Author’s Disclosure Block: Harshini Chakravarthy, none; Jing Cui, none; David Granville, This author is a Co-Founder and serves as the Chief Scientific Officer for viDA Therapeutics.; Joanne Matsubara, none; Amir Hosseini, none; Cole Wagen, none 

Abstract Body 

Purpose: Age-related macular degeneration (AMD) is a multifactorial neurodegenerative retinal disease, and a leading cause of vision loss in the elderly. The Y402H polymorphism in the Complement Factor H (CFH) gene is recognized as an important risk factor for AMD. We previously showed higher levels of complement activation product C5a in the Bruch’s membrane and choroid of human eyes with the high-risk CFH variant1. We also demonstrated that Granzyme B (GzmB), a serine protease is elevated in human eyes with neovascular AMD2,3. Here, we explore the relationship between CFH gene and GzmB immunoreactivity in choroidal mast cells in genotyped human eyes. Further, we assessed GzmB gene expression in human retinal pigmented epithelial (RPE) cultures stimulated with complement C5a. Study Design:  To study the relationship between CFH genotype and GzmB expression in choroidal mast cells, we used formalin-fixed, paraffin-embedded 6 µm cross-sections of human donor eyes genotyped for protective variant or high-risk variant of CFH gene (N=5, each group). To explore the effects of complement C5a on GzmB gene expression, adult human primary RPE cultures or ARPE-19 cell line were grown to confluency in 12-well plates or chamber slides and stimulated with 0.1-0.5 µg/mL of recombinant human C5a for 24-48 hours (N=3). Methods: In donor eye sections, expression levels of tryptase (mast cell marker) and GzmB were determined by double immunofluorescence labeling. Images were captured using a Zeiss LSM 800 Zen 3.7 Blue confocal microscope. Tryptase and GzmB-labelled cells were quantified per field for 8-10 f ields at 20x magnification. RPE cultures were stimulated with C5a, mRNA was extracted, converted to cDNA and GzmB gene expression was studied by RT-PCR using gene-specific primers. RPE cells in chamber slides were also immunostained for GzmB, and quantified using Image J. Results: A significant increase in the total number of mast cells (p<0.05), and GzmB-expressing mast cells (p<0.05) was observed in the inner choroid of eyes with high-risk CFH gene variant. A trend of increased degranulating mast cells and GzmB-expressing RPE in high-risk donor eyes was also observed. C5a stimulation of RPE cells resulted in elevated GzmB gene expression at both mRNA and protein levels. Conclusions: Our data shows a significant increase in infiltration of GzmB+ mast cells in the inner choroid of human eyes with high genetic risk of AMD. Further, complement activation product C5a stimulates GzmB gene expression in cultured RPE cells. These results suggest that genetic risk at the CFH gene may trigger early events in AMD pathogenesis4, with a key role for extracellular GzmB.