It is appreciated that plaques that cause clinical events, so called “vulnerable plaques”, have a high macrophage content2
. 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 regression8,9
. 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 inflammation27,28
. 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 states18,29-31
. 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 tissues18-20,32
. 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 tissue18
. By contrast, netrin-1 is upregulated on gut epithelial cells during transient ischemia and attenuates neutrophil recruitment to protect from hypoxia-induced inflammation20
. 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 atherosclerosis33
, 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 transcription19,20,34
. Hypoxic stress is intimately linked to atherosclerosis35
and these transcription factors are activated in lesional macrophage. Induction of netrin-1 by oxLDL involved activation of NF-κB via CD3623,24
, a scavenger receptor previously implicated in the retention of macrophages in atherosclerosis22,36
. Macrophages that infiltrate the arterial intima are thought to exit via lymphatic vessels or egress into the bloodstream, or undergo apopotosis and efferocytotic clearance37-39
. 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 humans40
. 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/CCL215-9
. 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.