Targeting Unc5b in macrophages drives atherosclerosis regression and pro-resolving immune cell function

Abstract

Atherosclerosis results from lipid-driven inflammation of the arterial wall that fails to resolve. Imbalances in macrophage accumulation and function, including diminished migratory capacity and defective efferocytosis, fuel maladaptive inflammation and plaque progression. The neuroimmune guidance cue netrin-1 has dichotomous roles in inflammation partly due to its multiple receptors; in atherosclerosis, netrin-1 promotes macrophage survival and retention via its receptor Unc5b. To minimize the pleiotropic effects of targeting netrin-1, we tested the therapeutic potential of deleting Unc5b in mice with advanced atherosclerosis. We generated Unc5b^fl/flCx3cr1^creERT2/WT mice, which allowed conditional deletion of Un5b (∆Unc5b^MØ) in monocytes and macrophages by tamoxifen injection. After inducing advanced atherosclerosis by hepatic PCSK9 overexpression and western diet feeding for 20 wk, Unc5b was deleted and hypercholesterolemia was normalized to simulate clinical lipid management. Deletion of myeloid Unc5b led to a 40% decrease in atherosclerotic plaque burden and reduced plaque complexity compared to Unc5b^fl/flCx3cr1^WT/WT littermate controls (Ctrl^MØ). Consistently, plaque macrophage content was reduced by 50% in ∆Unc5b^MØ mice due to reduced plaque Ly6C^hi monocyte recruitment and macrophage retention. Compared to Ctrl^MØ mice, plaques in ∆Unc5b^MØ mice had reduced necrotic area and fewer apoptotic cells, which correlated with improved efferocytotic capacity by Unc5b-deficient macrophages in vivo and in vitro. Beneficial changes in macrophage dynamics in the plaque upon Unc5b deletion were accompanied by an increase in atheroprotective T cell populations, including T-regulatory and Th2 cells. Our data identify Unc5b in advanced atherosclerosis as a therapeutic target to induce pro-resolving restructuring of the plaque immune cells and to promote atherosclerosis regression.

Keywords: Treg; atherosclerosis; efferocytosis; inflammation; regression.

Fig. 1.

Myeloid Unc5b expression impairs atherosclerosis regression. (A and B) Uniform Manifold Approximation and Projection visualization of scRNA-seq of CD45⁺ cells isolated from the aortic arch of male Ldlr^–/– mice fed a WD for 16 wk (A), with dot plot showing Unc5b normalized gene expression (B). DN, double negative; DP, double positive; NK, Natural Killer; n = 13,397 cells. (C) Violin plot showing normalized Unc5b expression in Unc5b⁺ myeloid cells at baseline and 4 wk after mice were switched to chow diet. (D) Pathway enrichment analysis of genes differentially expressed in Unc5b⁺ and Unc5b^– myeloid cells. (E) Schematic of interventional study design in mice; Unc5b^fl/fl-Cx3cr1^WT/WT(Ctrl^BL) and Unc5b^fl/fl-Cx3cr1^CreERT2/WT (Unc5b^BL) mice were injected with PCSK9-AAV and fed western diet for 20 wk to establish atherosclerosis (baseline), and then injected with tamoxifen and put on chow diet for 4 wk (TAM + chow) to induce Unc5b deletion (∆Unc5b^MØ) or not (Ctrl^MØ). (F) Unc5b mRNA levels in thioglycolate-elicited peritoneal macrophages (Left, pMØ) and bone marrow–derived macrophages (Right; BMDM) isolated from ∆Unc5b^MØ and Ctrl^MØ mice (n = 3 per group). (G) Representative hematoxylin and eosin (H&E) staining of aortic root plaques (outlined in dashed-line; Scale bar, 200 µm), and quantification of plaque area along the aortic root of mice at baseline and after TAM + chow diet. (H and I) Representative images of aortas En Face (H), and quantification (I) of plaque area in the whole aorta, aortic arch, and abdominal aorta of mice at baseline and after TAM + chow diet. Scale bar, 2 mm. Data are mean ± SEM. P-values by Student’s T-test (C and F) or one-way ANOVA with Tukey’s multiple comparison test (G and I).

Fig. 2.

Macrophage-specific deletion of Unc5b reduces plaque macrophage and lipid content. (A) Representative immunofluorescence images of aortic root plaque of Ctrl^MØ and ∆Unc5b^MØ mice at baseline and after TAM + chow diet costained for macrophages (CD68; red) and neutral lipid content (BODIPY, green), SMA (ACTA2, white). White dashed-line indicates plaque area and yellow dashed-line outlines plaque 40 µm-subendothelial space (Scale bar, 200 µm.) (B–F) Quantification of CD68⁺ (B); BODIPY⁺ (C); total ACTA2⁺ (D); fibrous cap ACTA2⁺ (E); and collagen (F) content of plaques shown in A. Data are mean ± SEM. P-values by one-way ANOVA with Tukey’s multiple comparison test (B, C, E, and F) or Kruskal–Wallis test with Dunn’s multiple comparison test (D).

Fig. 3.

Unc5b controls macrophage dynamics within the plaque. (A and B) Quantification EdU⁺ Ly6C^hi monocyte recruitment (A) and green bead-labeled Ly6C^lo monocyte recruitment (B) in the aortic root plaques of Ctrl^MØ and ∆Unc5b^MØ mice at baseline and after TAM + chow diet intervention. (C) Flow cytometric quantification of Ly6C^hi (Left) and Ly6C^lo (Right) monocyte accumulation in aortic arch plaques of Ctrl^MØ and ∆Unc5b^MØ mice at baseline and postintervention. (D) Representative immunofluorescence staining and quantification of Ki67⁺CD68⁺ proliferating macrophages (Ki67, green; CD68, red) in aortic root plaques. Arrows indicate colocalization and white dashed-line indicates lesion border. Scale bar, 100 µm, Inset Scale bar, 20 µm. (E) Quantification of red bead-labeled macrophages retained in aortic root plaques of Ctrl^MØ and ∆Unc5b^MØ mice at baseline and postintervention. (F) Quantification of TUNEL⁺ cells in aortic root plaques of of Ctrl^MØ and ∆Unc5b^MØ mice at baseline and postintervention. Data are mean ± SEM. P-values by one-way ANOVA with Tukey’s multiple comparison test (A–D and F) or Kruskal–Wallis test with Dunn’s multiple comparison test (E).

Fig. 4.

Deletion of Unc5b increases macrophage efferocytosis and reduces plaque complexity. (A) Quantification of the ratio of phagocytosed-to-free TUNEL⁺ apoptotic cells (AC) in aortic root plaques of Ctrl^MØ and ∆Unc5b^MØ mice at baseline and after tamoxifen + chow diet intervention. (B) Representative imaging of latex bead engulfment and quantification of the number of beads engulfed per cell in Unc5b-deleted (∆Unc5b^BMDM) or WT (Ctrl^BMDM) BMDMs. Scale bar, 40 µm. (C) Schematic of experimental design. Cells were pulsed with green-labeled apoptotic Jurkat T cells for 1 h, washed, and incubated for an additional 24 h chase. (D) Representative images and quantification of phagocytosed AC uptake at 1 h and AC-derived fluorescence intensity (relative fluorescence units) remaining at 24 h in ∆Unc5b^BMDM and Ctrl^BMDM. Scale bar, 40 µm. (E) Quantification of plaque necrotic area (outlined in black) in H&E stained aortic root plaques. (F) Histological classification of aortic root plaques according to Stary’s grading (38); II, moderate lesions (foam cells, SMCs, intracellular lipid accumulation); III, preatheroma (foam cells, SMCs, pools of extracellular lipids); IV, atheroma (foam cells, SMCs, large pools of extracellular lipids, necrotic core); V, fibroatheroma (foam cells, SMCs, large pools of extracellular lipids, large irregular necrotic core). (B) Dots are average data per experiment (pool of n = 3); and (D) dots are individual fields of view from three individual experiments (n = 3). Data are mean ± SEM. P-values by one-way ANOVA with Tukey’s multiple comparison test (A and E), two-way ANOVA with Sidak’s (B) or Tukey’s (F) multiple comparison test, or Student’s t test (D).

Fig. 5.

Myeloid Unc5b deletion increases accumulation of pro-resolving T cells in plaques. (A–D) Flow cytometric quantification of aortic arch CD4⁺ T cells (A), T-bet⁺ (Th1) cells (B), GATA3⁺ (Th2) cells (C), and FOXP3⁺ (Treg) cells (D) in Ctrl^MØ and ∆Unc5b^MØ mice at baseline and after TAM + chow diet intervention. (E–G) Representative immunofluorescence staining and quantification of CD3⁺GATA3⁺ Th2 cells (E); and FOXP3⁺ Treg (F and G) in aortic root plaques of Ctrl^MØ and ∆Unc5b^MØ mice at baseline and postintervention. Arrows indicate positive cells and white dashed-line indicates lesion borders. Scale bar, 100 µm, Inset Scale bar, 20 µm. (H–J) Flow cytometric quantification of FOXP3⁺ Treg abundance in the deep cervical and mediastinal lymph nodes (H), intestinal lymph nodes (I), and spleen (J). Data are mean ± SEM. P-values by one-way ANOVA with Tukey’s multiple comparison test (A–I), or Kruskal–Wallis with Dunn’s multiple comparison test (J).