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

Abstract

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.

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.

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.

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.

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.

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.