ST-elevation myocardial infarction (STEMI,
= 20) were included. PBMC was isolated from blood samples obtained at admission to hospital and mRNA isolated. Anaphylatoxin-receptor-expression was analyzed with qPCR using mRNA from whole blood and PBMC, respectively.
Our main findings were (i) Tocilizumab decreased C5aR1 and C5aR2 mRNA expression significantly (
< 0.001) and substantially (>50%) at day 2 and 3, whereas C3aR expression was unaffected. (ii) Tocilizumab did not affect complement activation. (iii) In analyzes of different CAD entities, C5aR1 expression was significantly increased in all CAD subgroups compared to controls with the highest level in the STEMI patients (
< 0.001). For C5aR2 and C3aR the expression compared to controls were more moderate with increased expression of C5aR2 in the STEMI group (
< 0.05) and C3aR in the NSTE-ACS group (
< 0.05).
Expression of C5aR1 and C5aR2 in whole blood was significantly attenuated by IL-6R-inhibition in NSTEMI patients. These receptors were significantly upregulated in PBMC CAD patients with particularly high levels of C5aR1 in STEMI patients.
BackgroundTocilizumab improves myocardial salvage index (MSI) in patients with ST-elevation myocardial infarction (STEMI), but its mechanisms of action are unclear. Here, we explored how cytokines ...were affected by tocilizumab and their correlations with neutrophils, C-reactive protein (CRP), troponin T, MSI and infarct size.MethodsSTEMI patients were randomised to receive a single dose of 280 mg tocilizumab (n=101) or placebo (n=98) before percutaneous coronary intervention. Blood samples were collected before infusion of tocilizumab or placebo at baseline, during follow-up at 24–36, 72–168 hours, 3 and 6 months. 27 cytokines were analysed using a multiplex cytokine assay. Cardiac MRI was performed during hospitalisation and 6 months.ResultsRepeated measures analysis of variance showed significant (p<0.001) between-group difference in changes for IL-6, IL-8 and IL-1ra due to an increase in the tocilizumab group during hospitalisation. IL-6 and IL-8 correlated to neutrophils in the placebo group (r=0.73, 0.68, respectively), which was attenuated in the tocilizumab group (r=0.28, 0.27, respectively). A similar pattern was seen for MSI and IL-6 and IL-8 in the placebo group (r=−0.29, –0.25, respectively) in patients presenting ≤3 hours from symptom onset, which was attenuated in the tocilizumab group (r=−0.09,–0.14, respectively).ConclusionsTocilizumab increases IL-6, IL-8 and IL-1ra in STEMI. IL-6 and IL-8 show correlations to neutrophils/CRP and markers of cardiac injury in the placebo group that was attenuated in the tocilizumab group. This may suggest a beneficial effect of tocilizumab on the ischaemia-reperfusion injury in STEMI patients.Trial registration numberNCT03004703.
ObjectiveBeyond reducing inflammation and troponin T (TnT) release, the interleukin-6 receptor antagonist tocilizumab reduces neutrophil counts in patients with non-ST segment elevation myocardial ...infarction (NSTEMI). It is unclear if this is related to formation of neutrophil extracellular traps (NETs), carrying inflammatory and thrombotic properties.MethodsIn a placebo-controlled trial, 117 patients with NSTEMI were randomised to a single dose of tocilizumab (n=58) or placebo (n=59) before coronary angiography. The NETs related markers double-stranded DNA (dsDNA), myloperoxidase–DNA (MPO–DNA) and citrullinated histone 3 (H3Cit) were measured at five consecutive time points during hospitalisation (days 1–3).ResultsOur major findings were: (1) H3Cit levels were significantly higher in the tocilizumab compared with the placebo group at all time points (all p<0.05), and H3Cit area under the curve (AUC) was 2.3 fold higher in the tocilizumab compared with placebo group (p<0.0001). (2) MPO–DNA and dsDNA did not differ between the groups. (3) In both treatment arms, dsDNA AUC was associated with TnT AUC. (4) Neutrophil count AUC correlated inversely to H3Cit AUC (p=0.015) in the total population.ConclusionsIn patients with NSTEMI, treatment with tocilizumab is associated with increased circulating H3Cit levels, suggesting that tocilizumab enhances NETosis. Further studies should clarify whether NETosis is a relevant side effect of tocilizumab. Regardless of tocilizumab, dsDNA associated with TnT release, indicating a link between extracellular nuclear material and myocardial injury.
Prompt myocardial revascularization with percutaneous coronary intervention (PCI) reduces infarct size and improves outcomes in patients with ST-segment elevation myocardial infarction (STEMI). ...However, as much as 50% of the loss of viable myocardium may be attributed to the reperfusion injury and the associated inflammatory response.
This study sought to evaluate the effect of the interleukin-6 receptor inhibitor tocilizumab on myocardial salvage in acute STEMI.
The ASSAIL-MI trial was a randomized, double-blind, placebo-controlled trial conducted at 3 high-volume PCI centers in Norway. Patients admitted with STEMI within 6 h of symptom onset were eligible. Consenting patients were randomized in a 1:1 fashion to promptly receive a single infusion of 280 mg tocilizumab or placebo. The primary endpoint was the myocardial salvage index as measured by magnetic resonance imaging after 3 to 7 days.
We randomized 101 patients to tocilizumab and 98 patients to placebo. The myocardial salvage index was larger in the tocilizumab group than in the placebo group (adjusted between-group difference 5.6 95% confidence interval: 0.2 to 11.3 percentage points, p = 0.04). Microvascular obstruction was less extensive in the tocilizumab arm, but there was no significant difference in the final infarct size between the tocilizumab arm and the placebo arm (7.2% vs. 9.1% of myocardial volume, p = 0.08). Adverse events were evenly distributed across the treatment groups.
Tocilizumab increased myocardial salvage in patients with acute STEMI. (ASSessing the effect of Anti-IL-6 treatment in Myocardial Infarction ASSAIL-MI; NCT03004703)
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Interleukin-6 (IL-6) contributes to atherosclerotic plaque destabilization and is involved in myocardial injury during ischaemia-reperfusion. Interleukin-6 is therefore a potential therapeutic target ...in myocardial infarction (MI). We hypothesized that the IL-6 receptor antagonist tocilizumab would attenuate inflammation, and secondarily reduce troponin T (TnT) release in non-ST-elevation MI (NSTEMI).
In a two-centre, double-blind, placebo-controlled trial, 117 patients with NSTEMI were randomized at a median of 2 days after symptom onset to receive placebo (n = 59) or tocilizumab (n = 58), administered as a single dose prior to coronary angiography. High sensitivity (hs) C-reactive protein and hsTnT were measured at seven consecutive timepoints between Days 1 and 3. The area under the curve (AUC) for high-sensitivity C-reactive protein was the primary endpoint. The median AUC for high-sensitivity C-reactive protein during hospitalization was 2.1 times higher in the placebo than in the tocilizumab group (4.2 vs. 2.0 mg/L/h, P < 0.001). Also, the median AUC for hsTnT during hospitalization was 1.5 times higher in the placebo group compared with the tocilizumab group (234 vs. 159 ng/L/h, P = 0.007). The differences between the two treatment groups were observed mainly in (i) patients included ≤2 days from symptom onset and (ii) patients treated with percutaneous coronary intervention (PCI). No safety issues in the tocilizumab group were detected during 6 months of follow-up.
Tocilizumab attenuated the inflammatory response and primarily PCI-related TnT release in NSTEMI patients.
Genetic and experimental studies support a causal involvement of IL-6 (interleukin-6) signaling in atheroprogression. Although trials targeting IL-6 signaling are underway, any benefits must be ...balanced against an impaired host immune response. Dissecting the mechanisms that mediate the effects of IL-6 signaling on atherosclerosis could offer insights about novel drug targets with more specific effects.
Leveraging data from 522 681 individuals, we constructed a genetic instrument of 26 variants in the gene encoding the IL-6R (IL-6 receptor) that proxied for pharmacological IL-6R inhibition. Using Mendelian randomization, we assessed its effects on 3281 plasma proteins quantified with an aptamer-based assay in the INTERVAL cohort (n=3301). Using mediation Mendelian randomization, we explored proteomic mediators of the effects of genetically proxied IL-6 signaling on coronary artery disease, large artery atherosclerotic stroke, and peripheral artery disease. For significant mediators, we tested associations of their circulating levels with incident cardiovascular events in a population-based study (n=1704) and explored the histological, transcriptomic, and cellular phenotypes correlated with their expression levels in samples from human atherosclerotic lesions.
We found significant effects of genetically proxied IL-6 signaling on 70 circulating proteins involved in cytokine production/regulation and immune cell recruitment/differentiation, which correlated with the proteomic effects of pharmacological IL-6R inhibition in a clinical trial. Among the 70 significant proteins, genetically proxied circulating levels of CXCL10 (C-X-C motif chemokine ligand 10) were associated with risk of coronary artery disease, large artery atherosclerotic stroke, and peripheral artery disease, with up to 67% of the effects of genetically downregulated IL-6 signaling on these end points mediated by decreases in CXCL10. Higher midlife circulating CXCL10 levels were associated with a larger number of cardiovascular events over 20 years, whereas higher
expression in human atherosclerotic lesions correlated with a larger lipid core and a transcriptomic profile reflecting immune cell infiltration, adaptive immune system activation, and cytokine signaling.
Integrating multiomics data, we found a proteomic signature of IL-6 signaling activation and mediators of its effects on cardiovascular disease. Our analyses suggest the interferon-γ-inducible chemokine CXCL10 to be a potentially causal mediator for atherosclerosis in 3 vascular compartments and, as such, could serve as a promising drug target for atheroprotection.
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