Plasmacytoid Dendritic Cells Maintain Corneal Heme-Angiogenic Privilege Through Secretion of Anti-Angiogenic Molecules

Abstract

Purpose : Resident plasmacytoid dendritic cells (pDCs) have been recently identified by our group to accompany limbal vessels under homeostatic conditions. Although the role of various immune cells in the induction of heme-angiogenesis is well studied, the significance of pDCs remains to be elucidated. The aim of this study is to unravel the role of pDCs in preserving corneal heme-angiogenic privilege during steady state as well as following angiogenic stimuli.

Methods : Corneal pDCs were locally depleted by subconjunctival injections of 30ng Diphtheria toxin (DT) in 6-8 week male BDCA2-DTR mice. Wild-type C57BL/6 mice receiving DT and BDCA2-DTR mice treated with PBS served as controls. Corneal neovascularization (NV) was evaluated via immunofluorescent staining of corneal whole-mounts with CD31 (pan-endothelial marker) followed by confocal microscopy. NV area was measured by ImageJ. Relative mRNA levels of Endostatin and Thrombospondin-1 in the corneal stroma were assessed via real-time PCR. Naïve and sutured corneas underwent flow cytometry for CD45 (pan-leukocyte marker), Siglec-H, PDCA-1, B220 (pDC markers), Endostatin, and Thrombospondin-1. ANOVA with Scheffe post hoc test was used to assess statistical significance.

Results : On day 7 following local depletion of pDCs, corneal angiogenic privilege was lost, demonstrating NV in pDC-depleted corneas (NV area=32.0±3.1%) in comparison with PBS (12.5±1.9%) and DT treated controls (12.7±1.3%; p<0.001). In order to assess if pDC repopulation may lead to regression of NV, mice were kept for 14 days after initial 7-day depletion. Upon pDC repopulation, NV was reduced to 23.3±0.9% (p=0.02). Similarly, we observed greater NV on day 7 after suture placement in pDC-depleted corneas (84.7±10.3%) versus PBS (32.3±2.1%) and DT treated controls (29.7±5%; p=0.001). Relative Endostatin and Thrombospondin-1 mRNA levels were decreased to 45.2% and 56.6% of controls on day 7 and to 36.7% and 54.9% of controls on day 14 after pDC depletion (p<0.01). Flow cytometry showed that Endostatin and Thrombospondin-1 co-stained with pDCs in both naïve and suture-induced inflamed corneal single cell suspensions.

Conclusions : Resident corneal pDCs harbor anti-hemeangiogenic properties through secretion of Endostatin and Thrombospondin-1 and are therefore crucial for the maintenance of corneal heme-angiogenic privilege in steady state and upon angiogenic stimuli.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.