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. Netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated macrophage migration to chemokines implicated in their egress from plaques. Acting via its receptor UNC5b, netrin-1 inhibited CCL2and CCL19-directed macrophage migration, Rac1 activation and actin polymerization. Targeted deletion of netrin-1 in macrophagesseverely diminished atherosclerosis progression in Ldlr−/− mice and promoted macrophage emigration from plaques. Thus, netrin-1 promotes atherosclerosis by retaining macrophages in the artery wall and 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 Correspondence should be addressed to K.J.M. (kathryn.moore@nyumc.org), Kathryn J. Moore, Ph.D., New York University School of Medicine, 522 First Avenue, Smilow 705, New York, NY 10016, tel: 212.263.9259, fax: 212.263.9115. AUTHOR CONTRIBUTIONS J.vG. performed migration and atherosclerosis studies; M.D. performed smooth muscle studies and fetal liver cell transplantation; K.R and L.F. performed mouse atherosclerosis studies, J.A-L., S.P. and B.R. performed microscopy; T.R., A.R. and J.F performed biochemical assays; T.M. and K.O. performed immunohistochemical studies of human atheroma; E.D. and E.F. assisted in bead labeling experiments, L.S and A.L-H. contributed to experimental design, data analysis and provided helpful discussion; K.M. designed, analyzed and interpreted the studies and wrote the manuscript with J.vG. The authors have no competing financial interests. NIH Public Access Author Manuscript Nat Immunol. Author manuscript; available in PMC 2012 August 01. Published in final edited form as: Nat Immunol. ; 13(2): 136–143. doi:10.1038/ni.2205. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript oxidative modification of these lipoproteins, which triggers an influx of monocytes1. 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 content2. 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 vessels3. 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 phenotype4. Recent studies in transplant-based mouse models of atherosclerosis regression have shown that reducing plasma non-HDL cholesterol and/or increasing highdensity lipoproteins (HDL), promotes emigration of macrophages from lesions to regional and systemic lymph nodes5-9. Macrophage expression of the chemokine receptor CCR7 was shown to be essential for decreasing the macrophage content of plaques8,9, 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 midline10. Conversely, co-expression of the UNC5b receptor with DCC converts netrin-1 attraction to repulsion, whereas expression of UNC5b alone mediates short-range repulsion10,11. Previous studies have uncovered instructional roles for netrin-1 and its receptors outside the nervous system in organogenesis12,13, angiogenesis14,15 and tumorigenesis16,17, 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 aureus18. 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 accumulation18-21. 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 van Gils et al. Page 2 Nat Immunol. Author manuscript; available in PMC 2012 August 01. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript netrin-1 in myeloid cells severely reduced atherosclerosis lesion size and complexity in Ldlr−/− mice and was associated with macrophage emigration from plaques.