Ocular Microbiology and Immunology Group
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2016 OMIG Abstract 16

Human Cornea Hosts Resident Plasmacytoid Dendritic Cells That May Mediate Corneal Angiogenic Privilege
Arsia Jamali, Maria J Lopez, Victor G Sendra, Deshea L Harris, Pedram Hamrah
Center for Translational Ocular Immunology, Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine Boston, MA, Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA

Purpose: To study the presence and functions of plasmacytoid dendritic cells (pDCs) in preserving corneal heme-angiogenic privilege.

Methods: Initially, murine corneal pDCs were locally depleted by subconjunctival injections of 30ng Diphtheria toxin (DT) in 6-8 weeks male BDCA2-DTR mice. Wild-type C57BL/6 mice receiving DT and BDCA2-DTR mice treated with PBS were controls. Corneal neovascularization (NV) was assessed on confocal micrographs of corneal whole-mounts stained with CD31 (pan-endothelial marker) using ImageJ. Relative mRNA levels of Endostatin and Thrombospondin-1 (TSP-1) in the corneal stroma were measured via real-time PCR. Naïve and sutured corneas underwent flow cytometry for CD45 (pan-leukocyte marker), Siglec-H, PDCA-1, B220 (3 murine pDC markers), Endostatin, and TSP-1. Next, human corneas underwent flow cytometry for CD45, BDCA2, and BDCA4 (two human pDC markers) to assess presence of pDCs in human corneas. ANOVA with Scheffe post hoc test was used to assess statistical significance.

Results: Seven days following local pDC depletion, 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). To assess if pDC repopulation may lead to regression of NV, mice were assessed 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). Endostatin and TSP-1 mRNA levels were decreased to 45.2% and 56.6% of controls 7 days after pDC depletion (p<0.01). Murine corneal pDCs were co-stained with Endostatin and TSP-1. Novel population of resident pDCs were identified in normal human corneas.

Conclusions: Resident corneal pDCs are present in human and murine corneas and mediate anti-angiogenic properties through secretion of Endostatin and TSP-1 and are therefore crucial for the maintenance of corneal angiogenic privilege.

S: NIH K08-EY020575 (PH), NIH-R01-EY022695 (PH), Research to Prevent Blindness Career Development Award (PH), Falk Medical Research Trust (PH).

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