gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

Expression of IL17A in human atherosclerotic lesions is associated with increased inflammation and plaque vulnerability

Meeting Contribution

  • corresponding author C. Erbel - Department of Cardiology, University of Heidelberg, Germany
  • S. Wangler - Department of Cardiology, University of Heidelberg, Germany
  • T. J. Dengler - Department of Cardiology, University of Heidelberg, Germany
  • F. Bea - Department of Cardiology, University of Heidelberg, Germany
  • N. Wambsganss - Department of Cardiology, University of Heidelberg, Germany
  • M. Hakimi - Department of Vascular Surgery, University of Heidelberg, Germany
  • D. Böckler - Department of Vascular Surgery, University of Heidelberg, Germany
  • H. A. Katus - Department of Cardiology, University of Heidelberg, Germany
  • C. A. Gleissner - Department of Cardiology, University of Heidelberg, Germany

Deutsche Gesellschaft für Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Düsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf03

DOI: 10.3205/10dgaf03, URN: urn:nbn:de:0183-10dgaf035

Published: March 23, 2011

© 2011 Erbel et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

Introduction: A chronic (auto)immune response is the critical mechanism in atherosclerosis. Interleukin-17A is a pivotal effector cytokine, which modulates immune cell trafficking and initiates inflammation in (auto)immune and infectious diseases. However, expression of IL-17A in the context of human atherosclerosis has not been studied so far.

Methods: Carotid artery plaques were collected from 79 patients undergoing endarterectomy. Patients were grouped according to their symptomatic status (TIA, stroke), plaque morphology and medication. Quantitative RT-PCR was used to analyze tissue inflammation and immunohistochemistry to assess lesion morphology.

Results: Carotid plaques from patients with ischemic symptoms were characterized by a highly activated inflammatory milieu including accumulation of T cells (p=0.04) and expression of IL6 and VCAM1 (p=0.02, p=0.01). Expression of IL17A and its positive regulators IL21 and IL23 was present in atherosclerotic lesions, significantly upregulated in atheromas of symptomatic patients (p=0.03, p=0.004, p=0.03) and expression of IL17A and IL21 showed a strong correlation (p=0.002, r=0.52). Vulnerable plaque characteristics such as thrombotic or lipid-rich lesions were significantly associated with IL17A expression levels (p=0.03, p=0.02), but IL17A expression was not associated with decalcification, represented by Osteopontin. In addition, IL17A showed a marked negative correlation with the potent anti-inflammatory/atheroprotective cytokine IL10 (p=0.0006, r=–0.46). Furthermore, treatment with a HMG-CoA reductase inhibitor or acetylsalicylic acid showed reduced levels of IL21, IL23 and VCAM1, (all p<0.05), but did not influence IL17A.

Conclusion: The association of IL-17A with ischemic symptoms and vulnerable plaque characteristics suggests that the pro-inflammatory cytokine IL-17A may contribute to atherosclerosis und plaque instability.


Introduction

Complications of atherosclerosis, especially acute coronary syndromes, have been linked to rupture of vulnerable lesions, causing atherothrombosis and vessel occlusion [1]. Atherogenesis and the development of an vulnerable lesion is based on a chronic (auto)immune process including predominantly macrophages, T-lymphocytes and dendritic cells (DC) [2], [3]. Activated leukocytes contribute to plaque destabilization by secreting pro-inflammatory cytokines like IFN-γ, TNF-α and IL-1, various chemokines like MCP-1, and matrix metalloproteinases that degrade extracellular matrix proteins [2].

IL-17A is a recently discovered pro-inflammatory cytokine predominantly expressed by CD4+ T cells (Th17), but also by γ/δ T cells and NK cells [4]. IL-17A has various effects, including induction of expression of TNF-α, IL-1β and MCP-1 as well as adhesion molecules like ICAM-1 in inflammatory cells such as macrophages and endothelial cells [4]. The expansion of IL-17A secreting Th17 cells is specifically triggered by IL-23 and IL-21. IL-23 plays a pivotal role in organ-specific inflammatory autoimmune diseases [5] and is a potent growth and stabilization factor for the development of Th17 cells. IL-21, a member of the IL-2 cytokine family known to promote B-cell activation and differentiation during humoral immune responses, represents an alternative pathway to induce pro-inflammatory Th17 cells [6]. Previously, our group has demonstrated that the inhibition of IL-17A by anti-IL17A mAb treatment leads to reduced atherosclerotic lesion development in Apoe-/- mice [7]. In addition, the study shows that IL-17A induced various pro-inflammatory mediators and chemoattractants in monocytes/macrophages, vascular smooth muscle cells and dendritic cells, which play a crucial role in atherogenesis.

Major aims of the current study: First to test whether IL-17A is expressed in human lesions and if the expression correlates with ischemic symptoms and other clinical parameters; Second to test whether IL-17A expression is associated with other inflammatory molecules; Third to investigate whether IL-17A expression is correlated to morphological and biochemical indexes of plaque vulnerability such as thrombotic, lipid-rich or ruptured lesion as well as plaque decalcification, represented by osteopontin (OPN).


Methods

Patients

79 human carotid artery plaques from patients undergoing endarterectomy were studied. Clinical data were recorded for each patient, including age, sex, neurologic symptoms, cardiovascular risk factors, medication (HMG-CoA reductase inhibitor (statin) or acetylsalicylic acid (ASA)) as well as laboratory parameters. Patients with a stroke or a transitory ischemic attack (TIA) due to carotid stenosis within the last 6 weeks were defined as symptomatic. The grade of stenosis was assessed by duplex ultrasound or computed tomography. Approval for this study was given by the University Clinic Heidelberg Institutional Review Board, and appropriate informed consent was obtained from all patients included.

Plaque tissue processing and histological definition

Intraoperatively, carotid plaques were removed en bloc to preserve the plaque structure as described previously [3]. Specimens were cut along the length into equally sized pieces, 3 mm intervals. The tissue samples were shock frozen in liquid nitrogen in order to extract cDNA or were embedded in OCT for immunohistochemistry staining and stored at –80°C until use. For immunohistochemistry, specimens were serially sectioned (5 µm) and a section was stained every 75 µm for each staining. Frozen tissue sections were fixed with acetone, air dried and stained with hematoxylin for the assessment of the presence of calcification, a rupture of the fibrous cap or thrombotic formation. Oil red O staining was used to identify lipid-rich lesions.

Assessment of plaque composition was done in accordance to the American Heart Association classification and the oxford plaque study.

Risk factor definition

Clinical records were reviewed for all cases in order to determine the vascular risk factor profile such as hypertension, diabetes mellitus, hypercholesterolemia and hypertriglyceridemia.

Real-time polymerase chain reaction

Total cellular RNA was isolated from carotid plaques using the RNeasy kit (Qiagen) and the Boehringer cDNA kit (Roche Diagnostics) for reverse transcription according to manufacturer’s instructions. For quantitative PCR (qPCR) the Roche real-time PCR kit with SYBR Green (Roche Diagnostics) was used as described previously [7]. Primer sequences are given in the online resource (Table A). Data were analyzed on the basis of the relative expression method with the formula relative expression 2-ΔCT, where ΔCT is the difference in threshold cycle between the gene of interest and the housekeeping gene (β-actin) as a control.

Statistical analysis

Statistical analysis was performed using Prism software (Graphpad, USA). Continuous and categorical variables for the groups were compared with the nonparametric Mann-Whitney U test or Chisquare/Fisher exact test. A level of p<0.05 was considered as statistically significant.


Results

Patient characteristics

The patients studied had a mean age of 72.8±8 years and 61 (77%) patients were male. Among the 79 patients, 35 (44%) were asymptomatic while 44 (56%) had ischemic symptoms such as stroke (n=14) or TIA (n=30). Hypertension, hypercholesterolemia, hypertriglyceridemia and diabetes mellitus was observed in 55 (69%), 48 (61%), 32 (41%) and 30 (38%) patients respectively. Mean serum concentrations of LDL-cholesterol was 138±60.72 mg/dL, of triglycerides 194.50±133.42 mg/dL and of fasting glucose level 136.06±51.88 mg/dL. Indices of inflammation such as leukocytes and of C-reactive protein were 8.01± 2.13/nL and 8.74±11.63 mg/L (upper limit of normal: 5 mg/L). Among the 46 patients receiving statin therapy before surgery, 11 (23.9%) patients were on atorvastatin (22±8.7 mg/d), 11 (23.9%) patients were on simvastatin (23±10.7 mg/d), 21 (45.7%) patients were on pravastatin (32±11.5 mg/d) and three (6.5%) patients had fluvastatin (53.3±23.1 mg/d). Among the 79 patients, 7 received clopidogrel and 7 vitamin K antagonist (predominantly phenprocoumon), while 56 were treated with ASA.

Plaques from symptomatic patients are characterized by a prominent inflammatory environment

Expression of markers of atherogenic cell types was assessed in plaques from asymptomatic and symptomatic patients by qPCR. Specimens obtained from symptomatic patients were characterized by significantly higher expression of CD3E, the gene coding for the epsilon chain of the CD3 receptor on T cells, which is exclusively expressed in this cell type (p=0.03). IL6 expression, a pro-inflammatory cytokine mainly expressed by activated VSMC and macrophages, was significantly higher in plaques from symptomatic than asymptomatic patients (p=0.02). Similarly, expression of VCAM1, a marker of endothelial and monocyte/macrophage cell activation, was significantly higher in symptomatic patients (p=0.01). The expression levels of OPN (SPP1) (a marker of decalcification) were significantly higher in carotid atheroma from symptomatic patients (p<0.001). The pro-inflammatory cytokine LIGHT (TNFSF14), mainly expressed by activated platelets and T cells, was significantly higher expressed in carotid plaques of patients with clinical symptoms (p=0.01).

Symptomatic patients display increased expression of IL17A and related cytokines within atherosclerotic lesions

Using qPCR, IL17A expression was present in atherosclerotic lesions. Over 75% of the carotid plaque samples contained IL-17A cDNA transcripts. By grouping atheromas according to the symptomatic status of the patients IL17A showed only low expression levels in asymptomatic, but significantly higher levels in symptomatic patients (mean±S.D.-7.65±9.41 vs. 28.92±25.72, p=0.03). In atheromas of symptomatic patients a large percentage of IL17A expression levels were above median (or maximum) levels of the asymptomatic group. By investigating the expression of the important IL-17A inducers IL-21 and IL-23 both were also significantly higher in symptomatic compared to asymptomatic patients' plaques (p=0.004 and p=0.03 respectively). Evaluation of a possible association between IL-17A promoters and IL-17A demonstrated that expression of IL17A and IL21 showed a strong correlation (p=0.0002, r=0.52). By contrast, the levels of IL17A and IL23 were not significantly correlated.

IL17A expression correlates with plaque morphology and vulnerability

Thirty four plaques were further analyzed for histological lesion composition. Twenty-three out of 34 plaques showed significant calcification. Eleven out of 34 plaque samples included a lipid-rich lesion. Plaque rupture as well as thrombus material was found in 10 out of 34 tissue samples. To test whether the cDNA transcript levels of IL17A are associated with plaque vulnerability, expression of IL17A was correlated with plaque morphology including the presence of lipid-rich lesions, plaque rupture or a thrombotic formation. Very low levels of IL17A were found in plaques without lipid accumulation, but lipid-rich lesions contained significantly higher IL17A message levels (p=0.02). IL17A expression was low in plaques without a thrombus, but reached significantly higher levels in lesions containing thrombotic material (p=0.03). Interestingly, IL17A expression was not correlated with the presence of plaque rupture.

Decalcification of atherosclerotic lesions is an important step of plaque destabilization and OPN is a key regulator of this process. The current study supports the concept by showing that cDNA transcript levels of OPN (SPP1) were not only upregulated in atheromas of symptomatic patients, but also in ruptured lesions (p=0.01). In order to investigate a relation of IL-17A with (de)calcification, the study evaluated the expression levels of IL-17A in (non) calcified lesions. The data demonstrate that expression of IL17A showed a strong trend for increased expression in decalcified lesions (p=0.08). By investigating a possible association between IL-17A expression and decalcification cDNA transcript levels of IL17A showed no relation to OPN (SPP1) levels.

In order to evaluate the relation of IL17A with anti-inflammatory cascades, the levels of IL17A and IL10 were correlated. The expression level of IL17A showed a highly significant inverse correlation to IL10 in atherosclerotic lesions (p=0.0006, r=–0.45.

Effects of medication on IL17A expression and the inflammatory environment

Statin therapy led to significantly reduced expression levels of VCAM1, IL21, OPN (SPP1) and IL23 (p=0.03), but did not have any effect on CD3E, IL6 and IL17A in asymptomatic and symptomatic patients. ASA medication resulted in distinctly lower expression levels of VCAM1, IL21, OPN (SPP1), but not of IL6, IL17A and IL23.


Discussion

Inflammation plays a critical role in the progression of atherosclerosis and plaque vulnerability, eventually resulting in acute events [1], [2]. In the current study, we demonstrate that atherosclerotic plaques of symptomatic patients accumulate highly activated inflammatory cells which express various pro-inflammatory cytokines including IL17A. The expression levels of IL17A correlate with the levels of IL21, a promoter of IL-17A secretion. Higher IL17A expression was associated with characteristics of plaque vulnerability such as lipid-rich plaques or lesions with a thrombus formation. This is in line with the finding of inversely correlated mRNA expression levels of IL17A with the potent anti-inflammatory/atheroprotective cytokine IL10. Treatment with statins or ASA was associated with less inflammation as demonstrated by lower expression levels of CD3E, IL21, IL23 and VCAM1, but not of IL17A.

The crucial effector cytokine IL-17A, mainly expressed by CD4+ T cells, is a potent inducer of a pro-inflammatory milieu in various diseases by the induction of cytokines like IL-6, chemokines such as IL-8, MCP-1 and Groα as well as induction of adhesion molecules [4], [7]. These molecules are also important contributors to the progression of atherosclerosis [4], [7]. Moreover, IL-17A expression is known to be promoted by IL-21, released by natural killer and CD4+ T cells, as well as by IL-23, a cytokine related to IL-12 and secreted by macrophages and dendritic cells [5], [6]. The present study demonstrates that >75% of carotid endarterectomy samples contain cDNA transcripts of IL17A. In carotid artery lesions, accumulation of IL17A expression clearly correlated with the clinical status of the patient. Endarterectomy tissues from asymptomatic patients contained only low levels of IL17A cDNA transcripts, but were significantly higher in atheromas of symptomatic patients, which are characterized by a highly activated inflammatory milieu. In atheromas of symptomatic patients a large percentage of IL17A expression levels are above median levels of asymptomatic group. Furthermore, the expression of the important inducers of the IL-17A release IL21 and IL23 are in line with IL17A cDNA transcript levels resulting in the presence in atherosclerotic lesions and in an accumulation in atheromas of symptomatic patients. In addition, the present study demonstrates that the mRNA expression levels of IL17A and IL21 correlate in atherosclerotic lesions. These findings suggest that the IL17A cascade is a component of pro-inflammatory milieu in atherosclerotic lesions, which is upregulated in inflamed atheromas of symptomatic patients. In addition, tissue levels of IL-17A may represent a good discriminator of symptomatic plaques.

The morphological characteristics of a vulnerable plaque are a large lipid core, thrombus formation, thin fibrous cap, loss of collagen content as well as apoptosis and necrosis of various intramural cells such as macrophages and with a high risk for plaque rupture [8]. By investigating the relationship between plaque morphology and cytokine expression, we found that IL17A expression was associated with plaques containing a thrombotic formation or lipid-rich lesions. But IL17A was not upregulated in ruptured lesions. Thus, it can be suggested that IL17A expression is associated with plaque vulnerability. The finding of an inverse correlation of IL17A and IL10, an anti-inflammatory, atheroprotective cytokine, expression levels may underline the findings.

By contrast, calcification of lesions represents an element of biomechanical stability of atherosclerotic plaques. Thus decalcification, represented by OPN, in atherosclerotic lesions seems to be an important factor of plaque instability [9]. The current study supports the concept by showing that cDNA transcript levels of OPN (SPP1) were not only upregulated in atheromas of symptomatic patients, but also in ruptured lesions. By investigating the influence of cytokine expression with calcification, the study demonstrates that IL-17A expression levels were higher in decalcified (unstable) plaques. However, no correlation could be seen between the expression levels of IL17A and OPN (SPP1). Thus, IL-17A does not seem to influence characteristics of a stable lesion towards a vulnerable plaque by influencing the expression levels of OPN and thus the grade of calcification.

Treatment with statins and ASA has been reported to be effective for primary and secondary prevention of vascular diseases by affecting atherogenesis and stabilization of atherosclerotic lesions. Our study underlines these findings, showing that treatment with statins and/or ASA was associated with a modulation of the inflammatory response in atherosclerotic lesions. While both statin and ASA treatment were associated with reduced expression levels of IL21 (statins also IL23), neither affected the expression of IL17A. Interestingly, these findings were independent of the patient characteristics or their lipid profile. The fact that IL17A expression was not affected by medication may indicate that anti-inflammatory effects of statins and ASA are independent of the IL-17A pathway.

In conclusion, this study demonstrates the presence of IL17A in atherosclerotic lesions. The association of IL17A with clinical symptoms and lesion characteristics indicates a possible role of IL-17A in plaque vulnerability. The possible influence of IL-17A in atherosclerosis are strengthened by our findings of reduced atherosclerotic lesion progression and stabilization of the lesion by IL17A inhibition in Apoe-/- mice [7]. Based on a large percentage of IL17A expression levels above median (or maximum) levels of asymptomatic group IL-17A appears to be a sensitive markers of the “symptomatic” phenotype, more so than T cells, VCAM1 or IL6.


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