Inflammatory cytokine profiling identifies elevated G-CSF protein in human age-related macular degeneration (AMD) donor choroid.

Abstract

Purpose : Complement deposition, inflammation and alternations in systemic cytokines are involved in AMD pathology. The inflammatory signals regulating immune cell recruitment and activation in the choroid of AMD patients remains to be determined. We performed cytokine profiling on clinically phenotyped human donor tissue to identify inflammatory molecules upregulated in AMD donor RPE/choroid tissue.

Methods : Protein was isolated from AMD and control donor 8 mm RPE/choroid macular punches (n>20 per group), obtained within 8 hours of death. Protein concentration was quantified (Bio-Rad DC assay) and 50 µg total protein was assayed for expression of 40 cytokines using the human inflammation Quantibody array (Raybiotech QAH-INF-3). To investigate the cellular source of Granulocyte colony stimulating factor (GCSF), the most upregulated cytokine in AMD, we assayed for changes in CSF3 gene expression in human RPE-choroid organ cultures (treated with monomeric CRP for 12 hours) by RNA-Seq and human choroidal endothelial cells (treated with normal human serum (NHS) or heat inactivated NHS for 4 hours to activate the complement MAC followed by RNA isolation and qPCR for CSF3). Human donor tissue sections were labeled by immunohistochemistry for the GCSF receptor.

Results : GCSF is elevated in AMD donors compared to age matched controls (Figure 1). No effect of Y402H CFH genotype was observed on G-CSF levels. We assayed whether pro-inflammatory CRP or complement activation, two pathological features of AMD, alter CSF3 expression. Human donor RPE/choroid organ cultures treated with mCRP for 12 hours have 13-fold increased CSF3 expression (p=0.0016) compared to controls. In vitro, choroidal endothelial cells injured with complement membrane attack complex have 2-fold increased CSF3 expression compared to control stimulated cells (p=0.02). The G-CSF receptor is expressed primarily by inflammatory choroidal macrophages (singlecell-eye.org) and anti-G-CSFR colocalizes with anti-CD45 by immunofluorescence in human donor choroid tissue.

Conclusions : Increased G-CSF expression in AMD donor choroid endothelial cells as a result of complement or pro-inflammatory CRP may be involved in immune cell recruitment and activation in AMD, contributing to the inflammatory stress of the choroid early in AMD progression.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1. Protein levels of G-CSF in 23 control and 25 AMD donor macular RPE/choroid.