CX₃CR1 is required for monocyte homeostasis and atherogenesis by promoting cell survival

Chemokines are a family of chemotactic cytokines that activate specific G-protein-coupled 7-transmembrane receptors¹  and have been categorized into C, CC, CXC, and CX₃C families. The only known CX₃C chemokine, CX₃CL1, also known as fractalkine,^1-3  is expressed by activated vascular endothelial cells,³  neurons,⁴  epithelial cells,^5,6  smooth muscle cells,⁷  dendritic cells (DCs),⁸  and macrophages.⁹  The single known CX₃CL1 receptor, CX₃CR1,¹⁰  is expressed by T-cell and natural killer (NK) cell subsets,^10,11  brain microglia,^4,12,13  DC subsets^13-15  as well as blood monocytes.^10,13 

Classical small-molecular-weight chemokines are secreted proteins considered to form gradients by binding to extracellular matrix proteoglycans. In contrast, CX₃CL1 is synthesized as a transmembrane protein with its chemokine domain presented on an extended mucin-like stalk.^2,3  In this form, CX₃CL1 promotes tight, integrin-independent adhesion of CX₃CR1-expressing leukocytes.^7,16  In addition, constitutive and inducible cleavage by metalloproteases can result in release of a soluble CX₃CL1 entity from the cell membrane.^17-19  CX₃CL1 thus potentially acts as an adhesion molecule and a chemoattractant; albeit the differential importance of these activities for the physiologic role of CX₃CL1 remains unknown. In cell lines and cultured microglia, CX₃CR1 engagement triggers the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway resulting in cell survival and proliferation.^20-23  However, the significance of this activity for the in vivo role of the CX₃C chemokine system remains to be determined.

Atherosclerosis is characterized by the accumulation of lipids and fibrous elements in the large arteries and involves diverse factors, including components of the immune system.²⁴  Human CX₃CR1 gene polymorphisms were shown to be genetic risk factors for coronary artery diseases and atherosclerosis.^25,26  Moreover, mice deficient for either CX₃CR1 or CX₃CL1 display a relative resistance to atherosclerosis development in the respective murine disease models.^27-30  However, the mechanistic explanations for these phenotypes remain under debate.³¹ 

Monocytes are mononuclear phagocytes that are generated in the bone marrow (BM) and released to the bloodstream.^32,33  From there they enter peripheral tissues, where they can give rise to macrophages and DCs.^15,32-35  Undifferentiated monocytes represent a short-lived transitional state, with an estimated circulation half-life of approximately 71 hours in human³⁶  and approximately 17 hours in mice.^32,37  They are heterogeneous and can be divided into at least 2 main subpopulations,³⁸  with human monocytes comprising CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ cells.³⁹  Murine monocytes have been divided into Gr1^hi (Ly6C^hi) and Gr1^low (Ly6C^low) subsets,^35,40  which are functionally distinct with respect to both their migration ability and differentiation potential.^15,35,41,42  Although all monocytes express CX₃CR1, the 2 subsets differ by distinct surface CX₃CR1 levels, with human CD14⁺CD16⁺ and murine Gr1^low monocyte subsets displaying significantly higher CX₃CR1 amounts.³⁵  The role of this prominent CX₃CR1 expression for monocyte physiology remains unknown, although our own studies and reports by others suggest that CX₃CR1-CX₃CL1 interactions might control monocyte levels^35,43  and be involved in monocyte migration.^44,45 

Here, we investigated the role of CX₃CL1-CX₃CR1 interaction during steady state and atherogenesis, using mutant mice as well as human blood monocyte isolates. We show that mice deficient for either CX₃CR1 or CX₃CL1 display a specific reduction in the frequency of circulating Gr1^low blood monocytes, whereas Gr1^hi monocytes remain unaffected. Using forced Bcl2 expression, we provide genetic evidence that this phenotype results from enhanced cell death. The protective CX₃C signal is direct and evolutionary conserved, as addition of soluble CX₃CL1 inhibits cell death of cultured human monocytes. Altered CX₃CR1 function was reported to affect atherosclerosis progression in both humans and mice. Here, we show that the absence of the CX₃C signal results in increased apoptosis of plaque-resident cells, including macrophages, whereas introduction of a Bcl2 transgene allows disease progression in the absence of CX₃CR1. We suggest, therefore, that CX₃CR1 plays a central role during monocyte homeostasis and atherogenesis by promoting essential cell survival.

This study involved the use of the following C57BL/6 mouse strains: Cx₃cr1^gfp mice¹³ ; cx₃cl1^−/− mice⁴⁶ ; hMRP8bcl2 mice⁴⁷  and apoe^−/− mice (B6.129P2-Apoe^tm1Unc/J; The Jackson Laboratory, Bar Harbor, ME). Wild-type (wt) C57BL/6 mice were purchased from Harlan Laboratories (Rehovot, Israel). Mice used were 7 to 10 weeks of age. All mice were maintained under specific pathogen–free conditions and handled under protocols approved by the Weizmann Institute Animal Care Committee according to international guidelines.

For blood cell analysis, wt or cx₃cl1^−/− recipient mice were lethally irradiated with a 950-rad dose and 1 day later were intravenously (IV) injected with 5 × 10⁶ BM cells isolated from donor femora and tibiae. BM recipients were allowed to rest for 8 weeks before analysis. For atherosclerosis studies, apoe^−/− recipient mice were lethally irradiated 2 times with 650-rad doses, injected IV 1 day later with 5 × 10⁶ donor BM cells, and allowed to rest for 4 weeks before being placed on high-fat diet.

Apoe^−/− mice were fed an atherogenic diet containing 21% fat (0.15% cholesterol, 19.5% casein; Altromin, Lage, Germany), and after 12 weeks were fixed by in situ perfusion. The extent of atherosclerosis was quantified by Oil Red O staining of thoracoabdominal aortas prepared en face and in serial sections through the aortic root by computerized image analysis (MetaMorph version 6.0; Universal Imaging-Molecular Devices or Diskus software, Downingtown, PA), as previously described.⁴⁸ 

Mice were bled from their tail veins, and blood was subjected to a Ficoll density gradient (Amersham Biosciences, Sunnyvale, CA) for erythrocyte and neutrophil removal. BM cells were isolated from mouse femora, followed by erythrocyte lysis using ACK buffer (0.15 M NH₄Cl, 0.01 M KHCO₃). Cells were suspended in phosphate-buffered saline (PBS) supplemented with 2 mM ethylenediaminetetraacetic acid (EDTA), 0.05% sodium azide, and 1% fetal calf serum (FCS).

Human blood monocytes were isolated from white blood cell (WBC)–enriched blood (Israeli Blood Bank, Tel Hashomer, Israel). After erythrocyte and neutrophil removal through a Ficoll density gradient, monocyte subsets were magnetically separated using the Monocyte Isolation Kit II and CD16⁺ Monocyte Isolation Kit (both Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocols. For serum deprivation studies, the cells were washed with PBS to remove serum residues, and 5 × 10⁴ cells per well were plated in RPMI supplemented with nonessential amino acids. RPMI was further supplemented with FCS (10%; Beit Haemek Industries, Israel) or recombinant mouse CX₃CL1 (amino acids [aa] 1-337; R&D Systems, Minneapolis, MN). Cells were incubated for 4 hours before harvesting. For pertussis toxin (PTx) studies, cells were incubated 1 hour with PTx (Calbiochem, San Diego, CA) in serum supplemented medium, washed with PBS, and incubated as previously described. For the oxysterol studies 5 × 10⁴ cells/well were incubated with serum-supplemented RPMI either further supplemented or nonsupplemented with rCX₃CL1 for 6 hours, followed by incubation with 7-β-hydroxycholesterol (Sigma-Aldrich, St Louis, MO) for an additional 16 hours.

The following monoclonal antibodies and staining reagents were used according to the manufacturers' protocols: human-specific biotin-conjugated anti-CD14 (Caltag Laboratories, Burlingame, CA), fluorescein isothiocyanate (FITC)–conjugated anti-CD16 (Caltag Laboratories), and phycoerythrin (PE)–conjugated anti-Bcl2 (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse-specific PE-conjugated anti-CD115 (eBioscience, San Diego, CA) and allophycocyanin (APC)–conjugated anti-Gr1 (Ly6C/G; eBioscience). Propidium iodide (Sigma-Aldrich) was added to 5 minutes before sample acquisition. Cells were analyzed on a FACSCalibur cytometer (Becton Dickinson, Franklin Lakes, NJ) using CellQuest software (Becton Dickinson).

Plaque cellular composition was analyzed in transverse sections through the aortic root. Sections were stained with anti-MOMA-2 (AbD Serotec, Raleigh, NC) and anti–human Bcl2 (BD Biosciences, San Jose, CA) monoclonal antibodies, detected by alkaline phosphate substrate and the Vector Red Substrate Kit (Vector Laboratories, Burlingame, CA). Apoptotic nuclei were detected by terminal deoxynucleotidyl nick-end labeling (TUNEL) kit (Roche Applied Science, Indianapolis, IN), and nuclei were counterstained by 4′,6-diamidino-2-pheylindol (DAPI) when indicated. Images were recorded with a Leica DMLB fluorescence microscope and CCD camera (JVC).

To study the physiologic role of CX₃CR1, we took advantage of knockin mice that carry a targeted replacement of the cx₃cr1 gene by a gene encoding green fluorescent protein (GFP).¹³  All circulating blood monocytes, as defined by expression of CD115 (also known as MCSF [CSF1] receptor, Figure 1A), express CX₃CR1 and are, therefore, GFP-positive in cx₃cr1^gfp mice.¹³  The comparative analysis of leukocytes of wt and cx₃cr1^gfp/gfp mice revealed a slight, but consistent and significant, reduction of the absolute monocyte numbers in the absence of CX₃CR1 (Figure 1B). This was also reflected in their reduced percentage among total nongranular WBCs (ngWBC; Figure 1C). Blood monocyte levels of heterozygote mutant mice were comparable with wt mice (Figure 1C).

Murine blood monocyte subsets differ in their level of CX₃CR1 surface expression with Gr1^hi monocytes expressing intermediate amounts of CX₃CR1 and Gr1^low monocytes being CX₃CR1^hi.³⁵  Interestingly, Gr1^hi monocyte levels remained unaffected by the CX₃CR1 deficiency (Figure 1C). In contrast, Gr1^low monocytes of CX₃CR1 KO mice were found to be 3-fold reduced, as compared with wt and cx₃cr1^gfp/+ mice (Figure 1C). To avoid indirect effects on these quantifications due to a potential CX₃CR1 requirement by other leukocytes,^10,11,13  we plotted the fraction of Gr1^low monocytes among the total monocyte population in individual mice (Gr1^lowCD115⁺ cells out of total CD115⁺ cells). While in wt and cx₃cr1^gfp/+ naive mice approximately one-third of blood monocytes are Gr1^low, in cx₃cr1^gfp/gfp mice the latter comprise only 15% of the total monocyte population (Figure 1C′). This way of presentation will be used throughout the remainder of this study.

Mice deficient for CX₃CL1, the single known CX₃CR1 ligand,^3,10  have fewer F4/80⁺ blood monocytes than their wt littermates.⁴⁶  We therefore next tested whether also the cx₃cl1^−/− phenotype is restricted to the Gr1^lowCX₃CR1^hi monocytes. Indeed, cx₃cr1^gfp/gfp and cx₃cl1^−/− mice showed similar fractions of the Gr1^low subset, which were significantly lower than in wt controls (Figure 1D).

The observed Gr1^low monocyte reduction could result from a systemic defect in CX₃CR1- and CX₃CL1-deficient mice or be a direct outcome of the absence of the receptor on Gr1^low monocytes. To test these options, we generated mixed BM chimeras by reconstituting lethally irradiated wt mice with a 1:1 mixture of wt and cx₃cr1^gfp/gfp BM cells. Eight weeks after transfer, the recipients' blood contained both GFP-positive (cx₃cr1^gfp/gfp) and GFP-negative (wt) monocytes (Figure 2A), allowing the comparison of cells of distinct genetic background in the same environment. As shown in Figure 2A, compared with their wt counterpart, CX₃CR1-deficient Gr1^low monocyte numbers were reduced 3-fold. Next, we transferred wt and cx₃cr1^gfp/gfp BM cells into irradiated cx₃cl1^−/− and wt recipients. In the absence of CX₃CL1 expression by host cells, wt Gr1^low monocyte levels were significantly lower compared with wt > wt chimeras and similar to those found in cx₃cr1^gfp/gfp > wt mice (Figure 2B). To conclude, CX₃CR1- and CX₃CL1-deficient mice display a specific reduction of their Gr1^low monocytes, which is a direct result of CX₃CR1 requirement by BM-derived cells.

Next, we investigated the cause for the decrease of Gr1^low monocytes in the absence of CX₃CL1-CX₃CR1 interactions. Comparison of cx₃cr1^gfp/gfp and cx₃cr1^gfp/+ BM by us and others did not reveal any differences in Gr1^low BM monocyte levels (Figure 2C).⁴³  This indicates that, as opposed to other chemokine receptors such as CCR2,⁴⁹  CX₃CR1 is not required for BM egress.

The adoptive cotransfer of wt and CX₃CR1-deficient Gr1^low monocytes allows the comparison of monocyte fate, bypassing differences in monocyte production. We previously reported such cotransfers, 2 days after which we analyzed the recipients for the presence of grafted cells.³⁵  We observed a 2.5-fold reduction in the level of retrieved grafted cx₃cr1^gfp/gfp monocytes as compared with cotransferred cx₃cr1^gfp/+ monocytes in recipient's blood and peripheral organs.³⁵  The overall reduction in the level of grafted cx₃cr1^gfp/gfp monocytes suggested impaired survival.

To directly address increased monocyte apoptosis as the cause of the CX₃CR1 knockout (KO) phenotype, we took advantage of mice transgenic for the human antiapoptotic factor Bcl2 under the control of the neutrophil- and monocyte-specific MRP8 promotor (hMRP8bcl2 mice⁴⁷ ). As reported, enforced Bcl2 expression resulted in increased blood monocyte levels⁴⁷  (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). Interestingly, although intracellular staining for human Bcl2 revealed that both monocyte subsets expressed comparable transgene levels (Figure S1A), the observed increment was significantly more pronounced for the Gr1^low subset (Figure 1B). This might indicate that Gr1^low monocytes are more prone to die than Gr1^hi monocytes, a notion also recently invoked by others.⁴³ 

To test the influence of enforced Bcl2 expression on the CX₃CR1 KO phenotype, we generated double transgenic cx₃cr1^gfp/gfp;hMRP6bcl2 and cx₃cr1^gfp/+;hMRP6bcl2 mice. Interestingly, analysis of the blood of these animals revealed that Bcl2 transgenic CX₃CR1 knockout mice displayed Gr1^low monocyte levels comparable with Bcl2-transgenic cx₃cr1^gfp/+ mice, levels which comprised approximately 70% of their respective total blood monocytes (Figure 2D). This was in contrast to the significant reduction of monocyte levels observed in nontransgenic cx₃cr1^gfp/gfp mice (Figures 1C′ and 2D). Hence, enforced monocyte survival restores Gr1^low cell numbers of CX₃CR1-deficient mice to the levels observed in CX₃CR1-proficient mice, thus rescuing their phenotype.

To conclude, our results suggest that the specific reduction of Gr1^low monocytes in cx₃cr1^gfp/gfp mice reflects impaired survival due to the absence of antiapoptotic CX₃CR1-CX₃CL1 interactions.

CX₃CL1 has been shown to act as a survival factor for transformed cell lines,^20,22  as well as cultured brain microglia.²¹  To directly study the role of CX₃CR1-CX₃CL1 interactions in monocyte survival, we resorted to an ex vivo model. In this system, we studied the ability of recombinant soluble CX₃CL1 to rescue cultured isolated human blood monocytes from cell death induced by serum deprivation or exposure to oxysterol,⁵⁰  a cytotoxic oxygenated cholesterol derivative known to accumulate in atherosclerosis lesions.⁵¹  Cultured monocytes underwent major apoptosis after 4 hours of serum deprivation (Figure 3B), which led us to choose this time point for our analysis. Independent repeats of these experiments resulted in distinct percentages of serum-deprived dying cells (compare Figure 3B,D, and G), presumably as a consequence of genetic variation among human donors and different blood bank storage periods. Addition of recombinant soluble CX₃CL1 (full length without transmembrane anchor, aa 1-337) to monocytes either incubated with serum-free medium (Figure 3B) or with 7-β-hydroxycholesterol (Figure 3C) resulted in a significant reduction of the percentage of PI⁺ dying monocytes, as compared with nonsupplemented media. Decreased percentage of dead cells with the rising concentration of supplemented CX₃CL1 (Figure 3D) indicated dose-dependency of its activity. Since CX₃CR1 is a member of the 7-transmembrane G-protein coupled receptor (GPCR) family,¹⁰  its activity should be blocked in the presence of Pertussis toxin (PTx), a Gα-protein inhibitor. Indeed, addition of PTx to the cultured monocytes reversed the antiapoptotic effect of CX₃CL1 (Figure 3E). This indicates that Gα-protein–dependent signaling by CX₃CR1 is required for CX₃CL1-mediated monocyte survival.

Similarly to those of mouse, human blood monocytes comprise 2 subsets that are distinct in their CX₃CR1 expression levels.³⁸  CD14⁺⁺CD16⁻ monocytes express intermediate levels of CX₃CR1, and CD14⁺CD16⁺ monocytes express high levels of CX₃CR1.³⁵  In contrast to those of mouse, human CX₃CR1^high monocytes comprise only 5% of the total monocyte population in healthy individuals.³⁸  We next compared the effect of CX₃CL1 on the 2 human monocyte subsets (Figure 3F) upon serum depravation. Interestingly, both subsets responded similarly to 10 nM chemokine addition, as indicated by comparable levels of surviving cells (Figure 3G). In addition, no significant differences between monocyte subsets could be observed upon incubation with additional CX₃CL1 concentrations (1, 5, or 100 nM, not shown). Therefore, in contrast to the murine in vivo data, the protective effect of CX₃CL1 on cultured human monocytes is not restricted to CX₃CR1^hi cells.

To conclude, CX₃CL1 specifically acts as a human blood monocyte survival factor. However, at least in vitro, it has similar effect on both monocyte subsets. Importantly, this activity does not require cell-cell interactions.

Mice deficient for apolipoprotein E (ApoE) develop atherosclerosis if subjected to a Western high-fat diet.⁵²  However, when on a CX₃CR1-deficient genetic background, apoe^−/− mice display reduced atherosclerotic plaque formation.^27,28  Moreover, CX₃CL1-deficient mice also are relatively protected from atherosclerosis.^29,30 

To probe for a potential connection between the above-described prosurvival effect of CX₃CL1 and the reported atherosclerosis phenotype of CX₃CR1- and CX₃CL1-deficient mice, we sublethally irradiated apoe^−/− mice and reconstituted them with cx₃cr1^+/+, cx₃cr1^gfp/+, or cx₃cr1^gfp/gfp BM cells. Four weeks after BM transfer, the chimeras were exposed to a high-fat diet, and 3 months later their arteries were analyzed for the formation of atherosclerotic plaques. No differences in plaque areas between recipients of wt and cx₃cr1^gfp/+ BM were detected (Figure S2). Notably, the presence of ApoE-expressing donor cells in ApoE-deficient hosts alters disease progression in these mixed chimeras,⁵³  potentially preventing the detection of subtle differences detected by others. However, importantly and in agreement with the published data,^27,28,31 apoe^−/− recipients of cx₃cr1^gfp/gfp BM cells developed significantly reduced atherosclerotic plaque area levels in both thoracoabdominal aorta and aortic roots, as compared with apoe^−/− recipients of cx₃cr1^+/+ BM (Figure S2 and data not shown).

Atherosclerotic plaques display a complex composition of mononuclear phagocytes, including monocytes, DCs, and unique plaque-associated macrophages, the so-called foam cells.^24,31,54  Immunohistochemical analysis of atheroma of the cx₃cr1^gfp/+ > apoe^−/− BM chimeras revealed abundant CX₃CR1/GFP expressing cells (Figure 4A); among them were cells displaying the marker MOMA-2, which is known to be expressed by monocytes and foam cells,⁵⁵  as well as Oil Red O–positive fat deposits, a foam cell characteristic (Figure 4B). Interestingly, the number of both CX₃CR1/GFP-positive and MOMA-2–positive cells was significantly reduced in apoe^−/− recipients of cx₃cr1^gfp/gfp BM (Figures 4C,D non-tg bars).

Similar to steady-state conditions (Figure 1), analysis of apoe^−/− recipients of wt or cx₃cr1^gfp/gfp BM cells subjected to high-fat diet revealed that the CX₃CR1 deficiency caused a significant reduction of blood monocytes levels also under systemic inflammatory conditions (Figure 5A non-tg bars). Furthermore, this reduction was again more pronounced for the Gr1^low monocyte population (Figure 5B non-tg bars).

The observed decrease of CX₃CR1/GFP-positive cells in atherosclerotic plaques could thus result from impaired monocyte recruitment. Alternatively, but not mutually exclusively, CX₃CL1-CX₃CR1 interactions might be required locally for cell survival within the atheroma. The latter is an inherently hostile environment due to the abundant presence of oxidized cholesterols.⁵⁶  Of note, both activated endothelium and intimal smooth muscle cells express CX₃CL1^3,7,57,58  and, therefore, could serve as a source of survival signals for plaque-resident CX₃CR1-expressing cells.

To probe for this local scenario, we analyzed plaque apoptotic cell content using a TUNEL assay on tissue sections of the various apoe^−/− BM chimeras. As seen in Figure 4E, atheromas of (cx₃cr1^gfp/gfp > apoe^−/−) BM chimeras displayed a significantly increased frequency of TUNEL⁺ apoptotic cells, as compared with atheromas of (wt > apoe^−/−) BM chimeras. Notably, we could detect apoptotic cells, which were also CX₃CR1/GFP⁺ in (cx₃cr1^gfp/gfp > apoe^−/−) chimera, further indicating a CX₃CR1 requirement during cell survival (Figure 4F).

To investigate the involvement of cell death in the reduced plaque cell levels in the absence of CX₃CR1, we took advantage of the hMRP8bcl2-transgenic mice, which express human Bcl2 tg under monocyte- and macrophage-specific promotors.⁵⁹  To this end, we sublethally irradiated apoe^−/− mice and reconstituted them with hMRP8bcl2-transgenic cx₃cr1^+/+, cx₃cr1^gfp/+, or cx₃cr1^gfp/gfp BM. After recovery the mice were subjected to high-fat diet as described earlier. As in steady state (Figure 2D), introduction of the hBcl2 transgene increased the frequency of total monocytes (Figure 5A) and restored the ratio of the monocyte subsets in CX₃CR1 BM-deficient atherosclerotic mice (Figure 5B). Immunostaining for hBcl2 protein indicated that in addition to the blood, the transgene is also expressed in the atheroma (Figure 4G). Furthermore, hBcl2⁺ plaque cells were large in size, reminiscent of foam cells.

Next, we assessed the influence of enforced cell survival on plaque cellular composition. Immunohistochemical analysis revealed that the level of TUNEL⁺ apoptotic cells in apoe^−/− recipient of cx₃cr1^gfp/gfp;hMRP8bcl2-transgenic BM was restored to that found in CX₃CR1-proficient mice (Figure 4E). Importantly, the frequency of plaque-resident CX₃CR1/GFP (Figure 4A,C) and MOMA-2 (Figure 4D) expressing cells was increased to wt levels, rescuing the cx₃cr1^gfp/gfp phenotype.

Taken together, our data indicate that atherosclerotic plaques contain CX₃CR1-expressing cells, among them foam cells, that depend on CX₃CL1-CX₃CR1 interaction for their survival.

We have shown requirement of CX₃CR1 for the survival of blood and atheroma cells, including monocytes and foam cells. To test whether the absence of CX₃C-mediated cell survival signals could provide the mechanistic explanation for the reduced atherosclerosis susceptibility of CX₃CR1-deficient mice, we asked whether enforced cell survival would affect their protection from disease progression. To this end, we sublethally irradiated apoe^−/− mice and reconstituted them with hMRP8bcl2-transgenic BM, either cx₃cr1^+/+ or cx₃cr1^gfp/gfp. Plaques of CX₃CR1-deficient mice showed a decreased acellular area, also known as necrotic core (Figure 4H non-tg bar), indicating less progressed disease,⁵⁶  a phenotype that was restored upon hBcl2 expression (Figure 4H). As seen in Figure 6, Bcl2 transgenesis raised total plaque area levels in both the aorta and the aortic roots of apoe^−/− recipients even in the presence of CX₃CR1-expressing BM cells. Most importantly, recipients of both cx₃cr1^+/+;hMRP8bcl2 and cx₃cr1^gfp/gfp;hMRP8bcl2 BM cells showed similar disease scores (Figure 6A′,B′). Thus, expression of the Bcl2 transgene, leading to enforced monocyte and foam cell survival (Figure 4), overrode the protection imposed by the CX₃CR1 deficiency in cx₃cr1^gfp/gfp BM recipients.

To conclude, our results suggest that increased cell death in absence of CX₃CR1-CX₃CL1 interactions is the cause for the reduced atherogenesis observed in mice deficient for this chemokine receptor/ligand pair. Our results thus provide a mechanistic explanation for the well-established requirement for CX₃CR1 during atherogenesis, in both mice and humans.

The physiologic function of the CX₃C chemokine family, as represented by the membrane-tethered ligand CX₃CL1 (fractalkine) and its sole receptor, CX₃CR1, remains poorly understood. Here, we establish a novel role for CX₃CR1-CX₃CL1 interactions in mononuclear phagocyte survival. First, we show that in the blood of both CX₃CL1- and CX₃CR1-deficient mice, Gr1^low monocyte levels are specifically reduced. Enforced monocytic expression of the antiapoptotic factor Bcl2 reverted the phenotype, providing genetic evidence that this reduction results from impaired monocyte survival. In support of the in vivo data, recombinant CX₃CL1 specifically rescued cultured human monocytes from serum deprivation and oxysterol-induced death. This indicates a direct and evolutionary conserved role for CX₃CR1-CX₃CL1 interaction in cell survival. Finally, we demonstrate that CX₃C survival signals can provide the mechanistic explanation for one of the few known phenotypes of CX₃CR1- and CX₃CL1-deficient mice (ie, their relative protection from diet-induced atherosclerosis).^27-30  Thus, enforced survival of monocytes and foam cells in CX₃CR1 knockout mice restored atherogenesis scores to those found in wt mice, suggesting that CX₃CL1-CX₃CR1–mediated survival signals are critical for atheroma formation and progression.

CX₃CR1-proficient mice expressing the human Bcl2 transgene show higher blood monocyte levels as well as increased atherosclerosis compared with nontransgenic controls (Figures 2,5). The Bcl2 phenotype could, thus, mask the CX₃CR1 KO phenotype rather than rescuing it. However, if those 2 phenotypes are not linked, CX₃CR1-deficient mice would be expected to have, at least to some extent, lower monocyte levels and decreased atherosclerosis even on a Bcl2-transgenic background. Yet, in both cases, despite dramatic discrepancies between CX₃CR1-proficient and -deficient mice, no difference could be observed between groups of Bcl2-transgenic mice, supporting an antiapoptotic role for CX₃CR1. Moreover, the direct involvement of CX₃CR1 in cell survival control is also implied by the increased level of apoptotic cells in the plaques of atherosclerotic CX₃CR1-deficient mice (Figure 4E).

Although chemokines were originally defined as chemoattractants, a growing body of evidence indicates their additional involvement in the control of cell survival.⁶⁰  Interestingly, addition of the Gα-protein inhibitor PTx in our study increased apoptosis of serum-cultured monocytes (Figure 3E), which is compatible with the notion that serum contains chemokines that trigger survival promoting GPCRs. CX₃CL1 itself was reported to promote the survival of cancer cell lines,^20,22  as well as cultured microglia.²¹  Other chemokines, such as CXCL12/SDF1 and CXCL9/MIG protect CXCR4- and CXCR3-expressing cultured tumor cells from death.^61,62  Furthermore, CCL5/RANTES was shown to contribute to the in vivo survival of pulmonary macrophages during viral infection.⁶³  Interestingly, our recent finding of impaired foam cell levels and atherosclerotic lesion formation in ccr5^−/− mice^48,64  might suggest a potential role of CCR5-mediated survival also under these conditions.

We show that CX₃CL1 can protect both human monocyte subsets from cell death in vitro. In contrast, in vivo survival of the murine Gr1^hi monocytes subset did not depend on CX₃CR1 engagement (Figure 1). Of note, Gr1^hi monocytes, as well as their human CD14⁺⁺CD16⁻ counterpart, have an extensive chemokine receptor repertoire, as compared with Gr1^low and CD14⁺CD16⁺ cells.^35,38  Therefore, their survival in vivo could be ensured by other factors, including chemokines, to which Gr1^low and CD14⁺CD16⁺ monocytes might not respond. The differential sensitivity to CX₃CR1 deficiency of the monocyte subsets might also result from their distinct propensity to die, a notion supported by their distinct response to Bcl2 overexpression in this study as well as in other reports.⁴³ 

Monocytes are critical for the development, maintenance, and resolution of atherosclerosis.^65,66  In the plaque, they are believed to give rise to foam cells, a special macrophage subset found in those structures.⁵⁴  Foam cells were shown to play a key role in the development of early atherosclerotic plaques and their maintenance.⁶⁵  Accordingly, foam cell death during early disease stages has been shown to correlate with decreased atherogenesis, whereas their death during late stages leads to thrombosis and disease progression.⁵⁶  CX₃CL1 is prominently expressed by multiple nonhematopoetic cell types in the lesions, including muscle and endothelial cells.^5,7  Here, we suggest that in absence of its sole receptor, CX₃CR1, monocytes and foam cells undergo increased cell death, which together with potential effects on recruitment leads to a reduced foam cell load and the inhibition of plaque progression.

Our results suggest that inhibition of CX₃CR1-CX₃CL1 interactions during early atherogenesis stages might be beneficial in controlling this disease. However, since CX₃C signals are likely to be required for foam cell survival also during later atheroma stages, such inhibition could result in disease exacerbation. The development of therapeutic protocols based on interference with the CX₃CR1-CX₃CL1 axis will, therefore, require an in-depth understanding of the system.

We thank members of the Jung laboratory for critical reading of the manuscript and are grateful to Y. Chermesh and O. Amram for animal husbandry.

This work was supported by the MINERVA Foundation (Munich, Germany; S.J.) and the Israeli Science Foundation (Jerusalem, Israel; S.J.) as well as the Deutsche Forschungsgemeinschaft (FOR809; Bonn, Germany; A.Z. and C.W.). S.J. is the incumbent of the Pauline Recanati Career Development Chair.

Contribution: L.L., L.B.-O., and A.Z. designed and performed research; K.K., R.K., and E.S. provided critical technical assistance; S.A.L. and I.L.W. contributed vital reagents; and S.J., L.L., and C.W. designed research and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Steffen Jung, PhD, Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel; e-mail: s.jung@weizmann.ac.il.