CLINICAL PERSPECTIVE

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(Circulation. 2008;117:1649-1657.)
© 2008 American Heart Association, Inc.

Coronary Heart Disease

Combined Inhibition of CCL2, CX3CR1, and CCR5 Abrogates Ly6C^hi and Ly6C^lo Monocytosis and Almost Abolishes Atherosclerosis in Hypercholesterolemic Mice

Christophe Combadière, PhD^*; Stéphane Potteaux, PhD^*; Mathieu Rodero, MBSc; Tabassome Simon, MD, PhD; Adeline Pezard, MBSc; Bruno Esposito, BSc; Régine Merval, BSc; Amanda Proudfoot, PhD; Alain Tedgui, PhD; Ziad Mallat, MD, PhD

From the Institut National de la Santé et de la Recherche Médicale, Unit 543 (C.C., M.R., A. Pezard) and Unit 689 (S.P., T.S., B.E., R.M., AT., Z.M.), Université Pierre et Marie Curie, Paris 6 (C.C.), Paris, France; and Serono Pharmaceutical Research Institute, Geneva, Switzerland (A. Proudfoot).

Correspondence to Ziad Mallat, MD, PhD, Inserm U689, Centre de Recherche Cardiovasculaire Lariboisière, 41, Bd de la Chapelle, 75010 Paris, France. E-mail mallat{at}larib.inserm.fr

Received October 9, 2007; accepted February 1, 2008.

	Abstract

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Background— Monocytes are critical mediators of atherogenesis. Deletion of individual chemokines or chemokine receptors leads to significant but only partial inhibition of lesion development, whereas deficiency in other signals such as CXCL16 or CCR1 accelerates atherosclerosis. Evidence that particular chemokine pathways may cooperate to promote monocyte accumulation into inflamed tissues, particularly atherosclerotic arteries, is still lacking.

Methods and Results— Here, we show that chemokine-mediated signals critically determine the frequency of monocytes in the blood and bone marrow under both noninflammatory and atherosclerotic conditions. Particularly, CCL2-, CX3CR1-, and CCR5-dependent signals differentially alter CD11b⁺ Ly6G^– 7/4^hi (also known as Ly6C^hi) and CD11b⁺ Ly6G^– 7/4^lo (Ly6C^lo) monocytosis. Combined inhibition of CCL2, CX3CR1, and CCR5 in hypercholesterolemic, atherosclerosis-susceptible apolipoprotein E–deficient mice leads to abrogation of bone marrow monocytosis and to additive reduction in circulating monocytes despite persistent hypercholesterolemia. These effects are associated with a marked and additive 90% reduction in atherosclerosis. Interestingly, lesion size highly correlates with the number of circulating monocytes, particularly the CD11b⁺ Ly6G^– 7/4^lo subset.

Conclusions— CCL2, CX3CR1, and CCR5 play independent and additive roles in atherogenesis. Signals mediated through these pathways critically determine the frequency of circulating monocyte subsets and thereby account for almost all macrophage accumulation into atherosclerotic arteries.

Key Words: atherosclerosis • chemokines • inflammation • leukocytes • monocytes

	Introduction

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Vascular inflammation caused by the accumulation of modified lipids induces the recruitment of leukocytes into the subendothelial space and initiates atherosclerosis.^1–4 Monocytes in particular are critical mediators of this process. Osteopetrotic mice with spontaneous deficiency in monocyte colony-stimulating factor (M-CSF) show a profound reduction in atherosclerosis resulting from a marked decrease in macrophage accumulation within the lesions.⁵ Two major monocyte subsets show differential expression of CCR2 and CX3CR1 receptors. Ly6C^hi monocytes represent the inflammatory subtype. They express high levels of CCR2 but low levels of CX3CR1 (Ly6C^hi CCR2⁺ CX3CR1^lo) and are actively recruited to inflamed tissues, where they give rise to macrophages or antigen-presenting cells.^6–9 This subset dominates hypercholesterolemia-associated monocytosis^10,11 and appears to be recruited into atherosclerotic arteries primarily through CCR2.¹¹ However, atherosclerosis is not abrogated in CCR2-deficient (CCR2^–/–) mice.^12,13 Two potential nonexclusive explanations may be offered for this finding. The first is that, in addition to CCR2, other chemokine receptors significantly contribute to the recruitment of Ly6C^hi CCR2⁺ CX3CR1^lo monocytes into atherosclerotic arteries; this explanation is suggested by a recent report showing that CX3CR1 may contribute to their recruitment.¹¹ CX3CR1^–/–14,15 or CX3CL1^–/–16 mice show a significant reduction in atherosclerosis. However, whether combined CCR2 and CX3CR1 inhibition has additive effects on the trafficking of Ly6C^hi CCR2⁺ CX3CR1^lo monocytes in atherosclerosis remains unknown. The other hypothesis is that other monocyte subset(s) significantly contribute to atherogenesis. Ly6C^lo monocytes represent the noninflammatory subtype. They express high levels of CX3CR1 (Ly6C^lo CCR2^– CX3CR1^hi) and give rise to resident macrophages and myeloid dendritic cells in noninflamed tissues, including liver, spleen, lung, and brain.⁶ Although CX3CR1 is required for the accumulation of Ly6C^lo CCR2^– CX3CR1^hi monocytes in noninflammatory tissues,⁶ recent data unexpectedly indicated that CX3CR1 was dispensable for their recruitment into atherosclerotic arteries,¹¹ suggesting that the mechanisms of the CX3CL1/CX3CR1 pathway to promote lesional macrophage accumulation have yet to be clearly defined. In addition, whatever the monocyte subset and its response to specific chemokines, the current prevailing paradigm accounting for macrophage accumulation in atherosclerosis highlights the role of chemokines in the promotion of local monocyte trafficking between the blood and vessel wall.⁴ Yet, it remains elusive whether particular chemokine signals could mediate atherosclerosis-associated Ly6C^hi and/or Ly6C^lo monocytosis and thereby promote lesion development. In the present study, we hypothesized that different chemokine pathways cooperate to promote the accumulation of different monocyte subsets in atherosclerosis, possibly through the modulation of both local trafficking and systemic monocytosis.

Clinical Perspective p 1657

	Methods

Top Abstract Introduction Methods Results Discussion Conclusions References

 
Mice
CX3CR1-deficient mice (CX3CR1^–/–), apolipoprotein E/CX3CR1–deficient (Apoe^–/–/CX3CR1^–/–) mice, and CCL2–deficient (CCL2^–/–) mice were generated as previously described. All deficient mice were bred onto the B6 background (6 to 8 backcrosses). The CX3CR1^–/–/CCL2^–/– mice were generated by crossing CX3CR1^–/– with CCL2^–/– mice and by subsequent intercrossing of heterozygous F1 animals; the Apoe^–/–/CX3CR1^–/–/CCL2^–/– mice were generated by crossing CX3CR1^–/–/CCL2^–/– mice with Apoe^–/–/ CX3CR1^–/– mice and by subsequent intercrossing of heterozygous F1 animals. The mice were maintained at the Centre d’Exploration Fonctionnelle animal facility (Pitié-Salpétrière, Paris, France) under pathogen-free conditions with food and water available ad libitum. Only male mice were analyzed in this study. Treatment with Met-CCL5 was performed by intraperitoneal injection (100 µg in PBS) twice a week for 12 weeks as previously described¹⁷ starting at 14 weeks of age. To assess the effect of CCL2 on bone marrow and blood monocyte number, treatment with murine CCL2 (PeproTech, Rocky Hill, NJ) was performed by intraperitoneal injection (400 ng in PBS) 3 times over 1 week. Animal experiments were approved by the local Institutional Animal Care and Use Committee of Faculté de Médecine Pitié-Salpétrière (Paris).

Flow Cytometry
Cell surface staining used the standard procedures and the following monoclonal antibodies: antibody to CD11b (anti–CD11b)-(clone M1/70)-Alexa Fluor 488, anti–Ly-6G (1A8)-phycoerythrin, anti–Ly-6C (AL-21)-biotin, anti–CD4 (clone L3T4)-phycoerythrin, anti–CD3 (clone 145-2C11)-Alexa Fluor 488, anti–CD8a (53-6.7)-Alexa Fluor 647, anti–CD11c-allophycocyanin, and streptavidin-peridin chlorophyll protein (all from BD Pharmingen, San Diego, Calif). Anti–mouse neutrophil 7/4-Alexa Fluor 647 and F4/80-biotin were from Serotec (Oxford, UK). Cell suspensions were incubated with appropriate fluorochrome-conjugated monoclonal antibodies, run for 4-color fluorescence staining on a cytofluorometer (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ), and analyzed with Cell Quest Pro (Becton Dickinson) software.

Extent and Composition of Atherosclerotic Lesions in Aortic Sinus
Mice were anesthetized with isoflurane before being euthanized at 25 weeks of age. Plasma cholesterol and high-density lipoprotein levels were measured with a commercial cholesterol kit (bioMerieux, Marcy l’Etoile, France). The heart was taken out, fixed in 4% paraformaldehyde for 2 hours, and placed in a PBS sucrose 30% solution overnight at 4°C before being included in a cutting medium and frozen at –70°C. Successive 10-µm transversal sections of aortic sinus were obtained. Lipids were detected with Oil Red O as previously described.¹⁸ Plaque composition was determined by use of a monoclonal rat anti-mouse macrophage antibody (clone MOMA-2 MAB1852 Chemicon, AbCys, Paris, France), a polyclonal anti-CCR2 antibody (M-50, Santa Cruz Biotechnology, Inc, Santa Cruz, Calif), or a polyclonal anti-CCR5 antibody (Santa Cruz). The specificity of the last 2 antibodies was tested in tissue recovered from CCR2^–/– (Figure I of the online-only Data Supplement) or CCR5^–/– mice.¹⁹ At least 4 sections per mouse were inspected for each immunostaining, and appropriate negative controls were used. Lesion size in aortic sinus represents the whole intimal surface. The thoracic aorta was available from some animals for analysis of the extent of lipid accumulation with Oil Red O staining as previously described.¹⁸

Peritoneal Inflammation
Peritonitis was induced by intraperitoneal injection of sterile thioglycolate (3% wt/vol in 1 mL sterile saline, Sigma-Aldrich, St Louis, Mo). Cells were quantified by flow cytometric analysis of the peritoneal lavage 72 hours after injection.

Cells
Blood was drawn via retroorbital puncture with heparin as an anticoagulant. Bone marrow cells were collected from femurs and tibias by insertion of a needle into the bone and flushing with Hanks’ buffered salt solution supplemented with 0.2% BSA and 1% FCS as previously described. Total viable leukocyte number was determined with the trypan-blue exclusion method. Leukocyte subpopulation numbers were calculated as total leukocytes multiplied by percent cells within the selected population gated by flow cytometry analysis.

Statistical Analysis
Data are expressed as mean±SEM. Statistical significance was determined by use of 2- or 3-factor (monocyte number) ANOVA. A value of P<0.05 (Bonferroni test) was considered statistically significant. The relation between circulating monocyte number and lesion size was determined through a simple linear regression analysis.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

	Results

Top Abstract Introduction Methods Results Discussion Conclusions References

 
CCL2 and CX3CR1 Play Nonredundant Roles in Atherosclerosis
We first examined whether simultaneous deletion in CCL2/CCR2 and CX3CL1/CX3CR1 has independent and additive protective effects in atherosclerosis. To this end, we generated Apoe^–/–/CCL2^–/–/CX3CR1^–/– triple-knockout mice and compared their susceptibility to atherosclerosis with Apoe^–/–/ CCL2^–/– and Apoe^–/–/CX3CR1^–/– littermates. The mice were maintained on chow diet, allowing the development of spontaneous moderate hypercholesterolemia, and were euthanized at 25 weeks, an age at which Apoe^–/– mice develop advanced lesions in the aortic sinus.²⁰ The 4 groups of mice had comparable weight (data not shown). Total plasma cholesterol levels were similar between Apoe^–/–, Apoe^–/–/CCL2^–/–, and Apoe^–/–/CX3CR1^–/–mice (Figure 1). However, unexpectedly, Apoe^–/–/CCL2^–/–/CX3CR1^–/– mice showed an 75% increase of total but not high-density lipoprotein cholesterol levels (P<0.0001) compared with the other groups (Figure 1). Lesion size was significantly different among the studied groups (P<0.0001). Lesion size was significantly altered by CCL2 (P<0.0001) or CX3CR1 deficiency (P=0.0009), and no interaction was found between these 2 variables (P=0.86), indicating independent effects of CCL2 and CX3CR1 on lesion size. Interestingly, despite important hypercholesterolemia, triple-knockout mice exhibited a profound 67% decrease in lesion size at the level of the aortic sinus (95 777±17 047 versus 287 999±20 113 µm² in Apoe^–/–/CCL2^–/–/CX3CR1^–/– and Apoe^–/– mice, respectively; P<0.0001; Figure 1). This marked inhibition of plaque development was significantly more pronounced than the 28%, 36%, or 48% reduction in lesion size observed in Apoe^–/–/CX3CR1^–/– (P=0.0012 versus Apoe^–/–/CCL2^–/–/CX3CR1^–/– and P=0.039 versus Apoe^–/–), Apoe^–/–/CCL2^–/– (P=0.0082 versus Apoe^–/–/CCL2^–/–/CX3CR1^–/– and P=0.0072 versus Apoe^–/–), or Apoe^–/–/CCL2^–/–/CX3CR1^+/– mice (P=0.024 versus Apoe^–/–/CCL2^–/–/CX3CR1^–/– and P<0.0001 versus Apoe^–/–), respectively. These results clearly suggest that CX3CR1 and CCL2 play independent and complementary roles in atherosclerosis.

View larger version (50K): [in this window] [in a new window]   Figure 1. Combined CCL2 and CX3CR1 deficiency markedly inhibits atherosclerotic lesion development in Apoe–/– mice. A, Representative photomicrographs of Oil Red O staining of the aortic sinus of Apoe–/– (n=16), Apoe–/–/CCL2–/– (n=8), Apoe–/–/CX3CR1–/– (n=7), and Apoe–/–/CCL2–/–/CX3CR1–/– (n=15) mice showing the development of atherosclerotic lesions. Quantita- tive analysis of atherosclerotic lesion size (B) and serum cholesterol levels (C) in the different groups of mice. Values represent mean±SEM. Not shown in this Figure is the lesion size of Apoe–/–/CCL2–/–/CX3CR1+/– mice (149 221 µm2; n=12; P=0.024 vs Apoe–/–/CCL2–/–/CX3CR1–/– and P<0.0001 vs Apoe–/–). *P<0.05; **P<0.01.

Modulation of Local Monocyte/Macrophage Accumulation by CCL2 and CX3CR1
We next examined potential atheroprotective mechanisms associated with CCL2 and/or CX3CR1 deficiency. We found significant and similar inhibition of macrophage accumulation in lesions of Apoe^–/–/CCL2^–/– and Apoe^–/–/CX3CR1^–/– mice compared with Apoe^–/– mice (Figure 2A and 2B). Reduced macrophage accumulation in Apoe^–/–/CCL2^–/– mice was associated with a marked decrease in the accumulation of CCR2⁺ macrophages within the lesions (Figure 2C). Unexpectedly, but in agreement with the results of 1 recent study,¹¹ we observed a reduction in the accumulation of CCR2⁺ macrophages in lesions of Apoe^–/–/CX3CR1^–/– mice (Figure 2C). However, accumulation of CCR2⁺ macrophages was less affected by CX3CR1 compared with CCL2, and combined CCL2 and CX3CR1 deficiency showed no additive effect (Figure 2C). These results suggest a predominant role for CCL2/CCR2 pathway in the recruitment of CCR2⁺ monocytes into atherosclerotic arteries compared with CX3CR1. Interestingly, lesional macrophage accumulation was lowest in Apoe^–/–/CCL2^–/–/CX3CR1^–/– mice (Figure 2A and 2B) despite higher cholesterol levels (Figure 1C). This could result from an additive reduction in the recruitment of the other major monocyte subset (ie, CCR2^– CX3CR1^hi Ly6C^lo). However, a recent study clearly showed that neither CCR2, the only CCL2 receptor, nor CX3CR1 contributes to recruitment of CCR2^– CX3CR1^hi Ly6C^lo monocytes in atherosclerosis.¹¹ Thus, we hypothesized that the additive protection from lesion development conferred by the simultaneous deletion of CCL2 and CX3CR1 may be related to systemic rather than local effects of chemokine signaling, which may affect the numbers of monocytes in the bloodstream and their interaction with the vessel wall.

View larger version (53K): [in this window] [in a new window]   Figure 2. Combined CCL2 and CX3CR1 deficiency in Apoe–/– mice alters macrophage accumulation into atherosclerotic arteries. A, Representative photomicrographs of MOMA-2 staining (brown-red) in the aortic sinus of Apoe–/–, Apoe–/–/CCL2–/–, Apoe–/–/ CX3CR1–/–, and Apoe–/–/CCL2–/–/CX3CR1–/– mice showing macrophage accumulation within the lesions. Examples of the most advanced lesions (the intima is circled by a black line) in Apoe–/–/CCL2–/–, Apoe–/–/CX3CR1–/–, and Apoe–/–/CCL2–/–/CX3CR1–/– mice are shown to compare with the advanced lesions that develop in Apoe–/– mice. B, Quantitative analysis of MOMA-2 staining (total macrophage accumulation) and (C) CCR2-positive cells within atherosclerotic lesions of Apoe–/–, Apoe–/–/CCL2–/–, Apoe–/–/CX3CR1–/–, and Apoe–/–/CCL2–/–/CX3CR1–/– mice. D, Quantitative assessment of the adherence of bone marrow–derived monocytes recovered from wild-type, CCL2–/–, CX3CR1–/–, or CCL2–/–/CX3CR1–/– mice to carotid arteries of Apoe–/– mice in vitro. Values represent mean±SEM of 5 to 9 mice per group. *P<0.05; **P<0.01; ***P<0.001.

A recent study suggested that in response to bacterial infection, CCR2-mediated signals in bone marrow determine the emigration of immature Ly6C^hi monocytes into the circulation rather than promoting monocyte trafficking between the blood and peripheral tissue.²¹ We therefore examined the effect of CCL2 and/or CX3CR1 deficiency on the frequency of the 2 major monocyte subsets in the bone marrow and bloodstream.

Differential Modulation of Bone Marrow and Circulating Monocyte Subsets by CCL2 and CX3CR1
In the present study, we defined monocytes as side scatter–low, forward scatter–high cells expressing the myeloid antigen 7/4 (high and low populations) and high levels of CD11b but showing no expression for the neutrophil marker Ly6G (Figure 3A). In addition, side scatter–low, forward scatter–high, CD11b-high, 7/4-high cells showed almost no staining for NK cell marker NK1.1, and <20% of 7/4-low cells were contaminated by NK cells (online-only Data Supplement Figure II). CD11b⁺ Ly6G^– 7/4^hi cells correspond to Ly6C^hi monocytes, and CD11b⁺ Ly6G^– 7/4^lo cells correspond to Ly6C^lo monocytes (online-only Data Supplement Figure III). Monocyte number and phenotypes were significantly different among the various groups (P<0.001). Apoe background and CCL2 deficiency significantly affected monocyte number (P<0.001). In agreement with recent data,¹⁰ we observed increased bone marrow and blood monocytosis in Apoe^–/– mice (Figures 3 and 4, solid bars) and a shift toward the CD11b⁺ Ly6G^– 7/4^hi subset in the blood compared with Apoe^+/+ mice (Figure 3, open bars). However, in the present study, both CD11b⁺ Ly6G^– 7/4^hi and CD11b⁺ Ly6G^– 7/4^lo subsets contributed to blood monocytosis under moderate hypercholesterolemia (Figure 3B). The difference between these studies may be due in part to differences in the phenotyping of the 7/4^lo monocyte subset. We observed a significant reduction in the total number of circulating monocytes in CCL2^–/– mice under normocholesterolemic conditions (Apoe^+/+ background) because of a reduction in both CD11b⁺ Ly6G^– 7/4^hi and CD11b⁺ Ly6G^– 7/4^lo monocytes (Figure 3B), supporting similar findings reported in CCR2^–/– animals.^21,22 The significant reduction in circulating monocyte numbers was maintained under the hypercholesterolemic and atherosclerotic Apoe^–/– background (Apoe^–/–/CCL2^–/– compared with Apoe^–/– mice in Figure 3B). However, in contrast to CCR2 deficiency, which led to accumulation of Ly6C^hi (here, CD11b⁺ Ly6G^– 7/4^hi) monocytes in the bone marrow,^21,22 CCL2 deficiency did not, and it even led to a reduction in bone marrow monocyte number under the Apoe^–/– background (Figure 4B), suggesting that other chemokines compensate for the absence of CCL2 to mediate emigration of CD11b⁺ Ly6G^– 7/4^hi monocytes into the circulation.²² Short-term administration of CCL2 to C57Bl/6 mice led to an increase in monocyte number in the blood and bone marrow (online-only Data Supplement Figure IV), suggesting that CCL2 may control monocyte number. Whether this effect requires CCR2 signaling in monocytes is currently unknown. Our results clearly show that CCL2 deficiency inhibits bone marrow CD11b⁺ Ly6G^– 7/4^hi monocytosis in hypercholesterolemic mice and controls circulating monocyte number, which may have contributed, at least in part, to the marked inhibition of CCR2⁺ macrophage accumulation in the atherosclerotic lesions of Apoe^–/–/CCL2^–/– mice (Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 3. CCL2 and CX3CR1 differentially control the number of monocytes in the circulating blood under both noninflammatory and atherosclerotic conditions. A, Representative examples of analysis of blood myeloid populations using flow cytometry defining monocytes as CD11b+ Ly6G– 7/4+ cells (populations 1 and 2) and neutrophils as CD11b+ Ly6G+ 7/4+ cells (population 3). CD11b+ Ly6G– 7/4+ monocytes consist of distinct CD11b+ Ly6G– 7/4hi (population 1) and CD11b+ Ly6G– 7/4lo (population 2) populations, which correspond to the previously described Ly6Chi and Ly6Clo monocyte subsets, respectively (see online-only Data Supplement Figure I). B, Quantitative analysis of circulating total or individual monocyte subsets (number of cells per 1 mL blood) in the different groups of mice. Values represent mean±SEM of 10 to 15 mice per group euthanized between 10 and 15 weeks of age. Asterisks give probability values vs chemokine/chemokine receptor–sufficient mice within the same Apoe+/+ or Apoe–/– background (*P<0.05; **P<0.01; ***P<0.001); crosses give probability values vs corresponding gene-deficient Apoe+/+ background (P<0.05; P<0.01; P<0.001).

View larger version (19K): [in this window] [in a new window]   Figure 4. CCL2 and CX3CR1 control the number of monocytes in the bone marrow under atherosclerotic conditions. A, Representative examples of analysis of bone marrow myeloid populations using flow cytometry, defining monocytes as CD11b+ Ly6G– 7/4+ cells (rectangle) and neutrophils as CD11b+ Ly6G+ 7/4+ cells. Note that the CD11b+ Ly6G– 7/4hi population predominates in bone marrow. CD11b+ Ly6G– 7/4lo monocytes are barely detectable. B, Quantitative analysis of monocyte number in the bone marrow (number of cells per 2 femurs and 2 tibias) of the different groups of mice. Values represent mean±SEM of 10 to 15 mice per group euthanized between 10 and 15 weeks of age. Asterisks give probability values vs chemokine/chemokine receptor–sufficient mice within the same Apoe+/+ or Apoe–/– background (*P<0.05; **P<0.01; ***P<0.001); crosses give probability values vs corresponding gene-deficient Apoe+/+ background (P<0.05; P<0.01; P<0.001).

CX3CR1 deficiency was associated with a nonsignificant trend toward a lower number of monocytes in the bone marrow (Figure 4) but did not affect total blood monocyte number (Figure 3). Our observation that CX3CR1 deficiency resulted in reduced accumulation of CCR2⁺ cells within the lesions without affecting the number of CD11b⁺ Ly6G^– 7/4^hi (CCR2⁺) monocytes in the circulating blood suggests a role for CX3CR1 signaling in the recruitment of this monocyte subset from the blood into the atherosclerotic lesions, which is in agreement with previous findings.¹¹ Interestingly, CX3CR1 deficiency induced a specific reduction in the number of circulating CD11b⁺ Ly6G^– 7/4^lo (Figure 3C), consistent with the high level of CX3CR1 expression on this monocyte subset in Apoe^+/+ and Apoe^–/– mice. Furthermore, combined CCL2 and CX3CR1 deficiency in the Apoe^–/– background resulted in a significant reduction in bone marrow and circulating monocytes as a result of the additive reduction in CD11b⁺ Ly6G^– 7/4^hi and CD11b⁺ Ly6G^– 7/4^lo subsets (Figures 3 and 4). Of note, individual deficiency of either CCL2 or CX3CR1 was not sufficient to inhibit the ability of mice to increase total monocyte number in the bone marrow and circulating blood when switched from a normocholesterolemic nonatherosclerotic Apoe^+/+ to a hypercholesterolemic atherosclerotic Apoe^–/– background (Figures 3 and 4). However, combined CCL2 and CX3CR1 deficiency totally abrogated Apoe^–/–-associated bone marrow and blood monocytosis, which returned to levels observed in Apoe^+/+ mice, despite higher levels of plasma cholesterol (Figures 3 and 4). We found no effects of CCL2 and/or CX3CR1 deficiency on neutrophil, CD4⁺, or CD8⁺ lymphocyte count in the Apoe^–/– background (data not shown). These results identify critical, independent, and complementary roles for CCL2- and CX3CR1-mediated signals in bone marrow and blood (CD11b⁺ Ly6G^– 7/4^hi and CD11b⁺ Ly6G^– 7/4^lo) monocytosis, which could explain the additive roles of these pathways in promoting lesion development in Apoe^–/– mice.

In contrast to its role in atherosclerosis, we found that specific inhibition of the recruitment of CD11b⁺ Ly6G^– 7/4^lo monocytes in CX3CR1^–/– mice was not sufficient to alter monocytic peritonitis in response to intraperitoneal thioglycollate (online-only Data Supplement Figure V), confirming the prominent role of CD11b⁺ Ly6G^– 7/4^hi monocyte recruitment through CCL2/CCR2²³ in this setting.

Combined Inhibition of CCL2, CX3CR1, and CCR5 Almost Abolishes Atherosclerosis
Finally, we wanted to identify the chemokine pathway(s) responsible for the residual atherogenesis that showed resistance to combined CCL2 and CX3CR1 inhibition. In contrast to the marked reduction in CCR2⁺ macrophages, we found persistent accumulation of CCR5⁺ cells in lesions of Apoe^–/–/CCL2^–/–/CX3CR1^–/– mice (Figure 5A). Interestingly, treatment of the triple-knockout mice from week 14 to 25 with Met-CCL5, an antagonist of CCR5 signaling, induced an additional 75% reduction in lesion size (Figure 5B) despite persistent high plasma cholesterol levels (5.7±0.5 g/L; P=NS versus Apoe^–/–/CCL2^–/–/CX3CR1^–/– mice). This result is consistent with the role of CCR5 in monocyte recruitment and atherosclerotic lesion development.^11,17,19 The reduction in atherosclerosis after Met-CCL5 treatment was associated with a marked additional reduction in the number of circulating CD11b⁺ Ly6G^– 7/4^hi and CD11b⁺ Ly6G^– 7/4^lo monocytes (Figure 5C), again suggesting a critical role for chemokine signals in the modulation of the frequency of circulating monocytes in atherosclerosis. Met-CCL5 treatment did not affect blood monocyte number in Apoe^+/+ mice (data not shown), ruling out any toxic effect of Met-CCL5 on blood monocytes. Overall, we found that combined inhibition of CCL2, CX3CR1, and CCR5 pathways almost abrogates macrophage accumulation and atherosclerosis in Apoe^–/– mice (90% reduction). Of note, under this moderate hypercholesterolemia, lesion size highly correlated with the total number of circulating monocytes (r²=0.97, P=0.0016; Figure 5D) and with the number of CD11b⁺ Ly6G^– 7/4^hi (r²=0.90, P=0.01) or CD11b⁺ Ly6G^– 7/4^lo monocytes (r²=0.99, P=0.0003).

View larger version (13K): [in this window] [in a new window]   Figure 5. CCR5-mediated signals account for CCL2- and CX3CR1-resistant macrophage accumulation within atherosclerotic lesions. A, Quantitative analysis of CCR5-positive cells within atherosclerotic lesions of Apoe–/– (n=5), Apoe–/–/CCL2–/– (n=5), Apoe–/–/CX3CR1–/– (n=5), and Apoe–/–/CCL2–/–/CX3CR1–/– (n=9) mice. Values represent mean±SEM. *P<0.05; **P<0.01. B, Quantitative analysis of atherosclerotic lesion size in Apoe–/–/CCL2–/–/CX3CR1–/– mice with (n=5) or without (n=15) treatment with Met-CCL5 for 12 weeks. Values represent mean±SEM. *P<0.05 vs untreated Apoe–/–/CCL2–/–/CX3CR1–/– mice. Lesion size in Apoe–/– mice (n=16) is shown for comparison. C, Quantitative analysis of circulating monocyte subsets (number of cells per 1 mL blood) of Apoe–/–/CCL2–/–/ CX3CR1–/– mice with or without treatment with Met-CCL5. Values represent mean±SEM. **P<0.01 vs untreated Apoe–/–/CCL2–/–/CX3CR1–/– mice. D, Correlation between lesion size and circulating total monocyte number (mean±SEM are represented) in the different strains of mice (r2=0.97, P=0.0016).

	Discussion

Top Abstract Introduction Methods Results Discussion Conclusions References

 
The present study presents 3 major findings: (1) Combined activation of CCL2, CX3CR1, and CCR5 pathways accounts for most of the macrophage accumulation within atherosclerotic lesions; (2) CCL2, CX3CR1, and CCR5 markedly and differentially alter the number of monocyte subsets within the bone marrow and the circulating blood and are required for blood and bone marrow monocytosis in hypercholesterolemic atherosclerotic mice; and (3) control of systemic monocyte number by these chemokine pathways is highly correlated with their ability to modulate atherogenesis.

Taken individually, several chemokines and chemokine receptors play a significant role in the development of atherosclerosis.⁴ However, direct evidence of additive roles for these pathways in experimental models of the disease is still lacking. A recent study examined the effect of combined inhibition of CCR2 and CXCR3 signaling on lesion formation in Apoe^–/– mice.²⁴ The authors showed that CCR2 and CXCR3 play differential roles during atherogenesis, with CCR2 promoting lesion development within the aortic root and CXCR3 affecting lesion formation within the abdominal aorta.²⁴ Thus, CCR2 and CXCR3 differentially influence the site of lesion formation but play no additive role in promoting arterial leukocyte accumulation at a given atherosclerosis-prone site.

When we initiated the present study >3 years ago, we hypothesized that 2 particular pathways, CCL2/CCR2 and CX3CL1/CX3CR1, could play independent, complementary roles to promote a high level of macrophage accumulation within the atherosclerotic artery and to accelerate plaque development. Our hypothesis was based on several observations. Deletion of either the CCL2/CCR2 or CX3CL1/CX3CR1 pathway reduces monocyte accumulation within the vascular lesions and leads to reduced susceptibility to atherosclerosis in murine models.^12,14,16,25 CCL2/CCR2 interaction favors rolling and diapedesis of circulating monocytes through the endothelial layer^3,26,27 and requires integrin activation on monocytes.²⁸ On the contrary, CX3CL1 and its specific receptor CX3CR1 allow firm adhesion of monocytes on endothelial and smooth muscle cells independently of integrin activation.²⁹ These 2 receptors not only use different methods for monocyte adhesion/infiltration but also may recruit various subsets of monocytes. CX3CL1 preferentially mediates the adhesion of CD16⁺ monocytes, whereas CCL2 acts on CD16^– monocytes.³⁰ In addition, recent studies suggest that CCL2 enhances the adhesion of monocytes to CX3CL1,³¹ which may enhance the retention of monocytes on CX3CL1-expressing smooth muscle cells,³² suggesting that CCL2 and CX3CL1 act in unison to recruit monocytes and enhance their accumulation into vascular lesions. In the present study, we show that combined inhibition of 2 different chemokine pathways, CCL2 and CX3CR1, leads to additive reduction in lesion development within the aortic root of male Apoe^–/– mice fed a chow diet. The effect probably extends to other atherosclerosis-prone sites because we observed a trend toward a more profound inhibition of atherogenesis in the thoracic aorta after combined deletion of CCL2 and CX3CR1 (online-only Data Supplement Figure VI) despite higher plasma cholesterol levels. In addition, in the complementary work by Saederup et al³³ (see the companion article), the authors nicely show an additive reduction in atherosclerosis (within both the thoracic aorta and the aortic root) after combined deficiency in CCR2 and CX3CL1 using Apoe^–/– mice fed a Western diet. Taken together, these 2 studies clearly show for the first time that specific chemokine pathways, here CCL2/CCR2 and CX3CL1/CX3CR1, play nonredundant, complementary roles in vivo in a chronic inflammatory disease, here atherosclerosis.

Interestingly, we found that macrophage accumulation within atherosclerotic arteries was lowest in Apoe^–/–/CCL2^–/–/ CX3CR1^–/– mice but was not significantly different from that observed in Apoe^–/–/CCL2^–/– or Apoe^–/–/CX3CR1^–/– mice. It could be argued that this suggests no substantial additive effect of combined inhibition of CCL2 and CX3CR1 on macrophage accumulation within the vessel wall. However, it is important to note that Apoe^–/–/CCL2^–/–/CX3CR1^–/– mice showed the smallest reduction in lesion size despite a very important and significant increase in plasma cholesterol levels, a major atherogenic stimulus. Indeed, lesion size was significantly correlated with plasma cholesterol levels in the triple-knockout mice (online-only Data Supplement Figure VII), suggesting that if plasma cholesterol levels had been equivalent to those found in the other groups of mice, the reduction in lesion size would have been much more profound in the triple-knockout animals. Thus, taken together, these results clearly reveal a major inhibitory role of combined CCL2 and CX3CR1 deficiency on arterial inflammation.

An important finding in the present study is that the major role of chemokines and chemokine receptors in atherosclerosis may relate to their role in the modulation of monocyte number in both bone marrow and circulating blood. Our results clearly show that deletion of CCL2 and CX3CR1 abolishes hypercholesterolemia-associated blood monocytosis, identifying a critical role for chemokine signaling in this process. In addition, we found that chemokine pathways differentially affect the number of monocyte subsets, with CCL2 having major impact on both Ly6C^hi and Ly6C^lo monocytes and CX3CR1 specifically affecting Ly6C^lo monocytosis. Unexpectedly, the control of circulating monocyte number by CCL2 and CX3CR1 was associated with a reduction in, not accumulation of, monocytes within the bone marrow, suggesting that their role in the modulation of monocytosis goes beyond the control of monocyte emigration from the bone marrow into the circulating blood, as recently suggested for CCR2-mediated signaling.^21,22 It will be important in future studies to identify in detail the precise signals and mechanisms responsible for the chemokine-dependent increase in monocyte number, which may include increased monocyte differentiation, proliferation, and/or survival. A previous study¹⁰ suggested a predominant role for Ly6C^hi monocytes in atherogenesis under high-fat feeding. However, given the strong correlation between the number of Ly6C^lo monocytes and lesion size reported in the present work, we believe that this monocyte subset may significantly contribute to lesion formation, at least under moderate hypercholesterolemia.

An intriguing finding in the present study is that inhibition of CX3CR1 signaling resulted in a relative increase in the accumulation of CCR5⁺ cells within the lesions, suggesting an exclusive interaction between these 2 pathways in the recruitment of CCR5⁺ leukocytes. The latter may include macrophages, T cells, and smooth muscle cells. However, CCR5⁺ staining most likely represented macrophages given the very small size of T cells (relative to macrophages) and the absence of smooth muscle cells within the lesions of the triple-knockout mice (data not shown). Interestingly, inhibition of CCR5 signaling in CCL2/CX3CR1-deficient mice almost abrogated arterial macrophage accumulation, identifying 3 major and complementary pathways that control lesion development in Apoe^–/– mice. Again, inhibition of CCR5 signaling led to a marked reduction in the number of circulating monocytes, further supporting an important role for chemokines in the modulation of systemic monocyte number. The effect of Met-CCL5 on atherosclerosis could be related, at least in part, to inhibition of CCR5 signaling in T cells. However, it is remarkable that the reduction in lesion size in mice with defective CCL2, CX3CR1, and/or CCR5 signaling strongly correlated with the reduction in circulating monocyte number. Similar results were reported by Smith et al⁵ in Apoe^–/– mice with osteopetrotic mutation. It will be important to examine in future studies whether chemokine-mediated regulation of bone marrow and blood monocyte number requires an intact M-CSF pathway. Finally, it could be argued that modulation of bone marrow and blood monocytosis is not a primary effect of chemokine inhibition but is secondary to atherosclerosis reduction. However, our results show that short-term administration of CCL2 enhances bone marrow and circulating monocyte number, suggesting a direct effect of chemokines on monocytosis. In addition, the 99% reduction in atherosclerosis in severely hypercholesterolemic ldlr^–/– mice with heterozygous osteopetrotic mutation was not associated with significant changes in the percentage of circulating monocytes,³⁴ suggesting that a reduction in atherosclerosis is not a prerequisite for a reduction in circulating monocyte number.

The mechanisms responsible for the increase in total and non–high-density lipoprotein plasma cholesterol levels in Apoe^–/–/CCL2^–/–/CX3CR1^–/– mice were not explored. This increase could occur as a direct or indirect consequence of combined CCL2 and CX3CR1 deletion. Interestingly, Smith et al⁵ reported similar findings in Apoe^–/–/M-csf^–/– mice that showed reduced monocyte number, suggesting a potential role for monocytes/macrophages in the modulation of lipid metabolism.

	Conclusions

Top Abstract Introduction Methods Results Discussion Conclusions References

 
In addition to their roles in leukocyte recruitment from the blood into the vessel wall, which is the prevailing paradigm to explain the proatherogenic effects of several chemokine/chemokine receptor pathways, we show that CCL2-, CX3CR1-, and CCR5-dependent signals differentially alter CD11b⁺ Ly6G^– 7/4^hi and CD11b⁺ Ly6G^– 7/4^lo monocytosis in the blood and bone marrow and cooperate to promote full macrophage accumulation within atherosclerotic vessels. These results identify critical independent and complementary chemokine pathways in atherosclerosis and shed new light on the mechanisms operated by chemokines to enhance monocyte accumulation into inflamed tissues.

	Acknowledgments

 
The authors wish to thank Arnaud Cangialosi for technical assistance and Myriam Gort from the Centre d’Exploration Fonctionnelle animal facility (Faculté de Médecine Pitié-Salpétrière, Paris). We also thank Dr B.J. Rollins (Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Mass) for sharing the CCL2 mice.

Sources of Funding

This work was supported by grants from Inserm, ANR Cardiovasculaire, obésité et diabète (AO5088DS), and European Grant Innochem 518167. Drs Combadière, Tedgui, and Mallat are recipients of Contract Interface from Assistance Publique-Hopitaux de Paris, and M. Rodero is supported by a scholarship from the Canceropole Île de France. Dr Potteaux was supported by Groupe de Réflexion sur la Recherche Cardiovasculaire, France.

Disclosures

None.

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CLINICAL PERSPECTIVE

Monocytes are critical mediators of atherogenesis and are recruited into atherosclerotic arteries in response to chemokine/chemokine receptor signaling. Evidence that particular chemokine pathways may cooperate to promote monocyte accumulation into inflamed tissues, particularly atherosclerotic arteries, is still lacking. Here, we show that combined inhibition of CCL2, CX3CR1, and CCR5 in apolipoprotein E–deficient mice almost abolishes lesion formation, indicating that these pathways play independent and additive roles in atherosclerosis. Another important finding is that the major role of chemokines and chemokine receptors in atherosclerosis may relate to their role in the modulation of monocyte number in both the bone marrow and circulating blood. Our results clearly show that inhibition of CCL2, CX3CR1, and CCR5 abolishes hypercholesterolemia-associated blood monocytosis, identifying a critical role for chemokine signaling in this process. It is remarkable that the reduction in lesion size in mice with defective CCL2, CX3CR1, and/or CCR5 signaling strongly correlated with the reduction in circulating monocyte number, particularly the CD11b⁺ Ly6G^– 7/4^lo subset. Thus, in addition to their roles in leukocyte recruitment from the blood into the vessel wall, which is the prevailing paradigm to explain the proatherogenic effects of several chemokine/chemokine receptor pathways, signals mediated through CCL2, CX3CR1, and CCR5 critically determine the frequency of circulating monocyte subsets and cooperate to promote full macrophage accumulation within atherosclerotic vessels. It will be important to examine in future studies whether particular blood monocyte subsets are associated with the extent of atherosclerosis in humans.

	Footnotes

 
*Drs Combadière and Potteaux contributed equally to this work.

The online Data Supplement, which contains figures, can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.107.745091/DC1.

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