Circulation ORIGINAL RESEARCH ARTICLE Oxidized Low-Density Lipoprotein Receptor in Lymphocytes Prevents Atherosclerosis and Predicts Subclinical Disease BACKGROUND: Although the role of Th17 and regulatory T cells in Katerina Tsilingiri, PhD* the progression of atherosclerosis has been highlighted in recent years, Hortensia de la Fuente, their molecular mediators remain elusive. We aimed to evaluate the MD, PhD* association between the CD69 receptor, a regulator of Th17/regulatory T Marta Relaño, MSc cell immunity, and atherosclerosis development in animal models and in Raquel Sánchez-Díaz, PhD patients with subclinical disease. Cristina Rodríguez, PhD Javier Crespo, PhD METHODS: Low-density lipoprotein receptor–deficient chimeric mice Fátima Sánchez-Cabo, PhD expressing or not expressing CD69 on either myeloid or lymphoid cells Ana Dopazo, PhD were subjected to a high fat diet. In vitro functional assays with human José L. Alonso-Lebrero, T cells were performed to decipher the mechanism of the observed PhD phenotypes. Expression of CD69 and NR4A nuclear receptors was Alicia Vara, BSc evaluated by reverse transcription–polymerase chain reaction in 305 Jesús Vázquez, PhD Downloaded from http://ahajournals.org by on November 6, 2020 José M. Casasnovas, PhD male participants of the PESA study (Progression of Early Subclinical Fernando Alfonso, MD, PhD Atherosclerosis) with extensive (n=128) or focal (n=55) subclinical Borja Ibáñez, MD, PhD atherosclerosis and without disease (n=122). Valentín Fuster, MD, PhD RESULTS: After a high fat diet, mice lacking CD69 on lymphoid cells José Martínez-González, PhD developed large atheroma plaque along with an increased Th17/regulatory Pilar Martín, PhD T cell ratio in blood. Oxidized low-density lipoprotein was shown to Francisco Sánchez-Madrid, bind specifically and functionally to CD69 on human T lymphocytes, PhD inhibiting the development of Th17 cells through the activation of NR4A nuclear receptors. Participants of the PESA study with evidence of *Drs Tsilingiri and de la Fuente subclinical atherosclerosis displayed a significant CD69 and NR4A1 mRNA contributed equally. downregulation in peripheral blood leukocytes compared with participants Key Words: atherosclerosis ◼ CD69 without disease. The expression of CD69 remained associated with the risk antigen ◼ oxidized low density lipoprotein ◼ regulatory T lymphocytes of subclinical atherosclerosis in an adjusted multivariable logistic regression ◼ Th17 cells model (odds ratio, 0.62; 95% CI, 0.40–0.94; P=0.006) after adjustment for Sources of Funding, see page 254 traditional risk factors, the expression of NR4A1, the level of oxidized low- density lipoprotein, and the counts of different leucocyte subsets. © 2018 The Authors. Circulation is published on behalf of the American Heart Association, Inc., by Wolters CONCLUSIONS: CD69 depletion from the lymphoid compartment promotes Kluwer Health, Inc. This is an open a Th17/regulatory T cell imbalance and exacerbates the development of access article under the terms of the Creative Commons Attribution atherosclerosis. CD69 binding to oxidized low-density lipoprotein on T cells Non-Commercial-NoDerivs License, induces the expression of anti-inflammatory transcription factors. Data from which permits use, distribution, and reproduction in any medium, provided a cohort of the PESA study with subclinical atherosclerosis indicate that that the original work is properly cited, CD69 expression in PBLs inversely correlates with the presence of disease. the use is noncommercial, and no The expression of CD69 remained an independent predictor of subclinical modifications or adaptations are made. atherosclerosis after adjustment for traditional risk factors. https://www.ahajournals.org/journal/circ Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 January 8, 2019 243 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis nary artery atherosclerosis, with a significant increase in Clinical Perspective ORIGINAL RESEARCH Th17 and a decrease in Treg cells.12,13 The early lymphocyte activation antigen CD69 regu- What Is New? ARTICLE lates Th17 and Treg cell differentiation. CD69-deficient mice display enhanced Th17 differentiation and defec- • This study identifies CD69 as an oxidized low- density lipoprotein receptor in T lymphocytes that tive Treg cell function,14–16 resulting in an inability to contributes to the regulatory action of the adap- resolve inflammation or to maintain immune tolerance tive immune system, preventing atherosclerosis in diseases such as arthritis, asthma, contact dermati- development. tis, or myocarditis.17–19 However, no differences were • CD69 controls the onset and progression of athero- observed in the atheroma plaque formation in CD69−/− sclerosis, and its mRNA expression is an indepen- ApoE−/− mice.20 dent marker of early subclinical disease in humans. A key event in the process of atheroma plaque for- • Data from a cohort of asymptomatic individuals mation is low-density lipoprotein (LDL) peroxidation, indicate that CD69 expression in circulating T cells which generates highly inflammatory and immunogen- correlates inversely with the presence and exten- ic oxidized LDL (oxLDL).21 However, oxLDL can also elicit sion of subclinical atherosclerosis. anti-inflammatory responses by activating peroxisome • The expression of CD69 remained associated with the risk of subclinical atherosclerosis after adjust- proliferator-activated receptor-γ22 and liver X receptor ment for traditional cardiovascular risk factors. or by upregulating transcription factors with anti-in- flammatory activity such as NR4A nuclear receptors.23,24 What Are the Clinical Implications? The main objectives of this work were to analyze the role of CD69 lymphocyte expression in atherosclerosis • Oxidized low-density lipoprotein binding to CD69 development, the immune mechanisms involved, and confers a regulatory phenotype to human and its relation with human disease. Using chimeric ldlr−/− mouse T cells, dampening Th17 responses and ameliorating atherosclerosis. mice subjected an HFD as atherosclerosis model, we • Expression of CD69 in circulating cells might serve show that CD69 deficiency specifically on lymphocytes as a new biomarker for the presence of subclinical leads to an altered Th17/Treg equilibrium and a conse- atherosclerosis. quent increase in atheroma plaque size during an HFD. In vitro assays in human T cells showed that the interac- tion between oxLDL and CD69 activates the expression Downloaded from http://ahajournals.org by on November 6, 2020 A lthough lipid deposition in the vessel wall is the of NR4A transcription factors, skewing T cells toward hallmark of atherosclerosis, for many years, it a regulatory phenotype and dampening Th17 and Th1 has been recognized that inflammation also responses. Thus, we describe an unexpected regulatory plays a crucial role in the genesis and progression of the mechanism of the adaptive immune system that delays disease and the appearance of clinical manifestations atherosclerosis development in hyperlipidemic condi- (infarction, stroke).1 Recently, it has been demonstrated tions. Remarkably, our data from participants in the that the modulation of the inflammatory response is PESA (Progression of Early Subclinical Atherosclerosis) a valid target to reduce the risk of atherothrombotic cohort25 were in agreement with these results in that events in high-risk patients.2 The interplay between lip- downregulated CD69 expression in peripheral blood id metabolism and immune response is not completely leukocytes (PBLs) is associated with subclinical athero- understood. In the last decade, the contribution of lym- sclerosis in an adjusted multivariable logistic regression phocytes to atherosclerosis clinical manifestations has model. been highlighted, and effector T-cell responses seem to be exacerbated in hyperlipidemia.3,4 METHODS In vivo models of atherosclerosis suggest that regu- The data, methods, and study material will be available to latory T (Treg) cells suppress inflammatory responses other researchers for purposes of reproducing the results or and attenuate atherosclerosis.5 Experimental therapies replicating the procedures by contacting the corresponding aimed to increase Treg cell population under high fat authors. diet (HFD) conditions have conclusively demonstrated an important role for this T-cell subset in atherosclerosis Study Design attenuation.6,7 The role of Th17 lymphocytes is more To analyze the role of CD69 during atherosclerosis develop- controversial. Although genetic or pharmacological in- ment, we used chimeras from ldlr−/− CD45.1+ mice (the line hibition of interleukin (IL)-17 significantly ameliorates was obtained in house by crossing and subsequent selection atherosclerosis,8,9 concomitant increases in both IL-17 by genotype of B6;129S7-Ldlrtm1Her/J with B6.SJL-PtprcaPepcb/ and IL-10 lead to smaller plaques.10 Another study re- BoyCrl, from Jackson and Charles River, respectively) and ports bigger plaques in ApoE−/−IL-17−/− mice.11 A Th17/ CD69−/− or CD69+/+ B6 double reporter (dRep) for Treg cells Treg imbalance has been reported in patients with coro- (FIR mice, Foxp3–monomeric red fluorescent protein [mRFP]) 244 January 8, 2019 Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis and Th17 cells (IL-17A–IRES–enhanced green fluorescent Monitoring Th17 and Treg Cells in the In ORIGINAL RESEARCH protein [eGFP]), hereafter cd69−/−dRep and cd69+/+dRep, respectively. dRep mice allow us to monitor the presence of Vivo Model To assess the immune response, the percentages of IL-17– live Treg cells and Th17 cells throughout the experiment. To ARTICLE eGFP+ or Foxp3-RFP+ cells in peripheral blood CD4+ T cells evaluate the influence of immune cell CD69 expression dur- were monitored throughout the experiment by flow cytom- ing atherosclerosis development, Ldlr−/− mice were irradiated etry. The presence of Th17 and Treg cells was also evaluated and reconstituted with bone marrow (BM) from cd69−/−dRep at the end point of the experiment in aortic arch, spleen, non- mice or cd69+/+dRep. Next, atherosclerosis development was draining (axillary) LNs, and draining (para-aortic) LNs. evaluated in mice proficient or deficient for CD69 only in the myeloid compartment (MC; hereafter MC cd69+/+ and MC cd69−/−) or lymphoid compartment (LC; hereafter LC cd69+/+ OxLDL Binding Assays and LC cd69−/−). For a detailed description of chimeric mice Jurkat T (JK) cells or rat basophilic leukemia (RBL) cells sta- design, see the online-only Data Supplement. After 6 weeks bly transfected or not with CD69 were incubated with of BM reconstitution, mice were placed on an HFD (SSNIFF, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindo-carbocyanine per- S9167-E010) for 10 to 16 weeks. Immune response was chlorate–labeled (Dil) LDL in their native and oxidized form evaluated in peripheral blood, draining (para-aortic) lymph in the presence or not of unlabeled lipoproteins. Lipoprotein nodes (LNs), nondraining (inguinal, axillary, mesenteric) LNs, binding was determined with flow cytometry. In some cases, and spleen by flow cytometry and quantitative polymerase blocking anti-CD69 antibody was added. chain reaction (PCR). All animal procedures were approved by the ethics committee of the Comunidad Autónoma de Madrid and conducted in accordance with the institutional Human CD4+ T Cell Polarization guidelines that comply with the European Institutes of For human Th1, Th17, and Treg cell polarization, CD4+ T Health directives (2010/63/EU of the European Parliament cells were purified from peripheral blood of healthy donors and the Council on the Protection of Animals Used for and incubated with a specific cocktail of recombinant cyto- Scientific Purposes [Official Journal of the European Union, kines. After the indicated days of culture, cells were ana- 2010:53:33–79]). lyzed in a FACSCanto Flow Cytometer. When indicated, oxLDL (50 µg/mL) and anti-CD69 (20 µg/mL) were added to the cultures. Quantification of CD69 and NR4A Gene Expression in the PESA Study Assessment of CD69 and NR4A Expression of CD69 and nuclear receptors NR4A1 was eval- Transcripts Downloaded from http://ahajournals.org by on November 6, 2020 uated by reverse transcription PCR with Taqman probes in PBLs of a subset of the participants from the PESA study.25 Expression of mRNA levels of CD69 and NR4A nuclear This study prospectively enrolled 4184 asymptomatic par- receptors was analyzed in PBLs from patients with subclini- ticipants 40 to 54 years of age to evaluate the systemic cal atherosclerosis and healthy subjects with reverse tran- extent of atherosclerosis in the carotid, abdominal aortic, scription PCR with Taqman probes (Applied Biosystems). and iliofemoral territories by 2-dimensional/3-dimensional Expression of NR4A receptors was also assessed in Jurkat T ultrasound and coronary artery calcification by computed cells and human primary lymphocytes in the presence or not tomography at baseline and 3 and 6 years after enrollment of oxLDL. When indicated, blocking anti-CD69 antibodies for follow-up studies. Participants were then defined as free were also added. NR4A and CD69 mRNA expression was of atherosclerosis (no disease; no presence of plaque and a also determined in para-aortic LNs and peripheral blood lym- coronary artery calcium score of zero) or with evidence of phocytes from mice. focal (1 site affected) or generalized (4–6 sites affected) sub- clinical atherosclerotic disease.25 Following a general study Tissue Processing and strategy (ie, not for this specific analysis), 480 individuals Immunohistochemistry of the whole cohort were retrospectively selected on the For plaque area assessment, 5-µm-thick sections at 100-µm basis of the extent of subclinical atherosclerosis to perform intervals were collected starting at the origin of the aortic molecular tests. Individuals were included in this subcohort, valve cusps. Sections were stained with Oil Red O staining, prioritizing those with more territories with evidence of (Sigma-Aldrich) and hematoxylin, and lesion size was ana- plaque at baseline. For those presenting a tie, individuals lyzed with ImageJ software. For Masson trichrome stain- with coronary artery calcification score ≥1 were included. ing, 7-µm-thick sections at 100-µm intervals were collected. Control subjects were then selected from those individuals Sections were stained with the Masson-Goldner staining kit without plaques and matched with the chosen cases on the (Merck). For specific staining, anti-F480 antibody was pur- basis of age, sex, family history of cardiovascular disease, chased from Abcam (ab6640) and anti-CD3 from Santa Cruz dyslipidemia, and hypertension. From this subpopulation of Biotechnology (sc-1127). OxLDL was detected by immunoflu- 480 individuals, we selected 305 male participants classified orescence with rabbit anti-mouse oxLDL from Abcris. as without disease (n=122) or with focal (n=55) or gener- alized (n=128) disease to test the expression of CD69 and NR4A1 by reverse transcription PCR. General characteristics Statistical Analysis of this specific subcohort are shown in Table I in the online- In vivo experiments were performed according to a ran- only Data Supplement. domized complete block design (treatments and different Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 January 8, 2019 245 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis time points have been taken into account) or a fully ran- RESULTS ORIGINAL RESEARCH domized design. To determine significant differences between 2 means, P values were calculated by unpaired t Lack of CD69 on Lymphocytes test or Mann-Whitney U test according to normality test, Exacerbates Atherosclerotic Plaque ARTICLE and differences among >2 means were analyzed by 1-way ANOVA or the Kruskal-Wallis test. To account for multiple Formation comparisons, the Tukey or Bonferroni posttests were used To evaluate the role of CD69 expression on BM-derived to compare selected pairs of means and all pairs of means, cells in the development of atherosclerosis, male Ldlr−/− respectively. When indicated, the Wilcoxon signed-rank test mice were irradiated and reconstituted with BM from was used to analyze paired data. For differences in human cd69−/− dRep for Foxp3-mRFP and IL-17A–eGFP26 mice T-cell differentiation assays, data were analyzed with the (BM Cd69−/−) or WT-dRep littermates (BM Cd69+/+; Fig- Friedman test and the Dunn multiple-comparison test. In ure IA in the online-only Data Supplement). From week kinetic experiments with the same mice, 2-way repeated- measures ANOVA with the Sidak multiple-comparison post 13 on, we found increased Th17/Treg cells ratio in PBLs hoc test was performed. Differences were considered sig- of chimeric mice (Figure IB in the online-only Data Sup- nificant at P<0.05. plement), indicating a polarization toward proinflam- Table I in the online-only Data Supplement shows demo- matory phenotype in the absence of CD69. After 10 graphics and cardiovascular risk factor statistics of the PESA weeks of HFD, mice reconstituted with cd69−/−dRep BM subcohort selected for the quantitative PCR assay. Statistics displayed a significantly high increase in IL-17–eGFP+ were calculated with Stata (StataCorp, College Station, TX). cells in para-aortic LNs, with higher absolute numbers Data are expressed as mean±SD, median and interquartile compared with BM Cd69+/+ mice (Figure IC and ID in range, or number (percent). P values are derived from ANOVA the online-only Data Supplement). In agreement, the for log-transformed continuous variables and χ2 for categori- percentage but not cell numbers of Foxp3mRFP+ cells cal variables, except for those variables with n<6, for which the Fisher test was used. was significantly reduced in the absence of CD69 (Fig- The Cuzick extension of the Wilcoxon rank-sum test was ure ID in the online-only Data Supplement), indicating used to assess the significance of the trend in the quantita- that Treg cell recruitment to para-aortic LNs is not com- tive PCR data across the 3 ordinal groups allowing for ties. promised. The percentage of CD4+ IL-17–eGFP+ cells After that, 1-way ANOVA followed by the Tukey posttest was increased in the aortic arc of BM Cd69−/−chimeras, was used to identify significant differences between each whereas there is a tendency for decreased CD4+ and stage of subclinical atherosclerosis and baseline. Expression FoxP3+ cells (Figure IE and IF in the online-only Data Downloaded from http://ahajournals.org by on November 6, 2020 levels measured by quantitative PCR were normalized and Supplement). Collectively, flow cytometry data indi- considered in the log2 scale to fulfill the normality assump- cate that hyperlipidemia induces high proinflamma- tion. We then focused on the 2 most extreme groups (no tory activity and a disrupted Th17/Treg cells balance in disease versus generalized disease) and used a multivariable logistic regression model to assess the association of CD69 the absence of CD69. Histochemical studies revealed with subclinical atherosclerosis independently of known more extensive lesions and necrotic cores in aortic cardiovascular risk factors (age, smoking, family history of valves from BM Cd69−/− chimeras compared with the cardiovascular disease, dyslipidemia, hypertension, diabetes BM Cd69+/+ control group (Figure IG and IH in the on- mellitus), the expression of NR4A1, oxLDL levels, and vol- line-only Data Supplement). Atheroma plaques in both ume of different leukocyte subsets. These variables included groups consisted mainly of F4/80+ foam cells, although in the model were found to be statistically significant with we found a higher infiltration of CD3+ lymphocytes a value of P<0.1 in a univariable logistic regression model. into atheroma plaques in the BM Cd69−/− group (Figure Odds ratio, 95% CI, and P value are reported. Traditional II and IJ in the online-only Data Supplement). Peripher- cardiovascular risk factors were determined from blood sam- ples and interviews as follows: (1) diabetes mellitus: fasting al blood leukocytes were analyzed during an HFD with plasma glucose ≥126 mg/dL or treatment with insulin or oral no significant changes between BM Cd69+/+ and BM hypoglycemic medication; (2) arterial hypertension: systolic Cd69−/− mice (Figure IIA and IIB in the online-only Data blood pressure ≥140 mm Hg, diastolic blood pressure ≥90 Supplement). Because CD69 has been also related to mm Hg, or use of antihypertensive medication; (3) hypercho- the maintenance of T-cell helper memory,27 the CD44hi lesterolemia: total cholesterol ≥240 mg/dL, LDL cholesterol CD62Llo memory T-cell subset was also analyzed. Naïve ≥160 mg/dL, high-density lipoprotein cholesterol <40 mg/dL, T cells decrease with an HFD whereas memory T cells or use of lipid-lowering drugs; (4) smoking: current smoking increase in both chimeric mice, suggesting that CD69 status and a lifetime consumption of >100 cigarettes; and is not playing a role in the maintenance of T-helper (5) family history of cardiovascular disease: first-degree rela- memory cells in this model (Figure IIC in the online- tive diagnosed with atherosclerosis before 55 years of age in men and 65 years of age in women. Linear regression only Data Supplement). Moreover, we found that the models were used to assess the correlation between the ratio between dendritic cells and Treg cells during HFD expression levels of CD69 with NR4A1 receptors. All these remains equal in both chimeric mice (Figure IID in the analyses were implemented with the R statistical software online-only Data Supplement). These data suggest that (www.r-project.org). Th17 and Treg cell proportions are altered under the 246 January 8, 2019 Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis course of HFD in BM Cd69−/− chimeric mice, whereas dimers (Figure 2A, left), unlike other C-type lectins such ORIGINAL RESEARCH the other leukocyte subsets remained unaltered. as dectin-1 or the macrophage mannose receptor (Fig- To address whether the observed phenotype in BM ure V in the online-only Data Supplement). In LOX-1, ARTICLE Cd69−/− mice is specific to the LC or MC, we generated the oxLDL-binding surface is located at the top of the mixed BM chimeras proficient or deficient for CD69 in dimer and contains a unique basic “spine” formed by either the LC (LC Cd69+/+ and LC Cd69−/− groups) or MC the diagonal arrangement of arginine residues across (Figure 1A and Figure III in the online-only Data Supple- the dimer surface (Figure 2A, top right).30,31 Electrostatic ment). The specific deletion of CD69 on lymphocytes surface representation of the CD69 C-type lectin-like and myeloid cells was confirmed by flow cytometry domain dimer indicated that 4 arginine residues cluster analysis in the blood of hyperlipidemic mice (Figure 1A). at the center of the dimer and form a basic spine, simi- We observed a significantly enhanced plateau of Th17 lar to that of LOX-1 (Figure 2A, bottom right). Because response in the peripheral blood of the LC Cd69−/− group this structural feature is proposed to be important for after HFD, whereas percentages of Foxp3 cells were sig- oxLDL recognition,30 we hypothesized that CD69 bind- nificantly decreased compared with the Cd69+/+ group ing to oxLDL particles could account for the phenotype (Figure 1B and 1C). On observation that peripheral observed in vivo. Th17 responses started to diminish in the LC Cd69−/− Transfected Jurkat T cells stably expressing CD69 group after week 16 on HFD (Figure 1C), mice were eu- (JKCD69) on their surface bound oxLDL-DiI much more thanized, and immune responses and plaque formation efficiently than untransfected JK (JKwt) cells (Figure 2B were assessed. IL-17–eGFP+ cells in para-aortic LNs from and 2C). JKCD69 cells bound oxLDL in a dose-depen- LC Cd69−/− mice were once again increased, whereas dent manner and showed a weaker binding of native the percentages of Foxp3mRFP+ cells were comparable LDL (Figure 2D). This binding was able to induce CD69 in the 2 groups (Figure 1D). However, the absolute internalization in JKCD69 cells (Figure 2E). The specific- number of Treg cells was significant decreased, with a ity of oxLDL binding to CD69 was confirmed in blocking significant increase in the number of Th17 cells (Fig- assays with anti-CD69 antibodies (Figure 2F). ure 1D). Finally, atherosclerotic lesions were significantly more advanced with more extensive necrotic cores in the LC Cd69−/− group as assessed by Oil Red O and Mas- CD69/oxLDL Binding Controls Th17/Treg son trichrome staining (Figure 1E and 1F). Equilibrium and Expression of NR4A Downloaded from http://ahajournals.org by on November 6, 2020 Myeloid cells are pivotal for atherosclerosis develop- Nuclear Receptors ment.28,29 We next performed mixed BM chimeras pro- ficient or deficient for CD69 in the MC (MC Cd69+/+ OxLDL exposure significantly reduced the mRNA levels and MC Cd69−/−; Figure IIIA in the online-only Data of IL-8 and interferon-γ (IFN-γ) produced by JKCD69 Supplement). We did not detect differences during cells but not in JKwt cells after activation (Figure 3A). HFD in Th17 or Treg cell dynamics in the periphery, at Because of the role of CD69 in effector T-cell differ- the site of inflammation (para-aortic LNs; Figure IIIB entiation,16 we evaluated human T-cell differentiation through IIID in the online-only Data Supplement), or in to effector phenotypes. Challenge of human CD4+ T atheroma plaque formation (Figure IIIE and IIIF in the cells with oxLDL diminished the percentage of IL-17+ online-only Data Supplement). All the CD69-proficient and IFN-γ+ cells generated in response to Th17- or Th1- and -deficient groups gained similar amounts of weight polarizing stimuli (Figure 3B and 3C) and favored Treg throughout the experiment. However, the circulating differentiation (Figure 3D). This effect was dependent levels of lipids (free fatty acids, triglycerides, high-den- on CD69, as demonstrated by the blockade with anti- sity lipoprotein, LDL, and cholesterol) were lower in LC CD69 antibodies (Figure 3B through 3D). Cd69−/− compared with LC Cd69+/+, suggesting that the The NR4A orphan nuclear receptors have emerged enhanced plaque formation in LC Cd69−/− was not at- as key regulators of the immune response, controlling tributable to a metabolic defect (Figure IVA through IVC the magnitude of the inflammatory processes; NR4A1 in the online-only Data Supplement). and NR4A3 are crucial for Treg cell development.23,24 We conclude that specific CD69 deletion in the LC We assessed whether oxLDL regulates the expression of accounts for the increased proinflammatory phenotype NR4A nuclear receptors (NR4A1 and NR4A3) in human and the enhanced atheroma plaque formation. CD4+ T cells. As shown in Figure 3E, oxLDL enhanced NR4A1 and NR4A3 mRNA expression in T-cell receptor– activated human primary CD4+ T cells. An early induc- OxLDL Binds to CD69 on T Lymphocytes tion of NR4A3 was also evoked by oxLDL in JKCD69 The main receptor of oxLDL on vascular cells is lectin-like cells but not in JKwt cells (Figure 3F), which was blocked oxLDL receptor-1 (LOX-1), located in the same chromo- by preincubation with anti-CD69 antibodies (Figure 3G) somal locus immediately upstream to CD69. The LOX-1 or shRNA, confirming the CD69-dependent effect of and CD69 C-type lectin-like domains form very similar oxLDL (Figure 3H). Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 January 8, 2019 247 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis ORIGINAL RESEARCH ARTICLE Downloaded from http://ahajournals.org by on November 6, 2020 Figure 1. CD69 deficiency in lymphoid cells aggravates atherosclerosis. A, Schematic illustrating the generation of lymphoid chimeras (LCs). Ldlr−/− (CD45.1+) mice were lethally irradiated and reconstituted with mixed bone marrow (BM) from Rag2−/−γc−/− plus BM from C57BL/6-Cd69+/+ (LC Cd69+/+) or C57BL/6-Cd69−/− (LC Cd69−/−) (double-reporter [dRep]; interleukin [IL]-17–green fluorescent protein [GFP]+/Foxp3–monomeric red fluorescent protein [RFP]+) mice at a 3:1 ratio, respectively. Reconstitution of the lymphoid and myeloid compartments of CD45.2+ cells was assessed by fluorescence-activated cell sorting. Peripheral blood mononuclear cells (PBMCs) after 8 weeks of reconstitution were >90% CD45.2+. Histograms show CD69 expression on CD4 and CD8 T cells, B cells (B), macrophages (Mac), monocytes (Mono), dendritic cells (DCs), and natural killer cells (NKs). PBMCs were stimulated overnight with anti–CD3/CD28 for lymphoid cells and lipopolysaccharide for myeloid cells of the indicated groups. B, Flow cytometry analysis of IL-17–GFP+ and Foxp3-RFP+ CD4 T cells in peripheral blood of LC CD69+/+ and LC CD69−/− mice after 14 weeks of a high fat diet (HFD). C, Percentage of Th17 (IL-17–GFP+), regulatory T (Treg; Foxp3-RFP+) CD4+ T cells, and Th17/Treg ratio in peripheral blood leukocytes of LC Cd69+/+ and LC Cd69−/− mice at the indicated time points after HFD initiation. n=15 mice per group (pooled from 3 independent experiments; error bars show SEM). P values were calculated by 2-way repeated-measures ANOVA (Sidak post hoc test). *P<0.05. **P<0.01. **** P<0.0001. D, Percentage and absolute numbers of IL-17–GFP+, Foxp3-RFP+ CD4 T cells in para-aortic lymph nodes (LN). n=12 mice per group. E. Oil Red O staining and quantification of plaque and necrotic core surface in aortic valves from LC Cd69+/+ and LC Cd69−/− mice after 16 weeks of HFD. n= 8 mice/group. F, As in E, Masson trichrome staining and fibrosis quantification. n=7 mice per group Original mag- nification ×4. In D through F, error bars show SEM. *P<0.05, **P<0.01 as determined by unpaired t test or Mann-Whitney U test. 248 January 8, 2019 Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis ORIGINAL RESEARCH ARTICLE Downloaded from http://ahajournals.org by on November 6, 2020 Figure 2. CD69 binding to oxidized low-density lipoprotein (oxLDL). A, The LOX-1 and CD69 dimeric structures and their ligand-binding surfaces. Ribbon representations of the LOX-1 (Protein Data Bank code 1YXK) and CD69 (Pro- tein Data Bank code 1E8i) C-type lectin-like domain (CTLD) structures (left; side views, prepared with PyMOL). Top views of the dimers with their surface charge distributions are shown on the right (prepared with GRASP). Arginine residues that form a basic “spine” across the dimer surface are labeled. B, Histograms show the binding of DiI-labeled oxLDL (solid line) to Jurkat T cells stably expressing CD69 (JKCD69) or rat basophilic leukemia cells (RBLCD69). Dotted line indicates the displacement of DiI-OxLDL by unlabeled lipoprotein. C, OxLDL binding to untransfected Jurkat T (JKwt) and JKCD69 cells (mean±SD from 10 independent experiments). *P<0.05 by Mann Whitney U test in both cell lines. D, Dose-response curves of oxLDL and native low-density lipoprotein (LDL) binding to JKwt and JKCD69 cells. E, CD69 internalization induced by oxLDL. JKCD69 cells were incubated with unlabeled oxLDL, and membrane-surface CD69 expression was ana- lyzed by flow cytometry at the indicated times. Graph is representative of 1 of 3 experiments. F, Anti-CD69 antibodies inhibit the binding of oxLDL to JKCD69 cells (mean±SD from 4 experiments; *P<0.05). IgG1 indicates immunoglobulin G1; and MFI, mean fluorescence intensity. In the in vivo model of atherosclerosis, the reporter atheroma plaque in the main arteries of healthy individ- GFP+ Th17 cells and oxLDL localized closely at the ather- uals. Having observed a significant downregulation of oma plaque. Moreover, CD3+ T cells and oxLDL colocal- CD69 as plaque formation progressed in mice, we com- ized at atheroma plaque (Figure VIA and VIB in the on- pared CD69 and NR4A1 mRNA expression in PBLs from line-only Data Supplement). NR4A1 and NR4A3 mRNA PESA participants with focal (1 affected site, n=55) or expression was decreased in para-aortic LNs from BM generalized (4–6 affected sites, n=128) subclinical ath- Cd69−/− mice after 13 weeks of HFD (Figure 3I). More- erosclerosis with that of PESA participants without any over, PBL expression of CD69 and NR4A1 transcripts evidence of subclinical atherosclerosis (n=122; Table gradually declined in ldlr−/− mice during HFD adminis- I in the online-only Data Supplement). The extent of tration (Figure 3J), indicating that these receptors are subclinical atherosclerosis was assessed with advanced dynamically regulated in PBLs under these conditions. imaging techniques.25 There is a gradient in CD69 ex- pression across atherosclerosis extension stages; that is, CD69 expression decreases as disease progresses (trend CD69 Expression Is an Early Predictor of test, P=0.006; Figure 4A). The same is true for NR4A1 Subclinical Atherosclerosis (trend test, P=0.003; Figure 4A). These differences were The PESA study is a prospective study that uses ad- particularly significant for the generalized subclinical vanced imaging techniques to assess the presence of disease group (Figure 4B and 4C). Thus, we focused on Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 January 8, 2019 249 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis ORIGINAL RESEARCH ARTICLE Downloaded from http://ahajournals.org by on November 6, 2020 Figure 3. Oxidized low-density lipoprotein (OxLDL) binding to CD69 regulates the expression of NR4A receptors and cytokines in T cells. A, OxLDL effect on phorbol 12-myristate 13-acetate/ionomycin-induced expression of interferon-γ(IFN-γ) and interleukin (IL)-8 in untransfected Jurkat T (JKwt) and Jurkat T cells stably expressing CD69 (JKCD69) cells. Data correspond to fold induction of mRNA levels (IFN-γ, n=6; IL-8, n=4) analyzed with the Wilcoxon signed-rank test. *P<0.05. B through D, Binding of oxLDL to CD69 blocks Th1 and Th17 differentiation and promotes regulatory T (Treg) cells. Human CD4+ T-cell differentiation was carried out in the absence (control) or presence of oxLDL (50 µg/mL) or oxLDL+anti-CD69. After corresponding days of culture, the percent of IFN-γ+ T cells (B), percent of IL-17+ T cells (C), and percent of CD25+Foxp3+ T cells (D) were determined by flow cytometry. Data correspond to 4 independent experiments, and bars rep- resent mean±SEM of percent of positive cells. Data were analyzed with the Friedman test and Dunn posttest. *P<0.05 vs control. E, OxLDL induces NR4A1 and NR4A3 expression in activated human CD4+ T cells. Wilcoxon signed-rank test, *P<0.05 (n=4). F, Expression of NR4A3 in JKCD69 and JKwt cells treated (Continued ) 250 January 8, 2019 Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis ORIGINAL RESEARCH ARTICLE Figure 3 Continued. with oxLDL. Fold induction vs baseline (t=0). Data are mean±SD (n= 6) and were analyzed with 2-way ANOVA. ** P<0.0001, * P<0.01 vs JKwt at each time. G, Anti-CD69 inhibits oxLDL-induced NR4A3 expression in JKCD69 cells. Data are mean±SD (n= 6). Differences among groups were tested with the Kruskal-Wallis test. **P<0.001, *P<0.01 vs control. #P<0.02 vs oxLDL. H, shRNA silencing of CD69 expression blocks oxLDL-induced NR4A3 expression in JKCD69 cells. Data are mean±SD (n= 6). Data were analyzed with 2-way ANOVA. ** P<0.0001 vs shRNA CD69. I, mRNA levels of the indicated nuclear receptors in para-aortic lymph nodes from total bone marrow (BM) chimeras at 13 weeks after a high fat diet (HFD). n=5 mice per group. Error bars show SEM. *P<0.05, ***P<0.001 as determined by unpaired t test. J, CD69 and NR4A1 mRNA relative expression in blood lymphocytes from ldlr−/− mice at different weeks after an HFD. Error bars show SEM. */&P<0.05, **/&&P<0.01, ***/&&&P<0.001 as determined by 1-way repeated-measures ANOVA. The Dunnett multiple-comparison test was performed between values at 5 to 11 weeks after an HFD vs 3 weeks after an HFD for CD69 (*) and NR4A1 (&) individually. AU indicates arbitrary units. the control group (no disease) versus generalized dis- downregulation of CD69 expression on T lymphocytes ease group and examined whether CD69 can be used in mice on exposure to HFD. Despite a suggested role as independent marker for subclinical atherosclerosis. for oxLDL in adaptive immune responses, a putative re- Downloaded from http://ahajournals.org by on November 6, 2020 After a univariable logistic regression analysis (Table II ceptor on T lymphocytes has remained elusive.32 Our in the online-only Data Supplement) of relevant vari- study demonstrates that binding of oxLDL to CD69 in ables (known cardiovascular risk factors: age, smoking, human T cells has a protective effect against the in- BMI, hypercholesterolemia, hypertension, diabetes mel- flammatory response through the expression of NR4A litus, family history of cardiovascular disease; peripheral nuclear receptors, downregulating proinflammatory cy- count of different subsets of leucocytes; expression of tokines and promoting Treg differentiation. NR4A1 and the levels of oxLDL and C-reactive protein), Although the classic view is that oxLDL induces the a multivariable logistic regression analysis determined recruitment of inflammatory cells to the subendo- that CD69 expression remained an independent predic- thelial space, cells and tissues also respond to oxLDL tor of atherosclerosis at an early stage (odds ratio, 0.62; through the inhibition of proinflammatory signaling P=0.0056; Table). Furthermore, CD69 levels correlated pathways.33,34 The NR4A subfamily of human nuclear significantly with NR4A1 levels (Figure 4D and Table III receptors (NR4A1 [Nur77], NR4A2 [Nurr1] and NR4A3 in the online-only Data Supplement), supporting the [NOR-1]) can be induced in endothelial and smooth notion of a common regulation pathway. muscle cells by a range of stimuli (including oxLDL), regulating the expression of different molecules in- volved in the immune response.35 NR4A overexpres- DISCUSSION sion decreases the levels of IL-1β, IL-8, and monocyte Our findings indicate that the absence of CD69 in the chemoattractant protein-1 inflammatory cytokines. LC results in larger atheroma plaque formation in ldlr−/− Furthermore, NR4A1 and NR4A3 have been impli- deficient mice subjected to an HFD. We identify the cated in Treg differentiation.23,24 Our data show that functional interaction between CD69 and oxLDL as the binding of oxLDL to CD69 in human T cells induces the mechanism responsible for the observed phenotype. expression of NR4A receptors. Moreover, the absence Our clinical data support this concept because CD69 of CD69 during atherosclerosis development results in expression in peripheral leucocytes of subjects with sub- a lower expression of NR4A1 and NR4A3 in both PBLs clinical atherosclerosis is downregulated compared with and para-aortic LNs. The observation of the regulatory individuals without atherosclerosis. This finding was in effect of CD69/oxLDL interaction in human Treg dif- agreement with the experimental evidence showing a ferentiation, together with the loss of Treg responses Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 January 8, 2019 251 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis ORIGINAL RESEARCH ARTICLE Figure 4. Individuals with subclinical atherosclerosis express low levels of CD69 and NR4A1. A, mRNA expression of CD69 and NR4A1 is presented as mean±SEM in linear scale. Significance of the trend across groups was calculated with the Cuzick extension of the Wilcoxon rank-sum test to assess the significance of the trend across the 3 ordinal groups allowing for ties. B, mRNA levels of CD69 in peripheral blood leukocytes from healthy subjects (no disease [ND], n=122) and individuals with subclinical atherosclerosis (focal disease [FD], n=55; generalized disease [GD], n=128). C, mRNA levels of NR4A1 as in B. Data are expressed in log2 scale and were analyzed with 1-way ANOVA followed by Tukey posttests. * P<0.05. **P<0.005. D, Correlation between the expression of CD69 and NR4A1. The Pearson correlation test was used. Downloaded from http://ahajournals.org by on November 6, 2020 observed in lymphocyte CD69-deficient mice during through CXCL1 upregulation,11,40 the former seems not atherosclerosis development, suggests that the modu- to be the case in our model. Increased amounts of IL-17 lation of NR4A nuclear receptors could participate in seem to stabilize plaque formation when levels of IL- CD69 signaling/Treg differentiation. The more exten- 10 are also higher, namely in the presence of a proper sive atherosclerosis developed in the absence of CD69 could be associated, at least in part, with defects in Table. Multivariable Logistic Regression Comparing Individuals Treg cell differentiation. Treg cells exert an atheropro- With Generalized Subclinical Atherosclerosis and Individuals With No tective function through the suppression of T-cell pro- Disease liferation and secretion of anti-inflammatory cytokines Odds Ratio (95% CI) P Value and are protective during the initial phases of athero- log2(CD69) 0.62 (0.40–0.94) 0.0056 sclerosis not only by reducing atherosclerotic plaque log2(NR4A1) 1.12 (0.80–1.58) 0.0441 formation but also by improving stabilization of the atherosclerotic lesions.36–38 Age 1.21 (1.11–1.33) 0.0000 Besides promoting Treg cell function, CD69 controls Familial hypercholesterolemia 2.06 (0.90–4.87) 0.0630 Th17 differentiation through the association of its cy- Hypercholesterolemia 1.13 (0.58–2.22) 0.1168 toplasmic tail with the Jak3/Stat5 signaling pathway, Smoking 2.16 (1.00–4.87) 0.0027 regulating RAR-related orphan receptor-γ transcription Monocytes 0.06 (0.00–1.61) 0.4733 and differentiation toward the Th17 lineage.15 Recent Lymphocytes 1.57 (0.66 –3.82) 0.0025 evidence for the role of IL-17 in atherosclerosis has shed Leukocytes 1.61 (1.11–2.41) 0.0079 little light on the subject in both proatherogenic and antiatherogenic roles.39,40 Under HFD, ldlr−/− mice defi- Diabetes mellitus* Not applicable 0.0246 cient for CD69 in the LC developed exacerbated Th17 Oxidized low-density lipoprotein 1.02 (1.00–1.04) 0.0457 responses and more severe atherosclerotic lesions, sup- All covariates with a value of P<0.1 in the univariable analysis (Table II in the porting the role of IL-17 as a proatherogenic molecule. online-only Data Supplement) were considered. Despite previous reports supporting that IL-17 can ei- *P values were calculated with the likelihood ratio test to accommodate groups with 0 counts (diabetes mellitus), for which odds ratio estimates could ther stabilize the plaque through collagen production not be calculated. Odds ratio and CIs were estimated with a generalized linear or enhance the recruitment of proatherogenic cells model. 252 January 8, 2019 Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis regulatory response.39 In our model, however, we have Recent data emphasized the link between the in- ORIGINAL RESEARCH a concomitant increase of IL-17 and defective Treg de- flammatory response and atherosclerotic risk in the velopment and function. clinical arena.2 However, the complex interplay be- ARTICLE Previous results indicated that the regulatory action tween lipid metabolism and immune responses re- of CD69 during atherosclerosis was lost in ApoE−/− mains to be fully disclosed. Collectively, our in vivo mice because, compared with the double-knockout data strongly indicate an important role for CD69 ex- group (Cd69−/−ApoE−/−), no significant difference was pression on lymphocytes during atherosclerosis devel- observed in plaque formation.20 LDLr−/− and ApoE−/− opment. To validate this new paradigm in humans, we mice have been extensively used to study the mecha- assessed the expression of CD69 and NR4A in PBLs nisms of atherosclerosis development but feature im- from a cohort of subjects with thoroughly character- portant differences, for example, in plasma proteins. ized subclinical atherosclerosis. It is important to point The major accumulating lipoprotein in the plasma of out that all PESA participants included in the study LDLr−/− mice fed a high-cholesterol diet is LDL. Con- were asymptomatic and free of events. The profile of versely, ApoE−/− mice accumulate cholesterol mostly in CD69 expression detected in human samples was very the very-low-density lipoprotein and chylomicron frac- similar to that observed in the in vivo model. If we con- tions.41,42 This is a very important issue to be consid- sider focal disease and generalized disease as differ- ered that could account for the differences observed ent stages with the same pathology, our data indicate between CD69−/−/apoE−/− and CD69−/−/Ldlr−/− models. that expression of CD69 gradually declines as disease Our data show that oxLDL binding to CD69 exerts an progresses. Hence, we focused on the generalized dis- immune-regulatory function during atherosclerosis ease group, which is still preclinical, to assess the as- development. In the ApoE knockout model, the levels sociation of the expression of CD69 with subclinical of LDL may not have reached the threshold that is re- atherosclerosis, accounting for the effect of traditional quired for signaling through CD69. An additional dif- risk factors, peripheral counts of different subsets of ference that should be taken into consideration is that leucocytes, expression of NR4A1, and levels of oxLDL. Ldlr−/− lesions have higher T-cell density than the ApoE The proof of clinical study more closely resembling the model,43 meaning that Ldlr−/− mice have more T cells animal experiments was to compare subjects free of that could express CD69 to exert their function. Finally, disease or no disease versus those with established ApoE−/− mice on an HFD develop plaques more rapidly but yet subclinical atherosclerosis or generalized sub- and exhibit larger aortic lesions with larger necrotic clinical disease. CD69 remained significantly associ- Downloaded from http://ahajournals.org by on November 6, 2020 cores than Ldldr−/− mice41,42; therefore, this high-inten- ated with the extent of the disease after adjustment sity model could be masking the differences between for risk factors. This finding underscores the putative the CD69-expressing and CD69-deficient animals. We role of CD69 as a potential marker for the detection of found a significant decrease in the blood lipid profile atherosclerosis at a preclinical stage. of free fatty acids, triglycerides, high-density lipopro- The fact that atherosclerosis presence is well identi- tein, LDL, and cholesterol in the Cd69−/− lymphoid chi- fied in a context different from acute conditions (eg, meras. This “dissociation” of the immune profile of myocardial infarction) supports the causative role of the organism from the metabolic parameters is quite CD69 expression on atherosclerosis development rather intriguing; it seems to occur via mechanisms related than a consequence of an acute event. mainly to the adaptive immune responses (in this case, lack of CD69 and Th17 propensity). Our data pave the way for further research on mechanisms that could ARTICLE INFORMATION contribute to atherogenesis. Received February 13, 2018; accepted July 20, 2018. The online-only Data Supplement is available with this article at https://www. Our results describe for the first time an oxLDL re- ahajournals.org/doi/suppl/10.1161/circulationaha.118.034326. ceptor on lymphocytes with an important function in the regulation of the adaptive immune response Correspondence and atheroma plaque formation during an HFD. The Francisco Sánchez-Madrid, PhD, Immunology Department, Hospital Universita- chimeric ldlr−/− mouse models used shed light on the rio de la Princesa, Diego de León 62, 28006 Madrid, Spain, or Pilar Martin, Vas- role of CD69/oxLDL functional interaction in lympho- cular Pathophysiology Area, Centro Nacional de Investigaciones Cardiovascu- lares, Melchor Fernández Almagro 3, 28029 Madrid, Spain. Email fsmadrid@ cytes and on the maintenance of immune homeo- salud.madrid.org or pmartinf@cnic.es stasis to protect medium and large arteries from se- vere atheroma plaque formation over time. Further Affiliations studies of the new regulatory oxLDL/CD69 pair in Vascular Pathophysiology Area (K.T., M.R., R.S.-D., V.F., P.M., F.S.-M.), Bioinfor- human lymphoid cells during atherosclerotic disease matics Unit (F.S.-C.), Genomics Unit (A.D.), Proteomics Unit (J.V.), and Myocar- progression will provide novel insight into targeting dial Pathophysiology Area (B.I.), Centro Nacional de Investigaciones Cardiovas- culares Carlos III, Madrid, Spain. Department of Immunology (H.d.L.F., J.L.A.-L., these pathways for the prognosis/treatment of car- A.V., F.S.-M.) and Department of Cardiology (F.A.), Instituto de Investigación diovascular diseases. Sanitaria Hospital de la Princesa, IIS-IP, Madrid, Spain. Instituto de Investigacio- Circulation. 2019;139:243–255. DOI: 10.1161/CIRCULATIONAHA.118.034326 January 8, 2019 253 Tsilingiri et al CD69/oxLDL Regulates Atherosclerosis nes Biomédicas de Barcelona, IIB-Sant Pau, Spain (J.M.-G.). Institut de Recerca 8. Ge S, Hertel B, Koltsova EK, Sörensen-Zender I, Kielstein JT, Ley K, del Hospital de la Santa Creu i Sant Pau-Programa ICCC, IIB-Sant Pau, Barcelo- Haller H, von Vietinghoff S. Increased atherosclerotic lesion formation ORIGINAL RESEARCH na, Spain (C.R., J.C.). Centro Nacional de Biotecnología, Madrid, Spain (J.M.C.). and vascular leukocyte accumulation in renal impairment are medi- IIS-Fundación Jiménez Díaz University Hospital, Madrid, Spain (B.I.). Cardiovas- ated by interleukin-17A. Circ Res. 2013;113:965–974. doi: 10.1161/ ARTICLE cular Institute, Icahn School of Medicine at Mount Sinai, New York, NY (V.F.). CIRCRESAHA.113.301934 CIBER de Enfermedades Cardiovasculares, Madrid, Spain (H.d.L.F., R.S.-D., C.R., 9. Smith E, Prasad KM, Butcher M, Dobrian A, Kolls JK, Ley K, Galkina E. J.V., B.I., J.M.-G, P.M., F.S.-M.). Blockade of interleukin-17A results in reduced atherosclerosis in apoli- poprotein E-deficient mice. Circulation. 2010;121:1746–1755. doi: 10.1161/CIRCULATIONAHA.109.924886 Acknowledgments 10. Taleb S, Romain M, Ramkhelawon B, Uyttenhove C, Pasterkamp G, Herbin The authors are grateful to Irene García Fernández for technical assistance and O, Esposito B, Perez N, Yasukawa H, Van Snick J, Yoshimura A, Tedgui A, Belén Oliva for statistical support. The authors thank the group of Prof Almude- Mallat Z. Loss of SOCS3 expression in T cells reveals a regulatory role for na Ramiro for constructive comments and technology transfer. Finally, they interleukin-17 in atherosclerosis. J Exp Med. 2009;206:2067–2077. doi: thank Dr Richard Flavell for the dRep mice and Dr Miguel Vicente-Manzanares 10.1084/jem.20090545 for critical reading and editing of the manuscript. 11. Danzaki K, Matsui Y, Ikesue M, Ohta D, Ito K, Kanayama M, Kurotaki D, Morimoto J, Iwakura Y, Yagita H, Tsutsui H, Uede T. Interleukin-17A deficiency accelerates unstable atherosclerotic plaque formation in apoli- Sources of Funding poprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2012;32:273– Funding was provided by the Spanish Ministry of Economy and Competi- 280. doi: 10.1161/ATVBAHA.111.229997 tiveness: Plan Nacional de Salud SAF2017-82886-R to Dr Sánchez-Madrid, 12. Potekhina AV, Pylaeva E, Provatorov S, Ruleva N, Masenko V, Noeva E, Kras- SAF2015-64767-R to Dr Martínez-González; Instituto de Salud Carlos III nikova T, Arefieva T. Treg/Th17 balance in stable CAD patients with differ- (AES 2016): PI16/01956 to Dr Martin, Centro de Investigación Biomédica en ent stages of coronary atherosclerosis. Atherosclerosis. 2015;238:17–21. Red de Enfermedades Cardiovasculares; European Research Council, ERC- doi: 10.1016/j.atherosclerosis.2014.10.088 2011-AdG294340-GENTRIS to Dr Sánchez-Madrid; Proyecto Integrado de 13. Li Q, Wang Y, Wang Y, Chen K, Zhou Q, Wei W, Wang Y. Treg/Th17 ratio Excelencia PIE13/041 and Fundació La Marató TV3 (20152330 31); and Co- acts as a novel indicator for acute coronary syndrome. Cell Biochem Bio- munidad Autónoma de Madrid CAM (S2017/BMD-3671) to Drs Martin and phys. 2014;70:1489–1498. doi: 10.1007/s12013-014-9993-5 Sánchez-Madrid. Dr Tsilingiri is cofunded by the European Union Marie Curie 14. Sanchez-Diaz R, Blanco-Dominguez R, Lasarte S, Tsilingiri K, Martin-Gayo Program. M. Relaño is supported by a Contratos Predoctorales Severo Ochoa E, Linillos-Pradillo B, de la Fuente H, Sanchez-Madrid F, Nakagawa R, To- para la formación de doctores (BES-2015–072625) from the Spanish Ministry ribio ML, Martin P. Thymus-derived regulatory t cell development is regu- of Economy and Competitiveness. This research has been cofinanced by Fondo lated by C-type lectin-mediated Bic/microRNA 155 expression. Mol Cell Europeo de Desarrollo Regional. Centro Nacional de Investigaciones Cardio- Biol. 2017;37: e00341–e00346. vasculares (CNIC), Madrid, Spain, is supported by the Ministerio de Ciencia, In- 15. Martín P, Gómez M, Lamana A, Cruz-Adalia A, Ramírez-Huesca M, Ursa novación y Universidades, and the Pro CNIC Foundation and is a Severo Ochoa MA, Yáñez-Mo M, Sánchez-Madrid F. CD69 association with Jak3/Stat5 Center of Excellence (SEV-2015-0505). The PESA study is cofunded equally by proteins regulates Th17 cell differentiation. Mol Cell Biol. 2010;30:4877– the Pro CNIC Foundation and Banco Santander, Madrid, Spain. 4889. doi: 10.1128/MCB.00456-10 16. Cortés JR, Sánchez-Díaz R, Bovolenta ER, Barreiro O, Lasarte S, Matesanz- Marín A, Toribio ML, Sánchez-Madrid F, Martín P. Maintenance of immune Disclosures tolerance by Foxp3+ regulatory T cells requires CD69 expression. J Auto- Downloaded from http://ahajournals.org by on November 6, 2020 None. immun. 2014;55:51–62. doi: 10.1016/j.jaut.2014.05.007 17. 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