Macrophage-specific deletion of neuropilin-2 inhibits inflammatory signalling and attenuates atherosclerotic plaque development in apolipoprotein e-deficient mice

Abstract Background Atherosclerosis is the underlying pathology in most cases of ischaemic heart disease, which itself remains the leading cause of mortality worldwide. Atherosclerosis is a multifaceted vascular disease characterised by arterial wall inflammation resulting from the subendothelial retention, and modification, of lipoproteins by macrophages. Macrophages exhibit a high degree of plasticity, and undergo polarisation to a pro-inflammatory phenotype in response to pro-inflammatory cytokines and other environmental stimuli, making them an attractive therapeutic target. Neuropilin-2 (NRP2) is a cell-surface co-receptor with essential roles in angiogenesis and axonal guidance, and is also expressed by macrophages. However, little is known about the role of NRP2 in macrophage function or atherosclerosis. Purpose To investigate whether NRP2 is important for macrophage involvement in atherosclerosis, we characterised the effects of conditional NRP2 deletion on macrophage function and atherosclerotic plaque development. Methods We generated mice with macrophage-specific (LysM-Cre) deletion of Nrp2, and lineage tracing via EYFP transgene expression, on the pro-atherogenic Apolipoprotein E-deficient background (Nrp2-KOMac, Apoe−/−, EYFP). These mice were fed a high-fat diet (HFD) for 16 weeks before their aortas were stained with Oil-red-O to assess plaque coverage. Plaque content was then characterised by sectioning and staining the aortic roots. The role of NRP2 in modulating pro-inflammatory macrophage polarisation and signalling, was assessed using functional assays and transcriptome analysis in Nrp2-KO bone marrow-derived macrophages (BMDMs). Results HFD-induced plaque development was significantly reduced in Nrp2-KOMac, Apoe−/−, EYFP mice, with 17.4% total aortic plaque coverage in Nrp2-KOMac, Apoe−/−, EYFP mice, compared to 24.9% in WTApoe−/−, EYFP mice (p=0.0021, n=10). Plaques from Nrp2-KOMac, Apoe−/−, EYFP mice also displayed features consistent with increased plaque stability, including reduced necrotic core area and plaque lipid content, and increased cap thickness. NRP2 was significantly upregulated upon M-CSF-mediated differentiation of bone marrow progenitors into BMDMs, and further upregulated by pro-inflammatory polarisation using LPS and IFN-gamma. Furthermore, upregulation of Il1β, Tnfα and Il6, induced by LPS and IFN-gamma, was significantly reduced in Nrp2-KO BMDMs, and MCP-1 induced migration was also decreased in these cells. Transcriptome analysis revealed that NF-kappaB signalling pathway genes, and genes regulating monocyte chemotaxis, were downregulated in Nrp2-KO BMDMs. Conclusion Macrophage-derived NRP2 is pro-atherogenic, likely resulting from its role in positively regulating pro-inflammatory signalling and macrophage migration. Targeting NRP2 expressed on the surface of macrophages could therefore offer a novel therapeutic approach for reducing the disease burden associated with atherosclerosis. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation (MB PhD Studentship)