Characterization of Cytokine Gene Expression Associated With Non-Infectious HIV Retinopathy in Human Autopsy Eyes

Abstract

Purpose: : To determine cytokine-related pathogenesis of human immunodeficiency virus (HIV) retinopathy in human autopsy eyes.

Methods: : Fresh autopsy eyes (<24h) were procured from clinically diagnosed AIDS patients who died as a result of disease related complications. The globes were immediately placed in 50 mL of RNA later. The anterior segment and remaining vitreous were removed and the retina and posterior segments were flattened. PCR clean 2mm trephines were used to punch individual pathologic retina in areas of cotton wool spots (CWS) or intraretinal hemorrhages as well as control punches from six areas of the posterior pole and peripheral retina. The retinal biopsy tissues were placed into 1mL of TRIzol solution and frozen. Primer/probe sequences were designed using Primer Express 1.5 software. Total RNA was extracted using the TRIzol extraction protocol. 1µL of total RNA was directly added to the spectrophotometer and the optimal density of the RNA at OD260nm was measured. For quantitative real-time RT-PCR reactions (TaqMan), equal amounts of cDNA were run in duplicate and amplified in a 25µL reaction containing 12.5µL of 2X universal PCR master mix, 7.5µL of primers/probe mix (900nM/900nM/200nM final concentration), and 5µL of cDNA template (25ng) (qRT-PCR machine: ABI Prism 7700 Sequence Detection System).Amplification efficiencies were validated and samples were normalized to human GAPDH. Delta Ct values were calculated using the comparative Ct analysis method. The results are expressed as a mean fold modulation controlling for retinal areas without a lesion in the same eye.

Results: : Tissues from areas of CWS had a marked elevation in fold modulation of the following cytokines: IL-8 (20.8x), RANTES (7.9x), MIP1-b (4.4x), IL-10 (3.9x) and MIP1-a (3.6x). IL-4 and BAX-a were not noticeably expressed in CWS. Tissues from areas of intraretinal hemorrhages had a marked elevation of the following cytokines: RANTES (11x), IL-8 (3.8x), IL-10 (2.6x); IL-4, BAX-a, MIP1-a and MIP1-b were not upregulated in intraretinal hemorrhages.

Conclusions: : Certain inflammatory chemokines are expressed in retinal lesions in eyes with HIV retinopathy; these are primarily cytokines elevated by HIV infection. We did not find an increased expression of apoptotic chemokines, nor did we observe an elevation in the chemokines present in intraocular lymphoproliferative diseases.

Keywords: AIDS/HIV • cytokines/chemokines • inflammation