Interleukin 6 trans-signalling : cardiovascular risk marker or therapeutical target?

Abstract

Background: Interleukin (IL) 6 signals in two pathways. In classical signalling, essential in antimicrobial defence and tissue regeneration, IL6 binds the membrane-bound IL6 receptor (IL6R) which associates with the signal transducing receptor gp130. The pro-inflammatory effects of IL6 on the other hand, are governed by IL6 trans-signalling via the binary complex of IL6 and the soluble IL6R (sIL6R) through gp130 binding. The binary IL6:sIL6R complex is neutralised by the soluble gp130 (sgp130) thereby preventing IL6 trans-signalling. IL6 trans-signalling is associated with chronic inflammatory conditions.

Overarching aim: To analyse the association of IL6 trans-signalling, estimated by a ratio between the active binary IL6:sIL6R complex and the inactive ternary IL6:sIL6R:sgp130 complex (the binary/ternary complex ratio [B/T ratio]), with the risk of cardiovascular event (CVE) and investigate the presence of IL6 signalling in manifest atherosclerosis.

Methods and results: Study I. In a prospective cohort of 60-year-olds (60YO) without prevalent cardiovascular disease (CVD) (n=3645), the binary IL6:sIL6R and ternary IL6:sIL6R:sgp130 complex levels, expressed in nanomole/litre, were derived from baseline serum concentrations of IL6, sIL6R, and sgp130. IL6 trans-signalling was estimated by a ratio between the pro-inflammatory binary complex and the inactivated ternary complex, the B/T ratio. Cox regression was used to assess the risk of CVE (myocardial infarction, angina pectoris and ischaemic stroke), expressed as hazard ratio (HR) with 95% confidence interval (CI), associated with increasing circulating levels of IL6, sIL6R, sgp130 and with the B/T ratio, the latter analysed both as a continuous variable and dichotomised at the median. Estimates were adjusted for the common cardiovascular risk factors. The discriminatory ability of the B/T ratio as predictor was assessed by the area under the curve (AUC) and net reclassification index (NRI) in relation to the Framingham risk score (FRS) and IL6. The B/T ratio >median associates with increased CVE risk (adjusted HR 1.44, 95% CI 1.21–1.72). The prediction of CVE improved by adding the B/T ratio to the FRS and IL6 and 10% of subjects were re-classified. Study II. In the 60YO cohort, CVE risk associated with B/T ratio >median was investigated in subjects with low-intermediate cardiovascular risk defined by LDL ≤ / >4.0 mmol/L or according to the FRS. The difference in time to event (years; 95% CI) was analysed with quantile regression. In secondary analyses, risk of coronary and cerebrovascular events and time to event was analysed. Biological interaction between LDL and B/T ratio was estimated on the additive scale and the incremental discriminatory value of the B/T ratio with FRS and IL6 was compared in subjects with LDL≤ and >4.0 mmol/L. B/T ratio was associated with an increased risk of CVE primarily the LDL≤4.0 group (adjusted HR 1.59; 95% CI 1.24-2.05) but also in FRS <20% 10-year risk. The highest risk and earliest events were seen for ischemic stroke. No interaction between LDL and the B/T ratio was seen and the B/T ratio improved prediction in the LDL ≤4.0 group. Study III. Carotid artery plaques were obtained during endarterectomy in patients with high-grade carotid artery stenosis in the Biobank of Karolinska Endarterectomies (BiKE) study. Oligoprimers were designed to selectively amplify IL6R, sIL6R, GP130 and sGP130 genes. Using cDNA reverse transcribed from RNA extracted from plaques quantitative real time-PCR was performed and the relative difference in expression between groups was estimated using the ∆CT method (n=78). Correlations between plaque gene expression and plasma levels were tested using Spearman’s correlation coefficient. Gene expression of IL6, IL6R, sIL6R, GP130 and sGP130 were detected in all plaques. A pattern of differential plaque expression depending on disease severity was seen as well as a trend towards positive correlations between IL6 and sIL6R plaque expression and corresponding protein levels in the circulation. Study IV. In the 60YO cohort, risk of incident ischemic stroke associated with the B/T ratio >median was analysed using Cox regression and stratified by prevalent or incident atrial fibrillation (AF). In secondary analyses, the risk of first-time diagnosis of AF associated with the B/T ratio >median was analysed. The B/T ratio was associated with ischemic stroke risk only in subjects without AF (adjusted HR 1.49; 95% CI 1.08-2.06). In addition, no association between the B/T ratio and risk of first-ever AF (HR 0.96; 95% CI 0.75-1.24) was seen albeit an indication of an association with IL6.

Conclusions: Pro-inflammatory IL6 trans-signalling, estimated by B/T ratio>median mirroring a relative excess of the binary complex (IL6:sIL6R) in relation to the inactive ternary complex (IL6:sIL6R:sgp130), is associated with an increased risk of future CVE in subjects without prevalent CVD, primarily in individuals at low-intermediate risk of CVE. IL6 signalling is present in carotid artery plaques and the B/T ratio is associated with an increased risk of atherothrombotic ischemic stroke and early stroke events albeit no association was established for ischemic stroke in relation to AF or for AF per se.