Silencing Myeloid Netrin-1 Induces Inflammation Resolution and Plaque Regression

Abstract

Rationale: Therapeutic efforts to decrease atherosclerotic cardiovascular disease risk have focused largely on reducing atherogenic lipoproteins, yet lipid lowering therapies alone are insufficient to fully regress plaque burden. We postulate that arterial repair requires resolution of a maladaptive immune response, and that targeting factors that hinder inflammation resolution will facilitate plaque regression. Objective: The guidance molecule netrin-1 is secreted by macrophages in atherosclerotic plaques, where it sustains inflammation by enhancing macrophage survival and blocking macrophage emigration. We tested whether silencing netrin-1 in advanced atherosclerosis could resolve arterial inflammation and regress plaques. Methods and Results: To temporally silence netrin-1 in myeloid cells, we generated genetically modified mice in which Ntn1 could be selectively deleted in monocytes and macrophages using a tamoxifen-induced CX3CR1-driven cre-recombinase ( Ntn1 fl/fl Cx3cr1 creERT2+ ) and littermate control mice ( Ntn1 fl/fl Cx3cr1 WT ). Mice were fed western diet in the setting of hepatic PCSK9 overexpression to render them atherosclerotic, and then treated with tamoxifen to initiate deletion of myeloid netrin-1 (Mø ΔNtn1 ) or not in controls (Mø WT ). Morphometric analyses performed 4 weeks later showed that myeloid Ntn1 silencing reduced plaque burden in the aorta (-50%) and plaque complexity in the aortic root. Monocyte-macrophage tracing experiments revealed lower monocyte recruitment, macrophage retention, and proliferation in Mø ΔNtn1 compared to Mø WT plaques, indicating a restructuring of monocyte-macrophage dynamics in the artery wall upon netrin-1 silencing. Single cell RNA-sequencing of aortic immune cells prior to and after netrin-1 silencing revealed upregulation of gene pathways involved in macrophage phagocytosis and migration, including the Ccr7 chemokine receptor signaling pathway required for macrophage emigration from plaques and atherosclerosis regression. Additionally, plaques from Mø ΔNtn1 mice showed hallmarks of inflammation resolution, including higher levels of pro-resolving macrophages, interleukin-10, and efferocytosis, as compared to plaques from Mø WT mice. Conclusions: Our data show that targeting netrin-1 in advanced atherosclerosis ameliorates atherosclerotic inflammation and promotes plaque regression.

The Relationship Between Serum Netrin-1 Expression Levels and Prognosis in Revascularized Patients with Acute Ischemic Stroke

“…However, Rosenberger et al [17] found that netrin-1 was strongly induced during hypoxia and seemed to steadily increase over time. The results are similar to those of various chronic inflammatory models [18]. Therefore, netrin-1 levels might be time de-pendent.…”

Alternative adenosine Receptor activation: The netrin-Adora2b link

“…Subsequent studies suggest that netrin-1 can also function to promote atherosclerosis by entrapping macrophages in plaques (van Gils et al, 2012). In line with these findings, a recent study demonstrates that silencing of netrin-1 in the myeloid lineage promotes the resolution of inflammation and plaque regression (Schlegel et al, 2021). Netrin-1 signaling can also function as a macrophage retention signal for the promotion of chronic inflammation and insulin resistance in adipose tissue (Ramkhelawon et al, 2014) or in the pathogenesis of abdominal aortic aneurism in vascular smooth muscle cells (Hadi et al, 2018).…”

Single-cell RNA sequencing in atherosclerosis: Mechanism and precision medicine

“…Furthermore, cellular communication analysis revealed enhanced interactions between ApoE −/− outer membrane mesenchymal clusters and inflammatory macrophages. Schlegel et al (2021) conducted scRNA-seq on mouse aortic immune cells before and after Netrin-1 (Ntn1) silencing and detected one monocyte population and two macrophage populations (Cx3cr1 − macrophages and TREM2 hi macrophages). Ntn1 is described as an axon guidance molecule, which also plays an important role in atherosclerosis by regulating inflammation and the migration of immune cells such as macrophages (Serafini et al, 1996;van Gils et al, 2012).…”