Immunohistochemical Assessment of Proteins Associated with Complement Activation and Inflammation in Human Maculas with Homozygous Risk of AMD at Chromosome 1

Abstract

Purpose: To determine the effects of chromosome 1 genotype and cigarette smoking on levels of complement activation and inflammation in the human macula.

Methods: Eye tissue was obtained from consenting human donors. Donors were stratified into two groups based on diplotype at the AMD-associated CFH/CFHR locus: (i) homozygous risk diplotype (tagged by CC at rs1061170) (n=9, ages 56-78 years) and (ii) homozygous ‘protective’ diplotype (tagged by AA at rs1410996) (n=6, 61-78 years). Importantly, all donors were homozygous non-risk at the ARMS2/HTRA1 locus on chromosome 10. Immunohistochemistry was performed on macular tissue from all donors, using 26 antibodies against markers of inflammation, complement regulation and activation, followed by confocal microscopy and immunofluorescence quantification (all masked to donor status).

Results: Donors homozygous risk at the CFH/CFHR locus exhibited significantly higher levels of the complement membrane attack complex (MAC) in macular retinal pigment epithelium (RPE; p=0.003), Bruch’s membrane (BM; p=0.002), choriocapillaris (CC; p=0.001), CC intercapillary septa (IS; p=0.0001) and choroidal stroma (CS; p=0.001) compared to homozygous protected donors. Smoking was also associated with increased levels of MAC (e.g. p=0.006 in CC IS) and CRP (e.g. p=0.0002 in CC), but only in the risk group. Smoking, but not diplotype, was associated with higher levels of the oxidative stress marker 4-hydroxynonenal in macular retina (p=0.02) and RPE (p=0.05).

Conclusions: Genetic risk at the CFH/CFHR locus (without risk at the ARMS2/HTRA1 locus) is associated with higher levels of complement- and inflammation-associated proteins at the RPE-choroid interface. Levels of these proteins are higher yet in CFH/CFHR risk donors with histories of cigarette smoking, which is also associated with increased oxidative stress in both risk and non-risk donors. Examination of human macular tissue from donors with ‘pure’ diplotypes allows assessment of AMD-associated pathways that are driven solely by the CFH/CFHR gene locus. These findings have important implications related to the identification of chromosome 1-directed pathways and therapeutic targets.