The neuroimmune guidance cue netrin-1 promotes atherosclerosis by inhibiting the emigration of macrophages from plaques.

Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophages in the artery wall. The mechanisms by which these cells become trapped, thereby establishing chronic inflammation, remain unknown. Here we found that netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated the migration of macrophages toward chemokines linked to their egress from plaques. Acting via its receptor, UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of the actin-remodeling GTPase Rac1 and actin polymerization. Targeted deletion of netrin-1 in macrophages resulted in much less atherosclerosis in mice deficient in the receptor for low-density lipoprotein and promoted the emigration of macrophages from plaques. Thus, netrin-1 promoted atherosclerosis by retaining macrophages in the artery wall. Our results establish a causative role for negative regulators of leukocyte migration in chronic inflammation.

Atherosclerosis is a disease of chronic inflammation that is distinguished by the persistence of cholesterol-engorged macrophages in arterial plaques. Arterial inflammation is initiated by the subendothelial retention of plasma low density lipoprotein (LDL), and enhanced by oxidative modification of these lipoproteins, which triggers an influx of monocytes[1]. Unlike other inflammatory states, atherosclerotic inflammation does not readily resolve and cholesterol-laden macrophages persist in the arterial wall. These macrophage “foam cells” cause expansion of the plaque though recruitment of additional leukocytes and vascular smooth muscle cells, and contribute prominently to plaque instability through the secretion of extracellular matrix-degrading proteases and cytotoxic factors. Notably, atherosclerotic plaques that cause clinical events (ie. myocardial infarction and stroke) are characterized by a high macrophage content[2].

While macrophage retention in the artery wall has long been recognized as a fundamental step in creating the chronic inflammatory milieu underlying atherosclerosis, the mechanisms regulating this process are not well understood. Resolution of acute inflammation typically involves emigration of monocyte-derived cells out of the inflamed site through nearby lymphatic vessels[3]. This process appears to be impaired in atherosclerosis and has been attributed, in part, to the cholesterol loading of macrophages which shifts these cells to a more sessile phenotype[4]. Recent studies in transplant-based mouse models of atherosclerosis regression have shown that reducing plasma non-HDL cholesterol and/or increasing high-density lipoproteins (HDL), promotes emigration of macrophages from lesions to regional and systemic lymph nodes[5-9]. Macrophage expression of the chemokine receptor CCR7 was shown to be essential for decreasing the macrophage content of plaques[8,9], implicating the CCR7-specific ligands CCL19 and CCL21 in promoting the egress of these cells from the artery wall. These studies indicate that macrophage emigration from the plaque is actively inhibited during hypercholesterolemia, although the regulatory signals that impair this process remain largely unknown.

A paradigm for inhibitory guidance cues exists in the developing nervous system, where axonal migration relies on the integration of both chemorepulsive and chemoattractive signals to steer the axonal growth cone. One such guidance molecule netrin-1, a secreted laminin-related molecule, mediates both chemorepulsion and chemoattraction of axons navigating the spinal cord midline. This context-dependent response to netrin-1 is regulated by differential receptor expression by the target cell. For example, neurons expressing the Deleted in Colon Cancer (DCC) receptor or Neogenin are attracted by a diffusible gradient of netrin-1 secreted at the midline[10]. Conversely, co-expression of the UNC5b receptor with DCC converts netrin-1 attraction to repulsion, whereas expression of UNC5b alone mediates short-range repulsion[10,11]. Previous studies have uncovered instructional roles for netrin-1 and its receptors outside the nervous system in organogenesis[12,13], angiogenesis[14,15] and tumorigenesis[16,17], suggesting that netrin-1 regulates cell migration in a broader context.

Work from our group identified netrin-1 as a leukocyte guidance cue expressed by the endothelium that is downregulated during acute infection with Staphylococcus aureus[18]. These studies established that netrin-1 inhibited migration of monocytes, neutrophils and lymphocytes via its receptor UNC5b. Recent studies in models of hypoxia and reperfusion injury have extended these findings to show that expression of netrin-1 by epithelial cells also attenuates leukocyte accumulation[18-21]. Given its role in inhibiting leukocyte migration, we sought to determine whether netrin-1 contributed to the retention of macrophages in the chronic inflammatory milieu of the atherosclerotic plaque. Our studies show that netrin-1 was abundantly expressed by macrophage foam cells formed in vitro and in vivo, and in atherosclerotic lesions. In functional studies we demonstrate that netrin-1 expressed by foam cells differentially regulated the cellular constituents of atheroma. Netrin-1 inactivated macrophage migration and supported chemoattraction of coronary artery smooth muscle cells. Thus, expression of netrin-1 in plaques would be predicted to simultaneously prevent inflammatory cell egress and induce smooth muscle cell recruitment into the intima, thereby promoting lesion progression. In support of thishypothesis, we demonstrate that deletion of netrin-1 in myeloid cells severely reduced atherosclerosis lesion size and complexity in Ldlr−/− mice and was associated with macrophage emigration from plaques.

RESULTS

Macrophage foam cells express the guidance cue netrin-1

Given its role in attenuating leukocyte migration[18], we first investigated whether netrin-1 is expressed in atherosclerotic plaques. Immunostaining of serial sections of human coronary artery atherosclerotic plaques showed expression of netrin-1 and its chemorepulsive receptor UNC5H2 (also called UNC5b) in lesional cells that express the macrophage marker HAM56 (Supplementary Fig 1a,b). By contrast, DCC, the chemoattractive netrin-1 receptor, was not detected in these plaques (data not shown). A similar pattern of lesional netrin-1 staining was seen in the Ldlr−/− mouse model of atherosclerosis (Fig. 1a). In aortic sinus plaques of Ldlr−/− mice fed a western diet (WD) for 12 weeks, double staining for netrin-1 and the macrophage marker CD68 showed netrin-1 expression by lesional macrophages. In addition, extracellular netrin-1 staining was apparent in macrophage-rich regions of the plaque, consistent with netrin-1 being a secreted protein that can bind to extracellular matrix components. Analysis of the aortic arch, a second site of lesion predilection in mice, showed that Ntn1 mRNA was increased in Ldlr−/− mice compared to wild-type C57BL/6 mice, and netrin-1 expression was further upregulated by feeding these mice a WD (Fig. 1b). Similar results were obtained in the Apoe−/− mouse model of atherosclerosis (Supplementary Fig. 1c), indicating that netrin-1 expression by lesional macrophages is a common characteristic of mouse and human atheroma.

Figure 1

Netrin-1 and it receptor UNC5b are abundantly expressed by macrophage foam cells in atherosclerotic lesions

(a) Immunofluorescent staining of netrin-1 (green) and CD68 (red), and their colocalization (yellow, arrows), in aortic sinus atherosclerotic plaques of Ldlr−/− mice fed a WD. Dashed line indicates lesion border (scale bar= 50 μm). Staining is representative of plaques from 4 mice. (b) qPCR analysis of Ntn1 mRNA isolated from the aortic arch of C57BL/6 or Ldlr−/− mice fed a chow or WD. (c) qPCR analysis of Ntn1, Unc5b, Cd68 and Abca1 mRNA in pMø isolated from Ldlr−/− mice fed a chow or WD. (d) qPCR analysis of Ntn1 and Unc5b in pMø treated with 50 μg/ml oxLDL, and corresponding expression of netrin-1 protein measured in (e) cell lysates by immunoblot or (f) conditioned media by ELISA. (g) qPCR analysis of Ntn1 mRNA in wild-type (WT) or Cd36−/− pMø stimulated with 50 μg/ml oxLDL for 6 h. (h) Ntn1 promoter-luciferase reporter activity in HEK293 cells treated with oxLDL in the presence or absence of the NF-κB inhibitor BAY 11-7082 (20 μM). (b-h) Data are mean ± s.d. of triplicate samples in a single experiment and are representative of 3 independent experiments. *P<0.05.

To understand the molecular mechanisms regulating netrin-1 and UNC5b expression, we isolated peritoneal macrophages (pMø) from Ldlr−/− mice fed either a chow or WD. In this commonly used model of in vivo foam cell formation, WD-feeding markedly increased Ntn1 and Unc5b mRNAs, whereas expression of CD68 was unchanged (Fig. 1c). Similar results were observed in hypercholesterolemic Apoe−/− mice (Supplementary Fig. 1d), suggesting that increased cellular lipid accumulation may upregulate expression of netrin-1 and UNC5b. To test this, we treated pMø with native LDL, or LDL that had been oxidized (oxLDL), a modification that promotes cholesterol loading of macrophages.

Consistent with our in vivo data, oxLDL, but not LDL, increased macrophage expression of Ntn1 and Unc5b mRNA (Fig. 1d, Supplementary Fig. 1e). Immunoblot analysis confirmed increased cell-associated netrin-1 protein in oxLDL-treated macrophages (Fig. 1e), which was paralleled by an increase in netrin-1 in cell culture supernatants (Fig. 1f). Notably, the induction of Ntn1 mRNA (Fig. 1g) and netrin-1 protein (Supplementary Fig. 1f) by oxLDL required CD36, a scavenger receptor previously implicated in macrophage retention in atherosclerotic plaques[22]. Because we and others have shown that oxLDL binding to CD36 induces NF-κB activation[23,24] and the Ntn1 promoter contains an NF-κB binding site[20], we investigated whether NF-κB contributes to the upregulation of Ntn1. Using a Ntn1 promoter-luciferase reporter gene, we demonstrate that Ntn1 promoter activity was induced by oxLDL and this was reduced by the NF-κB inhibitor BAY 11-7082 (Fig. 1h). Collectively, these data demonstrate that loading of macrophages with cholesterol under physiologic conditions, or by oxidized lipids via CD36 in vitro, increases expression of netrin-1 and its receptor UNC5b.

Netrin-1 blocks the directed migration of macrophages

We next assessed the impact of netrin-1 on macrophage chemotaxis using transwell Boyden chambers and a real-time detection method (xCelligence). Using both methods, recombinant netrin-1 potently inhibited the chemotaxis of the macrophage cell line RAW264.7 to CCL2, also known as monocyte chemoattractant protein-1 (MCP-1), but had little effect on macrophage migration in the absence of chemokine (Fig. 2a-b). The inhibitory effects of netrin-1 on chemotaxis were dose dependent (Fig. 2a), with 250 ng/ml netrin-1 inhibiting CCL2-induced migration by >90% (Fig. 2b). Furthermore, pre-treating RAW264.7 cells with netrin-1 for 1 h rendered the cells refractory to CCL2 (Fig 2c). Similar to its effects on RAW264.7 macrophages, netrin-1 potently inhibited chemotaxis of pMø to CCL2 (Fig 2d). This effect was not chemokine-specific, as netrin-1 also blocked migration of RAW264.7 and pMø to the CCR7 ligands CCL19 and CCL21 (Fig 2e, Supplemental Fig. 2a-g), chemokines implicated in the egress of CD68+ cells from plaques[8,9]. To gain insight into the mechanisms by which netrin-1 inhibits macrophage chemotaxis, we measured its effect on organization of the actin cytoskeleton. Stimulation of pMø with CCL2 or CCL19 induced a marked reorganization of actin, characterized by the appearance of membrane ruffles, lamellipodia and filapodia (Fig. 2f (arrows), Supplementary Fig. 3a), and rapid cell spreading (Fig. 2g, Supplementary Fig. 3b). By contrast, pMø pre-treated withnetrin-1 prior to stimulation with CCL2 or CCL19 maintained a rounded morphology (Fig. 2f (arrowheads), Supplementary Fig. 3a) and showed no increase in mean cell area (Fig. 2g, Supplementary Fig. 3b), consistent with a non-motile status. Quantification of phalloidin-stained actin filaments by flow cytometry confirmed that netrin-1 blocked the increase in polymerized actin-1 associated with CCL2 stimulation (Fig. 2h). As the Rho GTPase Rac1 plays a key role in the reorganization of actin in macrophages, we next investigated whether netrin-1 altered Rac1 activation following chemotactic stimulation using glutathione-S-transferase (GST) beads conjugated to the PAK1-PBD to detect the activated GTP-bound form of Rac1. Whereas netrin-1 pretreatment of macrophages modestly increased basal amounts of activated Rac1, it inhibited CCL2-induced Rac1 activation (Fig. 2i) and phosphorylation of the focal adhesion kinase FAK (Fig. 2j), which cooperates with Rac1 to link the actin cytoskeleton to the extracellular matrix during cell spreading and migration. Collectively, these data indicate that netrin-1 inhibits the directional migration of macrophages by disrupting the Rac1 signaling cascade, re-organization of the actin cytoskeleton and cell polarization. As both netrin-1 and UNC5b were upregulated in cholesterol-loaded macrophages we postulated that netrin-1 secreted by macrophages in atherosclerotic plaques may act in an autocrine or paracrine manner via UNC5b to immobilize these cells in the artery wall. To test the role of UNC5b in the response to netrin-1, we pre-incubated macrophages with a recombinant UNC5b-Fc fusion protein or an antibody that binds to the extracellular domain of UNC5b. Both the recombinant UNC5b-Fc (Fig. 3a) and Unc5b antibody (Fig. 3b), but not control IgG, reversed the inhibitory effect of netrin-1 on CCL19-induced migration. By contrast, no change was seen with an inhibitor of the A2B adenosine receptor, another netrin-1 receptor implicated in the attenuation of neutrophil migration (Fig. 3c). Furthermore, consistent with the secretion of netrin-1 by macrophage foam cells, conditioned medium from oxLDL-but not LDL-treated macrophages, inhibited cell migration to CCL19 (Fig. 3d), and this was reversed by recombinant UNC5b-Fc (Fig. 3e). To confirm that netrin-1 is the active component secreted by oxLDL-treated Mø, we used pMø isolated from Ntn1−/− bone marrow chimeric mice, which do not express netrin-1 in response to oxLDL (Fig. 3f). Whereas conditioned medium from oxLDL-treated Ntn1+/+ pMø inhibits migration of naïve macrophages to CCL19 by 80%, conditioned medium from similarly treated Ntn1−/− macrophages reduces migration by only 25% (Fig. 3f). Furthermore, Ntn1+/+ conditioned medium incubated with recombinant UNC5b-Fc inhibited migration to a similar extent as Ntn1−/− conditioned medium (≈25%). By contrast, the effects of Ntn1−/− conditioned medium were unchanged by the addition of recombinant UNC5b (Fig. 3f). Together, these data suggest that netrin-1 secreted by cholesterol-laden macrophages would promote their accumulation in atherosclerotic plaques by inhibiting their emigration from this site of inflammation.

Figure 2

Netrin-1 inhibits macrophage migration to CCL2 and CCL19, via its receptor UNC5b

(a) Migration of RAW264.7 cells to CCL2 (100 ng/ml) was measured in the absence or presence of increasing concentrations of recombinant netrin-1 placed in the lower compartment of a Boyden chamber. (b) Real-time measurement of migration of RAW264.7 cells to 250 ng/ml netrin-1, 100 ng/ml CCL2, or both. (c) Migration of RAW264.7 cells pretreated (PT) with 250 ng/ml netrin-1 and exposured to CCL2 (100 ng/ml). (d-e) Migration of mouse pMø to (d) CCL2 (100 ng/ml) or (e) CCL19 (500 ng/ml), in the absence/presence of 250 ng/ml netrin-1. (f) pMø stained with phalloidin to detect polymerized actin after treatment with 100 ng/ml CCL2 with or without 250 ng/ml netrin-1. Arrows indicate membrane ruffles, scale bar 10 μm. (g) Mean cell surface area of pMø in (f). (h). Quantification of actin polymerization by flow cytometric analysis of phallodin staining. (i) Amount of activated Rac1 in pMø treated for 5 min with 100 ng/ml CCL2 with and without 250 ng/ml netrin-1. (j) Immunoblot of phospho- and total FAK in pMø incubated with 100 ng/ml CCL2 with or without 250 ng/ml netrin-1 pretreatment. Data are the mean ± s.d. of triplicate samples in a single experiment and are representative of 3 independent experiments. P < 0.05, **P < 0.01.

Figure 3

Foam cell secreted netrin-1 blocks macrophage migration

(a-c) Migration of pMø to CCL19 (500 ng/ml) with/without netrin-1 (250 ng/ml) in the presence of (a) recombinant UNC5b-Fc or IgG control, (b) anti-UNC5b or isotype-matched control antibody, or (c) an inhibitor of the A2B adenosine receptor (10 uM 8-PT). (d-e) Migration of pMø to CCL19 (500 ng/ml) in (d) the presence or absence of conditioned medium (CM) from macrophages treated with LDL or oxLDL (50 μg/ml; 48 h) and (e) with/without recombinant UNC5b-Fc or IgG control. (f) Migration of pMø to CCL19 (500 ng/ml) as in (e) except using conditioned medium (CM) from WT or Ntn1−/− macrophages treated with oxLDL. Inset: immunoblot of netrin-1 in lysates of WT and Ntn1−/− pMø. (a-f) Data are the mean ± s.d. of triplicate samples in a single experiment and are representative of 3 independent experiments. *P < 0.01. (g) Effect of netrin-1 (500ng) or PBS pretreatment on LPS-induced leukocyte emigration from the peritoneum of mice with established thioglycollate-induced peritonitis. Data are the mean number (± s.e.m) of leukocytes in the peritoneum of mice (n = 6/group) before and 4h after injection of 400ng LPS ip., and are representative of 2 independent experiments. *P < 0.05.

Studies in a transplant mouse model of atherosclerosis regression have demonstrated that macrophages in plaques exit via nearby lymphatics upon aggressive cholesterol lowering[6]. A similar pathway for macrophage clearance has been reported during resolving peritonitis where inflammatory macrophages emigrate to the draining lymphatics of the omentum[3,25]. To test the effect of netrin-1 on macrophage emigration in vivo we used a well-characterized thioglycollate-induced peritonitis model in which administration of lipopolysaccharide (LPS) induces the rapid egress of recruited leukocytes from the peritoneum[22]. As previously reported, i.p. injection of LPS reduced the number of leukocytes in the peritoneum by 75% in control mice (Fig. 3g). By contrast, mice pretreated with netrin-1 (i.p.) 45 min prior to this inflammatory stimulus showed no significant change in peritoneal leukocytes. Flow cytometric analysis of the macrophage marker F4/80 confirmed that macrophages were retained in the peritoneum of netrin-1 pre-treated mice compared to control mice (0.4 × 106 ± 0.1 × 106 control vs. 1.1 × 106 ±0.3 × 106 netrin-1). These data demonstrate that netrin-1 can inhibit the emigration of macrophages from a site of active inflammation.

Netrin-1 is a chemoattractant for smooth muscle cells

During the progression of atherosclerosis, smooth muscle cells from the underlying medial layer are recruited to the plaque and participate in promoting plaque growth. To investigate whether netrin-1 expression in plaque affects other cellular components of the atherosclerotic lesion, we measured its effect on migration of human coronary artery smooth muscle cells (CaSMCs). Unlike its inhibitory effect on macrophages, netrin-1 dose-dependently induces migration of CaSMCs (Fig. 4a). Furthermore, conditioned medium from oxLDL-, but not LDL-treated macrophages induces migration of CaSMCs (Fig. 4b). To understand how Netrin-1 mediates chemoattraction of CaSMCs, we measured expression of its known receptors. Whereas CaSMCs had low expression of Dcc and Unc5b, these cells abundantly expressed the DCC-related receptor neogenin (Fig. 4c-d), as previously reported for vascular smooth muscle cells13. Notably, pre-treatment of CaSMCs with a neogenin antibody abrogated the chemoattractive effect of conditioned medium from oxLDL-treated macrophages (Fig. 4e), supporting a role for foam cell derived netrin-1 in inducing SMC migration. Immunohistochemical staining of atherosclerotic plaques of Ldlr−/− mice showed colocalization of neogenin with smooth muscle actin (Fig. 4f), but not the macrophage marker CD68 (Supplementary Fig. 4a). Furthermore, neogenin was expressed in medial smooth muscle cells (arrows) and in smooth muscle cells that had migrated into the intima (arrowheads), as well as some cells that were not smooth muscle actin positive (Fig. 4f). Together these data indicate that through distinct mechanisms netrin-1 modulates CaSMC and macrophages migration.

Figure 4

Netrin-1 acts as a chemoattractant for smooth muscle cells via the receptor neogenin

(a) Migration of human CaSMCs in the presence of recombinant netrin-1. (b) Migration of CaSMCs in the presence of conditioned media from macrophages treated for the indicated times with LDL or oxLDL (50 μg/ml). (c) qPCR analysis of Neogenin, Unc5b and Dcc mRNAs in CaSMC. (d) Immunofluorescent staining for neogenin, DCC or isotype-matched control antibody in CaSMCs. Cells were co-stained with DAPI nuclear stain. Scale bar, 10 μm. (e) Migration of CaSMCs pre-treated with neogenin or isotype control antibody prior to exposure to conditioned media from macrophages treated as indicated for 24 h. (f) Immunofluorescent staining of alpha smooth muscle actin (SMA, green), neogenin (red) and DAPI (blue) in atherosclerotic plaques of Ldlr−/− mice fed a WD. Co-localization of SMA and neogenin (yellow in the merged image) was detected in the media (arrows) and in SMC that had invaded the intima (arrowheads). Staining is representative of plaques from 5 mice. Scale bar, 50 μm. (a-c, e) Data are the mean ± s.d. of triplicate samples in a single experiment and are representative of 3 independent experiments. *P < 0.05.

Hematopoietic deficiency of netrin-1 reduces atherosclerosis

Based on the above data we postulated that expression of netrin-1 by macrophage foam cells may promote atherosclerotic plaque growth by both blocking macrophage egress and inciting smooth muscle cell recruitment in the intima. To test this hypothesis, we used fetal liver cells from day E14 Ntn1−/− or Ntn1+/+ (WT) pups to reconstitute the bone marrow of lethally irradiated Ldlr−/− mice, thereby generating Ldlr−/− mice with either Ntn1−/− or WT macrophages. Four weeks post-transplantation, the chimeric Ntn1−/− →Ldlr−/− and WT →Ldlr−/− mice were challenged with a WD for 12 weeks. Analysis of Ntn1 and Ldlr gene expression in both circulating leukocytes and pMø of recipient mice confirmed a complete change in the genotype to the donor types (Supplementary Table 1, Supplementary Fig. 4b). There were no differences in serum total cholesterol and triglyceride concentrations, or cholesterol distribution in VLDL, LDL, and HDL in chimeric Ntn1−/− →Ldlr−/− and WT →Ldlr−/− mice (Supplementary Table 1, Supplementary Fig. 4c). Despite these similar serum cholesterol profiles, Ldlr−/− mice lacking expression of Ntn1 in bone marrow-derived cells show a striking reduction in atherosclerosis (Fig. 5).

Figure 5

Targeted deletion of netrin-1 in immune cells reduces atherosclerosis burden

(a) Quantification and representative photographs of atherosclerosis in the aorta en face of WT →Ldlr−/− and Ntn1−/− →Ldlr−/− chimeric mice (n=10/group). *P < 0.01. Scale bar, 5 mm. (b-c) qPCR analysis of Cd68 (macrophage), Cd3 (T cell), Cd11c (dendritic cell), Elane (neutrophil) and Ntn1 mRNA in the aortic arches of WT →Ldlr−/− and Ntn1−/− →Ldlr−/− chimeric mice (n=3/group). Data are the mean ± s.d. of triplicate samples in a single experiment and are representative of 2 independent experiments. *P < 0.05. (d-e) Lesion area of atherosclerotic plaques of the aortic root of WT →Ldlr−/− and Ntn1−/− →Ldlr−/− mice expressed as the mean (d) of individual mice and (e) of each genotype across the 400 μm of the aortic root. *P < 0.005. (f) Representative photographs of hematoxylin and eosin stained aortic sinus lesions of WT →Ldlr−/− and Ntn1−/− →Ldlr−/− mice. Scale bar, 200 μm.

Analysis of the aorta en face showed that Ntn1−/− →Ldlr−/− mice had a 55% reduction in mean atherosclerotic lesion area compared to WT →Ldlr−/− mice (Fig 5a) that was present throughout the aorta (Supplemental Fig. 4d). Isolation of mRNA from the aortic arch of the Ntn1−/− →Ldlr−/− mice showed reduced expression of the macrophage marker CD68 compared to WT →Ldlr−/− mice (Fig. 5b), and correspondingly, lower expression of Ntn1 (Fig. 5c). Ntn1−/− →Ldlr−/− mice also had reduced expression of markers for other leukocyte subsets in the aortic arch, including CD3 (T cell) and CD11c (dendritic cell) (Fig. 5b). Furthermore, analysis of atherosclerotic plaques in a second anatomical site, the aortic root, showed markedly smaller lesion area in Ntn1−/− →Ldlr−/− mice compared to WT →Ldlr−/− mice (Fig. 5d-f). Quantification of lesion burden by cross-sectional analysis of the aortic root established that this decrease in lesion area in Ntn1−/− →Ldlr−/− mice was consistently present throughout the 400 μm of the aortic root (Fig. 5e).

Histological characterization of the aortic sinus plaques using the Stary method revealed that WT →Ldlr−/− mice had more advanced atherosclerosis, typified by a greater proportion of complex lesions (stage 3-4), whereas Ntn1−/− →Ldlr−/− plaques tended to be less progressed (stage 1-3) (Fig. 6a). Immunohistochemical staining of aortic sinus plaques confirmed a reduction in both macrophage (MOMA-2, Fig. 6b) and smooth muscle cells (alpha smooth muscle actin, Fig. 6c) in Ntn1−/− →Ldlr−/− compared to WT →Ldlr−/− mice. During the progression of atherosclerosis, macrophage persistence contributes to changes in plaque morphology, particularly the formation of the necrotic core. This critical feature of dangerous plaques arises from the combination of apoptosis of cholesterol-laden macrophages and defective phagocytic clearance of the apoptotic cells by the surrounding macrophages (efferocytosis). Notably, TUNEL staining revealed that plaques in Ntn1−/− →Ldlr−/− mice contained significantly fewer apoptotic cells compared to WT →Ldlr−/− mice (Fig. 6d) and this correlated with a 46% decrease in necrotic area in Ntn1−/− →Ldlr−/− plaques (Fig. 6e). Together these data suggest that netrin-1 expression by lesional macrophages promotes atherosclerotic plaque growth and complexity by enhancing macrophage retention in the intima.

Figure 6

Targeted deletion of netrin-1 in immune cells reduces plaque complexity and promotes macrophage emigration

(a) Classification of aortic sinus plaques of WT →Ldlr−/− and Ntn1−/− →Ldlr−/− mice (n = 10) according to the Stary method. I. Early (foam cells), II. Moderate 1 (foam cells, SMC), III. Moderate 2 (foam cells, SMC, clefts), IV. Advanced (necrotic core). (b,c) Immunohistochemical staining of aortic sinus plaques for (b) macrophages (MOMA-2) and (c) SMC (α-smooth muscle actin). Scale bar, 100 μm. Staining quantified using IP Lab Spectrum software is presented at right. n = 6-9. (d) Immunofluorescent staining of apoptotic cells (green) in aortic sinus plaques. TUNEL positive nuclei are indicated by arrowheads TUNEL and quantification is presented at right. Scale bar, 50 μm. n = 9. (e) Quantification of necrotic areas of aortic sinus plaques. n = 10. *P = 0.07. (f) In vivo analysis of macrophage recruitment and retention in atherosclerotic plaques of WT →Ldlr−/− and Ntn1−/− →Ldlr−/−mice using a monocyte bead-tracking model. The mean number of bead-labeled macrophages per plaque is shown 3 days (baseline) and 14 days after monocyte labeling (n = 3-4/group). . Representative image of plaques stained for CD68 (red) and cell nuclei (blue). Arrows indicate the presence of the cells containing fluorescent beads (green) within the lesion. Data in a-f are the mean ± s.e.m., *P < 0.05, unless otherwise noted.

To test whether netrin-1 expression in plaques inhibits macrophage emigration, we used a macrophage tracking technique in which monocytes can be selectively labeled in vivo with fluorescent beads so that their movement into[26] and out of[7] plaques can be tracked. WT →Ldlr−/− and Ntn1−/− →Ldlr−/− mice were harvested 72 h after labeled bead injection, a time point previously shown to have peak recruitment of labeled cells to atherosclerotic plaque (“baseline”)[26], and after 14 days to measure the number of labeled macrophages remaining in plaques. The number of bead-labeled macrophages in WT →Ldlr−/− plaques was similar at baseline and 14 days, indicating that macrophages did not emigrate during this time (Fig. 6f). By contrast, in Ntn1−/− →Ldlr−/− plaques there was a 40% decrease in beads after 14 days compared to baseline, indicating that fewer macrophages were retained in the plaque in the absence of netrin-1. As the beads cannot be degraded, and any labeled macrophages undergoing apoptosis are phagocytosed by surrounding macrophages thereby transferring the bead label, the reduction in beads in the Ntn1−/− →Ldlr−/− mice was indicative of macrophage movement out of the plaque. Collectively, our data support the retention of macrophages in plaques by netrin-1 and demonstrate that upon the removal of this retention signal, macrophages emigrate from this site of chronic inflammation.

DISCUSSION

It is appreciated that plaques that cause clinical events, so called “vulnerable plaques”, have a high macrophage content[2]. Thus, strategies targeted at reducing macrophage accumulation and/or directing emigration of these cells from the plaque offer promise as a complement to standard lipid lowering therapies. However to date, the signals responsible for mediating macrophage retention in the artery wall have been poorly understood. Our data establish that netrin-1, a neuronal guidance molecule with recently ascribed immunomodulatory functions, and its receptor UNC5b, are expressed by macrophage foam cells of human and mouse atherosclerotic plaques. Notably, recombinant netrin-1 and netrin-1 secreted by in vitro formed foam cells potently block the directed migration of macrophages to CCL19, a chemokine implicated in the emigration of monocyte-derived cells from plaques in a transplant model of regression[8,9]. Moreover, netrin-1 also blocked migration of macrophages to CCL2, which along with its receptor CCR2, has been shown to play roles in myeloid cell trafficking to the lymph node during inflammation[27,28]. Thus, netrin-1 in plaques may act to immobilize macrophage foam cells and prevent their egress to the lumen or lymphatic system. Supporting this hypothesis, we find that the selective deletion of netrin-1 in bone marrow derived cells markedly reduces atherosclerotic lesion size and complexity in Ldlr−/− mice, and is associated with macrophage emigration from plaques. These data establish a causative role for netrin-1 in the persistence of inflammation in atherosclerosis, and highlight the importance of such negative regulators of leukocyte migration in chronic inflammation.

There have been an increasing number of reports that guidance molecules characterized in the developing nervous system can modulate leukocyte migration in inflammatory states[18,29-31]. Such studies have shown that members of the netrin, slit, semaphorin and ephrin families of guidance cues can have both chemoattractive and chemorepulsive effects on leukocyte trafficking. Recent studies from our group and others have shown that netrin-1 is expressed on endothelial and epithelial cells, where it appears to function in limiting leukocyte transmigration into tissues[18-20,32]. For example, endothelial expression of netrin-1 is downregulated in the lung during acute Staphylococcus aureus induced inflammation, coincident with recruitment of neutrophils to the tissue[18]. By contrast, netrin-1 is upregulated on gut epithelial cells during transient ischemia and attenuates neutrophil recruitment to protect from hypoxia-induced inflammation[20]. These studies showing that netrin-1 inhibits migration of circulating leukocytes into tissues, and thus is anti-inflammatory in this capacity, have provided insight into the homeostatic barrier functions performed by netrin-1. In this regard, a recent study reported that intravenous viral delivery of netrin-1 to Ldlr−/− mice reduced atherosclerosis[33], presumably by increasing its expression on endothelial cells, although no determination of the cell types expressing netrin-1 were made. Unfortunately, this study was underpowered and the traditional atherosclerosis measurements of cross-sectional lesion area and en face aortic lesion burden were not assessed, limiting its conclusions. By contrast, our data indicate that the expression of netrin-1 by macrophage foam cells within plaques is pro-atherosclerotic, and its inactivation of macrophage emigration from this inflamed site likely inhibits the resolution of inflammation. Thus, as in the nervous system where netrin-1 can have both positive and negative effects on axonal migration, it may play multifunctional roles in regulating inflammation depending on the site of its expression.

While expressed at low levels in monocytes and most tissue macrophages, netrin-1 is highly expressed by macrophage foam cells in human and mouse atheroma. The mechanisms of netrin-1 upregulation in this context may be similar to those in hypoxia, where HIF1-α and NF-κB regulate netrin-1 transcription[19,20,34]. Hypoxic stress is intimately linked to atherosclerosis[35] and these transcription factors are activated in lesional macrophage. Induction of netrin-1 by oxLDL involved activation of NF-κB via CD36[23,24], a scavenger receptor previously implicated in the retention of macrophages in atherosclerosis[22,36]. Macrophages that infiltrate the arterial intima are thought to exit via lymphatic vessels or egress into the bloodstream, or undergo apopotosis and efferocytotic clearance[37-39]. All of these mechanisms appear to be impaired in atherosclerosis, leading to the progressive accumulation of macrophages in plaques. However, recent studies in mouse models suggest that these processes can be restored with aggressive lipid management, providing hope that atherosclerosis regression can be achieved in humans[40]. Genetic manipulations that reduced plasma non-HDL cholesterol and/or increased HDL cholesterol, promoted macrophage emigration from plaques to regional and systemic lymph nodes via a mechanism involving CCR7, the receptor for CCL19/CCL21[5-9]. Notably, as netrin-1 blocked macrophage chemotaxis to CCL19 and CCL21, it would be expected to inhibit the normal migratory processes that bring about resolution of inflammation. Consistent with this, deletion of netrin-1 in lesional macrophages in Ldlr−/− mice promotes macrophage emigration and reduces plaque size. Although the exact means by which netrin-1 inhibits migration remains to be elucidated, our data indicate that netrin-1 inhibits Rac-mediated re-organization of the actin cyctoskeleton, preventing cell spreading and the migration of macrophages out of plaques.

In addition to its effects on macrophages, netrin-1 is a potent chemoattractant for CaSMC, a process dependent on the neogenin receptor. The recruitment of smooth cells by netrin-1 represents a second mechanism by which this guidance molecule could promote atherosclerosis. During plaque progression, the internal elastic lamina can become breached and smooth muscle cells migrate into the intima to participate in lesion growth. In plaques of Ntn1−/− →Ldlr−/− chimeric mice, we see a reduction in the accumulation of smooth muscle cells, consistent with the notion that loss of netrin-1 both allows macrophage emigration from the plaque, and curtails the recruitment of SMC into it. Furthermore, Ntn1−/− →Ldlr−/− plaques show fewer apoptotic cells and reduced necrotic area indicating that the mechanisms regulating tissue homeostasis are enhanced in the absence of netrin-1. Together these data underscore the important role that negative guidance cues may play in the persistence of inflammation, and indicate that local inhibition of such factors may have therapeutic value for the resolution of inflammation in atherosclerosis, and other chronic inflammatory diseases.