Author + information
- Received April 26, 2017
- Revision received October 15, 2017
- Accepted October 30, 2017
- Published online January 8, 2018.
- Lynda Zeboudj, MSa,b,
- Mikael Maître, MSa,b,
- Lea Guyonnet, PhDa,b,
- Ludivine Laurans, MSa,b,
- Jeremie Joffre, MD, PhDa,b,
- Jeremie Lemarie, MD, PhDa,b,
- Simon Bourcier, MDa,b,
- Wared Nour-Eldine, MSa,b,
- Coralie Guérin, PhDc,
- Jonas Friard, MSd,
- Abdelilah Wakkach, PhDd,
- Elizabeth Fabre, MDe,
- Alain Tedgui, PhDa,b,
- Ziad Mallat, MD, PhDa,b,f,
- Pierre-Louis Tharaux, MD, PhDa,b and
- Hafid Ait-Oufella, MD, PhDa,b,g,∗ ()
- aInserm U970, Paris Cardiovascular Research Center, Paris, France
- bUniversité René Descartes, Paris, France
- cLuxembourg Institute of Health, Department of Infection and Immunity, Strassen, Luxembourg
- dCNRS, LP2M, UMR 7370, Faculté de Médecine, Université de Nice Sophia Antipolis, Nice, France
- eDepartment of Medical Oncology, Hôpital Europeen G. Pompidou, AP-HP, Paris, France
- fDivision of Cardiovascular Medicine, Department of Medicine, University of Cambridge, Cambridge, United Kingdom
- gService de Réanimation Médicale, Hôpital Saint-Antoine, AP-HP, Université Pierre-et-Marie Curie, Paris, France
- ↵∗Address for correspondence:
Dr. Hafid Ait-Oufella, Inserm U970, Paris Cardiovascular Research Center, Université René Descartes, 56, rue Leblanc, Paris 75012, France.
Background Several epidermal growth factor receptor (EGFR) inhibitors have been successfully developed for the treatment of cancer, limiting tumor growth and metastasis. EGFR is also expressed by leukocytes, but little is known about its role in the modulation of the immune response.
Objectives The aim of this study was to determine whether EGFR expressed on CD4+ T cells is functional and to address the consequences of EGFR inhibition in atherosclerosis, a T cell–mediated vascular chronic inflammatory disease.
Methods The authors used EGFR tyrosine kinase inhibitors (AG-1478, erlotinib) and chimeric Ldlr-/-Cd4-Cre/Egfrlox/lox mouse with a specific deletion of EGFR in CD4+ T cells.
Results Mouse CD4+ T cells expressed EGFR, and the EGFR tyrosine kinase inhibitor AG-1478 blocked in vitro T cell proliferation and Th1/Th2 cytokine production. In vivo, treatment of Ldlr–/– mice with the EGFR inhibitor erlotinib induced T cell anergy, reduced T cell infiltration within atherosclerotic lesions, and protected against atherosclerosis development and progression. Selective deletion of EGFR in CD4+ T cells resulted in decreased T cell proliferation and activation both in vitro and in vivo, as well as reduced interferon-γ, interleukin-4, and interleukin-2 production. Atherosclerotic lesion size was reduced by 2-fold in irradiated Ldlr–/– mice reconstituted with bone marrow from Cd4-Cre/Egfrlox/lox mice, compared to Cd4-Cre/Egfr+/+ chimeric mice, after 4, 6, and 12 weeks of high-fat diet, associated with marked reduction in T cell infiltration in atherosclerotic plaques. Human blood T cells expressed EGFR and EGFR inhibition reduced T cell proliferation both in vitro and in vivo.
Conclusions EGFR blockade induced T cell anergy in vitro and in vivo and reduced atherosclerosis development. Targeting EGFR may be a novel strategy to combat atherosclerosis.
Epidermal growth factor receptor (EGFR) is a cell membrane–bound receptor with tyrosine kinase activity involved in the control of major signaling pathways, including cell survival, proliferation, and migration. EGFR overexpression, autocrine ligand stimulation, or constitutively active receptor mutants (1,2) can lead to dysregulation of this fine-tuned signaling system, resulting in a variety of pathophysiological disorders and promoting cancer development. Six EGFR ligands have been described, including epidermal growth factor (EGF), Heparin Binding-EGF, amphiregulin, and transforming growth factor–α. Extracellular ligand binding causes dimerization of EGFR, which becomes autophosphorylated at distinct tyrosine residues. In addition, EGFR could be transactivated in the absence of a specific ligand through G protein–coupled receptor activation (3).
EGFR has been extensively explored in cancer. Human and experimental studies have shown that EGFR activation on tumor cells ultimately leads to cell proliferation, invasion, and migration, as well as promoting angiogenesis and inhibiting apoptosis (4). Targeting of EGFR by either neutralizing monoclonal antibodies or small-molecule tyrosine kinase inhibitors (TKIs) have been shown during the past 10 years to be a successful therapeutic strategy in cancer setting (5,6). However, EGFR expression and function have been poorly investigated in nontumoral cells. Some investigators have described expression in circulating leukocytes (7,8), but little is known about EGFR’s role in modulation of the immune response.
Atherosclerosis is an inflammatory disease driven by innate and adaptive immunity, in which CD4+ T cells play a pathogenic role. Interestingly, EGFR ligands, including heparin-binding EGF, have been detected in human atherosclerotic plaques (9). The aim of this study was to ascertain the expression of EGFR in human and mouse CD4+ T cells and to investigate the effects of EGFR blockade on CD4+ T cell functions using pharmacological inhibitors and cell-specific genetic deletion in mouse models of atherosclerosis.
Experiments were conducted according to the guidelines formulated by the European Community for experimental animal use (L358-86/609EEC) and were approved by the ethics committee of INSERM and the French Ministry of Agriculture (agreement A75-15-32). To generate a cell-specific knockout of Egfr in CD4+ T cells, we crossbred mice carrying a Cd4Cre allele with mice carrying a floxed Egfr allele. All animals have been backcrossed more than 10 generations on C57bl/6 background. Ten-week-old male C57BL/6 Ldlr−/− mice were put on a high-fat diet for 8 weeks and were treated orally (daily gavage) with the specific EGFR TKI erlotinib (15 mg/kg/day). For bone marrow transplantation experiments, 10-week-old male C57bl/6 Ldlr−/− mice were subjected to medullar aplasia by lethal total body irradiation (9.5 Gy). The mice were repopulated with an intravenous injection of bone marrow cells isolated from femurs and tibias of sex-matched C57BL/6 Cd4Cre Egfr+/+ mice or Cd4Cre Egfrlox/lox littermates. After 4 weeks of recovery, mice were fed a proatherogenic diet containing 15% fat, 1.25% cholesterol, and 0% cholate for 4, 6, and 12 weeks.
Extent and composition of atherosclerotic lesions
Plasma cholesterol was measured using a commercial cholesterol kit (Biomérieux, Marcy-l’Étoile, France). The heart was removed, and successive 10-μm transversal sections of aortic sinus were obtained. Lipids were detected using Red Oil staining. The presence of T cells was studied using specific antibodies as previously described (polyclonal anti-CD3, Agilent, Santa Clara, California) (10). Egfr was detected in cells and lesions using rabbit polyclonal anti-phospho-Egfr (Cell Signaling, Boston, Massachusetts). For human staining, an anti-EGFR antibody (clone 31G7, AbCys, Paris, France) was used. At least 4 sections per mouse were examined for each immunostaining, and appropriate negative controls were used. Morphometric studies were performed using HistoLab software (Microvisions, Evry, France) (10).
Spleen cell recovery and purification
Spleen cells were purified according to standard protocols as follows. CD4+ T cells were negatively selected using a cocktail of antibody-coated magnetic beads from Miltenyi Biotec (Bergisch Gladbach, Germany) (anti-CD8a, anti-CD11b, anti-CD45R, anti-DX5, and anti-ter 119), according to the manufacturer’s instructions, yielding CD4+ cells with >95% purity. CD11c+ cells were positively selected with biotin-conjugated anti-CD11c monoclonal antibody (7D4, BD Pharmingen, Franklin Lakes, New Jersey), streptavidin microbeads (Miltenyi Biotec), followed by 2 consecutive magnetic cell separations using LS columns (Miltenyi Biotec), yielding CD11c+ cells with >80% purity.
CD4+ T cell culture and cytokine assays
Cells were cultured in RPMI-1640 supplemented with GlutaMAX (Thermo Fisher Scientific, Waltham, Massachusetts), 10% fetal calf serum, 0.02 mmol/l β-mercaptoethanol, and antibiotics. For cytokine measurements, CD4+ T cells were cultured at 1 × 105 cells/well for 48 h in anti-CD3-coated microplates (10 μg/ml) or with concanavalin A (10 μg/ml; Sigma-Aldrich, St. Louis, Missouri). In some experiments, CD4+ T cells were stimulated with purified soluble CD3-specific antibody (1 μg/ml; BD Pharmingen) in the presence of antigen-presenting cells purified on CD11c-coated magnetic beads (Miltenyi Biotec). Interleukin (IL)–2, IL-4, IL-10, and interferon (IFN)–γ productions in the supernatants were measured using specific enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, Minnesota).
Other methods (cell culture, proliferation assays, cytosolic calcium recording, flow cytometry, and western blot) are available in the Online Appendix.
Values are expressed as median (interquartile range [IQR]). Differences between values were evaluated using the nonparametric Mann-Whitney U test or the Kruskal-Wallis test. A p value of <0.05 was considered to indicate statistical significance. All of these analyses were performed using GraphPad Prism version 5.0b for Mac (GraphPad Software, La Jolla, California). No adjustments were made for multiple pairwise comparisons.
On the basis of preliminary experiments, we assumed that EGFR blockade induces a 40% reduction of atherosclerosis. With a standard deviation of plaque size estimated at 30%, the inclusion of 8 mice per group was sufficient to detect a significant difference between groups with 80% power.
EGFR in mouse CD4+ T cells: Expression and signaling pathways
Using immunocytostaining, we detected EGFR expression by splenocytes and found that EGFR colocalized, nonexclusively, with CD4+ T cells (Figure 1A). In addition, EGFR was present in mouse atherosclerotic lesions and colocalized with CD4+ T cells (Figure 1B). In vitro, anti-CD3-induced and concanavalin A–induced activation of purified CD4+ T cells caused EGFR phosphorylation after 60 min of stimulation (Figure 1C). AG-1478, a pharmacological inhibitor of tyrosine kinase, blocked EGFR phosphorylation in stimulated T cells (Figure 1C) and significantly reduced ERK1/2 phosphorylation (Figure 1D, Online Figure 1) but had no effect on AKT (Online Figure 1). In addition, we found that EGFR inhibition blocked cytoplasmic calcium increase following anti-CD3/CD28 stimulation (Figure 1E). In summary, anti-CD3 stimulation of CD4+ T cells led to EGFR transactivation that was efficiently inhibited by AG-1478.
Effects of pharmacological inhibition of EGFR on atherosclerosis
To address the role of EGFR on T cell functions, splenic CD4+ T cells were purified and stimulated in vitro. EGFR activity was inhibited using AG-1478 at different concentrations. EGFR inhibition significantly reduced T cell proliferation following non-antigen-specific (Figure 2A) and antigen-specific (Figure 2B) stimulation in a dose-dependent manner, without any effect on T cell apoptosis (Figure 2C) or nucleus-cytoplasm organization (Online Figure 2). Pharmacological inhibition of EGFR significantly reduced intracellular IFN-γ production by CD4+ T cells in response to anti-CD3 (Figure 2D) and concanavalin stimulation (Figure 2E). This was confirmed by enzyme-linked immunosorbent assay in the supernatants of cultured splenic CD4+ T cells, showing that EGFR inhibition significantly reduced the production of IFN-γ (Figures 2F and 2G) and reduced the production of IL-2 (Online Figure 3), as well as that of IL-4 (Figure 2H), in a dose-dependent manner but had no effect on IL-10 production, which was very low (Figure 2I).
To investigate the in vivo consequences of EGFR pharmacological inhibition, Ldlr−/− male mice were put on a high-fat diet for 8 weeks and were orally treated with erlotinib (15 mg/kg/day) (Figure 3A). At sacrifice, animal weight (Online Figure 4A) and plasma cholesterol level (Figure 3B) were not different between groups, but there were significant reductions in spleen size (Online Figure 4B) and splenocyte number (Figure 3C) in the erlotinib-treated group. Splenic CD4+ T cells from erlotinib-treated group were characterized by significant decreases in CD25 (Online Figure 5A) and CD44high expression (Online Figure 5B), suggesting reduced in vivo activation. Erlotinib had no effect on the CD4+CD25highFoxp3+ regulatory T cell population (Online Figure 5C). Splenic purified CD4+ T cells from the erlotinib-treated group were characterized by a reduction of proliferation ex vivo (Figure 3D) and a reduction of T helper (Th) 1 (IFN-γ) and Th2 (IL-4) cytokine production, with no effect on IL-10 (Figure 3E). Immunohistochemistry analysis revealed that erlotinib treatment induced a 70% reduction of T cell infiltration within atherosclerotic lesions (p = 0.01) (Figure 3F), which was associated with a 37% reduction in atherosclerotic lesion size in the aortic sinus (median 167 [IQR: 91 to 190] × 103 in treated mice vs. 267 [IQR: 180 to 333] × 103 μm2 in control mice, p < 0.05) (Figure 3G).
In the vast majority of cases, patients in need of antiatherosclerotic therapy already have established atherosclerotic plaques. Thus, we examined the effects of EGFR blockade on the progression of established atherosclerotic plaques in mice. Six-week-old Ldlr−/− female mice were put on a high-fat diet for 8 weeks and then were orally treated with a placebo or erlotinib (15 mg/kg/day) for 8 weeks (Figure 3H). At sacrifice, animal weight (data not shown) and plasma cholesterol levels (Online Figure 6C) were not different between groups. In the erlotinib-treated group, we observed a marked reduction in atherosclerotic lesion size both in the aortic sinus (median 176 [IQR: 159 to 218] × 103 vs. 269 [IQR: 193 to 343] × 103 μm2, p < 0.05) (Online Figures 6A and 6B) and along the thoracic aorta (median 14.0% [IQR: 12.4% to 14.9%] vs. 20.7% [IQR: 15.2% to 22.3%], p < 0.05) (Figure 3I). More important, atherosclerosis plaque size increased between baseline 14-week-old mice and 22-week-old mice in the placebo group but did not progress in the group receiving erlotinib (Figure 3I, Online Figure 6), suggesting that erlotinib treatment blocked atherosclerosis progression.
EGFR in human CD4+ T cells: Expression and functions
To evaluate the clinical relevance of our findings, we investigated the expression of EGFR in human blood T cells. We performed immunocytostaining of purified blood CD4+ T cells and found that EGFR was expressed and clustered in the membrane of CD4+ T cells after concanavalin-induced activation (Figure 4A). To investigate the functions of human EGFR, we purified circulating CD4+ T cells from healthy donors and performed in vitro proliferation tests. CD3/CD28-coated beads stimulation induced T cell proliferation and EGFR inhibition using AG-1478 or cetuximab, an EGFR-neutralizing monoclonal antibody, significantly decreased T cell proliferation (Figures 4B and 4C). This result was confirmed in vivo in patients with lung cancer. Circulating T cells were isolated from 3 patients before and 1 month after oral erlotinib treatment. Interestingly, T cell proliferation was lower after erlotinib treatment (Figure 4D).
Cell-specific genetic invalidation of Egfr in CD4+ T cells
Erlotinib administration induced T cell anergy and reduced atherosclerosis development. As the expression of EGFR is ubiquitous, we next assessed the specific role of EGFR activation in CD4+ T cells in erlotinib-induced atheroprotection. We bred mice carrying a Cd4-Cre allele with mice carrying a floxed Egfr allele and generated Cd4-Cre/Egfrlox/lox mice. The deletion of EGFR specifically in CD4+ T cells was confirmed by immunocytostaining (Online Figure 7). We purified splenic CD4+ T cells from control Cd4-Cre/Egfr+/+ and Cd4-Cre/Egfrlox/lox mice and performed functional tests. In vitro, in agreement with experiments using AG-1478, proliferation of CD4+ T cells from Cd4Cre Egfrlox/lox mice was significantly decreased compared with wild-type cells (Figure 5A), and their production of Th1 (Figure 5B) and Th2 (Figure 5C) cytokines was reduced. There was no difference in IL-10 production (Figure 5D). Similar reduction in cell proliferation and cytokine production was observed when EGFR-deficient CD4+ T cells were coincubated with CD11c+ antigen–presenting cells (Figures 5A to 5D). Intracellular staining by flow cytometry confirmed the specific reduction of IFN-γ production by CD4+ (Figure 5E) but not by CD8+ T cells. Apoptosis susceptibility was significantly lower in EGFR-deficient CD4+ T cells (Online Figure 8). To address the in vivo role of EGFR in CD4+ T cell proliferation, we transferred into Apoe−/−/Rag2−/− mice 20.106 CD4+ T cell–depleted splenocytes resupplemented with 8.106 purified carboxyfluorescein succinimidyl ester–labeled CD4+ T cells from either Cd4-Cre/Egfr+/+ or Cd4-Cre/Egfrlox/lox mice. The proliferation of adoptively transferred cells was visualized by flow cytometric analysis of carboxyfluorescein succinimidyl ester–labeled CD4+ T cells. At day 10 after transfer, we found that CD4+ T specific deletion of EGFR limited T cell proliferation in the spleen and lymph nodes (Figure 5F).
Impact of CD4+ T cell–specific invalidation of Egfr on atherosclerosis
To address the consequences of these findings in the context of atherosclerosis, we performed bone marrow transplantation experiments using either Cd4-Cre/Egfr+/+ or Cd4-Cre/Egfrlox/lox littermate bone marrow to repopulate lethally irradiated Ldlr−/− mice. After 4 weeks of recovery and additional 4 weeks on a high-fat diet, animals were sacrificed. We did not observe any difference in animal or spleen weights, but a 32% reduction was seen in the number of splenocytes in the chimeric Cd4-Cre/Egfrlox/lox group (p = 0.05). Leukocyte populations (neutrophils, monocytes, B cells, CD8+ T cells) were not different between groups, either in the blood or in the spleen (data not shown). Splenic CD4+ T cell subset was not different between chimeric Cd4-Cre/Egfr+/+/Ldlr−/− and Cd4-Cre/Egfrlox/lox/Ldlr−/− mice (Figure 6A), but the CD4+CD25highFoxP3+ regulatory T (Treg) cell population was slightly reduced in the group with CD4-specific deletion of EGFR (−14%, p < 0.05) (Figure 6B). Splenic CD4+ T cells from Cd4-Cre/Egfrlox/lox/Ldlr−/− were characterized by a significant decrease in CD69 (Figure 6C) and CD44high expression (Figure 6D), suggesting reduced in vivo activation. Functional tests were performed ex vivo in purified splenic CD4+ T cells. The suppressive function of Treg cells was preserved in Cd4-Cre/Egfrlox/lox/Ldlr−/− mice (Figure 6E), but the proliferation of CD4+ T cells in response to CD3 stimulation was reduced compared with cells from the control group (Figure 6F). Finally, we also observed a >2-fold decrease in IFN-γ and IL-4 production in the supernatant of anti-CD3-stimulated CD4+ T cells from Cd4-Cre/Egfrlox/lox/Ldlr−/− mice compared with Cd4-Cre/Egfr+/+/Ldlr−/− mice (Figure 6G).
As shown in Figure 7A, after 4 weeks on a high-fat diet, Egfr invalidation in CD4+ T cells led to a 36% decrease in atherosclerotic lesion size in the aortic sinus compared with controls (median 25 [IQR: 16 to 30] × 103 vs. 34 [IQR: 31 to 57] × 103 μm2 in Cd4-Cre/Egfrlox/lox/Ldlr−/− and Cd4-Cre Egfr+/+/Ldlr−/−, respectively, p = 0.03). The reduction of atherosclerosis was confirmed after 6 weeks (−39%, p = 0.02) and 12 weeks (−43%, p = 0.02) of a high-fat diet (Figure 7A). There were no significant differences in plasma cholesterol levels between the groups (Online Figure 9).
We finally analyzed T cell infiltration within the lesions. We found a decrease in T cell number in lesions of Cd4Cre Egfrlox/lox → Ldlr−/− compared with Cd4Cre Egfr+/+ → Ldlr−/− mice (Figure 6B) but no difference in macrophage infiltration (Online Figure 10).
Using several complementary approaches, we identified a critical role of EGFR in CD4+ T cell homeostasis, in both mice and humans. EGFR genetic invalidation or pharmacological blockade impaired T cell activation, proliferation, cytokine production and reduced atherosclerosis development (Central Illustration).
Using immunocytostaining and immunohistochemistry, we found that splenic CD4+ T cells and blood human T cells express EGFR, especially in response to CD3 or concanavalin A stimulation. Immunofluorescent staining showed, after anti-CD3 stimulation, that EGFR clustered on the cell membrane and was phosphorylated. In addition, tissue T cells within atherosclerotic plaques express EGFR. Our results are in line with those of Zaiss et al. (8) who detected both Egfr messenger ribonucleic acid expression and EGFR protein in purified CD4+ T cells.
Our findings indicate that EGFR signaling is crucial in CD4+ T cell homeostasis in both humans and mice. Pharmacological inhibition using TKIs (AG-1478 or erlotinib), neutralizing antibodies, or CD4+ T cell–specific genetic invalidation of Egfr markedly reduced in vitro and in vivo cell proliferation and Th1/Th2/Th17 cytokine production. A similar observation has been reported in a mouse model of graft versus host disease, with a reduction of Th1 and Th2 cytokine production in erlotinib-treated animals (11). In addition, we observed that EGFR inhibition and invalidation reduced T cell infiltration within atherosclerotic lesions, suggesting a modulation of T cell migration. This might account for reduced chemokine production, as described in a model of skin inflammation, in which TKI treatment decreased Ccl-17, Ccl-21, and Ccl-27 production (11). Reduction of T cell infiltration in the vascular wall of Cd4Cre Egfrlox/lox animals might also be due to altered cell motility resulting from impairment of cytoskeleton reorganization (12). A large body of evidence in cancer highlighted the role of EGFR signaling in epithelial-mesenchymal transition and invasion or migration of normal and malignant epithelial cells (13). Recently, Tai et al. (14) showed that EGFR/Src-signaling triggers the tyrosine phosphorylation of β4 integrin, which in turn activated focal adhesion kinase, which is involved in cytoskeleton reorganization. In our study, we did not observe any side effect due to EGFR blockade, including survival of erlotinib-treated or Cd4-Cre Egfrlox/lox mice. In addition, we did not find any difference in weight or infection susceptibility between groups. These observations suggest that EGFR inhibition modulates the immune response but does cause full immunosuppression.
EGFR inhibition and invalidation did not affect cell death susceptibility but induced a global CD4+ T cell anergy. The mechanisms of anergy induced by EGFR inhibition likely involved MAP kinase signaling pathway as suggested by reduced Erk phosphorylation in CD4+ T cells treated with AG-1478. This is in agreement with studies by Luo et al. (15) showing that erlotinib caused G0/G1 arrest and suppressed the phosphorylation of c-Raf and Erk in activated T cells. We also showed that EGFR pharmacological blockade negatively affected intracellular calcium signaling in T cells, confirming previous reports on cancer context. Bryant et al. (16) showed on glioma tumor cell lines that TKIs including erlotinib and gefitinib limited the acute cytoplasmic release of calcium from the endoplasmic reticulum in response to EGF.
A recent study suggested that EGFR and 1 of its ligands, amphiregulin, play a specific role in Treg suppressive functions. In mouse models of atherosclerosis, Treg deficiency, obtained by Foxp3, Cd28, or Cd80/86 genetic invalidation, increased T cell activation and accelerated vascular disease (17–19). However, in our study, the genetic invalidation of Egfr in Ldlr−/− chimeric mouse model had no effect on the suppressive function of Treg cells. We observed only a slight reduction in the Treg pool in chimeric Cd4-Cre/Egfrlox/lox/Ldlr−/− compared with Cd4-Cre/Egfr+/+/Ldlr−/− mice. Our results show that EGFR inhibition had a predominant effect on T cell anergy and reduced atherosclerosis development. In vitro, we challenged T cells with anti-CD3 antibody or concanavalin A and observed EGFR phosphorylation, probably through transactivation. Transactivation of EGFR is well documented for a G protein–coupled receptor such as ATR-1, the receptor for angiotensin II (20). This transactivation is mediated by metalloproteinase-dependent release of EGFR ligands, including EGF, transforming growth factor–α, and heparin-binding EGF, from their cell membrane–bound precursors and intermediary signaling molecules, including intracellular Ca2+, protein kinase C, and cytosolic tyrosine kinases such as Src kinases (21).
The expression of EGFR and its ligands has been detected in experimental and human atherosclerosis (9,22). Wang et al. (23) recently reported that EGFR engagement is important for macrophage proatherogenic activity, as its pharmacological inhibition reduced proinflammatory cytokine production, lipid uptake, and oxidative stress. In the present study, we report that global EGFR inhibition and CD4+ T cell–specific deletion of EGFR reduced both atherosclerosis development and progression and induced a less inflammatory plaque phenotype.
Our findings indicate that EGFR inhibitors, widely used in patients with cancer, are unlikely to worsen the risk for cardiovascular disease and further suggest that EGFR may constitute a novel therapeutic target in atherosclerotic disease. The recent positive results of CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study) (24) highlighted that modulating the immune system could be a promising approach to treat atherosclerosis-related cardiovascular diseases.
In this study, we showed that EGFR inhibition reduced both atherosclerosis development and progression in mice. Further studies are required to address the cardiovascular effects of EGFR tyrosine kinase inhibitors in cancer patients.
EGFR is expressed in human and mouse CD4+ T cells. EGFR pharmacological blockade or CD4+ T cell–specific invalidation induced T cell anergy and reduced both atherosclerosis development and progression in mice.
COMPETENCY IN MEDICAL KNOWLEDGE: EGFRs are critical regulators of CD4+ T cell activity in both mice and humans. Pharmacological EGFR blockade by erlotinib or selective invalidation in CD4+ T cells limits the development and progression of atherosclerosis in an experimental model.
TRANSLATIONAL OUTLOOK: More studies are needed to assess the potential therapeutic utility of pharmacological EGFR inhibition as an immunomodulatory approach to preventing complications of atherosclerosis.
This work was supported by Institut National de Santé et de la Recherche Médicale, research grants ANR-08-EBIO-003 from l'Agence Nationale de la Recherche and from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/ ERC grant agreement n°107037 (to Dr. Tharaux) and the British Heart Foundation (to Dr. Mallat). The authors have reported that they have no relationships relevant to the contents of this paper to disclose. Drs. Zeboudj and Maître contributed equally to this work. Drs. Ait-Oufella and Tharaux contributed equally to this work.
- Abbreviations and Acronyms
- epidermal growth factor
- epidermal growth factor receptor
- interquartile range
- low-density lipoprotein
- T helper
- tyrosine kinase inhibitor
- regulatory T
- Received April 26, 2017.
- Revision received October 15, 2017.
- Accepted October 30, 2017.
- 2018 American College of Cardiology Foundation
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