Toll-Like Receptor 4 Mediates Endothelial Cell Activation Through NF-κB but Is Not Associated with Endothelial Dysfunction in Patients with Rheumatoid Arthritis

Toll-Like Receptor 4 Mediates Endothelial Cell Activation Through NF-κB but Is Not Associated with Endothelial Dysfunction in Patients with Rheumatoid Arthritis

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  Toll-Like Receptor 4 Mediates Endothelial Cell ActivationThrough NF- k B but Is Not Associated with EndothelialDysfunction in Patients with Rheumatoid Arthritis Rossella Menghini 1 , Umberto Campia 2 * , Manfredi Tesauro 1 , Arianna Marino 1 , Valentina Rovella 1 ,Giuseppe Rodia 1 , Francesca Schinzari 3 , Barbara Tolusso 4 , Nicola di Daniele 1 , Massimo Federici 1,5 ,Angelo Zoli 4 , Gianfranco Ferraccioli 4 , Carmine Cardillo 3 1 Department of System Medicine, University of Tor Vergata, Rome, Italy,  2 Division of Cardiology, MedStar Heart Institute, Washington, DC, United States of America, 3 Department of Internal Medicine, Catholic University Medical School, Rome, Italy,  4 Department of Rheumatology, Catholic University Medical School, Rome, Italy, 5 Center for Atherosclerosis, Policlinico Tor Vergata, Rome, Italy Abstract Objective:   To investigate the effects of TLR4 antagonism on human endothelial cells activation and cytokine expression,and whether the Asp299Gly TLR4 polymorphism is associated with better endothelial function in patients with rheumatoidarthritis (RA). Methods:   Human aortic endothelial cells (HAECs) were treated with lipopolysaccharide (LPS), OxPAPC, and free fatty acids(FFA) at baseline and after incubation with the TLR4 antagonist eritoran (E5564). Cytokine expression was assessed byquantitative real-time PCR. In vivo endothelial function was assessed as brachial artery flow-mediated dilation (FMD) in RApatients with the wild type gene (aa) and with the Asp299Gly TLR4 polymorphic variant (ag). Results:   In HAEC, TLR4 antagonism with eritoran inhibited LPS-induced mRNA expression of IL-6, IL-8, TNF a , CCL-2, VCAMand ICAM (P , 0.05 for all) and inhibited Ox-PAPC-induced mRNA expression of IL-8 (P , 0.05) and IL-6, albeit not to astatistically significant level (p=0.07). In contrast, eritoran did not affect FFA-induced mRNA expression of IL-6 (P . 0.05). In30 patients with RA (15 with the ag allele) undergoing measurement of FMD, no differences in FMD and plasma levels of IL-6, IL-8, VCAM, and ICAM were found between the aa and the ag phenotype (P . 0.05 for all). Conclusions:   TLR4 signaling in endothelial cells may be triggered by LPS and oxidized phospholipids, leading to endothelialactivation and inflammation, which are inhibited by eritoran. Our in vivo investigation, however, does not support anassociation between the Asp299Gly TLR4 polymorphism and improved endothelium-dependent vasodilator function inpatients with RA. Further study is needed to better understand the potential role of TLR4 on endothelial dysfunction in thisand other patient populations. Citation:  Menghini R, Campia U, Tesauro M, Marino A, Rovella V, et al. (2014) Toll-Like Receptor 4 Mediates Endothelial Cell Activation Through NF- k B but Is NotAssociated with Endothelial Dysfunction in Patients with Rheumatoid Arthritis. PLoS ONE 9(6): e99053. doi:10.1371/journal.pone.0099053 Editor:  Angelo Scuteri, INRCA, Italy Received  January 28, 2014;  Accepted  May 9, 2014;  Published  June 11, 2014 Copyright:    2014 Menghini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Funding:  This work as supported by an intramural grant of Tor Vergata University to Dr. Menghini. The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript. Competing Interests:  The authors have declared that no competing interests exist.* E-mail: Introduction Chronic inflammation represents a pivotal mechanism in thepathogenesis of atherosclerosis [1]. Interestingly, recent evidencesuggests that innate immunity may also contribute to thedevelopment of vascular damage by interacting with inflammatorypathways [2]. In particular, toll-like receptors (TLRs) areincreasingly being recognized as a link between the innateimmune system, inflammation, and atherogenesis. This family of innate immune receptors is expressed by endothelial cells, in whichthey trigger various signaling pathways and lead to cell activation,increased expression of inflammatory cytokines and adhesionmolecules, and endothelial dysfunction [3,4]. While initiallyidentified as sensors of microbial invasion, TLRs are now knownto be activated also by endogenous ligands produced in inflamedtissues, potentially leading to further inflammation and perpetu-ating an inflammatory milieu [3]. Among them, TLR4, a receptorfor lipopolysaccharide (LPS) from Gram negative bacterial cellwalls, also exhibits affinity for fatty acids [5], extracellular matrixcomponents, fibrinogen, and various heat shock proteins [6]. Of note, TRL4 signaling leads to activation of NF- k B [4], a pathwayassociated with andothelial injury [7], and TRL4 expression isincreased in human atherosclerotic plaques [8]. Additionally, lack of TLR4 reduces atherosclerosis and alters plaque phenotype inapoE-deficient mice fed a high-cholesterol diet [9]. In agreementwith these data, clinical evidence indicates that the Asp299GlyTLR4 polymorphism, a functional variant in the TLR4 gene(896A R G) that attenuates receptor signaling and diminishes the PLOS ONE | 1 June 2014 | Volume 9 | Issue 6 | e99053  inflammatory response to LPS [10], is associated with decreasedatherosclerotic risk  [11]. However, whether antagonism of TLR4prevents TLR4-induced expression of inflammatory cytokines andadhesion molecules in human macrovascular endothelial cells hasnot been investigated in detail.Rheumatoid arthritis (RA) is one of the most prevalent systemicautoimmune diseases [12] and is associated with endothelialdysfunction [13] and increased cardiovascular risk [14]. This risk is attributed to the presence of both traditional and non-traditionalrisk factors, including inflammation and immunologic abnormal-ities [15]. A growing body of knowledge indicates that TLR4 mayplay a relevant role of in the pathogenesis of autoimmune damagein RA [3]. In line with this evidence, the Asp299Gly TLR4polymorphism is associated with decreased RA disease suscepti-bility and lower baseline disease activity [16]. However, whetherthe presence of the Asp299Gly TLR4 polymorphism is associatedwith better endothelial function compared with the wild typegenotype in patients with RA has not been studied.The current investigations were therefore designed to test thefollowing hypotheses: 1) antagonism of TLR4 with eritoran(E5564) inhibits the expression of inflammatory cytokines andadhesion molecules in human endothelial cells; and 2) the presenceof the Asp299Gly TLR4 polymorphism is associated with betterendothelium-dependent vasodilation compared with the wild typegenotype in patients with RA. Materials and Methods In-Vitro Experiments: Cell Culture and Treatment Human aortic endothelial cells (HAECs) were purchased fromLonza (Basel, Switzerland) and cultured according to themanufacturer’s instructions. All experiments were performed using HAECs between the 2 th and the 5 th passage. HAECs were treatedwith: LPS (Sigma Aldrich, St. Louis, MO) at a concentration of 100 ng/mL for 6 hours; ox-PAPC (oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine, Hy-cult Biotech, Uden, The Netherlands), an antigenic epitope of oxidized LDL, at a concentration of 100  m g/mL for 6 hours; orlong chain free fatty acids (FFA, oleic acid 500  m M + palmitic acid500  m M) for 24 hours. Cells were incubated with 10 nM eritoranfor 30 minutes prior to treatments where indicated. Eritoran (EisaiInc., Woodcliff Lake, NJ) is a synthetic analog of lipid A and apotent and specific antagonist of LPS action, which inhibits lipid Abinding to MD2 and terminates MD2/TLR4-mediated signaling [17]. At the end of treatments, HAECs where collected and usedfor molecular analysis. In-Vitro Experiments: Gene Expression Analysis Total RNA was isolated from HAECs using Trizol reagents(Invitrogen Corp, Eugene, OR). A total of 2  m g of RNA wasreverse-transcribed into complementary DNA (cDNA) using theHigh Capacity cDNA Archive kit (Applied Biosystems, FosterCity, CA). Fifty nanograms of cDNA was amplified by real-timepolymerase chain reaction (RT-PCR) using an ABI PRISM 7500System and TaqMan reagents (Applied Biosystems) and normal-ized to 18S ribosomal RNA as an endogenous control. Eachreaction was performed in triplicate, and the relative gene copynumber was calculated as 2 2 DD Ct as previously described [18]. In-Vitro Experiments: Western Blots Total protein was isolated from HAECs and western blots wereperformed as previously described [19]. The following antibodieswere used: anti-P65, anti-phosphoSer 536 P65, anti-I k B a , and anti-phosphoSer 32/36 I k B a , (Cell Signaling Technology Inc., Danvers,MA). Clinical Study: Study Population Nonsmoker patients with a diagnosis of RA according to the ACR revised criteria [20] and age- and sex-matched healthycontrols were enrolled in the study. None of the patients hadhistory or presence of hypertension, diabetes, hypercholesterol-emia, cardiovascular disease, vasculitis, or any other systemiccondition. All RA patients were on chronic treatment with eitherdisease modifying antirheumatic drugs (DMARDs), monoclonalantibodies, or their combination. When used, aspirin, coxibs orother nonsteroidal anti-inflammatory drugs were withdrawn atleast one week before the study; patients were allowed to useacetaminophen (paracetamol) or tramadol as needed. The Disease Activity Score 44 (DAS 44) was calculated for each patient. Thisscore assesses disease activity by including tender and swollen jointcount and the erythrocyte sedimentation rate. The level of diseaseactivity can be interpreted as low (DAS # 2.4), moderate (2.4 , DAS # 3.7), or high (DAS . 3.7) [21]. A DAS , 1.6 correspondswith being in remission according to the American Rheumatism Association (ARA) criteria [22]. The study protocol was conductedaccording to the principles expressed in the Declaration of Helsinki, was approved by the institutional Ethics Committee of Tor Vergata University, and all participants gave written informedconsent. Clinical Study: Laboratory Methods EDTA-collected blood samples were drawn on the study dayafter an overnight fast, immediately centrifuged for 15 min at1000 6 g and stored at  2 80 u C until analysis. Detection of autoantibodies.  Rheumatoid factor (RF)-IgMand RF-IgA (Orgentec Diagnostika GmbH, Mainz, Germany) andanti-CCP antibodies (Axis Shield Diagnostics, Dundee, UK) weremeasured using commercially available ELISA kits Equal volumesfor each sample were loaded according to each specific kit’sinstructions. The suggested cut-off levels were 20 U/mL for RF-IgM and RF-IgA and 5 U/mL for anti-CCP antibodies.  Soluble biomarkers:   plasma levels of IL-6, IL-8, ICAM, VCAM and MCP-1were measured using commercially available ELISA kits (R&DSystems, Minneapolis, MN, USA). Equal volumes for each samplewere loaded according to each specific kit’s instructions. Quan-titative levels of cytokines were determined by comparison withstandard curves and reported as picograms or nanograms per mL(pg/mL or ng/mL). The sensitivity of the test was of 0.7 pg/mLfor IL-6, 1.5 pg/mL for IL-8, 0.1 ng/mL for ICAM, 0.6 ng/mLfor VCAM and 5.0 pg/mL for MCP-1. Genomic DNA for TLR4genotyping was prepared from frozen whole blood with the use of a blood DNA isolation kit (Genomic Prep, Amersham PharmaciaBiotech, Piscataway, NJ). Subsequent allele-specific PCR ampli-fication for the TLR4 allele Asp299Gly was performed according to a previously described protocol [23]. Clinical Study: Endothelial Function Testing  Assessment of endothelial function was conducted in the fasting state using a standardized ultrasound procedure [24]. Brachialartery reactivity, a test of endothelium-dependent vasodilation,was assessed as previously reported [25]. Briefly, participants laysupine on a bed and were allowed to rest for at least 10 minutes.During the rest period, participants were connected to acontinuous ECG monitor and a pressure cuff was applied aroundthe upper forearm. The left brachial artery was then visualized onthe anterior aspect of the arm, 2–15 cm proximal to theantecubital fossa, using a Logiq E ultrasound machine (GE TRL4, Endothelium and Rheumatoid ArthritisPLOS ONE | 2 June 2014 | Volume 9 | Issue 6 | e99053  Healthcare Italia, Milan, Italy) with a high-resolution probe (12-MHz linear array transducer). After baseline images and flowmeasurements were obtained, the pressure cuff applied on theforearm was inflated at 250 mmHg for 5 minutes. Blood flow wasmeasured during the first 15 seconds after cuff deflation, andarterial image acquisition for diameter measurements wasperformed between 60 and 90 seconds after cuff deflation. Arterialdiameter was measured from the anterior to the posterior interfacebetween the lumen and the endothelium at end diastole, incidentwith the R wave on the ECG. Images were analyzed by aninvestigator, different from the sonographer, blinded to imagesequence and clinical data of study participants. Statistical Analysis For the clinical study, sample size calculation was based ondifferences between the values of FMD in the three groups. Using a 2-sided paired t test, a sample of 12 subjects in each group wascalculated to be necessary to detect a 2% difference in FMD with80% power and  a , 0.05. With anticipation of up to 3 (i.e. 20%)technically inadequate vascular studies, 15 participants per groupwere enrolled to yield 12 evaluable participants in each group. Allgroup data are reported as mean  6  SD. Group differences wereanalyzed by one way ANOVA, Fisher exact test, and unpairedStudent t test, as appropriate. All calculated p values are two-tailed, and a value of p , 0.05 was considered to indicate statisticalsignificance. Statistical analyses were performed using commer-cially available software. Results In-Vitro Experiments: Eritoran Inhibits LPS-Induced mRNAExpression of Inflammatory Cytokines in HAECs To determine the effects of TLR4 receptor antagonism on LPS-induced expression of inflammatory cytokines in HAECs, weassessed mRNA levels of IL-6, IL-8, TNF a , and CCL-2 mRNAafter treatment with LPS, alone and following pretreatment witheritoran for 30 minutes. Incubation with eritoran did not lead tosignificant changes in mRNA levels of these cytokines compared tocontrol. Treatment with LPS alone for 6 hours induced asignificant increase in cytokine expression. In contrast, whenHAECs were treated with LPS following pretreatment witheritoran, cytokine mRNA levels were similar to control (figure 1,top and middle panels). Figure 1. Effects of eritoran on LPS-induced mRNA expression (arbitrary units) of TNF a  (top left panel), CCL-2 (top right panel), IL-6(middle left panel), IL-8 (middle right panel), VCAM (bottom left panel), and ICAM (bottom right panel).  Values reported as mean 6 SD(n=5 per group). CT: control; ER: eritoran; LPS: lipopolysaccharide; ER/LPS: eritoran/lipopolysaccharide. *p , 0.05 vs CT, ER, and LPS/ER.doi:10.1371/journal.pone.0099053.g001TRL4, Endothelium and Rheumatoid ArthritisPLOS ONE | 3 June 2014 | Volume 9 | Issue 6 | e99053  In-Vitro Experiments: Eritoran Inhibits LPS-Induced mRNAExpression of Adhesion Molecules in HAECs To assess whether TLR4 receptor antagonism impacts LPS-induced expression of adhesion molecules in HAECs, wemeasured mRNA levels of VCAM and ICAM after treatmentwith LPS, alone and following pretreatment with eritoran for 30minutes. compared with control, eritoran alone did not lead tosignificant changes in VCAM and ICAM mRNA levels. Treat-ment with LPS alone for 6 hours caused a significant increase inmRNA levels of the adhesion molecules. In contrast, when HAECwere treated with LPS following pretreatment with eritoran,mRNA levels of VCAM and ICAM were similar to control(figure 1, bottom panels). In-Vitro Experiments: Eritoran Inhibits ox-PAPC-InducedmRNA Expression of IL-8 in HAECs To explore the effects of TLR4 receptor antagonism on ox-PAPC-induced expression of inflammatory cytokines in HAECs,we assessed the effects of ox-PAPC treatment on mRNA levels of IL-6 and IL-8, alone and following pretreatment with eritoran for30 minutes. Eritoran did not affect cytokine mRNA levelscompared with control. ox-PAPC alone for 6 hours significantlyincreased cytokine mRNA levels. In contrast, when ox-PAPCtreatment followed pretreatment with eritoran, mRNA levels of IL-8 were significantly reduced when compared with ox-PAPCalone (figure 2, top panel). Similarly, mRNA levels of IL-6 werereduced; however, they failed to reach statistical significance(p=0.07) (figure 2, middle panel). In-Vitro Experiments: Eritoran Does Not Affect FFA-Induced mRNA Expression of IL-6 in HAECs To confirm that eritoran’s effects on inflammatory cytokines aremediated by TLR4, we assessed the effects of FFA treatment onmRNA levels of IL-6, alone and after incubation with eritoran for30 minutes. As in the previous experiments, eritoran did not affectIL-6 mRNA levels compared with control. FFA alone for 6 hoursled to significantly increased IL-6 mRNA levels. Consistent withour hypothesis that eritoran’s effects on inflammatory cytokinesare mediated by TLR4, pre-treatment with eritoran did not affectFFA-induced inflammatory cytokine expression (figure 2, bottompanel). In-Vitro Experiments: Eritoran Reduces LPS-InducedPhosphorylation of NF- k B p65 Subunit and I k B- a  inHAECs To confirm that the effects of TLR4 antagonism on theexpression of inflammatory cytokines are mediated by a modula-tion of the NF- k B pathways, we assessed phosphorylation of NF- k B p65 subunit and of I k B- a  by western blotting (figure 3, toppanel). Compared with control, treatment with eritoran for 30minutes did not induce phosphorylation of NF- k B p65 subunitand of I k B- a . In contrast, treatment of HAECs with LPS for 6hours caused an increase in the phosphorylation of NF- k B p65subunit and I k B- a . Pretreatment with eritoran prevented the LPS-induced phosphorylation of NF- k B p65 subunit (figure 3, bottomleft panel) and I k B- a  (figure 3, bottom right panel). Clinical Study: Study Population Thirty patients and 15 age- and sex-matched healthy controlstook part in the study. Their baseline clinical characteristics arereported in Table 1. The mean DAS 44 score of all the patientswas 2.2 (range: 0.5–3.9). The majority of the patients were treatedwith a disease-modifying anti-rheumatic drug (27/30) and/or abiologic agent (19/30). No significant differences were observed inthese parameters, disease activity, and drug treatment between thetwo patient groups (all P . 0.05). Figure 2. Effects of eritoran on OxPAPC-induced mRNAexpression (arbitrary units) of IL-6 (top panel) and IL-8 (middlepanel) and on FFA-induced mRNA expression (arbitrary units)of IL-6 (bottom panel).  Values reported as mean 6 SD (n=5 pergroup). CT: control; ER: eritoran; OxPAPC: oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine; FFA: freefatty acids; OxPAPC/ER: oxidation products of 1-palmitoyl-2-arachido-noyl-sn-glycerol-3-phosphatidylcholine/eritoran. Top panel: *p , 0.05 vsCT and ER; NS: p=0.07 vs OxPAPC/ER. Middle panel: *p , 0.05 vs CT, ER,and OxPAPC/ER. Bottom panel: *p , 0.05 vs CT and ER; NS: p . 0.05 vsFFA/ER.doi:10.1371/journal.pone.0099053.g002TRL4, Endothelium and Rheumatoid ArthritisPLOS ONE | 4 June 2014 | Volume 9 | Issue 6 | e99053  Clinical Study: Brachial Artery Reactivity Baseline brachial artery diameter was similar in the three groups(3.7 6 0.6 mm, 3.3 6 0.5 mm, and 3.4 6 0.7 mm in the control, aa,and ag group, respectively, p=0.18). Flow-mediated dilation wassignificantly higher in the controls compared to the RA groups(p=0.014). However, no significant differences in FMD wereobserved between the two patient groups (p . 0.05) (figure 4 andfigure 5, top left panel). Clinical Study: Plasma Cytokines and Adhesion Moleculesin RA Patients Plasma levels of IL-6 and IL-8, of MCP-1, and of VCAM andICAM were similar between the aa and ag groups (figure 5). Discussion The main results of our in-vitro investigations are that, inHAEC, TLR4 antagonism with eritoran: 1) inhibits LPS-inducedmRNA expression of the inflammatory cytokines IL-6, IL-8,TNF a , and CCL-2, and of the adhesion molecules VCAM andICAM; 2) inhibits ox-PAPC-induced mRNA expression of IL-8and of IL-6, albeit to a borderline significant level; and 3) reducesLPS-induced phosphorylation of NF- k B p65 subunit and I k B- a .These findings indicate that TLR4 induces activation of humanmacrovascular endothelial cells through NF- k B-dependent path-ways, leading to the expression of genes regulating the productionof inflammatory mediators and adhesion molecules. Importantly,TLR4-dependent activation of these pathways is not restricted toexogenous ligands such as LPS, as mRNA expression of IL-6 andIL-8 was also triggered by Ox-PAPC. These phospholipidoxidation products are present in sites of chronic inflammation,in apoptotic cell membranes, and in oxidized LDL [26], suggesting a potential pathophysiologic role of Ox-PAPC-dependent LTR4activation in atherosclerosis. Finally, as FFA-induced IL-6expression was not affected by eritoran, our data suggest thatTLR4 activation in macrovascular endothelial cells is possiblyrestricted to specific lipid ligands such as Ox-PAPC.Our results are in agreement with the recent findings from Luand colleagues. These authors reported that, in HAEC and indermal microvascular endothelial cells, LPS induced an increase inmRNA expression and synthesis of IL-6, as well as a more robustgene expression of IL-8 and other inflammatory cytokines, ICAM,VCAM, chemokines, growth factors, and adhesion molecules [27].Our study shows that also Ox-PAPC, an endogenous byproduct of phospholipid peroxidation, triggers TLR4-dependent expression Figure 3. Representative Western blots (top panel) and bar graphs showing the effect of eritoran on LPS-induced phosphorylationof NF- k B p65 subunit (bottom left panel) and I k B- a  (bottom right panel) (n=5 per group).  Bottom left panel: *p , 0.05 vs CT; NS: p . 0.05vs LPS/ER. Bottom right panel: **p , 0.005 vs CT; NS: p . 0.05 vs LPS/ER.doi:10.1371/journal.pone.0099053.g003TRL4, Endothelium and Rheumatoid ArthritisPLOS ONE | 5 June 2014 | Volume 9 | Issue 6 | e99053
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