Assessment of Proteins Associated With Complement Activation and Inflammation in Maculae of Human Donors Homozygous
Tiarnan D L Keenan1, Marc Toso2, Chris Pappas2
1Center for Translational Medicine John A. Moran Eye Center, Department of Ophthalmology and Visual Sciences, University of Utah, Salt Lake City, Utah, United States 2Centre for Ophthalmology & Vision Science, Institute of Human Development, Faculty of Med.
Insights
Genetic risk at the CFH-to-F13B locus and smoking increase complement activation in the macula. Smoking also elevates C-reactive protein (CRP) and oxidative stress, highlighting key pathways for AMD therapies.
Area of Science:
- Ophthalmology
- Immunology
- Genetics
Background:
- Age-related macular degeneration (AMD) is a leading cause of vision loss.
- Genetic and environmental factors, including smoking, influence AMD pathogenesis.
- The complement system and inflammation play critical roles in AMD.
Purpose of the Study:
- To investigate the impact of chromosome 1 genotype at the CFH-to-F13B locus and cigarette smoking on macular complement activation and inflammation.
- To examine AMD-associated pathways driven solely by the CFH-to-F13B locus.
Main Methods:
- Analysis of human macular tissue from donors stratified by CFH-to-F13B diplotype (risk, neutral, protective) and ARMS2/HTRA1 genotype (nonrisk).
- Immunohistochemistry using 14 antibodies targeting complement and inflammation markers.
- Confocal microscopy and immunofluorescence quantification to assess protein levels.
Main Results:
- Homozygous risk genotype at CFH-to-F13B was linked to significantly higher terminal complement complex (TCC) levels in the macula.
- Cigarette smoking correlated with increased TCC, C-reactive protein (CRP), and oxidative stress markers in macular tissue.
- Elevated CRP levels were particularly noted in risk donors with a smoking history.
Conclusions:
- Genetic predisposition at the CFH-to-F13B locus and cigarette smoking both contribute to increased complement activation in the human macula.
- These findings identify specific chromosome 1-directed pathways implicated in AMD.
- The study highlights potential therapeutic targets for AMD by examining the interplay of genetic risk and smoking.
Purpose:
To determine the effects of chromosome 1 genotype and cigarette smoking on levels of complement activation and inflammation in the human macula.
Methods:
Donor macular tissue was stratified into three groups by diplotype at the AMD-associated CFH-to-F13B locus: homozygous "risk" (n = 9, 56-78 years), homozygous neutral (n = 2, 64-79 years), and homozygous "protective" (n = 6, 61-78 years) diplotype. Importantly, all donors were homozygous nonrisk at the ARMS2/HTRA1 locus, so that purely chromosome 1-directed pathways were examined. Immunohistochemistry was performed by using 14 antibodies, mostly against markers of complement and inflammation, followed by confocal microscopy and immunofluorescence quantification (all masked to donor status).
Results:
Donors homozygous risk at CFH-to-F13B exhibited significantly higher levels of terminal complement complex (TCC) in macular Bruch's membrane (BM; P = 0.03), choriocapillaris (CC; P = 0.04), and choriocapillaris intercapillary septa (CC IS; P = 0.03), compared to homozygous protected donors. Smoking was associated with increased TCC in BM (P = 0.05), CC IS (P = 0.03), and choroidal stroma (CS; P = 0.01), and with substantially elevated C-reactive protein (CRP) levels in RPE (P = 0.04), BM (P = 0.01), CC (P = 0.05), and CS (P = 0.05). Smoking was associated with higher levels of oxidative stress in macular RPE (P = 0.04) and CS (P = 0.01).
Conclusions:
Genetic risk at the CFH-to-F13B locus was associated with higher levels of complement activation at the human macular RPE-choroid interface, as was cigarette smoking. Levels of CRP were substantially elevated in risk donors with smoking history. Examination of human macular tissue from donors with "pure" diplotypes allows assessment of AMD-associated pathways driven solely by CFH-to-F13B. These findings have important implications for identifying chromosome 1-directed pathways and therapeutic targets.
