Neuropilin-1 Deficiency on Myeloid Cells Promotes a Pro-inflammatory Phenotype while Inhibiting Choroidal Neovascularization.

Abstract

Purpose : Neuropilin-1 (NRP1) is a receptor that binds diverse ligands and influences cell signalling. A genetic variation in NRP1 is associated with decreased response to ranibizumab, suggesting that modulation of NRP1 function has clinical potential to treat the neovascular (NV) disease wet age-related macular degeneration (wAMD). Accumulation of myeloid cells that express high levels of NRP1 at sites of choroidal neovascularization (CNV) prompted us to determine the function of myeloid-resident NRP1 in the pathogenesis of wAMD.

Methods : We ablated NRP1 in myeloid cells by crossing LysMcre mice with Nrp1 floxed mice. LysMCreNrp1^+/+ (Nrp1^+/+), LysMCreNrp1^fl/fl (Nrp1^fl/fl) and C57BL/6J WT (B6) mice were subjected to laser-induced CNV. CNV and mononuclear phagocyte (MP) aggregation were evaluated ex vivo in flat-mounted sclera-choroid-RPE cell complexes (SCRC). qPCR served to analyze inflammatory profiles (Il1β, Il6 and Tnfα) and macrophage (mφ) polarization markers (iNos, Cxcl11, Il10, Arg1, Cd163 and Cd206) in SCRC. Peritoneal mφ (pmφ) from Nrp1^+/+, Nrp1^fl/fl and B6 mice were stimulated in vitro with LPS or Il4 and mφ polarization was investigated. Transcriptomic analysis by RNA sequencing (RNAseq) and gene set enrichment analysis (GSEA) of Nrp1^fl/fl pmφs was performed. Finally, an NRP-based trap was administered intravitreally and efficacy monitored for CNV.

Results : CNV was decreased by 30% in Nrp1^fl/fl mice compared to control, while the number of MPs was similar in both groups. Remarkably, lasered choroids from Nrp1^fl/fl mice had a significantly increased inflammatory profile compared to controls, while mφ polarization markers indicated a pro-inflammatory phenotype. Cultured pmφs showed a bias towards pro-inflammatory phenotype in the Nrp1^fl/fl mice compared to control after stimulation with LPS or Il4. Transcriptomic analysis by RNAseq and GSEA revealed a decrease in alternative mφ activation-related genes compared to Wildtype, suggesting that the presence of NRP1 tempers their pro-inflammatory potential. Intravitreal injection of an NRP-based recombinant trap reduced CNV by ~40%.

Conclusions : We demonstrate that myeloid-resident NRP1 significantly influences pathological CNV. Ablation of NRP1 from myeloid cells promotes a pro-inflammatory phenotype yet decreases the formation of CNV. NRP1-derived Traps may be promising therapeutic entities for wAMD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.