Elevation of Granulocyte Colony Stimulating Factor in Human AMD Donor RPE-Choroid

PURPOSE . Choroidal inflammation, complement deposition, and accumulation of Creactive protein (CRP) are involved in age-related macular degeneration (AMD) pathology. The pro-inflammatory signals that regulate immune cell recruitment in the choroid of patients with AMD remain to be determined. We performed cytokine profiling of human AMD and age-matched control donor tissue to identify inflammatory molecules upregulated in AMD tissue.<br /> METHODS . Protein was isolated from 25 AMD and 21 control donor RPE/choroid macular punches. Total protein was quantified, and 50 mu g assayed for expression of 40 cytokines using an inflammation array. We validated the elevated expression of granulocyte colony stimulating factor (G-CSF) protein by ELISA in a second cohort of 22 control and 26 AMD donors. To identify an AMD associated stressor responsible for upregulating G-CSF we assayed for changes in G-CSF protein secretion in RPE/choroid organ cultures treated with the monomeric (m)CRP, an inflammatory protein elevated in AMD.<br /> RESULTS . Using a multiplex array, we identified elevated G-CSF protein in the choroid of AMD donors compared to age-matched non-AMD controls. Differential expression of GCSF was confirmed via ELISA in an independent cohort of samples (P = 0.01). The mCRP, which is deposited in AMD choroids, increased G-CSF protein secretion in RPE/choroid organ cultures. Single nuclei RNA sequencing identified choroidal endothelial cells and fibroblasts as the primary cell types responsible for increased G-CSF secretion in response to mCRP. The G-CSF receptor is expressed primarily by choroidal macrophages and dendritic cells and anti-G-CSFR colocalizes with anti-CD45 and anti-CD68 in human donor choroid tissue.<br /> CONCLUSIONS . Elevated G-CSF expression in AMD donor tissue as a result of increased levels of mCRP may be involved in immune cell recruitment in AMD contributing to inflammatory stress in the choroid.