Simvastatin inhibits the pro-inflammatory and pro-thrombotic effects of IL-17 and TNF-α on endothelial cells
- Department of Immunology and Rheumatology and the Immunogenomics and inflammation, Hospices Civils de Lyon, University of Lyon 1 mixed research unit, Hospital Edouard Herriot, Lyon, France
- Correspondence to Professor Pierre Miossec, Clinical Immunology and Rheumatology Unit, Hospices Civils de Lyon, University of Lyon 1 mixed research unit, EA 4130, Hospital Edouard Herriot, 69437 Lyon Cedex 03, France;
- Accepted 20 July 2012
- Published Online First 21 August 2012
Objectives Statins are widely used for primary and secondary prevention of coronary atherosclerosis. Simvastatin, besides its lipid lowering properties, has various anti-inflammatory effects. The aim of this study was to assess whether simvastatin modulates the vascular effects of interleukin (IL)-17, an emerging actor in atherosclerosis.
Methods The effect of simvastatin was assessed in human umbilical vein endothelial cells treated by IL-17 alone or combined with tumour necrosis factor (TNF)-α, with or without mevalonate, an inhibitor of simvastatin. Its effects on IL-17-induced cytokine or chemokine expression were assessed at the mRNA level using qRT-PCR or protein level by ELISA. Its effect on the IL-17-induced pro-thrombotic state and cell invasion was assessed using a lumi-aggregometer and a Matrigel assay, respectively.
Results Simvastatin decreased IL-17-induced IL-6, IL-8, CX3CL-1, RANTES mRNA and CX3CL-1 and CCL20 production. Simvastatin restored the level of IL-33 mRNA which was decreased by IL-17. It reduced the expression of IL-17-induced pro-thrombotic genes such as tissue factor. Simvastatin restored the level of platelet aggregation to normal levels. Simvastatin enhanced the expression of CD39 and thrombomodulin mRNA initially reduced by IL-17 and TNF-α combination. Simvastatin suppressed IL-17-induced endothelial cells invasion. All these effects were reversed by the addition of mevalonate. Finally, simvastatin had an additive effect with infliximab to decrease the effect of the combination of IL-17 and TNF-α on IL-6 mRNA expression. Similar conclusion was obtained with rosuvastatin.
Conclusions Statins inhibit the pro-inflammatory, thrombotic and pro-aggregation effects of IL-17 on vessels. This provides a new understanding of the beneficial effects of statins in blood vessel inflammation.
Statins are inhibitors of hydroxy-methylglutaryl (HMG) coenzyme A reductase used as cholesterol-lowering agents to treat hypercholesterolaemia. Furthermore, statins have also antithrombotic and antioxidative properties.1 ,2 Large-scale clinical trials have shown their benefits in primary and secondary prevention of atherosclerosis and its complications.3 ,4 Their immunosuppressive effect has recently been confirmed in various immune-mediated disease models.5 ,6 For instance, reduced rates of graft rejection were observed in statin-treated patients after heart transplantation.7 Furthermore, beneficial effects in autoimmune encephalomyelitis and multiple sclerosis have been recently suggested.8
Interleukin (IL)-17 (also known as IL-17A) is produced by Th17 cells.9 IL-17 is involved in inflammatory tissue destruction like in rheumatoid arthritis (RA). Furthermore, chronic inflammatory diseases such as RA are associated with enhanced cardiovascular risk and subclinical vascular disease. IL-17, considered pathogenic in RA, has recently shown to be involved in vascular dysfunction.10–12 Furthermore, IL-17 specifically when combined with tumour necrosis factor (TNF)-α induced in vitro pro-inflammatory cytokines in endothelial cells (EC), leading to an invasive and pro-thrombotic state.13–15
Several studies have described some of the mechanisms by which statins exert immunomodulatory effects beyond their cholesterol-lowering effects. They alter the functions of T cells and antigen-presenting cells with inhibition of pro-inflammatory mediator production such as TNF-α, IL-10, IL-6, IL-8, RANTES and MCP1 with decreased expression of class II major histocompatibility complex.16 ,17 Simvastatin inhibits EC activation induced by TNF-α and IL-1.18 ,19 In addition to the statin-mediated effect on the production of cytokines regulating Th17 cell differentiation, simvastatin directly inhibits IL-17 production in CD4 cells.20
The aim of our study was to extend our recent results14 and to investigate the effects of simvastatin on EC treated with IL-17 alone and in combination with TNF-α. Here, we show that simvastatin, the most commonly used statin drug inhibits the pro-inflammatory and pro-thrombotic phenotype effects of IL-17 and TNF-α on EC. Furthermore, it enhances the effects of anti-TNF inhibition.
Material and methods
Human umbilical vein endothelial cells (HUVEC) were collected from umbilical cords by collagenase perfusion of umbilical veins. The cells were maintained in endothelial cell basal medium (EBM2) with gentamycin, amphotericin-B and 10% foetal calf serum (all from Lonza, Cologne, Germany). A total of 500 000 confluent EC were treated or not with IL-17 (100 ng/ml), or TNF-α (1 ng/ml) or their combination for 6 h for mRNA studies and 24 h for protein quantification and functional assay. These concentrations were determined after dose course experiments (data not shown). TNF-α was added 2 h after pretreatment with IL-17. These conditions were selected because IL-17 has a priming effect on TNF-α, leading to the synergistic effect of their combination.21 All cells used in this study were between passages 3 and 6. All human materials were obtained according to the recommendations of the hospital's ethics committee.
Cytokine and reagents
Human recombinant TNF-α and IL-17A were purchased from R&D systems (San Diego, California, USA). Simvastatin, rosuvastatin and mevalonate were obtained from Sigma (St Louis, Missouri, USA) and used as described.22 CCL20 and CX3CL-1 levels were quantified in supernatants by ELISA (eBioscience; Diaclone Minneapolis, Minnesota, USA, and R&D Systems, respectively).
To the effect of simvastatin or rosuvastatin on cell viability, a trypan blue dye exclusion test was performed. Simvastatin and rosuvastatin at the indicated concentration were added to 70%–80% confluent HUVECs in a Petri dish, and incubated for up to 24 h. After incubation, the cells were detached with trypsin, and suspended in 1 ml of EGM-2 containing bovine serum albumin. Next, trypan blue solution (Gibco, Grand Island, New York, USA) was added to the cell suspension at a final concentration of 0.2% and incubated for 1 min. The trypan blue positive cells were counted as dead cells.
mRNA purification and quantitative real-time reverse transcriptase-PCR
Total RNA was isolated using RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA was obtained from cells 6 h after treatment with IL-17 and/or TNF-α. The procedures and conditions for real-time reverse transcriptase-PCR using the QuantiTect SYBR Green PCR kit (Roche, Meylan, France) have been described previously.23 Briefly, total RNA was denatured by incubating for 5 min at 65°C with 4 μM of oligo (dT) primer and then reverse transcribed by using in a final concentration of 0.5 mM dNTP, 40 μl RNase OUT, 0.01 M dithiothreitol and 10 U/μ of ThermoScript (Saint Aubin, France) reverse transcriptase. Reverse transcription was performed by incubation at 50 °C for 60 min followed by 85 °C for 5 min. The cDNA obtained was diluted 1 in 10 with distilled water and 10 μl used for amplification. Primers (list of primers is available in online supplementary material) were designed using the Primer Express software package (Applied Bio systems, Foster City, California, USA) and obtained from LC search Biotech (Ebersberg, Germany) or Eurogentec (Liege, Belgium). Gene expression was normalised with respect to the endogenous housekeeping control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative expression differences of respective genes were calculated using the comparative threshold cycle method as described by the manufacturer. mRNA expression of target genes was normalised with GAPDH mRNA expression and data are expressed as the fold induction compared with untreated controls.
Platelet aggregation was studied in platelet rich plasma (PRP) containing 250.106 platelets/ml by Born's method with a lumi-aggregometer (ChronoLog Corporation, Lille, France). PRP was preincubated at 37°C in an aggregometer cuvette. Platelet activation was started by addition of supernatants of EC stimulated or not by cytokines, and treated or not by simvastatin or mevalonate or their combination. Addition of ADP served as positive control and platelet aggregation was monitored and quantified by the increase of light transmission.
The effect of IL-17A alone or combined with TNF-α, with or without simvastatin on the invasiveness of HUVEC, was examined using the BD Bio-Coat Matrigel invasion assay system (BD Biosciences, Bedford, Massachusetts, USA) according to the manufacturer's instructions. VEGF (10 ng/ml) was used as chemo attractant. HUVEC (5×104 cells) were suspended in a medium containing 2% foetal calf serum and seeded into the Matrigel precoated transwell chambers. The transwell chambers were then placed into 24-well plates, in medium with or without cytokines. After 16 h, the upper surface of the transwell chambers was wiped with a cotton swab. Transwell filter was fixed in 1% gluteraldehyde for 15 min. Following fixation, the filters were stained using Hoechst solution before washing. Representative fields were captured digitally and cells in five random high-powered fields for each well were counted in order to assess the average number of migrating cells. Each condition was assessed in triplicate.
Data are expressed as mean and SEM. Means were compared by using the Mann–Whitney U test after Kruskal–Wallis and ANOVA. A p value <0.05 was considered significant. The statistical analysis was performed using SPSS for windows 7 (Chicago, Illinois, USA).
Simvastatin reduces IL-17-induced pro-inflammatory cytokines and chemokines in HUVEC
As first shown for synoviocytes, IL-17 and TNF-α induced IL-8 and IL-6 with a synergistic effect in EC.21 Simvastatin reduced IL-6 mRNA expression in a dose related fashion. In this experiment, IL-6 mRNA expression after activation increased by 2.5-fold (figure 1A). Simvastatin reduced IL-6 mRNA expression by 75% at 100 with a full inhibition at 400 ng/ml. Simvastatin effect on IL-6 mRNA expression was completely reversed by mevalonate (500 µM) (figure 1B). Combination of TNF-α with IL-17 had a massive and synergistic effect on IL-8 mRNA expression. Simvastatin inhibited completely this IL-8 induction. This effect was also inhibited by mevalonate addition (figure 1C).
Since IL-33 can reduce the development of atherosclerosis and IL-17 was shown to inhibit the expression of IL-33, we assessed whether simvastatin affected the effects of IL-17 on IL-33 mRNA expression.24 Simvastatin enhanced the IL-33 mRNA expression in resting cells by twofold and inhibited the effect of IL-17 on IL-33 mRNA expression. Mevalonate restored the inhibitory effect of IL-17 on IL-33 mRNA expression (figure 1D).
To further examine whether simvastatin affected the expression of chemokines other than IL-8, the expression of RANTES, CCL20 and CX3CL-1, all involved in atherosclerosis and produced by EC, was tested in its presence. Simvastatin reduced the cytokine-stimulated gene expression of RANTES and CX3CL-1 by 66% (p=0.0025) and 63% (p=0.0015), respectively (figure 2A,B). Furthermore, simvastatin potently reduced the production of CX3CL-1 and CCL20 in EC (figure 2C,D). All these inhibitions were completely reversed by the addition of mevalonate.
To extend the effects observed with simvastatin to other statins, similar experiments were performed with rosuvastatin. Similar effects were obtained as shown for the inhibition of IL-6 production measured by ELISA, although rosuvastatin was possibly more potent (50% inhibition with 100 ng/ml rosuvastatin vs 400 ng/ml simvastatin (data not shown)). In addition, no toxic effect on cell survival was observed with the two molecules (see online supplementary figure S1).
Simvastatin reduces the effects of pro-inflammatory cytokines on genes involved in thrombosis
After EC incubation with the TNF-α and IL-17 combination, tissue factor mRNA expression was enhanced with a clear synergistic effect. While IL-17 and TNF-α alone had no effect, the level was increased by 13-fold compared with resting cells. This effect was completely inhibited by simvastatin and restored by the addition of mevalonate (figure 3A). Among the genes involved in the antiaggregation phenotype of EC, CD39 is an ATPDase known to be downregulated in EC by inflammation.25 Simvastatin enhanced the expression of CD39 mRNA expression by 30% compared with resting cells. CD39 mRNA expression was reduced only by the combination of IL-17 and TNF-α. Simvastatin restored CD39 mRNA expression to control levels. This effect of simvastatin was also inhibited by mevalonate addition (figure 3B). Among the anticoagulant genes, thrombomodulin is predominantly synthesised by EC and inhibits coagulation and fibrinolysis. It functions as a cell surface receptor and is an essential cofactor for active thrombin, which in turn activates protein C, a powerful anticoagulant.26 Combination of IL-17 and TNF-α reduced the expression of thrombomodulin, and simvastatin restored that level almost to control levels. This effect was completely reversed by mevalonate addition (figure 3C).
Simvastatin controls the effects of IL-17 on platelet aggregation
As mentioned above, EC are known to have antithrombotic properties with inhibitory activity against platelet adhesion and aggregation. Using a lumi-aggregometer, the antiplatelet aggregating activity of EC was evaluated. Aliquots of 300 μl of cytokine-treated EC supernatants, supplemented with ADP and ATP, were added to 1 ml of PRP and aggregation was measured. The same experiments were performed with supernatants of EC treated with simvastatin alone or with mevalonate.
IL-17 and TNF-α alone reduced the antiplatelet aggregating activity of EC and induced platelet aggregation from 25% to 45% (p=0.03). Their combination increased platelet aggregation from 25% to 50%. Treatment with simvastatin decreased platelet aggregation from 50% to 30% (p=0.04). This inhibitory effect was more significant for platelet aggregation induced by the combination of IL-17 and TNF-α (p=0.001). Treatment with mevalonate restored the effect of cytokines and led to an increase of platelet aggregation (figure 4).
Simvastatin inhibits the effect of IL-17 combined with TNF-α on EC invasion
To evaluate the effect of simvastatin on EC invasion, an invasion study was performed using the Matrigel invasion chamber system. IL-17 and TNF-α alone have no clear effect but their combination had a positive effect to promote EC invasion (from 5±1 to 24±3×102 cells per field, p=0.02). Simvastatin reduced the level of cell invasion from a mean of 24±3 to 10±1×102 cells per field (p=0.03, figure 5).
Simvastatin enhances the effects of infliximab on IL-6 production induced by IL-17 combined with TNF-α
TNF-α antagonists were suggested as a therapeutic strategy to attenuate the cardiovascular risk in RA patients, but studies have yielded conflicting results.27 If IL-17 and TNF-α combination is synergistic to activate EC, adding simvastatin to the treatment regimen of RA patients could lead to a more potent reduction of the vascular risk than blocking TNF-α alone. To assess the effect of the combination of infliximab with simvastatin, the level of IL-6 mRNA was quantified after treatment with IL-17 combined with TNF-α.
This combination induced an increase of IL-6 mRNA in EC in a synergistic manner. Simvastatin inhibited the expression of IL-6 mRNA induced by IL-17 combined with TNF-α. Infliximab had a lower effect but the addition of simvastatin led to a complete inhibition of IL-6 mRNA expression as compared with infliximab alone in a dose dependent fashion (p=0.03, figure 6A). Dose course experiments have confirmed these results and have shown that a low dose of simvastatin has a potent inhibitory effect when combined with a low dose of infliximab. Similar experiments were performed with rosuvastatin which showed a synergistic effect when combined with infliximab. A control antibody had no effect (see online supplementary figure S2).
IL-17 has been recently implicated in the pathophysiology of cardiovascular diseases.28 In this study, we showed that simvastatin as well as rosuvastatin reduced the expression of pro-inflammatory cytokines and chemokines in EC stimulated by IL-17 alone or combined with TNF-α. Simvastatin inhibited the expression of tissue factor and restored the level of CD39 and thrombomodulin mRNA, which are two natural inhibitors of platelet aggregation localised at the EC surface. Furthermore, simvastatin reduced the effect of IL-17 and TNF-α combination on platelet aggregation. All these effects were reversed by mevalonate, indicating the specificity of these effects on HMG coenzyme A reductase inhibition. Finally, simvastatin had an additive effect with infliximab to reduce the expression of inflammatory cytokines such as IL-6. We demonstrate that simvastatin had opposite effects on cytokines involved in EC biology: it reduced the expression of IL-6, a key cytokine involved in vascular inflammation, but enhanced that of IL-33, with a vascular protective effect.24
Regarding chemokines, our results extend the results already obtained with cytokines other than IL-17.29 ,30 CX3CL1 or fraktalkine acts as an adhesion protein promoting the retention of monocytes and T cells in the atherosclerotic plaque. This chemokine and its receptor CX3CR1 are involved in blood vessel inflammation, as shown in mouse models of atherosclerosis and in vasculitis.31 The effects of simvastatin on CX3CL-1 mRNA expression and its production were inhibited by mevalonate indicating a specific effect on HMG coenzyme A reductase. Furthermore, simvastatin modulates the effects of IL-17 on RANTES expression and CCL-20. These two chemokines are clearly involved in atherosclerosis as recently shown in vivo and in vitro.32 ,33
We used the combination of IL-17 and TNF-α because as shown for RANTES, such a combination is often synergistic.12 The cooperation between these two cytokines creates a pro-inflammatory microenvironment promoting the development of atherosclerosis. In these studies, it is critical to use a low concentration of TNF-α. Indeed, opposite results have been observed with a higher dose of TNF-α (dose of TNF-α, usually 10–20 ng/ml).34 The fact that simvastatin reduces the effects of this cytokine combination could explain why this drug is effective during low-grade inflammation.4 The cooperation between both cytokines could explain that the effects of TNF inhibitors on the cardiovascular risk remain controversial. For instance, such treatment reduced the prevalence of myocardial infarction in the British society for rheumatology biologics register in responders, while it did not change the vascular events incidence in the non-responders.35
Using a functional assay, we show that simvastatin restored the antithrombotic phenotype of EC.15 One effect is the restoration of the expression of CD39, a natural antiaggregation molecule on EC and of thrombomodulin, a natural anticoagulant. This hypothesis was confirmed for the effect of thrombin on CD39 activities and its modulation by simvastatin.36 As expected, simvastatin decreased tissue factor mRNA expression, a procoagulant gene induced by IL-17 combined with TNF-α.36
Finally, IL-17 and TNF-α act in synergy to promote EC invasion. The increase of cell invasion means the ability to produce metalloproteases, able to destroy the basal membrane, as observed in atherosclerosis and cancer. In our study, simvastatin reduced cytokine-induced EC invasion, as already shown for tumour cells and myocardial fibroblasts.37 ,38
Cardiovascular events remain the main cause of mortality in inflammatory diseases such as RA.39 Among the complex cytokine network involved in RA, TNF-α and now IL-17 are seen as key actors.9 Many authors have suggested the use of statins in all RA patients,40 but this remains controversial. In addition, it is unclear if TNF-α blockers have truly a protective effect.41 Moreover, IL-17 has recently been described as a marker of vascular risk in RA patients.12 Our results suggest that simvastatin could have an additive effect when combined with infliximab. These results should be validated in clinical studies, but our data give another reason to prescribe statins in RA patients, including patients treated with biologics. It could be an indirect method to reduce the vascular effect of IL-17 and other cytokines on EC consequences of persistent low-grade inflammation. The dose of infliximab used here corresponds to the serum concentration observed between two infusions, meaning that statins could have an additive effect to counteract the EC activation on the long term.42
These results indicate that simvastatin inhibits the pro-inflammatory, thrombotic and pro-aggregation effects of IL-17 on vessels. They provide a new understanding of the beneficial effects of statins in blood vessel inflammation.
Contributors AH and PM designed the research, analysed data and wrote the paper. VLN and FL performed the research and analysed data.
Funding This work was supported by the Hospices Civils de Lyon and a Merieux research grant. Arnaud Hot was supported by a grant from the French society of internal medicine (SNFMI). Pr Miossec is a senior member of the Institut Universitaire de France (IUF). There are no affiliations with any organization or entity which have a direct financial or personal interest in the subject matter or materials discussed in this article.
Competing interests None.
Provenance and peer review Commissioned; externally peer reviewed.