IL-6 signalling biomarkers in hospitalised patients with moderate to severe SARS-CoV-2 infection in a single centre study in Sweden.

BACKGROUND: COVID-19 disease severity and need for intensive care has been associated with profound immune disturbances in which interleukin 6 (IL-6) is central. IL-6 signals through two pathways: classical IL-6 signalling with C-reactive protein (CRP) as a product is pivotal in the acute immune response against pathogens while IL-6 trans-signalling is involved in prolonged inflammation. We measured biomarkers of the IL-6 classical and trans-signalling pathways in patients with moderate or severe COVID-19 in the first wave of the COVID-19 pandemic. METHOD: In a longitudinal cohort study including patients admitted to Danderyd hospital, Stockholm, Sweden, with COVID-19 (n = 112), plasma IL-6 mirroring activity in both pathways, CRP as marker of classical signalling and the soluble IL-6 receptor (sIL-6R) and soluble glycoprotein 130 (sgp130) as markers of trans-signalling were analysed at baseline. Potential differences in biomarker levels between groups of moderate and severe COVID-19 defined by care level, level of respiratory support and one-month mortality was analysed, as was correlations between biomarkers. In addition, levels 4 months after hospital admission were compared to those at baseline. RESULTS: Levels of IL-6 and CRP were increased in severe COVID-19 whereas IL-6 trans-signalling markers (sIL-6R, sgp130) did not differ between the groups. CRP correlated positively with IL-6 in all patients while correlation with IL-6 could not be demonstrated for sIL-6R and sgp130 in either group. Levels of IL-6, CRP and sIL-6R were significantly decreased after 4 months whereas sgp130 levels increased. CONCLUSION: Classical signalling is the dominating IL-6 pathway in moderate-severe COVID-19.

Soluble angiotensin-converting enzyme 2 is transiently elevated in COVID-19 and correlates with specific inflammatory and endothelial markers.

The main entry receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is angiotensin-converting enzyme 2 (ACE2). SARS-CoV-2 interactions with ACE2 may increase ectodomain shedding but consequences for the renin-angiotensin system and pathology in Coronavirus disease 2019 (COVID-19) remain unclear. We measured soluble ACE2 (sACE2) and sACE levels by enzyme-linked immunosorbent assay in 114 hospital-treated COVID-19 patients compared with 10 healthy controls; follow-up samples after four months were analyzed for 58 patients. Associations between sACE2 respectively sACE and risk factors for severe COVID-19, outcome, and inflammatory markers were investigated. Levels of sACE2 were higher in COVID-19 patients than in healthy controls, median 5.0 (interquartile range 2.8-11.8) ng/ml versus 1.4 (1.1-1.6) ng/ml, p < .0001. sACE2 was higher in men than women but was not affected by other risk factors for severe COVID-19. sACE2 decreased to 2.3 (1.6-3.9) ng/ml at follow-up, p < .0001, but remained higher than in healthy controls, p = .012. sACE was marginally lower during COVID-19 compared with at follow-up, 57 (45-70) ng/ml versus 72 (52-87) ng/ml, p = .008. Levels of sACE2 and sACE did not differ depending on survival or disease severity. sACE2 during COVID-19 correlated with von Willebrand factor, factor VIII and D-dimer, while sACE correlated with interleukin 6, tumor necrosis factor α, and plasminogen activator inhibitor 1. Conclusions: sACE2 was transiently elevated in COVID-19, likely due to increased shedding from infected cells. sACE2 and sACE during COVID-19 differed in correlations with markers of inflammation and endothelial dysfunction, suggesting release from different cell types and/or vascular beds.

IL6 trans-signaling associates with ischemic stroke but not with atrial fibrillation.

BACKGROUND: Pro-inflammatory processes underlie ischemic stroke, albeit it is largely unknown if they selectively associate with the risk of atherothrombotic or cardioembolic ischemic stroke. Here we analyze whether pro-inflammatory interleukin (IL) 6 trans-signaling, is associated with the risk of ischemic stroke and underlying atrial fibrillation (AF). METHODS: During a 20-year follow-up, 203 incident ischemic strokes were recorded from national registers in the cohort of 60-year-old men and women from Stockholm (n = 4232). The risk of ischemic stroke associated with circulating IL6 trans-signaling, assessed by a ratio between the pro-inflammatory binary IL6:sIL6R complex and the inactive ternary IL6:sIL6R:sgp130 complex (B/T ratio), was estimated by Cox regression and expressed as hazard ratio (HR) with a 95% confidence interval (CI) in the presence or absence of AF. Risk estimates were adjusted for cardiovascular risk factors and anticoagulant treatment. In a secondary analysis, the association of IL6 trans-signaling with the risk of incident AF (n = 279) was analyzed. RESULTS: B/T ratio > median was associated with increased risk of ischemic stroke in study participants without AF (adjusted HR 1.49; 95% CI 1.08-2.06), while an association could not be demonstrated in the presence of AF. Moreover, the B/T ratio was not associated with the risk of AF (HR 0.96; 95% CI 0.75-1.24). CONCLUSIONS: Pro-inflammatory IL6 trans-signaling, estimated by the B/T ratio, is associated with ischemic stroke in individuals without AF. These findings suggest that the B/T ratio could be used to assess the risk of non-AF associated ischemic stroke.

Expression of Interleukin 6 signaling receptors in carotid atherosclerosis.

Interleukin (IL) 6 contributes to atherosclerotic plaque development through IL6 membrane-bound (IL6R and gp130) and soluble (sIL6R and sgp130) receptors. We investigated IL6 receptor expression in carotid plaques and its correlation with circulating IL6 and soluble receptor levels. Plasma samples and carotid plaques were obtained from 78 patients in the Biobank of Karolinska Endarterectomies study. IL6, sIL6R, and sgp130 were measured in plasma and IL6, IL6R, sIL6R, GP130, and sGP130-RAPS (sGP130) gene expression assessed in carotid plaques. Correlations between plaque IL6 signaling gene expression and plasma levels were determined by Spearman's correlation. Differences in plasma and gene expression levels between patients with (n = 53) and without (n = 25) a history of a cerebral event and statin-treated (n = 65) and non-treated (n = 11), were estimated by Kruskal-Wallis. IL6 and its receptors were all expressed in carotid plaques. There was a positive, borderline significant, moderate correlation between plasma IL6 and sIL6R and the respective gene expression levels (rho 0.23 and 0.22, both p = 0.05). IL6R expression was higher in patients with a history of a cerebrovascular event compared to those without (p = 0.007). Statin-treated had higher IL6R, sIL6R, and sGP130 expression levels and plasma sIL6R compared to non-treated patients (all p < 0.05). In conclusion, all components of the IL6 signaling pathways are expressed in carotid artery plaques and IL6 and sIL6R plasma levels correlate moderately with IL6 and sIL6R. Our data suggest that IL6 signaling in the circulation might mirror the system activity in the plaque, thus adding novel perspectives to the role of IL6 signaling in atherosclerosis.

Temporal trends in incidence, recurrence and prevalence of stroke in an era of ageing populations, a longitudinal study of the total Swedish population.

BACKGROUND: Stroke incidence has declined during the past decades. Yet, there is a concern that an ageing population together with improved survival after stroke will result in a raised proportion of the population who have experienced a stroke, as well as increasing incidence rate of recurrent strokes, and, absolute numbers of strokes. The objectives of this study were to investigate how the age specific incidence rates of recurrent strokes have developed in relation to the incidence rates of first strokes and how the postponement in age look like, and to see how the prevalence proportion of stroke as well as the absolute number of incident strokes has changed over time. METHODS: This study includes the total Swedish population born 1890-1954 living in Sweden from 1987. Stroke was identified through hospital admissions and deaths in national health registers (mandatory for all hospitals in Sweden). Age specific incidence rates were calculated for first, second, all recurrent, and all strokes for each calendar year between 1994 and 2014 for each age between 60 and 104 years. The proportion in the population with a history of stroke up to 7 years back in time was also calculated for different age groups and for different calendar years. RESULTS: Not only the incidence rate of first stroke but also of recurrent strokes have declined. The declines are evident in all ages up to 90 years of age, but not in ages above 90 years. Despite improved survival in stroke, the prevalence proportion has declined over the period and was around 3% in 2014 (somewhat higher for men than women). Even incident cases of stroke in absolute number has declined. CONCLUSIONS: Decreasing incidence rates of stroke have offset an increase in both absolute and relative numbers of stroke that otherwise would have taken place due to improved survival and an ageing population. The decline in stroke recurrence has been as strong as the decline in first strokes.

A novel biomarker of interleukin 6 activity and clinical and cognitive outcomes in depression.

BACKGROUND: Inflammatory cytokines like interleukin-6 (IL-6) are implicated in depression, but most studies have hitherto focused on circulating levels of IL-6 rather than its activity. IL-6 trans-signalling is thought to be responsible for most of the pathogenic effects of IL-6 and is implicated in autoimmune diseases like rheumatoid arthritis. We tested the association between a multi-protein-derived measure of IL-6 trans-signalling and clinical and cognitive outcomes in patients with depression. We hypothesised that this novel measure of IL-6 activity/bioavailability would be associated with clinical and cognitive measures previously reported to be associated with inflammation in depression. METHODS: Using data from 86 patients with International Classification of Diseases-10 diagnosis of depression, we calculated a ratio score representing IL-6 activity/bioavailability using serum IL-6, soluble IL-6 receptor (sIL-6R), and soluble glycoprotein 130 levels. We tested the relationship of this novel biomarker with 12 cytokines using correlation analyses and with cognitive and clinical measures using multivariable linear regression, following z-transformation of all immune exposures. RESULTS: The novel measure of IL-6 activity/bioavailability was correlated with IL-6 (r=0.42, P=0.03), C-reactive protein (CRP) (r=0.42, P=0.03), sIL-6R (r=0.91, P<0.01), and tumour necrosis factor alpha (r=0.43, P=0.03). The IL-6 activity/bioavailability measure was associated with higher somatic symptoms of depression (ß=1.09; 95% CI 0.30, 1.88; PFDR=0.01), fatigue (ß=4.34; 95% CI 1.26, 7.42; PFDR=0.03), depression severity (ß=3.06; 95% CI 0.71, 5.40; P=0.02), poorer quality of life (ß=-0.07; 95% CI -0.13, -0.01; PFDR=0.045), and decreased psychomotor speed (ß=-5.46; 95% CI -9.09, -1.84; PFDR=0.01),. There was little evidence of associations with reaction time, anhedonia, anxiety, emotional perception and recall, executive function, and sustained attention (Ps>0.05). The effect estimates for the associations of the novel measure with depression outcomes were comparable to those for individual immune proteins (i.e., IL-6, CRP, sIL-6R). CONCLUSION: A novel multi-protein-derived measure of IL-6 activity/bioavailability shows robust associations with various inflammation-related clinical and cognitive outcomes in depression and performs well in comparison to single inflammatory proteins. We need replication of these findings in other samples, experiments for mechanistic validity of this novel biomarker, and clinical studies to assess its usefulness as a marker of illness risk and prognosis.

Time trends in atrial fibrillation-related stroke during 2001-2020 in Sweden: a nationwide, observational study.

Background: Great efforts have been made to improve stroke prevention in atrial fibrillation (AF) patients. Meanwhile, incidence of AF is increasing, which may affect the share of AF-related stroke on all strokes. We aimed to examine the temporal trends in the incidence of AF-related ischemic stroke between 2001 and 2020, if it varied by use of novel oral anticoagulant drugs (NOAC), and if the relative risk of ischemic stroke associated with AF changed over time. Methods: Data from the total Swedish population aged ≥70 years during the period 2001-2020 were used. Annual incidence rate (IR) was calculated for overall and AF-related ischemic stroke which was defined as first-ever ischemic stroke with AF diagnosed up to 5 years before, on the same day, or within 2 months after the stroke event. Cox regression models were performed to examine if the hazard ratio (HR) between AF and stroke changed over time. Findings: While IR of ischemic strokes declined during 2001-2020, IR of AF-related ischemic stroke remained stable between 2001 and 2010 but showed a consistent decline between 2010 and 2020. The HR of ischemic stroke within 3 years from an AF diagnosis came down from 2.39 (95% confidence interval: 2.31-2.48) to 1.54 (1.48-1.61) over the study period, which was largely explained by a substantial increase in the use of NOAC among AF patients after 2012. Yet, by the end of 2020, 24% of all ischemic strokes had a preceding or concurrent AF diagnosis, which is slightly higher than in 2001. Interpretation: Even though both the absolute and relative risk of AF-related ischemic stroke declined over the past 20 years, every fourth ischemic stroke in 2020 still had a preceding or concurrent AF diagnosis. This represents a great potential for future gains in stroke prevention among AF patients. Funding: Swedish Research Council and Loo and Hans Osterman Foundation for Medical Research.

A machine learning based approach to identify carotid subclinical atherosclerosis endotypes.

AIMS: To define endotypes of carotid subclinical atherosclerosis. METHODS AND RESULTS: We integrated demographic, clinical, and molecular data (n = 124) with ultrasonographic carotid measurements from study participants in the IMPROVE cohort (n = 3340). We applied a neural network algorithm and hierarchical clustering to identify carotid atherosclerosis endotypes. A measure of carotid subclinical atherosclerosis, the c-IMTmean-max, was used to extract atherosclerosis-related features and SHapley Additive exPlanations (SHAP) to reveal endotypes. The association of endotypes with carotid ultrasonographic measurements at baseline, after 30 months, and with the 3-year atherosclerotic cardiovascular disease (ASCVD) risk was estimated by linear (ß, SE) and Cox [hazard ratio (HR), 95% confidence interval (CI)] regression models. Crude estimates were adjusted by common cardiovascular risk factors, and baseline ultrasonographic measures. Improvement in ASCVD risk prediction was evaluated by C-statistic and by net reclassification improvement with reference to SCORE2, c-IMTmean-max, and presence of carotid plaques. An ensemble stacking model was used to predict endotypes in an independent validation cohort, the PIVUS (n = 1061). We identified four endotypes able to differentiate carotid atherosclerosis risk profiles from mild (endotype 1) to severe (endotype 4). SHAP identified endotype-shared variables (age, biological sex, and systolic blood pressure) and endotype-specific biomarkers. In the IMPROVE, as compared to endotype 1, endotype 4 associated with the thickest c-IMT at baseline (ß, SE) 0.36 (0.014), the highest number of plaques 1.65 (0.075), the fastest c-IMT progression 0.06 (0.013), and the highest ASCVD risk (HR, 95% CI) (1.95, 1.18-3.23). Baseline and progression measures of carotid subclinical atherosclerosis and ASCVD risk were associated with the predicted endotypes in the PIVUS. Endotypes consistently improved measures of ASCVD risk discrimination and reclassification in both study populations. CONCLUSIONS: We report four replicable subclinical carotid atherosclerosis-endotypes associated with progression of atherosclerosis and ASCVD risk in two independent populations. Our approach based on endotypes can be applied for precision medicine in ASCVD prevention.

Circulating Biomarkers in Lower Extremity Artery Disease.

Lower extremity artery disease (LEAD), a chronic condition with disturbed lower extremity circulation due to narrowing of the arteries, is predominantly caused by atherosclerosis and is associated with the presence of cardiovascular risk factors and an increased risk of cardiovascular events. LEAD is prevalent among older individuals and predicted to rise with the ageing population. In progressive disease, the patient experiences symptoms of ischaemia when walking and, in advanced critical limb-threatening ischaemia, even at rest. However, LEAD is asymptomatic in most patients, delaying diagnosis and treatment. In this setting, circulating biomarkers may facilitate earlier diagnosis in selected individuals. This review provides a broad overview of the circulating biomarkers investigated to date in relation to LEAD and discusses their usefulness in clinical practice.

Interleukin 6 trans-signalling and the risk of future cardiovascular events in men and women.

OBJECTIVE: Pro-inflammatory interleukin 6 (IL6) trans-signalling is associated with increased risk of cardiovascular events (CVEs). Whether this association exists for both sexes is, however, uncertain. Hence, we analysed the risk of CVE associated with IL6 trans-signalling in men and women and investigated if potential interaction between IL6 trans-signalling and sex affects the risk. METHODS: In a prospective cohort of 60-year-old men and women without cardiovascular disease (men=2039, women=2193), subjects were followed for 20 years. To assess the IL6 trans-signalling activity, the proportion between the active binary and inactive ternary IL6 complexes, the binary/ternary ratio (B/T ratio), was estimated. CVE (myocardial infarction, angina pectoris and ischaemic stroke, n=629) risk was analysed with Cox regression, presented as HRs with 95% CIs. B/T ratio was dichotomised, with levels >median representing IL6 trans-signalling. Interaction was analysed on the additive scale and expressed as the synergy index (S). Analyses were adjusted for cardiovascular risk factors. RESULTS: B/T ratio >median was associated with increased CVE risk in men (HR 1.63; 95% CI 1.32 to 2.01), but not in women (HR 1.21; 95% CI 0.93 to 1.57). There was a significant synergistic interaction (S=1.98; 95% CI 1.15 to 3.42) between the B/T ratio and male sex, the combination increasing the risk by 88%. CONCLUSIONS: Our results suggest differential susceptibility to inflammation mediated by IL6 trans-signalling and subsequent CVE in men and women. The B/T ratio could be considered as a novel biomarker for cardiovascular risk in men, but not in women.

The predictive role of interleukin 6 trans-signalling in middle-aged men and women at low-intermediate risk of cardiovascular events.

BACKGROUND: Interleukin 6 trans-signalling is independently associated with the risk of cardiovascular events. The aim of this study was to investigate if interleukin 6 trans-signalling can identify individuals at risk for cardiovascular events (coronary artery disease and ischaemic stroke) among those at-low-intermediate risk. METHODS: In a cohort of 60-year-olds (n = 4232, incident cardiovascular events n = 525), interleukin 6 trans-signalling was estimated by a ratio between the pro-inflammatory interleukin 6: soluble interleukin 6 receptor binary receptor complex and the inactivated interleukin 6: soluble interleukin 6 receptor: sgp130 ternary complex (B/T ratio). Risk associated with B/T ratio >median was investigated in individuals with low-density lipoprotein cholesterol ≤4.0 (mmol/l) and in those at low-intermediate risk according to the Framingham risk score (FRS) using Cox regression and expressed as hazard ratio and 95% confidence interval. Difference in time to event (years; 95% confidence interval) was analysed with quantile regression. The interaction between low-density lipoprotein cholesterol and B/T ratio was estimated on the additive scale. Incremental discriminatory value of the B/T ratio if low-density lipoprotein cholesterol ≤4.0 was compared to that of the FRS and interleukin 6. RESULTS: B/T ratio >median was associated with increased cardiovascular event risk when low-density lipoprotein cholesterol ≤4.0 (hazard ratio 1.59; 95% confidence interval 1.24-2.05) or FRS ≤ 10%, >10-≤20% (hazard ratio 1.27; 95% confidence interval 1.00-1.61 and hazard ratio 1.78; 95% confidence interval 1.36-2.34, respectively). B/T ratio >median and low-density lipoprotein cholesterol ≤4.0 were associated with early cardiovascular events, particularly ischaemic stroke. No interaction was observed between low-density lipoprotein cholesterol and the B/T ratio, both factors increasing cardiovascular event risk by 60%. In the presence of low-density lipoprotein cholesterol ≤4.0, the B/T ratio slightly improved discrimination measures. CONCLUSIONS: Interleukin 6 trans-signalling increases cardiovascular event risk in middle-aged men and women otherwise classified at low-intermediate cardiovascular risk.

Interleukin 6 trans-signalling and risk of future cardiovascular events.

Aims: The pro-inflammatory response to interleukin 6 (IL6) trans-signalling in atherosclerosis is driven by the IL6 and soluble IL6 receptor (sIL6R) binary complex. The binary IL6:sIL6R complex is inactivated by sgp130 through the formation of the ternary IL6:sIL6R:sgp130 complex. The aim of this study was to investigate if IL6 trans-signalling, estimated by a ratio between the binary and ternary complexes, associates with the risk of future cardiovascular events (CVE) in a Swedish cohort of 60-year-old men and women (n = 4232). Methods and results: Binary and ternary complex levels expressed in nanomol/Litre were derived from serum concentrations of IL6, sIL6R, and sgp130. Cox regression models were used to assess the risk of CVE (myocardial infarction, angina pectoris, and ischaemic stroke, n = 525), expressed as hazard ratio (HR) with 95% confidence interval (CI), associated with increasing circulating levels of the three molecules and with the binary/ternary complex ratio. Estimates were adjusted for the common cardiovascular (CV) risk factors. To assess the level of IL6-trans-signalling, we estimated the binary/ternary complex ratio and then analysed the association with CVE risk. A ratio higher than the median, representing a relative excess of the active binary complex was associated with increased CVE risk (adjusted HR 1.44, 95% CI 1.21-1.72). Conclusion: The ratio between the functional moieties of IL6 trans-signalling, IL6:sIL6R, and IL6:sIL6R:sgp130, was associated with CVE risk indicating that it could be a promising marker of CV risk and possibly be used in selecting patients for anti-inflammatory therapy.